A2 Mating Type Research Articles

Tipos de compatibilidad A1 y A2 de Phytophthora capsici y P. drechsleri coexisten en plantas ornamentales de vivero

<p>No se ha reportado la presencia de ambos tipos de compatibilidad de especies de Phytophthora en plantas hospedantes sembradas en una misma maceta en viveros comerciales de México. En 2015, se colectaron aislados de <em>Phytophthora</em> en viveros comerciales en la Ciudad de México y en el estado de Morelos. Se detectaron los tipos de compatibilidad A1 y A2 de <em>Phytophthora capsici</em> y <em>P. drechsleri</em> en plantas marchitas de <em>Capsicum annuum</em> (tallos) y <em>Petunia x hybrida</em> (rizósfera), respectivamente. Los aislados de <em>Phytophthora</em> coinoculados <em>in planta</em> formaron oosporas en condiciones de invernadero. <em>Phytophthora</em> fue re-aislada de plantas inoculadas e identificada mediante herramientas morfológicas y moleculares. Los tipos de compatibilidad A1 y A2 de <em>Phytophthora capsici</em> y <em>P. drechsleri</em> se presentan simultáneamente en plantas de invernadero, lo cual sugiere que están ocurriendo recombinación sexual y variación genética. Los tipos de compatibilidad A1 y A2 de <em>Phytophthora capsici</em> y <em>Phytophtora dechsleri</em> fueron detectados por primera vez en <em>Capsicum annuum</em> y <em>Petunia x hybrida</em> respectivamente, en viveros mexicanos.</p>

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.

Virulence and Mating Type of Phytophthora infestans Isolates in the Czech Republic

Abstract To evaluate the frequency and stability of the occurrence of P. infestans races and mating types in the Czech Republic, 338 monosporic isolates were collected from 31 sites in different potato-growing areas from 2012 to 2014 and in 2016. In total, 142 isolates were evaluated for virulence and race structure using the detached leaflet assay on Black’s differential set, supplemented with cultivar Sarpo Mira and somatic hybrid REG 46F. With the exception of virulence for resistance genes R9 and Rpi-blb-1, all virulence genes were detected among isolates, with a predominance of genes R1, R3, R7, R10, and R11. Most isolates were virulent to five or more R-genes, with a mean virulence complexity of 7.1. Among the 38 races detected, the most commonly occurring races were 1.2.3.4.6.7.10.11 and 1.2.3.4.7.10.11. Of the 338 isolates tested by the pairing test and the cleaved amplified polymorphism sequence (CAPS) marker, 40% were of the A1 mating type and 60% were of the A2 mating type, with an A1 : A2 isolate ratio demonstrating the predominance of the A2 mating type each year of the survey.

Characterization of the Black Shank Pathogen, Phytophthora nicotianae, Across North Carolina Tobacco Production Areas.

Black shank disease of tobacco, caused by the oomycete Phytophthora nicotianae, is a major threat to production in the United States and tobacco-producing areas worldwide. In a statewide survey of North Carolina, the rapid shift from race 0 to race 1 was documented. Collected pathogen isolates were characterized phenotypically for mating type and mefenoxam sensitivity, and genotypically by comparing sequences from three cytoplasmic and two nuclear regions. Both the A1 and A2 mating types were found throughout the state. When both mating types were recovered from the same field, pairings of isolates yielded viable oospores, indicating for the first time the potential for sexual sporulation by P. nicotianae in natural populations. Because the loss of complete resistance required a renewed use of the fungicide mefenoxam, a subset of the survey isolates was screened for sensitivity to the fungicide. All isolates were sensitive, with a mean effective concentration to inhibit 50% of hyphal growth of 0.4 μg/ml that was similar across mating types and races. Molecular characterization of 226 isolates revealed that the pathogen exists as multiple clonal types within the state. Genetic diversity among the pathogen population and the potential for sexual recombination may help explain the ability of the pathogen to rapidly adapt to host resistance genes.

Regulation of mating type switching by the mating type genes and RME1 in Ogataea polymorpha

Saccharomyces cerevisiae and its closely related yeasts undergo mating type switching by replacing DNA sequences at the active mating type locus (MAT) with one of two silent mating type cassettes. Recently, a novel mode of mating type switching was reported in methylotrophic yeast, including Ogataea polymorpha, which utilizes chromosomal recombination between inverted-repeat sequences flanking two MAT loci. The inversion is highly regulated and occurs only when two requirements are met: haploidy and nutritional starvation. However, links between this information and the mechanism associated with mating type switching are not understood. Here we investigated the roles of transcription factors involved in yeast sexual development, such as mating type genes and the conserved zinc finger protein Rme1. We found that co-presence of mating type a1 and α2 genes was sufficient to prevent mating type switching, suggesting that ploidy information resides solely in the mating type locus. Additionally, RME1 deletion resulted in a reduced rate of switching, and ectopic expression of O. polymorpha RME1 overrode the requirement for starvation to induce MAT inversion. These results suggested that mating type switching in O. polymorpha is likely regulated by two distinct transcriptional programs that are linked to the ploidy and transmission of the starvation signal.

Incidence of the potato late blight pathogen, Phytophthora infestans, in Syria and its mating type

Late blight, caused by Phytophthora infestans (Mont.) de Bary, is a devastating disease found worldwide. During 2011, 2013 and 2014 cropping seasons, prevalence of potato late blight in all survived areas throughout the interior of Syria was examined and the disease incidence varied and ranged from 0 to 85.71%. Potato leave samples showing late blight symptoms were collected from five different regions known to grow potato intensively. Based on their morphological characteristics, 22 isolates were distinguished as P. infestans. Using two fungal specific primers, ITS3 and PINF2 in a PCR assay, all isolates were confirmed as P. infestans, while they produce ~ 450 bp band. Similarly, all confirmed P. infestans isolates were able to produce a 170-bp band when INF-1 and INF-2 primers were deployed indicating that studied isolates had merely mating type A1 and no isolate with mating type A2 was found which might explain the absence of oospores in considered areas.

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.

Intra- and Intergenomic variation of Ploidy and Clonality characterize Phytophthora capsici on Capsicum sp. in Taiwan

Phytophthora capsici is a devastating disease of pepper (Capsicum sp.) in Taiwan causing complete loss of commercial fields. The objective of this study was to characterize genetic diversity for 38 newly collected isolates and three historical isolates. Analysis of data includes whole genome sequence for two new isolates and for two isolates collected previously in 1987 and 1995. In addition, 63 single nucleotide polymorphism loci were genotyped using targeted-sequencing, revealing 27 unique genotypes. Genotypes fell into three genetic groups: two of the groups contain 90% (n = 33) of the 2016 isolates, are triploid (or higher), are exclusively the A2 mating type and appear to be two distinct clonal lineages. The isolates from 2016 that grouped with the historical isolates are diploid and the A1 mating type. Whole genome sequence revealed that ploidy varies by linkage group, and it appears the A2 clonal lineages may have switched mating type due to increased ploidy. Most of the isolates were recently race-typed on a set of differential C. annuum, and although there was no direct correlation between virulence and ploidy, many of the triploid isolates were less virulent as compared to the historical diploid isolates. The implications for breeding resistant pepper and conducting population analyses are discussed.

Universality of the Phytophthora mating hormones and diversity of their production profile

Sexual reproduction of the plant pest Phytophthora is regulated by two mating hormones α1 and α2, which are acyclic oxygenated diterpenes first isolated from P. nicotianae A1 and A2 mating types, respectively. A previous report suggested the universality of these factors within this genus. To confirm this concept, we investigated 80 strains (19 species) of Phytophthora and a related genus, not only for the responsiveness to mating hormones but also for their productivity. The results indicated that among the 55 heterothallic strains, 24 (44%) responded to a mating hormone and 40 (73%) produced one or both hormones. These findings demonstrate the interspecies universality of mating hormones within the genus Phytophthora. Hormone productivity was found to be highly diverse and dependent on the strains used. Although the A2 mating type has been regarded as the α2 producer, 19 (59%) of the 32 A2-type strains produced both the hormones and two A2-type strains exclusively produced α1 in high yields. These results indicate that hormone biosynthesis in Phytophthora is universal but highly diverse and complex, and varies with culture conditions, providing us valuable information for future studies on the mechanism of mating hormone biosynthesis of Phytophthora.

Resurgence of cucurbit downy mildew in the United States: Insights from comparative genomic analysis of Pseudoperonospora cubensis.

Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew (CDM), is known to exhibit host specialization. The virulence of different isolates of the pathogen can be classified into pathotypes based on their compatibility with a differential set composed of specific cucurbit host types. However, the genetic basis of host specialization within P. cubensis is not yet known. Total genomic DNA extracted from nine isolates of P. cubensis collected from 2008 to 2013 from diverse cucurbit host types (Cucumis sativus, C. melo var. reticulatus, Cucurbita maxima, C. moschata, C. pepo, and Citrullus lanatus) in the United States were subjected to whole‐genome sequencing. Comparative analysis of these nine genomes confirmed the presence of two distinct evolutionary lineages (lineages I and II) of P. cubensis. Many fixed polymorphisms separated lineage I comprising isolates from Cucurbita pepo, C. moschata, and Citrullus lanatus from lineage II comprising isolates from Cucumis spp. and Cucurbita maxima. Phenotypic characterization showed that lineage II isolates were of the A1 mating type and belonged to pathotypes 1 and 3 that were not known to be present in the United States prior to the resurgence of CDM in 2004. The association of lineage II isolates with the new pathotypes and a lack of genetic diversity among these isolates suggest that lineage II of P. cubensis is associated with the resurgence of CDM on cucumber in the United States.

A PCR-based Assay for Distinguishing between A1 and A2 Mating Types of Phytophthora capsici

Sexual reproduction in the plant parasite Phytophthora capsici Leonian requires the interaction of two distinct mating types, A1 and A2. Co-occurrence of these mating types can enhance the genetic diversity of P. capsici and alter its virulence or resistance characteristics. Using an intersimple sequence repeat (ISSR) screen of microsatellite diversity, we identified, cloned, and sequenced a novel 1121-base pair (bp) fragment specific to the A1 mating type of P. capsici. Primers Pcap-1 and Pcap-2 were designed from this DNA fragment to specifically detect the A1 mating type. Polymerase chain reaction (PCR) using these primers amplified an expected 997-bp fragment from known A1 mating types, but yielded a 508-bp fragment from known A2 mating types. This PCR-based assay could be adapted to accurately and rapidly detect the co-occurrence of A1 and A2 P. capsici mating types from field material.

Population Structure and Genetic Diversity of Phytophthora nicotianae from Tobacco in Georgia.

Black shank, caused by Phytophthora nicotianae, occurs worldwide and is responsible for significant yield loss in tobacco production in Georgia. Management of the disease has primarily relied on utilization of tobacco cultivars with resistance to race 0 of the pathogen and application of the fungicide mefenoxam. Races of P. nicotianae currently prevalent in tobacco production in Georgia, their sensitivity to mefenoxam, and genetic diversity of the pathogen are largely unknown. To determine population structure and genetic diversity of the pathogen, simple sequence repeat (SSR) markers were used. Three races of P. nicotianae (races 0, 1, and 3) were isolated from infected tobacco plants, with race 3 identified in Georgia for the first time. The majority of isolates were identified as A2 mating type and all isolates were sensitive or intermediately sensitive to mefenoxam at 1 or 10 μg/ml, with effective concentration of mefenoxam for 50% mycelial growth reduction values ranging from <0.01 to 0.12 μg/ml. Bayesian and unweighted pair group method with arithmetic means analyses of 59 isolates using SSR markers grouped the isolates in two major groups. Group I contained 20 isolates, of which 19 isolates were collected from Berrien County. Group II contained 39 isolates collected from Bacon, Cook, Tift, and Toombs Counties as well as one sample from Berrien County. Genetic diversity of the isolates was associated with geographical location of collection, and isolates in group I were primarily (75%) race 1, whereas isolates in group II were primarily (69%) race 0. The presence of a single pathogen mating type at most of the locations implies low probability of sexual recombination that may have contributed to the low genetic diversity at a particular geographical location. Sensitivity of the isolates to mefenoxam indicates that the fungicide remains to be a potent tool for growers to combat the disease. Information generated in the study advances our knowledge about diversity and population structure of P. nicotianae, which facilitates development and implementation of effective disease management programs.

Phytophthora cinnamomi A1: An ancient resident of New Guinea and Australia of Gondwanan origin?

SummaryThis article re‐examines the hypothesis, first proposed by Shepherd (Search, 6(11‐12), 1975, 484), that Phytophthora cinnamomi is an ancient organism in Australia and New Guinea. It further evaluates data that suggest the A1 mating type is Gondwanan in origin and may have been present in New Guinea for up to 10 million years. It is postulated that there has been a mating type change in P. cinnamomi from A1 to A2 in relatively recent times as a result of genetic transformation of the A1 mating type.

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.

Complete mitogenome mapping of potato late blight pathogen, Phytophthora infestans A2 mating type

Complete mitochondrial genome of Phytophthora infestans, A2 mating type (MT) with a size of ≅37,767 bp was sequenced. A total of 53 protein-coding genes are predicted on both strands, including 25 tRNA, 2 rRNA, and 18 respiratory proteins. Gene order of A2MT was consistent with that established in A1, despite high level of polymorphism in both coding and non-coding regions. The mtDNA of A2MT was found to have 99.5% and 99.4% homology with Ia and Ib, whereas 94.7% and 94.3% with IIa and IIb, respectively. Study of repeats revealed a dinucleotide (AT)9 specific to A1 and homology of cox1 gene sequence revealed the relationship among 50 Phytophthora species.

DNA Markers for Selection of Late Blight Resistant Potato Breeding Lines

Potato late blight, caused by the oomycete Phytophthora infestans, is one of the most devastating diseases in the agricultural sector around the world. Many genes (R genes) conferring resistance to late blight have been identified in various potato species and most of these R genes have been used in potato breeding. The aim of this study was to develop and validate PCR-based assays for the R genes Rpi-blb1, Rpi-blb2, Rpi-blb3 and Rpi-bt1, to distinguish between late blight resistant and late blight susceptible potato progeny in the given breeding background. A total of 100 breeding progeny were screened for the presence of these R genes and tested for resistance against P. infestans mating type A2, genotype US-8 strain, using detached leaf and tuber rot assays. PCR products for the Rpi-blb1 and Rpi-bt1 resistance genes were identified in the resistant progeny but were absent in the susceptible ones; therefore these PCR assays could differentiate between late blight resistant and susceptible plants. Genotypic data from the DNA markers derived from the Rpi-blb1 and Rpi-bt1 genes was found to correlate with the phenotypic data for foliar late blight but not with data for tuber rot. Our results demonstrate that markers derived from these two R genes could be useful for marker-assisted selection (MAS) for foliar late blight resistance in potato breeding programs.

Occurrence and Distribution of Mating Types of Pseudoperonospora cubensis in the United States.

During the past two decades, a resurgence of cucurbit downy mildew has occurred around the world, resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and several hypotheses, including introduction of a new genetic recombinant or pathotype of the pathogen, have been suggested as potential causes for this resurgence. Occurrence and distribution of mating types of Pseudoperonospora cubensis in the United States were investigated using 40 isolates collected from cucurbits across 11 states from 2005 to 2013. Pairing of unknown isolates with known mating-type tester strains on detached leaves of cantaloupe or cucumber resulted in oospore formation 8 to 10 days after inoculation. Isolates differed in their ability to form oospores across all coinoculation pairings, with oospore numbers ranging from 280 to 1,000 oospores/cm2 of leaf tissue. Oospores were hyaline to golden-yellow, spherical, and approximately 36 μm in diameter. Of the 40 isolates tested, 24 were found to be of the A1 mating type, while 16 were of the A2 mating type. Mating type was significantly (P < 0.0001) associated with host type, whereby all isolates collected from cucumber were of the A1 mating type, while isolates from squash and watermelon were of the A2 mating type. Similarly, mating type was significantly (P = 0.0287) associated with geographical region, where isolates from northern-tier states of Michigan, New Jersey, New York, and Ohio were all A1, while isolates belonging to either A1 or A2 mating type were present in equal proportions in southern-tier states of Alabama, Florida, Georgia, North Carolina, South Carolina, and Texas. Viability assays showed that oospores were viable and, on average, approximately 40% of the oospores produced were viable as determined by the plasmolysis method. This study showed that A1 and A2 mating types of P. cubensis are present and the pathogen could potentially reproduce sexually in cucurbits within the United States. In addition, the production of viable oospores reported in this study suggests that oospores could have an important role in the biology of P. cubensis and could potentially influence the epidemiology of cucurbit downy mildew in the United States.

Six new Phytophthora species from ITS Clade 7a including two sexually functional heterothallic hybrid species detected in natural ecosystems in Taiwan.

During a survey of Phytophthora diversity in natural ecosystems in Taiwan six new species were detected. Multigene phylogeny based on the nuclear ITS, ß-tubulin and HSP90 and the mitochondrial cox1 and NADH1 gene sequences demonstrated that they belong to ITS Clade 7a with P. europaea, P. uniformis, P. rubi and P. cambivora being their closest relatives. All six new species differed from each other and from related species by a unique combination of morphological characters, the breeding system, cardinal temperatures and growth rates. Four homothallic species, P. attenuata, P. flexuosa, P. formosa and P. intricata, were isolated from rhizosphere soil of healthy forests of Fagus hayatae, Quercus glandulifera, Q. tarokoensis, Castanopsis carlesii, Chamaecyparis formosensis and Araucaria cunninghamii. Two heterothallic species, P. xheterohybrida and P. xincrassata, were exclusively detected in three forest streams. All P. xincrassata isolates belonged to the A2 mating type while isolates of P. xheterohybrida represented both mating types with oospore abortion rates according to Mendelian ratios (4-33 %). Multiple heterozygous positions in their ITS, ß-tubulin and HSP90 gene sequences indicate that P. xheterohybrida, P. xincrassata and P. cambivora are interspecific hybrids. Consequently, P. cambivora is re-described as P. xcambivora without nomenclatural act. Pathogenicity trials on seedlings of Castanea sativa, Fagus sylvatica and Q. suber indicate that all six new species might pose a potential threat to European forests.

In-vitro characterisation of the four lineages of Phytophthora ramorum

Phytophthora ramorum is a plant pathogen with a wide host range including many ornamental hosts and tree species. In Ireland and the UK P. ramorum is known to cause sudden larch death. There are four distinct genetic lineages of P. ramorum, with the fourth lineage (EU2) described in 2012 and only present in Northern Ireland and Scotland. In this work, experiments that compare all four lineages of P. ramorum using several phenotypic characters are described. A total of 166 isolates (EU1: 116, EU2: 40, NA1: 8, NA2: 2) from several EU countries and the United States and several hosts were amassed, and a selection of isolates were compared according to standard phenotypic tests. The EU1 and EU2 isolates tested were all A1 mating type. Regarding linear growth rate, we found the isolates ranked as follows EU2 > NA2 > EU1 > NA1, with all lineages growing fastest at 20 °C. The lineages ranked as NA2 > EU1 > EU2 > NA1 based on their in-vitro aggressiveness on detached wounded Rhododendron leaves, all lineages most aggressive at 20 °C. At 20 °C, we found that there was no significant difference between the EU1 and EU2 lineage based on their linear growth rate or in-vitro aggressiveness. Temperature, host ramet and P. ramorum lineage all had statistically significant effects on the observed aggressiveness of the isolates. From an experimental point of view, our results are broadly in agreement with other phenotypic studies of P. ramorum, finding variation between the lineages, but also variation within the lineages. From an applied perspective, our work on Rhododendron indicates that the EU1 and EU2 lineages pose similar levels of threat to plant health in Ireland and the UK, however, how these results transfer to other hosts (e.g. Larix kaempferi) needs more study.

Evaluation of the Ph‐3 gene‐specific marker developed for marker‐assisted selection of late blight‐resistant tomato

AbstractLate blight caused by Phytophthora infestans is one of the most destructive diseases of tomato (Solanum lycopersicum L.) that mainly occurs in cool and wet environments. With the spread of the A2 mating type and new clonal lineages, fewer fungicides provide effective control of the disease, which has increased its worldwide threat. Host resistance could contribute significantly to sustainable disease control. Ph‐3 is a race‐specific late blight resistance gene commonly used in commercial tomato breeding. Availability of precise and easy to use gene‐based markers would facilitate selection. In this study, a Ph‐3 on‐gene cleaved amplified polymorphic sequence (CAPS) marker, Ph3.gsm/HincII, was developed based on the published gene sequence of Ph‐3. The effectiveness of the marker was evaluated along with other published Ph‐3 markers using an F9 recombinant inbred line (RIL) population derived from NC 23E‐2(93) × L3708. Markers Ph3.gsm/HincII and TG328/BstNI accurately genotyped the RIL population for Ph‐3. In addition, Ph3.gsm/HincII was able to differentiate variable susceptible alleles. This reliable codominant DNA marker would be very useful in marker‐assisted selection, particularly for resistance gene pyramiding.