Asexual Recombination Research Articles

Strain Degeneration in White Button Mushroom (Agaricus bisporus) Using Amplified Fragment Length Polymorphism

Background: The growing population and limited agricultural land have pushed the demand for higher food production per land area. Edible mushrooms are a rich source of protein. Agaricus bisporus is the most common edible mushroom with high economic value. One of the most important steps in the production process of white button mushroom is the production of spawn. It is highly important to make pure high-quality mycelium during the production of spawn. Studies have shown that at this stage of production, a high level of morphological changes occurs in some mushrooms, which causes the formation of fluffy-shaped mycelium. This phenomenon is a kind of strain degeneration in edible mushrooms and is one of the biggest concerns of the producers of white button mushroom. Objectives: The purpose of this study was to investigate the nature of stroma (strain degeneration) in white button mushroom, and to identify the causes of genetic changes. Methods: In this experiment, ten successive subcultures were produced from the mother mycelium on potato dextrose agar medium (pH: 6.2), and the genetic changes of mycelium were examined after ten successive subcultures. DNA extraction was performed after sampling of the mother, normal, and fluffy mycelia. Six pairs of designed primers and propagated parts were loaded into six polyacrylamide gels. DNA fragments were visualized using silver staining. Genetic comparisons were made between the mother, normal, and fluffy mycelia by the amplified fragment length polymorphism (AFLP) method, and the results were evaluated using POPGENE software. Results: The AFLP marker produced 137 fragments for the H2 strain, of which 57 fragments had polymorphism between the mother, normal, and fluffy mycelia. For the 737 strain, 149 fragments were produced, of which 73 fragments had polymorphism between the mother, normal, and fluffy mycelia. On average, the mother, normal, and fluffy mycelia had about 42 and 49% polymorphism for the H2 and 737 strains, respectively. The degree of polymorphism among the samples confirmed genetic differences between the mother, normal, and fluffy mycelia. Conclusions: Based on the results of the present study, we concluded that strain degeneration, which manifests as the fluffy mycelium of white button mushroom, has a genetic origin. These genetic differences can be attributed to diverse factors, such as the phenomenon of heterokaryosis, asexual recombination, and the formation of mitosis crossing over. These factors and their effects require further evaluations.

Genetic diversity of plant pathogen valsa sordida using microsatellite markers

The genetic diversity of Valsa sordida isolates from different geographical regions and hosts was investigated using MP-PCR markers. PCR amplifications were done using eight primers. Of them, only four primers [(ATC)7, (ACTG)4, (CGA)5, and (AAC)8] produced polymorphic bands. At least 88.5% polymorphism was revealed by four primers and the maximum polymorphism (97%) was generated by (ACTG)4 primer and three diagnosable groups (1, 2 and 3) were resolved in the resulting dendrogram constructed by the UPGMA algorithm. The results showed high polymorphism among the isolates and confirmed the merit and accuracy of the MP-PCR markers for studying the genetic variability of V. sordida isolates at the intra-species level. We have not found any correlations between observed genetic diversity and the geographical region or host plant of the isolates, unless in limited cases. The abundant formation of the sexual state of the fungus in the infected parts of trees, as well as possible asexual recombination during asexual reproduction, are suggested as influencing factors of high genetic variability among the individuals.

Investigating variability and behaviour of Colletotrichum gloeosporioides strains from lesions of coffee blister spot

AbstractCoffee blister spot has been associated with species from the Colletotrichum genus, but there is no information on the variability of isolates present on leaf lesions. This study evaluated a population of Colletotrichum gloeosporioides strains from blister spot lesions in Coffea arabica. Colletotrichum spp. isolates were collected from blister spot lesions on leaves of coffee trees from Catuaí and Topázio cultivars (Coffea arabica). Monosporic cultures were obtained from colonies with sporulation. A pathogenicity test was carried out by inoculation of pathogens on the leaves of young coffee plants. C. gloeosporioides strains were characterized by morphologial, cytological and physiological analyses. The molecular analysis was carried out using Inter‐Retrotransposon Amplified Polymorphism (IRAP) markers. C. gloeosporioides strains showed no pathogenicity on coffee plants and presented a wide variability in all traits evaluated. The presence of sexual strains, formation of CATs (conidial anastomosis tubes) among conidial strains and high mycelial compatibility among strains observed suggest the occurrence of sexual and asexual recombination. The role of these C. gloeosporioides strains on the lesions of coffee plant leaves is unclear.

Genotyping-by-Sequencing Reveals Fine-Scale Differentiation in Populations of Pseudoperonospora humuli.

Pseudoperonospora humuli is the causal agent of downy mildew of hop, one of the most important diseases of this plant and a limiting factor for production of susceptible cultivars in certain environments. The degree of genetic diversity and population differentiation within and among P. humuli populations at multiple spatial scales was quantified using genotyping-by-sequencing to test the hypothesis that populations of P. humuli have limited genetic diversity but are differentiated at the scale of individual hop yards. Hierarchical sampling was conducted to collect isolates from three hop yards in Oregon, plants within these yards, and infected shoots within heavily diseased plants. Additional isolates also were collected broadly from other geographic regions and from the two previously described clades of the sister species, P. cubensis. Genotyping of these 240 isolates produced a final quality-filtered data set of 216 isolates possessing 25,227 variants. Plots of G'ST values indicated that the majority of variants had G'ST values near 0 and were scattered randomly across contig positions. However, there was a subset of variants that were highly differentiated (G'ST > 0.3) and reproducible when genotyped independently. Within P. humuli, there was evidence of genetic differentiation at the level of hop yards and plants within yards; 19.8% of the genetic variance was associated with differences among yards and 20.3% of the variance was associated with plants within the yard. Isolates of P. humuli were well differentiated from two isolates of P. cubensis representative of the two clades of this organism. There was strong evidence of linkage disequilibrium in variant loci, consistent with nonrandom assortment of alleles expected from inbreeding and/or asexual recombination. Mantel tests found evidence that the genetic distance between isolates collected from heavily diseased plants within a hop yard was associated with the physical distance of the plants from which the isolates were collected. The sum of the data presented here indicates that populations of P. humuli are consistent with a clonal or highly inbred genetic structure with a small, yet significant differentiation of populations among yards and plants within yards. Fine-scale genetic differentiation at the yard and plant scales may point to persistence of founder genotypes associated with planting material, and chronic, systemic infection of hop plants by P. humuli. More broadly, genotyping-by-sequencing appears to have sufficient resolution to identify rare variants that differentiate subpopulations within organisms with limited genetic variability.

Molecular Characterization of Australian Isolates of Puccinia graminis f. sp. tritici Supports Long-Term Clonality but also Reveals Cryptic Genetic Variation.

Long-term surveys of pathogenicity in Puccinia graminis f. sp. tritici in Australia have implicated mutation as a major source of virulence, at times leading to the demise of stem-rust-resistant wheat cultivars and substantial yield losses. Since 1925, these surveys have identified at least four occasions on which exotic isolates of P. graminis f. sp. tritici appeared in Australia, with each acting as a founding isolate that gave rise sequentially to derivative pathotypes via presumed single-step mutation. The current study examined the relationship between virulence and molecular patterns using simple-sequence repeat (SSR) markers on selected isolates of P. graminis f. sp. tritici collected in Australia during a 52-year period in order to propose an evolutionary pathway involving these isolates. Studies of SSR variability among this collection of isolates within a putative clonal lineage based on pathotype 21-0, first detected in 1954 (the "21/34 lineage"), provided compelling evidence of clonality over the 52-year period, coupled with single-step acquisition of virulence for resistance genes. It also supported the postulation that two triticale-attacking pathotypes (34-2,12 and 34-2,12,13) detected in the early 1980s were derived from pathotype 21-0 via stepwise sequential acquisition of virulence for Sr5, Sr11, Sr27, and then SrSatu. Some of the isolates examined that were regarded as members of the race 21/34 lineage based on pathogenicity differed significantly in their SSR genotypes, indicating that they may have originated from processes more complex than simple mutation. This included two isolates of pathotype 21-0, which were collected in 1994 and 2006. Given that sexual recombination in P. graminis is rare or absent in Australia, the cryptic complexity observed could indicate that one or more of these isolates arose as a consequence of asexual recombination.

Suppressor of fusion, a Fusarium oxysporum homolog of Ndt80, is required for nutrient-dependent regulation of anastomosis

Heterokaryon formation is an essential step in asexual recombination in Fusarium oxysporum. Filamentous fungi have an elaborate nonself recognition machinery to prevent formation and proliferation of heterokaryotic cells, called heterokaryon incompatibility (HI). In F. oxysporum the regulation of this machinery is not well understood. In Neurospora crassa, Vib-1, a putative transcription factor of the p53-like Ndt80 family of transcription factors, has been identified as global regulator of HI. In this study we investigated the role of the F. oxysporum homolog of Vib-1, called Suf, in vegetative hyphal and conidial anastomosis tube (CAT) fusion and HI. We identified a novel function for an Ndt80 homolog as a nutrient-dependent regulator of anastomosis. Strains carrying the SUF deletion mutation display a hyper-fusion phenotype during vegetative growth as well as germling development. In addition, conidial paring of incompatible SUF deletion strains led to more heterokaryon formation, which is independent of suppression of HI. Our data provides further proof for the divergence in the functions of different members Ndt80 family. We propose that Ndt80 homologs mediate responses to nutrient quality and quantity, with specific responses varying between species.

Anastomosis groups and molecular variation in Pseudocercospora griseola.

The fungus Pseudocercospora griseola is the causal agent of angular leaf spot, a common bean (Phaseolus vulgaris L.) disease. The successful development of angular leaf spot-resistant cultivars depends on understanding the levels of variability in pathogen populations. P. griseola shows great pathogenic and genetic variation using inoculation on differential bean cultivars and molecular markers. Nevertheless, how this variability develops is not clearly understood. Parasexuality has been presented as a mechanism used by asexual fungi to increase variation. Hyphal fusion is the first step for the parasexual cycle, and it can be considered an additional trait for population studies. The aim of this study was to identify hyphal fusion (anastomosis) among P. grisola isolates and to evaluate the variability of isolates using analyses of anastomosis groups and RAPD markers. Hyphal anastomosis was observed in all isolates. Three isolates showed 85.0% compatibility and were compatible with 17 isolates. This is the first report of the occurrence of anastomosis between P. griseola isolates. Sixteen anastomosis groups were observed and only one group was formed by five isolates (Pg-01, Pg-02, Pg-07, Pg-08, and Pg-12). There was a large number of anastomosis groups and absence of clustering among isolates for hyphal fusion, demonstrating the existence of genetic variability for loci involved in the control of this trait. High genotypic variability by RAPD markers was detected, but there was no relationship between genotype and anastomosis group. More studies are necessary to elucidate further the process of asexual recombination in this phytopathogen.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Recovery and characterization of asexual recombinants ofMagnaporthe grisea

Eleven nitrate non-utilizing (nit) mutants were recovered from six field isolates ofMagnaporthe grisea that had different degrees of sensitivity to the blasticide kitazin P (iprobenfos, IBP). Allnit mutants were resistant to chlorate and there were no significant differences in hyphal growth rate, conidial production, sensitivity to IBP, and pathogenicity betweennit mutants and their parent isolates.nit phenotypes and IBP-resistance were used as two independent genetic markers to study asexual recombination inM. grisea. Asexual recombinants were recovered from the heterokaryotic mycelium of three inter-strain pairings, namely, DY2-3(nitl-LR) + A7-3 (nitM-S); A7-3 (nitM-S) + F4-2 (nitl-MR); and F4-2 (nitl-MR) + DY2-4 (nitA-LR), at a frequency of 7.31%, 14.00% and 8.63%, respectively. The growth rate, conidial production and pathogenicity of asexual recombinants were similar to those of parental strains. We concluded that asexual recombination resulting from hyphal fusion might contribute to variability inM. grisea.

Population structure and mating-type genes of Colletotrichum graminicola from Agrostis palustris.

Eighty-seven isolates of Colletotrichum graminicola, mostly from Agrostis palustris, were collected in grass fields, most of which were in Ontario, Canada. Specific primers were designed to amplify the mating-type (MAT) genes and, among 35 isolates tested, all yielded a band of the expected size for MAT2. For six isolates, the MAT2 PCR products were sequenced and found to be similar to that reported for MAT2 of C. graminicola from maize. Based on 119 polymorphic bands from 10 random amplified polymorphic DNA primers, analyses of genetic distances were found to generally cluster isolates by host and geographic origin. Among 42 isolates from a grass field in Ontario, significant spatial autocorrelation was found to occur within a 20-m distance, implying that this is the effective propagule dispersal distance. Although clonal propagation was observed in the 87 isolates with 67 unique genotypes, the extent of genetic variation in local populations implies some occurrence of sexual or asexual recombination.

Characterisation of Isolates of Phytophthora infestans From Hungary

A total of 36 single-lesion isolates were collected from 9 crops of potato and 13 of tomato in different regions of Hungary in the past decade, particularly in 1998. These were analysed for mating type, sensitivity to metalaxyl, allozyme genotype at glucose-6-phosphate isomerase and peptidase loci and genotype at 24 loci detected using the multilocus RFLP probe RG57. The ratios of the mating types A1 to A2 were 8 : 9 and 4 : 15 among isolates recovered from potato and tomato, respectively. Resistance to metalaxyl was found more frequently among isolates from potato and in the A1 mating type. The populations were not clearly differentiated on the basis of host origin. All isolates were homozygous (100/100) at the locus for glucose-6-phosphate isomerase. Unlike in other European countries, the most common peptidase allele was 96. Genotypes at the peptidase locus were 96/96 (50%), 96/100 (27.7%) and 100/100 (16.6%). In addition, one isolate from 1993 and another from 1998, were defined as 83/96, a genotype that had not been described elsewhere. The 18 RG57 fingerprints that were observed among 36 isolates, with one exception, seem to be unique to Europe. On the basis of combined genotypic traits, 20 multilocus genotypes were designated. These data, which reveal a remarkable variability with unique genotypes of the late blight pathogen, suggest that migration and sexual and/or asexual recombination have a role in the recent evolution of the pathogen in Hungary.

Characterization of Pyricularia grisea in the United States Using Independent Genetic and Molecular Markers

ABSTRACT A total of 540 isolates of Pyricularia grisea from rice in the United States were examined for vegetative compatibility, MGR586 DNA fingerprint diversity, and mating type based on hybridization with the mat1-1 and mat1-2 sexual mating type alleles. The collections contained both archived and contemporary field isolates representative of the known MGR586 lineages and races that occur throughout the United States. Complementary nitrate nonutilizing (nit) or sulfate nonutilizing (sul) mutants were used to assess vegetative compatibility in P. grisea. There was a complete correspondence between vegetative compatibility groups (VCGs), MGR586 lineage, and mating type among 527 contemporary isolates (collected between 1991 and 1997) from Arkansas, Louisiana, Missouri, Mississippi, and Texas; all isolates in MGR586 lineages A, B, C, and D belonged to VCGs US-01, US-02, US-03, and US-04, respectively. In addition, all isolates tested in VCGs US-01 and US-04 had the mat1-1 mating type allele whereas those in VCGs US-02 and US-03 had the mat1-2 allele. The strict association of independent markers during this sample period was consistent with a strictly asexual mode of reproduction. However, examination of archived isolates collected in the 1970s and 1980s and contemporary isolates revealed an incongruent relationship between the independent markers. MGR586 C and E isolates were vegetatively compatible which indicated that multiple robust MGR586 delineated lineages could be nested within certain VCGs. Although isolates in lineages C and E were vegetatively compatible, they were of opposite mating type. Several hypotheses, including recombination, could account for the incongruence between the various markers. Among the eight MGR586 lineages (A through H) that occur in the United States, all isolates in lineages A, D, E, G, and H had the mat1-1 allele, whereas isolates in lineages B, C, and F had the mat1-2 allele. Nit mutants can be recovered relatively easy from P. grisea and should allow large numbers of individuals within a population to be assessed for vegetative compatibility. VCGs may prove to be an effective multilocus marker in P. grisea. Thus, VCGs should be a useful means for characterizing genetic structure in populations of the rice blast fungus worldwide, provide a useful genetic framework to assist in interpreting molecular population data, and may provide insight into potential sexual or asexual recombination events.

Population structure ofGibberella pulicaris (AnamorphFusarium sambucinum) from potato tuber dry rot in North America and Europe

Gibberella pulicaris is the sexual stage ofFusarium sambucinum, the major cause of potato tuber dry rot in North America and Europe. In a previous study 42 strains from dry-rotted tubers in North America were analyzed for genetic diversity by mating type and vegetative compatibility group (VCG) tests. In the present study, an additional 96 strains from North America, 21 strains from Europe, and six strains from other geographic areas were analyzed. Both mating types, MAT1-1 and MAT1-2, occur widely outside North America. In contrast, all but two, Canadian, strains of the 138 North American strains analyzed to date are MAT1-1. VCG analysis provides additional evidence that genetic diversity ofG. pulicaris from potato tubers is high in Europe but low in the United States. These results suggest that the potential for pathogen change due to sexual recombination is low in the United States, but may be higher in Europe. Limited genetic diversity ofG. pulicaris in the United States indicates that there are likely to be few barriers to heterokaryon formation and asexual recombination in the field. Thus, the potential exists for rapid spread and persistence of advantageous mutations throughout theG. pulicaris population, which may have contributed to the recent proliferation of strains resistant to the fungicide thiabendazole which has been widely used to control tuber dry rot.

Genetic structure of the scald pathogen (Rhynchosporium secalis) in South East Australia: implications for control strategies

A survey of electrophoretic variation in 89 isolates of Rhynchosporium secalis collected from cultivated and wild barley grass in Victoria, New South Wales and Tasmania detected an average of 2.5 alleles at each of 11 loci in 5 enzyme systems. At five loci, two alleles each occurred at frequencies exceeding 0.2. Comparison of the frequency of alleles at these loci showed little association with host or geographic origin, pathogenicity of the isolates or with the allelic state at other loci. Given that this pathogen reproduces asexually only, this lack of association confirms previous suggestions that some means of asexual recombination is important in this pathogen. Furthermore, the results suggest that the most effective means whereby long-term control over R. secalis will be achieved is through the use of both pathotype-specific and field resistance complemented by other strategies like the use of fungicides and possibly varietal mixtures.

Evidence of asexual recombination in Rhynchosporium secalis

Three single‐spore isolates of Rhynchosporium secalis that differed in their α‐esterase and β‐glucosidase isozyme patterns were inoculated as two mixtures, each of two isolates, on to seedlings of the susceptible barley cultivar Maris Mink. Approximately 100 single‐spore isolates were taken from mature lesions produced by each mixture. These were subjected to polyacrylamide gel electrophoresis and the gels were stained for α‐esterase and β‐glucosidase. Parental types only were produced by one of the isolate mixtures. However, one of the 10 lesions examined for the second mixture produced nine isolates with a novel combination of isozymes, indicating that some form of asexual recombination had occurred. The use of isozymes as a natural marker system for the detection in vivo of asexual recombination in pathogenic fungi is discussed.

Mutants of Puccinia graminis avenae induced by Ethyl Methane Sulphonate

SPONTANEOUS mutations affecting uredospore colour in cereal rusts have been observed by several investigators. The mode of inheritance of the mutant colours in the wheat stem rust fungus Puccinia graminis tritici E. and H. and oat stem rust fungus P. graminis avenae E. and H. has been shown to follow a simple Mendelian pattern1,2 and such mutants serve as particularly useful markers in investigations of the role of asexual recombination in this group of fungi. In an attempt to produce uredospore colour mutants in the oat stem rust organism for this purpose, the mutagenic effect of the alkylating agent ethyl methane sulphonate (EMS) was investigated following its success in this respect in such widely diverse organisms as bacteriophage4 and barley5.

Parasexual recombination in the banana-wilt fusarium

Three strains of F. oxysporum capable of wilting the banana cultivar ‘Gros Michel’ (Musa-AAA group) were irradiated with ultraviolet light to produce genetic markers that allowed them to be identified in culture by nutritional deficiencies. Heterokaryons that formed between the strains produced a few prototrophic spores with wild-type growth rates when compared with the parent mutant cultures. Progeny from 3036 subcultures of the prototrophic, and presumed diploid, conidia could be divided into three classes: daughter diploids (99·3%), haploid parentals (0·2%) and recombinant haploids (0·5%). Further recombination occurred with additional genetic markers that differentiated the strains by their differences in haemagglutinating properties with human blood group O and by differences in pathogenicity for M. balbisiana. The banana-wilt Fusarium can undergo asexual recombination by a method similar in every respect to the parasexuality that has been demonstrated in certain other imperfect fungi. The implications of this in relation to banana breeding for wilt resistance and to problems of cultural variation in Fusaria from banana are discussed.