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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Infection of Alfalfa (Medicago sativa L.) Pollen by MycoparasiticFungi Coniothyrium minitans Campbell and Gliocladium

catenulatum Gilmon and AbbottHuang Hung-Chang, Eric G. Kokko, Agriculture and Agri-Food Canada, LethbridgeResearch 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

Hung-Chang, H., Kokko, E.G., and Jenn-Wen, H. 2003.Infection of alfalfa (Medicago sativa L.) pollen bymycoparasitic fungi Coniothyrium minitans Campbell andGliocladium catenulatum Gilmon and Abbott. RevistaMexicana de Fitopatología 21:117-122.Abstract. Studies using scanning and transmission electronmicroscopy revealed that pollen grains of alfalfa (Medicagosativa) are susceptible to infection by mycoparasitic fungi,Coniothyrium minitans and Gliocladium catenulatum. Themode of infection was similar between the two mycoparasites.The infection process involved direct hyphal penetration ofpollen cell walls, most frequently through germ pores. Growthand proliferation of the invading hyphae in the cell lumenresulted in plasmolysis, as well as degradation anddisintegration of pollen cytoplasm. The impact of infectionof alfalfa pollen grains by C. minitans and G. catenulatum ondispersal of these mycoparasites and their potential for controlof blossom blight of alfalfa, caused by Sclerotiniasclerotiorum, is discussed.

Additional keywords: Medicago sativa, blossom blight ofalfalfa, Sclerotinia sclerotiorum.

Resumen. Por medio de estudios con microscopio de barridoy de transmisión electrónica se determinó que los granos depolen de alfalfa (Medicago sativa) son susceptibles a lainfección por los hongos micoparasíticos Coniothyriumminitans y Gliocladium catenulatum. El modo de infecciónentre los dos micoparásitos fue similar. El proceso deinfección involucró la penetración directa de las hifas delhongo en las paredes celulares del polen, por lo general através de los poros del germen. El crecimiento y proliferaciónde las hifas que invadieron el lumen celular causó plasmólisisasí como degradación y desintegración del citoplasma delpolen. Se discute el impacto de la infección de los granos depolen de alfalfa por C. minitans y G. catenulatum, sobre ladispersión de estos micoparásitos y su potencial para controlarel tizón de la flor de la alfalfa causado por Sclerotiniasclerotiorum.

Palabras clave adicionales: Medicago sativa, tizón de la florde alfalfa, Sclerotinia sclerotiorum.

Pollen grains are rich in protein, aminoacids and sugars(Yamakawa, 1984), and are an important source of nutrientsfor insects (Crompton and Wojtas, 1993) and fungi (Huanget al., 1998a). Numerous reports indicate that pollen diffusatesstimulate spore germination and/or hyphal growth of fungi,including Retiarius superficiaris sp. nov. and Retiariusbovicornutus sp. nov. (Olivier, 1978), Verticillium dahliaeKleb. (Ma et al., 2000), Helminthosporium sativum Pamm.,King and Bakke (Fokkema, 1968), Phoma betae Frank(Warren, 1972), Botrytis cinerea Pers. ex Fr. (Chou andPreece, 1968), and Sclerotinia sclerotiorum (Lib.) de Bary(Hartill, 1975). Ultrastructural studies have revealed thatpollen grains are susceptible to infection by plant pathogenicfungi, such as S. sclerotiorum on pollen grains of alfalfa(Medicago sativa L.) (Huang et al., 1997), canola (Brassicanapus L.) (Huang et al., 1998b), and pea (Pisum sativum L.)(Huang and Kokko, 1993); V. dahliae on pollen grains ofcotton (Gossypium hirsutum L.) (Ma et al., 2000) andVerticillium albo-atrum Reinke and Berth. (Huang andKokko, 1985), and B. cinerea (Huang et al., 1999) on pollengrains of alfalfa. Huang et al. (1998a) suggested that pollenmay play a significant role in the dissemination of fungalpathogens. Coniothyrium minitans Campbell (Campbell,1947) and Gliocladium catenulatum Gilmon and Abbott(Huang, 1978) are mycoparasitic fungi which attack S.sclerotiorum, an important pathogen causing seriouseconomic losses of numerous crops (Purdy, 1979; Huang,2000). Application of C. minitans was effective in reducingsclerotinia wilt of sunflower (Helianthus annuus L.) (Huang,1980; McLaren, et al., 1994), drop or wilt of lettuce (Lactucasativa L.) (Budge and Whipps, 1991), and white mold ofbean (Phaseolus vulgaris L.) (Huang et al., 2000b) causedby S. sclerotiorum. A commercial product Contans WG® basedon C. minitans has been registered for use for biocontrol ofsclerotinia diseases of crops in Germany and other countries,including Austria, France, and the USA (Luth, 2001). The

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

Revista Mexicana de FITOPATOLOGIA/ 117

objective of this ultrastructural study was to determine themode of infection of alfalfa pollen by the mycoparasitic fungi,C. minitans and G. catenulatum.

MATERIALS AND METHODSInoculation 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, alfalfaflowers were removed from racemes and each floret wastripped open by touching the keel petal gently using a woodentoothpick. Fresh pollen grains were collected and mixed withsuspensions of pycnidiospores of C. minitans or conidia ofG. catenulatum. The isolates used for the study were C.minitans DAOM 149432 and G. catenulatum DAOM 149586,both originating from infected sclerotia of S. sclerotiorumfrom sunflower fields near Morden, Manitoba, Canada. Sporesuspensions of mycoparasites were prepared by adding steriledistilled water to 21-day-old cultures on potato-dextrose-agar,and adjusting the concentration to 103 spores/ml. Foruninoculated controls, fresh pollen grains were mixed withsterile distilled water without fungal spores. The suspensions/mixtures, pollen mixed with C. minitans, pollen mixed withG. catenulatum or pollen alone, were individually transferredusing 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, andeach dish was sealed with Parafilm™. After incubation at

20°C for 5 days under continuous fluorescent light, sampleson cover slips were processed for SEM. Samples fortransmission electron microscopy (TEM) were prepared bythe same culture method described for the materials used inSEM, except that, instead of using cover slips, thesuspensions/mixtures, pollen mixed with a mycoparasite orpollen alone, were placed on sterile cellophane strips (6.0H1.5cm, L x W), 0.2 ml per strip.Scanning Electron Microscopy (SEM). For SEM, the coverslips containing 5-day-old samples, pollen and C. minitansor pollen and G. catenulatum, or pollen alone (control), wereimmersed in 2% glutaraldehyde fixative in 0.05 M sodiumphosphate buffer, pH 7.0, at 4°C overnight (16 h) and thenbrought to room temperature. Samples were washed 3 times,10 min each, with the sodium phosphate buffer solution, andthen dehydrated in a graded ethanol series and critical pointdried (Polaron E3100) with liquid carbon dioxide as thetransitional fluid. The material was adhered onto aluminumspecimen mounts with colloidal silver paste, air-driedovernight and sputter-coated (Denton Vacuum Desk-1) withgold (approximately 15 nm thickness). The specimens wereexamined and photographed on a Hitachi S-570 SEM.Transmission Electron Microscopy (TEM). For TEM, the5-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 0.05Msodium 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.

Figs. 1-2. Electron micrographs of healthy alfalfa pollen. 1) A scanning electron micrograph showing a pollen grainwith 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 theother region. x 7,000.

118 / Volumen 21, Número 2, 2003

example, S. sclerotiorum is an important pathogen of blossomblight 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). WhetherC. minitans-infected pollen could enhance biocontrol ofblossom blight of alfalfa warrants further investigation.Several pollinating insects, including alfalfa leafcutter bees(Megachile rotundata (Fabricius)) (Huang et al., 1986) andhoneybees (Apis mellifera L.) (Stelfox et al., 1978), werereported to spread pathogen-infected and/or pathogen-contaminated pollen grains. Huang et al. (1986) reported thatleafcutter bees carried V. albo-atrum-infected pollen grainsof alfalfa by foraging in an alfalfa field with high incidenceof verticillium wilt. Stelfox et al. (1978) reported thathoneybees spread S. sclerotiorum-contaminated pollen grainsresulting in the development of head blight of rapeseed(Brassica spp.). Alfalfa is a cross-pollinated crop that reliesupon the use of leafcutter bees for commercial production ofalfalfa seeds (Goplen et al., 1980). Since alfalfa pollen grainsare 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 enhancingbiocontrol 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 ResearchCentre, Lethbridge, Alberta, Canada for the Spanishtranslation of the abstract. The authors would also like tothank B. Lee and R. S. Erickson for their technical assistance.

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