玉米大斑病菌交配型组成鉴定及其有性生殖条件优化

为进一步探究玉米大斑病菌Setosphaeria turcica有性生殖规律,自我国12个省区采集不同玉米种植区的大斑病样,分离后获得664株菌,采用PCR分子检测方法对其交配型进行鉴定;在室内对影响该病原菌有性生殖的诱导介质、Fe~(3+)浓度、碳源、光照等条件进行优化。结果表明,自然条件下玉米大斑病菌存在A交配型(携带StMAT1-1基因)、a交配型(携带StMAT1-2基因)和Aa交配型(同时携带StMAT1-1基因和StMAT1-2基因)菌株,其中A、a、Aa交配型菌株数量分别为46、585和33株,所占比例分别为6.93%、88.10%和4.97%;在温度25℃、相对湿度55%、玉米叶片为诱导介质、外源添加0.12 g/L FeCl_3和0.85 g/L CaCO_3、光照培养条件下,有利于有性态子囊壳的产生,同等条件下黑暗培养更利于有性态子囊壳成熟。     

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。     

粤、桂、苏、皖四省稻瘟病菌有性态的研究

应用国际稻瘟病菌标准交配型菌株KA3和THl6对2000年从广东、广西、江苏和安徽4省采集的145个稻瘟病菌菌株的育性和交配型测定结果表明,广东、广西和安徽3省稻瘟病菌群体中存在着Matl.1和Matl.2两种交配型,江苏省的23个可交配菌株均为Matl.1。各省间稻瘟病菌群体的可交配率差异不大,总的可交配率为48.3%。有9个菌株与标准菌株交配产生了子囊孢子,可育率为6.2%。检测到2个两性菌株,不同地区稻瘟病菌群体雌雄性别的比例有显著性差异。用不同交配型的可育菌株进行互交,14个组合中只有1个组合产生子囊壳,但没有形成成熟的子囊孢子。     

小麦白粉病菌有性生殖与自然群体交配型检测

 本研究根据MAT1-1-1(GenBank登录号HQ171902)和MAT1-2-1(GenBank登录号HQ171899)的部分基因序列设计和筛选其特异性引物,使用PCR方法对2012年度采自四川、陕西、云南、贵州、河南、安徽、江苏、河北、山东和甘肃10个省30个采集地点共516个小麦白粉病菌菌株的交配型基因进行检测。结果显示30个采集地点两种交配型基因都能检测到,其中28个采集地的两种交配型菌株比例(MAT1-1-1 :MAT1-2-1)符合1:1。选择具不同交配型基因的小麦白粉病菌株配对接种,16个随机杂交组合都能产生闭囊壳。对获得的闭囊壳进行子囊孢子释放,均可获得子囊孢子。该研究结果为深入研究有性生殖对小麦白粉病菌株毒性多样性的作用奠定了基础。     

稻瘟病菌群体育性分析的主要影响因子研究

 将稻瘟病菌10个两性可育菌株,包括GUY11、KA3、KA9、KA7、6023、2539W、8773R-19、8773R-27、8113R-2和8113R-10相互交配,结果表明,10个菌株育性好,交配型明确;2539W已丧失雌性功能,不再是两性菌株。原先由6023/2539W测为可育的24个福建省田间稻瘟病菌菌株分别与另外8个两性菌株交配,结果得出不同两性菌株测得同一组稻瘟病菌菌株的育性差异很大,这可能是由于两性菌株的交配能力强弱差异以及两性菌株与待测菌株之间的亲和性不同引起的。分析认为,GUY11和KA3最适于测定稻瘟病菌群体的育性和交配型,并用其测定了227个来自福建省田间稻瘟病菌菌株的育性。结果表明,福建省稻瘟病菌群体可育菌株率为81.1%,其中81.0%为交配型MAT1-2,19.0%为MAT1-1。研究结果将有助于进一步认识稻瘟病菌的群体结构特点及其演化规律。     

云南省稻瘟病菌的有性世代研究初报

为了解云南省稻瘟病菌有性世代的形成状况,用云南省14个地区采集的约300个稻瘟病菌株和分离自龙爪稷上的A.a交配型标准菌株,在燕麦片培养基上作了交配。交配结果是:西双版纳州、曲靖地区的分离菌中A,a两交配型菌都有;楚雄州、大理州的分离菌中仅有A交配型菌:昆明、保山、玉溪、文山、昭通、迪庆、临沧及怒江等地的分离菌中仅有a交配型菌,红河州和丽江地区的分离菌都末形成子囊孢子,无法确定其交配型。此外,曲靖地区龙爪稷上分离10个梨孢属菌株及日本茨城县糠稷上分离的7个梨孢属菌株,分别与上述标准菌株、云南省稻瘟病菌株、日本秋田县稻瘟病菌株作了交配,都末形成有性世代。     

球孢白僵菌有性世代培养及子代毒力稳定性鉴定

在利用球孢白僵菌Beauveria bassiana有性态改良球孢白僵菌菌株的研究中,菌株的选择非常重要,其中利用交配型基因选择菌株是必要手段。而且虫生真菌的有性态能够使菌株间产生基因交流,促进其进化的过程。为更好地研究球孢白僵菌的遗传规律,以获得避免菌种衰退的方法,本研究建立了分别以柞蚕蛹为活体寄主和糙米培养基为基质的2种人工培养有性世代繁殖体系。结果表明,交配型为MAT1-1-1型和MAT1-2-1型混合型菌株BbDPSD5,经过自交在2种基质上均能够产生有性态。而具有2种单一交配型的菌株BbOFDH1-5和BbDPSD2进行配伍试验,BbOFDH1-5×BbDPSD2、BbOFDH1-5×BbDPSD5和BbDPSD2×BbDPSD5在2种基质上均没有有性态产生。对获得的有性态子囊孢子经无性繁殖产生的菌株进行r DNA-ITS片段以及交配型基因检测,并对测序结果进行比对分析。结果显示,扩增得到的r DNA-ITS序列和MAT1-2-1交配型基因与亲本序列同源性为100%,而没有检测到MAT1-1-1型。2种培养方式获得的结果完全一致。对有性态子代子囊孢子产生的株系进行毒力测试结果表明,所有子代菌株与原始菌株BbDPSD5的毒力差异不显著,子代交配型基因虽然缺失了MAT1-1-1,但是毒力没有衰退,未出现菌种退化现象。     

稻瘟菌对水稻品种梅雨明的无毒性的遗传分析和分子标记

 将交配型为a、并对水稻品种梅雨明不致病的稻瘟菌菌株S1522与对该品种致病的、交配型为A的稻瘟菌菌株P131和S159杂交,分别获得了24和56个单子囊孢子后代。致病性测定结果表明:这些后代菌株在梅雨明上的致病个体与不致病个体的比例近等于1:1。由此推测:稻瘟菌对水稻品种梅雨明的无毒性是由1个基因控制的。进一步通过RAPD比较了致病后代与不致病后代的DNA多态型,获得了1个与该无毒基因连锁的、长度为1000 bp的RAPD标记,两者间的遗传距离为3.7cM。     

KMnO4和H2O2刺激掘氏疫霉卵孢子萌发对单卵孢株生物学性状的影响

 本文对经KMnO₄、H₂O₂刺激掘氏疫霉(Phytophthora drechsleri)卵孢子萌发建立的单卵孢株群体应用于掘氏疫霉遗传研究的可行性做了研究。2个掘氏疫霉菌株(A₁、A₂交配型各1株)经聚碳膜间隔配对产生的卵孢子用KMnO₄、H₂O₂以及不用上述氧化剂处理分别建立K、H、C 3个单卵孢株群体(卵孢子萌发率8-16%)。在群体水平上对来自同一亲本的上述3个群体生物学性状比较结果表明,H₂O₂处理刺激卵孢子萌发不影响生长速度、菌落形态、耐35℃高温生长、致病力及交配型的遗传稳定性,所建立的单卵孢株群体可用于上述性状的遗传研究;KMnO₄处理导致掘氏疫霉A₁和A₂交配型在自交后代各自分离为A₁、A₂、A₁A₂和A₂、A₁A₂,但不影响其他生物学性状的遗传稳定性,所建立的单卵孢株群体可用于除交配型性状以外的其他生物学性状的遗传研究,不宜用于交配型正常遗传规律的研究。用KMnO₄处理掘氏疫霉A₁或A₂交配型菌株单株自交产生的卵孢子,从A₁菌株自交后代获得A₂和A₁A₂以及从A₂菌株自交后代获得A₁A₂交配型单卵孢株的结果说明,掘氏疫霉异宗配合菌株体内可能同时包含控制A₁和A₂交配型的遗传因子,在正常条件下有1种交配型遗传因子不表达。KMnO₄处理可望作为揭示交配型遗传本质的有用试验手段。     

KMnO_4和H_2O_2刺激掘氏疫霉卵孢子萌发对单卵孢株生物学性状的影响

本文对经KMnO_4、H_2O_2刺激掘氏疫霉(Phytophthora drechsleri)卵孢子萌发建立的单卵孢株群体应用于掘氏疫霉遗传研究的可行性做了研究。2个掘氏疫霉菌株(A_1、A_2交配型各1株)经聚碳膜间隔配对产生的卵孢子用KMnO_4、H_2O_2以及不用上述氧化剂处理分别建立K、H、C 3个单卵孢株群体(卵孢子萌发率8—16%)。在群体水平上对来自同一亲本的上述3个群体生物学性状比较结果表明,H_2O_2处理刺激卵孢子萌发不影响生长速度、菌落形态、耐35℃高温生长、致病力及交配型的遗传稳定性,所建立的单卵孢株群体可用于上述性状的遗传研究;KMnO_4处理导致掘氏疫霉A_1和A_2交配型在自交后代各自分离为A_1、A_2、A_1A_2和A_2、A_1A_2,但不影响其他生物学性状的遗传稳定性,所建立的单卵孢株群体可用于除交配型性状以外的其他生物学性状的遗传研究,不宜用于交配型正常遗传规律的研究。用KMnO_4处理掘氏疫霉A_1或A_2交配型菌株单株自交产生的卵孢子,从A_1菌株自交后代获得A_2和A_1A_2以及从A_2菌株自交后代获得A_1A_2交配型单卵孢株的结果说明,掘氏疫霉异宗配合菌株体内可能同时包含控制A_1和A_2交配型的遗传因子,在正常条件下有1种交配型遗传因子不表达。KMnO_4处理可望作为揭示交配型遗传本质的有用试验手段。     

Mating system complexity and cryptic speciation in the seed bank pathogen Pyrenophora semeniperda

Mating strategies contributing to a balance between inbreeding and outcrossing are understudied in all but a few model fungal pathogens. This study examined factors that influence the occurrence of the sexual state of Pyrenophora semeniperda. It was consistently found to have functional copies of the MAT1-1 and MAT1-2 idiomorphs essential for sexual reproduction, despite considerable polymorphism in both single nucleotide polymorphisms (SNPs) and number of 18-base minisatellite repeats. The two idiomorphs occurred at approximately equal frequencies across 25 populations on Bromus tectorum seeds in the western United States, suggesting maintenance of sexual reproduction. The putative mating system is described as facultative pseudohomothallism, with only one of the two MAT1 idiomorphs found in a nucleus. Unikaryotic strains of opposite mating type can potentially mate, as can nuclei of opposite mating type in a thallus that results from anastomosis between vegetatively compatible unikaryotic strains. Strains shown to be dikaryotic using simple sequence repeat (SSR) markers may contain either or both MAT1 idiomorphs. Most populations consist of a mixture of MAT1-1, MAT1-2 and MAT1-1/MAT1-2 genotypes. A possible constraint on recombination is the presence of multiple strain groups characterized by ITS haplotype. These are apparently vegetatively incompatible, as even dikaryotic strains are invariably composed of a single ITS haplotype. Different ITS haplotypes also have unique combinations of MAT1-1 and MAT1-2 allelic variants, suggesting that perhaps these strain groups are also sexually incompatible. Phylogenetic analysis using both genome-wide SNP/indel polymorphisms and SSR markers demonstrated genetic divergence among ITS haplotypes, supporting the hypothesis that these strain groups may represent cryptic species.     

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.     

蝉棒束孢交配型基因的分析和检测

蝉棒束孢Isaria cicadae为药用真菌蝉花的无性型,在防治同翅目害虫上具有重要作用。在自然状态下蝉棒束孢多以孢梗束状态存在,其子实体阶段极为少见。交配系统是真菌有性生殖过程中的决定因素,因此可从交配型的角度探究蝉棒束孢有性子实体稀少的原因。本研究运用RACE技术首次从蝉棒束孢中克隆出MAT1-1-1与MAT1-2-1全长cDNA序列,并进行生物信息学分析。根据cDNA序列设计并优化得到蝉棒束孢交配型鉴定引物,并对采集自安徽地区40株蝉棒束孢交配型进行鉴定,结果表明交配型MAT1-1菌株14株、MAT1-2菌株26株,并没有检测到同时含有两种交配型或两种交配型全部缺失的现象,因此蝉棒束孢有性型子实体几无发生的现象并不完全归因于交配型因素。     

Disease severity,incidence and races of <Emphasis Type="Italic">Setosphaeria turcica</Emphasis> on sorghum in Uganda

In order to understand the underlaying causes of new severe turcicum leaf blight outbreaks in East Africa, a survey was undertaken in Uganda to examine the sorghum—Setosphaeria turcica interaction in terms of disease severity and incidence, the overall fungal population structure, and new resistant resources. Highest disease severities were recorded on caudatum accessions, whereas kafir genotypes were most resistant. The disease was more severe in the most humid farmlands compared to moderately dry agro-ecologies. In districts with wide adoption of the Epuripur variety a very high incidence (100%) of turcicum leaf blight was found. The two S. turcica mating type genes MAT1-1 and MAT1-2 assessed on fungal isolates deriving from both sorghum and maize diseased leaves were found in 20 of 23 districts sampled and in equal proportions. Upon cross inoculation on maize differential lines, four S. turcica isolates were identified as race 1, two as race 2, and one isolate corresponded to race 0 and race 3, respectively. The remaining 10 S. turcica isolates did not cause any disease symptoms on the maize lines assessed. Highly resistant accessions originating from a regional collection were found among the five sorghum races (kafir, guinea, caudatum, bicolor and durra), and are now implemented in new sorghum disease resistance programs.     

Bipolar heterothallism in B-group isolates of Leptosphaeria maculans

Pairwise combinations were carried out between the eight isolates originating from each of three asci of the B-group of Leptosphaeria maculans . Some of the pairings produced mature pseudothecia when incubated on wheat straws at 18°C and under a 12-h photoperiod of blue light. Isolates within a given tetrad could be attributed to one of two sexual compatibility groups, on the basis of combinations of isolates leading to fertile crosses. Within each tetrad, mating type segregated in a 1:1 ratio, suggesting the existence of one mating type gene ( MAT1 ) with two alleles MAT 1–1 and MAT 1–2. Under these same conditions, each individual isolate from an A-group ascus originating from Brassica juncea produced pseudothecia with one or other of two compatible A-group testers originating from B. napus . However, attempts to mate these eight A-group single-ascospore isolates with two B-group single-ascospore isolates of opposite mating types remained unsuccessful. This work is the first successful report of in vitro sexual reproduction between B-group isolates, and provides evidence for bipolar heterothallism in B-group isolates of L. maculans . The implications of this mating protocol for genetic studies of B-group isolates and the consequences of this work for the taxonomy of L. maculans are discussed.     

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.     

Distribution of Mating Types and Diversity in Virulence of <Emphasis Type="Italic">Didymella rabiei</Emphasis> in Israel

The distribution of mating types and diversity in virulence of Didymella rabiei populations were studied in Israel from 1997 to 1999. Forty-one monoconidial D. rabiei isolates from 18 commercial fields distributed among all the chickpea production areas of the country were paired with MAT1-1 and MAT1-2 mating type tester isolates of D. rabiei. Both mating types were found in all chickpea production areas of the country. Of the 18 fields sampled, MAT1-1 was observed in 44%, and MAT1-2 in 88% of the sites. In some sites both mating types were present in close proximity, suggesting that sexual reproduction of the pathogen was feasible. The contribution of sexual reproduction of the fungus to virulence diversity was tested on detached leaves of six differential chickpea cultivars. Nine isolates were derived from different well separated foci (derived from ascospores as inoculum) and eight isolates were derived from a single, well defined infection focus (derived from sister conidia). In the analyses of variance the cultivar × isolate interaction showed no significant (P of F>0.09) effect on disease incidence; the chickpea cultivars differed significantly (P of F<0.0001) in their response to D. rabiei; and the isolate effect was highly significant (P of F = 0.0007) for the conidial population, but not significant (P of F>0.1) among isolates of the ascosporic population. Nevertheless, when comparing a cultivar at a time, the ascosporic and conidial populations did not differ significantly (P of F>0.1) in their virulence diversity. Virulence of 41 isolates collected from the different chickpea fields was tested on detached leaves of four Israeli cultivars that differ in their field response to D. rabiei. The cultivar × isolate interaction showed no significant effect (P of F = 0.95) on disease incidence. The main effects of cultivar and isolate on disease incidence were highly significant (P of F<0.0001). Accordingly, our data do not support the hypothesis that there is pathogenic specialization in the D. rabiei–C. arietinum pathosystem in Israel.