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Morphological development and
interactions of Gliocladium roseum and
Botrytis cinerea in raspberry
a
Hai Yu & John C. Sutton
a
a
Department of Environmental Biology , University ofGuelph , Guelph,
Ontario, NIG 2WI
Published online: 23 Dec 2009.
To cite this article: Hai Yu & John C. Sutton (1997) Morphological development and interactions of
Gliocladium roseum and Botrytis cinerea in raspberry, Canadian Journal of Plant Pathology, 19:3,
237-246, DOI: 10.1080/07060669709500518
To link to this article: http://dx.doi.org/10.1080/07060669709500518
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Canadian Journal of Plant Pathology
Revue canadienne de phytopathologie
Published by
Publiée par
The Canadian Phytopathological Society
La Société Canadienne de Phytopathologie
Volume 19(3):237-336
September
1997
septembre
ISSN 0706-0661
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CANADIAN JOURNAL OF PLANT PATHOLOGY 19:237-245, 1997
Morphological development and interactions of Gliocladium roseum
and Botrytis cinerea in raspberry
Hai Yu and John C. Sutton
Department of Environmental Biology, University ofGuelph, Guelph, Ontario NIG 2WI.
Accepted for publication 1997 04 28
Morphological development and interactions of Gliocladium roseum and Botrytis cinerea on leaves, stems, and stamens of raspberry were examined by light microscopy. Tissues were inoculated with conidial suspensions of the antagonist, the pathogen, or
both, and kept in continuous high humidity at 21-23°C. In the absence of B. cinerea, G. roseum produced germ tubes and superficial hyphae with short side branches that penetrated the host. No symptoms developed, but numerous conidiophores and conidia of G. roseum were observed on tissues at 40-72 h after inoculation. In the absence of G. roseum, B. cinerea germinated
slowly on leaves but rapidly on stems and stamens. The pathogen produced a few single-lobed appressoria on leaves, various
kinds of appressoria on stems, and a range of appressoria and infection cushions on stamens. After coinoculation, G. roseum
strongly suppressed germination and germ tube growth of B. cinerea on leaf surfaces. On stems the antagonist moderately suppressed germ tube growth and intensely parasitized the pathogen. Hyphae of G. roseum grew on, coiled around, penetrated, and
developed within hyphae and conidia of B. cinerea. On stamens, G. roseum reduced colonization incidence of the tissues but did
not suppress germination, growth, or formation of appressoria and infection cushions by B. cinerea, or intensely parasitize the
pathogen. Available nutrients are postulated to determine the mode of antagonism on the various organs. G. roseum suppressed
sporulation and, by inference, infection and colonization of B. cinerea in raspberry tissues more effectively when applied before
or at the same time as the pathogen than after the pathogen. It is concluded that G. roseum is a nonpathogenic parasite of raspberry with flexible modes of antagonism towards B. cinerea in this host.
Additional index words: Rubus idaeus, gray mold, biological control, antagonism, nutrient competition, mycoparasitism.
Yu, H., and J.C. Sutton. 1997. Morphological development and interactions of Gliocladium roseum and Botrytis cinerea in raspberry.
Can. J. Plant Pathol. 19:237-246.
Le développement morphologique et les interactions entre Ie Gliocladium roseum et le Botrytis cinerea sur les feuilles, tiges et
étamines de framboisier ont été examines par microscopie photonique. Les tissus ont été inoculés avec des suspensions de conidies de l'antagoniste, de l'agent pathogène ou des deux a la fois, et conserves a 21-23°C dans des conditions constantes
d'humidité élevée. En l'absence du B. cinerea, le G. roseum a produit des tubes germinatifs et des hyphes superficielles avec des
embranchements latéraux courts qui ont pénétré l'höte. Aucun symptöme n'est apparu, mais de nombreux conidiophores et conidies du G. roseum ont pu être observes sur les tissus 40 a 72 h après 1'inoculation. En l'absence du G. roseum, le B. cinerea a
germé lentement sur les feuilles mais rapidement sur les tiges et les étamines. L'agent pathogène a produit quelques appressoria
a lobe unique sur les feuilles, divers types d'appressoria sur les tiges et une gamme variée d'appressoria et de coussins d'infection sur les étamines. Après les inoculations mixtes, le G. roseum a fortement réprimé la germination et la croissance des tubes
germinatifs du B. cinerea a la surface des feuilles. Sur les tiges, l'antagoniste a modérément réprimé la croissance des tubes germinatifs et a fortement parasite l'agent pathogène. Les hyphes du G. roseum ont cru sur, se sont enroulées autour, ont pénétré et
se sont développées a l'intérieur des hyphes et des conidies du B. cinerea. Sur les étamines, le G. roseum a reduit le taux de
colonisation des tissus, mais n'a pas réprimé la germination, la croissance ou la formation des appressoria et coussins d'infection
du B. cinerea, et n'a pas non plus parasite intensément l'agent pathogène. Il est propose que c'est la disponibilité des nutriments
qui determine le mode d'antagonisme sur les différents organes. Le G. roseum réprimé la sporulation, en consequence, l'infection et la colonisation des tissus de framboisier par le B. cinerea, avec plus d'efficacité lorsqu'il est appliqué avant ou en même
temps que l'agent pathogène plutöt qu'après. En conclusion, le G. roseum est un parasite non pathogène du framboisier avec des
modes flexibles d'antagonisme envers le B. cinerea sur eet höte.
Mots clés supplémentaires: Rubus idaeus, moisissure grise, lutte biologique, antagonisme, competition nutritionnelle,
mycoparasitisme.
Gliocladium roseum Bainier is a powerful antagonist
of Botrytis cinerea Pers.: Fr. in at least 15 hosts of
economie importance, and is considered to have major
potential for biocontrol of the pathogen in cropping
237
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997
systems (Sutton et al. 1997). The antagonist is able to
control B. cinerea in leaves, stems, flowers, and fruits
of various plants under a diversity of crop conditions.
Wide adaptability of G roseum as a biological control
agent against B. cinerea might be related to strong
ecological adaptation of the antagonist to host plants
and to varied modes of antagonism of the fungus
towards the pathogen (Sutton et al. 1997). Knowledge
of relationships of G. roseum with hosts, and of interactions between the antagonist and B. cinerea in host
tissues is sparse, yet fundamental for developing rational strategies for biocontrol of the pathogen.
Evidence indicates that diverse associations exist
between G. roseum and higher plants. The antagonist
was reported on the phylloplane of strawberry
(McLean & Sutton 1992) and the mycorrhizoplane of
European silver fir (Mosca & Marchisio 1985), and is
known to colonize, without symptom production,
apparently healthy roots, stems, pods, and seeds of
soybean (Mueller & Sinclair 1986), roots of red
clover (Skipp & Christensen 1990), and leaves of
strawberry (Sutton & Peng 1993b) and black spruce
(Zhang et al. 1996b). It is also common in stressed,
senescent, and dead roots and foliage of numerous
plants (Sutton et al. 1997).
Mycoparasitism and competition for nutrients or
substrate are presumed modes of antagonism of G.
roseum towards B. cinerea in plants (Sutton & Peng
1993a, 1993b; Sutton 1994, Zhang et al. 1996a).
Mycoparasitism of B. cinerea by G. roseum has not
been described in plants, but studies on agar media
indicated that the fungus is a mycoparasite of hyphae,
spores, sclerotia, and fruiting bodies of numerous
fungi including B. cinerea (Barnett & Lilly 1962,
Walker & Maude 1975, Lim & Chan 1986, Fravel
1988, Deacon & Berry 1992). Whipps (1987) reported
mycoparasitism of another sclerotiniaceous fungus,
Sclerotinia sclerotiorum, by G. roseum on plant tissue
segments. Peng (1991) observed that mutants of G.
roseum that produced high or intermediate levels of
an antifungal metabolite, or none at all, did not differ
in biocontrol effectiveness against B. cinerea in strawberry leaves, and suggested that antibiosis was not a
key mechanism of biocontrol. From observations on
an agar medium and in water culture, Pachenari &
Dix (1980) concluded that antibiosis through toxins
and cell-wall degrading enzymes is a mode of antagonism by G. roseum towards Botrytis allii.
In the present study, morphological development
and interactions of G. roseum and B. cinerea were
investigated in leaves, stems, and flowers of raspberry
{Rubus idaeus L.). Gray mold caused by B. cinerea is
of worldwide economic importance in raspberry, and
considerable information exists on development of the
pathogen in this host (Ellis et al. 1991, Yu 1996).
Gliocladium roseum is known to strongly suppress B.
cinerea in raspberry but relationships of the antagonist
with raspberry and modes of biological control are
poorly understood (Yu 1996). A preliminary report
was published (Yu & Sutton 1995).
Materials and methods
Raspberry plants. Primocanes of raspberry cv.
Boyne were dug from research plots near Guelph,
Ontario, in November, stored with adhering soil
beneath plastic film in a cold room (1°C) for 6 wk to
satisfy rest requirement (Crandall & Daubeny 1990),
and planted in 25-cm diameter plastic pots (1 cane
per pot) containing a soilless mix (Promix®, Plant
Products Ltd., Brampton, Ont.). The plants were
grown in a climate-controlled greenhouse where air
temperature averaged 18° to 24°C during the day
(0800 to 2000 hours) and 13° to 17°C at night (2000
to 0800 hours). White shades in the greenhouse roof
were retracted when irradiance was < 800 uE s~' m"2
and drawn when irradiance exceeded 1200 uE s'1 nr2
and at night to reduce heat loss. The plants were supplied with a soluble N:P:K (20:8:20) fertilizer (5 g/L
water) once a week. Raspberry shoots, each with several leaves and an inflorescence with 4-5 freshlyopened flowers and several flower buds, were used
for biocontrol studies.
Inoculum production. Isolate PG-A-Fr-88-710 of
G. roseum from strawberry (Peng & Sutton 1991) and
isolate HYU-92-1 of B. cinerea from a raspberry fruit
at the Cambridge Research Station near Cambridge,
Ontario, were used for inoculation. Inoculum of the
antagonist and the pathogen were produced on potato
dextrose agar medium under cool-white fluorescent
lamps (12-h photoperiod) at 20° to 22°C for 20 days
and 14 days, respectively. Conidia of G. roseum and
B. cinerea were suspended in sterile distilled water
plus surfactant (0.05 mL Triton X-100/100 mL), filtered through three layers of cheesecloth, counted
with the aid of a hemacytometer, and diluted to 107
and 106 conidia/mL water plus surfactant respectively.
Conidial germination of the fungi on PDA during 16 h
at 21-23°C consistently exceeded 98%.
Development and interactions of G. roseum and
B. cinerea on the host. One-cm-diameter leaf disks,
stem segments each 1.5 cm long, and freshly-opened
flowers were cut from raspberry shoots. Leaf disks
were placed on fibreglass screen (1 mm mesh) that
overlaid moist paper towels in petri dishes, and each
was inoculated near the centre of the upper surface
with a 20-uL droplet of conidial suspension of G.
roseum, B. cinerea, or G. roseum plus B. cinerea.
Stem segments and flowers were immersed in the
conidial suspensions for 30 s and arbitrarily selected
groups of 10 segments or flowers were each placed in
a petri dish as described for leaf disks. Inoculated tissues were incubated in the laboratory at 21-23°C.
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Development and interactions of G. roseum and B.
cinerea on inoculated tissues were investigated
microscopically. A leaf disk, stem segment, and
flower were removed from each of four replicate petri
dishes of each treatment at 4, 8, 12, 16, 20, 24, 32,
40, and 72 h after inoculation. A portion of epidermis
(15 x 3 mm) was removed from each stem segment,
and a cluster of 12-14 stamens was taken from each
flower. Leaf disks, pieces of stem epidermis, and stamen clusters were mounted in lactophenol containing
0.05% trypan blue on microscope slides, gently heated over a flame for 2 min to clear the tissues, and
examined on a compound light microscope (Saha et
al. 1988). Germination incidence of G. roseum and of
B. cinerea was estimated on each kind of host tissue
based on observations of 50 to 100 conidia. Germ
tube length was measured in 10 germinated conidia,
selected arbitrarily on each tissue piece, except when
long tubes became intermingled and not easily distinguishable. Incidence of stamens in which hyphae of
B. cinerea were found was estimated from observations of all stamens in each replicate cluster.
Verticillate conidiophores of G. roseum were counted
at 100 x magnification in four microscope fields
(each 2.43 mm2) on each leaf disk, piece of stem epidermis, and stamen cluster. Morphological development of G roseum and of B. cinerea on each kind of
host tissue and interaction between the antagonist and
the pathogen were observed and recorded on colour
negative film (Kodak Ektachrome® tungsten ASA
64) using a Nikon AFX-II exposure control unit.
Time of application of G. roseum and B. cinerea.
Time of inoculation of the antagonist and of the
pathogen in relation to biocontrol was examined in
leaf disks, stem segments, and flowers of raspberry.
Tissues were inoculated with B. cinerea only, or with
B. cinerea plus G. roseum, which was applied at 32 h
before, at the same time as, or 32 h after the
pathogen. For inoculation of leaf disks, one 20-uL
inoculum droplet of G. roseum, B. cinerea, or both
fungi was placed near the centre of each disk. Stem
segments and flowers were inoculated by immersion
in inoculum for 30 s. There were four replicate
groups of 10 leaf disks, 10 stem segments, and five
flowers in each treatment. Immediately after inoculation, each group of leaf disks, stem segments, and a
group of 16 clusters of 10-12 stamens that were
removed from the five flowers of each replicate were
placed on paraquat-chloramphenicol agar medium
(PCA; Peng & Sutton 1991), incubated at 20-22°C
for 7-10 days, and examined for incidence of conidiophores of B. cinerea.
Data analysis. Statistical computations were performed using the Statistical Analysis System (SAS
Institute Inc., Cary, NC). Data on the incidence of
conidial germination and germ tube length of B.
239
cinerea and G. roseum, of B. cinerea hyphae in stamens, and numbers of G. roseum conidiophores were
examined using analysis of variance (ANOVA).
Observations in repetitions of application timing
studies were subjected to analysis of homogeneity of
variance and were pooled accordingly. Treatment
means of sporulation incidence of B. cinerea were
separated by protected least significant difference test
(PLSD; Snedecor & Cochran 1989).
Results
Morphological development of G. roseum.
Conidia of G. roseum germinated and developed
germ tubes and superficial hyphae, all 1 to 1.5 |jm in
diameter, on leaves, stems, and stamens that were
inoculated with the antagonist only. Short branches
on some germ tubes, observed chiefly at 16 and 20 h
after inoculation, penetrated into the epidermis of the
various tissues (Fig. 1A). Post-penetration development of the branches was not investigated. At 24 and
32 h after inoculation, numerous hyphae of the antagonist were present on each type of inoculated tissue
and portions of the hyphae were thickened to about
4.5 \im in diameter. Verticillate conidiophores were
observed developing from thickened hyphae on stems
and stamens at 32 h after inoculation, and were abundant on all inoculated tissues and bore conidia at 40 h
(Fig. 1, B and C). Penicillate conidiophores of G.
roseum bearing abundant conidia were observed on
all tissues at 72 h after inoculation (Fig. 1, D and E).
Morphological development of B. cinerea.
Conidia of B. cinerea germinated and produced
mainly germ tubes on leaf disks and germ tubes plus
superficial hyphae on stem segments and stamens.
Germ tubes and superficial hyphae of the pathogen
were 2.4 to 4.9 urn in diameter. Appressoria formed
at apices of many germ tubes and on short to long
superficial hyphae. On leaf disks, simple appressorium-like swellings (protoappressoria, not delimited by
a septum; Emmett & Parbery 1975) and single-lobed
appressoria developed on about 80-90% of germ
tubes by 32 h after inoculation. On stem segments,
appressorium-like swellings and single-lobed appressoria were present on most germ tubes, and single
and multilobed appressoria were present on superficial hyphae at 32-40 h after inoculation. Hyphae
were frequent in the superficial stem tissues at 72 h
after inoculation (Fig. 2A). On stamens, appressorium-like swellings and single-lobed appressoria
developed on about 50% of germ tubes, and an infection peg was observed on some of the appressoria
within 8 h of inoculation (Fig. 2B). At 16 h, germ
tubes in many instances were long (> 250 urn) and
abundant superficial hyphae were present. At 16-24
h, single-lobed and multilobed appressoria, and
complex, dome-shaped infection cushions with lobate
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997
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YU, SUTTON: RASPBERRY/GLIOCLADIUM/BOTRYTIS
24]
Figure 2. Morphological development of Botrytis cinerea in detached raspberry tissues maintained in high relative humidity at 21-23°C.
A) Hyphae in stem tissues at 72 h after inoculation (bar = 10 urn). B) Germinated conidium with a simple appressorium and infection peg
(indicated by arrow) on a stamen filament at 8 h after inoculation (bar = 10 um). C) Bilobed hyphal appressorium on a stamen filament at
16 h after inoculation (bar = 10 um). D) Multilobed appressorium on a stamen filament at 24 h after inoculation (bar = 10 um). E) Hyphae
(indicated by arrow) in a stamen filament at 32 h after inoculation (bar = 10 um).
appressoria were numerous on filaments of stamens tubes, or hyphae of G. roseum. By 32 h after inocula(Fig. 2, C and D). At 24 h, hyphae of B. cinerea were tion, hyphae of G. roseum had grown towards germigrowing into stamen filaments from infected anthers; nated conidia of B. cinerea and developed numerous
at 32 h, endophytic hyphae were present in 95% of short branches. Some branches contacted and grew
stamen filaments (Fig. 2E ).
on conidia, germ tubes, and hyphae of the pathogen.
Morphological interactions of G. roseum and B. By 72 h, hyphae of G. roseum coiled around and pencinerea. Relationships between G. roseum and B. etrated about 10% of conidia and 50% of hyphae of
cinerea differed on the coinoculated leaves, stems, B. cinerea (Fig. 3, B and C). Invaded conidia and
and stamens. On leaf disks, conidia of B, cinerea usu- hyphae were empty, and some hyphae had collapsed.
ally failed to germinate in the presence of conidia, No hyphae of B. cinerea were found in the epidermis
germ tubes, and superficial hyphae of G. roseum, and of stem segments at any time of observations. On stathe antagonist rarely parasitized conidia and germ mens, superficial hyphae of G roseum and B. cinerea
tubes of the pathogen (Fig. 3A). On stem segments, were abundant at 24 h after inoculation. A few short
conidia of B. cinerea germinated and germ tubes and branches developed on hyphae of G. roseum but the
superficial hyphae of the pathogen developed in the antagonist was not observed to grow on, coil around,
presence and in the absence of nearby spores, germ or penetrate hyphae of the pathogen. Endophytic
Figure 1. Morphological development of Gliocladium roseum on detached raspberry tissues maintained in high relative humidity at
21-23°C. A) Conidium with a germ tube bearing a short lateral branch (indicated by arrow) penetrating stem epidermis at 16 h after inoculation (bar = 10 um). B) Verticillate conidiophores bearing conidia on a stamen at 40 h after inoculation (bar = 100 urn). C) Verticillate
conidiophores bearing conidia on a symptomless leaf disk at 40 h after inoculation (bar = 30 um). D) Dense cluster of penicillate conidiophores bearing conidia on the adaxial surface of a symptomless leaf disk at 72 h after inoculation (bar = 10 um). E) Penicillate conidiophores and conidia at the edge of a symptomless leaf disk at 72 h after inoculation (bar = 10 um).
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997
Figure 3. Development of Gliocladium roseum (Gr) and Botrytis cinerea (Be) on detached raspberry tissues maintained in high relative
humidity at 21-23°C. A) Hyphae of G. roseum and ungerminated conidia of B. cinerea on a leaf disk at 32 h after coinoculation (bar =
10 um). B and C) Hyphae of G. roseum growing on and within partially vacuolated hyphae of B. cinerea on stem segments at 72 h after
coinoculation (bar = 10 (jm).
hyphae of B. cinerea were observed in about 5-10%
of stamens.
Germination incidence of G. roseum and B.
cinerea. Conidial germination of G. roseum on tissues inoculated with the antagonist only was not
observed at 4 h, but at 8, 12, 16, and 24 h after inoculation, respectively, was 19, 67, 74, and 85% on leaf
disks, 23, 80, 93, and 98% on stem segments, and 30,
92, 98, and 100% on stamens.
Conidia of B. cinerea germinated rapidly on tissues inoculated with the pathogen only. At 4 and 8 h
after inoculation, respectively, germination incidence
was 15 and 17% on leaf disks, 53 and 88% on stem
segments, and 78 and 98% on stamens. Maximum
germination was much lower on leaf disks (42% at 24
h) than on stem segments (88% at 8 h) and stamens
(100% at 12 h).
In coinoculated tissues, G. roseum suppressed germination of B. cinerea chiefly on leaves. The antagonist suppressed B. cinerea on leaves by 69 to 93% at
4 to 40 h after coinoculation, and suppressed the
pathogen on stems and stamens by 14 to 24% and 0
to 7%, respectively, at 4 to 8 h after coinoculation but
not at later times. The pathogen significantly reduced
germination of G. roseum by 51, 39, and 11% respectively at 8, 12, and 16 h after inoculation on leaves,
and on stems by 43% at 12 h, but did not affect the
antagonist on stamens.
Germ tube growth of G. roseum and B. cinerea.
Growth of germ tubes of G. roseum on tissues inocu-
YU, SUTTON: RASPBERRY/GLIOCLADIUM/BOTRYTIS
300 -|
200
Gliocladium roseum
Inoculations
G roseum
G roseum + B. cinerea
Botrytis cinerea
O
•
Inoculations
B. cinerea
B. cinerea + G. roseum
\///\
100 -I
_
Leaves
0 -I
Inoculations
G. roseum
G. roseum + B. cinerea
32 h
e
3
X
300-j
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■Jl
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200 A
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Sterns
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243
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40 h
300
200 -1
100 H
Stamens
JL^
o H
T
16
24
32
40 0
8
16
24
32
Time after inoculation (h)
Figure 4. Estimated length of germ tubes of Gliocladium roseum
and of Botrytis cinerea as a function of time after inoculation in
leaf disks, stem segments, and stamens of raspberry that were inoculated with the antagonist only, the pathogen only, or with the
antagonist and the pathogen. Curves show mean values of germ
tube length with standard error bars.
lated only with the antagonist was slow for at least
24 h after inoculation on leaf disks, for 16 h on stem
segments, and for 12 h on stamens (Fig. 4). However,
germ tubes grew rapidly on stem segments at 16-24 h
and on stamens at 12-24 h after inoculation.
In tissues inoculated with B. cinerea only, germ
tubes of the pathogen grew slowly on leaf disks for at
least 40 h after inoculation, and on stem segments for
24 h, but subsequent growth on stem segments was
rapid (Fig. 4). Growth of germ tubes on stamens was
rapid for at least 16 h after inoculation.
In tissues inoculated with G. roseum and with B.
cinerea, the pathogen had little or no effect on germ
tube growth of the antagonist, but the antagonist suppressed germ tube growth of B. cinerea almost completely on leaf disks, and slightly on stem segments at
32^10 h after inoculation (Fig. 4). G. roseum failed to
suppress germ tube growth of B. cinerea on stamens.
Conidiophore production by G. roseum. G.
roseum produced numerous verticillate and penicillate conidiophores on inoculated tissues, all of which
remained symptomless. Verticillate conidiophores
were observed on stem segments and stamens at 32
and 40 h after inoculation, and on leaf disks at 40 h
(Fig. 5). Penicillate conidiophores were observed on
oH
[
1
Leaves
m
Stems
Stamens
Figure 5. Estimated density of verticillate conidiophores of
Gliocladium roseum on leaf disks, stem segments, and stamens of
raspberry 32 h and 40 h after the tissues were inoculated with G.
roseum only or with G. roseum plus Botrytis cinerea.Data bars are
mean values each with a standard error bar.
leaves, stems, and stamens at 72 h but not at 40 h or
earlier. Density of verticillate conidiophores was
greatest (31 conidiophores/mm 2 ) on stamens. In
tissues inoculated with B. cinerea and G. roseum, the
pathogen reduced production of verticillate conidiophores on leaf disks, stem segments, and stamens by
21, 100, and 90%, respectively.
Colonization of stamens by B. cinerea. At 24, 32,
and 40 h after inoculation, respectively, hyphae of B.
cinerea were observed in tissues of 17, 95, 100% of
stamens inoculated with the pathogen only, but in 0,
5, and 10% of stamens inoculated also with G.
roseum. Reduced incidence of B. cinerea in stamens
treated with G. roseum was significant (P - 0.0001)
at each time of observation.
Time of application of G. roseum and B. cinerea.
G. roseum strongly suppressed sporulation incidence
of B. cinerea in leaf disks, stem segments, and stamens when applied 32 h before, or at the same time
as, the pathogen, but it was only slightly or moderately suppressive when applied at 32 h after the
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997
Inoculations
B. c. only
V772 G. r. 32 h before B. c.
E^^CvM G. r. at same time as B. c.
^
G.r.32hafter5.c.
a
a
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b
c c
Leaves Stems Stamens
Inoculation treatments
Figure 6. Effect of time of application of Gliocladium roseum
(G.r.) in relation to time of inoculation of Botrytis cinerea (B.c.) on
sporulation incidence of the pathogen in leaf disks, stem segments,
and stamens of raspberry. Data bars are mean values with standard
error bars. Treatment means for a given host organ with same letter
are not significantly different (protected LSD P > 0.05).
pathogen (Fig. 6). Biocontrol activity of G. roseum
applied 32 h after application of the pathogen was
greatest on leaf disks.
Discussion
The microscopic observations clarified the association of G. roseum with raspberry tissues and supported the concept that the antagonist is a nonpathogenic
parasite of raspberry. G. roseum developed abundant
germ tubes, superficial hyphae, conidiophores, and
conidia on green leaves, green stem segments, and
stamens of the host, each of which remained symptomless. Patterns of germination incidence of the
antagonist over time were generally similar on the
various host organs, but germ tubes became longer on
stems and stamens, possibly in response to greater
availability of nutrients. Observations of penetration
hyphae were consistent with previous inferred evidence that the antagonist infected and colonized
leaves, stems, and stamens of raspberry (Yu 1996).
Colonization of apparently healthy host tissues by G.
roseum was reported in soybean by Mueller and
Sinclair (1986), who concluded that the fungus is a
nonpathogenic systemic parasite in that host; similar
reports have been made for red clover, strawberry,
and black spruce (Skipp & Christensen 1990, Sutton
& Peng 1993b, Zhang et al. 1996b). Profuse production of verticillate and penicillate conidiophores and
of conidia of G. roseum on symptomless leaves,
stems, and stamens of raspberry underscored the
potential for population increase of the antagonist on
raspberry and for sustained biological control of B.
cinerea. Evidence from temporal relationships of
infection and colonization with production of conidiophores and conidia, indicated that sporulation probably depended at least in part on nutrients obtained by
G. roseum from within the host tissues. To our
knowledge, this is the first report of a fungal biological control agent producing inoculum on living
foliage and flowers of a host plant.
In contrast to G. roseum, development of B. cinerea
differed markedly on the various raspberry organs. On
leaves, germination of conidia and germ tube growth
were slow, and appressoria infrequent, whereas on
stems germination was rapid, germ tubes elongated
rapidly after 24 h, and various kinds of appressoria
formed. On stamens conidia germinated rapidly, germ
tubes grew rapidly during 4-16 h, and a range of
appressoria and infection cushions formed, observations that agreed with previous findings on stamens of
strawberry, plum, and nectarine (Bristow et al. 1986,
Fourie & Holz 1994). Simple and complex appressoria and infection cushions similar to those observed in
raspberry were reported in other hosts (Emmett &
Parbery 1995, Sharman & Heale 1977, Garcia-Arenal
& Sagasta 1980, Van den Heuvel & Waterreus 1983,
Fourie & Holz 1995). Developmental differences of
B. cinerea on various raspberry organs possibly were
functions of availability of endogenous and exogenous nutrients on the host surface, which can be
expected to be low in leaves, intermediate in stems,
and high in stamens (Tukey 1971, Blakeman 1980).
Numerous reports indicated that exuded nutrients,
pollen, and other materials stimulated germination,
germ-tube growth, and appressorium formation of the
pathogen on host surfaces (Blakeman 1980, Verhoeff
1980). In the present study, nutrients leaking from cut
surfaces of tissues also might have influenced development of B. cinerea. Possible antifungal substances
in leaf exudates (Blakeman 1980) and their relationships with B. cinerea and G. roseum have not been
investigated in raspberry.
Raspberry organs markedly affected the mode of
antagonism of G. roseum towards B. cinerea. The
chief mode apparent on leaves was suppression of
conidial germination and germ tube growth, while on
stems antagonism was manifested as moderate suppression of germ tube growth and, later, as intense
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YU, SUTTON: RASPBERRY/GLIOCLADIUM/BOTRYTIS
parasitism of B. cinerea by G. roseum. On stamens,
G. roseum reduced colonization incidence of the tissues but generally did not suppress germination,
germ tube growth, or, from microscopic observations,
appressoria and infection cushions of B. cinerea.
From the collective observations it is postulated that
mode of antagonism was conditioned by different
kinds and concentrations of available nutrients on the
various raspberry organs. Strong competition for
scarce nutrients might account for the reduced germination of B. cinerea in the presence of G. roseum on
leaves, but not on stems and stamens on which the
pathogen germinated and developed abundantly (Elad
1996). Levels of available nutrients, known to influence hyperparasitism by necrotrophic hyperparasites
(Barnett & Binder 1973), possibly were favorable for
parasitism of B. cinerea by G roseum on stems, but
inadequate on leaves and excessive on stamens.
Kinds and concentrations of nutrients might also have
affected production of antifungal metabolites and
wall-degrading enzymes by G roseum (Pachenari &
Dix 1980, Al-heeti & Sinclair 1988, Peng 1991,
Deacon & Berry 1992), as in other fungi (Griffin
1994), but evidence of antibiosis of G. roseum other
than that associated with hyperparasitism is lacking.
Although deleterious morphological changes were
not found in B. cinerea when the pathogen developed
in proximity to G. roseum, the studies did not preclude ultrastructural changes (Bélanger et al. 1995).
Necrotrophic mycoparasitism of B. cinerea by G.
roseum on stem tissues of raspberry was similar to
that observed on various Botrytis spp. on agar media
(Barnett & Lilly 1962, Ricard et al. 1974, Walker &
Maude 1975, Lim & Chan 1986, Berry & Deacon
1992). Hyphae of the antagonist grew towards and on,
coiled around, penetrated, and developed within
hyphae and conidia of B. cinerea. Vacuolation and
wall lysis observed in hyphae of the pathogen that
were or were not penetrated by G. roseum were
attributed in previous studies to low molecular weight,
short range toxins, and wall-degrading glucanases and
chitinases secreted by the mycoparasite (Pachenari &
Dix 1980). Whether or not G. roseum penetrated only
hyphae that were killed by toxic metabolites of the
antagonist, or whether the antagonist also penetrated
living hyphae and spores, as suggested by Walker &
Maude (1975) in studies of B. alii and reported by
Lim & Chan (1986) in Phytophthora palmivora, were
not ascertained in the present study.
The susceptibility of G. roseum to antagonism by
B. cinerea underscored that antagonism between
these fungi is mutual and warrants recognition in
development of biocontrol programs against the
pathogen. Antagonism by B. cinerea was manifested
as reduced germination of G. roseum on leaves and
stems, chiefly at 12 h after inoculation, and reduction
245
or elimination of conidiophore production of the fungus on stems and stamens. Mode of the 'reverse
antagonism' was unclear, but observations were consistent with nutrient competition and antibiosis.
Antagonism of B. cinerea towards G roseum might
contribute to the requirement for inoculum concentration of G. roseum to be the same or greater than that
of the pathogen for biocontrol to be effective, and
reduction in biocontrol when inoculum concentration
of the pathogen is extraordinarily high (Yu 1996).
Observations that G. roseum suppressed sporulation of B. cinerea in raspberry tissues more effectively when applied before or at the same time, as
opposed to after, the pathogen can be interpreted in
terms of infection, colonization, and substrate competition by these fungi. Application of G. roseum before
B. cinerea probably allowed the antagonist to infect
and colonize the tissues before B. cinerea whereas
the reverse could be expected when G. roseum was
applied after the pathogen. From observations on
strawberry (Sutton & Peng 1993a, b), B. cinerea
might colonize raspberry tissues chiefly, or at least
more rapidly, when they senesce, die, or are killed, as
when placed on PCA. Strong suppression of B.
cinerea by G. roseum in tissues inoculated with the
two fungi simultaneously pointed to competitive
advantages of the antagonist on the host, which could
have resulted from antagonism of the pathogen
before and after infection. Early or rapid colonization
of tissues by G. roseum might be a major advantage
for the antagonist in raspberry as in strawberry
(Sutton 1994), and could counter any disadvantage of
slow germination relative to that of the pathogen. By
inference from sporulation incidence of B. cinerea,
G. roseum strongly reduced infection and colonization of raspberry tissues by the pathogen when
applied before or at the same time as the pathogen.
From observations of development of G. roseum
and of interaction of the antagonist with B. cinerea,
we conclude that the fungus is well adapted to raspberry and flexible in mode of biocontrol on this host.
Although a presumed nonpathogenic parasite with
epiphytic and endophytic growth and development,
G. roseum apparently did not cause stress or otherwise deleteriously affect the tissues. This was in
keeping with a previous report that the antagonist did
not alter chlorophyll level, photosynthetic rate, or
electrolyte leakage from foliage of black spruce
seedlings (Zhang et al. 1996b). Adaptation to the host
and flexible biocontrol are attributes of potential
importance in control of gray mold by G. roseum in
raspberry cropping systems.
We acknowledge financial support provided by the Natural
Sciences and Engineering Research Council of Canada (grant
OGP0006119 to J.C. Sutton) and thank Yangdou Wei for valued
advice.
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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19. 1997
Al-heeti, M.B., and J.B. Sinclair. 1988. Antagonism between
Gliodadium roseum, Trichoderma harzianum, or Trichothecium
roseum and Phytophthora megasperma f. sp. glycinea.
Mycopathologia 103:135-140.
Barnett, H.L., and F.L. Binder. 1973, The fungal host-parasite
relationship. Annu. Rev. Phytopathol. 11:273-292.
Barnett, H.L., and V.G. Lilly. 1962. A destructive mycoparasite,
Gliodadium roseum. Mycologia 52:72-77.
Bélanger, R.R, N. Dufour, J. Caron, and N. Benhamou. 1995
Chronological events associated with the antagonistic properties
of Trichoderma harzianum against Botrytis cinerea: indirect
evidence for sequential role of antibiosis and parasitism.
Biocontrol Sci. Technol. 5:41-53.
Berry, L.A., and J.W. Deacon. 1992. Video-analysis of
Gliodadium roseum in relation to mechanism of antagonism of
plant pathogens. Pages 64—66 in D.F. Jensen, J. Hochehull, and
N.J. Fokkema, eds., New Approaches in Biological Control of
Soil-Borne Diseases. IOBC / WPRS Bulletin. Wageningen.
222 pp.
Blakeman, J.P. 1980. Behaviour of conidia on aerial plant surfaces. Pages 115-151 in J.R. Coley-Smith, K. Verhoeff, and
W.R. Jarvis, eds., The Biology of Botrytis. Academic Press,
London. 318 pp.
Bristow, P.R., R.J. McNicol, and B. Williamson. 1986.
Infection of strawberry flowers by Botrytis cinerea and its relevance to grey mould development, Ann. Appl. Biol.
109:545-554.
Crandall, P.C., and H.A. Daubeny. 1990. Raspberry management. Pages 157-213 in G.J. Galletta and D.G. Himelrick, eds.,
Small Fruit Crop Management, Prentice Hall, Inc. Englewood
Cliffs, New Jersey. 602 pp.
Deacon, J.W., and L.A. Berry. 1992. Modes of action of mycoparasites in relation to biocontrol of soilborne plant pathogens.
Pages 157-167 in E.C. Tjamos, G.C. Papavizas, and R.J. Cook,
eds., Biological Control of Plant Disease; Progress and
Challenges for the Future. Plenum Press, London. 462 pp.
Elad, Y. 1996. Mechanisms involved in the biological control of
Botrytis cinerea incited diseases. Eur. J. Plant Pathol.
102:719-732.
Ellis, M.A., R.H. Converse, R.N. Williams, and B. Williamson
(eds.). 1991. Compendium of Raspberry and Blackberry
Diseases and Insects. APS Press, St Paul, MN. 100 pp.
Emmett, R.W., and D.G. Parbery. 1975. Appressoria. Annu.
Rev. Phytopathol. 13:147-167.
Fourie, J.F., and G. Holz. 1994, Infection of plum and nectarine
flowers by Botrytis cinerea. Plant Pathol. 43:309-315.
Fourie, J.F., and G. Holz. 1995. Initial infection processes by
Botrytis cinerea on nectarine and plum fruit and the development of decay. Phytopathology 85:82-87.
Fravel, D.R. 1988. Role of antibiosis in the biocontrol of plant
disease. Annu. Rev. Phytopathol. 26:75-91.
Garcia-Arenal, F., and E.M. Sagasta. 1980. Scanning electron
microscopy of Botrytis cinerea penetration of bean {Phaseolus
vulgaris) hypocotyls. Phytopathol. Z. 99:37^12.
Griffin, D.H. 1994. Fungal Physiology. 2nd Edition. Wiley Liss, New York. 458 pp.
Lim, J.K., and L.G. Chan. 1986. Parasitism of Phytophthora
palmivora
by Gliodadium
roseum. Z. Pflanzenkr.
Pflanzenschutz 93:509-514.
McLean, M.A., and J.C. Sutton. 1992. Mycoflora of strawberry
in Ontario. Can. J. Bot. 70:846-852.
Mosca, A.M.H., and V.F. Marchisio. 1985. Mycorrhizoplane
fungal populations of Abies alba seedlings. Allionia 27:30-39.
Mueller, J.D., and J. Sinclair. 1986. Occurrence and role of
Gliodadium roseum in field-grown soybeans in Illinois. Trans.
Br. Mycol. Soc. 86:677-680.
Pachenari, A., and N.J. Dix. 1980. Production of toxins and wall
degrading enzymes by Gliodadium roseum. Trans. Br. Mycol.
Soc. 74:561-566.
Peng, G. 1991. Biological control of grey mould {Botrytis
cinerea) on strawberries. Ph.D. Thesis, Univ. of Guelph,
Guelph, Ontario. 220 pp.
Peng, G., and J.C. Sutton. 1991. Evaluation of microorganisms
for biocontrol of Botrytis cinerea in strawberry. Can. J. Plant
Pathol. 13:247-257.
Ricard, J.L., C. Grosclaude, and C. Ale-Agpa. 1974.
Antagonism between Eutypa armeniacae and Gliodadium rose­
um. Plant Dis. Rep. 58:983-984.
Saha, D.C, M.A. Jackson, and J.M. Johnson-Cicalese. 1988. A
rapid staining method for detection of endophytic fungi in turf
and forage grasses. Phytopathology 78:237-239.
Sharman, S., and J.B. Heale. 1977. Penetration of carrot roots
by the gray mould fungus Botrytis cinerea Pers. ex Pers.
Physiol. Plant Pathol. 10:63-71.
Skipp, R.A., and M.J. Christensen. 1990. Selection for persistence in red clover: influence of root disease and stem nematode. N. Z. J. Agric. Res. 33:391-333.
Snedecor, G.W., and W.G. Cochran. 1989. Statistical Methods,
8th ed. The Iowa State University Press. 503 pp.
Sutton J.C. 1994. Biological control of strawberry diseases. Adv.
Strawberry Res. 13:1-12.
Sutton, J.C, and G. Peng. 1993a. Biosuppression of inoculum
production by Botrytis cinerea in strawberry leaves. Pages
47-52 in N.J. Fokkema, J. Köhl, and Y. Elad, eds.. Biological
Control of Foliar and Post-Harvest Diseases. IOBC/WRRS
Bulletin Vol. 16., Montfavet, France. 246 pp.
Sutton, J.C, and G. Peng. 1993b. Biocontrol of Botrytis cinerea
in strawberry leaves. Phytopathology 83:615-621,
Sutton, J.C, D.W. Li, G. Peng, H. Yu, P.G. Zhang, and R.M.
Valdebenito-Sanhueza. 1997. Gliodadium roseum: a versatile adversary of Botrytis cinerea in crops. Plant Dis.
81:316-328.
Tukey, H.B. 1971. Leaching of substances from plants. Pages
67-80 in T.F. Preece, and C. H. Dickinson, eds., Ecology of
Leaf Surface Micro-organisms. Academic Press, London.
640 pp.
Van den Heuvel, J., and L.P. Waterreus. 1983. Conidial
concentration as an important factor determining the type of
penetration structures formed by Botrytis cinerea on leaves
of French bean {Phaseolus
vulgaris).
Plant Pathol.
32:263-272.
Verhoeff, K. 1980. The infection process and host-pathogen
interactions. Pages 153-180 in J.R. Coley-Smith, K. Verhoeff,
and W. R. Jarvis, eds., The Biology of Botrytis. Academic
Press, London. 318 pp.
Walker, J.A., and R.B. Maude. 1975. Natural occurrence and
growth of Gliodadium roseum on the mycelium and sclerotia of
Botrytis allii. Trans. Br. Mycol. Soc. 65:335-337.
Whipps, J.M. 1987. Effect of media on growth and interactions
between a range of soilborne glasshouse pathogens and antagonistic fungi. New Phytol. 107:127-142.
Yu, H. 1996. Relationships of epidemiological factors,
Gliodadium roseum, and bee vectors to grey mold of raspberry
caused by Botrytis cinerea. Ph.D. Thesis, Univ. of Guelph,
Guelph, Ontario. 252 pp.
Yu, H., and J.C. Sutton. 1995. Interactions between the biocontrol
agent Gliodadium roseum and the pathogen Botrytis cinerea on
raspberry. Phytopathology 85:1165. (Abstr.)
Zhang, P.G., J.C Sutton, and A.A. Hopkin. 1996a. Inoculum
concentration and time of application of Gliodadium roseum in
relation to biocontrol of Botrytis cinerea in black spruce
seedlings. Can. J. For. Res. 26:360-367.
Zhang, P.G., J.C. Sutton, W. Tan, and A.A. Hopkin. 1996b.
Gliodadium roseum reduces physiological changes associated
with infection of black spruce seedlings by Botrytis cinerea.
Can. J. Plant Pathol. 18:7-13.