110
CHAPTBR
,O
DEVELOPI.IENT
7,1
INTRODUCTION
7
OF SELECTIVE }flDIA
7
FOR RAMICHLORIDIUM
PINI
Necessities of investigators having diverse interests in fungi have
leci to Ehe formulation of new, and modifications of existing, media to suit
specific need of the rvorkers over the past years. The results of these
have been that as many as 185 fungal culture media have been listed by
Ehe
Booth iL971). Tsao (1970) has extensively revier^,ed a large nuurber of
\
selective nedia and has stressed the ever increasing need for evolving
more and more selective medig, Most of the selective media so far evolved
have dealE
nuurber
of
with the isol-ation of fungi from soil.
only a very
smal1
deal with isolation from infected plants. These
media
include Ehose of Schneider (1956), Russel (1956), Eckert and rsao
(l_960,
L962), Tsao and Menyoea (1966) and Vaarraja (1968).
From the work
sclerophoma
carried out on the isolation of fungi (chapcer
pff!:gphi&.
was found
assoeiates of Ramichioriditrn pini.
eausal pathcgen
to be the most f requent fungus
The
6)
among the
latter has been shor,,n to be the
of lodgepole pine shoot dieback (vide chapter 1o).
iherefore- any medium which r+i11 suppress S. pythicphila while allowing
rormal grorrrth of R. pini under investigation is like1y to be very useful
:o
subsequent
workers. Moreoverr-[, pythiophi-la being sc ubiquitous
:ungus on coniferous shoots and neeclles, aDy medium on which
this
a
ftrngus
-"'.' be suppressed while allowing grovrth of some other pathogenic fungi
: a'-uralIy deserves to be t.reated as a very usefur medir:m.
secondly, during isolation work, B. pini was found noE to produce
::rr.'
or only a very few conidia on 2z malt agar, but
mass production of
:--e fungus and preferably its spores, is necessary for any extensive
111
rnocul-aEion experiments
During isolation of fungi on a nurnber of niedia
(Chapter 6, table 6.7) ir was found that
Czape.k Dox agar (Oxoid) afforded
some
sort of
.
suppression to S. pythiophila.
Hence, attempts to modify
Czapeli Dox Agar (CDA) ro,
further suppress the growth of S" pythiophile
and to find a medium or natural substrate on which
mass productr.on of
spores of R. pini could be obtained was consiciered
fully justified.
7
.2
MATERIA]. AND
I'TETHODS
Five culture media, czapek Dox Agar i.e. cDA (oxoid),
zT. r,IaLt Extract
Agar i'e' zz MA and oat Meal Agar 'i.e. oI'1A (Booth
tgTr), potaro
,
i.e.
(oxoid) and vegetable Agar i.e. vA (viae chapter
5, sub-section 1.5) were first tested. to assess the
relative growth of
S' pythiophila and R. pig on them. The media were
autoclaved at
Dextrose Agar
1089 gm/sq cm (15
pDA
p.s.i.) for
20 minures, rhe pH adjusred
ro 6.G before
plating, b), the use of either a LOZ solution of sodium
hl,droxicie or lactic
acid in sterili-sed r,+ater, and aoproximatery 20 mr
medium
r.ras poured
into
standard 85 rrn diameter petri-dishes. 7 rm
diameter myceriar agar plug
inocura from one rnonth o1d cultures of the
fungi were used as stanciar<i
inocula. One inocur-um,,+as praced in the centre of
a dry piate with the
rycelial surface in direct contact i+ith the medium. A standard
replication
;f 5 pLates was used. The plates wego-incubate<,
at lgoc in dark and
::served after 5, 1o and 2o days. Linear grorrTth
on agar (Ryan, Beadle and
-3tum' 1943; Fawcett , lg25), being the least laborious method
of estiurating
::cwth' was used. Two records of diameter growth
at right angles for
at every time of observations were taken for regutar
colonies.
;ere growth was irregular, maximum and minimum diameters
LTere measured
:: get an average. Measurement was taken to the nearest
ffin to avoid
=ach colony
112
._____rru. These
ruere Lr're
the sfandard
s
practict :s for
.:'::ies. rn ar.
the present and
..:-. - exper.
the first
subsequent
-4Perlment as a conve
nient
:::;:il perfoaman^^
and doubje check
r
t'anc€'r dry
on
,leigrtt of agar
col0nies by removal
0f agar
:
-':-f
ttdter
pal
and ilerye)r,
-.:: :ried on previously
.
in
1g4c ras also used. ?he co.{onies
foil at
r,reighed aluminium
:-..--:c inmediately after removal from the
1O5oC
for
so removed
24 hours, and
oven.
:rom the results of the aforementioneo experiment Czapek Dox Agar
(Oxoid),
gfowth
furtlaer
for
cllosen
y-u
co
invesc-igarion
was due xo xo><icify
of S. PJill5p$la
see
Lhe
vlreihe-
-edu-ed
it
and whether
ar <ieficiencsr
merjir:m is as follows:
could be furrher manipul aLed. The compositi-on of the
Sodium
2.o p
nitrate
Magnesium glYceroPhosPhate
Ferrous sulPhate
Poxassium chloride
Pot,assium sulphate
O.5
gm
50
ng
25O
nB
250
mg
Sucrose
30
gn
Agar
12
gm
Distilled water
1000
cc
Using this basic composition a nurnber of modifiei media were gsmpsssd
- each lacking
and F were
one
of
che
first six ingredients.
Thus inedia
A, B, C, D,
prepared. Bricish Drug Houses reagenu grade chenicals
E
were
used except magnesium glpeophosphalewhich was obtained f rom Oxoid Limited
(originally supplied through Zia',merm"l-rurfb" Ltd.,
Dawson Road,
Bletehley,
)Iilton Keynes, England). #edia preparation, culturing etc. were as before.
ihe twc fungi were grown on media A to F aad CDA (Oxoid) and at 18oC.
From
the above experimenE, presence of
nagnesium glyeerophosphate and
::-storted and irregular growth of l. pythiophila colon:Les were found to
::r-related.
Then changing
the concentratior:. of tiris salt modifi-ed CDA
-:dia G, H, I, J, K and L were prepared conEaining O.2, 0.5, 1.0, 1.5,
4.0 g of
magnesium
be
glycerophosphate/litre of medium respeciivelv.
2.O
The
LL2
fractions.
These r{ere t.he standard
practices for the presert
studies. In the first experiment as a
ancl subsequent
convenienE and doubie check on
growth performance, dry weight
of agar colonies by removal of agar in
boiling water
Lg45) r,ras
(Day and
iiervey,
also used. The cotcnies so
were <lried on previously rueighed aluminium
weighed inrrediately
From
after removal from the
foil at
105oC
for
removed
24 hours, and
oven.
the results of the aforementione<i experimenE Czapek Dox Agar
(Oxoid), was chosen for further investigation to see vrhether the redueed
growth
of
-S. U.chlgphil.a was <iue
to toxicity or <ieficiency
and whether
it
could be further manipulated. The composici-on of the meciiurr is as follows:
Sodium
nitrate
2.0 p
Magnesium glycerophosphate
O.5
gm
Ferrous sulphate
50
mg
Potassium ehloride
25O
mg
Pot,assium sulphate
25O
mg
Sucrose
30
gn
Agar
12
gm
Distilled water
iOOO cc
Using this basic cornposition a nurnber of modifiei media were
- each lacking
one
of the first six ingredients.
Thus media
composed
A, B, C, D, E
'nd F were prepared. Brieish Drug Houses reagenc grade chenicals
were
:sed exceDt magnesium g$neophosphalewhich was obtained from Oxoid Limited
ioriginally supplied through Zimmelpari-Hobbs Ltd.,
)'ilton
Keynes,
Englandt
rre two fungi were
From
Media preparation,
grown on media A
Davrson Road,
cutturing etc. were as before.
to F and cDA (oxoid)
the above experiment, presence of
Blerchley,
and
at
18oc.
ruagnesium glycerophosphate and
::sLorted and irregular growth of !" pythiophila colonj-es were found to
::::re1ated. Then changing the concentraEion of this salt modifi_ed CDA
-cia G, H, I, J, K and L were prepared containing O,2, 0.5, 1.O, 1.5,
:::4.0 g of magnesium elveeroDhosohate/1itre of mediurn resnecfiv,.'1 v^
be
Z.A
The
1l-3
two fungi were then
grovrn
on CDA (Oxoid) and modified media G, H,
I'
J, K and L.
7.3
RESULTS
of l. pythiophila but normal growth of l. pini
The results of diameter growth of S. pythiophila and R. pini at lBoC
for 2O days on MA, CDA, OMA, PDA and Veg A (table 7.1) and variance analyses
on the data (appendices 7.L a1,.d 7.2) show very reduced growth of
7.3.L
Media f-or suppression
t. pyrhiophila on CDA (fig. 7.L)
and a
highly significant T-ratio
respectively. Diameter growth of R. pini on Ehe five media showed slight
variations (fig . 7.2) but the differences were found to be i-nsignificant.
On
the other hand, on CDA diameter growth of S. pythiophila was highly
significantly lower than any of the remaining nedia (table 7.2). The
iimits of the *".. (-- t t.se) shor,red very high variability in the case of
of S. pythi-ophil.a on CDA. In rnycelial dry mass production
(tab]e 7.2) on CD,!9. pythiophila maintained the sane pattern, but 5. piE
attained the highest dry weight. This was due to the fact that on CDA
Ciameter growth
of pycnidia like fruiting structures (see figs.
:.3 and 6.4) with gelatinous exuda-uion which largely eontributed to Ehe
i.pini
produced plenty
:ry weighi. This was almost absent on the remaining fledia.
114
TABLq_
}EAN DIAMITER
S.
(20 DAYS)
GROWTI{
PYTHIOPIIILA AliD
P..
PINI
i.
AND MYCBLTAL
Rep
a
t t.
1i-
DpJ r^rErcHT (9C DAYS) 0r
ON FIVE CULTURE }MDIA
Iiean diarneter in
Iledi
l
l'lean <iry weight in
nuir
+ t.se
se
mg
cation
t. pythioptriia
X. p."i
1.
CDA
5
7.A*
53.4!L6 . t+
24.4!O.B
2,
27" t{A
5
83.3t
1.9
29
3.
Ol,lA
5
72.4t
2
4.
PDA
5
85.Ol
.).
VA
5
71.li
s. :f-" :_*ni*e
7.O
7
6c)
.2!\1.9
E. Pini
266.O!L7 .6
E
139
.4t\2.6
23.010.8
248.6iL7.7
185
.0114.5
O
28.6tO,';
2L5.6xL2.3
219. Btl_1 .6
0.8
28. 1tO.
84.otLg .2
85.Otr3.1
"3
I'cte: *7 - diameter of agar plug
206.6r19.
,4!O.5
B
inoculum.
IgBijE 7.4
:-l'liARY OF
DLTNCAIi?S
IruLTIPLE k.Al{cE TEST Oi{
-. S. pythj.ophta Dianeier
il
:. R.. pini
-. s. lrlE:elile Dry weight
. R. pini.
li:t.es:
DATA
IN TASLE 7.1
Ilean growih (Ciameter 5S ary weight)
on five media
fungi
:
THE
lt
ti
Means rrot. under seored
85.0(41 ._86.3.(2) l2.g.r__J_ii!.(r_ s3.6(1)
29./1(2) 28.6(4) 28.1(-5) 24.4(L) 23.0(3)
248{673) 2L5.6(4)
206 .6
(2)
84 . o (s
)
6e .2
(1)
26e,.o(t) 219.8(4) 18s.0(3) :139.4(2) Bs.O(5)
are significantly cliffereni (P = O.01)
Figure within bracket represenEs the mediirn.
t
115
t
FIG.7,|DLA'METERGR0I^ITHOFS.PYT}II0PHILACN2zUALTAGAR
ixxtnoss ace,n (P)l o^rlr3ai I'c*lt (o) - YEGETABLE
rN DARK
A].iO CZeprK DOX AGAR (C) nlrun 15 DAYS ii reoc
ffir,rro
(V)
AC,AR
o
c*t
C
.i
::G.T.2DIAMETERGRohlIHoFR.PINIoNTHEFI\rECUl,TijRE}EDIA
,ffiG .- i.u AFTER ts tavs nr rd"c rt'] DARK
,-
tL6
Therefore,
was possible tcr
CDA (Oxoid) i.ras chcseri
for manipularj.crn to sre vrhether it
altain further significant reduction in grorvth of
!. 2aEhfgp!.t:. withouc any narked redrrction in .growth of R. pini. The
diameter grorvth of ihe f ungi- were measured for -5, iO and 20 da.vs on media
CDA,
A, B, C, D, E and F.
in table 7.3.
r,rir..u se s standarrl error, Sd=
The resull-s are girrgn
t.se equais t. $
StanCar:d devi.ation, [ = nurber of obenrations
The expression
2.776
t, with 4 degrees of
freedom and
derive<l
was
5
at 5Z 1evel cf probability
was
and
.
0n all the media excepE C
and
of t is
table. in the present experiment j-n a!-L cases n
from Ehe t
hence
and thc value
arr.d
D, wiEhin iO days groruth, irregular
largely variable colonies of l. pythiophila developed. This is
represented by the
fact thaf the
rvere much more pronouneed
in
st.andard deviaticns
case
of the colony
of the rernaiaing media.
diameters
Thus because of
higher standard deviation, higher t.se values were obtained (vide table
7.3).
On medir:u D
the colonies of S. pytiil3g|ile were verlr? faint
expecied as the carbo::r. souree
C lacki-ng
in
(i.e. sucrose) r.;as absent, bui
magnesium glycerorhr:sphaie
ru'hich
is
on medium
S. lyl}fggLUg pro<iueed profuse
rrycelial growth on uni-form and less variable colonies up to 10 days.
Subsequentll', although variation increased to scne exient, profuse
-ycelia production continued. fhis iadicated that the fungus did not
suffer from the deficiency of mangeaium glvcerophosphate. 0n the other
:.and, except
in
case
of
medium
Driri aii other
cases, presence
of this salt
=d very irregula:: aaffmalformed colonies were associaced. This suggested
:::at the salt was toxic to s. plthiophila. To test tilis the fungi r+ere
:ext
gro\&'n
on
CDA and
modified media G, H, I, J, K'and L. The results
-:l ,liameter grorvth for 5, 1o and 2o days at l8oc (tab1e 7.4)
-; to lO days, tii;irneter
groruth
of S. pyttri-qphflg
was
re.,,ea-l.
signrficanrly
itrat,
7)7
J
iAq!91.3.
(il'{
DIA},IETER GP.OifTli
L,
I0.1) 01.- s. PYTHIOPHILA ,{ND R.
s. Ert!1sbltg
Ilean coloay Ciameter
ci f ica
7.Ar.
7
Grand
20
l.O I
l.O
c
.Ot O | 16. 6x4.6 | S:. +rro . o
8. 2t0 .7 123. Ot B. I
lt
+
.ot
7
.2
25.7
io.
20
10
ie.6l 24.51 18.1
3
.41 L4.9
35.
1
7.7
))
1
7.2 10.6118.o1 11.9
11
.6 i 2s
CDA-Sodium
CDA-Iiagne
glycero-
pho
_..r
I
l-o
CDA-Ferrous
nitraie
(^
Mean colony
ciiarneter
mean
(CoA)
sulphate
D
t t- se
Grand
Czapek Dox
1{
R. pini
tion
5
agar
0N cDA, AND IfiDIA
C, D, E I,ND F r\FIER 5, 10 AND 20 DAYS
]3,
Culiure media
Code Spe
Fltif
9
s
CDA-Sucrose
sul-pirate
s
3|
i4. 2t5 . 2i 43.9:L2.3
i_
sphate
CDA-Potas
.9tO.
i
10.8=i.Ol 19.311. 7 l44.Sr 9.6
25.O
Q1
Il
25
"7
8.1 10.3119.61 L2.7
11 0
7.4 11.4 I 25.61 14.8
"
61O,
31
8.8r l.4J
22.2it.7li,+1.tt
i-1
2.7
.9=4.5160.9i 3.7
1i.1119.91 13.0
CD.{-Potassir
chl-oride
s.4=o.sf rz .4tB'6l€L e16.3
,oo
12.
I
I
I
- diameter of agar plug
inoculum.
BI
27
.Ll ls. I
LL7
rAq!g_L.3
DIAI'IETER GROi!'Tl{
L2
(
B,
lli l0.t) 0l:
S.
PYTHIOPHILA i\ND R.
PINI
C, D, E z'ND F AFIER 5, 10 AND 20
ON CDA, AND ISDIA
DAYS
Culiure media
Mean colony Ciameter
t t. se
Mean colony
<ii arne te r
Specificarion
Grand
mean
Czapek Dox
agar
(CDA)
7.Art I
7.oi
l.O
7.O
7.A
o116.6!-4.6 )J.4flb.b
io.:
2s.?
I
3s.1
I t.t
i9.6 24.51 18.1
CDA-Ferrous
sulphate
B.
2t0 .7 l23. ct B. 7
74.o!
7
,2
11" 5 2s
.41
14
.9
i
CDA-Sodiurn
nitraie
9.9io.3li.t.zt5,z 43.9tL2.3
11
LLt
io.8rl.Olt_9.311.7 44.& 9.6
25
]3.6i0 .llzz.2i2.7 41.11 2,7
25.i I E.i
10. 3
8.8t1.4ll:-J.9=4.5 60.9t 3.7
)7
LL.4 25.61 14.8
9. =o.Sl r2.4t8,6 63.ft 16.3
29.e
-t I
i
I
-r.:-
n
10. 6
lS.ol
r1.9
CDA-Iiagnes i-
glycero-
phosphate
CDA-Suerose
CDA-PoEas
sul-phate
s
i
CDA-Potassi
ch 1or i de
=- - diameter of agar pl_ug inoeuLum.
.C' t1 E. 1
q
I
li.1 19.91 13.0
1e.61 L2.7
7.5 LZ. 6 2i
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1s.8
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L^r
NU
(JV
-
E
po
q-{
E
q
)
r..l
.lJ
'a
E
119
(P = 0.02) lower on medium L as cornparecl with
cDA or dny of the otrrer
media. From a separaie tria1, mean rlry in'eight of 10 days"
oJ.tJ col0ny of
,. pythiophila- an zT" v"A, cDA and medium L (ar tgoc) was fou'd ro be
154'B' 55'2- enc 15'2 mg respectivel.v. This showed tliat
in eotar mycelia
production by
-l' plthiophila. medir:m L differeci from cDA highly significantly
(P = o'oo1) ' Thus tr-he new nnodified medium L
is considereri to be very
useful for isoration of R. pi-ni froru tissue which is
also coloni-sed by
!-. u.thi.u:f1, because before the latter is able to grow our of infected
inoeula' there wil' be a well
10 days, growth
of R.
.3
of R. pirri. secondly, up to
ejn-L \,r'as somer,rhat lower on nediurn
reverse was true (tebLe 7.4)
7
gror^/n corony
L trut later on the
.
.2
R. pini
During the process of developing a se,-ecrive uredium
to suppress
s' pythiophila, the cultures were also exanined to
see r+hetirer, on any
of
the media, developnent cf eonidia or other Eypes
of spores occurred. rt
r+as found tirat, oa czapek Do.x agar, although
mycelia bore onJ-y a few small
sterile conioicphores! the black pycnidia like aiructues (bui
not pyenidia)
developed racher r,rcfuse conid.iophcres which
bore soue
examine wh;'r this happeaed andr,'hecher producticn
conidia.
To
of conidia coul<i be further
inpro'ed, a basic nediun, i.e. BII (LiIly and BarneEt,
1951)
r,'ith indi,ridual ingredients of CI)A srr6 media M, l.J,
0, p
in the usuai way.
vras supplemented
ar:d Q were prepare,C
The composition
of the'hedia i,iere as
fo.r-rows;
120
Medium
Basic mcdium (BM):
Susrose
Asparagine
KH2Po4
MgSo4, 7H20
Fe
+++
++
Zrr
(as ferrie oxide)
(as zinc powder)
++
Mn
g
o.5
g
0.2
mg
0.2
mg
i mg
5mg
lOO mg
Lzg
0.
Biotin (as D Biotin crysralline)
Thi."rni ne
Agar (oxoid No.
3)
Dis
rilled
M
tsM
+ 5e6ium nitrate
2g
N
BI1
+ Potassir:m chlcride
0.5 g
o
P,1"1
+ Magnesir:re gl)rcerophosphate
o.5
P
BIl + Ferrous sulphate
0.01g
a
Bll +' Potassium sulphate
o.35g
3- piti
=--
"1o
2g
1g
was grolrn on
10OO cc
\^rater
5 repLicate plates on ea.ch of tire
g
above medis
- exaiuined for conidia pr:oducrion.
i'Ioreo1'e.r,
natural subsiraEes such as lodgepoie pine bark, shoots,
::=:les, hay, whoi.e corn and processeci s\t'oGt corn i.iere separately
&oclaved and effect
lriameter growth of
-:--t
:
3i1, M, N, O.
P
sporul-atf a;d'of.
on
P,,.
pini
and Q,
X. pini
was
studied,
was very comparable on CDA (oxoid) and
after 20 days.
Hence
this is not included
r: , Pycnidia-type fruiting bodies, as noriccci on COA (oxoid), werc found
-
='.-=
lcp on
arecliuin
P containing ferrous sulphate in aciclitio, to thar in
121
rhe basie necliuin (BM). This indicatetl why in CIA (ov.oid) black pycnidia
type struct-ures dg1;sf6J.:ed. Furthernore, exarnination of the mycelia from
meclia BLI, !1,
N, O, P and
Q
for the
presence
of conidiophores and conidia
re.realed ti"rat in all the medi-a the rryce1-ia mostly clustered into myceliaL
developed from such mycelia
"orriai"pirores
(fig. 7.3). DevelcpmenL of conidia was, however, observed in the case of
aggregaLes and profuse snal1
to a lesse.t: ext"ent in Q.
meciia P anC
Hence
CDA
not very profuse.
in t}:e aext stage, the concentraEion of ferrous sulphate in
was var:ied an<i rneciia
2OO,3CO and 4OO mg of,
Then
Coni<lia \r7ere
R, S, T and ij were preparei'Oy addiCional
1OO,
ferrous sulphate respecEively/liEre of media.
R. pini was graw:r for 20 days in 5 replicate piates. Black pycnidia-
type fruit bodies vrere more profuse in media T and U which developed
mainly larger conidiophores on undifferentiated iryphae
slighCly more than that on
CDA
an<i
conidia,
a1one. However, mycelia in general- bore
only a few conidiophores. Therefore, because of practical inconveniencet
such inocula were eonsi<iered inadequate
inoculation experiments
in the next
fcr in vitro stuCies
arrd
artificial
.
stage- a number
natural substrates
sr.rch
as lodgepole piae
bark, shoois, needles, hay an<l whole corn, bciied j.n r*ater, partly
drie-d,
separately autoclaved an<i inoculated with R. pinl were tri-ed. Itlhen the
fungus was established, some
under near
and.
?Z
IA
of
Ehe
flask cuLtures were also treated
ultraviol-et tblack iight! along with plate cul-tures on CDA
(Couuronwealth
}lycologi-ca].Insti.tute,
exam"ination revealegoniy scanty production
1968)
'
Subsequent
of conidia on CDA plates
an<i
on corn. The fungus also p::cduced conidia to some exlenc on corn even
simply kept under
when the flasks hrere not trea.ted in rbLack lightt
lrra
laboratory conditions. The growth of, fungus \das not, hotlever, very
prolific.
Gialt
Therefore in ihe nexi stage Green Cia:rtNibl-ets Corn (Green
Foods
Ltd,23
WoodsiCe Road, A:nersham, Bucks, England), processei
sugar and salr, were
usecl
. First the corns rrere washed in tap \rrater,
in
122
partiydr;-ed, 50 g taken in 250
1OB9 gm/sq cn
rri"l-
flasks,
27, stcrose arided, autociaved at
pressrrre for 3O minrites, inoeulatr:C r;ith a"uiycelial- agar
plug cuiture of 3" pjg1 and incubated. in alterna.te light/darkness
conditions of the laboratcry and temperatr:::e of ab,ou:lBoC-2OoC for
month.
On such corn more
prolific
grorvth
of the fungus took place
a
and
within 15-20 days growth the cob surfac€s r{er€ eovered by rnvc.elial growth
of R. pini (fig. 7.4)
aud haci
prolific production of conidia.
Thus
at last,
a souree of mass production of inocula of E. gir1i under investigation ruas
established. The fungus propagated in this \,/B'i wBS the standard sor:rce of
inocuLum
7.4
in the subsequent inoculation exueriuen:s
(Cha-pter 1O).
SU}O4ARY
The diameter growth
of S. p:rthiophila in pure culture ot
is about 2.5 times faster tiran R. pini (table 7"5). This
suppression
27"
malt agar
causes quick
of mycelial growth by R. pini from an infecteC tissue if that
is also inhabited by S. p,y_thio-phiia
and thereby
results in undesirably 1-ow
recovery of 5. pir,i unless Lhe material is really in an early stage of
coLonisaiion.
isolation of E. ?.*:. Czapek Do:< a.ga:: (O:^:oi.cl) was found
to suppress S. pyth.iophila signifi.cantly (P.= O.Ol) in ihe first few days
es compared to 27" yf-. It a11owe<i cunparable growih up to i0 days, bui
To improve
subsequently grorvth
of S. pyririophilg
Through manipulation
glyce:ophosphate
in
$p-A
of
was
the
became
_iD"gtedients
signifieantly
it
was found
(P -= O.O1) higher.
that
magnesium
primarily responsible fcr the suppression of
S. pythiophila, the rnachanism of rvhich has not beeri studied. Further work
;:i ihe concentration of this ingredient revealed that
r,rhen
its concentration
-s increased from 0.5 g to 4.59/Li.tre of medium (i.e. mediurn L) not only
:;;ther sigiri.ficant (P = 0.02) suppressior: of s. pyrhlolh.iia as ccmpared
::
CDA
occurred but also E. pinl was al1or,,red to develop a significantl-y
: = tl.O1) higher diameter gr:owth as
compar:ed
to S. pythiophil-a on ihe
same
L23
TABLE.
DIAJ\fiTER. GRoWTH (rN
(oxorD)
OF
I4I"1)
S.
.7.5
PYTHIOPIIILA AND R.
AND I,EDIUM
!4!r
oN 2Z yLA,
CDA
L AT'TER 5, 10 AND 20 DAYS
S. pythiophila
R. pini
Media
10
5
2Z
l{alrt
7
agar
Czapek Dox agar
s
24.9
(i.e.
7.A
CDA)
+ 4.g magnesi ,m
glyce rophosphate/ 1 it re
7.4
44.t
15
20
7.A
83. 3
.6
5
10
20
7.O
7.O
10.5
7.O
27 .7
29.4
10. 3
19.6
24.5
tJ.b
26.8
CDA
7.A
9.4
40. o
9 5
x,liameter of agar plug inoculur
It is therefore obvicus thaE on medium L more preferential isol_ation
:: R. pini under iavestigation wiil be possibLe than CDA (oxoid) afforded
.table 7.L). Figure 7.5 shorvs rhe grows of S. pythiophi-la an 27" tIA, CDA
,ard nedium L.
secondly R. pi-ni der,'eloped only
agar.
sterile myeelial grou'rh on 22 raalt
czapek Dox Agar was found
to suppori produetion of, pycnidia-like
:ruiting structures (see fig. 6.4) whieh contained prcfuse ccnicliophores
:: undifferentiated hyphae (see fig. 6.7) but conidia only to soge extent.
;: uediun P (Basic
- '01
medium
by Li11y and Barnett (1951) + ferrous sulphate
g/litre) prof::se conidiophores
5leve1-ope<i
:- pycnidia-1ike strucrures, but loniaia
r,rere
both on ordinary mycelia
and
not prcfuse.
Fina11y, canneci eorn (Green Giant liiblets ccrn) was found
to support
:::fuse mycelial gror,ath as'*rerr as prolific production of ccnidia of
_{. pin..
r:j s was used as ihe standard medium for mass production of eonidia
of the
: -:;s
and
for use in vilro sturlies
and
in artificial
inoculetion experimer:.ts.
L24
*:
r
a
FIG. 7.3 R. PINI I,vTlEN GROi4t{ 0N
M,I;_bl P_A\D Q PRODUCED THESE
SI'{ALL
COI{
IDIOPHORT
,*
FIG. 7.4
THE BASIC I'fiDIT]]4 OR },SDIA
HYPHAL CLUSTERS A].]D PROFUSE
S
*^
l.OTilOD OF }1}.SS PRODUCTION OF COIJIDIA OF R, PINI ONsr,uErT5ruu pLUS 2i; sucRosE. TliE pHorocRApli slioirs Fnorusr
GROWIH OF TH]i FU}']GUS A}tD SO}G UNCOLONISED CORNS
125
FIG. 7.5
IffiTl
COLOI{Y DIA}GTER OF
czApEK DoX AGAR
AFTER 10 DAYS AT
1BOC
S. PYT}IiOPI]ILA ON 22 }IAJT AGAR
(lnftulf
,altrj lrenrur,r i,
(
si,ralLE.sT)
L26
7
.5
DISCUSSION
of agar inedia for the selecti.re isolatio* c,f specific
fungi or ariy micro-organisms is genera-r1-y based on the pri,ncipr.e of
selective Exclusir:n of undesired rnicro-organisrns, thus p*rmitting the
Development
preferenti-al establisirnent of the desired fungi on the isolation
(Tsao, L97O). In the present stud), the same basie principJ,e has
medium
been
followed io suppress rhe growrh of rhe undesirecl fasr gror,ring S.
lylliggilg
(as was obrrious from the::esu1Es of conpreheasive isoiation
of fungi, in
chapter 5) r'hile provi,ring
f
rom the patt.ern
no::maL growth
of iscLation, chapter
of
R..
gir!
(suspecced as parhogenic
6, and sr.rbsequently proved as the
rathogen, chapter 1o). This was achieved by increasing the concentration
o; Magnesiun glycerophosphare in czapek Dox Agar (0xcid) frcm 0.5 g/lirre
to 4.5 e/tLtre in the new modified ruedium L._
rt may be bc'rne in rnind that although magnesium is generally used
-agnesium sulpi:aie aild phosphate as
salts of
potassium.
generally
as
a
:cncedtration cf 0.5 to tr-.0 g,lliare cf medium is of more coilnion use in
-'-:rgai culrure ne.lia (ioorh. 1"gjL). Crabil_i (1912) and Scheffer anci
-i'lker (1953)
usecl a uuch
higher concen"tration of 5.0 g/1j.tre of potassium
::'-srogen phosphate and 2,5 g/tLcre of Magnesium su1phate to
suit their
.::cific
needs.
The use af ne-gnesi,;:n gll,cerophosphaEe
cor:1cl
:unga1 culrure rnedi-a lisred by Booth (197l).
-zapek Dox Agar (O:<oid)
not be traced i"n any of the
The ontrlr use found
was
.
-he nevr modi-fie*'mecium L has the attr:ibutes of not only si-gnificantly
^-:_^
l. p:'llrioi;hita further as
-.::essing
a,
con:p;rred
io the growih of ihe
fungus
: =:ec to s. pythioph.ila. It is iherefore cc,nsi-dcresj thai the higher
- -=:':ration of Magnesium glycerophospirate
is rrrl.ikely to natter irnci
-
-:= i.-o::kers on shoot dieback of loCgepole oirre alrs6gio;uri r,.ittr R" pini
1r7
Secondly, canned corn with 22 sucrose as a
meditrrn
producti.on of co:riCia is,-rndoubtedi-i' ve::y valuable.
for
rnass
r28
CTi.,TPTER 8
B.
O
8.1
rl'l VITR0 STUIIIIS OF IIAMICIILORIDUI'I PINI
E!-FECT CF TEIPERATURE,
8.1.1
pl{ t'rln DI}'I'SUED DlrY LIGIIT CN }{YCELUTL
GROI^ITH
INTRODUCTION
It has already
been evident rhar Rami_chlcridium
is a nels species" It
comes under
class
sub-division
Hyphorlvcetes,
, !971)
and
plg!
fanily l-{oniliaceae, or,ier' Hyphcmycetales,
Deu'ce.romycotina
is probably ihe pathogen of
(afterAinsworth
lodgepol-e pine shoot <iieback
(Chapter 6).
!l
\,'Knowledge
of the cardinal- (i.e.
minimum, optimum and :naximal)
temperaiures, pH, relative hurni-dity ete. of a fungus in vitro ccnstitutes
a very useful basis fo:: dealing rvith the behaviour of that particuiar
fungus
in vivo.
oPcima o.f
Togashi. (1949) from a compilation
temperature
a iarge nunl:er of plant pathogenic fungi inCicared rhar the
cotimun ienperature
for
of the
fot spore germination is geaerailv
in cultrt*.,
gror,rth
1
Har,rker (1950) noted
:iten takes place over-a broacle-c range of
:,
ver3" close
pH than
that peraritting
spor-e
1
:'rt so far i11-defined, infiueaces
'::d' formation
roi_r,
exei:ts undoubted,
on mycelial growth (coch::ane, 1g5B)
of conidia of various groups of fungi
incl-udin-g rnembers of
the Deuteromycotina (Fikry, Lg32i..-H{ston and Oswald,
ihe most preva!-effr fulgal associate
1946:l
of It. p_1"! i" shoot Ciel:ack of
-: :;:pole uncler in'vestigarion is I. I1,t:I1clg!.!1g (chapter 6) .
-. :-.:'te fast grot,'ing r.lith a
-: 7.5) .
thaE
that uycelial growth in fungi
:;oducti-on. l_Ligirt of r.he visibLe range, 4co-Eoo
: =-
to
Xenr:poulo
rnean
The larrer
dianeter growth of abr:ut 4 irrn/,3ay (vide
s (1974) recoriled 5 mr/riay rnean dia:::reter grcr,"iir '
- - ' )1a1t Agar and found 25oC as the optinnilir teirDerature for n:';,.celi-al
- - ':.'- anc germination of biastospores. Bc,ttr myceiiill gr(1:!'th and
128
gl!:gB
]T !fT{g
B.O
STU}I]:S 0F
8
RA]'IICIiL ORIDU],{
P
IN
I
I
8.1
EI'FECT CF TEIPERATURE, pI{ t_,tn DIF5,SUED DAy LIG1T ON }IyCELIAL
8.1.1
GROWTH
INTRODUCTION
It has already
been evidenr rhat
is a ner'r species" rt
}ggigh!1ilgtut 3;-nl
comes under feroil-y Moniliaceaeo or,ler. Hyphomycetai
es,
class Hyphorlrcetes, sub-divisioa Deuteronycotina (after
, 1971) and is probably rhe pathogen of 1or1gepo1-e pine
Ainsworth
shoret <iieback
(Chapter 6).
r'- Knowlecge
of the eardinal (i.e.
mi*imum, opt.imum and naximal)
relalive huraiclity etc, of a fungus in vitro ccnstj.iutes
: very useful basis fo:: dealing rvi.th the behaviour
of that particular
temPeratures, PH,
:ungus
i* vivo.
Togashi (r94g) from e compilation of rhe temperature
:pE,.'ra of a large nunber of plant pathogenie fungi
i*cicaced thet the
::tinum ierrDerature for spore ge::rinaticn is geireral-1v verl*
crose to that
t
:lr
in eulture.
cu l ture . Har'rker (1950) noied that nrycelial
-',-: growth "n
groin,th ir: fungi
H
:::en takes place over*a broacler ra:1qe
ra:1ge cf
of nH
pH ih.ari
than ,-h:ithat *o-*.i+r.i-permitting spore
.,
- :::.:ct j"on. Ligirt of ihe visibl-e rair.ge, 4co-EOo
rni_r, exei:ts
undoubt-ed,
--: so far ill-cefined, infiueaces
on nyceliai- growth (coch::ane, 1958)
- :or-mation of eonidia of various groups of fungi inc1udi3g rnembers of
'. : --: i.rre-ro,lcotin.r (Fikr-y ..g32;
Ururto, anri Oswald, 19,i6).
,
-ee most prevalenffulgal associate nf
p3.nr in si:.oot ciieback of
-'-
-8.
- :-=:;ie ,ncler in'vestigarion is
-{.. !L.}:_tpLil3 (ciraprer 6}. The larter
-: - -::e fast grort'ing r,rith a o,ean diarreter growth of abr:ut 4 mm/day (vide
-,
.: . .5) . Xenoptiul.os (19 74) recorried b nunlday mer.n dianeter
!:l:ch:ti:
- :::it Agar aird found 25oc as the opiimuur temDerature for
n:.;iceri-a1
- ':'rr Sernri.ratirin of blastospores.
Botrr
myceiiill
grc:,;.ch ani
.
129
blastospr:res germination vs ternperatli:es irad sharp aucfit* on either side
of tire optinurn. A knowl-c,dge of the optimum concii-tions fsr nr-v-ce"lial gro&'ti}
of R". pjini is thus very importarrt- in derreloping mo,e exaciing isolation
methods as
vrell as in. decidi.ng up,:n
optimum conditions rviticir would have
to be providerj during inoeul.ation experinents. Ior the abc.re realsonS,
the effects of temperature, pH and diffused day light on mycelial gror'rth
and production
B. 1 .2
of conidia in cultu::e by 3. !1n! were
studie<i.
}L{TERIAI AND }TETHODS
Unless oEhengise mentioned , 27 MaLt Extract Agar (.27 llA) was used
as the culture medium as this was fcund to supporE the best uycelial growth
of R. pi-ni as compared to the other nedia tested (table 7.1). This was
nixing 2o g mat-t extract (oxcid)
,r;r;
in
1OOO m1
of dtstilieC water.
anci 12 g
oxoic agar no.
The mediurn vas autoc1a.,'ed
at
LO89
3
g/sq
cm
(15 p.s.i.) for 20 minutes. The plt of the mediurn was 5.8 and 5.2 before
and
aiter autoclavi-ng respectiveiy.
The plates
prepareC b.v orocuring appro-xiriately 25
ul
for inocuLation
medium
in
9O nu:
were
(oute:: diarcete-r)
petri-Cishes and leaving ir to solidify.
8. 1.2.
1
Ef f
eqt. o-f._!-eneerg*re
Determination of the effecc of tenperature- on lhe aryceLial growth
of R. g:4
was based on
linear diameter growlh on agar (Faraceti,
Ryan, Beadle and Tatnoa, 1943) an*.b5i"mtcelia1 clry rveight
by renoval of agar in"Soiling water
The inoculun cousisred
(Da5. and
1925;
of agar colonies
l{ervey, l9li6).
of a plug, 7 um in
diameEer
cut from the
periphery of a 20 days olri subcul-ture on 2X },lA, by means of an agar plug
cutter.
The small- coLuru:
of agar plug with
thp- inyceliuro on
the top
tras
:raasferrecl u'ith rhe help of, a sterilised scalpel on tc the cenlre of
-r,v 2Z l4/r,
pll 7,2 (deeiried from a prr:liminary ev,perirent ariil adjusted
a
I
-=El
130
just befr:re pouring the plates) arrd place*
that the
in.,,erte<l so
growth came in direct contact rgith :he n:,e,lj.rrm. The pet::i
sealed
with SeIlotape.
incubaEed
at
Ooc
The incubators
Each
rnycelial-
cli shers rser:e
replicate of 5 ptates, thus inoculated,
u/as
, zoc, 4oc, 7oc, l1oc, 1soc, l-goc, 21oc, 25oc and 3ooc.
for lloC,
15oC, l-8oc and 2loC were maintained
in cc,ld
room
enabling the selected temperature to be kept virtually constanE. Other
incubators also had fairly good control, and f luctuaEi.cns, if any, \^rere
limited Lo + loC at the nost. The diaureters r.rere neasured. after 10, 2A,
30, 4ar 50 and 60 days to the nearest nriliimetre. At each ti-ine every
colony was IEeasured along tr{o perpendicular iines passing through the
cenEre
of che inoculum plug and thus an average diameter for a particular
colony was obtained.
8.t
,2
.2
Ef f
ect of pI
A serious problem to get solidified agar medi-un, even r,rith 4,2 agar
aE
a pH 3.5 and lor.rer'$Ias recorded by Dianandis (Lg77). He noted that
RhizosLhaera
E$:qlfi
Birbak could
grorn,
f,e properly measured as ctre ge1
-,,r&s
: rob lem r+as sought,.
I{A was
27"
at DH 3.5 but diamerer coul<l not
not firm. Fi::st a soi.rrion to this
first auroclavei
potringthe plates, at about 4Oo- 45oC, the
sterilised lactic aeid
(LOZ
aad
pH was adjusted
solution). By this
arethod a
just before
by adding
firm gel r^ras
2.6, using only 27" of the sarre agar (oxoid agar no. 3).
From a prelirniaary rrial E. Ets3.tas found ro grow down to pH 3.2.
obtained
dor,m
to pH
serefore, plates 9f. zLW having pH 2.G,2.8, 3,0, 3.2r 4.2r 5.2,6.2,
7-2' 8-2, g.2, ta.z, 11.2 and 12.2 were prepared by adjusring Lhe pH before
grcuring. Five replic.ate prates were inocurated in the
same way as
described for the cenperature experiment. The plates were ineubated in
the dark at iBoC. i)iametet's were reeorded i-n the way already ciescri.bed
after 1o, 20, 30, 40, 50
and 6o
days. Dry weights were
al-so deterrrined
)-
131
as before.
:.-i;h it,
The pH change afcer fun5lai grovilh was also measured. To cornpare
anoEher series of 2Z
i,I,A
plates with pH 2.6, 2"8, .).o, 3.Z-LZ.Z
was
:repareci, autoclave,l and pI{ values after autoclaving were recorded.. Ttris
;as dcne merety for interest to see rghat would iiave happened if
an
:':toclaved series had been used.
5.t
2.3
Eff
From
ect of light (EJ day)
previous experience it was
develop any
Agar
it
and darhness (by nigh!_:S_-""1g_tanr
knor^rn
that on 27" MA n. piql
<i_arkne-sl
does not
fruicing bodies or conidia in light or dark, i:r:t on Czapek
does develop black- pyenidia
like structures,
Dox
aad conidia only rarely
(chapter 7). Therefore, to conpare the growth of the fungus, to quantify
the effecl of light on diameter growth and to obsenre for spclre production
the fungus was grown on these
to 7.2 after auioclaving
t-wo
media. The pH of the
meriia was adjusted
anci approximately 25 m1 medium was poured per
plate. A myceliaT- agar plug inocuLurn 7 nun in diameter,
was then
transferred fron one month olci cr-rl"ture of g. gag| t.c the centre of each of
the plates. Five plaies of each meCillm were covered with al-uriiiniun foil
Ehat the plates vere
The second
in the dark but eera,tion
set r:f plates
was
\,r3s
so
not totallv cut off.
not coverec. These plates were then
incubated aE rcon teni)erature (1Bo- 22oc) in a transpare.fli perspex chanber.
fhe Ciameter growt.h was reccrde<i
4L
iO day intervals up to 60 days
:ry r..rsltht.s were also determined
t_n
the way alreaciy described.
.
]-.3
.
i.3. t IIEgL of
BJSIILTS
t_enperaru.re
The mean diameter growth
.'-:! on lrear
- - :s
when
mycei
j-ai d::.i rveight in table E.2,
trated in f igs .
:::crature for
of 3. pitl i-s rabulaced in table 8.1 while
8.
i,
B.
These ar-e frrrther
2 and 8. 3 r^;hicir show tirat tire
n,vce1ia1- growth
at
pH
oprimum
7.2, is i5oC',,'ith nc gr6r.,ti1 at
L37.
,,;f,
,o
30
#2s
\
5?1
qoo,,
-..*7-.
r5
8.1 EEFEqT 0F TElfEzurTURrs oF ooc, 2oc, 4oc, 7oc, i1oc,
i5crq 18oc, 21oc, 2-5oc mm 3c,tc oN TiiE DTAI,fiTER" Gp,ol,f?n oF
R. PII.]I AFTER. 50 DAYS OF INCUBATION ON 2Z MN-\ AGAN pH 7.2
FrG.
(J
o
@
t{
lt
()
o
F{
()1)L.:()i.)
L)
ccoco
r4.fc.io(}
o
rl
N
F.l
.,-) <)
tI
\
I
I
\
a
z
H
TJ
O
H
O&
ca f:l
P{
H
FI
zl-{
\
*
t
i
I
t
ii
i
\tti
rl
)l
I l.'
\\
OCC)
-j'
'
:Y)
:I
al
"ilr{
Z
o
O
\t
c..t o:
N
@
F{
tii itiriStl
OO..OOOO
\o -.i
a\
rl
-i
r-l
C)
,r.iifi .\}.i0T03 lrf3w
co
'J
\i
\t
r-.1
I
(rl
t.)
-2.
I
I
:
-
P,,
:
g
--
rlf
/
I
,l
I
@
J
o
Z
H
L
ct6
L!
=
F
zH
FrJJ
LJJ=
-
i:q
2_
oo
)
t!) Crl
f-.{ i:)
(J
x.
r-1
t9
t;
lr
Ir
b.
:
:
ll
(t,.
.3
tsl
ts
€t
e\
\\
U
\
O()O
\a
ca
(\
'i{'i NI }1i{,1:tl,"t\,/Ie .i.ft0T{i3 tiriiit
p
H
\
135
TABLE
DIj,METIiR
-8I
E. 3!g AT DlFFiiP,llNT Tiit{PER.i!Itri:r.!:,S rfN
,") AFTIIR 10, 20, 30, 4A, 50 ;:rrd 60 DAYS
G,\.OWTIJI OF
ur
fiean coiony dianete-r in
Tenperature
2Z id\LT
Mean dia.
groi.rth/day
um
oC
in
10
20
30
40
50
60
7
7
7
7
7
TJ
1
7
7
7
7
0
8.4
o2
o
7
2
7.O
4
8.0
9.8
7
9.4
14.3
i9.3
11
10. 9
20.
1
27
15
1) I
.)tr
t.
l_8
15. 3
ZL
..i_i. c
25
8.6
30
mm
LA.2
it_.
B
0.1
L4.L
16.
3
18.9
a.2
L).
29.8
37 .O
0.5
J).U
+J. I
49
.5
4.7
JO.J
48.2
61 .9
69.2
i.o
30.5
41 .5
53.
53.
68.2
1.O
25.5
31r.5
45.2
5s .6
i1
')
L5.B
18.
11
7.O
7.0
1)
)
.5
t
I
2
7.4
O
1
AGAtr{,
0.9
25.6
(1. 3
o
1.O
liote: * Diairreter of agar plug irroculum.
ooc and 3ooc. Liirea.r ezrension at 18oc was mcre than that at r5oc up
;o 5o days but subsequentiy growtrr ar 15oc excee<led that at 1goc. Mean
:;.,ce1iatr dry r,eighr was, however, -,gf
:rowth at
-ry
zLaC was coffsiderab,
mass producti.oa
I"r"r ar
15oC (f
ig.
g" 3)
.
Diameter
I.v greater than thai at 1loc, br:t in mycelial
the differenee was just rrarginal . A par:aI-le-1 situation
exisied at 7oc an.d 25oc. The growth curves a::e almost syrnileirical .
,LJO
IAELL-9-a
IIEAN MYCELIAL DRY WEIGHT OF
R.
60
zuI
AT DrI'FHRENT
TEr.rpERA:rUB.llS AFTER
DAYS GROWIH
Incubation
Replicatiqn
Temperature
PH
oc
Period in
llean Cry weighE
(in mg) t t.se
days
7.2
o
-7n
2
.0*
60
5
60
5
1.8.2t 2.L
4
4
t./
60
5
32.8! 7.2
'1
7.2
60
5
97
1t
7.2
60
15
7'
1B
.6!LL.2
5
L49.4!L2.8
60
5
220.8!1t.3
7.2
60
5
199.ztLA.8
2L
7.2
60
5
155.61 8.13
25
1.,
6A
30
7.2
60
Mean
94.A! 6.9
4. O*
5
u'eight of mycelia of 7 sm di-ameter inoculr:m plug
= 2.776 ai P = O.O5; se - standard error
-lt all the ternperatures u,here gror.rth cccurred, the central part of
'
--
-trrrle
s, rncluding the orig"inai iaoculum, v/as paie mouse grey, 15,
: .:iayner, 197or; on the uppe.r surface. rn case of the colonies
. -: lloc, l5oc, lEoc and 2roc, except ttre ceritral area, most parts
'--= cclonies i,sere dull green (29,7O n) and
in these c.eses the reverse
-1:': green (33, 21 m). In most c:ases the coion.v rnargins rere eati.re,
-
,
-:::rcst
-
f
ringe of abcut I
run
was l^rtritish,
-' L .5 mmr'iay r{as obtainr:,r a[ ]-iioC
-f
r:ora
A, r.irilximuril incar) <li,:raeter
1i-rc ria.rs,
aner.
tiren
137
gradually declined (fig. s.4), A similar paEtern was also recorded at
l1oc,
21oC and
25aC. The re,erse \das true at 2oC, 4oC and 7oC where
diameter growth rate gradual-ly increased. At. 15oC there seemed to be two
peaks. At no Eemperature did any spores deveiop. This
r17as
ascerrained by
microscopic examinations.
8,1,3.2 Fitting the tsrmperature/mycelial grovrA s;1qeJ@
In order to gain the ad.rantages of a mathenatical, predictive
atEempts wer:e also made Eo
model,
fit the daia on Ciameter growth (table 8.1)
and
raycelial dry weight (table 8.2) to predictive regressicn models.
Considering the cun'ed nature
of the data, the polyncmial- regression
:rom the Minitab programne (R.yan, Joiner and Ryan, 1976) \t,as consi-dered
as a more appropriate tool and was therefcre utilised for arralysing the data.
The resulEs
:ig. 8.5.
of
Ehe analyses are presented
in appendix 8.1
and
is:
Y:2.2O + 5.67 Xl - 0.0360 X2 - O.OO51 X3 The F*raEi_o
:igh1y signi-f icant. The R - squareri value of (C.9566)Z : O.9i.5O
The regression equation
L76.2_9
is
:epresents that 91.5 per cent of tire variation in the mycelial diameter
:lcwth can be explaine<i or predicted through che knoroledge of temperature.
I::.us, the mycelial diameter gror,rth and Lemperature data fit a third order
:: l,vnomial regression model (f ig. 8.5) with reasonable accuraey.
Similarly, the mycelial dry weight
:-: a third order polynomial
and temperature data are seen to
regr-e.s.s{on model
(fig. 8.6
and appendix
a.l) reasonably well*
:.
-,3.3 Effect of
pH
The mean diameter growth
.--.-.:.r
of R. prni at 12 levels of
pH
of
27. I"1A
is
in table 8.3, while that on mean mycelial dry weight is in table
-,-r l\tqtq SIg [Ulthqf ilhiltrote( in f,igs. 8,7) 8.8 and 8.9 vhich shcw
r38
prc.- B.l
GITOWTH
RATE
Or
R..
qrNr
oN
2U I,IAI,T AGAR AT i]iI.F!]i{ENT
2.O
2.O
1q
1.5
1.O
1.O
o.5
0.5
TEI4PERATURHS
U
^o^
ZV
2.A
2.A
1.5
1.5
1.O
1.O
0.5
o.5
0
-o^
iL
rroc
2.A
2.O
1.5
1.5
l.o
1.0
o.5
o.5
o
15oc
tSoc
2.O
2,O
1.5
-'"- 1.5
1.O
1.0
0.5
0.5
10
2Loc
20
30
40
_q0
60
,o
lo
25oc
DAYS OF INCUBAT]ON
?_o
30
40
50
60
1:lB
ITIG.. 8.4
GROI{TH RATN
0F
il. lINr o*
i,p.i,T AGAR AT ,,ir,irEREr,{T Tri*pliiv\TiJR}ts
:,
2.0
2.O
1q
1.5
1.O
1.O
0.5
0.5
0
2"c
2.O
2.o
1.5
1.5
1.0
1.0
o.5
o.5
o
7oc
2.A
2.A
1.5
1.5
l.o
1.O
o.5
0.5
0
15oc
18oc
2.A
2.O
1.5
- """t.s
1.0
1.0
0.5
0.5
10
2L'c
40 50
o
60
DAYS OE INCUBATION
30
40
50
60
139
ITIG. 8.5
DIA}IET}iIT GIiOI{TH
OF
R. PI}iI
ON 'IJJ}fIERATURN OI I}JCUBATION SHOWING
it
THII DATi\ POINTS, TITTED CURVIi AND RliGRilsslol{
EQUATION
l. t111
REGRESSTON
aquarrorv rs
Y= 2.?A * 5.67 W - 0.0360 x2 - 0.0051 X3 ...
EQUAT.TON
NO,
3
Y
80"
3+
2a
68.
*
4
*
50.
rt
:0.
8.0
TE},{I]EP"ATTJRJ]
16.0
24"0
OF INCUI]ATION IINOC
32rO
40" 0
140
FIG. 8.5 I{YCELIAL DI{Y IIEIGIIT
OF
R. Lryl 0N TEIIPER\TIIRE 0F
INCUBATION SHOWING
EQUATION
TI{E Dr\TA I']OINTS, FITTED CURVE A}iD }ILGII.}.,SSION
,
THE RNGRESSION EQUATIO}I IS
Y = -53.2 + 28.6 Xl - 0.768 X2 = 0"0043
...
X3
EQUATTOTi
NO.
4
Y
e40.
)
Z
dq
-.1
{Iz
2
180 "
-:: 120.
:-:
_-1
=
a"
:
=
)-
6fl.
=
:
=
O"
L
0c
T.O
21.0
14.0
TE}iPERATURE QF INCUBATIO-N
-./
IN
OC
x
e8"0
3.5.0
L4L
a @
t tot
t
,.- /
I
_/
*
ryG. B. 7 EFFECT ,oI B-H_OF 2 . B, 3. o, 3 .2 , 4 .2 (TOp ROw)
z^lulgoru nowl', e.2, 1o.2, 11. 2 AND ,
l;,
; u,;?,1."I;r;^.u.:
(EorroM Ror^r) o' zz-uar.i"oqn*'o*,r,il.i;#,i,io^filo*,
L2.2
oF R. prNr AFTER 60 DAys ar radc
L42
FrG. B.B I{LAtr DiAr,ffTEri
2Z I,L\LT AGAR AT
GROTJTH
Or q. PIIiI
AT
12 LEVELS 0F INITIAL pH Oti
1BOC
,-H
Y
u
a
|
7.2
Q)
2.1
1O.2
lL.2
3.2
30
12.2
20
3.o
2.8
'10
20
?n
INCUBAT]ON
PERIOD
40
IN D/TYS
143
FIG. 8.9
OPTI}IL]M DIAMETER GROWTH A*I{D :I'OTAL }fYCE].IU}I PRODUCTION AS TI]]ASURED
BY DRY I,ILIGHT OF
at
R. PINI
ON 2Z I4ALT AGAR AT 11 LEVE,LS OF' PH AITER 60 DAYS
l8oc
o- _._
(F%O
--
_
€
DRyt4EIGHT OF MYCELIU,t/COi,Owy
COLONY DIAMETER
220
200
I
I
- -a'
8.- -'e' - -'S1BO
160
te
o
-/-za'.\a..
\
140
\r\\
120
'21
-ob \1\\
b\
ll
z
BO
40
c
4
2.
H
IEI
(J
E
fri{
o
H
--l
1
FJ
o
(J
Ff
il
2o
H
o
100
60
*
21
\r\r
\r\r
\r
il
()
(}
fl
20
I
L.2 2.2 3,2 4.2 5.2 6.2
./"
7.2
rNrTrAL pH oF ZZ'"il,o.lt AcAn
*:_
8.2
9
.2 10.2
11
.2
0
12.2
H
F
&
a
rd
74/t
rAlILt
DIA},IETER CROh'T}I CF
l
R. PINI AT DII'}'ER[NT
DH
lBoc arruR 10, 20, 30, 4A,
the
time of
culturing)
Replicate
plates
(
2.6
60
50 AND
Ap
2'/" Mr\LT AGI,R AT
DAYS
Mean colony diarneter
pH
(ar
LEVELS
in
Mearr
mm
diameter
growrh/
10
20
7x
7
i.o
30
40
50
7
1
60
day
7
I
7.O
7.O
7.o
7.A
7.O
7,O
7.O
7.C)
7.O
7.O
7.A
7.O
/.o
.4
44.9
o.6
o
I
2.8
5
7,C
3.0
5
7.O
7.O
7.O
3.2
5
7.O
l4.c
L9.4
28.
4.2
5
L9.9
34.7
43.O
53. 8
6L.4
68.0
1.0
5.2
5
2L,8
35.9
48.
1
57
.9
67.s
72
.7
1.1
6,2
5
2A.2
37 .O
/,o
1
60. r
7,.2
5
20. z
52.O
64.7
77 .O
84.
I
1.3
8.2
q
L9.2
33.
47.5
56.8
oo.y
7/,
1
1.1
17.
30.2
52.3
r,
)t.)
65
.9
9.2
O
I
O
10. 2
5
i5.8
)t-
LL.2
5
11.1
2A.L
1)
5
7.O
10. 3
15
'
-- Diarnter
of agar piug
A
11
a
O
36
t'O
.8
a
I
o
o
ta
76.7
1.O
I
o.-j
55 .5
64
27.O
36.2
42.5
50. 6
0.7
.3
r9.B
24.1
10
.)
o.4
.i
.4
1.0
inoculurf.
:-:at pH 7.2 is optimuu for diameter growth while at pH 6.2 iiighest rnycelial
-:,.- mass was produced. The effect of autoclaviug and fungal g::owth on the
:--3nge
:-
of original
l -:re 8, 10.
pH values
is
summarised
in table 8.5
and presented in
145
sql'jg
ciiANcjis
oli pl{ oF 2z }IALI iti:1i:i. AFTrin Aulcci.Avuic
ANif .\.i..rfriit
iiUNGi\L GR0l,,'Tlt FOR'6O Di\yS
+
EF"--_-a
0-*--4
ORIGINAL pH tEI,Or,ii CULTURING
R. PINI
FrNAr, ,H 4p1pB TuNGAL GROWTH io*?o
&_______o pH
,orc
CI1ANGE AFTER AUTOCLAVING
i2.2
lt.2
Lo.2
Q)
Q.)
7.2
6.2
<,
1.2
?)
'). L)
J'
/,
1.L
.)
5.2 5.2 7 .2 8.2 9.2
pH op 2z II,\LT
AGAR
?'rl
_.f
LO.2 LL.,2 L2,2
L46
TABLE 8.4
llr.-CELIAI DRy hEIGHT 0F R.
3r*.1
***o*,
o,
pti l,rivut s or,
tjl
r,rALT AGAR
Incubation
ReplicaEioris
I{ean colony drlr rveighr
TernpcraEure I period
oc
| (aays;
3.O
t.se(atP=0.O5)
tgo
60
5
3.2
1B
4.2
.
4.4 t
1.39
60
tr
)
L42.6
!
7.68
1B
60
5
184.8
!
L4.'1L
5.2
1B
60
5
L85,6
!
23.32
6.2
18
60
5
LgO.4
I
9.16
7.2
1B
60
5
L9i.4 !
L4.44
8.2
1B
60
5
zLL.2
!
26.37
9.2
1B
60
5
18O.8
r
3, 88
IB
60
5
L52.6
t
13. E8
1B
6C
5
L29.4
1B
OU
5
62.&
! 24.t5
t L1.99
I0.2
LL.2
..-,2
-::::I
0F AUTOCLAVING
AND
IA}!I.-gal
GROL'TH 0F R. PI.NI ON TIIE
2Z I{.tLT
-.-j:t ^H (i)
': =::er fungal growth
'- ,:. autoclaving*
q,
CIIAIJGIS OF pH
(in
OF
AGAR
6
)
1.,
o.z q)
10
.2 lL.2 L2.2
4.8 5.4 s.5 5.5 5.6 6.1 8.0
B.B
9,L
9.5
3.6 +.2 4.6 5,4 .5.8 5.9 qo
6.1
5.6
8.8
(1) - pH values were adjusted after
autoclaving.
* These values are from
a separate test
mg)
L46
I
}IYCELIAI DRY WEIGI]T
A,T.].L
I.1
U
.4
PINI AT DIiIET}1I]I{:i, pll r,rrcls
OIT R.
or i;x IlrrLT ACIAR
Incubation
Repl icat iorrs
l'{ean colony dry r,'eigl-rt
t.se(atP=0.O5)
Tenpcrature
Period
(days)
3.O
tBo
60
5
3.2
18
60
)
L42.6
!
4.2
1B
60
5
184.8
x
14.7L
5.2
1B
60
5
L86.6
x
23.32
6.2
18
6A
5
L9A.4
t f .i6
7.2
1B
60
5
L93.4
8.2
1B
60
5
= L4.44
zLL.2 t 26.37
9.2
1B
60
5
180.8
LO.2
l_8
60
5
L52
.6 t
13. E8
LL.2
1B
60
5
129
.4 L
2t+.15
L2.2
1B
60
5
^11
v
oc
I
4.4 t:
E
62.&
1
(in
1.39
t-
.69
3.
BB
! \4.99
TABLE 8.5
:ai::i
0F
AUTOCLAVING AliD GROII'TH
0F R. PINI
ON TItE CI{A}IGIS OF
pH
OF
2Z MALT AGAR
--:-aipH
'
(1)
=::er fungal growth
: :. uto clavinc*
=
(1)
*
).
/,a
5.2
/.
6 a
o.1 9.2 70 .2
5.4 5.5 5.5 5.6 5.1
1) )
B.O
8.8
9.1
9.5
3.6 4.2 4.6 5.4 5.8 s.9 s.9
6.L
6,6
B.
- pli ,rrl.res were adjusted aft,er autoclaving.
These values
tL.2
are from
a
se?arate test
B
mg).
L47
The diameter grou/th
Ehose
at
pH
5.2, 6.2
and
at
pH
4.2, 9.2
and 10.2 were
very comparable
an6
8.2 were cLose lo each other. At pH 2.2 markeclly
higher diameter growth took plac.e. The clecli-ne in diaireter gror^rth and
mycelial dry r,,'eight was rnore graduar in the alkaline range of pH but it
was
dramatie in the strongly acidic pll range. Mycelial grorvth was very comparable
at
;t
pH
tt.Z
and pH
3.2.
The growth
of the fungus at
pH l-2.2 r,;as
restricted.
3.0, only a few outgrowths, z-3 arn in diameter, (fig. g.1r) appeared
efterabouE 9o days of incubation from the origina! 7 nm.r inoeula,
but myceiia
pH
3rowth did not extend on
to the uredir:m outsitie the inoculum. 0n the
:riginal inocula, profuse pycnidia like struciures deveroped (rig. g.12).
-.-: pH 2.8 mycelium was
killed.
colonies gror,ring at pH 3.2 to pH 6.2 had more raised mycelial gror.+th.
-,-e upper surface of such colonies was of pale mouse grey (L7, 117d) while
black (5, 84 m). Colonies at pH 7.2 and pH 8.2 were
-: the central part sonei.rhat raised, mouse grey in cc10ur (17, 117i) and
:.--e reverse was fuscous
-=''-eioped profuse
pycnidia like f ruiting strirctures similar to those already
--:iced in figure r:.4, the remaining part of upper surface r^ras
rather smoo th
.,-: coloured fuscous b1ack, The reverse vias also cf the samL? col_our. At
:-= pH 9.2 to pH t:..2 coionies were smooth. compact and with yery little
-_ r-1 pycnidia type fruiting sErucrures, mouse grey (15, l18i) on the upper
':- :ark mouse grey (15, 1f9k) on the reverse. The pycnidia like structures
'::r 7.2 and' pH 8.2 had developed profuse, sma11 conidiophores on
--:rerentiated hyphae, but. ve.ry-. ;f6w or no coniciia. A very
'r::-::rion of conidi.a*on unusually thick conidiophores
-:
sparse
occurred at the
'": _ -
and pH L2.2
(fie. 8.13).
The hyphae became highly vacuolated at
u
- Ll
ilaximum mean diameter growth
.
of 1.8 runlday
was obtained
at
pH 7,2.
--.=::l-, at the more favourable range of pH 4.2 - pi{ 9.2, af ter an initial
- :: growth, the ,,te of growth, witir slight excepiions, gradually
---
(fig.
8.14).
At the
148
I
TUFTS OF I'IYCELIAL OUTGROWTH, OF R. PINI, 2-3
-8.11 TWO
rlr DiA.i'l-l-ER
DEVELOPiID Oli TiiE / lt'I rN Dri$tETER. IttcELrAL AGAR
PLUG INOCULIII, hHEN INCUBATED Oi't 22 MALT AGAR, pH 3.0, AT
iS"c rN THE DARK FOR 40 DAYS,16X,
TIG.
iIG. B.I2 PYCNIDiA LIKI FRUITING STRUCTUITES DEVEI,C}PED ON THE
:I'CET,TAI, AGAR PLIJG INOCULI}{, 7 }O{ IN DIAMETER; Oir R, PINI
,;{EN INCUBATED ON 2Z },{.\LT AGAR, PH 3.0, AFTER 40 DAYSIEX
Eqrr
t49
lrc. 8.13
PRODUCTTON 0! coNrDrA oti RELATrrvrsLy rHrcKER
coNtntopHoREs By R. prNr oN 27" MAL,T AGAR,, pH iZ.i,-ai--r8.c
NOTE THE OenfUNno rasat*SC,Ln OF CONIDIA, OZOX.
150
tr'Tn
r iu.
a 1/
urI{
GROITITI1
;;-
IL\TE 0Ir R.
rul
AT Dli,Fllltrltl pli t,lrvn,s ort zZ
ILALT
2.O
1.5
1.0
0.5
o
2.o
1.5
1.0
0.5
o
2.o
1.5
i.0
0.5
0
t.0
-i
-.c
'
:
10 20
LL.2
30
10
1'>
n
ACAR
L51
8.1.3.4 Fir-uing the
m.v_celial g::owrh/pH dara ro
re<1i-
cti-v models
As in the case of temperature <lata, iire data on tlianeter gror+th and
dry weight at pH 3"0 to pH t2.2 were analyserl . Ihe suilmiar)/ of anal.ysis for
diameter grovrl-h/pH data is given in appendix 8.3.
The regression equation
is:
Y = -77.L + 4Z.O X1 -2.75 x2
-he F-ratio L52.78 is highly significant.
The R-squared vatue
of
(0.9149)2
= 0.3370 explains 8.3.7 per cent
of the variation" The T-ratio of the
-:efficients :ire higirry significant. Thus the mycerial di-arneter growth
fit the regression model (rig. 8.15) ::eascnably r.re11. Simil.trly,
---e rryeelial dry weight and pH data fi.t the regression equation:
':d
pH data
: -305 + L74 xi -17.8 X2 + O,4g1 X3
---'e results of analysis and the fitted curve are given in appenciix
8.4 and
Y
16 respectiyely.
--
--ure
:
-.3.5 -Ej{S5:-5_rug-h./d".k".""
"" ".
"
B.
The mean eolony dia&eters reccrcie<i (taUte a.O) and
suqmary of
-:-ance analysis (Appen<lix 8.5) reveal that on both the
media, plates which
,"::: exposed co day lighr/night darkness i-n March _ I,Iay, J-ggZ,
aiEained
---:iicantly higher (p = 0.01) <liameter growth onr^rard from the third
:'=:i'ation (on L9.4.sz) Lhan those kept i, constant darkness. Again,
:'- ;he 4o <iay!s observation onr,rardr,.on czapek Dox Agar (cDA)., diameter
:-;:::
icantry higher 1p-='o.os; as compared to 22 MA, rn
the differenbes were not significanL.
was signif
.".::,.-=_:ss,
I
.T
l
I
.i
J
t't
L52
IrIS-:-8._15. IIIAMLTER
sHOI^IINC THE DATA
GROI^ITH
0!' R.
P}Nr
ol; INITTAL
pH OF
p0INTS, FITTr,lll clrttv[ Al.tD RnGRlrssION
C1TT,TURE -r.lEDILrM
EQUATION
THE RNGRESSION EQUATION IS
{ =-
77.L + 42.O Xt
-
2.75
X2
EQUI\TION NO.1
100.
80.
50r
I
Y
lt3
5.0
INITIAI
1''
pH
OI. CULTUR-E ]'{EDIUM
H
"18, o
r-5 3
FIG._-B
JO
I"ryCELIAL D1t}' htriGHT OF
R. PfIJ
0:\ pH
olt zr" uAi:l
AGAR, S}IO\^IING
THX DATA POINTS, FITTEJ) CURVE AND RECRNSSION EQUATICN
THE REGRESSION EQUATION IS
Y = -305. + L74. x1
-
17.8 xZ + 0.497
X3
EQUATiOi't
NO.
2
Y
$
s
ts
,
l1
?
+
2
2
+
t
4
5.5
3.O
plt oii
I
.O
CULTURE }EDIUI,I
a'o.5
x
13.0
15" 5
1s4
ffi
r4ELI*9._5-
DIAWTER GROI,II}I OF R. I']INI
DARKNESS ON
2Z }.'A
AND CDA,
GRO'IJN
IN
]JII.-I|USED DAY
pH 7.2 AT l8oc
50 AND 60
2zoc AFTER Lo,
diameter (in
mm)
LIGHT
(days )
i\I,iD
20,30,
40,
DAYS
I,iean colony
Period of
incub ation
-
LIGII'I
1 t.se
DAP.KNESS
27" MA
vua
7x
7
27.
I"tA I
co;-
7
7
lo
20.gto.27
L9
.2tA,56
20 ,7 lO
2A
31-
. 7tO.56
31
.510.43
30. 9ro. 52
30.5r0 .43
30
44
.4!L.34
44,1!3.45
4L.2!1.99
39. 710.33
40
53.6!2.L2
56.9!2,79
49
.9t2.O2
50.711.04
50
61.012.88
64,O!2.20
56.7!2.O4
5
60
79
.LlL .34
68.411.48
68.812.
.9!1
,7 3
7
4
.56
20.9lO.2
t-
7.910 .81
73
-:ameter of agar plug inocultutr
of the resurts of r.nycelial dry weights of B. piu
-=--e 8.7) and variance analysis (^rppendix g.6) reveal rhar on cDA pi ates
Exarnination
--:::aie'L in dark highly significanrl;r (p = o.or) higher mycerial
dry Itrass
']:::oduced as compared to pud" in diffused day light and darkness at
-=-,:. This rn'as irue co the fact that R.. pini in continuous darkness
:-:ed plenty of pycnidi.a-like f ruicing srrucrures (see fig. 6.4) r.ri Eh
:-irrrous exudations which increased dry rveight considerably. But on 2Z
:-
: r -:r3S tl-re f ruiting structures did not develop, therefore.
::.:
-. --:i-cant increase in dry weight took place.
on1_v an
155
rABIE
}IYCELIAL DRY WEIGIIT ON R.
DARKNESS
8_.7
PINI IN DIFFUS}iD DAY LIGHT A]'ID COIiI'J.}IUOUS
AT 18OC - 22OC AFTER 60
DAYS
lfean Cry weight/colony
(mg)
TreaE,ment
Day light/night
a
Grand
darkness
27" MA
CDA
123.2
305.2
2L4.2
383. B**
260.7x
344.5***
237 .5
Continuous darkness
137
)lean
130.4
.
2
mearl
(me)
.6
EFI-llCll O!' :|EIPERATURE, pH eNO pGLATIVE H111IIDITy ON THE SERI11\IATION
OF
CONIDIA AND }ILONGATIOi\I oF CI;PJ{ TUBES
r.
l. t
INTP.ODUCTION
Spores, specialised, self-contained, uicroscopic structures capable
:: initiating n.ew growth are the principal agent of dispe,rsal of fungi.
---::equently any consideration of ecolcgy or of the spread. of
::-::cmically ir,iportant fungi must take spore germination into accouni.
I'= eontroL of plant pathogens by protectant fungicides is in essence the
::::-en of inhibiting the germinat.igrr of spores (cochrane, 1958).
Ihe most commcnly usad criterion cf spore gerinination is the fracLion
:: ::lres thai in a given tirne form a germ tube; the cine period is
'*:
-=---,' so chosen so as
to
aI
low all viable spilres to germinate. The tirree
---':-:-:uishing morphological events of typical spore germination are:
r
-:-=:: division, srelling of the spore,
and emergence of, the gerrn tube.
L56
Ten:perature
is
one
of the major factors in the longevity of
spores
is the second major factcr in spore longevirl- andrin
:.iture, is probably more often lirniting Ehan is tcmperature (cochrane,
3.elacive hurnidity
.958).
The optimun temperature
for spore germination is generally very
:iose to that for growth in culture
(Togashi
, Lg4g).
The
principal effect
:f sub-optimal temperature is to delay the onset of germination i.e. to
l:rcrease i.he latent period. Once germj.nation has begun, it is almost as
:apid at low temperature as at high. rn general, gerin tube growth is
,ioser in its temperature response to mycelial grcwi.h than it is co spore
:ermination (Cochrane,
195B).
Available water is one of the primary determinants of spore
::i:ioination. spores of few fungi are capable of germinating at as 1ow
.s 65 per eent relative humidity (snow, L945); several fungi, including
-:ecies of Aspergillus, Penicillirlm.
an<l
a few other genera, have spores
;--ich germinate at relative humidities near
BO
per:
--ckson, L956; Bonner, l94B and swayer, L9z9). A
cent (Armolik and
somer,'hat
larger
group
:: species, such as Fomes cll}.o$ls (sisier and cox, lg54), Fusarium spp. and
"=:ticillium albo-atrun (Schneider, Lg54), gernrinate
at hurridities of 90-95
:=: cent but do not require liquid water; and there are nany fungi whose
::-:res are founrl to germinate only in liquid water, such as conidia of
.--erotinla spp. (Claylon, L942). Spore gerrnination occurs over a narro\{er
:=::erature range as humidity becomes moue unfavourable t,r: ge.rnination
-
--
, 195 B)
Infection of a plant by a pathogen is
-rrane
depencient, usua11,rr, on
at least
::-. extension of the germ tube, if this process is more sensitive to
-:-::"'ourable conditions than is total germination, A false picture of
.::=::tialities of the
organism
will be
deduced from observations ou the
-:'- germination alone (cochrane, 1958). Delp (1954) fron str,idies of
'-- --:ia1 germination of grapr.l powdery milder^r, Uncinul-a necator (Schw.)
:
the
t57
Burr, on dry slides and host leaves at diffe::ent temperaEures showed that
the behaviour of the fungus was essenti"ally the same at comparable
temperatures. Moreover, ic is cften possible to correlate the ecological
niche oi a parasitic fungus with the response of i-ts spores to temperatures.
Most plant pathogens, but with some exceotions, grow best in media
with an initial
As a rule the pH -range for sporulation
pH of 5.0 - 6.5.
is narrower tl-lan lhat for vegetative growth alrd the range for spore
narrower (ting and Yang, L944).
germinaEion is still
;f external pH are probably on permeability
an<l
TLre
principal effects
other surface
phenornena.
Other factors such as available oxJ'gen l-eve.1., carbon dioxide
:oncentration, age of spores, light, presence of nutrients, stimulating
s-ibst-ances, effect of the presence of other micro-organisms are known to
-,-ariously inf luence spore germination (Cochrane, 1958; Hawker, 1950). But
:3cause of practical limitations,
the present studies were, therefore,
-r=iced to the effect of temperature, pH and relative humidity cn the
:=rmination of conidia and germ tube elongation (except pH) of R. piE.
.t:ecver, during these studies dat.a were so colleeted that the effect of
:::re density on germination could also be examineci as, in certain cases
--.-.-estigated, too much crowding of spores has been shovm to inhibit
-=:-ination (Boyd, L952; Yarwood, 1956).
r - .2
YTATERIAL AND
METHODT
-_
I -'-.1
Effect.of
of
F,emperature on
thg ge_lni-nation ?f*c_oniriia anjl elongation
germ tubes
Cochrane (1958) emphasised
that in the study of spores in relation
I :=:lerature it is impor:tant to control variables such as age cf
- -::1,'and pH. In the Dresent experirnent
27.
spores,
water Agar, pH 7.2 was usi:d
-: --cating rnedium. Conidia to be tested for germi-nation \rerc obtained
- .-'.'ut a one month old culLure on coru (vi<ie Ctrapter 7) as f o.!,lr:r.rs.
158
First, mycelial growth with profuse coniciia was remol'ed
wiEh sterile- needles so that no corrr tissues \,/ere
cy shaking the mycelia in
roEtle, coniCia lrere
1O
fr:om the corn surfaces
with fhe nycelium.
Then,
ml steriiised distilled water in a McCartney
suspendeC
in water.
L,,rtren
lhe bottle
r,ras allorved to
stand for a while most of the mycelial mass floated, then the conidial
suspension was
carefully rvithdrawn r^rith a steril-ised syringe and the
:ontents transferred to another steril-ised I'{cCartney bottle, shaken Iightly
=ed again all-owed
to stand so that the rernaining mycelial- fragments floated.
Iee conidial suspension was again withdralrn and by repeating the
- to 3 times it
r+as
same process
possible to get a suspension of conirlia viriually free
::orn hyphae. A small drop
of the conidial
suspension r^,as placed on
a 7 um
-:ameter agar block and examined microscopically for the density of coniciia
-',r microfield. If too few conidia for easy counting were noticed, more
--'-celia were put in the suspension and the above process repeated, but if
:--e density of conidia per microfieLd was found to be too high it
:-iuted with
:-: thereby
more
lrater. fhis separation process ensured uniform mixing
randcrn
distribution of the conidia of various
Five agar blocks, 7 nun in diameter,
.-
:-5
was
age classes,
r^/ere place,J on each
of a series
steril-ised slides. The iatter were then singly placed over a thin
:--- of distiiled
waEer
with the help of "Blue tac'r, a reusable
inerE
.---esil,e, ir,.45 petri dishes. rn the next stage, one smal1 drop of conidial
.-=:ension was placed at the centre of each of the agar blocks, the lids of
- =:etri dishes replaced
and se1- ed'with
sellotape (see fig. S.17). Five
r:--rr- dishes, so treated, were then incubated at each of 2ocr 4oc, 7oc,
--'a,15oc, l8oc, zirocr
:=-':ively thin
25cc and 3ooc
and uniforrn colururs
in incubators. Thus, alchough trre
of air in the petri disires, in
::: :iides with agar plugs bearing eonidia
were
which
kept, theoretically had
-:-:l1e capacity to reiairr water vapour at the various temperatures of
., --:'iion,
the presence of distilied \rater in all the petri <iishes ensured
.\r
,
159
l
Sellotape sealing
Petri dish
Microscope slide placed over r,nater by Blue
tac support,s
Agar plug cn wliich conidial
suspens ion was placed
Distilled
\^rater
Blue tac support
.. :.=3:.1] SC]]}I].{ATTE* PRESENTATION O}' A PETRI DIS}i
SHOI.IING A MICROSCOPE
---: SUSPENDED OVER A THIN FIL}I OiT I,VATER WITH 'BLUI] TAC, SUPPONT, ON
: -_ SLIDE ARE PLACED 5 AGAR PLUGS AIID ON THESE CoNIDIAI
SL]SPENSIoN oF
hIAS PLACED. Ti{E AT}ICSPHERE OF THE PETRI
DISH
IS
THUS AT
-- _, R.H
=I
L60
tirat the air atmospheres r^rere saturatecl . llherefore the resultant
relative
humidicy (i'e' existing water vapour pressure of tire atinosi:i-ierc
expressed
:. e percentage of the saturated wat.er vapour pressure
at the same
:tiperature) at all the temperatures of incrrbation was reascnably and
safely considered to be
Examination
100
per cent.
of singl-e plate frorn
each
of the temperatures of incubation
snowed no germination
afcer 24 hours of incubation, but after 48 hours of
:;:cubation profuse germination of conidia L,iEh clearly neasurable
germ tubes
;as noticed' Therefore all the plates \,rere removecl frcni
the incubators
-::er 48 ho.urs, the germinating spores an<1 germ tubes rnrere kilLed
by soaking
--- ethanol for about 5 minuEes and then mounted in lactophenol cotton blue
-layton, L942) and covered with eover slips. Frorn each of the five
:::1:-cate plates at any of the temperatures 1oo conidia
were
count.ed
-==rination and 50 germ
tr.rbes were
for
measured. For this a nurnber of
-'':rofields were taken at random and all the germinated
::iidia were couilted from ar-1 the microfields (40 x 8.5
and ungerminated
nag.) except the
-=st one from which just enough to conplete the ioo rvere counted from
. -:e of the mic::r:f i.eld.
one
A ccnidiuin was consi<iered germinated'^'hen lhe gerin
tube was disti,ctly
--=-;gnisable (see figs' 8.18 and 8.19). New growth from
snrall fragments of
-: ryphae, if any, r,ras recognised easily by the presence of darker
:-:3 distincr
walrs
shape as compared
to the conidia (figs. B.1g and g.19).
tubesr-Ji{d conidial lengths we::e excluded.
-- --re &easurenent of germ
The
:::a \rere analysed usir-rg a polynomial regression
as already referred to in
: -:-section 8.2.3.L.
161
I
t
FIG. B.18 A PIECE 0F HYPIIA (DlnLER),
( :-l,if;;V )PrNI. t{$Ii
GERIII}LATIIn
A-ND UNGHRMII'TATED (DARKER) CONT-DIA 0F R.
lll.tirT
LTNGER}iINATND I]CNIDIA ARE EASILY DIEFERINTIATED TTIC}.] ?IIJJ
GERMINATED ONES, E\rEN THOSE WITH SMALL GERI'I TUB:IS
I (:j$i
1ffi
rl
_s;,--
I
i
.1
a
t
IIG_q.U srx colrrDrA oF E. SIxl: Ar
runr
UToNGATION
oN 2z ILALT .\cAR.
48 HO]JRS OF INCUBAT'ION, 36CX.
)IINIltutI AT OPTIMUM pH
pH
NOTE
DIIFER-ENT Lirl',iC'l:i *7 ryr,:.lttl
7.2 ,\T tOO7, :.i1. lFTtrq
Ti:iAT
SI^JELL
jj'I
1.?11:
,\r
L62
3.2
.2.2
Pl-ates
on
af
of
coni<iia
waaer agar having pl{ 2.6 to p}l
12.2 .,;ere preparecl in
:he u,ay alreaciy describe<i (see sub_section
B.L.2.Z). Tuua agar plugs were
:aken on sterilised slides, then
fixed over <iistilled water in petri
dishes
::rd inoculated with a coni<lial suspension
(see S.Z.2.i), The piates
\{e re
sealed and a replicaEe of 5 plates
for each of the 12 pH levels was
l::cubated at l8oc in an incubator.
The conidia r,;ere assessed
for germination
:---ier 48 h'urs' The necessa*rydetails of trrese metirods are as
arready
:=seribed uncler terperature.
Germ iube length rras rot
measure. as availabre
::=e became 1i*iting because of
other engagenente. secondly, because
27.
-:..'ted practical va,ue, this
of
re i
ative
of
was also
not repeated subsequently.
humidi ty_ on the
elongation of germ tubes
rmi.nation of eonidia
rn stridying the relative humidities,
both stacic air
'--stem for simuitaneous control 0f temperatul:e
ancr
and
and uoving
air
humiciity have been
-=:crted (clayron, 7g42; Delp, tg54).
ccchrane (1g58) emphasised
rhar the
=:'iic air pri*ciple - incubation in a closed
system over a r-ruaridity
:=.--.'lacing soi.ution - is bcth
simple and accurate. ?he::efore
in ihe
:=sent study ihe static air
principle was followeri.
An incubation charnber
-":gned by Mr J.S. Murray and partly
modified by the author was
used in
'-:-iaining constant relative humid:i-ty. This
apparatus i"s de.scribed below.
perspsx,
of
-:-:s
6.5 cm x 2.-5 cin x o.g cu (depth),
first a concave caviEy,
' - : in diarneter and 7"irm
in depth, ruas cut. Then, usi,g
tire same centre,
.
a
- -- --Jrr orameter. and
^^)
2
r'ua in depth ringwas
removed. This created a srrarl0w
:,i: around the edge of the first
cavity. fn tire next stage frou
cover
-::::s, r-3 nrm in diameter, about
3 mm were removed f::om rwo
cpp'sire sicies
--': when such a cover glass was placed
on the ledge o! the cavity, the
i::ce be10w in the smalr-er cavity
ancr the air space abou*
ii: rhe
rn
163
larger shallcrv crepression remained partly unintcrrupted.
I,rext, by gently
touching the rnycelial grorvtrr of about a month
o1cl culture of
r. gilrl
grown on corn (chapter 7) plenty of
conidia were collec.eci on the upper
surface of the cover glasses. These were individuarly
placed at the top
of Ehe lower cavity in ihe chanber. 54 such chamr.rers
w€.re prepared. rn
sets of 9' a hurnidity regulating solution (table g.B)
r^ras inserted using a
syringe' to partially fill the cavity so that
about 2 nn air space was
left under the cover glass . 54 larger cover grasses
irere
then used to
cover the mouths of the wider caviiies
anci then sealed with -rraseline.
rhus in the small air sPace abo';e aad
bel-ora the 13 urn cover glass bearing
conidia, a uniform vapour pressure was obtained
by incubating
't the required temperature.
the set
The perspe>r chambers as used
.*:<periment
up
in the present
is schematically shown in figure 8.20.
Germination of conidia of R. pi,,. was
tested at si:< leve,s of
:elative humidity which r,sere maint.ained by
using saturated solutions of
:eagent grade chemicals selected mainl-y
from the list by winston anc Bates
-960). Tnese are shown in table B.B.
TABLE 8. J
RELATIVE HUJ.IIDITY LEVELS AND SUBS?ANCES
USED
I
Saturated solutions
I"Iake
Relative h,.:midity
(Z) at
Glucose
Potassium
Sodium
Sodium
tartrate
sulphate, deeahydrate
sulphite,
heptahycirate
Potassium sulphate
Distilled
r^rater
BDH
55
BDH
75
BDH
93
BDH
95
BDH
9B
21oC
100
I
L64
Outer f{roove
fnne r ;i::oif v€ cont aining
humidity
regulating solution
13 mm ccver glass
25
mm
cor./er glass
Vaseline sealing
O.l\)
_
SCI]EMTIC
PR-ESENTATiON OF
THE TOOL USED
.,-:INAT ION ON DRY COVER
GLASS AT DIFFEREI{T
RH
IN
,LEYELS
CONIDIA
r65
For each of the six relative h'.rmiclity levers, a replicate
of 9 sets
of rspore-ehauberst
incubated;it 2, loc in an incubator naintained in a
cooled room. This temperature was chosen as iL riras found
to be the optirnum
:emperature for the germination of conidia and gern tube
el-ongation (vide
vras
spore germination temperature
stucly). A replicate of .3 rvas removed after
2*, 48 and 72 hours of incub.ation, for exarnination.
Before starting the replicate<l experirnent, from
a trial examination
-t was found that when the sealed experinental r-init of perspex r,ras
incubated
the conidia did not <iie even after one hour but when
the top cover
3'ass and humicity reguiating solution were remotr.ed, the coniclia
z'- 75oc'
and germ
:trDes die<i
in lO minutes
exposure.
the conidia were praced on a dry cover glass, trying
to kil1
::am by ethanol or mounting fluid, was liker_y
to r,rash them away and hence
:-'-cided. rather dry heat at 75oc for 15 minutes
after remo-,,ing the top
---'-er glass and humidity solution, was use,J. Then the
cover glass bearing
-:= conidia was transferred directly on to a slide and examined
Be'cause
-:roscopicalry' A total 0f 200 conidia at random i{as couniecl from each
-: che three replicate sets for each of the six trumidity ievels
at 3 Limes
:: :cservaEions. No gerraination ioor: place after
24 hours, and only
:-:;ot germination after 48 hours, but it was profuse
after 72 rrours of
--::'cation' Therefore germ tube lengths were measured after 72 hours
-: -;bation.
The anaiyses on the data frdm temperature
and pH st,.rdies \.sere carried
-: _sing polynomial iLgression and data from relative
humidity studies
_.-. :.ra1-ysed using the t_test.
166
8.2,2.4 EffecE of density of conidilper
mi_c.rof ie_1C ori*,fgryUlCr_]on
For studing Lhe effect of densifl, of coniciia per microrield, data
were taken fro;n conidia gerrninat.ion ternperature and ci:iridia germination
pH studies.
In collecting data for these studies a standard 4O x 12.5
nicrofield was used throughout. and 1OO conidia were counted for each of
:ive replicates and for each temperature of incubation or pH of culture
-edium used. In counting 100 conidia, a1-1 conidia of a number of randomly
:elect.ed microfields were counted, except the last microf iel-d frorn which
:-:st enough coni<iia from one side were counted to make the total of 1OO.
To determine the effect of densitlz, the microfields examined were
:-assified into spore density groups and the tot.al nuriber of ger.rninated
::aidia of a particular density group
rnras
calculated from the 5 replicates
=:d expressed as a percentage of Ehe total conidia.
:.r.3
R-ESULTS
:.1.3.1 l!f""t
rf***p.f.tq.
r" rn
genn tubes
The mean
1. :ihi
af
per cent germination and gerrn tube length of the conidia of
ter 48 irours of lncubation
aE ZaC, rrag, 7oC,
_-:c,25oc and 30oc are presente<l irr tables 8.9
lloc,
15oC, lBoC,
anci 8.i-0 respectively.
I:=se are also graphically presented irr figure 8.21. The results of analyses
:: :hese data are
-:
summarised
in
appendi-ces
-=igures 8.?-2 atd 8.23 ::espectivelf.
8.7 and B.B and the.fitted
curves
/6'F
rrG..
8..el
oPTrlru}l TET{PEMTURE FOR GERI.IINATION OF CONIDIA AND ULONGATION
OF GERM TUBE OIT R.
PINI
AFTBR
48
llOUPrS
OF INCUBATION ON 2Z MALT r\Gi\R,
I
^H:c
*-----a
PER CENT GERMI}]ATION
&s-----o
CER\I TUBE LENGTH
100
100
90
90
BO
70
60
q
,\,--
50
l
:+O
I
,\
/\
t\
:
_JU
:
40=
H
t\
I
e
-20
:
:
z\
/1
,\u/\
/\
30-8
IjI
_1
'1,t)
t{
;)
H
LU
10*
a
rd
-
oq
7
10
TE}P.ERATLIRE
!5n'
LB
OF INCUBAT ION
2L
IN
25
OC
168
FTG.
B
.22
GER],fI}IATION PI]RCENTAGE OF CONrilrA
OF INCU]]ATION
STiOWING
oF
3. g[r
THE DATT\ POINTS, FITTED CURVE
THE REGRESSION EQUATION
AT{D
0N
TEI'fPERATURE
RIiCRISSiON EQUATION
IS
Y= 23.4 + 2.25 xL + 0.591
X2
-
0.0215
X3
EQUATTON
N0.
7
Y
10O
o
*+*
4
)
80"
?
6O.
4Oo
24.
0.
Y
7"0
1q.0
31"0
TE}IPEMTUNI -Of".iIICUBATION
.?8* S
IN gC
0
169
FIG.
B. 2.]
GI]It}i TUBE LENGTII oF cONrDrA
oF R.
INCUBA].'ION
SHOWING THE DATA
POINTS, TITTED
llryI oli
TI]}fPER{TURE
CURVE AN'D RXGIi-ES s rON
EQ'UAT ION
THN REGRESSIO}i EQUATION
IS
Y = -1.61
-
3.93 x1 + 0.669 x2 _ 0.0180 x3
.
EOLIATION
NO.
I
ES"
*
-
60"
*
+
?.
-
:
:
-
40n
20.
=
:
O.
LS*O
TEI'IPEMTURE
oF
OF
2$
3CI.o
"g
INCUBATION
iN
og
x
B
170
TABJ.E 8.9
0!' I
EFFECT
LEYELS OF TE}IPEMTURF] ON THE GERUINATION
OF THE CO}.IIDIA
E. PINI AFTER 48
HOURS
OF INCUBATION ON 2Z
Incubation
Replication
Ternperatur.
oC
pH
R.
H.
I4IATER
OF
AGAR, pH 7.2
No. of
coni di a/
rep 1 i cate
7"
Iulean Ll
germination
t t.se (ar p =
4
l7 )
t'''
100
5
100
7
7.2
100
5
100
5.20!2.69
11
7.2
100
5
100
36 . 6Ot3. 33
15
7.2
100
5
100
83.20!2.83
100
5
100
84.2O11 . 83
I
O.O5)
o
j
1B
1 .l
21
,
.rl
100
5
100
90.4Or1.89
25
,.rl
100
5
100
52. 80r5 .36
30
, .21 100
5
100
o
The cptimum temperature
for germination of the conidia and e1-ongation
:: rhe gern tubes is 21oc (see fig. s.21). corridia
at various stages of
:=rm tube development
at the optimum temperature are
shor,-n i-n
figure s.24.
-:e difference in per cent germination of conidia at
15oc and l8oc rnras
:-:f small and only slightly lower than that
at the optimum temperature.
:-: gern tube growth at 15oc and l'fc was
consi.derabiy less than that at
:---= opLimun temperature. c"r,
i,]u" growth at r5oc and 25oc was virtually
:--= saEre, but gur*irrition of conidia
at the former was siguificantly higher
L77
l
i
j
1
i
I
I
i
,t\f
{
FrG. 8.24 srx coNrDrA oF R. PrNr AT DTFFEP.ENT srAGEs oF GERM
TueE ELONGATToN oN 2z l,rAtr*Au'r,, pu-z.r-il"Iilz R.H. AI.TEI.
48 I{ouRS oF rNCuBATroN, 360x. NorE THAT swELLrNc tr{AS
)IINIMUI,I AT THE OPTIMUM pli
T7L
,l
I
l
l
l
\+
,.\-,i"&.-
F'IC.
9.24
SIX CONIDIA OF X.. PI}JI AT DIFFEF.NNT
STAGES OF GER}I
2z MArr AfrrL pH 7.2 ar locz R.H. AI.TEB.
48 HOURS oF TNCUBATTON, 360x. morr rnar
SWELLTNG tr{As
MINlMUl,l AT TirE OpTIMUM pH
TUBE ELoNGATToN oN
172
_rAuLE*_A.lg
EFFECT
0F 8 LEVEL' oF
c.NrDrA oF
r.
Incub
prNr
TEr,pE&4.TuRE
AFTER /+8 lrouRS
oN THE EL'NGATT.N oI.
GEp,u rul,irs
OF
0F rNcutsATioN 0N 2z
pH
ation
No. of
conidia/
replicate
Replica tion
Iemperature
oC
wATriR AGAR,
Mean.
in
germ tube length
pm
t t.se
4
t.z
100
5
2A
7*
11
100
5
2A
100
5
20
B.O112.08
100
5
20
23.45t2.86
100
5
20
45.3016 . 83
11
1.1
15
7.2
LB
-l
')
2L
7.2
100
5
20
25
55.41r13.16
1a
100
5
2A
23.3114.50
100
5
20
30
7.2
l.z
t at 5Z level of probability
and4df=2"716
Germ tubes were
very snal1 and germi.nated
cr:nidia very rare,
therefor-e, no measurenents
(i.e. --) raken.
Although afrer 4g hours,
at 40c and 70c there were oz
and.5.2AZ
respectivelyn after 1L days
at locugl.6a ! Z,Be, per cent
germination of
ecnidia was observed in
a separatu,_ru{.
This suggested
that
at 1ow
t*-*lerature germinatiorrvas
delayed by way of extendingthe
period of laEency,
The regression equations
for 1) conidia germination
temperature data
(table 8'9) and 2) grr*
tube length and temperature
data (table g.10) are
ar follows:
" :
- :
= 23.4
_r 2.25
= _1.61 _3.93
XI+ 0.591 x2 _o .a2L5 x3
XI+ o.669 x2 _o.o1BO x3
L73
F-rarios 1) 208.31 wirh 36 <lf and 2)
45.31 wirh 3l df are highly
significanr. The R-squared values of
l) (O.97Of)2 = A.94tCr i.e. 94.LO
per cenr' and 2) (o'8g27)2 = o-7g70
i.e. 79.70 per cenr are highly
significant. The t-ratios are also mostly
significant (appendices g.7
and B.B). Therefore, it is reasonably
safe io say that trre data on per
cent germination of conidia and germ
tube length ter,perature data fit
the
regressiou models with acceptable
accuraey.
The
:o.^z.J.
),
Z Effect
Th" ;;r
cenr germination of eonidia of
R. pin:- after 48 hours
:'' incubation on 2Z Water Agar at pH
2.g, 3.0, 3.2, 4.2, 6.2, 6 .2, 7 .2,
i.2, 9.2, 1O,2, 11.2 anci LZ,2 are
summarised in table B. 11 ar:d
graphically
):lo\^rn in figure 8.25. The
results of analysis on the data are
given in
-:re appendix g.9 and the fitted curve
in figure g.26.
t74
rtrTa
!v.
.)tr
Q LJ
a.
cPTIMUII pli FOR TIl]:, GERMTi'iATrOi,i oF
S. tINr
c0tirDrA A!.rER 48
f-iouRs 0N 2Z ]\{ALT ACAR AT t8o(l
I
3.O
r.2
4.2 5.2
-H
Y
OF
o.t /.1 8,2
2Z }T\LT
ACAR
9.,2
ta.2 LL.2
L2.2
175
FrG. .8.2q
GERMINATIO}J PEII.CINTTTGI'
INCUI]ATION SIIOWII.IG TI1E
O]i CONIDIA O]1 R. PII\.I
ON PIl Or
DATA POINTS, TITTED
CUR\iE AND RNGR.ESSION
EQUATION
THE RncRnssIoN EQUATION
IS
Y
: -156. + 79.9 XI 100"
7.36 X2 + s.157 x3
EQUATION
NO.
Y
*
80.
6CI.
:Z
4Or
?0.
Or
5.0
pH
or
lo5
cuLTuRE }fEDIUM
10"0
L
2.5
x
5
L76
TABLE 8.11
EF}'ECT OF L2 LEVELS oF pH OI' 27. WATER
AGAR ON iTl]i CERI,IIII*AT i 0t{
OF
R. PINI
A!'TER
48
Incubation
DH
Temperature
oC R.
H.
7.
HOUP.S
CF INCUBATIO}I AT
0I
IA
1BOC
llo. of
Replicarion conidia/ llean Z gernination
rep 1 icate t t.se (at p : O.O5)
2.8
1B
100
5
100
3.0
18
100
5
100
o
L
,4otl .42
I
3.2
18
100
4.2
1B
100
5
100
80
1B
100
5
100
78.8Ot4.69
6.2
1B
100
5
100
82 . 8014 .5 1
7)
18
100
5
100
9A.80!6.77
8.2
1B
100
5
100
86
.6016. 83
9.2
1B
100
5
100
77
.4Ot5.72
LO.2
18
100
5
100
69
.8015.
LL.2
L8
100
5
100
41 . 80113.55
1) )
l.B
100
5
100
q,
C)iit ID
The optimum germination
ioo
tock place
ar. pE
I
58.6015 . 39 :
,40!2 .42
16
o
7.2 (fig. 8.24).
-'=:mination percentages over the
1ln€i pH 4.2 - pH 9.2 were Ligrr and
:=ir:ly close to each other. Drop in germination
was quite rnarked from
;:' !,2 ia
3.2, and pH 1o.2 to pH LL.Z, but became 1initing
as the pH
-.-:reased either towarrls more acid (pH 3.0) or
alkaline 1pH tz.z) side.
- .= germination curve is more or less symnetrieal
with the exception of
..- -.2. The unusuaily higher gernination
at pH 4.2 may tre clr:e to fault in
:::-lng, Ehe pH value or Some other unknovm
causes. At pH 3.2 the coni,.1ia
,
"
=
-
pH
-ed very colrsiCerablv
ancl bgCarne alrroqf
rnrr-,i
t,^.F^-,. &r^^--
L77
3 Effect of relativejgi-qii):-gr@,f*:g*idig
8' 2' 3'
and
S*lg1eari:Ljl €grm rubes
The mean per cent germinaiion
of the coni<lia of R. i1". af various
levels of relative humidity is given in
table 8.12 and represenLed in
:igure 8.27. The mean germ tube length
is presented in table 8.13. The
--ptimum relaEive humidity for germination
of conidia and eiongation of
:=:': tubes on dry cover glass is LAOZ (fig.
B.2g).
TABLE 8.13
::::CT
-. :..
OF
6
LEVELS OF PGLATIVE HUMIDiTY
PINI ON DRY
COYER GLASS AFTER
AT
Saturated
Incubation
solution
used
.i
ON
THE GERI,III\]ATION oF CONIDIA
24, 4B AND 72 HCURS OF INCUtsATION
ZLOC
No: ?f . fuean Z germinarion
Replication conidia/
replicate
UCCSe
: ,. tas s ium
:.rtrate
jcdium
ulphate
s
,;^
_Egd^
^
.-,.-drace)
3
j:dium
=:Iphite
septa
.-.-.-:
ra te )
.:;aSSium
.:_phate
--:
-srilled
_af
**
*r(
S
*
3
o.57
9B
3
5.17*x 13.50n*
100
J
i-gnif i canr ofP
Signif :-cant ofP
o. 01
o. oo1
29
.50,k*
3. 83
5
7.5o**rr
178
FIG. 8.27 lFrjjc:t 0F ItEL;\Trvli irullr,Drr\' (li.H.) 0N .r-Iiia
Glr{ir,rr\rro}J orr c.NrDrA
O}- R. PINI ON DITY COVEIT GLASS
4
('**^--_o
1O0Z R. H.
9BZ R.H.
50
952 R.H.
40
30
z4
48
PERIOD OF INCUtsATION
.rG.
IN HOURS
72
OPTI}IU}{ P'.ELATIVE Hi}{IDITY FOR TiiE
GER}IINATION OF CONIDIA AND
:-0NGATI0N OF GERM TUtsE OF R. PINI
AFTER 72 HOURS ON DRY COVER GLASS
B-.
28
:l
60
o--*o
p[R
et--- ----{,
GER}I TUEE tEi{GTH
CENT GER]"IINATION
1
.
30
zo
H
H
I
204
rI]
(J
H
10s
l!]
95R.ELATII,IH liil}fT]]T.I"Y 7
,I
j
{
L79
After 48 irours, z ge,nination at
significantiy (p = 0.01)
higher than z gerrnination at 9Bz RH, which
r.ras in ttrrn sig,ificantiy
(P = o.o1) higher Ehan z germination
ar g5z. RJr. AfLer 72 hours, rhe z
germination at 1OOZ RH, r.ras highly significanriy
(p = O.001)
Z ger:rnination at 9gZ
1002 RH,"ras
higher than
RH.
TABLE 8. ].3
EFT'ECT OF
3
LEVELS
OF CONIDIA OF
OF
R. PII{i
P.EII.TII/E HUMIDITY ON THE L]i.ONCATION
OF
AT'TER
72 iiOURS OF. II,JCUBATION AT 21OC
GLASS
Tnocul
Saturaied sol-utions
ation
R. H. Tempe ratr:re
-oc
Sodiun
sulphite, l[z
?otassium su1-phate
listilled water
.. - Dignrt].canE
- _,e &a
95
/t
9B
2L
100
2L
at P = O.O5
GER}f TUBES
OU IRY COVER
:
No. of
Replicate conidia/ i'lean germ tube
repi i cate length (prai t
+
^^
L . JU
t
3
25
-1
25
25.28*tL4.42
3
25
43.z3xl-lg .19
7.BB
7.75
180
8' 2' 3'
4 E"gr%=per
qrgr"q&-era-s.,_ge,*i,..,_i..
cilstribution of per cent gernrinaeior-i
of co.riclia in <lifferent
conidi-a densiEy Sroups for data
from conidia germinati,n
The
terrrperature
studies and conidia gerrrination
pH strrdies are sunrmarised
i, the tables
B'14 and 8.15, and their graphic
presentaticns are shovrn in the
figures
B .29 and 8.30 ::espectively.
TABLE 8.14
?ER CENT DISTRIITUTION Clr,
0F COI\.IDIA OF R.
'ERUINATION
AT DIFFEREN'I TE}fELATURES
OF INCUBATION
Incubation
PINI
PER I.{ICRO}IELD
Per cent germination of conidia
umber
of
conidia
Ilaximum aurnber
of conidia per microfield
Temp,
oc
7
500
5,20
12
.50 LO.29
5.20
3. 37
1i
500
35
15
5CO
83. 20 80.00 77.78 87.86
1Y.J/
18
500
.5A J/.)U
3.5.29
5.26
?q 10
82.O2
84.73
8.25
46.88
84.20
82.81 oo.
o/.05 90.32 90.5c 100.00
b'+
tr, ,i\
21
500
90.40
25
_500
52.801 25 .00 57 .A2 50.41
47.67
7
56.76 52.73
1Bt
r rG,
_-..l--:..l+
g.
.29
EFIIECT O}JDENSITY
OF CO}{IDIA O},
I. ],.INI ON :ITiE GIIRMINATION AT
]IFI'TRENT ?E}II,ERATURXS
OF INCUBATION ON*Z
I{ALT AGAR pH l.z
;_€_.
zloc
rBoc
o.--k-*.a6
a. . . . ! ...
El*
. r. . o
-m
tr:*-...*-E
15oc
11oc
25oc
7oc
100
90
80
70
6C
50
40
"*/--.-o-----s*
30
__*_O
d/
20
10
o
-J
--*
20
I'IAXIIfl,TI }IU]'fBER
OF CONIDIA IN EACH
}.{ICROFIELD
(4o.oo x
1
2.
5 l!{AGrvIPIcATroN)
25
L82
triG.
g.3r_l
si)TErrourlr,
r L a Liuil
I
U!'FECT
pH
0F DENSI?Y
or
CONrDIA
or 3. IILr
valurs ol.zz II\LT AGAR AFTER
48 HouRS
TI{E CEITi,IINATION /t'I
l1f
1
Boc
.
--O--.---+
-.-*n--'11*--=-*-JI>-o
L:^={,.
%-
-
t.
*''\
,/
l._-\e"
-_
":g:_i"g
(t
/
--t
a
z
\\/ ,/
/
l--
\-.'
P
-r--- -*o-----O ,/ I
o-"
/-
s
/
A/
/\/
\.,'
/ / '.
./
'o----*-G*{
c/
6.------*__p
P
O--..--*O
O*-----O
a
._=_-**_.-*
a
C---
-
-.-*43
o*----_-.- o
Z
:<
--*- -*'-+r
o__'-
e
=
-Z
3.o
3.2
4.2
5.2
6.2
-l)
8.2
.2
9
-.__-_a 10. 2
_._'n LL,z
s_-___*
=
:%+--.**-::--
2A
30
MA-{I}ruM N[n:,mER.
40
50
olr CONIDIA IN
60
70
BO
EACH ]IICROFIELD
90
1&3
IAqLp-*q":-i:.
PER CENT DISTRIBUTIOI,I
AT
Incub
ation
R. PINI
PER }IICROFIELD
Per cent germination of conidia
Mean
/"
of
6L r rrr
conidia ina- Ilaximum number of conidia per microfi,eld
tion
T
ao
Giizu,iINATION OF CONIDIA OF
pH LEVELS oF INCUEATIOI.I I{EDIA
DIFFEP.EIJT
Ntimber
pE
OF
DLI
/o
10
20
30
40
50
a 1i
L.
.L)
i.61
1
3.O llB
100
)UU
?,
18
100
500
58.60
4,2
1B
100
500
80.40 78.05 80.68 81.98 79.53
78.80 90. oo 75 .00 79.75 8A.74 86.
1
.40
60
70
.50
33. 33 54.L7 58.33 55 .68 53.70 69 .35
I
5.2
1B
100
500
6.2
18
100
500
i82.So BB. OO
7.2
1B
ioo
500
90. 80 84.2L 9L.99 9L.7
8,2
ia
100
500
86.60
9.2
1E
10c
500
77.40 83. 33
500
69. 80 7L.43 53,12 72.15 69.23 68.
500
41. 80 33. 33 46.99
10.2
lOO
LL.2
100
Data
.-
.
l
of the tables g.14
75
BB. 19
4
B1
r-7.06 81.82
90. oo
.00 89 .52 87.74 89.71 82.OB 82.69
1a a.
I J. LJ
and g.15
as
75.36 82.01
3t+
.56
31+
.7 5
l
l_3
31.37
illusrrared in the figures
:
a
8.29
- i.3tt respectively clearly revep,l that density of con:dia of R.
pini
--=:tLy do not play an i.mportant role, if any, in governing
the rate of
{
1
:
--_-::ation.
)
I
',1
i
I
I
184
8.
3
SU}.{NIARY
I" r,1j:g. studies were carried out to understand the
E. pi"r-with respect to
Eemperature, pH,
such information could be
in artificiai
be-haviour of
relative humidity etc. so that
ucilised in viyo studies
and
particularly
inoculation experiments.
Ttre optimum temi)erature
for diameter gr:owth'and mycelial dry
?roduction is 15oC-1BoC. Mj,celial growth r.ras cornparable cver the
mass
pH
iange 4.2 to 9.2, with optirnun for diameter growth at pH 7.2 whLle
:ry r,reight at
pH
8.2. Diarneter growth
and rnyceli.al dry roeight
obtained by dissolving agar in boiling rorater) showed positive
significant association. Both mycelial growth/teurperature
;:orvth/pH data r+ere
and mycelial-
firted to prediccive regression nodels.
ef previous rnorkers found it difficutt to get solidifiea
agar at pH S.5 bur I found that by adjusring
=:ar ge1 even with 3-42
A number
:::aineci up to pll 2.6.. This is ccnsicered as useful advantage.
Diameter growthof IL. pini-.,r6s
significantly higher in diffused
::v lighr es colrpared tc that ia total
;as true on boEh of
darlcness
at about 18oc.
2Z l"Ialt Agar and czapek Dox Agar. on
This
I
the latter
-=iium, profuse production of pycnidia like fruiting bodies in dark
:=sulted significantly higher rnycelial_ dry weight.
Gerrnination
:=:rerature cf
of conidia \{as coii}pe.rable in the incubation
15oC
to
2'!,oC,
but 2loC clearly supported nore
::r:lounced growth of germ tubes. Low temperature seemed to extend
:-= ?eriod of lateney. similar to myeelial growth, conidia germination
)
184
8.
3
SU},ftIARY
In viiro studies were carried out
Eo undersLand the behaviour of
E. pi"]. with respect to tenperature, oH, relative humidity etc. so that
such information coul<i be utilis"d g vi.,"o- studies and particularly
in artificiai
inoculaticn experiments.
Ttre optimur temperaLure
for diametel' growth'and nrycelial dry
production is 15oC-1BoC. M),ce1ial growth
range 4.2
r,.?as
cornparable
to 9.2, with optinun for dianeter growth at
pH
cver the
j.Z
mass
pH
whLle
3ry r;eight at pH 8.2. Diarneter grorvth and ir,yceli.al dry weight
riobtained by dissolving agar
ia boiling uzater)
showed posieive
significant association. Both myeelial growth/temperature
firted to predictive regression nodels.
;rowEh/pH data r+ere
A number
of previous
=;ar gel even with
and mycelial
r^,orkers found
it difficult to get solidified
agar at p.1J^ 3.5 but I found that by ar3justing
3-42
of 2z Malt agar medium just before plating, solidified gel rras
::taine<i up lc pll 2.6.. This is ccnsicered as usefur advantage.
-ne pH
lliameter
g::owEh
of
Ii.. pini-.vas signif
icantly higher in dif fused
:a;- light as colrpared tc that ia total darkness at about 18oc. This
;as true on both af
27"
l"Ialt Agar and czapek Dox Agar. on the latter
-=cium, profuse producEion of pycniclia like fruiting bodies in dark
:.sulted significantly higher
Gerrnination
of conidia
arycelial_ dry weight.
lnas conp;a.J:able
in the incubation
:.::perature of 15oc to,_2loc, but 21oc clearly supported rnore
-:--;:ounced growth
of
germ
tubes.
Low temperature seerned
to
extend
:-= period of latency. SimiLar to mycelial growth, conidia germination
185
was conparable over the pll
range 4.2 to 9.2. Nearly
germinated at pH 3.2 in 48
hours as comp.rred to
pH
7
.2 Conidia
regression
9OZ
gei:minariony'pH 63sg
60,"1
clraidia
et the
optimurn
fitted well to predictive
model..
Free r'rater was noE found
to be essential for thc.: germinatio,
of {' pini coni.lia; but presence
of f::ee water enhanced germination.
At 1002 RH in 4g hours nearly
502 conidia germinated
on dry cover
giass' Density of conidia per
microfield did not affect per
cenE
3ermination of conidia.
)
t86
8.4
DISCUSSION
cochraire (1958) concluded that
raciial growth of fungi on agar
rnedium
is adequate for studies of the en.rironrnentar
factors (e.g. remperature)
:ut inadequate for nutritional studies,
Chauclhuli (Lg23i found. excellent
:orrelation of railial growth on agar
with <lry ruei.6;ht cf riry-celial growth
':ron liquic culture) in tenperature stuciies of
vertic*rl1iuur
_el.!_o-g,u..*
:'einke and Berth' rn the present
ca.se hi-ghly significant (p
= o.oo1)
::i-relation of radial gro.urth on agar
an<1 ciry r,reight at va::ious
temperatures
-- incubation (r = o'gg43, l'iith 6 df) anci
r:i{ of culLirre rnedium (r - o.9690,
xn
7 cf) were obtained' These ir:dicaie
"'.
that in case of a non-sporurati,g
-r culture) fungus like R. g:qi dry,weighr of
col0ny obtained by rernoving
'-:=r in boilirtg v/ater seems to give a simple, inexpensive
and reasonably
:=-iab1e methoci of getcing mycelial
dry weight.
a rarlge of plt values of agar media
are needeci, acijusting the pH
=-ues before autoclaving, even when
a suiiable buffer is useci, resrrlts
in
: '= -final pH 1evels after autoclaving
being remarkabi-y ,.wer. Diamandis
-: 7) anri obviously othei:s rrarze experi,:r:.ced
this. rn suci:. a situation,
.::-''-r'nt a fungus on such medi:l cioes
no. present a true measure of
the
'---'ty of the fungus to Srow at lhe pH 1e-re1s initially
deci<Ied. presence
: '...Fn:1:
rei '!s
rikely to hinder the normal changes
of oH by the fungal activity.
:=rr':er' sone of the cornnonly
s."6 0rganic acid buffers are
known to be
- -:itory tr: fungi (Munro,
1970). oi, the ot.her hand, ivhen
pH revels of
--:-:e rnedia are adjusired shortly 6.ro.*
plating aL about 40ac_ 450c,
as
' :.--' present case of
i7, M,rtt Agar, a firm ge1 r.-as
obtai'ed even ai pH 2.6
- : only 2Z agar, whereas by adjusti.ng
pH before autocLaving Diamandis
'-.. failed to ger solidifie
d. 2Z MA below pH 2.5
even v;ich 42 agar.
-'=:i:iore
::
' avoidance of any buf f er seems to be rnore usef ul since it
al lor.rs
: :: geE a more precise measure
of the al.riliiy of a gi'en fungus
to ch;:nge
- ='.': is of culiure me<j:-a
during grorrrtil .
Irrhen
l
l
I
L87
Fitting the growth data (i. e. <iiameter, <ir)- neighr)
for
:actors such as temperature and pli of eulture media,
lcr
environmental
predictive
regression modets, as in the presen' case, girre
a clearer w;ly of weighing
the data and ailows one to predict values on gror6,th clorrespcnding
to
a
given value of temperature or pH. This is certainry
useful in comparing
ihe grortzth ciata of any particul-ar fungus by a nun-,ber
of workers as Lhey are
unlikely Eo use the sartre tenperatures or PH ie,,,eIs jn Eheir srudies.
The temperature-gror,rth curve tends
to
become mcre nearly
.-.---retrical as the opti:num becomes ior,rer (Cochrane, 1958). In the present
--:vestigation, the optirnu- temperature is fairiy lorE (i..e" 15oC) and both
:re temperature-diameter
,-=ry near:ly synmetrical
and temperature.-dry weight curves
(fig. 8.3)
are
4.2 to 9.2
of.
.
The growth responses
of R. pini. over the pH range
:--.* culture meciia were very
sinilar (fig. 5.7).
pH
Maxinum gror.vth
!
LOT"
:-:urreC within the range pH 6.2 - 8.2 (table 8.3). Very comparable pH
-:-gas haye been recorded for a nunber of fungi and these reinforce the fact
.-::
rnosE
plant patliogens grow best ic
media
lriih initial pil 5.rl -
6.5
-:;hrane, 1958). I"lunro (1970) recorded that many mic::o-organisms have an
:::,rum pH for growth around 7 w:'-ih most favouring fhe pH range 5 to B.
The pronounced ef.fect
of low temperat.ure such as 4oc and 7oc in
:.-.r'ing the onset of germi-nation as observe<i in the present case has also
r:=- recorded for other fungi such as !t"fqrpt tbgfg glg!-!.g!:. (Monr.) de
'::. , Fusarium mog]-l-t-ornae. sheldsn-and scler-oti.nia f rucLicola (wint.)
r'-:. (cochrane, 1958I'. Per cent germinatior: of the conidi.a of B. I}gi
,"
i-- ::-e temperatures 15o -
21oC were
very comparable giving the gernination
:-:-.-: rather a flat centre with steep ends whereas the curve for germ tube
:::::s
'nad
a sherp peak at 27oc (rig. s.2L). A sin:ilar sitr.racion was noted
=-ier (1950) for
Cr-rnninghamell_a
C_t";ig".
187
Fitting the growth data (i.e.
<liame.ter, dr)'r,,eight)
for
environmental
ractors such as temperarure and pti of cuLtlrre media, tcr predictive
regression nodels, as in the present case, girre a clearer r.r;ay of weighing
:he data and ailows one to predict values on growth correspcr"rding to a
3iven value of temperature or pH. This is certainly useful in comparing
::ie gror{th data of any part.icular fungus by a number of workers as they are
:::likely to use the same temperatures or pH l-er,,els in their studies.
The tempereture-grslrrrl. s11.1:e tends
:'.-=retrieal as the cptimum
to
become mcre nearry
becomes ioruer (cochrane,
195g). rn
tire present
-:-r"estigation, the opti**rn tempereture is fairr_y 1ou, (i.e" 15oc) and both
::e temperature-dianeter and temperature.-dry weighc curves (fig. 8.3) are
-,-;ry nearly synunetrieal .
The growth responses
of B. pi"i over the pH range pH 4.2 to 9.2 of
:-e culEure me<iia were very sirnilar (fig. 5.7). Maxiniim gror.+th t LOy,
:::urrec within rhe range pH 6.2 - g.2 (table g.3). Very comparable pH
:a:1ges have been recorded
for a nuuber of fungi and these reinforce the fact
:-'-i nost plant pathogens grow best in ne,lia uith initial pil 5.o 6.5
-:chrane, 1958). I'lunre (tolo1 record.ed Ehat many mic::o-organisms have an
:-:ii.um pH for gror+rrr around. 7 with most favouring tiie pH range
5 to B.
The pronounced
effect of low temperature such as 4oC and 7oC in
:=-a'-'ing the onset
of germination as obser.,ued in the present case has also
::=: recorded for orher fungi such as jEy_fgl*lgre
lgfej.11anl (Monr.) ae
l'r--: r Fusarium gg*tfIor."e
r'-:.
sle1don-and scl-erorinia
(Cochrane, 1958)':' per cent germination
fructicola (I^Iint,)
of the conidia of E. pin]
r: :::e temperatures 150 - 210c were very comparable giving the germination
1--'-3 rather a flat centre with steep en<ls whereas the curve
for germ tube
-:-i::rs had a sharp peak at 21oc (tig. 8.21). A siniilar situation was noted
; ;=_ker (1950) for
Cunninghamel_1g S_Lgjjr"S_.
.'
lBB
inlith the exceplion of pH 4.2 tine f all in the per cent gerlnination f rom
the optimum towards the acid. ::ange \,ras rather rapid whereas that tr>...rarcls
the alkaline range of pE r^ras more gradual (rig. g.25), in the present
investigation. Walker (i950) on airalysing the clata of fungi which germinate
best in slightly acid media (e.g. OsIgliE
"glgglr_"
Tu1.), in slighrly
(e.e. !g-Us:gifl"h* gg:-lyi:, and on bcth (a Fusarium spp.
isolated from cotton) remarked that the fall- in germi.nation is usually more
rapid on Ehe acid side than on che a1ka1ine. Like other fungi, includ.ing
alkaline
:iant
medi-a
parhogens such as Fr*"_ annos-gx
!'r (r\.ishbech,
l_g51),
$!i.1at9. spp.
'Clayton' L942), free waEer was not essential for the germination of the
:cnidia o-f R. pini. but germination was significantly iurproved r,rhen the
:;;ridia
in
with liquid. water on an agar surface as compared. to
:: a dry cover glass ai 1o0Z relative humiC:i,ty. Yarwooci (1950) suggeste6
:-:at fungus spores of low rrater conteni must absorb water to abouE the
vrere
conEacE
of that i-n che powdery u.iId.e-'s corridia (70 per cent) before they can
'=:ninate. Ic is obvious that the tirne required by R. pini conidia Eo a6sorb
-='.'el-
;'--er in presence cf free '"rater on agar biock is clearly shorter than for
'-:lrbing the sane amoutt of wat.e:: fron water vapour of a saEurated elvironrrent.
---s explains r^rhy in
preserrce
of free
rcatej: on agar block 902 conidia
:=:::nated in 48 hours as comlared to 302 on dry cover glass at
:: -'dit:y, birt ir 72 hours germination on cover glasses r^las 5gz.
L}OZ
relative
l
Prof.Dr.Mohammad Abdur Rahman