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BACKGROUND: Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. RESULTS: In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. CONCLUSIONS: These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Objective: Heifer development is crucial for the optimization of reproductive efficiency in beef production. Heifer development is largely influenced by nutrition. Nutritional status of these heifers can influence immunological responses that are crucial for reproduction. Commercial Angus heifers (n = 9) were utilized, in a pilot study, to elucidate the effects of sampling time (days 0, 7, 14, 21, 28, and 35), pregnancy status, and type of mating on individual cytokine concentrations and cytokine profiles in the vagina following breeding. Results: Cytokine profiles were analyzed using MetaboAnalyst 5.0 and one-way ANOVAs were performed in R Studio to identify differences in individual cytokines based on sampling time, pregnancy status, and type of mating. Cytokine profiles were different (P = 0.05) 7 days after either mating type. Cytokines, IL-1β, IL-17a, MCP-1, and TNFα were different based on the mating type and pregnancy status. Multiple cytokines, IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17a, VEGFa, and MIP1α, were different between days regardless of pregnancy status. In conclusion, vaginal cytokines differ based on pregnancy status, type of mating, and time which may be indicative of vital pathways that need to be activated for pregnancy. [ABSTRACT FROM AUTHOR]

Abstract Objective Heifer development is crucial for the optimization of reproductive efficiency in beef production. Heifer development is largely influenced by nutrition. Nutritional status of these heifers can influence immunological responses that are crucial for reproduction. Commercial Angus heifers (n = 9) were utilized, in a pilot study, to elucidate the effects of sampling time (days 0, 7, 14, 21, 28, and 35), pregnancy status, and type of mating on individual cytokine concentrations and cytokine profiles in the vagina following breeding. Results Cytokine profiles were analyzed using MetaboAnalyst 5.0 and one-way ANOVAs were performed in R Studio to identify differences in individual cytokines based on sampling time, pregnancy status, and type of mating. Cytokine profiles were different (P = 0.05) 7 days after either mating type. Cytokines, IL-1β, IL-17a, MCP-1, and TNFα were different based on the mating type and pregnancy status. Multiple cytokines, IL-1α, IL-1β, IL-6, IL-8, IL-10, IL-17a, VEGFa, and MIP1α, were different between days regardless of pregnancy status. In conclusion, vaginal cytokines differ based on pregnancy status, type of mating, and time which may be indicative of vital pathways that need to be activated for pregnancy.

Abstract Background Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. Results In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. Conclusions These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Colletotrichum fructicola shows morphological and genetic differences in plus and minus strains. However, the mechanism of the differentiation between two types of strains is still largely unclear. Our early transcriptome analysis revealed that CfHMG expression differed in plus and minus strains. To define the functions of the CfHMG gene, we constructed gene deletion mutants by homologous recombination. We found that a CfHMG deletion mutant of the minus strain, CfHMG-M, could lead to a reduction in perithecium sizes and densities on media and sterile perithecium formation compared with the minus wild type (WT), whereas there was no effect for the plus mutant CfHMG-P. In co-cultures between CfHMG-P and minus WT, CfHMG-M and plus WT, or CfHMG-P and CfHMG-M, the quantities of perithecia were all reduced significantly. When conidial suspensions were inoculated on non-wounded apple fruit, it was found that the virulence of the minus mutant decreased significantly but not for the plus one. Further, we found that the virulence decrease in minus mutants was caused by a decrease in the conidium germination rate. Our results indicate that CfHMG of C. fructicola plays an important role in the mating line formation between the plus and minus strain for both strains and differentially regulates the perithecium size, density, fertilization, and virulence of the minus strain. The results are significant for further detecting the differentiated mechanisms between the plus and minus strains in Colletotrichum fungi. [ABSTRACT FROM AUTHOR]

Phytophthora infestans is the causal agent of late blight in potato. The occurrence of P. infestans with both A1 and A2 mating types in the field may result in sexual reproduction and the generation of recombinant strains. Such strains with new combinations of traits can be highly aggressive, resistant to fungicides, and can make the disease difficult to control in the field. Metalaxyl-resistant isolates are now more prevalent in potato fields. Understanding the genetic structure and rapid identification of mating types and metalaxyl response of P. infestans in the field is a prerequisite for effective late blight disease monitoring and management. Molecular and phenotypic assays involving molecular and phenotypic markers such as mating types and metalaxyl response are typically conducted separately in the studies of the genotypic and phenotypic diversity of P. infestans. As a result, there is a pressing need to reduce the experimental workload and more efficiently assess the aggressiveness of different strains. We think that employing genetic markers to not only estimate genotypic diversity but also to identify the mating type and fungicide response using machine learning techniques can guide and speed up the decision-making process in late blight disease management, especially when the mating type and metalaxyl resistance data are not available. This technique can also be applied to determine these phenotypic traits for dead isolates. In this study, over 600 P. infestans isolates from different populations—Estonia, Pskov region, and Poland—were classified for mating types and metalaxyl response using machine learning techniques based on simple sequence repeat (SSR) markers. For both traits, random forest and the support vector machine demonstrated good accuracy of over 70%, compared to the decision tree and artificial neural network models whose accuracy was lower. There were also associations (p < 0.05) between the traits and some of the alleles detected, but machine learning prediction techniques based on multilocus SSR genotypes offered better prediction accuracy. [ABSTRACT FROM AUTHOR]

Studying the signatures of evolution can help to understand genetic processes. Here, we demonstrate how the existence of balancing selection can be used to identify the breeding systems of fungi from genomic data. The breeding systems of fungi are controlled by self-incompatibility loci that determine mating types between potential mating partners, resulting in strong balancing selection at the loci. Within the fungal phylum Basidiomycota, two such self-incompatibility loci, namely HD MAT locus and P/R MAT locus, control mating types of gametes. Loss of function at one or both MAT loci results in different breeding systems and relaxes the MAT locus from balancing selection. By investigating the signatures of balancing selection at MAT loci, one can infer a species breeding system without culture-based studies. Nevertheless, the extreme sequence divergence among MAT alleles imposes challenges for retrieving full variants from both alleles when using the conventional read-mapping method. Therefore, we employed a combination of read-mapping and local de novo assembly to construct haplotypes of HD MAT alleles from genomes in suilloid fungi (genera Suillus and Rhizopogon). Genealogy and pairwise divergence of HD MAT alleles showed that the origins of mating types predate the split between these two closely related genera. High sequence divergence, trans-specific polymorphism, and the deeply diverging genealogy confirm the long-term functionality and multiallelic status of HD MAT locus in suilloid fungi. This work highlights a genomics approach to studying breeding systems regardless of the culturability of organisms based on the interplay between evolution and genetics.

Recombination is often suppressed at sex-determining loci in plants and animals, and at self-incompatibility or mating-type loci in plants and fungi. In fungal ascomycetes, recombination suppression around the mating-type locus is associated with pseudo-homothallism, i.e. the production of self-fertile dikaryotic sexual spores carrying the two opposite mating types. This has been well studied in two species complexes from different families of Sordariales: Podospora anserina and Neurospora tetrasperma. However, it is unclear whether this intriguing association holds in other species. We show here that Schizothecium tetrasporum, a fungus from a third family in the order Sordariales, also produces mostly self-fertile dikaryotic spores carrying the two opposite mating types. This was due to a high frequency of second meiotic division segregation at the mating-type locus, indicating the occurrence of a single and systematic crossing-over event between the mating-type locus and the centromere, as in P. anserina. The mating-type locus has the typical Sordariales organization, plus a MAT1-1-1 pseudogene in the MAT1-2 haplotype. High-quality genome assemblies of opposite mating types and segregation analyses revealed a suppression of recombination in a region of 1.47 Mb around the mating-type locus. We detected three evolutionary strata, indicating a stepwise extension of recombination suppression. The three strata displayed no rearrangement or transposable element accumulation but gene losses and gene disruptions were present, and precisely at the strata margins. Our findings indicate a convergent evolution of self-fertile dikaryotic sexual spores across multiple ascomycete fungi. The particular pattern of meiotic segregation at the mating-type locus was associated with recombination suppression around this locus, that had extended stepwise. This association between pseudo-homothallism and recombination suppression across lineages and the presence of gene disruption at the strata limits are consistent with a recently proposed mechanism of sheltering deleterious alleles to explain stepwise recombination suppression.

Pyrenophora teres f. teres (Ptt) is a severe pathogen to spring barley in Northern Europe. Ptt with relevant mutations in fungicide target proteins, sterol 14α-demethylase (CYP51A), cytochrome b (Cyt b), and succinate dehydrogenase (SDH) would put efficient disease control at risk. In the growing seasons of 2021 and 2022, 193 Ptt isolates from Estonia were analysed. In this study, mutation detection and in vitro fungicide sensitivity assays of single-spore isolates were carried out. Reduced sensitivity phenotype to mefentrifluconazole was evident in Ptt isolates with a F489L mutation in CYP51A or with 129 bp insert in the Cyp51A gene-promoter region. However, sensitivity to a prothioconazole-desthio remained high regardless of these molecular changes. The Ptt population was mostly sensitive to bixafen, fluxapyroxad, pyraclostrobin, and azoxystrobin. The sensitivity of fluxapyroxad and bixafen has been affected by two mutations, C-S135R and D-H134R, found in SDH subunits. The F129L mutation in Cyt b influenced azoxystrobin but not pyraclostrobin sensitivity. In total, 30 isolates from five fields had relevant mutations in three target protein genes simultaneously. Most of these isolates had a reduced sensitivity phenotype to mefentrifluconazole, fluxapyroxad, and azoxystrobin, while sensitivity to other tested fungicides remained high. Furthermore, possible sexual reproduction may enhance the pathogen's fitness and help it adapt to fungicides. [ABSTRACT FROM AUTHOR]

Thielaviopsis paradoxa sensu lato is a soilborne fungal pathogen that causes Thielaviopsis trunk rot and heart rot in palms. The loss of structural integrity resulting from trunk rot can cause the palm trunk to collapse suddenly and poses a serious threat to life and property. Even though rudimentary knowledge about the Thielaviopsis infection process in palms is available, nothing is known about the T. paradoxa species complex in the US. The aim of this study was to characterize T. paradoxa s. lat. isolates collected from diseased palms grown in Florida. Multi-locus phylogeny using three genes, ITS, β-tubulin, and tef1-α, revealed that the isolates separate into two distinct clades with high bootstrap support. The majority of the isolates clustered with the species T. ethacetica, while two isolates formed a separate clade, distinct from T. musarum, and might represent an undescribed Thielaviopsis species. One representative isolate from each clade, when grown on three distinct media and at four different temperatures, showed differences in gross colony morphology, as well as growth rates. The T. ethacetica isolate TP5448 and the Thielaviopsis sp. isolate PLM300 grew better at opposite ends of the temperature spectrum tested in this study, i.e., 35 °C and 10 °C, respectively. In pathogenicity assays on whole plants, the T. ethacetica isolate proved to be more aggressive than Thielaviopsis sp. isolate PLM300, as it produced larger lesions when inoculated on wounded leaflets. An unequal distribution was observed for the mating-type locus of T. ethacetica, as 12 isolates carried the MAT1-1-1 allele, while the status for four isolates remained undefined. Variation in mycelial growth in response to different fungicides was also observed between the two clades. These results demonstrate the existence of two Thielaviopsis clades that can infect palms in Florida and underscore the need for targeted sampling to help uncover the diversity of Thielaviopsis species across palm-growing regions in the US. [ABSTRACT FROM AUTHOR]

In China, Fusarium pseudograminearum has emerged as a major pathogen causing Fusarium crown rot (FCR) and caused significant losses. Studies on the pathogen's properties, especially its mating type and trichothecene chemotypes, are critical with respect to disease epidemiology and food/feed safety. There are currently few available reports on these issues. This study investigated the species composition, mating type idiomorphs, and trichothecene genotypes of Fusarium spp. causing FCR in Henan, China. A significant shift in F. pseudograminearum-induced FCR was found in the present study. Of the 144 purified strains, 143 were F. pseudograminearum, whereas only 1 Fusarium graminearum was identified. Moreover, a significant trichothecene-producing capability of F. pseudograminearum strains from Henan was observed in this work. Among the 143 F. pseudograminearum strains identified, F. pseudograminearum with a 15ADON genotype was found to be predominant (133 isolates), accounting for 92.36% of all strains, followed by F. pseudograminearum with a 3ADON genotype, whereas only one NIV genotype strain was detected. Overall, a relatively well-balanced 1:1 ratio of the F. pseudograminearum population was found in Henan. To the best of our knowledge, this is the first study that has examined the Fusarium populations responsible for FCR across the Henan wheat-growing region. [ABSTRACT FROM AUTHOR]

Estrus synchronization is the process of controlling or guiding a cow/heifer estrous cycle so that they can mate at roughly the same time. It may be used to increase the probability of detecting estrus, increase the rate of calving in response to feed availability and market demand for dairy products, and increase the possibility of pregnancy in dairy cattle. The aim of this study was to assess breeding practices, evaluating of hormonal estrus synchronization and mass artificial insemination of dairy cow/heifers in Andracha and Masha districts of Southwestern Ethiopia. Three hundred eighty-nine (389) synchronized cows were selected from record of artificial insemination center (logbook). For experimental (action) study a total of 210 (103 from Andracha and 107 from Masha) dairy cows/heifers were selected and injected single dose of prostaglandin. Cow/heifers which do not respond by single injection were re-injected another single dose of prostaglandin. The data were analyzed using SPSS version 21 software. The study revealed that cows or heifers treated with single dose of prostaglandin 157(74.8 %), and 53(25.2 %) responded to heat and none response respectively in the study location. The study also revealed that overall conception rate (CR) and number of services per conception (NSPC) for single dose of prostaglandin injection in the researched areas were 68 (43.3 %), and 2.3 respectively. It was concluded that the effectiveness of PGF2α used to synchronize estrus in dairy cows/heifers was good and the overall conception rate 43.1 higher than national level conception rates (7.14–40.23) to first inseminations.

The prevalence and distribution of pathotypes and mating types of Ascochyta rabiei (Pass.) Labr. were evaluated during the winter (October to March) seasons of 2017-18 and 2018-19. Forty-five A. rabiei isolates were obtained from six different states. From the survey results, the state of Punjab recorded the highest disease rating (Rating 9) compared with other chickpea-growing areas. The β-tubulin gene was amplified (330 bp) from all the isolates, and sequence analysis was performed. The phylogenetic tree produced by cluster analysis of β-tubulin gene sequences differentiated the 44 A. rabiei isolates. Using multiplex PCR, mating types were identified. From the mating type (MAT) study of all the A. rabiei isolates, amplification was observed at the 490-bp size, which corresponds to mating type 2 (MAT1-2). The four pathotypes (I, II, III, IV) and six physiological races were delineated based on the differential reaction of the disease. The most widely distributed pathotype was pathotype III (52.27%) among the surveyed regions of North Indian states, except Uttarakhand (where only pathotype II was recorded), followed by the least aggressive pathotype I (22.7%), highly aggressive pathotype IV (15.9%), and aggressive pathotype II (9.1%). Among the six physiological races identified, the most common races were race 5 (25%), followed by race 1 (22.73%), race 6 (18.18%), race 4 (18.18%), race 3 (9.09%), and race 2 (6.82%). Because of the prevalence of a single mating type among the surveyed locations in India, we opine that there may be less diversity in A. rabiei compared with isolates reported from other countries. However, to substantiate this conclusion, evaluation of more isolates of A. rabiei is required. [Figure: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Aims: Farnesol was identified 20 years ago in a search for Candida albicans quorum sensing molecules (QSM), but there is still uncertainty regarding many aspects of its mode of action including whether it employs farnesol transport mechanisms other than diffusion. Based on the structural similarity between farnesol and the farnesylated portion of the MTLa pheromone, we explored the effects of ploidy and mating type locus (MTL) on the antifungal activity of exogenous farnesol. Methods and results: We approached this question by examining five MTLa and five MTLα haploid strains with regard to their farnesol sensitivity in comparison to six heterozygous MTLa/α diploids. We examined the haploid and diploid strains for percent cell death after exposure of exponentially growing cells to 0-200 µM farnesol. The heterozygous (MTLa/α) diploids were tolerant of exogenous farnesol whereas the MTLa and MTLα haploids were on average 2- and 4-times more sensitive, respectively. In the critical range from 10-40 µM farnesol their cell death values were in the ratio of 1:2:4. Very similar results were obtained with two matched sets of MATa, MATα, and MATa/α Saccharomyces cerevisiae strains. Conclusion: We propose that the observed MTL dependence of farnesol is based on differentially regulated mechanisms of entry and efflux which determine the actual cellular concentration of farnesol. The mechanisms by which pathogens such as C. albicans tolerate the otherwise lethal effects of farnesol embrace a wide range of physiological functions, including MTL type, ubiquinone type (UQ6-UQ9), energy availability, and aerobic/anaerobic status. [ABSTRACT FROM AUTHOR]

Candida albicans, a normal member of the human microbiome and an opportunistic fungal pathogen, undergoes several morphological transitions. One of these transitions is white–opaque switching, where C. albicans alternates between 2 stable cell types with distinct cellular and colony morphologies, metabolic preferences, mating abilities, and interactions with the innate immune system. White-toopaque switching is regulated by mating type; it is repressed by the a1/α2 heterodimer in a/α cells, but this repression is lifted in a/a and α/α mating type cells (each of which are missing half of the repressor). The widely used C. albicans reference strain, SC5314, is unusual in that white–opaque switching is completely blocked when the cells are a/α; in contrast, most other C. albicans a/α strains can undergo white–opaque switching at an observable level. In this paper, we uncover the reason for this difference. We show that, in addition to repression by the a1/α2 heterodimer, SC5314 contains a second block to white–opaque switching: 4 transcription regulators of filamentous growth are upregulated in this strain and collectively suppress white–opaque switching. This second block is missing in the majority of clinical strains, and, although they still contain the a1/α2 heterodimer repressor, they exhibit a/α white–opaque switching at an observable level. When both blocks are absent, white–opaque switching occurs at very high levels. This work shows that white–opaque switching remains intact across a broad group of clinical strains, but the precise way it is regulated and therefore the frequency at which it occurs varies from strain to strain. [ABSTRACT FROM AUTHOR]

The Tremellomycetes are a species-rich group within the basidiomycete fungi; however, most analyses of this group to date have focused on pathogenic Cryptococcus species within the order Tremellales. Recent genome-assisted studies of other Tremellomycetes have identified interesting features with respect to biotechnological applications as well as the evolution of genes involved in mating and sexual development. Here, we report genome sequences of two strains of Filobasidium floriforme, a species from the order Filobasidiales, which branches basally to the Tremellales, Trichosporonales, and Holtermanniales. The assembled genomes of strains CBS6241 and CBS6242 are 27.4 Mb and 26.4 Mb in size, respectively, with 8314 and 7695 predicted protein-coding genes. Overall sequence identity at nucleic acid level between the strains is 97%. Among the predicted genes are pheromone precursor and pheromone receptor genes as well as two genes encoding homedomain (HD) transcription factors, which are predicted to be part of the mating type (MAT) locus. Sequence analysis indicates that CBS6241 and CBS6242 carry different alleles for both the pheromone/receptor genes as well as the HD transcription factors. Orthology inference identified 1482 orthogroups exclusively found in F. floriforme, some of which were involved in carbohydrate transport and metabolism. Subsequent CAZyme repertoire characterization identified 267 and 247 enzymes for CBS6241 and CBS6242, respectively, the second highest number of CAZymes among the analyzed Tremellomycete species. In addition, F. floriforme contains five CAZymes absent in other species and several plant-cell-wall degrading CAZymes with the highest copy number in Tremellomycota, indicating the biotechnological potential of this species.

Abstract Sexual reproduction in fungi promotes genetic diversity and helps the fungus to adapt to environmental stresses. Fusarium verticillioides is a heterothallic filamentous ascomycete that is a major cause of maize ear and stalk rot worldwide, while also posing a threat to human and animal health by producing various mycotoxins. Sexual reproduction in F. verticillioides is controlled by the MAT-1 and MAT-2 loci, which mandate that only strains of opposite mating types can mate to yield perithecia and ascospores. Nevertheless, there exists a phenomenon called ‘spore killing’, in which only four typical ascospores appear in the asci following a cross between a strain carrying the spore killer allele (Sk K ) and one with the spore killer sensitive allele (Sk S ). In this study, 31 isolates of F. verticillioides collected from eight provinces in China during the maize growing season from 2014 to 2020 were compared based on their mating type, spore killing genotype, and pathogenicity. To determine the mating types and spore killing genotypes of these isolates, partial sequences were amplified from the MAT loci and the SKC1 gene, respectively. The PCR results showed that out of the 31 isolates, 18 were MAT-1 and 13 were MAT-2, and that 25 had Sk K genotypes and 6 had Sk S genotypes. Genetic crosses between LNF15-11 (MAT-2) and 18 MAT-1 isolates produced normal perithecia with varying numbers. However, crosses between LNF15-11 and the 3 isolates (SDF18-36, HNF14-8, and GSF19-6) produced only four ascospores per ascus, while the remaining isolates except SDF18-28, yielded eight ascospores per ascus. These findings suggest that the SKC1 amplicon variation can be used to differentiate Sk K and Sk S genotypes in the field and that the 3 isolates are truly Sk S genotypes with the MAT-1 allele. Altogether, this study contributes to our knowledge of the mating type and spore killing genotype of F. verticillioides in China and offers valuable strain resources for investigating heterothallic sexual reproduction.

Colletotrichum fructicola shows morphological and genetic differences in plus and minus strains. However, the mechanism of the differentiation between two types of strains is still largely unclear. Our early transcriptome analysis revealed that CfHMG expression differed in plus and minus strains. To define the functions of the CfHMG gene, we constructed gene deletion mutants by homologous recombination. We found that a CfHMG deletion mutant of the minus strain, CfHMG-M, could lead to a reduction in perithecium sizes and densities on media and sterile perithecium formation compared with the minus wild type (WT), whereas there was no effect for the plus mutant CfHMG-P. In co-cultures between CfHMG-P and minus WT, CfHMG-M and plus WT, or CfHMG-P and CfHMG-M, the quantities of perithecia were all reduced significantly. When conidial suspensions were inoculated on non-wounded apple fruit, it was found that the virulence of the minus mutant decreased significantly but not for the plus one. Further, we found that the virulence decrease in minus mutants was caused by a decrease in the conidium germination rate. Our results indicate that CfHMG of C. fructicola plays an important role in the mating line formation between the plus and minus strain for both strains and differentially regulates the perithecium size, density, fertilization, and virulence of the minus strain. The results are significant for further detecting the differentiated mechanisms between the plus and minus strains in Colletotrichum fungi.

Phytophthora infestans is the causal agent of late blight in potato. The occurrence of P. infestans with both A1 and A2 mating types in the field may result in sexual reproduction and the generation of recombinant strains. Such strains with new combinations of traits can be highly aggressive, resistant to fungicides, and can make the disease difficult to control in the field. Metalaxyl-resistant isolates are now more prevalent in potato fields. Understanding the genetic structure and rapid identification of mating types and metalaxyl response of P. infestans in the field is a prerequisite for effective late blight disease monitoring and management. Molecular and phenotypic assays involving molecular and phenotypic markers such as mating types and metalaxyl response are typically conducted separately in the studies of the genotypic and phenotypic diversity of P. infestans. As a result, there is a pressing need to reduce the experimental workload and more efficiently assess the aggressiveness of different strains. We think that employing genetic markers to not only estimate genotypic diversity but also to identify the mating type and fungicide response using machine learning techniques can guide and speed up the decision-making process in late blight disease management, especially when the mating type and metalaxyl resistance data are not available. This technique can also be applied to determine these phenotypic traits for dead isolates. In this study, over 600 P. infestans isolates from different populations—Estonia, Pskov region, and Poland—were classified for mating types and metalaxyl response using machine learning techniques based on simple sequence repeat (SSR) markers. For both traits, random forest and the support vector machine demonstrated good accuracy of over 70%, compared to the decision tree and artificial neural network models whose accuracy was lower. There were also associations (p < 0.05) between the traits and some of the alleles detected, but machine learning prediction techniques based on multilocus SSR genotypes offered better prediction accuracy.

Aims: Farnesol was identified 20 years ago in a search for Candida albicans quorum sensing molecules (QSM), but there is still uncertainty regarding many aspects of its mode of action including whether it employs farnesol transport mechanisms other than diffusion. Based on the structural similarity between farnesol and the farnesylated portion of the MTLa pheromone, we explored the effects of ploidy and mating type locus (MTL) on the antifungal activity of exogenous farnesol.Methods and results: We approached this question by examining five MTLa and five MTLα haploid strains with regard to their farnesol sensitivity in comparison to six heterozygous MTLa/α diploids. We examined the haploid and diploid strains for percent cell death after exposure of exponentially growing cells to 0–200 µM farnesol. The heterozygous (MTLa/α) diploids were tolerant of exogenous farnesol whereas the MTLa and MTLα haploids were on average 2- and 4-times more sensitive, respectively. In the critical range from 10–40 µM farnesol their cell death values were in the ratio of 1:2:4. Very similar results were obtained with two matched sets of MATa, MATα, and MATa/α Saccharomyces cerevisiae strains.Conclusion: We propose that the observed MTL dependence of farnesol is based on differentially regulated mechanisms of entry and efflux which determine the actual cellular concentration of farnesol. The mechanisms by which pathogens such as C. albicans tolerate the otherwise lethal effects of farnesol embrace a wide range of physiological functions, including MTL type, ubiquinone type (UQ6-UQ9), energy availability, and aerobic/anaerobic status.

Bipolaris maydis is the pathogenic microorganism of southern corn leaf blight, a persistent biotic constraint responsible for substantial yield losses of corn worldwide. In the present study, 96 isolates from six representative fields growing single and multiple sweet corn cultivars in Pingnan, Fuqing, and Jian'ou in Fujian Province, which are characterized by different geographical characteristics and cropping patterns, were genetically analyzed using inter-simple sequence repeat (ISSR) markers to assess the impact of geographical origins and corn cultivars on B. maydis population differentiation. B. maydis isolates originated from diverse regions possessed higher genetic variety than those from single and multiple sweet corn cultivars. Phylogenetic analysis showed that the isolates from single and multiple sweet corn cultivars were randomly grouped into different clusters, with those from the same location tending to form clusters. A greater genetic differentiation among different geographical populations than between those from single and multiple sweet corn cultivars was observed by pairwise comparison. Hierarchical analysis indicated that among-population variation was higher when comparatively analyzed B. maydis populations from different locations than in those from single and multiple sweet corn cultivars. In conclusion, these results suggest that geographical origin acts a more considerable role in genetic differentiation of B. maydis than corn cultivar. Two divided genetic clusters were detected in the B. maydis populations from single and multiple sweet corn cultivars at the three locations in Fujian Province, with major genetic variation being derived within populations. The high haplotypic diversity and expected mating type ratio of 1:1 in combination with significant linkage disequilibrium suggested that a mixed reproductive strategy occurs in the B. maydis population in Fujian Province. This study will enrich the information on the role that geographical origins and corn cultivars play in the population structure of the pathogen as well as the reproductive strategies in B. maydis population in Fujian Province. [ABSTRACT FROM AUTHOR]

Trichophyton quinckeanum, the causative agent of mouse favus, has been responsible for several infections of animal owners in recent years and showed an infection peak around 2020 in Jena, Thuringia. The isolated T. quinckeanum strains from Thuringia differ in some positions of the ITS region compared to strains from the IHEM collection as well as to Trichophyton schoenleinii. All T. quinckeanum strains of the new genotype show up to a 100-fold increased itraconazole resistance as measured by microplate laser nephelometry (MLN) assays. Analysis of genes involved in Trichophyton indotineae azole resistance, such as Erg1, which encodes squalene epoxidase, and Erg11B, one of two copies of the sterol 14-α demethylase gene, show a 100% identity between the two T. quinckeanum genotypes. In contrast, Erg11A fragments differ in 15-nucleotide positions between both T. quinckeanum genotypes, resulting in the unique amino acid substitution Ala256Ser in resistant strains. The new T. quinckeanum genotype may have evolved through interspecies mating. Mating type analysis showed a nearly 100% identity of the minus type MAT1-1-1 fragment for all T. quinckeanum isolates. The closely related Trichophyton schoenleinii belongs to the plus mating type and has 100% identical fragments of Erg1 and Erg11B. Erg11A protein sequences of T. schoenleinii and T. quinckeanum showed increased diversity. [ABSTRACT FROM AUTHOR]

Colletotrichum fructicola is a devastating fungal pathogen of diverse plants. Sexually compatible plus and minus strains occur in the same ascus. However, the differentiation mechanism of plus and minus strains remains poorly understood. Here, we characterized a novel Cys2-His2-containing transcription factor CfCpmdl. The plus CfCpmdl deletion mutant (is+CfCpmdl) resulted in slow hyphal growth and a fluffy cottonlike colony, and the minus deletion mutant (¿s-CfCpmdl) exhibited characters similar to the wild type (WT)./\+CfCpmdl led to defective perithecial formation, whereas A-CfCpmdl produced more and smaller perithecia. The normal mating line was developed by pairing cultures of h-CfCpmdl and plus WT, whereas a weak line was observed between is+CfCpmd 1 and minus WT. Conidial production was completely abolished in both plus and minus mutants. When inoculated on non-wounded apple leaves with mycelial plugs, is-CfCpmdl was nonpathogenic because of failure to develop conidia and appressoria, while is+CfCpmdl could infect apple leaves by appressoria differentiated directly from hyphal tips, even though no conidia formed. Collectively, our results demonstrate that CfCpmdl of C. fructicola is an important gene related to plus and minus strain differentiation, which also affects hyphal growth, sporulation, appressorium formation, and pathogenicity. [ABSTRACT FROM AUTHOR]

Emergence of pathogens with decreased sensitivity to succinate dehydrogenase inhibitor fungicides is a global agronomical issue. Analysis of Didymella tanaceti isolates (n = 173), which cause tan spot of pyrethrum (Tanacetum cinerariifolium), collected prior to (2004 to 2005) and after (2009,2010,2012, and 2014) the commercial implementation of boscalid in Tasmanian pyrethrum fields identified that insensitivity developed over time and has become widespread. To evaluate temporal change, isolates were characterized for frequency of mutations in the succinate dehydrogenase (Sdh) B, C, and D subunits associated with boscalid resistance, mating type, and SSR genotype. All isolates from 2004 and 2005 exhibited wildtype (WT) Sdh alleles. Seven known Sdh substitutions were identified in isolates collected from 2009 to 2014. In 2009,60.7% had Sdh substitutions associated with boscalid resistance in D. tanaceti. The frequency of WT isolates decreased over time, with no WT isolates identified in 2014. The frequency of the SdhB-H277Y genotype increased from 10.7 to 77.8% between 2009 and 2014. Genotypic evidence suggested that a shift in the population structure occurred between 2005 and 2009, with decreases in gene diversity (uh; 0.51 to 0.34), genotypic evenness (E5; 0.96 to 0.67), genotypic diversity (G; 9.3 to 6.8), and allele frequencies. No evidence was obtained to support the rapid spread of Sdh genotypes by clonal expansion of the population. Thus, insensitivity to boscalid has developed and become widespread within a diverse population within 4 years of usage. These results suggest that D. tanaceti can disperse insensitivity through repeated frequent mutation, sexual recombination, or a combination of both. [ABSTRACT FROM AUTHOR]

Faba bean ascochyta blight, caused by Ascochyta fabae Speg. (teleomorph: Didymella fabae Punith.), is one of the most devastating diseases of the crop. It can cause yield losses that reach 95% in conducive weather conditions. Surveys were carried out in five regions of Tunisia: Beja, Bizerte, Jendouba, Kef and Tunis-Cap Bon. A total of 513 fungal isolates were collected from 2011 to 2013. A molecular characterization was conducted to identify the mating type of each individual using a mating type specific PCR. Results revealed that the two mating types MAT1-2 and MAT1-1 coexisted in all surveyed regions. An imbalance in favor of MAT1-2 was observed particularly in Bizerte and Jendouba regions (sex ratio was 18:85 and 32:80, respectively). Moreover, morphological and pathogenic characterization of 122 isolates among the collection revealed a significant variability in conidia type (one celled or two celled conidia) frequency, in conidia mean size and in aggressiveness toward Badii faba bean cultivar (incubation period, IP; percentage necrotic leaf area, S; and area under disease progression curve, AUDPC). A principal component analysis (PCA) performed on morphologically studied parameters (frequency of conidia cell number and conidia mean size) identified three groups of isolates based on morphological traits: one celled (1C) and two celled (2C) conidia rates, one celled and two celled conidia length and width (1L, 1W, 2L and 2W, respectively). A second PCA using aggressiveness parameters (IP: Incubation period, S1, S4 and S9: percentage of necrotic leaf area respectively 5, 20 and 45 days after inoculation) identified three distinct pathogenic groups: poorly pathogenic AG1, moderately pathogenic AG2 and highly pathogenic AG3. Morphological and pathogenic groups and mating type data were used to conduct a multiple factorial correspondence analysis (MFCA) which revealed a correlation between the variables studied. Five groups were identified, each associated with a morphological and pathogenic trait and mating type. The most pathogenic group belonged to MAT1-2 suggesting that in locations where MAT1-2 is prevalent the epidemic risk is more important. [ABSTRACT FROM AUTHOR]

Sexual reproduction in fungi promotes genetic diversity and helps the fungus to adapt to environmental stresses. Fusarium verticillioides is a heterothallic filamentous ascomycete that is a major cause of maize ear and stalk rot worldwide, while also posing a threat to human and animal health by producing various mycotoxins. Sexual reproduction in F. verticillioides is controlled by the MAT-1 and MAT-2 loci, which mandate that only strains of opposite mating types can mate to yield perithecia and ascospores. Nevertheless, there exists a phenomenon called 'spore killing', in which only four typical ascospores appear in the asci following a cross between a strain carrying the spore killer allele (SkK) and one with the spore killer sensitive allele (SkS). In this study, 31 isolates of F. verticillioides collected from eight provinces in China during the maize growing season from 2014 to 2020 were compared based on their mating type, spore killing genotype, and pathogenicity. To determine the mating types and spore killing genotypes of these isolates, partial sequences were amplified from the MAT loci and the SKC1 gene, respectively. The PCR results showed that out of the 31 isolates, 18 were MAT-1 and 13 were MAT-2, and that 25 had SkK genotypes and 6 had SkS genotypes. Genetic crosses between LNF15-11 (MAT-2) and 18 MAT-1 isolates produced normal perithecia with varying numbers. However, crosses between LNF15-11 and the 3 isolates (SDF18-36, HNF14-8, and GSF19-6) produced only four ascospores per ascus, while the remaining isolates except SDF18-28, yielded eight ascospores per ascus. These findings suggest that the SKC1 amplicon variation can be used to differentiate SkK and SkS genotypes in the field and that the 3 isolates are truly SkS genotypes with the MAT-1 allele. Altogether, this study contributes to our knowledge of the mating type and spore killing genotype of F. verticillioides in China and offers valuable strain resources for investigating heterothallic sexual reproduction. [ABSTRACT FROM AUTHOR]

In an attempt to determine the mating type of different Sporothrix schenckii sensu stricto isolates that remained viable after a long period of preservation in a culture collection and to correlate them with the degree of virulence/pathogenicity, a PCR technique using primers designed for the sequences of MAT1-1-1 and MAT1-2-1 genes and a murine experimental model were used. The results showed that there was no correlation between the mating type and virulence among the isolates. Furthermore, different degrees of virulence/pathogenicity, ranging from high to low, were found among them based on different virulence parameters. It was assumed that the long period of preservation favored the changes, yielding the isolation of variants. Thus, we believe that new technologies for studies on factors can improve our knowledge of the pathogenesis of sporotrichosis. [ABSTRACT FROM AUTHOR]

Morels are one of the most highly prized edible and medicinal mushrooms worldwide. Therefore, historically, there has been a large international interest in their cultivation. Numerous ecological, physiological, genetic, taxonomic, and mycochemical studies have been previously developed. At the beginning of this century, China finally achieved artificial cultivation and started a high-scale commercial development in 2012. Due to its international interest, its cultivation scale and area expanded rapidly in this country. However, along with the massive industrial scale, a number of challenges, including the maintenance of steady economic profits, arise. In order to contribute to the solution of these challenges, formal research studying selection, species recognition, strain aging, mating type structure, life cycle, nutrient metabolism, growth and development, and multi-omics has recently been boosted. This paper focuses on discussing current morel cultivation technologies, the industrial status of cultivation in China, and the relevance of basic biological research, including, e.g., the study of strain characteristics, species breeding, mating type structure, and microbial interactions. The main challenges related to the morel cultivation industry on a large scale are also analyzed. It is expected that this review will promote a steady global development of the morel industry based on permanent and robust basic scientific knowledge. [ABSTRACT FROM AUTHOR]

Candida albicans is a fungal component of the human gut microbiota and an opportunistic pathogen. C. albicans transcription factors (TFs), Wor1 and Efg1, are master regulators of an epigenetic switch required for fungal mating that also control colonization of the mammalian gut. We show that additional mating regulators, WOR2, WOR3, WOR4, AHR1, CZF1, and SSN6, also influence gut commensalism. Using Calling Card-seq to record Candida TF DNA-binding events in the host, we examine the role and relationships of these regulators during murine gut colonization. By comparing in-host transcriptomes of regulatory mutants with enhanced versus diminished commensal fitness, we also identify a set of candidate commensalism effectors. These include Cht2, a GPI-linked chitinase whose gene is bound by Wor1, Czf1, and Efg1 in vivo, that we show promotes commensalism. Thus, the network required for a C. albicans sexual switch is biochemically active in the host intestine and repurposed to direct commensalism.

Verticillium dahliae is a soil-borne plant pathogenic fungus that causes Verticillium wilt on hundreds of dicotyledonous plant species. V. dahliae is considered an asexually (clonal) reproducing fungus, although both mating type idiomorphs (MAT1-1 and MAT1-2) are present, and is heterothallic. Most of the available information on V. dahliae strains, including their biology, pathology, and genomics comes from studies on isolates with the MAT1-2 idiomorph, and thus little information is available on the MAT1-1 V. dahliae strains in the literature. We therefore evaluated the growth responses of MAT1-1 and MAT1-2 V. dahliae strains to various stimuli. Growth rates and melanin production in response to increased temperature, alkaline pH, light, and H2O2 stress were higher in the MAT1-2 strains than in the MAT1-1 strains. In addition, the MAT1-2 strains showed an enhanced ability to degrade complex polysaccharides, especially starch, pectin, and cellulose. Furthermore, several MAT1-2 strains from both potato and sunflower showed increased virulence on their original hosts, relative to their MAT1-1 counterparts. Thus, compared to MAT1-1 strains, MAT1-2 strains derive their potentially greater fitness from an increased capacity to adapt to their environment and exhibit higher virulence. These competitive advantages might explain the current abundance of MAT1-2 strains relative to MAT1-1 strains in the agricultural and sylvicultural ecosystems, and this study provides the baseline information on the two mating idiomorphs to study sexual reproduction in V. dahliae under natural and laboratory conditions.

Eukaryotes utilize distinct mitogen/messenger-activated protein kinase (MAPK) pathways to evoke appropriate responses when confronted with different stimuli. In yeast, hyperosmotic stress activates MAPK Hog1, whereas mating pheromones activate MAPK Fus3 (and MAPK Kss1). Because these pathways share several upstream components, including the small guanosine-5'-triphosphate phosphohydrolase (GTPase) cell-division-cycle-42 (Cdc42), mechanisms must exist to prevent inadvertent cross-pathway activation. Hog1 activity is required to prevent crosstalk to Fus3 and Kss1. To identify other factors required to maintain signaling fidelity during hypertonic stress, we devised an unbiased genetic selection for mutants unable to prevent such crosstalk even when active Hog1 is present. We repeatedly isolated truncated alleles of RGA1, a Cdc42-specific GTPase-activating protein (GAP), each lacking its C-terminal catalytic domain, that permit activation of the mating MAPKs under hyperosmotic conditions despite Hog1 being present. We show that Rga1 down-regulates Cdc42 within the high-osmolarity glycerol (HOG) pathway, but not the mating pathway. Because induction of mating pathway output via crosstalk from the HOG pathway takes significantly longer than induction of HOG pathway output, our findings suggest that, under normal conditions, Rga1 contributes to signal insulation by limiting availability of the GTP-bound Cdc42 pool generated by hypertonic stress. Thus, Rga1 action contributes to squelching crosstalk by imposing a type of "kinetic proofreading". Although Rga1 is a Hog1 substrate in vitro, we eliminated the possibility that its direct Hog1-mediated phosphorylation is necessary for its function in vivo. Instead, we found first that, like its paralog Rga2, Rga1 is subject to inhibitory phosphorylation by the S. cerevisiae cyclin-dependent protein kinase 1 (Cdk1) ortholog Cdc28 and that hyperosmotic shock stimulates its dephosphorylation and thus Rga1 activation. Second, we found that Hog1 promotes Rga1 activation by blocking its Cdk1-mediated phosphorylation, thereby allowing its phosphoprotein phosphatase 2A (PP2A)-mediated dephosphorylation. These findings shed light on why Hog1 activity is required to prevent crosstalk from the HOG pathway to the mating pheromone response pathway.

In China, Fusarium pseudograminearum has emerged as a major pathogen causing Fusarium crown rot (FCR) and caused significant losses. Studies on the pathogen’s properties, especially its mating type and trichothecene chemotypes, are critical with respect to disease epidemiology and food/feed safety. There are currently few available reports on these issues. This study investigated the species composition, mating type idiomorphs, and trichothecene genotypes of Fusarium spp. causing FCR in Henan, China. A significant shift in F. pseudograminearum-induced FCR was found in the present study. Of the 144 purified strains, 143 were F. pseudograminearum, whereas only 1 Fusarium graminearum was identified. Moreover, a significant trichothecene-producing capability of F. pseudograminearum strains from Henan was observed in this work. Among the 143 F. pseudograminearum strains identified, F. pseudograminearum with a 15ADON genotype was found to be predominant (133 isolates), accounting for 92.36% of all strains, followed by F. pseudograminearum with a 3ADON genotype, whereas only one NIV genotype strain was detected. Overall, a relatively well-balanced 1:1 ratio of the F. pseudograminearum population was found in Henan. To the best of our knowledge, this is the first study that has examined the Fusarium populations responsible for FCR across the Henan wheat-growing region.

Thielaviopsis paradoxa sensu lato is a soilborne fungal pathogen that causes Thielaviopsis trunk rot and heart rot in palms. The loss of structural integrity resulting from trunk rot can cause the palm trunk to collapse suddenly and poses a serious threat to life and property. Even though rudimentary knowledge about the Thielaviopsis infection process in palms is available, nothing is known about the T. paradoxa species complex in the US. The aim of this study was to characterize T. paradoxa s. lat. isolates collected from diseased palms grown in Florida. Multi-locus phylogeny using three genes, ITS, β-tubulin, and tef1-α, revealed that the isolates separate into two distinct clades with high bootstrap support. The majority of the isolates clustered with the species T. ethacetica, while two isolates formed a separate clade, distinct from T. musarum, and might represent an undescribed Thielaviopsis species. One representative isolate from each clade, when grown on three distinct media and at four different temperatures, showed differences in gross colony morphology, as well as growth rates. The T. ethacetica isolate TP5448 and the Thielaviopsis sp. isolate PLM300 grew better at opposite ends of the temperature spectrum tested in this study, i.e., 35 °C and 10 °C, respectively. In pathogenicity assays on whole plants, the T. ethacetica isolate proved to be more aggressive than Thielaviopsis sp. isolate PLM300, as it produced larger lesions when inoculated on wounded leaflets. An unequal distribution was observed for the mating-type locus of T. ethacetica, as 12 isolates carried the MAT1-1-1 allele, while the status for four isolates remained undefined. Variation in mycelial growth in response to different fungicides was also observed between the two clades. These results demonstrate the existence of two Thielaviopsis clades that can infect palms in Florida and underscore the need for targeted sampling to help uncover the diversity of Thielaviopsis species across palm-growing regions in the US.

Abstract Objectives Candida albicans, a polymorphic yeast, is one of the most common, opportunistic fungal pathogens of humans. Among the different morphological forms, opaque form is one of the least-studied ones. This opaque phenotype is essential for mating and is also reported to be involved in colonizing the gastrointestinal tract. Considering the significance of the clinical and sexual reproduction of C. albicans, we have investigated the morphophysiological modulations in opaque form using a proteomic approach. Data description In the current investigation, we have used Micro-Liquid Chromatography-Mass Spectrometry (LC-MS/MS) analysis to create a protein profile for opaque-specific proteins. Whole-cell proteins from C. albicans (ATCC10231) cells that had been cultured for seven days on synthetic complete dextrose (SCD) medium in both as an opaque (test) and as a white (control) form cells were extracted, digested, and identified using LC-MS/MS. This information is meant to serve the scientific community and represents the proteome profile (SWATH Spectral Libraries) of C. albicans opaque form.

Faba bean ascochyta blight, caused by Ascochyta fabae Speg. (teleomorph: Didymella fabae Punith.), is one of the most devastating diseases of the crop. It can cause yield losses that reach 95% in conducive weather conditions. Surveys were carried out in five regions of Tunisia: Beja, Bizerte, Jendouba, Kef and Tunis-Cap Bon. A total of 513 fungal isolates were collected from 2011 to 2013. A molecular characterization was conducted to identify the mating type of each individual using a mating type specific PCR. Results revealed that the two mating types MAT1-2 and MAT1-1 coexisted in all surveyed regions. An imbalance in favor of MAT1-2 was observed particularly in Bizerte and Jendouba regions (sex ratio was 18:85 and 32:80, respectively). Moreover, morphological and pathogenic characterization of 122 isolates among the collection revealed a significant variability in conidia type (one celled or two celled conidia) frequency, in conidia mean size and in aggressiveness toward Badii faba bean cultivar (incubation period, IP; percentage necrotic leaf area, S; and area under disease progression curve, AUDPC). A principal component analysis (PCA) performed on morphologically studied parameters (frequency of conidia cell number and conidia mean size) identified three groups of isolates based on morphological traits: one celled (1C) and two celled (2C) conidia rates, one celled and two celled conidia length and width (1L, 1W, 2L and 2W, respectively). A second PCA using aggressiveness parameters (IP: Incubation period, S1, S4 and S9: percentage of necrotic leaf area respectively 5, 20 and 45 days after inoculation) identified three distinct pathogenic groups: poorly pathogenic AG1, moderately pathogenic AG2 and highly pathogenic AG3. Morphological and pathogenic groups and mating type data were used to conduct a multiple factorial correspondence analysis (MFCA) which revealed a correlation between the variables studied. Five groups were identified, each associated with a morphological and pathogenic trait and mating type. The most pathogenic group belonged to MAT1-2 suggesting that in locations where MAT1-2 is prevalent the epidemic risk is more important.

Volvocine green algae are a model for understanding the evolution of mating types and sexes. They are facultatively sexual, with gametic differentiation occurring in response to nitrogen starvation (-N) in most genera and to sex inducer hormone in Volvox. The conserved RWP-RK family transcription factor (TF) MID is encoded by the minus mating-type locus or male sex-determining region of heterothallic volvocine species and dominantly determines minus or male gametic differentiation. However, the factor(s) responsible for establishing the default plus or female differentiation programs have remained elusive. We performed a phylo-transcriptomic screen for autosomal RWP-RK TFs induced during gametogenesis in unicellular isogamous Chlamydomonas reinhardtii (Chlamydomonas) and in multicellular oogamous Volvox carteri (Volvox) and identified a single conserved ortho-group we named Volvocine Sex Regulator 1 (VSR1). Chlamydomonas vsr1 mutants of either mating type failed to mate and could not induce expression of key mating-type-specific genes. Similarly, Volvox vsr1 mutants in either sex could initiate sexual embryogenesis, but the presumptive eggs or androgonidia (sperm packet precursors) were infertile and unable to express key sex-specific genes. Yeast two-hybrid assays identified a conserved domain in VSR1 capable of self-interaction or interaction with the conserved N terminal domain of MID. In vivo coimmunoprecipitation experiments demonstrated association of VSR1 and MID in both Chlamydomonas and Volvox. These data support a new model for volvocine sexual differentiation where VSR1 homodimers activate expression of plus/female gamete-specific-genes, but when MID is present, MID-VSR1 heterodimers are preferentially formed and activate minus/male gamete-specific-genes. [ABSTRACT FROM AUTHOR]

The fungal species Candida albicans is both a member of the human microbiome and a fungal pathogen. C. albicans undergoes several different morphological transitions, including one called white-opaque switching. Here, cells reversibly switch between two states, white and opaque, and each state is heritable through many cell generations. Each cell type has a distinct cellular and colony morphology and they differ in many other properties including mating, nutritional specialization, and interactions with the innate immune system. Previous genetic screens to gain insight into white-opaque switching have focused on certain classes of genes (for example transcriptional regulators or chromatin modifying enzymes). In this paper, we examined 172 deletion mutants covering a broad range of cell functions. We identified 28 deletion mutants with at least a fivefold effect on switching frequencies; these cover a wide variety of functions ranging from membrane sensors to kinases to proteins of unknown function. In agreement with previous reports, we found that components of the pheromone signaling cascade affect white-to-opaque switching; however, our results suggest that the major effect of Cek1 on white-opaque switching occurs through the cell wall damage response pathway. Most of the genes we identified have not been previously implicated in white-opaque switching and serve as entry points to understand new aspects of this morphological transition.

Candida albicans is a major human fungal pathogen, capable of switching among a range of morphological types, such as the yeast form, including white and opaque cell types and the GUT (gastrointestinally induced transition) cell type, the filamentous form, including hyphal and pseudohyphal cell types, and chlamydospores. This ability is associated with its commensal and pathogenic life styles. In response to pheromone, C. albicans cells are able to form long mating projections resembling filaments. This filamentous morphology is required for efficient sexual mating. In the current study, we report the genetic regulatory mechanisms controlling the development of mating projections in C. albicans. Ectopic expression of MTLα1 in "a" cells induces the secretion of α-pheromone and promotes the development of mating projections. Using this inducible system, we reveal that members of the pheromone-sensing pathway (including the pheromone receptor), the Ste11-Hst7-Cek1/2 mediated MAPK signalling cascade, and the RAM pathway are essential for the development of mating projections. However, the cAMP/PKA signalling pathway and a number of key regulators of filamentous growth such as Hgc1, Efg1, Flo8, Tec1, Ume6, and Rfg1 are not required for mating projection formation. Therefore, despite the phenotypic similarities between filaments and mating projections in C. albicans, distinct mechanisms are involved in the regulation of these two morphologies.

AbstractThe diversity of A mating type in wild strains of Lentinula edodes was extensively analyzed to characterize and utilize them for developing new cultivars. One hundred twenty-three A mating type alleles, including 67 newly discovered alleles, were identified from 106 wild strains collected for the past four decades in Korea. Based on previous studies and current findings, a total of 130 A mating type alleles have been found, 124 of which were discovered from wild strains, indicating the hyper-variability of A mating type alleles of L. edodes. About half of the A mating type alleles in wild strains were found in more than two strains, whereas the other half of the alleles were found in only one strain. About 90% of A mating type combinations in dikaryotic wild strains showed a single occurrence. Geographically, diverse A mating type alleles were intensively located in the central region of the Korean peninsula, whereas only allele A17 was observed throughout Korea. We also found the conservation of the TCCCAC motif in addition to the previously reported motifs, including ATTGT, ACAAT, and GCGGAG, in the intergenic regions of A mating loci. Sequence comparison among some alleles indicated that accumulated mutation and recombination would contribute to the diversification of A mating type alleles in L. edodes. Our data support the rapid evolution of A mating locus in L. edodes, and would help to understand the characteristics of A mating loci of wild strains in Korea and help to utilize them for developing new cultivars.

Many filamentous ascomycetes develop three-dimensional fruiting bodies for production and dispersal of sexual spores. Fruiting bodies are among the most complex structures differentiated by ascomycetes; however, the molecular mechanisms underlying this process are insufficiently understood. Previous comparative transcriptomics analyses of fruiting body development in different ascomycetes suggested that there might be a core set of genes that are transcriptionally regulated in a similar manner across species. Conserved patterns of gene expression can be indicative of functional relevance, and therefore such a set of genes might constitute promising candidates for functional analyses. In this study, we have sequenced the genome of the Pezizomycete Ascodesmis nigricans, and performed comparative transcriptomics of developing fruiting bodies of this fungus, the Pezizomycete Pyronema confluens, and the Sordariomycete Sordaria macrospora With only 27 Mb, the A. nigricans genome is the smallest Pezizomycete genome sequenced to date. Comparative transcriptomics indicated that gene expression patterns in developing fruiting bodies of the three species are more similar to each other than to nonsexual hyphae of the same species. An analysis of 83 genes that are upregulated only during fruiting body development in all three species revealed 23 genes encoding proteins with predicted roles in vesicle transport, the endomembrane system, or transport across membranes, and 13 genes encoding proteins with predicted roles in chromatin organization or the regulation of gene expression. Among four genes chosen for functional analysis by deletion in S. macrospora, three were shown to be involved in fruiting body formation, including two predicted chromatin modifier genes.

Several Phyllosticta species are known as pathogens of Citrus spp., and are responsible for various disease symptoms including leaf and fruit spots. One of the most important species is P. citricarpa, which causes a foliar and fruit disease called citrus black spot. The Phyllosticta species occurring on citrus can most effectively be distinguished from P. citricarpa by means of multilocus DNA sequence data. Recent studies also demonstrated P. citricarpa to be heterothallic, and reported successful mating in the laboratory. Since the domestication of citrus, different clones of P. citricarpa have escaped Asia to other continents via trade routes, with obvious disease management consequences. This pathogen profile represents a comprehensive literature review of this pathogen and allied taxa associated with citrus, focusing on identification, distribution, genomics, epidemiology and disease management. This review also considers the knowledge emerging from seven genomes of Phyllosticta spp., demonstrating unknown aspects of these species, including their mating behaviour.TaxonomyPhyllosticta citricarpa (McAlpine) Aa, 1973. Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Botryosphaeriales, Family Phyllostictaceae, Genus Phyllosticta, Species citricarpa.Host rangeConfirmed on more than 12 Citrus species, Phyllosticta citricarpa has only been found on plant species in the Rutaceae.Disease symptomsP. citricarpa causes diverse symptoms such as hard spot, virulent spot, false melanose and freckle spot on fruit, and necrotic lesions on leaves and twigs.Useful websitesDOE Joint Genome Institute MycoCosm portals for the Phyllosticta capitalensis (https://genome.jgi.doe.gov/Phycap1), P. citriasiana (https://genome.jgi.doe.gov/Phycit1), P. citribraziliensis (https://genome.jgi.doe.gov/Phcit1), P. citrichinaensis (https://genome.jgi.doe.gov/Phcitr1), P. citricarpa (https://genome.jgi.doe.gov/Phycitr1, https://genome.jgi.doe.gov/Phycpc1), P. paracitricarpa (https://genome.jgi.doe.gov/Phy27169) genomes. All available Phyllosticta genomes on MycoCosm can be viewed at https://genome.jgi.doe.gov/Phyllosticta.

Carrillo, J.D., Rugman-Jones, PF., Husein, D., Stajich, J.E., Kasson, M.T., Carrillo, D., Stouthamer, R., and Eskalen, A. 2019. Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan. A number of ambrosia beetles have come to prominence in recent years because of the damage they inflict on a variety of trees within invaded habitats across the globe. Ambrosia beetles rely on symbiotic microorganisms, mainly fungi, as a dedicated food source and carry those microorganisms around with them within specialized organs termed mycangia. Investigation of members of the Euwallacea fornicatus species complex and their fungal symbionts in Taiwan revealed promiscuous symbioses with ambrosial Fusaria clade (AFC) members, Graphium spp., and Paracremonium spp. based on co-phylogenetic analyses. For AFC members, a novel diagnostic PCR assay targeting mating type genes MAT1-1-1 and MAT1-2-1 was developed and validated by amplicon size and sequencing. Mating type screening of AFC members revealed the isolates screened are all heterothallic (self-sterile), with both MAT types represented and recovered from fungi vectored by E. fornicatus (tea shot hole borer), E. kuroshio (Kuroshio shot hole borer), and E. whitfordiodendrus (polyphagous shot hole borer) in Taiwan. Members of the Euwallacea fornicatus species complex and the variety of ambrosia fungi they utilize further confirms that their relationship with these fungi are more likely promiscuous in native areas, as opposed to strictly obligate to a specific combination of fungi as observed in invaded areas.

Phytophthora colocasiae is an important pathogen that causes great economic losses in taro production in tropical and subtropical regions, especially in Japan. Understanding the genetic variations in P. colocasiae populations and their transmission patterns in Japan is essential for effective disease control. Here, the genetic diversity of 358 P. colocasiae isolates, including 348 from Japan, 7 from China, and 3 from Indonesia, was assessed using 11 simple sequence repeat (SSR) primer pairs with high polymorphism. The phylogenetic tree of the SSR locus showed that the isolates from Japan could be divided into 14 groups, with group A being the dominant group. Among foreign isolates, only six from mainland China were similar to those from Japan and clustered in groups B and E. Analysis of molecular variance (AMOVA), principal components analysis (PCA), and cluster analysis (K = 3) results revealed a moderate level of genetic diversity, mainly within individuals. Populations showed high heterozygosity, a lack of regional differentiation, and frequent gene flow. Analysis of mating types and ploidy levels revealed that A2 and self-fertile (SF) A2 types and tetraploids were dominant across populations. Explanations and hypotheses for the results can provide more effective strategies for disease management of taro leaf blight. [ABSTRACT FROM AUTHOR]

Summary: The Closterium peracerosum–strigosum–littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium.We sequenced genomes representing the two MTs, mt+ and mt−, in Closterium and identified CpMinus1, a gene linked to the mt− phenotype. We analyzed its function using reverse genetics methods.CpMinus1 encodes a divergent RWP‐RK domain‐containing‐like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt− strain, while CpMinus1‐knockout mt− strains were phenotypically mt+.We propose that CpMinus1 is the major MT determinant that acts by evoking the mt− phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg–sperm anisogamous mating system. mt− specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum L.) and tomato (S. lycopersicum L.) are the most economically important vegetable crops in Egypt and worldwide. The winter crop in Egypt is particularly prone to late blight caused by Phytophthora infestans. A total of 152 P. infestans isolates were isolated from the 2013, 2014, 2016 and 2018 winter crops with 82 isolates from potato, 69 from tomato and one isolate from eggplant (S. melongena L.). All isolates belonged to the A1 mating type with no evidence of A2 or self-fertile strains. The majority of isolates (53%) were sensitive to metalaxyl, 32% were intermediate and 15% were resistant. Variation in aggressiveness between three P. infestans isolates EG-005 (13_A2) and EG-276 (23_A1) from potato, and EG-237 (23_A1) from eggplant was determined on tuber slices and leaflets of 10 potato cultivars. The eggplant isolate EG-237 showed higher sporulation capacity compared with the other tested isolates and was able to infect potato (Lady Rosetta cv) and tomato (Super Strain B cv). The simple sequence repeat (SSR) genotyping data showed that in contrast to our previous work (3-year period 2010-12) in which the proportion of 13_A2 lineage was 35%, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. The data have been submitted into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. The AVR2 and AVR2-like RXLR effector genes were amplified and sequenced. In the avirulent AVR2 gene, only one heterozygous SNP was detected at position 31 in the N terminus in six isolates out of eleven, whereas two heterozygous SNPs were detected at position 29 in the N-terminus and ninety-two in the C- terminus of the AVR2-like gene. This suggests that changes in the previously reported virulence profile of 23_A1 are not related to commercial cultivars carrying the R2 gene. In addition, this is the first report of P. infestans on eggplant in Egypt. [ABSTRACT FROM AUTHOR]

Phytophthora infestans is the oomycete responsible for late blight disease of Solanaceae that causes both yield and economic losses. With the aim of reducing plant wilt and high management costs mainly due to wide fungicide applications, alternative eco-sustainable control strategies are needed. RNA interference (RNAi) is a powerful tool for gene function studies that can be accomplished by constitutive transformation or transient expression such as virus-induced gene silencing (VIGS) experiments. VIGS makes use of viruses to deliver sequences homologous to a target gene fragment and trigger RNAi. Indeed, a P. infestans ortholog of plant loricrin-like protein (LLP), named PiLLP, has been silenced using the direct infection of a recombinant vector based on the plant virus tobacco mosaic virus (TMV-PiLLP-1056), aiming to reduce the oomycete sexual reproduction. For this purpose, the gene coding for the green fluorescent protein (GFP) present in the TMV-GFP-1056 vector has been replaced with an antisense construct obtained by fusion PCR of the PiLLP 5′-UTR and 3′-UTR sequences. Here, we show that RNAi can be expressed in the A1 mating type of P. infestans strain 96.9.5.1 by VIGS using the direct infection of TMV-PiLLP-1056. We provide evidence that the recombinant vector can enter, replicate, and persist in mycelia of P. infestans where it induces the partial downregulation of the PiLLP transcript. Compared with the wild-type, the PiLLP-silenced A1 mating type had slower colony growth and a diminished virulence in detached tomato leaflets. This seems to be the first evidence of a constitutive gene downregulation of P. infestans using a recombinant vector based on a plus-sense RNA plant virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However, S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineered the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats with Cas9)-mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits-thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.IMPORTANCE The yeast Kluyveromyces marxianus grows at high temperatures and on a wide range of carbon sources, making it a promising host for industrial biotechnology to produce renewable chemicals from plant biomass feedstocks. However, major genetic engineering limitations have kept this yeast from replacing the commonly used yeast Saccharomyces cerevisiae in industrial applications. Here, we describe genetic tools for genome editing and breeding K. marxianus strains, which we use to create a new thermotolerant strain with promising fatty acid production. These results open the door to using K. marxianus as a versatile synthetic biology platform organism for industrial applications.

Blackleg (Phoma) disease, caused by the ascomycete fungi Plenodomus biglobosus and P. lingam, threatens oilseed rape (OSR; Brassica napus) crops internationally. In many parts of the world, both species co-occur, but in China only P. biglobosus has so far been reported. Plenodomus biglobosus reproduces asexually (pycnidiospores), but also sexually (pseudothecia-yielding ascospores), via a heterothallic mating system requiring MAT1-1 and MAT1-2 genotypes. However, the roles of airborne ascospore inoculum in driving blackleg disease outbreaks in China are less well understood compared to elsewhere in the world. This is despite the very different agronomic cropping practices in parts of China, in which paddy rice and OSR are often grown in rotation; OSR stubble is often submerged under water for long periods potentially affecting pseudothecial development. Here, we indirectly investigate the potential role of sexual reproduction by developing new polymerase chain reaction (PCR) -based mating-type diagnostics for P. biglobosus and subsequently screening an international collection of 59 European and 157 Chinese isolates. Overall, in both Europe and China, P. biglobosus mating types did not deviate from a 1:1 ratio, such as is generally thought to occur under frequency-dependent selection in sexually reproducing pathogen populations. Both mating types were balanced in all the individual European countries tested (Austria, France, Poland, UK). Conversely, in China, mating types were only balanced in the eastern region; in the northern and southwestern regions there were skewed ratios, more typical of predominantly asexual reproduction, towards MAT1-1 and MAT1-2, respectively. The implications of these findings and future research directions for improved understanding of P. biglobosus epidemiology on OSR, particularly in China, are considered. [ABSTRACT FROM AUTHOR]