禾谷镰孢转化体系的优化

禾谷镰孢是小麦赤霉病和玉米茎腐病的主要病原真菌。试验优化了聚乙二醇介导的原生质体转化法,建立了禾谷镰孢高效转化的稳定体系,并总结了影响转化成功的主要因素。以此方法对禾谷镰孢进行活体荧光标记,并用转化菌株侵染小麦胚芽鞘,在荧光显微镜下追踪病原真菌在宿主植物体内的生长及扩展过程。     

桃蛀螟为害对春玉米镰孢穗腐病发生及产量损失的影响

【目的】桃蛀螟Conogethes punctiferalis(Guenée)已经成为黄淮海玉米穗期的重要害虫,本研究的目的是探究桃蛀螟发生危害与玉米镰孢穗腐病发病程度和玉米产量损失之间的关系。【方法】在玉米吐丝、灌浆和乳熟期,通过人工接种法将桃蛀螟初孵幼虫及拟轮枝镰孢菌Fusarium verticillioides单接或复合接种到玉米果穗上,收获时调查果穗上桃蛀螟为害级别、镰孢穗腐病发病率及病情指数,测定玉米产量。【结果】在玉米果穗的不同发育阶段,镰孢穗腐病病情指数均以复合接种处理最高,单接虫次之,单接菌最低。在单接虫处理中,镰孢穗腐病的发生程度及桃蛀螟为害级别均以吐丝期最重。拟轮枝镰孢菌与桃蛀螟复合侵染危害后,使得玉米产量性状发生改变,导致产量下降,以吐丝期危害影响最大,单穗损失率为33.09%,灌浆期和乳熟期分别为22.50%和10.13%。【结论】玉米穗期桃蛀螟的为害明显会加重镰孢穗腐病的发生,从而导致了更严重的玉米产量损失。     

内生真菌印度梨形孢诱导小麦抗根腐病作用研究

【目的】明确印度梨形孢(Piriformospora indica)诱导小麦对根腐病产生抗性的作用机制。【方法】用印度梨形孢悬液浸种,以无菌培养液为对照,用病原菌禾谷镰孢菌(Fusarium graminearum)侵染小麦,对其相关生理生化指标及转录组变化进行分析。【结果】禾谷镰孢菌能诱导小麦产生过氧化氢,降低细胞内水含量,破坏细胞膜的稳定性;根部定殖印度梨形孢的小麦细胞内抗氧化酶活性增强,活性氧自由基含量降低,胞内水含量提高,细胞膜稳定性增强;印度梨形孢定殖能改变由于病原菌引起的mRNA转录组变化,抗性相关基因的表达增强。综合表明印度梨形孢定殖能有效地提高小麦对禾谷镰孢菌的抗性。【结论】研究结果为深入理解植物与微生物互作、开发新型高效环保抗根腐病生物制剂提供理论和实验依据。     

内生真菌印度梨形孢诱导小麦抗根腐病作用研究

【目的】明确印度梨形孢(Piriformospora indica)诱导小麦对根腐病产生抗性的作用机制。【方法】用印度梨形孢悬液浸种,以无菌培养液为对照,用病原菌禾谷镰孢菌(Fusarium graminearum)侵染小麦,对其相关生理生化指标及转录组变化进行分析。【结果】禾谷镰孢菌能诱导小麦产生过氧化氢,降低细胞内水含量,破坏细胞膜的稳定性;根部定殖印度梨形孢的小麦细胞内抗氧化酶活性增强,活性氧自由基含量降低,胞内水含量提高,细胞膜稳定性增强;印度梨形孢定殖能改变由于病原菌引起的mRNA转录组变化,抗性相关基因的表达增强。综合表明印度梨形孢定殖能有效地提高小麦对禾谷镰孢菌的抗性。【结论】研究结果为深入理解植物与微生物互作、开发新型高效环保抗根腐病生物制剂提供理论和实验依据。     

尖孢镰孢菌果胶裂解酶基因家族鉴定及侵染表达模式分析

【背景】番茄枯萎病是番茄生产中常见的土传真菌病害。【目的】为鉴定番茄枯萎病基因组果胶裂解酶基因家族,明确该基因家族在侵染过程表达模式。【方法】采用生物信息学方法鉴定了番茄枯萎病尖孢镰孢菌(Fusarium oxysporum f. sp. Lycopersici)基因组内PEL基因家族,并分析了基因结构、染色体定位及三级结构,同时利用荧光定量PCR分析了FoPEL1-16基因在接种番茄根系的表达情况。【结果】番茄尖孢镰孢菌基因组内PEL基因家族成员有16个。氨基酸序列长度在163-548个氨基酸,信号肽长度在16-21个氨基酸。染色体定位分析表明16个基因在染色体上分布不均,分别定位在7条染色体上。根据基因结构和保守基序分析结果 16个基因可分为4类。进化分析表明该基因家族成员可聚成4支。三级结构预测结果显示同一家族存在相似结构域。荧光定量PCR分析结果表明Fo PEL基因在侵染过程表达水平明显上升。【结论】番茄尖孢镰孢菌基因组内果胶裂解酶以基因家族形式存在,其基因结构存在差异暗示了其功能多样性;FoPEL基因在侵染过程表达明显增强,说明其参与病原菌的致病性。本研究为解析尖孢镰孢菌致病基因功能分析及寄主病原互作提供了重要理论基础。     

甘肃大葱贮藏期镰孢菌腐烂病病原鉴定

【目的】大葱在贮藏期频繁发生镰孢菌腐烂病,损失严重。明确该病害病原种类对病害防治具有重要意义。【方法】利用组织分离法对采集自甘肃省兰州市(区)蔬菜市场的16份大葱贮藏期镰孢菌腐烂病病样进行病原物的分离、纯化培养,经单孢分离后根据形态学特征,再结合r DNA-ITS、EF-1a(tef)基因序列分析的方法进行鉴定。【结果】共分离得到80株镰孢菌,经鉴定分属3个种,即层出镰孢菌(Fusarium proliferatum)、尖孢镰孢菌(F.oxysporum)和燕麦镰孢菌(F.avenaceum),其中层出镰孢菌为大葱镰孢菌腐烂病的优势致病菌,分离频率为52.50%。对兰州白葱不同部位进行致病性测定,结果表明层出镰孢菌对大葱鳞茎的致病力最强,而燕麦镰孢菌对大葱鳞茎的致病力最弱。【结论】3种镰孢菌作为该病害的病原,属国内首次报道。     

玉米茎基腐病生防菌的筛选及应用

【目的】拟针对禾谷镰孢进行生防菌的筛选和应用研究,以期为玉米茎基腐病生防菌剂的研制奠定基础。【方法】通过对峙培养法对玉米茎基腐病的主要致病菌禾谷镰孢进行玉米内生生防细菌的筛选,从玉米主栽品种九单48幼苗内部获得一株具有较强抑菌效果的内生生防细菌(简称48SJ7-1);基于传统鉴定、16S rRNA基因序列测定及系统发育分析,对48SJ7-1进行鉴定;并通过盆栽试验测定该生防菌株的防效。【结果】菌株48SJ7-1经鉴定为甲基营养型芽孢杆菌,GenBank登录号为KU377993,48SJ7-1盆栽防效为68.47%,与对照药剂2%戊唑醇悬浮种衣剂差异不显著。【结论】48SJ7-1对玉米茎基腐病的主要致病菌禾谷镰孢有较好防效,对玉米生长还有明显的促进作用,而且表观上无药害发生。     

美国玉米自交系对4种病原茎腐病的抗性鉴定及遗传多样性分析

对外引玉米种质进行抗病鉴定是对其充分利用并选育抗病品种的基础和前提。本研究于2017-2018年连续2年采用人工接种技术评价了149个美国自交系对分别由禾谷镰孢(Fusarium graminearum)、拟轮枝镰孢(Fusarium verticillioides)、层出镰孢(Fusarium proliferatum)和芒孢腐霉(Pythium aristosporum)4种病原菌引起的茎腐病的抗性表现,并选择30个抗性较好的与6个骨干自交系进行遗传多样性分析。结果表明,149个美国自交系中,对禾谷镰孢、拟轮枝镰孢、层出镰孢和芒孢腐霉表现中抗及以上抗性的自交系分别有62个、44个、41个和54个,其中对4种茎腐病全部表现高抗的自交系有23个,全部表现中抗及以上抗性的自交系有32个,对3种镰孢菌茎腐病全部表现高抗的自交系有27个;所有供试材料中,88个自交系对4种茎腐病的抗性表现一致,61个自交系对4种茎腐病的抗性存在差异;共筛选出20条多态性引物,每个引物可检测出5～11个等位基因,平均等位变异数为7.7个,聚类分析将36个材料划分为6个类群,其中2FACC、J8608、黄早四分别被单独聚为一类,剩余自交系被划分为3个类群,分别包含自交系个数为26、5和2,第Ⅰ类群又分为3个亚群。本研究结果明确了外引美国自交系对不同病原菌引起的玉米茎腐病的抗性差异,为抗病育种中抗源的选择提供参考。     

小麦禾谷镰孢菌茎基腐病抗源的筛选与评价

为了发掘我国小麦种质资源中具有禾谷镰孢菌茎基腐病(crown rot)抗性的材料,采用禾谷镰孢菌苗期接种试验的方法,研究了82份小麦种质对禾谷镰孢菌茎基腐病的抗性.结果表明,在鉴定的材料中没有发现高抗材料;中抗材料13份,占总数的15.8%,包括CI12633、红蚰子、FHB143、Tiszataj和紫秆子等;大多数材料为感病材料.值得注意的是不同小麦材料对禾谷镰孢菌赤霉病和茎基腐病具有不同的抗性水平,两种病害没有正相关性,暗示小麦茎基腐病和赤霉病的抗性机制可能不同,因此,需要广泛挖掘具有茎基腐病抗性的小麦资源.     

弯曲平脐蠕孢拮抗细菌的筛选、鉴定及拮抗作用

【背景】玉米弯曲平脐蠕孢是引起玉米叶斑病的主要病原,严重危害农业生产。利用细菌防治弯曲平脐蠕孢是目前研究的热点。【目的】筛选对玉米弯曲平脐蠕孢有高拮抗性的菌株,对其进行鉴定并探究其拮抗机制。【方法】采用平板对峙法,从玉米田地表下土壤分离获得22株细菌,再经过复筛得到一株对玉米弯曲平脐蠕孢具有较高拮抗活性的菌株L-14。【结果】筛选发现,菌株L-14对层出镰孢(Fusariumproliferatum)、禾谷镰孢(Fusariumgraminearum)、大斑凸脐蠕孢(Exserohilum turcicum)、弯曲平脐蠕孢(Bipolaris papendorfii)和灰霉病菌(Botrytis cinerea) 5种植物病原真菌均有抑制作用,是一株广谱拮抗作用的生防菌株。通过对该菌株进行形态观察、16S rRNA基因序列分析以及生理生化特性检测,鉴定其为枯草芽孢杆菌。对菌株L-14的抗菌机制进行研究,发现该菌发酵液的蛋白粗提物对弯曲平脐蠕孢气生菌丝形态无影响,但可致基内菌丝畸变,对弯曲平脐蠕孢分生孢子萌发有抑制作用。【结论】筛选的拮抗菌在防治植物病害上具有广谱性及较高的拮抗活性,能有效防治玉米叶斑病。     

玉米感染禾谷镰刀菌后PAL、POD活性和同工酶谱的变化

为了明确苯丙氨酸解氨酶活和过氧化物酶活与玉米对镰刀菌茎腐病的抗性关系,探讨玉米对镰刀菌茎腐病的抗病机制及其在玉米抗病性鉴定中的利用,用禾谷镰刀菌孢子悬浮液对抗病品种陕单931和感病品种西农11号在抽雄初期进行接种,并于接种后测定茎秆髓部组织内的PAL,POD活性变化以及POD同工酶谱的变化。结果表明,玉米植株受镰刀菌侵染后,抗病品种的PAL酶活上升快,活性强,且形成两个活性高峰,高活性时间持续长;感病品种PAL酶活上升慢,活性相对较弱,且只形成一个峰,高活性持续时间短。抗病品种POD酶活峰值高,感病品种峰值低;抗病品种高酶活持续的时间长,感病品种高酶活持续的短。POD同工酶谱研究表明,抗、感品种POD同工酶带都有增多。抗病品种增5条,感病品种新增2条。PAL活性变化、POD活性变化及同工酶谱酶活变化与其对茎腐病的抗性有密切的关系,可作为抗病育种的生理生化指标。     

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-alpha gene (Translation Elongation Factor-1 alpha) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-alplha gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1 alpha gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex (F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.     

Isolation and evaluation of a Bacillus methylotrophicus strain for control of corn stalk rot

Bacteria were isolated from corn plants and efficacy of a selected isolate for control of corn stalk rot caused by Fusarium graminearum was evaluated. The bacterial isolate provided the greatest suppression of F. graminearum in lab studies and reduced corn stalk rot significantly in 2013 and 2014 field experiments.     

Genetic diversity and trichothecene chemotypes of the Fusarium graminearum clade isolated from maize in Nepal and identification of a putative new lineage

On smallholder farms in the foothills of the Himalayan Mountains in Nepal, fungi of the Fusarium graminearum clade cause Gibberella ear rot of maize and contamination with the 8-ketotrichothecenes nivalenol and deoxynivalenol. Previous DNA marker analyses of the F.?graminearum clade from maize in Nepal found a high level of genetic diversity but were limited in detail or scope. The present study incorporated a collection of 251 field strains from a wide geographic distribution in Nepal and utilized sequencing of the MAT1-1-3 gene of the mating type locus to determine the number and frequency of lineages and species of the F. graminearum clade. The frequency of nivalenol and deoxynivalenol chemotypes was determined by chemical analysis and by TRI13 deletion-marker analysis. We found that Gibberella ear rot of maize in Nepal is associated with a complex of species of the F. graminearum clade - mainly Fusarium asiaticum and Fusarium meridionale, but also Fusarium boothii and a putative new lineage, which we have designated the 'Nepal lineage'. Fusarium graminearum sensu stricto, which dominates in maize elsewhere in Asia and worldwide, was not detected in Nepal. Although nivalenol production has been associated experimentally with lower virulence in maize ear rot and wheat head blight, this collection of the F. graminearum clade from maize in Nepal is dominated (4:1) by nivalenol producers, suggesting that traits other than crop plant pathogenesis affect population structure in this complex agroecosystem.     

小麦抗茎腐病种质筛选及鉴定新方法的建立

为筛选针对我国黄淮麦区小麦茎腐病抗病新种质,建立可区分小麦其他茎基部病害的茎腐病抗性鉴定方法,本文采用室内苗期鉴定方法对主要来自黄淮麦区的108份小麦品种和高代品系进行茎腐病抗性评价,对其中45份小麦材料同时采用荧光定量PCR方法测定基部茎秆的禾谷镰刀菌DNA含量并与其茎腐病平均病级进行相关分析。共筛选到中抗茎腐病材料22份,未发现高抗和免疫品种(系);相关分析结果表明,小麦基部茎秆禾谷镰刀菌DNA含量与其茎腐病平均病级呈极显著正相关(r=0.73**),小麦基部茎秆禾谷镰刀菌DNA含量可以作为小麦茎腐病抗性的重要参考。抗病新种质的筛选和荧光定量PCR抗性评价方法的建立将为今后黄淮麦区小麦抗茎腐病品种的培育提供帮助。     

Microbiological and SYBR green real-time PCR detection of major Fusarium head blight pathogens on wheat ears

Fusarium head blight (FHB) caused by several Fusarium species is one of the most serious diseases affecting wheat throughout the world. The efficiency of microbiological assays and real-time PCRto quantify major FHB pathogens in wheat ears after inoculation with F. graminearum, F. culmorum, F. avenaceum and F. poae undergreenhouse and field conditions were evaluated. The frequency of infected kernel, content of fungal biomass, disease severity and kernel weight were determined. To measure the fungal biomass an improved DNA extraction method and a SYBR Green real-time PCR were developed. The SYBR Green real-time PCR proved to be highly specific for individual detection of the species in a matrix including fungal and plant DNA. The effect of Fusarium infection on visible FHB severity, frequency of infected kernels and thousand-kernel mass (TKM) significantly depended on the Fusarium species/isolate. F. graminearum resulted in highest disease level, frequency of infected kernels, content of fungal biomass, and TKM reduction followed by F. culmorum, EF avenaceum and F. poae, respectively. The comparison of frequency and intensity of kernel colonization proved differences in aggressiveness and development of the fungi in the kernels. Only for F. graminearum, the most aggressive isolate, application of microbiological and real-time PCR assays gave similar results. For the other species, the intensity of kernel colonization was lower than expected from the frequency of infection.     

Molecular mapping of QTLs for resistance to Gibberella ear rot, in corn, caused by Fusarium graminearum.

Gibberella ear rot, caused by the fungus Fusarium graminearum Schwabe, is a serious disease of corn (Zea mays) grown in northern climates. Infected corn is lower yielding and contains toxins that are dangerous to livestock and humans. Resistance to ear rot in corn is quantitative, specific to the mode of fungal entry (silk channels or kernel wounds), and highly influenced by the environment. Evaluations of ear rot resistance are complex and subjective; and they need to be repeated over several years. All of these factors have hampered attempts to develop F. graminearum resistant corn varieties. The aim of this study was to identify molecular markers linked to the genes for resistance to Gibberella ear rot. A recombinant inbred (RI) population, produced from a cross between a Gibberella ear rot resistant line (CO387) and a susceptible line (CG62), was field-inoculated and scored for Gibberella ear rot symptoms in the F4, F6, and F7 generations. The distributions of disease scores were continuous, indicating that resistance is probably conditioned by multiple loci. A molecular linkage map, based on segregation in the F5 RI population, contained 162 markers distributed over 10 linkage groups and had a total length of 2237 cM with an average distance between markers of 13.8 cM. Composite interval mapping identified 11 quantitative trait loci (QTLs) for Gibberella ear rot resistance following silk inoculation and 18 QTLs following kernel inoculation in 4 environments that accounted for 6.7%-35% of the total phenotypic variation. Only 2 QTLs (on linkage group 7) were detected in more than 1 test for silk resistance, and only 1 QTL (on linkage group 5) was detected in more than 1 test for kernel resistance, confirming the strong influence of the environment on these traits. The majority of the favorable alleles were derived from the resistant parent (CO387). The germplasm and markers for QTLs with significant phenotypic effects may be useful for marker-assisted selection to incorporate Gibberella ear rot resistance into commercial corn cultivars.