离体水稻叶片划伤接种鉴定稻瘟菌的致病型

抗瘟品种的培育和抗瘟基因布局需要快速、准确、大规模地定性水稻抗源及其后代的抗瘟基因型和稻瘟菌致病型。为此, 本研究建立了水稻离体叶片划伤接种方法。该方法依据主效抗瘟基因抵抗稻瘟菌在寄主体内扩展的特点, 通过针刺在水稻叶片上造成伤口, 避免了寄主侵入抗性的干扰, 从而有利于抗扩展性的定性鉴定。作者利用活体喷雾接种、叶片无划伤接种和本研究建立的离体叶片划伤接种等3种接种方法, 在秧苗4~6叶龄期, 对菌株12-DG-68在24个水稻抗瘟单基因系上的致病反应进行了测定, 结果显示：叶片划伤接种的检测结果稳定、一致; 而叶片无划伤接种和活体喷雾接种的检测结果假抗性比例分别为12.5%和4.2%, 不同叶龄期的叶片间反应型不一致率达7%。此外, 离体叶片划伤接种还可利用菌丝块接种, 以鉴定分生孢子产量低的菌株的致病型。因此, 水稻叶片划伤接种是一种准确、稳定和方便的稻瘟菌接种方法, 可用于大规模定性测定水稻抗源及其后代的抗瘟基因型和稻瘟菌的致病型。     

一个与稻瘟病菌无毒基因AVR-Pik~m连锁的SCAR标记的分离

 本研究将以前在稻瘟病菌菌株S1522获得的与决定对水稻品种梅雨明无毒性的基因(AVR-Pik^m)相连锁的1个RAPD标记OPO12₁₀₀₀进行了克隆和鉴定。核苷酸序列测定与分析结果表明:OPO12₁₀₀₀的大小为946个碱基,不含有与已报道的稻瘟病菌Mg-SINE、Fosburry、Magyy、Grasshopper、Pot2以及Pot3等同源的重复序列。根据OPO12₁₀₀₀的核苷酸序列,设计了1对24个核苷酸的特异引物,对无毒表型亲本S1522和毒性表型亲本S159、无毒表型群体基因池、毒性表型基因池以及有性杂交后代108个菌株进行了PCR扩增,所有无毒表型的菌株均能特异性地扩增出1条与OPO12₁₀₀₀大小相同的DNA条带,而毒性表型的菌株除5个重组个体外,均不能扩增出这条特异带。此结果表明,与稻瘟病菌无毒基因AVR-Pik^m连锁的RAPD标记OPO12₁₀₀₀被成功地转化为SCAR标记,为进一步通过染色体步移克隆该无毒基因奠定了基础。     

辽宁省稻瘟病菌对稻瘟灵的抗性监测及其对稻瘟病的田间防治效果

为明确辽宁省水稻稻瘟病菌Magnaporthe oryzae对稻瘟灵的抗性水平及变化动态，本研究于2017—2019年自辽宁省6个市的6个稻区采集并分离获得187株稻瘟病菌，利用菌丝生长速率法测定这187株稻瘟病菌菌株对稻瘟灵的敏感性及抗性，并采用茎叶喷雾法测定稻瘟灵对水稻稻瘟病的田间防治效果以及该药剂与其它药剂的交互抗性。结果表明：供试187株稻瘟病菌菌株对稻瘟灵的EC₅₀范围为0.94~34.96 μg/mL，左侧正态分布峰内EC₅₀平均值为5.05 μg/mL，稻瘟病菌对稻瘟灵的敏感性分布频率不符合正态分布，表明菌群中已经出现对稻瘟灵敏感性下降的亚群体。187株稻瘟病菌菌株对稻瘟灵的平均抗性倍数为3.70，抗性菌株出现频率为55.61%，平均抗性指数为0.40，抗性水平呈逐年增加趋势。2017—2019年稻瘟灵对水稻叶瘟病的防治效果分别为82.97%、78.86%和81.36%，对水稻穗颈瘟的防治效果分别为79.81%、77.68%和80.20%，说明在现有防控手段下未产生高水平抗性群体。稻瘟灵与肟菌酯、戊唑醇之间无交互抗性。与不同药剂轮换使用的前提下，稻瘟灵仍可作为主要药剂防治稻瘟病。     

水稻地方品种‘月亮谷’纯系对田间稻瘟病菌的选择

云南元阳哈尼梯田稻作模式是水稻可持续生产模式之一。尽管梯田地理条件适合稻瘟病的发生,但地方品种‘月亮谷’在超百年的种植历史上未有稻瘟病大发生的记载,其原因值得探索。为了解‘月亮谷’不同抗性品系对环境中稻瘟病菌的选择作用,以‘月亮谷’单粒传纯系为材料,通过孢子捕捉法和常规病组织分离法采集稻瘟菌株,进行人工培养、表型观察,接种测定了这些稻瘟菌菌株对25个抗稻瘟病单基因近等基因系的致病型。结果表明,梯田环境空气中采集的稻瘟菌孢子菌落形态呈放射状,菌落疏松,生长在培养基浅表,产孢量总体差异不明显,黑色素颜色较浅;从水稻感病组织上分离到的稻瘟菌菌落呈地毯状,菌落紧密,匍匐在培养基表面,产孢量个体差异较大(P0.05),黑色素颜色较深。测定的稻瘟菌菌株对25个近等基因系都有致病性,联合致病性在12.0%~56.0%之间。来自环境空气中的菌株的平均联合致病性(24.8%)低于分离自‘月亮谷’纯系的菌株(38.4%)。进一步分析显示,稻瘟菌株联合致病性与其菌丝生长速率、产孢量相关性不明显;但与黑色素合成存在一定关联,颜色越深,致病性越强,来源于‘月亮谷’不同品系上的菌株致病性强于环境空气中的菌株。上述结果表明,元阳哈尼梯田环境中的稻瘟菌群体与‘月亮谷’纯系上分离到的稻瘟菌群体存在较大差异。     

江西省稻区稻瘟病菌遗传宗谱与致病型的关系

为了探寻稻瘟病菌无性世代DNA水平的变异,明确江西省稻区稻瘟病菌遗传宗谱与致病型之间的对应关系,利用rep-PCR(repetitive element-based PCR)分子指纹分析技术,对稻区稻瘟病菌的群体结构和遗传多样性进行分析,并用41株代表性菌株对35个水稻品种进行了致病性测定。结果表明,以相似度75%为界,可以将不同稻区采集的99个菌株划分为14个遗传宗谱,其中,宗谱4、1和10为优势宗谱,分别包含37、18和12个菌株,占总数的37.37%、18.18%和12.12%;稻瘟病菌遗传宗谱与致病型间存在复杂的关系,同一宗谱的菌株对应多个致病型,而同一致病型的菌株,分属于不同的遗传宗谱,两者之间不存在简单的对应关系。     

稻瘟菌群体中dsRNA的多样性及稻瘟菌菌株QSP5中病毒对寄主生物学性状影响的研究

 真菌病毒在真菌中广泛存在,一些真菌病毒可以影响病原真菌营养生长、产孢和色素形成等性状。本文研究了真菌病毒在水稻稻瘟菌中的多样性以及其对寄主生物学性状的影响。采用单孢分离的方法从发病水稻叶片上分离获得了120个稻瘟菌菌株,其中约52%携带dsRNA片段。菌株QSP5被2个基因组为dsRNA的病毒即产黄青霉病毒1-C(Magnaporthe oryzae chrysovirus 1-C, MoCV1-C)和稻瘟菌病毒3(M. oryzae virus 3, MoV3)所侵染。对菌株QSP5进行单个的分生孢子纯化,获得了仅携带MoV3的菌株QSP5-9。菌株QSP5与QSP5-9的产孢量、菌丝形态以及菌落形态存在明显差异,经研究推测MoCV1-C与菌株QSP5 的产孢能力衰退、菌丝生长和产色素异常相关。同时对MoV3的稳定性进行了初步的研究。从稻瘟菌菌株QSP5获得了100个单孢分离物,通过对随机挑选的40个单孢分离物进行检测,结果表明这些分离物100%携带MoV3,只有部分单孢分离物携带MoCV1-C。另外,从稻瘟菌菌株QSP5-9获得了94个单孢分离物,对随机挑选的45个单孢分离物进行检测,结果表明这些分离物中均含有与菌株QSP5-9大小相同的dsRNA片段。根据以上结果推定MoV3在稻瘟菌菌株QSP5和QSP5-9及其无性后代中具有一定的稳定性。     

水稻纹枯病拮抗细菌的筛选与利用

稻种是生防菌的主要来源之一,为在浙江稻区获得对水稻纹枯病具良好防治效果的生防菌,有必要明确该稻区不同栽培方式下稻种拮抗菌的分布及其特性.从3种不同栽培方式的稻种上分离并测定水稻纹枯病拮抗细菌,发现撒播稻稻种上拮抗菌比率显著高于条播和移栽稻.从1000个细菌分离物中筛选出13个拮抗能力强的菌株,经致病性测定发现S270262在水稻植株上产生病斑;对12个非致病拮抗菌进行稻种细菌化、盆栽测定和田间小区试验及Biolog鉴定,结果表明10个拮抗菌对稻苗具促生作用,2个有抑生作用;盆栽和田间防治效果好的菌株为T300363、S300265和S90132,它们分别被鉴定为Salmonella subsp.1 G、Pseudomonas aeruginosa和Pseudomonas spp..     

福建省稻瘟病菌对主要抗瘟基因及主栽品种的致病性分析

为明确福建省各稻区的主要抗瘟基因及主栽品种的利用价值,2012—2014年采用喷雾法测定了丽江新团黑谷上的193个菌株对30个水稻抗瘟基因及93个主栽水稻品种的致病性。结果表明,供试稻瘟病菌对30个抗瘟基因表现为强致病力和较强致病力的出现频率分别为13.47%和52.85%,对93个主栽品种表现为强致病力和较强致病力的出现频率分别为1.55%和11.40%;供试稻瘟病菌对抗瘟基因Pi-k~m、Pi-7(t)、Pi-k~p和Pi-9(t)的毒力频率均低于20%;供试稻瘟病菌对谷优2329、谷优5138、昌优964等37个品种的毒力频率均低于20%,对谷优系列、全优系列、深优系列、泰丰优系列及天优系列水稻品种的毒力频率均低于20%。研究表明,在福建地区抗瘟基因Pik~m、Pi-7(t)、Pi-k~p和Pi-9(t)可作为抗源使用,且谷丰A、全丰A、深97A、泰丰A和天丰A仍是抗瘟性较好的育种亲本材料。     

稻瘟菌双分病毒MoPV2特性研究

稻瘟病是稻瘟菌引起的一种世界性病害,每年造成水稻大幅减产。真菌病毒是指能够在真菌体内进行复制和繁殖的病毒,可以作为生物防治的资源。本实验从发病的水稻叶片上采用单孢分离的方法获得纯化培养的稻瘟菌菌株YC13。菌株YC13携带多条dsRNA片段,其中片段F1与Magnaporthe oryzae virus 2(MoV2)的Coat Protein(CP)和RNA-dependent RNA polymerase(RdRp)的氨基酸序列均具有99%的同源性,确定为MoV2的基因组;片段F4、F5序列与双分病毒Penicillium stoloniferum virus F(Ps V-F)的RdRp和CP的氨基酸序列分别具有69%和63%的同源性,命名为Magnaporthe oryzae partitivirus virus 2(MoPV2)。MoPV2的基因组含有2条dsRNA:dsRNA 1编码RdRp,dsRNA 2编码CP。根据MoPV2的RdRp编码的氨基酸序列将MoPV2归属于双分病毒科(Partitiviridae)G ammapartitivirus属,是一种新的真菌病毒。通过病毒粒子转染,将MoPV2转到稻瘟菌菌株RB11。与菌株RB11相比,带毒菌株RB11T25的分生孢子产生量显著减少,并且对大麦活体的致病性也有所降低,但其他生物学特征均无明显的影响。本研究在稻瘟菌中发现了一种新的真菌病毒,该病毒能够使稻瘟菌致病力下降,为稻瘟病的防治提供了潜在的病毒资源。     

不同稻区稻瘟病菌致病型变异及基于MAS的品种对稻瘟病的抗性鉴定

稻瘟病是影响水稻生产的重要病害。监测田间稻瘟病菌菌群致病型的组成是有效推行利用抗病品种抗瘟策略的必须环节。本研究采集2015年湖北省10个稻作区的穗颈瘟标本,以单基因鉴别品系对分离得到的菌群进行致病型的测定,并分区进行品种的抗瘟性鉴定,结果表明,不同稻作区菌群致病型的组成存在差异,品种对不同稻作区菌群的抗性明显不同,单基因品系中9个抗性基因在湖北稻区受侵染程度也存在明显差异。     

一个新的与稻瘟病菌无毒基因AVR-Pikm紧密连锁的SCAR标记

 无毒基因是病原物中决定寄主抗病性表达的功能基因,其功能的丧失导致毒性小种的产生。本研究利用随机引物扩增DNA多态性技术,从稻瘟病菌菌株S1522中新筛选到1个与无毒基因AVR-Pik^m紧密连锁的DNA标记OPE12₁₄₀₀。根据OPE12₁₄₀₀的核苷酸序列,设计了1对含有17个核苷酸的特异性SCAR引物,并利用该引物对无毒表型亲本S1522和毒性表型亲本S159及其有性杂交后代的108个菌株进行了PCR扩增。结果表明:所有无毒表型的菌株均能特异性扩增出1条与OPE12₁₄₀₀大小相近的DNA片段,而毒性表型的菌株除2个重组体外,均不能扩增出此片段。根据计算,这一SCAR标记与目标无毒基因AVR-Pik^m之间的遗传距离为1.89 cM,与本研究小组先前报道的另一个标记OPO12₁₀₀₀位于目标基因的同一侧,但与OPO12₁₀₀₀相比,距目标基因近了2.86 cM。本标记的获得将有助于确定AVR-Pik^m在染色体上的位置,有助于确定用于进一步筛选位于相反一侧的连锁标记的重叠群区域。     

Genetic Analysis of Avirulence/Virulence of an Isolate of Magnaporthe grisea from a Rice Field in Texas

ABSTRACT An isolate of Magnaporthe grisea, Tm4, from a rice field in Texas was crossed with a fertile laboratory strain, 70-6. The progenies showed segregation of avirulence/virulence on rice cvs. Newbonnet, Lemont, Lebonnet, Leah, and Katy. The avirulent/virulent segregation ratios were 29:6 on Newbonnet, Lemont, and Lebonnet; 28:7 on Leah; and 33:2 on Katy. There was cosegregation on the first three cultivars. Several avirulent progenies were backcrossed to virulent parent 70-6. Three generations of backcrossing avirulent progenies to 70-6 led to segregation ratios that suggested certain strains had only one avirulence gene. Strains avirulent only on cv. Katy or only on cvs. Newbonnet, Lemont, and Lebonnet were test crossed with virulent siblings. Strains that gave progeny ratios approximating 1 avirulent:1 virulent when crossed with virulent siblings were selected for further test crossing. Intercrosses between strains with possible single avirulence genes were made to determine whether these strains had the same or different avirulence genes. Many lines still segregated two genes for avirulence after three generations of backcrossing. This is based on the recovery of virulent progenies from crossing two avirulent siblings.     

抗稻瘿蚊品种多抗1的抗性遗传分析及抗性基因定位

稻瘿蚊是亚洲稻区主要害虫,采用抗虫品种进行防治是最理想的方法。1993～1995年,广东省农科院与国际水稻研究所有关专家紧密合作,对能抗华南4个稻瘿蚊生物型的品种多抗1作进一步抗性遗传分析,确认多抗1对中国稻瘿蚊生物型1和4的抗性受显性单基因控制,这个基因暂定名为GM—6(t)。以多抗1×丰银占1组合的F3代160个家系作基因标记,据DNA库分离个体分析(BSA)原理,用随机扩增多态性DNA(RAPD)标记物OPM6(1.4kb),首次成功地标记了这个抗性基因。随后多态性扩增产物经~(32)p标记,用作探针,检测另一个参考作图群体IR64×Azucena,将这个抗性基因定位在水稻第4条染色体上,位于RG214和RG163两个DNA限制性片段长度多态性(RFLP)标记之间。应用这些分子标记辅助选择有可能不必通过稻瘿蚊的直接筛选,快速准确地选育抗稻瘿蚊品种或进行抗性基因累加。     

Identification of an Avirulence Gene in the Fungus Magnaporthe grisea Corresponding to a Resistance Gene at the Pik Locus

ABSTRACT A rice isolate of Magnaporthe grisea collected from China was avirulent on rice cvs. Hattan 3 and 13 other Japanese rice cultivars. The rice cv. Hattan 3 is susceptible to almost all Japanese blast fungus isolates from rice. The genetic basis of avirulence in the Chinese isolate on Japanese rice cultivars was studied using a cross between the Chinese isolate and a laboratory isolate. The segregation of avirulence or virulence was studied in 185 progeny from the cross, and monogenic control was demonstrated for avirulence to the 14 rice cultivars. The resistance gene that corresponds to the avirulence gene (Avr-Hattan 3) is thought to be located at the Pik locus. Resistance and susceptibility in response to the Chinese isolate in F(3) lines of a cross of resistant and susceptible rice cultivars were very similar to the Pik tester isolate, Ken54-20. Random amplified polymorphic DNA markers and restriction fragment length polymorphism markers from genetic maps of the fungus were used to construct a partial genetic map of Avr-Hattan 3. We obtained several flanking markers and one co-segregated marker of Avr-Hattan 3 in the 144 mapping population.     

A Molecular Marker Correlated with Selected Virulence Against the Tomato Resistance Gene Mi in Meloidogyne incognita, M. javanica, and M. arenaria

ABSTRACT Root-knot nematodes of the genus Meloidogyne are economically important pathogens of a wide range of crops. The tomato resistance gene Mi typically confers resistance to the three major species, M. incognita, M. javanica, and M. arenaria. However, virulent populations completely overcoming the Mi resistance still occur. In an attempt to develop molecular markers for virulence against Mi and gain insights into the genetic relationships among virulent populations of different species and origins, random amplified polymorphic DNA (RAPD) analyses of laboratory-selected virulent, field virulent, and avirulent populations of M. incognita, M. javanica, and M. arenaria were carried out. A RAPD marker, specific for selected virulent populations, was identified, and subsequently, converted to a sequence characterized amplified region (SCAR). Sequence characterization of the SCAR locus showed that alleles from laboratory- and field-selected virulent populations were highly similar to each other and clearly different from alleles from natural virulent and avirulent populations. This result suggests that the genetic mechanism for virulence against Mi may be similar among selected virulent populations of the three Meloidogyne spp., but different between selected and natural virulent populations. Based on the nucleotide polymorphisms at the SCAR locus, codominant and dominant polymerase chain reaction-based markers were developed enabling rapid diagnosis of selected virulent genotypes in M. incognita, M. javanica, and M. arenaria.     

Genetic Analysis of Host Species Specificity of Magnaporthe oryzae Isolates from Rice and Wheat

ABSTRACT A Triticum isolate (pathogenic on wheat) of Magnaporthe oryzae was crossed with an Oryza isolate (pathogenic on rice) to elucidate mechanisms of their parasitic specificity on wheat. When the pathogenicity of their F (1) cultures (hybrids between a Triticum isolate and an Oryza isolate) was tested on wheat, avirulent and virulent cultures segregated in a 7:1 ratio. This result suggests that three loci are involved in avirulence of the Oryza isolate on wheat. One of the three loci conditioned papilla formation, whereas the others conditioned the hypersensitive reaction. Allelism tests revealed that the locus conditioning papilla formation is Pwt2 while one of the two loci conditioning the hypersensitive reaction is Pwt1. The other locus conditioning the hypersensitive reaction was different from any other known loci and, therefore, was designated as Pwt5.     

Evidence of Genetic Exchange by Parasexual Recombination and Genetic Analysis of Pathogenicity and Mating Type of Parasexual Recombinants in Rice Blast Fungus, Magnaporthe oryzae

ABSTRACT A selectable marker gene conferring resistance to bialaphos (BI) was introduced into rice blast isolate Y90-71BI and another conferring resistance to blasticidin S (BS) into isolate 3514-R-2BS of Magnaporthe oryzae to demonstrate exchange of DNA. Colonies obtained from co-cultures of these two isolates were resistant to both BI and BS and had both resistance genes as shown by Southern blot analysis of their genomic DNA. Conidia from these BI-BS-resistant isolates had only one nucleus per cell after staining with 4',6-diamidino-2-phenylindole (DAPI). Using flow cytometry, however, these BI-BS-resistant isolates were found to be haploid. Segregation of BI-BS-resistant isolates for pathogenicity (avirulence to virulence) on rice line K59-1 was consistent with a 1:1 ratio, as was segregation for mating type. These BI-BS-resistant isolates were thus apparently derived from parasexual exchange of DNA and the segregation of pathogenicity and of mating type of the parasexual recombinants might correspond to that of the progeny of the offspring of the sexual cross.     

Evidence of Cytoplasmic Inheritance of Virulence in Cronartium ribicola to Major Gene Resistance in Sugar Pine

ABSTRACT Tests for Mendelian segregation of virulence and avirulence in Cronartium ribicola, causal agent of white pine blister rust, to a major gene (R) for resistance in sugar pine were made using haploid basidiospore progenies from single diploid telia as inoculum on resistant genotypes. The telia were sampled from a small deme in the Siskyou Mountains of northern California, where a few mature sugar pines known to be Rr genotypes had become infected after withstanding the chronic blister rust epidemic for several decades and where intermediate frequencies of virulence in the ambient basidiospore population were subsequently measured. Infection type on inoculated seedlings with R was qualitative: all progenies of 81 single telia tested over 3 different years were either virulent (compatible) or avirulent (inducing hypersensitive necrosis), never a mixture of both reactions. The complete absence of heterozygotes in the telia population is strong evidence that virulence is not controlled by a nuclear gene. The data are consistent with earlier tests showing that basidiospore inoculum derived from aeciospores isolated from infected Rr trees produced mostly (>90%) virulent reactions on R- seedlings. The evidence indicates that transmission of virulence is uniparental via the cytoplasm of aeciospores. Exchange of spermatia between haploid thalli does not appear to be involved.     

Virulence and Molecular Polymorphism in International Collections of the Wheat Leaf Rust Fungus Puccinia triticina

ABSTRACT Collections of Puccinia triticina, the wheat leaf rust fungus, were obtained from Great Britain, Slovakia, Israel, Germany, Australia, Italy, Spain, Hungary, South Africa, Uruguay, New Zealand, Brazil, Pakistan, Nepal, and eastern and western Canada. All single-uredinial isolates derived from the collections were tested for virulence polymorphism on 22 Thatcher wheat lines that are near-isogenic for leaf rust resistance genes. Based on virulence phenotype, selected isolates were also tested for randomly amplified polymorphic DNA (RAPD) using 11 primers. The national collections were placed into 11 groups based on previously established epidemiological zones. Among the 131 single-uredinial isolates, 105 virulence phenotypes and 82 RAPD phenotypes were described. In a modified analysis of variance, 26% of the virulence variation was due to differences in isolates between groups, with the remainder attributable to differences within groups. Of the RAPD variation, 36% was due to differences in isolates between groups. Clustering based on the average virulence distance (simple distance coefficient) within and between groups resulted in eight groups that differed significantly. Collections from Australia-New Zealand, Spain, Italy, and Britain did not differ significantly for virulence. Clustering of RAPD marker differences (1 - Dice coefficient) distinguished nine groups that differed significantly. Collections from Spain and Italy did not differ significantly for RAPD variation, neither did collections from western Canada and South America. Groups of isolates distinguished by avirulent/virulent infection types to wheat lines with resistance genes Lr1, Lr2a, Lr2c, and Lr3 also differed significantly for RAPD distance, showing a general relationship between virulence and RAPD phenotype. The results indicated that on a worldwide level collections of P. triticina differ for virulence and molecular backgrounds.