Population Genetics

116. Development of a genus-specific genetic marker for identification of a phytopathogenic fungus.

Elisa M. Becker and William E. Hintz, University of Victoria.

The basidiomycete fungus Chondrostereum purpureum is being developed as a biocontrol for forest weed species. To fulfill registration requirements we are conducting provincial and national population surveys, environmental fate studies and efficacy trials. During these studies C. purpureum as well as many other fungi are routinely isolated from wood. The mycelia of many fungi are morphologically similar on agar media and traditional biochemical tests are not specific enough to resolve fine differences, hence a PCR-generated marker that could be used to distinguish C. purpureum from other fungi was desired. Our recent work has revealed relatively little variation in the intergenic spacer region (IGS) of the ribosomal DNA (rDNA) of isolates of C. purpureum collected from several regions of the world. We hypothesized that a small portion of this region would be a good target for the design of diagnostic primers specific for C. purpureum. A 500 base-pair fragment of the rDNA IGS was subcloned and the terminal sequences used to design sequence-characterized oligonucleotide primers for amplification of specific regions (SCAR). This SCAR primer pair has been used to successfully amplify a 500 base-pair fragment from every C. purpureum isolate screened to date. Furthermore, this fragment was not amplified from any other fungal species tested including those which are closely related to C. purpureum and/or found in a similar ecological niche. These diagnostic primers are now being used to routinely screen DNA from all putative C. purpureum cultures collected.

117. Molecular population genetics of the pathogenic fungus, Coccidioides immitis: recombination, distinct populations, and cryptic speciation.

A. Burt, V. Koufopanou, G.L. Koenig, B. M. Dechario, T.J. White and J.W. Taylor. University of California, Berkeley; Imperial College, Silwood Park; Roche Molecular Systems, Alameda, CA.

Understanding the reproductive mode (clonal or recombining), and the population structure of fungi is important to studies of their identification, molecular development, epidemiology, and control. With the ascomycete pathogen, Coccidioides immitis, we used phylogenetic and population genetic analysis of 14 loci (defined as base subsituations or small length mutations in usually arbitrary DNA), to show that Arizona C. immitis recombine in nature. Using 11 of these loci and population genetic analysis (Wright's Fst), we now show that gene flow among C. immitis from Arizona, California and Texas is significantly reduced, particularly to and from California. Subsequent analysis of ca. 2400 nucleotides from parts of five genes in 17 widespread C. immitis confirms recombination among C. immitis individuals in-California and out-of-California, and demonstrates genetic isolation of the two groups over an estimated 8 myr. Eight fixed nucleotide differences between the groups facilitate group-specific identification and make it desirable to include representatives of both groups (species?) in efforts to control this fungus by drugs or vaccines.

118. Multiallelic molecular markers for strain typing and epidemiology studies of fungal populations.

Dee A. Carter and Nai Tran Dinh. Microbiology Department, University of Sydney, NSW, Australia.

Multiallelic molecular markers such as microsatellites and short repetitive sequences are being widely applied to population and genetic studies of many eukaryote species, but their application to the study of fungi has been relatively limited. As these markers are analysed by directed PCR amplification they can be detected in minute amounts of even extensively degraded DNA, making them ideal for the study of hazardous pathogens or organisms that are difficult to culture. In addition, these markers may be amplified from impure DNA samples so they can be applied to obligate pathogens and commensals without the need to eliminate host tissue. Here we report the development of multiallelic markers from the genomes of Histoplasma capsulatum and Aspergillus parasiticus, and show how these markers can be used to distinguish isolates of these fungi at the individual or clonal level.

119. Mitochondrial DNA haplotypes in clonal and sexual populations of Phytophthora infestans.

Pia D. Gavino and William E. Fry, Dept. of Plant Pathology, Cornell University, Ithaca NY.

The diversity in nuclear DNA (nDNA) and mitochondrial (mtDNA) was assessed within clonal and sexual populations of the late blight fungus, Phytophthora infestans. Every isolate tested (n=60) from central Mexico, the center of origin of P. infestans, had a different nDNA fingerprint. Surprisingly, all these isolates had the same mtDNA haplotype (Type A). The high nDNA diversity and low mtDNA diversity was consistent with the absence of recombination in mtDNA and the uniparental inheritance of mtDNA in sexual populations of P. infestans. In northern Mexico, however, two mtDNA haplotypes (Types A and B) were identified within clonal and sexual populations (n=30). Type B appears to have evolved from Type A through an insertion of about 2 kb and rearrangement in the mitochondrial genome. Types A and B were also found in other clonal and sexual populations of P. infestans worldwide. Type A was found in most of the old clonal lineage (US- I genotype) and in recombinant isolates recently found in British Columbia (n= 18). Both haplotypes were likewise present in recent clonal lineages in Latin America (n=33) and in sexual populations in Europe (n=14) and in the Pacific northwest of the United States (n=26). These findings support the hypothesis that the mtDNA haplotype B of P. infestans may have originated from northern Mexico.

120. Wright's fixation index analysis reveals the probable mating system for 16 species of Phytophthora.

Stephen B. Goodwin, USDA-ARS/Purdue University, West Lafayette, Indiana.

Approximately half of the 67 species of Phytophthora are heterothallic; the rest are homothallic. If hetero- or homothallism dictates the mating system, there should be almost no heterozygosity in populations of homothallic Phytophthora species due to self fertilization. In contrast, heterothallic species should contain high levels of heterozygosity. However, levels of heterozygosity within species of Phytophthora so far have not been analyzed. To test whether there are differences in mating system, Wright's fixation index [F = 1 - (HObs / HExp), where HObs is the observed heterozygosity and HExp the expected heterozygosity assuming random mating] was calculated for 16 species of Phytophthora by reanalysis of previously published data. As expected, fixation indices were near 1.0 for four of the six homothallic species. Fixation indices for four of the ten heterothallic species were between zero and 0.3, as expected for random mating populations. The remaining species could be divided into three groups. One group, consisting of three hetero- and one homothallic species, probably had a mixed mating system with intermediate fixation index values near 0.5. A second group of heterothallic species had high fixation index values similar to those for homothallic species, probably due to asexual reproduction or inbreeding. A third group contained two heterothallic species with negative fixation index values. Deviations from expectation probably were due to asexual reproduction, incorrect scoring of some isozyme data, or possibly a Wahlund effect. Many Phytophthora species probably have a mixed mating system in nature that cannot be predicted on the basis of hetero- or homothallism. However, this conclusion is preliminary and must be confirmed by analyses of larger samples from carefully defined populations.

121. Mathmatical and computer modeling of the spread hypovirulence in fungal populations.

Susan Kelling, Keith Klein and Marty Wolf, Mankato State University, Mankato MN, 56002.

We employed a multiple population growth model to simulate the effect of the release of hypovirulence factors into a population of a plant pathogen. The model followed logistic growth kinetics with discrete generations for ease of analysis. The following variables were tested for their effect on the predicted spread of hypovirulence: reproductive rate of infected and uninfected individuals (rate for infected individuals < rate for uninfected individuals),number of genotypes (vegetative compatibility groups, VCG), transmission rate of hypovirulence within VCG and between VCG's (rate within>rate between), migration rate of individuals between populations, and recovery rate from hypovirulence infection. The model was realized on a massively parallel processor and run as a simulation for many generations. The simulations predict that recovery rate is the single best predictor of the success of hypovirulence as a control method.

122. Classical and molecular genetic analyses of Colletotrichum spp.

R Lardner1, KM Plummer1, MN Pearson1, PR Johnston2, MD Templeton3. 1University of Auckland, 2Manaaki Whenua-Landcare Research , 3HortResearch, Auckland, NZ.

The genus Colletotrichum contains plant pathogens which cause disease on a number of economically important crops worldwide. C. acutatum f sp pineum is capable of killing Pinus radiata seedlings. This pathogen was first noted in New Zealand (NZ) in the 1960's. In the mid 1980's, a Colletotrichum species caused widespread dieback of tree lupin (Lupinus arboreus) in NZ. Tree lupins are cocultivated with P. radiata, providing nitrogen for young plantations. Tree lupin dieback had not been previously recorded in NZ, however the causal fungus had similar biology to the pine pathogen. It was proposed that the lupin pathogen may have arisen from the NZ pine pathogen population. Sequence analysis of the D2 region of rDNA of various NZ Colletotrichum taxa grouped the pine and lupin pathogens with three fruit rotting taxa. We have used morphological characters, as well as vegetative compatibility (VC) groups, RAPD analysis, and pathogenicity on lupin and pine, to investigate taxonomic relationships between the five taxa. All five taxa could be differentiated on the basis of colony morphology and RAPD banding patterns. All isolates from lupin had identical RAPD patterns. Isolates from pine and the three fruit rotting taxa were able to complement each other, however the isolates ex-lupin were all in one, separate VC group. Pathogenicity tests showed host specificity of the isolates from lupin and pine.

123. Differentiation between physiological races of Fusarium oxysporum f.sp. dianthi by pathogenicity assay, random amplification of polymorphic DNA (RAPD) and distribution of the Fot1 transposable element.

Quirico Migheli1, Elena Briatore1, Marie-Josee Daboussi2, Thierry Langin2 1 Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali (Di.Va.P.R.A) - Patologia vegetale, Universit di Torino, Via Leonardo da Vinci 44, I-10095 Grugliasco, TO (Italy) 2 Institut de Genetique et Microbiologie, Universite Paris-Sud, Bat. 400, F-91405 Orsay (France)

RAPD fingerprinting was used in combination with pathogenicity assay on differential cultivars and analysis of the distribution of the Fot1 transposon to characterize a representative collection of Fusarium spp. isolates from diseased carnation. In F. oxysporum f.sp. dianthi, isolates were clustered in three RAPD groups: group 1 included isolates of race 1 and 8; group 2 was formed by isolates of race 2 and single representatives of race 5 and 6; group 4 included isolates of race 4 only. No correlation was found between RAPD data and geographic origin of the tested Fod isolates, as representatives of pathotype 2 isolated in Italy, Israel and Japan presented the same amplification profile. Four isolates showing a low level of pathogenicity on all tested carnation cultivars shared an identical amplification pattern and are probably saprophytic F. oxysporum. Finally, two F. redolens isolates from Japan and seven non-pathogenic isolates of F. proliferatum collected from diseased carnation in Italy, Israel, Spain and The Netherlands were clearly recognized according to their RAPD fingerprint. The distribution of the transposable element Fot1 was determined by Southern hybridization on DNA restricted with XhoI, which has no sites in the Fot1 sequence. Fot1 copy number varied between 1 and 6, with insertion sizes comprised between 2 and 12 kb. The Fot1 hybridization patterns led to the same clustering based on RAPD fingerprinting. Inverse PCR experiments are being carried out to clone flanking genomic DNA from different races and design race-specific primers.

124. Genetic Variation in Rhizoctonia solani AG1 using DNA-Based Genetic Markers.

Rodney E. Pettway, U. Liane Rosewich, H. Corby Kistler, University of Florida, Gainesville, and Bruce A. McDonald, Texas A&M University, College Station.

To study the population genetic structure and genome organization of Rhizoctoni solani AG1, a PstI library was constructed from the DNA of an isolate that originated from Texas. Using these clones as probes we have screened the DNA from two sets of isolates. The first set consisted of 14 isolates from five fields each located in a different county in Texas. EcoRI-digested DNA was hybridized to 27 probes, whereby 88% of the probes were polymorphic and 40% were highly repetitive (>50 bands). Only two isolates, collected from the same field had the same multilocus haplotype. The clonal nature was confirmed by using one of the repetitive clones as a DNA fingerprinting probe. The second set of isolates consisted of seven isolates from different states (Arkansas, Mississippi, Alabama, Texas and Louisiana). These were tested with a subset of nine probes. In addition to EcoRI, PstI, HindIII and XhoI were used to determine optimal probe-enzyme combinations. Among the 27 probe-enzyme combinations tested,19 detected RFLPs among the seven isolates, whereby HindIII and XhoI seemed to detect RFLPs most efficiently. Even though alleles were shared between isolates originating from different states, a sharing of haplotypes was not detected. Probes developed in this study will be used to test for heterozygosity and random mating.

125. Genetic analysis of Cantharellus cibarius populations in rain forests of the pacific northwest.

Rusty Rodriguez, Regina Redman, Judy Ranson, and Roger Hoffman, NBSC, Biological Resources Division, United States Geological Survey, Seattle, WA 98115

Five clusters of Cantharellus cibarius, that occurred within a 30 meter diameter, were analyzed by PCR amplification to determine the genetic structure of this population. The number of fruiting bodies varied from 15 to 54 per cluster and the location of each fruiting body relative to a benchmark placed in each cluster was recorded for Geographic Information System (GIS) mapping. A small non-lethal sample was collected from each fruiting body for DNA extraction. Arbitrarily primed PCR (apPCR) was used to identify 15 products that were polymorphic in a subset of these samples. The apPCR products were cloned and sequenced in order to design marker-specific PCR primer sets for dual primer PCR (dpPCR) of all individuals in the five clusters. These analyses indicated that each cluster of fruiting bodies was genetically distinct and representing five different populations. In addition, each population expressed varying levels of genetic diversity indicating that, although each population represented highly related individuals, the individuals were not clonal.

126. Morphological and molecular characterization of isolates of Fusarium oxysporum f.sp. radicis-lycopersici from Florida.

U. Liane Rosewich, Rodney E. Pettway, H. Corby Kistler, Univ of Florida, and Talma Katan, Volcani Center, Bet Dagan, Israel.

Work is in progress to elucidate the population genetic structure of the tomato pathogen Fusarium oxysporum f.sp. radicis-lycopersici. Isolates have been collected from all major tomato growing counties in Florida. Characterization of vegetative compatibility groupings (VCG) of 102 isolates revealed, that most isolates (81.4%) fell into VCG 0094 which previously has been only reported for Belgian isolates. A previously undescribed VCG, tentatively assigned 0098, is also widespread in Florida. In addition, molecular markers are being developed and tested to characterize the collection. Single-copy probes developed from a genomic library have yielded to date only a limited amount of polymorphic markers. Only 17 out of 42 probe-enzyme combinations were polymorphic, whereby most polymorphisms were caused by a single isolate. Clonal relationships were better visualized by using a moderately repetitive fingerprinting probe (pEY10). Seven different haplotypes could be differentiated among 19 isolates, selected from VCG 0094. Electrophoretic karyotyping also revealed polymorphisms. Visualizing chromosomes < 1.5 Mb, four karyotypes were found among the eight strains examined, all belonging to VCG 0094.

127. Influence of geographic distance and mating strategy on the genetic population structure of wood decay basidiomycetes.

Jan Stenlid, Nils Hogberg, Hanna Skoldberg, and Rimvydas Vasiliauskas. Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, S-750 07 Uppsala, Sweden.

The extent to which populations of wood inhabiting basidiomycetes differentiate gentetically on various geographical scales is not fully known. In Heterobasidion annosum, a widespread pathogenic species, and Fomitopsis pinicola, a common saprotrophic decay fungus, evidences from spore dispersal gradients and DNA fingerprinting indicate that distances above 500 km may be needed in order to find strong differentiation among populations. In H. annosum we have indications of relative frequent rematings to occur within single substrate units. Expansion of the geographical range of the fungus following the last ice age as well as host species preferences are likely to contribute to present day population structure. In the largely non-outcrossing Stereum sanguinolentum, near-clonal vc-group lines can be isolated from widely seperated locations in Northern Europe. Data from DNA markers are compatible with strong inbreeding within the vc-groups of S. sanguinolentum.

128. Sexual reproduction of Botrytis cinerea and population structure in Champagne: two sibling species?

Giraud Tatiana, Fortini Dominique, Leroux Pieffe and Brygoo Yves. INRA.

B.cinerea is a haploid, filamentous, heterothallic ascomycete. It attacks a wide range of plants in temperate regions and causes grey mould on many economically important crops, such as grapes. Previous studies have shown that this species has a great genetic diversity and morphological variability, for which the usual explanations are heterokaryosis and aneuploidy. Sexual reproduction is not commonly invoked as a cause for genetic diversity because, although apothecia are produced in laboratory for any strain, its sexual organs have rarely been observed in field. We used 15 markers (RFLP markers, sensitivity to fungicides and presence of two transposable elements, Boty and Flipper), to analyze 356 field isolates, from four different places in Champagne, collected in 1994 and 1995, from three different types of wineyard. The markers revealed that B. cinerea contained a large amount of intra-population genetic variation. The two transposable elements were found to be associated : one group of isolates had both transposable elements, the other had none. These groups were subsequently shown to be different for all the other markers using tests of difference of allelic frequencies. Morphological characters (e.g., spore length) corroborated this structure of the population of Champagne. RFLP markers showed genetic recombination in both groups of isolates, using the number of haplotypes, and tests of gametic desequilibrium. We conclude that there are two sympatric populations of B. fuckeliana in Champagne. One species seems to be local and well-adapted, while the other one is likely to be a migrant population, less homogeneous and less fit on grapes in Champagne.

129. Spore-killing in Podospora anserina: an overview.

Marijn van der Gaag, Fons Debets, Jessica Oosterhof and Rolf Hoekstra. Dept. of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.

Spore-killers are segregation distorters found in several ascomycete fungi. In Podospora anserina spore-killing can be identified by the abortion of two of the four dikaryotic spores within the ascus. In the post-meiotic phase the spore-killer allele prevents the formation of spores not containing the spore-killer allele. In case of a cross-over event, ascospores end up with a 'killer' and a 'sensitive' allele. The ascus contains then the normal amount of four spores, the sensitive allele is 'saved'. The further the spore-killer allele is localised from the centromere on a chromosome, the higher percentage of four-spored asci within a fruiting body, and the less efficient the killer-gene. Backcrosses of the heterokaryotic spores from four-spores asci with the parental strains reveal that the sensitive alleles remain undamaged by the spore-killing action. At the moment seven different types of spore-killer genes can be identified in P.anserina, of which 6 recently isolated from nature. The newly isolated Psk7 killer is apparently identical to a French killer strain originating from 1969. All spore-killer types can be distinguished from each other by killing percentage, localisation on linkage groups, and killing interaction among each other. Some sort of linear killing order or dominance seems to exists, when different killer types are crossed with each other. Still most of the natural population consists of strains sensitive to spore-killing, coexisting with the spore-killer strains. No resistant strains have yet been found nor fixed killer genes. Meiotic drive elements only work within an outcrossing population, however P. anserina is a secondary homothallic fungus, and mainly reproduces by selfing.

130. The dynamics of linear plasmids in Podospora anserina.

Marijn van der Gaag and Rolf Hoekstra. Dept. of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.

A natural population of recently isolated strains of the ascomycete Podospora anserina were screened for homologues of the linear longevity inducing plasmid pAL2-1. Of the 78 wild-type isolates 14 hybridised with a pAL2-1 specific probe, however only one of the plasmid inhabiting strains showed the long-lived phenotype. Also some strains were found which contained a related plasmid family, instead of a single plasmid. The inheritance of plasmid ladder containing strains was also investigated. The plasmid was inefficiently sexually transferred to the next generation. Not only a loss of the plasmid family members could be observed, also about 20% plasmid-free ascospores were produced. No significant difference in amount of plasmid-free spores between normal binucleate and uninucleate ascospores was found. The linear plasmids are transmitted maternally. Furthermore, horizontal transfer experiments showed that the linear plasmid could easily infect plasmid-free strains, in both vegetative compatible and incompatible situations. The ascospores produced by the 'plasmid-free' strains in the horizontal transfer experiments, also showed presence of the linear plasmid, indicating that infected maternal tissue directly leads to the production of plasmid containing spores.

131. Poulation structure of the black Aspergilli.

Anne D. van Diepeningen, Alfons J.M. Debets, Klaas Swart and Rolf F. Hoekstra. Department of Genetics, Agricultural University Wageningen, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.

A survey was made of natural populations of the soilborne fungus Aspergillus niger. Representatives of the whole range of black Aspergilli were found to be able to utilise media containing 20% tannin, showing the utility of these media for exclusive selection of black Aspergilli. We made use of this unique characteristic to selectively isolate A. niger from nature. 642 Strains were isolated from soil samples of different countries throughout the world and some sites were sampled over a range of years. All isolates were classified according to their mitochondrial restriction fragment length polymorphisms. A majority of mitochondrial types occurred all over the world among populations and similar frequencies of mitochondrial patterns were observed. More endemic -possibly recent characters can also be found. Infections with mycoviruses occur in 10% of all isolated black Aspergilli, irrespective of sampling site, mitochondrial background or year of sampling.

132. Heterokaryon incompatibility blocks virus transfer among natural isolates of black Aspergilli.

Anne D. van Diepeningen, Alfons J.M.Debets and Rolf F. Hoekstra. Department of Genetics, Agricultural University Wageningen, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands.

Somatic (also heterokaryon or vegetative) incompatibility in black Aspergillus strains was examined using nitrate-nonutilizing mutants selected on chlorate medium. Pairings of complementary mutants showed that in natural populations of the asexual black Aspergilli, somatic incompatibility between different strains is the rule. Among strains that appear related on basis of mitochondrial RFLP classification and (sometimes) mycovirus patterns, even isolated from the same site, rarely somatic compatibity was observed. Moreover, even heterokaryon self-incompatibility occurs. Mycoviruses are present in a considerable fraction of the sampled natural population, but surprisingly horizontal transfer of mycoviruses is strongly limited - at least under laboratory conditions - to the rare compatible combinations of strains. Thus, unlike in other fungal species, somatic incompatibility in black Aspergilli efficiently blocks virus transfer. Viruses present in black Aspergillus isolates are stably transmitted to asexual progeny.

133. Trans-species genetic polymorphism at the vegetative incompatibility locus(het-c) in Neurospora.

Jennifer Wu, Sven Saupe and N. Louise Glass, University of British Columbia, Canada

In Neurospora crassa, genetic control of vegetative incompatibility results from genetic differences at one or more het loci. Eleven het loci have been identified so far in Neurospora crassa. The het-c locus has been molecularly characterized and encodes a 966 aa glycine-rich polypeptide. Specificity at het-c is mediated by at least three different allelic types (het-COR - type, het-cPA - type and het-CEM - type). A variable domain of 27 - 34 aa in HET-c, HET-CPA and HET-CEM was sufficient to confer allelic specificity. Sequences of the specificity region from isolates of eight species of Neurospora and four strains of Gelasinospora have been determined and analyzed. Pairwise and maximum-likelihood comparison of these alleles reveals two interesting features: (I)These alleles are highly conserved over a range of divergent taxa. Ancient allelic polymorphisms have been maintained among contemporary species. (2) Interspecific similarities at het-c are greater than intraspecific similarities. The data suggest that in Neurospora, the existence of genetic polymorphisms associated with the specificity region of het-c predates speciation and also the origin of genus.