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An optimized method for mycelial compatibility testing in Sclerotinia sclerotiorum

     Biology Department, University of Toronto at Mississauga, 3359 Mississauga Road North, Mississauga, ON, L5L 1C6 Canada

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Classification of isolates into mycelial compatibility groups (MCGs) is used routinely in many laboratories as a quick marker for genotyping Sclerotinia sclerotiorum within populations. Scoring each new sample requires optimization of standardized conditions to support adequate growth of all paired isolates. Appropriate conditions for growth are especially important because diverse compatibility reactions are difficult to categorize and score (e.g. in samples from populations with high genetic diversity, such as those that receive immigration from genetically diverse sources or those that deviate from strict clonality). The current standard medium for MCG testing can be inhibitory to isolates from some samples, confounding scoring of compatibility. We identified two foci for optimization: (i) choice of medium, in this experiment, Patterson’s medium amended with red food coloring (termed modified Patterson’s medium, MPM, the current standard medium) versus potato dextrose agar (PDA) and (ii) amount of McCormick’s red food coloring amended to the growth medium. The red food coloring often yields a red reaction line in incompatible interactions; alternative incompatible reactions are a line of thick or thin hyphae. Based on results to date, self-self pairings of S. sclerotiorum are compatible and are a reliable standard for scoring compatible self-nonself mycelial interactions. PDA amended with 75 µl/L of McCormick’s red food coloring was identified as optimal for isolates inhibited by MPM from a highly diverse, recombining population sample. This precisely amended PDA was also suitable for isolates from highly clonal populations that were not inhibited by MPM or by higher concentrations of red food coloring. Under the optimized, standardized conditions all paired isolates grew together and produced interactions that could be scored in repeatedly identifiable categories, compatible or incompatible. Workers are advised to optimize conditions before screening a new population sample.

Key words: MCG, VCG, vegetative compatibility

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Mycelial compatibility, the ability of two strains of filamentous fungi to anastomose and form one continuous colony, is synonymous with vegetative compatibility. A distinction must be maintained between vegetative and heterokaryon compatibility unless it is known that two strains not only anastomose but also form a stable heterokaryon. As an easy test for self-recognition, vegetative compatibility has been extremely useful in intraspecific strain comparisons. The deployment of vegetative and heterokaryon compatibility testing, as well as the mechanisms behind these phenomena, have been amply reviewed (Leslie 1993, Glass and Kuldau 1992, Glass et al 2000, Saupe 2000) and continue to be lively areas of research.

In an initial description of mycelial interactions in Sclerotinia sclerotiorum we reported relatively high mycelial incompatibility, with 21 strains incompatible with all other isolates and the remaining 10 strains forming four mycelial compatibility groups (MCGs) of two to three strains each (Kohn et al 1990). All self-self pairings were compatible. All MCGs were transitive (i.e., isolates A, B and C represented one MCG, and isolate A was compatible with isolate B, isolate B was compatible with isolate C, isolate A was compatible with isolate C). From each apothecium produced from four strains, all sibling ascospores were mycelially compatible with each other and with the parent homothallic isolate. This is consistent with selfed (homothallic), sexual reproduction, the results of which are indistinguishable from asexual reproduction in a haploid organism such as S. sclerotiorum. MCG determination was on a defined medium originally developed for vegetative compatibility testing in S. minor, Patterson’s medium (Patterson 1985), with the modification of 6 drops of McCormick’s red food coloring per liter of medium (termed modified Patterson’s medium, MPM). After acclimatization 7 d on MPM blocks of inoculum cut from the growing margin were confronted, two to a 9 cm Petri dish, 3.5 cm apart and incubated in the dark at room temperature. Pairings were evaluated 4, 7 and 14 d after inoculation. Compatible pairings formed one confluent colony. Incompatible pairings produced a visible reaction in the interaction zone, such as a red line visible on the colony reverse, or a line of fluffy, aerial mycelium or thin mycelium on the colony surface. Microscopically, challenging hyphae in compatible interactions did not necessarily anastomose but were able to overgrow each other. In incompatible interactions that resulted in macroscopic red reaction lines, deterioration of hyphae was observed within and adjacent to the interaction zone. Genetic regulation of vegetative compatibility is not yet elucidated in S. sclerotiorum, but if other ascomycetes such as Neurospora crassa and Podospora anserina are a guide, it is expected to be multigenic (Glass et al 2000).

After demonstrating that MCG and DNA fingerprint were linked in clonal populations of S. sclerotiorum, we, and others, have deployed MCG typing in population studies of both S. sclerotiorum and S. minor (Kohn et al 1991, Kohli et al 1992, Kohli et al 1995, Cubeta et al 1997, Carpenter et al 1999, Carbone and Kohn 2001, Hambleton et al 2002, Phillips et al 2002, Durman et al 2003, Hollowell et al 2003, Kull et al 2004, Atallah et al 2004, Sexton and Howlett 2004). A population sample should be a repeatable design, such as random or nonrandom sampling in transects or quadrats, that will entirely depend on the objectives of the study. At least 30 isolates from a site are advised. The sample is efficiently genotyped by pairing subsamples of 10–12 isolates in all combinations including self-self, then pairing representatives of MCGs in the subsamples until all isolates in the sample have been placed in MCGs. In clonal populations, MCGs are transitive and each MCG is associated with one DNA fingerprint or with fingerprints that differ by fewer than five hybridizing bands; no fingerprint is associated with more than one MCG. In clonal populations a few MCGs are frequently sampled with many additional less frequent MCGs or singleton genotypes (Kohli et al 1995, Hambleton et al 2002). Even with some evidence of recombination MCGs still can be identified and each might be associated with a single fingerprint or microsatellite or AFLP genotype (Atallah et al 2004, Sexton and Howlett 2004, Kohn et al unpubl). In a highly recombinant population, except for isolates from adjacent plants, the expectation is that each isolate sampled is either incompatible with all other isolates or is part of an intransitive MCG and each isolate either has a unique fingerprint or a fingerprint associated with more than one MCG. An apothecium resulting from outbreeding, rather than self-fertilization, should show segregation of three or more MCGs among sibling, single-spore progeny. Although some clones have been sampled over wide geographical areas and repeatedly since 1989 (Kohn et al 1991, Hambleton et al 2002), there is population subdivision. Population subdivision has been identified with population genetic, phylogenetic and coalescent analyses of multilocus DNA sequences. Each population is isolated genetically in that it receives limited immigration from other populations and members of a population share a common evolutionary origin. Each population includes clonal (frequency > 1) and singleton genotypes identified by MCG typing, DNA fingerprinting and microsatellite screening that are different from genotypes in other populations (Carbone and Kohn 2001, Phillips et al 2001, Malvarez unpubl). In our experience the only isolates presenting difficulties in MCG typing have been those from South America and areas in the western United States (Malvarez unpubl). We were able to use the original, standard MCG protocol without difficulty to group a sample of uniformly slower growing isolates from a divergent, apparently inbreeding population of the wild plant, Ranunculus ficaria, from Norway (Kohn 1995). There were no difficulties using the standard protocol in MCG typing isolates from a population with some recombination from North Carolina (Cubeta et al 1997, Kohli and Kohn 1998).

Under the standard protocol for MCG typing, inoculum is cut from 1 cm behind the growing hyphal front because hyphal tips were shown to provide less repeatable results. As a rule, self-self pairings are the basis for determining a compatible interaction in self-nonself pairings. Scoring of reactions as compatible or incompatible in highly to moderately clonal populations has been repeatable among several workers across different laboratories, the litmus test for any method. One caveat is that unusually slow growing or parasitized isolates must be eliminated from screening. With sampling of more geographically diverse populations, however, several problems became apparent, especially when we began to screen populations with greater genotypic diversity, a lack of transitivity within MCGs and disassociation of DNA fingerprint and MCG, consistent with outbreeding and recombination, rather than clonality. Although reactions were repeatable, three main problems were encountered: (i) inconsistency in medium preparation as some laboratory personnel were generous with the 6 drops of red food coloring while others were parsimonious; (ii) apparent inhibition by MPM of hyphal growth in some isolates; leading to (iii) difficulty in scoring as even self-self confrontations could fail to grow together, obscuring the distinction between compatible and incompatible interactions. In incompatible reactions a wide diversity of interactions were observed, including three types of reaction that could occur without the easy-to-score red reaction line: a zone of sparse hyphae between paired isolates, a zone of abundant aerial hyphae, or a zone demarcated by sclerotia. The key to scoring always has been comparison with self-self pairings.

This report presents criteria for optimizing mycelial compatibility testing in S. sclerotiorum. The features studied were the composition of the medium and the concentration of McCormick’s red food coloring. While an optimized procedure is provided we hasten to point out that each new geographical region or host sampled might yield isolates that differ from the phenotypic norms for agricultural isolates of S. sclerotiorum from the soybean, bean, lettuce and canola growing areas of North America. With each new sample one should determine that the medium, food coloring concentration and potentially other factors such as temperature and distance between inoculum blocks are optimal for ease and repeatability of scoring. The experiment presented here offers a framework for optimization of MCG testing in any sample of isolates.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Three isolates of S. sclerotiorum were used to compare treatments for optimization of the MCG testing protocol: 1027, 1029, 1047, from lettuce, from the San Joaquin Valley, Kern County, California, 17 Mar 2004 (J. Nunez, UC Cooperative Extension, Bakersfield). These isolates were selected to represent a population composed of strains that exemplify problems with the MCG screening method. These isolates represent a divergent, recombining population, and not a cryptic species, on the basis of high genetic diversity, lack of association of markers, as well as evidence of recombination and subdivision in population genetic, phylogenetic and coalescent analyses of multilocus DNA sequences. (Malvarez et al unpublished).

Eleven isolates were used to validate the optimized procedure and demonstrate repeatability among isolates from the highly clonal population on soybean and canola characterized in previous studies (Kohn et al 1991, Kohli et al 1995, Carbone and Kohn 2001, Hambleton et al 2002, Kull et al 2004) and a moderately recombining pea and lentil population from Washington state (Kohn et al unpublished, Mavarez et al unpublished). From the clonal population were four isolates that had been easily scored with the original MCG protocol employing MPM amended with 6 drops of food coloring: 1980 from bean in Lincoln, Nebraska, the S. sclerotiorum genome sequence isolate, clone 2 (Kohn unpublished); LMK 211 from canola in Harriston, Ontario, in 1989, clone 2 (Kohn et al 1991) and used as a standard in all DNA fingerprinting in the Kohn laboratory; LMK 199. from canola in Harriston in 1989, clone 1(Kohn et al 1991); 19–434 from soybean in Harriston in 2002 (Hambleton et al 2002), clone 1. From Quincy, Washington, seven isolates from pea were used: SS02-5, –12, –33, –41, –57, –87 and –22.

To compare treatments and optimize the protocol, isolates 1027, 1029 and 1047 were paired in all combinations. All pairings were repeated with each of six treatments. The test media were PDA (Difco) and modified Patterson’s medium (MPM: 0.68 g/L KH₂PO₄, 0.5 g/L MgSo₄·7H₂O, 0.15 g/L KCl, 1.0 g/L NH₄NO₃, 18.4 g/L D-glucose, 0.5 g/L yeast extract, 15.0 g/L agar, 1.0 L glass distilled H₂O, desired concentration of McCormick’s red food coloring, 200 µL M.P.F.Y.E. trace element solution (Vogel 1964) contains: 95 mL glass distilled H₂O, 5.0 g citric acid monohydrate, 5.0 g ZnSO₄·7H₂O, 1.0 g Fe(NH₄)₂ (SO₄)₂·6H₂O, 0.25 g CuSO₄·5H₂O, 0.05 g MnSO₄·1H₂O, 0.05 g H₃BO₄, 0.05 g Na₂MoO₄·2H₂O, optional: add 1 mL CHCL₃ as a preservative to store at room temperature, 20–23 C). Treatment 1 was Patterson’s medium, that is 0 µL/L McCormick’s red food coloring (McCormick Corp., Dallas, Texas) and treatments 3–4 were MPM with each of three different concentrations of red food coloring: 75 µL/L, 150 µL/L, and 275 µL/L. Treatments 5–6 were PDA with two different concentrations of McCormick’s red food coloring: 0 µL/L and 75 µL/L.

Isolates were grown on PDA 5 d at room temperature (20–23 C) in the dark. Isolates were acclimated on each of the six treatment media an additional 5 d at room temperature in the dark. Inoculum was a 4 mm³ block cut from at least 1 cm inside the growing edge of the colony. Inoculum blocks were confronted 3.5 cm apart on the test medium in all self-self and non-self combinations. Reactions were scored 4–7 d after inoculation on test media.

To validate the optimization protocol and test repeatability, the 11 isolates first were classified in MCGs with the above procedure on MPM with 6 drops of McCormick’s red food coloring. Each isolate was grown in serial culture in 32 cm race tubes in the dark at room temperature (20–23 C) with two replicates. Subcultures were made at three points, after 2, 4 and 6–9.5 m (395–481 d depending on growth rate) to the end point of the experiment. To type MCGs the progenitor isolates and each subculture were confronted in pairs in all combinations on PDA amended with 75 µL/L McCormick’s red food coloring as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Representative results of the comparison of treatments are shown (FIG. 1). In the comparison of MPM with PDA, even self-self interactions on Patterson’s medium (without red food coloring) produced a visible interaction zone, demarcated by a line of aerial hyphae. In contrast, on PDA self-self interactions filled the Petri dish and grew together without a demarcation line. On MPM with increasing concentrations of red food coloring, the self-self pairings resembled the incompatible interactions, with the two confronting strains failing to grow together. The self-self interactions were reliably scored as compatible only on PDA, with or without an amendment of 75 µL/L red food coloring.

View larger version (87K): [in this window] [in a new window]   FIG. 1. Comparison of six treatments on self-self and self-nonself mycelial compatibility pairings among three isolates of Sclerotinia sclerotiorum (1027, 1029, 1047). Treatments were Patterson’s medium (PM) without McCormick’s red food coloring and modified Patterson’s medium (MPM) with McCormick’s red food coloring, for a total of 4 concentrations of red food coloring (µL/L), and potato dextrose agar (PDA) with 2 different concentrations of McCormick’s red food coloring.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
An inhibition of growth on Patterson’s medium was observed when compared with growth on PDA. Concentrations of McCormick’s red food coloring of 150 µL/L and 275 µL/L in Patterson’s medium further inhibited growth such that self-self pairings, in which two pieces of inoculum from the same strain were confronted, failed to grow together and likely would have been scored as incompatible. In contrast, the same self-self pairings were easily determined to be compatible on PDA, with or without red food coloring. Because the amendment of red food coloring might yield a red reaction line on the colony reverse in incompatible interactions, for ease of scoring, our optimized test for mycelial compatibility uses PDA amended with 75 µL/L of McCormick’s red food coloring. For isolates from clonal populations that were not inhibited by the standard method with MPM with 75 µL/L of red food coloring, scoring was consistent and repeatable with both the standard method and the optimized method. For all isolates tested, scoring was consistent and repeatable with the optimized protocol.

We strongly recommend that if self-self interactions appear to be incompatible, i.e. if a reaction line is produced in the interaction zone, one should suspect inhibition by the food coloring and repeat the pairings on PDA without red food coloring. Because self-self interactions are likely to be fully compatible, they offer the standard against which self-nonself pairings can be evaluated.

	ACKNOWLEDGMENTS

 
This research is part of ongoing projects supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Soybean Growers-NCRSP White Mold Group, and the USDA-ARS Sclerotinia Initiative.

	FOOTNOTES

 
Accepted for publication June 27, 2006.

¹ Corresponding author. E-mail: kohn{at}utm.utoronto.ca

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This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Schafer, M. R. Articles by Kohn, L. M. Search for Related Content PubMed Articles by Schafer, M. R. Articles by Kohn, L. M. Agricola Articles by Schafer, M. R. Articles by Kohn, L. M.