Isolates Of Mating Type Research Articles

Sex Pheromone Receptor Ste2 Orchestrates Chemotropic Growth towards Pine Root Extracts in the Pitch Canker Pathogen Fusarium circinatum

In ascomycetous fungi, sexual mate recognition requires interaction of the Ste2 receptor protein produced by one partner with the α-factor peptide pheromone produced by the other partner. In some fungi, Ste2 is further needed for chemotropism towards plant roots to allow for subsequent infection and colonization. Here, we investigated whether this is also true for the pine pitch canker fungus, Fusarium circinatum, which is a devastating pathogen of pine globally. Ste2 knockout mutants were generated for two opposite mating-type isolates, after which all strains were subjected to chemotropism assays involving exudates from pine seedling roots and synthetic α-factor pheromone, as well as a range of other compounds for comparison. Our data show that Ste2 is not required for chemotropism towards any of these other compounds, but, in both wild-type strains, Ste2 deletion resulted in the loss of chemotropism towards pine root exudate. Also, irrespective of mating type, both wild-type strains displayed positive chemotropism towards α-factor pheromone, which was substantially reduced in the deletion mutants and not the complementation mutants. Taken together, these findings suggest that Ste2 likely has a key role during the infection of pine roots in production nurseries. Our study also provides a strong foundation for exploring the role of self-produced and mate-produced α-factor pheromone in the growth and overall biology of the pitch canker pathogen.

Temporal and Spatial Variation in the Population Structure of Spanish Fusarium circinatum Infecting Pine Stands

Fusarium circinatum is an introduced fungal pathogen extended to the northern regions of Spain that causes Pine Pitch Canker (PPC) disease. In this work, we analyzed the pathogen's genetic diversity to study changes over time and space since the first outbreak occurred in Spain. Using six polymorphic SSR markers, 15 MLGs were identified in 66 isolates, and only three haplotypes were found with frequencies higher than one. In general, genotypic diversity was low and decreased shortly over time in the northwestern regions while maintained at País Vasco, where only one haplotype (MLG32) was detected 10 years. This population also included isolates of a single mating type (MAT-2) and VCGs identified in only two groups, while isolates from NW regions were of both mating types and VCGs represented in 11 groups. The existence of haplotype MLG32 maintained on time and widely distributed suggests its good adaptation to the environment and the host. Results showed that the pathogen in País Vasco remains clearly differentiated from other northwestern populations. This fact was supported with no evidence of migration among regions. Results are explained by the asexual reproduction, but also selfing at least to a lesser extent that leads to identification of two new haplotypes.

Phytophthora × cambivora as a Major Factor Inciting the Decline of European Beech in a Stand within the Southernmost Limit of Its Natural Range in Europe.

The objective of this study was to investigate the role of the oomycete Phytophthora × cambivora in the decline affecting European beech (Fagus sylvatica) in the Nebrodi Regional Park (Sicily, southern Italy). In a survey of a beech forest stand in the heart of the park, Phytophthora × cambivora was the sole Phytophthora species recovered from the rhizosphere soil and fine roots of trees. Both A1 and A2 mating type isolates were found. Direct isolation from the stem bark of trees showing severe decline symptoms and bleeding stem cankers yielded exclusively P. gonapodyides, usually considered as an opportunistic pathogen. The mean inoculum density of P. × cambivora in the rhizosphere soil, as determined using the soil dilution plating method and expressed in terms of colony forming units (cfus) per gm of soil, the isolation frequency using leaf baiting, and the percentage of infected fibrous roots from 20 randomly selected beech trees with severe decline symptoms (50 to 100 foliage transparency classes) were 31.7 cfus, 80%, and 48.6%, respectively. These were significantly higher than the corresponding mean values of 20 asymptomatic or slightly declining trees, suggesting P. × cambivora is a major factor responsible for the decline in the surveyed stand.

Phylogenetic Analysis of Phaeosphaeria Species Using Mating Type Genes and Distribution of Mating Types in Iran.

Phaeosphaeria species are pathogenic on wheat, barley and a wide range of wild grasses. To analyze mating type loci of the Phaeosphaeria species and investigate mating type distribution in Iran, we sequenced mating type loci of 273 Phaeosphaeria isolates including 67 isolates obtained from symptomatic leaves and ears of wheat, barley, and wild grasses from two wheat-growing region in Iran as well as 206 isolates from our collection from other regions in Iran which were isolated in our previous studies. Mating type genes phylogeny was successfully used to determine the species identity and relationships among isolates within the Phaeosphaeria spp. complex. In this study, we reported seven new host records for Phaeosphaeria species and the Phaeosphaeria avenaria f. sp. tritici 3 group was first reported from Iran in this study. Mating type distribution among Phaeosphaeria species was determined. Both mating types were present in all sampling regions from Iran. We observed skewed distribution of mating types in one region (Kohgiluyeh va Boyer-Ahmad) and equal distribution in the other region (Bushehr). However, when considering our entire dataset of 273 Iranian Phaeosphaeria isolates, the ratio of mating types was not deviated significantly from 1:1 suggesting possibilities for isolates of opposite mating type to interact and reproduce sexually, although the sexual cycle may infrequently occur in some regions especially when the climatic conditions are unfavorable for teleomorph development.

Mating Type and Aggressiveness of Phytophthora infestans (Mont.) de Bary in Potato-Growing Areas of Punjab, Pakistan, 2017\u20132018 and Identification of Genotype 13_A2 in 2019\u20132020

Samples of blighted potato leaves were collected from fields in six potato-growing districts of the Punjab Province of Pakistan in 2017–2018. A total of 149 isolates of Phytophthora infestans were obtained from six potato cultivars (Asterix, Barsenna, Burana, Caroda, Mazika, Sante). Of these isolates, 73% were A1 mating type, 23% were A2 mating type and 4% were self-fertile. Both mating types of P. infestans occurred in all six districts sampled, but in every case, the A1 mating type predominated. The foliar aggressiveness of 104 of these isolates (weakly pathogenic isolates were excluded) was tested on detached leaflets of potato cv. Caroda, and a composite aggressiveness index (CAI) calculated from lesion area (measured after 10 days), latent period and infection frequency was used to compare isolates. There was no difference in CAI between isolates obtained from different districts or cultivars. The A2 mating type isolates had significantly greater CAIs than A1 isolates but this does not imply a genetic linkage nor a general association between mating type and aggressiveness. It may be that the A2 isolates belonged to an aggressive clonal lineage such as 13_A2 that has been reported in other countries in the region. While it was not possible to test the isolates collected in 2017–2018, genotyping of samples collected in 2019–2020 showed the widespread occurrence of the EU_13_A2 clonal lineage in the same districts of Pakistan and supported this hypothesis. This is the first report of EU_13_A2 from Pakistan. The implications for potato late blight management in the Punjab are discussed.

Characterization of Natural Isolates of Bipolaris maydis Associated with Mating Types, Genetic Diversity, and Pathogenicity in Fujian Province, China.

Due to the natural destructiveness and persistence of the southern corn leaf blight (SCLB) fungus Bipolaris maydis (Nisikado et Miyake) Shoem, the characterization of B. maydis field isolates is essential to guide the rational distribution of resistant materials in corn-growing regions. In the present study, 102 field isolates collected from seven locations covering the entire region of Fujian Province, China, were assessed for mating type distribution, genetic diversity, and pathogenicity toward local sweet corn cultivars. Mating type detection via polymerase chain reaction indicated that 36.3 and 63.7% of isolates were MAT1-1 and MAT1-2, respectively; more than 80% of these isolates were confirmed using cross assays with known mating type isolates. Thirteen intersimple sequence repeat (ISSR) markers within and among two mating type populations revealed a high level of DNA polymorphism for all combined isolates and between MAT1-1 and MAT1-2 populations. The MAT1-2 population was more diverse based on DNA polymorphism than the MAT1-1 population. The value of GST was 0.0070, ranging from 0.0399 to 0.3044 based on analysis of combined isolates and individual regional populations, respectively, suggesting the presence of genetic differentiation in the two mating type populations from different locations. Pathogenicity assays revealed that both MAT1-1 and MAT1-2 populations were pathogenic to all 11 local sweet corn cultivars tested in this study. The potential of sexual reproduction, existence of genetic diversity in the two mating type populations, and pathogenicity suggest that B. maydis populations have independently clonally adapted under natural field conditions during corn cultivation.

A Microsatellite Analysis Used to Identify Global Pathways of Movement of Phytophthora cinnamomi and the Likely Sources of Wildland Infestations in California and Mexico.

The genetic structure of a sample of isolates of the oomycete plant pathogen Phytophthora cinnamomi from natural and agricultural outbreaks and the long-distance movement of individual genotypes were studied using four microsatellite markers to genotype 159 isolates of Californian, Mexican, and worldwide origins. Allelic profiles identified 75 multilocus genotypes. A STRUCTURE analysis placed them in three groups characterized by different geographic and host ranges, different genic and genotypic diversity, and different reproductive modes. When relationships among genotypes were visualized on a minimum spanning network (MSN), genotypes belonging to the same STRUCTURE group were contiguous, with rare exceptions. A putatively ancestral group 1 had high genic diversity, included all A1 mating type isolates and all Papuan isolates in the sample, was rarely isolated from natural settings in California and Mexico, and was positioned at the center of the MSN. Putatively younger groups 2 and 3 had lower genic diversity, were both neighbors to group 1 but formed two distinct peripherical sectors of the MSN, and were equally present in agricultural commodities and natural settings in Mexico and California. A few genotypes, especially in groups 2 and 3, were isolated multiple times in different locations and settings. The presence of identical genotypes from the same hosts in different continents indicated that long-distance human-mediated movement of P. cinnamomi had occurred. The presence of identical genotypes at high frequencies in neighboring wildlands and agricultural settings suggest that specific commodities may have been the source of recent wild infestations caused by novel invasive genotypes.

Population Genetic Analysis of Phytophthora parasitica From Tobacco in Chongqing, Southwestern China.

Tobacco black shank, caused by Phytophthora parasitica, is one of the most notorious tobacco diseases and causes huge economic losses worldwide. Understanding the genetic variation of P. parasitica populations is essential to the development of disease control measures. In this research, 210 simple sequence repeat (SSR) markers for P. parasitica were identified, 10 of which were polymorphic among nine reference strains. We further performed population genetic analysis of 245 P. parasitica isolates randomly collected from tobacco fields in Chongqing for mating type, molecular variation at 14 SSR loci (four of which were identified previously), and sensitivity to the fungicide metalaxyl. The results showed that the A2 mating type was dominant and no A1 mating type isolate was discovered. SSR genotyping distinguished 245 P. parasitica isolates into 46 genotypes, four of which were dominant in the population. Low genotypic diversity and excess heterozygosity were common in nearly all of the populations from Chongqing. Population analysis showed that no differentiation existed among different populations. All isolates tested were highly sensitive to metalaxyl. Taken together, our results showed that the P. parasitica populations from tobacco fields in Chongqing belonged to a clonal lineage and were highly sensitive to metalaxyl.

REACTION OF POTATO CULTIVARS AND CLONES TO Phytophthora infestans

ABSTRACT The in vitro and field reactions of 23 potato cultivars and clones to two isolates of Phytophthora infestans were tested. The in vitro test used the detached potato leaflets method, and disease severity was evaluated 6 days after inoculation. Field experiments were conducted in autumn of 2013, in two locations in Pelotas City, Rio Grande do Sul State, Brazil. Seven evaluations of the disease severity were performed. From the severity values, the area under the disease progress curve (AUDPC) was calculated. Tubers were harvested to determine the yield (g plot-1). The A2 mating type isolate was the most aggressive for the majority of the potato genotypes. It was possible to group the different reactions of the potato genotypes based on the AUDPC values. Clones C2553-1-06, C2573-4-06, C2550-4-06, C2551-2-06, and CIP392,617.54 showed moderate-to-high resistance to the two isolates of P. infestans. The values of severity and AUDPC obtained in vitro and field tests were positively correlated for both P. infestans isolates, but in some interactions, inconsistencies were observed. Tuber yield was negatively correlated with AUDPC.

Race Characterization of Phytophthora Root Rot on Capsicum in Taiwan as a Basis for Anticipatory Resistance Breeding.

Peppers (Capsicum sp.) are an increasingly important crop because of their use as a vegetable, spice, and food colorant. The oomycete Phytophthora capsici is one of the most devastating pathogens to pepper production worldwide, causing more than $100 million in losses annually. Developing cultivars resistant to P. capsici is challenging because of the many physiological races that exist and new races that are continuously evolving. This problem is confounded by the lack of a universal system of race characterization. As a basis to develop a global anticipatory breeding program, New Mexico recombinant inbred lines (NMRILs) functioned as a host differential for Phytophthora root rot to characterize the race structure of P. capsici populations in Taiwan. Using the NMRILs, 24 new races were identified, illustrating the utility and usefulness of the NMRILs for anticipatory breeding. Virulence of P. capsici was observed to be geographically specific and in two virulence clusters. Interestingly, all but two isolates collected in 2016 were the A2 mating type, which is a shift from the predominantly A1 mating type isolates collected prior to 2008. The NMRILs host differential provides an approach for scientists to work together on a global scale when breeding for resistance as well as on a local level for regional gene deployment. Additionally, we propose that the current race numbering system, which has no biological meaning, be supplemented with the virulence phenotype, based on the susceptible NMRILs to a given isolate. This work provides insights into the population dynamics of P. capsici and interactions within the highly complex Capsicum-Phytophthora pathosystem, and offers a basis for similar research in other crops.

Whole-Genome Analysis Illustrates Global Clonal Population Structure of the Ubiquitous Dermatophyte Pathogen Trichophyton rubrum.

Dermatophytes include fungal species that infect humans, as well as those that also infect other animals or only grow in the environment. The dermatophyte species Trichophyton rubrum is a frequent cause of skin infection in immunocompetent individuals. While members of the T. rubrum species complex have been further categorized based on various morphologies, their population structure and ability to undergo sexual reproduction are not well understood. In this study, we analyze a large set of T. rubrum and T. interdigitale isolates to examine mating types, evidence of mating, and genetic variation. We find that nearly all isolates of T. rubrum are of a single mating type, and that incubation with T. rubrum “morphotype” megninii isolates of the other mating type failed to induce sexual development. While the region around the mating type locus is characterized by a higher frequency of SNPs compared to other genomic regions, we find that the population is remarkably clonal, with highly conserved gene content, low levels of variation, and little evidence of recombination. These results support a model of recent transition to asexual growth when this species specialized to growth on human hosts.

Evaluation\xa0of PCR markers for Phytophthora infestans mating type determination

Late blight is a devastating potato disease caused by Phytophthora infestans. This organism can reproduce asexually and sexually between the strains of two mating types named A1 and A2. Mating type is an important strain characteristic affecting the pathogen’s population structure. We validate different PCR markers for P. infestans mating type determination by comparison of the results obtained with the markers (W16, S1, PHYB) with the pairing test results for 26 isolates collected worldwide and for a group of 146 Polish isolates. This study identifies an interesting feature of the isolates of genotype US-1. For all these A1 mating type isolates, the product specific for A2 isolates is amplified using the marker W16. Analysis of sequences of W16 PCR products indicates high similarity of the US-1 isolates with modern A2 mating type isolates. When US-1 isolates are excluded from analysis, 95 and 96% of isolates are correctly assigned by markers W16 and S1, respectively, when compared with the pairing test results. Marker PHYB produces 14% of discrepant results with the pairing test. Our results show that molecular markers can be useful tools for P. infestans mating type determination, but their application should be preceded by validation in each local population since their efficiency may vary depending on a pathogen’s genotype.

First Report of Phytophthora infestans Self-Fertile Genotypes in Southern Brazil

During the autumn of 2011 and 2012, leaf and stem samples of potato plants infected with P. infestans were collected in southern Brazil. Monosporangial isolates of P. infestans were obtained from the samples and mating types were determined. Each isolate was paired with the A2 and A1 mating types of P. infestans and self-paired on Petri plates containingV8 juice agar. After this, the plates were kept at 18°C in darkness for 20 days. Three isolates collected in Parana State (municipalities of Ponta Grossa 25°12′26″S, 50°07′11″W; Contenda 25°38′52″S, 49°34′26″W; and Castro 24°51′50″S, 49°53′50″W) formed oospores when paired with both mating type isolates and also when self-paired.

English

In this study, the genetic diversity and structure of 90 monosporidia mating-type isolates of Sporisorium scitamineum collected from China were comprehensively evaluated based on the DNA extracted from mating-type haploid sporidias using inter-simple sequence repeat (ISSR) polymorphism molecular marker technique. Fifteen selected ISSR primers produced 162 amplified DNA fragments, of which 115 (71.0%) were polymorphic. Polymorphic information content (PIC) values ranged from 0.12 to 0.92 with an average of 0.82, indicating that a moderate degree of genetic diversity exists among these 90 monosporidia mating-type isolates of S. scitamineum collected from China. Genetic differentiation coefficient (Gst) was calculated to be 0.320, indicating that the total genetic variation between populations was 32%, while 68% of the total genetic variation was from within populations. Meanwhile, gene flow (Nm) was 1.063, indicating lower rate of gene flow between populations. Based on the ISSR marker data set, the results of clustering by the unweighted pair group method of arithmetic average and principal component analysis were similar and could divide these 90 S. scitamineum isolates into two main groups. The isolates collected from the same province tend to cluster in the same main groups. This study shows that the genetic differentiation of these 90 monosporidia mating-type isolates of S. scitamineum was highly correlated with the ecological environment of collection sites, and the heterogeneity of ecological environment was the main driving force for the differentiation of S. scitamineum.   Key words: Sugarcane, Sporisorium scitamineum, mating-type haploid sporidia, inter-simple sequence repeat, genetic diversity.

Temporal Genetic Dynamics of an Experimental, Biparental Field Population of Phytophthora capsici.

Defining the contributions of dispersal, reproductive mode, and mating system to the population structure of a pathogenic organism is essential to estimating its evolutionary potential. After introduction of the devastating plant pathogen, Phytophthora capsici, into a grower’s field, a lack of aerial spore dispersal restricts migration. Once established, coexistence of both mating types results in formation of overwintering recombinant oospores, engendering persistent pathogen populations. To mimic these conditions, in 2008, we inoculated a field with two P. capsici isolates of opposite mating type. We analyzed pathogenic isolates collected in 2009–2013 from this experimental population, using genome-wide single-nucleotide polymorphism markers. By tracking heterozygosity across years, we show that the population underwent a generational shift; transitioning from exclusively F1 in 2009–2010, to multi-generational in 2011, and ultimately all inbred in 2012–2013. Survival of F1 oospores, characterized by heterozygosity excess, coupled with a low rate of selfing, delayed declines in heterozygosity due to inbreeding and attainment of equilibrium genotypic frequencies. Large allele and haplotype frequency changes in specific genomic regions accompanied the generational shift, representing putative signatures of selection. Finally, we identified an approximately 1.6 Mb region associated with mating type determination, constituting the first detailed genomic analysis of a mating type region (MTR) in Phytophthora. Segregation patterns in the MTR exhibited tropes of sex-linkage, where maintenance of allele frequency differences between isolates of opposite mating type was associated with elevated heterozygosity despite inbreeding. Characterizing the trajectory of this experimental system provides key insights into the processes driving persistent, sexual pathogen populations.

Characterization of Phytophthora infestans populations in northwestern Algeria during 2008–2014

A total of 161 Phytophthora infestans isolates, collected from infected potato and tomato plants during 2008–2014, were characterized based on mating type, metalaxyl sensitivity and polymorphism at 12 simple sequence repeat (SSR) loci, in order to investigate the population of P. infestans in the north-west of Algeria, an emerging potato production region. The majority of isolates were of A2 mating type (112 isolates). A high percentage (89 %) of resistance to metalaxyl among isolates was detected. The metalaxyl resistant phenotype was present in both mating types with a higher percentage in A2 mating type isolates. SSR-based genotypic analysis of P. infestans population showed a low diversity. Genotype 13_A2 was the predominant in the population with a frequency of 67 % followed by 2_A1 (21 %) and 23_A1 (5 %). Genotype 23_A1 was detected only in tomato and potato isolates collected in 2013 and 2014.

Genetic diversity of Sporisorium scitamineum in mainland China assessed by SCoT analysis

The genetic relationships among 90 mating-type isolates of Sporisorium scitamineum collected from mainland China were evaluated based on the start codon-targeted (SCoT) marker technique. Fourteen selected SCoT primers amplified 135 DNA fragments, of which 98 (72.6 %) were polymorphic. The polymorphic information content values ranged from 0.649 to 0.910 with an average of 0.820, indicating that a high degree of genetic diversity exists among these Chinese S. scitamineum isolates. Cluster groups identified using the unweighted pair-group method with arithmetic mean (UPGMA) and principal component analysis (PCA) were similar and divided the 90 S. scitamineum isolates into three main groups (G1, G2, and G3) based on a Jaccard’s similarity coefficient of 0.74. The S. scitamineum isolates collected from the same province tended to cluster in the same main groups. This study showed that the genetic diversity of the 90 S. scitamineum isolates was associated with their geographic origin, with no evidence of coevolution between the host and S. scitamineum. The knowledge of genetic diversity of S. scitamineum is useful in breeding programs for development of smut-resistant sugarcane genotypes. The results of this study demonstrate that SCoT markers are informative and can be used to evaluate the genetic diversity of S. scitamineum.

High genotypic diversity found among population of Phytophthora infestans collected in Estonia

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most important diseases of potato worldwide. This is the first study characterising Estonian P. infestans population using the SSR marker genotyping method. 70 P. infestans isolates collected during the growing season in 2004 from eight potato fields in three different regions of Estonia were characterised with nine polymorphic SSR markers. A1 and A2 mating type isolates were detected from every studied field indicating the high potential for sexual reproduction, which raises the genotypic diversity in P. infestans population. Results revealed highly diverse P. infestans population in Estonia resembling the Northern European populations. Most of the multilocus genotypes were detected only once among the collected isolates. Subpopulations collected from different geographical regions of Estonia showed no differentiation from each other but instead formed one highly diverse group.

Mating Types Analysis of Magnaporthe oryzae Populations by Molecular Methods

Rice blast is a devastating disease which is caused by the heterothallic fungus Magnaporthe oryzae. Compatible sexual recombination which occurs between two M. oryzae strains of different mating types, can enhance genetic variability. Assessment of mating type alleles is used as a marker to measure population diversity. Forty six isolates of M. oryzae were collected from infected rice leaves from various ecosystems of coastal Odisha, India, and the mating type analysis using molecular markers was carried out. MAT1-1 mating type was dominating in all the ecosystems and MAT1-2 was found to be present in uplands as well as in irrigated fields. Both mating types could be found in the same field in irrigated ecosystem. The disease spread was very fast vertically as well as horizontally in those fields resulting in blast lesions looking as ‘green islands (gi) produced in senescence leaves’, and MAT1-2 was found to be associated with all gi lesions. Consequently, the management of the disease in those plots was very difficult. Interestingly, ribosomal RNA IGS region could not be amplified in MAT1-2 isolates but consistent amplification was obtained in MAT1-1 mating type isolates.

Two clonal lineages of Phytophthora citrophthora from citrus in South Africa represent a single phylogenetic species

Phytophthora citrophthora from citrus in eastern Corsica and Spain consists of distinct clonal lineages. In South Africa the extent of genetic variation among citrus-associated P. citrophthora isolates is unknown. This was investigated with isolates from South Africa (n =60), Spain (n =10) and six isolates representing three P. citrophthora groups CTR1, CTR2 and CTR3 previously identified with isozyme polymorphisms (Mchau and Coffey 1994). South African and Spanish isolates belonged to two lineages (G1, G2) based on an internal transcribed spacer (ITS) phylogeny, random amplified microsatellites (RAMS) and random amplified polymorphic DNA (RAPD) profiling. Although combined RAMS and RAPD data identified 14 genotypes, unweighted pair group method with arithmetic mean (UPGMA) analyses grouped the isolates into two clusters corresponding to lineages G1 and G2. Lineage G1 predominated among isolates from South Africa (92%) and Spain (100%). Phylogenetic analyses of the β-tubulin, cytochrome c oxidase subunit I (COX1) and ITS regions did not support the hypothesis that the two lineages represent distinct phylogenetic species but suggested that isozyme group CTR2 and possibly CTR3 are species distinct from P. citrophthora sensu stricto. Mating-type analyses, using tester strains from groups CTR2 and CTR3 revealed that most G1 lineage isolates (n =57) were sterile but that some were of the A1 mating type (n =8) whereas all G2 lineage isolates were A2 (n =5). The mating-type designation was confirmed with P. capsici tester strains. However, when A1 (G1 lineage) and A2 (G2 lineage including CTR1 reference isolates) mating-type isolates were paired in all possible combinations, no oogonia or antheridia were produced. This suggests that only tester strains P. capsici, CTR2 and CTR3 were able to produce sexual structures and that lineages G1 and G2 are sterile and reproductively isolated, which is supported by molecular data.