Academia de Ciencias de la URSS

Instituto de Microbiología

N. A. KRASIL'NIKOV

EL SUELO
LOS MICROORGANISMOS
Y
LAS PLANTAS SUPERIORES

Publicado por la Academia de Ciencias de la URSS

Moscú 1958
Editado para:
LA FUNDACIÓN DE LA CIENCIA NACIONAL, WASHINGTON, D.C., y LA
SECCIÓN DE AGRICULTURA, EE.UU. por EL PROGRAMA DE ISRAEL PARA
LAS TRADUCCIONES CIENTÍFICAS en 1961

El título de Original ruso: El pochvy de Mikroorganizmy el i vysshie rasteniva

Traducido por: Dr. Y. Halperin
Impreso en Jerusalén por: S. MONSON

Prologo de la edicion original :

Este libro se consagra al estudio del problema de la interacción entre los

microorganismos del suelo y las plantas superiores. El material presentado
incluye información básica sobre la estructura, el desarrollo, la variabilidad y
clasificación de las bacterias, actinomicetes y los hongos a la luz de los
descubrimientos científicos recientes. Tambien presenta información sobre la
importancia de los microorganismos en la nutrición de las plantas, el papel de
la actividad microbiana en la nutrición complementaria de las plantas, el efecto
de microbios en la satisfacción de las demandas nutritivas de las especies
superiores, su importancia en el desarrollo de dichas especies y su influencia
en la fertilidad del suelo.
Además, se suministra información sobre la importancia de los antibióticos
como un medio de terapia y prevención de enfermedades en la práctica
agrícola.

Este libro se diseñó para el uso de microbiologos, fisiólogos de los vegetales,

especialistas del suelo, fitopatólogos, micólogos, agrobiólogos y agrónomos.
También puede servir como libro de texto para estudiantes en las facultades de
Biológia general o biología agrícolas de universidades o institutos de
Silvicultura
INTRODUCCION

Nota de la traducción : este libro fue editado en 1958. Las repetidas referencias temporales deben
considerarse referidas a esa fecha de escritura de este libro.

La base científica de la ciencia del suelo como ciencia natural, fue establecido
por los trabajos clásicos de Dokuchaev. Previamente, el suelo se había
considerado como un producto de las transformaciones fisicoquímicas de las
rocas. Un substrato muerto del que las plantas obtienen los elementos
minerales para su nutrición. El suelo y sustrato se igualaban en los hechos.

Dokuchaev considera al suelo como un elemento natural que tiene su propia

génesis e historia de desarrollo. Como un cuerpo en el que tienen lugar
complejos y multiples procesos. Asi el suelo se considera como diferente del
lecho de roca o sustrato. Este último se vuelve suelo o tierra, bajo la influencia
de una serie de factores creadores del suelo : el clima, la vegetación, la
geografía, el sustrato y la edad. Según él, la tierra debe llamarse el "diario de
hoy" u horizonte exterior de las rocas, mas alla del tipo que sean ellas. Y las
rocas cambian naturalmente por el efecto conjunto del agua, aire y diversos
tipos de organismos vivos y muertos.

El excelente especialista de la ciencia del suelo, P.A. Kostychev (1892), al

desarrollar la teoría del mismo, concede una importancia especial a los factores
biológicos. Él considera la tierra como un botánico y no como un geólogo. En el
estudio del origen de los “suelos negros” o chernozem, Kostychev atribuye un
papel esencial a las plantas y a los microbios. Él escribe que en esa formación
del “suelo negro” no solo está involucrada la geografía y fisiología de plantas
más evolucionadas sino tambien de las inferiores que realizan la
descomposición de la materia orgánica.

La acumulación del humus del suelo depende de la intensidad e integridad de

la descomposición de los residuos de las plantas, de las raíces y de las partes
que estan sobre el suelo.
En estos procesos Kostychev identifica a las criaturas microscópicas, a los
hongos y a las bacterias, con el rol más importante de todos. Siendo un
excelente microbiólogo llevó a cabo interesantes experimentos en la
descomposición de la materia orgánica y la formación de humus. Dichos
experimentos mostraron que, en distintos casos, la descomposición de los
residuos de las plantas empezó de una manera diferente. A veces las bacterias
habitan primero la materia en descomposición y otras veces los hongos surgen
primero.
Las diversas partes de una misma materia en degradacion se descomponen
diferentemente, en una parte un tipo de organismo se multiplica y cerca de alli
puede aparecer un organismo completamente diferente. Se concluye,
consecuentemente, que las distintas formas de descomposición cambian,
según los cambios en las propiedades de la substancia en descomposición.

Kostychev (1889) por primera vez estableció que el humus está formado por
hongos del suelo. Asimismo debe serle atribuido el descubrimiento de la
regularidad de la relación entre carbón y nitrógeno (C:N) en el suelo y de su
importancia en el desarrollo de plantas y microbios. Kostychev descubrio la
esencia del enriquecimiento del humus por el nitrógeno. A lo largo del proceos
de descomposicón de los residuos de una planta, que sabemos contiene no
más del 1,5 al 2 % de N, se obtiene humus con el 4 a 5 % de nitrógeno. Esta
transformación de la materia orgánica que surge de la observación de los datos
de Kostychev, ocurre con la ayuda de microorganismos (Kostychev, 1951).

Con sus trabajos Kostychev creo los cimientos de la microbiólogia de suelos.

Tambien delineo un vasto programa de investigación, cuyos problemas estan
siendo resueltos actualmente por los microbiólogos sovieticos. Vernadskii
(1927), expuso la teoria de los cuerpos naturales “bio-inertes”’. El postulo que
el suelo en su totalidad , es un cuerpo “bio-inerte”.
Todas las propiedades fisicoquímicas del suelo aparecerían
considerablemente diferentes, si no tomaramos en cuenta la sustancia viva
presente en él. En este sentido Vernadskii planteo la interdependencia entre las
propiedades fundamentales del suelo y los organismos presentes en el.
Vil'yams (cientifico del suelo ruso (1863-1939) cuyo nombre se escribe asi al
traducirse del ruso, aunque puede aparecer como Williams en publicaciones
extranjeras), al desarrollar las enseñanzas de Dokuchaev and Kostychev,
introdujo muchos nuevos elementos importantes de la ciencia del suelo.
El considero al suelo como un cuerpo natural y fundamento de la producción
agricola. Y le atribuyo al mismo la nueva cualidad productiva llamada fertilidad :
la habilidad de producir especies cosechables.
En su criterio, la noción de suelo y su fertilidad eran inseparables. Consideraba
a la fertilidad como una propiedad esencial, un indicador de calidad del suelo,
independientemente de la expresion cuantitativa de ella. O sea que oponemos
la idea de suelo fertil a la idea de piedra esteril, otra denominación de la roca
grosera (Vil'yams, 1949).

Sin embargo, debemos señalar que la roca posee algun tipo de fertilidad.
Existen investigaciones que demuestran que hasta la roca mas dura esta
poblada de algunos organismos. Se desarrollan líquenes en sus superficies, y
cubren grandes areas en las cumbres de las montañas. La capa superior de los
macizos rocosos, llamada corteza de erosión, esta saturada de bacterias y
algas, asimismo como hongos, actinomicetes, protozoarios y otros organismos.
De acuerdo a nuestros datos, hay en la capa superior de las rocas basálticas,
entre miles y millones de bacterias por grano de sustrato. Dato corroborado por
otros investigadores (Novogrudskii, 1950; Glazovskaya, 1950; Parfenova, 1955;
Krasil'nikov, 1949 and others).

Hay diversidad en la fertilidad de las rocas. Algunas estan densamente

pobladas por líquenes y microorganismos, mientras que otras lo estan solo
parcialmente. Se trata de rocas, o para ser mas preciso, partes de la misma
roca, que no estan pobladas por líquenes aunque contienen solamente algunas
formas microbiológicas : bacterias, actinomicetes y hongos (Krasil'nikov,
1949b).
Incluso bajo condiciones articas, en las islas del oceano polar Artico (Franz
Josef Land, Novaya Zemlya, Severnaya Zemlya y otras areas) las rocas y los
suelos calcareos sueltos contienen un considerable numero de
microorganismos. Se cuentan de diez mil a cientos de millones por gramo. Mas
aún, estos organismos viven activamente y desarrollan transformaciones
químicas y bioquímicas (Krasil'nikov and Artarnonova, 1958).

Para estudiar las propiedades que determinan las capacidad de producción de

cosechas de un suelo, y aumentar su fertilidad mediante nuestra influencia en
el mismo; " lo primero que se necesita es conocer estas propiedades,
enumerarlas, y elegir, del abanico de propiedades y cualidades de los suelos,
justo aquellas que determinan la capacidad del suelo de producir los productos
necesarios para la humanidad " (Vil'yams, 1949, p 138).
Por lo tanto, Vil'yams relaciona en forma inseparable, la teoria del suelo con la
teoria de la fertilidad y su capacidad de producción.

La propiedad principal del suelo esta determinada por factores biológicos,

fundamentalmente por los microorganismos. El desarrollo de la vida en el
suelo, conduce a la fertilidad. "La noción del suelo esta inseparablemente
ligada a la noción del desarrollo en él, de organismos vivos ". Los
microorganismos crean el suelo. "Cuando muere la vida o esta se detiene, el
antiguo suelo se convierte en un objeto de la geologia" (Vi'lyams, 1950, p 204).
Kostychev and Vil'yams transfirieron a la ciencia del suelo de un capitulo de la
geología, a un capitulo de la biología.

El conocimiento nuevo de la esencia biológica del proceso de genesis del

suelo, que fue establecido por Kostychev and Vil'yams, le ha dado a la mayoria
de los microbiólogos sovieticos un faro en la ruta para sus investigaciones.
La microbiología tambien ha contribuido considerablemente al desarrollo de
esta nueva orientación en la ciencia del suelo, y ha resuelto satisfactoriamente
muchas de las preguntas sobre fertilidad del suelo. Durante las dos ultimas
decadas, la microbiología ha demostrado que los procesos vitales que tienen
lugar en el suelo, son mucho mas importantes y complejos de lo que se habia
supuesto. Donde anteriormente se consideraba que habia cientos de miles,
incluso millones de celulas microbianas por gramo de suelo, hoy, gracias a
mejores metodos de investigación, dicha cifra ha crecido hasta cientos de
millones o billones.
La masa total bacteriana que existe en la capa de suelo fértil de una hectarea
asciende de 5 a 7 toneladas. Esta masa se compone de celulas individuales
que estan vivas, se desarrollan y se multiplican.
Ademas de las bacterias, hay un gran numero de hongos, actinomicetes, algas,
ultramicrobios, phages, protozoarios, insectos, gusanos y otras criaturas vivas,
que viven en al suelo.
En un gramo de suelo hay cientos de miles, millones de hongos y
actinomicetes; algas por miles o cientos de miles. Bastante seguido su numero
asciende a cientos de miles por gramo de suelo. La masa total de todos estos
organismos en la capa superficial del suelo llega a 2 o 3 toneladas por
hectarea. Los analisis indican que hay en el suelo una gran masa de phages--
actinophages y bacteriophages, que tienen una intensa actividad.

No menos importante es la fauna del suelo. De acuerdo a diferentes autores,

hay cientos de miles de amebas, ciliatas, y otros protozoarios por gramo
(Brodskii, 1935, Nikolyuk, 1949; Dogel', 1951). En un metro cuadrado de
superficie de suelo hay de 10 a 100 invertebrados grandes : gusanos de tierra,
miriapodos, larvas de distintos escarabajos, etc. La poblacion de pequeños, no
microscópicos. artropodos (ticks, Collembola, y otros) se cuenta por diez a
cientos de miles en un 1 m2 de capa de suelo cultivado. Y en los suelos de la
jungla este numero aumenta a millones de individuos.
Los nemátodos a veces se encuentran por millones en 1 m 2. De acuerdo a los
recuentos de Gilyarov (1949, 1953), la masa total de esta fauna llega a varias
toneladas (3-4) por hectárea de suelo..
Como se ve por todos los datos citados, cada partícula de suelo esta saturada
de criaturas vivas. La enorme mayoría de ellas esta en estado de actividad
continua durante el periodo vegetativo completo. Los individuos aislados o
criaturas unicelulares se multiplican vertiginosamente y llegan a cifras
astronómicas. En todo este proceso de actividad vitál, la poblacion completa
del suelo desarrolla un trabajo de importancia cósmica. Transforma enormes
cantidades de componentes orgánicos y minerales, y sintetiza nuevas
sustancias orgánicas e inorgánicas. Se encuentran también en el suelo, varios
catalizadores biológicos : metabolitos de microbios y otras criaturas, incluyendo
enzimas, vitaminas, auxiones (cualquier tipo de hormonas que regulan el
crecimiento de las plantas), antibioticos, toxinas y muchos otros compuestos.
Todas estas sustancias en su conjunto con los organismos vivos, le dan
sus propiedades particulares al suelo, diferenciandolo de un cuerpo geologico o
una roca mineral.

La biogénesis del suelo es el mas significativo indicador de su fertilidad. Tan

pronto como la actividad de una población microbiana empieza en una roca, se
manifiestan los primeros síntomas de fertilidad. El grado de fertilidad de un
suelo esta determinado por la intensidad de los procesos vivos de su poblacion
microbiana. Las manifestaciones cuantitativas de estos procesos biológicos son
diversas y dependen del clima, la geografía, la topografía; tanto como de las
estaciones del año y otros factores externos.
El conocimiento de la vida del suelo, la investigación de los procesos biológicos
y bioquímicos que tienen lugar en el, esta conectada inseparablemente con el
conocimiento de los organismos vivos que lo habitan. La investigación del
mundo biológico en un suelo, en consecuencia, es uno de los problemas
básicos que presenta la poblacion viva del suelo; y debe ser uno de los
problemas mas importantes de pedologia (palabra de origen ruso que se
refiere al estudio de los suelos desde el punto de vista de la agricultura) y de la
misma agricultura.

Este trabajo gira en torno al conocimiento actual de los microorganismos,

principalmente las bacterias, actinomicetes y parcialmente de los hongos, que
habitan el suelo. Y de su vinculación con las especies botánicas superiores; de
la importancia de los distintos grupos y especies microbianas en la vida de las
plantas superiores. Y tambien del efecto de las sustancias metabólicas en el
crecimiento y producción de los cultivos agrícolas.
Debemos consignar que en los ultimos años se le esta prestando mayor
atención a los factores biológicos cuando se aborda el tema de la fertilidad de
los suelos. Tanto los agricultores como los cientificos del suelo estan
demostrando un interes creciente en la población microbiana. Esto no es
ninguna sorpresa, si tenemos en cuenta que, es imposible resolver los
problemas de la pedologia sin tener en cuenta la microflora del suelo. Ni que
hablar de los problemas de la agricultura y del crecimiento de los cultivos.

Como se ve a la luz de los datos citados, la importancia de los

microorganismos en la vida de las plantas es muy grande. Sin embargo todavia
esta poco investigado. ¨Contrario sensu¨, es menor la importancia de las
plantas en el desarrollo y la vida activa de los microbios del suelo.
La interacción de los microorganismos del suelo y las plantas superiores, es
muy compleja y multifacética. El efecto de los primeros sobre las segundas
puede ser positivo o negativo. La composición de la microflora cambia
dramaticamente según el porcentaje de cobertura de un suelo por las plantas,
bajo las mismas condiciones climáticas externas . Las plantas son un factor
ecologico muy poderoso en la selección de ciertas especies de bacterias,
hongos , actinomicetes y otros habitantes del suelo. Como resultado de
prácticas agricolas equivocadas en la rotación de cultivos, el suelo se infesta de
formas microbianas contraproducentes. Sin embargo, como resultado del uso
de especies adecuadas en la rotación de cosechas, uno puede cambiar la
microflora del suelo en una dirección deseada y eliminar los organismos
dañinos. En otras palabras, se puede restaurar la salud del suelo.

La influencia de los organismos del suelo en el crecimiento y desarrollo de las

plantas superiores es muy diversa. Y no se limita, de ninguna manera, a la
mineralización de las sustancias naturales. En la actualidad , no debemos
considerar a los microorganismos solamente como un eslabón en la
circulación de las sustancias, ni como agentes de distribución de recursos
minerales para la nutrición de las plantas. Los microorganismos del suelo
desarrollan una influencia directa y muy esencial en las plantas, que puede ser
negativa o positiva según las especies y las condiciones ecológicas. Por
ejemplo, el rol positivo de las bacterias en la simbiosis raiz-nodulo y otros
casos, es bien conocida aunque poco investigada.
Se esta prestando una gran atención a los organismos libres fijadores de
nitrogeno--Azotobacter, Clostridium y otros. Tambien se estan usando
fertilizantes que combinan bacterias y hongos (azotogeno, nitrogeno, silice,
phosphorobacterin y otros).
Un capitulo importante de la ciencia esta dedicado a la biologia de las bacterias
y hongos fitopatógenos. Pero ha sido poco investigado y dilucidado el rol de la
microflora viva, particularmente aquella que habita la zona de las raices.
Comparativamente la investigación de la microflora rizoesférica (la del area
circundante a las raices de las plantas) se ha iniciado hace poco. Los datos
obtenidos demuestran que su efecto sobre las plantas es muy considerable.
Existe un gran numero de especies dentro de la microflora risoesférica, que
afectan el crecimiento de las plantas via productos de su metabolismo. Algunas
especies microbianas son activas productoras de distintas sustancias bióticas
como vitaminas , auxiones, aminoacidos y otras sustancias esenciales para el
crecimiento de las plantas. Otras especies son antagónicas a las bacterias,
hongos o protozoarios fitopatogénicos. Los microorganismos de estas especies
favoceren las propiedades de inmunidad de los tejidos, protegiendo a las
plantas de las infecciones.
Hay muchos organismos, dentro de la microflora del suelo, que producen
sustancias tóxicas y perjudican a las plantas, inhibiendo su crecimiento y
desarrollo. Se debe concluir, que deben existir otras sustancias microbianas,
todavia a descubrir, que se supone que ejercerian un efecto potencialmente
positivo o negativo en las plantas.

Distintos representantes de especies de la microflora, útiles o dañinas, viven en

la rizoesfera. La cantidad promedio de ellas, varia según los distintos tipos de
suelos y bajo las diversas condiciones ecológicas. Por lo tanto, no es el mismo
su efecto total sobre la actividad de las bacterias en la zona de las raices.
Puede ser positivo o negativo dependiendo de cuales sean las especies
microbianas predominantes. En este trabajo se suministrara las ultimas
informaciones de importancia disponibles.

Es evidente que en estas investigaciones de la actividad de la microflora del

suelo, se necesita un conocimiento exacto de las especies presentes, del
mismo modo que se necesitan conceptos precisos sobre el estado deI suelo,
de sus condiciones ecológicas, de su desarrollo y de su acumulación.
Actualmente, las investigaciones de la biologia de suelos en general, y las
preguntas sobre la interacción de los microorganismos con las plantas
superiores en particular, no pueden ser resueltas con un recuento simple de los
microbios y de su composición. Es necesario establecer el grado de
diseminación de las diferentes especies, investigar su biología, determinar su
interacción con las plantas y con las otras especies de la microflora del suelo.
Esto último sera en el futuro, un problema importante en microbiologia de
suelos.

El antagonismo microbiano representa un factor ecológico importante en el

desarrollo y la formación de las asociaciones microbianas en general y de la
diseminación de las especies individuales en particular.
Debemos puntualizar que es muy dificil determinar las especies particulares de
las bacterias . La estructura similar y la falta de marcas externas para
identificarlas, no les permite a los investigadores reconocer inmediatamente y
con precisión, las formas y las especies que se presentan en el analisis de los
suelos y otros sustratos naturales.

No seremos capaces de reconocer el tipo de microflora de la zona radicular

hasta que no aprendamos a identificar y diferenciar a las especies bacterianas.
Sin este conocimiento es imposible determinar el tipo de transformaciones
bioquímicas presentes en el suelo.
Y por lo tanto el lugar donde este suelo sera fértil, se vuelve no determinable.

Parte III
FACTORES BIOLÓGICOS DE LA FERTILIDAD DEL SUELO
La diversidad y abundancia de la población viva del suelo fue discutida en el
capítulo anterior. La masa viviente de microbios: bacterias, hongos,
actinomicetes y algas, por sí misma constituye más de 10 toneladas de la capa
arable de 1 Ha de un suelo fértil bien cultivado. Además, en el suelo habitan un
gran número de protozoos, insectos, gusanos y otras criaturas representativas
del mundo vivo.
Toda esta población realiza un inmenso trabajo, procesando considerables
cantidades de diferentes substancias minerales y orgánicas, descomponiendo
los residuos de plantas y animales, participando en la transformación de sus
productos de descomposición.
Los microbios sintetizan y excretan diferentes productos metabólicos, que al de
entrar en el suelo, le confieren fertilidad.
Gracias a la población microbiana, la formación geológica muerta, sin agregar,
cobra vida y se convierte en un cuerpo productivo. Así se crean las condiciones
para la nutrición y crecimiento de las plantas superiores. Los microorganismos
constituyen el vínculo indispensable y el más importante en la nutrición de las
plantas.
Los microorganismos del suelo, no solamente crean las condiciones necesarias
para el crecimiento de las plantas superiores, también tienen un efecto directo
sobre ellas a través de sus productos metabólicos. Este capítulo trata del efecto
de los micro organismos como agentes de mineralización de los compuestos
orgánicos de los residuos de plantas y animales y como un factor biológico
necesario para la normal nutrición y crecimiento de las plantas.

Nutrición de las Plantas

Los fertilizantes se han usado desde tiempos antiguos en agricultura, para
aumentar los rendimientos. El hombre ha usado los fertilizantes mucho antes
que la ciencia planteara y resolviera los principales problemas de la ciencia del
suelo, agricultura, agroquímica y fisiología vegetal. Las reglas de la fertilización
se elaboraron empíricamente, pero como ha puntualizado Pryanishnikov,
muchas de esas reglas alcanzaron gran precisión.
Los romanos, por ejemplo, sabían de las valiosas propiedades fertilizantes de
los excrementos animales y de algunas substancias minerales como ceniza,
yeso, calcio y marga. Más aún, sabían que el valor fertilizante de los
excrementos de distintos animales es diferente, siendo el de aves, el más
estimado.
Los romanos también conocían acerca de los fertilizantes verdes. Así, ellos
enterraban con el arado abono verde en las pendientes del Vesubio para
aumentar la fertilidad del suelo.
La teoría de nutrición vegetal aún no había sido elaborada. Se hacían vagas
suposiciones sobre una “gordura” de la tierra (terrae adeps). Esta “gordura” era
responsable dela fertilidad del suelo. Para aumentarla era necesario el
excremento animal.
Estas suposiciones contenían el núcleo de la teoría del humus, que
subsecuentemente se hizo general. Esta teoría asume que los compuestos
orgánicos son de la máxima importancia en la nutrición vegetal.
Ecos de estas ideas se pueden oír en el lenguaje de diferentes naciones.
Permítasenos recordar que la palabra “TUK” (fertilizante) alguna vez tuvo dos
significados, uno de ellos “gordura” (Pryanishnikov, D. N., Nutrición Vegetal,
Trabajos Reunidos, Vol. 1, 1952, p 58).
La teoría de nutrición vegetal del humus establecía que las plantas se
alimentan de substancias orgánicas presentes en los excrementos de animales
y en general en el humus. Esta teoría, difundida en el siglo 18 era preferida por
los criadores y agrotécnicos de la época, ya que estaba bien confirmada por la
práctica agrícola.
Las explicaciones del efecto favorable de las substancias orgánicas sobre el
crecimiento y fertilidad de las plantas diferían. La concepción más difundida
asumía que la planta tomaba el carbono de las substancias orgánicas,
incorporándolo a su cuerpo. (Davy, 1813).
Otros autores pensaban que los excrementos contenían ciertas substancias
especiales. Así, por ejemplo, Prof. Vallerius en 1766 asumía que sólo las
substancias orgánicas o substancias de la “gordura” del humus o del suelo
jugaban algún papel en la nutrición vegetal, mientras que otros compuestos del
suelo actuaban como meros solventes de la “gordura”.
Algunos científicos de los siglos 16 al 18 y en particular Olivier de Serres,
(1600), suponían que la causa de la acción del fertilizante está en su calor.
Bernard Palissy en su trabajo (1563) adelanta la idea que el contenido de sal
del fertilizante desempeña un papel en el crecimiento de las plantas.
Los primeros intentos experimentales para dilucidar el problema de la nutrición
vegetal estuvieron a cargo de Van-Helmont. En su trabajo (1629) presentó los
resultados de cinco años de experimentos sobre cultivo de ramas de hiedra en
suelo que recibió sólo agua de lluvia. En cinco años la rama creció y logró un
peso final que era 33 veces mayor que el peso inicial. El suelo donde creció no
perdió peso. Como no se conocía entonces la composición del aire, Helmont
concluyó que la planta utilizó sólo agua y que eso fue suficiente para que la
planta construyera su cuerpo.
El investigador francés Duhamel llegó a la misma conclusión. Cultivó plantas
con agua del Sena. El no tenía agua destilada a su disposición para
experimentos de control. Los resultados positivos obtenidos por él no son
confiables dado que el agua del Sena contiene varias substancias orgánicas e
inorgánicas.
Woodword mostró que el punto de vista de Van.Helmont estaba equivocado. En
sus experimentos demostró que la menta crece mejor en agua de río que en
agua de lluvia y crece mejor aún si se agrega suelo al agua. Presenta la
siguiente información: la ganancia de peso de la planta en grains* durante 77
días fue como sigue: en agua de lluvia, 17, en el agua de cloacas de Hyde
Park, 139, la misma, con adición de suelo, 284 [*grains es una unidad de peso]
Woodword concluyó que no sólo el agua, sino algo más que está presente en
el suelo es necesario para el crecimiento de las plantas.
La práctica agrícola de esos tiempos no poseía base científica alguna para la
dilucidar el vínculo observado entre el crecimiento de las plantas, su
rendimiento y el suelo. Cuando Lomonosov y luego Lavoisier descubrieron la
ley de la conservación de la materia, recién quedó establecido un sólido
fundamento científico del conocimiento.
Lavoisier descubrió la composición del aire y la esencia del proceso de
oxidación, combustión y respiración. No mucho antes de su muerte (1794)
escribió, acerca de la nutrición y crecimiento de las plantas, que las plantas
obtienen los materiales necesarios para su organización del aire circundante y
generalmente del reino mineral (de acuerdo con Pryanishnikov, 1952).
Los nuevos métodos de investigación química elaborados por Lavoisier fueron
aplicados por Priestley, Senebe y Sossur, al estudio del metabolismo de las
plantas. Estos científicos demostraron que las plantas obtiene el carbono del
aire y que además de carbono, las plantas requieren de sales.
Mientras reconocía la importancia del carbono atmosférico, Sossur también
pensaba que el humus del suelo es de gran importancia. El humus contiene
una cierta substancia indispensable para las plantas. La experiencia diaria de
los agricultores apuntaba a un vínculo muy fuerte entre la fertilidad del suelo y
la presencia de substancias orgánicas en él.
El científico inglés Davy (1813) llamó la atención sobre varias substancias
extractables azucaradas, pegajosas, aceitosas, pastosas y sobre el dióxido de
carbono del suelo. Estos elementos, según él, contienen todos los elementos
necesarios para la vida de las plantas.
La teoría del humus se hizo muy popular después de los conocidos trabajos de
Teer (1752-1828), fundador de la primera escuela agrícola y el propagador de
la rotación de cosecha en lugar del sistema de los tres campos. Escribió que la
fertilidad del suelo depende enteramente del humus. Dado que además del
agua, el humus es la única substancia del suelo que puede servir como
nutriente para las plantas (de acuerdo con Pryanishnikov, 1952).
Estas ideas sentaron las bases para una teoría de agotamiento y regeneración
de la fertilidad del suelo. Se suponía que mientras las plantas absorbieran más
substancias húmicas, mejor crecerían y mejor sería la cosecha. Diferentes
plantas absorben diferentes substancias orgánicas. Por ejemplo, el trigo
requiere más humus que el centeno.
Teer y sus adherentes consideraban que el humus es un producto importante
de las plantas y una substancia de la máxima importancia para la vida de las
plantas. El humus se forma a expensas de las plantas. Algunas plantas
excretan más humus del que absorben. En otras palabras, algunas plantas
enriquecen el suelo en humus y otras agotan sus reservas. A la primera
categoría pertenecen el trébol y las leguminosas.
Los que proponían la teoría del humus no tomaban en cuenta a las substancias
minerales. Estas, según ellos, mejoran la descomposición del humus y lo
transforman en asimilable para las plantas.
La teoría del humus era muy popular inclusive en los treinta del siglo pasado en
diferentes países como Alemania, Inglaterra, Francia y Rusia.
De los investigadores rusos de la última parte del siglo 18 y principios del 19,
quienes llamaron la atención sobre la importancia de los fertilizantes orgánicos,
deben ser mencionados A. Bolotov, I. Komov y A. Poshman. Los trabajos de
estos científicos mostraron que los fertilizantes tienen una inmensa influencia
sobre las cosechas. Komov Supuso que las substancias orgánicas son
necesarias para las plantas en la misma medida que lo son para los animales y
recomendó usarlas en la práctica. De acuerdo con él, los fertilizantes orgánicos
no pueden ser reemplazados por sales.
El papel de los compuestos nitrogenados en la nutrición vegetal empezó a
entenderse mejor al principio del siglo 19.
Liebig asumió que las plantas obtienen nitrógeno a expensas del amoníaco
atmosférico y que su presencia en la atmósfera es suficiente para ellas. Sin
embargo los experimentos lo refutaron. Se encontró en cambio, que esta
substancia es harto insuficiente para las plantas.
Los bien conocidos estudios de Bussengo revelaron las fuentes de nutrición por
nitrógeno de las plantas. En 1837-38, desarrolló su teoría sobre fertilizantes
nitrogenados y recomendó el uso de fertilizantes ricos en nitrógeno. Vinculó la
extenuación del suelo con el agotamiento de las fuentes de nitrógeno. En este
proceso le adjudicó diferente importancia a diferentes plantas. Algunas plantas
absorben más nitrógeno del suelo, otras menos. Adjudicó a ciertas plantas un
activo papel en el mejoramiento de la fertilidad, por ejemplo, el trébol “Uno
debe pensar”, dijo, “que los cultivos que mejoran el suelo no se limitan a
enriquecerse ellos mismos con C, H y O2 solamente, sino que también
enriquecen el suelo con nitrógeno” (de acuerdo con Pryanishnikov, 1945).
Hellriegel (1889), descubrió la razón de la peculiar acción de las leguminosas
sobre el suelo. Con sus experimentos encontró que las leguminosas asimilan
nitrógeno del aire. Voronin (1886) estudió los nódulos de las raíces de las
leguminosas y encontró microorganismos dentro de los mismos, que en sus
palabras son “los causantes de la formación de nódulos”. Más tarde, Hellriegel,
mediante profunda experimentación mostró que estos organismos simbióticos
son la causa de la fijación de nitrógeno por las leguminosas.
Así, las conclusiones de los viejos investigadores, por ejemplo Teer, han sido
confirmadas por los experimentos posteriores de Bussengo, Hellriegel y otros,
quienes encontraron que las plantas no sólo agotan el suelo, sino que también
lo enriquecen con substancias nutrientes orgánicas.
La teoría del humus de la nutrición vegetal del siglo 18 y primera mitad del 19
era muy popular. Se consideraba esencial la fertilización con substancias
orgánicas, no sólo para aumentar los rendimientos, también para, en general,
aumentar la fertilidad del suelo.
La actitud hacia la teoría del humus en la nutrición vegetal y en general hacia
los fertilizantes cambió abruptamente después que Liebig propuso su teoría
sobre la nutrición mineral de las plantas. Criticó severamente la teoría del
humus y la consideró básicamente equivocada. Consideró todos los estudios
realizados antes que los suyos por fisiólogos y agrónomos, inconsistentes y sin
valor para la solución del problema de la nutrición vegetal. Criticó la teoría del
humus: “Tomándola”, en las palabras de Pryanishnikov,"en su extrema
expresión y llevándola al absurdo"
Liebig rechazó totalmente inclusive la posibilidad de asimilación de substancias
orgánicas por las plantas. En su opinión, sólo los compuestos inorgánicos
pueden servir como fuente de nutrición de las plantas. Consideró a l humus
como fuente de CO2 que promueve el proceso de erosión de los silicatos y
prepara el alimento mineral para las plantas
Liebig no reconoció el papel de las plantas en el enriquecimiento del suelo.
Severamente negó y criticó la noción de “enriquecimiento del suelo en fuentes
de nutrición”, Las plantas, según él, agotan el suelo, llevándose elementos de
nutrición mineral con las cosechas. Pero el agotamiento del suelo se lleva a
cabo por diferentes plantas a diferentes tasas y en diferentes direcciones.
Algunas solamente toman principalmente calcio del suelo (por ejemplo, las
arvejas), otras, potasio y silicio (granos de trigo). Por lo tanto, la rotación de
cultivos, recomendada por investigadores anteriores, sólo hace más lento el
agotamiento del suelo.
De acuerdo con esta teoría, Liebig recomendaba la introducción en el suelo de
fertilizantes minerales. La cantidad de estos fertilizantes aplicados debería
tener en cuenta la utilización por las plantas.
Debido a la autoridad de su autor, Liebig, la teoría mineral de nutrición vegetal
fue aceptada por sus contemporáneos, prácticamente sin crítica alguna. La
autoridad de la escuela química de Liebig suprimió todas las ideas previas y
teorías de nutrición vegetal orgánica. Liebig, dice Ressel (1933) le dio el golpe
de gracia a la teoría del humus. Solo los más atrevidos podrían mantener que
las plantas toman el carbono, no del CO2, sino de otras fuentes. Aunque uno
debe admitir que no tenemos pruebas que las plantas obtengan todo el CO2
necesario de esta forma.
La teoría de Liebig fue desarrollada y sujeta a cambios en correspondencia con
la información fáctica recientemente adquirida. En nuestro país la teoría de
Liebig fue profundamente revisada por el académico Pryanishnikov y sus
seguidores. Pryanishnikov tomó su propio camino en agroquímica, él y sus
estudiantes elaboraron una serie de valiosos postulados, sentando la base para
medidas prácticas en la agricultura. Tiene le honor de haber resuelto el
problema de obtener mejoras básicas en el balance de nitrógeno de la
agricultura de la URSS. En oposición a la teoría de Liebig, adjudicó una mayor
importancia al proceso biológico del suelo y especialmente a la acumulación de
nitrógeno Pryanishnikov no negó la posibilidad de asimilación de substancias
orgánicas por las plantas y él mismo lo demostró experimentalmente.
No obstante, la teoría de Liebig todavía se refleja en los estudios de muchos
especialistas. En la teoría de nutrición vegetal uno observa una obvia
subestimación del papel de las substancias orgánicas del suelo. Su importancia
en la nutrición de las plantas es, a menudo, totalmente negada. Como regla, el
significado de las substancias orgánicas del suelo se formula básicamente en
dos postulados.
1. Las substancias húmicas son reservas de elementos nutrientes para las
plantas que sólo quedan disponibles al ser mineralizadas.
2. El humus mejora las propiedades fisicoquímicas del suelo, aumenta su
capacidad de absorción y por lo tanto, promueve la acumulación de
substancias nutrientes. Refuerza la estructura del suelo y con eso
mejora muchas otras propiedades. Los fertilizantes orgánicos, estiércol,
compost, etc, preparan el suelo para recibir los fertilizantes minerales,
aumentan su capacidad de amortiguación (tampón), etc.
Todo esto es bien cierto y está confirmado por antiguas prácticas y por muchos
experimentos. Sin embargo, en nuestra opinión, es inadecuado reducir la
importancia de estos fertilizantes sólo a los dos postulados aludidos.
La acción positiva del humus y fertilizantes orgánicos en el crecimiento de las
plantas no puede ser explicada por el contenido de elementos minerales de
nutrición presentes en ellos. Russel (1933), resumiendo la información
experimental de 60 años de trabajo de Rothamstead Station, dijo que aunque
las plantas pueden crecer satisfactoriamente y alcanzar el desarrollo completo
con sólo substancias nutritivas inorgánicas, bajo condiciones naturales, la
nutrición se realiza en la presencia de substancias orgánicas. La pregunta, si
estas substancias juegan algún papel en este proceso ha sido muy discutida.
Los datos experimentales no son concluyentes. En los experimentos de campo
de Rothamstead, ninguna de las combinaciones de fertilizantes artificiales es
tan efectiva como el estiércol para mantener las cosechas año a año.
Los extensos experimentos de los investigadores alemanes Gerlach, Hansen,
Schultz, Wagner, Schneidewind, y otros, nos llevan a la conclusión que
cualquiera sea la cantidad de fertilizante mineral, usando sólo fertilizantes
minerales, no se puede alcanzar rendimientos tan altos como con la
introducción simultánea de estiércol (Schneidewind, 1933).
Resumiendo los experimentos de muchos años en la estación Lauchstadt,
Schneidewind nos da un ejemplo con la siguiente información de rendimientos
de remolacha azucarera, en quintales por Ha con sólo fertilización mineral,
raíces 487,6; contenido de azúcar 77,66; tops [parte aérea?] 291,7. Bajo
fertilización mineral y orgánica (estiércol): raíces 533,6; contenido de azúcar,
88,11 y tops [parte aérea?], 366.6,
Similar información nos da Schneidewind para cultivos de papas. Usando sólo
fertilizantes artificiales no pudo obtener más que 240 quintales por Ha, mientras
que con el agregado de fertilizantes minerales y estiércol, el rendimiento
aumentó a 306-312 quintales por Ha. Este efecto del estiércol se repitió año
tras año y fue independiente del monto de las precipitaciones, ya fuera un año
seco, húmedo o promedio.
Russel (1933) y sus colaboradores han mostrado que las substancias
orgánicas del suelo estimulan el crecimiento de las plantas y aumentan las
cosechas. Concluye que no hay mezcla de fertilizantes artificiales que pueda
ser tan efectivo como el estiércol, en mantener constantemente elevadas
cosechas año tras año.
El académico V. I. Palladin (1924), escribió respecto del problema la nutrición
vegetal, que las plantas verdes se pueden alimentar de compuestos orgánicos
ya listos. Esa nutrición puede ir en paralelo con la asimilación de carbono de la
atmósfera. De acuerdo con las observaciones del autor, hojas verdes aisladas
cultivadas con nutrientes artificiales conteniendo azúcar siempre acumularon
más substancias orgánicas en sus tejidos y tuvieron mayor turgencia que las
cultivadas con nutrientes minerales. El incremento de carbohidratos con la
nutrición con azúcar alcanza a 5 g por m2 de superficie foliar.
Lebedyantsev (1936) en una anotación a su traducción del libro de Liebig
(Química Aplicada a la Agricultura y la Fisiología) menciona el trabajo de
Sossur sobre nutrición de plantas y escribió: “Sossur consideró que el CO 2 del
aire es la principal fuente de nutrición de carbono, sin negar, no obstante, la
posibilidad de utilizar carbono de compuestos orgánicos del suelo, ya que no
contaba con hechos que permitieran negarlo. Hoy debemos destacar que no
contamos con esos hechos y la cuestión de la asimilación de compuestos
orgánicos del suelo permanece en gran medida abierta, aunque, sin duda, la
principal fuente de carbono para las plantas verdes e, después de todo el CO 2”
(página 306)
Como puede verse, de acuerdo a lo que antecede, la cuestión de la nutrición
vegetal, a pesar de los numerosos estudios realizados, permanece no resuelta
en muchos aspectos.
Es difícil estar de acuerdo con el concepto según el cual, durante toda la
historia de su evolución, las plantas, a pesar de haber estado en contacto con
las substancias orgánicas del suelo, no hubieran adquirido la habilidad de
asimilarlas de uno u otra manera.
No existe base para negar el bien conocido hecho y experimentos que indican
que las plantas utilizan compuestos minerales para su nutrición. Numerosas
observaciones hablan a favor de esta forma de nutrición vegetal y es la más
importante bajo condiciones naturales. Pero ¿es esa nutrición suficiente para
obtener altos rendimientos y semillas totalmente viables año tras año? Para
nosotros, esta cuestión no está del todo resuelta.
No hace mucho se decía que las raíces de plantas no asimilaban el CO 2 del
medio, pero ahora esto puede ser considerado una realidad. Las plantas no
sólo absorben CO2 de la atmósfera, sino también del suelo (Samokhvalov,
1952; Kursanov, 1953, 1954).
Se debe asumir que las plantas que experimentan falta de CO 2 en la atmósfera,
gustosamente lo asimilan del suelo. Bajo distintas circunstancias desfavorables
la actividad fotosintética de las plantas puede disminuir considerablemente. Así,
por ejemplo, durante la sequía, las estomas se cierran y el flujo de CO 2 se
detiene o se debilita. La respiración de las plantas, bajo estas condiciones, sin
embargo no se detiene e inclusive puede incrementarse. Se produce la
privación de alimento, en mayor o menor grado. La debilitación de la
fotosíntesis puede ser causada por otros factores. En todos tales casos, es
evidente que las plantas pueden cambiar a una alimentación heterótrofa,
asimilando compuestos orgánicos del suelo para suplementar su nutrición.
 Heterotrofismo de las Plantas
En muchos representantes del reino vegetal, desde las bajas formas, tales
como las algas azul verdosas y algas verdes hasta las plantas superiores que
dan flores, se ha observado la habilidad para asimilar compuestos orgánicos ya
listos y usarlos como nutrientes.
Se sabe que muchas (si no todas) las algas pueden, bajo ciertas condiciones,
crecer en medios sintéticos con fuentes minerales de nutrición, tanto como en
medios orgánicos que contienen diferentes compuestos complejos
nitrogenados y carbonáceos. Se ha demostrado por experimentos especiales,
que estos organismos, especialmente los verdes unicelulares, que usualmente
crecen en medios puramente minerales, crecen mucho mejor con la adición de
substancias orgánicas a la solución. Ellos pueden asimilar substancias
carbonáceas y nitrogenadas (Gollerbach y Polyanskii, 1951).
Las algas azul verdosas Cyanophyceae--Oscillaria, Nostoc Anabaena,
Lyngbya, y otras, tampoco las algas verdes de los grupos Chroococcum,
Spirogyra, etc, no pueden cultivarse indefinidamente en medios orgánicos
artificiales sin debilitar notoriamente su viabilidad. En nuestro laboratorio hemos
mantenido un cultivo puro del alga verde Chlorella por ya más de 25 años. No
observamos ninguna disminución de sus funciones. Esta alga asimila nitrógeno
de la peptona y amino ácidos y crece muy bien en agar y también en agar de
carne-peptona (MPA) y en gelatina.
De acuerdo con las observaciones de muchos investigadores, las algas verdes
Chroococcum y Spirogyra, las algas azul verdosas Phormidium, Nostoc,
Anabaena, Gloeocapsa, y otras, fácilmente asimilan el azúcar, amino ácidos,
alcoholes, ácidos orgánicos, urea, caseína y otras substancias orgánicas.
Estudios con cultivos puros de algas realizados primero por Pringsheim (1913)
y luego por muchos otros (Harder y Humfeld, 1917; Cataldi, 1941; Gerloff,
1950) han mostrado que estos organismos crecen en medios orgánicos en la
oscuridad tanto como en la luz. Chlorella asimila oxalatos sodio y potasio y
ácido tartárico, málico y algunos otros ácidos orgánicos en la oscuridad.
Allen (1952) estudió 26 cultivos de algas azul verdosas y algunas algas verdes
pertenecientes a 11 géneros, Anabaena, Nostoc, Oscillatoria, Lyngbya.
Phormidium. Gloeocapsa. Aphanocapsa, Plectonema Cladothrix,
Chroococcum y Synechococcus. Estos cultivos fueron criados en medios
minerales con diferentes fuentes de nutrientes orgánicos, extracto de levadura,
varios azúcares (glucosa, sacarosa, lactosa, maltosa, etc) manitol, glicerol,
etanol, ácidos orgánicos, amino ácidos, caseína, urea, hidrolizado de caseína,
etc. En presencia de estas substancias orgánicas, muchas algas crecieron
mejor que en los medios con nutrición mineral solamente. La asimilación de las
substancias orgánicas se produce en muchas especies con la misma
intensidad tanto en la luz como en la oscuridad. Con el cultivo prolongado de
las algas en medio orgánico, algunas algas pierden su pigmento y continúan
viviendo como típicos saprofitos (Fogg y Wolff, 1955).
Los autores que han estudiado las algas concluyen que ellas pueden vivir como
heterótrofas utilizando compuestos orgánicos, como substancias carbonáceas
y nitrogenadas, de la misma manera que ordinarios saprofitos. Esto es
comprensible, porque las dos clases de organismos viven en un entorno rico en
substancias orgánicas, en suelos y en cuerpos de agua, donde los residuos de
plantas y animales sirven de fuentes de nutrientes orgánicos después de su
descomposición.
El musgo verde del género Splachnum y Getrapladon, se establece y crece
sobre los excrementos de animales, utilizando substancias orgánicas para su
nutrición.
Entre las plantas superiores con flores, hay un grupo conocido como “plantas
del humus”. Se puede observar a distintos representantes de dicho grupo en
diversos estadios de degradación del aparato de la clorofila. Las plantas del
humus son llamadas así porque crecen en substratos ricos en humus y en
residuos en descomposición de plantas y animales.
El carácter de la nutrición y las condiciones de la existencia de estos
organismos han dejado una cierta impronta en su estructura y apariencia.
Algunos pierden su coloración verde, las hojas se reducen (pero sólo en la
parte inferior del tallo) y pierden la capacidad de asimilar el dióxido de carbono
de la atmósfera. A este grupo pertenecen las especies de los siguientes
géneros: Monotropa Dum., Zymodorum, Neottia Sw., Corallorhiza Rale,
Epipogon S. G. Gmel., Voyria Aubl, Fletia, Pogonia Less, Voyriella Miq., y otros
(ver abajo).
En otros representantes de las plantas del humus, se han preservado las hojas
verdes y ellas permanecen autótrofas, asimilando el dióxido de carbono de la
atmósfera, pero , no obstante, ellas pueden utilizar substancias orgánicas
listas. Estos representantes pertenecen a las especies: Dentaria L., Pyrola L.,
Goodyera R. Br., Cephalanthera Rich., Epipactis Adans,Platanthera Rich., y
otras. Todas estas plantas, en mayor o menor grado viven y se alimentan como
heterótrofos, asimilando compuestos orgánicos junto con fuentes minerales de
nutrición.
Es bien conocido el grupo de plantas insectívoras. En nuestras latitudes tales
plantas con flores se encuentran en Drosera rotundifolia L., y Utricularia
vulgaris L. A este grupo pertenecen plantas de sur como Cephalotus follicularis
Labill, muchas especies de of Nepenthe L., Dionaea muscipula Ellis,
Drosophillum Link, Aldrovanda Monti, Sarracenia L., y otras.
En la literatura se describen cerca de 500 especies de estas plantas
insectívoras. Estas plantas tienen un complejo aparato para cazar insectos y
glándulas especiales para su digestión.
Los pequeños animales que caen en la trampa de estas plantas son
descompuestos por enzimas a formas solubles que son entonces absorbidas
por las plantas y utilizadas para su nutrición.
Es característico de estas plantas insectívoras claramente heterótrofas el no
perder su capacidad de asimilar el CO2 atmosférico. Muchas especies que
obtienen substancias orgánicas nutrientes listas, del cuerpo vivo de otras
especies de plantas, también pertenecen a las plantas heterótrofas. Estas son
las plantas parásitas. Están divididas en parásitas obligadas y facultativas.
Entre las parásitas obligadas, las trepadoras del género .Cassytha L. (fam.
Lauraceae L.) y Cuscuta L. (fam. Convolvulaceae L.) son las especies más
conocidas.
La biología de Cuscuta L. es conocida en gran detalle (se han reconocido hasta
50 especies) Son parásitas de los cereales, arbustos y árboles. Penetran los
tejidos del huésped con sus haustorias y succionan nutrientes del huésped. Las
hojas de estas plantas están débilmente desarrolladas y son difíciles de ver.
Los tallos carecen de clorofila.
Orobanche L. son también plantas etioladas con hojas levemente desarrolladas
(en forma de escamas) carentes de clorofila. Son parásitas delas raíces de
girasol, lino, repollo y otros. Se conocen cerca de 180 plantas parásitas de este
género. Los tallos de Orobanche se confunden tanto con las raíces de la planta
huésped, que es imposible distinguir entre las células del huésped y el parásito.
Lathraea squamaria también pertenece al grupo de plantas parásitas. En
contraste con las anteriores no trepa. Consiste en un tallo incoloro, acuoso. Las
hojas de esta planta están levemente desarrolladas y tienen la forma de
escamas incoloras. El sistema radicular está junto a la raíz del huésped, de la
que la planta parásita chupa la savia del huésped con ayuda de haustorias. La
planta al principio crece bajo la tierra, luego sus tallos jóvenes emergen a la
superficie. Las partes aéreas son de un color violeta rojizo.
Las Balanophoraceae son plantas tropicales parásitas que viven en las raíces
de varios árboles Se conocen cuarenta especies de estas parásitas (14
géneros) Estas plantas, como la Orobanche, crece en tal intimidad con las
raíces del huésped que no se puede discernir un borde visible entre ellas.
Plantas parásitas relacionadas a Balanophoraceae son las Rafflesiaceae Dum.,
que parasitan árboles de las regiones tropicales y subtropicales. Estas plantas
penetran la corteza del árbol huésped, abrazan su tallo y raíces y succionan la
savia de las raíces.
Las plantas parásitas de la familia Loranthaceae D. Dow incluyen más de 300
especies. Viven exclusivamente en los tallos y ramas de diferentes árboles. Un
representante típico de este grupo es nuestro ordinario muérdago blanco
Viscum album L., que vive en árboles de hojas caducas y en pinos. Esta planta
penetra las ramas del huésped con sus raíces y haustorias. El tallo de Viscum
album que tiene la forma de un arbusto ramificado dicotómicamente presenta
hojas carnosas, de un color verde amarillento.
Las plantas de la familia del muérdago están caracterizadas por el hecho que
sus representantes arbóreos no han perdido completamente su capacidad de
fotosíntesis y preservan al apariencia normal de sus tallos. Son un grupo
intermedio ubicado entre los parásitos obligados, carentes de clorofila, que se
nutren de substancias orgánicas listas y los parásitos facultativos, que
preservan su estructura normal en todas sus partes como su capacidad para
asimilar el CO2 del aire.
A los parásitos facultativos pertenecen unas 100 especies de la familia
Santalaceae R. Br., y más de 500 especies de la familia Scrophulariaceae R.
Br. En nuestra flora se encuentran especies del género Thesium L. (familia
Santalaceae) y especies del género Euphrasia L., Alectorolophus Boehm.,
Melampyrum L., Pedicularis Boehm., Odontites Pers y otros (familia
Scrophulariaceae).
Son todos pastos que crecen en praderas o bosques. Poseen hojas verdes y
capacidad fotosintética bien definida. En el estadio inicial se desarrollan como
autótrofos típicos sin la menor inclinación a volverse parásitos. Sin embargo,
cuando sus raíces alcanzan un tamaño determinado (1-2cm) aparecen las
haustorias, con ayuda de las que se adhieren a la raíz de otra planta parásita
que encuentren durante su desarrollo. Desde ese momento en más, la planta
semi parásita comienza a suplementar su alimentación a expensas de la planta
huésped. Pueden, sin embargo, crecer sin el huésped en el caso de no
encontrarlo. También pueden parasitarse entre sí.
Hay en la naturaleza muchas plantas epifitas que se desarrollan sobre plantas
como substrato. Se asume que se nutren independientemente, a expensas de
substancias minerales presentes en la corteza de las plantas. Sin embargo, la
posibilidad de usar substancias orgánicas como tejidos muertos o sus
productos de descomposición no está excluida. Este problema aún no se ha
estudiado adecuadamente.
Muchas plantas asimilan substancias orgánicas, productos metabólicos de
microbios que viven sobre y dentro de sus raíces. Las plantas mejor conocidas
son las que en sus tejidos tienen microbios simbiontes, actinomicetes y hongos
La simbiosis mejor conocida es la de plantas con micorriza. De acuerdo con la
información de la literatura, hay micorriza en las raíces de casi todas las
plantas, que son en mayor o menor medida, simbiontes. La simbiosis es tal,
que algunas especies de plantas no pueden crecer sin el hongo. Esas plantas
son aparentemente, parásitos obligados, heterótrofos, cuya nutrición depende
de los productos metabólicos de los hongos. Algunas orquídeas sirven como
ejemplo de ese parasitismo. Requieren hongos para su normal crecimiento y
desarrollo. Crecen mal sin hongos y ciertas especies como Neottia nidusavis
Rich., que se encuentran en nuestros robles y pinos no crecen para nada si
carecen del hongo simbiótico. Esta orquídea crece y se desarrolla bajo la
superficie del suelo y sólo emerge para florecer y dar fruto. No tiene color verde
y es pálida, los gruesos tallos tienen brotes débilmente desarrollados, algo
amarillentos con hojas escamosas. Neottia nidusavis Rich. Ha perdido
totalmente la capacidad de nutrición con compuestos minerales. Está casi
completamente desprovista de clorofila. Su nutrición es a expensas de los
productos de descomposición del hongo que crece dentro de su tallo y raíces.
Hay una teoría, según la cual, estas orquídeas pueden asimilar compuestos
directamente del suelo y el hongo meramente sirve para el procesamiento de
esos compuestos hacia una forma en que pueden ser asimilados más
fácilmente. El cuadro citológico del crecimiento del hongo dentro de los tejidos
de la orquídea muestra que el micelio crece hasta un determinado tamaño,
seguido de enrollamiento y lisis. Los productos de la lisis son asimilados por la
planta.
Otras especies de orquídeas que han perdido su color verde y son enteramente
dependientes de hongos, se pueden encontrar en nuestros bosques. A estas
orquídeas pertenecen: Epipogon aphyllus Sw. y Corallorhiza trifida Chat., que
crecen en los bosques del cinturón moderado. Tales orquídeas parásitas se
encuentran también en las selvas tropicales (Burgeff, 1932). En la literatura se
describen orquídeas cuyo parasitismo se hace manifiesto solo en una cierta
fase de su desarrollo. Las especies del género Myrmechis (Myrm. glabra Bl.,
Myrm. gracilis Bl. ) Cuando jóvenes crecen en forma de una raíz, que obtiene
su nutrición enteramente a expensas de hongos. Luego forman órganos
aéreos, brotes que florecen con hojas verdes y empiezan a nutrirse por sí
mismas. Cuando los brotes se caen se hacen parásitas nuevamente.
De acuerdo con la información de Bernard, las semillas de orquídea no pueden
germinar sin el hongo que forma la micorriza. Bernard ha mostrado que las
orquídeas no pueden germinar sin el hongo y ha elaborado métodos para la
infección artificial de estas plantas con la micorriza.
Las plantas parásitas que han perdido su coloración verde se encuentran entre
los otros representantes de las plantas verdes. Tal, por ejemplo es Monotropa
hypopithys L., de la familia de Pirolaceae Drude. Están desprovistas de la
coloración verde y sus hojas se han transformado en escamas marrones. Esta
y otras plantas del género Monotropa L., reciben sus nutrientes enteramente a
expensas de hongos que crecen dentro de sus tejidos. Gordyagin (1922)
describió un helecho, Ophioglossum symplex cuya generación de esporas no
sexual, se nutre a expensas de hongos, absorbiendo productos orgánicos del
su metabolismo y descomposición.
De acuerdo con las observaciones de aquel autor, en los bosques de la
república autónoma de Tatar se han encontrado helechos y otras plantas
verdes que asimilan el CO2 de la atmósfera, pero no pueden existir sin la
simbiosis con hongos. Esas plantas están muy difundidas en la naturaleza. Las
plantas superiores de este tipo, preservan la coloración verde y la capacidad de
asimilación del CO2 atmosférico pero no pueden desarrollarse normalmente y
alcanzar el estadio de floración y fructificación en ausencia del hongo.
La mayoría de las plantas tiene hongos en sus raíces. Son útiles aunque no
indispensables. Los hongos , en tales casos promueven su crecimiento y
nutrición. Las plantas, según muestra la experiencia, crecen mejor, se adaptan
mejor a nuevos lugares y dan mayores incrementos de masa (Lobanov, 1953,
Reiner y Nelson-Jones, 1949).
Keller (1948) y Lyubimenko (1923) al comparar el grado de simbiosis de
diferentes plantas con hongos y el grado de parasitismo sobre el hongo,
consideró estos fenómenos como subsecuentes estadios de evolución, como
estadios de transición entre autotrofismo, a saprofitismo y parasitismo. O sea a
heterotrofismo. En su libro “Los Fundamentos de la Evolución de las Plantas”
Keller escribe: “En el curso de la evolución, las plantas superiores de diferentes
familias pasan por este estadio, y, bajo la presencia de condiciones naturales
apropiadas, cambian a nutrición a expensas de hongos. Así las hojas pierden
su importancia como órganos de asimilación y quedan subdesarrolladas,
permaneciendo sólo en la forma de escamas desprovistas de coloración
verde.”
Lyubimenko (1923) destacó que no hay marcada diferencia entre plantas
autotróficas y heterotróficas. El saprofitismo, según él, es la consecuencia
natural de la síntesis de compuestos orgánicos. Los organismos que son
capaces de sintetizar compuestos orgánicos a partir de minerales,
preferentemente usan compuestos orgánicos listos y son capaces de recibir su
nutrición en la misma forma que los organismos saprofitos. El saprofitismo no
es por sí mismo una propiedad específica de cierto grupo de organismos, por el
contrario, es una característica de todos los organismos, con la posible
excepción de las bacterias nitrificantes. Por lo tanto aparece en la naturaleza
en diferentes grados. Por un lado encontramos plantas que sólo
accidentalmente asimilan compuestos orgánicos del entorno, estas son las
saprofitas facultativas. Para ellas, la alimentación saprofítica no es
indispensable. Por otro lado encontramos plantas que son verdaderas
saprofitas, que han perdido completamente la capacidad de sintetizar
compuestos orgánicos de los inorgánicos. Entre estas dos categorías existe
una transición gradual de formas que atenúa la división y en realidad,
eliminando la división (Lyublmenko, 1923, pagina 186).
Como puede verse de la cita anterior, Lyubimenko considera que todas las
plantas son capaces de nutrición orgánica en mayor o menor medida. La única
excepción de acuerdo con él, son las nitrificadoras. Podemos agregar, que
recientemente se ha acumulado material que muestra que aún esos
organismos pueden utilizar nutrientes orgánicos y crecer en medios nutrientes
orgánicos de la misma manera que muchas bacterias saprofitas.
Es bien conocido que las plantas pueden estar en cerrada simbiosis, no sólo
con hongos, sin también con bacterias. Las bacterias formadoras de nódulos,
que penetran en la raíz de las leguminosas, con la formación de colonias en la
forma de nódulos. Los nódulos son protuberancias de la raíz llenas de células
bacterianas. Estas bacterias crecen abundantemente, se multiplican en los
nódulos y producen substancias nitrogenadas que son utilizadas por las
plantas.
Análogos nódulos se forman en las raíces de otras plantas (no leguminosas)
Por ejemplo se han descripto nódulos en raíces de Alnus Gaertn., Myrica L.,
Podocarpus L'Her., representantes de la familia Elaeagnaceae Lindl.; E.
multiflora Thunb., E. angustifolius L., en Shepherdia canadensis Nutt., en varias
especies de Ceanothus L., en Coraria japonica Gray, y otros.
Los nódulos en estas plantas se forman por diferentes representantes de los
microbios, como bacterias, micobacterias, actinomicetes, y, de acuerdo con
nuestras investigaciones, también por proactinomicetes.
Estos organismos tienen un definido efecto positivo en las plantas, muy
parecido al de las bacterias formadoras de nódulos en las leguminosas. Bajo
condiciones en que las bacterias y por consiguiente, los nódulos están
ausentes, las plantas crecen débiles y a menudo no alcanzan el estadio de
floración y fructificación (Fred, Baldwin y McCoy, 1932; Jongh, 1938).
Hay muchos simbiontes microbianos que viven en las hojas de las plantas,
formando particulares protuberancias –nudos- en los tejidos. Esos nudos son
formados por bacterias y micobacterias. Estos organismos, al multiplicarse
llenan las células de los nudos de la misma forma que las bacterias dela raíz
forman nódulos en los tejidos de la raíz. Estos microorganismos, de acuerdo
con nuestras investigaciones, ejercen un efecto positivo en el crecimiento de
las plantas Sin ellos, las plantas crecen débilmente y algunas especies no
llegan al estadio de floración y fructificación, como puede verse en Ardisia
Thunb. La plantas permanecen enanas, pero una vez infectadas con la
correspondiente bacteria, aparecen los nudos en sus hojas, las plantas
mejoran, asumen la apariencia normal, empiezan a florecer y a fructificar
(Jongh, 1938).
Alrededor de 400 especies de plantas con los nudos arriba descriptos se
mencionan en la literatura, incluyendo 30 especies del género Ardisia Thunb.,
244 especies del género Pavetta L., 42 especies del género Psychotri L., 5
especies de los géneros Amblyanthopsis y Amblyanthus D. C., 1 especie del
Dioscorea L., y otras. Las bacterias que forman los nudos pasan de planta a
planta a través de la semilla.
De acuerdo con la información de muchos investigadores, todos estos
microbios simbiontes, que viven en los tejidos de las plantas verdes, afectan a
las plantas a través de sus productos metabólicos. Algunos forman y excretan
compuestos bióticos que activan el crecimiento de las plantas, otros, en
adición, fijan nitrógeno atmosférico y se lo pasan a la planta en el estado fijado.
Habiendo adjudicado una gran importancia a los arriba aludidos bacterias y
micorrizas que proveen de nutrición orgánica a las plantas verdes, debemos
dirigir la atención a otras especies de microorganismos que están fuertemente
vinculados con el sistema radicular de las plantas, aunque este vínculo es de
diferente carácter, Tenemos en mente la flora de la rizosfera.
Un inmenso número de microorganismos vive alrededor de las raíces de las
plantas. Los más numerosos entre ellos son las bacterias. ¿Tienen algún efecto
en la nutrición de las plantas? ¿Qué efecto tienen sus productos metabólicos
sobre la planta? ¿Son ellos asimilados por las plantas y qué papel juegan en el
crecimiento de ellas? ¿Si las plantas utilizan los productos metabólicos de los
simbiontes microbianos, por qué entonces no usarían análogas substancias
producidos por organismos de vida libra?
Aparentemente, la diferencia reside en que en el caso de la simbiosis entre la
planta y el microbio, los productos metabólicos permanecen dentro del tejido,
mientras que en el último caso, estos compuestos se forman y acumulan fuera
de la planta, en el entorno que la rodea y para poder estar disponibles para la
planta, deben encontrar un camino hacia la planta a través de las raíces o por
otros medios.
Muchos autores piensan que la permeabilidad del sistema radicular para las
substancias minerales difiere de aquél para los compuestos orgánicos. El poder
de succión de las raíces es diferente en estos dos casos. El poder de succión
de la planta heterótrofa parásita (Lathraea squamaria) es 22.7 atm, y el poder
de succión de la planta huésped (Prunus padus L. ) es sólo 3.7 atm (Kostychev,
1933).
Muchos autótrofos típicos cuyo poder de succión no supera el promedio,
asimilan fácilmente compuestos orgánicos, Hutchinson y Miller (1912),
estudiando la absorción de nitrógeno por la plántula de arveja, encontraron un
fenómeno, que desde el punto de vista de la opinión prevalente, que ellas
absorbían compuestos nitrogenados más fácilmente que nitrógeno en forma de
nitrato, fue paradojal.
Este resultado fue confirmado por numerosos investigadores (ver más tarde)
Como puede verse, por lo anterior, la subdivisión de las plantas de acuerdo con
su forma de nutrición en autótrofas y heterótrofas debe considerarse como
relativa. Cada una de las llamadas plantas autótrofas es capaz, en mayor o
menor medida, de asimilar compuestos orgánicos.
Ciertamente, el significado de “nutrición” debe ser diferenciado del término
“síntesis de substancias orgánicas” Nutrición, en su real sentido debe
reservarse a todos los procesos que están directamente involucrados con la
asimilación de substancias por las partes vivas del organismo desde el
momento en que el compuesto orgánico es preparado (Lyubimenko, 1923, pag.
180). Este proceso ocurre similarmente en todos los organismos, ya sea que
pertenezcan al reino vegetal o animal. Todos se alimentan de compuestos
orgánicos. Para construir un cuerpo vivo, todas las células, ya sean
microbianas, animales o vegetales, deben contar con compuestos orgánicos
elementales listos, o sea sus bloques de construcción de compuestos
nitrogenados y no nitrogenados que participan en el proceso general.
La diferencia entre la nutrición vegetal y la animal reside en que la última
obtiene compuestos orgánicos listos, sintetizados del exterior, mientras las
plantas sintetizan para ellas.
El proceso de nutrición vegetal consiste entonces de dos elementos: a) síntesis
de substancia orgánica desde elementos inorgánicos, Lyubimenko este
proceso como preparativo, dispensable, desde el punto de vista de la nutrición
en el sentido estricto de la palabra y b) nutrición propiamente dicha, o sea la
asimilación de partículas elementales orgánicas para la formación de un
organismo vivo. Este proceso ocurre de la misma manera en plantas y
animales.
La nutrición de plantas y microorganismos puede ocurrir de acuerdo con el
primero o el segundo de los procesos, pero cada proceso se puede manifestar
en diferente grado, de acuerdo con las condiciones de existencia y diferencias
genéticas.
Vinogradskii, en sus brillantes estudios sobre quimiotrofismo de los
microorganismos trazó una línea definida entre estos dos tipos de nutrición,
dividiendo a todos los microorganismos en autótrofos y heterótrofos. Lebedev
(1921), contrariamente, asumió que estos dos tipos de microorganismos
difieren entre sí, no por su forma de nutrirse, sino por la forma en que obtienen
su energía. Lebedev piensa que ambos, autótrofos y heterótrofos asimilan el
CO2, pero mientras los primeros obtienen su energía a expensas de la
oxidación de substancias inorgánicas, los últimos lo hacen a expensas de la
oxidación de compuestos orgánicos. Este científico fue el primero que mostró
experimentalmente que los heterótrofos utilizan CO 2. Su información fue
confirmada por muchos otros investigadores.
Al presente, se ha confirmado que muchos microorganismos, inclusive
heterótrofos pueden asimilar CO2 y Werkman inclusive asume que todos los
organismos en general asimilan CO2 y que esto es una función fisiológica
importante que asegura la síntesis de productos metabólicos intermedios
indispensables (Werkman y Wilson, 1954).

Parte III, continuación:

Efecto del humus en el crecimiento de las plantas
El efecto positivo del humus sobre el crecimiento y metabolismos de las
plantas ha sido probado por numerosas observaciones y los resultados de
exactos experimentos de laboratorio.
Bottomley y sus colaboradores (1914) cultivaron lenteja de agua Lemna
minor L., en la solución nutritiva de Knopp suplementada con pequeñas dosis
de extractos acuosos de turba bien compostada. Obtuvo los siguientes
resultados: Después de 35 días crecieron 30 individuos a partir de los 10 que
había originalmente en la solución, en ausencia de humus; mientras que en
presencia de humus, crecieron 132 individuos. Los que crecieron en presencia
de las substancias orgánicas fueron mucho mayores y su color verde más
intenso. Su peso seco fue 50,5 mg por 100 individuos, mientras que el peso
seco del mismo número de plantas de control fue de sólo 29,4 mg. En su último
experimento Bottomley (1920) mostró que el extracto de turba tiene un efecto
positivo también sobre otras plantas tales como Lemna minor L., Salvinia
natans All., Limnobium stolonifer L. El rinde de masa seca, cuando se cultivaron
en presencia de extracto de turba fue mayor que en los controles.
La turba, después de ser procesada por microorganismos, estimuló el
crecimiento de las plantas más marcadamente de lo que lo hizo la turba
natural, sin compostar. Sobre esta base, Bottomley preparó un fertilizante
especial: humogen. Esta preparación dio resultados positivos, tanto bajo
condiciones de laboratorio como a campo. Fue patentada y recomendada para
su uso en agricultura, con varias plantas. De acuerdo con lo establecido en la
patente, se agregaba peptona a la turba para mejorar el crecimiento y
metabolismo delos microorganismos. Para acelerar la descomposición de la
parte orgánica de la turba se agregaban Azotobacter, Rhizobium, y otras
bacterias. La turba inoculada se mantenía a temperatura de 25º C por 3-4
semanas. A continuación, la preparación estaba lista para usar.
Mockeridge (1917, 1924) repitió los experimentos de Bottomley y confirmó
totalmente su data, Ella agregó extractos de turba compostada y no
compostada a la solución nutritiva donde cultivó la lenteja de agua. Después de
9 semanas, a partir de 10 individuos, crecieron 249 plantas en el control, 3.134
en presencia de turba procesada y 1.080 en presencia de turba sin procesar El
peso seco de la lenteja de agua fue de 6,5 mg en el control y de 19,5 mg en la
presencia de humus de turba.
En nuestros experimentos hemos usado composts bien procesados,
preparados con distintos residuos de plantas, tales como paja de Euagropyrum,
trigo, avena, heno de alfalfa, y también de estiércol bien procesado. Se agregó
a la solución nutriente el extracto concentrado de estos composts y de
estiércol. En cantidades de 5-10 gotas por 100 ml del medio, lo que resulta en
0.005-0.01 mg de la substancia seca. Esta solución nutriente se usó como
substrato para el cultivo de Lemna minor y para la fertilización del substrato
arenoso en el que cultivamos cereales y leguminosas.
Lemna minor L. se cultivó bajo condiciones estériles en vasos de vidrio
ubicados en la ventana del laboratorio. En los vasos de control, el medio no
contenía los extractos arriba mencionados. La cosecha fue contada después de
85 días Se contó el número de individuos y se determinó su peso seco. Se
eligieron plantas que eran similares en su apariencia. Los resultados están en
la tabla 30.
Tabla 30
Efecto de los composts de plantas en el crecimiento
(después de 85 días)

Número de
Peso seco (mg) Peso seco (mg)
Composts especímenes en el
total de 100 plantas
vaso
Control (sin humus) 180 29 16
Compost de
2,500 800 32
Euagropyrum
Compost de trigo 1,200 324 27
Compost de avena 1,800 540 30
Compost de trébol 2,200 726 33
Lavado de estiércol 1,800 540 30

Como puede observarse, de esos datos, las plantas (Lemna minor) crecen
mucho mejor en contenedores con la substancia orgánica. Las plantas fueron
mayores, se colorearon más intensamente y su sistema radicular se desarrolló
mejor.
Los diferentes composts tienen diferente efecto sobre el crecimiento de Lemna
minor. El mayor efecto se obtuvo con el uso del compost de Euagropyrum y
con lavado de estiércol. Las plantas que crecieron en ellos fueron las que más
desarrollaron. El extracto de compost de paja de avena favoreció el crecimiento
de las plantas en número similar a aquéllas obtenidas con el extracto de lavado
de estiércol, pero el tamaño fue menor. El menor efecto se obtuvo usando el
compost de paja de trigo..
Voelcker (1915) ensayó el efecto de la preparación de Bottomley en macetas y
en condiciones de campo sobre arvejas, avena y trigo sarraceno. El mayor
incremento de lo cosechado fue para el caso de trigo sarraceno, que llegó al
407%; El efecto sobre la avena fue más débil, con un incremento del 131%. En
el experimento con arvejas sólo se observó el incremento de la masa verde,
que llegó al 231%. El rendimiento en grano, por el contrario fue menor (87%)
que en el control (100%). Clark y Roller (1924) obtuvieron resultados similares.
Esta planta creció mucho menos en un medio nutriente bajo condiciones
estériles que en la presencia de humus. El crecimiento más abundante de la
lenteja de agua se observó en vasos conteniendo estiércol y turba
compostados. El extracto de compost de alfalfa tuvo el efecto menos
destacado. Estos autores habían encontrado (1931) que los composts son más
efectivos cuando están contaminados con bacterias que cuando son estériles.
Aschby (1929) destaca que las substancias orgánicas de los residuos de
plantas procesados, aunque no sean indispensables para el crecimiento de las
plantas (Lemna minor L.), tienen, sin embargo, un considerable efecto sobre su
crecimiento. Saeger (1925) introdujo una solución de extracto alcalino de
humus en el medio nutriente de cultivo de lenteja de agua. El rendimiento
duplicó el control. Encontró que la acción sobre el crecimiento del humus y
extracto de levadura era igual
Hillitzer (1932) sobre la base de estos experimentos y el análisis de la
información disponible, concluyó que el humus del suelo y los composts
estimulan el crecimiento de las plantas. Los componentes del humus actúan
específicamente sobre el sistema radicular, impulsando el crecimiento.
Olsen (1930) realizó una completa serie de experimentos sobre el crecimiento
de lenteja de agua y girasol en presencia y en ausencia de composts en el
medio nutriente. Mediante estos experimentos comprobó los resultados de
Bottomley y Voelker. En presencia de pequeños contenidos de humus
(extractos acuosos de turba procesada) la lenteja de agua , tanto como el
girasol crecieron considerablemente mejor que en su ausencia. Empleó la
solución de Dettmer como medio nutriente. El peso seco de la lenteja de agua
cultivada en presencia de humus fue de 482 mg y en el vaso de control, de 189
mg; El peso del girasol, en presencia de humus fue de1.33 g y en el vaso de
control, de 0.80 g. En presencia de citrato de hierro, la acción positiva del
humus fue menor o inexistente (de acuerdo con el autor). Sobre la base de esta
última observación concluye que el principio activo en el humus y composts,
son algunas formas de compuestos de hierro.
Nuestros experimentos para aumentar las cosechas agrícolas con los
composts arriba mencionados dieron similares resultados positivos. Hemos
cultivado trigo, centeno, rye gras y trébol sobre pura arena de cuarzo,
humedecida con la solución nutriente mineral, en presencia y en ausencia de
extractos acuosos de composts. Las cantidades de compost agregadas fueron
las mismas que en los experimentos con lenteja de agua.
Los experimentos fueron conducidos en condiciones de esterilidad. Las plantas
se cultivaron por 20-40 días, arrancadas en conjunto, con sus raíces y
analizadas. Se registraron su peso total, altura y apariencia general. Los
resultados más espectaculares se obtuvieron con trigo y rye gras (Tabla 31)
Tabla 31
Efecto del humus sobre el crecimiento de las plantas
(peso seco en g)

Rye gras:
Trigo: altura de Trigo: peso de Rye gras: peso
Composts altura de
planta, cm planta, g de planta, g
planta, cm
Control (sin
compuestos 14.9 0.95 10.2 0.64
orgánicos)
compost de
18.5 1.63 12.6 0.98
Euagropyrum
Estiércol 17.1 1.52 12.1 0.71

Como puede observarse en la tabla, el efecto del compost de Euagropyrum y

el del estiércol sobre el crecimiento del rye gras y del trigo fue de la misma
magnitud. Todas las plantas tienen mayor masa cuando se cultivan en
presencia de substancias orgánicas que en vasos de control. El mayor
incremento de masa se obtuvo con cereales. El trébol reaccionó menos a la
introducción de compuestos orgánicos.
En la segunda serie de experimentos ensayamos el efecto de estiércol bien
procesado y de paja fresca. Usamos un gramo y medio de estiércol y 20 g de
paja, junto con la solución nutriente de Knopp aplicados a cada kg de substrato
arenoso. Los experimentos se realizaron en condiciones de esterilidad. Se
cultivaron trigo, avena y arvejas. Los resultados obtenidos en presencia de
estiércol fueron similares a los del experimento previo. En presencia de humus,
el rendimiento de la avena fue 35% mayor, del trigo 25% mayor y de las arvejas
(peso fresco) 42% mayor. En presencia de extractos de paja fresca, la cosecha
fue similar a la de los vasos de control.
En una serie de experimentos comparamos el efecto de los extractos acuosos
de Euagropyrum y de un extracto de paja compostada de la misma planta
sobre el crecimiento del rye gras. El peso seco de las plantas en el vaso de
control (sin fertilizante orgánico) fue de 0,5 g, en presencia del extracto de paja
fresca, de 0,7g y en presencia de Euagropyrum compostado, fue de 1,1 g.
En experimentos con plántulas de pino se encontró que pequeñas dosis de
compost de Euagropyrum estimuló marcadamente el crecimiento de las
plántulas experimentales. Las tratadas crecieron a mayor altura que los
controles, sus tallos fueron más gruesos, sus agujas más largas y de color más
brillante (figura 61) Krasil'nikov y Raznitsyna, 1946, Raznitsyna, 1942).
Chester y Street (1948) cultivaron lechuga en una solución completa de
nutrientes con y sin el agregado de substancias orgánicas. Ensayaron extractos
de humus del suelo, extracto acuoso de caseína e hidrolizados de levadura.
Todas las soluciones contenían 6.05 mg N por 10 m3 del medio de cultivo. Los
rendimientos de las plantas fueron como sigue::

Control (ausencia de compuestos orgánicos); peso seco (en g) 0.157

con el agregado de humus del suelo; peso seco (en g) 0.199
Con el agregado de hidrolizado de caseína; peso seco (en g) 0.158
con el agregado de extracto de levadura, peso seco (en g) 0.172

Figura 61. Efecto estimulante del compost de Euagropyrum sobre el crecimiento de

plántulas de pino
Swaby (1942) no obtuvo incrementos en el rendimiento de leguminosas
cultivadas en presencia de substancias orgánicas, pero pudo notar el efecto
estimulante del os microbios, cuando los experimentos se hacen bajo
condiciones no estériles
De acuerdo con Schaffnit y Neumann (1953), la turba compostada tiene un
efecto estimulante sobre el crecimiento de las papas y sobre la germinación de
las semillas de alfalfa. Ellos atribuyen el efecto estimulante a la presencia de
microorganismos que crecen abundantemente en estos composts.
Andreyuk (1954) estudió el efecto de composts de turba especialmente
preparados en el crecimiento de cultivos de granos. Los composts se
prepararon con turba, 25-50% (en peso) estiércol y 1,5-2,0 fluor fosfato. Luego
se incubaron bajo condiciones de laboratorio o en el campo por 4-7 meses o
más. En algunos experimentos se introdujo Azotobacter, bacterias de la
celulosa y otras bacterias. El rendimiento del centeno de invierno en presencia
de estos composts fue de 2-2,5 veces mayor que en el control (5,3 quintales)
Muchos otros autores han notado el efecto positivo de pequeñas dosis de
humus (Nikishkina, 1948; Logvinova, 1939; Street, 1950, y otros). Los
detallados estudios de Khristeva (1948) habían mostrado que las soluciones
de ácidos húmicos ejercen un efecto directo sobre las plantas superiores. En
concentraciones extremadamente bajas (0.001 % y 0.0001%) impulsaron el
crecimiento y mejoraron el rendimiento del trigo, avena, cebada, remolacha
azucarera, tomates y otras plantas. De las plantas ensayadas, las papas y
remolacha azucarera tuvieron la mejor reacción a la aplicación de humus.
Buena reacción del trigo, cebada, avena, mijo, maíz, arroz, trigo sarraceno.
Euagropyrum y alfalfa. El humus tuvo poco efecto sobre arvejas, porotos,
lentejas, maní, algodón y sésamo y casi ningún efecto sobre girasol, planta del
aceite de castor, zapallo, hibiscos, etc. El mayor incremento de rendimiento por
la influencia de las substancias húmicas excede lo obtenido por aplicación de
cantidades equivalentes de fertilizantes minerales por más o menos 10-50%.
La acción de los fertilizantes húmicos ha sido ensayada por el autor en
diferentes suelos tales como podzoles, serozems, chernozems, castañozems y
otros. En todos los casos el efecto fue positivo.
De acuerdo con Khristeva, las substancias húmicas encuentran su camino
dentro de las plantas en pequeñas cantidades. Allí estimulan el sistema fenol-
oxidasa y participan en el metabolismo general de la planta. La función
fisiológica de las substancias húmicas es la promoción de la respiración de la
planta. Como resultado, un incremento del flujo de nutrientes, activación de los
procesos de síntesis y así se produce un mayor crecimiento de la raíz y partes
aéreas.
La mayor reacción a las substancias húmicas se observa en plantas jóvenes. El
peso de la raíz y consecuentemente el crecimiento de toda la planta se
incrementa.
Kock (1955), destacando el efecto positivo de las substancias húmicas sobre la
planta, lo explica por la acción de hierro que está presente en el humus. Esto,
sin embargo no ha sido confirmado por los estudios de otros investigadores.
Tovarnitskii y Rivking (1937), Tovarnitskii y Statkovskaya (1938), Thiman, Lane
y otros (1933, 1939) usaron orina y extractos de levadura para tratamientos de
presiembra de avena, trigo y otras plantas para estimular su crecimiento.
En Italia del sur se usa ampliamente la orina del ganado para tratar las semillas
antes de la siembra de los cereales (Zeding, 1955). Virtanen y Hausen (1933-
1934) agregaron extracto de levadura a cultivos en agua y en arena de arvejas.
La floración ocurrió 5-10 días más temprano y rindió un 50% más que el
control. Estos autores también observaron que esos efectos no podían
reproducirse cuando se cultiva las plantas en suelos ricos en humus. Esto
puede explicarse porque en los suelos ricos en humus ya hay una gran
cantidad de substancias bióticas
A la información de este capítulo se le debe agregar una gran cantidad de
material sobre fertilizantes bacterianos.
La práctica de emplear fertilizantes conteniendo bacterias da, en ciertos casos,
resultados positivos. El agregado de “azotogen” de turba produce un
incremento de 10-25% en las cosechas. Este incremento puede resultar aún
superior en muchos casos.
Las preparaciones bacterianas usadas como fertilizantes bajo el nombre de
“azotogen” son extensamente usadas en nuestro país. Están preparadas de
cultivos de Azotobacter en terrones de turba. La turba seleccionada debe ser
de calidad adecuada. No debe ser ácida y debe estar bien procesada. Durante
la producción de la preparación, se agregan fuentes de nutrición fácilmente
asimilables, como azúcares, alcohol, jugo de remolacha, etc. La turba
inoculada con Azotobacter se incuba a temperatura y humedad óptimas;
Frecuentemente se la mezcla para asegurar una mejor aireación.
La incubación dura 10-20 días. Durante este período, la turba queda bien
compostada. No sólo Azotobacter, sino también muchas otras bacterias,
hongos y actinomicetes, crecen en abundancia en la masa dela turba.
El número de bacterias no formadoras de esporas al momento de su máxima
acumulación, en una buena preparación, alcanza varios billones de células por
gramo. El número de Azotobacter alcanza 100-200 millones por gramo (Tabla
32).
Tabla 32
Composición cuantitativa de la microflora en el azotogen de turba
(número de células en miles por g, en el período de máxima acumulación)

Bacterias, Bacterias, no
Mico- Actino-
Preparación formadoras formadores de Hongos
bacteria micetes
de esporas esporas
Turba no
100 40,000 1,000 1,500 750
compostada
Turba
compostada sin 1,500 5,500,000 1,000,000 3,500,000 150,000
Azotobacter
Turba
compostada
1,700 7,500,000 1,500,000 3,200,000 250,000
con
Azotobacter

Como puede verse en la tabla, la turba compostada, ya sea con presencia o

ausencia de Azotobacter, contiene la misma cantidad de microbios. Su número
total excede considerablemente el de la turba inicial.
La composición de la microflora del compost varía con el incremento de la
madurez del compost. En los primeros días de incubación, crecen
abundantemente los géneros no formadores de esporas Bacterium,
Pseudomonas, y los hongos. Los hongos crecen sólo en la superficie. Las
micobacterias se pueden encontrar en grandes cantidades en la turba
compostada. Al final de la incubación (madurez) de la preparación, las
bacterias y hongos decrecen en número, siendo su lugar ocupado por
actinomicetes. Alcanzan tal desarrollo que los agregados de turba quedan
cubiertos por éstos. Esta cobertura tiene la apariencia de una harina blanca
visible a ojo desnudo. La madurez del compost puede determinarse entonces
por la intensidad del crecimiento de los actinomicetes.
Los análisis muestran que este desarrollo consecutivo y cambio de microflora
se observa en aproximadamente las mismas relaciones cuantitativas en turba
compostada sin Azotobacter.
La introducción de ésta última, sin embargo, puede causar un cambio en la
composición de las especies de las bacterias no formadoras de esporas, pero
ello puede ser marcado sólo para ciertas especies. La composición general del
grupo no cambia.
Hemos realizado ensayos de turba (compostada o sin compostar, en presencia
o en ausencia de bacterias) durante una cantidad de años. Han estado
involucrados diferentes plantas, granos y cultivos de cosecha. El carácter
general de la eficacia de estos preparados bajo condiciones de campo se
indica en la tabla 33.

Tabla 33
Comparación de la acción de preparados fertilizantes sobre el rendimiento

Inoculación Remola-
Maíz: Papas: Remo- Trigo:
bacteriana Maíz: Papas: cha: Trigo:
quintales quintales lacha: quintales
del % % quintales %
por Ha por Ha % por Ha
preparado por Ha
Control 32.0 100.0 230 100 559 100 -- --
Azotobacter
33.9 106.0 255 110.8 610 109 -- --
cepa 54
Turba
37.1 116.0 261 113.7 649 116 -- --
azotogen
Turba
36.7 114.7 263 114.3 639 114 -- --
compostada
Turba no
34.1 108.7 254 110.4 615 110 -- --
compostada

Como puede verse en la tabla, el cultivo puro de Azotobacter (cepa de

colección. No 54) es menos efectivo que la turba azotogen preparada con
turba. La turba compostada sin Azotobacter produce aproximadamente el
mismo aumento de rendimiento que el azotogen preparado con turba. Como ya
fuera puntualizado, la turba no compostada es menos efectiva que la
compostada.
Hemos obtenido data similar de experimentos conducidos bajo diferentes
condiciones en suelos podzoles, cerca de Moscú i en los serozems de Kigirz
URSS y Tadzhik URSS (Valle Vakhsh). En la mayoría de los casos, el azotogen
preparado de turba y la turba bien compostada con Azotobacter produjo
similares incrementos de rendimiento.
En algunos casos, los composts bacterializados fueron más efectivos que los
preparados sin bacterias (Tabla 34)

Tabla 34
Crecimiento de la lenteja de agua en agua en presencia de pequeñas
cantidades de
compost de turba inoculado con bacterias
Peso seco de 100
Condiciones de los experimentos Total de muestras
muestras en mg
Control (sin compost) 106 26
Compost no inoculado con
450 37
bacterias
Compost inoculado con bacterias:
Az. Chroococcum 510 39
Ps. flourescens No. 14 580 38
Ps. flourescens No. 15 85 27

Muchos investigadores asumen que el efecto favorable del humus, estiércol y

composts reside en su contenido de cenizas, de las que el nitrógeno es el
constituyente de mayor importancia. Esto último, de acuerdo con estos
investigadores determina la eficacia de la masa compostada y del humus del
suelo. A mayor contenido de nitrógeno en el compost, más efectivo resulta. De
acuerdo con los adherentes a este punto de vista, después de la mineralización
de los compuestos orgánicos, las substancias nitrogenadas se transforman en
substancias inorgánicas y así quedan disponibles para las plantas.
De acuerdo con nuestras observaciones y la información disponible en la
literatura, los principios activos del humus y composts no son los nutrientes
minerales presentes en los mismos, sino las substancias orgánicas y los
metabolitos activos de los microbios. Las substancias minerales aplicadas en
cantidades equivalentes a las contenidas en los composts no han tenido un
efecto comparable.
Los elementos minerales obtenidos mediante el quemado de estiércol, turba
compostada o humus del suelo no producen un efecto comparable al obtenido
por aplicación de las substancias orgánicas. Hemos hecho experimentos con
lenteja de agua en soluciones nutrientes suplementadas con pequeñas
cantidades de extractos preparados con estiércol, compost y humus. En otros
experimentos se agregaron cenizas a la solución de nutrientes. Las cenizas se
obtuvieron quemando cantidades equivalentes de estas substancias. Los
resultados se dan en la tabla 35.
 Tabla 35
Efecto de la ceniza de humus sobre el crecimiento de Lemna minor L.
(al día 50 de crecimiento)
El número de Peso seco de 100
Experimento
especímenes en el vaso especímenes, mg
Control 750 27
Estiércol 1,800 39
Ceniza de estiércol 850 33
Turba compostada con
1,150 40
bacterias
Ceniza 900 31
Humus de suelo 1,100 33
Suelo quemado 700 29

Lochhead y Thexton (1952) obtuvieron resultados similares. De acuerdo con su

data, la ceniza obtenida de composts tiene un efecto menor sobre el
crecimiento de los microbios que los composts mismos.
Todo esto habla a favor de asumir que la estimulación del crecimiento de las
plantas por el humus observado, está causada, no por la fracción mineral, sino
por otras substancias.

Efecto del humus en el contenido de vitaminas de las plantas

Los composts de vegetales, estiércol y substancias húmicas no sólo activan el
crecimiento de las plantas e incrementan sus rendimientos, también mejoran su
valor nutritivo, que es materia de gran importancia. Las plantas cultivadas en
campos fertilizados con estiércol son más ricas en vitaminas y otras
substancias valiosas que las cultivadas en campos no fertilizados. McCarrison
(1926) encontró que las semillas de mijo y trigo cosechadas de campos
fertilizados, contienen más vitaminas, que semillas de campos bajo fertilización
mineral. Los animales alimentados con forraje de campos fertilizados con
estiércol, fueron más resistentes a las infecciones y su apariencia más
saludable que los alimentados con forraje de campos no fertilizados.
Roulands y Wilkenson (1930) determinaron vitaminas del complejo B en el
heno de trébol cultivado en campos fertilizados y no fertilizados. En el primer
caso, el trébol tuvo más contenido de vitaminas. Las ratas de laboratorio
alimentadas con trébol de campos fertilizados ganaron peso más rápidamente
que las alimentadas con trébol de campos no fertilizados. En los 30 días del
experimento, las primeras ganaron 110 g y las segundas 60 g.
Clark (1935) encontró más vitamina B y C en cultivos de lenteja de agua en
solución nutriente suplementada con humus que en un cultivo similar sin
suplemento de humus.
Nath y colaboradores (1927, 1932) han mostrado que la substancia orgánica
del estiércol y composts, como el aceite de hígado de bacalao y algunas otras
substancias mejoran el crecimiento de las plantas y aumentan su contenido de
vitaminas. Las semillas de cereales cultivadas en campos fertilizados tienen
mayor viabilidad y el porcentaje de germinación es mayor que en las semillas
de campos fertilizados con fertilizantes minerales.
De acuerdo con Graff (1928) la hierba timotea, festuca e hierba de las
praderas (Phleum pratense L., Festuca rubra L., F. pratensis Huds. y Poa.
pretensis L.) tuvieron más contenido de vitaminas del grupo B cuando se
cultivaron en suelo fertilizado con urea que cuando se cultivaron en suelos en
los que se habían aplicado fertilizantes minerales

Antoniani y Monzini (1950) analizaron plantas que crecieron en suelos irrigados

con aguas cloacales y plantas que crecieron en campos irrigados con agua
suplementada con sales nutrientes minerales. El contenido de vitamina B fue
mayor en las primeras plantas.
Leong (1939) comparó el efecto del estiércol y fertilizantes minerales en el
contenido de vitamina B1 de tejidos de trigo y cebada. El contenido de vitamina
B1 del trigo fue similar en ambos casos , pero la cebada contenía el doble de
vitaminas cuando se cultivó en presencia de estiércol que en presencia de
fertilizante totalmente mineral (Tabla 36)

Tabla 36
Contenido de vitamina B1 en plantas en relación con la fertilización
(µg por 1g masa de planta)
Fertilizante Trigo Cebada
Sin fertilizante (control) 1.0 1.1
Estiércol 1.2 2.0
Fertilizante totalmente mineral P, K,
1.2 1.1
Mg, NO3

Hurni (1944, 1945) cultivó plantas en arena en presencia de un fertilizante

totalmente mineral y encontró que en esas condiciones, la formación de tiamina
fue menor que durante el cultivo en presencia de humus.
En los experimentos de Lebedev (1953), la alfalfa cultivada en campos
fertilizados con estiércol o composts tuvo un contenido de 81 mg/g de caroteno
durante el período de formación de pimpollos, mientras que las plantas no
fertilizadas contenían sólo 27 mg/g.
Ott (1937) notó un incremento en el contenido de vitaminas de las plantas
cultivadas en campos fertilizados con una mezcla de fertilizantes orgánicos y
minerales.
Hammer y Maynard (1942), resumiendo la literatura, notaron que el contenido
de vitaminas en las plantas varía de acuerdo con las condiciones climáticas y
del suelo, estación, edad de las plantas, etc. Ellos adjudicaron la mayor
importancia al valor nutritivo del suelo y especialmente la presencia de humus y
fertilizantes en general.
Al presente, la mayoría de los investigadores concentran su atención en las
fuentes minerales de nutrición como los factores que afectan el contenido de
vitaminas de las plantas. Las vitaminas A, C, B1 y B2 fueron medidas en los
tejidos de varias plantas (leguminosas y cereales) cultivadas en campos
fertilizados con varias sales de potasio, fósforo y nitrógeno (Scheunert y
Wagner, 1938; Scharer y Preissner, 1954, y otros).
Luego de la aplicación al suelo de un fertilizante totalmente mineral, la
zarzaparrilla negra dio un rinde de bayas de 5.158 kg/Ha (Stepanova, 1950).
Su contenido de vitamina C total fue de 1.827 mg/kg. De acuerdo con Dyakova
(1945), la aplicación de una porción de nitrógeno aumentó el contenido de
caroteno de la avena de 10 a 28 mg y una aplicación del triple de nitrógeno
aumentó el caroteno de 10 a 53 mg (en el estado de encañazón). La aplicación
de calcio al suelo también incrementó el contenido de caroteno de las plantas.
La falta de calcio en el suelo disminuye la síntesis de tiamina en las plantas de
tabaco (Ovcharov, 1955).
Algunos autores estudiaron la variación del contenido de vitaminas en las
plantas en conexión con la aplicación de micro elementos al suelo. Mc Harque
(1924) notó un efecto positivo del manganeso en la acumulación de vitamina
B1 en las semillas de trigo, arroz, tomates y frutas cítricas. Hammer (1945)
aporta información sobre micro elementos en la formación y acumulación de
ácido ascórbico y caroteno en las plantas.
Scharer y Preissner (1954), sobre la base de sus experimentos, llegaron a la
conclusión que mientras más completa sea la mezcla fertilizantes usados,
mayor contenido de vitaminas de las plantas. La cantidad de vitaminas no
siempre se corresponde con el rendimiento de la cosecha. No es infrecuente
observar que el contenido de vitaminas sea alto para una cosecha de
rendimiento relativamente bajo. Por ejemplo en una cosecha promedio de
cebada de 4.3 g*
La vitamina B1 fue 440 µ g y en una cosecha de 11.93 g, 390 µ g por 100 del
grano. *[La unidad usada no está clara en ruso, probablemente se refiere a un
índice arbitrario de rendimiento]
Se observó un considerable aumento en el contenido de vitamina B 1 de las
plantas, en experimentos en que se aplicó fósforo granulado. En un suelo
arcilloso, débilmente ácido se obtuvieron los siguientes resultados: control,
rendimiento de grano 7,12 g, contenido de vitamina 558,7 µ g/100g; con la
aplicación de N, K y superfosfato, el rendimiento fue de 24,5 g y el contenido de
vitamina 798 µ g/100 g de grano.
La mayor acumulación de vitaminas se encontró en las plantas leguminosas.
Esto puede explicarse por la presencia de las bacterias noduladoras de la raíz.
Estas, al crecer en la raíz, forman vitaminas que encuentran su camino de los
nódulos a la planta.
Schounert y Wagner (1939, 1940) estudiaron el contenido de vitamina B 1 y B2
en las semillas de cebada y centeno, cultivados en campos que fueron
fertilizados por muchos años, como en campos que no tuvieron ningún
fertilizante. La comparación de los análisis no mostró ninguna diferencia
marcada en el contenido de vitamina entre plantas cultivadas en campos con y
sin fertilizante.
La falta de efecto alguno de los fertilizantes minerales o inclusive de
fertilizantes orgánicos también fue notada por varios otros investigadores
(Hornemann, 1925; Harris, 1934), Estos autores no han dado una explicación.
Debemos asumir que sus experimentos fueron conducidos en condiciones
desfavorables para la síntesis de vitaminas.
Existe información que muestra variaciones en el contenido de aminoácidos de
los tejidos de las plantas en presencia de diferentes fertilizantes. De acuerdo
con Sheldon, Blue y Albrecht (1948), la alfalfa y algunas otras plantas
cultivadas en campos fertilizados tienen una diferente composición cuantitativa
y cualitativa de amino-ácidos que las cultivadas en campos no fertilizados. El
mayor contenido se encontró en plantas cultivadas con estiércol.
Se debe hacer notar que la mayoría de los investigadores que estudiaron el
efecto de los fertilizantes minerales, no tuvieron en cuenta la microflora, y
especialmente la que habita en la rizosfera.
Se sabe que los fertilizantes minerales y orgánicos producen un gran efecto
sobre la vida de los microbios del suelo. Estos crecen y sintetizan distintas
substancias biológicamente activas, incluyendo las vitaminas. Por ejemplo,
para muchos suelos, se sabe que los fosfatos y nitratos aumentan
considerablemente el crecimiento y acumulación de bacterias, hongos y
actinomicetes. Azotobacter, bacterias noduladoras de la raíz ciertas especies
del género Pseudomonas y otras formas microbianas crecen bien sobre
terrones de superfosfato.
El material presentado muestra que los residuos de plantas y animales y las
substancias orgánicas en general tienen un efecto favorable sólo después de
su descomposición y procesamiento por los microorganismos. Esto es,
después de haber sido transformados en humus. Las substancias activas del
humus no son los residuos de plantas o animales, sino los productos del
metabolismo microbiano, los productos de una síntesis secundaria.
Asumimos que los principios activos de los composts, estiércol y del humus en
general, no son los elementos nutritivos minerales, o más estrictamente, no
tanto ellos, como algunos compuestos especiales de carácter orgánico. Estos
compuestos varían en su naturaleza y comprenden un grupo especial de las
llamadas substancias bióticas.

Substancias Bióticas del Suelo

De la información precedente, puede observarse que las pequeñas dosis de
substancias orgánicas presentes en los composts y en el estiércol, son
necesarias para la mejora del crecimiento de las plantas.
Surge la pregunta, ¿cuáles de estas substancias orgánicas son las activadoras
del crecimiento de las plantas?
Se ha dicho más arriba, que la parte orgánica del suelo cosiste en una multitud
de compuestos complejos de proporciones específicas y no específicas. La
mayoría de estos compuestos están relacionados con los ácidos húmicos.
Además de los ácidos húmicos se han encontrado muchas otras substancias
en la parte orgánica del suelo. Se incluyen substancias con propiedades bio
catalíticas. Entre estas, enzimas, vitaminas, auxinas, ciertos aminoácidos y
otras substancias bióticas.
Bottomley (1914-1920), Mockeridge (1924), Voelcker (1915), Clark (1935),
Saeger (1925), y otros han asumido que el efecto activador de los fertilizantes
húmicos, estiércol procesado y diferentes composts, con o sin bacterias está
condicionado por substancias especiales presentes en ellos, las llamadas
“auximones”
Su acción es similar a la de la biología de las levaduras. El análisis de la turba
infectada con bacterias mostró la presencia de bases purínicas. Las mismas
también se encontraron en células de Azotobacter. Esta fue la razón para la
conclusión de Mockeridge de que el compuesto activante de los composts y del
Azotobacter es uno y la misma substancia.
Estudios posteriores demostraron que la acción del humus es causada por
substancias especiales producidas por microbios (Nath, 1932, McCarrison,
1924, y otros).
Anstead (1935) propuso que estas substancias fueran llamadas fitaminas.
Según él, las fitaminas son producidas solamente por los microorganismos.
Luego de entrar en las plantas son transformadas en vitaminas. Estas últimas,
al entrar en el cuerpo humano y de los animales están sujetas a
transformaciones y se convierten en hormonas. La hormonas y vitaminas son
excretadas del cuerpo animal con la orina y los excrementos, encuentran su
camino al suelo o estiércol y allí son transformadas por los microorganismos en
fitaminas.
De esa forma, de acuerdo con Anstead, las fitaminas, vitaminas y hormonas
son producto de una y la misma substancia.
La misma visión fue sostenida por (1939). Asumió que no hay diferencia entre
las hormonas animales y las vitaminas y que están interrelacionadas.
El vínculo entre los bio catalíticos vegetales y animales es destacado por
muchos investigadores. Sin embargo no hay bases para hablar de un ciclo de
estas substancias en la naturaleza.
Lochhead y Chase (1943) intentaron dilucidar la naturaleza de la substancia
activadora del humus. Prepararon extractos de humus de suelo y lo sometieron
a varios procesos, tales como la extracción con solventes orgánicos, absorción
con carbón y otros absorbentes, seguido de elución. Los experimentos
mostraron que la ceniza de humus y composts no tiene efecto alguno sobre el
crecimiento de las plantas y de microorganismos de 63 cultivos microbianos,
sólo uno creció en presencia de cenizas obtenidas de composts. Los extractos
obtenidos de composts y del humus del suelo, tuvieron, sin embargo un efecto
positivo en el crecimiento. El extracto con acetona fue el más efectivo. En su
presencia, 26 cultivos crecieron bien, 26 cultivos lo hicieron adecuadamente y
11 cultivos no crecieron. Los extractos con alcohol y éter también dieron
buenos resultados. Los filtrados y eluados de carbón, cada uno tuvo un
pequeño efecto y su efecto fue mayor si se los combinaba.
Allison y Hoover (1936) notaron un efecto positivo de los ácidos húmicos en el
crecimiento y actividad de las bacterias noduladoras de la raíz. Ellos
descubrieron la presencia en la humina de una substancia especial que ellos
llamaron “Factor R” o “Coenzima”
Robinson y Endington (1946) encontraron una substancia biótica en los suelos
que llamaron “fluorina” Según ellos, esta substancia es absorbida en
considerables cantidades por las plantas.
Parker-Rodes (1940) encontraron auxinas en el suelo. La cantidad de auxinas
variaba de acuerdo con las propiedades del suelo. En suelos fertilizados con
estiércol, la cantidad de auxinas era 0,200 µ g/ kg y en suelos pobres, no
fertilizados sólo 0,06 g/ kg de suelo. Estas substancias se encuentran en
menores cantidades (0,09-0,106 µ g/ kg) en suelos bajo fertilización mineral. El
suelo es enriquecido en auxinas en el curso del compostado. El suelo
esterilizado contenía 0,042 µ g auxinas por kg de suelo y después de 6 días de
incubación en invernadero, bajo temperatura y humedad favorables, se
incrementaron a 0.146 µ g/ kg de suelo.
Williams, Stewart, Kejes y Anderson (1942) encontraron mayores cantidades de
auxinas en los suelos que Parker-Rodes. De acuerdo con su información, el
horizonte A de suelos fértiles contienen hasta 175 µ g auxinas por kg de suelo
Suelos no fértiles contienen 40-60 µ g de auxinas por kg de suelo. En el
horizonte B no se detectaron auxinas.
Hamense (1946) con el uso de métodos de análisis mejorados encontró 100
veces más auxinas en los suelos que los investigadores anteriores. De acuerdo
con su información, el contenido de auxina fue de 160-450 µ g por kg de suelo
(Schmidt, 1951, y Hamense, 1946).
Stewart y Anderson (1942) han mostrado que la cantidad de auxinas en los
suelos fértiles es totalmente suficiente para la estimulación del crecimiento
vegetal. Hamense (1946) encontró en diferentes suelos 0,16-0,045 µ g
equivalentes de ß-ácido indolacético en 1 g de suelo seco. El contenido de
auxina se incrementa después de la aplicación de fertilizantes orgánicos,
entonces cae debajo del nivel inicial y más tarde sube nuevamente al nivel
normal, característico del suelo en particular. De acuerdo con las
observaciones del autor, las preparaciones químicas puras de auxinas no se
preservan en la suelo por largos períodos.
De acuerdo con Matskov (1954), los preparados químicos de 2,4-acido
diclorofenoacético se preservan en el suelo por un invierno completo (5-6
meses). Otro estimulante del crecimiento, la heteroauxina (ß-ácido indolacético)
no se preserva en e suelo tanto tiempo.
Las auxinas también fueron detectadas en el suelo por Roberts I. y Roberts E.
(1939) y algunos otros investigadores
Además de auxinas, los suelos contienen otras substancias bióticas tales como
vitaminas, biotina, ácido nicotínico, ácido pantoténico, ácido fólico, amino
ácidos, varios factores de crecimiento R, Z, X y otros. Estas substancias se han
encontrado en las siguientes cantidades:

Roulte y Schopfer, 1950;

Tiamina 0,29-1,93 µ g
Schopfer, 1943
Schmidt y Starkey, 1951;
Riboflavina 9,0-980 µ g
Carpenter, 1943
Biotina 23,0-62,0 µ g Roulet y Schopfer, 1950
Vitamina B6 Cantidades no indicadas
Robbins, Hervey y
Vitamina B12 0,2-1,5 µ g
Stebbing, 1951, 1952
Inositol Cantidades no indicadas
Acido nicotínico Cantidades no indicadas Roulet, 1948
Ácido para amino Cantidades no indicadas
Roulet, 1948
benzoico
Acido pantoténico Cantidades no indicadas Roulet, 1948
Acido fólico Cantidades no indicadas Roulet, 1948
Factor X Cantidades no indicadas Lochhead y Texton, 1950
Factor desconocido Cantidades no indicadas Lochhead y Texton, 1950

Además de moléculas de vitaminas completas, se han encontrado en la

naturaleza, fracciones de moléculas, que tienen el mismo efecto sobre ciertos
organismos que las moléculas enteras. Por ejemplo la pirimidina, tiazol y otros
(Lilly y Leonian, 1939).
Hemos encontrados vitaminas en diferentes suelos, como regla, estaban en
mayores cantidades en los lugares donde los procesos microbianos eran más
intensos. . Así , ellas se presentan en mayor cantidad en los suelos chernozens
que en los podzoles (Tabla 37).
Tabla 37
Contenidos de Vitaminas y de Bacterias en Distintos suelos

Riboflavina Tiamina Biotina Bacterias

Suelos
µ g/100 µ g/100 g µ g/100 g millones /g
Chernozem (Moldavia
98,0 4,5 45,0 1,500
USSR)
Podsol
5,0 1,2 25,0 0,5
(región de Moscú)
 Los suelos cultivados contienen más vitaminas que los suelos vírgenes.
El contenido de vitaminas del suelo está influido no sólo por el grado de cultivo,
sino también por la naturaleza de la cobertura vegetal. Las plantas que
favorecen abundante crecimiento de microorganismos, como regla, asimilan
más substancias en el suelo. En los Seozems de Asia Central, las mayores
concentraciones de substancias bióticas se encontraron después de 2-3 años
de alfalfa. Su concentración fue menor bajo algodón y muy poca se encontró en
suelos vírgenes (Tabla 38)
Tabla 38
Contenido de Vitaminas y Microbios de los suelos de Asia Central, bajo diferentes plantas

Tiamina Biotina Microorganismos

Suelo
µ g/100 g µ g/100 g millones /g
Suelo virgen, valle del río
1,5 10.0 0,5
Vakhsh
Suelo virgen 0 + 0,1
Suelo cultivado (2 años
6,5 38,0 4,500
de alfalfa)
Suelo cultivado (mucho
3,0 18,0 1,500
tiempo de cultivo)

De acuerdo con Shavlovskii (1954, 1955), el suelo bajo cultivo de papas,

contiene 0,5 µ g/kg biotina, y el suelo bajo trébol 1,3 µ g/kg. En la región de
Lvov, en suelos serozem de bosques y en podzol chernozem, el autor obtuvo la
información suministrada en la Tabla 39.
Tabla 39
Contenido de vitaminas en suelos bajo distintos cultivos, en µ g/kg
Acido
Suelos Biotina Riboflavina
nicotínico
Bosque serozem, bajo trigo 0.3 4,0 100.0
El mismo, bajo pasturas 0,7 7,0 230,0
Podzol chernozem, bajo
0,8 10,0 280,0
trigo
El mismo, bajo pasturas 1,5 14,0 350,6

En el suelo que rodea al sistema radicular hay más substancias bióticas que
fuera de la rizosfera. En un kg de suelo de la rizosfera de trigo cultivado en
campos fertilizados de la estación experimental Dolgoprudnoe (región de
Moscú) encontramos por 100 g de suelo, 10 µ g de tiamina, 150 µ g
riboflavina, 35 µ g biotina; Fuera de la rizosfera, 1.2 µ g tiamina, 25 µ g
riboflavina, 3 µ g biotina; En la rizosfera de tabaco, 10-15 µ g tiamina, y fuera
de la zona radicular 1,5-4,0 µ g por 100 g de suelo.
De acuerdo con Shavlovskii, la cantidad de vitaminas en la rizosfera del trigo
sarraceno es el doble que fuera de la zona de la raíz. En el vigésimo día de
cultivo encontró:

Fuera de la zona de
En la rizosfera,
raíz
µ g / kg
µ g / kg
Acido nicotínico 600 260
Biotina 2 0.5
Vitamina B6 8 --

Roulet (1954). Encontró mayores concentraciones de vitaminas en la rizosfera.

La cantidad de substancias bióticas en la capa superior del suelo es mayor que
en las capas inferiores. Las mayores concentraciones se encuentran el la capa
superior (0-20 cm) (Tabla 40)
Tabla 40
Distribución de las vitaminas en las capas del suelo (µ g/kg)
Suelo Capa, cm Tiamina Biotina
Podsol de la región de
Moscú bajo 2 años de 0-20 2,3 14
trébol
20-30 0 3,0
50-70 1,9 8,0
80-100 0 0
Serozem del valle de
Vakhsh bajo 3 años de 0-20 5,6 42
alfalfa
. 30-40 1,8 14
45-60 2,4 21
70-90 1,1 4,2

En algunos suelos, se nota un pequeño aumento de la concentración de

substancias bióticas a la profundidad de 60-70 cm.

Otros investigadores han notado la disminución de la concentración de

vitaminas en capas profundas del suelo. Roulet y Schopfer (1950) dan la
siguiente información sobre la distribución de vitaminas de acuerdo con la
profundidad de las capas (por 100 g de suelo):

capa, cm tiamina, µ g Biotina, µ g

10 1,93 62
20 0,86 39
30 0,62 27
50 0.29 23
Estos autores encontraron tiamina y biotina inclusive a profundidades de 2,0 a
8,5 m.
Lilly y Leonian (1939) encontraron considerables cantidades de tiamina y
biotina como también B1 y sus componentes tiazol y pirimidina en el suelo.
Estaban concentrados en la capa superior del suelo. A la profundidad de 60 cm
no fueron detectados. West y Wilson (1938, 1939) encontraron tiamina y biotina
en la zona radicular de tabaco y lino.
De acuerdo con Schmidt y Starkey (1951), se puede encontrar riboflavina en el
suelo en cantidades variables. De acuerdo con la fertilidad del suelos, cubierta
vegetal, etc. En suelos bajo bosques, los autores encontraron 500 µ g de
riboflavina y en suelos bajo labranza y fértiles, cerca de 10 µ g por 00 g de
suelo.
Después de la aplicación al suelo de fertilizantes orgánicos tales como paja,
pasto o azúcar, la cantidad de riboflavina aumentó. A mayor cantidad de
substancias orgánicas aplicadas, mayor fue la concentración de riboflavina en
el suelo. Por ejemplo, después de aplicar pasto 15%*, se encontró cerca de
200 µg de riboflavina. Si la cantidad de pasto agregado era 10%, se
encontraban 120 µg de riboflavina. En presencia de pasto de 5%, sólo se
encontraron 60 µg de riboflavina. Las cantidades de riboflavina están dadas por
100 g de suelo (figura 62) *[El 15% se refiere al pasto, no está claro a qué se
refiere el porcentaje]

Figura 62. Formación de riboflavina en el suelos, en presencia de distintas cantidades de

substancias orgánicas (pastura de alfalfa) 1--15%; 3--5%

Para el final del período de crecimiento, en agosto-octubre, se encontró más

vitaminas que en la primavera. La cantidad de riboflavina encontrada en la
primavera fue de 80-300 µg, en e otoño alcanzó a 600-980 µg por 100 g de
suelo.
Roulet (1954) encontró que la cantidad de biotina en la capa superior del suelo
de un jardín botánico aumenta en el otoño y decrece en invierno y primavera.
De acuerdo con este autor, los suelos de bosques y pantanos contienen aún
más vitaminas que los suelos de praderas.
La cantidad de substancias bióticas en el suelo cambia constantemente de
acuerdo con las condiciones externas, tales como temperatura, humedad,
estación , etc.
Las vitaminas se conservan en el suelo por distintos períodos, la duración de
los cuales dependen del suelo y las condiciones climáticas. De acuerdo con
Stewart y Anderson (1942), las substancias estimulantes del crecimiento,
pueden persistir en suelo seco por 3-4 años. De acuerdo con Schmidt y
Starkey, 50% de la riboflavina puede detectarse en suelo fresco después de 3
años. El ácido pantoténico se descompone totalmente en un día.
Se pueden listar algunos aminoácidos como factores de crecimiento de las
plantas inferiores y superiores. Las plantas sintetizan estos amino ácidos, de la
misma manera que sintetizan vitaminas, pero, no obstante, la adición de
pequeñas dosis de amino ácidos tiene un efecto positivo en el crecimiento de
las plantas.
Nielsen y Hartelius (1938) ensayaron amino compuesto. Seis de ellos, ß-
alanina, asparagina, ácido aspártico ácido glutámico, lisina y arginina,
mejoraron marcadamente el crecimiento de organismos inferiores; la ß--alanina
tuvo el mayor efecto. Cinco µg de ß-alanina por 50 ml de medio fue suficiente
para incrementar el crecimiento y rendimiento en un 66% en peso seco. La
máxima estimulación se encontró a la concentración de 1:100.000. La arginina
actúa en una concentración de 1:20.000 y la lisina a una dilución de 1:4.000. La
glutamina ejerce una acción positiva en el crecimiento de los organismos a una
dosis de 1:1.000. Las substancias en tales dosis no se pueden considerar
como fuentes de nutrición. Otros amino ácidos tales como la aspargina y otros
pueden actuar a concentraciones todavía mayores y no pueden ser
considerados factores de crecimiento, sino nutrientes.
La cantidad de aminoácidos en los suelos varía; depende de las propiedades
del suelo y de las condiciones climáticas. Mientras más fértil sea el suelo, más
amino ácidos contiene. La concentración de aminoácidos esta determinada por
la tasa de entrada al suelo y por su tiempo de preservación.
Esta lista no incluye todas las substancias bióticas de suelo. SE debe asumir
que el suelo, lo mismo que otros substratos naturales, contiene muchas otras
substancias desconocidas para nosotros, las que actúan como bio
catalizadores, mejorando el metabolismo de los organismos.
Lochhead y Texton (1940, 1950, 1952) detectaron substancias activantes en el
suelo, de naturaleza desconocida y que no podían ser reemplazadas por
ninguno de los factores conocidos.

El Origen de las Substancias Bióticas del Suelo

Todas las diferentes substancias bióticas tienen su origen en la actividad
metabólica de las plantas y microbios.
Formación de substancias bióticas por las plantas: Se ha mencionado arriba
que durante su vida, las raíces de muchas plantas excretan substancias que
estimulan el crecimiento de organismos, por ejemplo, las semillas de
Orobanche ramosa germinan sólo en la vecindad de las raíces de girasol, lino,
maíz soja y algunas otras plantas. Las secreciones de las raíces de estas
plantas activan el crecimiento delos brotes de Orobanche ramosa. El factor
activador es termoestable y no se descompone por hervirlo o por prolongado
secado. (Bartsinakii, 1935; Beilin, 1941).
De acuerdo con Golubinskii (1950), la campanilla estimula la germinación de
las semillas de melón. Los granos de polen de las angiospermas mutuamente
se estimulan para la germinación excretando substancias activantes
(Golubinskii, 1946).
Timonin (1941) encontró tiamina y biotina en las secreciones radiculares del
lino. Según él, estas vitaminas segregadas por la raíz considerablemente
promueven el crecimiento y proliferación de microorganismos en la rizosfera.
West (1939) encontró tiamina y bio substancias en las secreciones radiculares
de lino y tabaco
Meshkov (1952) encontró biotina y tiamina en las secreciones del maíz y
arvejas. Según él, mientras más de estas substancias se segregan, mayor es el
crecimiento de las plantas. El maíz segrega más biotina y las arvejas más
tiamina. Las secreciones radiculares contienen vitaminas en las siguientes
cantidades por g de peso seco de las plantas: maíz, .0,5402 µg de tiamina y
0,2388 µg de biotina; las arvejas, 0,6634 µg de tiamina y 0,2658 µg de biotina.
De acuerdo con nuestras observaciones las arvejas, el trigo y el maíz segregan
más substancias bióticas en su período de desarrollo temprano que en el
período de fructificación (Tabla 41).

Tabla 41
La presencia de vitaminas en las secreciones de las raíces de distintas plantas
(µ g por ml de substancia nutriente)
Después de 10 Después de 10 Después de 45 Después de 45
días de días de días de días de
Plantas
crecimiento: crecimiento: crecimiento: crecimiento::
Tiamina Biotina Tiamina Biotina
Trigo 0,1 0,6 0 0,1
Maíz 0,2 0,5 0 0
Arvejas 0,5 1,5 0,1 0,7

Las plantas cultivadas en soluciones aireadas segregan más substancias

bióticas que las cultivadas en soluciones con pobre aireación. Por ejemplo, el
trigo cultivado con buena aireación segregó 0,21 µg de tiamina y el trigo
cultivado en presencia de pequeñas cantidades de oxígeno segregó 0,11 µg de
tiamina. Las respectivas cantidades de biotina segregada fueron: 0,8 y 0,6 µg
(por cm3 del medio).
Hay indicaciones en la literatura que las semillas de varias plantas, al germinar
segregan pequeñas cantidades de vitaminas al medio (Meisel, 1950; Schopfer,
1943).
Los compuestos bióticos pueden encontrar su camino al suelo junto con los
residuos en descomposición. Se sabe que las plantas contienen considerables
cantidades de varios compuestos que estimulan el crecimiento y desarrollo de
los organismos Por ejemplo, de acuerdo con Burcholder y otros (1944), las
siguientes cantidades de tiamina están presentes en los tejidos de: soja, 47-
61 x 10-7; cebada 28-51.8 x 10-7; maíz 17,6-31,2 x 10-7 (en moles por kg de
peso seco). Las partes aéreas contienen más tiamina que las raíces: En las
hojas del maíz se observaron 17,6-30, 2 x 10 -7; y en las raíces, 4,06-9,78 x 10-7
moles por 1 kg de masa seca.
Auxinas , vitaminas del grupo B, bios, vitaminas D, K C, H y P, ácido
pantoténico, ácido para amino benzoico, ácido nicotínico, derivados purínicos y
varias hormonas se han encontrado en las plantas (Zeding, 1955; Ovcharov,
1955, y otros.
Muchas substancias bióticas son excretadas en las heces de los animales y
seres humanos. Por ejemplo, los humanos excretamos diariamente en la orina,
600 µg de tiamina, 600 µg de riboflavina, 620 µg de inositol y grandes
cantidades de ácido pantoténico, como también otros compuestos (Meisel,
1950). En las heces de los seres humanos y los animales se encuentran
vitaminas del grupo B, ácido pantoténico, ácido nicotínico, ácido fólico y otras
substancias. Los investigadores muestran que estas substancias son
sintetizadas por la microflora de los intestinos (Perets, Gryazno y Agibalova,
1948; Nepomnyashchaya, 1950; Najjar y otros, 1943-1950; Perets, 1955), Las
vitaminas y otras substancias entran al suelo con el estiércol. De acuerdo con
Bonner y otros (1938), 1kg de estiércol contiene 130 µg de tiamina. La
riboflavina, el ácido pantoténico, el ácido nicotínico y otras substancias
activadoras están presentes en el estiércol. Sauerland (1948) encontró
considerables cantidades de substancias bióticas en el estiércol, heces y orina
del ganado. Las cantidades de estas substancias fluctúan con las estaciones.
Las mayores cantidades se encuentran en el verano, menos en el invierno. La
cantidad de factores de crecimiento en el estiércol y heces animales también
varían con la calidad del forraje.
Todas estas substancias encuentran su camino hacia el suelo y globalmente
constituyen sólo una pequeña fracción del total de substancias bióticas en el
suelo. Los microorganismos son el principal factor de enriquecimiento del suelo
con estas substancias.

Los microorganismos Sintetizan Substancias Bióticas

Hace mucho tiempo que se conoce la capacidad de los microorganismos para

sintetizar substancias bióticas.

Wildiers (1901) mostró la presencia de substancias activadoras en cultivos de

levadura. Estas substancias fueron llamadas bio substancias por el autor.
Quince o veinte años más tarde, varios especialistas pusieron atención a estas
substancias. Se encontraron en distintos substratos orgánicos, en tejidos de
plantas y en cultivos de muchos microbios.

Las investigaciones mostraron que las substancias bióticas son sintetizados

paro distintos microorganismos, bacterias, hongos, levaduras, actinomicetes y
otros. (Meisel, 1950, Ierusaimskii, 1940 Kudryashov, 1948; Bukin, 1940,
Stephenson. 1951, Schopfer, 1943).

Los organismos se dividen de acuerdo con sus capacidades de sintetizar

substancias bióticas en auxoautótrofos y auxoheterótrofos. Los primeros
sintetizan todos los compuestos necesarios para su crecimiento y por lo tanto
crecen en medios sintéticos avitamínicos. Los segundos no sintetizan, o mejor
dicho, no sintetizan todos las substancias bióticas necesarias y por lo tanto no
pueden crecer en medios avitamínicos.

La capacidad para sintetizar distintos factores de crecimiento está presente en

muchas, sino en todas las especies de bacterias del suelo.

Las bacterias quimio sintéticas son las más activas en sintetizar substancias
bióticas. Pueden crecer en medios puramente minerales, completamente
avitamínicos. Son capaces de sintetizar substancias orgánicas a partir del CO 2
atmosférico. Por ejemplo, se encontró, tiamina, riboflavina, ácido pantoténico,
ácido nicotínico, vitamina B y algunos otros compuestos, en los cultivos de
Thiobact. thioxidans (O'Kane, 1943). Estas y otras bacterias quimio sintéticas –
las nitrificadoras y las bacterias oxidantes del metano y el hidrógeno- sintetizan
su cuota completa de substancias bióticas. Sin esta habilidad no podrían
haberse desarrollado en el medio mineral.

Las bacterias incapaces de asimilar el CO2 pero que crecen bien en medios
sintéticos avitamínicos con fuentes orgánicas de carbono, también son capaces
de sintetizar substancias bióticas. A tal grupo pertenecen la mayoría de la
microflora del suelo: Azotobacter, bacterias noduladoras de la raíz,
representantes de los géneros Peoudomonas, Bacterium, oligonitrofilos,
micobacteria, y otros.

Boysen-Jonsen (1931) encontró que la heteroauxina es sintetizada por 16

especies de bacterias entre las cuales estaban Ps. radiobacter, Bact.
denitrificans, Bac. mycoides, Bac. subtilis, Bacterium sp., y otras. Raznitsyna
(1938) empleando el método del coleóptilo ha detectado la formación de este
compuesto por varios representantes de las bacterias y micobacterias. Ella
dividió los organismos en tres grupos, de acuerdo con su habilidad para
sintetizar auxinas: a) organismos que no las sintetizan (o las sintetizan en
pequeñas cantidades) Mycobacterium rubrum, Az. vinelandii, Bac. mycoides, y
otras, b) bacterias de mediana actividad, Az. agile, Az. chroococcum cepas 31,
35, Bact. coli, Myob. luteum, Ps. flourescens, cepas F. 24 y otras, c) bacterias
que sintetizan grandes cantidades de auxinas, Bact. proteus, Ps. fluorescens
cepa 21, Az. chroococcum cepa 54, Mycob, album, y otras.
Roberts I, y Roberts E. (1939) estudió la capacidad de las bacterias, hongos y
actinomicetes para sintetizar heteroauxinas. Este compuesto fue sintetizado por
99 cultivos de un total de 150 estudiados. De acuerdo con los autores, los
productores de auxinas más activos fueron las bacterias y actinomicetes. La
heteroauxina se encontró en diferentes especies de bacterias no formadoras de
esporas. Az. chroococcum, Pseudomonas, Bacterium, Vibrio, Mycobacterium, y
muchas otras bacterias.

Además de auxinas se pueden detectar muchos otros compuestos en los

cultivos de bacterias, tales como tiamina, riboflavina, ácido para amino
benzoico, ácido nicotínico, ácido pantoténico, ácido fólico, vitamina C, vitamina
K, vitamina B3. vitamina B12, inositol, biotina, pro vitamina D2, alfa y ßcarotenos,
factor R, factor Z, y otros (Detinova, 1937, Leo y Burris, 1943, Jones y Grooves,
1943; Burton y Lochhead, 1951, Lochhead, 1952; Lochhead y Burton, 1955),
La habilidad de las bacterias formadoras de nódulos de la raíz para sintetizar
tiamina, riboflavina, ácido pantoténico, vitamina B 12 y otras fueron descubiertos
por Burton y Lochhead (1951), Went y Wilson (1938, 1939). Estas y otras
vitaminas se encontraron en cultivos de Az. chroococcum, en varias especies y
cepas del género Pseudomonas y en micobacterias. En años recientes se
encontró vitamina B12 en muchas bacterias y especialmente en actinomicetes.
Algunos de estos organismos, tales como Mycob. propionicum, A. rimosus, A.
aureofaciens, y otros se emplean en la industria para la producción de esta
vitamina. De acuerdo con nuestras observaciones, 90-95% de los
actinomicetes asilados de suelos sintetizan vitamina B 12 (Krasil'nikov, 1954 c),
De acuerdo con Darken, 64-66% de los actinomicetes del suelo sintetizan esta
vitamina, (Darken, 1953).

Yamagutschi y Usami (1930) encontraron vitamina B 2 en 15 cultivos de

bacterias (Bac. subtilis, Bac. Mesentericus, Bac. mycoides, Bact. prodigiosum,
varios micrococos, y otros).

Landy, Larkum y Oswald (1943) encontraron ácido para amino benzoico en

cultivos de 35 especies de bacterias y actinomicetes. Entre estos se
encontraban: Bact. proteus , Ps. pyogenes, Bac. aerogenes, Bac. subtilis, Bac.
megatherium, Mycob. diptheriae, Mycob. stereosis, y otros.

Herrick y Alexopoulus (1943) encontraron tiamina en cultivos de 22 especies de

bacterias y actinomicetes.

De acuerdo con Schmidt y Starkey (1951), 99 especies de bacterias y

actinomicetes, de un total de 150 especies estudiadas sintetizaron
heteroauxina. Veintidós cultivos de bacterias de un total de 75 cultivos
estudiados sintetizaron vitaminas en medios sintéticos.

Nosotros hemos estudiado 192 cultivos bacterianos aislados de distintos suelos

de la URSS. Estos cultivos fueron ensayados para determinar su capacidad de
sintetizar vitamina B1 y heteroauxina. Los resultados están en la Tabla 42. Se
puede ver que más del 50% de las bacterias estudiadas sintetizan B 1 y casi el
40% sintetiza heteroauxina.
 Tabla 42
Compuestos Bióticos en Bacterias
No de Número de Número de
Bacterias cultivos cultivos que cultivos que
ensayados sintetizan B1 sintetizan B2

Bac. Subtilis 18 10 10

Bac. Mesentericus 15 12 5

Bac. Sp 13 3 1

Ps. Flourescens 8 8 8

Ps. Denitrificans 12 10 12

Ps. Mycolytica 3 3 3

Bact. Coli 2 0 0

Bact. Proteus 2 0 0

Bact. Liquefaciens 8 5 3

Bact. sp. 13 6 8

Rhizobium trifolii 15 10 0

Rhizobium phaseoli 15 12 0

Rhizobium leguminosarum 8 6 8

Az. Chroococcum 16 16 10

Az. Vinelandii 4 4 2

Mycob. Album 12 7 6

Mycob. Citreum 10 10 8

Mycob. Rubrum 3 0 0
Microc. Albus 3 0 0

Microc. Flavus 5 1 1

Microc. Aureus 6 0 0

Shavlovskii (1954, 1955) estudió cultivos de Ps. fluorescens, Ps. aurantiaca,

Bact. herbicola y Ps. radiobacter de la rizosfera de plantas. Estas bacterias se
cultivaron en medios sintéticos de una determinada composición y después de
8 días, se determinó el contenido de vitaminas de las células bacterianas y del
filtrado del cultivo. Los resultados están en la Tabla 43.
Tabla 43
Cantidad de Bacterias Sintetizadas por Distintas Bacterias en µ g,
(después de 8 días de cultivo)
Acido
Tiamin Biotina
Acido nicotínic Riboflavin Biotin
Tiamin a en en
nicotínic o en Riboflavin a en a en
a en 10 células células
Bacterias o en 10 células 1 a en 10 ml células 1 10 ml
ml de 1g 1g
ml de g de de medio g peso de
medio peso peso
medio peso seco medio
seco seco
seco

Ps.
Aurantiac 4,0 203 7,0 355 1,8 91 3,2 162
a
Ps.
flourescen 0,2 23,3 4,4 511 0,14 162 0,18 20,9
s
Ps.
radiobacte 0,4 6,2 5,2 80.2 2,8 43 3,0 46
r II
Ps.
radiobacte 0,3 13,2 4,0 176 3,6 158 0,6 26
r III
Bact. 0,05 14,7 1,7 470 0,04 11,7 0,03 8,8
herbicola

Muchos hongos producen substancias bióticas. Así, por ejemplo, se detectó

tiamina en cultivos de Aspergillus niger, A. oryzae, Penicillium glaucum, Mucor
mucedo, Mucor racemosus, en algunas especies de Phytophithora, Rhizopus,
Fusarium y otras; También en cultivos de levaduras como Torula utilis, Sacchar.
Cereviseae, Sacchar. logos, Endomyces vernalis, Willia anomala (Bilai, 1955;
Goinman, 1954; Bukin, 1940, y otros),

Se han encontrado hongos del suelo que sintetizan vitamina B 2. Algunos de

ellos sintetizan esta vitamina en tales cantidades, que son usados en la
industria. Estos son Candida (Oidium), Guilliermondella, Eremothecium ashbyi
(Dikanskaya, 1951). En muchos hongos se han encontrado biotina, ácido
pantoténico, ácido nicotínico, ácido para amino benzoico, vitaminas C y K y
muchas otras (Meisel, 1950).

Rogosa (1943) estudió 114 diferentes cultivos de levaduras tales como Torula
sphaerica (26 cepas) T. cremoris (20 cepas), Sacch. fragilis, Monilia
pseudotropialis, Mycotorula lactis, Sacch. anamensis, Torulaopsis kefyr, Torula
lactosa, Zygosaccharomyces lactis y otras. En todos los cultivos encontró
vitamina B2 en cantidades dentro del rango de 0.6 a 0.11 µ g por ml de medio
sintético

Las substancias bióticas se han encontrado también en los hongos micorriza.

De acuerdo con Schaffstein (1938), los hongos micorriza de las orquídeas
sintetizan factores de crecimiento requeridos por la planta huésped.

Los compuestos bióticos también son sintetizados por algas en el suelo. Ya fue
mencionado antes, que estos organismos están ampliamente distribuidos enel
suelo. Frecuentemente las algas crecen sobre la superficie de la tierra,
formando una cobertura azul verdosa visible a ojo desnudo.

Se sabe que las algas segregan distintas substancias orgánicas –productos

metabólicos (Goryunova, 1950). Dentro de estas substancias se encuentran
substancias bióticas.

Ondratschek (1940) encontró ácido ascórbico (vitamina C) en las secreciones

de tales algas como Hormidium borlowi, H. flaccidum, H. nitens and H.
stoechidium. El alga verde Chlorella sintetiza heteroauxina (Lilly y Leonian,
1941).

La mayoría de los microorganismos del suelo son parcialmente auxoautótrofos,

esto es, requieren sólo algunas substancias bióticas. Por ejemplo, Clostridium
butyricum requiere sólo biotina y sintetiza todos sus demás requisitos Algunas
especies de bacterias requieren sólo tiamina o ácido pantoténico. De acuerdo
con Burcholder, McWeigh y Mayer (1944) de 163 cultivos de levaduras 87%
requirieron biotina, 35% tiamina y ácido pantoténico y 12 % requirieron sólo
inositol.

Hay muchas bacterias que pueden sintetizar sólo una fracción de una molécula
de vitamina. Por ejemplo, algunos organismos sintetizan sólo parte de la
molécula de tiamina, ya sea tiazol o pirimidina, componentes de la vitamina B 1.
Consecuentemente, la primera sólo requeriría pirimidina y la segunda sólo
tiazol.
Hay muchos microbios del suelo que requieren ß-alanina, destiobiotin. Ácido
pimélico y algunos otros compuestos –compuestos de una u otra molécula de
una substancia biótica (Meisel, 1950; lerumalimskii, 1949; Stephenson, 1951, y
otros).

Thompson (1942) ha demostrado que las bacterias sintetizan vitaminas en

cantidades que exceden lo requerido para su metabolismo. El exceso de
vitaminas es liberado al entorno. El enriquecimiento del substrato con
vitaminas, tiene lugar, no sólo a expensas de las células en descomposición,
sino también, por medio de la secreción de vitaminas por los organismos vivos.
No se excluye la posibilidad que algunas substancias bióticas sean productos
de desperdicio de las células en crecimiento.

De acuerdo con Thompson, cerca del 50% de la tiamina sintetizada permanece

ligada dentro de las células de las bacterias. Esta fracción encuentra su camino
al suelo sólo después de la muerte y descomposición de las células.

La tabla 44 da una idea sobre el aspecto cuantitativo de la síntesis de

substancias bióticas por microorganismos. Esta información se ha compilado
de diversas fuentes
Tabla 44
Síntesis de Vitaminas por Microorganismos Cultivados en Medios Nutrientes
(1 por µ g de peso seco de células)
Acido Acido Ino- Acido
Microorganismos B1 B2 B6 Biotina
Nicotínico Pantoténico sitol Fólico

Bact. aerogenes 19,9 154 630 780 26,8 47,9 1.400 105

Ps. Flourescens 74 377 560 311 75.7 68,1 1.700 74,8

Bact. Proteus 23 95 330 130 16,4 21,4 1.000 42

Clostr. butyricum 39,3 235 1.930 318 23,2 0 870 18,8

Az. vinelandii 96 351 593 184 -- 4,2 -- --

Penicill 2,6 47 212 212 23,0 1,5 -- 14,6

chrysogenm
Sacchr. 360 42 1.000 100 100 1,2 5.000 31,2
cerevisiae

Torula utiis 52,8 62 535 180 1,9 35 3.500 31,2

De acuerdo con West y Wilson (1938, 1939) hay 19,6 µg de tiamina y 0,37 µg
de riboflavina por g de peso seco de la bacteria noduladora de la raíz del trébol
cultivada en medio sintético. Clostridium (Clostr. butyricum) sintetiza 0,9 µg/g
de riboflavina y Micro. ochraceus, Micr. citreus, Ps. pyocyanea, cerca de 10-15
µg.

Yamagutschi y Usami (1939) encontraron cerca de 1,5 µg de tiamina por g de

peso seco de células en cultivos de Ps. fluorescens, Ps. alba, y Bact.
prodigiosum. En cultivos de Bact. Proteu ellos encontraron 9-14 µg de tiamina
por 1 g de peso seco de células.

Muchas especies de micobacterias sintetizan considerables cantidades de

substancias bióticas. Por ejemplo, Mycob. smegmatis sintetiza cerca de 135 µg
de vitamina B2 por ml de medio sintético y 36 µg por g de peso seco de células
(Mayer y Rodbart, 1946). Cuarenta a ochenta µg por ml de medio de la
substancia activa micobactim se encontró en cultivos de Mycob. phlei (Francis
at al., 1953).

Ostrowsky y otros (1954) estudiaron las vitaminas en distintos

microorganismos representativos. Sus resultados están indicados en la Tabla
45.
Tabla 45
Contenido de Vitaminas en Células Bacteriales
( µg por g de peso seco de células)
Acido
Acido Acido
Microorganismos B1 B2 Biotina Peteroyl-
Nicotinico Pantoténico
glutámico

Thiobact. 21 31 92 0,77 0,46 0,75

thioparus
Thobact 23 60 15 0,64 1,89 57,0
thiooxydans

Ps. pyocyanea 15 43 240 2,4 1,0 140,0

Ps. chroococcum 96 -- 590 -- -- --

Propionibact. 6,4 -- -- -- -- 93,0

pentosaceum

Clostr. butyricum 9,3 55 250 1,7 0,5 92,0

Bact. proteus 21,0 -- 250 3,4 4.2 100,0

Ps. flourescens 26,0 68 210 7,1 1,8 90,0

Bact. 27,0 35 240 4,1 3,2 120,0

prodigiosum
Las cantidades indicadas arriba no son estrictamente constantes. Pueden
variar, dependiendo de la fase de crecimiento y condiciones del cultivo de las
especies individuales de bacterias, hongos o actinomicetes. En algunos casos,
las células viejas contienen menos vitaminas que las células jóvenes, mientras
que en en algunas especies se observa el cuadro inverso: las células viejas
contienen más vitaminas que las células jóvenes. Por ejemplo, algunos cultivos
de Ps. radiobacter contiene más vitamina B1 después de 8 días de crecimiento
que después de 2 días de crecimiento.

En algunos medios, los microbios sintetizan muchos factores de crecimiento,

en otros sólo unos pocos o ninguno.

La intensidad de la formación de vitaminas por los organismos del suelo está

muy influida por los microbios simbióticos. Algunos de ellos suprimen la
síntesis, mientras que otros estimulan este proceso. De acuerdo con Smalii
(1954), Azotobacter (Az. chroococcum) en cultivo puro sintetiza 173 µg de
heteroauxina (por masa celular 1 Placa de Petri en agar de Ashby) y en la
presencia de los siguientes microorganismos, sintetiza esta substancia en las
cantidades que se indica:
Bact. mycoides, 220 µg
Bact. denitrificans, 196 µg
Ps. radiobacter, 243 µg
Torula rosea, 234 µg
Act. Coelicolor, 188 µg
Penicill. Nigricans, 149 µg

La capacidad de sintetizar auxinas o vitaminas no caracteriza a una especie.

Diferentes cepas de una y la misma especie difieren marcadamente entre sí.
Por ejemplo, de más de 100 cepas de Az. chroococcum que hemos aislado en
diferentes suelos y lugares de la URSS, algunas de ellas sintetizaron grandes
cantidades y otras sólo pequeñas cantidades, o nada de nada. La formación
de heteroauxina por los diferentes representantes de estos cultivos se muestra
en los fotogramas de coleóptilo (Figure 63).

Figure 63. La formación de heteroauxina por distintos cultivos de

Azotobacter chroococcum. La curvatura del coleóptilo después de
la inmersión en el cultivo, expresado en grados:
a) cepa de museo 54, ángulo de desviación: 32°; b) cepa aislada
de suelo de huerta en la vecindad de Moscú, ángulo de
desviación: 10°; c) cepa aislada de los suelos de Kara Kum,
ángulo de desviación: 8°; d) cepa aislada de suelo podsol
cultivado (Estación Experimental Chashnikovo, Región de Moscú,
ángulo de desviación: 0°; 1: coleóptilo de control; 2: experimento
sumergido en el cultivo bacteriano.
Se obtuvo similar información al estudiar otras especies bacterianas y no sólo
para heteroauxina, sino también para biotina, tiamina, riboflavina y otras
substancias bióticas.
Aunque estrictamente no existe especificidad de especie para la síntesis de
substancias bióticas, el análisis masivo muestra diferencias grupales en esa
cuestión. Más cepas de Azotobacter sintetizan vitaminas y auxinas que
bacterias del género Bacterium.
Sólo algunas especies de bacterias noduladoras de la raíz son capaces de
sintetizar heteroauxina y éstas, son inclusive, formas débiles. Hemos
investigado 12 especies de bacterias noduladoras de trébol, alfalfa, porotos,
vicia, Lathyrus vermus, pulmonaria, arvejas, Onobrychis, soja, lupino, acacia y
astragalus. Todas estas especies o no sintetizaron heteroauxina para nada o la
sintetizaron sólo en pequeñas cantidades (Figura 64).

Figura 64, Formación de heteroauxinas por distintas especies de

bacterias noduladoras de la raíz. La magnitud de la curvatura por
inmersión en cultivos de:
a) trébol rojo, el ángulo de curvatura: 6°; b) soja, ángulo de
curvatura: 6°; c) habas, ángulo de curvatura: 3°; d) arvejas,
ángulo de curvatura: 4°; e) vicia; 4°; f) trébol dulce: 2°; g)
chauchas: 2°; h) proactinomicetes de nódulos de aliso: 3°; 1--
control coleóptilo; 2—experimento inmerso en bacterias.
De 60 cepas de bacterias noduladoras de la raíz de la alfalfa, sólo 9 cepas
sintetizaron este compuesto en cantidades suficientes para apenas provocar
una curvatura perceptible del coleóptile. Se examinaron 15 cepas de
Rhizobium trifolii, 8 cepas de Rhizobium leguminosarum y 15 cepas de Rh.
phaseoli. En todos los casos el cuadro fue el mismo.
No hemos detectado síntesis alguna de heteroauxinas por los
proactinomicetes que forman nódulos en las raíces del aliso. Los actinomicetes
y proactinomicetes del suelo sí sintetizan heteroauxina en mayor o menor
medida.
De acuerdo con Starkey (1944), el contenido de ácido nicotínico de los
residuos de plantas está en el rango de 2,4 a 85 µg/g. La misma substancia,
en células microbianas es de 150-1.920 µg/g. Esto es, aproximadamente 25-
60 veces más.
Se debe destacar que los estudios de substancias bióticas se llevaron a cabo
sobre relativamente pocas especies de microorganismos. La elección de
organismos fue hecha al azar y los estudios fueron restringidos a unas pocas
vitaminas solamente, en la mayoría de los casos a tiamina y riboflavina.
Se debe asumir que en realidad, muchos y posiblemente todos los
microorganismos del suelo, sintetizan estas y otras substancias bióticas, que
juegan un papel esencial en la vida y metabolismo de los organismos inferiores
y superiores.
Es obvio que bajo condiciones naturales (vida en el suelo) el metabolismo
microbiano y la síntesis de substancias bióticas va a diferir de aquél bajo
condiciones de laboratorio (en medios nutrientes artificiales).
Schmidt y Starkey (1951) han demostrado que si se introducen al suelo
residuos de plantas que no contienen vitaminas, estas aparecen y se
acumulan en mayor o menor medida debido a la descomposición de los
residuos por los microbios. El aumento del contenido de riboflavina del suelo
es concomitante con la intensificación del metabolismo microbiano (figura 65).
Mientras más residuos de plantas se introduzcan al suelo, más intenso el
crecimiento microbiano y la formación de riboflavina (Tabla 46)

Figura 65. formación de riboflavina en el suelo como producto del

metabolismo de los microorganismos, la actividad de los cueles
es determinada por la evolución del CO2 en mg por 100 g de
suelo:
1--riboflavina, en µg/100 g; 2--CO2 en mg/100 g.

Tabla 46
Formación de Riboflavina en el Suelo Durante la
Descomposición de Paja de Avena
(µg por 100 g de suelo)
Acumulación de
56
riboflavina en 0 día 1 día 3días 4 días 7 días
días
días:
1,25 gramos de
11 19 26 27 26 13
paja aplicados
2,5 gramos de
20 22 60 55 38 19
paja aplicados

Se obtienen resultados similares si se introduce glucosa o sacarosa al suelo

en lugar de paja. Las bacterias inoculadas en el suelo que carecen de
vitaminas empiezan a crecer a expensas de los azúcares y se acumulan
riboflavina, biotina, heteroauxinas y otros en el suelo.
De acuerdo con los cálculos de Meisel (1950), cerca de 400 g de vitamina B 1,
300 g de vitamina B6 y 1 kg de ácido nicotínico son sintetizados por los
microbios en la capa superficial de 1 hectárea de suelo fértil de las regiones
del sur, durante una estación (9 meses)
Las substancias bióticas se conservan en el suelo por diferentes períodos de
tiempo. Una preparación pura de una vitamina introducida en el suelo puede
ser detectada durante varios días. De acuerdo con Schmidt y Starkey (1951),
la riboflavina y el ácido pantoténico persisten en el suelo de 3 a 20 días o más
(Tabla 47)

Tabla 47
Conservación de la Riboflavina y del Acido Pantoténico en el Suelo
(µg/100 g de suelol)
Cantidad Present Present Present Present Present
Present
introducid e luego e luego e luego e luego e luego
Vitamina Suelo e luego
a en el de 0 de 2 de 3 de 6 de 21
de 1 día
suelo días días días días días

Riboflavin
a
Estéri
40 38 33 -- 40 34 34
l

No
40 36 36 -- 43 16 12
estéril

Estéri
80 69 68 -- 81 67 64
l

No
80 65 68 -- 81 49 13
estéril

Acido
pantoténic
o
Estéri
50 34 34 35 35
l

No
50 32 34 10 10
estéril

Estéri
100 72 77 80 73
l

No
100 68 64 18 10
estéril

La riboflavina persiste más en el suelo que el ácido pantoténico. Ambos

compuestos duran más en suelo estéril que en suelo contaminado, ya que las
substancias bióticas, como todos los demás compuestos están sujetos a la
descomposición microbiana.
Si el metabolismo microbiano se detiene artificialmente, las vitaminas
introducidas en suelo contaminado persisten por los mismos períodos que en
suelos estériles. Se encontró que los compuestos bióticos (vitaminas y
heteroauxina) persisten en muestras de suelos secos tomadas de campos
cultivados y fertilizados, de 3 a 4 meses a 4 años, dependiendo de la clase de
suelo y sus propiedades como también de las propiedades de las mismas
vitaminas (Stewart y Anderson, 1942).
Las vitaminas y otras substancias bióticas que entran al suelo por una u otra
ruta son descompuestas y sintetizadas nuevamente por microorganismos.
Algunas vitaminas desaparecen, otras aparecen. Hay un continuo reciclado de
estas substancias en el suelo. Se puede encontrar substancias bióticas en el
suelo durante todo el período vegetativo, mientras vivan los microbios, se
reproduzcan y exhiban actividad metabólica. La cantidad de substancias
bióticas está determinada por la tasa de su síntesis e introducción al suelo y
también por su tasa de destrucción, o su estabilidad.

The effect of biotic substances on plants

It was noted above that green plants synthesize for themselves the necessary biotic
substances or phytohormones. Under conditions favorable for their growth this
synthesis meets all their requirements for normal growth. In certain, not infrequent,
circumstances, apparently under some unfavorable conditions, the plant synthesizes
inadequate amounts of these substances. Then specific avitaminoses develop which are
expressed to a greater or lesser degree in the form of certain physiological disturbances
and diseases.

Different plants react variously to the addition to the substrate of growth factors and
vitamins. Some respond by enhanced growth or by changes in the course of biochemical
processes, others react weakly and still others do not react at all. This permits us to
assume that the first produce only minimal amounts of the active substances which are
insufficient for their normal metabolism, the second synthesize them quite actively but
in amounts still insufficient to satisfy all their needs, and the third synthesize them in
adequate quantities.

Investigations show that even the last group of plants by no means always synthesize
adequate amounts of biotic substances. The vitamin content of plants varies within a
wide range, depending on external conditions of growth. It varies according to the soil
and climate conditions (Murry, 1948; Rakitin, 1953). Fertilizers have a great effect on
the quantity of vitamins present in plants.

In all cases of avitaminosis the vitamins from the substrate are absorbed by the plant.
Even under normal conditions of growth, plants utilize ready-made biotic substances, if
available.

The utilization of vitamins, auxins and other compounds from the soil has been
confirmed in many experiments. Many plants and biotic substances were studied under
laboratory and field conditions, in sterile and nonsterile experiments.
 The plants' requirements for vitamins and auxins has been thoroughly studied in
experiments with isolated organs and tissues, and especially with isolated roots.

It is known that excised roots of many plants will not grow in synthetic media in the
absence of biotic substances and a carbon source. If a root 2-3 mm long is excised from
a plant which grew under sterile conditions and placed in a synthetic artificial medium
(Bonner's medium or other) it will grow in length to reach considerable dimensions and
will form lateral roots, etc, only if the necessary biotic substances are present in the
medium. In the absence of the latter, or if their concentration is insufficient. the roots
will not grow at all or the growth will be weak.

Investigations show that roots of different plants demand different growth factors. For
example, roots of flax require vitamin B1, roots of peas, horse-radish, lucerne, clover
and cotton require vitamins B1 and B6; roots of tomatoes. thorn apple, and sunflower
require vitamins B1, B6 and pantothenic acid (Bonner et al., 1937; Robbins and Bartley,
1922-1938; Robbins and Schmidt, 1939, 1945). Excised roots of many plants, growing
on synthetic media, synthesize all the required growth factors. Some of them synthesize
them in amounts sufficient for their normal growth, others form too little. The first grow
well in artificial media, the latter require the addition of the missing factors (Bonner,
1942). Bonner and Bonner (1948) give the following data on the vitamin requirements
of isolated roots (Table 48).

Table 48
Vitamin requirements of isolated roots of various plants
(according to Bonner T. and Bonner H., 1948)
Vitamin B1 Nicotinic acid Vitamin B6
Plants
requirement requirment requirement
Linum usitatissimum Boenn stimulates - -
Raphanus sativus L. + + -
Medicago sativa L. + + -
Trifolium repens L. stimulates + -
Gossypium hirsutum L. + + -
Crepis rubra L. + + -
Cosmos sulfureus + + -
Pisum sativum Gov. + + -
Daucus carota L. + - +
Lycopersicum esculentum Mill + - +
Lycopersicum esculentum
+ stimulates +
pimpinellifolium
Dun + stimulates +
Helianthus annuus L. + stimulates +
Acacia melanoxylon R. Br. + stimulates +
Datura stramonium L. + + +
 The following data show the effect of vitamins, on the growth of isolated roots. The
roots of flax in the presence of vitamin B1 elongated by 185 mm. and in its absence by
31 mm in one week. The roots of flax are calculated to synthesize vitamin B1 at a rate of
0.02 µ g per week. Their vitamin B1 requirement for normal growth is 2 µ g, i.e., 100
times more than they synthesize.

The roots of white clover grow well in a medium containing vitamins B1 and PP*.
*[ The correct designation of this vitamin is unclear.] They increase in length with each
successive transfer into a fresh nutrient medium. In the first 5 weeks the roots elongate
by 84 mm, the increment in the next 5 weeks amounts to 109 mm, in the third five-week
period the increment amounts to 129 mm, in the fourth five-week period--136 mm, and
in the following 5 weeks 151 mm. The increment becomes uniform upon subsequent
transfer amounting to about 22 mm per week.

The roots of sunflower in the absence of the vitamin complex or in the presence of
only one of the vitamins PP or B6 cease to grow after 7 consecutive resowings. Roots
which were supplied with all three vitamins, PP, B1 and B6 grew well for along period of
time allowing for many transfers into fresh media. In the first five weeks the increment
was 74 mm, in the next 5 weeks it amounted to 96 mm, in a further 5 weeks--120 mm,
and in the following fortnight--150 mm.

The roots of plants belonging to diverse varieties of one and the same species react
differently to vitamins. For example, one variety of tomatoes requires vitamin B6 and
does not respond to vitamin PP and on the contrary another variety requires vitamin PP
and does not react to vitamin B6 (Bonner and Bonner, 1948).

Ovcharov (1955) introduced vitamin PP into a medium in which he grew cotton plants
whose leaves were cut off. He observed enhanced formation of new roots on the old
roots.

Went, Bonner and Warner (1938) had shown that thiamine stimulates the growth of
roots of peas, lemons and camellia. The results were more markedly pronounced when a
mixture of thiamine and heteroauxin was employed. Positive results were also obtained
in these cases with a mixture of vitamin B1 and indoleacetic acid (Grebenskii and
Kaplan, 1948) and also with vitamin K, biotin and pantothenic acid with biotin
(Scheurmann, 1952).

Psarev and Veselovskaya (1947) noted the stimulating effect of thiamine on the
formation and growth of wheat roots.

In some cases roots of certain plants required only parts of the vitamin molecule, for
example only thiazole or pyrimidine (components of vitamin B1).

Some roots require unknown biotic compounds and cannot, therefore, be grown in
vitro.

Robbins (1951) in his review, brings a list of plant species the roots of which can grow
on nutrient media. There are 22 such species. Roots of 27 species could not be grown in
isolation despite the addition of various vitamins, auxins, amino acids and other biotic
substances.

It should be noted that even the roots which can grow in vitro do not grow in the same
manner as when attached to the plant. They grow in length and branch, but do not get
thicker or if they do thicken, then only very slightly. The activity of the cambium is
completely or almost completely suppressed. Consequently, no entirely adequate
medium has as yet been found for isolated roots.

The requirement for biotic substances is well pronounced in seedlings. The need
embryos of some plants develop better and quicker in the presence of certain vitamins
added to the substrate. For example, the growth of pea seedlings separated from the
cotyledons considerably increases in the presence of thiamine and biotin (Kögl and
Haagen-Smit, 1936). Pantothenic and ascorbic acids also act favorably on pea embryos
(Bonner T. and Bonner H. , 1948).

Plant embryos do not synthesize biotic substances, they utilize the food reserves
present in the seeds. Even the green sprouts of many plants in the early growth period
synthesize vitamins weakly or not at all (Bonner et al., 1939). Ripe embryos of thorn
apple are easily grown on artificial media without vitamins while the nonripe embryos
require vitamins PP, B1, B6, C and others.

Pantothenic acid also has a favorable effect on lucerne sprouts. Treating pea seeds with
vitamin C enhances their growth by 213% as compared to the controls. Sprouts of
meadow grass react positively to the addition of vitamins B1, PP, H and pantothenic acid
to the medium. The grape seeds germinate quicker in the presence of an 0.01 % solution
of vitamin PP and, moreover, the formation of roots and the growth of aerial parts is
more intense (Flerov and Kovalenko, 1947).

The presowing treatment of cotton seeds with vitamins B1 and PP considerably

enhances their germination and the subsequent growth of the sprouts, Seventy-five per
cent of the seeds germinated after treatment with the vitamin as compared to 45 % in the
control. The length of the sprouts in the former case was on the average 1.35 cm and
1.65 in the latter Zakharyants, Gorbacheva and Zglinskaya, 1950).

An increase in the growth and subsequent yield of bean seeds after treatment with
vitamins B1 and PP was observed. The height of the plants (from the treated seeds) was
greater by 18%, the increment of the vegetative mass was greater by 39%, and the yield
of seeds was 28% higher then that of the control plants (Dagis, 1954).

Bonner et al. reached the conclusion that the lower the vitamin content of plant leaves,
the stronger they react to the addition of these substances. According to them, peas and
tomatoes, contain 13-18 µ g of vitamin B1 per kg of dry leaves and do not react to its
addition. Cabbage, cosmos, Japanese camellia and others contain small amounts of
vitamins in their leaves and react positively to the addition of these substances.
However, this does not hold for all plants. There are species or even varieties of one and
the same species which contain a small amount of vitamins in their leaves and react less
to their addition than plants with higher vitamin content.

The addition of vitamins has a favorable effect even on mature plants. Tung trees after
the addition of 0.5 mg of vitamin B1 grew in 70 days twice as much as the controls. The
application of low concentrations of vitamin B1 to poppies increased the weight of their
bolls as well as the crop in general. Application of vitamin B1 together with water had a
favorable effect on the growth of spinach. The weight increment during the 63 days of
the experiment exceeded many times that of the control plants (Table 49).

Table 49
Dry weight of spinach leaves (in mg) at the end of 63 days of the experiment, after 13
irrigations with a solution of vitamin B1
Treatment 1* 2* 3* 4* 5* 6* 7* 8* 9*
Control 32.4 58.1 56.2 35.8 9.6 -- -- -- --
Vitamin
28.5 103.9 255.6 390.0 504.1 534.9 375.9 205.0 45.7
B1

*This is unexplained in Russian text. Probably refers to position of leaves on plants.

Denisov introduced vitamin B2 into a substrate where egg plants were grown and
obtained a marked increase in yields. After 77 days growth the control plants had stems
7.1 cm long, the weight of the tops was 48 5 g and the weight of the roots 12.5 g. The
corresponding figures for plants grown in the presence of the vitamin B2 were 12.2 cm
121 g and 22.9 g (Ovcharov, 1955).

The growth of vine grafts, soya and other plants in increased under the influence of
vitamin PP. Lemon seedlings react markedly to the addition of this vitamin (Table 50).

Table 50
The growth rate of lemon seedlings under the influence of vitamin PP
(according to Kocherzhenko and Snegirev, 1946)
Average height Average height Average height Average height
Treatment of plants on 15/ of plants on 15/ of plants on 15/ of plants on 15/
VIII IX X XI
Control 27.2 29.2 35.5 38.3
Vitamin PP 23.8 34.7 44.5 47.7

Analogous data were obtained by Matveev and Ovcharov (1940) in their experiments
with a Bukhara almond. The plants were sprayed with an aqueous solution of vitamin
PP and adenine. Earlier opening of buds and more rapid development of' leaves were
observed. The number of leaves was 4 times greater than in the control plants.

Vitamins play a considerable role in the development of orchids. These plants, as

already mentioned, grow badly, or do not grow at all without the micorhizal fungi. It
was found that the seeds of orchids contain only small amounts of vitamin PP, which are
not sufficient for normal germination. This shortage is remedied thanks to the
mycorhizal fungi. Treating the seeds with vitamin PP secures their normal germination
in the absence of the fungi. The dry weight increment was 3 times higher than that in
control plants. It was shown that orchids of the group Vanda grow well in the presence
of substances obtained from the mycorhizal fungi. These substances resemble, in their
action, bios II (according to Kelly, 1952). According to Noggle and Wynd (1943), some
orchids grow well in the presence of nicotinic acid. Henrikson (1951) noted the positive
effect of thiamine, vitamin B6 and nicotinic acid on the germination and subsequent
growth of Thunia marschaliana Rchb. f. (Table 51).

Table 51
The effect of vitamins on the growth of the orchid Thunia marschaliana Rchb. f.
(according to Henrikson, 1951)
Height of plants, Number of Length of roots,
Vitamins Dry weight, mg
mm leaves mm
Control 48.0 5 60.5 30.8
B1 83.5 6 92.5 59.1
B6 48.0 6 58.5 59.1
C 55.5 6 55.5 30.1
PP 101.0 8 176.5 86.6

Rakitin and Ovcharov (1948) employed vitamin PP and adenine for increasing the
growth of cotton plants in their early growth stages. Thereby, not only growth, but also
fruiting was increased. The number of bolls was increased, and the cotton yield (raw
material) was considerably higher than that of the controls. Similar data were obtained
by Zakhar'yants, Gorbacheva and Zglinskaya (1950). They sprayed cotton plants with
solutions of vitamin PP and thiamine, The cotton crop increased by 34.1% as compared
to the control.

The fat-soluble vitamins, A, E, K, in contrast to vitamins of the B-group suppress the

growth and diminish the crop of plants. Carotene suppresses the growth of safranin
which is itself rich in carotene. Vitamin K suppresses the growth of fungi, some bacteria
and the roots of higher plants. Vitamin PP antagonizes the action of vitamin K. Vitamin
E, according to Schopfer (1950), arrests the growth of certain plants. The height of
plants in the control was 35.75 cm and in the presence of vitamin E--6.14 cm; the
number of flowers in the former was 80.4 and in the latter, 10.

Ovcharov (1955) immersed the seeds of plants in a solution of thia ine and yeast
extract, Such procedure markedly stimulated the growth and increased the crops, the
seeds too were larger.

Söding, Bömke and Funke (1949) obtained 30% higher yields of carrots after treating
their seeds with nicotinic acid, vitamin B1, vitamin C and other substances.

Experiments with vitamins under sterile conditions are worthy of mention. McBorney,
Bollen and Williams (1935) tested the action of pantothenic acid on the growth of
lucerne under sterile conditions in sand cultures, in a medium which did not contain
nitrogen. Pantothenic acid was added in high concentrations. Plants under these
conditions grew in the presence of pantothenic acid ( in high concentrations of
pantothenic acid) much better and the yield was higher.
 Magrau and Mariatt, (1950) showed that a number of vitamins, such as thiamine,
nicotinic acid, biotin and pantothenic acid had a positive effect on the growth of Poa
annua L. under sterile conditions. Swaby (1942) tested the effect of certain organic
substances including some containing vitamins, on the growth of cereal and leguminous
plants, in the presence and absence of microorganisms. The experiments showed that in
the presence of microorganisms organic substances rich in vitamins have a favorable
effect on the growth of plants.

Shavlovskii (1954) tested the effect of pantothenic acid, vitamin B1, nicotinic acid and
vitamin B6 on the growth of lucerne. The latter was grown on agar medium under sterile
conditions for 30 days. The results are given in Table 52.

Table 52
The effect of vitamins on the growth of lucerne
(vitamin concentration in the medium = 0. 1 µ g/ml)
Dry-mass weight of
Dry-mass weight of Dry-mass weight of
Vitamins 20 plants in mg:
20 plants in mg: Tops 20 plants in mg: Total
Roots
Control (without
38.6 8.0 46.6
vitamins)
Pantothenic acid 36.4 11.6 48.0
Vitamin mixture 37.2 12.2 49.4

Analogous experiments were carried out by Shavlovskii with buckwheat. The plants
were grown in sand wetted with the nutrient solution of Hellrigel. containing 1 µ g of
the vitamin per ml. Other containers were supplemented with yeast extract and
vitaminless casein hydrolysate. In one series of experiments the bacterial culture of Ps.
aurantiaca--vitamin producers were introduced. Plants were grown for 2 days and then
analyzed. The results are given in Table 53.

Table 53
The effect of biotic substances on the growth of buckwheat
Dry mass Dry mass Dry mass Dry mass
weight of 10 weight of 10 weight of 10 weight of 10
Biotic compound
plants in mg: plants in plants in mg: plants in mg:
Cotyledons mg: Stems Roots Whole plant
Control without vitamins 73.0 64.0 32.0 168.0
Bacteria Ps. aurantiaca 76.0 64.0 41.0 181.0
Vitamin B1 82.0 63.0 39.0 184.0
Vitamin B12 79.0 64.0 32.5 175.5
Vitamin mixture 80.0 65.0 36.0 181.0
Yeast extract 0.01% 80.0 66.0 40.0 186.5
Yeast extract 0.1% 90.0 65.0 40.0 195.0
Casein hydrolyste 0.1% 80.0 66.0 43.0 189.0
 It can be seen from the given data that the substances tested, markedly increase the
increment of the roots and aerial parts of the plants.

The biological role of vitamins has been little studied, but, according to the available
data, it is important. It is well known that many of them are components of various
enzymatic systems. The so-called coenzymes which enter into chemical interaction with
the substrate include many vitamins, It has been found experimentally that vitamin B1 in
a compound together with phosphoric acid is the coenzyme of carboxylase-
cocarboxylase.

Carboxylase is an enzyme participating in transformations of carbohydrates. It is

widely distributed in plants, animals and microbes. Without it the various
transformations of carbohydrate compounds, including pyruvic acid, are not feasible.
The latter is the key intermediate in the metabolism of living cells, which links the
metabolism of carbohydrates, proteins and fats.

In the absence or shortage of vitamin B1 the synthesis of cocarboxylase is slowed

down or arrested and, consequently, carbohydrate metabolism is slowed The latter is
frequently arrested at the stage of pyruvic said, which leads to the accumulation of
pyruvic acid in the cell and the complete cessation of metabolism.

Vitamin B1 participates not only in decarboxylation of pyruvic acid but also in the
reverse reaction--the fixation of CO2 in pyruvic acid. The role of vitamins in the fixation
of CO2, as the investigations of recent years have shown, is very great.

Vitamins also play a considerable part in the formation and transformation of proteins.
It has been shown that vitamins B2, B6, B12, PP and H participate in the formation of
amino acids and their transaminations. The shortage of vitamin B6 leads to a decrease in
the formation of amino acids from organic acids and ammonia. Vitamin Be takes part in
the formation of amino acids from organic acids and ammonia. Transamination, i.e.,
transfer of an amino group (NH2) from one acid to another, takes place in the presence
of vitamin B6.

In fat synthesis from sugars, vitamins B1, B2, PP and pantothenic acid participate, and
the transformation proteins into fats also requires vitamin B6.

Vitamins play an immense role in respiration. It was shown that enzymes participating
in respiration consist of proteins and a coenzyme. The latter consists of vitamin B2 and
phosphoric acid. Vitamin B2 in enzymatic systems plays a role in oxidation-reduction
processes. Folio acid is of great importance in respiration. The germination of seeds and
the respiration of sprouts increases under the action of this acid (Stephenson, 1951,
Schopfer, 1943, Zeding, 1955).

Growth stimulators--auxins and heteroauxins--have a positive effect on the colloidal

and chemical properties of protoplasm. According to some authors, they participate in
the general metabolism of the cell as separate components. By increasing the
metabolism they influence the growth of the cells of the aerial parts and especially of
the roots. Under the influence of heteroauxin the influx of plastic substances increases
which leads to the formation of now roots in greater quantities. During the rooting of
grafts, hydrolysis of starch and fats increases in the cells of the latter. The activity of
peroxidase is also increased and the tissues are better hydrated (Maksimov, 1940,
Turetskaya, 1955, Zeding, 1955, and others). The action of these substances does not
affect the turgor of cells only, as previously assumed, but affects the general metabolism
of the plant. In this respect they resemble other biotic substances. Data exist which show
that heteroauxin stimulates the formation of auxins, (Zeding, 1955).

Kuhn (1941) has shown that carotene and carotenoids have a great effect on the
formation of sexual cells and on conjugation of a Chlamydomonas alga. According to
him, there exist carotenoids with specific properties of male and female hormones. He
found a carotenoid--safranol--with properties of a male hormone and a carotenoid--
picrocrocin--with the properties of a female hormone.

Vitamins have a favorable effect on the fertilization of plants. It was found that the
sexual organs are rich in vitamins especially the pollen. For example, the pollen of the
pea tree contains 2,300 mg of carotene and that of sunflower, 1,460 mg per kg. Pollen of
some plants do not contain large amounts of carotene. Vitamins decompose under the
action of light and pollen decolorizes and loses its activity. Processing of such pollens
with carotene increases its capacity to germinate. Thus, according to Lebedev (1952),
without the addition of carotene the percentage of germinated hemp pollen was 39%,
the length of the pollen tubes was on the average 100 µ ; in the presence of carotene the
percentage of germinated pollen was 53.5 and the length of the pollen tubes 312 µ . The
lower the vitamin content, the sharper the reaction to the addition of carotene. Pollens
rich in this vitamin stimulate the germination of pollen which contains small amounts of
the provitamin if they are left to germinate together.

Other vitamins (C1, B6, B1, B2, PP) also have an effect on the germination of pollen.
Pollens of different species and also of different varieties of the same species do not
give the same reaction to the addition of vitamins. For example, pollens of one variety
of tobacco require 0.0002 mg of vitamin B1, and pollens of another variety of the same
plant require 0.005 mg per liter of the solution. Thirty one per cent of pine pollens
germinated in a medium vitamin PP and in the presence of this vitamin 54% of the
pollens germinated. About 10-12% of the pollen grains of one variety germinate in the
presence of vitamin B1 and in another variety--52% germinate (Polyakov, 1949).

Part III, continued:

The assimilation of biotic and other substances by plants

The problem of plant absorption of biotic substances-vitamins. auxins, and other

organic substances, has interested investigators for a long time.

Assimilation of vitamins
 Many investigators have studied the uptake of vitamins and auxins through the root
system or leaf surface. Carpenter (1943) introduced riboflavin by spraying the crowns
of decapitated plants such as tomatoes, tobacco, fuchsia and carrots, which were
subsequently kept in a dark room. The analysis of their sap showed the presence of
riboflavin in much higher concentrations than that in the control plants, sprayed only
with water. Plants sprayed with a thiamine solution contained thiamine in higher
concentrations than the control plants (Hurni, 1944; Schopfer, 1943).

Bonner et al., (1939) analyzed plant tissues grown in a solution containing vitamin B1.
The results of these experiments are given in Table 54.

Table 54
Thiamine concentration in leaves of plants after its artificial application, in mg/kg of
dry weight
(according to Bonner at al., 1939)
Plants Treated Plants Control Plants
Brassica alba Schmalh. 15.8 6.0
Brassica nigra Koch 6.4 3.9
Agrostia tenuia L. 8.6 5.8
Poa trivalis L. 7.2 4.4
Cosmos Gay 6.0 5.0

Radioactive vitamins are being used in recent years for the detection of their uptake by
plants.

Shavlovskii (1954) employed vitamin B1 containing radioactive sulfur S35. The plants
were grown under sterile conditions on an agar medium of Knopp, supplemented with
the mentioned radioactive vitamin at a concentration of 0. 1 y/ ml. The activity of this
medium was 4,400 counts / minute (cpm) per 1 ml. The amount of vitamin in the tissues
of the plant was determined after various periods of time. The number of cpm showed
the amount of absorbed vitamin. The results are given in Table 55.

Table 55
Absorption of radioactive vitamin B1 by plants, from sterile agar substrate
(according to Shavlovskii, 1954)
Buckwheat: Buckwheat: Peas: cpm Peas: cpm Corn: cpm per
Plant organs cpm per plant cpm per plant per planlt per plant plant after 11
after 11 days after 38 days after 11 days after 38 days days
Leaves 3,458 4,920 1,110 1,326 6,993
Stems 1,655 13,288 1,486 2,912 --
Roots 505 1,601 455 3,360 783

These results show that the radioactive (S35 ) vitamin B1 enters the plant is the roots, is
initially concentrated in the leaves, and then gathers in the stem and in the roots.
Apparently, the increase of the vitamin concentration in the roots and stems in the late
growth phase of the plant may be explained by the fact that the vitamin is absorbed in
the early phase of the growth when the vitamin synthesis by the shoot is inadequate.
Later on the plant begins to synthesize the required amount of the vitamin. and the
latter, under favorable conditions, enters the stem and roots. It has been shown that
plants can obtain vitamins from microorganisms. This was demonstrated by the use of
radioactive isotopes. If bacteria or yeasts were saturated with an amino acid or vitamin
labeled with phosphorus P32 or sulfur S35 and inoculated into a medium where plants
were being grown, the radioactive substances were soon detected in the tissues of
plants. Shavlovskii (1954) grew buckwheat in sand with a culture of Ps. aurantiaca or
yeasts (Torlopsis utilis, T. latvica and Rhodotorula rubra). All the cultures were
previously saturated with radioactive vitamin Bl (S35). After 11 days the plants were
analyzed for the S35 content of their tissues. Simultaneously the excretion of the
radioactive vitamin by the bacteria was measured. The analyses showed that buckwheat
takes up the vitamin excreted by the microbes in detectable amounts. The greatest
amount went to, the plant (8.5 % of the total activity) from Ps. aurantiaca (Table 56).

Table 56
Transfer of vitamin B1 from microbes to buckwheat
(according to Shavlovskii, 1954)
S35 (of vitamin S35 (of vitamin S35 (of vitamin S35 (of vitamin
B1) cpm/plant B1) cpm/plant B1) cpm/plant B1) cpm/plant
Part of plants
Ps. aurantiaca T. latvica Rh. rubra T. utilis
8,100 cpm 45,000 cpm 9,100 cpm 10,230 cpm
Cotyledons 294 252 144 150
Stems 217 161 96 114
Roots 180 109 77 77
Total per 1 plant 691 522 317 341
Given up by
microbes to 8.5 1.2 4.0 3.3
plant, in percent

Assimilation of amino acids

The capacity of roots to absorb amino acids has been proven experimentally. Petrov
(1912) gives data on the absorption of asparagine by plants (corn). Shulov (1913),
Pryanishnikov (1952) and Byalosuknya (1917) confirmed these data. According to
them, asparagine is a good source of nutrition for peas, corn, cabbage, mustard, and
other plants.

Hutchinson and Miller (1911) showed that pea plants can assimilate leucine, glycine,
aspartic acid and tyrosine. Klein and Kisser (1925) have shown that during growth in
sterile conditions, oats can assimilate arginine not less than they can assimilate nitrates.
According to Virtanen and Lathe (1937, 1946), peas and clover assimilate aspartic acid
well, while wheat and barley react negatively to this substance, Steinberg (1947)
showed that isoleucine has a harmful effect on the growth of tobacco. Tanaka (1931) has
shown that the plant Sysirinchium bermudianum utilizes asparagine, glycine, and
cystine. Miller (1947) followed the absorption of dimethionine by tobacco and tomatoes
growing under sterile conditions. The sap of roots and aerial parts contained 1.0- 2.5 mg
methionine per 5 ml. Miller is of the opinion that amino acids are assimilated by plants.

Riker and Gütsche (1948) have shown that some amino acids suppress the growth of
isolated tissues of sunflower. The authors think that the deleterious action of the amino
acids is caused by their excess, which interferes with the metabolism. Sanders and
Burkholder (1948) noted that a mixture of amino acids has a more favorable effect than
each of them separately.

Virtanen and Lincol ascribe a stimulating role to the amino acids. According to them,
small concentrations of alanine and phenylethylamine (decarboxylation product of
phenylalanine) strongly affect the growth of peas, in the same way as the heteroauxin.
The plant parts change markedly, becoming stronger and greener.

In order to demonstrate the uptake of amino acids by plants, Shavlovskii (1955)

studied the absorption of, radioactive methionine S35 by buckwheat, corn, and peas.

This culture excretes more vitamin than the others. Yeasts excreted various amounts of
the vitamin depending on the species. The data given below should be considered as
approximate and possibly lower than in reality. This amino acid was added to the
medium of Knopp in which these plants were grown. After 11 days of growth the plant
tissues were examined for the presence of radioactive sulfur. The results are given in
Table 57.

Table 57
The absorption of radioactive methionine S35 by plants (cpm per plant)
(according to Shavlovskii, 1955)
Activity, Activity,
Specific Specific Specific Activity, (dry
(dry (dry
Plants activity: activity: activity: weight) of:
weight) of: weight) of:
Leaf Stem Root Root
Leaf Stem
Buckwheat 56 81 625 389 759 1,389
Corn 65 -- 124 5,814 -- 3,452
Peas 46 31 628 828 992 7,530

As can be seen from the table, the absorption of this amino acid by plants is quite
vigorous. The highest concentration of the amino acid in in the roots. The intensity of
absorption varies according to the composition of the medium and external conditions.

The absorption of methionine is more rapid and more pronounced in the presence of
vitamins B1 and B6.

Ratner and Dobrokhotova (1956) have shown the activating effect of thiamine
pyridoxine and pantothenic acid on the synthesis of glutamic acid and alanine in the
roots of sunflower.
 The distribution of the radioactive sulfur of methionine is different from that of
radioactive inorganic sulfur. In the former came sulfur is concentrated in the roots and
in the latter, in the aerial parts (Thomas and Hendricks, 1950, and others). This gives
grounds for the assumption that methionine as well as other amino acids are assimilated
by plants without any change in their molecules and are being used by them for protein
synthesis. Shavlovskii extracted protein from the roots of peas which had grown in the
presence of radioactive methionine and showed that they contained the major part of the
methionine absorbed by the roots. One gram of fresh roots gave 23,171 cpm, and the
proteins isolated from them--13,220 cpm.

Plants absorb the amino acids synthesized and excreted by microbes. This was shown
by Shavlovskii who used methionine containing radioactive sulfur S35. As in
experiments with vitamins, Shavlovskii grew bacteria Ps. aurantiaca and yeasts
Sacchar. cerevisiae, in a medium containing radioactive sulfur S35 in the form of (Na2
S3504). The bacteria were then autolyzed and the autolysates containing radioactive (S35)
amino acids were added to the medium where plants were grown, Radioactive sulfur
was then found in the plant tissues; the roots showed more radioactivity than the aerial
parts. In the presence of autolysate of Ps. aurantiaca the activity in the cotyledon tissues
was 137 cpm, in the stems--356 cpm, and in the roots--720 cpm; in the presence of
yeast autolysate the corresponding figures were: 43 cpm, l99 and 569 cpm.

In his subsequent experiments Shavlovskii grew buckwheat in a medium with living

cultures of Ps. aurantiaca previously grown in a medium containing radioactive sulfur.
Seeds of the buckwheat were infected with these cells before sowing (700 million cells
per seed, with a total activity of 125,000 cpm).

The following activity was found on the second day of growth in the plant:
cotyledons--228, stems--181, roots--132 cpms. Consequently, on the second day the
plants had taken up about 0.4 % of the bacterial radioactivity. A direct relationship
between the amount of bacteria and the absorption of radioactivity was noted. Upon
introduction of 3.45 billion cells per seed, about 1% of the total radioactivity of the cells
was detected in the plants (after 7 days growth).

The capacity of microorganisms to transfer their metabolic products to the plants was
shown in a paper by Akhromeiko and Shestakova (1954). These authors, in their studies,
employed radioactive phosphorus P32. They grew Az. chroococum, Ps. fluorescens and
yeasts (isolated from soil) in media containing P32 as a source of phosphorus nutrition.
The cultures of microorganisms thus grown were carefully washed with water and
inoculated into the sand in which saplings of oak and ash trees were grown.

Experiments had shown that radioactive phosphorus is taken up by the plants in

considerable amounts; it is emitted at a different rate and in different amounts by the
various microbial species. The greatest effect is obtained in experiments with yeasts.
About 43 % of the radioactive phosphorus from yeast was taken up by plants in the first
days of their growth. Oak saplings absorbed more radioactivity than ash-tree saplings.

These experiments show that biotic substances formed by microbes, in addition to the
amino acids and other metabolites, are excreted into the substrate, and from the
substrate are absorbed by plant roots.
 Assimilation of antibiotics

Higher plants absorb not only vitamins, auxins and amino acids but also many other
organic compounds present in the soil and formed by microorganisms.

Of all the metabolites which can serve as indicators of absorption by plants,

antibiotics, in our opinion, are the most outstanding.

Antibiotics are very specific. They are not present in plant tissues and are not formed
by them. They are easily detected and differentiated from other organic substances
including phytocides. In our experiments we have employed antibiotics in their native
state as well as in the form of chemically pure preparations. Antibiotics produced by
different representatives of soil microorganisms were used: penicillin (mold product),
streptomycin, globisporin, aureomycin, terramycin, and others (products of
actinomycetal metabolism), subtilin, gramicidin, and others (of bacterial origin).

The crude as well as the chemically pure preparations were added, in various
concentrations, to substrates where plants were grown. Experiments had shown that
antibiotics were taken up by roots rapidly and in considerable amounts and were more
or less uniformly distributed in all plant tissues and organs.

In a manner similar to that of biotic substances and amino acids the antibiotics
concentrate in the root system more than in other parts. From there they enter the aerial
parts.

Antibiotics are absorbed by plants directly from the soil where they are formed by
microorganisms. The latter, as it will be shown below, developing in soil under certain
conditions, produce and accumulate considerable amounts of these active metabolites,
These naturally formed substances enter the plant tissues via the root system in the same
way as the chemically pure antibiotics.

The antibiotics are known to be rather complex organic substances of high molecular
weight. For example, streptomycin consists of three basic groups: N-methyl, S-methyl
and also carbonyl group. Its formula is C21H39O12N7. Its molecular weight is more than
500. No less complex are aureomycin, terramycin, penicillin and other antibiotics.

If such complex organic compounds as antibiotics, amino acids and vitamins are
absorbed it may be assumed that many other carbon and nitrogen compounds present in
the soil are also absorbed by plants.

A voluminous work was performed in the laboratory of the famous French botanist
Bonne to determine the absorption of organic compounds by plants. Laurent J. and
Laurent J. (1903). studied assimilation of many organic compounds by plants.
According to their data, peas, lentils. corn, rice, and wheat utilize glucose, saccharose,
glycerol, dextrin, starch and potassium humate. Lefevre (1905, 1906) noted the capacity
of plants to assimilate amino acids and other nitrogenous compounds. Ravin (1913),
experimenting with horse radish came to the conclusion that plants can assimilate the
organic acids--succinic, citric, malic, tartaric and oxalic.
 Maze and Perrier (1904) grew corn in a methanol-sugar solution. In 30 days of growth
these plants absorbed 10-14 g of saccharose, their dry weight was 14-21.9 g.
Approximately the same amount of glucose was absorbed by plants in another series of
experiments.

According to Pryanishnikov, 1952; Shulov, 1913; Byalosuknya, 1917; and others,

peas, corn, cabbage, buckwheat and other plants assimilate well glucose, saccharose and
lactose. Especially good growth of plants was obtained in the presence of levulose.

Klein and Kisser (1925) had shown that oat cultures assimilate organic compounds
well without preliminary clevage of their molecules. Arginine is assimilated less than
nitrate nitrogen. Similar data were obtained by a Japanese investigator Tanaka (1931).
This author studied the assimilation of organic nitrogenous co pounds by higher plants
under conditions of sterile growth and obtained positive results.

Seliber (1944) brings results of his own studies and those of others on the growth of
potatoes at the expense of starch and other substances present in the tubers. The sprouts
of potatoes grow worse if the mother tubers are removed. The author describes cases of
formation of sprouts and tubers inside the mother tuber. The growth of sprouts in those
cases proceeds completely at the expense of food reserves of the mother nodule.
Organic compounds of the latter pass into the embryo and into the daughter tuber.

Numerous experimental data as well as agrobiological observations exist which

confirm the possibility of heterotrophic nutrition of plants.

Effect of bacteria on the assimilation of nutrients by plants

The assimilation of mineral and organic nutrients by plants proceeds at various

intensities and depends not only on the composition and properties of the compounds in
question but also on the quantitative and qualitative composition of the microflora
around the root system.

Studies show that under conditions of sterile growth of plants these substances are not
absorbed to the same extent as in the presence of microorganisms. We have grown
wheat, peas and corn in a sterile nutrient solution and followed the absorption of
penicillin, streptomycin, aureomycin and other antibiotics in the presence and absence
of various bacterial species.

Bacterial cultures were chosen which did not inactivate or decompose the above-listed
antibiotics and were at the same time resistant to them for the inoculation of the nutrient
solution. We have tested and chosen more than 10 cultures belonging to different
species: 3 cultures of root-nodule bacteria (Rh. trifolii, Rh. meliloti and Rh. phaseoli), 2
strains of azotobacter (Az. chroococcum), 4 strains of Ps. fluorescens, isolated from the
rhizosphere of different plants, and 2 strains of the genus Bacterium (Bact. denitrificans
and Bact. sp.) also isolated from the rhizosphere of wheat a nd peas. The effect of the
bacteria was determined by the rate of disappearance of the antibiotics from the solution
and their uptake by the plants. The presence of antibiotics in the plants was determined
by the conventional microbiological tests. The indicator microbes were cultures of the
sporiferous bacillus Bact. subtilis and staphylococcus--Staph. aureus.

Tables 58 and 59 bring data on the uptake of streptomycin and penicillin by wheat and
peas in the presence of 5 bacterial cultures and their mixture. Experiments with corn and
other plants gave similar results.

Table 58
The effect of bacteria on the uptake of streptomycin by wheat
(units per g of plant tissue; the initial solution contained 2,500 units-100 units/ml)
Roots
Stems after Leaves Roots after Stems after 3 Leaves
Bacteria after 1
1 day after 1 day 3 days days after 3 days
day
Az.
100 20 10 160 20 75
chroococcum
Rh. trifolii 150 30 40 200 20 100
Ps flourescens
30 0 0 50 20 30
No 4
Bacterium sp.
20 0 0 50 10 10
25
Oligonitrophil 100 30 50 150 20 50
Bacterial
200 50 100 200 30 120
mixture
Control
(without 20 10 0 100 10 60
bacteria)
Table 59
The effect of bacteria on the uptake of penicillin by peas
(units per g of plant tissue; initial solution in the vessel contained 5,000 units--200
units/ml)
Roots Leaves
Stems after Roots after Stems after 2 Leaves after
Bacteria after 10 after 10
10 hours 2 days days 2 days
hours hours
Az.
120 30 40 200 120 180
chroococcum
Rh. trifolii 100 20 40 250 100 150
Ps
flourescens 10 0 0 50 10 20
No 4
Bacterium sp.
5 0 0 30 5 10
25
Bacterial
250 50 100 250 120 210
mixture
Control
(without 50 5 0 150 50 100
bacteria)

As can be seen from the table, the bacteria have a considerable effect on the uptake of
antibiotics by tissues of plants. The various bacterial species have different effects.
Some of them enhance the uptake (Az. chroococcum and Rh. trifolii) others (Ps.
fluorescens No 4 and Bacterium sp. No 25) inhibit the uptake of antibiotics by the
plants. The largest amounts of streptomycin and penicillin were absorbed in the
presence of a bacterial mixture and the least absorption was in the vessels containing the
culture of Bacterium sp. No 25.

The analyses have shown that the increased concentration of antibiotics in the plants is
accompanied by decreased concentration of antibiotics in the solution.

Analogous data were obtained in experiments with plants growing in sand. The uptake
of antibiotics was highest in the presence of Azotobacter and root-nodule. bacteria and
the smallest in the presence of the nonsporiferous bacillus Bacterium sp. No. 25 (Figure
66).

Figure 66. The effect of bacteria on the uptake of streptomycin (from sand) by plants
(peas). The analysis was performed 10 hours after the introduction of the antibiotic into
the vessel:

I--bacteria of the group of oligonitrophils (strain 25 and others); II--Azotobacter (the

same results with Rhizobium); III--control without bacteria. 1--roots, 2--lower part of
stem, 3--middle part of stem, 4--upper part of stem, 5--leaves.

Gerretsen (1948) described the enhancement of nutrient uptake by plant roots under
the influence of the microflora. He grew oats, sunflower, buckwheat and other plants in
vessels with sterile and nonsterile soil, in the presence and absence of bacteria.
Phosphates, soluble. insoluble and sparsely soluble in water, were introduced into the
soil (bi- and tricalcium, phosphate, phosphoric powder, etc).

The experiments have shown that under sterile conditions, without bacteria, the uptake
of phosphates proceeds at a lower rate than in the presence of bacteria. The greatest
effect was obtained in the experiments with buckwheat. in the presence of specially
chosen bacteria.

Kotelev and Garkovenko (1954) studied the uptake of inorganic radioactive

phosphorus by bacteria. Under sterile conditions the radioactive phosphorus was taken
up at a slower rate and in smaller amounts. The plants showed only 4% of the total
radioactivity when grown under sterile conditions, in the presence of bacteria the
radioactivity of the plants increased to 13%.

Experiments with labeled (P32) lecithin gave analogous results. Lecithin was added to
the solution used for wetting sand in which barley was grown. The experiments were
carried out in the presence and in the absence of bacteria. In the absence of bacteria
1.9% of the radioactive phosphorus was taken up, while in the presence of bacteria
16.6-18.6% of the phosphorus was taken up, i. e., 8-9 times more.

Bacteria markedly increase the rate of phosphorus uptake by plants from granules
prepared with radioactive superphosphate. Barley sprouts grown under sterile conditions
gave 400--500 cpm, but 1,000-1,500 cpm, when grown under nonsterile conditions
(cpm. per 10 mg of plant dry weight) (Kotelev, 1955).

We employed the tracer-atom technique to carry out a number of experiments on the

uptake of phosphorous compounds from a substrate in the presence and in the absence
of different bacterial species. Plants (barley) were grown in a nutrient substrate
(solution, sand and soil), previously sterilized and then inoculated with pure cultures of
bacteria. Radioactive phosphorus (P32) was then added to the substrate. After 15-20 days
of growth the plants were analyzed. We determined the presence of organic and
inorganic phosphorus, as well as sparsely soluble (in water) phospholipides, and
phosphoproteins. In addition, amino acids were determined (by the use of paper
chromatography) in the fraction containing organic water-soluble compounds
(Krasil'nikov and Kotelev, 1956). Bacteria isolated from the rhizosphere of corn, grown
in Moldavia, were used in these experiments. All of them belonged to the
nonsporiferous bacteria of the genera Bacterium and Pseudomonas. One culture (10-A)
was isolated from podsol soil of the Moscow Oblast'. Each vessel filled with sand was
supplemented with 465 mg P205 (P32). The total activity per vessel was 6,400,000 cpm.
Counting was performed after 17 days growth. The results of the first series of
experiments are given in Table 60.

Table 60
The effect of soil bacteria on the uptake of radioactive phosphorus by plants
Fraction of
Fraction of
organic P, Fraction of
cpm/100g of organic P,
Uptake of cpm/100g organic P,
Experiment dry plant cpm/100g
P32, % plant mass in cpm/100g plant
substance plant mass in
aqueous mass in proteins
lipides
solution
Control 3,000 0.7 375 450 1,700
(without
bacteria)
Infected with
3,830 1.4 475 575 2,300
Bacterium sp.
Infected with
4,300 1.3 578 675 2,300
Bacterium sp
Infected with
-- 1.2 650 700 2,900
Bacterium sp
Infected with
-- 0.6 300 400 2,000
Bacterium sp

Figure 67. Radiochromatograms of organic and inorganic phosphorus compounds in the

soil and in plant tissues. The plants were grown in sterile and nonsterile soil (in the
presence of natural microflora).

1--phosphorus compounds in the soil (sterile soil); 2--phosphorus compounds in plants

(barley), grown in sterile soil; 3--phosphorus compounds in plants grown in nonsterile
soil; a--organic phosphorus; b--inorganic phosphorus.

The radiochromatogram, (Figure 67) represents the process of accumulation of organic

phosphorus in the tissues of barley grown under sterile conditions, and in the presence
of bacteria. It can be seen from the figure and Table 58 that bacteria have a considerable
effect on the uptake and accumulation of phosphorous compounds in the tissues of
plants. Under the influence of the bacteria a formation of quite different phosphorous
substances takes place.

The results of the determination of amino acids are not loss demonstrative. We have
determined the amino acids present in extracts of 70% alcohol by paper
chromatography. Five hundred-mg samples of the dry mass of plants were extracted for
4 hours at 45° C with 25 ml of alcohol. The extracts were then filtered, the filtrate was
evaporated to 1-2 ml and subjected to chromatography. The results are given In Table
61.

Table 61
The effect of bacteria on the composition of amino acids in plant tissues
Dry
Uptake
weight Glu-
Experimen of P32 Aspara- Aspartic
of Lystine Serine Glycine tamic Alanine Valine
t per gine acid
plants, acid
100 g
mg
Control
(without 545 465 ++ + +/- - - - - -
bacteria)
bacteria 1p 573 700 ++++ +++ +++ +++ +/- +/- ++ -
bacteria 2p 710 786 +++ ++ ++ ++++ + +/- +++ -
bacteria
-- -- +/- - - +++ ++++ ++++ +/- +
125
bacteria
-- -- + ++ ++ ++ - +++ +/- +/-
151-A

Legend: 4 pluses--very intense color; 3 pluses--intense color; 2 pluses--moderate color;

1 plus--weak color; plus--minus-color hardly detected, on the border of accuracy;
minus--absence of the amino acid.

Figure 68. Radiochromatograms of the amino acid composition of plants grown in

sterile conditions after artificial contamination with bacteria:

A) amino acids of plants grown in sand; B) amino acids of plants grown in the soil
(chernozem). a-- amino acids of plants grown in the absence of bacteria; b--amino acids
of plants grown in the presence of Bacterium sp 1; c--amino acids of plants grown in the
presence of a strain of Bacterium sp. 2; 1--lysine; 2-3--asparagine and aspartic acid; 4--
serine; 5--glycine; 6--glutamic acid; 7--alanine; ?--unknown compound.

The radiochromatogram (Figure 68) shows the distribution of amino acids in plants
grown in the presence of 2 bacterial cultures (lp and 2p). It can be seen from the figure,
that the presence of bacteria in the substrate causes formation and accumulation of
amino acids of a different nature to those formed in the absence of bacteria. Some
amino acids (serine, glycine, alanine, valine, cysteine) cannot be detected, in the control
sprouts of the barley grown under sterile conditions, only their traces can be found.
These amino acids are present in considerable amounts in plants grown in the presence
of bacteria. Different bacterial cultures have a varying effect on the formation and
concentration of amino acids in plant tissues. In our experiments cultures 125 and 151-
A favored the accumulation of glutamic acid, and bacteria lp and 2p favored the
accumulation of alanine, lysine and asparagine.

The effectivity of the same bacteria to plants growing in soil (heavy loam chernozem),
although considerable. was somewhat different to that noticed when the plants were
grown in pure sand. The quantitative and qualitative relationship of amino acids was
different. Neither lysine nor alanine could be found in the barley sprouts grown in the
presence of lp and 2p bacteria, asparagine and glycine were found only in traces (Figure
68 B).

According to Akhromeiko and Shestakova (1954), microorganisms inhibit the uptake

of phosphorus (P32) by woody plants. Saplings of oak and ash tree were grown in sterile
and nonsterile soil and the uptake of radioactive phosphorus was determined. The
authors have noticed that bacteria of the rhizosphere at first take up the tracer
phosphorus and subsequently release it.

Ratner and Kozlov (1954) grew corn under sterile conditions, according to the method
of Shulov, in the presence and in the absence of bacteria in the solution. They found that
when bacteria were present in the solution, organic compounds of phosphorus and
nitrogen were found in larger amounts than when the plants were grown without
bacteria. In the sterile vessels the plants contained the following: amides and amines--
1.300 mg; organic nitrogen--1.191 mg (32.33%); in nonsterile vessels the respective
figures were 1.562 mg and 1.960 mg (44.22%).

The difference in composition of the amino acids is well defined on the chromatogram.
The addition of microbial metabolic products, or a dead culture, to plants in their early
growth stage on sterile medium, causes an increase in the phosphorus and nitrogen
content of their exudate. The microbial metabolic products increase not only the uptake
of these substances by the roots but also the synthetic capacity of the roots. The
increased incorporation of the radioactive phosphorus P32 into the lipides and
nucleoproteins, as well as the increased content of amide and amine nitrogen in the
exudate confirms these assumptions.

Microorganisms and their metabolic products affect the process of nitrogen

transformation in the root. In the presence of microorganisms the rate of metabolism of
amino acids in the roots increases as well as the process of transformation of the
inorganic to organic nitrogen. In the presence of microorganisms the uptake of inorganic
and organic compounds--microbial metabolites--is increased.

Besides, microorganisms promote the transport of nutrients in the soil. They are
carriers of nutrients, supplying the root system with various nutrients.

Numerous papers have stressed the point that mere contact of roots with the soil is not
sufficient to secure the nutrient requirements of the plant. Mediators between nutrient
sources of the soil and the root system exist in soils. Such mediators are the microbes.

Khudyakov (1953 b) has shown that molds can transport nutrients in their hyphae. It is
known that protoplasm moves along the hyphae at high speed. In mucor fungi it is as
much as 50-80 µ and more per minute. Various inorganic and organic compounds,
including nutrients move together with the protoplasm from the site of their
concentration to the site of demand in the growing mycelium of the fungus. It is well
known that many fungi grow abundantly on roots and around them, forming the
mycorhiza.

Not only fungi but also bacteria promote the transportation of nutrients in the soil.
Kotelev (1955) employed tracer techniques in the following manner. He introduced
grains of radioactive P32 in the form of superphosphate into the soil and followed the
diffusion of the phosphorus in the presence and absence of bacteria. The diffusion of
phosphorus in sterile soil was very slow and uptake by roots was either lacking or
negligible. The diffusion of phosphorus in the presence of bacteria was much more
rapid.

The above indicates that it is not permissible to consider the problem of plant nutrition
only from the point of view of autotrophy. Alongside the assimilation of inorganic
compounds, plants also assimilate various organic-carbonaceous and nitrogenous
substances. Some of these are utilized to meet the plant's energy requirements, others
serve as biocatalysts. The latter are taken up by the roots and aerial parts of the plant
and increase the intensity of the biochemical and biological processes in the cells and
tissues. They enhance the growth of plants and increase the absorption capacity of the
roots the assimilation of absorbed substances and other vitally important functions.

The biologically active substances of the soil not only enhance the growth and increase
the yield of plants but also confer on the plant better nutritional qualities.

Plants which obtain vitamins and other organic compounds from the soil in adequate
amounts, yield crops of higher quality and their seeds are of a higher vitality.

The fact that plants can grow in pure mineral nutrient media in the absence of
microorganisms cannot serve as proof of the uselessness of the latter in the nutrition of
plants.

Plants can indeed be grown in mineral media and yield seeds without the participation
of microbes. Is it possible, however. to secure the vitality of such plants in subsequent
generations?

We have presented our observations on the growth of green algae and duckweeds.
Grown under sterile conditions, in mineral media without the addition of composts or
metabolic products of bacteria, these plants lose their viability and eventually die out.
Plants grown in the same media but supplemented with composts or metabolic products
of bacteria, as well as plants grown in the presence of living bacterial cells (nonsterile
conditions) have been kept in our laboratory for more than 20 years without any visible
decrease of vitality.

The assumptions of some authors concerning the fact that soil contains only small
amounts of organic compounds of phosphorus and nitrogen (in the form of vitamins,
amino acids and phytin) which cannot, therefore, be considered as nutrients of any great
importance, are also groundless. Our knowledge of forms of organic compounds and of
the dynamics of their transformations in the complex microbial coenoses, is inadequate.
The knowledge we do possess, however, allows us to assume that the processes of
synthesis of various organic compounds proceed incessantly in the soil. Owing to such
uninterrupted synthesis (be as it may in small amounts) the total production of these
compounds may be sufficient to meet the needs of plants.

We cannot understand the assumption of certain authors that microorganisms by their

assimilation of inorganic substances preclude the utilization of the latter by plants. This,
according to some authors, leads to plant starvation. The saprophytes of the soil are
looked upon as a harmful factor. They do such harm to plants that, according to some
specialists, they should be separated from plants because of their competition for
soluble salts (Peterburgskii, 1954).

Bacterial life in the soil is known to be very short; it is counted in hours. Even during
their life, the dying cells are subjected to autolysis. At the end of the enzymatic lytic
processes, processes of solubilization of their residues by enzymes of other microbes
ensue. The process of bacterial cell destruction is rapid, The metabolism of living cells
is an endless sequence. The elements absorbed from the substrate are soon excreted.
Experiments with radioactive elements have shown that phosphorus (P32), for example,
appears in the substrate after a few minutes.

The microflora of the root zone is of great importance in plant nutrition. Growing near
or on the roots, microorganisms, together with the plants, create a special zone--the
rhizosphere. Soil in this zone differs in its physical, chemical and biological properties
from the soil outside the root zone. It possesses different conditions, for the absorption
and excretion of substances by the roots.

The interaction between microorganisms and plants on one hand, and between
individual microbial species and their metabolites, on the other hand, is the basis for the
different transformations of inorganic and organic compounds. As a result, compounds
which serve as nutrients for plants are formed. These are absorbed by the roots.
 Substances present in the soil are subjected to a greater or lesser extent of processing
before their absorption by the roots. The plants do not absorb those compounds which
are characteristic for this or other soil, but metabolic products of the rhizosphere. The
rhizosphere microflora prepares various organic and inorganic nutrients for the plants.

The role of the rhizosphere microflora reminds one of the digesting organs of animals.
Microorganisms in the final account serve the same function in the plant nutrition as the
digestive system of animal organisms (Krasil'nikov, 1940).

The same point of view is held by the American specialist Prof. Clark (1949). He
considers that microorganisms living in the rhizosphere perform the same work as do
the intestines of animals. Academician Lysenko (1955) is even more definite in this
respect. He thinks that the microflora of the root zone acts as the digestive organ of
plants. We can agree with such a comparison if we recall the function of the microflora
of the intestines. In recent years, the microflora of the intestines has more and more
come to be considered as a factor of supplementary nutrition for animals and human
beings. Many intestinal bacteria are known to produce substances which enter the
organism of the animal and play an essential role in biological processes as biocatalysts.

In many animals, microorganisms of the digestive tract participate directly the

digestion of food. For example, the cellulose bacteria in the intestinal tract of ruminants,
decompose cellulose into digestible products.

Part IV

INTERACTION BETWEEN SOIL MICROORGANISMS AND

PLANTS

We have already noted the importance of microorganisms in the life of plants as being
among the biological factors of soil fertility. Between the two there exist special
interrelationships which express themselves to some degree by the total productivity of
the soils.

Investigations show that the vegetative cover is a powerful factor in the life of
microorganisms. The peculiarities of the plant species leave their imprint on the
quantitative and qualitative composition of the microflora biocoenoses of soils. Plants
create and form microbial societies, which effect the microbial population of the entire
root system and harvest remains. During their life, plants excrete through their roots
various organic and mineral substances which attract microorganisms. During the
growth cycle of the plant, roots constantly form and shed root hairs, lose necrotic
epidermal cells, etc. All these elements are then taken up by the microbes and become
the source of their nourishment.

The species composition of the microflora, just as the soil, has a definite and
considerable influence on the growth and development of plants, and consequently on
the crop yield. The study of the nature of the interaction between microorganisms and
plants is one of the main and most interesting problems, not only of microbiology, but
also of the study of soil and plant cultivation.

Effect of Plants on the Soil Microflora

The effect of the vegetative cover on the microflora of the soil was studied long ago by
various investigators. Plants not only have an influence on living microorganisms
(through their root system) but also influence them after their death and after the
harvesting of the crops (in cultivated fields), In the former case, this effect is caused by
root excretion and by the dying particles of the roots themselves. In the latter case, the
active factors are the remnants of the roots and the aerial parts. In both cases, great
changes take place in the composition of the soil microflora. In addition, the roots exert
a beneficial effect on the physical and chemical properties of the soil, improving the
conditions for the existence of microorganisms. One can judge the importance of plant
roots in the biology of soils by the great bulk of the root mass and its extension.

Root mass of plants

The studies made by Kachinskii (1925), Chizhov (1931), and others showed that the
root system is a great mass by weight with a vast surface. This was observed by Tol'skii
(1904-1911) when he studied the root system of forest plantations in the Buzuluk forest.
The author gives numerical data on the development of the root system and its
dispersion in woods on different soils and with differing densities of planting. At a
density of 77 trees per 0.2 hectares, the length of the roots of one plant is 0.21 km, and
at a density of 260 trees per 0.2 hectares, it is 0.42 km.

L. P. and L. L. Golodkovskii (1937), on the basis of observations made on the

development of the root system of lucerne under differing soil-climatic conditions of its
growth, obtained the following data (Table 62).

Table 62
Development of the root system of a 3-year-old lucerne
under various soil and climatic conditions
(Weight of the air-dried mass of roots in 1 cubic meter of soil, in grams)
serozem, meadow, weak typical southern
Horizon, cm
watered watered podsolized podsol chernozem
0-10 343.1 253.7 315.0 348.0 692.0
10-20 149.2 157.0 386.0 386.0 181.3
20-30 95.1 57.0 100.0 95.0 47.3

According to data obtained by Dittmer (1937, 1938), one plant of winter rye has a total
root length (without hairs) of 623.4 km. The thinner the roots, the greater their length.
The main roots have a length of 0.07 km; the secondary roots 5.4 km; the roots of the
third order, 175 km, and those of the fourth order, 442.8 km. The largest mass consists
of the active part of the root system.

The area of all the roots of one plant is 237.4 m2; the area of the roots of the first order,
0.1 m2 ; the root area of the second order 4.2 m2; the root area of the third order, 70.5
m2; and the area of the roots of the fourth order, 162.8 m2.

The root hairs on the roots of one plant number to 14.3 billions. They are mainly
located on the roots of the third and fourth orders (14.1 billions). The length of all the
hairs of one plant is more than 10,000 km. Their total area (one hair is 700-1,000 µ in
length) is double the surface area of the root, i. e., more than 400 m2. Consequently, the
roots of one rye plant in the fourth month of growth have a total length of 11,250 km
and an area of 6,388 m2.

In the textbooks written by Kursanov, Keller, and Golenkin, smaller figures are given
for the length of roots. For instance, according to their data, the total length of melon
roots without hairs is 25 km; in wheat, about 20 km; and in spring rye, 623 km. The
surface of the root system of one rye plant is 237 m2. The surface of the roots of rye is
130 times greater than the surface of the aerial organs (Kursanov, 1940).

According to Pavlichenko (1938), the length of roots of the wild oat (Avena fatua) is
87.8 km, and of wheat, 71.2 km. In one cubic decimeter of soil, the total length of roots
in as follows: in oats, 6 5 cm; in rye, 90 m; and in meadow grass, 553 m. The length of
the root hairs is as follows: in oats, 11.6 km; in rye, 24.3.km; in meadow grass, 73 km.

Detailed studies of the root system of fescue plants, conducted by Savvinov and
Pankova (1942) in the Volga steppes, showed the following: in a two-meter 2 layer of
soil there are 1.75 kg dry mass of roots per m2, of which one third to one quarter are
living. The weight of the aerial part of these plants comprises 0.48 kg, i. e., about one
quarter of the total weight of the roots.

The nature of the distribution of the roots in the soil is illustrated in Table 63.

Table 63
Distribution of roots in soil layers, per 1 m2
(according to Savvinov and Pankova, 1942)
Weight of dry
Type of Weight of dry
Root length, Root surface, root mass,
plants and Depth, cm root mass, gm,
cm m2 gm, living
soils total
roots
Fescue
vegetation, 0-25 18.5 12.3 278 1,112
chestnut soil
0-50 28.0 18.09 344 1,419
0-100 32.0 20.77 418.4 1,735
0-300 36.0 22.40 436.3 1,757
Cereal-grass
vegetation,
chestnut soil
0-25 61.6 10.3 186.8 1,547
0-50 80.4 13.96 251.1 1,792.5
0-100 94.5 16.97 310.9 2,002.3
0-200 100.0 18.10

Shalyt and Kalmykova (1935) studied the formation of the roots of steppe vegetation
in chernozem soils ("Askaniya-Nova" National Reserve). According to their data, the
air-dry mass of roots of the Festuca-Stipa vegetative association is 3,002.5 g (30
tons/hectare). and in the Basaltic solonets of the same reserve 1,175.8 g per 1m3 (11.75
tons/hectare). The total surface of the Festuca roots is 225 m2, and for the Stipa, 126.2
m2. Savvinov and Pankova consider these numbers to be somewhat exaggerated. One
can assume that the cited differences have a purely ecological cause and are not the
result of a methodical error. It is obvious that under the conditions of the southern
Ukraine, which has chernozem soils, the relationship a between roots and their aerial
parts will differ from those of the chestnut soils near the Volga. Moreover, the
differences discussed by the authors are not very striking and fundamental. As in the
data submitted by Savvinov and Pankova, as wen as that of Shalyt and Kalmykova, the
numerical indicators are of about the same order.

Muntz and Girard (1888) measured the length and diameter of the roots of plants
grown on the experimental fields of the Paris Agricultural Institute and obtained the
following data. In 1 m3 of soil, there was a 5.18 m2 total area of clover roots; in the case
of meadow grasses, 7.58; oats, 10.70; winter wheat, 11.30; and poppies, 2.17m2.

According to Belyakova (1947), the root mass of the dry roots of lucerne in the soils of
the Vakhsh valley is as follows: in the first year of growth, 11.12 tons, in the second
year of growth, 22-24 tons, and in the third year, 30 tons and more per hectare.

Nad"yarnyi (1939) found that mixtures, several years old, of leguminous and cereal
grasses accumulate more roots than pure cultures. In the upper layer of the soil (0-20
cm), over a two-to three-year period, up to 40-75 centners* roots per hectare were
found. [*One Russian centner equals 100 kg.] A greater accumulation of root mass was
observed by Belyakova and Parishkura (1953) in soils having mixed crops of grasses. In
other studies, many tons of dry root mass per hectare are given.

The main mass of roots is concentrated in the surface layer (0-25 cm) or somewhat
deeper, depending on soil-climatic conditions and on the type of flora. Sometimes, in
the deeper layers of soil, a second maximum (less pronounced) of root concentration is
observed. The nature of the distribution of the root mass along the horizons of the soil is
given in Figure 69.
Figure 69. Distribution of wheat roots in the soil (according to Kachinskii, 1950)

According to their distribution, the importance of the roots will be greatest in the upper
horizons. The biological significance of the root system is determined by its activity, i
e., by its ability to absorb elements of nutrition from its surroundings and excrete
products of metabolism into the environment, Studies have shown that the active part of
the root system is its largest part. According to some date, it comprises 50 to 75 percent
of the total root mass.

The substances which are excreted by the root system are utilized by microorganisms
as nutrient sources. A great number of microbes concentrate around the roots, growing,
multiplying, and excreting their metabolites, many of which are assimilated by the plant
roots. The whole root system during the life of plants, as well as after their death, exert
an immense influence on the growth and development of microorganisms.

The effect of the root system on the composition of the microflora may be direct or
indirect, positive or negative.

The utilization of the nutrient elements by the plants is connected to some degree with
the metabolism of microorganisms. A greater influence of plants on microorganisms is
that the former enrich the soil with organic substances.

Root excretions

Roots are no longer looked upon as mere suctorial organs through which plants absorb
various nutrient elements from the soil. As early as the 18th century, the ability of roots
to excrete certain substances, which affect the properties of the soil and determine its
fertility, was noted.
 The presence of CO2 in the excretions of roots was noted by many authors including
Sossur (1804), Trevisan and Meis (1839), Pollaci (1858), and others. Sachs in 1860-
1865 experimentally demonstrated that the root systems of various plants excreted CO2
(Pryanishnikov, 1952; Konstantinov, 1950).

Lundergardh (1924) determined the amount of CO2 liberated by the roots of wheat
grown in sterile sand and in sand containing bacteria. He obtained the following results:
one gram of a dry mass of roots excretes 3.05 mg of CO2 per hour in sterile sand and
5.57 mg in the presence of bacteria.

The considerable excretion of CO2 by the roots of plants was observed by Zaikovich.
According to his observations, the roots of well-developed corn excreted 0.24 g per day,
and, according to Knopp, 0.25 g. In the experiments carried out by Kossovich, mustard
roots excreted, on the average, 27.3 mg of CO2 per day. Barakov observed the excretion
of CO2 by the roots of different plants and concluded that the maximum amount of CO2
excretion occurs during the period of the most active metabolism of the plant, during its
flowering (according to Konstantinov, 1950).

Chesnokov and Bazyrina (1934) grew flax in vessels with podsol soil or sand and
determined that the respiration of the soil with the plants growing in it greatly exceeded
the sum of the respiration of the same roots and soil taken separately.

The more bacteria present in the rhizosphere, the more intense the formation of CO2 by
the roots of plants (Table 64).

Table 64
The influence of plants on the formation of CO2 in soil at a temperature of 20i C
(according to Waksman, 1952)
Number of
CO2, mg per
Plants bacteria, millions / pH of soil
kg, per day
g
Triticum vulgare L. 49 6.75 69.4
Secale cereale L. 42 6.44 68.2
Avena sativa L. 45 6.42 79.0
Beta vulgaris L. 78 6.89 74.3
Medicago sativa L. 120 6.89 86.8
Trifolium pratense L. -- 6.66 82.4

The intensity of CO2 formation depends on the species of the plants, their age, the
season of the year, and other factors.

The approximate volume of the root respiration of grain cultures under conditions of
growth in the field comprised 25-30 percent of the respiratory volume of the soil as a
whole (Konstantinov, 1950).

During their life, plants excrete different mineral and organic compounds via their
roots. Compounds of phosphorus, potassium, calcium, sodium and other elements have
been found in root excretions.

Sabinin (1940, 1955) and his associates (Minina, 1927) have shown that the excretion
by roots of elements of mineral nutrition is accomplished by exosmosis and is regulated
by the concentrations of these substances in the external substrate. Tueva (1926)
established that the exosmosis of calcium and potassium from roots takes place until an
equilibrium state of these elements is established in the surrounding medium. Such a
regularity was found by Osipova and Yuferova (1926) in relation to the absorption and
excretion of sulfur and phosphorus by the roots of corn and wheat.

Avdonin (1932) observed the loss of ash elements in cultivated oats under field
conditions, These losses differ in quantity depending on the conditions of the growth of
the plant.

Akhromeiko (1936) decided that some plants excrete mineral substances via their
roots, while others do not. He observed phosphoric acid in the root excretions of lupine,
peas, buckwheat, mustard, and rape. The amount of phosphorus excreted attained 14-34
per cent of all the phosphoric acid taken up by the plant.

There are some writers who deny that it is possible for roots to excrete mineral
compounds. The authors of these works assume that the substances found are the
decomposition products of root residues.

Of the root excretions, the organic substances are of the greatest importance, The
presence of these substances was observed for the first time at the end of the last
century. Dyer (1894) established the presence of acidic compounds in the root excretion
of plants of barley, wheat, oats, foxtail, and others. Acids were detected in root
excretions by Lemmerman (1907), Künze, (1906), Schreiner and Reed (1907),
Doyarenko (1909), and others.

Stoklasa and Ernest (1909) found that plants excrete acetic, formic, and oxalic acids
through their roots. Maze (1911) and Shulov (1913) found organic acids and sugar in
root excretions. Organic substances were found by Kostychev (1926), Truffaut and
Bessonoff (1925, 1927), and others.

Mashkovtsev (1934) found that the roots of germinating seeds of rice excrete sugars,
aldehydes, ethyl alcohol, and other compounds precipitating with lead acetate.

Minina (1927) detected organic substances in root excretions of lupine, beans, corn,
barley, oats, and buckwheat, upon cultivating them in Knop's nutrient solution, The
excretion in most of these cultures reached its maximum during the fourth week of
growth, and in buckwheat, at a somewhat earlier period. Upon the ripening and aging of
the plants, the amount of root excretions decreases and toward the end of the growth
period stops altogether.

Lyon and Wilson (1928) found nitrogenous and nonnitrogenous organic compounds in
the root excretions of corn. The amount of nitrogenous substances in root excretions,
according to these authors, decreases with the age of the plant.
 Winter and Rümker (1952) observed phosphatides, amino acids, thiamine, biotin,
meso-inositol, paraaminobenzoic acid, carbohydrates, tannins and alkaloids in the root
excretions of plants. Harley (1952) found sugars, amino acids, vitamins, and other
organic compounds in root excretions.

Virtanen and his associates (Virtanen and Laine, 1937) observed in the root excretions
of young sprouts of leguminous plants; peas, clover, etc, aspartic and glutamic acids,
tryptophan and ß-alanine.

Cereals, oats and barley, grown in the same vessel with leguminous plants in the
complete absence of nitrogen sources in the medium, grew normally and developed at
the expense of the nitrogen excreted by the leguminous plants. Similar experiments
were conducted by Lipman as early as 1912.

The possibility of transferring metabolic products from certain plant species to others
was confirmed by the experiments of Preston and his associates (Preston, Mitchell and
Reeve, 1954). Plants sprayed with methoxyphenylacetic acid were grown in the same
vessel with plants which were not sprayed, After some time, the given substance was
detected in all the tissues of the unsprayed plants, in larger or smaller quantities: the
nonsprayed plants absorbed the methoxyphenylacetic acid excreted by the roots of the
sprayed plants.

Many investigators have detected a considerable amount of nitrogenous organic

compounds in the roots of cereal plants when they were grown together with legumes
(Scholz, 1939; Wyss and Wilson, 1938; Madhok, 1940; Nicol, 1934; Isakova and
Andreeva, 1938).

Sabinin (1940) found that the roots of pumpkins excrete from nine to eleven different
amino acids. These acids were determined and differentiated by paper chromatography
(Kursanov, 1953).

Other investigators deny the presence of nitrogenous substances in the root excretions
of leguminous plants (Bond, 1937, and others).

Wilson, Wyss, and others (1937, 1938) assumed the possibilty of the excretion of
nitrogenous compounds by roots. However, these compounds, according to these
authors, are metabolic products of the nodular tissues and not of the roots of the
leguminous plants themselves.

Engel and Roberg (1938) in order to verify Virtanen' s data, cultivated alder which was
inoculated with cultures of proactinomycetes, forming nodules, and observed in the
substrate (sand) a considerable quantity of organic nitrogenous substances excreted by
the roots.

Virtanen, in reply to Wilson, Bond, and others, noted that the process of the excretion
of organic substances is closely linked with external conditions--sunshine, aeration,
nutrition, and the pH of the medium. Confirming earlier data by new experiments, the
author stated that the detected nitrogenous compounds are products of the fixation of
free nitrogen, which were not utilized in the formation of protein and plant tissues and
not products of protein decomposition (Virtanen and Torniainen, 1940).

The organic compounds excreted by the roots of various plants are not identical. In
leguminous plants, one detects more nitrogenous compounds--amino acids, amide
compounds, and others (Virtanen and others, 1937, 1938, 1940). In cereals. the root
excretions are richer in carbon substances--sugars, organic acids, and others. According
to our observations, peas, broad beans, beans, lupine, and other leguminous plants
excrete substances having a neutral or weakly alkaline reaction, and cereals--corn and
wheat--secrete substances having an acid reaction, According to the data of some
investigators, the roots of peas excrete nucleotides and flavins (Lundegardh and Stenlid,
1944).

West and Wilson (1939) observed biotin and thiamine in the root excretions of flax and
sugar in the excretions of certain cereals, Brown and others (1949) proved the presence
of pentoses or closely related compounds (alpha-ketoxylose) in the root excretions of
grasses.

Brown and Edwards (1944) found special substances which stimulate the growth of
other plants in root excretions.

Groh (1926) studied the root excretions of lupine, broad beans, wheat, oats, barley, and
rye. In some plants, substances were detected which have an acid reaction, and others
which have an alkaline one. On the basis of these findings, the author divides plants into
two groups: the acid group, including peas, broad beans, lupine and wheat; and the
alkaline group, including oats, rye, barley, and mustard. According to Pryanishnikov
(1905), lupine excretes substances of an acid nature. Due to these excretions, this plant
dissolves the highly soluble phosphates, transforming them into an easily assimilated
form. Other plants, like mustard and buckwheat, are not able to excrete substances of an
acid nature and cannot dissolve the mineral compounds essential for nourishment.

The studies made by Fred (1918, 1919), conducted under strictly sterile conditions,
clearly showed the presence of substances of an acid nature in root excretions, which
dissolve marble plates. The author pointed out in this connection that, in the presence of
bacteria, the process of the dissolution of marble is considerably faster.

The roots of Italian rice excrete a substance which fluoresces with a blue light upon
ultraviolet irradiation. This substance is so characteristic that it may, according to the
author, serve as an indicator of the given plant (from Audus,1953)

The difference in chemical composition between the root excretions of different

varieties of the same species of plant was also noted by other investigators. Timonin
(1941) established the presence of substances in a variety of flax resistant to fusariosis
(Bizon var.), which activate the growth of the fungus Trichoderma viridis an antagonist
of the organism causing fusariosis. In the strain which was sensitive to fusariosis (Novel
var. ) substances were found in the root excretions which stimulate the development of
the fungus Fusarium, the cause of fusariosis of flax.

Chemical analysis has shown that in the root excretions of the resistant variety of flax,
there is a great amount (25-30 mg per plant) of hydrocyanic acid, which possesses
antimicrobial properties. In the excretions of the roots of the sensitive variety of flax,
this acid is either absent or present only in traces.

Eaton and Rigler (1946) observed an analogous situation in the root excretions of the
cotton plant. In the variety resistant to root decay more carbon compounds were found
than in the sensitive variety. According to the authors, the given substances attract
microbe antagonists, which inhibit the development of the organism causing root rot.

It should be noted that the problem of plant-root excretions only comparatively

recently became a subject of thorough study. Therefore, we possess only scant
information on the qualitative composition of root excretions. In addition, our
knowledge of the quantities of the substances excreted by the roots in even more scant.

The few studies available show that roots excrete considerable amounts of organic
substances. Dyer (1894), in determining the amount of acids excreted by the roots of
plants, established that 100 ml of nutrient solution from barley contained 0.38 mg of
acids; from wheat, 0.58; oats 0.65, foxtail, 0,86; timothy grams, 0.80; orchard grass.
0.81; white clover, 1.28; red clover, 1.55 and from broad beans, 1.11 mg of acids.

According to Maze (1911), one corn plant in a sterile nutrient solution excretes 57 mg
of sugar and 84 mg of acids in twenty days of growth. Shulov (1913) found in the root
excretions of this plant, after a two-month period of growth in a nutrient solution, 94 mg
of nonreducing and 34 mg of reducing sugars and 80 mg of malic acid. The roots of
peas excreted, during the same period, 140 mg of sugar. According to the observations
of the author, when plants were cultivated on ammonium nitrate, there were more root
excretions than when the plant was given calcium nitrate.

Pfeifer, (1912, 1917) investigated the root excretions of wheat and buckwheat.
According to this author, 0.27 g of wheat roots excreted 0.134 mg of organic acids and
0.110 g of buckwheat roots excreted 0.155 mg of organic acids, which comprises 1.3 per
cent of the total weight of the plants. According to Shulov (1913), the root excretions of
corn comprise 0.6 per cent of the plant's weight.

Demidenko (1928) grew corn and tobacco in solutions which were either changed or
unchanged. The corn roots of one plant, grown in a solution which was not changed
excreted 486 mg of organic substances during the whole vegetation period and, when
the solution was changed seven times. the roots excreted 1,136 mg of organic
substances, Roots of tobacco, for the same period, excreted 158 mg in an unchanged
solution, while in a changed one, it excreted 439 mg of organic substances. In
summarizing these observations, the author concluded that the total root excretion
comprised 27 per cent of the plant mass.

Mashkovtsev (1934) found that seeds of rice upon germination lose 20-30 per cent of
dry weight, with about one fourth of the loss consisting of root excretions of organic
compounds.

Virtanen and his associates (1933) found that the roots of peas grown in vessels along
with cereals excrete 126.4 mg mineral nitrogenous compounds in 58 days, of which
77.4 per cent is comprised of the nitrogen of amino acids, 3.3 per cent amide
compounds, 2.05 per cent of melanin*, and 2.73 per cent of other nitrogenous
compounds. *["Melanin" appears in Russian text but it may erroneously refer to
"humin."]

When barley was grown together with peas, it grew normally and developed, although
no nitrogen was introduced into the vessel with sand; in the tissues of experimental
plants of barley, 32.3 mg of nitrogen were found, and in the tissues of the control plants.
which were grown without peas, the nitrogen found amounted to only 0.7 mg and these
plants developed very poorly.

The barley in these experiments did not utilize all the nitrogen excreted by the peas. A
considerable part of it, up to 89.0 mg, remained in the substrate in the form of these or
other organic nitrogen compounds. (Virtanen, Synnöve Karström, 1933; Virtanen,
1937).

Virtanen and Laine (1936, 1937) found that in the root excretions of clover and other
leguminous plants in the period preceding flowering, mainly (75 per cent of the total
bound nitrogen) aspartic acid, gluconic acid, tryptophan and ß-alanine were detected.
During flowering, the major part of the nitrogenous root excretions consisted of
tryptophan.

Lyon and Wilson (1921) calculated that for the whole vegetative period, the roots of
plants excrete up to 5 per cent of the total weight of the plants' organic substances.

Engel and Roberg (1938) determined that, during a two-month period of growth, the
roots of one alder plant, inoculated with proactinomycetes, excreted 27.7 mg of
nitrogenous compounds and uninoculated plants excreted 23.6 mg of nitrogenous
compounds (Table 65).

Table 65
Excretions of nitrogenous substances by alder roots, mg
(from data supplied by Engel and Roberg, 1938)
Nitrogen in the Nitrogen in substrate
Increase in
Experiment initial substrate after 2 months growth of
nitrogen
(sand) alder
Inoculated (with nodules) 3.3 31.0 27.7
Uninoculated 2.5 26.1 23.6

Meshkov (1953) investigating the root excretions of peas and corn grown in a sterile
nutrient solution, obtained the following results: during twenty days of growth, the roots
of peas excreted into the solution 2.87 mg of reducing sugars in experiments performed
during 1946, and 4.28 mg in the experiments carried out during 1947. The weight of the
dry mass of the vegetable crop comprised 1.92 g in 1946 and 1.85 g in 1947. The roots
of corn for the same period excreted into the solution 8.4 mg in 1946, and 8.17 mg in
1947. The weight of the dry mass was 3.69 and 2.35 g respectively. According to the
observations of the author, the amount of root excretions depends to a considerable
degree on the weight of the roots, rather than on the weight of the green parts, leaves
and stems. The total weight of the latter amounts to 2 per cent in the case of peas and
1.3 per cent in the case of corn, of the total weight of the mass of the plants.

In our investigations (1934 b) we determined the growth of microorganisms in the

media to which these substances were added. For this purpose, of a great number of
species tested the following cultures were chosen: two cultures of yeast; Torula rosea
and Sporobolomyces philippovi, and two bacterial cultures, Pseudomonas fluorescens
and Ps. denitrificans.

These microorganisms were grown in a solution in which wheat and corn were grown,
and also in a pure nutrient solution with various concentrations of glucose. After certain
time intervals, the cells were counted in a Thoma counting cell and plated on liquid and
solid nutrient media. The results are given in Tables 66 and 67.

Table 66
Growth of microorganisms in a rhizosphere solution of wheat
(in thousands per 10 ml of medium)
Time of action of solution---
3 days 8 days 15 days 25 days 40 days
>
Torula rosea 75 1,500 1,000 1,500 1,100
Sporobol. philippovi 150 2,200 2,000 2,000 1,500
Ps. flourescens 1,200 100,000 150,000 7,000 1,000
Ps. denitrificans 1,800 150,000 160,000 7,500 1,500
Table 67
Growth of bacteria and yeasts in a pure initial solution with various concentrations of
glucose (in thousands per 10 ml on the eighth day of growth)
Glucose
Sporobolom. Pseudomonas Pseudomonas
concentration, Torula rosea
philippovi flourescens denitrificans
mg per 100 ml
5 400 650 20,000 1,500
10 680 900 35,000 2,500
20 1,000 1,500 60,000 50,000
50 2,000 3,000 150,000 100,000
100 3,500 5,000 250,000 200,000

In comparing the maximum numbers of microbial calls which had grown on the
rhizosphere solution with the corresponding numerical indicators of growth in glucose-
containing medium, we obtained the following results: the maximum number of Torula
rosea cells, which attains 1,500,000 in the rhizosphere solution, is equal to the same
number in the case of a glucose concentration which slightly exceeds 20 mg.
Approximately the same amount is also necessary for Sporobolomyces philippovi. In
order to accumulate 150 million bacterial cells in the rhizosphere solution, we evidently
require approximately 50 g of glucose or some other equivalent substance.

Consequently, according to the data of this analysis, the roots of wheat (the vessel
contained three plants) excreted in 15 days of growth about 50 mg of organic
substances, utilizable by bacteria, and about 20 mg of substances utilizable by yeast.

In experiments with corn, similar results were obtained. Substances utilizable by

bacteria were excreted in a larger amount than substances suitable for the nourishment
of yeast.

It became known recently that the roots of vegetating plants excrete various enzymes
into the medium. The presence of enzymes in root excretions had already been
suspected when the problem of the saprophytism of higher plants and the problem of
their growth and nutrition on organic media was investigated (Kamenskii, 1883;
Lyubimenko, 1923, 1935; Keller, 1948).

Eckerson (1932) has shown that plant roots are able to reduce nitrate to nitrite with the
aid of excreted nitrate reductases. Thus, the roots formed up to 2 mg of nitrite nitrogen
during 17 hours at 37° C. Klein and Kisser (1925), growing plants in a sterile nutrient
solution, detected after some time in this solution an enzyme which reduces nitrate to,
nitrite.

Kuprevich (1949) investigated the root excretions of 23 species of plants, belonging to

16 families: oats, wheat, barley, vetch, clover, flax, heather, camomile, willow herb,
dandelion, nettle, knotweed, sorrel, tea, oak, birch, poplar, willow, pine, spruce, the
common brake, and others. Various enzymes were detected: catalase, tyrosinase,
phenolase, asparaginase, urease, invertase, amylase, cellulase, protease, and lipase.

The amount of excreted enzymes and their activity varies among the different species
of plants. For example, the activity of amylase was expressed by indices from one to
four, i. e., from barely detectable activity to the full decomposition of the substrate.
Lipase was detected in traces in only four species of plants (dandelion, touch-me-not,
nettle, and pine).

We studied amylase (1952 a) in the root excretions of wheat, corn, and peas, grown
under sterile conditions. It was observed that when small samples of roots were placed
in a vessel with starch, the latter was comparatively quickly decomposed (Table 68). For
example, 0.2 g of wheat roots decomposed 20 mg of starch in 60 minutes.

Table 68
Decomposition of starch by enzymes excreted by plant roots
Root Reaction Reaction Reaction Reaction Reaction Reaction
Plants sample, for 0.5 for 1 for 2 for 4 for 8 for 24
grams hours hour hours hours hours hours
Wheat
0.5 - - - - - -
0.2 + - - - - -
0.1 + + - - - -
0.05 + + + - - -
Corn
0.5 + + + + - -
0.1 + + + + + +/-
Peas
0.5 + + + + + -
0.1 + + + + + +

A plus stands for the presence, and a minus designates the absence of starch, plus-minus
designates an undetermined reaction.

Roots of plants grown in the field decomposed starch even more intensively. In one
hour, 25 mg were decomposed by a suspension of 0.1 g of wheat roots and by a
suspension of 4.5 g of corn roots.

Starch was most quickly decomposed by roots which were not detached from the
plant. Young wheat plants, extracted from the soil and washed with water, when
submerged in a starch solution, decomposed 25 mg of starch in 30 minutes, while corn
plants decomposed 5 mg.

The enzyme amylase was also detected in the water in which wheat plants which were
taken from the soil were immersed for some time.

One wheat plant, two years old and grown in the field, excreted into the solution an
amount of amylase which decomposed, on the average, 20-25 mg of starch in one hour
at room temperature.

It can be seen from the above that starch is most actively decomposed by the root
excretions of wheat and to a lesser degree by the excretions of peas and corn.

Ratner and Samoilova (1955) detected in the root excretions of corn and sunflower,
enzymes which break down glycerophosphate and saccharose. The amount of these
enzymes, according to these writers, changes with the growth phase of the plant. The
maximum excretion of enzymes by corn is observed at the period preceeding flowering
and during the period of the formation of the spadix (Table 69).

Table 69
Excretion of enzymes by corn roots during various phases of growth, per gram of roots
(according to Ratner and Samoilova, 1955)
Phosphorus liberated
Reducing sugars,
Phases of growth from
mg
glycerophosphate
Initial vegetation period 0.06 4.06
Middle vegetation period 0.106 4.28
Formation of pseudo-ears 0.052 3.66
Beginning of formation of spadices 0.102 6.06
Formation of spadices 0.124 4.32
Ripening of seeds 0.068 0.51

The roots of these plants form enzymes which, in addition to glycerophosphate and
saccharose, also split glucose phosphate and ribonucleic acid. Thus, one gram of
sunflower roots splits 0.338 mg of ribonucleic acid in three hours, while one gram of
corn roots splits 0.048 mg of ribonucleic acid.

A similar picture is observed in relation to the splitting of glycerophosphate and

glucose phosphate. Sunflower roots split 0.375 mg of glycerophosphate and 0.208 mg
of glucose phosphate in three hours, while corn roots split 0.129 mg and 0.095 mg,
respectively.

The authors concluded that, due to the enzymatic activity of the roots, the latter can
supply the plants demand for phosphorus at the expense of the organic phosphorous
compounds if they are present in the medium.

In addition to enzymes, the plants excrete into the soil a number of other biologically
active compounds--various biotic substances (vitamins, auxins), toxins, etc. The amount
of these substances in soils may be quite considerable.

All these substances are sources of direct or supplementary nutrition for soil
microorganisms and enhance their growth and accumulation in the soil.

Root residue

In addition to root excretion, microorganisms utilize, as sources of nutrition, dead root

cells, hairs, epidermis, etc.

The chemical composition of roots varies in different plants. The roots of some plants
contain more water-soluble substances (proteins, sugars. etc), the roots of other plants
contain more hemicellulose, cellulose, and lignin. Belyakova and Parishkura (1953)
found the, following chemical composition of roots (Table 70).

Table 70
Chemical composition of various roots of plants, in per cent of dry weight
(Belyakova and Parishkura, 1953)
Dactylis
Labium
glomerata
Substance Lucerne Eragrostis multiflorum
(orchard
(rye grass)
grass)
Ash 5.19 11.67 12.89 13.69
Carbon 43.75 42.42 41.19 43.05
Nitrogen 2.38 0.75 1.22 1.89
C:N ratio 18.4 56.56 38.8 22.7
Protein 14.87 -- -- 11.81
Water-soluble part in percent
2.36 3.29 4.62 1.39
of carbon
Fats 3.01 9.0 1.3 2.0
Hemicellulose 18.21 11.26 15.82 12.58
Cellulose 20.67 20.19 18.21 19.04
Lignin 20.0 30.0 20.0 31.0

It is, obvious that the roots of different plants may attract different types of microflora
and may be decomposed by the various species of this microflora.

Upon the decomposition of roots of different plants by microorganisms, different

products of intermediate decomposition and different products of the metabolism of the
microbes themselves are formed. The latter are also sources of nutrition for other
species of microorganisms and, as such, together with the decomposition of roots,
attract another population of microbial biocoenoses. The shift of the microbial
population continues until the organic substances of the plant, animal, and microbial
residues are decomposed into their final products. These final products may be quite
versatile, depending on the composition of the organic residues, on the microflora
forming them, on soil and climatic conditions, and on the processes of synthesis taking
place in the soil outside the cells.

Rhizosphere

The role of roots in the life of microorganisms is not only limited to the supply of
nutrient substances. Around the roots more favorable physicochemical and biological
conditions for the existence of microbes, as well as for the plants themselves are
created.

In regions where there is an abundant accumulation and development of roots the

physical properties of the soil improve. The soil particles have more structure in the
rhizosphere of plants (see chapter on the structure of soils). With the structure of the soil
particles, the respiration process of roots and microorganisms improves, moisture is
better conserved, temperature is kept at a more constant level, etc.

The soil around roots is distinguished by a higher moisture content. According to our
observations, during the vegetative period, the soil in the rhizosphere of wheat under
conditions of the Volga steppes had a higher (by one-two per cent) moisture content, and
its moisture capacity was three to five per cent higher, than that of soils outside the root
region (Krasil'nikov, 1940), This is evidently connected with the change in the structure
and composition of the rhizosphere soil and with the capacity of the root systems of
plants to actively change the moisture content of the surrounding soil.

Breazeale and McGeorge (1953) have shown that when soil dries out beyond a certain
threshold, the plants moisten it in the vicinity of their roots with water transported from
their aerial parts. The latter utilize the atmospheric moisture.
 The authors grew tomatoes in soil which was gradually dried. When the plants began
to wither, the vessels were transferred to a room with a high humidity (80-90 per cent of
full humidity). The plants soon recovered, turgor was reestablished in the leaves, and
the soil around the roots became more moist.

The soil in the vicinity of roots also varies considerably with respect to acidity. Around
the roots of clover, lupine, and certain other plants, the strongly acidic podsol soils
became less acidic. If the control soil had a pH of 4.5. then the pH in the region of the
lupine roots increased to 5-5.4. In less acidic soils, the neutralization in the zone of the
roots is much more noticeable. (Table 71).

Table 71
Change in the acidity of the soil in the root area of different plants
Clover; Lupine; Wheat;
Clover; Lupine; Wheat;
Soil rhizo- rhizo- rhizo-
control control control
sphere sphere sphere
Strongly podsolized
deforested, the first year after 4.5 4.9 4.7 5.4 4.5 4.7
plowing
Cultivated, 15 years 5.1 5.8 4.9 5.8 4.9 5.1
Intensively cultivated garden
5.6 6.4 5.6 6.5 5.3 5.9
soil

The change which takes place in the environment of the root zone of plants was
observed by Kaserer (1940) and Eklunde (1923, 1930). According to Thom and
Humfield (1932), the neutralization of acidic as well as alkaline soils takes place in the
root zone. For instance, acidic clay soils have a pH of 4.5 and, in the vicinity of roots--
6.1. Alkaline soils of Colorado with a pH of 7.9 have a pH of 7.5 in the vicinity of the
roots of cereals.

Heller (1953) has shown that plants reduce the redox potential of the soil around the
roots. This lowering of the potential, according to his data, is caused by the presence of
root excretions and the microorganisms attracted by them. Intense photosynthesis of the
green parts of beets lower the rH2 value (redox potential) of the soil at a distance of one
cm from the root surface. Cessation of photosynthesis is immediately accompanied by
an increase of the rH2 in the soil of the root zone. The introduction and the growth of
bacteria in the zone of the roots lowers the rH2 of the best tissues.

The soil around the roots is richer in organic substances. As noted above, it possesses
greater quantities of various products of microbial metabolism, products of the
decomposition of root hairs, epidermal cells, and root excretions. In this zone, one also
notices higher concentrations of enzymes, vitamins, auxins, certain amino acids and
other biotic compounds,

Nitrate nitrogen is absent from the root zone or is only present in small quantities. We
analyzed the soil around roots of different plants during the whole vegetation period in
the fields of the Volga area. Nitrates have only been detected in the rhizosphere during
the early stages of the growth of plants and at the end of vegetation (Table 72).

Table 72
Nitrate content in the rhizosphere of plants
(in mg per kg of soil)
Wheat; Sunflower;
Date of Wheat; Soy; rhizo- Soy; Sunflower;
rhizo- rhizo-
analysis control sphere control control
sphere sphere
31 May 11.4 29.86 0 12.3 4.65 21.4
5 June 15.9 35.22 0 12.68 4.85 21.1
12 June 22.5 33.9 0 14.2 4.12 22.1
15 June 7.7 37.7 0 11.03 3.1 18.5
19 June 0 24.34 0 9.67 0 15.9
26 June 0 23.96 2.9 14.19 0 6.1
2 July 0 22.54 0 2.3 0 3.5
5 July 0 16.34 0 4.58 0 13.33
10 July 0 9.11 0 12.65 0 12.45
16 July 0 11.27 3.0 13.54 0 11.4
22 July 0 10.8 0 8.65 0 19.4
27 July 0 10.3 0 9.54 0 12.54
31 July - - 0 7.24 0 7.86
6 August - - 3.4 6.9 0 5.7
10 August - - 5.4 6.4 0 4.2
15 August - - 3.6 4.2 3.4 5.2
19 August - 0 5.4 4.7 4.6 7.2
25 August - 0 4.1 4.2 3.6 5.2

Katznelson and Richardson (1943) have found that the soil in the root area is less
subject to the sterilizing effects of chemical substances. On processing soil with
formalin and chloropicrin, the authors detected a much greater decrease in the number
of microorganisms outside the zone of the root system. In the root region of certain
plants (tomatoes and others), the microbes did not react at all to these chemicals and
their number did not decrease. Living organisms, the root region and the root system of
plants seem to be less accessible to chemical action.

In our experiments, plants of corn and beans were grown under sterile and unsterile
conditions, in growth containers (9 kg) filled with garden soil from the Moscow area.
When the plants reached the stage of flowering or bud formation, antiseptic substances
were introduced into the soil: 0.5 liter of a 30 per cent solution of formalin and two g of
chloropicrin per vessel. In the sterile soil the plants perished and, in the unsterile soil,
they continued to grow normally,
 It can be assumed that, in the rhizosphere of plants, a protective barrier is formed in the
form of metabolic products of microbes, which are much more numerous here than
outside the rhizosphere. Evidently, the chemical substances are directly decomposed in
the rhizosphere by microbial organisms.

The metabolism of microorganisms is more intense in the root region, as are many
chemical and biochemical processes, as well as the transformations of various organic
and mineral substances. In the rhizosphere, various minerals, rocks, limestone, marble,
etc are decomposed at a faster rate. This process is not only caused by root excretions
(CO2 and other acids) but also by the microflora of the rhizosphere. The more intense
the growth of microbes, the faster the decomposition process of substances. Certain
compounds, for instance, tricalcium phosphate, do not dissolve in the sterile rhizosphere
of plants, but when soil microbes are added to the vessel the substance becomes
available to the plants (Gerretsen, 1948). One of the tasks of agricultural microbiology
is the enrichment of the root region with microbes, which transform nonsoluble
phosphorus compounds into the soluble compounds available to the plant.

Under the influence of the microflora in the rhizosphere, one notices an increase in the
solubility of iron and manganese compounds. According to Starkey (1955), this increase
is caused by the change in the redox potential, which in quite different here than outside
the rhizosphere. In the rhizosphere, iron, manganese. and other metals occur in
combination with organic compounds formed by microbes. According to the author,
amino acids, organic acids, and other metabolites of microorganisms form stable
complex compounds, which are preserved in the soil for a long time. They are utilized
by the plants and used as a source of iron, manganese, and other elements. The
quantities of these organometallic compounds are greater in the rhizosphere than outside
this region.

Weinstein and others (1954) experimentally confirmed this data. They grew plants
(sunflowers) in solutions both with and without the addition of microbial metabolites
and they followed the absorption of the mineral salts of iron. In the presence of
metabolites or ethylenediaminetetraacetic acid, the uptake of iron was faster, while in
the absence of these substances and of microbes, the applied elements were not taken up
by the plants. These observations showed that plants evidently take up iron, not in the
form of mineral compounds, but in the form of organomineral substances formed under
the influence of microorganisms.

All the above data show that in the vicinity of the roots of vegetating plants, a special
zone is formed in which more favorable conditions prevail for the existence, not only of
microorganisms, but also of the plants themselves.

Part IV, continued:

The microflora of the rhizosphere

Increased accumulation of microbes in the root soil was first observed by Hiltner in
1904. He proposed the term "rhizosphere." In investigating the root system of various
plants, Hiltner came to the conclusion that the accumulation of microbes in this area
was not accidental and that it was caused by the biological activity of the roots.

It should be noted that some investigators before Hiltner observed the accumulation of
certain species of bacteria in the root region, but they looked upon this phenomenon
from a narrowly specialized point of view.

Epstein (1902) observed the development of special bacteria on the roots of beets,
which differ from those of the soil. Welich (1903), Grüber (1909), and Maassen (1905)
found large numbers of Clostridium gelatinosum cells in the rhizosphere of the sugar
beet.

After Hiltner, the phenomenon of the accumulation of microorganisms around roots

was observed by Layon (1918). He pointed out that the root systems of plants strongly
change the natural microbial associations of the soil. The ratios between species in the
rhizosphere microflora, according to this author's observations, differ from the ratios
prevailing in the microbial biocoenoses of nonrhizosphere soil. Hoffman (1914).
Maaasen and Behn (1923), Stoklasa (1926) and Richter and Werner (1931) observed the
increased development and accumulation of fungi in the rhizosphere.

A more detailed and systematic study of root-region microflora began in the 19301's.

Detailed studies made by Starkey (1929, 1934) showed that the roots of plants have a
considerable influence on the accumulation of microorganisms in the soil. According to
his observation, the number of microorganisms in the rhizosphere is several times
higher than that in ordinary soil outside the root area. For example, on the rhizosphere
of beets, 427 million bacteria per gram were found, while in the control soil only 8.2
million per gram were found; in the rhizosphere of clover, he found 11,320 million per g
and in the control soil only 6.6 million per g; in the rhizosphere of wheat, 653.4 million
per g were found; and in the control soil, only 22.8 million bacteria per g were found.
Pochenrider (1930) studied the microflora of the soil in the root region of cruciferous
plants and also observed the intensified development of certain bacterial species
(Azotobacter and others).

The most numerous and detailed investigations were performed by the Soviet
specialists, Isakova (1934-1940), Krasil'nikov and others, (1934, 1945), Sidorenko
(1940), Obraztsova (1936), Berezova (1941, 1945), Korenyako (1942), and many
others. Berezova published data on her study of the microflora of the rhizosphere of
flax; Isakova on that of rubber plants, tangerines, tung trees, and the tea plant, and
Obraztsova, on the tea plant.

Large accumulations of microbes in the region of the roots of forest plants were found
by Samtsevich and his associates (1949) and by Kozlova (1953).

Romeiko (1954) observed the development and accumulation of microflora in the

rhizosphere of hops. Noting the concentration of microbes in the root region the author
emphasized the connection between this phenomenon and the developmental phase of
the plant.
 Popova (1954) obtained data on the intensified accumulation of microbes on the root
systems of vines which grow in the serozem soils of Uzbekistan.

Our investigations (1934-1945) of the soil of the root area have shown that plants in
any soil under different climatic conditions, regardless of the zone, possess the capacity
of concentrating soil microorganisms in the vicinity of their roots, We studied the
microflora of cereals (wheat, rye, oats, barley) grasses (orchard grass, rye grass, etc),
leguminous plants (beans, peas, vetch, clover, lucerne, etc), commerical crops (cotton,
tobacco, flax, hemp, etc), and several varieties of fruit trees and decorative plants
(acacia, poplar, lime tree, oak, apple, plum, pear, etc). Studies were performed in
various parts of the Soviet Union: in the South--in Crimea, in the Caucasus, Armenia,
and Georgia, on the coast of the Black Sea, in Central Asia, and on the fields of the
Tadzhik, Uzbek, and Kirgiz Republics, in Kazakhstan, in the lowlands of the Volga area
and in the Astrakhan steppes; in the Ukraine, Moldavia, Siberia, on the Sakhalin; in the
northern regions of Yakutiya, Igarka, and the Kola Peninsula. The microflora of plants
of the moderate region was also studied: Leningrad, Moscow, Yaroslavl', Kuibyshev,
and other regions, and the plants of Belorussia, Latvia, Estonia, and other places,

According to our observations, the total number of microorganisms in the zone of the
plant roots exceeds the number of microbes in ordinary soil by tens, hundreds, and
thousands of times. The differences in the quantitative relationships depend on the
species of plant and the soil-climatic conditions (Table 73).

Table 73
Total number of bacteria in soils
(in thousands per gram)
Rhizo- Rhizo-
Soil and plants Control Soil and plants Control
sphere sphere
Kola Peninsula Central Asia, Serozem
Potatoes 12,500 320 Cotton 3,500,000 74,000
Clover 21,000 400 Lucerne 7,100,000 60,000
Oats 7,800 270 Orchard grass (Dyctylis) 3,800,000 35,000
Oat grass
Mixture of grasses 19,500 350 4,200,000 47,000
(Arrhentherum)

Severnaya Zemlya Armenia, Serozem

Saxifrage 7,600 120 Wheat 1,800,000 12,000
Poppies 5,200 210 Lucerne 3,600,000 14,000
Cereals 12,000 400
Chestnut Soil
Moscow Oblast'
Wheat 2,400,000 10,000
Podsol
Wheat 750,000 1,500
Flax 500,000 1,150 Georgia, Krasnozem
Clover 1,000,000 1,200 Tobacco 500,000 500
Tea plant 800,000 600
Trans-Volga region,
Chestnut soil
Wheat 2,100,000 14,000 Crimea, Southern shore
Lucerne 3,700,000 13,800 Tobacco 1,200,000 8,000
Vineyards 1,700,000 3,000

Note: The numerical data given in the table was obtained from the study of soils by the
dilution method, by inoculation into synthetic media (Chapek, Gil'taya).

In recent years, the microflora of the rhizosphere has gained more of the attention of
many foreign specialists. In addition to the above-mentioned studies, made by Starkey,
others have been made by Lockhead and his associates, and by Jensen, Katznelson,
Timonin, and others, on the bacteria of the rhizosphere.

All of these investigators obtained data on the considerable accumulation of

microorganisms in the root region. Katznelson (1946) found 100-1,000 times more
microbes in the rhizosphere of beets than were found outside the root zone. Thom and
Humfield (1932) obtained the following data: in the rhizosphere of corn, there are 136
million bacteria per one gram of soil, while outside the rhizosphere (control) only five
million.

According to Starkey ( 1955), in the rhizosphere of beans, there are 200 million
bacteria per gram of soil; in beets, 427 million per gram; and in grain crops, 653 million
per gram. The control samples of soil contained one to five million bacteria per gram of
soil.

The poorer the soil in organic substances, the less fertile it is, and the weaker is the
influence of the root system on the quantitative composition of the microflora of the
rhizosphere. The quantitative relationship of the microflora of the rhizosphere (M.R.) to
that of the control (M.C.) is most strongly expressed in poor soils. It is more marked in
the primary, slightly fertile podsol soils of the Kola Peninsula than in the chernozem
soils of Moldavia or Kuban,.

In the primitive noncultivated or slightly cultivated soils of the Kola Peninsula the total
number of bacteria reaches from 50,000 to 300,000 cells per gram. In the rhizosphere of
clover in its first year of life, there are 150-400 million per gram. The M.R. to M.C.
ratio is 1000:1. In the well-cultivated chernozem soils of the Khar'kov Oblast, the
number of bacteria in the rhizosphere of Lucerne is from 2,500 to 5,000 million per one
gram, and outside the rhizosphere, from 150 to 300 million per gram. The M.R. to M.C.
ratio is 17:1.

Central Asia's slightly cultivated serozem soils contain comparatively small numbers
of bacteria, within the range of five to ten million per gram. In the root region of lucerne
in its first year of life, we counted up to 1,000 million bacteria per gram. This same soil,
after five to eight years of cultivation and the introduction of the corresponding
fertilizers, contained 7,000 million bacteria in the rhizosphere of one-year-old lucerne,
and, outside the roots, 60 to 100 million organisms per gram. The M.R. : M.C. ratio was
100 to 200) in the first case, and 70 to 100 in the second.

Such a shift in the M.R. : M.C. ratio was observed by us in chernozems, podsols,
serozems, chestnuts, and other soils, regardless of the geographical region, Only the
quantitative expression differed.

The quantitative ratios between the number of microbes in the rhizosphere and outside
it increase, with the depth of the penetration of the root system. In the lower layers, the
numerical indices are expressed more strongly (Table 74).

Table 74
Number of microorganisms in the rhizosphere and outside it at different soil horizons
(in thousands per 1 g of soil)
MR:MC
Soil Plant Depth, cm Rhizosphere Control
ratio
Moscow Oblast'
podsol
Rye 0-25 350,000 1,200 300
40-60 250,000 300 800
80-100 5,000 3 1,700
Clover 0-25 950,000 1,500 630
50-70 300,000 300 1,000
90-110 10,000 5 2,000
Moldavia
chernozem
Lucerne 0-25 5,000,000 100,000 50
40-60 700,000 3,500 200
80-100 80,000 300 270
120-150 10,000 20 500
Wheat 0-25 1,500,000 75,000 20
40-60 300,000 2,000 150
80-100 30,000 100 300

At a depth of 90-110 cm in the rhizosphere of clover, growing in podsol soil, the

number of bacteria is 2,000 times higher than outside the rhizosphere, and in the
rhizosphere of lucerne in chernozem soil at the same depth, it os 270 times higher.
Approximately the same ratios are observed in the case of wheat.

In the deep layers of soil there are usually very few bacteria, while in the rhizosphere
of plants, even at the depth of two to three meters, they grow abundantly.
 Under conditions of deep growth, the root systems of plants create favorable
conditions for the growth of microorganisms, not only by their excretion of nutrient
substances, but also evidently due to such factors as the improvement of their conditions
of respiration and metabolism.

The number of microbes growing in the root area varies with the ago of the plant. As
should be expected, the maximal number of bacteria is observed during the period of the
most active growth of the plant. The more intense are the life processes, the more
organic substances are excreted by the root, and the more intense the multiplication of
microbes in the rhizosphere. Observations show that an abundant growth of
microorganisms takes place in the early stages of the plant's growth; however, the most
vigorous growth of microbes ensues during the period of flowering and in the period
directly preceding it. (Figure 70). Sometimes one also observes three elevations in the
growth curve of microbes: the first small elevation is seen in the early stage; a second
great elevation ensues before and during flowering; the third elevation occurs before
ripening. The last is usually barely noticeable.

Figure 70. Quantitative composition of the microflora of the root area during different
phases of the plants' growth

Under conditions of irrigation, small rises in the growth curve of microbes are
observed after each irrigation (Krasil'nikov, Kriss, and Litvinov, 1936b). Under these
conditions, they are caused by the increase in soil moisture. The question of whether the
moisture was the direct cause of the enhanced growth of the microbes or whether this
growth was strengthened as a result of an increased amount of nutrient substances
excreted by the roots due to the higher intensity of metabolism in the plant may
probably be answered as follows: both mechanisms are operative.

When soils in the rhizosphere are analyzed after harvest, one can observe that the
activity of microorganisms does not cease. In the presence of moisture and higher soil
temperatures, a sharp increase in the number of microbes in the root zone is observed.
In this case, the dead roots are subjected to intense decomposition. The number of
microbes during this period may quite often exceed that of the rhizosphere of living
plants during rapid growth.

In cases when there is little moisture in the soil (in arid regions), the root residues after
harvest decompose slowly and there is no noticeable increase in the number of
microbes. The roots remain in the soil undergoing no major changes for long periods.

Differences in the quantitative composition of microbes in the rhizosphere of different

plants are slight. In our investigations we could not detect regular and significant
differences in the numbers of bacteria in the rhizospheres of different plants of the
cereal and leguminous families. In the same field sector, under the same agrotechnical
and soil-climatic conditions, the total number of bacteria in the rhizospheres of wheat,
oats, and barley are approximately the same. When comparing the microflora of the
rhizospheres of cereal and of leguminous plants, one notices a slightly higher number of
microbes in the leguminous type. However, this difference is not always noted.

As to the nature of microbe distribution in the rhizosphere of vegetating plants, we

noticed regular diffuse growth of microbes, as well as the existence of more or less
isolated colonies.

In the literature, one finds indications that microbial cells are present not only on the
surface of roots but also inside them, having penetrated the tissue of the epidermis, the
intercellular substance, and also the protoplasm of the cells themselves (Berezova,
1953; Rempe, 1951; Hennig and Villforth, 1940). According to the data obtained by
Schanderl (1939, 1940), the cells and tissues of plants are not sterile and always contain
bacteria. In his last paper, the author claims that plant cells grow in symbiosis with
bacteria. The latter are present in the protoplasm in greater or smaller amounts.

Our investigations did not confirm Schanderl's data. Neither in the tissues nor in the
cells of healthy plants could we detect bacteria, fungi, or actinomycetes. Upon
microbiological analysis, the plant tissues always remained sterile. Even the roots of
leguminous plants did not have bacteria in their tissues outside the nodules. Other
scientists also, (Burcik, 1940; Schaede, 1940) did not detect bacteria in tissues of
healthy plants. Detailed studies were recently made by Stolp (1952). This author
attempted to verify Schanderl's data. He investigated various plants, leguminous,
cereals, and others. in not one case did he find microbial cells in plant tissues. Bacteria
can penetrate into the dying tissues and cells when the re sistance of the latter is
weakened. For example, the root hairs, upon dying, are completely filled with bacterial
cells. Obviously, Schanderl and others studied such dying cells and tissues, or they
accepted as bacteria the intracellular structures.

In order to elucidate the nature of the distribution of microorganisms on and in the

vicinity of roots, we employed the method of growing vegetation on glass plates
mounted on special vegetative frames, set up in such a way that the roots of the growing
plant spread on the glass, leaving their imprints on them. For these frames we used flat
boxes with glass walls, 5-6 cm wide, 20-25 cm high and 50 cm long.

The frames were filled with soil or sand and the plants were grown in them. In order to
make sure that the roots would stick to the glass, the frames were arranged at a slope of
40-50° (Figure 71). The roots, due to geotropism, grew in a downward direction and
touched the glass, sticking tightly to its surface.

Figure 71. Frames for growing plants:

A--frame in a sloping position, for obtaining imprins of roots and microflora on glass;
B--nature of the growth of roots on the lateral side of the frame (glass).

For the sake of the convenience of microscopic examinations, we placed microscopic

slides on the inner side of the glass surface of the vegetation frame. After certain time
intervals, the glass with the roots growing over it was taken out and examined
microscopically after having been previously stained or left unstained,

When the growth of microorganisms was abundant, one could see with the naked eye a
zone of film, measuring three to five mm in radius, around the root, This zone was
especially noticeable on the glasses of overgrowth which had been immersed in sand
(Figure 72). The sand was easily removed from the surface of the glass while the
microbial cells remained. One found by simple microscopy that the microorganisms
grow diffusely in the zone of the roots in the form of isolated colonies or small foci. The
colonies are located between the hairs on the surface of the roots, or in their vicinity
(Figure 73). In cases where there was a great deal of moisture covering the roots and the
root hairs, the bacterial cells spread. often occupying considerable segments along the
roots (Figure 74).
Figure 72. Imprints of root branches on glass with surrounding microflora:

A--natural size of the slide; imprints of roots in the form of hairs (a) are seen; B--the
same preparation magnified 1:100; branch of the root overgrown by hyphae of fungi
and actinomycetes.

Figure 73. Colonies of bacteria and actinomycetes on the surface of roots:

a--colonies of actinomycetes (marked by +), b--bacterial colonies, c--colonies of

mycobacteria (our own observations); d and e--bacterial colonies (after Linfold, 1942).
Figure 74. Diffuse distribution of bacteria on root surfaces:

a--nonsporiferous bacteria; b--mycobacteria; c--nonsporiferous bacteria and

mycobacteria.

Isolated colonies, as wen as profuse areas of microflora, mainly consist of

nonsporeforming bacteria and mycobacteria. One also finds colonies of actinomycetes
and mycelia of fungi. Very seldom does one see small colonies or single cells of
sporeforming bacteria.

Nonsporeforming bacteria and mycobacteria form well-defined, compact colonies,

located most often on the surface of the roots, between the root hairs, and also at a
certain distance from them (Figure 75). Colonies of actinomycetes often consist of
proactinomycete elements, (Figure 75), or of entangled mycelia. Fungi also grow
around roots in the form of single hyphae and mycelia.

Figure 75. Colonies of actinomycetes around roots. The zone of massive growth of
bacteria, at a certain distance from the root, is clearly seen
 If one prepares imprints of the root area of microflora growing in sand, and not in soil,
one does not encounter this picture. Upon the microscopic examination of these
imprints, one can observe only mycelial threads of fungi and actinomycetes,
occasionally with sporangiophores on branches. One seldom observes single colonies of
actinomycetes, Around the root branches and hairs bacterial cells are also encountered,
but in limited numbers and, as a rule, they appear as single cells or in pairs and very
seldom in colonies. When such a preparation is being grown, by covering it with a thin
layer of an agar medium, a considerably larger number of bacteria and mycobacteria
appear than can be observed under direct microscopy. The great majority of cells are not
detected by microscopy. This in due to the fact that the cells of bacteria and
mycobacteria, and possibly certain actinomycetes, exist in a fragmented state in the
form of small granules hardly discernible from soil particles. These granular elements,
stained with erythrosin, are encountered in the soil of the root zone in great numbers.
They probably form the major part of the mass of the rhizosphere microflora. These
elements, when transferred to a nutrient medium, grow out, giving rise to cells of
normal size, which are detectable under laboratory conditions.

The methodof overgrowing the root zone ofplants on glass was employed by Linfold
(1942). He grew seeds of corn, lettuce, pineapple, and the cowpea (Vigna sinensis) in
soil in a special chamber. The imprints obtained on glass plates were microscopically
examined. The author observed an abundant growth of bacteria in the form of large
colonies around the roots and root hairs; colonies were often located at the hair tips
(Figure 76). At a certain distance from the roots, according to the author, amoebae,
Infusoria, and nematodes grew.

Figure 76. Bacterial colonies on the tips of root hairs (according to Linfold, 1942)

Starkey (1938) employed the method developed by Rossi-Kholodnii for the study of
root-area microflora. He found that the bacteria grew on the roots in clusters, and fungi
and actinomycetes, in the form of threads. The author buried the slides in the soil. under
the root system. Of course, he could not see the greater part of the bacteria on the
overgrown glass. As in our experiments, the bacteria were probably in a fragmented
state and could not be detected among the numerous soil particles without being
cultured.
 Stille (1938), using this method, found an abundant growth of bacteria around the root
hairs.

These data show that, in the root zone, the microflora probably grows in the same way
as it generally does in the soil, in colonies or in aggregates. Only when there is a high
moisture content in the substrate do bacterial cells grow in extensive areas.

Group composition of the microflora of the root area. Studies have shown that around
and on the roots of vegetating plants one finds various representatives of
microorganisms--bacteria, actinomycetes, fungi, algae, yeasts, protozoa, phages, and
other living organisms.

However, the prevailing group in the rhizosphere, regardless of the conditions of

growth and the age of the plants, are the nonsporiferous bacteria,

The second place (largest group) among rhizosphere microflora is occupied by

mycobacteria.

The remaining forms of microbes, actinomycetes, fungi, sporiferous bacteria, etc are
encountered in much smaller numbers. The quantitative ratios of these microorganisms
can be found in any plating of rhizosphere soil on agar media containing protein or on
synthetic media. In Table 75 data are given on the of different groups of microbes
growing in the rhizospheres of various plants immeditately before flowering. They were
obtained by plating one drop (0, 05 ml) of a 1:1,000 dilution.

Table 75
Quantitative relationships between microorganisms in the rhizosphere of plants
(number of colonies per plate)
Total
Nonspore- Spore-
number myco- actono-
Soil Plant forming forming fungi
of bacteria mycetes
bacteria bacteria
colonies
Trans-Volga
Wheat 1,200 950 10 200 38 2
region
Chestnut soil Oats 1,800 1,450 5 300 35 10
First
Moscow Oblast' year 2,200 1,675 8 500 15 2
clover
Podsol Rye 2,200 1,675 6 50 20 4
Central Asia Lucerne 3,200 2,700 15 450 34 1
Serozem Cotton 2,500 2,150 12 305 30 3

Approximately the same group-composition ratio of rhizosphere microflora is also

found in other plants. Nonsporeforming bacteria are the prevailing group among the
rhizosphere microflora of wheat, oats, clover, lucerne, cotton, and other plants either
growing in the southern regions on chernozem, chestnut, and serozem soils, or in the
northern regions in podsol or other soils. In some cases, deviations in the number of
bacteria of the different groups may exist, the percentage of this or that microbial group
may change, but the general nature of the ratio between the different groups of
microbial biocoenoses in preserved.

The group relation of the microflora in the root area varies considerably with the age
of plants.

It was noted above, that upon the ripening of plants, the total number of
microorganisms in the rhizosphere decreases. During this period the quantitative ratio
between the different representatives and groups changes; the number of sporeforming
bacteria, fungi, and actinomycetes increases, and new organisms appear. At the same
time, the total amount of nonsporeforming bacteria decrease, some species disappearing
altogether, etc.

On the diagram in Figure 77, data are given on the analysis of the rhizosphere
microflora of oats during different periods of its growth, obtained in studies of the
plants of the Trans- Volga region (Krasil'nikov, Rybalkina, Gabrielyan, Kondratleva,
1934).

Figure 77. Quantitative relationship between microorganisms at different stages of the

plant's growth (oats):

A--flowering period; B--period of ripening.

In later works we noted the same changes in the ratios of the representatives of the
rhizosphere microflora of many other plants, growing in different areas on differing
soils (Krasil'nikov, Kriss, and Litvinov, 1936).

The change in the quantitative composition of rhizosphere microflora with the age of
the plant was also noted by Starkey (1938), Isakova (1939), and some other
investigators.

Nonsporeforming bacteria comprise the main, and the most numerous and versitile
group of soil microflora, in general. This group embraces representatives of various
families, genera, and species; these include such organisms as Azotobacter, rhizobia,
thiobacteria, photobacteria, Azotomonas, Sulfomonas, nitrifying bacteria, denitrifying
bacteria, and others.

All these representatives are encountered in the rhizosphere of plants. Most frequently,
organisms of the genera Bacterium and Pseudomonas reach the largest numbers, These
organisms, comprise the main microflora of the root area,

The species composition of these organisms is scarcely known. We have no adequate

methods for the diagnosis and differentiation of these bacteria and we are not in a
position to distinguish between the nonsporeforming bacteria in the rhizosphere of one
genus of plants and those of another. Only a few genera can be accounted for:
Azotobacter, Rhizobium, and some others.

Kostychev and others (1926) were of the opinion, that Azotobacter adapted itself to the
rhizosphere of certain plants: tobacco, rice. and others and is their essential companion.

Later, many investigators found Azotobacter in the rhizosphere of plants. Sidorenko

(1940b, c) studied its growth in the root region of wheat, barley, beets, lucerne, sudan
grass, soybean and other plants grown on the chernozem soils of the Khar'kov Oblast'.
Popova (1954) found Azotobacter in the rhizosphere of grape vines: Obraztsova (1936)
and Isakova (1935) found it in the root zone of subtropical plants: lemon, tangerine, and
tung tree; Daraseliya (1950) found it in the root zone of the tea plant. Raznitsyna (1947)
and Tanatin (1949, 1953) observed the distribution of Azotobacter in the soils of Central
Asia under cotton, lucerne, wheat, and various vegetable crops. Pavlovich (1953)
studied the distribution of Azotobacter in the rhizosphere of various plants in the
Latvian Republic; Banak (1956), in Moldavia; Gerbart (1952), in the western Ukraine
districts, Petrosyan and his associates (1949) and Afrikyan (1953) in Armenia.

According to our observations, the growth of Azotobacter takes place in the root area
of various plants under differing climatic conditions and in different geographical
regions, from the northern regions to the southernmost points, on mountain tops and in
valleys. The growth of Azotobacter is observed in the rhizosphere of forest trees
(Samtsevich and others, 1952); Krasil'nikov, 1945), fruit trees, bushes, and in other
plantations (Kanivets, 1951; Kulikovskaya, 1955, and others).

The number of Azotobacter organisms in the rhizosphere of plants may reach

considerable dimensions. We counted from 0 to 200 million Azotobacter cells in 1 g of
soil in the root area. Tanatin (1953) counted tens of thousands and hundreds of
thousands in a gram of rhizosphere soil. Approximately the same numbers were found
by other investigators (Pavlovich, 1953; Babak, 1956; Raznitsyna, 1947).

No less frequent are the rhizobia. Their identification in the rhizosphere is not only of
diagnostic. value but also of practical significance, Depending on the abundance of their
growth in the root area of leguminous plants, their effectiveness will differ under the
same conditions of activity.

Root-nodule bacteria, as is well known, grow abundantly on the roots of those plants
on which they can form nodules. However, they can also grow on roots of other plants.
For instance, the nodule bacteria of lucerne grow luxuriously on the roots of lucerne and
also in the root zone of cotton; the nodule bacteria of clover also grow well in the
rhizosphere of lucerne, peas, and certain other plants. The nodule bacteria of peas grow
well in the root zone of peas, clover, wheat, etc. The total number of nodule bacteria in
the rhizosphere of plants may be considerable. Korenyako (1942) counted them in
hundreds of thousands and in millions per gram of soil. Similar data is given by
Raznitsyna (1947), Petrosyan and his associates (1949), Petrosyan (1956), and others.

Ammonia-producing bacteria, denitrifying bacteria, and certain other groups of

nonsporeforming bacteria grow abundantly in the rhizosphere. Of the first two groups
many millions and billions are present per gram of soil in the rhizosphere (Krasil'nikov,
Rybalkina, and others, 1934; Krasil'nikov, Kriss and Litvinov, 1936, Raznitsyna, 1947;
Starkey, 1928; and others).

Nitrifying bacteria, cellulose bacteria, nitrogen-fixing anaerobic bacteria of the genus

Clostridium, and certain other groups do not grow well, comparatively speaking, in the
rhizosphere (Rempe, 1252; Krasil'nikov, 1934, 1936).

The second place, with respect to their quantitative growth in the rhizosphere, is
occupied by mycobacteria. Their number in the root area reaches hundreds of thousands
and millions. Their species composition was not studied. Most often one encounters the
nonpigmented forms of Mycob. album, M. mucosum, and others.

One finds sporeforming bacteria in the rhizosphere of plants much less frequently and
in smaller numbers. In general, they comprise only a fragment of one percent of the
microflora. They are especially scarce during the period of vigorous vegetative growth
of plants. Usually these bacteria begin growing abundantly at the end of vegetation,
especially on dead, decomposed roots.

Actinomycetes also occupy a small place among root-area microflora during the early
stages of the development of plants. This group of organisms is very widespread and
versatile in its species make-up. In the rhizosphere, there are various representatives of
actinomycetes. Toward the end of vegetative development their number increases
considerably. They multiply with special intensity on semidecayed dead roots. One
often sees rootlets covered completely with the mycelia of these organisms in the form
of a fluffy or a mealy-white coating.

Fungi are detected in the rhizosphere by the conventional analyses of small quantities.

It is known that certain plants have a well-developed fungal coating on their roots,
coalescing with the root tissue. This: fungal coating is called mycorhiza. Depending on
the nature of its relation to the roots one can distinguish between endotrophic and
ectatrophic mycorhiza. Mycorhiza fungi are widespread in the root systems of many
plant species, both woody types and grasses. Some investigators are of the opinion that
all plants have mycorhiza. The fungi participating in the mycorhiza belong, according to
their systematic positions, to different classes and orders, families and genera. It is
supposed that these organisms are of great importance to the plants. However, this
problem has been only slightly studied (Lobanov, 1953; Reiner and Nelson-Jones, 1949;
Kelly, 1952).
 Many plants do not grow well without mycorhiza fungi, and some do not grow at all,
However, one seldom encounters roots with an abundant growth of these organisms in
grassy field crops.

It should be noted that upon an ordinary microbiological analysis of the rootlets, one
observes, as a rule, single mycelial hyphae. Fungi are detected by special studies with
the use of special analytical methods.

The question of algal growth in the root area of vegetating plants has been only
slightly studied. The research done by Katznelson (1946) and Shtina (1953, 1954 b)
showed that various algae live in the rhizosphere of plants in considerable quantities.,
Their total number reaches tens and hundreds of thousands in one gram of soil.

Shtina studied the growth of algae in the rhizosphere of rye, timothy grams, clover,
lupine, potatoes, barley, and oats. Among some of these plants, the number of algae in
the root area was two to three times higher than outside it (rye, timothy grass, clover,
lupine). For example, in the root zone of clover, 149,000 cells were found in one gram
of soil, and in the control area (outside the rhizosphere), only 99,000 cells per gram
were detected.

Qualitatively, the algae composition within the rhizosphere is approximately the same
as outside it. It consist mainly of diatoms, green and blue-green algae (Shtina 1954 a
and b). They probably also have a certain importance in the life of root-area
biocoenoses.

In the rhizosphere of plants, one observes invertebrate animals: protozoa, nematodes,

insect larvae, etc. The number of these organisms in the root area of healthy plants is
small and does not exceed a few thousands per 1 m2 under various plants. Shilova
(1950) studied the soils under grasses, rye, and lupine, in long fallow, and fallow soils.
According to her calculations, in 1 m of the surface horizon (0-10 cm) of soil, there are
from 7,000 to 187,000 invertebrates, and the number of certain members of Apterygota
in this area, under grasses, reached 760,000.

The composition of this fauna is extremely diverse. One finds in the rhizosphere
members of Acarina (mainly under forest cultures), and representatives of Apterygota.
Enchytraeidae, and others (Shilova, 1950; Gilyarov, 1949, 1953). Katznelson (1946).
Linfold (1942), and Brodskii (1935) have described the distribution of the protozoa,
amoebae, ciliates, flagellates, and others in the soil of the root area. These organisms are
frequently encountered when studying the soil of the root area in ordinary laboratory
studies.

Nikolyuk (1949) has established that there are two to three times more protozoa in the
root zone of lucerne than outside it.

The greatest number of these organisms are accumulated in the rhizosphere of cotton
during its first year of cultivation (up to 100,000 in 1 g soil). The author ascribes this
accumulation of protozoa to the abundant growth of bacteria in the rhizosphere, which
serves as nutrient material for the protozoans.
 Ressel (1955), Brodskii (1945). Nikolyuk (1949), and others ascribe great importance
to this group of organisms, as a factor affecting the composition of microbial
biocoenoses in the soil.

Quite often one encounters worms and nematodes in the root zone of plants. Certain
nematodes, as is well known, grow well on roots and, in penetrating into plant tissues,
cause diseases. Arkhipov (1954) noticed the death of nematodes under certain plants.

Pathogenic microbial forms--bacteria, actinomycetes and fungi--often grow and

accumulate in the root area. Under certain conditions, these organisms penetrate the
tissues of plants. in agricultural practice, cases of mass infections of plants, as a result of
the growth of pathogenic fungi and bacteria in the root zone are not infrequently
encountered. The development of microbial antagonists inhib iting phytopathogenic
organisms in the rhizosphere is also possible.

In general, many different forms of organisms may grow in the rhizosphere of plants,
both useful and harmful; those which facilitate the nourishment and develop ment of
plants, and, an the contrary, those which inhibit and poison them. The prevalence of
these other organisms depends on soil-climatic conditions, on the manner in which the
farm is handled, and on the whole agrobiological complex.

Part IV, continued:

The specificity of the microflora of the root zone

As indicated above, the chemical composition of root excretions, as well as that of the
dying root hairs and cells of the root epidermis, varies in different plants. Consequently,
root production will attract different soil microflora. Plants which excrete carbon
compouonds attract to their roots a microflora which differs from the microflora
attracted by plants which mainly excreted nitrogenous substances. With the presence of
sugar in root excretions one kind of bacterial and fungal species will develop and, in the
presence of organic acids, others will develop. These microorganisms which utilize the
available nutrient substances more quickly and fully will predominate in the plant
rhizospheres.

The qualitative composition of root excretions and dying root residues determines the
characteristics of the quantitative and species makeup of the microflora in the soil of the
root region.

The first to have observed the capacity of plants to affect the microflora of soils was
the writer, S.T. Aksakov.

In 1896 he wrote: "The mushroom is the child of the forest. . . . As is well known, if
one sows, plants-- in a word, if one plants a forest in a bare field--the mushroom types
characteristic of the varieties of the planted forest will undoubtedly start to grow there.
However, the shadow (as many have thought) cast by the branches of trees is not the
only secret force by which trees cause mushrooms to grow around them. It is true that
the shadow is one of the primary reasons for this phenomenon. It protects the soil from
the burning rays of the sun, and it create a moisture in the soil and even the dampness
which is essential for the forest, as well as for the mushrooms. However, the main
reason for the formation of the mushrooms, in my opinion, are the tree roots which, in
their turn, having moistened the surrounding soil, give it the sap of the tree. This, in my
opinion, is the secret of mushroom growth." (Notes and Observations on how to Collect
Mushrooms, vol. V, 1896).

Later, data on this subject were published In the scientific literature (Galakhov 1929;
Danilov, 1943, 1949; Vasil'kov, 1953, and others). Detailed information on the
interrelationship between edible mushrooms and higher plants in the forest
phytocoenoses of Latvia is given in the dissertation presented by Mazelaitis (1952), and
information on the distribution of mushrooms in the forests of the Moscow area in given
in the book by Shiryamov "The Search and Collection of Mushrooms in the Forests of
Moscow Area," (1948).

An analysis of the obtained observation and studies shows that the distribution of
edible mushrooms is closely connected with the composition of phytocoenoses. When
the forest and plant species of an area are known, one can determine beforehand which
species of mushroom will grow in the area and the distribution of the mushroom types
of interest to us. For instance, the white mushroom is encountered in heather, mountain-
cranberry, spruce-sorrel, and oak-bilberry forest. The ordinary brown mushroom is
found in pine forests with birch and heather; Boletus lutens is found in young pine
forests having a certain grassy vegetation, etc (Vasil'kov, 1953).

We demonstrated experimentally the selective action of plants. Different plants were

grown under sterile conditions, in sand or cotton, and soaked with Knop's nutrient
solution. Bacteria were introduced into the medium, either separately, or in the form of
mixture. The results are given in Table 76.

Table 76
The effect of plants on the qualitative composition of bacteria
(number of cells in thousands per ml on the 20th day of growth)
Root- Root-
Pseudomonas Pseudomonas
nodule nodule Az.
Plant flourescens flourescens
bacteria-- bacteria-- chroococcum
strain No 1 strain No 2
clover lucerne
Wheat 10 100 0 200,000 1,000
Corn 0.1 200 0.01 15,000 4,000
Cotton 10,000 100,000 3.0 100,000 30,000
Sugar beets -- 1,000 1.5 100 100,000
Flax 0.1 10 0.01 300 100
Clover 100,000 10,000 6,000 1,000 1,000,000
Lucerne 1,000 100,000 5,000 1,000 100,000
Peas 10,000 1,000 3,000 1,000,000 1,000

As can be seen from the given data, some bacteria grow well, others grow only
slightly, while others show intermediate growth on the same plant. Azotobacter, for
instance, did not grow at all or grew very poorly in vessels in which wheat, corn, and
flax were planted, showed intermediate growth on cotton, and abundant growth on
clover, lucerne, and peas.

The reaction of two strains, Nos. 1 and 2, of the same species of nonsporeforming
bacteria. Ps. fluorescens, to the root excretions of plants differed. Strain No. 1 grew
most abundantly in the rhizosphere of wheat, corn, and especially cotton, while strain
No. 2 grew much better in the presence of the root excretions of clover and lucerne. The
root-nodule bacteria reacted positively toward the roots of cotton, clover, lucerne, and
peas, and less so to the action of the root system of sugar beets.

We also grew a series of other sporeforming and nonsporeforming bacteria,

mycobacteria and yeasts. The sporeforming bacteria Bac. mesentericus, Bac. subtilis,
Bac. megatherium, as a rule, did not grow in a medium with the root excretions of the
experimental plants, but did not die in it. The mycobacterium, Mycob. album, grew
fairly well in the rhizosphere of corn and wheat and somewhat better in that of clover
and peas. Brewer's yeast did not grow at all with corn, wheat, and beans, but wild yeast
of the genus Torula, on the other hand, grew beautifully and multiplied.

Similar data were obtained by Metz (1955) in experiments with sterile cultures,
nonsporeforming bacteria and, on other plants, their growth stopped. According to his
data, the growth of Azotobacter is strongly suppressed in the rhizosphere of celandine,
buttercups (Ranunculus acer L., Ran. repens L), peonies (P.officinalis L.), and fumitory,
(Fumeria officinalis L.), and is less suppressed in the rhizosphere of Viola tricolor
Wittr., Allium schoenoprasum L., Rumex patientia L., and Epillobium montanum L.

Plants such as Crepis virens K., Hieracium pilosella L., Armoracia rusticana Gaertn,
and others, which strongly suppress the growth of sporeformong bacteria, do not have
any deleterious effect on the growth of Azotobacter. On the contrary, many of them
stimulate the growth of this microbe.

A similar effect on the growth of Azotobacter by these or other plant species prevails
under conditions of natural growth (Table 77).

Table 77
Accumulation of azotobacter in soil with different plants
(number of cells per gram of soil)
Region Plant Cultivated soil Fallow soil
Moscow Oblast',
podsol
Wheat 0-10 0-10
Rye 0-10 0-10
Barley 50 60
Oats 70 50
Potatoes 100 50
Clover 1,000 80
 Flax 0 10
Kola Peninsula,
podsol
Clover 1,000 0
Cow parsnip 50 0
Barley 10 0
Kuibyshev Oblast'
Serozem
Wheat 200 200
Barley 500 200
Lucerne 5,000 250
Moldavian SSR,
Serozem
Wheat 500 800
Lucerne 600 800
Sudan grass 1,200 500
Central Asia Uzbek
SSR, Serozem
Wheat 20 100
Corn 50 100
Lucerne 3,500 250
Vakhsh Valley,
Serozem
Cotton 450 400
Lucerne 10,000 800
Rye grass 6,000 600
Orchard grass 300 400
Rice 12,000 1,200
Kirgiz SSR, Serozem
Wheat 380 250
Beets 500 300
Potatoes 180 150
Cotton 10 50
Trans-Volga region,
Chestnut soil
Wheat 0-10 0
Millet 0 0
Sunflower 40 50
Corn 0-20 20
 Lucerne 2,000 200
Sweet clover 1,500 200
Crimea, Southern
coast
Tobacco 250 200
Vineyards 150 180

The effect of grass plants on the growth and accumulation of Azotobacter in the soil is
especially well demonstrated in monocultures. The longer a plant is cultivated, the more
bacteria and fungi accumulate in the soil. Such a long-term accumulation of microbes
was observed by us in the soils of Central Asia, in the lucerne-cotton crop rotation,
Usually, after lucerne is grown for three years whether in the pure form or in a grass
mixture, cotton is cultivated for several years (six to nine). After this type of crop
rotation, the microflora changes considerably in relation to Azotobacter, and to other
species as well. (Figure 78). Under lucerne, the number of Azotobacter cells increases
and, under cotton, it decreases. However, Azotobacter does not completely vanish under
cotton.

Figure 78. Growth of Azotobacter in soils with a crop rotation of lucerne-cotton in the
fields of the Vakhsh valley, Tadzhik SSR:

1-- cotton, 2--lucerne.

'This regularity of change in microbial forms is observed in monocultures of plants, for

example, under lucerne and wheat in the chestnut soils of the Trans-Volga region, or
under clover-flax and oats in the podsol soils of the Moscow Oblast' (Figure 79).
Figure 79. Growth of root-nodule bacteria of clover on various plants:

1--clover; 2--wheat; 3--oats (monoculture) on the experimental fields of the Agricultural

Academy im, Timiryazev.

In the crop rotations of an ordinary farm with industrial crops, one observes thesame
results, but with less markedly expressed numerical indices (Krasil'nikov, 1940a).

Sheloutnova (1938) and Wenzl (1934) observed the growth of Azotobacter under a
culture of tobacco and vineyards; Mashkovtsev (1934) and Uppal and coworkers (1939)
observed its growth under rice.

We have studied the growth of Azotobacter in forest plantations in the fields of

Kirgizia, Moldavia, Ukraine, and the Moscow Oblast'. The arboretum of a seven- to ten-
year-old plantation, in sectors previously under lucerne or other vegetation possessing a
considerable number of Azotobacter cells was investigated. The growth of Azotobacter
was quantitatively measured on strips sown with different tree varieties: maple, ash,
poplar, acacia, spruce, pine, oak, etc (Table 78).

Table 78
Growth of Azotobacter in soil under forest trees
(number of cells in 1 g of soil)
Azotobacter in Azotobacter in
Soil Plant
forest strip plowed-up strip
Moldavian SSR,
Chernozem
Oak forest 80 2,700
Acacia 150 2,800
Lime 0 3,000
Moscow Oblast', Podsol
Spruce 0 100
Birch 0 250
Oak 0 100
Kirgiz SSR, Serozem
Maple 60 6,000
Ash 500 5,000
Poplar 0 4,200
Acacia 1,500 4,000
Elm 0 6,000
Birch 0 3,500
Grass mixture -- 100,000

As can be seen from the data given, afforestation, as a rule, removes Azotobacter from
the soil. Only under certain species in it preserved in small numbers,. Under certain
plants, the acacia and the ash tree, Azotobacter grows moderately well, although it does
not reach the numbers found in plowed-up soils.

Artificially planted birch, aspen, oak, and especially spruce and pine trees in podsol
soils quickly remove Azotobacter. Azotobacter also perishes in Southern chernozem
soils, if the latter are afforested with oak, pine, hornbearn, etc.

Fruit trees, such am apple, pear. plum, cherry, and others, do not suppress the growth
of Azotobacter and many of them are even favorable to its accumulation in the soil. The
soils of the orchards investigated by us in Moldavia (chernozem). in the central belt of
RSFSR (podsol), in Crimea, and in other regions of the USSR contain larger quantities
of Azotobacter than the soils of fields which are intensively cultivated (Table 79).

Table 79
Growth of Azotobacter in soils under fruit trees
(number of cells in 1 g soil)
Azotobacter in
Region and soil Azotobacter in orchard
field
Moldavia, Chernozem
Kishinev region 2,500 450
Kalarash region 7,500 1,200
Slobodzei region 7,000 1,500
Rezinski region 5,000 650

Moscow Oblast', Serozem

Chashnikovo, experimental field 1,200 120
Snegiri 2,400 60
Kragnyi Mayak 1,400 0

Crimea
Alupke, schist 1,500 0
Yalta, humus 3,700 450

Sudak valley
Redish-brown, sepia brown 12,000 1,500
Steppe zone 4,200 540
Koktebel'. Heavy aluvium loam 2,100 250
Old Crimea. Gravel chernozem with low
1,800 80
humus content
Perekop region. chestnut solonchak 3,600 240

As can be mean from the above-mentioned, certain species of plants enhance the
growth of Azotobacter in soil, others suppress it, and others neither enhance nor
suppress its growth.

This type of plant classification is only relative and only holds true for sterile cultures,
where the microbes are subjected to a one-sided action by root excretions, with the
exclusion of other external factors.

Under conditions of natural growth in the field, the effect of root excretions is to a
large extent annulled by many factors and, first of all, by microorganisms. Therefore,
the summary effect in such cases will express itself in a weaker form and sometimes
will not be observed at all. A comparison of the effectiveness of plants should be
performed under the same soil-climatic conditions.

The same plant in different soils and in differing climatic and geographical zones may
act differently on Azotobacter. As was noted above, wheat acts negatively on the growth
of Azotobacter under conditions of sterility, in open ground in the Trans-Volga region on
chestnut soils, and in the central belt on podsol, but does not suppress the growth of
Azotobacter under the conditions prevailing in Kirgizia on chernozem soil, on the
chernozem soil of the Kuibyshev Oblast', and in other places. Even in the same region
and in the same soil, the effect of wheat may differ, depending on the extent of the
cultivation of the soil. Soils of the podsol zone, where cultivated, often contain a
sufficient number of Azotobacter under wheat and other plants. Of great importance for
the growth of Azotobacter is the agrotechnical cultivation of soil.

The root excretions of young plants differ from those during the period of ripening
and, therefore, the microflora during these periods of growth will also differ.

Only by consideration of a multitude of factors affecting the growth of Azotobacter

can the contradictory indices, obtained by different investigators, on the distribution of
this microorganism be explained.

These considerations not only pertain to Azotobacter but to all other microbial species
in the rhizosphere of plants.

The selective effect of plants is well demonstrated in the case of root-nodule bacteria.
Korenyako (1942) tested three species of bacteria, Rh. trifoli. Rh. meliloti and Rh.
leguminosarum, by introducing them into containers in which clover, lucerne, peas,
wheat, corn, and cotton were grown on sand. Root-nodule bacteria of lucerne grew
equally well under lucerne and under cotton; Rh. trifolii grew abundantly under clover,
lucerne, peas, and wheat, less well under flax, and hardly at all under corn. Root-nodule
bacteria of peas were found in large numbers under peas, clover, and wheat.

These data were confirmed by experiments performed in open soil (podsol). Root-
nodule bacteria of clover were introduced under clover, lucerne, peas, wheat, and corn.
The growth of the bacteria in the rhizosphere was followed through the entire vegetative
period, The results are given in Table 80.

Table 80
Growth of Rh. trifolii in the rhizosphere of various plants
(in thousands in 1 g soil)
Date of analysis Clover Lucerne Peas Wheat Corn
23 June 100 10 10 1 1
2 July 100 100 10 0.1 0.1
23 July 100 1,000 10 10 1.0
30 July 100 100 100 100 0.1
21 August 10,000 100 1,000 1,000 10
20 September 1,000 100 190 100 10
20 October 1,000 10 100 0.01 0.1

The intense growth of root-nodule bacteria under leguminous plants was also noted by
other investigators (Wilson and Wagner, 1936, and Lewis, 1938).

Rudin (1956) noted the activating action of corn sap, and especially that of pea sap, on
root-nodule bacteria. According to his observations, the sap of inoculated peas having
nodules on their roots is more active than the sap of noninoculated peas, The sap of corn
roots enhances the virulence of the root-nodule bacteria of peas.

According to our observations, the root-nodule bacteria of lucerne grow well under
timothy grass, cotton, and rye grass. Their number in the serozem soil of Central Asia,
reached hundreds of thousands in one gram of soil, i. e., almost the same as under
lucerne. In the chestnut soils of the Trans-Volga region, these bacteria in the rhizosphere
of timothy grass amount to tens of thousands in one gram of soil, and under orchard
grass, oats, and millet, their number was considerably smaller.

The number of root-nodule bacteria in the soil, after leguminous plants, decreases, if
the subsequent culture in the crop rotation is not favorable to their growth this decrease
in the number of bacteria in the soil takes place at different rates, depending on the soil
and on the plant species. Under flax and wheat, the number of root-nodule bacteria of
clover in the podsol of the Moscow Oblast, decreases comparatively rapidly.

The experimental data obtained by Chailakhyan and Megrabyan (1955) on the specific
action of the roots of leguminous plants on root-nodule bacteria are of interest. The
authors found that the ground roots or the root sap of leguminous plants do not
negatively affect the growth of root-nodule bacteria of their own species, but suppress
the growth of bacteria of other foreign species (Table 81), The maximum expression of
this selective action by the roots of leguminous plants is observed during budding and
flowering stages, becoming less marked later.

Table 81
Suppressing effect of the roots of leguminous plants on the growth of root-nodule
bacteria.
Zones of inhibition of growth around the roots, in mm
(according to Chailakhyan and Megrabyan, 1955)
Root- Root- Root- Root-
Root- Root- Root- Root-
nodule nodule Root- Root- nodule nodule
nodule nodule nodule nodule
Roots of bacteri bacteri nodule nodule bacteri bacteri
bacteri bacteri bacteri bacteri
plants a, a, bacteri bacteri a, a,
a, a, a, a,
Ono- Lucern a, Peas a, Soy Broad Trigo-
Vetch Clover Beans Lupine
brychis e beans nella
Vetch 0 4 3 4 6 3 3 7 4 4
Onobrych
4 0 3 5 7 4 4 8 5 3
is
Lucerne 2 5 0 4 6 6 4 6 3 3
Clover 5 6 4 0 6 5 5 5 4 4
Peas 2 2 3 3 0 3 2 3 3 3
Beans 6 5 5 6 6 0 3 6 7 5
Soy 5 4 6 5 5 4 0 7 6 5
Broad
3 3 2 2 3 3 2 0 3 3
beans
Trigonell
4 4 3 7 4 3 4 6 0 6
a
Lupine 4 5 4 4 7 4 3 7 4 0

In our investigations we were unable to find such specificity in the selective

antibacterial action of either the roots or the aerial parts of leguminous plants.

Torne and Brown (1937) tested the bacterial effect of the sap of a great number of
plants, leguminous and others. According to their data, the extracts of leaves of many
plants inhibit the growth of root-nodule bacteria. These plants include clover, cabbage,
carrots, turnips, and others. The authors did not observe any specificity in the inhibitory
action of the sap. Lucerne sap inhibited the bacteria of its own species to the same
extent as it inhibited that of clover, beans, and other plants. Root sap was less
bactericidal than the sap of the aerial parts and, in many plants, the root extract had no
toxicity for bacteria whatsoever.

Among the other microbes which grow in the root zone of vegetating plants we also
studied mycolytic bacteria. These bacteria are characterized by their ability to dissolve
the mycelia, of fungi (Khudyakov, 1935; Novogrudskii, 1936). They differ in
classification and comprise a mixed group of bacteria. They also possess a more or less
defined specificity. Each species in this group of bacteria dissolves certain forms of
fungi--saprophytes and phytopathogenic forms.

According to Korenyako (1939). Raznitsina (1942), and Kuzina (1951, 1955),

mycolytic bacteria grow abundantly and accumulate under certain plants. Under the
conditions prevailing in Central Asia, in serozem, soils under lucerne, their number
varies from 100,000 to one million in one gram. Under cotton, on the other hand, their
number decreases. In soil which has been plowed for six to nine years, under this
culture [cotton], the number of mycolytic bacteria is minimal, and under three-year-old
lucerne it is maximal (Figure 80).

Figure 80. Accumulation of mycolytic bacteria in soil under lucerne and cotton (Central
Asia)

1--under lucerne; 2--under cotton.

Under the conditions prevailing in the central belt of the Soviet Union, many
mycolytic bacteria grow well and accumulate in podsol soils under clover and under
certain other leguminous plants. Some of these bacteria dissolve fungi of the genus
Fusariam, and others dissolve fungi of the genus Helminthosporium. Mycolytic bacteria
do not grow uinder wheat and especially under flax, and they are removed from the soil
relatively quickly.

Daraseliya (1949) found a large accumulation of mycolytic bacteria in the rhizosphere

of the tea plant. According to the author, the limited distribution of phytopathogenic
fungi of the genus Fusarium in the soils of tea plantations is caused by the abundant
growth of these bacterial antagonists.

Nitrifying bacteria also grow in the rhizosphere of plants, where, under certain species,
for instance under leguminous and certain nonleguminous plants, their number is
considerably higher than under cereals and certain vegetable cultures. In studying the
species characteristics of denitrifying bacteria, which were isolated from the rhizosphere
of lucerne, wheat, and millet at the Ershov Station (Saratov Oblast'), we successfully
determined some of their characteristics. The strains which were isolated from the root
zones of wheat were in the most cases more active than the strains growing under
lucerne and millet. Nitrate reduction was completed by the former in three to five days,
while the latter completed this reaction under the same conditions in five to ten days. In
the former case, the process was accompanied by the violent evolution of gas (nitrogen),
while in the latter, the formation of gas was not observed or was negligible.

This example of the growth of denitrifying bacteria is probably not general and was
observed only under the soil-climatic conditions of the given region. In the serozem
soils of Central Asia and in the podsol soils of the Moscow Oblast' we have not
observed such specificity. Certain differences in the properties of denitrifying bacteria,
growing in the rhizosphere of lucerne and wheat, were observed in the chernozem soils
of Moldavia.

Among other species of nonsporulating bacteria growing in the rhizosphere of plants,

specific forms also probably exist which have better adapted themselves to the
conditions of life under certain species of plants. Unfortunately, with the methods
available we are unable to detect specialized forms under these or other plants. Certain
investigators have observed different concentrations of physiological groups of bacteria
under different plant cultures.

According to observations made by Starkey (1929, 1931), the process of the formation
of nitrate from ammonium sulfate in the rhizosphere of certain plants, was more
intensive than in others.

According to our observations, the root system of peas activates the process of the
decomposition of cellulose. In our experiments we used special growth containers, filled
with turfy podsol soil. One wall of the container (made of glass) was covered with filter
paper. The plant grew from the container, which had been placed in a sloping position,
and the roots were established so that they grew on the surface of the paper. The
consecutive stages of the process of the destruction of the paper could be followed
through the glass. Wheat, corn, peas, vetch, and beans were planted. The experiments
showed that the roots of cereals do not exert a noticeable effect on the process of
cellulose decomposition. Among the leguminous plants, beans proved to be ineffective,
vetch only slightly stimulatory, while the roots of peas strikingly enhanced the process
of the decomposition of paper (Figure 81).
Figure 81. Decomposition of the cellulose in soil under the influence of the root system
of plants

a) destruction of paper around the roots of peas; b) destruction of paper in control soil,
outside the root area.

Lockhead and his associates (1950, 1955) demonstrated that the composition of the
microflora of the rhizosphere of various plants differs with respect to vitamin
requirements. In the root area of some species of plants, bacteria prevail which require
vitamin B1 or B2 and, in the rhizosphere of other plants, bacteria requiring biotin,
vitamin B12, cysteine, methionine, etc are prevalent.

There are indications in the literature, that algae also differ in their growth in the root
area of plants. According to data obtained by Shtina (1954a, 1955), rye, timothy grass,
and potatoes mainly enhance the accumulation of diatom algae; in the rhizosphere
clover and lupine enhance the growth of green algae and perennial grasses, and potatoes
partially enhance the growth of blue-green algae (Table 82).

Table 82
Growth of algae in the root zone of plants
(in thousands per 1 g of soil)
Blue-
Green algae Blue-green Green
Diatoms in Diatoms green
Plant in algae in algae in
rhizosphere in control algae in
rhizosphere rhizosphere control
control
Ry e 42.0 79.8 8.2 18.0 65.0 8.0
Timothy grass, 43.6 63.6 8.6 28.6 78.1 6.6
first year
Timothy grass,
73.2 147.6 8.6 28.6 78.1 6.6
second year
Clover, first year 15.4 90.0 11.0 28.6 40.0 6.0
Clover, second
37.4 105.0 6.8 39.5 58.4 1.1
year
Lupine 19.2 93.6 2.4 37.2 69.6 1.0
Potatoes 27.6 46.8 7.2 15.6 45.6 3.6

There are many indications that if unfavorable conditions prevail during the growth of
the plant, more or less great numbers of phytopathogenic organisms grow and
accumulate in their rhizosphere.

Timonin (1941) showed that under a culture of flax considerable numbers of the
following fungi often develop: Fusarium lini, Alternaria, Cephalosporium, and certain
others causing plant diseases. Sanford and Broadfoot (1951) observed the growth of the
phytopathogenic fungi Helminthosporium sativum and Fusarium culmorum in soil
under wheat and oat monocultures. The fungus Ophiobolus graminis, under the
conditions which prevail in certain localities in Canada, is suppressed by oats and clover
and, according to Winter (1940), it grows better in the rhizosphere of wheat, than in the
soil outside the root zone. Martin (1950) found an accumulation of the fungi Fusarium
solani, Pyrenochaeta sp., and other species in the soil under citrus plantations. The
author is of the opinion that the weak growth of the seedlings and saplings of citrus
plants in the soil of citrus groves is caused by the deleterious effect of the microflora.
Cotton is favorable to the accumulation in the soil of the phytopathogenic fungi
Verticillium dahliae and Fusarium vasinfectum, causing it to wither, At the same time,
lucerne suppresses the growth of these fungi. The accumulation of phytopathogenic
fungi in soil under the influence of vegetative cover was also noted by other
investigators (Lockhead and others, 1940-1950; Weindling, 1946; Eaton and Rigler,
1946; Timonin, 1946; Grammer, 1955, and others).

Under certain conditions, plants can accumulate microbial antagonists in the soil,
which inhibit the growth of such useful species of microorganisms as Azotobacter, root-
nodule bacteria, and mycorhizal fungi, which are the producers of various biotic
substances: vitamins, auxins, amino acids, and other products of microbes.

Plants may also favor the growth and accumulation in the soil of the microbial
antagonists of phytopathogenic bacteria, fungi, actinomycetes, and even viruses.

Hildenbrand and West (1941) observed a lower rate in the root-rot disease in
strawberries when they were sown after soybean, and an increased incidence of the
disease when they were sown after clover. According to data obtained by Cooper and
Chilton (1950), in the root zone and on the roots of sugar cane, actinomycetes,
antagonists of the fungus Pythium arrhenomonas, which is the causative agent of the
root disease of this plant, grow abundantly. Their number in the rhizosphere reaches
77,000 and more, while outside the rhizosphere, away from the root, it does not exceed
2,000 in one gram of soil.
 Similar data wam given by Sanford (1946, 1948), Weindling (1948), Fallings (1954),
and others.

Plants have a beneficient effect on soil. not only in relation to phytopathogenic

microbes, but also in relation to pathogenic microbes affecting men and animals.

Bogopol'skii (1948, 1950) studied the effect of the root system of various plants on the
viability of bacteria of the colon group in the soils of urban plantations, in the parks and
squares of Kiev. According to his observations, certain grasses used for lawns
considerably hastened the death of these bacteria. For instance, in a plantation of sweet
clover, after 60 days, 25 organisms out of one and a half million originally introduced
were found, 45 were found under clover, and 110 were found under oats, of the total
number originally introduced in the soil. Under orchard grass the colon bacillus almost
totally disappeared (10 organisms per gram), while In the control zone without plants,
180 bacteria per gram were observed.

According to our observations, the colon bacillus and a pyogenic staphlococci die in
the soil under some plants quicker than under others (Table 83).

Table 83
Death of Staph. aureus and Bact. coli under the influence of plants
(number of cells per one gram of soil)
Time of stay in Two-year-old
Bacteria Fallow soil Grass mixture
soil, days clover
Staph aureus
0 2,500,000 2,500,000 2,500,000
5 100,000 10,000 50,000
10 10,000 100 10
20 1,000 0 0
30 100 0 0
50 0 0 0
Bact. coli
0 1,500,000 1,500,000 1,500,000
5 500,000 20,000 30,000
10 25,000 1,000 5,000
20 1,500 100 600
30 200 0 10
50 10 0 0

On the tenth day after the introduction of the staphylococci, it could not be found
under a grass mixture of lucerne and rye grass, nor under clover with timothy grass.
Under clover it disappeared after 20 days and in fallow soil, after 50 days. The colon
bacillus disappeared more quickly under clover than under a grass mixture or even
under fallow soil. The same results were obtained by Mishustin (1954) in vegetation
experiments with orchard grass, rye grass, brome grams, clover, and fescue grass. Two
cultures were introduced into the soil, Bact. coli and Bact. coli aerogenes. They died
sooner under clover and fescue grass. Under other plants these bacteria died much later.

Arkhipov (1951, 1954) studied the extent of the growth in soils of the bacterium which
causes anthrax under different plants, under conditions of vegetation experiments, and
under field conditions. Wheat, rye, clover, vetch, lucerne, barley, Euagropyrum,
potatoes, buckwheat, millet, lupine, flax, garlic, onions, etc were grown. Experiments
showed that certain plants (garlic, winter wheat, rye, onions, rhubarb, and vetch)
completely remove the anthrax bacillus from the soil. Lucerne, spring wheat, hemp, and
the castor-oil plant exert a weak inhibitory effect. Ornithopus sativus, carrots, radishes,
rape, water cress, and others have no effect whatsoever. Finally, such plants as potatoes,
Eusgropyrum, horseradish, radishes, and turnips stimulated the growth and
accumulation in the soil of the above microbe. On the basis of the results of his own
studies and data obtained from the literature, V. V. Arkhipov recommended the sowing
of winter wheat, rye, vetch, clover, rhubarb, garlic, and onions for the more rapid
removal of anthrax bacilli from the soil.

The selective action of plants on the microflora is not only caused by the specificity of
the nutrient substances excreted by the root system, but also by special antimicrobial
compounds. In the chapter on toxicity of soils we shall give data showing that many
plants form and excrete different toxic substances into the environment. Among them
there are many compounds which strongly inhibit the growth of certain species of
microbes.

Earlier we noted (1934b, 1939) that the root excretions of wheat, corn, flax, and some
other plants visibly supress the development of certain kinds of bacteria--Azotobacter,
sporiferous bacteria, and other groups both under sterile experimental conditions and
directly in the soil in their natural surroundings. It was shown that the root excretions of
corn act differently to those of wheat. Under the influence of these excretions, the cells
of the bacteria undergo a considerable deformation, degeneration, involution, and
subsequently die. Sometimes this degeneration of the culture is accompanied by the
birth of new forms and species.

The presence of antimicrobial substances in the root excretions of plants was observed
by Sidoranko (1940b), Meshkov (1953) and others. The soil studies carried out for
many years in the Wareham forests (in England) by Rayner and Nelson-Jones (1949)
showed that the obstacle standing in the way of afforestation of certain soil zones is not
the lack or insufficiency of nutrient elements but the presence of special organic
substances. These substances, according to these authors, retard the growth not only of
young saplings of various wood varieties but also that of many microorganisms. Rayner
has shown that by introducing organic fertilizer manure, or compost into these poisoned
soils, the toxicity diminishes or even vanishes. According to her observations, this
decrease in toxicity takes place due to the activation of the microflora in the presence of
fresh organic substance.

Extensive studies of the antimicrobial properties of toxins excreted by plants were

made by Metz (1955). The root excretions and root sap of 100 species of plants were
studied. It was shown that 10 species out of 100 (Armoracia rusticana Gaertn,
Chelidonium majus L., Crepis viren K., Hieracium. pilosella L., Hypericum perforatum
L.., Lampsana communis L., Ranunculus acer. L., Viola silvatica Schm., V. tricolor
Wittr., Pulmonaria officinalis L.) greatly inhibited the growth of sporiferous bacilli or
micrococci; 15 species--Brassica campestris L., B. napus L., Campanula rapunculoides
L., Capsella bursa pastoris Mad., Fumaria officinalis L., Ranunculus repens L.,
Paeonia officinalis L., Sinapis alba L., Galium verum L., Allium schoenoprasum L., and
others inhibited their growth moderately; 31 species: Achillea millefolium L., Aethusa
cynapium L., Avena sativa L., Chrysanthemum lencanthemum L., Cichorium intybus L.,
Datura stramortium L., Hordeum sativum Gessen and others inhibited the growth of
these bacteria only slightly. The remaining species of plants did not have any inhibitory
effect.

Stiven, (1952) found that in the soil under the plants Tragopogon plumosus L., and
Pentanisia variabilis Harw., the processes of nitrification and the growth of Bac.
subtilis, Bac. coli, and others are inhibited considerably. Pure substances obtained from
the root excretions of these plants have the same effect.

The action of root toxins is not specific, according to Metz. The roots inhibit the
growth of bacteria, both when they were isolated from the rhizosphere of the given plant
and when they were isolated from that of another species. For example, the roots of
Chelidonium majus L. inhibit, to the same degree. the growth of bacteria isolated from
their own root zone and those from the root zones of goutweed, violet, hawkweed, and
others, and also bacteria from fallow soil. However, the author notes, the root-zone
microflora is less sensitive to the action of the roots than organisms from outside this
zone. The growth of bacteria and mycobacteria, isolated from fallow soil or from soil
outside the root zone, in the majority of cases, is inhibited by the roots of many plants,
while most of the bacteria of the rhizosphere are not inhibited at all.

According to our observations, the roots of lucerne and peas, under conditions of
growth in a sterile nutrient solution. excrete substances which inhibit the growth of
Azotobacter chroococcum and Pseudomonas fluorescens, certain species of rootnodule
bacteria, and others.

The growth rate of the bacteria from two-month-old plants grown in a solution was
determined (Table 84).

Table 84
Effect of root excretions of peas and lucerne on growth of bacteria
Bacteria Peas Lucerne
Az. chroococcum
strain No 54 ++ -
strain No A ++ -
halophylic strain - -
garden strain - +
strain No 6 - -
Az. Vinelandii ++++ +++
Ps. flourescens No 8a + -
Ps. aurantiaca +++ +++
Root nodule bact. of:
kidney beans +++++ ++
peas + -
vetch + -
lucerne + -
Lathyrus + _
broad beans +++++ +++
soy - +
lupine - -
sweet clover +++++ +++
clover ++++ -

The longer plants have been growing in a solution, the more strongly the solution
affects the bacteria. After peas grew for three months in the solution, Azotobacter strain
No 54 and strain A did not grow at all, nor did the root-nodule bacteria of clover and
sweet clover.

Antibacterial substances are also excreted by isolated roots if the latter are grown in an
artificial nutrient medium. We cultivated the roots of peas. lucerne, lupine, and certain
other plants in Bonner's nutrient solution for one to three months and, after various
periods of time, we determined the presence of antibacterial substances in it. The
bacteria were introduced into the solution and, by means of plating, their viability and
degree of multiplication were established, Cultures of Azotobacter strain No 54, a
halophilic strain and a garden strain, and, in addition, root-nodule bacteria of peas,
vetch, lucerne, and others, were sown into the solutions.

The results of the experiments showed that the excretions of roots grown in an isolated
form act on the same species of bacteria as do the excretions of non-isolated roots. Only
the degree of their inhibition was weaker. The antibacterial spectrum of root excretions
was the same in both cases. This indicated the fact that isolated and nonisolated roots
excrete the same antimicrobial substances.

Thus, by this experiment, it was observed that certain antimicrobial substances

excreted into the medium, are directly synthesized in the root system of the vegetating
plant.

Part IV, continued:

Microflora of decomposing roots

Investigations have shown that plants not only determine the microflora of the soil
during their growth but also determine them by means of their dead residues, especially
those of roots. It was established that these residues, depending on the species of plant
or, more accurately, on their chemical composition, are decomposed by various forms of
microbes. The qualitative composition of the microflora of the decaying roots of wheat
and clover, cotton and lucerne, differ considerably.

We performed microbiological studies of the root flora and of the flora of the area
around roots which were rotting in the soil after the harvesting of crops. Studies have
shown that around the roots of wheat, corn, sunflower, soybean, and other plants, the
number of microorganisms is considerably higher than in the soil outside the root zone.
If by ordinary calculation (plating on an agar medium) in the soil outside the root zone
one finds four to eight million bacteria in one gram, then around decaying roots there
are 20 to 140 million. The number of bacteria varies according to the degree of the
decomposition of the root tissue.

The group composition of the microflora of decaying roots is given in Table 85, which
shows that the increase in the total number of microorganisms paralleled the
multiplication of the cellulose bacteria.

Table 85
Qualitative composition of the microflora of corn roots at various stages of decay
(number of cells in millions per gram of soil)
Time of
Nonspori- Spori-
sampling after Coccoid Myco- Actino- Cellulose
fireous ferious Fungi
harvesting of forms bacteria mycetes bacteria
bacteria bacteria
crops, in days
5 52 6.5 3.5 17.0 9.0 0.3 6.8
12 46 8.5 3.0 12.0 12.0 0.8 5.0
18 120 12.5 2.5 17.5 4.0 0.3 13.0
22 180 15.0 2.8 15.0 5.0 0.6 12.0
27 120 14.0 12.0 27.5 15.0 0.3 8.0
32 100 10.0 20.0 30.0 25.0 1.5 4.0
38 60 5.0 20.0 23.0 22.0 1.6 3.0

Actinomycetes, mycobacteria, and coccoid forms appeared somewhat later, after the
development of the nonsporiferous bacteria. Coccoid forms are basically mycobacteria,
actinomycetes. and, to a certain degree, mycococci, i.e., organisms belonging to the
group of actinomycetes.

Our subsequent studies were made with root residues which were introduced into the
soil. We studied the microflora of the decaying roots of lucerne, clover, wheat, corn, and
Euagropyrum, introduced in podsol soil in glass vessels.

At the same time, the microflora of a root-mass compost was studied under the
conditions of a laboratory experiment. The results are given in Table 86.

Table 86
Quantitative and qualitative composition of the microflora of decaying roots
 (number of cells in thousands per one gram of soil)
Root- Nonspori-
Azoto- Mycolytic Actino- Sporiferous
Roots nodule ferous Fungi
bacter bacteria mycetes bacteria
bacteria bacteria
IN
COMPOSTS:
Lucerne 1,000,000 0 1,000,000 1,000,000 0.05 1 0.01
Clover 1,500,000 0 1,000,000 10,000 0.1 5 0.001
Wheat 0 0 100,000 0.05 15 35 35
Corn 0.03 0.3 400,000 0.1 250 15 150
Euagropyrum 0.05 0.01 5,000,000 1,000,000 150 20 0.001

IN SOIL:
Lucerne 500,000 2,500 1,200,000 10,000 150 40 0.5
Clover 150,000 1,700 750,000 1,000 300 30 0.8
Wheat 0 0 15,000 0 1,500 360 280
Corn 0 0 13,000 0 4,500 120 340
Euagropyrum 10 0 70,000 100,000 2,800 400 450

In the table, data are given on a number of microbes during the initial stage of root
decay. During the subsequent stages of decomposition, the quantitative ratios of
microorganisms changed; the number of actinomycetes increased considerably, while
the number of sporiferous and nonsporiferous bacteria and fungi decreased. In a half-
decayed mass of roots, actinomycetes often cover the root particles with a white coating
of aerial mycelium.

Nonsporiferous bacteria prevail during all stages of root decay, but their species
composition varies. The number of cellulose bacteria during the root decay of various
plants differs. Filter paper spread on a soil plate containing the roots of clover or lucerne
decomposes more quickly and more effectively than on soil containing the roots of
wheat. In the former case, 500-700 eroded spots were counted on the paper and in the
latter case, only 130-270 spots were found. A compost of the roots of Euagropyrum
contains fungi which are absent in the decaying roots of clover, and vice verse. In some
cases, there is a mass multiplication of mycolytic bacteria and, in other cases, these
bacteria are scarce or not detected at all (Krasil'nikov and Nikitina, 1945).

Bodily (1944) introduced residues of clover and wheat straw into the soil and he
observed that in the presence of clover the number of microorganisms in soil was
greater than in the presence of wheat straw.

Other plant residues, in addition to roots, possess, to a certain degree, a determining

effect on the composition of the soil microflora. Birch leaves decompose differently and
possess a different microflora than leaves of aspen and oak. The composition of the
microflora of Euagropyrum straw and wheat straw also differ. Consequently, all the
plant residues of the roots, stems and leaves upon decomposition, enhance the growth of
different species of microorganisms in the soil.

Al'bitskaya (1954) obtained data on fungi from her study of the decomposition of plant
residues of forest and steppe vegetation. According to these data, the roots of steppe
vegetation are mainly decomposed by fungi of the genera Penicillium,
Cephalosporium , Fusarium, and also, to a limited extent, by members of the genus
Mucorales. Roots of oak are decomposed by fungi of the genus Trichoderma--T.
lignorium, T. koningii, and rarely, by members of the genus Penicillium.

Plant residues of both steppe and forest vegetation are more intensively decomposed
after being inoculated by a mixture of the natural microflora where, in these cases, the
greatest loss in water-soluble organic substances is observed, which indicates a most
complete decomposition of the residues. Upon the decomposition of steppe vegetation,
there is a greater CO2 evolution and a decrease in water-soluble substances and in
lignin. Upon the decomposition of the roots and leaves of oak, the water-soluble
substances are depleted in carbon, and upon the decompo sition of steppe vegetation,
their car bon content increases.

Thus, plants, while alive, differentiate, select, and accumulate certain compounds. In
other words, the vegetative cover, as a whole, is a powerful determining factor in the
microbial biocoenoses of soils. In the zone of the root system, only those organisms
which assimilate the root excretions of the plant in question more quickly, can develop,
supplanting other, less well adapted, species.

In different plants, the dominating microbial forms differ in their systematic position as
well as biologically. It may be said that each species of plant, or group of closely related
species, concentrates a more or less specific microflora.

Unfortunately we are not yet in the position to determine this specificity in a precise
way. Our methods of recognizing and classifying microbes, especially bacteria, are far
from being completely developed. We cannot exactly say in what manner the
nonsporiferous bacteria, which dominate in the rhizosphere of wheat differ from the
bacteria in the rhizosphere of clover, oats or potatoes. By their appearance, cell size,
motility, colony structure, and nature of growth on media, they do not differ in the
majority of cases, nor do they differ in their generally accepted physiological properties.

Only a more thorough study of the biochemical activity of microorganisms will enable
us to differentiate between them. However, such a method of study has not yet found
wide use in laboratory investigations of rhizosphere microflora.

The selective action of the vegetative cover may be directed toward the selection not
only of useful, but also of harmful microflora. Under unfavorable conditions, when
agrotechnical rules are not observed, with an incorrect choice of crop rotation, the fields
are contaminated by phytopathogenic bacteria and fungi and other harmful microbes--
weeds. Especially, after the prolonged repeated cultivation of plants on the same field in
monocultures, one observes this effect. The accumulation of an undesirable microflora
under monocultures is most often caused by the insufficient growth of microbial
antagonists in the rhizosphere, which are characteristic of the given plant under
conditions of normal growth.

Many outbursts of epiphytotic diseases, as for example fusarial infections of cereals,

cotton, saplings of woody plants, as well as cotton wilt are, in our opinion, caused by
the above phenomenon. This was proved by microbiological studies made of cotton
fields which were afflicted by the fungi Verticillium dahliae and Fusarium vasinfectum.
These fungi are removed as soon as mycolytic bacteria begin to grow in the soil. These
bacteria, as was shown above, grow under lucerne and certain grass mixtures.

In agricultural practice from time immemorial, crop rotation has been used as one of
the methods of increasing crop production. It was empirically found that with certain
crop rotations, not only crop production increased, but disease decreased. It is known
that lucerne ameliorates the soil in cotton farms and inhibits the growth of the organisms
causing cotton diseases. On this basis, one can select plants for rational crop rotation.

Noting the great influence of the vegetative cover on the formation of microbial
biocoenoses in soil, one should not forget the importance of the soil itself as a substrate
and the effect of the activity of man on external conditions. The physicochemical state
of soil determines to a great extent the direction of the microbiological processes, and to
a similar extent the development of different microbial species. The distribution of
Azotobacter in soil not only depends on the plants. on their root excretions and
decomposition products, but also on soil acidity and the presence of phosphorus,
calcium, molybdenum, and other nutrient elements.

The cultivation of soil, the liming of soils, the use of fertilizers and other factors favor
the growth and accumulation of Azotobacter. In arid regions the growth and
accumulation of Azotobacter in soils depends to a considerable degree on irrigation.

Effect of Soil Microorganisms on Plants

In the foregoing chapter, the considerable influence of the microbial population of the
soil, and the accumulation of individual species and groups of microorganisms in the
root zone, was shown. The importance of root microflora for the life of plants has been
studied only a little and the information available on the action of different microbes on
the growth of plants is meager. We will not dwell here on the activity of root-nodule
bacteria, Azotobacter, and mycorhizal fungi, since data on this subject are abundant in
scientific literature. In this chapter, data are given only on those organisms which exert a
beneficial or harmful effect on plants, due to the products of their metabolism; these are
microbial antagonists, activators, inhibitors, etc.

Microbial activators

It was noted above that certain soil microorganisms are capable of producing various
biotic substances--vitamins, auxins, amino acids, and other biocatalysts. Such
microorganisms activate the biological processes and, therefore, we named them
microbial activators.

There is much data in the literature on the positive effect of pure cultures of bacteria,
fungi, and actinomycetes on the growth and development of plants. Microbial activators
increase the percentage of germinating seeds, enhance the growth of the young plants,
and often change the nature of the biochemical processes.

Already toward the end of the last century, Geier (1882), and later Zimmermann
(1902), described the bacteria living in the tissues of plants which had a certain
activating effect on their growth. Such bacteria could form special nodules in the leaves
of subtropical and tropical plants.

According to their systematic position, these bacteria differ from each other. In
members of the genus Ardisia Thunb., the nodules in the leaf tissues are formed by
nonsporiferous bacteria of the genera Bacterium and Pseudomonus. In Pavetta L.,
Chomelia L,, Psychotria L., and certain other genera, mycobacteria were isolated from
the nodules, in Dioscorea L., and others, bacteria of the Rhizobium type were isolated
(Krasil'nikov, 1940 a, b).

Miehe (1911, 1918) studied in detail the bacteria which are members of the genera
Ardisia Thunb. and Pavetta L. According to his data, they formed special substances
which cause the stimulation of the tissues (Reizwirkung). They do not fix nitrogen.

Jongh (1938) found that Ardisia does not grow or grows poorly without bacterial
symbionts, and does not bloom nor bear fruit. When bacteria were introduced into the
tissue of the plant, its growth and development improved drastically, the growth of
branches was enhanced, leaves acquired normal form, and flowers and fruits appeared.

The role of the symbionts of the root-nodule bacteria group is widely known. Forming
nodules on the roots of leguminous plants and on certain nonleguminous ones, under
certain conditions they considerably improve the growth of plants and increase crop
yields.

The biological role of root-nodule bacteria for plants is widely known. However, the
mechanism of the action of these organisms is still obscure. It is assumed that root-
nodule bacteria fix molecular nitrogen and supply it to the host plant. There is no clear-
cut experimental data to support this assumption.

There are reasons to believe that root-nodule bacteria, as well as the bacteria from the
nodules on the leaves of the above-mentioned plants, act favorably through their
metabolites. According to our data, leguminous plants, in symbiosis with the nodule
hacteria, fix molecular nitrogen for themselves from the air. The bacteria, due to their
metabolic products, act as biocatalysts, activating the nitrogen-fixing ability
(Krasil'nikov and Korenyako, 1946a).

The positive action of mycorhizal fungi has already been mentioned. These fungi are
widespread in nature. One view has been expressed that all plants have mycorhizae, but
differ as to the nature of the co-habitation. In some plants the mycorhiza is endotrophic
and in others ectotraphic. In the former, the fungal hyphae grow almost exclusively in
the root tissues and only a few extend into the soil, outside the root, The endotrophic
mycorhiza, in its turn, includes two types of mycorhizae the phycomycetal and vesicular
type, wore often encountered in grassy and woody plants, and the orchid mycorhiza
found in the plants of the orchid family These two types of mycorhiza differ in the
nature of the structure and development of their mycelial hyphae. In phycomycetal
mycorhizas, the hyphae are not septate and often form characteristic swellings in the
root tissues--vesiculae. The mycelium in the orchid mycorhiza is septate; the hyphae
form characteristic entanglements only within the root cello. As a rule, they do not have
vesicles.

In the endotrophic mycorhiza of both types, the hyphae develop only in the cortex of
roots in the intercellular space, or penetrate into the cells. The mycelia of the fungus do
not penetrate into the central part of the root.

Ectotrophic mycorhiza is characterized by the growth of mycelial hyphae on the

surface of the root tips, which surrounds them with a thick and quite dense cover. From
this cover the hyphae extend into the soil. There are no root hairs on this part of the root.
A small part of the hyphae penetrates inside the root, but not very deeply, their growth
being usually limited to the intercellular space of the epidermis, where the hyphae
interweave, forming he dense Hartig net. The hyphae seldom penetrate into the upper
two to three layers of the cortical cells. The hyphae do not penetrate the cortical cells,
and, in the few cases where this does happen, they soon die inside the cells. Entotrophic
mycorhizae are most often encounter ed among woody coniferous and leaf-bearing
plants.

Between the endotrophic and ectotrophic mycorhizae, there are intermediate

formations--the ecto-endotrophic mycorhizas.

Some authors (Lobanov, 1953) are of the opinion that an absolutely ectotrophic
mycorhiza does not exist at all. They maintain that in woody plants, especially during
the early stages of their development, there is always an endotrophic mycorhiza. Only
later the external cover on the root tip develops.

The systematic distribution of the mycorhiza fungi varies. Fungi-forming vesicular

mycorhiza probably belong to Phycomycetes of the Endogenaceae family, The
endotrophic mycorhizas in orchids are formed by the Fungi Imperfecti of the genus
Rhizoctonia, while in certain orchids, the mycorhiza is ferried by higher fungi,
Basidiomycetes with well-developed fruiting bodies--Armillaria mellea, Xerotus
javanicus, and Marasmius coniatus.

Ectotrophic and endotrophic mycorhizae of woody plants are mainly formed by

mushrooms, most often by the imperfect fungi of the genus Phoma. There are
mycorhiza-forming fungi, among the Ascomycetes and other groups of fungi. For
instance, in beech, 12 different species of fungi have been described as participants in
the mycorhizae in pine 17 species were described; and, in spruce, up to nine species, etc
(Kursanov, 1940; Yachevskii, 1933; Kelly, 1952; Magru, 1949; Lilly and Barnett, 1953,
Goimann, 1954, Lobanov, 1953 and others).

These data clearly show that there is no strict specificity among mycorhizal fungi, as in
root-nodule bacteria. The first to observe the positive effect of mycorhizal fungi on the
growth of plants was Kamenskil (1880) and after him, Voronin (1886), Vysotskii (1902),
and others. They all looked upon mycorhizal fungi as symbionts, which exert a great
influence on the growth of plants. Baraney (1940) presents extensive data confirming
this point of view. One of his tables is given below (Table 87).

Table 87
Effect of mycorhizas on the growth of pine seedlings
Indexes of growth With mycorhizas Without mycrohizas
Length of shoots of seedlings, cm 35.5 17.5
Increase in length after 2 years, cm 18.0 3.0
Length of sprouts of 2nd order, cm 10.0 0.3
Weight of shoots part, gm 17.0 3.1
Weight of roots, gm 11.0 4.5
Number of leaves 42 12
2
Total area of leaves, cm 591.0 96.0

The essence of the action of fungi consists in supplying the plants with nitrogenous
and carbonaceous elements of nutrition in some cases and, in others, in the supply of
auxiliary nutrients or biotic substances, and more correctly with both. There is a great
deal of data in the literature on the significance of mycorhizal fungi in the nutrition of
plants, which was already mentioned In the previous chapters of this work. Recently, by
the use of direct experiments with labeled atoms, it was shown that mycorhizal fungi
take up find transmit various nutrient elements.

Kramer and Wilbur (1949) and Mellin and Nilson (1950, 1952) have shown that fungi
transmit P32 and N15 from the external solution into the tissues of the roots and stems of
pine (Pinus taeda L., P. resinosa, P. silvestris L.). Morrison (1954) found that in the
presence of the mycorhizas, there in enhanced transfer of P32, not only to the roots and
atoms of Pinus Rediata, but also to the leaves. Herley and MacCready (1950, 1952)
have shown that mycorhizal fungi take up labeled phosphorus, accumulating up to 90%
in their mycelia.

The data on studies made with labeled atoms does not disclose the nature of the
compounds by way of which the labeled phosphorus and nitrogen are transmitted to the
mycorhizal fungi, one must assume that these elements when entering the cell of the
fungus, take part in the general process of building its substance in the form of one of
the organic compounds of metabolic products, The labeled elements are released from
the cell as metabolites which enter into the medium and, from there, into the roots and
green parts of the host plant. Such it process was demonstrated in Shavlovskii's
experiment with rhizosphere bacteria (see above).

The free-living soil bacteria also have a considerable effect on plants. Clark and Roller
(1931) studied the action of pure cultures of the following bacteria on the growth of
duckweed: Bact. coli, Clostridium sporogenes, Clastrid welchii, Ps. fluorescens
liquefaciens, Bact. aerogenes, Staph. aureus, Bac. subtilis, Bact. prodigiosum etc. Some
of these bacteria stimulated the growth of buckwheat, while others had no visible effect
on it.

Kozlowski (1935) observed the action of pure bacteria cultures on the growth of barley
and apples, The tested cultures of the sporferous bacteria, Bac. cereus, Bac. mycoides,
and Bac. subtills and of the nonsporiferous bacteria, Bact. denitrificans, Bact. putidium,
and Ps. pyocyanea and also cultures of fungi. The sporiferous bacteria had no effect on
the growth of plants. Of the nonsporiferous bacteria, Ps. pyocyanea and Bact. putidum
inhibited their growth, while Bact. denitrificans, stimulated the growth of barley, but not
that of apples.

We tested 130 different bacteria, mycobacteria, and actinomycetes isolated from

various soils. Among them were the following; 32 strains of Azotobacter, 33 strains of
root-nodule bacteria, 40 of Pseudomonas, 10 of Bacterium, four of mycobacteria, six of
actinomycetes, three of Bac. mycoides, and two of Bac. subtilis. Cultures of these
organisms and products of their metabolism were added to the medium in which wheat
seedlings were grown in one series of experiments, and to that of isolated roots of peas
and wheat in another series of experiments.

Isolated roots are a convenient object for the study of the requirements of biotic
substances, since they are incapable of the independent synthesis of the whole gamut of
these substances.

We grew isolated roots of peas, wheat, rye and other plants on Bonner's synthetic
medium of the following composition:

Distilled water 1,000 ml

Ca (N03) 2 . 4H2 O, 0.23 g
MgSO4 . 7H2O, 0.36 g
KNO3,,0.81 g
KCl, 0.65g
KH2PO4,0.12 g
Iron, Traces
Saccharose, 20 g

Extracts and filtrates of bacterial cultures were added in various amounts, as auxiliary
substances. The bacterial cultures were grown in liquid media and filtered with bacterial
filters. The results are given in Table 88.

Table 88
Influence of metabolic products of soil microorganisms on the growth of isolated roots
(increase in cm on the 30--40th day of growth)
Increase in length of Increase in length of
Microorganisms
roots of peas roots of wheat
Control (no metabolites of microbes) 0.3 0.1
Az. chroococcum
strain 54 15.0 17.0
strain 37 5.0 0.8
strain A 27.0 15.0
Rh. terifolii
leguminosarum 31.0 30.0
phaseoli 50.1 45.0
lupini 7.0 15.0
meliloti 0.0 5.0
sojae 3.0 10.0
Ps. aurantiaca 65.0 55.0
Ps. flourescens
strain 4 40.0 35.0
strain 15 10.0 0.5
strain 30 25.0 12.0
strain 69 38.0 12.0
Ps. denitrificans 6.0 38.0
Ps. mycolytica 10.0 31.0
Ps. nonflourescens
strain 3 4.0 30.0
strain 10 10.0 31.0
strain 12 23.0 11.0
strain 15 0.1 0.2
Bact. denitrificans 25.0 12.0
Bact. album 37.0 37.0
Bact. mycolyticum 6.0 25.0
Bact. vulgaris 0.0 0.0
Bac. mycoides 0.0 5.0
Bac. mesentericus
strain 3 0.0 3.0
strain 11 0.0 0
strain 27 5.0 0
strain 29 3.0 0
Bac. subtilis
strain 7 0.0 0
strain 17 2.0 0
strain 21 3.0 0
A. violaceus 0.0 0
A. aurantiacus 3.0 0
A. globisporus
strain 160 65.0 25.0
strain 187 37.0 30.0
strain 375 10.0 45.0
A. grisus
strain 17 0.0 0.0
strain 57 0.0 0.0
strain 1067 0.0 25.0
A. albus 37.0 56.0
A. alboflavus 71.0 45.0

As can be seen from the table, a large increase in the length of the roots was observed
in the presence of filtrates of Azotobacter, root-nodule bacteria, and bacteria of the
genera Pseudomonas and Bacterium (Figure 82) Metabolic products of certain
actinomycetes were quite active. Sporiferous bacteria often showed a negative action,
inhibiting the growth of roots.

Figure 82. Effect of products of bacterial metabolism on the growth of isolated roots of
peas:

1--metabolites of Ps. fluorescens ; 2--metabolites of Ps. aurantiaca; 3-control (no

metabolites added).

In the same microbial group and even in the same species, different strains showed
different effects on the roots. For example, among cultures of Ps. fluorescens, strain No
4 strongly activates the growth of wheat roots, while strain No 15 only slightly activates
or does not activate these roots at all; strainNo 15 of Ps. nonfluorescens is inactive,
while strains Nos 5 and 10 are active; the root-nodule bacteria of lucerne did not
promote the growth of these roots and even suppressed them, while the root-nodule
bacteria of peas and especially those of beans greatly enhanced root growth. The same
was observed in all other groups of organisms.

The roots of different plants reacted differently to the action of filtrates of the same
culture. The roots of wheat reacted more intensively than the roots of peas to the
metabolites of Ps. nonfluorescens, strain 10, while with the filtrate of strain 12, the
picture was reversed.

Filtrates of microbial cultures show a positive effect only when used in small amounts.
When the amounts are large, their effect on the growth of isolated roots is negative. The
roots do not grow or grow very poorly, deviating from the normal, they thicken, swell,
do not branch, become brown too soon, and die.

The metabolites of many organisms in small concentrations strongly suppress the

growth of roots. Such organisms which produce toxic substances are found among
various groups of microorganisms but they are especially numerous among the
sporiferous bacteria. This group of bacteria is in general the most toxic in relation to
plants and many microbes (see below).

Similar data were obtained in experiments with plant seedlings. The filtrates of certain
microorganisms noticeably activated the germination of seeds, while the filtrates of
others had an inhibitory or no effect.

As in the experiments with isolated roots, filtrates of Azotobacter, nonsporiferous

bacteria of the genera Pseudomonas, Rhizobium and Bacterium, and many species of
actinomycetes most actively enhanced the growth of seedlings, In the genus
Azotobacter, the strains of Az. vinelandii and Az. agile var. jakutiae were active. The
strains of Az. chroococcum differed considerably in activity. Some possessed strongly
and accentuated activating properties with respect to the growth of wheat, others had
only weak activating properties and still others had no effect whatsoever on the growth
of plants. Under the influence of certain strains, the suppression of wheat growth was
observed.

We studied the activating effect of bacteria on different plants under field conditions
for a period of three years (1945a). The seeds were treated with a culture of bacterial
activators and were sown on kolkhoz fields in various regions. Altogether more than
100 experiments were performed, not including those with Azotobacter. The summary
of the results is given in Table 89,

Table 89
Effect of bacterial activators on plant crop
number of positive increase in
Crop Bacteria
experiments experiments crops, %
Wheat
Ps. flourescens
strain No. 14 16 12 12-27
strain No. 30 9 7 15-25
strain No. 25 12 6 10-14
Bact. sp strain No. 106 10 8 15-29
Oligonitrophiles 10 7 12-18
Az. chroococcum strain
20 12 10-23
No. 103
Oat
Ps. flourescens
strain No. 14 10 7 14-28
strain No. 30 -- -- --
strain No. 25 5 5 10-16
Bact. sp strain No. 106 12 8 12-30
Oligonitrophiles 2 2 14-22
Az. chroococcum strain
25 15 13-19
No. 103
Clover
Ps. flourescens
strain No. 14 6 5 18-23
strain No. 30 4 2 12-25
strain No. 25 4 3 10-30
Bact. sp strain No. 106 6 4 18-26
Oligonitrophiles -- -- --
Az. chroococcum strain
12 8 14-27
No. 103

The data in Table 89 show that bacterial activators exert a similar effect on crops as do
azotogen*, nitrogen, and other bacterial compounds. In our experiments, Azotobacter
was used in the form of peat azotogen. *[*Azotogen--Russian commercial name for
azotobacter field-inoculating preparation.] In Table 89 are given only those cases where
a positive effect was obtained when Azotobacter was absent from the soil; it had
perished during the first few days after its introduction into the soil. Therefore, the
effect was caused, not by Azotobacter, but by other microbes.

Akhromeiko and Shestakova (1954) successfully tested bacteria, which had been
isolated from the rhizosphere, on the growth of oak and ash tree seedlings. With oak,
there was a 24-34 per cent increase in the increment of dry matter, and with ash a 40 per
cent increase. Samtsevich and others (1952) used an Azotobacter culture for the
inoculation of oak seedlings in a steppe zone. According to their observations, this
microbe increases the percentage of acorn germination and enhances the growth of oak
seedlings. Similar results were obtained by Runov and Enikeeva (1955), Mishustin
(1950b) Smali (1951) and others.

Shtern (1940 a, b) tested radiation strains of Azotobacter on oat seedlings, grown in

vegetation containers. Certain radiation strains were more active than the initial culture.

Afrikyan (1954a) studied a large collection (more than 200 strains) of sporiferous
bacteria isolated from Armenian soils. The wheat seeds which were inoculated with
these cultures were allowed to germinate either in Koch dishes on cotton or in sand in
containers. The experiments showed that there are very few activators among the
sporiferous bacteria of the Bac. subtilis and Bac. mesentericus group. More often one
finds bacterial inhibitors in this group which suppress seed germination and the growth
of plants. These data are in agreement with our observations (see below).

Popova (1954) employed cultures of bacteria isolated from the rhizospere of grape
vines for the enhancement of the germination of grape seeds and grape stalks. Certain
species of Ps. sinuosa increased the percentage of germinating seeds to 80% while in
the control plants, only 10-12% of the seeds germinated by the 45th day. These bacteria
also enhanced the growth of seedlings and roots. In the control plants, the buds swelled
on the 16th day and, in those treated with bacteria, on the fourth day. The highest
activity was shown by Azotobacter chroococcum and the nonsporiferous bacterium
Bact. album, strains 2 and 3.

Pantosh (1955) found that nonsporiferous bacteria of the Pseudomonas and Bacterium
groups have an activating effect on the growth of the plant from the rhizosphere of
which they were isolated. In experiments performed in vegetation containers, on quartz
sand, the inoculation of the seeds with bacteria before sowing increased a crop of wheat
by 20-65%above that of the controls, as follows:

Inoculated with Weight of dry plants, g Total nitrogen content, mg

In the control (uninoculated) 9.9 376
Bacterium sp. 11.1 430.7
Ps. radiobact. 16.5 466.5
Flavobacterium solare 11.2 408.9
Bact. parvulum 14.3 430.3

Petrosyan (1956) studied the effect of bacterial activators on leguminous plants, on the
formation of their nodules and on their accumulation of nitrogen. The experiments were
performed in containers and in plots under field conditions. In the former case, the
following results were obtained:

Weight of Number of Total

plants, g nodules nitrogen, %
In experiments with vetch
Control (no bacteria added) 34 (100%) 110 4.86
Inoculated with root-nodule bacteria 45 (132%) 121 5.11
Inoculated with rood-nodule bacteria and
65 (190%) 161 5.29
activator
Inoculated with a pure culture of activator 56 (163%) 129 5.88

In experiments with lucerne

Control (no bacteria added) 34 (100%) 87 4.39
Inoculated with root-nodule bacteria 38 (109%) 129 4.96
Inoculated with rood-nodule bacteria and 53 (153%) 177 5.24
activator
Inoculated with a pure culture of activator 48 (139%) 155 5.07

Similar results were obtained in field experiments.

The increase in crops due to bacterial activators was as follows: after vetch, 172.6%--
control crop of 11.6 kg; after lucerne, 141.3%--control crop of 10.2 kg; after
Onobrychis, 150%--control crop of 8.4 kg from one plot.

In our experiments, we obtained similar results. In vegetation containers certain

bacterial activators (Ps. aurantiaca No 1, Pseudomonas No 145, Bacterium sp. No 160
produced an increase in leguminous plants as follows: clover, beans, lucerne and lupine,
by 30-80% above that of the control plants. In field experiments the crop increase of
these plants after inoculating them with the activators, was 24-30% above the control
levels. The number of nodules increased considerably when activators were employed.

On the roots of one plant, the following number of nodules were found: in control
plants without inoculation of bacteria, 8; on plants inoculated with root-nodule bacteria
of lucerne, 9-12; and after inoculation of bacterial activators (Ps. aurantiaca), 28. On
the roots of beans, the number of nodules was 6. 8 and 16; on the roots of lupine, 0.2,
0.5 and 1.2 (Krasil'nikov and Korenyako, 1945c).

Dry mass of clover

Inoculation of seeds
obtained, g
Uninoculated 26
Active initial culture 28
Avirulent strain I 35
Avirulent strain II 21
Avirulent strain A 40
Avirulent strain D 42
Avirulent strain B 26
Avirulent strain A1 36
Avirulent strain A3 26
Avirulent strain E 18

Bacterial activators stimulate the activity of root-nodule bacteria, enhancing the

formation of nodules and, by means of the latter, the growth of the plants. Activators
also act positively on leguminous plants without nodule bacteria directly stimulating
their growth by their metabolic products.

In one series of experiments, we employed avirulent strains of the root-nodule bacteria

of clover and vetch, obtained experimentally. The seeds were inoculated with cultures of
these bacteria and sown in sterile sand in glass containers. Crops were obtained after
two months.
Figure 83. Organotropic action of bacteria on lucerne:

Exp. A: a--stimulation of root growth with the simultaneous suppression of the growth
of the aerial parts; b--control plants; exp. B: a--stimulation of the growth of aerial parts;
b--control plants.

Figure 84. Effect of metabolic products of bacteria on the growth of Phycomyces

blakeslseanus:

1--control growth of the fungus in the absence of metabolites; 2--mass formation of

zygotes (dark spots) in the presence of metabolites of bacterial stimulators (Bacter. sp.
No 2); 3--abundant growth of aerial mycelium with conidia in the presence of
metabolites of bacterial activators (Azotobacter chroococcum).

Similar results were obtained in experiments with vetch. Using avirulent experimental
strains of root-nodule bacteria, Nos 1, A, D, and A1, the crop was considerably greater
(123-161%) than when the inoculation was performed with the initial virulent culture
(107-115%). In experiments with avirulent bacteria, no nodules were found on the roots
of the plants while when the initial culture was used for Inoculation, 9-58 nodules were
found on each plant.

Certain chemically pure substances obtained from cultures of actinomycetes and other
fungi, as for instance gibberellins and gibberellin-like substances, activate the growth of
plants (Figure 85).

Figure 85. Effect of antibiotics on the growth of corn:

1--seeds treated with antibiotic solution before sowing, 2--control, seeds treated with
water.

Dorosinskii and Lazarev (1949), Dorosinskii (1953), Lazarev and Dorosinskii (1953)
grew oats in sterile, well-washed, loamy soil in the presence of bacteria and in their
absence.

The plant crop in the absence of bacteria but with full mineral fertilization was, on the
average, 2.6 g; in vessels with bacteria, 7.1 g; in vessels with bacteria, but without a
mineral fertilizer, 6.5 g.

Fomin (1951) grow certain melon cultures, fruit, and wood varieties of plants, treating
them with preparations of Azotobacter, Psaudomonas, and "silicate" bacteria. After such
treatment, the crops of all these plants increased.

Certain microorganisms exert an organotropic action on plants: they activate the

growth of individual organs or tissues. For instance, Ps. tumefaciens stimulates the
multiplication of the cells of the root tissue or stem tissue in tomatoes, carrots, and other
plants, as a result of which swellings are formed, There are microbes which, by their
metabolic products only, activate to a considerable extent, the growth of roots or serial
parts.
 In our investigations, we have observed bacterial cultures which, under the conditions
of vegetation experiments (in sand) only stimulated the growth of roots, not affecting
the aerial parts at all. In other variations of the experiment some bacteria enhanced the
growth of the aerial parts without influencing the root system (Figures 83 and 85).
Observations were also made of bacterial cultures which activate the process of
fertilization in fungi (Figure 84) and yeasts, In Table 90, data are given on the activity of
the substances which stimulate this process.

Table 90
Activity of substances stimulating the sexual process of fungi and yeasts
(number of units in 1 g of dry substance)
Phycomyces
Substrate Zygosacchar. sp.
blakesleanus
Compost inoculated with bacteria 150 80
Soil humus 60 60
Az. chroococcum strain A 180 120
Ps. flourescens strain 30 150 30
Extract from an aspen raceme 260 80

Molliard (1903) observed the stimulation of the formation of apothecia in the fungus
Ascobolus under the influence of the bacteria within. Sartory (1916) obtained perithecia
in aspergilli only in those comes where the fungi grow together with the sporiferous
bacillus Bac. mesentericus.

Stimulation of the copulation of the fungus Phycomyces blakealesanus when grown

together with bacteria or their metabolic products (factor Z), extracted from agar-agar,
was observed by Robbins and Schmidt (1939, 1945). This "factor", according to their
data, is present in the tissues of many plants and is also formed by microbes.

Nickerson and Thimann (1941, 1943) found that a certain substance among the
metabolic products of the fungus Aspergillus niger enhances the copulation of the yeast
Zygosaccharomyces. The active principle of this stimulant is soluble in water and 90%
ethyl alcohol and consists of two substances: an acid closely related to glutamic acid,
and riboflavin, Burnett (1956) caused an increased copulation and the formation of
zygotes in mucor fungi by the addition of ß-carotene to the medium.

In our collection, there are actinomycete-antagonists, the metabolic products of which

have a stimulating effect on the fruiting processes in higher plants. One such culture
enhanced flowering and fruition in the cotton plant. Under conditions of the experiment
in growth containers, plants treated with the native liquid of the given actinomycete had
10-12 bolls, while the control plants had 5-6 bolls on each bush. Correspondingly, the
cotton crop approximately doubled in quantity. We treated plants with nonpurified
substances. It should be assumed that chemically purified preparations will have an
even stronger activating effect on plants.

In recent years great interest has arisen over gibberellins and, especially, gibberellic
acid, as a stimulant to the growth and development of plants. These substances are
obtained from the fungus Gibberella fujikoroi (a conidial stage of Fusarium
moniliforme). This fungus was first isolated in Japan by Kurozava, in 1926, from the
tissues of diseased rice. The rice disease caused by this fungus is quite widespread in
Japan, and expresses itself in an extreme elongation of the stems, in the yellowing of
leaves, and in the death of the plant. Yabuta isolated the active substance from a culture
of the fungus and he found that it stimulated the growth of many plants. Later, this
substance was isolated in the crystalline form and studied in greater detail. In England
and in America, three substances, gibberellin A1, gibberellin A2, and gibberellic acid
(gibberellin A3) were obtained from the culture fluid. The last substance is the most
active and is of the greatest interest. It was, therefore, more thoroughly studied and
more fully elucidated, Chemically defined, it is a dihydroxylacetone acid, a tetracyclic
compound, with the general formula C19H22O6.

The activating effect of gibberellic acid expresses itself in extremely small

concentrations. Only 0.01 microgram/ml is enough to stimulate the growth of peas; at
higher concentrations the stimulation of growth increases. The greatest effect is
observed at a concentration of 10 micrograms/ml. According to the data obtained by
Brian (1957), the increment in the growth of peas exceeds by 10 times that of the
control plants. Its height was 42 cm after treatment with gibberellic acid, while in the
control, nontreated plants, it was 7 cm. This stimulating effect was also observed by
Brian in experiments with wheat, but to a lesser degree than in the experiments with
peas. Gibberellic acid does not show an activating effect on the growth of roots, neither
in peas nor in wheat.

Of special interest is the effect of gibberellic acid on the growth of biennial plants:
cabbage, rape, carrots, sugar beets, etc. It is known that these plants give off flower
shoots and bear fruit during their second year of life; during their fire, year of growth
only a rosette of leaves and roots is formed. The flowering and formation of seeds may
also be achieved during the first year of growth, but only after yarovization [a Russian
term for vernalization; translator].

Experiments have shown that these plants form flower shoots and flowers and produce
seed during their first year of growth after being treated with gibberellic acid, without
previous yarovization, This acid has the same effect as the one obtained by yarovization.
The enhancement of flowering and fruition is observed in longday plants. Lang (1956)
obtained flowers and fruit on henbane (Hyoscyamus niger) during the first year of its
growth. After the introduction of 300 micrograms/ml in the tissue, the plant soon
formed flowering, or main shoots, on which flowers developed.

During the last two or three years, a vast amount of material has accumulated showing
the strong activating effect of gibberellic acid on the growth and flowering of various
plants. The strongest effect is seen with the long-day plants.

It should be noted that the stimulating effect of gibberellic acid was so far only
obtained under experimental conditions in a hothouse. Under field conditions in soil,
there have either been no results, or a weak stimulation of only certain plants has been
observed. A small increase of crops, within the range of 11-25 per cent has been
observed in meadow grasses.
 In plants which react to gibberellic acids, one often observes that there is a decrease in
the amount of chlorophyll, that the leaves have a yellowish tint, that their total nitrogen
content decreases, and that in the tobacco leaves the percentage of nicotine decreases
and , in rice, a decrease in the amount of sugars. It is assumed that this is caused by a
lack of nutrition. With appropriate fertilization this has not been observed.

The mechanism of the action of gibberellic acid is not clear; however, all the
investigators note that it differs from that of auxins. The latter affects the processes of
growth in a different way. These substances also differ in their chemical composition.

The data given in this chapter show that there are species of microorganisms which
form very active substances, which stimulate the growth and certain processes and
functions in plants. Gibberellic acid is the first metabolic product of microorganisms
obtained in a chemically pure form. It should be assumed that in the near future many
other substances will be obtained which possess the capacity to activate the growth and
development of plants. Similarly, as takes place among microbial antagonists. The
stimulating substances of the microbial activators belong to various classes of
compounds. Here, a great research study lies ahead in the isolation of these substances
and in the study of their nature.

One must note that among the antibiotic substances and activating compounds, there is
often much in common in the way they act on organisms. Many antibiotics show a
stimulating effect on the growth of plants and animals; they enhance the increment of
live weight of the latter, and sometimes enhance the process of fruition in both higher
and lower plants. On the other hand, activating substances quite often possess clearly
expressed antimicrobial properties.

Distribution of microbial activators in the soil

Very little is known on the distribution and growth of microbial activators in soil. At
the same time, in daily laboratory practice, one very often encounters these bacteria, We
have in mind the auxoautotrophs, which have been mentioned before. These organisms
synthesize all the substances necessary for growth and development, and, therefore,
grow well on simple synthetic media. For the purpose of their study, we used the
following medium:

Twice distilled water 1,000 mm

KNO3 1.0 g
KH2PO4 1.0 g
MgSO4 . 7 H20 0.2 g
CaCl2 0.1 g
NaCl 0.1 g
FeCl3 traces
Glucose 20.0 g
 There were no vitamins or auxins in the medium.

The total number of auxoautotrophs growing on this medium was quite large. We
counted from several tens of thousands to many hundreds of millions of bacteria in one
gram of soil.

The number of auxoautotrophic bacteria changes, depending on the properties of the

soil and climatic conditions. As a rule, their total number is greater in fertile soils than in
nonfertile ones. When the auxoautotrophs from the chernozem soils of Moldavia,
Crimea, and Kuban are plated on an agar medium, 30 to 150 million appear in one
gram. In the turfy podsol soils of the nonchernozem belt of the Moscow and Leningrad
Oblast's and of Latvia, the number of these organisms varies within the range of
100,000 to 4.5 million per gram. In the soils of the Kola Peninsula (primary soils, loose
calcareous soils, etc) there are 5,000 to 30,000 cells in one gram,

In cultivated soils their number is greater than in noncultivated soils, and in gardens it
is greater than in fields (Table 91).

Table 91
Quantitative ratio of auxoautotrophic and auxoheterotrophic types in soils
(in thousands per one gram of soil)
On a meat-
On a vitamin-
Soil and region peptone agar
free medium
MPA
Podsol. Arctic Circle. Virgin soil 5 3.5
Podsol. Arctic Circle. Cultivated soil 300 360
Podsol. Moscow Oblast'. Virgin soil 200 180
Podsol. Moscow Oblast'. Cultivated soil 2,500 2,000
Podsol. Moscow Oblast'. Garden soil 45,000 60,000
Krasnozem. Caucasus. Cultivated soil 3,500 1,500
Podsol. Latvia. Virgin soil 150 100
Podsol. Latvia. Cultivated soil 1,800 1,500
Chernozem. Moldavia. Virgin soil 1,200 800
Chernozem. Moldavia. Cultivated soil 30,000 50,000
Chernozem. Crimea. Virgin soil 20,000 35,000
Chernozem. Crimea. Cultivated soil 160,000 200,000
Chernozem. Kuban'. Cultivated soil 220,000 280,000
Chernozem. Kuban'. Cultivated soil 450,000 600,000

Comparing the data on the number of bacteria growing on a synthetic vitamin-free

medium and on an ordinary protein medium, one can observe that the number of
auxoautotrophs and auxoheterotrophs is almost the same. In humus soil the ratio
between these two categories is approximately 1:1. In certain cases, there are even more
auxoautotrophs (see above).

According to Schmidt and Starkey (1951), about 30 per cent of soil bacteria synthesize
biotic substances and excrete them into the soil.

Lochhead and Chase (1943) found from 10-14 per cent of auxoautotrophs among the
microflora of soil. These authors divide soil microbes into seven groups, according to
their ability to grow on vitamin-free media with the addition of a few auxiliary
substances. To the first group belong the microbes growing on media completely devoid
of vitamins. In the second group are included organisms growing on a vitamin-free
medium with the addition of a few amino acids (cysteine, alanine, proline, asparagine,
arginine, leucine, glycine, lysine, etc). To the third group the authors relate those
microbes which require for growth certain vitamins: pantothenic and nicotinic acids,
thiamine, riboflavin or others. The fourth group includes organisms growing on a
medium to which both amino acids and vitamins were added. Those microorganisms
growing on a basal vitamin-free medium with an admixture of yeast extract comprise
the fifth group.

A basal vitamin-free medium with an admixture of soil extract reveals the bacteria of
the sixth group. This medium, with a small admixture of yeast and soil extracts, is
suitable for the bacteria of the seventh group, the most numerous one. Microbes of the
first group comprise approximately 10-14 per cent of all soil bacteria; the second group,
10 per cent; the third, 12-14 per cent; the fourth 16-17 per cent; the fifth, 18-20 per cent;
the sixth, 3-7 per cent; and finally, the seventh, 40-50 per cent of all soil bacteria. This
subdivision of bacteria is quite arbitrary.

A great number of vitamin-producing bacteria are found in the rhizosphere of plants.

According to our calculations, they comprise 40-80 per cent of all soil bacteria and their
number varies depending on the species of the plant, the stage of its growth, and upon
external conditions. In analyzing the rhizosphere of wheat and clover, growing in the
fields of the Moscow district Dolgoprudnoe), we obtained the data given in Table 92. In
the root zone of these two plants and in all other plants studied by us, the number of
auxoautotrophs was no smaller and often larger than the number of auxoheterotrophs.

Table 92
Number of auxoautotrophic bacteria in the rhizosphere of plants
(in thousands per gram of soil)
Autxoauto- Auxohetero- Auxoauto- Auxohetero-
trophs in trophs in trophs in the trophs iin the
Plant Soil
early phase early phase fruit-bearing fruit-bearing
of growth of growth phase phase
Wheat Rhizosphere 800,000 100,000 300,000 500,000
Wheat Control 5,000 3,500 3,000 3,000
Clover Rhizosphere 1,500,000 1,800,000 1,000,000 1,600,000
Clover Control 40,000 30,000 20,000 20,000

West and Lochhead (1940a, b), and Wallace and Lochhead (1949), in their detailed
studies also found that auxoautotrophs prevailed in the rhizosphere of flax and other
plants. The same was noted by Katznelson and Richardson (1943), Stolp (1952) and
others (Table 93).

Table 93
Quantitative ratios of auxoautotrophs in the rhizosphere and outside it, by percentage
(according to Wallace and Lochhead, 1949)
Plant and soil Early growth stage Flowering stage
Wheat / Rhizosphere 42.3 48.5
Wheat / Outside the rhizosphere 14.4 9.7
Oats / Rhizosphere 44.1 47.4
Oats / Outside the rhizosphere 14.8 9.7
Clover / Rhizosphere 55.5 42.8
Clover / Outside the rhizosphere 9.3 2.4
Lucerne / Rhizosphere 40.5 37.2
Lucerne / Outside the rhizosphere 9.3 2.4
Flax / Rhizosphere 35.8 32.9
Flax / Ouitside the rhizosphere 9.7 8.6
Timothy grass / Rhizosphere 25.3 15.4
Timothy grass / Outside the rhizosphere 9.3 2.4

As is well known, the ability of microorganisms to produce biotic substances is, in

most cases, not connected with their taxonomic division into groups. Auxoautotrophs
and heteroauxotrophs are found among various microbial groups: among sporiferous
and nonsporiferous bacteria, micrococci, mycobacteria, actinomycetes, fungi, etc. The
largest percentage is encountered among the bacteria of the genus Pseudomonas.
According to our calculations, the number of these bacteria reaches 0-60 per cent of all
the bacteria growing on an agar medium devoid of vitamins. A smaller, but quite a high
percentage (30-40 per cent) is encountered among the genus Bacterium and
mycobacteria. Considerably rarer are the auxoautotrophs among sporeforming bacteria
of the genus Bacillus (Table 94).

Table 94
Number of auxoautotrophs of different groups of microorganisms,
living in the rhizosphere of plants and outside the root zone
(by percentage)
In the
Outside the In the rhizosphere,
Bacteria rhizosphere,
rhizosphere wheat
clover
Pseudomonas 30.0 40.0 46.0
Bacterium 40.0 34.0 23.4
Mycobacterium 18.0 25.0 30.0
Bacillus 6.0 0.3 0.1

Oligonitrophiles are active producers of biotic substances. These organisms are widely
encountered in soils and their characteristic feature is that they grow abundantly in a
vitamin-free and nitrogen-free medium. Evidently, they all belong to the
auxoautotrophs.

Among the actinomycetes one rarely finds cultures which will not grow on Chapek' s
synthetic medium. They grow well on a vitamin-free medium.

Yeasts of the genera Torula, Mycotorula, and some others are widespread in the soil.
These organisms are powerful producers of vitamins and various other biotic
substances. Therefore, they grow well on mineral, vitamin-free media, forming large
slimy or semislimy colonies. Some of them, as, for instance, Torulopsis pulcherrima are
widespread in the soil and grow abundantly on the nitrogen-free medium of Ashby,
forming large colonies similar to Azotobacter colonies. They are of special interest as
activators of plant growth and of the life processes of microorganisms.

Part IV, continued:

Microbial inhibitors and their action on plants

These data show how large and versatile is the microflora which produces biotic
substances. With the aid of these substances, the microorganisms growing in the soil
activate the growth, nourishment, and many other vital processes of both higher and
lower organisms.

In soils, as well as in other natural substrate, there are microorganism-inhibitors.

which, in course of their metabolic activity, suppress the growth and development of
higher and lower plants. They form special substances which are toxic to plant tissues
and organs. Toxins, or phytotoxins formed by phytopathogenic fungi and bacteria, were
studied long ago by numerous investigators (see Kuprevich, 1947; Sukhorukov, 1952;
Bilai, 1953, Gorlenko, 1953; Goiman, 1954). However, the question of whether these
organisms produce toxic substances directly in soil remained unsolved in the literature.
It is known that many species of fungi and bacteria form toxins which act on animal
organisms. Growing on food products, fodder, and on various plant residues, they
excrete toxic substances. Upon feeding these products to animals, one often observes a
strong case of poisoning (Pidoplichko, 1953).

Investigations show that microbial inhibitors may poison plants with their toxins under
conditions of their natural growth in soil, if favorable conditions for such growth are
formed. They suppress germination of seeds, the growth of sprouts and plant growth in
general and decrease the total crop. Consequently, when there is a massive growth of
these organisms, they may become an important factor in determining the fertility of
soil and the crop yield of plants.

The suppressing action of microbial inhibitors is also manifested in the growth of

lower plants--fungi, bacteria, algae and others. In such cases, the microbes are called
antagonists.
 Microbial inhibitors are found among various groups of lower forms: bacteria, fungi,
and actinomycetes. Greig-Smith (1911) established the fact that toxic substances formed
by certain bacteria, suppress the growth of plants. Ressel (1933) ascribed great
importance to Protozoa, which consume microbial cells. Hutchinson and Thaysen
(1918), Lewis (1920), and Laudenberger (1952) noticed in certain nonsporiferous
bacteria of the genus Pseudomonas the ability to synthesize potent toxic substances.
Johnson and Murwin (1931), and later Braun (1950), discovered this ability in
Pseudomonas tabaci, the causative agent of tobacco disease. The ability to form toxic
substances was also found in other bacterial groups.

Among the group of nonsporiferous bacterial inhibitors, representatives of the genera

Bacterium and Pseudomonas, are comparatively often encountered and lose often those
of the genus Rhizobium. They are often found in the rhizosphere of vegetative plants.
We studied more than 300 cultures of these organisms isolated at different times from
different soils, from the chestnut soils of the Trans-Volga region, the serozem soils of
Central Asia, and the podsol soils of the Moscow and other regions.

Of this number of cultures which were studied, about 100 suppress to a greater or
smaller degree the growth of plants and the germination of seeds, Strongly expressed
herbicidal properties were possessed by certain strains of Ps. flourescens, Ps. pyocyanea
and Bacterium sp. They completely or almost completely inhibited the germination of
needs of clover, vetch, and wheat (Figure 86). The seeds merely sprouted and died, or
did not show any signs of germination.

Figure 86. Suppression of the germination process of clover seeds by nonsporiferous

bacterial inhibitors, Posudomonas sp.

a--control; b--in the presence of bacterial inhibitors.

The toxic properties of many sporiferous bacteria are sharply expressed, We studied
more than 350 cultures, isolated from various soils of the Soviet Union. The bacteria
were grown in liquid nutrient media. The seeds were treated by soaking them for several
hours in the culture fluid. Seeds of plants treated with culture fluid were germinated on
cotton or on paper which had been wetted with water.
 The toxic or herbicidal effect of the bacterial fluid revealed itself in the suppression of
growth and the lowering of the percentage of germinating seeds, Analyses have shown
that approximately 20-30 per cent of the cultures investigated possessed inhibitory
proportion. Among the bacteria isolated from turfy podsol soils, the number of
inhibitors was large (about 34-45 per cent).

The nature and strength of their effect vary in different cultures. Some organisms
completely or almost completely inhibit the germination of seeds (Figure 87), others are
less inhibitory, and still others do not show any inhibitory effect whatsoever.

Figure 87. Effect of sporiferous bacterial inhibitors on the germination of plant seeds.
The seeds were soaked in the bacteria-culture fluid and germinated on cotton or on
paper wetted with water:

A--effect of Bac. mesentericus (strain 50) on germination of wheat seeds: 1--control,

seeds soaked in water; 2--seeds soaked in culture fluid; B--effect of Bac. mesentericus
(strain 67) on the germination of seeds of peas: 3--seeds soaked in culture fluid; 4--
control, seeds soaked in water.

The species of the inhibitors studied by us mainly belonged to Bac. mesentericus and
Bac. subtilis.

The capacity to suppress the germination of seeds and the growth of seedlings is
revealed in various degrees among strains belonging to the same species. Among
cultures of the bacterium Bac. messentericus, we found more than 180 strains isolated
from various soils, including 100 strains from the podsol soil of the Chashnikovo
Experimental Station. Among these were some very strong inhibitors, while others did
not inhibit plants growth at all. Some of them inhibited the germination of wheat seeds,
others those of peas, vetch or clover, while some of thorn inhibited the germination of
wheat, peas, vetch and clover seeds. In Table 95 data are presented from an experiment
with vetch and clover.

Table 95
Effect of metabolic products of bacteria on the growth of plants
(calculated for the 30th day of growth, in cm)
Clover: height Clover: length Vetch: height Vetch: length
Bacterial cultures
of shoots of roots of shoots of roots
Control 5.0 4.0 24.5 12.0
Bac. subtilis strain 7 4.5 0 26.0 6.0
Bac. subtilis strain 15 5.5 0.5 22.0 3.5
Bac. brevis. strain 3 4.8 0-0.2 25.0 2.5
Bac. mesentericus 5.2 0 25.5 1.0

Among sporiferous and nonsporiferous bacteria, there are sometimes strains

possessing an organotropic or selective herbicidal action. They either suppress the
growth of only this root system or of only the aerial parts. We found cultures which
completely inhibited the growth of the roots of vetch and wheat. The seeds sprouted
without the formation of roots, while the latter were very much reduced (Figure 88).
The aerial part developed more or less normally as long as the seeds contained nutrient
substances.

Figure 88. Suppression of growth of wheat roots by bacterial inhibitors:

a--experiment; b--control.

Some bacterial strains in our collection (three nonsporiferous and four sporiferous
strains) inhibited the growth of the aerial parts, but did not affect the root system. The
needs germinated a root, while the aerial part was strongly reduced (Figure 89).

Figure 89. Inhibition of growth parts of vetch by cultures of bacterial inhibitors:

a--experimental, b--control plant.

Certain strains of bacteria suppress the sporulation process of lower organisms, the
formation of zygotes in phycomycetes and the formation of spores in yeasts
(Krasil'nikov, 1947 a). It is possible that there are microbes which inhibit the fruiting
process of higher plants as well.

In our collection of actinomycetes there are strains which cause chlorosis of higher
plants by the action of their metabolic products. Chlorosis appeared in corn and wheat
after treating the seeds before sowing with a culture fluid of certain species of
actinomycetes and, even more markedly, with purified preparations of antibiotics. If the
seeds of these plants are kept in a solution of an antibiotic for two, to four hours before
sowing, the seedlings are completely colorless, without the slightest sign of the
formation of chlorophyll. The growth of much plants is suppressed and, soon ceases
altogether. In some cases the plants, recover, become green, and continue to grow more
or less normally,

If the seeds are treated with weaker solutions of the antibiotic, one obtains seedlings
which are slightly green and somewhat etiolated. Strongly etiolated plants are obtained
upon the treatment of seeds with streptomycin. Soaking seeds for two hours, in a
solution of one microgram per ml causes the complete etiolation of the seedlings. The
latter do not become green for a period of 15-30 days and finally die. A suppression of
chlorophyll synthesis is caused by aureomycin, terramycin and other antibiotics.

We obtained the etiolation of duckweed by growing it in a nutrient solution to which

an antibiotic had been added. Depending on the concentration of the antibiotic, the
growth of the plants was inhibited to a greater or smaller degree. The extent of the
appearance of the green color also varies, from slight chlorosis to full colorlessness.

Certain toxins of microbial origin cause the phenomenon of chlorosis in grapevines,

According to our observations, this phenomenon may be due to fungi of the genus
Fusarium. We found certain strains, the toxins of which caused the etiolation of shoots,
of cuttings, and grape stock, when treated before planting in the soil. The plants that
grew from them had light green leaves with a yellowish hue, their development was
slow, and other deviations were observed which are characteristic of chlorosis of grape
vines (Krasil'nikov and Kublitskaya, 1956).

This picture of the etiolation of cuttings was observed by us after the treatment of the
vine with antibiotics of actinomycete origin. Certain strains of gray and pigmented
actinomycetes synthesized substances which inhibit the formation of chlorophyll in the
leaves of grapevines. Cuttings, when immersed with their basal ends in the crude fluid
culture and subsequently planted in the soil developed and showed obvious signs of
etiolation.

The inhibition by antibiotics of the formation of chlorophyll in plants has been

mentioned by certain other investigators. Provasoli, Huntner, and Schatz (1948)
obtained colorless cultures of Euglena sp. under the influence of streptomycin. The
antibiotic was added to the nutrient solution in small quantities; under its influence the
chloroplast of the cells was destroyed, as a result of which completely etiolated forms of
organisms were obtained.

The phenomenon of chlorosis as an effect of streptomycin was observed in cereals

(wheat, corn, etc) by Von Euler (1947) and Hagborn (1956). They wetted the seeds of
plants in the antibiotic solution and planted them in the soil. The seedlings were devoid
of green color.

Berezova and Sudakova found that the death of the growing tip of flax is not
connected with boron starvation, but is the result of poisoning by toxins formed by
bacteria.

Kugushova has shown that on the roots of lucerne, bacteria may grow which, by their
excretions, cause the failing off of the buttons (according to Berezova, 1953 a).

Inhibitors, suppressing the growth of plants and the germination of seeds, are
encountered in great numbers among actinomycetes. In this group of microorganisms,
cultures with strong herbicidal properties are most often found among the orange A.
aurantiacus, among the gray A. griseus, and among other species and groups (Table 96).
 Table 96
Effect of the culture fluid of actinomycetes on the germination of plant seeds
Number of germinated seeds by % of control------> beans corn clover lucerne wheat
A. aurantiacus, strain 1149 86 60 66 77 12
A. aurantiacus, strain 1306 44 60 50 88 12
A. griseus, strain 2283 142 60 100 100 100
A. griseus, strain 293 86 120 83 111 100
A. globisporus, strain 070 114 80 50 100 87
Control 100 100 100 100 100

Experiments with seedlings have shown a more or less similar picture. Some cultures
of actinomycetes strongly suppress growth, while others only slightly or not at all (Table
97).

Table 97
Effect of filtrates of actinomycetes on plant seedlings
(in length of plant parts in cm)
Wheat, Wheat, Corn, Corn, Beans, Beans,
Actinomycetes
rootlets sprouts rootlets sprouts rootlets sprouts
A. aurantiacus, strain 1149 1.5 2.0 12.0 7.0 15.0 13.7
A. aurantiacus, strain 1306 1.5 7.5 0.7 3.5 1.7 3.8
A. griseus, strain 2241 12.4 10.5 13.7 15.0 11.0 8.8
Control 14.0 15.0 15.0 12.0 18.5 14.0

Toxic substances of actinomycetes and other microorganisms exert a suppressing

effect on single isolated organs or parts of plants; on leaves, cuttings, etc. If one
immerses the cuttings, or cuts off the leaves, then, after a certain period of time, they
wither and die. By the speed of the withering and death of these parts one may judge the
strength of the action of the toxic substances.

In our experiments we used cuttings of various plants, of beans, peas and corn, and
branches of lemon, apple, pear, and apricot trees, etc.

If one puts on the surface of an uncut leaf a piece of cotton which has been wetted with
toxin, after a few hours spots appear of a necrotic nature. The stronger the poison, the
more sharply the necrotic spots on the leaf are expressed. This method was used by us
in testing the toxic substances formed by microorganisms.

Among the inhibiting factors of great importance are the phages: bacteriophages and
actinophages. The studies of Rautenshtein (1955), Khavina (1954), and certain others
show that these agents are widely distributed in soils, where they are detected in
considerable numbers. There is reason to believe that they suppress and lyse cells of
bacteria or actinomycetes as readily as under conditions of pure cultures.

For example. root-nodule bacteria become inactive when phages multiply abundantly
in the soil. Under conditions of the experiment, they multiply to a considerable extent.
We have counted tens and even hundreds of thousands in one gram of soil. According to
Demolon and Dunez (1934), phages of root-nodule bacteria of clover and lucerne, under
certain conditions, saturate the soil to such an extent that the soil becomes much less
fertile for these plants, root-nodule bacteria do not develop in it, and there is only a
slight or no formation of nodules on their roots, and when they do develop they have an
abnormal appearance. The authors are of the opinion that the observed clover-lucerne
soil exhaustion is caused by the accumulation of phages. According to certain data,
phages penetrate the plant, and, by interfering with plant metabolism, lower crop
production (Vandecaveye and others, 1940).

There is data in the literature on the formation of toxic substances by fungi. Leng
(1949) has shown the poisoning effect of the Penicillium fungi on the seedlings and of
cereals. The most active inhibitors in these experiments were P. notatum, and P.
oxalicum. Monnaci and Torini (1932) and Diachum (1934) note the formation of toxins
by fungi, which act on cereals under conditions of their growth in soil.

Producers of toxic substances are known among various groups of soil microflora. An
important place is occupied by representatives of the genus Fusarium. The substances
formed by them were obtained in a chemically pure form having a known structure; for
example, lateritin, C6H46O7N2 ; avenacein, C25H44O7N2; fructigenin, C26H44O7N2;
sambucynin, C24H42O7N2, and enniatins, lycomarasmin, yavanicin, etc.

These substances act differently on plants and animals. Some of them are specific
(Goiman, 1954).

Fusaria are very widespread in nature. The probably play an important role in the
toxicoses of soils. Their inhibitory effect on the growth of plants was observed by many
authors (Rehm, 1953; Laundoldt, 1952; Sukhorukov, 1952). The significance of these
fungi for the fertility of soils is not only determined by their ability to synthesize toxins
and excrete them into the soil but also by their phytopathogenic properties.

Bilai (1955) described in his monograph many strains of the genus Fusarium which
have a deleterious effect on the germination of seeds and on the growth of seedlings of
rye, oats, and barley. The products of their metabolism, obtained in the form of filtrates,
were tested under various conditions. The results of the author's experiments are given
in Table 98.

Table 98
Effect of filtrates of Fusarium cultures on the germination of plant seeds
(in length of plant parts in cm)
Rye, Rye, Barley, Barley,
Fungal culture
rootlets sprouts rootlets sprouts
Control 21.5 4.25 29.8 3.6
Fus. poal., strain 2 3.8 1.9 -- --
Fus. poal., strain 5 8.3 2.6 16.0 3.6
Fus. poal., strain 9 11.7 2.5 11.8 2.3
Fus. poal., strain 41 2.4 1.6 -- --
Fus. poal., strain 45 15.0 5.4 8.4 1.2
Fus. sporitrichioides, strain 28 6.1 1.4 18.4 2.1
Fus. sporitrichioides, strain 30 11.3 3.2 6.0 1.5
Fus. sporitrichioides, strain 51 15.3 6.3 11.2 1.5

As can be seen from the table, the filtrates of some strains affect the seedlings of rye,
while others act predominantly on the growth of barley. Certain strains suppress the
growth of rye and wheat to the same extent as that of barley or oats.

Klechetov (192 6) in studying the phenomenon of the flax exhaustion of soils found
the growth of the fungi Fusarium, Thielaviopsis basicola, Cladosporium herbarum,
Alternaria, and Macrosporium in these soils; these fungi, according to the author, form
toxic substances and are the reason for the death of the sown flax.

A considerable role in the exhaustion of soils and in the lowering of plant yields is
attributed in the literature to the fungi of the genus Fusarium. Kvashina (1938),
Kurtesova (1940), and Ioffe (1950).

Kublitskaya (1955) studied the degree of the distribution of fungi of the genus
Fusarium in the soils of Central Asia (Uzbek SSR) under grapes. She isolated 52
cultures and many of them proved to be toxic for grapevines, causing poisoning and
death to the cuttings and stock under the conditions of growth in soil. Certain strains
caused chlorosis under experimental conditions.

Strongly expressed herbicidal properties are exhibited by fungi of the genus Pythium.
According to Likais (1952), Pythium debaryanum forms toxins in the soil which inhibit
the root systems of plants.

Mirchink (1950) studied a large collection of fungi isolated from turfy podsol soils of
the Moscow district and found among them many toxigenic forms. The most toxic and
the most widespread fungi in these soils are representatives of the genus Penicillium
and, secondly, Fusarium and Trichoderma. Fungi of the genus Trichoderma (T.
lignorum) and certain representatives of the genus Fusarium strongly suppress the
germination of wheat seeds, as a result of which the number of germinating seeds
decreases by 68 per cent and more. The length of sprouts in the presence of the
metabolic products of Trichoderma is 3.5 cm; in the presence of the fungus Fusarium,
4.0 cm; and in the control, 4.6 cm. The Penicillia inhibitors are often found in the turfy
podsol soil in a great number of species. Some of them are very toxic for wheat, which
can be seen in Table 99 and in the photograph (Figure 90).

Table 99
Toxic effect of fungi of the genus Penicillium on wheat seeds
Per cent of Mean length of
Fungi
germinated seeds sprouts, cm
Control (nutrient medium) 100 4.6
Control (water) 100 4.6
P. cyclopium 0 --
P. paxilli 54 2.6
P. ochro-chloron 74 1.5
P. martensii 74 3.0
P. nigricans, strain II/14 100 1.0
P. nigricans, strain II/35 87 0.6
P. nigricans, strain VIII/8 90 1.0

Figure 90. Effect of the culture fluid of the fungus Penicillium nigricans on the
germination of wheat seeds:

a--control; b--treated seeds.

Active toxin producers in soil are fungi of the genera Trichoderma, Trichothecium,
Botrytis, and others. From cultures of Helminthosporium (H. victoriae), the toxin
victorin was isolated, which inhibits the growth of roots and seedlings of oats at a
dilution of 1:1,000,000. This substance is formed by the fungus directly in the soil
(Weeler Luke, 1954; Tyler, 1948). Toxic substances harmful to plants were found
among the representatives of Verticillium. The most well studied among them is V.
alboatrum. Its toxic substance was found by Bewley (1922), It causes the withering of
tomatoes, cotton, tobacco, and other plants. Green (1954) discovered two substances in
this fungus-- a protein and a polysaccharide. The former is excreted into the medium
and the latter enters the tissues of the plants, The poisoning effect of this fungus was
also noted by Sukhorukov (1952) and others,
 Among members of the genus Trichothecium were found the toxic substances
trichothecin and others, which inhibit plants and certain microbes, Similar substances
were found in Deuterophoma tracheiphilus, causing "malsecco" in citrus plants, They
were also encountered in many other fungi (Hossayon, 1953; Freeman and Morrison,
1949, Gelman 1954).

It is obvious that the importance of microbial inhibitors in soil toxicosis will be mainly
determined by the degree of their growth and activity.

The distribution of microbial inhibitors and their accumulation in the soil has been but
occasionally studied; as were microbial activators. Monnaci and Torni (1932) found
about 60 per cent of the soil fungi isolated and investigated by them, to be inhibitors.

According to our data, there are a great number of inhibitors among the fungi, bacteria,
and actinomycetes in soils. Out of 1,500 cultures of actinomycetes, more than 200
inhibited, to a larger or smaller degree, the germination of beet or wheat seeds and 16
strains completely suppressed their germination; 21 cultures strongly suppressed and 58
weakly suppressed the growth of clover and lucerne, The total number of inhibitors
among actinomycetes is comparatively small, on the average 5-15 per cent,

One finds inhibitors among sporiferous bacteria considerably more often, Out of 560
strains studied, belonging mainly to three or four species, Bac. mesentericus, Bac.
subtilis, Bac. cereus, and Bac. brevis. 178 strongly suppressed the germination of clover
seeds, more than 200 cultures suppressed to some degree the germination of peas.
According to our data, there are about 40 per cent inhibitors among the sporiferous
bacteria of Bac. mesentericus and Bac. subtilis isolated from the turfy podsol soils.

Inhibitors among nonsporiferous bacteria are encountered much less frequently than
among sporiferous bacteria, According to our calculations, their number can be
expressed in a tenth part of one per cent. Some species of the genus Bacterium and
Pseudomonas possess, however, strongly expressed toxic properties in relation to plants
and microorganisms.

It should be noted that certain microorganisms among bacteria and fungi react to toxic
substances in the same way as do higher plants, which enables us to use them as test
organisms in the screening for and the study of phytotoxins. Microbial tests have a
number of advantages, With them one can more quickly determine and solve a number
of problems related to the toxicosis of the soil and the poisoning of plants, In mass
studies we often use both tests; the microbiological and the plant test.

We carried out the quantitative evaluation of microbial inhibitors in different soils, but
went into greater detail in the turfy podsol soils of the Moscow Oblast', the Kola
Peninsula, and in other regions of the USSR. Virgin and cultivated soils, forest and
swampy soils, meadows, ate were investigated,

We counted from 5,000 to 450,000 inhibitors in one gram of soil depending on the
properties of the latter (Table 100). In slightly cultivated soils, the absolute number of
inhibitors is smaller, but its percentage may be higher than in wellcultivated soils.
 Table 100
Number of inhibitors in podsol soils of Moscow area
(Number of cells in 1 g of soil)
Actino-
Actino- Bacteria Fungi
Bacteria Fungi mycetes
mycetes inhibiting inhibiting
Soil inhibiting inhibiting inhibiting
inhibiting beet beet
azotobacter azotobacter beet
azotobacter seedlings seedlings
seedlings
Dolgoprodnoe
Virgin soil 15,000 23,000 1,300 8,000 3,000 500
Plowed fields 45,000 17,000 2,000 15,000 7,000 1,000

Agricultural
Academy
Timiryazev
Forest 40,000 80,000 17,000 25,000 10,000 4,000
Virgin soil 10,000 32,000 1,500 10,000 3,000 500
Plowed fields 120,000 150,000 2,300 50,000 35,000 3,000

Chashnikovo
Forest 120,000 82,000 12,000 20,000 12,000 7,000
Virgin soil 40,000 16,000 1,600 10,000 5,000 1,000
Plowed fields 450,000 160,000 1,400 150,000 60,000 500

Inhibitors which suppress the growth of Azotobacter in podsol soils are much more
numerous than microbes which suppress plant growth. Mirchink (1958) studied the
fungal flora of soils of the experimental station Chashnikovo (Moscow Oblast') and
found that 11-38% were inhibitors which suppress plant growth. They were distributed
in the following manner: in forest soil--13%, in glades--11%, in cultivated soils, 15-38%
of the total microflora, detected by by existing methods (Table 101).

Table 101
Number of fungi in podsol soils
(thousands in 1 g of soil)
Soils Total Inhibitors, %
Control soils without fertilizers 60 32
Fertilized with mineral nitrogen 138 38
Calcium-containing fertilizers + manure 36 24
Calcium-containing fertilizers + manure + P.K. 18 15

As can be seen from the data given, the greatest number of inhibitors was found in
soils cultivated to a limited extent. Mineral fertilizers do not diminish but, on the
contrary, they noticeably increase the content of inhibitors.
 It was experimentally established that microbial inhibitors form toxic substances
directly in the soil in which they grow.

If these organisms are introduced into nontoxic or inactivated soil and the soil is
incubated under certain conditions of humidity and temperature, then after a certain
time it will become toxic for these or other plants or for certain species of
microorganisms, depending on the peculiarities of the inhibitor.

Rybalkina (1938 a) observed the appearance of toxicosis in flax-exhausted soil upon

growth of the fungus (Fusarium lini).

Mirchink (1956) incubated soil (podsol) with fungi-inhibitors and she observed the
appearance of toxicosis. In soils in which the fungus Penicillium cyclopium grew
abundantly if artificially introduced, seeds of wheat did not germinate at all or
germinated in small numbers (Figure 91). Other species of fungi isolated from podsol
soils also poisoned the soil but to a lesser degree. On such soils germinating wheat
seedlings constituted 15-60% of the number of seedlings in normal control soil.

Figure 91. Poisoning of soil by cultures of fungi upon artificial infection. Germination
of wheat seeds:

a--in control (noninfected) soil; b--in soil in which Penicillium nigricans grew; c--in
soil infected with Penicillium cyclopium.

Clover-exhausted soil inactivated by heating regains its toxicity by growing the

appropriate microbial inhibitors in it. In such soil with regenerated toxicity, clover and
root nodule bacteria grew much more poorly than in normal soil. There was either no
nodule formation on the root of clover or it was considerably suppressed (Table 102).
Root-nodule bacteria in such soil became avirulent and lost the capacity to penetrate the
roots and form nodules in them. Cultures of some fungi of the genera Penicillium,
Fusarium, Trichoderma and some sporeforming bacteria, when growing abundantly in
inactivated forest or field soil restored the soil's original toxicity in relation to wheat and
Azotobacter (Table 103).

Table 102
Growth of clover on soil with restored toxicity
Number of
Number of
Number of nodules per
Expermiental conditions sprouts on the
sproutings plant
30th day
(average)
Inactivated soil not infected with inhibitors
46 46 23
(control)
Inactivated soil, infected with inhibitors:
Ps. pyocyanea 32 26 0.05
Ps. tumefaciens 39 22 0.0
Fusarium sp. 41 31 0.5
Mixture of all bacteria 30 19 0.0

Note: Each vessel contained 50 seeds. Inoculation was performed with active cultures of
Rhizobium trifolii.

Table 103
Accumulation of toxic substances in podsol soil an a result of growth of inhibitors
Length of Length of Survival of
Name of inhibitor wheat wheat Azotobacter
rootlets, cm sprouts, cm cells, hours
Control: inactivated soil (not infected) 12 8 240
Infected with:
Bacillus strain 12 3 5 6
Bacillus strain 23 6 2 12
Bacillus strain 8 4 4 16

Toxins produced by inhibitors may, under certain conditions, accumulate in

considerable quantities and endow the soil with toxic properties. The extent of
accumulation of toxic substances depends upon the intensity of their formation by
microorganisms, the rate of destruction and leaching, and also upon the degree of
adsorption.

PART IV, continued:

 Microbial Antagonists

Among the soil microorganisms there are forms that inhibit the growth of other
microbes. They are usually called antagonists.

There is no fundamental difference between inhibitors and antagonists. Both groups

act with the aid of special metabolites in their metabolic products: the inhibitors affect
cells of higher organisms and the antagonists act on lower organisms. Even this
distinction is not always sharp, since there are inhibitors, which also suppress microbes
and, on the other hand, there are antagonists that are also toxic to plants.

As was already mentioned, substances formed by inhibitors are called toxins or

phytotoxins, and substances produced by antagonists are called antibiotics. This
differentiation is of a purely formal or conventional nature. As is well known, there are
many substances among the antibiotics which are highly toxic to plants and animals.

Regardless of this relativity of concepts and designations the antibiotics and their
producers are a special branch of science and are considered as special substances with
specific manifestations.

Microbial antagonism has caught the attention of scientists for many years. Pasteur,
Mechnikov and their contemporaries noticed the ability of some species of microbes to
suppress the growth of others (cf. Nakhimovskaya, 1937; Waksman, 1947; Krasil'nikov,
1950 and others). Pasteur observed this ability in the anthrax bacillus in relation to the
microbe causing chicken cholera. Mechnikov found it in the lactobacilli in relation to
the putrefactive bacteria and certain colon-type bacilli. On this basis he devised a
method of changing the intestinal flora and sanitation of the human and animal
intestines. The phenomenon of antagonism was observed in various groups of
microorganisms, among bacteria, fungi, actinomycetes, algae, protozoa, etc. Microbial
antagonists acting upon various pathogenic bacteria against cocci (staphylococci,
streptococci, pneumococci and diplococci), against organisms causing intestinal
infections (dysentery, parathyphoid, typhoid and cholera) against the tubercle bacillus,
diphtheria, peat and anthrax brucellosis, tularemia and gas gangrene have been
described. A great number of antagonists were described acting against pathogenic
fungi, yeasts, protozoa, etc.

Recently, the efforts of investigation have been concentrated on the detection of

antagonists acting against viruses and malignant tumors, Antagonists of viruses and
tumors can be found among actinomycetes and bacteria (Waksman, 1953; Kashkin,
1952; Kurylowicz and Slopek, 1955).

Much data are available on the suppressing action of antagonists of phytopathogenic

bacteria, actinomycetes and fungi. Antagonists were found acting against various
organisms. The greatest attention is paid to antagonists--actinomycetes.

Among 20 cultures of actinomycetes isolated from rhizosphere soil, Lochhead and

Lauderkin (1949) found 11 cultures that suppressed the growth of phytophathogenic
strains of Actinomyces scabies.
 Meredith and Semeniuk (1945-47) found that among the actinomycetes which they
studied 21% inhibited growth of the fungus Pythium graminicola, which causes root
necrosis in a number of plants. The actinomycete-antagonists of Chalaria quercina
which cause wilt in the oak, and actinomycete-antagonists of Cerasto mella ulni, which
affect woody plants, such as the elm, etc have been described (Stallings, 1954).

From the soils of the southern shore of Crimea, Petrusheva (1953) isolated 31 cultures
of actinomycetes. Twenty-two of these inhibited the growth of the fungi Thielaviopsis
basicola which causes root rot of tobacco plants, and Fusarium sp. which causes the
"black foot" disease of citrus saplings, Leben and Keitt ( 1948) had a collection of
actinomycetes that inhibited 33 species of phytopathogenic fungi.

Cooper and Chilton (1950) have done much work on the detection of actinomycete-
antagonists of the phytopathogenic fungus Pythium arrhenomonas which is widely
distributed in the soils of Louisiana. Out of 8,302 strains which they isolated, 18.5 to
31.5% were antagonists. Kublanovskaya (1950) noted actinomycete -antagonists to the
agent of wilt of the cotton plant-- Verticillium dahliae and Fusarium vasinfectum.
Lechevalier et al., (19 5 3) tested 197 strains of actinomycetes for antagonism to
Cerastomella ulni and found only one active strain. Actinomycete-antagonists were
found in soils which were active against phytopathogenic fungi--Helminthosporium
sativum, H. victoriae, Coletotricum circinans, Verticillium albo-atrum and others
(Stevenson, 1954; Stessel et al., 1953).

In our studies we found actinomycetes in soil, which suppress phytopathogenic fungi.,

Fusarium lin, F. solani, F. vasinfectum, Helinthosporium sativum, Alternaria humicola,
Rhizoctonia solani, Botrytis alii, Deuterophoma tracheiphilus, Trichoderma lignorum,
Monila fructigena, and also fungi of the genera Penicillium, Aspergillus, Cladosporium,
Verticillium and others.

Among fungi there are many antagonists. Antagonists have been described against
agents of various diseases: Fusarium, Peziza, Rhizoctonia, Ophiobolus, Botrytis,
Monilia, Sporotrichum, Pythium, Phymatotricum, Phytophthora and Sclerotium.

Porter (1924) described antagonistic relations between fungi and bacterial antagonists,
and the phytopathogenic fungi Helminthosporium and Fusarium. Sanford and
Broadfoot (1931) isolated from the soil 6 species of fungi which inhibit the growth and
activity of the fungus Ophiobolus graminis. Weindling (1932, 1948) described a case of
parasitism of the fungus Trichoderma lignorum on the fungi of the genus Rhizoctonia
and others. The same was observed by Novogrudsk (1936). The latter given a long list
of fungi and bacterial antagonists, indicating the species of fungi and bacteria inhibited
by them.

In this list appear more than 30 fungal species which inhibit over 50 fungi belonging to
different genera and families. Many other investigators also found antagonists among
the fungi (Allen and Haenseler, 1935; Joseph, 1952; Vasudeva, 1950, 1953 and others).

Stemmel, Leben and Keitt (1953) isolated 170 fungi antagonistic to other fungi from
soils.
 Anwar (1949) found that among soil fungi and bacteria approximately half (out of 86
studied) suppressed the growth of the fungus Helminthosporium sativum, and about
12% inhibited Fusarium lini. From various soils, Gregory at al., (1952) isolated 14
cultures of fungi, 29 strains of actinomycetes and 31 strains of bacteria, which actively
suppressed the growth of the fungus Pythium debaryanum. Three actinomycete strains
and 1 bacterial strain suppressed growth of the root-nodule bacteria, Rh. meliloti and
Rh. trifolii.

Among the sporeforming and nonsporeforming bacteria, it is not uncommon to

encounter antagonists to phytopathogenic microbes. Among the sporeformers
antagonists to various phytopathogenic fungi have been described. Porter (1924)
mentions a number of bacteria which inhibit the growth of Helminthosporium. The
latter did not grow in the presence of Bac. capoulatus, Bac. mesentericus and Bac.
mycoides. Bamberg (1931) indicated inhibition of growth of the fungi Tilletia tritici,
Ustilago zeae, U. levis, U. avenae by the sporeforming bacillus Bacillus D.

The afore-mentioned bacteria showed similar action against phytopathogenic fungi

such as Penicillium sp., Helminthosporium, Ophiobolus, Acrostalagmus, Fusarium.
Sclerotinia gleosporium, Alternaria and others (Novogrudskii, 1936; Weindling, 1946;
Stessel et al., 1953; Stallings, 1954; Krasil'nikov, 1953 a and others).

Many antagonists have been described among the nonsporeforming bacteria. Studies
show that they are most frequently encountered among the Pseudomonas and
Bacterium species and also among myxobacteria. Bisby (1919) described the species
Pseudomonas phaseoli which inhibited the fungus Fusarium oxysporium. Fawcett
(1931) indicated Ps. juglandis as an antagonist of the fungus Dothlorella gregaris.
Johnson and Marvin (1931) detected the antagonistic action of certain nonsporeforming
bacteria (Bacterium C-1. Bacterium X, etc) on growth of the fungi Ustilago zeae, U.
avenae, Alternaria solani, A. brassicae and A. Tenuis.

Khudiyakov (1935) isolated and studied in detail bacteria which dissolve the mycelium
of the fungi Fusarium graminearum, F. culmorum, F. scirpi, F. lini. F. herbarum, F.
equiseti, Sclerotinia libertiana and others. These bacteria were called mycolytic
bacteria. Subsequently, many other investigators detected these bacteria in the soil
( Raznitsyna, 1942; Berezova, 1932; Korenyako, 1939; Kublanovskaya, 1953, etc).

Ark and Hunt (1941) had cultures of the bacteria Bac. vulgatus and Bac. sp. that
inhibited the growth of many phytopathogenic bacteria--Bact. amylovorum Bact.
aroideae, Bact. carotovorum, Bact. phytophthorum, Ps. campestris, Ps. lachrymans and
others. These bacteria also inhibited certain fungi--Fusarium graminearum, F.
lycopersici, Phytophthora sp. and others. Similar data are given by other authors
(Johnson, 1935, Weindling, 1946; Christensen and, Davis, 1940; Vasudea, 1952 and
Skinner, 1956).

Antagonists to phytopathogenic fungi have been described among the myxobacteria

(Johnson and Marvin, 1931; Kononenko, 1937).

Fungi which attack nematodes are described in the literature. They were first described
by M. S. Voronin in his work "Mycological Studies" published in 1869 and by Sorokin
in 1871. In a series of papers Soprunov (1954) showed that these fungi differ in their
species composition and are very widespread in soils. The majority of them belong to
the Hyphomycete, to the genera Trichothecium, Arthrobotrys, Dactylaria, Dactylella,
etc.

These fungi "catch" the nematode with their hyphae and poison it with their
metabolites. Attempts were made to use these fungi in the struggle against
phytopathogenic nematodes. The introduction of this fungus into the soil lowers the
incidence of plant disease. In the struggle with nematodes which affect cucumbers, the
fungi-antagonists, or an they are called the predatory fungi noticeably decrease the
incidence of disease; in the control group there were 23 galls per each plant and in the
treated plants, an average of 0.6 galls (Soprunov, 1954).

According to Gorlenko (1955), artificial enrichment of soil by predatory fungi lowers

the morbidity of cucumbers 1.5-7 times. Tendetnik (1957) used the predatory fungi for
exterminating pathogenic larvae, the ancylostomes in mines and also to destroy
strongyles in the manure of infected animals.

On the basis of existing evidence, one may say that there are no species of bacteria.
actinomycetes, proactimomycetes, micromonospores, protozoa, algae, etc against which
no antagonist can be found. In laboratory practice, one usually gives the name
antagonist only to a microbe which suppresses the widely used test organisms, which
usually comprise a narrow range of known cultures of bacteria or fungi. One does not
take in account the fact that the so-called inactive forms (nonantagonists) in these tests
might be active against other organisms. On the basis of our own experience and data
from literature, we can say that the property of antagonism is characteristic of all
species of microorganisms, but is expressed differently and to various degrees
depending on the natural properties of the antagonist on the one hand, and the
sensitivity of the test organism on the other hand, and also upon the type of substrate
and other external conditions.

The antimicrobial action of the antagonists manifesto itself not only under laboratory
conditions on artificial media but also under natural conditions of habitation, in the soil.
In sterile soil, where there are no antagonists the growth of microbes and the
biochemical processes which they cause take place at an intense rate. However, it is
sufficient, to introduce an antagonist in such soil in order to stop or to slow down the
growth and biochemical processes of microbe.

In his experiments, Afrikyan (1951) directed the action of antagonists of the group of
sporeforming bacteria: Bac. subtilis and Bac. mesenterics against the organisms: Bac.
mycoides and Az. chroococcum. The result are given In Table 110.

Table 110
Intensity of Azotobacter growth as depending on
growth of the antagonistic bacterium--Bac. mesentericus
(experiment with sterile soil; table shows number of cells, in thousands,
in 1 g of soil after passage of days)
 Initial
Experimental conditions 1 2 3 5 7 10
number
No antagonist (control) 80 150 380 520 760 800 1,000
With antagonist 80 100 30 0.05 0 0 0.05
No antagnist (control) 200 500 600 800 800 800 500
With antagonist 200 100 50 0.5 0 0 0.05
Antagonist 80 400 1,200 1,500 1,600 1,000 800

Bac. mesentericus was introduced into sterilized soil together with cultures of
Azotobacter to which it is an antagonist, The growth of Azotobacter was suppressed by
Bac. mesentericus. Under conditions of growth in sterilized soil when grown alone,
Azotobacter reached 1 million cells per 1 gram of soil and in the presence of Bac
mesentericus, Azotobacter grew very slowly and only during the first day; then the
number of its cells decreased rapidly, dropping practically to zero. Only at the end of the
experiment, after 10-15 days, when growth of the antagonist and formation of the
antibiotic stopped, did the Azotobacter resume growth although at a very slow rate.

The same was observed in experiments with Bac. mycoides. This microbe in very
sensitive to the antibiotic action of the potato bacillus. In an isolated condition, in
sterilized soil, its growth is excellent but it ceases to grow completely or almost
completely in a mixture with the antagonist (Figure 92).

Figure 92 Growth of Bac. mycoides in soil (sterile) in presence of the antagonist--Bac.

mesentericus:

1--growth of Bac. mycoides without antagonist; 2--growth of Bac. mesentericus in soil;

3--growth of Bac. mycoides in a mixture with Bac. mesentericus.

Mikhaleva (1951) studied the growth of the root-nodule bacteria of clover, peas,
kidney, beans, lupine, soy, lucerne, etc in the presence of antagonists and actinomycetes,
The most resistant, according to their data, were the soy bacteria In podsol soils these
bacteria are more strongly suppressed by actinomycetes than in chernozem soils. Root-
nodule bacteria of clover perish after 4 days in the soil in the presence of the antagonist.

The activity of the root-nodule bacteria decreases markedly under the influence of the
antagonist; the number of nodules on the roots is usually smaller than in the controls.
According to our observations, the root-nodule bacteria of clover vetch and peas react
noticeably to the action of antagonistic Bac. subtilis, Bac. mesentericus and others.
When the soil is artificially enriched with these bacteria the nodules do not develop on
the roots of the above-mentioned plants or they appear in small numbers only, with a
degenerated appearance, an unusual form and a small size.

Stolp (1952) studied the growth and activity of root-nodule bacteria of peas in the
presence of Pennicillium expansum. Robinson (1946) observed cultures of antagonists
among actinomycetes, bacteria and fungi, which actively suppressed the growth and
virulence of root-nodule bacteria in the laboratory and under field conditions as well.

The general importance of bacterial antagonists is determined not only by the nature
and strength of their activity but also by their number in the soil. The more intensely
they grow in the soil, the higher their concentration and the stronger the effect they
exert.

It is difficult or almost impossible to assess the total number of antagonists in the soil.
As was already mentioned, all microbes possess antagonistic proprties against some
microbe. However, it is practically impossible to detect antagonism against all existing
microorganisms.

In our studies we give quantitative indexes of distribution. of antagonists in relation to

certain species of bacteria or fungi. Among the bacteria, cultures of Azotobacter, root-
nodule bacteria, mycobacteria, micrococci and nonsporeforming bacteria (colon
bacillus, etc) were used.

According to our calculations, there are very large quantities of actinomycete-

antagonists with clearly expressed antimicrobial properties in different soils.

In the previous chapter (microbial inhibitors) data were presented on the number of
microbes which suppress the growth of Azotobacter. The number of bacterial
antagonists ranged between 10,000 and 450,000; fungi, between 1,300 and 17,000 and
actinomycetes, from 10,000 to 160,000 in 1 g of soil, depending on the properties of the
latter.

In relation to certain other species of bacteria (Bac. mycoides, Bac. subtilis) and fungi
(Fusarium lini, Fusarium sp., etc) the total number of antagonists is much higher. In our
analyses of various soils of the Soviet Union we found tens and hundreds of thousands
and often millions of them in 1 g of soil.

In the podsol soils of the Moscow area one may find 40,000- 1,000,000 actinomycetes-
antagonists or even more, per 1 g of soil; sporeforming bacteria in numbers of 20,000-
500,000 may be found in 1 g of soil. In the chernozems of the southern districts of the
USSR the total number of microbes is higher and, as a rule, the number of antagonists is
also higher. However the percentage of antagonists may be lower. For example, the soils
of the Crimean steppes contain a smaller percentage of antagonists than the northern
soils of the Kola Peninsula or the soils of the central districts. There are relatively few
antagonists in the red soils of the Caucasian coastal area (Table 111).
 Table 111
Number of actinomycetes-antagonists in soils (in thousand/1 g
soil)
Soil and region Actinomycetes Antagonists
Serozem, Tashkent 560 200
Serozem, Vakhah valley 1,300 360
Chernozem, Kuban' 2,000 400
Chernozem, Kar'kov 1,000 400
Chernozem, Kuibyshev 1,500 800
Chernozem, Crimean steppe 1,200 120
Krasnozem, Batumi 200 10
Chestnut soil, Armenia 600 200
Brown soil, Armenia 1,200 600
Chernozem, Armenia 800 200
Chernozem, Georgia 1,800 350
Podsol, Moscow, Oak forest 1,200 350
Podsol, Moscow, Spruce forest 360 0
Podsol, Moscow, Birch forest 1,200 420
Podsol, Moscow, tilled, a 1,500 800
Podsol, Moscow, tilled, b 600 200
Podsol, Leningrad, tilled 1,500 800
Podsol, Kola Peninsula, ferruginous 0.4 0.3
Humus, Kola Peninsula,
2.0 1.5
ferruginous
Tundra, Kola Peninsula, forest 0 0
Swampy soil, Kola Peninsula 0.2 0.2

In certain soils one may find 2,000-400,000 bacterial antagonists per 1 g of soil; they
belong to two groups--Bac. subtilis and Bac. mesentericus. Among the nonsporeforming
bacteria and especially among species of the genera Pseudomonas and Bacterium, there
are many antagonists. A special place in relation to their quantity among the antagonists
of this group is occupied by the mycolytic bacteria. These bacteria grow well in many
soils and in the rhizosphere of various plants. According to our figures, their total
number approaches tens of millions and more in 1 g of soil (Krasil'nikov, 1940 a,b, c;
Kusina, 1951; Kublanovskaya, 1953).

Rasnitsyna (1947) studied the soils of the Vakhah valley and counted 100,000 to 100
million mycolytic bacteria (which lyse the fungus Fusarium vasinfectum) in 1 g of soil,
depending upon the vegetative cover. The greatest number was found in the rhisosphere
of lucerne, spear grass and the Euagropyrum; they were less numerous under rye grass,
brome grass and peas. Kuzina (1955) gives more or less similar indexes for light
serozems of the Uzbek SSR.
 Novogrudskii (1949 a) isolated mycolytic bacteria from Kazakhstan soils which lyse
the fungi: Fusarium culmorum, F. graminiarum, Verticillum dahliae, Colietotrichum
lini, Alternaria tenuis, Amblyosporium botrytis, Pyronema confluens, and Mucor
racemosus.

In the same soils the author counted from 100 to 100 thousand mycolytic bacteria in 1
g of soil. These are active solely against Fusarium graminiarum and were more
numerous under lucerne than under oat or millet.

Above were given data on growth and accumulation of these bacteria in soils under
different plants.

In nature microorganisms do not live alone, but in associations which contain many
foreign species--competitors and noncompetitors. In these associations definite and
quite complicated relations are established among the species, of both a symbiotic and
an antagonistic nature.

In microbial populations or coenoses, each species in its struggle for survival

throughout a long history of evolution, elaborated certain means of struggle with its
competition. These means are versatile. Microbes may displace their competitors by
abundant multiplication or they may form during their metabolism various specific and
nonspecific substances which suppress microbial growth. These nonspecific substances
are, for example, organic acids, alcohols, peroxides and other compounds. These
metabolic products are characteristic of many microbial species.

Examples of the nonspecific metabolites are certain inorganic substances, such as

ammonia, hydrogen sulfide, ions of chlorine, SO3, aluminum, iron and other elements,
Plentiful emission of H2S, formed as a result of the vigorous metabolic activity of the
appropriate microbes, may cause the poisoning of the milieu, making it inadequate for
the growth of many foreign species of microbes and even for higher plants, as was
observed by us in soils with high ground-water level in the Trans-Volga region
(Krasil'nikov, Rybalkina and others, 1934).

Microbes producing acids, alcohols and other organic compounds suppress the growth
of organisms which are sensitive to these substances, regardless of the species to which
they belong.

The most characteristic and outstanding reactions are those which are caused by
particular specific substances, the so-called antibiotics, which act against microbes.
These substances have specific effects. The microbial antagonists which form these
substances suppress growth of certain specific species only. Some antagonists inhibit
only gram-positive bacteria and others, inhibit both grarn-positive and gram-negative
bacteria. Some of them act on cocci and others on bacilli.

Some antagonists have an inhibitory effect only on fungi or only on phages and
viruses, etc.

Antibiotic substances produced by antagonists are a potent weapon in the struggle with
competing microbes.
 A characteristic feature of such antagonists is the fact that as a rule, they act only upon
foreign species. A. streptomycini, the producer of streptomycin, does not inhibit cultures
of its own species. The producer of aureomycin, A. aureofaciens does not inhibit
cultures of its own species, regardless of from where they were isolated or under what
conditions they lived previously. Similarly, other antagonists which produce antibiotic
substances, terramycin, chloromycetin, actinomycin, sulfactin, etc do not suppress the
growth and development of strains which belong to the same species.

These characteristics of specific or selective action of antagonists determine to a large

extent the species makeup of microbial populations in natural substrates.

Experience shows, that there are microorganisms that form antibiotics under
conditions of solitary growth, on artificial nutrient media. Their ability to form these
antibiotic substances is hereditarily fixed and expresses itself in the absence of
competitors.

These organisms are looked upon as potent antagonists. They are often found among
various microbial species. They comprise the main group of producers of antibiotic
substances obtained to date in various laboratories and in the antibiotic industry. In other
species this property is not fixed by heredity and appears only in the presence of
competitors, i.e., under conditions of mixed populations. In pure isolated cultures these
organisms do not produce antibiotic substances. For example, the colon bacillus, Bact.
coli suppresses the growth of Bac. anthracis only in those cases where both organisms
are together. In a pure culture the colon bacillus does not produce antibiotic substances
which are active against the mentioned microbe. Apparently these microbes only
produce antibiotic substances under pressure, out of necessity.

Shiller in 1914 was the first to pay attention to the forced nature of the formation of
antibiotics by microorganisms. He was working in Mechnikov's laboratory on the
interrelations between the acidophilic bacilli, sporeforming bacteria, streptococci,
pneumococci and other species (Shiller, 1952).

The phenomenon of forced antagonism was also noticed by other investigators (Peretts
and Slavskaya, 1934; Izabelinskii and Soboleva, 1934 and others). Streshinskii (1949,
1950) has demonstrated the formation of antibiotic substances in mixed cultures of a
fungus and a sporeforming bacillus. According to his observations, Bac. subtilis forms
an antibiotic substance against the Penicillium fungus only when the two grow together.
The fungus too becomes a more active antagonist in the presence of the bacillus. We
observed forced antagonism in a number of actinomycetes. Cultures grown separately
on nutrient media, do not form antibiotic substances, but in the presence of certain
microbes (fungi or bacteria) these substances which suppress the growth of competitors
are formed. Two inactive species of actinomycetes when grown together, form
antimicrobial substances.

All such organisms possess a latent antagonistic capacity which is not fixed
hereditarily.

The majority of microbial antagonists readily lose their ability to produce antibiotics,
active strains turning inactive in the process of variation. This property is encountered in
many organisms used in industry and causes many difficulties in the antibiotic industry
and in laboratory practice.

Antibiotic substances should be classified an potent weapons in the struggle of

microbes with neighboring competitors; as a biologically important attribute formed in
complex populations during phylogenesis and as a property determining the degree of
development and distribution of the microorganism in nature. The biological role of
antibiotic substances and, therefore, the phenomenon of antagonism as a whole, is quite
important in the life of both higher and lower soil organisms.

Through use of their metabolic products antagonistic microbes suppress their

competitors, removing them from the substrate and thus exerting a definite selective
action. To a certain degree, microbial antagonists regulate the formation of microbial
coenoses (colonies) in the soil in general. They play an important role in the
improvement of soils, in the so-called process of self-purification of soils. The removal
of harmful pathogenic and phytopathogenic flora and fauna is accomplished by
microbial antagonists.

Microbial antagonists suppress not only the growth and propagation of competing
organisms, but also many of the functions of the latter. There are among
microorganisms those which inhibit certain processes which occur in microbes.

Inhibitors were observed to suppress nitrification, denitrification, nitrogen fixation,

decomposition of cellulose, etc. Appropriate cultures inhibit the decomposition of
organic substances, fermentation of sugars, etc. Microbial cultures are known which
inhibit the formation of certain metabolites, various acids, enzyme biotic substances,
auxins, vitamins, amino acids and other biocatalysts. There are organisms in the soil
which, with their metabolic products, suppress the formation of toxins and antibiotics
and often inactivate them in the medium, if they are formed there.

There are data in the literature dealing with the suppression by inhibitors and their
metabolites of processes of cell multiplication, spore formation, budding and the sexual
process. Inhibitors often inhibit the process of respiration.

In the presence of antagonists or their metabolic products many active substances:

biocatalysts, antibiotics, toxins, and enzymes lose their peculiar functions, and become
inactive. Toxins become harmless, antibiotics no longer suppress the corresponding
microbes, enzymes no longer cause the decomposition of organic matter, etc. In general,
one may say that for any metabolite, living nature creates an antimetabolite (Woolley,
1954). The metabolite penicillin becomes nonbactericidal for staphylococci and certain
other bacteria, which produce an antimetabolite penicillinase (Abraham and Chain,
1940; Woodruff and Foster, 1945). Sulfonamide compounds are inactivated by bacteria
which produce paraaminobenzoic acid (Woods, 1940; Sevag and Green, 1944, 1950) or
the factors H.P. etc (Moller and Schuerz. 1941; Green 1940). Mucin suppresses the
antibacterial action of tyrothricin; tannic acid neutralizes actinomycin (Waksman, 1947).
In our bacterial collection there were strains, which completely inactivated the antibiotic
substances, formed by the sporeforming bacteria: Bac. subtilis, Bac. mesentericus and
Bac. cereus. We also possessed bacterial cultures, which annuled the toxic effect of Ps.
pyocyanea metabolites. The toxin of the fungus Botrytis cinerea is neutralized by
metabolites of certain actinomycetes.

A solution of mycetin at a 1:10,000 dilution completely suppresses the growth of

Staph. aureus and in the presence of filtrate or cells of a Bac. proteus culture the
antibacterial effect is either nonexistant, or becomes very weak. A culture of the colon
bacillus and certain Pseudomonas strains had such an inactivating effect in our
experiments. An inactivating effect of microbes was also found in relation to other
antibiotics (Krasil'nikov and Nikitina, 1951).

As seen from the above-mentioned data, antagonists exert a definite suppressing effect
on various microorganisms. Due to their action, they have a considerable influence on
the formation of microbial coenoses in soil in general and determine, to a certain degree,
the distribution and accumulation of the various species of soil microflora.

Antagonists, therefore, can be considered as one of the powerful factors governing soil
fertility and plant-crop abundance.

It is only natural that this group of microorganisms attracts the attention of specialists
in many fields--microbiologists, phytopathologists, plant breeders and others. The
possibility of practical utilization of antagonists is one of the most important reasons for
the study of the phenomenon of antagonism.

The protective role of microbial antagonists

As was noted before, in soils in which antagonists grow abundantly (bacteria, fungi or
actinomycetes). microbes, sensitive to them, saprophytes as well as phytopathogens,
grow much more slowly, or not at all. This served as a basis for the use of microbial
antagonists in the struggle against harmful microflora, and against organisms causing
plant disease.

The first attempts in this direction were made by Porter (1924). He treated wheat seeds
with bacterial antagonists and then infected them with Helminthosporium fungi. The
seeds either did not become infected and germinated normally, or they were slightly
affected. Bamberg (1931) infected wheat seeds in order to protect them against smut.
Weindling (1940, 1948) used the fungus Trichoderma lignorum for the protection of
citrus saplings from Rhizoctonia. This fungus according to other authors, also protects
cucumbers and peas from Rhizoctonia and wheat from fusariosis (Allen and Haenseler,
1935; Bisbu, James and Timonin, 1933). Milliard and Taylor (1927) observed a
protective effect of the actinomycetes-antagonist---A. praecox in relation to the agent of
scab in potatoes A. scabies. No scab was observed upon prolific growth of the
antagonist.

A similar phenomenon was described by Kissling (1933). He experimented with

bacterial antagonists in subduing potato scab. Under conditions of a field experiment,
upon sufficient growth of the antagonist the disease either did not appear at all or had a
limited occurrence only.
 Gregory, Allen, Riker and Peterson (1952) demonstrated the protective role of
actinomycete-antagonists in their experiments with the phytopathogenic fungi Pythium
ultimum and Pythium debaryanum. These fungi were quite virulent for lucerne. The
introduction of certain species of actinomycotes-antagonists into the soil (Actinomyces
sp.) protected the plants from the disease. A positive result was also obtained through
use of antagonists--fungi Trichoderma lignorum and Penicillium patulum, and the
sporeforming bacteria, Bacillus sp. No 6.

Rehm (1953) treated wheat and rye seeds with actinomycetes-antagonists in order to
fight the organisms which cause fusarlosis (Fusarium nivale and Fus. culmorum). As a
result, seed germination Increased by 30 %.

Gorrard and Lockhead (1938) compiled a long list of microbial antagonists which
suppress the development of phytopathogenic organisms in soil. Among them there are
sporeforming and nonsporeforming bacteria, fungi, actinomycetes and protozoa. Cordon
and Haenseler (1939) have shown experimentally the inhibitory effect of the
sporeforming Bacillus simplex on the growth and development of the fungus
Rhizoctonia solani which affects cucumbers and peas. A culture of the antagonist was
introduced Into the soil in which the plants were grown. As a result, the morbidity of the
latter dropped. In the control without bacterial antagonists 65 % of the total number of
cucumber plants and 48% of peas were affected; after introduction of the antagonist the
mortality role was 35 and 45% respectively.

Ark and Hunt (1941) mention two cultures of sporeforming bacteria--Bac. vulgatus
and Bacillus sp. which suppressed the growth of many phytopathogenic bacteria in soil
and protected the plants from disease. Other investigators also speak of the antagonistic
action of microbes in soil against phytopathogenic bacteria and fungi (Johnson, 1935;
Eaton and Rigler, 1946; Allen and Haenseler, 1935; Feeney and Garibaldi, 1948;
Kenknight, 1941 and others).

In the Soviet Union, as already mentioned above, Khudyakov (1935) and

Novogrudskii (1936) established the lytic effect of mycolytic bacteria on
phytophatogenic fungi. These bacteria were subsequently exclusively tested fighting
plant infections under laboratory conditions, and in open fields as well. The results were
positive in many cases.

The percentage of mortality noticeably decreased. For example, the experiments of

Berezova (1939), performed on kolkhoz fields with flax, have shown, that mycolytic
bacteria lower the incidence of fusariosis by an average of 40% and in isolated cases
their action was even more effective.

Raznitsyna (1942) used mycolytic bacteria which are isolated from the soil against
fusariosis of pine saplings (Figure 93). Under open-field conditions on a sector strongly
affected by fusariosis, inoculation of bacteria gave an outright positive effect, and the
lowering of morbidity reached 80 % and more (Table 112). The plants looked
considerably healthier on sectors treated with mycolytic bacteria, than in the control.
The needles were longer, stronger and greener, the stem of the saplings thicker and taller
than in saplings not treated with bacteria (Figure 94).
Figure 93. Protective action of bacteria in fusariosis of pine saplings:

1--plants infected with the fungus Fusarium; 2--plants also infected with Fusarium, but
treated with mycolytic bacteria.

Table 112
Protective action of bacterial antagonists in fusariosis of pine saplings sown in May
1940
Number that Height of plants
Seeds germinated
Experimental conditions survived until in September,
per 100 planted
September cm
Control (not inoculated with
40 5 3.0
bacteria
Inoculated with bacterial
75 61 4.9
culture No. 30
Inoculated with bacterial
65 46 3.9
culture No. 77
Inoculated with
70 46 4.1
Euagroypyrum compost

Figure 94. Pine saplings growing on sectors affected by fusariosis:

a--not treated with bacteria; b and c--treated with mycolytic bacteria.

 Korenyako (1940) studied mycolytic bacteria in the soils of the Uzbek SSR for a
number of years. She isolated cultures of bacteria of the genus Pseudomonas, which
showed antagonistic properties against the agents causing wilt of the cotton plant
Verticillium dahliae. When these bacteria were tested on the experimental fields of
STAZRA SOYUZNIKHI (Tashkent) which were strongly affected by the mentioned
fungus, positive results were achieved. The mycolytic bacteria suppressed the growth of
the fungus and lowered the morbidity of the cotton plant by 60- 80%. Kuzina (1951)
and Kublanovskaya (1953) corroborated these results. They showed that mycolytic
bacteria in mixtures with other bacteria suppress phytopathogenic fungi more
vigorously.

Davydov (1951 used mycolytic bacteria against the mildew organism (Sphaerotheca
mors uvae) on the gooseberry shrub. The parasitic fungus disappeared upon introduction
of the bacterial antagonists and the plant recovered and developed normally.

Petrusheva (1953) used cultures of actinomycetes as antagonists against tobacco-

seedling rot caused by the fungus Thielaviopsis basicola, and against blackleg of citrus
cultures, caused by Fusarium sp. In soil treated with antagonists the plants grew
normally; when the actinomycetes were not introduced into the soil, the mortality of the
plants reached 70%.

Gurinovich (1953) used cultures of actinomycetes and bacterial antagonists in the

struggle against the vascular disease of cabbage caused by nonsporeforming bacteria
Pseudomonas campestris. The antagonists were introduced into the soil affected by the
soil microbe, and prevented the plants from becoming infected.

Seiketov (1951) tested four cultures of the fungus Trichoderma--T. glaucum T.

lignorum, T. koningii and T. album as antagonists of Rhizoctonia, which affects
potatoes. The percentage of mortality of the "early rose" variety was considerably lower
(7-8%) in the experimental plants than in the controls (54 %).

Kuzina (1955) used mycolytic bacteria against wilt of the cotton plant caused by
verticillium. She treated the seeds with bacteria before sowing. The morbidity in the
control sectors was 54% and after treatment with the bacterial antagonists it was 8--9%.
Correspondingly, the crop yield was: in the control- 1, 030 bolls and the total weight of
seed cotton was 342 g. In the experimental plants there were 1,630 bolls and the weight
of the seed cotton was 514 g, i.e., 57% higher than those of the control.

Kublanovskaya (1953) used actinomycetes as antagonists to fight cotton-plant wilt.

She prepared a special composted preparation of cotton cake with actinomycetes. This
was introduced into the soil in which the cotton seeds were sown. The actinomycetes
introduced in this manner proliferated abundantly in the soil and suppressed the growth
and activity of the fungi Verticillium dahliae and Fusarium vasinfectum, protecting the
plants from the disease. At the end of the growth period the number of plants in the
cotton plants of the 8517 variety affected by the wilt was: 52.6% of all control plants
and 18.1% in sections treated with actinomycetes; in the cotton plants of the 108-F
variety there were 23.3 % diseased control plants and 3.3 % treated plants. In the treated
sections all the plants looked stronger, the bushes were larger, the leaves wider and
flowering was more abundant, etc (Figure 95).

Figure 95. Protective effect of actinomycetes-antagonists against cotton-plant wilt

caused by Verticillium:

a) plants affected by wilt, not treated with the actinomycete: b) plants whose seeds were
treated with a culture of actinomycetes (according to Kublanovskaya (1953).

The crop in these experiments also increased noticeably in sectors treated with the
antagonists. The 8517 variety yielded: 9.4 centners/ha in the control sector and 22.6
centners/ha in the experiment. The variety 108-F yielded: 17.8 centners/ha in the control
sector and in the experiment 22.8 centners/ha.

Chinese scientists Yin, Chen, Yang et al., (1955) corroborated Kublanovskaya's data.
They prepared compost from soil with cotton cake and grew actinomycetes in it which
were antagonists of the wilt fungus Verticillium dahliae. The ripened compost was used
for the treatment of the seeds. The latter were sown together with the compost. The
incidence of the wilt disease decreased by 50-75 %, the cotton crop increased by 13-
45%.

Mitchell et al, (1948) observed vigorous growth of antagonists-actinomycetes and

bacteria, when those were introduced in the soil together with a composted plant mass.
The phytopathogenic fungus causing root rot in the cotton plant perished. The positive
role of composted preparations saturated with microbial antagonists was noted by
Sanford (1946-1948). He observed in his experiments a drop in the morbidity of potato
tubers, pine saplings, etc. Similar data are given by some other investigators (Weindling,
1946; Garret, 1946; Winter and Rümker, 1950). They all noted that the favorable action
of composted preparations was not due to an improvement in plant nutrition but to the
more intense development of antagonists in the soil.
 Schaffnit and Neuman (1953) used peat composts, enriched with bacterial antagonists
on the snowy mold caused by Fusarium nivale. The preparations were made from
various types of post. The best results were achieved with bog peat of mass origin. The
percentage of morbidity in wheat treated with composted peat was considerably lower
than in the control.

Wood and Tveit (1955), on the basis of data from the literature and their own
observations, came to the conclusion that microbial antagonists play an important role
in soil improvement. Microbes of local origin are much more effective. In order to
enhance their growth and activity the authors suggest the introduction of certain plant
residues into the soil as sources of nutrition.

There are many other studies in the literature which confirm the positive effect of
microbial antagonists in the struggle against phytopathogenic fungi, bacteria and
actinomycetes. All these studies show that microbial antagonists can be used in
agricultural practice for the improvement of soils. For this purpose the soil should be
enriched with the appropriate antagonists.

This can be achieved by various methods. As may be seen from the above data the
antagonists mentioned may be introduced into the soil in the form of pure cultures
(which is not very effective), or in the form of composts.

In the enrichment of soils with antagonists, the vegetative cover plays an especially
important role. It was noted above that the plants are a powerful selective factor. Some
of them, under certain conditions favor the growth and accumulation of
phytopathogenic microbes in the soil, and others favor the growth of the antagonists of
these microbes. By selecting, by means of special experiments, those plants in whose
rhizosphere the needed antagonists grow abundantly, and by using these plants in the
crop rotation, one may remove or suppress the growth and the harmful activity of the
pathogenic microbe.

If the selected plants are inoculated with microbial antagonists before sowing, their
accumulation in the soil can thus be markedly enhanced.

Upon introduction of pure cultures of antagonists, one must take into account their
adaptability, their growth in the soil, and their activity. If the antagonists lose their
activity in the soil or in a substrate which is not suitable for them which often happens),
or if they do not grow or grow but little, their effect will be small or will not express
itself at all.

Part IV, continued:

Significance of antagonists in plant immunity

Microbial antagonists, an noted above, suppress their competitors with their metabolic
products, among which a special place is occupied by the antibiotics. The latter may
accumulate not only in artificial nutrient media but also directly in the soil, being
concentrated there in smaller or greater amounts.

It is also known that plants can absorb various organic substances from the medium,
including antibiotics, through their roots. If the antibiotic substances enter the plants, it
should be assumed that they may produce a certain effect in the tissues, namely,
suppress the activity of microbes that have penetrated the cells and increase the toxicity
of the cell sap and, consequently, also increase the resistance of the plants to infections.
In other words, an assumption is made that the entry of antibiotics is reflected in the
immunobiological properties of the plants.

It should be noted that the problem of immunity in plants, regardless of the numerous
studies done on the subject, is still very little known.

Literature data show that immunity, nonsusceptibility to infections in plants is

determined by various, quite complicated internal and external factors.

One of such internal factors is the toxicity to bacteria of the call sap. It was noted long
ago that the sap of plants possesses the ability to suppress growth of bacteria and fungi.
Wagner (1915) observed death of bacteria in sap from the tubers and stems of potatoes.
Precipitating these juices with ammonium sulfate and washing the precipitate with
water, he obtained a substance of a protein nature with strong antibacterial properties.
The substance is thermolabile, decomposes quickly in light and under the influence of
oxidases and peroxidases. The toxicity of potato juice was observed by Cholodnyi
(1939), he found that the toxicity of the juice increases upon sprouting of potato tubers.

Antimicrobial properties were observed in the juice of onions, corn, tomatoes, cotton,
orchids and other plants. In the literature there are descriptions of the inactivation of
fungal toxins by the sap of plants resistant to the diseases (Yachovskii, 1935, Vavilov,
1919, Naumov, 1940, Carbone and Arnaudi, 1937). There are indications, that the cell
sap from varieties which are resistant to the infection is more toxic than that of
susceptible varieties (Kramarenko, 1949; Gorlenko, 1950).

The antimicrobial properties of vegetative juice are explained by the presence of

various substances in the cells.

An opinion was voiced that plant immunity is caused by the presence of elements of
mineral nutrition in the sap. Some of them, potassium copper, cadmium, etc create a
nonfavorable milleau in the plant tissues for growth of microbes. According to some
authors, they are favorable to the accumulation of organic acid in the cells. Other
investigators tried to explain the resistance to infections by the osmotic pressure of the
sap, by the presence of alkaloids, enzymes, agglutinins, and lysis, dissolving the
microbial cells, etc.

Israil'skii (1952) expresses the opinion, that the nonsusceptibility of plants to certain
infections is caused by bacteriophages which saturate the tissues of the plants.

Winter and Ruemker (1952) concluded that the nonsusceptibility of plants is caused by
the presence of a special "resistance factor" in the roots.
 Many investigators ascribe considerable importance in plant immunity to the redox
system (Rubin, 1050, et al.) or to the permeability of the protoplasm of the cells
Kuprevich, 194 7, Kokin, 1948; Sukhorukov, 1952), Recently, a connection was noted
between plant resistance to infections and the production of pigments of the anthocyanin
group.

On the basis of him extensive studies Tokin (1951) attaches great importance to the
phytocides in the immunity of plants. In phytocides are included different chemical
antimicrobial substances which are formed by plants. Essential oils and organic acids,
aldehydes, alcohols, phenols and also various specific compounds may be included
here.

One would assume that during the course of the history of their development plants
acquired various means of protection against infections, mechanical, chemical and
biological.

While ascribing a certain importance to all these, in our opinion, the investigators have
not yet touched one of the most important protective factor" the antimicrobial
antibiotics, formed in the soil by microbes and absorbed there from the plants.

Formation and accumulation of antibiotics in soil

It was recently established that in the soil there are antibiotic substances which are
formed by the microbial antagonists. The formation of antibiotic substances by
microbes in the soil was experimentally proved by many investigators. Gottlieb and
Siminoff (1952) have shown the presence in soil of chloromycetin, formed by
Actinomyces venezuelae. The substance was isolated from the soil and chemically
purified. Owing to favorable conditions of growth of the actinomycetes, 25.0-27.8
mg/kg of chloromycetin accumulate in the soil. The formation of chloromycetin in the
soil by A. venezuelae was also noted by Jefferis (1952). The greatest amount is formed
upon the addition of peptone or lucerne hay to the soil. In the presence of starch or oat
meal the antibiotic is not formed.

Gregory et al., (1952) observed the formation of antibiotic substances in soil by

cultures of bacteria--Bacillus sp. B-6, by actinomycetes--Actinomyces No 67 and by
fungi Pencillium patulum. In the presence of appropriate nutrient sources in soil (soy
meal) the following amounts of antibiotics were formed:

In sterile soil, units In nonsterile soil, units

Bacillus sp. B-6 300 30
Actinomyces sp. No 67 5-10 3
Penicillium patulum 100 10

Hessayon (1951, 1953) showed the formation of the antibiotic trichothecin by the
fungus Trichothecium roseum. Large quantities of the antibiotic were detected in loamy
soil, and smaller quantities in sandy soils.

According to Gottlieb (1952) the antibiotic actidione is formed in the soil in

considerable quantities (10 µg/g and more) in the presence of soy meal. Grossbard
(1940) has grown Penicillium patulum in the soil in the presence of various sources of
nutrition. The formation of patulin took place in the presence of beet cake, glucose,
fresh wheat straw, timothy grass and certain other plant residues. According to this
author, antibiotics are also formed in the soil by Aspergillus terreus and A. antibioticus
in the presence of wheat straw and certain other sources of nutrition.

In a later work Grossbard (1952) has shown the formation of antibiotics in the soil by
the fungi: Aspergillus terreus--70 units; Penicillium sp.--80 units; Aspergillus clavatus--
110 units and Penicillium clavatus--100 units in 1 g of soil.

On the third day of growth of the fungus Aspergillus clavatus, in the presence of
brown sugar, Gottlieb and Siminoff (1952) noticed the formation of clavacin in the soil
in the amount of 16 µg/g. Taking into account the extent of adsorption and inactivation
of the substance by soil particles, the authors determined its total production as not less
than 50 µ g/g.

Stevenson and Lochhead (1954) studied the formation of antibiotic substances by the
fungus Penicillium sp. and by the actinomycetes K-1 and V-27 in sterile as well as in
nonsterile soil. In sterile soil the fungus forms as much antibiotics as on Chapeks
medium (about 20--30 µ g/g and in nonsterile soil 3 times less (7 -l0 µ g/ g). The
actinomycetes form up to 16 units/g and more of the active substance. As sources of
nutrition both organisms use various plant residues--the green bulk of clover, orchard
grass, brome grass and wheat.

Wright (1955) observed the formation of griseofulvin in the soil by the fungus
Penicillium nigricans in the presence of other soil fungi. According to his data, a pure
culture of Penicillium nigricans forms the following amounts of the antibiotic (in sterile
soils), in podsol at pH 5.3--0.2 µ g on the 7th day and 2.4 µ g on the 14th day, in podsol
with Ca(OH)2--2.4 µ g on the 7th day and 20 µ g on the 14th day; in orchard soil at pH
6.2--0.6 µ g on the 7th day and 10 µ g on the 14th day--in l g of soil. Upon infecting the
soil with other fungi the production of griseofulvin either decreases or increases,
depending on the species of the fungi. On the 14th day of incubation griseofulvin was
detected in the soil in the following quantities:

Amount in µ g
Penicill nigricans at 40 µ g/g (Control) 100
Penicill nigricans + Trichod. viride 3.2
Penicill nigricans + Trichod. viride 3.2
Penicill nigricans + Trichod. viride 0.4
Penicill nigricans + Mucor ramannianus 12
Penicill nigricans + Cladosp. herbrum 100
Penicill nigricans + Penic. expansum 1.5
 Penicill nigricans + Penic. frequentans 0
Penicill nigricans + Penic. albidum 200
Penicill nigricans + Penic. stolonifer 200

In sterile soil which is contaminated with nonsterile soil griseofulvin accumulates

much less, namely about 6 µ g/g.

The author notes that in sterile and nonsterile soils antibiotics are also formed by many
other fungi. On the 14th day of incubation in soil infected by fungi he found the
following quantities of antibiotics:

Antibiotic In 1 g of soil, µ g
Trichoderma viride gliotoxin 40
Trichoderma viride viridin 0
Penicill. expansum patulin 200
Penicill. frequentans frequentin 20
Penicill. stolonifer mycophenolic acid 0.16
Penicill. nigricans griseofulvin 40

Our studies show that antibiotic substances are formed in the soil by various
antagonistic organisms--bacteria, actinomycetes and fungi, if the conditions are suitable.

In podsol soil in the presence of nutrient sources (soy meal, lucerne or clover straw,
sugar or other substances) bacterial antagonists form 40-180 units/g in sterile soil and
10-80 units/g in nonsterile soil. (Table 113). As can be seen from the table, the
formation of antibiotic substances is more intense in sterile soils. As an artificial
nutrient, here too, the presence of special substances is essential to the formation of
antibiotics, and often those are not the same substances as those necessary for the
nutrition and growth of the antagonistic bacteria. Of many organic substances tested
only some of them were suitable for the synthesis of antibiotics, although growth of the
antagonists occurred with any one of the nutrient sources used in the experiments.

Table 113
Formation of antibiotics in soil by bacteria
(Units/ g on the 5th through 7th day of growth)
Sterile Nonsterile
soil, soil, Nutrient source
Bacteria Nutrient source used
antibiotic antibiotic used
formed formed
Soy meal, meat-
Bac. mesentericus 180 Starch, soy meal 80
peptone
Meat-peptone broth, Meat-peptone
Bac. subtilis 120 40
soy meal broth
Ps. flourescens 40 Lucerne 10 Lucerne
 Meat peptone
Ps. nitrrificans 60 Meat-peptone broth 20
broth, lucerne

Similar data were also obtained upon testing the actinomycetes-antagonists. The latter,
when growing in soils where appropriate organic sources of nutrition are available, form
antibiotic substances in detectable amounts (Table 114). We detected these antibiotics
directly by the use of microbiological assay methods in the soil, and after extraction
with organic solvents. Extraction as a rule, causes lower quantitative results. Where the
microbiological assay method indicates 100units/g by extraction one succeeds in
isolating not more than 10-30 units/g, i.e., about 10-30% of the total amount present in
soil. Depending on the nature of the soil and the properties of the substance itself, the
amounts of the extracted substances may vary (Krasil'nikov, 1954c).

Table 114
Formation of antibiotics by actinomycetes under various soil conditions
(units / g)
Actino-
Actino- Actino-
Actino- Actino- mycete Actino-
mycete B, mycete
Soil mycete 290, mycete 290, 287, mycete
lucerne B, corn
soy meal corn extract lucerne 287, starch
straw extract
straw
Sterile podsol 80 120 80 10 100 100
Nonsterile
30 40 10 0 20 0
podsol
Sterile
0 20 20 0 10 0
serozem
Nonsterile
0 0 10 0 0 10
serozem
Sterile
30 60 20 0 50 80
chernozem
Nonsterile
0 20 10 0 20 20
chernozem

Korenyako, Artamonova and Letunova (1955) studied the formation of antibiotics in

soil by actinomycetes belonging to different species. In all more than 100 cultures were
studied, 13 of them in great detail. The actinomycetes were grown in soil with the
addition of various organic nutrients. All of them, except a very few, formed antibiotics
in soil in greater or smaller amounts. The most active ones are presented in Table 115.

Table 115
Formation of antibiotic substances by actinomycetes in various soils
(units / g)
Cherno- Krasno- Cherno- Krasno-
Actinomycetes Podsol Serozem Podsol Serozem
zem zem zem zem
Sterile Non- sterile
A. Aurantiacus, 1149 200 50 100 80 80 10 30 0
A. globisporus 81-B 120 100 60 60 60 60 40 20
A. globisporus 2302 80 30 100 20 30 0 20 0
A. globisporus 2570 150 20 60 50 50 20 20 0
A. griseus 2535 170 100 100 120 80 40 30 40

The data given in the table show that microbial antagonists form antibiotic substances
with different intensities depending on the type of soil. The largest amount of these
substances is formed in podsol soil, less in chernozem and chestnut soils and the
smallest amounts in krasnozem.

The antimicrobial properties of antibiotics express themselves differently in the soil

than in artificial media. Not all the antibiotics are active in the soil. Martin and Gottlieb
(1955) have shown, that circulin, neomycin and biomycin do not suppress the growth of
the sporiferous bacterium Bac. subtilis in the soil even at very high concentrations (500
µ g/g), whereas on laboratory nutrient media they inhibit its growth at 0. 1 µ g/g. At the
same time subtilin noticeably inhibits the growth of Bac. cereus in these very same
soils. In these author's experiments antinomycin was the most active. Five µ g/ g
actinomycin in soil was a sufficient amount for the suppression of Bac. subtilis growth.

The effectiveness of antibiotics in the soil is determined not only by their properties
but also by their concentration. In turn the latter depends on the rate at which these
substances are formed and enter, the soil on the one hand, and by the rate of their
inactivation on the other hand. As is known, the majority of antibiotics disappear from
the soil. Some are inactivated by the soil solution or destroyed by microbes, others are
washed out by water and a considerable portion is adsorbed by soil particles.

in our experiments (Krasil'nikov, 1954c) we followed the degree of activity of

antibiotic preparations, introduced artificially into various soils under different
conditions. The rate of inactivation of antibiotics, the extent of their being washed out
by water and their adsorption by the soil were studied. The average indexes chernozem
soil are presented in Table 116.

Table 116
Changes in the antibiotic content of soil (chernozem)
two hours after the introduction of the former
(in units/g)
Washed out with Remained in active
Antibiotics Inactivated
water and absorbed state
Streptomycin 850 30 1,120
Globisporin 800 120 1,080
Terramycin 850 250 800
Pennicillin 300 1,320 380
Preparation No 1609 10,000 0 0

Up to 2,000 units/g of chemically pure preparations were introduced into the soil.
Preparation 1609 was introduced at a concentration of 10,000 units/g.

As is seen from these data, streptomycin, globisporin and terramycin are inactivated in
chernozem to more or less the same extent (about 25%), penicillin, to a lesser extent and
preparation No 1609 is completely inactivated. In other soils the inactivation of these
antibiotics presents a different picture (Table 117). The least inactivation of these
antibiotics is observed in podsol soils and in krasnozem, and the greatest in chernozem.

Table 117
Minimal doses of antibiotics, at which the soil still exhibits antibacterial properties
(in units/ g)
Antibiotic Serozem Chernozem Krasnozem Podsol
Streptomycin 350 850 300 80
Globisporin 100 600 400 80
Terramycin 600 850 200 400
Preparation 1609 10,000 10,000 10,000 50,000
Penicillin 120 300 150 60

In order to determine what part of antibiotics are adsorbed by the soils, we washed the
latter with water. Upon introduction of antibiotics into podsol soil in the amount of
2,000 units/g the following portions could be washed out with water: penicillin, 1,650
units/g; streptomycin, 40 units/g; globisporin, 120 units/g; terramycin, 350 units/g, and
the antibiotic No 1609--none.

Penicillin more than other antibiotics can also be washed out of other soils; e, g.,
krasnozem, 1, 800 units/ g and from serozem, 1, 500 units/ g.

By comparing the numerical indexes of inactivation of the antibiotics and their

proneness to be washed out by water, the amount of antibiotics in the adsorbed state can
be determined and therefore also the adsorbing capacity of the soil. According to our
data in podsol soil the adsorption picture was as follows: globisporin--1,800 units/g;
streptomycin--1,880 u/g, terramycin--1,350 u/g, penicillin--280 u/g per gram. In
serozem the figures were 1,670, 1,600, 1,200 and 380 units/g respectively. Streptomycin
is adsorbed by chernozem at the rate of 1, 120 units /g and by krasnozem--1,650 units/g;
globisporin--1,080 and 1,540 units/g and terramycin--900 and 1,620 units/g
respectively.

As seen from this data the amounts of antibiotics detected are considerably smaller
than those introduced. In order to create antibacterial activity in 1 g of serozem soil for
example, it is necessary to use a minimum of 350 units streptomycin; 100 units
globisporin; 600 units terramycin; 130 units penicillin and more than 10,000 units
preparation No 1609. Upon introduction into the soil of smaller antibiotic doses than
those indicated, the antimicrobial properties of the soil will not be detected either by a
qualitative test or by extraction with various solvents (water, alcohol, ether, acetone,
etc). All these data show that to the numerical indexes obtained upon quantitative
determination of antibiotics formed in the soil by microbial antagonists, one should add
the amounts of antibiotics adsorbed and inactivated. If 20 units/g of antibiotic substance
were found in the soil, the actual amount produced by the antagonists would be
considerably higher.

The rate of destruction of antibiotics in the soil varies. Some of them are inactivated
within a few hours and others may be preserved for a few days or even weeks,
depending on the nature of the substance and the properties of the substrate.

Antibiotics with basic properties such as streptomycin are very quickly inactivated in
the soil, Neutral compounds (chloromycetin) are inactivated slowly, and substances of
the acid type occupy an intermediate position.

Adsorption and inactivation of antibiotic substances depend to a considerable degree

on soil acidity. Aureomycin and terramycin saturate neutral loamy soils at a
concentration of 30,000 µ g/g, while for the saturation of acidic loam 60,000) µ g/g and
more are required, i.e., twice as much.

At pH 3.2 soil rich in humus adsorbs 4,000 µ g/g of antibiotics and at pH 5.6-7.6 only
400 µ g/g, i.e., ten times less.

Consequently, the antimicrobial action of antibiotics will differ in these soils. In order
to inhibit growth of Bac. polymyxa in soil at pH 5.6 a concentration of 5,00 µ g/g of
terramycin is required, while at pH 6.2, 200 µ g/g suffice (Gottlieb and Siminoff, 1952;
Martin and Gottlieb, 1952).

The stability of antibiotics in the soil also varies with the acidity of the latter.

According to Gregory et al., (1952), the antibiotic actidione is preserved in alkaline

soil at pH 7.8 for 8 days and in an acidic soil at pH 5.2--for more than 14 days. Clavacin
to completely inactivated in the first day in alkaline soil and in acidic soil it is preserved
for 3-4 days. Ninety per cent of fradicin is preserved in alkaline soil for 14 days, in an
acid soil it is adsorbed and completely inactivated an the first day.

Chloromycetin is less strongly inactivated in the soil than are streptomycin and
terramycin. When chloromycetin is introduced into sterile soil, it remains for more than
14 days without change. In nonsterile soil, with a large number of various
microorganisms, the preparation is gradually inactivated; after 3 days only 70% of it is
left, after 7 days--about 30% and after 2 weeks only 20% are recovered.

Pramer and Starkey (1052) have found, that streptomycin introduced into the soil at a
concentration of 1,000 µ g/g is preserved in sterile soil for over 3 weeks and in
nonsterile soil for 2 weeks. About 50% of it is destroyed within the first week. In the
presence of glucose the antibiotic is preserved for a longer period of time.

Jefferis (1952) tested many antibiotic substances. He introduced them into various
soils and observed the rate of their destruction. The following data have been obtained
(Table 118).

Table 118
 Preservation time of antibiotics in different soils
(days)
Introduced Nonsterile Sterile Nonsterile Sterile
Antibiotic
µ g/g podsol podsol orchard soil orchard soil
Albidin 30 7 14 2 3
Frequentin 100 10 16 2 7
Gliotoxin 20 40 16 2 7
Griseofulvin 30 20 40 16 17
Patulin 2,000 32 32 2 2
Penicillin 50 3 2 2 2
Streptomycin 400 26 16 6 16
Viridin 100 8 16 1 1

An may be seen, in orchard soil there is a faster inactivation of antibiotics. According

to the author, certain substances are destroyed faster in sterile soil than in nonsterile soil,
which is puzzling and disagrees with the observations of other investigators.

According to our data (Krasil'nikov, 1954c), the preservation time of antibiotics varies
greatly and depends first of all, upon the properties of the substances, and secondly, on
the type of soil and external conditions (temperature, humidity, acidity, etc), The same
antibiotic is inactivated and parishes at different rates in different soils. For instance,
globisporin is preserved for 7 days in podsol, but for only 2 days in krasnozem.
Aureomycin is active 10 days in podsol but not more than 3 days in krasnozem (Table
119).

Table 119
Preservation time (days) of antibiotic substances
Prepar- Prepar-
Globi- Aureo- Terra-
Soils ation No ation No Penicillin
sporin mycin mycin
112 1609
Chernozem Sterile 25 15 30 30 2 10
Nonsterile 5 2 3 6 0.2 1
Podsol Sterile 90 80 60 50 5 20
Nonsterile 7 5 10 8 0.1 3
Serozem Sterile 35 20 40 30 3 15
Nonsterile 5 4 8 8 0.1 1
Red soil Sterile 22 12 20 15 2 5
Nonsterile 2 2 3 3 0.1 0.5

Antibiotics are most rapidly inactivated in krasnozem and podsol soils.

Preparation No 1609 disappeared immediately in nonsterile soils; other preparations

could still be detected after a few days. In sterile soils antibiotics are preserved for
considerably longer periods of time than in nonsterile soils. As can be seen from Table
119, the majority of active substances disappear in the first 2-3 days in nonsterile soils,
while in sterile soils they are preserved for 2 -3 months.

Similar data are obtained upon introduction of crude antibiotics in the soil. Korenyako,
Artamonova and Letunova (1955) introduced active actinomycetal substances in the
form of culture liquids into podsol, chernozem, krasnozem and serozem soils. About
700 crude preparations were examined, 12 of them, in greater detail. The results are
given in Table 120.

Table 120
Preservation time of crude antibiotics in soils
(days)
Ser- Cher- Kras-
Podsol, Ser- Cher- Kras-
Podsol, ozem, nozem, nozem,
Antibiotics non- ozem, nozem, nozem,
sterile non- non- non-
sterile sterile sterile sterile
sterile sterile sterile
A. violaceus strain
20 5 20 5 20 5 10 5
1806
A. aurauntiacus
180 8 180 8 180 8 100 8
strain 1149
A. globisporus
180 8 180 8 180 8 5 2
strain 81-B
A. globisporus
180 25 180 25 180 25 180 25
strain 76
A griseus strain
180 2 180 2 180 2 20 2
2535
A. griseus strain
120 20 120 20 120 20 120 20
2392
Control soils 0 0 0 0 0 0 0 0

The soil solution exerts a slightly inactivating effect. We tested a solution obtained by
using a strong press from incubated samples of podsol, serozem and chernozem soils,
with 4 antibiotics: penicillin, globisporin, preparation No 15 (grisein) and preparation
No 1609. The soil solution was added in various amounts to the antibiotic solution of a
known titer and after a certain period (1-5 hours or more) the activity of the preparations
were examined. The results were not always clear-cut, however, reliable data were
obtained in experiments with grisein and, preparation 1609 and in some cases also with
penicillin. The solution from incubated podsol soil inactivated antibiotics to a lesser
degree than solutions obtained from serozem and chernozem. One ml of solution
extracted from podsol neutralized 20-30 units of preparation No 1609, 5-7 units of
grisein and 2-5 of penicillin. Soil solution from incubated serozem inactivated 50-60
units of preparation No 1609, 7-10 units of grisein and 10-12 units of penicillin.
Solution of incubated chernozem inactivated 80, 15 and 25 units, respectively. In
addition it also inactivated approximately 5-7 units of globisporin.
 The inactivating force of soil solution is closely related to the species makeup and
metabolism of the microorganisms. If one incubates soil in the presence of' small
amounts of organic substance and in addition inoculates the soil with certain bacterial
species, the solution thus obtained would inactivate considerably more antibiotics. We
incubated podsol soil with various bacterial species (sporeformers and
nonsporeformers) with an admixture of various sources of organic nutrition (soy meal,
clover, hay, corn extract, peptone), The greatest effect was obtained in an experiment
with a nonsporeforming bacillus (strain No 6) which was incubated in soil with soy
meal. The solution obtained thereof (1 ml) inactivated 100 units of preparation No 1609
and up to 50 units of penicillin, but did not inactivate grisein or globisporin. The latter
was inactivated by solution from soil incubated with bacterium No 15 in the presence of
clover hay.

The inactivating action of the soil solution was observed by Waksman and Woodruff
(1942), They examined the effect of actinomycin in pure solution and with an admixture
of a humus extract. A culture of Bac. mycoides was killed in the former case by 1 µ g
and in the latter case, by 10 µ g or more of the substance. A similar effect was observed
by Skinner (1956) while studying the antibiotic obtained by him from Actinomyces
albido-flavus.

Inactivation of antibiotics in soil is probably mainly by products of microbial

metabolism. It has been shown, that with increase of the latter, destruction of antibiotics,
is enhanced. In the above-mentioned experiments, in soil incubated together with soy
meal, the growth of bacteria was very intense and their composition differed from that
in soil with clover or peptone.

Winter and Willeke (1951 a, b) introduced penicillin into composted soil rich in humus
and into loamy soil poor in organic substances. In humus soil, where there was abundant
growth of microbes, the antibiotic disappeared after 2-3 hours. In soil poor in humus the
same antibiotic was preserved for 12 hours. If the soil microflora is removed by
sterilization, penicillin in such soil is preserved for more than 3 days, while in nonsterile
soil it disappears on the second day.

We have demonstrated the inactivating role of the soil microflora in experiments with
pure cultures of bacteria and actinomycetes. It was found, that the antibiotics, penicillin,
mycetin and streptomycin are inactivated in different degrees by metabolic products of
various species of bacteria and actinomycetes. Some species or strains of bacteria
inactivate streptomycin more strongly, while others inactivate penicillin and mycetin
more strongly. There are cultures the metabolic products of which enhance the activity
of antibiotics (Krasil'nikov and Nikitina, 1951).

The data given here change our concepts on the preservation of antibiotics in soil.
Antibiotics are not always fully inactivated in the soil. Depending on the soil-climatic
conditions and also an the chemical properties of the antibiotics themselves, they may
be preserved and accumulate in the soil.

Part IV, continued:

 Entry of antibiotics into plants

The importance of antibiotic substances formed in the soil, for plants, is determined
primarily by the extent to which the former enter the plants via the roots and by their
activity within the plant.

The question of whether antibiotic substances are capable of entering the plants is now
answered in the affirmative. Vegetating plants absorb various organic substances
through their roots including antibiotics formed by bacteria, actinomycetes and fungi.
The absorption by plants of subtilin, gramicidin, pyocyanine, licheniformin, (antibiotics
of bacterial origin), as well as streptomycin, globisporin, aureomycin, terramycin,
grisein, griseofulvin, etc (substances formed by actinomycetes) has been experimentally
established.

Table 121 shows the degree of entrance of chemically pure antibiotic preparations,
from solutions into the plant, via the root.

Table 121
Assimilation of antibiotics by plants
(units per 1 g tissue)
Wheat, Wheat, Wheat, Peas, Peas, Peas,
Antibiotics
roots stems leaves roots stems leaves
Penicillin 150 60 60 150 80 100
Streptomycin 100 20 20 100 20 30
Grisein
(preparation No 120 30 30 150 20 40
15)
Mycetin 100 5 0 100 10 0
Subtilin 300 0 0 80 10 5

Penicillin enters the plant at the fastest rate and in the largest amounts, followed by
grisein and streptomycin. Mycetin subtilin and gramicidin enter in small amounts and
do not travel high up in the plant.

Aureomycin, terramycin, globisporin and many other active substances enter readily
into the plants.

Plants absorb from the substrate not only chemically pure preparations but also crude
antibiotics, in the form in which they are excreted by their producers. We added a liquid
containing antibiotics to a culture solution and grew plants in it. After a certain time one
could detect antibiotics in the tissues of the latter (Table 122).

Table 122
Uptake of crude antibiotic substances from culture fluids by plant roots
(units/ml)
Microorganisms producing Introduced Found in Found in Found in Found in
 antibiotic substances into the soil wheat roots wheat leaves pea roots pea leaves
A. streptomycini 250 60 30 80 40
A. aureofaciens 600 120 50 100 40
A. grisseus strain 80 100 20 25 50 25
Bact. nitrificans 120 45 10 40 10
Bact. fluorescens 200 40 5 40 35
Bact. denitfricans 90 10 10 15 5

Antibiotic substances enter plants from solid substrates, directly from soils.

In our experiments (Krasil'nikov 1951 a; 1952 b, c; 1953 c; 1954 a) we have tested

various antibiotics formed by bacteria, fungi and actinomycetes-antagonists. Chemically
pure preparations were introduced under adult plants in containers with sand or soil and
their concentration was determined after given time intervals on the root tissues and
aerial organs. The experiments were performed both under sterile and nonsterile
conditions of growth. In Table 123 are given the data from the experiment with the
sterile sand substrate. Streptomycin 500 units and grisein (preparation 15), 500 units
were introduced per 1 g of substrate.

Table 123
Entrance of antibiotics into plants from sterile substrates
(units/g of tissue)
Kidney Kidney
Plant Peas in Wheat in Peas in Wheat in
Antibiotics beans in beans in
origin sand sand soil soil
sand soil
Grisein Root 150 250 100 80 70 50
Grisein Leaves 80 120 60 50 40 20
Streptomycin Root 220 160 120 100 80 30
Streptomycin Leaves 100 80 40 40 40 40

As can be seen from the table, antibiotics enter into the plants via the roots in quite
large quantities. They are found in roots, stems and leaves for 10 days and more.

In experiments performed with sterile soil (Moscow area podsol) the same results were
obtained. The antibiotics, globisporin, 500 units and grisein, 800 units were introduced
per 1 g of substrate. Peas, kidney beans and wheat were grown on this soil. Analyses
have shown, that globisporin was preserved in soil for 30 days and grisein for 40 days.
During this time they penetrated through the roots and into the plants in smaller or
larger quantities (Table 123). The rate of entry of antibiotics from sterile soil was only
slightly less than the rate of entry from nutrient solutions or from the sand substrate.
After a few hours (6-10) the antibiotic substances could be found in the roots and in the
lower parts of the stem.

Our experiments have shown, that antibiotics are also absorbed by plants from natural
nonsterilized soil. The same antibiotics, grisein and globisporin, were introduced into
vessels with podsol soil under adult plants (wheat and kidney beans) in doses of 800
units/g. After 24 hours and sometimes even later we detected these antibiotics in the
tissues of the roots and aerial organs (Table 124).

Table 124
Entrance of antibiotics into plants from nonsterile soil
(units/g of tissue)
Kidney Kidney
Wheat, Wheat, Peas, Peas,
Antibiotics beans, beans,
roots leaves roots leaves
roots leaves
Grisein 20 10 30 10 30 20
Streptomycin 30 10 20 6 40 20
Aureomycin -- -- 20 4 20 6
Terramycin -- -- 30 10 20 6
Control plants 0 0 0 0 0 0

Plants also absorb crude antibiotics from the soil. We tested three native antibiotics
formed by the actinomycetes No 290, 287 and strain "B." In the presence of the
appropriate organic matter in the soil, these cultures, as is shown above, form 30-120
u/g of antibiotic substances. When we grew peas and wheat in such soil, we could detect
the antibiotics in the tissues in small, but sufficient quantities which were large enough
to inhibit growth of microbes sensitive to them (Table 125).

Table 125
Assimilation of crude antibiotics from the soil by plants
(units/g of tissue)
Peas, Peas, Wheat, Wheat,
Antibiotics
roots leaves roots leaves
No 290 10 2-3 4 +
No 287 15 3 10 2
"B" 2 + 4 +

The "+" sign designates small quantities detected only qualitatively.

Thus, in pea plants, 2-15 units/g antibiotics were obtained and in wheat somewhat less.
In some cases, when there is an abundant growth of antagonists which form large
amounts of antibiotics in the soil (100-150 units/g), large quantities of the latter enter
the plants-up to 20-30 units/g in the roots and 10-20 units/g in the leaves.

The zones of growth inhibition of the test organism around pieces of tissues of the
experimental plants may be seen in Figure 96. Around the shreds of tissue of the control
plants no such growth-inhibition zones are formed.
Figure 96. Entrance of antibiotics from the soil into plant tissues. Zones of lack of
growth of bacteria around pieces of tissues:

1--roots; 2 and 3--stems (lower and upper parts); 4--leaves; 5--roots of control plant,
grown in soil without antibiotics, the zone is missing.

It should be noted that plants may not only absorb the antibiotics that exist in the free
state from the soil but also those adsorbed by soil particles. It was stated earlier, that a
considerable part of the antibiotics are adsorbed immediately after their formation and
become tightly fixed. Adsorbed antibiotics cannot be washed,out with water or with a
number of organic solvents, even upon prolonged treatment. However, due to the
activity of their root systems, the plants, are in the position to sever this, bond between
the antibiotics and the soil particles and to desorb and take up the antimicrobial
substances.

We introduced streptomycin, up to 2,000 units/g and more into the soil (podsol,
garden) sometimes to full saturation, and then rinsed the soil with water until no more
antibiotic was found in the elutions.

After this treatment about 1,500 units of streptomycin per gram remained in the soil.
Plant seedlings of peas or wheat were planted in such soil and after some time their
tissues were analyzed for the presence of the antibiotic. Usually, after 3-4 days and often
even after 30-40 hours streptomycin was found in the roots, stems and leaves in
amounts up to 10-15 units/g and more. As a rule, soil analyses have shown the absence
of the free antibiotic; the latter was probably in the adsorbed state and was actively
absorbed by the roots.

Antibiotic substances entering plant tissues, enhance the bactericidal effect of their sap
and thus increase the resistance of the plants to diseases.

The more abundant the growth of antagonists in the soil, the more antibiotic
substances they produce, the more of the latter enters the plants and the stronger the
bactericidal effect of the sap becomes.
 The sap of plants which are grown in a sand substrate without microorganisms and
without humus is, according to our observations, less bactericidal than the sap of plants
which are grown in nonsterile soil rich in humus.

We enriched the soil artificially with actinomycetes--producers of streptomycin, and

we grew in this soil plants--peas and wheat. The sap of such plants was tested for its
bactericidal effect on Bac. mycoides and Staph. aureus. Death of the bacterial cells in
the sap of the experimental plants followed after 8-12 hours, and in the sap of the
control plants which were grown in soil not enriched with actinomycetes, there was only
suppression of growth, but death of the bacteria was not observed.

Plants grown in soil well fertilized with manure or compost had more active sap than
the sap of plants taken from unfertilized soil. The sap of plants grown in a greenhouse
(corn) was less bactericidal, than the sap of plants, grown in the open ground in the
same soil (Krasil'nikov and Korenyako, 1945 a). Eaton and Rigler (1946) observed
increased resistance of roots of cotton plant to Phymatotrichum ornnivorum upon
treatment of the seeds with carbohydrates. In these cases, according to the author,
intense growth of bacterial antagonists in the rhizosphere of the plants was observed.

Kublanovskaya and Brailova (1954), studied the bactericidal properties of the sap of
the cotton plant in relation to the fungus Fusarium vasinfectum and found that its
fungitoxicity toward the given fungus was less when the plants grew in soil with
antibiotic substances than the fungitoxicity of the control plants growing in soil without
antibiotics. The coefficient of multiplication of the fungus in the sap of the control
plants was: 13.6 in the germination phase and 11.8 in the phase of cotyledons; in the sap
of experimental plants: 7.8 in the phase of germination and 9.8 in the phase of
cotyledons. As is seen, the antifungal properties of cotton-plant sap are strengthened at
the expense of the antibiotic substances coming from the soil. Accordingly, the plants
morbidity due to wilt was less: in the control 96% of the plants were diseased, and
among the experimental plants--only 18.4%. Enhancement of antifungal properties of
plant sap was also observed by Kublanovskaya in field experiments on plots fertilized
with actinomycetal cotton-cake composts.

Stapp and Spicher (1954, 1955) observed the appearance of protective substance a in
the sap of the potato in relation to Bact. phytophthorum during the development of the
plant, when the soils were enriched with microbial antagonists.

The data given show that the plants absorb antibiotic substances from the soil.
Antibiotics may be absorbed by the plants not only from solutions of chemically pure
substances but also from a complex organic mixture of metabolites, the microbial
antagonist.

Actinomycetes, bacteria and fungi which produce antibiotic substances, grow in the
soil in the rhizosphere of plants. They saturate this zone or microfoci in the soil with the
products of their metabolism, including antibiotics. The latter enter the plants through
the roots and exert their action there. It is self-evident that the concentration of
antibiotics in soil, when formed under natural conditions, will be lower than the
concentrations created upon artificial introduction. However, under natural conditions,
these substances are constantly formed and therefore one would assume that their
entrance into plants is not stopped during the whole vegetative period.

Having entered the plant tissues the antibiotic substances protect them against the
penetration of microbial parasites, suppress the growth of those that have already
invaded, produce or elevate the toxicity of the plant sap and thus elevate to a larger or
smaller extent the immunological properties of the plant.

In other words, microbial antagonists are factors which increase the resistance and
insusceptibility of plants to infections.

Antibiotic substances as a therapeutic means in plant cultivation

Suggestions on the possible use of antibiotic substances for medical purposes were
made by Pasteur, Mechnikov and their contemporaries. Scientists attempted to use
bacterial cultures together with their metabolic products for curing the sick. Fehlesein
(1883) described a case of curing lupus by introduction of Streptococcus erysipelas into
the patient's skin. Colley (1893) used the same organisms for the treatment of a cancer
patient. Pavlovskii (1887) introduced a culture of Pneumococcus into the bodies of
animals and so prevented them from being infected with anthrax. Bourchard (1889)
used the metabolic products of Pseudomonas pyocyane against anthrax. Manasein
(1871) and Polotebnev (1872) used the green mold Penicillium in treatment of patients.
Many other specialists attempted to use microbial cultures for the purpose of medical
treatment. A special branch of medicine, bacteriotherapy even came into existence (cf.
Kashkin, 1952; Ermol'eva, 1946; Waksman, 1947; Waksman and Lechevalier, 1953;
Kohler, 1955; Korzybski and Kurylowicz, 1955 and others).

All these investigations had no proper success and recognition and were soon dropped.
Only after active substances--penicillin, streptomycin, etc were isolated and chemically
purified, did the antibiotic substances produced by microbial antagonists receive general
recognition.

Presently, many antibiotic substances, penicillin, streptomycin, aureomycin,

terramycin, subtilin, etc are widely used in therapeutical medicine and veterinary
science. This successful use of these preparations in medicine naturally served as a
stimulus for work on the use of antagonists and their metabolic products against
infections of plants.

In the foregoing chapter we have shown the beneficent role of microbial antagonists,
their inhibition of phytopathogenic organisms in the soil and then protection of plants
against fungal and bacterial infections. We noted there that microbial antagonists
remove phytopathogenic forms directly from the soil and by virtue of this alone protect
plants from diseases.

However, the antibiotic substances obtained from cultures of antagonists may be used
for the removal of phytopathogenic organisms not only from the soil but also within the
plant. In other words, these substances should be used as curative remedies.
 What then should be the requirements, in this case, from the antibiotics?

As in the case of treatment of human beings and animals, antibiotics used in plant
growing should: 1) be active against the agent causing the plant disease and have the
ability to inactivate toxins; 2) should penetrate easily into the plant tissues; 3) should
not be inactivated too rapidly; 4) should exhibit antibacterial activity within the plant
tissue; 5) should not be harmful to the plants at concentrations which are toxic to
bacteria.

In addition, the method of use should be technically possible and all the measures
should be economically profitable.

The first point was, in principle, proven experimentally. It was established, that
phytopathogenic bacteria and fungi are susceptible to the inhibitory action of antibiotics.

As was noted above, for each phytopathogenic microbe it is possible to choose its
corresponding antagonist and to obtain its antibiotic substances. Among the immense
variety of microorganisms existing in nature, producers of antibiotics against bacteria,
fungi, actinomycetes, viruses, etc can always be found.

Concerning the second postulate--the ability of antibiotic substances to penetrate the

plants, this question too was answered in the affirmative. In the previous chapter it has
been shown that these substances easily enter into the root system and proceed to the
aerial parts. Antibiotics can also be introduced through the aerial organs--stems, leaves
and seeds.

The possibility of introducing drugs into plants via the stem, was shown by Shevyrev
in 1903. He introduced various antiseptics into fruit trees with the aim of killing
parasites. The method developed by him is nowadays often used for extrarhizal
nourishment of plants. This method is as follows: a hole is bored in the trunk of the tree
and one end of a wick (of gauze or cotton) is placed in the hole; the other end is
immersed in a bottle which contains the antibiotic solution; the antibiotic enters the
trunk of the tree through the wick and spreads to all parts of the plant.

In grassy plants antibiotics may be introduced via the stem by simply wetting it or
smearing a paste containing the preparation on it. We used the first method. A wetted
piece of cotton or gauze was wrapped around the stem of the plant and covered with
wax paper, to prevent rapid desiccation.

We tested the method of introducing antibiotic substances through the trunks of trees
on various varieties of fruit-bearing and decorative plants and under different climatic
conditions--in Crimea, Caucasus and Moscow (Krasil'nikov and Kuchaeva, 1955).
Various antibiotics were introduced into the plants--penicillin, streptomycin,
globisporin, aureomycin, terramycin, grisein and other chemically purified preparations.
In a few cases we also introduced crude antibiotic substances in the form in which they
appear in the culture fluid.

Experiments have shown that all these antibiotics can enter the plant via the trunk, but
in different amounts and at different rates. Penicillin enters at the most rapid rate (see
above). Most of the experiments on absorbability were performed with it (Table 126).

Table 126
Entrance of penicillin into plants upon introducing it via the stem
(July-August 1954)
Time
of Detected Detected
Anti-
Diameter intro- Solution in lower in upper
Age, Height, biotic
Plants of trunk, duction adsorbed, branches branches
years meters units
cm of anti- ml and and
adsorbed
biotic, leaves leaves
days
Maple (acer
15 3 8 5 20 200,000 - -
platanoides K.)
Ash tree
(Fraxinus 8 4 7 5 15 150,000 - -
chinensis L.)
Lime tree (Tilia
5 25 7 5 10 100,000 - -
cordata Mill.)
White acacia
(Robina
8 2.8 7 5 10 100,000 - -
pseudo- acacia
L.)
(Halimodendro
n argenteum 15 1.8 6.3 5 0 -- - -
Fisch.)
Cherry
(Cerasus 9 4.5 6.5 4 430 4,300,000 + -
vulgaris Mill.)
Bird cherry
(Padus
15 4 5.8 5 210 2,100,000 + -
virginiana
Roem)
Apple (Malus
domestica 8 3.2 7.3 3 380 3,800,000 + +
Borkh)
Peach (Persica
8 1.5 6.0 5 560 5,600,000 + +
vulgaris Mill.)
Apricot
(Armeniaca 7 1.6 5.4 5 900 9,000,000 + +
vulgaris Lam.)
Sweet cherry
(Cerasus avium 7 2.1 6.1 5 165 1,650,000 + +
Moench.)
 As seen from the table, various woody plants absorb different amounts of penicillin.
Some of them, as for example, cherry, sweet cherry, apple, peach and apricot trees
absorb antibiotics in large quantities, others like maple, ash tree and lime tree absorb
little of it.

The distribution of the penicillin within the plant also differs. In some plants (cherry,
apple, peach, etc) it moves quickly to all parts, into the branches and leaves of the whole
crown; in other plants (bird-cherry tree and Halimodendron) it slowly reaches only the
lower parts of the branches and leaves and does not reach the upper part of the crown; in
still other trees (maple, ash tree and lime tree) it cannot be detected in the leaves at all.

The intensity of the uptake and the distribution of antibiotic substances in the plant
changes noticeably with changes in climatic conditions--temperature, air humidity, soil
moisture, etc. The lower the temperature and the higher the humidity of soil and air, the
slower is the uptake of antibiotics. For example, in May 1954 in the Nikitskii Garden,
when the average temperature of the month was 13.4° C the temperature of the soil
14.2°C and the relative air humidity 92%, peaches and apricots absorbed 45-50 ml
penicillin solution each on the first day and during 5 days--100-110 ml. In August, the
average daily temperature of the air was 24.3° C the soil temperature was 23° C and
relative air humidity was 41%, the same plants took up 200-210 ml on the first day and
during 5 days--about 1 liter of penicillin solution (Table 127). In May the absorption of
penicillin was 3-5 times less than that in August, although in the spring plant suction is
usually higher.

Table 127
Intensity of extrarhizal absorption of penicillin by plants under various weather
conditions
(the Nikitskii Botanical Garden, 1954) (in ml of absorbed solution of 10,000 unit / ml
activity)
Plant 1st day 2nd day 3rd day 4th day 5th day Total
May: temp of air 13.4° C, soil temp.
14.2° C, relative air humidity 92%
Apricot 45 30 20 10 5 110
Peach 50 30 10 5 0 95
Sweet cherry 35 15 5 1 0 56
August: sir temp. 24.3° C, soil temp.
23.0 C, relative air humidity 41%
Apricot 210 200 190 200 100 900
Peach 200 180 100 40 40 560
Sweet cherry 80 50 15 10 10 165

We obtained similar data in experiments with birch (10 years old) under the Moscow
climate. A globisporin solution was administered (activity 5,000 u/ml) to the same plant
via the stem on dry and on rainy days. Two repeated experiments were performed: the
first in June and the second in August. The amounts of antibiotic absorbed during 5 days
were: on dry days in June--1,500 ml, in August--900 ml and on rainy days of the same
months the corresponding absorption was 350 and 200 ml, i.e., 4.5 times less.

In experiments with lemon trees in the orchard of the Institute of Subtropical Cultures
(Anaseuli) in the rainy period in September 1952, the antibiotic grisein was absorbed to
such a low degree that the work had to be postponed until drier weather set in.

The rate of distribution of antibiotics within the plant corresponds to the intensity of its
absorption. The faster, and the more antibiotic enters, the sooner it is found in the
different parts of the plant. In experiments with plants of the Nikitskii Garden we
introduced a penicillin solution into trees and the rapidity of its appearance in the leaves
'was measured. Each day after the introduction of the solution 20-30 leaves were
removed from the tree and analyzed separately for the presence of the antibiotic in
them. Table 128 shows the percentage of leaves in which the antibiotic was detected.

Table 128
Rapidity of the distribution of the absorbed penicillin in the tissues of plants
(the figures designate the percentage of leaves saturated with the antibiotic)
Upper
Lower Upper Lower Upper Lower
Quantity part of
part of part of part of part of part of
adminis- crown
Plants crown crown crown crown crown
tered in after
after one after one after two after two after three
mg three
day day days days days
days
IN MAY:
Apricot 45 0 0 41.6 33.3 90 13
Peach 50 0 0 50 25 55 18
Sweet cherry 35 0 0 0 0 60 0
IN AUGUST:
Apricot 200 100 100 100 100 100 94
Peach 210 100 100 100 100 100 100
Sweet cherry 80 100 100 100 100 100 75

In August in dry warm weather, the antibiotic is rapidly distributed throughout the
whole tree. It can be found everywhere a few hours after its introduction. In May there
was cool rainy weather in Crimea. The uptake of the antibiotic and its distribution in the
tissues was very weak. Only 2 days after introduction could one detect the antibiotic in
the plant's leaves, and then only in some leaves.

The entrance of the said substances into the plants is connected with the physiological
conditions of the latter. The more intense the metabolic processes of the plants, the more
vigorous their growth, the faster are the antibiotics absorbed. When the external factors
slow down the growth of the plant, the inflow of antibiotics into the roots also slows
down. It was noted above, at a lowered air temperature the uptake of active substances
is much more sluggish than at a higher temperature in the summer. The same was
observed by Stokes (1954) in her work. She determined the rate of uptake of
griseofulvin by plants at different temperatures. At 25° C the substance enters the plant
5 times as fast and in larger quantities than at 10° C. She also observed the detrimental
effect of excessive humidity on the uptake of the antibiotic. At a 56% relative humidity
its concentration in the tissues is 4 times higher than that at a humidity of 91% and a
temperature of 25° C.

Antibiotic substances taken up by the root system, are transported via the xylem to the
aerial parts, the leaves. If, however, the antibiotics are introduced through the leaf
surface, their transportation is accomplished through the phloem, i.e., as in case of
substances synthesized in the leaf.

Antibiotic substances entering the plant, penetrate inside the cells and cause a certain
effect there. In order to follow the penetration of these substances into the cells we used
antibiotics which were luminescent in ultraviolet light. Mycetin and certain other
substances belong to these antibiotics.

We allowed the antibiotic solution to pass through the tissues of the plant, we then
performed microscopic analyses of microtone slices. Mycetin first enters into the
cytoplasm, staining the various granules and rodlike mitochondria and then enters the
nucleus, where it reaches higher concentrations than in the cytoplasm

Some of these substances at certain concentrations inhibit nuclear division upon

entering the cells.

Pramer (1955) followed the penetration of antibiotics--penicillin, streptomycin and

chloromycetin into the cells of the algae Nitella Clavata. These algae were immersed in
a solution of the antibiotic for some time, were then washed thoroughly, and the cell
juice which was squeezed out of the individual cells was collected with a micro-pipette.
In this juice the content of the antibiotic which was introduced into the algae was
determined.

It was found that streptomycin and chloromycetin penetrate the membrane, reach the
inside of the cells, and spread throughout the protoplast giving the latter bactericidal
properties.

Penicillin, according to the author, is not detected inside the cells. It either does not
penetrate them or, if it does, is immediately inactivated.

Nielsen (1955) found that antibiotics formed by the plankton of water reservoirs
suppress the photosynthetic activity of algae of the Chlorella group.

There are theories which state that upon introduction of various substances into the
trunk of a tree, they spread in a sectoral fashion, corresponding to the vascular transport
system.

Taking this in account, we paid special attention to the distribution of antibiotics in the
periphery of the bark of woody plants. The administered antibiotic was determined in
the leaves and branches located in various parts of the crownaccording to sectors and
circular rings-in the lower, middle and upper parts.
 Numerous analyses show quite clearly, that penicillin, streptomycin, grisein and other
antibiotics are distributed more or less evenly throughout the crown of the plant. We
observed no sectoral distribution of the substances introduced in fruit, ornamental or
forest trees.

Certain antibiotics enter the root system from above and travel downward. According
to our observations, grisein possesses this property. When it is introduced into the stem
or the trunk of a lemon tree it can be found after a certain time in the lower part of the
trunk and in the roots. The tissues contained: in the trunk, at the point where the
substance was introduced 120 units/g, near the root 60 units/ g and in the roots-30-50
units/g.

The method of administering antibiotics through the intact stem by the use of gauze
and cotton bandages, was employed by us in experiments with grassy plants and with
shrubs; it was also tested with woody plants. Young branches of garden roses, apple
trees and pear trees, stems of peas and wheat were wrapped with cotton (or gauze)
wetted with a solution of penicillin, streptomycin, grisein or another preparation; after a
lapse of some time, the plant tissues were subjected to analysis. As the investigations
have shown, these substances penetrated inside the plants, but never accumulated in
high concentrations. This method is thus hardly suitable for wide use. However, it may
be used for local therapy.

Introduction of antibiotics through the leaf surface has been performed in experiments
with woody and grassy plants. The leaves of the plant were either sprayed with a
sprayer or wetted with cotton.

The spraying of the crown of plants with antibiotic solutions, using a sprayer was
employed by us in experiments with fruit trees: peaches, apricots and apple trees and
with grassy plants; peas, corn, wheat, etc. Preparations of penicillin, streptomycin and
grisein in dilutions of 1:1,000-1:5,000 were used. After some time these antibiotics were
determined in the tissues of leaves, branches and stems. Before analysis the severed
leaves and branches were thoroughly washed in water.

Analysis showed the following amounts of antibiotics (in 1 gram tissue):

Streptomycin in units /
Penicillin in units / g
g
Apple tree up to 5 2-3
Sweet cherry 15 5-10
Peach 10 10
Apricot 40 20-40 (grisein)
Peas up to 20-50 10-20
Wheat 5-10 2-10

The greater the distance from the location of the antibiotic administration, the lower its
concentration in the organs of the plant.
 Upon wetting leaf surfaces with pieces of cotton soaked in a solution, even more
convincing results were obtained. Two to five hours after the application of the cotton
bandages with the antibiotic, the latter could be detected in the leaf tissue which were
quite removed from the spot of its application as well as in the petioles of leaves, and
even in the tissues of the branches which bear those leaves.

The American specialists use antibiotics in the form of dust, spraying them on the
crown of the plants. The dust particles reaching the surface of the leaves, dissolve and
penetrate into the tissues.

It should be pointed out that in all the experiments with the various methods of
introduction of antibiotics, the antibiotics move in the direction of the lower parts as
well the upper parts of the plant. Upon introduction of a solution of penicillin or grisein
through the trunk of an apricot tree, these substances were detected in the branches,
leaves and root tissues as well. The same was observed with peas. A preparation of
penicillin introduced through the stem surface, was subsequently found in leaves in the
upper parts in a concentration of 5-10 units/g and in the roots, in a concentration of 3-5
units/g (Table 129).

Table 129
Distribution of antibiotics in tissues of peas upon their introduction through
the stem
(u/g of tissue)
Antibiotics Roots Stems Leaves
Penicillin 3-5 10-20 5-10
Grisein 3-5 10-15 3-5
Streptomycin 1-3 10-30 3-5

Mitchell, Zaumeyer, Andersen et al., (1952, 1954) introduced antibiotics, applying

them with lanolin paste. The paste with the antibiotics was spread on the stem surface,
and after some time the active substance was determined in the tissues of branches and
leaves. Brian, Wright et al., (1951) observed the penetration of the antibiotic
griseofulvin into plants. Leben, Arny and Keitt (1953) introduced helixin and antimycin
into plants, and Hessayon (1951) introduced trichothecin--an antibiotic obtained from
the fungus Trichothecium.

Antibiotics can be used for the sterilization of infected seeds. It is known that in plant
seeds there are often phytopathogenic bacteria and fungi which are sources of plant
diseases. In order to get rid of these agents, various chemical substances are used--
antiseptics. However, the antiseptics which inhibit the growth of microbes also act
deleteriously on the seed tissues and decrease their ability to germinate.

The antibiotics, unlike the antiseptics, act selectively, inhibiting microbial metabolism
without causing any harm to the seed embryo. The sterilizing effect of antibiotics was
tested by us on cotton seeds. It is sufficient to immerse the seeds for 4-8 hours in an
antibiotic solution in order to kill the microbes in the seed tissues (Krasil'nikov,
Mirzabekyan and Askarova, 1951; Askarova, 1951: Mirzabekyan, 1952).
 Blanchard and Diller (1951) treated leguminous seeds with aureomycin, allowed them
to germinate and then determined the entrance of the aureomycin into the seedlings. The
authors noticed a larger accumulation of the antibiotic in the roots than in the upper
parts.

The effectiveness of antibiotics depends on their concentration in the tissues. In turn,

the concentration depends on the properties of the plants, especially on the properties of
the antibiotic and also on external conditions.

It was found that. when the solution of antibiotic is concentrated, more of it penetrates
the plant (Table 130).

Table 130
Degree of saturation of plants with antibiotics
(units/g of tissue)
Introduced into Wheat Wheat Pea Pea Corn Corn
Antibiotics
substrate units/ml roots leaves roots leaves roots leaves
Penicillin 5,000 3,500 3,000 4,000 3,800 5,000 4,000
1,000 600 500 500 300 800 300
500 200 100 300 160 180 80
100 70 40 50 30 60 25
50 80 40 50 40 80 70
Grisein No 15 1,000 800 600 500 400 950 600
500 250 160 300 180 300 100
100 70 40 80 50 80 50
50 50 40 60 40 80 85
10 30 20 50 30 50 30

Very high concentrations of antibiotics (penicillin--5,000 u/ml), grisein--1,000

units/ml) have a toxic effect on plants. They begin to wither and guttation stops. Smaller
concentrations are harmless; the plants develop normally.

Part IV, continued:

Winter and Willeke (1951, b) have shown that in tissues of lettuce, penicillin may
accumulate up to 500 units/g, and streptomycin, up to 100 units/g and cause no
noticeable pathological phenomena. When the penicillin concentration in the tissues
reaches 1,000-2,000 units/g, the plant suffers from poisoning.

Brian, Wright, Stubbs and Way (1951) point out that considerable concentrations of
griseofulvin in the tissues of oat and lettuce brought about no poisoning effect.
 If the concentrations of the antibiotics in the substrate are very low, the plants may
accumulate them in their tissues. For example, when the concentration of penicillin in
the medium is 50 units/ml, up to 80 units/g accumulates in the roots of or wheat and
corn; when the concentration of grisein is 10 units/ml in the medium, up to 20-50
units/g and more are accumulated in the roots of peas.

Pramer (1953-55) tested different antibiotics--streptomycin, aureomycin,

chloromycetin, terramycin, neomycin, etc. According to his observations, different
substances enter the plants and saturate their tissues to various degrees. In experiments
with cucumber seedlings, the most highly absorbed drug was streptomycin, At a
concentration in the solution of 500 µ g/ ml, its accumulation in the tissue reaches 100-
150 µ g/ g, 18 hours after treatment. Streptomycin penetrates less readily and in smaller
quantities the tissues of tomatoes and kidney beans. The author notes that sometimes the
concentration of streptomycin in leaves is higher than that in stems and roots.

Chloromycetin penetrates the cucumber seedlings much more weakly than does
streptomycin and reaches a concentration of 20-50 µ g/g. Aureomycin, terramycin and
neomycin do not penetrate this plant at all.

Pramer (1954), investigated the absorbability of streptomycin, chloromycetin and

penicillin into the cells of algae Nitella clavata and found that after the algae were
immersed for 12 minutes in a streptomycin solution which contained 8 µ g /ml, the
algae's cell sap contained the same concentrations of antibiotic as that of the external
solution. After immersion in the same solution for 18.5 hours the concentration of
streptomycin in the sap was 7 times higher than that of the surrounding solution.

Chloromycetin penetrated the cells of the algae with difficulty. Only after 24 hours was
it detected in the sap. Penicillin was not detected at all in the cells of the algae after 25
hours of immersion in a solution with 25 µ g/ml antibiotic concentration. The author
believes that penicillin penetrates the cells quickly, but is immediately oxidized.

The concentration of antibiotics in plant tissues depends not only on the amount of the
substances entering but also on the rate of their disappearance, or the time of their
preservation.

Antibiotics are known to be preserved in the body of animal organisms for a short time
only. They are excreted from the body during the first hours after their entrance, which
complicates the work with antibiotics in hospitals. Only with the aid of special
substances--prolongators--does one succeed in keeping the antibiotic in the animal or
human organism.

In plants the antibiotics which are introduced are preserved for much longer periods of
time. These very same substances -- penicillin, streptomycin, globisporin, aureomycin,
etc are preserved within the plants for several days or even weeks. For example in
tissues of sweet cherry, penicillin is preserved for 4 days and in tissues of the apricot
tree--16-17 days (Table 131).

Table 131
Time of preservation of penicillin inside woody plants
 Age
Antibiotic Time of
of
Plants indroduced, preservation Location where experiments were carried out
plant,
in units in days
years
Moscow, Central Botan. Garden Ac. Sci,
Cherry 8 2,400,000 8
USSR
Moscow, Central Botan. Garden Ac. Sci.
Apricot 5 2,200,000 16
USSR
Peach 8 4,800,000 15 Crimea, Nikitskii Botan. Garden
Apricot 7 6,000,000 17 Crimea, Nikitskii Botan. Garden
Sweet
7 1,450,000 4 Crimea, Nikiskii Botan. Garden
cherry

In grassy plants antibiotics are preserved for a similar length of time.

Grisein (No 15) when introduced into tissues of the cotton plant and peas, is preserved
there for 10-20 days (Table 132).

Table 132
Time of preservation of the antibiotic grisein No 15 in plants
(unit/g of tissue)
Time of analyses, number of Cotton, Cotton, Peas, Peas,
days after introduction roots leaves roots leaves
Initial amount 350 100 180 80
1 200 50 120 60
2 150 30 100 40
3 100 20 70 30
5 80 10 30 10
7 50 8 10 5
10 30 5 10 5
20 10 0 0 0
30 0 0 0 0

Askarova (1951) and Mirzabekyan (1953, 1955) found antibiotic substances inside the
cotton plant 20 days after their introduction.

Brian et al., (1951) detected griseofulvin in tissues of lettuce and oats during 3-4
weeks.

Antibiotics first disappear from the aerial parts of the plants and later from the root
system.

Analyzing a nutrient solution in which plants saturated with antibiotics have been kept,
we could establish that these substances were excreted by the roots. However, the
amount of the substances excreted was much smaller than that of the plant. If in the
tissues of one pea plant there were about 4,000 units of penicillin then about 600 units
were excreted into the solution.

In the experiment with streptomycin, single pea plants were immersed for one day in
streptomycin solution. The number of units of the antibiotic which were absorbed from
the solution were precisely determined, and the plant was removed from this solution.
The roots were washed with water and immersed in Hellriegel' s nutrient solution which
did not contain antibiotic. After certain time intervals the amounts of the antibiotic in
the solution and in the plant tissues were determined. The results are given in Table 133.

Table 133
Excretion of streptomycin from pea tissues into solution
Units
Units in in
Times of analyses, number of days Units in Units Units in
in the
after introduction solution inroots stems
leaves whole
plant
Initially 0 350 45 80 1,707
3 120 270 20 40 1,123
5 240 150 20 20 767
10 320 20 5 5 105.5
15 350 0 0 0 0

In these experiments one pea plant absorbed 1,800 units, after 10 days only 105 units
were found, and after 15 days nothing at all was left. During this time only 350 units
were excreted into the solution by the roots. Therefore, we assume that the remaining
1,357 units of antibiotic were assimilated by the tissues as sources of nutrition and
underwent biochemical changes.

Comparing the degree of absorption of the antibiotic with the nature of the distribution
of the latter in the plant, and with the time of its preservation in the tissues, one may
conclude that there exists a direct connection between these phenomena. The more the
antibiotic was absorbed, the sooner it was found in the leaves and upper branches, and
the longer it was preserved there.

However, this is not always so. Quite often, upon intense absorption of an antibiotic
the latter is not detected in the tissues or is found there in very small quantities. For
example, maple and bird cherry under similar conditions absorb the same amounts of
penicillin, but in the bird-cherry tree it penetrates into the upper parts and reaches the
leaves, while in the maple it is found neither in the leaves nor in the branches. In the
apricot, peach, sweet cherry and apple trees penicillin may be detected in the leaves
upon introduction of 350-500 thousand units per tree while in maple, ash tree, lime tree
and acacia it cannot be detected even when it is introduced in considerably larger
quantities.

We introduced 7-15 million units per tree of globisporin and penicillin into the trunks
of a 10-year-old birch and a 7-year-old willow--calculated on the basis of 300-600 units
per 1 g woody mass. After 36 hours penicillin and globisporin could be detected in the
leaves of the willow; the former was 1.5-2 times more concentrated than the latter
(Table 134). In birch the antibiotic was not found either in the leaves or in the branches.
However, traces of the antibiotic were detected in the wood of the trunk at a distance of
not more than 10-30 cm. upward and downward from the point of introduction.

Table 134
The uptake and distribution of penicillin and globisporin in tissues of birch and willow
(solutions introduced: penicillin--15, 000 units/ ml globisporin--10,000 units/ml)
Anti- Anti- Anti- Anti- Anti- Anti-
Units
Amount of biotic biotic biotic biotic biotic biotic
Type of per 1
anti- biotic Total units found found found found found found
Antibiotic/Type g of
solution per plant after after after after after after
of tree wood
introduced 10 20 36 48 72 120
mass
hours hours hours hours hours hours
Birch
Penicillin 1,000 15,000,000 600 0 0 0 0 0 0
Globisporin 800 8,000,000 250 0 0 0 0 0 0
Willow
Penicillin 850 13,600 500 0 trace 20 30 20 15
Globisporin 680 6,800,000 300 0 0 10 15 15 10

The absence of antibiotics in the leaves and branches of birch, maple, lime tree and
other plants may be explained by their inactivation or by the adsorption by the tissues
adjacent to the point of introduction, and, in certain cases, also by the weak uptake of
the solution. The last explanation does not apply in the case of the birch. As is seen from
the table, the birch absorbed more penicillin and globisporin solution than the willow,
but nevertheless, the antibiotic was not found either in the leaves or in the branches.

Investigation of the causes of this phenomenon has shown that the wood of the trunk
and branches and the leaf mass of birch possess a clearly expressed inactivating and
absorbing capacity in relation to the antibiotics tested. By specially devised methods we
have established, that one gram of wood of the trunk of birch absorbs 18,000 units of
penicillin and fully inactivates 6,000 units; it absorbs 6,000 units globisporin and
inactivates more than 3,000 units. A ground mass of green leaves absorbs 10,000 units
penicillin and inactivates 8,000 units; it absorbs 6,000 units globisporin and inactivates
5,000 units. In other words, the wood and especially the leaf mass of the indicated
plants almost completely inactivate the absorbed antibiotics--penicillin and globisporin.
In order to detect the antibiotics in the given tissues, it is essential that they be
administered in a higher concentration (more than 6,000 units globisporin and more
than 8,000 units penicillin per g).

Plant tissues probably inactivate all other antibiotics which enter them. We tested
streptomycin, penicillin, aureomycin and certain crude preparations of actinomycetal
origin on various plant tissues: apples, lemons, peaches, cherries, etc. In all cases a
different degree of inactivation was observed. Thus, tissues of lemon saplings (3-5 years
old) inactivated aureomycin within the limits of 50--100 units/g, the leaf tissue was a
stronger inactivator than the tissue of the trunk. The tissues of the apple tree and even
more so, those of ornamental woody plants inactivated aureomycin to the extent of 200-
500 units/g.

Inactivation of the crude preparation No 399 (from strain 399) in our experiments, was
as follows:

Tissue of the lemon-tree trunk; 70 units/g

Tissue of the lemon-tree leaves; 160 units/g
Tissue of the pear-tree leaves; 220 units/g
Tissue of the apple-tree leaves; 180 units/g

Antibiotic substances may be introduced into the plant through the aerial parts, not
only as chemically pure preparations, but also in their crude state, in the form of a
culture fluid which is diluted with water to a certain concentration. We introduced the
crude antibiotics via the trunk and through the leaf surface of woody plants and grasses.
Plant seeds were also soaked in crude substance seeds of wheat, clover, peas, etc.

Antibiotic substances introduced in the form of culture liquid are distributed in the
same manner as are chemically pure preparations, but at lower rates.

If seeds treated with antibiotics are immediately germinated, these substances may be
detected in the seedlings. This translocation of the antibiotics from the seeds to the
seedlings was observed by us in the cotton plant, peas and wheat. Special analyses have
shown that the antibiotics completely permeated the cotyledons of the leguminous
plants, as well as the endosperm of the cereals. Such a saturation of the food reserves
with antibiotics--penicillin, streptomycin, grisein and certain other substances, does not
cause any harm to the seedlings. The latter develop normally and utilize the reserves of
the endosperm or the cotyledons exactly as the control seedlings but only if the
antibiotic is not toxic.

Antibiotics introduced into plants have an antimicrobial action. If plants are artificially
infected with a phytopathogenic form of bacteria or fungus, and a corresponding
antibiotic is employed, the disease will not appear or will be weaker than it is in the
control. We have introduced many nonpathogenic bacteria inside plants--Bact. coli,
Bact. prodigiosum, Bact. album, Ps. fluorescens, Ps. sp., Rhizobium trifolii, etc. They all
died much more rapidly in the tissues of plants to which antibiotics were introduced.
For example, the root-nodule bacteria of clover, Bact. coli and Bact. prodigiosum,
introduced into the stem of peas or kidney beans, die there after 20-30 hours and later,
while the plants treated with streptomycin showed no bacteria after only 2-6 hours. The
phytopathogenic fungus Fusarium sp. spread on seedling of pine, grew well on them,
penetrated the inside and caused their death after several days. On plants that were
treated with the corresponding antibiotic (No 121), the given fungus did not grow, and
the growth of the plant was normal. There are many other observations which
demonstrate the antimicrobial action of antibiotics inside plant tissues.

The antibiotics used should not be toxic to the plants. It is known that among the
antibiotics there are various preparations, some of which are very toxic and cause the
poisoning of certain tissues or of the entire plant. To such antibiotics belong: gramicidin,
mycetin, clavacin, catenulin, magnamicyn, etc. Clavacinan antibiotic produced by the
fungus Asp. clavatus, inhibits the growth of cereal roots at a dilution of 1:1,000,000
(Wang 1948). Other antibiotics--penicillin, streptomycin, grisein, terramycin, etc--may
for all practical purposes be considered nontoxic. They may accumulate in tissues in
large quantities, do not cause any disturbances, and, at certain concentrations, even
stimulate the growth of plants (Barton and Mac Nab, 1954; Askarova, 1951), Scheffer
and Kloke (1954) introduced antibiotics into soil in which they later cultivated plants.
Only very high concentrations of the antibiotics caused the inhibition of the growth of
barley and rye.

There is no need to introduce over-large amounts of antibiotics in treatment. The

antimicrobial doses of the antibiotics of this group are considerably lower than the doses
which cause the poisoning of the plants. For example, penicillin inhibits the growth of
bacteria in wheat tissues at a concentration of 3-10 units/g, while the plant can
withstand a dose of more than 1, 000 u/ g. Streptomycin and globisporin inhibit growth
of bacteria in plants at concentrations of 5-10 units/g while the plants can withstand a
dose of more than 500 units/g, etc.

There are many antibiotics that occupy an intermediate position in relation to their
toxicity. Such antibiotics can also be successfully used in the healing practice.
Griseofulvin is one of them. Its therapeutic dose is 5-10 µ g/g. A dose of 20 µ g/g is
toxic for wheat and causes a burn and the swelling of the roots (Stokes, 1954).

One should also emphasize another feature of the action of antibiotics, namely their
ability to inactivate toxins formed by fungi and bacteria. Inactivation of toxic substances
by products of microbial metabolism was mentioned before (Krasil'nikov, 1947 a). It
has been shown, that the toxic effect of gramicidin can be eliminated by neutralizing it
with the metabolic products of bacteria. Actinomycetes inactivated the toxin which was
formed by the sporeforming bacteria, Bac. subtilis and Bac. mesentericus.

It is known, that in many infections (if not in all) the plants suffer from poisoning by
the toxins which are produced by microbes developing in the affected tissues.

Therefore, by selecting appropriate microbes these toxins can be rendered harmless

and thus the poisoning of the plant can be prevented. Clover seeds saturated with
bacterial toxin did not germinate in our experiments, or germinated to a limited extent;
their seedlings lagged in growth and soon perished. When the poisoned seeds were
treated at once with antitoxin, the percentage of germinating seeds increased
considerably and the growth of these seedlings was only slightly less than that of the
controls (Figure 97). Toxin, which was mixed with antitoxin in a certain ratio had no
toxic effect at all.
Figure 97. Antitoxic effect of metabolites (antitoxin) of bacteria on clover seedlings,
poisoned with bacterial toxin:

a--control; seeds of clover were not treated with toxin before sowing; growth normal;
b--seeds treated with toxic product, formed by bacterial inhibitors--there is no
germination or a weak one; seedlings soon perish; c--seeds treated with the same toxin
and then with the antitoxin substance produced by bacteria.

The inactivating effect of certain actinomycetes was observed by us in experiments

with toxins formed by the fungus Botrytis cinerea. This fungus grows abundantly on
leaves of certain plants--birch, oak, etc which are wetted by the carbohydrate excretions
of aphids. The toxic substances formed by the fungus affect the tissue of birch leaves.
Toxins of the given fungus were obtained by us in a culture in a nutrient medium with
honey produced from insects. Its application to the surface of a birch leaf causes burn
and necrosis of the tissue with subsequent yellowing and death of the leaf.

An antitoxin against this toxin which was formed by an actinomycete antagonistic to

Botrytis cinerea was found. The addition of a certain amount of antitoxin to the toxin
neutralizes it, and the mixture obtained is rendered nontoxic for birch. This antitoxin is
also beneficial when first symptoms of poisoning appear. The process of wilting and
formation of necrotic patches stops and the leaves recover from the injury and continue
to develop normally.

The antitoxic action of actinomycetes, or more exactly, the action of their metabolic
products, was observed by us in experiments with toxins formed by the fungi Fusarium
vasinfecturn, Fusarium sp., Trichothecium etc.

The phytopathogenic fungus--Deuterophoma tracheiphila causes the poisoning of

citrus plants by its metabolic products. An antagonist (A. griseus) of this fungus was
found among the actinomycetes, which produces antitoxic substances and inhibits the
growth of the fungus. The antibiotic grisein, obtained from a certain actinomycete, also
had suppressive effects on this pathogen, which is the cause of a disease of citrus plants.

It may be assumed that for any toxin of microbial origin an antitoxin can be found.

Among the microbes there are many species which form strong poisons not only for
plants but also for animals and human beings (botulin, tetanus toxin, etc). Under natural
conditions these toxins are inactivated by other microbes, which produce antitoxins. It is
tempting to use these antitoxins against food poisoning and other toxicoses of man and
animals.

As is seen from the above-mentioned data, antibiotic substances fulfill all the
requirements demanded of healing substances in plant breeding.

The possibility of using antibiotics for curative purposes was also proven by indirect
laboratory and field experiments.

The first experiments in this direction were performed with crude antibiotic
substances, obtained from bacteria and actinomycetes (Krasil'nikov, 1947 a). More
fundamental and systematic studies were performed with chemically purified
substances. Antibiotics were employed in the struggle against infections of woody and
grassy plants (Krasil'nikov, Mirzabekyan and Askarova, 1951; Askarova, 1951;
Mirzabekyan, 1952). Mirzabekyan employed the specially chosen antibiotic, grisein, in
treatment of apricot and peach trees suffering, from "bacterial wilt." This disease is
caused by Bact. armentaca. It is expressed in the withering of the crown first, and then
of the whole tree.

At first the experiments were performed on young one- to two-year-old saplings of

apricots and peaches. They were artificially infected with a culture of Bact. armeniaca
and a few days later, treated with antibiotics.

An aqueous solution of the preparation was introduced into the leaf surface by wetting
it. In all cases where the plants were treated with antibiotic immediately after
inoculation, the disease did not appear. In cases where the treatment was started after a
delay and when the symptoms of the disease, the wilting of leaves, were already
apparent the disease stopped developing, leaves and branches recovered, and the plant
continued to develop normally.

A hundred per cent of plants, that were not subjected to treatment, were infected and
perished (Figure 98).

Figure 98. Curative effect of antibiotics. Apricot seedlings infected with Bact.
armeniaca:
a and b--plants not treated with antibiotic; c--plants treated with antibiotic; d--control
plants (noninfected).

Experiments with fruit-bearing, 15- to 20-year-old plants were performed in the

experimental fruit section of the Academy of Sciences of the Armenian SSR and in one
of the fruit farms of Armenia.

The antibiotic grisein was introduced into the stem and was also sprayed on the crown.
The process of drying ceased after the treatment. The leaves and branches recovered and
continued to grow normally (Figure 99). In cases where the injury was a severe one and
where there were dying branches, an effect was also noticed. The antibiotic stopped the
further spread of the disease and new sprouts appeared on the still living parts.

Figure 99. Curative effect of the antibiotic grisein in "bacterial wilting" of apricots:

a--a tree not treated with the antibiotic; b--a treated tree.

Positive results were obtained upon the use of antibiotic substances in the struggle with
"malsecco" of citrus plants under laboratory conditions. The disease known as
"malsecco" is caused by the fungus Deuterophoma tracheiphila.

Young lemon trees artificially infected with the fungus easily succumbed to the
"malsecco" disease. In the struggle with this disease antibiotics were selected which
were later tested on experimental plants. The antibiotic solutions were introduced into
the trunk and through the leaf surface.

Among the preparations tested, grisein had a curative effect. The plants recovered
quickly or did not get sick at all, while the control plants which were not subjected to
the treatment, died,

In order to sterilize the grafts of lemons which were used as grafting material
Mirzabekyan (1955) saturated them with an antibiotic solution. Specialists have found,
that the infection is introduced with the grafting material, In the laboratory experiment it
was shown quite feasible to employ antibiotics in the practice of grafting plants; grafts
treated with streptomycin or grisein were sterile.

Among the grassy plants, antibiotics were most frequently used with cotton cultures,
affected by gummosis. This is a widespread disease and causes great losses to
agriculture. It is caused by the nonsporeforming Ps. malvacearum, which is distributed
and carried by the seeds. These often harbor the bacteria internally, thereby considerably
complicating the struggle against infection.

Askarova (1951-52) preliminarily chose a number of antibiotics which inhibit Ps.

malvacearum and she discovered that some of them (Nos 73/20, 160, 114, etc) penetrate
the seeds of cotton freely and kill the bacteria which cause gummosis. These substances
therein saturate the tissues of the entire seed and embryo but do not harm them. The
ability of such seeds to germinate does not decrease and in some cases (preparations
160, 265) it even increases. At first the experiments were performed in growth
containers and later on experimental plots and on industrial fields. Only crude
antibiotics in the form of culture fluid were employed. The activity of this fluid was
1,000-2,000 units/ml.

The results were positive. Seeds treated with antibiotics germinated better and their
shoots were taller and healthier. There were fewer diseased plants at the early stage of
growth and at the end of vegetation, than in the control (Table 135).

Table 135
Effect of antibiotics on the appearance of gummosis in the cotton plant
(experiment under field conditions on small plots)
(after Askarova 1951)
Morbidity, % in
Morbidity, % in the
Conditions of experiment stage of boll
cotyledon stage
formation
Control (no treatment of seeds) 72.4 19.1
Seeds treated with preparation No 114 8.1 0.0
Seeds treated with preparation No 117 10.2 0.4
Seeds treated with preparation No 86 18.4 2.0

Yield of seed cotton

From control plot 1,640 kg
From experimental plot, prep. No 114 3,040 kg
From experimental plot, prep No 160 3,480 kg
From experimental plot, prep No 86 2,250 kg

In the experiments, seeds treated with antibiotics germinated earlier which was quite
distinctly reflected in the subsequent development of the cotton plant (bud formation,
flowering, opening of the bolls); the vegetation time of the plants was shortened by 8-10
days.

Similar results were obtained in experiments performed under industrial conditions.

Pretreatment of seeds with antibiotics 105 and 114 before sowing lowered the disease
incidence of cotton-plant gummosis 5-6 times and because of this a higher cotton crop
was obtained. For example, in the kolkhoz "Kzyl-Argin" (Tashkent Oblast') twenty-five
per cent of the control cotton plants were affected by gummosis and on the treated fields
the morbidity decreased to 5.3%. The control cotton plants yielded 24 centners/ hectare
of seed cotton and the experimental plants--30.6 c/ha (Askarova, 1951-1952).

Similar experiments were performed by Mirzabekyan (1952-1953) in Armenia. She

employed grisein No 15 in the form of partially purified raw material in treatment of
seeds. The incidence of the gummosis disease of the cotton plant decreased by 67-84%,

Of interest were the experiments of Askarova (1952) against the secondary gummosis
infection of the cotton plant. In cotton-growing practice one often observes a secondary
massive infection of a given culture by gummosis during vegetation.

This is often facilitated by rainfall. Under conditions of a field experiment and on the
kolkhoz fields, application of antibiotic substances during this secondary infection also
yields positive results.

Bel'tyukova (1951) disinfected seeds with an antibiotic microcide before sowing. The
incidence of infection of the plants with pathogenic bacteria decreased considerably
after such treatment.

Bushes of garden rose affected by mildew were specially treated by us with an

especially chosen antibiotic of actinomycetal origin. After the leaves were washed three
times with a solution of the crude antibiotic, the symptoms of mildew began to
disappear and after some time the leaves acquired a normal or almost normal
appearance. (Figure 100).

Figure 100. Treatment of bushes of garden roses, affected by mildew:

A--bushes not treated with antibiotic; the leaves are covered with a white coating of
fungus; B--bushes treated with antibiotic; green leaves, free of fungus.

A positive effect of antibiotics was observed in all cases where treatment was started in
the early stages of the disease.

In strongly affected bushes the treatment had only partial results: the mildew cover
disappeared, but the leaves acquired a brownish-green color, which either disappeared
later, or as more often occurred, remained until the end of vegetation.

Positive results with antibiotics were obtained by Protsenko in floriculture, Gurinovich

in market gardening and Afrikan and others in experiments with field crops and
vegetable cultures.

Recently, foreign investigators extensively investigated antibiotics in their a struggle

against plant diseases under conditions of production, in orchards, gardens and fields.
For this purpose special preparations of antibiotics are produced--agrimycin 100,
agristrep, phytomycin, acco-streptomycin, etc. They are crude preparations of
streptomycin together with terramycin or some other antibiotic. Agrimycin contains
15% streptomycin sulfate and 1.5% oxytetracycline (terramycin) in a powderlike filling.
Agristrep is similar to agrimycin but differs from the latter in that it has a higher content
of commercial streptomycin (37%). Phytomycin--a liquid preparation, contains 20 %
streptomycin and is the most stable upon at storage. Accostreptomycin contains 45%
streptomycin is also qulte at stable, and can be preserved for 2 years. Of wide use in
plant growing is the antibiotic, actidione.

Treatment of fruit trees. The best results in the application of antibiotics were obtained
in horticulture in the treatment of fruit and nut trees infected with bacteria. Goodman
(1954 a, b, c) in Missouri, Young and Winter (1953) in Ohio, Hueberger and Poulos
(1953) in Delaware, Ark (1953 a, 1954) and Dunegan (1954) in California, Kienholz
(1954) in Oregon, Clayton (1955) in North Caroline, Kirby (1954) in Pensilvania and
Mills (1955) in New York obtained good results, upon spraying and dusting antibiotics
on plants infected with bacteria. Wherever such treatment was performed the morbidity
of apple and pear trees decreased or stopped altogether. According to Goodman,
Dunegan, Ark and others, 3-4 sprayings of agrimycin at a concentration of 30-100 µ
g/ml completely eliminated the infection of woody plants. Ark sprayed powderlike
crude streptomycin and a solution of a purified preparation. In treatment of apple and
pear trees afflicted by Bact. amylovorum or of walnut afflicted by Bact. juglandis the
pure preparation was superior. In treating nut trees, two sprayings of streptomycin
sulfate solution at a concentration of 10 µ g/ml were applied. Dye D. and Dye M. (1954)
successfully treated the seedlings of pear trees, infected by Bact. juglandis with a
solution of streptomycin sulfate and dihydrostreptomycin at a concentration of 100 µ
g/ml.

Good results are obtained upon treating fruit trees (cherry, etc) infected by Bact.
syringae, with actidione. One or two sprayings with a solution containing 1-2 µ g/ml is
sufficient to stop the disease. The characteristic spots on leaves cease to appear and
those already existing disappear. Today, actidione is used before fruit formation and
after the cherries ripened, although investigations show that this antibiotic does not
poison the fruits and may be used during fruit bearing (Hamilton and Szkolnik, 1953;
Cation, 1953).

In Germany, Klinkowski and Keller (1956) in their struggle against mildew of fruit
trees used crude antibiotics (filtrates of culture fluids) obtained from specially selected
actinomycetes. The preparations were applied to the trunks of the apple trees infected
with the disease "white ripening", The experiments performed in orchards on a large
scale yielded positive results.
 Treating leguminous plants. Mitchel et al., (1952) tested antibiotics--streptomycin,
terramycin, neomycin, aureomycin, patulin, subtilin, etc, (in all 12 preparations) in
treatment of leguminous plants artificially infected with bacteria. He introduced these
bacteria into the plants by the use of a paste, which he smeared on the stems. Under
laboratory experimental conditions, kidney beans and soy were totally protected against
bacterial wilt by treatment with streptomycin or dihydrostreptomycin. All control plants
perished.

After the successful experiments of Mitchell and co-workers, large-scale field

experiments were started for testing antibiotics on leguminous plants. On the
experimental fields of Beltsville in Maryland streptomycin was used in the struggle
against the fungal disease of Chilean beans, caused by Phytophthora phaseoli. Spraying
with an antibiotic solution at a concentration of 100 µ g/ml the morbidity decreased
markedly or even disappeared entirely. The application of crude streptomycin in
concentrations of 50 µ g/ml gave better results than the use of chemically pure
preparations even at higher concentrations. It is assumed that the crude preparation of
streptomycin contains some other substance with antifungal properties.

In fighting mildew of beans, agrimycin at a concentration of 25 µ g/ml in a mixture

with copper preparations of the same concentration, was used. This preparation was
more effective than the copper preparation and agrimycin applied separately (Zaumeyer
and Fisher, 1953; Zaumeyer, 1955).

Dekker (195 5) tested the action of a culture fluid of the actinomycetes-antagonists--A.

rimosus on pea seeds infected with Ascochyta pisi and Mycosphaerella pinodes. The
antibiotic substance penetrated the seeds and killed the disease agent therein. Such seeds
germinated normally and gave rise to healthy seedlings, while in the control massive
infection was observed.

Klinkowski, Kohler and Shroedter (1955) treated bean seeds with crude antibiotics
formed by Penicillium chrysogenum, and A. griseus in order to prevent infection of the
sprouts by the bacteria Ps. phaseolicola. The seeds were soaked in antibiotic substances
and thus relieved of infection.

Treatment of vegetable cultures. Brian et al., (1951) treated infected lettuce and
tomatoes with griseofulvin. This drug possesses strong antibiotic properties and inhibits
numerous species of fungi, including phytopathogenic ones. It does not affect bacteria.
Spraying lettuce, infected by the fungus Botrytis cinerea, with a solution of the
antibiotic yields quite satisfactory results.

Similar results were obtained in experiments with tomatoes infected by the fungus
Alternaria solani. Griseofulvin was either introduced into the substrate under the root
system, or it was sprayed on the leaves. In the control containers the morbidity
incidence was 100%, while upon treatment the disease was not apparent at all, or only a
small percentage of the plants contracted the disease.

In one of the experiments the number of spots that appeared as a result of the disease
an the leaves of the tomatoes, was counted. In cases where griseofulvin was introduced
in a dose of 10- 2 0 µ g/ml per 1 g of substrate there were no spots on the leaves or only
very few; in plants not treated with the antibiotic there were more than 1,250 spots on a
single plant.

The antiblotic thiolutin (obtained from A. albus) was used by Gopalkrishmann and
Jump (1952) against fusarium wilt of tomatoes (caused by Fusarium oxysporium
lycopersici. The authors treated tomato seedlings by dipping their roots in the antibiotic
solution before planting them in the soil. This procedure completely protected the plants
against the disease. The control plants all succumbed to the disease. Microbiological
analysis of the tissues of the plant showed that with small doses (10 µ g/ml) of the
antibiotic there were no outward signs of the disease but the mycelium of the fungus
could be found Iin the tissues. After treatment with large doses of the antibiotic (40-80 µ
g/ml) the tissues of the plants were sterile and no mycelia were observed in them.

In another series of experiments the authors soaked tomato seeds in the antibiotic and
planted them in the soil. After 12 days 100% of the control plants had fusariosis, while
the treated plants showed no signs of the disease or only a very small number of them
showed its symptoms.

No less effective results were obtained when antibiotics were used against bacterial
infections of tomatoes or other cultures. Conover (1954, 1955) reported good results in
treating tomatoes and pepper infected with Bac. vesicatorium, with agrimycin and
streptomycin. After 5 sprayings with a solution of the antibiotic at a concentration of
200 µ g/ml, 74% of the plants were completely healthy and only 0.4%, were seriously
affected. Among the control plants 12% were healthy and 34% were very sick. Ninety-
five per cent of the treated plants were suitable for replanting and among the untreated
plants only 27% could be replanted.

Cox et al., (1953) completely cured diseased pepper by spraying it 3 times with
streptomycin at a concentration of 500 µ g/ml. Similar data are given by Crossan and
Krupka (1955), who found that upon treatment with the antibiotic the disease agent in
the leaves of pepper is totally eradicated. Higher concentrations of the antibiotic,
according to Cox (1955), yielded a smaller effect and sometimes even caused an
increase in morbidity. The author noted the beneficial effect of a mixture of
streptomycin (100-200 µ g/ml) and copper preparations.

No less effective results are obtained upon treatment of celery infected with
Pseudomonas apii, a disease which is very common in Florida. The use of agrimycin
(300-600 µ g/ml) almost completely eradicates the disease. As in the case of treating
tomatoes and peppers, a mixture of streptomycin and a copper preparation gives better
results.

Sutton and Bell (1954) treated turnips infected by Pseudomonas campestris. They
treated the seeds with aureomycin solutions diluted 1:2,500 and 1:1,000 before sowing.
Short exposure of the seeds to such a solution completely eradicated the disease agent;
the germination of the seeds was normal and even a stimulation of growth of the
seedlings was noted. The plants were healthy, while in the control they were infected to
an extent of 30-76%.

The disease of the eyes of potato tubers caused by the bacteria Bact. atrosepticum and
Pseudomonas fluorescens is often the cause of serious injury to potatoes in the field and
storage. The application of antibiotics in such cases gives very good results. Bonds et
al., (1953-1955), at first in greenhouse experiments and later under field conditions,
found that treatment of cut tubers of diseased potatoes with a streptomycin sulfate
solution (25 µ g/ml) prevents the disease in 80-100% of plants. Dipping of the tubers for
a short time in the solution of the preparation not only diminishes the morbidity but also
increases the viability of the sprouts. According to Webb (1955), treatment of potato
eyes with agrimycin had little effect, however treatment with phytomycin had very good
results. The tubers produce more viable plants with abundant flowering and the tuber
crop was 15% higher that of the control. Heggested and Clayton (1954) had good
treatment results wit tobacco infected with Pseudomonas tabaci. The authors used a
streptomycin sulfate solution followed by agrimycin and agristrep in 200 µ g/ml
concentrations. These solutions were sprayed on the plants 2-3 times during the
summer. As a result, there was almost no plant morbidity, while more than 30% of the
control plants perished. The effectiveness of the antibiotic was higher than that of the
copper preparations. Beach and Engle (1955) in Pennsylvania achieved excellent results
in the treatment of tobacco with antibiotics. They used phytomycin at a 100 µ g/ml
concentration. Kirby (1955) treated diseased tobacco plants with agrimycin (100 µ
g/ml) mixed with febram. All the authors noted a decrease in morbidity, improvement of
growth, increase in number of leaves and also a greater development of the root system.

Antibiotics were successfully used in treating decorative plants. Robinson, Starkey and
Davidson (1954) treated chrysanthemums infected with bacteria with solutions of
streptomycin, terramycin, neomycin, chloromycetin and other substances. The best
results were obtained with the first three preparations. The antibiotics were introduced
into the roots or into the grafts. The treated plants either did not succumb to the disease
at all, or the incidence of disease was very low, while the control plants all perished. The
antibiotics eradicated the infection in the plant tissues (Pramer, 1955).

Treatment of grain cultures. Attempts have been made to use antibiotics in diseases of
cereals. Wallen (1955) used various antibiotics against wheat rust, caused by Puccinia
graminis var. tritici. The rust-effective antibiotic in his experiments was actidione.
Concentrations of 50-500 µ g/ml, although toxic to the plants, lowered the morbidity to
0-5 %. At a lower concentration (2 5 µ g/ml) no toxicosis manifested itself and the
percentage of morbid plants was within the range of 50-60%, while the morbidity in the
control was 100%.

The crop of the wheat treated with the antibiotic was higher than that of the untreated
plots. The percentage of germinating seeds was higher among the experimental plants
(more than 90%) than among the controls.

Leben, Army and Keitt (1953) applied the antibiotic helixin "B" against the disease of
oats, caused by Helminthosporium victoriae and against the disease of barley, caused by
Helminthosporium sativum. The antibiotic considerably lowered the incidence of
disease in the plants. In the control, under conditions of an experiment in growth
containers, there was 28-29% of diseased plants and under field conditions--2%; after
treatment with the preparation no diseased plant was observed. In the growth containers
and under field conditions diseased plants did not exceed 1%. Positive results were also
obtained in laboratory experiments, using antibiotics against wheat rust (caused by
Tilletia foctens), oat rust (caused by Ustilago avanae), and barley rust (caused by
Ustilago hordei).

Henry et al., ( 1952 -1953) achieved an almost complete eradication of the rust disease
of wheat by treatment with actidione mixed with a preparation called "Dixie clay." Even
better results were obtained when actidione was used as a dust mixed with "Dixie clay"
and a preparation called "Captan", which alone did not give good results in the struggle
against the mentioned diseases.

Antibiotics have not so far been effective in combating virus diseases of plants.
Attempts were made to use various antibiotic substances against tobacco mosaic
(Schlegel. David and Rawlins (1954) and certain other virus diseases (Leben and
Fulton, 1952); the small positive effect sometimes observed was not caused by
suppression of the virus particles but by the action of the antibiotics on the host plant,
which increased its growth and enhanced the resistance of the tissues (Zaumeyer, 1955).

The given data on the use of antibiotics in plant growing are as yet scanty. However,
there is a basis for hope that these substances will prove to be no less effective in the
treatment of plants than in the treatment of animals and human beings. Experiments
show that a number of antibiotics may already be widely applied in agriculture, in the
struggle against fungal and bacterial diseases of woody and grassy plants.

Antibiotics are in many cases not inferior in their action to modern antiseptics, and
often surpass them. It is possible that in the future, with the study of conditions and the
mechanism of action, and with improvement of the methods of their application,
antibiotics will become even more effective.

If the antibiotics, after their introduction into the plant stems are directed into the root
system, and accumulate there at a higher or lower concentration, then these preparations
would probably be effective against root diseases; this might be of great importance.

One also should not overlook the economic side of this enterprise.

At first one should assume that it will be advisable to apply antibiotics to the most
costly cultures, mainly in horticulture, in the struggle against diseases of fruit and
decorative plants.

In these cases, not only the cost of a given treatment of the tree is of importance, but
also the time necessary for growing a fruit-bearing plant, should be considered.
Antibiotics are less harmful to the health of man than antiseptics. A certain portion of
the chemical substances which enter the plant tissues, when they are treated with
antiseptics, concentrate there, and to some degree lower the nutrient qualities of the
plants both as food and fodder. The possibility is not excluded that certain elements may
prove to be harmful to man and animals.

Antibiotics are less dangerous in this respect. They cannot accumulate in

concentrations which are toxic to animals and man. One may choose antibiotics which
are altogether harmless. Certain antibiotics have an activating effect on plants and
increase their growth. All this illustrates the necessity of devoting more attention to the
antibiotics than has been done until the present.

Part IV, continued:

Concerning the Epiphytic Microflora

In discussing the problems of interaction between microorganisms and higher plants

one cannot ignore the epiphytic microflora. Microbial epiphytes are the organisms
which concentrate on the surface of the green parts of vegetating plants and are
nourished by the excretions of the latter.

The epiphytic microflora has been but little studied, especially its quantitative and
qualitative composition on various plants.

On the surface of the aerial parts of plants one finds different microorganisms--
bacteria, actinomycetes, fungi, yeasts, algae and protozoa. Their number may by very
high. Duggeli (1904) counted many thousands of microorganisms on the surface of
cereal seeds. From 80,000 to 25 million bacterial cells and from 4,000 to 7,200 fungi in
one gram of wheat seeds have been detected by Morgentaller (1918). The author points
out that on healthy seeds there are almost no fungi.

In germinating wheat seeds there are 60,000 bacterial cells per gram of grains, and in
nongerminating seeds--13 millions. Mack (1936), Kent-Jones and Amos (1930),
inspected 21 samples of wheat seeds from different countries, and found from 8,000 to
eight million bacterial cells on the surface of one gram of seeds. Gustafson and Parfeitt
(1933) in a similar study counted from 46,000 to 3,260,000 bacterial cells in one gram
of wheat seeds.

Rautenshtein (1939) studied the microflora of wheat seeds in the various stages of
ripening: milky, waxy, and full maturity stage. The results of his observations are given
in Table 136.

Table 136
Quantitative and group composition of microorganisms on ripening seeds of wheat
(number of cells in thousands in one gram of seeds)
Maturation Total No of Actino-
Wheat variety Bacteria Fungi Yeasts
stage microbes mycetes
Cesium 0111,
Milky 8,050 7,250 150 650 0
second class
Waxy 5,525 5,275 225 25 0
Full 17,650 17,050 500 100 0
Cesium 0111,
Milky 33,375 32,500 625 250 0
first class
Waxy 72,000 70,900 500 600 0
 Full 41,500 41,250 250 0 0

Bacteria are the most numerous among the microorganism groups which were found.
Yeasts are not always encountered.

With ripening of the seeds the number of microbes on their surface increases. James,
Wilson and Stark (1946) counted from 280,000 to 164 million microorganisms in one
gram of wheat seeds. The numbers of microbes found on the surface of the green parts
of plants are not smaller. Many investigators counted from 49,000 to 6,300,000
epiphytes in one gram of tissue (Khudiakov, 1953; Thomas and Hendricks, 1950;
Stirling, 1951; James, 1955, and others). According to Kroulik, Burkey and Wiseman
(1955). the number of epiphytes in one gram of tissue of green plants of corn, oats,
clover, lucerne, garden grass, and other plants varies from 1,540,000 to 99,200,000.
These numbers vary from species to species, in relation to the age of the plants, and also
with the soil-climatic conditions. As a rule, the number of epiphytes on the surface of
young plants is larger than the number on ripening ones. In relation to seeds, the
opposite picture was observed. Bacteria form the greatest part of the epiphytic
microflora. The species composition of the bacteria is quite diverse, but the dominating
part of it is considerably small. Almost all the investigators noted the predominance of
bacteria with a yellow pigment, classified as Ps. herbicola on plants. This bacterial
species was described by Duggeli (1904), He found that these bacteria were the
dominant species. Their total number reached 380,000 and more in one gram of tissue.

According to Weller (1929), Ps. herbicola comprises 90-100% of the total bacterial
flora on seeds of wheat and rye. Upon germination of seeds in the soil, these bacteria
soon disappear, reappearing toward the end of ripening. On growing plants, according to
the author, there is abundant growth of lactic-acid bacteria. On seeds of barley and oats
Weller found sporeforming bacteria. Rautenshtein (1939, a, b) found 75-98% of Ps.
herbicola among the bacteria populating the surface of wheat seeds. Cocci and Sarcina
are encountered in a few cases. He also found a great number of lactic-acid bacteria.
Among the fungal flora, Rautenshtein found the fungi Cladosporium herbarum,
Trichoderma koninglii and less often Dematium, Asperigillus, Penicillium, Oospora and
also the species A. globisporus and A. griseus and the yeast species of the genus
Torulopsis. Many heat-resistant and thermophilic bacterial forms were found. The
majority of these forms belonged to the sporeforming species of the type Bac.
mesentericus. They also grow at a temperature of 17-20° C.

James, Wilson and Stark (1946) distinguish between two types of epiphytic bacteria--
type A and type B. Type-A bacteria form yellow colonies and are all considered to be
cultures of the same species--Ps. herbicola. The other type belongs to the colorless
Pseudomonas species. Of the fungal flora these authors found the following on plants:
Acrostalagmus, Alternaria, Penicillium, Aspergillus, Botrytis, Cephalosporium,
Fusarium, Torula, Monilia and other fungi. There were also phytopathogenic species
among them Helminthosporium sativum, Hormodendron pallidum, H. viride, Alternaria
tennis, Fusarium culmorum, Cladosporium herbarum, Septoria nodorum, etc.

James (1955) gives the following data on the extent of distribution and accumulation
of Ps. herbicola. Out of 200 plants of oats, barley, and flax, growing in different regions
of Canada which were studied, more than half contained this microbe in amounts of
100,000 and more, about 15 % of the plants contained from 10,000 to 100,000 bacteria
and some samples were free of this bacterial species altogether.

Clark (1947) and others observed the yellow bacteria in great numbers on the green
parts of the cotton plant.

The predominance of these bacteria on other plants was noticed by many investigators
(Burri, 1903; Mack, 1936; Thomas and Hendriks, 1950; and others).

Wallace and Lochhead (1951) point out the connection between the epiphytic and
rhizosphere microflora. The latter, according to these authors, is intermediate between
the microflora of the soil and the epiphytic microflora.

Khudiakov studied a great number of plants (1953a). He investigated the migration of

bacterial epiphytes from the surface of the seeds during the germination of the latter, to
the seedlings and later to all the plant organs including fully formed and mature seeds.
More than 20 microbial species were isolated from different wheat varieties by this
author. Among these microbes there were three species of yeasts, two species of fungi
and the remaining were bacteria, among which three,. cultures produced a yellow
pigment, three others orange or red, one green, and all the others colorless. According to
the author's observations, each of these species is the predominant form on some seed
variety.

Upon analysis of preharvest seeds of wheat (Moscow variety, 2411) grown in the
Moscow Oblast', 97% of the bacteria found were Ps. herbicola; no yeasts or colorless
bacteria of the Ps. fluorescens group were observed. On another wheat variety which
was grown alongside the former and under the same conditions, there were 60% yeasts
and no Ps. herbicola bacteria were observed.

Reciprocal cross infection of the wheat varieties by the isolated cultures has shown
that the latter were not specific. Epiphytes from one wheat variety, when transmitted to
another variety, grew as well as they did on seedlings of their own host plant.
Khudiakov has shown that epiphytic microflora can be changed at will by treating the
seeds before sowing with the corresponding microflora. He treated sterilized oat seeds
with cultures of epiphytic yeasts and bacteria which he isolated, and sowed them in
open ground.

Those microbes that had been artificially introduced (Table 137) were found on these
plants. On the control plants the bacteria which usually concentrate on this species
predominated.

Table 137
Effect of bacterial inoculation of seeds on the composition of the epiphytic microflora
of oats
Number % % %
% yeasts, % %
Organisms introduced of yeasts, Bacteria, bacteria,
No 2 yeasts, bacteria,
with the seeds colonies No 1 Ps. yellow
mycelian White others
on plate red herbicola green
Red yeasts No 1 80 80.8 0 6.2 0 0 5.0
Mycelial yeasts No 2 107 1.8 90.6 2.8 0 0 4.6
Bacterium sp. yellow-
132 5.3 4.5 14.4 7.5 65.1 3.0
green
Control seeds (not
inoculated with 84 2.3 2.3 9.5 80.9 0 4.6
bacteria)

Kvasnikov and Sumnevich (1953) investigated woody plants in Central Asia--poplar,

apple, pear, cherry, maple, Greek nut, etc and grassy cultures: corn, lucerne, cotton,
Sorghum cernium, milo, potato, sugar beet, cabbage, etc. In all cases, lactic-acid
bacteria were found on all the plants in great quantities. According to the authors, on
wild plants the number of these bacteria is considerably smaller. The nearer the plants
are to places of human habitation, the more lactic-acid bacteria one finds on the surface
of plants.

Kroulik, Burkey and Viseman (1955) divide the bacteria into chromogenic and
colorless groups. Among the former, the Ps. herbicola type predominates and among the
latter--lactic-acid bacteria Lactobacterium plantarum.

In our studies we investigated various species of grassy and woody plants growing in
the central belt of the USSR. The number of bacteria and fungi on the surface of leaves
and branches has been determined. Similarly to other investigators, we also detected
hundreds of thousands and millions of bacterial cells per gram of tissue. In the different
plants the predominant epiphytic microflora varies in its species composition. In some
plants Ps. herbicola predominates and in others--other species of the genus
Pseudomonas and Bacterium, and sometimes--lactobacilli. Quite often one finds large
numbers of yeasts of the genus Torula (T. rosea, or T. alba) and of the genera
Sporobolomyces and Mycotorula.

Large numbers of microorganisms are found on the surface of fruits. Studies show that
on berries and fruits there are bacteria, fungi and yeasts, actinomycetes and even
protozoa. Epiphytes are encountered on wild as well as on cultivated fruits.

On berries as on other parts of the plants the most numerous group of microbes are the
bacteria with fungi and yeasts following. Often there are as many as hundreds of
thousands or even millions of them on 1 g of berries. The number of microbes varies
with the variety and species of the berries, with the degree of naturity, and with climatic
and other external conditions. As a rule, their number increases with the ripening of the
berries.

The quantitative ratio between bacteria, yeasts and fungi also change.

The microflora of the vine grapes has been the most thoroughly studied. According to
the data of Akhinyan (1952) the total number of microorganisms on the surface of vine
grapes of the "Kakhet" variety ranges between 3,000 and 4,000,000 per 1 g, depending
on the region and where the vine was grown (Table 138).
 Table 138
Distribution of microflora on the surface of vine grapes
(according to Akhinyan, 1952) (number of cells in 1 g of berry)
Region (Armenian SSR) Yeasts Fungi Bacteria
Ashtarak region
Oshakan village 20,850 4,100 650,000
Voskevaz village 75,000 -- 296,000
Artashat region
Aizestan village 4,008,600 123,000 7,500
Yuva village 3,500 -- 60,000

The presence of such a large microflora on the surface of plants cannot be explained
by their being carried over mechanically from the air. The accumulation of certain
specific species speaks against it. The latter evidently grow and multiply on the plant
surface. Consequently, they must find there sufficient quantities of food substances
necessary for mass reproduction.

Plants, as has been pointed out above, excrete various volatile and nonvolatile
substances--with the aid of special glands or by guttation. In the drops formed by the
guttation of rye grass glutamine was found (Chibnall, 1939), Genkell (1946) observed
the excretion of mineral salts together with the fluid excreted by plants of salty marshes.
Vigorov (1954) found in the guttation drops of 7-to 9-day-old wheat seedlings 1.8
mg/ml of dry substance, containing 5-10 mg/ml ammonium-nitrogen and 40-45 mg/ml
phosphorous compounds. The author observed, that the intensity of guttation depends
on illumination, soil humidity, on the presence of nitrogen and other nutrient elements.
Introduction of ammonium salts into the soil elevates the excretion of nitrogen
compounds in the guttation drop. One of the tests for the presence of organic substances
in the fluids is the growth of microorganisms in them. According to the author, fungi
grow abundantly in guttation drops.

According to the data of Kholodny (1944 a,b,c) all or many of the organic substances
excreted by plants are used by microbes as sources of nutrition.

The significance of the epiphytic microflora in the life of the plant is many-faceted.
Among the epiphytic microflora there are many activators (Ps. herbicola, yeasts, etc),
which form biotic substances--vitamins, auxins, folic acid, thiamine, riboflavin and
other compounds, and also organisms forming antibiotic substances with strong
antimicrobial properties.

On the surface of leaves and stems of plants there are microorganisms forming toxic
substances. In the epiphytic group there are also parasitic and phytopathogenic forms.

One should assume that the metabolic products of the epiphytic microflora behave in a
certain manner in the plant tissue, having a definite effect on them. The ability of leaves
to absorb various substances was known for a long time. On this basis methods of
extrarhizal feeding have been elaborated and also methods of introduction of substances
with the aim of changing certain physiological functions shedding of leaves, arresting of
flowering, etc.

It was also established that plants can absorb various microbial metabolites, vitamins,
antibiotics and other compounds through the leaf surface. As was indicated above, these
substances not only enter the plant through the leaves but they can be introduced by this
route in large quantities for the purpose of feeding as well as for fighting bacterial and
fungal infections. Among the epiphytic microflora there are numerous antagonists
which produce antibiotic substances which suppress their competitors, and among them
also phytopathogenic microbes. Growing abundantly on plants, such organisms may
fulfill a protective role removing or suppressing infectious agents originating from
without. If we were to change the composition of the epiphytic microflora on the
surface of the green parts of plants at will, and form certain coenoses of antagonists
there, this would prove to be of great value to plant and fruit growing.

Part IV, continued:

Toxicosis of Soils and Biological Factors Causing It

The phenomenon of toxicosis of soils has been known for a long time in agricultural
practice and has always attracted the attention of many investigators.

Toxicosis expresses itself in the suppression of the growth and development of higher
plants, and in the lowering of crop yields. The phenomenon of toxicosis is frequent
under monocultures. In such cases, one speaks of the soil exhaustion as the reason for
the suppressed growth of plants.

Tiring of soils was observed by agriculturists and scientific specialists. Plank (1795),
De Candol (1813), Daubeni (1845), Uzral (1852), and others, indicated the lowering of
soil fertility under monocultures and explained it by an accumulation of toxic
substances. Later more attention was devoted to this phenomenon by many
investigators: Kossovich (1905), Pryanishnikov (1928), Timiryazev (1941), Vorob'eva
and Shchepetil'nikova (1936), Krasil'nikov and Garkina (1946) and others (of
Krasil'nikov and Mirchink, 1955, Grummer, 1955).

Soils toxicosis expresses itself in relation to both higher plants and lower plants--
bacteria, fungi, actinomycetes, algae, etc. Much date has been accumulated on fatigue
and toxicosis of soils and the significance of this factor for the fertility of the latter.
However, the essence of this phenomenon remained obscure until now. There are
different points of view concerning its causes, but they can all be reduced to two basic
ones.

According to one opinion, soil toxicosis is caused by the accumulation of special toxic
substances as a result of growing plants against the rules of agrotechnique (Ishcherekov,
1910; Whitney and Cameron, 1914; Greig-Smith, 1913, 1918 and others). According to
the other point of view, the existence of toxins in the soil is denied, and the fatigue of
soils is explained by lack of nutrient substances, as a result of their unbalanced
withdrawal from the soils in case of monocultures (Ressel, 1933; Pryanishnikov, 1928;
Kossovich, 1905; Hutchinson and Thaysen, 1918).

Whitney and Cameron (1914) during their studies of soil fatigue found that plants in
such soils do not suffer from lack of food but from accumulation of large amounts of
toxins. Ishcherekov noted the possibility of removing toxic substances from the soil by
washing with water. After washing, the plants grow better and produce normal crops.
Thorough studies by Greig-Smith show that in Australian soils toxic substances
accumulate in considerable amounts. Their concentration depends upon the type of soil,
season of the year and other external factors. According to his observations, the toxins
are thermolabile and are destroyed upon boiling and by drying.

Hutchinson and Thaysen (1918) studied European soils and found that the toxic
substances which accumulate in them are thermostable, unlike those formed in
Australian soils.

Many investigations have shown that soil toxicosis in relation to microorganisms is

observed much more often and expressed more strongly than toxicosis in relation to
higher plants.

It has long been known that microorganisms, pathogens and saprophytes which enter
the soil do not grow and sooner or later perish. The bactericidity of soils was noted by
Garre (1887), Freudenreich (1889) and others, They showed that pathogenic bacteria of
the colon group, pyogenic cocci and diphtheria bacilli perish in the soil. Microbes such
as the tubercle bacillus, the bacillus of anthrax and many others also perish in the soil
(cf. Mishustin and Perteovskaya, 1954).

The soil possesses the ability to rid itself of pathogenic bacteria entering it. The rate of
autoliberation from these microbes differs in various soils (Table 104).

Table 104
Survival of pathogenic bacteria in various soils
(in days)
Bacteria minimum maximum
Bact. typhi 15-20 360
Bact. dysenteriae 6-10 270
Vibrio cholerae 6-12 120
Mocob. tuberculosis 60 210
Bact. necrosis 10 75
Mact. melitensis 3-10 90
Bact. pestis 3 30
Bact. tularense -- 75

Many pythopathogenic fungi and bacteria cannot remain in soils for prolonged periods
of time. The survival of certain bacteria of this group upon being introduced into the soil
is as follows: Bact. armeniaca --6-8 days, Bact. citri --6-40 days, Bact. aroideae --3-15
days and Bact. tabacum --7-14 days.
 Bact. malvacearum, Bact. citriputeale, Bact. amylovorum and others die relatively
quickly in the soil (cf. Gorlenko, 1950). The death of the fungus Pythium ultimum in
forest humus was observed by Peitsa (1952). According to this author, humus from
under various woody plants possessed different toxicity; the strongest toxicity was
found in extract of humus from under pine, next from under beech and the the weakest
of all, from under birch. The antimicrobial properties of soil are not less sharply
expressed in relation to saprophytic bacteria, fungi, actinomycetes and other
microorganisms. Members of the soil microflora, which come from other soils often
also perish in the soil.

Root-nodule bacteria introduced into clover-tired soil do not grow, and die
comparatively quickly. Thus, from 65,000 bacteria introduced per one gram of a soil:

after two days 15,000 bacteria remained

after three days 1,000 bacteria remained
after five days, 10 bacteria remained.

After ten days the bacteria disappeared almost completely and only a few cells were
found.

Kazarev (1907) observed, that the fungus Pyronema confluens grew well in sterilized
soil and did not grow in nonsterile soil. Extract of nonsterile soil added to the sterile soil
made the latter unsuitable for the fungus. A similar phenomenon was also observed by
Novogrudskii (1936 a). The toxic substance causing the death of the fungus is
thermolabile; it can be destroyed by heating to 120° C.

Numerous data are available concerning the adaptability of Azotobacter, rootnodule

bacteria and certain sporeforming bacteria to soil.

Most strongly expressed and most widespread toxicosis is observed in soils of the
podsol zone. According to our observations, there is either no Azotobacter growth in
these soils, or it dies fairly quickly.

During our many years of study of soil microflora, we have investigated thousands of
samples of podsol soils taken from various places of the Soviet Union.

Selected indexes of soil-toxicosis distribution in the various districts are given in Table
105.

Table 105
Toxicosis of turf-podsol soils
Total No of Samples
Soils and region samples toxic to
studied Azotobacter
Kola Peninsula
Forest 43 0
Humus-ferruginous 105 5
Swamps 22 0
Cultivated garden 215 87

Leningrad Oblast'
Fields, acid 28 0
Fields, neutral 25 5
Garden 17 15

Arkhangel'sk Oblast'
Virgin 35 0
Cultivated 40 8
Forest 27 0
Garden 23 16

Kaluga Oblast'
Forest 17 0
Virgin 25 0
Cultivated 32 10
Garden 35 28

Ryazan' Oblast'
Forest 13 0
Virgin 18 0
Cultivated 21 8
Garden 18 15

Yaroslavl' Oblast
Virgin 30 0
Cultivated 30 6
Garden 30 27

Karelo-Fin AASR
Forest 21 0
Field, virgin 30 0
Field, cultivated 50 10
Garden 20 18
 Kalinin Oblast'
Fields, virgin 47 0
Fields, cultivated 53 12
Garden 23 16

Gor'kii Oblast'
Virgin 20 0
Cultivated 20 6
Garden 20 12

Moscow Oblast', Volokolamak

Region
Forest 13 0
Virgin 33 0
Cultivated 40 5
Garden 50 42

Dmitrovsk Region
Forest 33 0
Virgin 53 0
Cultivated 67 12
Garden 67 53

State Farm "Krasnyi Mayak"

Virgin 150 0
Cultivated 200 12
Garden 70 52

Chashnikovo
Virgin 250 0
Cultivated 350 18
Garden 50 48
Forest 70 0

As can be seen from the data given, podsol forest and virgin field soils are not suitable
for Azotobacter. All the 717 fields soils and 237 forest soils were toxic. Very seldom
does one encounter samples of weakly-cultivated field soils (116 out 2,100 of studied
samples), where Azotobacter grows. Well-cultivated and fertilized garden soils are less
toxic or not toxic at all. Of 1,863 samples 1,244 contained Azotobacter at a greater or
lesser density and 23 samples proved to be toxic for it.

Of all the podsol soils studied by us, those which were studied in greatest detail were
the soils of the Moscow district on the fields of the experimental station in Chashnikovo
and the Academy of Agricultural Sciences im. Timiryazev. These soils are loams with
considerable leaching, Soils from under forests, with different woody species (spruce
grove, birch wood, aspen grove, oak grove, etc), and soils of glades covered by grassy
vegetation, soils weakly -cultivated which were plowed one or two years ago, soils
cultivated for a long time (15-20 years and more) and soils of a renewed forest were
studied.

In all cases the investigations were conducted all the year round; the samples for
analysis were taken at 6-10 day intervals during the summer months and once a month
during the winter. The soils were analyzed while fresh.

We determined the toxicity of soils by the viability of Azotobacter and by germination

of seeds of plants (wheat, beet, etc). At the same time a total count of the microflora was
made, including microbial inhibitors, which form toxic substances.

Studies have shown that many soils contain toxic substances. In forest soils, as a rule,
there are more of these substances than in forest-free soils, there are less in plowed soils
and still less in well-cultivated ones.

The toxicity of forest soils is determined by the varieties of trees growing in it. The
greatest amount of toxic substances is found under spruce grove, and in a smaller
amount, under pine and aspen grove. Soils under birch wood and oak grove are weakly
toxic or nontoxic at all.

In Table 106 data are given on the toxic action of soils on germination of seeds of beet
and wheat and on Azotobacter.

Table 106
Toxic action of forest podsol soils of the Moscow Oblast'
Time of death
Germination of Germination of
Soils of Azotobacter
beet seeds, % wheat seeds, %
cells (in hours)
From under a spruce-grove 1 5 2-4
From under a pine-grove 5 25 20
From under a birch-grove 50 80 72
From under an oak-grove 80 90 82
Fallow cultivated soil 72 90 30 days

After clearing a forest and plowing, the soil becomes less toxic; Azotobacter does not
perish for several days or even weeks. With renewal of the forest, the soil's toxicity is
also restored (Krasil'nikov, Mirchnik and others, 1955).
 The formation of toxic substances in chernozem soils under an artificially planted
forest in the region of the southern steppes was observed by Runov (1953).

Plots covered by forests in the chestnut-soil zone of the Trans-Volga region, lose their
Azotobacter. We observed a similar picture upon afforestation of soils in Central Asia,
Kirghizia, Vakhsh valley of the Tadzhik SSR, Moldavia and other places. Soils rich in
Azotobacter, lose them as soon as certain varieties of trees start growing in them.

Formation of toxic substances in soils is observed under certain grassy plants,

particularly often in monocultures.

While studying the clover-exhausted soils of the experimental fields of the Agricultural
Academy im. Timiryazev, we observed that they were obviously toxic for Azotobacter
and for root-nodule bacteria, as well as for plants (Krasil'nikov and Garkina, 1946).

Toxicity changes considerably with the seasons of the year, as do many other
properties of soil. It is most apparent during the summer-autumn months (July-
September); in the late autumn and in winter it decreases and approaching spring it
reaches its minimum. Azotobacter perishes more quickly in summer soils than in winter
ones, while in spring soils, it even grows (Table 107).

Table 107
Toxicity of soils in relation to the season of the year
(Survival of Azotobacter in different months, hours)
Soils July Aug. Sept. Oct. Dec. Jan. Feb. April June
Forest 2 6 2 2 24 24 72 216 96
Weakly cultivated 24 24 24 72 72 120 -- 216 96
Well cultivated: under
24 24 2 6 24 40 24 24 --
Timothy grass
Well cultivated: under
72 96 96 96 120 216 -- 216 --
clover

Similarly, seeds of beet germinate with greater energy and in greater numbers in spring
soils (April-May) than in summer-autumn ones (August-October).

According to Rybalkina, toxicity of soils in relation to Bac. mycoides is lost after cool
rainy weather.

According to the observations of Reiner and Nelson-Jones (1949), the greatest toxicity
in the forest fields of Wareham (England) is detected in the autumn-winter months, with
a decrease beginning in March.

One may assume that the increase and decrease of soil toxicity is caused by
quantitative fluctuations in the toxin content. Toxic substances are either washed out by
rain waters in the autumn and thaw waters in the spring, as it was assumed by Reiner
and Nelson-Jones, or they are inactivated by the low temperature in the winter. For the
verification of these assumptions we conducted special experiments.

In one series of experiments the soil (forest) was thoroughly washed with water and
studied for toxicity. In washed soil Azotobacter perished at the same rate as in
nonwashed soil. As can be seen, the toxic substances present in the soil which we
studied are not washed out with water or only a part of them is washed out, as may be
assumed, the part that is not adsorbed by the soil particles. The majority of the toxic
substances are probably in the adsorbed state. Therefore, the decrease of the soil toxicity
in the spring is not caused by washing out by rains and thawing snow but, one may
assume, by the action of winter frosts.

In another series of experiments we subjected forest soil to freezing at minus 15-20° C

for two months, and after thawing, an Azotobacter culture was introduced into it and the
soil was incubated at 25° C. The results showed that in control soil, maintained at room
temperature, Azotobacter died after one and a half hours while in soil which had been
subjected to freezing, it did not die for 96 hours.

The soils investigated by us were not inactivated by heating at 100° C for 30 minutes,
Inactivation was not attained even after autoclaving at 120° C for 30 minutes. In
"exhausted" soils as we have shown earlier, the toxic substances are destroyed and
disappear, at a temperature of 100° C maintained for 30 minutes, Obviously, the nature
of the inhibitory substances in these soils is different.

Cultivation of podsol soils exerts a large influence on their toxic properties.

Azotobacter grows better in chalked soils than in nonchalked soils (cf. Sushkina, 1940).

Many investigators (Christenson, 1915; Gainey, 1918, 1940 and others) ascribe the
absence of Azotobacter in podsol soils to their acidity. They think that Azotobacter
cannot grow in soils which have a pH of 5.5 or lower, If one even occasionally finds this
microbe, it is considered to be of a special acidophilic species (Az. indicum). According
to these authors, as soon as one neutralizes acid soils, the ordinary Azotobacter (Az.
chroccoocum) will start to grow and accumulate in them.

There exists another opinion, according to which proliferation of Azotobacter is

conditioned, not by acidity, but by the ratio of the anions CO3 : PO4 (Niclas, Poshenrider
and Mock, 1926), by the condition of the oxide and suboxide salts of metals (aluminum,
iron, etc).

There are indications that, Azotobacter does not grow in many acidic soils after
chalking, when the pH comes close to neutral (6.5--7.0).

Levinskaya and Malysheva (1936) observed that introduction of CaCO3 into acidic soil
(podsol of Murmansk Oblast') does not improve the growth of Azotobacter.

Brenner (1924) chalked acid soils of Finland and introduced Azotobacter. This measure
did not decrease the soil toxicity. The introduced microbe perished as fast as in the
nonchalked soils. According to the author. toxicosis of podsol soils is caused by special
toxic substances. formed upon decomposition of plant residues (moss, etc) and also by
toxic iron compounds.

Katznelson (1940) studied the viability of Azotobacter in acidic soils of America and
tested various organic and mineral fertilizers with and without neutralization of the soil.
The author reached the conclusion that there was no strict correlation between soil
acidity and viability of Azotobacter. At pH 5.9 its number may be higher than at pH 6.6.
At the same pH value of soil, Azotobacter proliferates in some cases and does not grow
in others.

In our experiments on neutralization of soils by CaCO3, MgO, NaOH the conditions of

growth of Azotobacter in podsol forest soil improved considerably, but its toxicity was
not completely eliminated. Under field conditions chalking also failed to reduce
toxicosis of soil. As in the experiments of Brenner, we did not find Azotobacter in
podsol, sparsely cultivated and well-chalked soils. We did not find it either in the year
when chalking was applied, or 1-3 years later. Azotobacter was not detected in these
soils even after a single introduction of manure. Azotobacter introduced in such soil
died out at a slower rate than in the control, surviving 6-10 days and more, however its
multiplication was not observed. Upon introduction of potassium nitrate or potassium
phosphate the death of the introduced Azotobacter was speeded.

Therefore, the suppressing factor consists, not only of soil acidity, but also of many
agents: physical, chemical and mechanical ones. For instance suboxide salts of
aluminum, iron and other compounds, which, by the way, are in direct relationship to
the pH of the environment, may inhibit growth of Azotobacter.

However, the main factors which cause soil toxicosis are, in our opinion, in many (if
not in all) cases, excretion products of plants and microbial metabolites.

Formation of toxic substances by plants

Schreiner and Reed (1907) found that roots of certain plants excrete toxic substances.
When they grew wheat repeatedly in the same vessel containing sand or soil, they
observed a decrease in crops after each new sowing; this differed in various soils (Table
108).

Table 108
Wheat crops upon repeated sowing in the same soil (after Schreiner and Reed)
(in % of the first sowing)
First Second Third Fourth
Soil and region
sowing sowing sowing sowing
Clay--Cecille 100 68 57 44
Loamy--Leonardstown 100 30 37 23
Clay--Tacoma 100 53 53 46
Sandy--Portsmouth 100 64 30 --

Application of fertilizers causes a certain increase in crops but only after the first
sowing. The authors obtained the strongest effect after the application of lime and
manure. The crop of the first sowing was as follows (per cent of control):

Control (without fertilizer), 100

Mineral fertilizer, 130
The same plus lime, 205
Manure, 200
Manure plus lime, 238

Upon repeated sowing with the same amount of fertilizers the crop decreased. The
solution from under wheat was not suitable for this plant: seeds did not germinate well
in it, and seedlings were clearly retarded. The inhibition of root growth of flax seedlings
was especially strong in this solution. When nutrient substances were added to this
solution, only the growth of the aerial parts improved, but the roots remained
undeveloped.

Toxic substances obtained from the substrate (from the solution or from the soil or
sand) in which wheat grew, are thermolabile, inactivated by boiling, and absorbed by
charcoal and chalk. If a toxic solution In filtered through charcoal or chalk, or subjected
to heating, plants grow normally in it. The addition of pyrogallol to the soil extract
removes its toxic properties. The same effect is obtained with the use of naphthylamine.
These substances differ in their chemical composition and belong to the picolinic acid,
salicylaldehyde, vanillin, and dihydroxystearic acid.

The experiments of Schreiner and Reed were repeated by Periturin (1911, 1912) with
the same results. According to his data, the root excretions of wheat suppress not only
the growth of the wheat seedling but also that of the oat. Schmuck (1911) grew cereals
in sand after wheat; in some cases he cut the wheat at the root, while in others he let it
grow to the end. After the harvest of crops, wheat or oats were sown again. In some
containers roots of wheat were introduced into the sand. These experiments showed that
the presence of wheat roots lowered the crop of oats to 76.8% and that of wheat to
45.2%.

Molliard (1915) tested the effect of root excretions of peas and corn, grown under
sterile conditions, on seedlings of the same plants. The data obtained by him agree with
those of Schreiner and Reed.

Hedrick (1905) observed an inhibitory effect of root excretions of oats on the growth
of young apricot trees, The effect of root excretions of potatoes and tomatoes was less
strongly expressed. A still smaller effect was that of roots of mustard and rape. Root
excretions of beans and clover did not suppress growth of the above-mentioned trees.
Similar phenomena were observed in the horticulture department of the Experimental
Station of Woburn (USA), where it was shown that root excretions of grass suppressed
the growth of young peripheral root tips of apples and pears which constitute the most
active part of the root system.
 Jones and Morse described the inhibitory action of the nut tree (gray nut--Jualans
cinerea L.) on the growth of the creeping cinquefoil shrub. The latter does not grow in
the vicinity of this tree to a distance of approximately twice the diameter of the foliage.
Jensen has experimentally established that the root excretions of maple, cornel, cherry,
tulip, and pine suppress the growth of wheat; the strongest suppression was observed in
the summer, during the period of plant growth, when root excretions were more
abundant than in autumn (after Schreiner and Reed, 1907).

Pickering (1903-1913) observed the deleterious effect of grasses on growth of fruit

trees. He grew grasses in baskets with soil, hung under the foliage of an apple tree. The
water coming through during downpours irrigated the soil around the apple tree. The
roots of the latter were poisoned and the plants perished.

Fletcher (1912) observed the high sensitivity of Sesamum indicum L. to root excretions
of Andropogon sorghum Brot. According to his observations, this plant cannot ripen in
the vicinity of sorghum. Sewell (1923) found that roots of sorghum are toxic to wheat.
Shull (1932) did not confirm the results of Fletcher and Sewell. According to his data,
sorghum has no toxic effect on plants.

Mashkovtsev (1934) observed the thinning out of rice plantations after 2- 3 years of
monoculture. The author thinks that the reason for this was the presence of toxic
substances in the soil.

Ahlgren and Aamodt (1930) studied the interaction between different plants by
growing them separately and together in containers. When Timothy grass was grown
together with spear grass, or meadow grass with spear grass or with Timothy grass,
much lower yields were obtained than when they were grown separately. The dry
weight in grams of the plants in isolated cultures was as follows:

Spear grass, 0.396

Meadow grass, 0.343
Flat meadow grass, 0.450
Timothy grass, 0.577
Mixture:
Spear grass, 0.244
Timothy grass, o.360
Mixture:
Meadow grass, 0.195
Spear grass, 0.311
Mixture:
Flat meadow grass, 0.260
Meadow grass, 0.264

Waks (1039) found toxic substances in the root excretions of Robinia pseudoacacia L.

Many investigators connect the formation of toxic substances in soil with the growth
of plants (Jakes, 1937; Rippel, 1936, Winter and Bublitz, 1953, Dimond and Waggoner,
1953, Nutman, 1952; Grümmer, 1955 and others).
 It has been found that in many plants there are special substances--cholines and
blastocholines (blastanein--germination and cholycin--to prevent), inhibiting
germination of their own seeds and also of seeds of other species of plants. The nature
of those substances differs in different plants. They may be excreted by the roots of
seedlings and suppress growth of neighboring plants. Root excretions of seedlings of
birch suppress the growth of rye grass and lychins: root excretions of seedlings of wheat
and rye grass suppress germination of moods of certain weeds of Anthemis arvensis L.
and Metricaria inodora L; seedlings of beans suppress germination of seeds of flax and
wheat and seedlings of violets inhibit germination of wheat seedlings (after Audus,
1953).

Benedict (1941) observed the death of Bromus inermis Leyss, after its repeated sowing
for many years in the field. The soil of such fields was toxic for the plant itself and for
certain other plants. an well. Introduction of fertilizers did not abolish the toxicosis but
only somewhat diminished it.

Bode (1940) described the poisonous effect of root excretions of Artemisia absinthium
L. on the growth of fennel, caraway, sage and other plants sown in its vicinity. The
height of the anise stem at a 70 cm distance from absinth was 5.7 cm at a distance of
100 cm--17 cm and at a distance of 130 cm--39 cm. A toxic substance was isolated,
which was found to be a glucoside. This glucoside, called absinthin, is formed in the
leaves of absinth and in easily extracted with water; it is washed out by the rains and
enters the soil under the plant crown. It is preserved for prolonged periods in the soil.

There are indications that accumulation of toxins in the soil also takes place under
fruit trees. Proebsting and Gilmore (1940) have shown that soil from under old peach
trees is toxic for young peach saplings. Martin (1950-1951) found the same to be true
for soils that have been under lemon trees for a long time. Plants planted on plots that
have never before been under citrus grove a grew 9% faster than plants planted on old
citrus-grove soils. Tomatoes and other vegetables grew quite satisfactorily on soils of
old lemon groves and showed no signs of repressed growth.

According to Martin, the toxicity of much "exhausted" soils was not eliminated by
washing with water for six weeks. Only by treatment with 2% sulfuric acid or 2% KOH
and subsequent saturation with calcium did he succeed in removing the toxicity and
restoring the fertility of the soil.

The inhibitory effect of root excretions of grassy plants, mustard, tobacco, tomato, and
others in noted by many authors. Their effect in expressed in grassy plants and woody
varieties as well. The degree of their inhibitory effect varies from 6 %to 97 %
depending on plant species and on external conditions (Livingston, 1023; Breazeal,
1924; Conrad, 1927 and others, cf. Grammer, 1955).

Guyot (1951) ascribes a decisive role in toxicosis of soil to root excretions. He

investigated a great number of plant species, and detected in many of them the ability to
form toxins and to excrete them into the soil. These plants can be divided into groups
according to the intensity of their toxic action. If one were to use Brachypodium
pinnatum P. B. as a control plant which does not form toxins, with an index of 100, the
remaining plants will have the following indexes:
Helianthemum vulgare Gärten., 85, weakly toxic
Barkhausia foetids Mönch., 81, weakly toxic
Thymus serpyllum L., 75, moderately toxic
Hieracium pilosella L., 70, moderately toxic
Origanum vulgare L., 70, moderately toxic
Asperula cyanchica L., 69, moderately toxic
Teucrium chamaedrys L., 68, moderately toxic
Picris hieraciodies L., 65, moderately toxic
Papaver rhoeas L., 61, moderately toxic
Achillea millefolium L., 59, moderately toxic
Hieracium umbellatum L., 39 strongly toxic
Solidago virga aurea L., 30, strongly toxic
Hieracium vulgatum Fr., 29, strongly toxic

Upon repeated sowings of the same plants, their seeds progressively germinate less
well, and the mature plants yield smaller and smaller crops. According to Guyot and
Massenot (1950), Hypericum perforatum L. under the same sowing conditions had in
the first year a density of 4,200 plants per plot, the year after--1,100 and on the third
year--500 plants on the same plot. Similar results are given by Curtis and Cottham
(1950) with various species of sunflower. Hurtis (1953) tested the effect of root
excretions of corn, peas, wheat. oat, rye, and lucerne, on the germination of mustard
seeds. Excretions of barley roots caused a strong inhibition of seed germination, root
excretions of other plants showed no such effect. According to Schilling (1951),
cauliflower grows better if there in celery in its vicinity. Piettre (1950) noticed a strong
poisoning of soils under many-year plantations of coffee plants. He isolated a substance
belonging to the fatty acids from these soils. The most ubiquitous among them is
lignoceric acid (C24H48O2) which strongly suppresses the growth of plant seedlings.

The Swedish scientist Oswald (1947) studied the toxicity of soils under certain grassy
plants. According to his data. seeds of rape--Brassica napus, and B. rapa L. germinated
very weakly or not at all in soil from under Agropyrum. repens P. B. or Festuca rubra L.
The author succeeded in isolating a substance inhibiting germination of rape seeds from
roots of the couch grass. The toxicity of soils on which couch grass or Festuca rubra L.
have been grown was removed by heating at 80-90° C (Oswald, 1949, 1950).

According to Schuphan (1948), lettuce suppresses growth of radish seed and radish is
toxic to the development of lettuce seeds. Lettuce excretes, according to this author,
saponin, and radish excretes mustard oil. The toxic substance was extracted from the
leaves with ether and obtained in crystaline form (0. 5 mg from 1 g of leaves). It proved
to be 3-acetyl- 6-methoxybenzaldehyde (Bonner 1950)

Lyubich (1955) tested the interaction between various woody plants. Planting various
species in pairs on the same hole it was found that certain species inhibit the growth of
others (Table 109).

Table 109
Interaction between woody plants
Plants English Green ash Box Indian Japanese Elm
 (Ulmus
(Fraximus pagoda
oak elder bean pinnato-
viridus) tree
ramosa
English oak + + + -
Green ash (Fraximus
+ + - + - -
viridus)
Box elder + - + -
Indian bean + - +
Japanese pagoda tree + -
Elm (Ulmus pinnato-
- -
ramosa)

Note; a plus sign means good growth (no suppression), a minus sign means suppression
of growth.

An is evident from the table, each of the tested plants suppressed the development of
shoots of any other type.

According to Guyot, root excretions of Hieracium pilosella L., even at low

concentrations, are toxic to many plants. Vigorov has found that by its root excretion
Agropyrum repens P. B. inhibits the growth of seedlings of pine and Caragana (after
Grümmer, 1955).

Rodygin (1955) observed a 40-80% cancer morbidity of the lime tree when it was
grown in the vicinity of asp. In the neighborhood of other plants (pine, spruce, fir) the
percentage of cancerous lime tree did not exceed 2-3%.

According to Bordukova (1947) the kind of plants that grow in the vicinity of potatoes
is important. In the vicinity of sunflower, tomatoes, apple, cherry, raspberry, pumpkin or
cucumber, the resistance of potatoes to Phytophthora was lowered. Potatoes grown in
the vicinity of a birch wood rot more easily than potatoes grown in the vicinity of pine.

One cannot combine narcissus and lily-of-valley flowers together in one bunch since
they would soon wither; similarly, mignonette increases the withering of flowers in a
vase.

The prolonged studies of Bonner and his co-workers (1938-1948) have shown that
guayule roots excrete a toxic substance--trans -cinnamic acid. Ten milligrams of this
acid in 1.5 kg of soil completely inhibits germination of guayule seeds. The higher the
concentration of this substance, the longer it remains in the soil. One milligram of
cinnamic acid endures 14 days in nonsterile soil and in sterile soil for an even longer
period of time.

The shrub Encelia parinosa Adana, (of the compositae) grows in deserts. It is
characterized by the fact that no grass grows for a certain distance around it.
Investigations have revealed that the soil under the crown of this shrub is poisoned by
the toxic substances, formed by its leaves. An extract of the leaves, or even better, the
leaves themselves when introduced into the soil arrest growth of other plants--tomatoes,
pepper and rye. These toxins do not act on the growth of Encelia, barley, oats and
sunflowers.

It is well known that a great number of plants produce various compounds possessing
toxic properties in relation to bacteria and to plants. Substances such as glucosides,
saponin and coumarin are very widespread among plants. Inhibitors of the saponin
group were found in hay stacks which suppress the germination of seeds and growth of
algae (Moewus and Bonnerjee, 1951, Lindahl, Cokk et al, 1954), These substances
reach the soil with plant residues. Upon decay of the latter they re liberated and may
exist in the soil, in active form for a certain length of time, affecting the microflora and
the higher plants. According to Benedict (1941). dead roots of brome grass release toxic
substances upon their decomposition which suppress germination of brome-grass seeds.
The above-mentioned absinth and Encelia release their toxins upon the decomposition
of their aerial parts.

Golomedova (1954) studied the effect of aqueous extracts of many grasses and shrubs
on plant growth. Tartar honeysuckle, maple, ash tree, buckthorn and amorpha inhibit
growth of fescue and Euagropyrum, Extracts of couch grass, root and green parts of
Austrian absinth inhibit oak saplings.

Feldmeier and Guttenberg (1953) obtained alcoholic and ether extracts from seeds and
seedlings of beans, which inhibited growth of oat coleoptiles.

Bublitz (1954) tested the effect of extracts of pine branches, decomposing in soil, on
the germination of Lepidium sativum L, seeds and on the growth of certain bacteria in
compost. The extracts noticeably inhibited growth of bacteria and germination of seeds,
more so in an acid medium (pH-5.6) than in a neutral one.

Lindahl, Cook et al, (1954) obtained a toxic substance of the saponin type from lucerne
hay, According to Mishustin (1956) these substances are excreted by the roots of lucerne
during vegetation,

Callison and Conn (1927) and McCalla (1948, 1949) found toxic substances of the soil
in the form of decomposition products of plant residues. They established the presence
of the following compounds among these substances: vanillin, coumarin, dehydrostearic
acid, salicylic acid and other compounds. Small doses of these substances, noticeably
inhibited growth of plants.

Toxic substances are present in plants of many species of the umbellate family: poison
hemlock, poisonous cicuta, water dropwort, marshwort, Anthriscus and others. In their
tissues one finds phthalides, various tars, esters, acids and other compounds. In
Cruciferae plants one finds mustard oils.

Various volatile substances are present in many odorous plants. In some plants they are
formed in the seeds and fruits, while in others in the leaves and stems or in the roots.
Essential oils of a series of plants: citrus plants clove, mint, Satureia, thymes,
germander, eucalyptus, etc and the resin of coniferous trees, poplar and others inhibit
the germination of seeds of various plants to various degrees (Weintraub and Pricae,
1948, Grümmer, 1955, Molisch 1937; Madaus, 1936, Clausen, 1932).

Lebodev (1948) has found that absinth inhibits growth of flax, peas, beans, sage and
clove. Roots of ash excrete volatile substances which inhibit growth of the oak.

Golubinski (1946) observed the stimulation of pollen germination by volatile

substances formed by plants.

Solov'ev (1954) found that volatile substances from certain plants (Agropyrum
pectiniforme R. et Sch.) stimulate the germination of lucerne pollen, those of other
plants (Bromus intermis Leyss, Phelum Pretense L.) inhibit and still others have no
effect at all.

The volatile substances excreted by onion, garlic and horse radish are well known (cf.
Tokin, 1951).

In agricultural practice plants forming volatile substances have, since ancient times,
been used as preservatives against spoilage of foodstuffs, For instance peasants put
pieces of garlic into the cornbins for protection against the weevil, and against Agrostis
segetum they use branches of the bird cherry tree (Grimm, 1950).

Volatile substances excreted by plants have a definite effect on phytopathogenic

microflora and may play a protective role, Fahlpahl (1949) observed a protective effect
of hemp, The volatile substances which it excreted inhibited growth of certain
pathogenic microorganisms, due to which plants growing in the vicinity of hemp were
less subjected to diseases. Schilling (1951) gives data on the protective effect of celery.
When cabbage grows in the vicinity of this plant it is less affected by microorganisms.

Pirozhkov (1950) noted the lethal effect of the volatile substances of tomatoes on
certain insects attacking the gooseberry shrub, as Tenthrodinodea and Pyralididae.
According to this author's observations gooseberry shrub in the vicinity of tomatoes do
not suffer from these insects.

Certain organic acids of plant origin are also toxic for seedlings of a number of plants,
They are often found in the fruits. Malic and citric acids, in apples; 3.4-
dihydroxycinnamic acid and 3-methoxy-4-hydroxycinnamic acid in tomatoes,
transcinnamic acid, in guayule, etc (Akkerman and Veldstra, 1947),

The alkaloids are very widespread among plants. Some of them inhibit growth of
plants. The most well known are cocaine, physiostigmine, aconite, caffein and quinine,

It should be noted, that the nature of the toxic substances excreted by plants is
unknown in the majority of cases. Grümmer (1955) relates these substances to a special
group of specific substances--the cholines,

Recently, artificially produced substances have penetrated more and more into
agricultural practice; these substances exert a certain inhibitory effect on plants,
Substances have been obtained that put put potato tubers "to sleep." To these belong a
series of compounds--methyl esters of alpha-naphthylaceatic acid, ß-naphtyldimethyl
ester, isopropylphenylcarbonate and certain other esters of phenylcarbonic acid (Krylov,
1954). Small doses of these substances (0.1% of aqueous solution) keeps tubers from
sprouting in storehouses (Moewus and Schader, 1951). Preparation M-1 (methyl ester of
ß-naphthylacetic acid) is offered for use in the preservation of potatoes, A dosage of 1.5-
3 kg of this substance is enough to protect 1 ton of potatoes from sprouting, increasing
their yield by 10-14%, and decreasing weight losses 2.5-5 times, it also protects the
starch and vitamin C, decreases the accumulation of the glucoside and solanin in the
tubers (Krylov, 1954).

Pteroylglutamic acid (4-amino-9-methylpteroylglutamic acid) and indolyl-3-acetic acid

suppress growth of roots and, to a lesser extent, inhibit the growth of the aerial parts of
the plants.

Maleic hydrazide causes a long-lasting inhibition of plant growth. At a concentration

of 0.01% this compound suppresses the growth of the raspberry for 24-38 days and
ripening of berries for 16-33 days. This substance is recommended for use on lawns, i.
e., it strongly checks the growth rate of grass.

Suppression of plant growth by chemical preparations in widely used in agriculture, In

a number of cases it is necessary to arrest the development of buds and especially the
beginning of flowering in fruit trees (apples, pears, aprocits, peaches, etc), These
substances can also be used in decorative plant breeding, when it is necessary to arrest
the growth of plants, etc.

It should be noted, that many substances of the auxin group may act as inhibitior or
herbicides and as stimulants as well. For example, the well-studied compound- 2.4-
dichlorophanoxyacetic acid (2.4-D) sometimes stimulates and sometimes suppresses
seed germination, depending on the concentration used. Para-aminobenzoic acid has a
stimulating effect at a concentration of 0.001% and strongly inhibits seed germination
and plant growth at a concentration of 0.05%. Nicotinic acid at a concentration of
0.01% is also a strong poison for plants.

The toxic substances differ in their stability. Some of them are quite stable, are not
destroyed upon prolonged stay in the soil, and may be concentrated to a greater or
smaller extent. Other substances are easily destroyed and vanish from the soil. In such
cases, plowing is enough to remove the toxicity of the soil. The vegetative cover,
fertilizers and other measures also change actively the toxic substances in the soil,

Certain substances are easily leached by rains and are removed from the soil, while
others are in the adsorbed state and are not eluted by water.

Studies show that there is a direct relationship between the concentration and length of
time of preservation of toxins in the soil, and the qualitative and quantitative
composition of the soil microflora,

In sterile soils the active substances are preserved for considerably longer periods of
time than in nonsterile soils. When sterile soil is inoculated with a particle of nonsterile
soil, the toxins in it soon become inactivated as in nonsterile soil (Brown and Mitchell,
1948, Audus, 1953, Jorgensen and Hammer, 1946),

Stapp and Spicher (1955) and Jensen and Peterson (1932) have described bacteria
which strongly destroy herbicides. Their activity aids in the liberation of soil from
toxins.

As can be seen from the above-mentioned data, the accumulation of toxic substances
under natural conditions may vary, depending on the type of soil, the nature of the
substance and on external conditions. Some substances accumulate in considerable
quantities, while others remain in small concentrations or are not accumulated at all.
The concentration of these substances determines the degree of toxicosis and fatigue of
soils.

Under natural conditions. in soils and other substrates into which toxic substances
constantly enter there is some degree of accumulation of the latter. At certain
concentrations of toxins in the soil, poisoning of plants may take place.

The question of whether toxins can themselves enter plants is answered in the
affirmative.

It was experimentally shown, that a number of toxic substances penetrate the plants via
the roots and spread to the tissues. The mere fact of the poisonous effect of inhibitors in
the above experiments show that these substances penetrate the plants. There are also
direct experiments with chemically pure substances obtained from bacteria, fungi and
actinomycetes. Gramicidin--a preparation obtained from the sporeforming bacterium--
Bac. brevis, possesses strongly toxic properties; it poisons the tissues of animals as well
an those of plants. Small doses of it in the nutrient medium cause rapid browning of
roots, withering of aerial parts, and death of the whole plant.

Pyocyanin, a substance formed by the blue pus bacillus--Ps. pyocyanea, is endowed

with strongly toxic properties. Plants-- wheat and clover--perished within a few hours
under its influence. Substances obtained from many actinomycetes are also toxic for
plants: mycetin, lavendulin, actinomycetin, longisporin, etc. Among the fungal
metabolites, notatin, glyotoxin, and some others possess herbicidal properties.

Toxins and antibiotics may enter the plants through their leaves. If a drop of the
solution of a toxic substance is placed on the surface of the plant, after some time one
observes symptoms of poisoning, not only in the tissues that have been in direct contact
with the solution, but in distant parts as well. Sometimes the whole branch withers
away. This phenomenon of poisoning of branches and leaves was observed by us in
birch, due to the action of the toxin produced by the fungus Botrytis cinerea
(Krasil'nikov, 1953b).