EXPERIMENTAL MYCOLOGY 14,255-267 (I!%))

Mycelial Interactions in Sclerotinia sclerotiorum LINDA M. KOHN, IGNAZIO CARBONE, AND JAMES B. ANDERSON

Department of Botany, University of Toronto, Erindale College, Mississauga, Ontario, Canada L.5L ZC6

Accepted for publication April 3, 1990

KOHN, L. M., CARBONE, I., AND ANDERSON, J. B. 1990. Mycelial interactions in Sclerotinia sclerotiorum. Experimental Mycology 14,255-267. Mycelial interactions were examined among 35 isolates of Sclerotinia sclerotiorum and two Asian species, Sclerotinia asari and an unnamed, Japanese species. Pairings were scored as compatible when strains merged to form one colony and incompatible when strains grew to form two distinct colonies. Incompatible mycelial pairings resulted in an interaction zone in which a distinct reaction line and abundant aerial mycelium or thin mycelium were observed with some variation among replicates. All pairings of a strain with itself were compatible. Ofthe 31 strains of S. sclerotiorum tested, 21 were mycelially incompatible with all others. Among the remaining 10 strains of S. sclerotiorum, there were four mycelial compatibility groups consisting of two or three strains each. Pairings of S. asari with all other strains resulted in a unique incompatible reaction, a mycelium-free interaction zone. Two of three strains of the Japanese species were intercompatible, but pairings of each of the three strains with all other strains were incompatible. Microscopically, mycelial interactions in pairings of strains were complex. Anastomosis between paired strains was not always observed. This may be due in part to the conversion of many hyphal tips, in both compatible and incompatible interactions, to sites of microconidiogenesis no longer capable of hyphal fusion. Incompatible pairings were fol- lowed by hyphal deterioration in one or both strains; hyphal deterioration was not observed in compatible interactions. Of the 31 strains tested, 4 strains of S. sclerotiorum produced apothecia. Pairings between single ascospore isolates within each strain were compatible, as were pairings with the parent isolate. Mycelial interactions of single ascospore isolates with other strains were identical to those of the parent isolate, indicating that the parent fruitbody was homozygous for any determinant(s) of mycelial incompatibility. The data from this study suggest that a high level of mycelial incompatibility exists among strains of S. sclerotiorum, comparable to levels of vegetative incompatibility reported in other ascomycetes, that the extent of mycelial incompatibility indicates that genetic heterogeneity exists within the species, and that mycelial compatibility/incompatibility reactions may be an effective way of categorizing intraspecific heterogeneity. B 1990 ~cad~rni~

press, Inc. INDEX DESCRIPTORS: mycelial incompatibility; vegetative incompatibility; Sclerotiniu sclerotio-

rum; Ascomycetes.

Mycelial incompatibility, as evidenced ity has been demonstrated in a wide range by the formation of a reaction line between of Ascomycetes and ascomycetous an- strains in pairings on agar medium, is an amorphs including Neurospora crassa indication of intraspecific heterogeneity (Garnjobst, 1953; Garnjobst and Wilson, that has been used effectively in population 1956; Perkins, 1988), Aspergillus nidulans studies of fungi such as Ophiostoma ulmi (Jinks et al., 1966), Podospora anserina (Brasier, 1983; as Ceratocystis ulmi) and Cryphonectria parasitica (Anagnostakis,

(Blaich ,and Esser, 1971; Fincham et al., 1979), Cochliobolis heterostrophus (Leach

1987). Mycelial incompatibility is one of and Yoder, 1983), Gibberellafujikuroi (Pu- several events associated with vegetative halla and Spieth, 1985), Fusarium oxyspo- incompatibility, the failure of different rum (Puhalla, 1985), Verticillium dahliae strains to form a heterokaryon as evidenced (Puhalla and Hummel, 1983), and Septoria by the lack of complementation of mutant nodorum (Newton and Caten, 1988). Vege- strains in pairings. Vegetative incompatibil- tative incompatibility can be a useful tool

255 0147-5975190 $3.00 Copyright 0 PI90 by Academic Press, Inc. All rig&s of reproduction in my form reserved.

256 KOHN, CARRONE, AND ANDERSON

for distinguishing intraspecific phenotypic variants, including those differing in viru- lence, as has been demonstrated in Fusu- rium oxysporum (Puhalla, 1985; Jacobson and Gordon, 1988; Katan and Katan, 1988; Ploetz and Correll, 1988).

Although the cosmopolitan plant patho- gen, Sclerotinia sclerotiorum (Lib.) de Bat-y harbors great diversity in its somatic phenotype (Purdy, 1979; LeTourneau, 1979; Steadman, 1979), it is a well-marked species based on morphological character- istics (Kohn, 1979) and restriction fragment length polymorphisms (RFLPs)’ in nuclear and mitochondrial DNA (Kohn et al., 1988). Finding criteria that systematically distinguish intraspecific variation in S. scfe- rotiorum has been a problem. What we term mycelial incompatibility has been re- ported in the other important species of Scferotinia, 5’. minor Jagger (Patterson, 1986) and S. trifoliorum Erikss. (Bjiirling, 1942; Loveless, 1951).

In this study, 35 strains of Sclerotinia sclerotiorum and two closely related Asian species, one undescribed (Saito, personal communication) from Japan, and the other, Sclerotinia asari Wu and C. R. Wang, from China, were tested for mycelial incompati- bility by macroscopic and microscopic ex- amination of pairings. These strains are as- sumed to be haploid and homokaryotic; molecular criteria provide no evidence of diploidy or heterokaryosis (Kohn et al., 1988; Kohn and Anderson, unpublished data). The primary objective of this study was to determine whether intraspecific het- erogeneity among a set of strains from di- verse localities and hosts could be identi- fied by mycelial incompatibility reactions. With the acquisition of appropriate mutant strains, mycelial incompatibility reactions could be tested for heterokaryosis. A sec-

1 Abbreviations used: RFLP, restriction fragment length polymorphism; PDA, potato dextrose agar; MPM, modified Patterson’s medium; PM, medium of Puhalla.

ondary objective was to compare the char- acteristics of interspecific mycelial reac- tions, which have been used in studies de- fining the two Asian species (Wong, personal communication; Saito, personal communication), with the intraspecific re- actions. The strain of S. asari is the only extant strain available from the locality in China and ours is the only laboratory with strains of both Asian species.

To acquire mutant strains for hetero- karyon complementation in compatible pairings (or lack of complementation in in- compatible pairings), strains were also screened on chlorate-amended medium for the production of non-nitrate-utilizing sec- tors. This efficient method for producing mutant strains exploits non-nitrate-utilizing mutants arising spontaneously on medium amended with chlorate, a toxic analog of nitrate (Puhalla, 1985; Correll et al., 1987; Newton and Caten, 1988).

MATERIALS AND METHODS

Strains. Strains used in this study are listed in Table 1. Strains were isolated from surface-sterilized sclerotia or from host tis- sue. Cultures were grown on Difco potato dextrose agar (PDA) in the dark at room temperature (20-22”(Z), and were main- tained under liquid Nz with glycerol (10% solution) as a cryoprotectant.

Vegetative pairings. Strains were con- fronted on modified Patterson’s medium (MPM) containing 0.68 g KH,PO,, 0.50 g MgS04 . 7H,O, 0.15 g KCl, 0.50 g yeast ex- tract (Difco), 1.00 g NH4N0,, 18.40 g D- glucose, 0.20 ml of a solution of trace ele- ments (Vogel, 1964), 15.00 g agar (D&o), and 6 drops of red food coloring (McCor- mack’s) per liter. While red food coloring was originally used to distinguish MPM from other media used in our laboratory, the accumulation of this red color in the interaction zone of many incompatible pair- ings was useful in scoring.

Each strain was grown on MPM for 7

MYCELIAL INTERACTIONS IN Sclerotinia 257

TABLE 1 List of Sclerotinia Strains

LMK No. Host Location

Sclerotinia sclerofiorum

2 28 34 44 57 58 59 71 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 91 98

121 122 123

Sclerotinia asari

124 Sclerotinia n. sp.

99 100 101

Lettuce LaSalle, Ontario ? Ontario Cabbage New South Wales, Australia Lettuce River Canard, Ontario Ranunculus jkaria Norway R . jicaria Norway R . jicaria Norway Soybean Greeley, Colorado Carrot Ontario Lettuce Yuma, Arizona Soybean Australia Bean cull Mitchell, Nebraska Snapbean New York Snapbean Hancock, Wisconsin Lima bean Westley, California Canada thistle Montana Canada thistle Montana Navy bean Harrow, Ontario Dry bean Alberta Charlevoix bean Michigan Alfalfa Touchet, Washington Soybean Manhattan, Kansas Lettuce Bradford Marsh, Ontario White bean Guelph, Ontario Bean cull Mitchell, Nebraska Canada thistle Montana Canada thistle Montana ? Presser, Washington Rapeseed Guangxi, China Soybean Heilongjiang, China Sunflower Heilongjiang, China

Asarum heterofropoides China

Burdock carrot Angelica acutiloba

Japan Japan Japan

- Collector/source

Jarvis (128) Jarvis (19) DAOM 138593A Wong (S 11) ATCC 34325 Jarvis (150) Schumacher Schumacher Schumacher Steadman (143) Kohn Steadman (144) Steadman (147) Steadman (152b) Steadman (155) Steadman (156) Steadman (160) Steadman (17Ob) Steadman (176) Steadman (182) Steadman (184) Steadman (194) Gilbert Jardine Kohn/Grenville Kohn/Grenville Steadman (152a) Steadman (153) Steadman (170a) Steadman (190) WonglWu Yusan WongiWu Yusan WongWu Yusan

WonglWu Yusan

Saito (SI-BAI) Saito (SI-CM-l) Saito (SI-ANG-T)

Note. ATCC, American Type Culture Collection; DAOM, National Fungus Collection, Ottawa, Canada.

days prior to pairing. For pairings, 2-mm3 blocks of inoculum taken from the growing

was performed at least twice. Pairings were examined 4, 7, and 14 days after inocula-

margin of the colony were placed 3.5 cm tion. apart on MPM in 9-cm petri dishes, one Each strain was prepared and incubated pairing per dish, and incubated in the dark to produce apothecia following the method at room temperature. Strains were con- fronted in all combinations; each pairing

of Russo et al. (1982), except that sclerotia were incubated on sterile, saturated glass

258 KOHN, CARRONE, AND ANDERSON

wool rather than vermiculite. When sclero- tia from a strain developed apothecia, one apothecium was harvested. A small piece was excised from the hymenium and exam- ined microscopically to check for normal development of asci and ascospores. Then, from a mass of spores from a single apo- thecium, 25 single ascospore isolates were selected at random with the aid of a dissect- ing microscope (50x magnification) and needle; germination was evaluated after 12 h. From these isolates, 10 were paired in all combinations with each other, with the pa- rental isolate (that produced the apothecia and underwent meiosis), and with a sample of three other strains, one of which had been compatible and the other two of which had been incompatible with the parental isolate. Pairings were performed and eval- uated as described for parental strains.

Microscopic studies of pairings. Glass microscope slides were coated with a thin layer of MPM by dipping sterile, 75 x 25- mm slides in molten (SOOC) MPM. Each coated slide was then placed in a sterile glass petri dish containing a piece of filter paper and a bent glass rod. The slide was inoculated with two l-mm3 blocks of inoc- ulum placed 1.5 cm apart, representing the two strains in a pairing. The inoculated slide was placed on the bent glass rod above the filter paper. The filter paper was then saturated with 5% sterile glycerol to main- tain adequate moisture in the dish during incubation. Slide cultures were incubated in the dark at room temperature. For each pairing, three slide cultures were prepared; slides were examined 4,7, and 14 days after inoculation. To examine a slide, a 22 X 50- mm glass coverslip was placed over the col- onies and pressed down lightly; slides were examined with Nomarski differential inter- ference contrast microscopy, using a Zeiss Universal photomicroscope.

Experiments to produce non-nitrate- utilizing mutants. Inoculum from l-week- old cultures was transferred to the minimal medium of Puhalla (PM) (Puhalla and

Spieth, 1985) without FeSO,, but amended with 1.5 g/liter L-asparagine and 0, 15, or 45 g/liter KClO,. After incubation at room temperature in the dark for 1 week, the col- ony diameter, hyphal density, and presence or absence of sclerotia were noted for each colony. Any putative, chlorate-resistant sectors were subcultured and transferred to unamended PM; growth characteristics of the original culture on unamended PM served as a control.

RESULTS

Macroscopic Studies of Pairings

Results of pairing the 35 isolates are shown in Fig. 1. Isolates were scored as either compatible (squares) or incompatible (circles); differences among the incompati- ble reactions are indicated as half-solid, crossed, or empty circles. The red reaction line observed in the interaction zone of some incompatible pairings was due to an accumulation of the red food coloring, or at least the coloring elements of it, in the cy- toplasm of hyphal tips. Pairings were scored as compatible when the two strains merged to form one colony, with no distinct interaction zone (Fig. 1, solid ‘squares; Figs. 2-4). Pairings were scored as incom- patible (i) when a red reaction line was vis- ible on the colony surface and reverse at Day 7 (Fig. 1, half-solid circles; Figs. 5-7); (ii) when a red reaction line was visible only on the reverse, with abundant aerial mycelium in the interaction zone on the col- ony surface (Fig. 1, empty circles); (iii) when a red line and a mycelium-free space between colonies were observed (Fig. 1, crossed circles; Fig. 8); (iv) when no red line was observed but when a distinct band of abundant, aerial mycelium occurred in the interaction zone (Fig. 1, empty circles; Fig. 9); and (v) when no red line was ob- served but when a distinctly thin band of mycelium was observed in the interaction zone (Fig. 1, empty circles; Fig. 10).

Of the 35 strains tested, 23 were myce-

MYCELIAL INTERACTIONS IN Sclerotinia 259

(D -a P NN D P

2 DO00

28 DO0

SE

3‘l DO

44

57

FIG. 1. Vegetative incompatibility reactions among 35 strains of Sclerotinia sclerotiorum and two other species of Sclerotinia. Solid square denotes compatible reaction. Circle denotes incompatible reaction; empty circle denotes incompatible reaction in which a band of aerial mycelium or thin mycelium was observed in the reaction zone, with red reaction line present or absent on colony reverse; half-solid circle denotes incompatible reaction in which red reaction tine was observed on colony surface and reverse; crossed circle denotes incompatible reaction in which a red reaction line on colony surface and reverse and a space between colonies were observed.

lially incompatible with all others. Among there was some variation in specific incom- the remaining 12 strains, there were five patibility reactions. For example, in some mycelial compatibility groups consisting of incompatible pairings scored with empty two or three strains each (57,58,59; 77,82, circles in Fig. 1, ah replicates produced a 83; 34,94; 86,98; and 99, 101). All self-self band of thickened aerial mycelium in the pairings were compatible. Scoring for com- interaction zone on the colony surface, but patibility versus incompatibility was con- some replicates produced a red line in the sistent through repeated pairings, although interaction zone visible on the colony re-

KOHN, CARRONE, AND ANDERSON

FIGS. 2-10. Pairings of strains of Sclerorinia scleroriorum demonstrating representative reactions 14 days after inoculation (with exception of Fig. 10).

FIGS. 2, 3. Strain 94 x 94, compatible, self-self reaction. Fig. 2. Colony reverse. Fig. 3. Colony surface.

FIG. 4. Strains 86 X 98, compatible reaction. FIGS. 5, 6. Strains 99 x 91, incompatible reaction. Fig. 5. Colony reverse; note reaction line. Fig.

6. Colony surface. FIG. 7. Strains 100 x 58, incompatible reaction. FIG. 8. Strains 124 x 100, incompatible reaction. FIG. 9. Strains 80 X %, incompatible reaction. FIG. 10. Strains 77 x 89, incompatible reaction, 7 days after inoculation.

MYCELIAL INTERACTIONS IN Sclerotinia 261

verse while some did not. The two other species of Sclerotiniu, one unnamed from Japan represented by strains LMK 99, 100, and 101, and the other, S. asari, repre- sented by LMK 124, each showed distinc- tive incompatibility reactions with all iso- lates of S. sclerotiorum and with each other. The Japanese isolates produced in- tense reaction lines with all other isolates and, with the exception of the compatible 99 x 101, with each other. S. asuri’(LMK 124) produced a unique, incompatible reac- tion with all other isolates, a red line along the periphery of the challenging isolate, and a mycelium-free space in the interaction zone.

Of 31 strains tested only LMK 80,83,88, and 91 produced apothecia. These apothe- cia developed normal hymenial elements with mature asci containing eight uniform ascospores. Germination of ascospores was 96-100% for each strain. Pairings between sibling, single ascospore isolates were com- patible, as were pairings with the parent isolate. Reactions with other strains were identical to those of the parent isolate; there was no difference in pairing reactions with isolates derived from a single asco- spore and in reactions with the parental, premeiotic isolate. The absence of any seg- regation for mycelial incompatibility indi- cates that the parent fruit bodies were ho- mozygous for any determinant(s) of myce- lial incompatibility.

Microscopic Studies of Pairings

Hyphae were traced from the center of a colony toward the interaction zone to de- termine that the anastomosis was actually between the two challenging strains and not an intracolonial fusion. In compatible inter- actions intercolonial anastomoses were not observed in all pairings; in some compatible pairings, one strain simply appeared to overgrow the other without obvious hyphal fusion. Anastomosis either was by a direct

fusion or was preceded by winding of one hypha around the other or by formation of a simple appressorium (Figs. 11-13) and a vesicle-like process in the penetrated cell (Fig. 14). In some pairings, anastomosis was followed by formation of a cluster of hyphal initials from the point of fusion (Fig. 12). Deterioration of hyphae after anasto- mosis was not observed in compatible pair- ings .

In incompatible pairings, anastomoses were not frequently observed. Where they were evident, they were similar to those in compatible interactions, preceded by wind- ing of one hypha around the other (Fig. 16) or by formation of a simple appressorium (Fig. 17), often with a subtending vesicle- like process. In pairings which had pro- duced a red line in the interaction zone in petri dish platings, there was usually hyphal deterioration between Days 6 and 12 in the interaction zone (Fig. 18) or on one side of the confrontation, just behind the zone; some incompatible pairings without red lines also demonstrated deterioration in one or both strains in a pairing.

In both compatible and incompatible in- teractions , ladder-like, intracolonial anas- tomoses were observed (Figs. 14,15). Also, in both compatible and incompatible inter- actions, hyphal tips in many strains became dedicated to microconidiogenesis (Figs. 19, 20), especially in the interaction zone, vis- ible macroscopically as zones of aerial mycelial growth (Fig. 9). The conversion of hyphal tips with the potential for anastomo- sis to microconidiogenous tips incapable of anastomosis was notable. Some strains pro- duced abundant appressorial pads (Fig. 21) and crystals and loops (Fig. 22) in pairings.

Screening for Non-Nitrate-Utilizing Mutants on Chlorate-Amended Medium

The strains of S. sclerotiorum showed a striking range of innate chlorate resistance. At 15 g/liter chlorate, there was only mod-

262 KOHN, CARBONE, AND ANDERSON

MYCELIAL INTERACTIONS IN Scleroriniu 263

erate inhibition of the growth of some strains. The growth of many strains was un- affected by this chlorate concentration. At 45 g/liter chlorate, the growth of all strains was inhibited, some completely and others only moderately. For several strains, the degree of inhibition was inconsistent between different experiments. Several possible chlorate-resistant sectors were subcultured from colonies showing strong inhibition at 45 g/liter chlorate. All of the subcultures grew as well on unmodified PM as the original cultures, indicating that the subcultures were still able to utilize nitrate as the sole nitrogen source.

DISCUSSION

Our data demonstrate that among a representative sampling of strains, most vegetative pairings of strains of Sclerotiniff sclerotiorum will be unable to fuse and form one colony. Mycelial incompatibility

among strains of Sclerotiniu indicates both that genetic heterogeneity exists within the species and that mycelial compatibility/ incompatibility reactions are an effective way of categorizing intraspecific heteroge- neity. We describe the macroscopic and mi- croscopic characteristics of these interac- tions. The high level of mycelial incompat- ibility among strains of S. sclerotiorum may reflect the generally high level of vegetative incompatibility observed in many asco- mycetes or ascomycetous anamorphs.

Macroscopic determinations of mycelial compatibility in S. sclerotiorum are simple. A compatible interaction results in two strains fusing to form one colony, while an incompatible interaction results in two strains growing to form two distinct colo- nies. Within the interaction zone of incom- patible pairings, however, variations in re- actions, such as the red reaction line, a strip of abundant aerial hyphae, a zone of thin, sparse hyphae, or a zone free of mycelium, were observed. A few strains produced dis-

FIGS. 1 l-22. Hyphal interactions in pairings of strains of Sclerotinia sclerotiorum. Bar = 20 Fm. FIGS. 11, 12. Strain 44 x 44, compatible, self-self interaction, 3 days after inoculation. Fig. 11.

Hyphal anastomosis between colonies, 3 days after inoculation. Fig. 12. Proliferation of growing hyphal tips from point of hyphal anastomosis, 3 days after inoculation.

FIG. 13. Strains 57 x 59, hyphal anastomosis between strains in compatible interaction, 3 days after inoculation.

FIG. 14. Strains 77 x 83, hyphal anastomosis between strains in compatible interaction; arrow indicates simple appressorium subtended by vesicle; intracolonial hyphal anastomoses are indicated by asterisks, 4 days after inoculation.

FIG. 15. Strains 101 X 121, intracolonial anastomoses within colony of 101, reaction between strains is incompatible, 10 days after inoculation.

FIG. 16. Strains 121 x 82, incompatible reaction, hyphal anastomosis between strains is preceded by winding, 10 days after inoculation.

FIG. 17. Strains 89 x 88, incompatible reaction, hyphat anastomosis between strains, 3 days after inoculation.

FIG. 18. Strains 124 x 91, incompatible reaction, hyphal deterioration in interaction zone, 14 days after inoculation.

FIG. 19. Strains 28 x 77, incompatible interaction, hyphal tip of 28 producing microconidia in interaction zone 14 days after inoculation; 77 did not microconidiate but produced appressorial pads.

FIG. 20. Strains 44 x 85, incompatible interaction, micorconidiating hyphal tip of 44 in interaction zone, 7 days after inoculation; 85 did not microconidiate but produced appressorial pads.

FIG. 21. Strains 99 x 91, incompatible interaction, appressorial pad produced by 91, 4 days after inoculation.

FIG. 22. Strains 57 X 59, compatible interaction, loop and crystals in interaction zone, 3 days after inoculation.

264 KOHN, CARBONE, AND ANDERSON

tinctive interactions in pairings with all other strains. For example, all pairings with LMK 124 (S. asari) resulted in a mycelium- free interaction zone not observed in any other pairings. In mycelial pairings of many ascomycetes, such as Cryphonectria para- sitica (Anagnostakis, 1987) and Ophios- toma ulmi (Brasier, 1983), a distinct inter- action line is the inevitable outcome of in- compatible mycelial pairings. A distinct line is not seen in all incompatible interac- tions in S. sclerotiorum, however, and is easily observed only on the colony reverse and only if the medium is amended with food coloring.

mycelium-free interaction zone (usually with a red line) in pairings with each of the other strains.

Do strains in the same mycelial compat- ibility group share other phenotypic char- acteristics? The intercompatible strains 77, 82, and 83 were isolated from soybean, bean culls, and snapbeans, respectively, but from different geographic areas. The correlation between host specificity and mycelial compatibility should be pursued among local populations, to minimize the added factor of geographic separation. In- tercompatible strains 57, 58, and 59 were prepared from each of three apothecia, col- lected at one site, of Sclerotinia jkariae, which has been synonymized under S. scle- rotiorum (Kohn, 1979) and has recently been shown to share RFLP phenotypes for nuclear ribosomal DNA with strains of S. sclerotiorum (Kohn et al., 1988). The isola- tion and possibly clonal nature of the pop- ulation in Norway offer reasonable expla- nations for the intercompatibility of these strains. That strains of the new, Japanese species (strains 98, 99, and 100, each iso- lated from a different host and site) are dis- tinct from strains of S. sclerotiorum was supported both in RFLP studies (Kohn et al., 1988) and in the incompatible reactions, with a distinct red line, between these iso- lates and all others; there is also incompat- ibility among strains of the Japanese spe- cies. Strain 124, S. asari, is further distin- guished from all other strains in this study by the unique incompatible reaction, a and the accretion of crystals.

Microscopic characteristics of mycelial compatibility are complex. In microscopic observations of pairings, incompatible re- actions were usually followed by hyphal de- terioration in one strain or in the interaction zone between both strains. In some pair- ings, anastomosis was followed by prolifer- ation from the point of fusion in a compat- ible interaction or deterioration at the point of fusion in an incompatible interaction. Significantly, anastomosis was not evident in all interactions, whether compatible or incompatible. In Neurospora, by compari- son, anastomosis is the inevitable result of hyphal confrontation, with hyphal prolifer- ation from fusion the outcome of a compat- ible vegetative reaction, and hyphal deteri- oration the outcome of an incompatible re- action (Garnjobst and Wilson, 1956). In S. sclerotiorum, it is possible that reactions could result from relatively few, easily overlooked anastomoses in a pairing. There are two explanations, however, for the pau- city of anastomoses. First, in all pairings with strain 124, S. asari, a clear space was observed between strains, without hyphal anastomosis and with extensive hyphal de- terioration evident microscopically. We speculate that a diffusible, inhibitory sub- stance may be produced by strain 124. Sec- ond, in most strains, many hyphal tips be- come incapable of hyphal growth and fu- sion when they develop microconidiogenic phialides. This conversion of hyphal tips into dedicated, microconidiogenous loci oc- curs earlier in most incompatible pairings than in compatible pairings. It is possible that such factors as nutrient depletion or the contact stimulus of reaching a foreign surface (e.g., another mycelium, agar that is too dry, the edge of a petri dish) may contribute to the induction of microconidi- ation, as well as to the production of ap- pressorial pads (Tariq and Jeffries, 1984)

MYCELIAL INTERACTIONS IN Sclerotinia 265

Although Patterson (1986) considered mi- croconidiation in the interaction zone to be a sign of “vegetative” incompatibility, he referred to microconidiating interactions as “sexually compatible,” even when these interactions occurred in pairings of strains that he had determined to be of the same mating type based on the production of apothecia. Since we observed microconidi- ation in compatible (both self-self and self- nonself pairings) and in incompatible vege- tative pairings, we conclude that micro- conidiation alone is a poor criterion for vegetative incompatibility in Sclerotinia and probably has no connection with sexual compatibility.

Another feature which should be inter- preted with caution is the ladder-like anas- tomosis often illustrated as a “compatible interaction.” In our study, when traced to the colony origin, these were inevitably peg-to-peg fusions within one colony, such as were illustrated by Buller (1933, Fig. 33). Gregory (1984) describes such fusions as a normal “mode” of hyphal growth within a colony. In our study, anastomoses between two colonies, even in self-self pairings, usually involved formation of simple ap- pressoria (Tariq and Jefferies, 1984) and vesicle-like processes, or direct fusion of one hypha to another.

Given the high level of mycelial incom- patibility among strains of S. sclerotiorum, it would be interesting to determine whether compatible mycelial pairings form a heterokaryon. Supporting evidence for heterokaryosis is the demonstration of the presence of genetically marked nuclei of different parental types within a common cytoplasm. Because S. sclerotiorum lacks naturally occurring uninucleate propagules, and protoplasts are predominantly large and multinucleate (Anderson, unpub- lished), selection for mutants is difficult. Non-nitrogen-utilizing mutants raised on chlorate-amended medium are especially useful in other ascomycetes because they arise spontaneously and frequently as col-

ony sectors. Strains of S. sclerotiorum, however, were generally only moderately inhibited even on high levels of chlorate and inhibition was inconsistent among tri- als. The few, putative chlorate-resistant sectors isolated in these experiments were capable of nitrate utilization. Chlorate re- sistance or absence of sectoring has been observed in other fungi such as Pyriculuriu oryzae and Schizophyllum commune (J. Leslie, personal communication), as well as Macrophomina phaseolina (Pearson et al., 1986), so it is now apparent that this tech- nique is not appropriate for the selection of non-nitrate-utilizing mutants in all fungi. In several other fungi, phenotypes similar to those that we observed among our Sclero- tinia strains have been associated with chlorate-resistant, nitrate-utilizing (cm) mutants and heterokaryons, which may have either altered nitrate reductase or al- tered uptake characteristics (Cove, 1976; Newton and Caten, 1988; Correll et al., 1987; Klittich and Leslie, 1989).

The level of mycelial incompatibility in S. sclerotiorum reflects genetic heterogene- ity within the species. The degree of myce- lial compatibility reported in this study sug- gests that if any genetic exchange occurs in the field, however, it is most likely through the ascogenous system in sexual reproduc- tion, since mycelial incompatibility among strains of S. sclerotiorum probably dimin- ishes the possibility for somatic heterokary- osis via hyphal fusions. Intraspecific ge- netic heterogeneity within a population can be identified and categorized by determin- ing mycelial compatibility groupings, espe- cially when combined with other criteria. In recent studies, we have mapped mycelial compatibility groups in transect studies of S. sclerotiorum in the field and have iden- tified a series of DNA probes and RFLPs for which each mycelial compatibility group in a local area (3 km) has a unique phenotype (Kohn and Anderson, unpub- lished). In future studies, we hope to deter- mine whether phenotypic characteristics

266 KOHN, CARBONE, AND ANDERSON

pertinent to pathogenesis, such as specific aspects of aggressiveness, virulence, and host specificity, correlate with groupings of strains based on mycelial incompatibility. The maintenance of such genetic diversity, particularly in local environs, is also in- triguing. Although S. sclerotiorum is known to produce apothecia from mono- sporous isolates (Drayton, 1934), it is not known whether heterothallic strains may exist or whether homothallic strains can outbreed. We hope to address this question by using molecular characterization of pa- rental strains in mating experiments.

ACKNOWLEDGMENTS

We thank Elida Stasovski for preparation of photo- graphic prints. This work was supported by Operating Grants from the Natural Sciences and Engineering Re- search Council of Canada to L.M.K. and to J.B.A., respectively.

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