07060669709500518

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Morphological development and
interactions of Gliocladium roseum and
Botrytis cinerea in raspberry
Hai Yu a & 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
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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
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 rasp-
berry 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 super-
ficial hyphae with short side branches that penetrated the host. No symptoms developed, but numerous conidiophores and coni-
dia 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 sup-
pressed 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 rasp-
berry 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 coni-
dies 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 coni-
dies 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'infec-
tion 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 ger-
minatifs 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'infec-
tion 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 economie importance, and is considered to have major
of Botrytis cinerea Pers.: Fr. in at least 15 hosts of potential for biocontrol of the pathogen in cropping
237
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238 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 inter-
actions between the antagonist and B. cinerea in host
tissues is sparse, yet fundamental for developing ratio-
nal 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 straw-
berry 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 antago-
nism 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 sup-
plied with a soluble N:P:K (20:8:20) fertilizer (5 g/L
water) once a week. Raspberry shoots, each with sev-
eral leaves and an inflorescence with 4-5 freshly-
opened 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), fil-
tered 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 tis-
sues were incubated in the laboratory at 21-23°C.
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YU, SUTTON: RASPBERRY/GLIOCLADIUM/BOTRYTIS 239
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 sta-
men clusters were mounted in lactophenol containing
0.05% trypan blue on microscope slides, gently heat-
ed 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 distin-
guishable. Incidence of stamens in which hyphae of
B. cinerea were found was estimated from observa-
tions 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 epi-
dermis, and stamen cluster. Morphological develop-
ment 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 inocula-
tion, 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 conid-
iophores of B. cinerea.
Data analysis. Statistical computations were per-
formed using the Statistical Analysis System (SAS
Institute Inc., Cary, NC). Data on the incidence of
conidial germination and germ tube length of B.
cinerea and G. roseum, of B. cinerea hyphae in sta-
mens, 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 develop-
ment of the branches was not investigated. At 24 and
32 h after inoculation, numerous hyphae of the antag-
onist 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 abun-
dant 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 appressori-
um-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 appres-
soria were present on most germ tubes, and single
and multilobed appressoria were present on superfi-
cial hyphae at 32-40 h after inoculation. Hyphae
were frequent in the superficial stem tissues at 72 h
after inoculation (Fig. 2A). On stamens, appres-
sorium-like swellings and single-lobed appressoria
developed on about 50% of germ tubes, and an infec-
tion 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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240 CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997
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