APPLIZD AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1976, p. 60-62 Copyright X 1976 American Society for Microbiology

Further Studies

on a

Vol. 31, No. 1 Printed in U.S.A.

Mycoparasitic Basidiomnycete Species1

R. F. CERRATO,2 R. E. DE LA CRUZ,3 AND D. H. HUBBELL* Department of Soil Science, University of Florida, Gainesville, Florida 32611

Received for publication 3 June 1975

An unidentified basidiomycete was found capable of parasitizing 37 of 50 isolates of fungi tested as hosts. All phytopathogenic fungi tested, as well as most of the saprophytic fungi, were susceptible to this mycoparasite. In some cases, reproductive structures as well as hyphae were infected. High glucose-yeast extract ratios in the test medium favored parasitism. The mycoparasite was able to utilize 14 of 27 compounds tested as carbon sources. observed microscopically in lactophenol without staining, thereby allowing an extended period of observations. Nutritional conditions. The influence of relative concentrations of glucose and yeast extract on the degree of parasitism by the mycoparasite was studied by incorporating varying amounts of these components in the modified Melin-Norkrans medium of Marx (8) minus carbon and nitrogen sources. Glucoseyeast extract ratios (grams/liter) tested were 10:5, 10:1, 5:10, 1:10, and 1:1. Twenty-five milliliters of each medium was placed in a petri dish and inoculated using agar plugs to test antagonistic effects of the mycoparasite against M. phaseolina, RhiMATERIALS AND METHODS solani Kuhn, Fusarium oxysporum Schlect, Host range studies. Potato-dextrose agar (PDA; zoctonia Rhizopus nigricans Ehrenberg, and Trichoderma Difco) was used to grow the various fungal hosts Rifai. Microcultures were also prepared (Table 1) and the mycoparasite. Tests for fungal harzianum were incubated at 28 C for 6 days. At the end of susceptibility to mycoparasitism were conducted in and incubation period macroscopic and microscopic the following manner. Agar plugs (1 cm in diameter) the observations were made. of the actively growing test fungus and the mycoCarbon sources. Utilization of different carbon parasite were placed 3 cm apart in a petri dish sources the mycoparasite was studied using the containing 25 ml of potato-dextrose agar. In all cases, method by Wickerham (11). The test medium conthe mycoparasite was incubated 4 to 5 days prior to sisted ofof yeast nitrogen base (Difco) plus the carbon inoculation of the host because of its lower growth rate; the plates were then incubated for an additional source to be tested. All sugars were sterilized separately by filtration (Millipore) except the polysac4 to 5 days at 28 C. Observations were made daily as the two colonies charides, which were autoclaved at 121 C and 15 pressure for 15 min. All media were inocuapproached each other, and antagonism was rated lb/in2 by loop with the aerial mycelia of the fungus visually after observation of fresh preparations by lated which had been incubated on potato-dextrose agar phase contrast microscopy at a magnification of 400 x. for 5 days at 28 C. Observations were made every 4 Mode of parasitism. Microcultures were prepared using the method of Riddell (9). The host and the days. Additionally, humic acid was tested at the parasite were inoculated simultaneously on opposite following concentrations: 1, 0.5, 0.25, 0.12, 0.06, and sides of the agar blocks. The slide cultures were then 0.03%. The pH of the media was adjusted to 5.5 to placed on top of spacers of glass tubing in separate 6.0 with NaOH prior to sterilization. sterile petri dishes. Eight milliliters of sterile 10% glycerol solution was added to each petri dish to RESULTS provide moisture to the system. After incubating the Host range studies. Thirty-seven of fifty 4 cultures for to 5 days at 28 C, the organisms were

The use of an unidentified basidiomycete mycoparasite for biological control of "dampingoff' in slash pine (Pinus elliottii Engelm var. elliottii) seedlings caused by Macrophomina phaseolina (Tassi) Goid. was studied by de la Cruz and Hubbell (4) under laboratory and field conditions. The objectives of this study were to investigate the host range, mode of parasitism, influence of nutritional conditions on the degree of parasitism, and the utilization of different carbon sources by the mycoparasite.

strains of fungi tested were susceptible to mycoparasitism (Table 1). Mycoparasitism was identified by coiling, invasion, and subsequent lysis of the host in areas of contact with the mycoparasite. Thirteen species showed resistance to the parasite (rating of 0), although the latter had the capacity to grow over these

Florida Agricultural Experiment Stations Journal Series no. 5939. 2 Present address: Centro de Investigacion del I.P.N., Departamento de Bioingenieria y Biotecnologia, Apartado I

Postal 14-740, Mexico 14, D. F. 3Present address: University of the Philippines at Los Banos, College of Forestry, Laguna, Philippines.

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MYCOPARASITIC BASIDIOMYCETE

VOL. 31, 1976

TABLE 1. Susceptibility of various fungi to mycoparasitism by an unidentified basidiomycete species ungus Test fungus (isolate no.)

ource Sourcea

Host ~~~reactionb

TYPec

Test fungus (isolate no.)

Sourcea

Absidia sp. Arachniotus ruber Arthrobotrys oligospora Aspergillus fumigatus Aspergillus niger (604) Aspergillus flavus Alternaria brassicicola Alternaria longipes Alternaria solani Aspergillus niger (Li) Aspergillus niger (FD) Blakeslea trispora Botryotinia camelliae Cochliobolus unicatus Curvularia oryzae Ceratocystis sp. Cylindrocladium floridanum Cephalosporium cinnamomeum Fusarium oxysporum (159) Fusarium oxysporum (175) Fusarium oxysporum (593) Fusarium solani Fusarium sp. Geotrichum candidum Helminthosporium sp.

JWK JWK JWK JWK DPI JWK DPI DPI DPI RFC FD JWK JWK JWK JWK

4 1 0 1 2 0 2 3 3 2 2 1 1

S S S S S P P P S S P

DPI DPI RG

2 4 4

P P P

JWK JWK

JWK DPI DPI

1 1 2 0 0

S P P

Helminthosporium maydis Macrophomina phaseolina Macrophomina phaseolina (10) Matarrhizium sp. Mucor racemosus Monilia sitophila Pestalotia sp. Penicillium javanicum Penicillium chrysogenum Pythium ultimum Pythium myriotylum Phoma sp. Pisolithus tinctorius Rhizoctonia solani (10) Rhizoctonia solani Rhizopus nigricans (135) Rhizopus nigricans (FD4)

S S S P S S P P P M P P

DPI DPI DPI DPI RG FD DPI

2 1 1 1 1 1 1

P P P P P S P

0 3 0 2 0 0 2 3 0 0 3 3 4 4 0 0 1 2 2 2 2 0

b

Saccharomyces cerevisiae Schizophyllum commune Stemphylium floridanum Sordaria fimicola Thamnidium elegans Trichoderma harzianum Trichoderma viride

Thelephora terrestris

JWK JWK JWK FD RG RG DPI DM RG RG JWK FD JWK JWK JWK JWK JWK DPI DPI DM

reactionb Host b |TYPe

S,P S,P P P P P S S S M

a FD = F. Dazzo, Department of Microbiology, University of Florida; JWK = J. W. Kimbrough, Department of Botany, University of Florida; RG = R. Garcia, Department of Plant Pathology, University of Florida; DM = D. Marx, U.S. Department of Agriculture, Forest Service, Athens, Ga.; DPI = U.S. Department of Agriculture, Division of Plant Industry, Gainesville, Fla.; RFC = R. Cerrato, Instituto Politecnico Nacional, Mexico. bInfection rated visually as extensive = 4 to none = 0. c S, Saprophytic; P, phytopathogenic; M, ectomycorrhizal.

colonies. All of the plant-pathogenic fungi tested were susceptible hosts. The degree of susceptibility ranged from slightly susceptible (rating of 1) to highly susceptible (rating of 4). Among the saprophytes, the most susceptible were the phycomycetes, R. nigricans, Absidia sp., Mucor racemosus Fres., and Thamnidium elegans Link. Mode of parasitism. The mode of parasitism varied. In the case of Trichoderma viride Per. and T. harzianum the parasite grew beside the host and coiled around it. Complete invasion often occurred as in M. racemosus and R. nigricans. In some instances, macroconidia of Fusarium sp., arthrospores of Geotrichum candidum Link, ascospores of Arachniotus ruber (Van Tieg.) Schrot., conidia of Alternaria brassicicola Wiltshire and Helminthosporium maydis Nisikado et Miyake, and sporangia, sporangiophores, and sporangiospores of Absidia sp. and R. nigricans were invaded. Resistance of the ectomycorrhizal fungi Pisolithus tinctorius Pers. and Thelephora terrestris Ehrh. to the mycoparasite was confirmed (4). Nutritional conditions. The combinations of 10 g of glucose plus 5 g of yeast extract and 10

g of glucose plus 1 g of yeast extract stimulated the parasitism (Table 2). The parasite colony grew over the host colony, and coiling plus invasion was observed under the microscope. When the glucose-yeast extract ratios were changed to 5:10 and 1:10 the host grew over the mycoparasite colony. Carbon sources. The compounds tested as possible carbon sources for the mycoparasite are shown in Table 3. The mycoparasite was able to utilize 14 of 27 carbon sources tested. DISCUSSION The mycoparasite was able to attack both plant-pathogenic fungi and saprophytic fungi. It is probable that the host range is still much wider than observed in these experiments. The mode of parasitism is similar to that previously reported (1-3, 5, 6). These authors observed coiling and growth of the mycoparasite inside the host as well as an affinity between the parasite and spores of certain fungi. Polyporus versicolor (L.) Fr. was frequently observed to destroy spores of Ceratocystis fimbriata All. and Halst. In this study, the mycoparasite attacked the macroconidia of Fusarium sp., ascospores of A. ruber, arthrospores of G. candidum, conidia

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CERRATO, DE LA CRUZ, AND HUBBELL

TABLE 2. Degree of parasitism of an unidentified basidiomycete species on different fungi grown on agar media containing varying concentrations of glucose and yeast extract Host reactiona

Fungi 10:5b 10:1 5:10 1:10 1:1

Macrophomina phaseolina Rhizoctonia solani Rhizopus nigricans Fusarium oxysporum Trichoderma harzianum a

la 2 1 0 0

3 3 3 0 1

0 0 0 0 0

Infection rated visually as extensive

0 0 0 0 0 =

0 0 1 0 0

Glucose-yeast extract ratio (grams/liter).

TABLE 3. Utilization of different carbon sources by an unidentified mycoparasitic basidiomycete species Sub-

SubCarbon source

strate

utiliza-

Carbon source

tiona

n-Glucose Galactose L-Fucose Mannitol Fructose Dulcitol D-(+)-Xylose D-(-)-Xylose L-( + )-Arabinose D(-)-Ribose L-(+)-Rhamnose Trehalose Sucrose Cellobiose Lactose Melibiose

strate

utilizationa

4 1 0 1 3 0 2 0 0 0 0 2 0

Raffinose D-( + )-Glucosamine N-acetylglucosamine Cellulose (powder) Cellulose (paper) Dextrin (bacteriological) Starch (potato) Amylopectin Polypectate, sodium Phytate, sodium Humic acid

0 0 0 2 3 2 2 2 2 2 0 0

3

Control (minus carbon source)

0

1 2

a Growth ranked visually as extensive

=

and parasitism was very low or not observed when the ratio was 1:10. The mycoparasite grew on 14 different carbon sources. Polysaccharides such as cellulose, starch, and dextrins were utilized. In view of this versatility it is possible that large amounts of inoculum for field studies could be obtained using crude, inexpensive carbon sources such as starch or molasses. The presence of extracellular enzymes such as cellulase, dextrinase, and amylase in the mycoparasite may be related to their parasitism of other fungi in the soil (7, 10).

4 to none

= 0. b

APPL. ENVIRON. MICROBIOL.

4 to none

=

0.

ofA. brassicicola and H. maydis, and sporangiospores, sporangiophores, and sporangium of Absidia sp. and R. nigricans. Some of the spores of the above fungi were destroyed. The relative concentrations of glucose and yeast extract significantly affected the degree of parasitism. The higher glucose-yeast extract ratios were most favorable to mycoparasitism. Lower degrees of parasitism were observed when the glucose-yeast extract ratio was 10:5,

ACKNOWLEDGMENTS We wish to thank F. Dazzo, R. Garcia, J. Kimbrough, D. Marx, and the Division of Plant Industry, Gainesville, Fla., for fungus cultures. We are indebted to R. Loeppert for the sample of humic acid.

LITERATURE CITED 1. Barnett, H. L. 1964. Mycoparasitism. Mycologia 56:119. 2. Barnett, H. L., and F. L. Binder. 1973. The fungal host-parasite relationship. Annu. Rev. Phytopathol. 11:273-292. 3. Butler, E. E. 1957. Rhizoctonia solani as a parasite of other fungi. Mycologia 49:354-373. 4. de la Cruz, R., and D. H. Hubbell. 1975. The biological control of the charcoal root rot fungus Macrophomina phaseolina on slash pine seedlings by a hyperparasite. Soil Biol. Biochem. 7:25-30. 5. Griffith, N. T., and H. L. Barnett. 1967. Mycoparasitism by basidiomycetes in culture. Mycologia 59:149154. 6. Haskins, R. H. 1963. Morphology, nutrition, and host range of a species of Pythium. Can. J. Microbiol. 9:451-457. 7. Lumaden, R. D. 1969. Sclerotinia sclerotiorum infection on bean and the production of cellulase. Phytopathology 59:653-657. 8. Marx, D. H. 1969. The influence of ecototrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59:153-163. 9. Riddell, R. W. 1950. Permanent, stained mycological preparations obtained by slide culture. Mycologia 42:265-270. 10. Rodriguez-Kabana, R. 1960. Enzymatic interactions of Sckerotium rolfsii and Trichoderma viride in mixed soil culture. Phytopathology 59:910-921. 11. Wickerham, L. J. 1951. Taxonomy of yeast. U.S. Department of Agriculture Technical Bulletin no. 1029. U.S. Government Printing Office, Washington, D.C.