Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.     

Mating type gene MAT1-2-1 is common among Japanese isolates of Verticillium dahliae

Mating type genes of Verticillium dahliae, a wilt pathogen affecting many plant species, were identified to examine sexual recombination between Japanese pathotypes. We amplified a DNA sequence encoding high mobility group (HMG) box from V. dahliae using PCR. A cloned genomic DNA fragment included a sequence homologous to MAT1-2-1 gene. Despite that sequence's presence in all V. dahliae isolates we used, MAT1-1-1 (an opposite mating type gene) was never amplified. We concluded that V. dahliae is potentially heterothallic. Furthermore, sexual bias practically obviates sexual recombination between Japanese pathotypes. This report describes, for the first time, a mating type gene of phytopathogenic Verticillium.     

玉米大斑病菌两性交配型菌株的出现频率及其育性分析

玉米大斑病菌属于异宗配合真菌,在自然界中存在A、a交配型菌株和Aa两性交配型菌株,为明确两性菌株的出现频率及其对有性生殖的影响,采用有性态诱导和交配型PCR鉴定的方法,对2011—2014年采集的野生菌株及室内诱导产生的有性杂交后代F1、F2代进行交配型组成鉴定,通过检测两性菌株子囊壳和子囊孢子的发育情况明确其有性生殖能力,结合RT-PCR技术对有性生殖过程中MAT1和MAT2基因的表达情况进行分析。结果表明,野生菌株和有性杂交后代中均存在两性交配型菌株,且出现频率相当,介于2.09%和6.25%之间;Aa两性菌株的育性与杂交时对应菌株的交配型组成相关,两性菌株与A交配型菌株或a交配型菌株杂交均可育,能产生成熟的子囊壳和子囊孢子,但其自交时败育,只产生子囊壳,不产生子囊孢子。两性菌株Aa自交和两性菌株Aa与A交配型或与a交配型菌株杂交时,Aa菌株中MAT1和MAT2基因表达量无明显差异,推测MAT基因在有性生殖产生子囊壳的过程中发挥了重要作用。     

Identification of pathogenic fungi and preliminary screening for resistance in <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. germplasm

Sexual reproduction in fungi is controlled by mating type genes, which are located at the MAT locus. In this study, we investigated the structure of this locus in the phytopathogenic fungus Phyllosticta citricarpa, the causal agent of citrus black spot disease. Despite intensive study, its sexual state has never been observed in single-spore culture. Through analysis of the genome sequences of two individual P. citricarpa isolates, the sequence of the DNA lyase gene was identified and, as previously reported in the literature, the mating type genes were located in the 3′ flanking region of this gene. The results suggested that P. citricarpa is heterothallic, owing to the exclusive presence of the MAT1–1 or MAT1–2 gene in individual strains. In order to characterize the MAT locus, we designed primers to amplify this region. P. citricarpa was found to have complete and apparently functional copies of MAT genes, containing α-1 and HMG domains, present in different isolates. In addition to MAT1–2-1 and MAT1–1-1 genes, the MAT1–1-4 gene was located in the 5′ flanking region of the MAT1–1-1 gene and the MAT1–2-5 gene was located in 5′ flanking region of the MAT1–2-1 gene. A multiplex PCR protocol was also developed to differentiate P. citricarpa idiomorphs, which can be used in distribution and incidence studies of mating type strains, in order to determine the occurrence of sexual reproduction and to facility crossing studies. Furthermore, in Brazil, the two idiomorphs occur in a 1:1 ratio, which is expected in sexually reproducing populations.     

Identification and distribution of mating‐type idiomorphs in populations of Podosphaera macularis and development of chasmothecia of the fungus

Podosphaera macularis, the causal agent of hop powdery mildew, is known to produce chasmothecia (formerly cleistothecia) in eastern North America and Europe. Ascocarps have not yet been reported from the Pacific Northwestern region of North America. Reasons for the apparent absence of chasmothecia in the Pacific Northwest were unknown. This study established that P. macularis is heterothallic and ascocarp ontogeny, maturation, dehiscence and ascospore infection proceed similarly to other powdery mildew fungi. Genome sequencing of a MAT1‐1 isolate revealed the structure of the MAT1 locus and presence of MAT1‐1‐3, demonstrating further similarities to other powdery mildew fungi. PCR assays with primers designed from conserved domains of the MAT1 idiomorphs were developed to characterize the frequency of idiomorphs in populations of P. macularis. Amongst 317 samples of P. macularis collected during 2012 and 2013 from the Pacific Northwest only the MAT1‐1 idiomorph was found. In contrast, among 56 samples from the eastern United States and Europe, MAT1‐1 and MAT1‐2 idiomorphs were detected at equivalent frequencies. At temperatures representative of late season conditions in the Pacific Northwest, chasmothecia formed readily when a Pacific Northwest MAT1‐1 isolate was paired with a MAT1‐2 isolate collected from outside the region. Although these findings do not encompass all climatic, geographic or temporal barriers that could inhibit the formation of chasmothecia, the current absence of the ascigerious stage of P. macularis in the Pacific Northwest could be explained by the absence of the MAT1‐2 mating type idiomorph.     

Both Mating types of Phaeosphaeria (anamorph Stagonospora) nodorum are Present in Western Australia

Phaeosphaeria (anamorph Stagonospora) nodorum is the most serious fungal pathogen of wheat in the West Australian (WA) wheat belt and is a diallelic heterothallic loculoascomycete. Its population genetics has received considerable attention. A recent study, which sampled isolates from diverse locations worldwide, has indicated that the mating-type idiomorph MAT1-1 is considerably more frequent than MAT1-2 in many populations. To investigate this, we developed PCR primers that amplify each idiomorph. In a sample of 23 isolates cultured from ascospores collected in the field, nine amplified DNA with the MAT1-1 primers and 14 amplified DNA with the MAT1-2 primers. The virulence of a MAT1-2 isolate was comparable with MAT1-1 isolates. Although these sample sizes are small, we suggest that this result is consistent with the presence of equal numbers of both mating types in populations of ascospores in WA.     

Mating Behaviour and Vegetative Compatibility in Spanish Populations of Botryotinia fuckeliana

Mating behaviour and vegetative compatibility were studied in Spanish populations of Botryotinia fuckeliana. Fifty-seven isolates out of the 61 tested were sexually fertile with one or more of the reference strains of known mating type (MAT1-1 or MAT1-2). Thirty-nine isolates were heterothallic, giving fertile crosses when mated with the MAT1-1 (24 isolates) or the MAT1-2 (15 isolates) reference strain. Eighteen isolates crossed successfully with both MAT1-1 and MAT1-2 reference strains, and were referred to as homothallic or MAT1-1/2. Both mating types were widespread, being represented in isolates from two regions, from the same and different greenhouses, from different hosts, and from different years of isolation. Isolates were paired on Malt Extract Agar + NaCl to evaluate vegetative compatibility. Most of the paired isolates were unable to fuse and showed a different reaction of incompatibility. Nitrate-non-utilising (nit) mutants were selected by growth on a medium amended with 30–50gl^–1 potassium chlorate. Over 600 chlorate-resistant sectors were recovered from 40 isolates at a mean frequency of 0.15–2.39 sectors per colony, but only 11% were identified as nit mutants by their thin growth with no aerial mycelium on minimal medium. However, most of these nit mutants reverted to wild type during six months of storage on chlorate-amended medium. Genetic complementation between nit mutants occurred only in two cases between mutants from the same isolate.     

黑龙江省稻瘟病菌育性及其交配型分析

为明确黑龙江省采集自不同年份、不同地区的稻瘟病菌Magnaporthe oryzae的育性能力和交配型分布,采用2株标准菌株GUY11(MAT1-2)和KA3(MAT1-1)对2016—2017年黑龙江省西部、东部、中部3个地区经单孢分离的241株稻瘟病菌进行育性测定,并利用PCR技术对其交配型进行检测。结果表明,黑龙江省西部、东部、中部的241株稻瘟病菌中可育性菌株比例为11.62%,其中雌性菌株、雄性菌株、两性菌株分别占1.66%、4.56%和1.25%,不能判断其性别的未知菌株占4.15%。采集自不同地区、不同年份的稻瘟病菌可育性差异均较大,西部、东部、中部地区可育性菌株出现频率分别为13.25%、7.27%和12.62%;2016年采集的稻瘟病菌可育性较高,可育性菌株出现频率为25.30%。黑龙江省稻瘟病菌群体中同时存在MAT1-1和MAT1-2两种交配型,主要以交配型MAT1-1占优势,出现频率为58.92%,交配型为MAT1-2的菌株出现频率为8.30%。不同地区稻瘟病菌的交配型亦有差异,交配型为MAT1-1的菌株在黑龙江省东部地区出现频率最高,为72.73%,在中部、西部地区的出现频率次之,分别为61.17%和46.99%。表明黑龙江省水稻种植区的稻瘟病菌同时存在2种交配型菌株,其交配型存在丰富的多态性,但其可育性及交配型分布不均衡。     

Microsatellite and mating type markers reveal unexpected patterns of genetic diversity in the pine root‐infecting fungus Grosmannia alacris

Grosmannia alacris is a fungus commonly associated with root‐infesting bark beetles occurring on Pinus spp. The fungus has been recorded in South Africa, the USA, France, Portugal and Spain and importantly, has been associated with pine root diseases in South Africa and the USA. Nothing is known regarding the population genetics or origin of G. alacris, although its association with root‐infesting beetles native to Europe suggests that it is an invasive alien in South Africa. In this study, microsatellite markers together with newly developed mating type markers were used to characterize a total of 170 isolates of G. alacris from South Africa and the USA. The results showed that the genotypic diversity of the South African population of G. alacris was very high when compared to the USA populations. Two mating types were also present in South African isolates and the MAT1‐1/MAT1‐2 ratio did not differ from 1:1 (χ² = 1·39, P = 0·24). This suggests that sexual reproduction most probably occurs in the fungus in South Africa, although a sexual state has never been seen in nature. In contrast, the large collection of USA isolates harboured only a single mating type. The results suggest that multiple introductions, followed by random mating, have influenced the population structure in South Africa. In contrast, limited introductions of probably a single mating type (MAT1‐2) may best explain the clonality of USA populations.     

Genetic diversity of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">spinaciae</Emphasis> in Japan based on phylogenetic analyses of rDNA-IGS and <Emphasis Type="Italic">MAT1</Emphasis> sequences

Twenty-eight isolates of Fusarium oxysporum f. sp. spinaciae (FOS; the causal agent of spinach wilt) collected from Japan were assessed for mating type and subjected to phylogenetic analysis. Mating type analysis revealed all isolates to be MAT1-2, suggesting that there is no sexual recombination within the population. Phylogenetic analyses based on nucleotide sequences of the ribosomal DNA intergenic spacer (IGS) and the mating type locus (MAT1) suggested that FOS is polyphyletic. The cluster analysis based on IGS showed four phylogenetic groups (S1–S4) among the isolates. Two distinct lineages, S1 and S3, included FOS isolates both of the vegetative compatibility group (VCG) types, 0330 and 0331, demonstrating that VCG differentiation in FOS may not necessarily reflect the phylogenetic relationships based on IGS and MAT1-2-1.     

Genomic characterization of mating type loci and mating type distribution in two apparently asexual plantation tree pathogens

The Eucalyptus stem canker pathogens Teratosphaeria gauchensis and T. zuluensis (Capnodiales, Teratosphaeriaceae) are found in many tropical regions of the world where their hosts are cultivated for plantation forestry. Population genetic analyses have suggested that some populations undergo recombination, even though their sexual states have never been observed. Against this background, the aim of this study was to characterize the mating type (MAT) locus of these species and thus to better understand the basis of their diversity. Known Mycosphaerellaceae MAT genes were used to identify and investigate the MAT locus in the T. gauchensis and T. zuluensis genomes. Both species were found to be heterothallic and primers were designed to amplify the opposite MAT idiomorphs as well as conserved regions within the MAT1-1-1 and MAT1-2-1 genes. Each Teratosphaeria MAT idiomorph was defined by either the MAT1-1-1 or the MAT1-2-1 gene, and an idiomorph-specific hypothetical protein (MAT1-1-10 and MAT1-2-12). Populations of T. zuluensis from Asia and southern Africa were dominated by a single mating type, whereas the proportions of the different idiomorphs for T. gauchensis in South America and southern Africa were similar. There was no physical evidence of sexual reproduction for either species and it is argued that although recombination may be possible, it is unlikely to form an important part of their life cycles in diseased Eucalyptus plantations. Instead, continuous human-mediated multiple introductions of these species have probably resulted in the current genetic structure of their populations, which holds risk for future disease outbreaks and interspecific hybridization.     

Identification mating-type locus structure and distribution of <Emphasis Type="Italic">Cochliobolus lunatus</Emphasis> in China

Cochliobolus lunatus (teleomorph: Curvularia lunata) is an important plant pathogenic fungus that causes the maize foliar spot, resulting in serious yield losses. In ascomycetes, a single mating-type (MAT) locus with two idiomorphs controls sexual development. The structure and arrangement of the MAT genes were examined to understand the MAT locus of C. lunatus. MAT loci were MAT1–1-1 or MAT1–2-1, flanked upstream and downstream by regions encoding GTPase activating protein, pyridoxamine phosphate oxidase domain, and β-glucosidase. A MAT1–1 or MAT1–2 idiomorph was identified in single isolate, and sexual reproduction in vitro indicated that the species was heterothallic. In vitro crossing between isolates with opposite MATs produced perithecia, asci, and ascospores. A multiplex MAT-specific PCR method was developed and used to test mating-type genes in 177 C.lunatus isolates collected from China. The ratio of isolates of each mating-type in China was consistent with a 1:1 ratio.     

Mating type and fungicide resistance in populations of Podosphaera xanthii in south Italy

Powdery mildew is one of the most common and severe diseases of cucurbits, causing heavy yield losses in all growing areas when not successfully controlled. Two different fungal species, Podosphaera xanthii and Golovinomyces orontii, are generally recognized as causal agents. The results of monitoring carried out in 2016 and 2018 confirmed that P. xanthii is the exclusive pathogen causing cucurbit powdery mildew (CPM) in southern Italy. P. xanthii is a bipolar heterothallic fungus; a PCR-based method for distinguishing MAT1-1 and MAT1-2 idiomorphs was applied for assessing mating type distribution in fungal populations present on cucurbits in different sites. The idiomorph MAT1-2 was prevalent over the MAT1-1 in 2016, whereas they were approximately in a 1:1 ratio in 2018; this finding corroborated the hypothesis that the MAT1-1 idiomorph was more recently introduced in the area. Cyflufenamid-resistant isolates were widespread in commercial greenhouses and field plantings even though use of this fungicide had been drastically reduced by the farmers 1 year before the monitoring due to the effectiveness losses observed in CPM control. Occurrence of cyflufenamid resistance and its impact on efficacy were evaluated in a field trial comparing different fungicide spray schedules. Cyflufenamid-resistant isolates were detected even at the first appearance of symptoms on leaves, increasing over time. Isolates resistant to cyflufenamid showed a resistance factor as high as 900. Generally, P. xanthii was better controlled when cyflufenamid was used in integrated strategies rather than in spray schedules based on the exclusive use of the fungicide.     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Genetic linkage map of <Emphasis Type="Italic">Phaeosphaeria nodorum</Emphasis>, the causal agent of stagonospora nodorum blotch disease of wheat

A genetic linkage map of the fungal pathogen Phaeosphaeria nodorum, the causal agent of stagonospora nodorum blotch disease of wheat, was created. A total of 152 ascospore-derived progeny from a single pseudothecium, which resulted from a cross of two opposite mating type isolates, Sn37-1 and S-81-B13B, was analysed with AFLP, RAPD, ISSR, expressed sequence tag (EST)-derived microsatellite primers and sequence tagged site markers developed from specific genes. The genetic linkage map consisted of 276 molecular markers, and included markers developed from five genes [Glyceraldehyde 3-phosphate dehydrogenase (gpd), malate synthase (Mls1), mannitol 1-phosphate dehydrogenase (Mpd1), mating type (MAT1) and RNA polymerase II (RPB2)], which were assigned to 21 major linkage groups (LGs). The total length of the 21 major LGs was 1,932.1 centiMorgans (cM) with an average spacing of 6.88 cM between loci. The idiomorph mating type gene (MAT1) loci was placed in LG 2 and was closely linked to RAPD marker A4-680. On the other hand, 24 molecular markers and four gene loci [β-glucosidase (bgl1), histidinol dehydrogenase (Hdh2), mannitol 1-phosphate dehydrogenase (Mpd2), and xylanase (Xyl 10-2)] were dispersed in 11 minor LGs. The segregation ratio of the xylanase (Xyl 10-1) locus was distorted and not mapped. This is the first genetic linkage map reported for this important foliar pathogen of wheat. In combination with the genomic sequence of P. nodorum strain SN15 (), the availability of a genetic linkage map of this organism would be an important tool to investigate quantitative trait loci (QTL) of biologically important phenotypes and for positional cloning.     

Mating Relationships between Isolates of Phaeosphaeria nodorum, (anamorph Stagonospora nodorum) from Geographical Locations

Mating types of Phaeosphaeria nodorum isolates (from North Africa, North America, Australia, Europe and Near East) were determined in laboratory conditions. Although both mating types were found, MAT1-1 and MAT1-2 were not evenly distributed among the isolates. Sexual compatibility pairings with standard mating type testers revealed that 56 of 101 isolates could be assigned to MAT1-1 and 5 to MAT1-2. Although the teleomorph occurred in different countries, the two mating types were observed only among isolates collected from France, Great Britain, Germany and Morocco. For Morocco, where no P. nodorum pseudothecia have been reported, this is the first report of the existence of the two opposite mating types. The remaining 40 isolates could not be designated to a specific mating type. However, in 17 crosses, pseudothecial initials or sterile pseudothecia were observed on wheat straw after two months. The implications of the predominance of one mating type are discussed.     

大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

The solar chamber: an innovative technique for controlling verticillium wilt of olive1

Control of olive tree wilt, caused by Verticillium dahliae, is not easy. A simple solar-chamber technique has been developed to achieve effective control. The treated tree is irrigated, the soil is covered by a plastic sheet, and a chamber made of a metal framework covered with plastic sheets is placed over the tree. Trees were exposed for 10, 15 and 20 days. Using this approach the temperature in the chamber reached 55°, and soil temperatures at 5 and 15 cm depth reached 55 and 45°, respectively. It was no longer possible to isolate V. dahliae from infected trees after 15–20 days. Growth of treated trees was improved as compared with the untreated ones. Laboratory studies on the efficacy of dry heat on the survival of V. dahliae in diseased branches showed that 50–55°C was lethal after 1 h. On PDA medium, the fungus was killed in 1 h at 50°C and in 45 min at 55°C.     

Genotypic and phenotypic characterization of the European A2 isolates of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis>

Phytophthora ramorum, a recently described North American and European pathogen, has three clonal lineages. The NA1 and NA2 lineages are found in North American forests and nurseries, while the EU1 lineage appears mainly in European nurseries. P. ramorum is heterothallic, having two mating types A1 and A2. All NA1 and NA2 isolates are of A2 mating type. When first collected, all EU1 isolates were of A1 mating type, with the exception of one A2 isolate collected in Belgium in 2002. Screening 410 other Belgian isolates for mating type revealed two additional EU1-A2 isolates collected in 2002 and 2003. PCR-RFLP, AFLP and SSR markers were used to determine the nature of the mating type change. The three isolates show no indications of sexual recombination or mitotic crossing over, indicating that mutation or mitotic gene conversion is the most likely explanation for the mating type change. We compared the pathogenicity and sporulation characteristics of the EU1-A2 isolates to those of EU1-A1 and NA1-A2 isolates on four host plants. Despite small differences in pathogenicity on some hosts, the EU1-A2 isolates were similarly aggressive to each other and to the EU1-A1 isolates and more aggressive than the NA1-A2 isolates. Sporulation characteristics were also comparable among EU1-A2 isolates and between EU1-A1 and EU1-A2 isolates, except for EU1-A2 isolate BBA 26/02. The limited genotypic and phenotypic differences between EU1-A2 isolates probably evolved after the mating type change, which may have occurred several years before the isolates were detected. There are strong indications that the EU1-A2 population has been eradicated from Belgium.