Pathotypes and Genetic Diversity of Blumeria graminis f.sp.hordei in the Winter Barley Regions in China

Barley powdery mildew caused by Blumeria graminis f. sp. hordei （Bgh） is one of the most destructive foliar diseases of barley in the winter barley region of China. The evaluation and assessment of the virulence and diversity of Bgh populations help to determine effective sources of resistance to the pathogen. 515 isolates were collected from seven populations of Bgh on cultivated barley in seven geographically distant locations in 2006. Their virulence was determined by inoculation onto 26 differential host lines. All of the isolates belonged to 58 pathotypes and 13 of which included 81% of these isolates. The most abundant pathotype was pathotype 0047 （18.3%）, the second most abundant was pathotype 0045 （11.8%） and the third most abundant was pathotype 0057 （7.8%）. Most of virulent genes investigated in this study showed similar frequencies in the seven different areas. These indicate that the seven locations may be in a uniform epidemiological region and barley cultivars in these areas may have similar genetic background. Diversities within these populations and distances between these populations measured by KOIND package were different. Correlations were not found between the genetic distance and the geographical distances between different locations. This suggested that long distance spread and local epidemics existed in the major winter barley growing regions in China.

Virulence and Diversity of Blumeria graminis f.sp.tritici Populations in China

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is an important disease in China. To characterize the virulence and diversity of the pathogen, 1 082 isolates were obtained from 8 major wheat-growing regions during the spring growing season in 2011. The virulence test was performed by inoculation on detached leaves of 22 differential lines with known Pm genes. Frequencies of virulence on these genotypes ranged from 0 to 97.4%. None of the 1 082 isolates was compatible to Pm21 and less than 20.0%were virulent to the genotype carrying Pm13. In contrast, the virulence frequencies of each population was more than 50.0%to differentials carrying Pm1a, Pm3b, Pm3c, Pm3f, Pm5a, Pm6 and Pm8. In total, 1 028 pathotypes were detected, of which 984 were unique. Phenotypic diversity indices revealed a high level of diversity within populations. Genetic distance between different populations correlated signiifcantly with geographical distance （R2=0.494, P 0.001）. In addition, isolates from Xinjiang appear to form a separate group. Signiifcant positive or negative associations between alleles at pairs of virulence loci were detected in 57 allele pairs to Pm genes. Virulence and diversity of the 8 populations suggested that varieties with effective resistance gene combinations should be developed at a regional level.

Population Genetic Analysis of Blumeria graminis f. sp. tritici in Qinghai Province, China

To gain more precise information about molecular genetic variation for wild populations of Blumeria graminis f. sp. tritici from Qinghai Province, China, 38 single-colony isolates were purified from samples collected from Haidong District, Xining City and Hainan Tibetan Autonomous Prefecture in 2010. The virulence of 21 isolates among them was tested at seedling stage on 34 wheat cultivars（lines） carrying known powdery mildew（Pm） resistant genes. The results showed that V1 a, V3 a, V3 c, V3 e, V5 a, V6, V7, V8 and V19 had high virulence frequencies（〉75%）, indicating a wide distribution; and V1 c, V5 b, V12, V13, V16, V21, VXBD, V2＋6, V2＋Mld and V4＋8, with less distribution, appeared to be lower in frequencies（0-20%）. The Nei＇s gene diversity（H）, Shannon＇s information index（I） and the percentage of polymorphic loci（P） were 0.23, 0.35 and 67.65%, respectively, which revealed a virulent diversity. The results from single nucleotide polymorphisms（SNPs） of 38 isolates showed that three housekeeping genes were found to contain a total of 9 SNP sites. 10 haplotypes（H1-H10） were inferred from the concatenated sequences, with 1 haplotype（H1） comprising of over 55% of Qinghai population. Phylogenic analysis did not show obvious geographical subdivision between the isolates. A multilocus haplotype network presented a radial structure, with H1 in the central as an inferred ancestor. Using analysis of molecular variance（AMOVA）, we found 1.63% of the total variation was among populations and 98.37% within populations, with a low fixations index（FST=0.01634, P〈0.05）. This revealed a relatively high genetic diversity but a low genetic divergence in Qinghai population. Moreover, the molecular data on gene flow（Nm=6.32） confirmed the migration of pathogen populations among areas in Qinghai Province.

Development of SSR Markers for a Phytopathogenic Fungus,Blumeria graminis f.sp.tritici,Using a FIASCO Protocol

Simple sequence repeats （SSR） have been widely used as molecular markers due to their abundance and high polymorphism, However, up to now, the SSR markers had not been developed in the obligate biotrophic phytopathogenic fungus, Blumeria graminis f.sp. tritici. From （AC）10 and （AG）10 enriched genomic libraries for Bgt, 25 primer pairs were designed using the FIASCO （fast isolation by AFLP of sequences containing repeats） protocol. Five primer pairs exhibited polymorphism with allelic diversity from two to seven alleles and produced 29 alleles in a survey of 90 isolates collected from six provinces （cities） in China, while the others displayed monomorphic. Levels of observed heterozygosity ranged from 0.000-0.044 （mean 0.025） and expected heterozygosity ranged from 0.297-0.816 （mean 0.538）. These molecular markers provide a novel source to genetic diversity assays and to genetic and physical mapping ofBgt. SSR markers of Bgt need to be further explored.

Mapping of powdery mildew resistance genes transferred to common wheat from wild emmer wheat revealed three functional Pm60 haplotypes

Powdery mildew(PM),caused by Blumeria graminis f.sp.tritici(Bgt),is one of the destructive wheat diseases worldwide.Wild emmer wheat(Triticum turgidum ssp.dicoccoides,WEW),a tetraploid progenitor of common wheat,is a valuable genetic resource for wheat disease resistance breeding programs.We developed three hexaploid pre-breeding lines with PM resistance genes derived from three WEW accessions.These resistant pre-breeding lines were crossed with susceptible common wheat accessions.Segregations in the F2populations were 3 resistant:1 susceptible,suggesting a single dominant allele in each resistant parent.Mapping of the resistance gene in each line indicated a single locus on the long arm of chromosome 7A,at the approximate location of previously cloned Pm60 from T.urartu.Sanger sequencing revealed three different Pm60 haplotypes(Hap 3,Hap 5,and Hap 6).Co-segregating diagnostic markers were developed for identification and selection of each haplotype.The resistance function of each haplotype was verified by the virus-induced gene silencing(VIGS).Common wheat lines carrying each of these Pm60 haplotypes were resistant to most Bgt isolates and differences in the response arrays suggested allelic variation in response.

小麦白粉病菌对氟唑菌酰羟胺的敏感基线建立

氟唑菌酰羟胺是一种新型琥珀酸脱氢酶抑制剂(succinate dehydrogenase inhibitor,SDHI)类杀菌剂,2019年在我国登记在小麦上防治赤霉病,为明确其对小麦白粉病的潜在防治效果,以及为监测小麦白粉病菌群体对其是否产生抗药性,本文采用离体叶段法测定了2000年前和2019年采自我国河南、山东等9省市的110株小麦白粉病菌菌株对氟唑菌酰羟胺的敏感性。结果表明,采自2019年的88株菌株,其EC_(50)范围为0.0102~0.7668μg/mL,平均EC_(50)为(0.2243±0.1796)μg/mL;采自2000年前的22株菌株,其EC_(50)范围为0.0521~0.8824μg/mL,平均EC_(50)为(0.3563±0.2796)μg/mL;两组菌株群体间的敏感性无显著差异。氟唑菌酰羟胺对110株菌株的EC_(50)范围为0.0102~0.8824μg/mL,平均EC_(50)为(0.2507±0.2103)μg/mL,敏感性频率分布呈单峰曲线,可作为小麦白粉病菌对氟唑菌酰羟胺的敏感基线。本研究结果可为氟唑菌酰羟胺防治小麦白粉病以及未来监测该病原菌对药剂的抗性提供参考依据。

白粉病菌(Blumeria graminis)胁迫下西藏青稞lncRNA全谱解析

白粉病是麦类作物中最具破坏性的病害之一,对谷物产量和品质造成严重损失。为明确西藏青稞对白粉病的抗病分子机制,本研究对两个青稞品种‘甘农大7号’(G7:高抗)和‘藏青13’(Z13:高感)进行白粉菌(Blumeria graminis f.sp.hordei,Bgh)接种,研究其基因的差异表达模式。对G7和Z13白粉菌接种前后的差异基因比较发现,钙依赖蛋白激酶、谷胱甘肽S-转移酶、纤溶酶原激活物抑制剂1、丝氨酸型内肽酶和MADS-box转录因子的编码基因在G7中表达水平较高,表明它们可能在抗Bgh中发挥作用。G7高表达的基因主要参与了离子通道、钙依赖蛋白激酶、植物激素途径等生物过程。另外,16个lncRNA及其21个候选靶基因表现出协同表达模式,这些表达模式丰富了植物激素信号转导和离子结合的过程。本研究结果对后续青稞及其近缘物种抗白粉病的分子机制研究具有一定的参考价值。

小麦白粉菌环介导等温扩增检测技术体系的建立与应用

为建立简便、快速和灵敏的小麦白粉菌Blumeria graminis f.sp.tritici(Bgt)分子检测技术体系,基于环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术,以BGT96224V316_LOCUS1725基因(GenBank:VCU40465.1)为靶标序列,设计并筛选特异性引物,建立小麦白粉菌的LAMP检测方法,并对其特异性、灵敏度和应用效果进行测定。结果表明,基于筛选出的1套特异性引物建立的LAMP方法能够从8种白粉菌属菌株和1种腐生菌中特异性地检测到小麦白粉菌;对小麦白粉菌DNA样品的检测灵敏度可达到300 fg/μL;对发病叶片的检测显示该LAMP方法能从人工接种的小麦叶片中准确检测出小麦白粉菌,检出时间为接种4 h及以上。综上,本研究建立了一种小麦白粉菌的LAMP检测方法,具有简便快捷、灵敏度高等特点,丰富了小麦白粉菌的检测体系。

新疆小麦白粉病菌群体遗传多样性分析

为了解新疆小麦白粉病菌群体多样性及其间关系,利用ISSR分子标记对2020年喀什、阿克苏、和田、昌吉、塔城共5个地区13个县市小麦白粉病菌群体进行遗传多样性研究。结果表明,喀什、阿克苏、和田、昌吉、塔城小麦白粉病菌群体的遗传距离为0.0317~0.0945,塔城与阿克苏群体的遗传距离最近,和田与喀什的遗传距离最远;总体Nei s遗传多样性指数为0.2571,群体Nei s内遗传多样性指数为0.2175,群体内的遗传变异占总体变异的84.60%,表明遗传变异主要来源于群体内。群体间基因流N m值为2.7490,表明新疆不同区域群体存在一定的基因交流。Normalized Mantel Statistic分析表明,小麦白粉病菌群体间遗传距离与地理距离相关性不大。

36个小麦审定和区试品种(系)的抗白粉病基因推导

明确我国当前小麦审定和区试品种含有的抗白粉病基因,可为这些品种在小麦抗病育种中的应用及品种合理布局和轮换提供依据。本研究采用24个不同毒性的小麦白粉菌菌株对36个小麦审定和区试品种(系)进行抗白粉病基因推导,参试品种(系)与46个已知抗病基因小麦品种(系)抗谱比较的结果表明,11个小麦审定品种中有5个品种对所有供试白粉菌菌株表现抗性,结合亲本溯源,推测其中有3个品种可能携有Pm21基因;另外6个审定品种中有5个品种可能含有抗病基因Pm2,且其对应的亲本或亲本组合中含有抗病基因Pm2。25个区试品种(系)中有3个可能含有Pm21,10个含有Pm2,1个含有Pm2+6,2个含有Pm 4b,1个含有Pm 8。另外,参试的36个品种(系)中还有9个品种(系)和已知基因品种抗谱存在一定差异。总体上,推导出已知基因的品种以含有Pm2基因的品种最多,Pm21基因的品种次之,建议在生产上加强对Pm21基因品种(系)特别是已审定的携有Pm21基因品种的推广和应用,应该注意一些省份在育种和生产上应慎用或少用含Pm2基因的品种(系)。

小麦不同营养型病原真菌的候选GPCR蛋白预测及对比研究

【目的】通过对小麦3种不同营养型病原真菌的候选GPCR蛋白进行找寻及性质对比分析,为进一步找寻新型农药作用靶标奠定理论基础。【方法】选择已经公布全基因组序列的3种小麦营养型病原真菌,基于GPCR蛋白所具有的7重跨膜结构典型特征,利用3种蛋白跨膜在线预测程序(TOPCONS、TMHMM、Philius)对上述病原真菌的候选GPCR蛋白开展找寻,以及候选GPCR蛋白序列长度、理论等电点、不稳定系数、最强亲(疏)水性残基等特征和遗传关系进行对比分析。【结果】小麦白粉病菌、叶枯病菌、叶斑病菌中分别含有候选GPCR蛋白11、32、69个,蛋白序列平均长度为396 aa。蛋白的理论等电点平均值在8.78左右,不稳定系数在40左右,亲水性在-2.70左右,疏水性在3.20左右,而最强亲(疏)水性氨基酸残基分布不一致。上述112个候选GPCR蛋白按遗传关系聚为三大类,分别具有71、36、5个蛋白。【结论】小麦不同营养型病原真菌的候选GPCR蛋白数量以活体型真菌数量最少,死体型真菌数量最多,半活体型真菌介于两者之间。不同候选GPCR蛋白在理论等电点、不稳定系数等理化性质及遗传关系等方面具有一定同质性特征,然而最强亲(疏)水性氨基酸残基具有较大差异。

一株内生细菌EA19的分离鉴定及其对小麦白粉病菌的抑制效果

从药用植物一年蓬茎组织中分离并筛选获得了1株内生生防细菌EA19。经形态学、生理生化和16 S rRNA编码基因序列分析等实验,EA19与解淀粉芽孢杆菌(Bacillus amyloliquefaciens)亲缘关系最近。采用菌丝生长速率法对EA19进行了10种植物病原真菌的抑菌活性测定。结果表明,EA19菌体对稻梨孢、镰孢菌、胶孢炭疽菌、稻平脐蠕孢、立枯丝核菌、核盘菌、链格孢和青霉菌等植物病原真菌具有拮抗活性,对稻梨孢和核盘菌的抑制率分别达到96.99%±1.10%和99.16%±0.65%。EA19菌体对小麦白粉病菌(Blumeria graminis f.sp.tritici)的分生孢子萌发和吸器形成具有极显著的抑制作用(P<0.01),并导致病菌附着胞发育畸形。

硅对小麦过氧化物酶、超氧化物歧化酶和木质素的影响及与抗白粉病的关系

以抗 /感白粉病的南农 99 18和苏麦 3号为试验材料 ,研究硅对小麦叶片过氧化物酶 (POD)、超氧化物歧化酶 (SOD)活性的动态变化和对木质素含量的影响。结果表明 ,在不接种的情况下 ,施硅与否对抗 /感品种植株叶片的POD和SOD活性及木质素含量均无影响。而在感病情况下 ,施硅能显著提高小麦感病品种的POD活性和木质素含量 ,但对抗病品种的POD活性和木质素含量影响不显著 ;可降低小麦感病品种的SOD活性 ,但提高了抗病品种的SOD活性。施硅能显著降低感病品种植株的白粉病病情指数 ,提高其对白粉病的抗病能力 ,相对免疫效果达38.79%,其中硅的浓度以 1.7mmol·L-1为最佳。

小麦/蚕豆间作条件下小麦的氮、钾营养对小麦白粉病的影响

通过小麦／蚕豆间作盆栽试验，研究比较了单作和间作条件下不同氮、钾营养水平对小麦氮、钾养分吸收和小麦白粉病发生的影响。结果表明：小麦蚕豆间作提高小麦子粒产量74．7％～133．9％，低氮条件下，间作提高小麦氮吸收量14．7％～169％；在高氮条件下，间作提高氮吸收量的优势降低；间作提高小麦钾吸收量32％～69％，增施钾肥提高小麦钾吸收量25．5％～57．3％。小麦间作蚕豆能明显减轻小麦白粉病的发生，间作平均防效达42．1％～83．1％；小麦白粉病的发生与小麦茎叶的氮吸收量呈显著正相关关系，r=0．623～0．702。

中国冬小麦区域试验品种抗病性评价

【目的】明确中国小麦主要后备品种和高代品系对条锈病、叶锈病、秆锈病、白粉病、赤霉病、纹枯病、黄矮病的抗性程度和水平,为培育抗病新品种、品种审定与推广、抗病品种合理布局、降低品种抗性"丧失"风险提供依据。【方法】以中国小麦条锈菌流行小种条中32(CYR32)、条中33号(CYR33)等、叶锈菌PHT、THT等、秆锈菌21C3、34C2等、对15个已知抗白粉病基因具有毒性的白粉菌混合菌系、赤霉病菌、纹枯病菌强毒力菌株和BYDV-GAV株系对1999—2014年度国家小麦区域试验品种(系)1 815份次试验材料进行成株期人工接种抗性鉴定,重要材料连续进行两年试验,以确保结果的重复和可靠。【结果】在所鉴定的品系中,具有3种抗病性以上的品种(系)共250个,通过农业部国家农作物品种审定委员会审定的品种共310个;中抗和高抗条锈病品种占参试品种的(39.8±21.7)%、中感和高感品种占(39.7±27.9)%;赤霉病、纹枯病、秆锈病和白粉病抗病品种所占比例分别为(31.5±27.5)%、(31.1±35.6)%、(48.2±25.6)%和(25.0±14.6)%、感病品种分别占(68.5±27.5)%、(62.2±38.6)%、(31.3±20.7)%和(70.7±14.6)%;抗叶锈病品种比例仅为(9.9±3.8)%,感病品种高达(70.4±15.1)%;除河农831表现中抗外,所有品种对黄矮病均表现感病,未出现高抗品种。【结论】抗源缺乏的病害未鉴定出抗性强的品种,抗条锈病和白粉病品种主要出现在甘肃、四川等地,抗叶锈小麦品种(系)较少。

山东省和河北省小麦白粉菌毒性与遗传多样性分析

由Blumeria graminis f.sp.tritici引起的白粉病是危害我国小麦安全生产的重要病害之一。分析菌株毒性结构和抗病基因有效性对于利用寄主抗性控制白粉病具有重要意义。本研究对2011年从山东和河北两省分离的41个菌株进行了毒性分析,并采用SSR标记对其遗传多样性进行了分析。测试菌株的毒性频率在0.35（Bg40-2,山东烟台）至0.74（Bg46-1,山东平度）之间。山东省菌株的平均毒性频率与河北省菌株没有显著差异。除别菌株外（例如Pm17）,山东省和河北省的菌株对大多数抗病基因的毒性差异不大。全部测试菌株对来自地方品种齿牙糙的Pm24基因都没有毒性。极少数菌株对Pm1c、Pm16、Pm20、PmH和Mlxbd的毒性频率低于0.1,在河北省邯郸市和黄骅市发现对Pm21基因具有毒性的菌株,但在山东省没有检测到对Pm21具有毒性的菌株。对Pm5e、Pm6、Pm12、Pm13、Pm17、Pm40、Pm2＋6和Pm5＋6的毒性频率在0.18~0.48之间,对Pm1a、Pm3a、Pm3c、Pm3g、Pm4a、Pm4b、Pm5a、Pm7、Pm8、Pm19、Pm33、Pm43、PmY39、PmPS5A和Pm1＋2＋9的毒性频率超过0.6。遗传多样性分析可见,小麦白粉菌群体的遗传变异主要发生在群体内部,菌株间具有一定程度的基因交流。同一地点采集的不同单孢分离菌株有些可聚为一类,但有些不能聚为一类,说明其遗传基础可能存在差异。供试菌株对不同抗病基因的毒性多态性与DNA多态性之间不存在一一对应的关系。

几种杀菌剂对小麦白粉病的防治效果

【目的】通过室内及田间试验,筛选对小麦白粉病防治效果较好的化学药剂及合理的使用方法。【方法】采取室内盆栽接菌试验及田间小区试验相结合的方式进行,在植株发病后喷雾防治,药后定期调查防治效果。【结果】药后10 d,250 g/L丙环唑乳油112.5 g a.i/hm2处理在室内及田间试验对白粉病的防效分别为82.02%和74.02%,防治效果优于其它药剂处理的防效;其次是25%三唑酮可湿性粉剂135 g a.i/hm2处理,室内及田间试验防效分别为81.84%和67.24%;400 g/L氟硅唑乳油48 g a.i/hm2处理的室内及田间试验防效分别为69.25%和67.99%,对白粉病的防效也较好。25%乙嘧酚水悬浮剂187.5 g a.i/hm2处理、250 g/L已唑醇悬浮剂18.75 g a.i/hm2处理、10%苯醚菌酯悬浮剂12 g a.i/hm2处理在室内试验中对白粉病防治效果较好,但田间小区试验中防效略低。【结论】250 g/L丙环唑乳油112.5 g a.i/hm2处理、25%三唑酮可湿性粉剂135 g a.i/hm2处理、400 g/L氟硅唑乳油48 g a.i/hm2处理可有效控制小麦白粉病的发生及危害,且对小麦生长安全,可在大田合理轮换使用。

2007年我国部分麦区小麦白粉菌对三唑酮的抗药性监测

采用拌种离体叶段法测定了我国9个省(市)的112个小麦白粉菌株对三唑酮的抗性。结果表明,供试菌株的平均EC50为57.25mg/L,平均抗性水平为27.39倍,其中86.61%供试菌株已经产生抗性,抗药性比敏感菌株高出10～40倍的菌株占47.32%,四川、山东、甘肃和贵州等地菌株的抗性高于其他地区。此结果可为三唑类杀菌剂在我国的推广应用和制定合理的抗药性治理措施提供依据。

我国部分麦区2011-2012年小麦白粉病菌小种及毒力分析

为了解我国部分春麦区和冬麦区小麦白粉病菌小种和毒力状况,2011-2012年对采自辽宁、黑龙江等东北春麦区及山东、湖北、四川等冬麦区的小麦白粉病菌标样进行生理小种鉴定和群体毒性频率测定。结果表明,2011-2012年东北春麦区优势小种均为411号小种,而山东、湖北等冬麦区的优势小种为377号小种,四川麦区的则为317号小种。小麦白粉病菌毒性基因V3b、V5、V7、V8、V1+2+9、V17毒力频率较高(>90.0%),而V2、V4a、V4b、V12、V13、V16、V18-V23、V5+6毒力频率较低(<31.4%),表明目前含有Pm2、Pm4a、Pm4b、Pm12、Pm13、Pm16、Pm18-Pm23、Pm5+6等抗病基因的品种在我国部分麦区育种中仍有较高的利用价值。