Revista Mexicana de Fitopatología

ISSN: 0185-3309
mrlegarreta@prodigy.net.mx
Sociedad Mexicana de Fitopatología, A.C.
México

Hung Chang, H.; Kokko, Eric G.; Jenn Wen, H.

Infection of Alfalfa (Medicago sativa L.) Pollen by Mycoparasitic Fungi Coniothyrium minitans Campbell
and Gliocladium catenulatum Gilmon and Abbott
Revista Mexicana de Fitopatología, vol. 21, núm. 2, julio-diciembre, 2003, pp. 117-122
Sociedad Mexicana de Fitopatología, A.C.
Texcoco, México

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 Revista Mexicana de FITOPATOLOGIA/ 117

Infection of Alfalfa (Medicago sativa L.) Pollen by Mycoparasitic

Fungi Coniothyrium minitans Campbell and Gliocladium
catenulatum Gilmon and Abbott
Huang Hung-Chang, Eric G. Kokko, Agriculture and Agri-Food Canada, Lethbridge
Research Centre, P.O. Box 3000, Lethbridge, AB T1J 4B1, Canada; and Huang Jenn-
Wen, Department of Plant Pathology, National Chung-Hsing University, Taichung, Taiwan.
LRC Contribution No. 387-02069. Correspondence to: huangh@agr.gc.ca

(Received: December 5, 2002 Accepted: January 27, 2003)

Hung-Chang, H., Kokko, E.G., and Jenn-Wen, H. 2003.
Infection of alfalfa (Medicago sativa L.) pollen by
mycoparasitic fungi Coniothyrium minitans Campbell and
Gliocladium catenulatum Gilmon and Abbott. Revista
Mexicana de Fitopatología 21:117-122.
Abstract. Studies using scanning and transmission electron
microscopy revealed that pollen grains of alfalfa (Medicago
sativa) are susceptible to infection by mycoparasitic fungi,
Coniothyrium minitans and Gliocladium catenulatum. The
mode of infection was similar between the two mycoparasites.
The infection process involved direct hyphal penetration of
pollen cell walls, most frequently through germ pores. Growth
and proliferation of the invading hyphae in the cell lumen
resulted in plasmolysis, as well as degradation and
disintegration of pollen cytoplasm. The impact of infection
of alfalfa pollen grains by C. minitans and G. catenulatum on
dispersal of these mycoparasites and their potential for control
of blossom blight of alfalfa, caused by Sclerotinia
sclerotiorum, is discussed.
Additional keywords: Medicago sativa, blossom blight of
alfalfa, Sclerotinia sclerotiorum.
Resumen. Por medio de estudios con microscopio de barrido
y de transmisión electrónica se determinó que los granos de
polen de alfalfa (Medicago sativa) son susceptibles a la
infección por los hongos micoparasíticos Coniothyrium
minitans y Gliocladium catenulatum. El modo de infección
entre los dos micoparásitos fue similar. El proceso de
infección involucró la penetración directa de las hifas del
hongo en las paredes celulares del polen, por lo general a
través de los poros del germen. El crecimiento y proliferación
de las hifas que invadieron el lumen celular causó plasmólisis
así como degradación y desintegración del citoplasma del
polen. Se discute el impacto de la infección de los granos de
polen de alfalfa por C. minitans y G. catenulatum, sobre la
dispersión de estos micoparásitos y su potencial para controlar
el tizón de la flor de la alfalfa causado por Sclerotinia
sclerotiorum.
Palabras clave adicionales: Medicago sativa, tizón de la flor
de alfalfa, Sclerotinia sclerotiorum.
Pollen grains are rich in protein, aminoacids and sugars
(Yamakawa, 1984), and are an important source of nutrients
for insects (Crompton and Wojtas, 1993) and fungi (Huang
et al., 1998a). Numerous reports indicate that pollen diffusates
stimulate spore germination and/or hyphal growth of fungi,
including Retiarius superficiaris sp. nov. and Retiarius
bovicornutus sp. nov. (Olivier, 1978), Verticillium dahliae
Kleb. (Ma et al., 2000), Helminthosporium sativum Pamm.,
King and Bakke (Fokkema, 1968), Phoma betae Frank
(Warren, 1972), Botrytis cinerea Pers. ex Fr. (Chou and
Preece, 1968), and Sclerotinia sclerotiorum (Lib.) de Bary
(Hartill, 1975). Ultrastructural studies have revealed that
pollen grains are susceptible to infection by plant pathogenic
fungi, such as S. sclerotiorum on pollen grains of alfalfa
(Medicago sativa L.) (Huang et al., 1997), canola (Brassica
napus L.) (Huang et al., 1998b), and pea (Pisum sativum L.)
(Huang and Kokko, 1993); V. dahliae on pollen grains of
cotton (Gossypium hirsutum L.) (Ma et al., 2000) and
Verticillium albo-atrum Reinke and Berth. (Huang and
Kokko, 1985), and B. cinerea (Huang et al., 1999) on pollen
grains of alfalfa. Huang et al. (1998a) suggested that pollen
may play a significant role in the dissemination of fungal
pathogens. Coniothyrium minitans Campbell (Campbell,
1947) and Gliocladium catenulatum Gilmon and Abbott
(Huang, 1978) are mycoparasitic fungi which attack S.
sclerotiorum, an important pathogen causing serious
economic losses of numerous crops (Purdy, 1979; Huang,
2000). Application of C. minitans was effective in reducing
sclerotinia wilt of sunflower (Helianthus annuus L.) (Huang,
1980; McLaren, et al., 1994), drop or wilt of lettuce (Lactuca
sativa L.) (Budge and Whipps, 1991), and white mold of
bean (Phaseolus vulgaris L.) (Huang et al., 2000b) caused
by S. sclerotiorum. A commercial product Contans WG® based
on C. minitans has been registered for use for biocontrol of
sclerotinia diseases of crops in Germany and other countries,
including Austria, France, and the USA (Luth, 2001). The
118 / Volumen 21, Número 2, 2003
objective of this ultrastructural study was to determine the
mode of infection of alfalfa pollen by the mycoparasitic fungi,
C. minitans and G. catenulatum.
MATERIALS AND METHODS
Inoculation of alfalfa pollen with mycoparasitic fungi.
Alfalfa plants, cultivar Barrier, were grown in Cornell peat-
lite mix (Boodley and Sheldrake, 1977) in plastic pots (15-
cm diam.) in a greenhouse. During the blossom period, alfalfa
flowers were removed from racemes and each floret was
tripped open by touching the keel petal gently using a wooden
toothpick. Fresh pollen grains were collected and mixed with
suspensions of pycnidiospores of C. minitans or conidia of
G. catenulatum. The isolates used for the study were C.
minitans DAOM 149432 and G. catenulatum DAOM 149586,
both originating from infected sclerotia of S. sclerotiorum
from sunflower fields near Morden, Manitoba, Canada. Spore
suspensions of mycoparasites were prepared by adding sterile
distilled water to 21-day-old cultures on potato-dextrose-agar,
and adjusting the concentration to 10 3 spores/ml. For
uninoculated controls, fresh pollen grains were mixed with
sterile distilled water without fungal spores. The suspensions/
mixtures, pollen mixed with C. minitans, pollen mixed with
G. catenulatum or pollen alone, were individually transferred
using a sterile pipette onto sterile cover slips (10 mm diam.),
0.1 ml per cover slip. For scanning electron microscopy
(SEM), samples on cover slips were placed on glass slides,
kept on moist paper in Petri dishes, one slide per dish, and
each dish was sealed with Parafilm™. After incubation at
Figs. 1-2. Electron micrographs of healthy alfalfa pollen. 1) A scanning electron micrograph showing a pollen grain
with three germ pores (GP). x 2,000. 2) A transmission electron micrograph of a pollen grain with a dense cytoplasm
(CYT), a thin wall in the germ pore region (GP), and the thick wall containing intine (IN) and exine (EX) layers in the
other region. x 7,000.
20°C for 5 days under continuous fluorescent light, samples
on cover slips were processed for SEM. Samples for
transmission electron microscopy (TEM) were prepared by
the same culture method described for the materials used in
SEM, except that, instead of using cover slips, the
suspensions/mixtures, pollen mixed with a mycoparasite or
pollen alone, were placed on sterile cellophane strips (6.0H1.5
cm, L x W), 0.2 ml per strip.
Scanning Electron Microscopy (SEM). For SEM, the cover
slips containing 5-day-old samples, pollen and C. minitans
or pollen and G. catenulatum, or pollen alone (control), were
immersed in 2% glutaraldehyde fixative in 0.05 M sodium
phosphate buffer, pH 7.0, at 4°C overnight (16 h) and then
brought to room temperature. Samples were washed 3 times,
10 min each, with the sodium phosphate buffer solution, and
then dehydrated in a graded ethanol series and critical point
dried (Polaron E3100) with liquid carbon dioxide as the
transitional fluid. The material was adhered onto aluminum
specimen mounts with colloidal silver paste, air-dried
overnight and sputter-coated (Denton Vacuum Desk-1) with
gold (approximately 15 nm thickness). The specimens were
examined and photographed on a Hitachi S-570 SEM.
Transmission Electron Microscopy (TEM). For TEM, the
5-day-old samples on cellophane strips, pollen and C.
minitans or pollen and G. catenulatum, or pollen alone
(control), were immersed in 2% glutaraldehyde in 0.05M
sodium cacodylate buffer, pH 7.0, at 4°C, overnight (16 h)
and then brought to room temperature. Samples were washed
(3 x 10 min) with the same sodium cacodylate buffer solution.
 Revista Mexicana de FITOPATOLOGIA/
example, S. sclerotiorum is an important pathogen of blossom
blight of alfalfa (Holley et al., 1995; Huang et al., 2000a)
and C. minitans is an important biocontrol agent of S.
sclerotiorum (Huang, 1980; Huang et al., 2000b). Whether
C. minitans-infected pollen could enhance biocontrol of
blossom blight of alfalfa warrants further investigation.
Several pollinating insects, including alfalfa leafcutter bees
(Megachile rotundata (Fabricius)) (Huang et al., 1986) and
honeybees (Apis mellifera L.) (Stelfox et al., 1978), were
reported to spread pathogen-infected and/or pathogen-
contaminated pollen grains. Huang et al. (1986) reported that
leafcutter bees carried V. albo-atrum-infected pollen grains
of alfalfa by foraging in an alfalfa field with high incidence
of verticillium wilt. Stelfox et al. (1978) reported that
honeybees spread S. sclerotiorum-contaminated pollen grains
resulting in the development of head blight of rapeseed
(Brassica spp.). Alfalfa is a cross-pollinated crop that relies
upon the use of leafcutter bees for commercial production of
alfalfa seeds (Goplen et al., 1980). Since alfalfa pollen grains
are susceptible to infection by C. minitans and G. catenulatum,
using leafcutter bees to deliver and spread mycoparasite-
infected pollen may be an effective strategy for enhancing
biocontrol of blossom blight of alfalfa caused by S.
sclerotiorum (Holley et al., 1995; Huang et al., 2000a).
Acknowledgements. The authors thank Dr. Héctor Cárcamo,
Agriculture and Agri-Food Canada, Lethbridge Research
Centre, Lethbridge, Alberta, Canada for the Spanish
translation of the abstract. The authors would also like to
thank B. Lee and R. S. Erickson for their technical assistance.
LITERATURE CITED
Bekesi, P. 1982. New inoculation method for infecting
sunflowers by Botrytis cinerea Pers. Acta Phytopathologica
Academiae Scientiarum Hungaricae 17:221-224.
Boodley, J.W., and Sheldrake, R., Jr. 1977. Cornell peat-lite
mixes for commercial plant growing. New York State
College of Agriculture and Life Science, Information
Bulletin 43:8.
Budge, S.P., and Whipps, J.M. 1991. Glasshouse trials of
Coniothyrium minitans and Trichoderma species for the
biological control of Sclerotinia sclerotiorum in celery and
lettuce. Plant Patholology 40:59-66.
Campbell, W.A. 1947. A new species of Coniothyrium
parasitic on sclerotia. Mycologia 39:190-195.
Chou, M.C., and Preece, T.F. 1968. The effect of pollen grains
on infections caused by Botrytis cinerea Pers. ex Fr. Annals
of Applied Biology 62:11-22.
Crompton, C.W., and Wojtas, W.A. 1993. Pollen grains of
Canadian honey plants. Agriculture and Agri-Food Canada
Publication 1892/E. 228 p.
Dillard, H.R., and Hunter, J.E. 1986. Association of common
ragweed with Sclerotinia rot of cabbage in New York State.
Plant Disease 70:26-28.
Fokkema, N.J. 1968. The influence of pollen in the
121
phyllosphere of rye on colonization by saprophytic fungi
and on infection by Helminthosporium sativum and other
leaf pathogens. Diss. Amsterdam. Meded. Phytopath. Lab.
Willie Commelin Scholten 87, 60 pp. Netherlands Journal
of Plant Pathology 77(1971): Suppl. 1.
Goplen, B.P., Baenzier, H., Bailey, L.D., Gross, A.T.H.,
Hanna, M.R., Michaud, R., Richards, K.W., and
Waddington, J. 1980. Growing and managing alfalfa in
Canada. Agriculture Canada. Publication No. 1705.
Ottawa, Canada. 49 p.
Hartill, W.F.T. 1975. Germination of Botrytis and Sclerotinia
spores in the presence of pollen on tobacco leaves. New
Zealand Journal of Agricultural Research 18:405-407.
Hartill, W.F.T., and Campbell J.M. 1974. Effects of flower
removal on the development of the of the Sclerotinia/
Botrytis complex of tobacco. New Zealand Journal of
Agricultural Research 17:147-152.
Holley, J., Linowski, R., Gossen, B., and Harrison, L. 1995.
Sclerotinia sclerotiorum causes blossom blight of alfalfa.
Proceedings of Annual Meeting of the Plant Pathology
Society of Alberta. November 6-8, 1995, Lethbridge,
Alberta.
Huang, H.C. 1978. Gliocladium catenulatum: hyperparasite
of Sclerotinia sclerotiorum and Fusarium species.
Canadian Journal of Botany 56:2243-2246.
Huang, H.C. 1980. Control of sclerotinia wilt of sunflower
by hyperparasites. Canadian Journal of Plant Pathology
2:26-32.
Huang, H.C. 2000. Economic Impact of Sclerotinia Diseases.
In: Economic Impact Module, Global Crop Protection
Compendium. CAB (Centre of Agricultural and
Biosciences) International, UK. 33 p. (in CD-Rom).
Huang, H.C., Acharya, S.N., and Erickson, R.S. 2000a.
Etiology of blossom blight of alfalfa. Plant Pathology
Bulletin 9:11-16.
Huang, H.C., Bremer, E., Hynes, R.K., and Erickson, R.S.
2000b. Foliar application of fungal biocontrol agents for
the control of white mold of dry bean caused by Sclerotinia
sclerotiorum. Biological Control 18:270-276.
Huang, H.C., and Kokko, E.G. 1985. Infection of alfalfa
pollen by Verticillium albo-atrum. Phytopathology
75:859-865.
Huang, H.C., and Kokko, E.G. 1993. Infection of pea pollen
by Sclerotinia sclerotiorum, Trends in Agricultural
Sciences. Plant Pathology 1:13-17.
Huang, H.C., Kokko, E.G., and Erickson, R.S. 1997. Infection
of alfalfa pollen by Sclerotinia sclerotiorum.
Phytoparasitica 25:17-24.
Huang, H.C., Kokko, E.G., and Erickson, R.S. 1999. Infection
of alfalfa pollen by Botrytis cinerea. Botanical Bulletin
Academia Sinica 40:101-106.
Huang, H.C., Kokko, E.G., Erickson, R.S., and Hynes, R.K.
1998b. Infection of canola pollen by Sclerotinia
sclerotiorum. Plant Pathology Bulletin 7:71-77.
Huang, H.C., Kokko, E.G., and Huang, J.W. 1998a.
122 / Volumen 21, Número 2, 2003
Epidemiological significance of pollen in fungal diseases.
Recent Research Developments in Plant Pathology 2:91-
109. Research Signpost.
Huang, H.C., Richards, K.W., and Kokko, E.G. 1986. Role
of the leafcutter bee in dissemination of Verticillium
albo-atrum in alfalfa. Phytopathology 76:75-80.
Iwanami, Y., Sasakuma, T., and Yamada, Y. 1988. Pollen:
Illustrations and scanning electronmicrographs. Kodansha
Scientific Books, Tokyo and Springer-Verlag, Berlin. 198
p.
Knox, R.B., and Heslop-Harrison, J. 1970. Pollen-wall
proteins: localization and enzymic activity. Journal of Cell
Science 6:1-15.
Kokko, E.G., Gowen, B., and Jahnke, C.R. 1990. An
inexpensive water volume control device for
ultramicrotomy. In: Electron Microscopy 1990:
Proceedings of the 12th International Congress for Electron
Microscopy. Seattle, Washington, 12-18 August, 1990. San
Francisco Press, San Francisco, CA, USA. pp. 732-733.
Luth, P. 2001. The biological fungicide Contans WG® - A
reparation on the basis of the fungus Coniothyrium
minitans. In: C.S. Young and K.J.D. Hughes (eds.).
Proceedings of Sclerotinia 2001 - The XI International
Sclerotinia Workshop. pp. 127-128. York, 8th -12th July
2001, York, England. Central Science Laboratory, York,
England.
Ma, P., Huang, H.C., Kokko, E.G., and Tang, W.H. 2000.
Infection of cotton pollen by Verticillium dahliae. Plant
Pathology Bulletin 9:93-98.
McLaren, D.L., Huang, H.C., Kozub, G.C., and Rimmer, S.R.
1994. Biological control of Sclerotinia wilt of sunflower
with Talaromyces flavus and Coniothyrium minitans. Plant
Disease 78:231-235.
Ogawa, J.M., and English, H. 1960. Blossom blight and green
fruit rot of almond, apricot and plum caused by Botrytis
cinerea. Plant Disease Reporter 44:265-268.
Olivier, D.L. 1978. Retiarius gen. nov.: Phyllosphere fungi
which capture wind-borne pollen grains. Transactions of
the British Mycological Society 71:193-201.
Purdy, L.H. 1979. Sclerotinia sclerotiorum: History, diseases
and symptomatology, host range, geographic distribution
and impact. Phytopathology 69:875-880.
Spurr, A.R. 1969. A low-viscosity epoxy embedding medium
for electron microscopy. Journal of Ultrastructure Research
26:31-43.
Stelfox, D., Williams, J.R., Soehngen, U., and Topping, R.C.
1978. Transport of Sclerotinia sclerotiorum ascospores by
rapeseed pollen in Alberta. Plant Disease Reporter 62:576-
579.
Warren, R.C. 1972. The effect of pollen on the fungal leaf
microflora of Beta vulgaris L. and on infection of leaves
by Phoma betae. Netherlands Journal of Plant Pathology
78:89-98.
Yamakawa, T. 1984. The effect of pollen on the infection of
fruit vegetables with conidia of Botrytis cinerea.
Proceedings of the Kansas Plant Protection Society 26:1-
8.