Brazilian Journal of Microbiology (2004) 35:292-294
ISSN 1517-8382
ISOLATION, MORPHOLOGICAL IDENTIFICATION AND PATHOGENICITY OF
CYLINDROCLADIUM SCOPARIUM AND C. CLAVATUM ISOLATES OBTAINED FROM PLANTS
RHIZOSPHERE CULTIVATED IN PERNAMBUCO STATE
Vitorina Nerivânia Covello Rehn1*; Maria Menezes1; Kurt Georg Rehn2; Rildo Sartori Barbosa Coêlho1
1
Departamento de Agronomia, Universidade Federal Rural de Pernambuco, Recife, PE, Brasil; 2Departamento de Bioquímica,
Universidade Federal de Pernambuco, Recife, PE, Brasil
Submitted: August 20, 2003; Returned to authors for corrections: July 12, 2004; Approved: December 20, 2004
SHORT COMMUNICATION
ABSTRACT
Twelve isolates of Cylindrocladium scoparium and 4 isolates of C. clavatum were obtained from the
rhizosphere of various species of plants by baiting with Ricinus communis leaves. The isolates of C. scoparium
developed conidia of 32-(45)-60 x 3 -(4)-5 µm, and pyriform to ellipsoidal vesicles. C. clavatum showed conidia
of 36-(44)-49 x 2-(4)-6 µm and clavate vesicles. All isolates induced necrosis on leaves and hypocotyls of
eucalypt seedlings, with varying expression of symptoms.
Key words: Ricinus leaf bait, morphological markers, eucalyptus pathogenicity
The genus Cylindrocladium Morgan (teleomorph:
Calonectria De Not) comprises species considered soil
inhabitants that are saprophytes and facultative parasites,
widespread in different environments all over the world (1).
Typical symptoms caused by the pathogenic species are root
rot, damping-off, wilt, leaf spotting, or necrotic lesions on fruits
(7).
The purpose of the present study was to isolate species of
Cylindrocladium from the rhizosphere of various plants by a
baiting method, to identify them by morphological criteria and
to check their pathogenicity on eucalypt seedlings under
greenhouse conditions.
Small pieces of Ricinus communis L. leaves were disinfected
(4), and placed upon soil samples of 20 g in sterile Petri dishes,
collected from the rhizosphere of various plants (Table 1) and
moistened with sterile distilled water (3). The baits were
incubated for two days under alternating light at a temperature
of 25ºC and mycelia grown on the leaf segments were transferred
to Petri dishes with PDA (Potato-Dextrose-Agar) to obtain pure
cultures. For morphological identification, segments of
disinfected Ricinus leaves were inoculated with plugs of 5mm
diameter from a young mold colony grown on PDA and leaves
were kept in a Petri dish on a cotton swab moistened with sterile
distilled water as above, until the formation of fruiting bodies.
These were stained with Amann’s blue (4) on microscope slides
and observed under a light microscope. Of each isolate, average
length and width of 20 conidia were determined with an ocular
micrometer. Shape and septation of conidia were also considered,
as well the shape of the vesicle (1,2,7). Seedlings of eucalypt
(Eucalyptus citriodora), 40 days after germination, were
inoculated on both hypocotyls and leaves. Plugs as above were
placed upon a slight incision in hypocotyls and fixed with
Scotch tape. With leaves, the plug was put on the upper surface
without wounding. Following inoculation, the plants were
incubated for 48 h in a moist chamber at a temperature of 25ºC.
The experiment was completely randomized using three
replications in each treatment. The size of the lesions was
measured four days after the inoculation. These data were
subjected to Duncan‘s test, comparing means at a 5% level of
significance.
*Corresponding author. Mailing address: Departamento de Agronomia, Área de Fitossanidade, Universidade Federal Rural de Pernambuco, Av. Dom
Manoel de Medeiros, S/N, Dois Irmãos, 52171-030, Recife, PE, Brasil. Tel.: (+5581) 3302-1209, Fax. (+5581) 3441-1711. E-mail: vickrehn@yahoo.com
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Cylindrocladium from plants rhizosphere
Table 1. Isolates, hosts, size of conidia and lesions (mm) induced by Cylindrocladium scoparium (Cs) and Cylindrocladium
clavatum (Cc) on hypocotyls and on leaves of eucalypt (Eucalyptus citriodora Hook) plants, two days after to inoculation.
Isolates/ host
C. scoparium (Cs)
Cs-Ara.1/ Annona crassiflora Mart.
Cs-Ara.2/ A. crassiflora
Cs-Hel.3/ Heliconia sp.
Cs-Pim.4/ Capsicum annuum L.
Cs-Euc.5/ Eucalyptus citriodora Hook
Cs-Euc.6/ E. citriodora
Cs-Euc.7/ E. citriodora
Cs-Euc.8/ E. citriodora
Cs-Pin.10/ Pinus caribaea var. hondurensis Barret & Golfari
Cs-Pin.11/ Pinus caribaea var. hondurensis
Cs-Pin.12/ Pinus caribaea var. hondurensis
Cs-Pin.13/ Pinus caribaea var. hondurensis
C. clavatum (Cc)
Cc-Euc.9/ E. citriodora
Cc-Pin.14/ Pinus caribaea var. hondurensis
Cc-Cel.15/ Pennisetum purpureum Schumacher
Cc-Uru.16/ Bixa orellana L.
C.V. (%)
Size of conidia (µm)1
Pathogenicity2
Length
Width
Hypocotyl lesion
Leaf lesion
33-(45)-59
33-(47)-60
32-(44)-57
37-(45)-58
32-(46)-59
32-(44)-59
32-(48)-57
34-(45)-60
33-(46)-59
33-(45)-58
33-(46)-58
33-(45)-58
3-(4)-5
3-(4)-5
3-(4)-4
3-(4)-5
3-(4)-5
3-(4)-4
3-(4)-5
3-(4)-5
3-(4)-5
3-(4)-4
3-(4)-4
3-(4)-5
19.66a
18.30a
18.16a
17.15a
16.92ab
16.92ab
15.72ab
15.62ab
15.30ab
14.93ab
13.63ab
12.52ab
17.61ab
24.67a
9.97bc
14.49abc
29.32a
25.36a
9.02bc
13.28abc
14.77abc
16.28abc
26.22a
24.96a
38-(45)-48
37-(45)-47
37-(44)-49
36-(43)-48
3-(4)-6
3-(4)-5
2-(3)-6
3-(4)-6
17.94a
14.55ab
10.62b
10.62b
8.03
7.57bc
6.69c
7.66bc
6.88c
15.66
1
Means ( χ ) of 20 conidia from each isolate cultivated on castor bean leaves. (Min.) Minimum and (Max.) maximum values are also shown;
Date are log (x+1) tranforms. Means of three replications;
Means followed by the same letter (vertical) are not signicantly different Ducan’s test at 5%.
2
Baits of Ricinus leaves as described permitted to obtain in
high yield, 16 isolates of Cylindrocladium. This method was
introduced by Orrego Fuente et al. (6), who used three different
baits, but Ricinus leaves gave the best results.
From 16 isolates obtained, 12 were identified as
Cylindrocladium scoparium (Cs), and four as C. clavatum (Cc).
Conidial lengths of C. scoparium varied between extremes of
32-60 µm, and C. clavatum 37-49 µm (Table 1). Crous and
Wingfield (1) found a higher range (40-66 µm) for C. scoparium
cultured on CLA (Carnation Leaf Agar), and Moreira et al. (5)
reported a lower range (24.5-43.5 µm) for this specie grown on
Ricinus leaves. For C. clavatum, there were also small
differences between the sizes of conidia observed and those
cited by other researchers (1,7). However, the data of Hodges
and May (2) were identical to the values described here. The
width of conidia was much alike in both species and the mean
of 4 µm was equal for all isolates except Cc-Cel.15 that seemed
slightly slimmer. A similar value was reported by others (1,2,5).
The species C. scoparium displayed hyaline and septate
hyphae, conidiophores with di- or trichotomic branches
composing phialides which vary between doliiform and reniform,
and a septate stipe with a pyriform or ellipsoidal vesicle in the
apical end; the conidia were cylindrical with rounded ends,
hyaline and bicelular. The colony grown on PDA was typically
light brown with a white border and a slightly irregular margin
(Fig. 1A). C. clavatum showed the same features of hyphae,
conidiophores and conidia as the other species; but had a clavate
vesicle on an also hyaline septate stipe. C. scoparium formed
on PDA a colony with a dark brown center and an irregular
white border (Fig. 1B). Both species developed also
microsclerotia, but the production of these resistant structures
and the spatial arrangement of the cells in their interior are
inadequate for taxonomy (1).
With eucalypt seedlings inoculated on hypocotyls or on
leaves, all isolates of Cylindrocladium were pathogenic (Figs.
1C, D), but not in the same degree. Cs-Ara.1 caused wilt as well
as collapse of the three target plants tested 48 h after their
inoculation and could thus be considered as the most aggressive
isolate, although its hypocotyl lesions did not look different
from the other isolates (Table 1). Patent pathology was seen at
least in two of three replications of the other isolates, after 96 h
of incubation, with exception of Cc-Pin.14 and Cc-Uru.16, which
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V.N.C. Rehn et al.
RESUMO
Isolamento, identificação morfológica e patogenicidade
de isolados de Cylindrocladium scoparium e
C. clavatum da rizosfera de plantas cultivadas no
Estado de Pernambuco
Doze isolados de Cylindrocladium scoparium e 4 isolados
de C. clavatum foram obtidos da rizosfera de diversas plantas
usando folhas de Ricinus communis como isca. A primeira
espécie mostrou conídios com dimensões de 32-(45)-60 x 3 -(4)5 µm e vesícula piriforme a elipsoidal. Os conídios de C. clavatum
apresentaram dimensões entre 36-(44)-49 x 2-(4)-6 µm e vesícula
clavada. Em plântulas de eucalipto todos os isolados induziram
necrose em folhas e hipocótilos, variando apenas na expressão
dos sintomas.
Figure 1. A - Cylindrocladium scoparium. 1-Schematic
structures: (a) conidiophore; (b) vesicles; (c) microsclerotia;
(d) conidia; length marker, 5 µm; 2, 3, 4-Microscopic aspects. 5Colony. B - Cylindrocladium clavatum. 1-Schematic structures:
(a) conidiophore; (b) vesicles; (c) microsclerotia; (d) conidia;
length marker, 5 µm; 2, 3, 4-Microscopic aspects. 5-Colony.
Aspect of lesions symptoms on leaves (C), and wilt symptom
(D) on Eucalyptus citriodora Hook plants induced by C.
scoparium after 72 hours.
Palavras-chave: isca de Ricinus,marcadores morfológicos,
patogenicidade em eucalipto
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in all plants of Eucalyptus camandulensis Denh., six days after
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