Limited vegetative compatibility following intra- and interspecific protoplast fusion inTrichoderma

EXPERIMENTAL

MYCOLOGY

13,364-371

(1989)

Limited Vegetative Compatibility following Intra- and Interspecific Protoplast Fusion in Trichoderma’ THOMAS E. STASZ,'GARY Department

of Horticultural

E. HARMAN,

AND M. LODOVICAGULLINO~

Sciences, New York State Agricultural Geneva, New York 14456

Experiment

Station,

Accepted for publication April 15, 1989 STASZ, T. E., HARMAN, G. E., AND GULLINO, M. L. 1989. Limited vegetative compatibility following intra- and interspecific protoplast fusion in Trichoderma. Experimental Mycology 13, 364371. A variety of auxotrophic mutants were prepared from several species and strains of Trichoderma by nitrosoguanidine (NNG) mutagenesis. Effective protopIasting from hyphae was achieved with the commercial enzyme preparation Novozym 234; however, pretreatment with 2-deoxy-o-glucose was required for several strains, Aggregation of protoplasts and subsequent fusion were monitored directly by complementary fluorescent staining and were effectively induced by polyethylene glycol and calcium regardless of the Trichoderma species, strains, or auxotrophs being fused. In all cases, about lo6 viable colony-forming units (CFUs) were formed from about 2 x 10’ protoplasts. However, subsequent recovery of somatic hybrid colonies was dramatically lower for interstrain fusions than for intrastrain (between two auxotrophs derived from one strain) fusions. Following intrastrain fusions, 2 to 10 x lo-’ of the viable CFUs grew under selective conditions regardless of the auxotrophs involved, indicating that induced heterofusions were frequent and nutritional complementation was functional. In interstrain fusions, however, only about 1 to 20 x lo-’ of the viable CFUs produced colonies under selective conditions, indicating a low level of postfusion compatibility. Restricted growth of these somatic hybrid colonies, which were not heterokaryotic, appears to result from fusion of heterologous protoplasts and vegetative incompatibility. No vegetatively compatible pairs of strains were resolved; all inter- and intraspecific protoplast fusions exhibited similarly limited compatibility. Limited compatibility may reduce the likelihood of parasexual recombination but does not preclude the possibility of genetic manipulation of Trichoderma strains by protoplast fusion. o 1989 Academic PESS, 1~. INDEX DESCRIPTORS: vegetative incompatibility; heterokaryon incompatibility; postfusion incompatibility; protoplast fusion; Trichoderma; mutagenesis; auxotrophic mutants; somatic hybridization.

Trichoderma spp. are being developed and utilized for biocontrol of plant pathogens in commercial agriculture (Chet, 1987; Harman and Hadar, 1983; Harman et al.,

1989; Papavizas, 1985) and for production of enzymes and other products in industrial microbiology (Everleigh, 1985; Ryu and Mendels, 1980). Methods of genetic manipulation are needed to produce more useful strains (Papavizas, 1987). Recently, protoplast fusion was utilized to produce improved biocontrol strains of Trichoderma harzianum (Harman et al., 1989; Stasz et al., 1988). The immediate result of protoplast fusion is the formation of heterofusant ce1l.s. These cells are heterokaryotic and also heterologous with respect to cytoplasmic organelles such as mitochondria. Subsequent growth of viable heterofusant cells results

’ Research was supported in part by Hatch Project 32494 and by grants from the Cornell Biotechnology Program, which is supported by the New York State Science and Technology Foundation and a consortium of industries; by the Eastman Kodak Company; and by U.S.-Israel Binational Agricultural Research and Development (BARD) Fund-Grant US-1224-86. ’ Present address: Life Sciences Research Laboratories, Eastman Kodak Co., Rochester, NY 146502122. 3 Present address: Istituto di Patologia Vegetale, Via Giuria, 15 10126 Torino, Italy, 364 0147-5975189 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved

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in the development of somatic hybrid hyphae and colonies. Genetic events can occur within heterofusant cells or within somatic hybrid cells produced from them, but hybrid cells are not necessarily either heterokaryotic or heterologous for organelles. Because protoplast fusion circumvents many barriers to naturally occurring plasmogamy, including barriers to hyphal anastomosis, interspecific and even intergeneric hybridization can be achieved. However, protoplast fusion does not avoid barriers to hybridization arising from postfusion vegetative incompatibility. A major advantage of protoplast fusion is the possibility of genetically manipulating asexual fungi to improve or combine phenotypic traits with complex or poorly understood genetic bases (Peberdy and Ferenczy, 1985). Recovery of somatic hybrids following protoplast fusion may be limited by postfusion vegetative incompatibility (Adams et al., 1987). Postfusion incompatibility could limit heterokaryosis to the heterofusant cells resulting from protoplast fusion, or could severely restrict growth of somatic hybrid colonies. Any reduction of the ex-

IN Trichoderma

365

tent of heterokaryosis would reduce likelihood of parasexual recombination cause the formation of heterodiploid nu as a result of karyogamy in a heterokar~~~ is infrequent (Hastie, 1981). In previous work (Stasz et al,, 1988), protoplast fusion of two selected strains was utilized to produce a wide variety of morphological and genetic variants in TrL$roderma harzianum. However, few ~eterof~sant cells regenerated to produce somatic hybrids, and these grew slowly even on permissive media, indicating a signi~ca~~ level of postfusion incomp~tibi~ity~ ‘The purpose of the present study was to mine the effects of postfnsion incompatibility on recovery of somatic hybrids in a range of protoplast fusions within tween species of Trichoderma and t mine if vegetatively compatible strains could be identified. MATERIALS

AND METHODS

Strains and auxotrophs. Prototrop strains and their sources are listed in Table 1. Anxotrophic mutants were prepared by

TABLE 1 Protoplasting Conditions and Yields for Various Species and Strains of Trichoderma Species

Strain

Source”

Hyphal ageb (h)

T. hamatum

52198 T12 T95 T35 T44 T8 417 1365 T105 gla

ATCC Harman Baker Chet & Sivan Chet Harman Vannacci Tronsmo Tronsmo Tronsmo

48 16 16 24 24 48 16 48 24 24

T. hrrrzianum

T. koningii

T. viride

2DGb (mgiliter) 200 0 0 200 200 200 0 200 200 150

Time (h) in enzyme’ 3.0 2.0 1.5 3.0 3.0 2.5 2.0 3.0 3.0 2.5

Protopiast giield’ 107-108 mloa lO8-109 w-209 I57-108

107-IO8 207-IO8 107-108 107-108 107-IO”

a American Type Culture Collection (ATCC), Rockville, MD: Dr. G. E. Harman, New York State Agricultural Experiment Station, Geneva, NY; Dr. R. Baker, Colorado State University, Ft. Collins, CO; Dr. IIan Chet and Alex Sivan, Hebrew University, Rohovot, Israel; Dr. Giovanni Vannacci, University of Pisa, Pisa, Italy; Dr. Ame Tronsmo, Agriculture University Norway, Oslo, Norway. b Hyphae for protoplasting were grown in potato dextrose broth amended with 0.1% yeast extract and with or without 2DG. ’ Protoplast yield from incubating about 10 g (wet wt) mycelium in 50 ml of solution containing 13 mpiml Novozyme 234 in 0.7 M NaCl at 30°C with shaking.

366

STASZ,

HARMAN,

treating suspensions of mature and immature conidia with iV-methyl-N1-nitro-Nnitrosoguanidine (NNG)4 at a concentration of 500 mg/liter for 2 h. Treated conidia were washed twice by centrifugation at 500g for 10 min and resuspension in an equal volume of distilled water to remove NNG. Treated conidia were incubated at 25°C for 5 days with shaking in a liquid basal medium (BM) (Toyama et al., 1984), and the cultures were filtered daily through four layers of cheesecloth to remove germinated prototrophic conidia. Diluted aliquots were spread on a solidified complete medium (CM) consisting of potato dextrose agar (Difco Labs, Detroit, MI) amended with 0.1% casein acid hydrolysate and 0.1% yeast extract and containing 0.1% (w/v) Igepal Co 630 (Alltech Associates, Deerfield, IL) as a colony restrictor (Norton and Harman, 1985). Well separated colonies were individually transferred to EM solidified with 1.5% agar to identify putative auxotrophs, and auxotrophy was diagnosed using differential media pools (Davis et al., 1980) added to BM. Auxotrophy was then confirmed by transferring the mutants to BM and to BM amended with the single needed nutrient. Once auxotrophs were found, single spore isolates were prepared to make them homogeneous and stable. Mutant stability was tested by plating spore suspensions on BM; reversion frequencies were one in lo7 or less. Leakiness of the auxotrophy was assessed by prolonged incubation of the mutants on BM. Stable, nonleaky mutants used in this study are listed in Table 2. Protoplast preparation and fusion. Protoplasts were prepared from young hyphae using Novozym 234 (Novo Laboratories,

4 Abbreviations used: NNG, N-methylN’-nitro-N-nitrosoguanidine; BM, basal medium; CM, complete medium; PDB-YE, potato dextrose broth with yeast extract; 2DG, 2-deoxy-D-glucose; PEG, polyethylene glycol; FITC, fluorescein isothiocyanate; BMS, basal medium osmotically stabilized with 0.6 M sucrose.

AND

GULLINO

Wilton, CT) and counted in a PetroffHausser bacterial counting chamber (Thomas Scientific, Swedesboro, NJ). Hyphae for protoplasting were grown in potato dextrose broth with yeast extract (PDBYE) as previously described (Stasz et al., 1988) except that this medium was amended with 150-200 mg/liter 2deoxy-D-glucose (2DG) for some strains (Table 1). For fusion, about 1 x 10’ protoplasts of each parent were combined in 1 ml buffered osmoticant. Fusion was induced by stepwise addition of polyethylene glycol (PEG) and calcium (Stasz et al., 1988). For controls, about 2 x 10’ protoplasts of each auxotrophic parent were fused independently (parentals selfed). Efficacy of induced fusion of protoplasts was tested in separate experiments using complementary vital fluorescent staining of the parental protoplasts (Harman and Stasz, 1988). Protoplasts of one parent were stained with rhodamine 6G, which fluoresces green, and protoplasts of the other parent were stained with hydroethidine, which fluoresces red. The size of fusion aggregates and the extent of heterologous fusion could be monitored by direct observation of the fusion mix in PEG by epifluorescence microscopy using filter combinations suitable for FITC. Formation of somatic hybrids. Fused protoplasts were recovered by centrifugation and serial dilutions were prepared in buffered osmoticant (Stasz et al., 1988). Aliquots of 0.5 ml were spread on plates containing basal medium osmotically stabilized with 0.6 M sucrose (BMS), BMS amended with the nutrient needed by one or the other of the parental auxotrophs, or BMS with both needed nutrients. Plates were sealed with parafilm to reduce dehydration and contamination and were incubated at 25°C. RESULTS

Protoplast formation and fusion. Protoplast release from young hyphae by Novozym 234 varied among strains. For some

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Somatic

Hybridization

between

TABLE 2 Auxotrophic

Parental strains (auxotrophy)

Species

367

IN Trichoderma

Mutants

of Trichoderma

Colonies from 2 x 10’ fused protoplasts~

spp.

Complement&m frequency (X 10-3b

Putative somatic hybrid colonies with nonparental appearance (%)’

Intrastrain (lys-) x 52198-408 (ars-) T95-1 (lys-) x T9S-3 (his-) TS-10 (ku-) x TS-445 (hi-) T105-227 (nit-) X TlOS-490 (asp-1

52198-349

hamaFum

harzianum koningii viride Intraspecitic harzianum

koningii

x

herzianum

x koningii

viride x viride Interspecific ha&mum x koningii

harzianum

harzianum koningii hamatum

x

viride

hamatum viride x

x

x

viride

T12-2 (his-) x T95-1 (lys-) T12-2 (hi-) x T44-75 (lys-) T12-2 (his) x T35-13 (arg-) T95-1 (lyss) x T3S-13 (argg) T8-85 (leu-) x 417-72 (arg-) TS-33 (trp) x 417-72 (at-g-) TS-33 (trp) x 1365-2 (leu-) TS-445 (hi-) x 1365-2 (lue-) T8-5 (ads-) x 1365-2 (leu-) TS-214 (arg-) x 1365-2 (leu-) 417-72 (argg) x 1365-2 (leu-) T105-490 (asp-) x gla-43 (met-) T12-2 (his-) x 417-72 (arg-) T12-2 (hiss) x T8-10 (leu-) T12-2 (hiss) x 1365-152 (argg) T95-1 (his-) x 1365-152 (arg-) T12-2 (his-) x T105-490 (asp-) T12-2 (his-) x T105-82 (leu) T95-1 (lys-) x Tl05-490 (asp-) T12-2 (his-) x gla-43 (med-) T95-1 (lys-) x gla-43 (met-) T95-1 (lys-) x 52198-349 (lys-) TS-10 (leu-) x gla-43 (met-) 417-88 (argg) x TlOS-82 (leu-) 417-72 (arg-) x T105-470 (asp-) 52198-349 (lys-) x T105-82 (leu-) 52198-349 (lys-) x gla-43 (met-)

19,000 2,200 200 2,000

10,wo 8,500 200

4,000

15 7 18 12 35

5 7

:i 15

9 5

100

11

100

13 20

100

10 -

20

22 8

10

6

8 25 25 25 14 16

10 2

1 16

16 1 13

10 9

a 0 0 0

-

6

21 12 20 2

1 10 19 8 6 5

87 95 2,7 22 71

13 67 100 100 :00 96

100 43

100 100 loo 100 100 100 89

100

a The total number of colonies per 0.5-ml aliquot of diluted fusion mix representing about 2 x 10’ protoplasts (before fusion) plated on osmotically stabilized basal medium. b The number of colonies on basal medium as compared with the number on complete medium. ’ Appearance of colonies that continued to grow when transferred from osmotically stabilized basal medium to basal medium.

strains, adequate release of protoplasts was obtained from young hyphae incubated in 13 mg Novozyme 234/ml in 0.7 h4 NaCl. For many strains, however, adequate yields were obtained only if hyphae for protoplasting were grown in the presence of 2DG. The optimum concentration of 2DG varied among strains, and the range of effective concentrations was narrow. Concentrations of 2DG and other conditions required for optimal release of protoplasts from prototrophic strains are given in Table 1. Hyphae grew more slowly in the presence of 2DG, but older cultures protopiasted adequately. In all cases, protoplast

release from auxotrophs was similar to that from corresponding prototrophic strains. Protoplasts of all strains responded similarly to the PEG fusion system. Complementary fluorescent staining revealed numerous aggregates of tightly adhered protoplasts. Most aggregates contained several protoplasts, and aggregates ranging from fused pairs of protoplasts to perhaps 1 more were observed. Few protoplasts remained nonaggregated. Most aggregates Included protoplasts of both parental types, and heterologous fusion between parental types, as indicated by mixing of the stained cytoplasms, was frequently observed.

368

STASZ,

HARMAN,

Intrastrain fusions. When protoplasts from two different auxotrophs of the same strain were fused, large numbers of prototrophic colonies were recovered within 3 days of plating on stabilized basal medium. Complementation frequencies S~Y~SUPeberdy (1980), calculated as the number of colonies on basal medium as a proportion of the number on complete medium, ranged from 2.0 to 10.0 X lop2 in most cases. In one intrastrain fusion between a leucine and a histidine auxotroph of T. koningii strain T8, the complementation frequency was only 0.2 x 10V2 (Table 2). In all cases, however, colonies isolated from intrastrain fusions grew rapidly when transferred to basal medium and were similar in appearance to the prototrophic strains from which the auxotrophic parentals were derived. In controls, fusion of about 2 x 10’ protoplasts of each auxotroph alone (selfed) resulted in about 10’ viable colony-forming units (CFUs), as determined by plating on complete medium. No colonies were recovered from controls following plating of protoplasts on minimal medium. Znterstrain fusions. Results of interstrain fusions differed markedly from those obtained with intrastrain fusions (Table 2). In 21 different interstrain fusions, low numbers of slow-growing somatic hybrid colonies developed under selective conditions. Complementation frequencies ranged from 1 to 20 x 10e5. Most colonies were capable of continued growth when transferred to fresh basal medium, and most of these prototrophs were morphologically dissimilar to either the auxotrophic parental strains or the corresponding wild-type strains. Colonies continued to appear on selective medium during prolonged incubation for as long as dehydration and contamination of the plates could be avoided. The numbers of colonies reported in Table 2 for interstrain fusions are approximate. During prolonged incubation of control plates, consisting of each parental auxotroph selfed, limited growth of hyphae from protoplast

AND

GULLINO

aggregates was observed for some mutants, e.g., auxotrophs for nicotinic acid. However, in contrast to protoplast fusions between strains, these colonies did not continue to grow when transferred to fresh basal medium. The initial growth was assumed to be due to utilization of stored nutrients or to nutrients released from degenerating protoplasts from the fusion mixture. Results of interspecific protoplast fusions were similar to those obtained with intraspecific fusions. No combination of strains was found to be even moderately compatible, although somatic hybrid colonies were recovered from all fusions (Table 2). DISCUSSION

Cell wall composition differs among strains of Trichoderma species as indicated by differences in protoplasting conditions required and yields obtained among strains. For several strains, adequate protoplasting by the commercially available enzyme preparation Novozym 234 was obtained only if hyphae were grown in the presence of 2DG. Incorporation of this glucose analog into cell walls increases their susceptibility to enzymatic degradation (van den Broek et al., 1978; Zonneveld, 1973). With other strains, native cell walls are degraded by Novozym 234, and incorporation of 2DG is not required for effective protoplasting. In Trichoderma, intrastrain protoplast fusions were compatible. In all intrastrain fusions, large numbers of rapidly growing prototrophic somatic hybrids were recovered under selective conditions. Thus, viable heterologous fusions occurred frequently, and nutritional complementation between the two parental genomes was functional, regardless of the nutritional auxotrophy involved. Interstrain protoplast fusions were relatively incompatible as compared with intrastrain fusions. Limited compatibility in

INCOMPATIBILITY

inter-strain fusions was demonstrated by the low numbers and poor growth of colonies recovered. That colonies from interstrain fusions resulted from somatic hybridization was indicated by their continued growth under selective conditions and by the lack of recovery of similar colonies from fusion of each auxotrophic parent with itself. Limited compatibility in interstrain protoplast fusions in Trichoderma was due to postfusion events and not to failure of induced heterologous fusion per se. Similar levels of protoplast aggregation and heterologous fusion were detected for all strains and species by complementary fluorescent staining and fluorescence microscopy. In intrastrain fusions, 2 to 10 X lo-* of all viable cells proved to be viable heterokaryons; thus the fusion process utilized was adequate. These data indicate, therefore, that the low levels of complementation (1 to 20 x 10m5)obtained in interstrain crosses were not due to failure of the fusion process. Also, it is unlikely that postfusion incompatibility is due to failure of nutritional complementation, because several different auxotrophs were used in interstrain fusions and because nutritional complementation was functional in intrastrain fusions. The genetic events subsequent to interstrain protoplast fusion have been partially described for a fusion between T. harzianum strains T12 and T95 (Staszand Harman, 1988; Harman and Stasz, 1989). The slow-growing progeny that originally developed were only partially prototrophic. They were unstable, and, upon culturing, sectors arose. These sectors usually grew more rapidly and were more fully prototrophic than the original progeny thallus. These variants frequently were themselves unstable, and continued to gave rise to further variants. As a consequence, families of progeny were obtained from single original thalli that varied markedly in morphotype and nutrient requirements. These variants were not a consequence of heterokaryosis, since these properties were stable through

IN

Trichoderma

369

single-conidial isolation. Isozyme analysis revealed no evidence of either heterokaryosis, diploidy, or recombination in any strain. Single-spore analysis revealed tbat some original thalli were extremely imbalanced heterokaryons) with nuclei giving rise to the isozyme profile of strain T12 being approximately 10,000 more prevalant than those giving rise of isozymes identical to T95. A complete genetic analysis of t progeny obtained-from the fusions report in this paper has been completed, and the same pattern of events was found in all cases. A report of these studies is in preparation. Therefore, the low level of compatibility reported in this work apparently occurs Just after fusion, and persists in the form S-F slow-growing progeny for an extended subsequent time. They do not arise as a consequence of unfavorable interactions witkin heterokaryotic hyphae, since they persist as homokaryotic strains isolated from si conidia. Likewise, isozyme analysis reveals that they do not arise as a consequence of the formation of diploid or recombinant aneuploid or haploid strains. Postfusion incompatibility resulting from heterokaryosis is common in fungi (e.g., Anagnostakis, 1977; Hastie, 1981; Perkins and Turner, 1988; P&alla and S~iet~? 1983), and difficulty or failure to isolate somatic hybrids following protoplast fusion has been reported for several fungi (e.g, Anne and Peberdy, 1985; Croft, 1985; Hastie, 1981) including T. harzianum (Stasz et al., 1988). Postfusion incompatibility i other fungi is heterogenic, and strains wit dissimilar alleles at any of several corn ibility loci are incompatible (Adams eb 1987; Leach and Yoder, 19S3; Perkins and Turner, 1988). A simi1a.rsystem may cagierate in Trichoderma. Thus, i~tras~ra~~fusions may be compatible because of ho gene&y at all compatibility loci. Limited compatibility in interstrain fusions may result from heterogeneity for at least some compatibility loci in the ~ete~~f~sa~~cells

370

STASZ,

HARMAN,

produced by protoplast fusion because these cells are heterokaryotic. In Trichoderma, degree of relatedness had no apparent effect on compatibility, and no compatible pair of strains was detected. In other fungi, degree of relatedness greatly influences postfusion compatibility (Kevei and Peberdy, 1985). Intraspecific fusions were no more compatible than interspecific fusions, and even fusions between genetically similar strains were incompatible, as compared with intrastrain fusions. For example, T. harzianum strains T9.5 and T35 have been shown to be isozymically identical at all 16 allozyme loci studied, whereas T. koningii strains 417 and 1365 differ at 13 of 16 allozyme loci (Stasz et al., 1988; unpublished). In the present study, however, fusion between strains T95 and T35 was no more compatible than fusion between strains 417 and 1365. Compatibility was limited in interspecific fusions, but viable somatic hybrids could be recovered. These differed in colony appearance from the parental strains and from each other. Thus, limited compatibility may reduce the likelihood of parasexual recombination but does not preclude the possibility of genetic manipulation of Trichoderma strains by protoplast fusion. ACKNOWLEDGMENTS

The authors thank Mary Catherine Matteson and Glenda Nash for technical assistance; Alex Sivan for helpful suggestions; and Ralph Baker, Ilan Chet, Alex Sivan, Ame Tronsmo, and Giovanni Vannacci for providing cultures of Trichoderma used in this study. REFERENCES

ADAMS, G., JOHNSON, N., LESLIE, J. F., AND HART, P. 1987. Heterokaryons of Gibberella zeae formed following hyphal anastomosis or protoplast fusion. Exp.

Mycol.

11: 33S353.

ANAGNOSTAKIS, S. L. 1977. Vegetative incompatibility in Endothia parasitica. Exp. Mycol. 1: 306316. ANNE, J., AND PEBERDY, J. F. 1985. Protoplast fusion and interspecies hybridization in Penicillium. In Fungal Protoplasts (J. F. Peberdy and L. Ferenczy, Eds.), pp. 259-277. Marcel Dekker, New York.

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CHET, I. 1987. Trichoderma-Application, mode of action, and potential as a biocontrol agent of soilborne plant pathogenic fungi. In Innovative Approaches to Plant Disease Control (I. Chet, Ed.), pp. 137-160. Wiley, New York. CROFT, J. H. 1985. Protoplast fusion and incompatibility in Aspergillus. In Fungal Protoplasts (J. F. Peberdy and L. Ferenczy, Eds.), pp. 225-240. Marcel Dekker, New York. DAVIS, R. H., BOTSTEIN, D., AND ROTH, J. R. 1980. Diagnosis of auxotrophs (auxanography). Appendix 2. In Advanced Bacterial Genetics, A Manual for Genetic Engineering (R. W. Davis, D. Botstein, and J. R. Roth, Eds.), pp. 207-210. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. EVERLEIGH, D. E. Trichoderma. 1985. In Biology of Industrial Microorganisms (A. L. Demain and N. A. Solomon, Eds.), pp. 487-510. Benjamin/ Cummings, Menlo Park, CA. HARMAN, G. E., AND HADAR, Y. 1983. Biological control of Pythium species. Seed Sci. Technol. 11: 893-906. HARMAN, G. E., AND STASZ, T. E. 1988. Fluorescent vital stains for complementary labelling of protoplasts from Trichoderma spp. Stain Technol., in press. HARMAN, G. E., TAYLOR, A. G., AND STASZ, T. E. 1989. Combining effective strains of Trichoderma harzianum and solid matrix priming to provide improved biological seed treatment systems. Plant Dis. 73, in press. HASTIE, A. C. 1981. The genetics of conidial fungi. In Biology of Conidial Fungi (G. T. Cole and B. Kendrick, Eds.), Vol. 2, pp. 511-547. Academic Press, New York. KEVEI, F., AND PEBERDY, J. F. 1985. Interspecies hybridization after protoplast fusion in Aspergillus. In Fungal Protoplasts (J. F. Peberdy and L. Ferenczy, Eds.), pp. 241-257. Marcel Dekker, New York. LEACH, J., AND YODER, 0. C. 1983. Heterokaryon incompatibility in the plant pathogenic fungus Cochliobolus heterostrophus. J. Hered. 74: 149-152. NORTON, J. M., AND HARMAN, G. E. 1985. Responses of soil microorganisms to volatile exudates from germinating pea seeds. Canad. J. Bot. 63: 1040-104.5. PAPAVIZAS, G. C. 1985. Trichoderma and Gliocladium: Biology, ecology, and potential for biocontrol. Annu. Rev. Phytopathol. 23: 23-54. PAPAVIZAS, G. C. 1987. Genetic manipulation to improve the effectiveness of biocontrol fungi for plant disease control. In Znnovative Approaches to P/am Disease Control (I. Chet, Ed.), pp. 137-160. Wiley, New York. PEBERDY, J. F. 1980. Protoplast fusion-New approach to interspecies genetic manipulation and breeding in fungi. In Advances in Protoplast Rem

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search (L. Ferenczy and G. L. Farkas, Eds.), pp. 63-71. Pergamon Press, Oxford. PEBERDY, J. F., AND FERENCZY, L. 1985. FungalProtoplasts. Application in Biochemistry and Genetics. Marcel Decker, New York. PERKINS, D. D., AND TURNER, B. C. 1988. NeurospoYU from natural populations: Toward the population biology of a haploid eukaryote. Exp. Mycol. 12: 91131. PUWALLA, J. E., AND SPIETH, P. T. 1983. Heterokaryosis in Fusarium moniliforme. Exp. Mycol. I: 328335. RYU, D. D. Y., AND MENDELS, M. 1980. Cellulases: Biosynthesis and applications. Enzyme Microb. Technol. 2: 91-102.

IN Trichoderma

371

STASZ, T. E., HARMAN, G. E., AND WEEDEN, N. F. 1988. Protoplast preparation and fusion in two biocontrol strains of Trichoderma harzianum. MyccZogia 80: 141-150. TOYAMA, H., YAMAGUCHI, K., SHINMYQ, A., AND OKADA, H. 1984. Protoplast fusion of Trichoderma reesei, using immature conidia. Appl. Environ. Microbiol. 47: 363-368. VAN DEN BROEK, H., STUNNENBURG, H. G., AND WENNEKES, L. M. J. 1978. Protoplasts from Aspergillus nidulans. Microbios 26: 115-128. ZONNEVELD, B. J. M. 1973. Inhibitory effect of cc-deoxyglucose on cell wall B-1,3-glucan synthesis and cleistothecium development in Aspergillus nidulans. Dev. Biol. 34: 1-8.