禾谷镰刀菌蛋白激酶研究进展

由禾谷镰刀菌引起的小麦赤霉病是小麦生产最重要的真菌病害之一,除了造成严重的产量损失外,其病原菌还会产生多种真菌毒素危害人畜健康。蛋白激酶在禾谷镰刀菌生长发育、植物侵染和胁迫应答等方面具有重要作用。综述了禾谷镰刀菌主要蛋白激酶在生物学功能和分子作用机制等方面的研究进展,并对未来禾谷镰刀菌蛋白激酶的研究趋势进行了展望,以期为今后禾谷镰刀菌蛋白激酶的研究与小麦赤霉病的防治提供理论参考。     

抗病小麦对脱氧雪腐镰刀菌烯醇的脱毒及产物的生物活性

抗赤霉病小麦品种苏麦３号、繁９能转化镰刀菌毒素脱氧雪腐镰刀菌烯醇（ＤＯＮ）成产物Ｘ,而感病小麦品种宁麦６号、徐州２１无转化能力。产物Ｘ对小麦黄花芽鞘的伸长生长无抑制作用,而对禾谷镰刀菌分生孢子的萌发有明显抑制。说明抗性小麦品种对赤霉病菌毒素的脱毒是小麦重要的抗赤霉病机制。     

抗病小麦对脱氧雪腐镰刀菌烯醇的脱毒及产物的生物活性

抗赤霉病小麦品种苏麦3号、繁9能转化镰刀菌毒素脱氧雪腐镰刀菌烯醇(DON)成产物X,而感病小麦品种宁麦6号、徐州21无转化能力。产物X对小麦黄化芽鞘的伸长生长无抑制作用,而对禾谷镰刀菌分生孢子的萌发有明显抑制。说明抗性小麦品种对赤霉病菌毒素的脱毒是小麦重要的抗赤霉病机制。     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

单端孢霉烯族毒素的产生及分子调控机制

禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的赤霉病是小麦生产上危害最为严重的病害之一。赤霉病除了造成减产外,感病籽粒中含有多种镰刀菌毒素,如单端孢霉烯族的呕吐毒素,可引起人畜中毒和重大疾病,给食品安全构成严重威胁。过去20年,随着禾谷镰刀菌全基因组序列的公布和遗传转化体系的成熟,禾谷镰刀菌Fusarium graminearum的功能基因组学的研究取得了较大进展,单端孢霉烯族毒素的产生、调控机制及网络研究成为热点。本文综述国内外单端孢霉烯族毒素的生物合成和分子调控机制,包括合成基因簇及决定不同产毒化学型的基因、产毒调控元件、环境因子调控产毒的分子机制,可为小麦抗赤霉病的育种提供新思路,为新型药剂的研发提供分子靶标,为赤霉病的持续防控和毒素污染的有效治理提供理论依据。     

禾谷镰刀菌粗毒素对不同抗性小麦品种成熟胚脱分化与再分化的影响

以5个赤霉病抗性水平不同的小麦品种为材料,研究了禾谷镰刀菌粗毒素对小麦成熟胚脱分化与再分化的影响.结果表明,小麦成熟胚愈伤组织的诱导率和分化率在品种间、毒素浓度间、毒素浓度×品种间差异极显著.低浓度（5 g/L）禾谷镰刀菌粗毒素能够促进抗性较强品种的出愈速度,高浓度禾谷镰刀菌粗毒素（10 g/L和15 g/L）对成熟胚愈伤组织的出愈速度起到抑制作用,随着浓度提高,抑制作用增强.成熟胚愈伤组织的诱导率和分化率随着禾谷镰刀菌粗毒素浓度升高而下降,品种的抗性越差下降幅度越大.10 g/L毒素浓度,5个品种成熟胚愈伤组织的SOD活性均比对照（0 g/L）增加;20 g/L毒素浓度,5个品种成熟胚愈伤组织的SOD活性均有所下降,抗性品种苏麦3号和郑9023的SOD活性仍高于对照,其它3个感性品种低于对照,SOD活性与小麦品种的赤霉病抗性水平存在一定关系.研究结果为小麦赤霉病抗性育种提供了一定的技术支撑.     

小麦DON毒素研究进展

近年来,由于全球气候急剧变化以及栽培、耕作制度的转变,导致禾谷镰刀菌引起的赤霉病频繁发生,不仅使小麦大面积减产,严重影响其品质,也造成经济上的重大损失。受赤霉菌侵染的小麦会在籽粒中产生脱氧雪腐镰刀菌烯醇（deoxynivalenol, DON）毒素,该毒素广泛污染小麦及其制品、粮油食品和饲料等,是世界上污染面积最大、污染量最高,对小麦危害最严重的真菌毒素之一。DON可以产生广泛的毒性效应,具有很强的细胞毒性,有明显胚胎毒性和一定致畸作用,严重威胁食品安全、人和动物健康。因此,对DON毒素调控机理及防控的研究已成为当前高度关注的热点问题之一。通过综述DON的理化性质、毒性机理、污染特征、分布规律、检测方法以及小麦抗DON毒素积累机制等方面的进展,以期为小麦抗DON毒素育种提供参考依据。     

我国小麦赤霉病成灾原因分析及防控策略探讨

小麦是世界上三大粮食作物之一,是全球30亿以上人口的主粮。近年来,由于各种病虫害危害,全球小麦生产和粮食安全受到严重威胁,其中由禾谷镰刀菌引起的小麦赤霉病是小麦生产上重要的病害之一。此外,病菌会产生多种真菌毒素对人畜生命健康构成严重威胁。化学药剂的使用以及抗病品种的种植可以有效地控制小麦赤霉病的发生。但是,由于高产优质抗病品种匮乏、气候变暖等因素影响,小麦赤霉病在我国小麦主产区频繁暴发;同时,赤霉病菌抗药性产生致使化学农药的防控效果大大降低。从气候变化、耕作制度改变、小麦品种抗性及病菌抗药性等方面,分析了赤霉病暴发成灾的主要原因。在此基础上,结合当前赤霉病防控研究进展以及存在的科学问题,探讨该病害持续绿色防控的对策建议,以期为我国小麦赤霉病的防控研究提供参考。     

禾谷镰刀菌cAMP受体类GPCR生物信息学分析

禾谷镰刀菌(Fusarium graminearum)是引起小麦赤霉病的主要致病菌。G蛋白偶联受体(G protein coupled receptors,GPCRs)是一类重要的细胞表面受体,其介导的cAMP信号通路可能参与了禾谷镰刀菌的致病和毒素合成,因此分析cAMP受体类型的GPCRs蛋白的结构及其理化性质对了解GPCRs的功能及其与赤霉病致病的关系具有重要意义。本研究运用生物信息学方法,对禾谷镰刀菌全基因组序列中cAMP类GPCR基因进行了生物信息学分析。发现禾谷镰刀菌中存在5个典型的cAMP受体类型GPCRs:FgcAR1、FgcAR2、FgcAR3、FgcAR4和FgcAR5,均含有7个跨膜结构域,并定位于细胞膜上。除FgcAR1外,其余为疏水性蛋白。蛋白质二级结构分析表明,均含有大量α螺旋,比例在60%左右,FgcAR4和FgcAR5没有β转角,FgcAR1、FgcAR2和FgcAR3也只有较少比例的β转角。这些GPCRs中含有较多的Ser和Thr磷酸化位点。遗传分析表明,禾谷镰刀菌cAMP受体类型的GPCR蛋白与假禾谷镰刀菌及F.langsethiae同源性最高,亲缘关系最近。本研究明确了禾谷镰刀菌中cAMP类GPCR蛋白的理化性质、定位、二级结构、磷酸化位点及进化关系,为了解小麦赤霉病发病机制及以GPCRs为靶标的新型杀菌剂研发奠定了基础。     

小麦赤霉病拮抗菌JB7的拮抗活性及鉴定

由禾谷镰刀菌(Fusarium graminearum, Fg)引起的赤霉病是限制小麦生产的主要病害之一。生物防治是一种高效且可持续的防治方法。【目的】从小麦种子内筛选具有抑制禾谷镰刀菌的菌株并对其生防潜力进行评估,为小麦赤霉病生防制剂的开发与利用提供菌种资源及理论支撑。【方法】采用平板对峙、孢子萌发法和无菌上清液抑菌试验筛选小麦种子内对禾谷镰刀菌具有拮抗活性的内生菌株;利用扫描电镜(scanning electron microscope, SEM)和共聚焦扫描电镜(confocal laser scanning microscope, CLSM)观察并分析无菌上清液对Fg的分生孢子形态、膜完整性以及胞内活性氧的影响;通过盆栽试验验证内生菌对小麦赤霉病的生防效果;应用二代Illumina HiSeq测序平台进行全基因组测序。【结果】从小麦种子中分离出一株高效抑制Fg生长的内生菌株JB7,其衰亡期无菌上清液对Fg孢子萌发抑制率高达85.23%。菌株JB7的无菌上清液使Fg孢子表面凹陷,破坏其细胞膜,造成核酸和蛋白质的渗漏,诱导Fg菌丝活性氧的累积,引起Fg菌丝可溶性蛋白和丙二醛含量的显著升高。该菌株具有分泌蛋白酶、纤维素酶、葡聚糖酶和产铁载体的能力。盆栽试验表明菌株JB7能显著降低小麦赤霉病的病情指数(P<0.05)。经全基因组学鉴定为甲基营养型芽孢杆菌(Bacillus methylotrophicus) JB7,该菌株基因组中含有12个抑菌功能的次级代谢产物合成基因簇。【结论】菌株JB7能抑制禾谷镰刀菌的生长,对小麦赤霉病有较强的防效,可作为生物防治小麦赤霉病的候选菌株。     

Contamination of malt barley and wheat by Fusarium graminearum and Fusarium culmorum from the crop years 2001-2003 in eastern Croatia

This study investigated infection levels with Fusarium graminearum and Fusarium culmorum in malt barley and wheat in eastern Croatia. The contamination was surveyed over three consecutive crop years (2001–2003) on five locations for barley and three wheat cultivating locations. F. graminearum loads reached levels of potentially serious threat for the commercial production of malting raw materials in both cereals (up to 29.1%). On the other hand, the mean percentage of kernels infected with F. culmorum was low to medium (up to 6.1%). The fungal invasions for years and locations were affected by meteorologic and other environmental factors and the pattern seemed to be consistent with species-specific optimal conditions reported by other authors.     

Potential for control of seedling blight of wheat caused by Fusarium graminearum and related species using the bacterial endophyte Bacillus mojavensis

Fusarium-infected wheat seed decreases germination, seedling emergence, and causes post emergence seedling death, and can contribute to wheat scab and ear rot of maize, with consequent production of mycotoxins such as deoxynivalenol and zearalenone. Current seed treatments have proved ineffective in controlling seedling blight and scab. A patented endophytic bacterial strain, Bacillus mojavensis RRC 101, and several other strains of this species were studied to determine in vitro antagonism to some Fusarium species and to assess the potential of this bacterium to serve as an endophytic biocontrol for seedling blight of wheat produced by species within the F. graminearum complex, as well as other species of Fusarium. Seedling emergence and seed germination were two tests used as indicators of seedling blight. These tests were conducted in growth rooms with two wheat cultivars highly susceptible to scab, Norm and Pioneer 2552, and other cultivars with varying resistance to scab. The results indicated that all strains of this bacterium were antagonistic in vitro to the strains of F. graminearum and its seven related species, as well as four strains of F. pseudograminearum and the two strains of F. verticillioides. Germination of the highly scab susceptible cultivar 2552 was increased from 77 to 97% when planted in soil containing a mixed inoculum of F. graminearum and related species. Seedling emergence in the very susceptible wheat cultivar Norm increased from 20 to 82% when treated with the bacterium. The data indicated that inoculating wheat kernels with B. mojavensis reduced seedling blight of wheat produced by F. graminearum and related Fusarium species indicating the potential for this bacterium as a biocontrol under field condition.     

Potential of fungal antagonists for biocontrol of Fusarium spp. in wheat and maize through competition in crop debris

Pathogenic Fusarium spp. cause head blight in wheat or ear rot in maize leading to yield losses and also a reduction in quality due to mycotoxin contamination of the grain. Infected crop residues are the main inoculum source for epidemics. Saprophytic fungi, obtained from cereal tissues or necrotic tissues of other crops, were screened for their ability to colonise wheat straw and maize stalks and to suppress sporulation of pathogenic Fusarium spp. Results of bio-assays conducted under controlled conditions were variable among Fusarium spp. and host substrates for most antagonists tested, such as yeasts, Trichoderma spp. and non-pathogenic Fusarium spp. Isolates of Clonostachys rosea consistently suppressed sporulation of F. culmorum and F. graminearum on wheat straw, and of F. culmorum, F. graminearum, F. proliferatum and F. verticillioides on maize stalks. Isolates of C. rosea, C. cladosporioides and F. equiseti were applied to pieces of maize stalks or flowering ears in preliminary experiments conducted under field conditions. The colonisation of stalk pieces by pathogenic Fusarium spp. was assessed after 9 months. Colonisation of stalk pieces by pathogenic Fusarium spp. was significantly reduced at several sampling dates. However, results obtained with the antagonists were not consistent for all sampling dates and between experiments.     

禾谷镰孢菌β2微管蛋白与苯并咪唑类杀菌剂互作研究

小麦赤霉病是小麦上的主要病害之一,在全世界范围内引起该病害的致病菌主要是禾谷镰孢菌Fusarium graminearum。目前,使用杀菌剂是生产上防治小麦赤霉病发生和危害的主要手段,常用的杀菌剂主要有苯并咪唑类杀菌剂(benzimidazoles)等,苯并咪唑类杀菌剂的作用靶标是β2微管蛋白。本研究旨在探究小麦赤霉病菌中β2微管蛋白与苯并咪唑类杀菌剂的互作机制,通过同源建模的方法获得了禾谷镰孢菌β2微管蛋白的三维结构,并在此基础上将β2微管蛋白与4种苯并咪唑类杀菌剂(多菌灵、苯菌灵、噻菌灵、甲基硫菌灵)进行分子对接。分子对接结果显示β2微管蛋白第198位苯丙氨酸和第236位缬氨酸与4种苯并咪唑类杀菌剂直接互作形成氢键,第50、134、165、167、198、200、236、237、239、240、250、253、257、314位氨基酸形成药剂结合口袋。通过比较β2微管蛋白与4种苯并咪唑类杀菌剂结合自由能,发现与其他3种杀菌剂相比,β2微管蛋白与多菌灵的结合自由能最小(-5.72 kcal/mol),说明其与多菌灵互作亲和力更强。采用菌丝生长速率法测定了禾谷镰孢菌对4种苯并咪唑类杀菌剂的EC₅₀值,禾谷镰孢菌对多菌灵、苯菌灵、噻菌灵、甲基硫菌灵的EC₅₀值分别为0.772、0.862、1.088、13.266 mg/L,该结果表明禾谷镰孢菌对多菌灵的敏感性强于其他3种杀菌剂,与分子对接结果相吻合。     

Field Evaluation of Fungal Competitors of Fusarium culmorum and F. graminearum, Causal Agents of Ear Blight of Winter Wheat, for the Control of Mycotoxin Production in Grain

Thirty-seven antagonists, from a collection of 113 pre-screened microorganisms, were tested in field experiments on winter wheat for their ability to control ear blight and production of mycotoxins, particularly deoxynivalenol (DON), in grain by Fusarium culmorum and F. graminearum. Ears were spray-inoculated during anthesis with spore suspensions, first of test fungi and then of the pathogen. Isolates of F. equiseti were the most effective treatments. A strain of F. equiseti (G9) decreased DON consistently on wheat inoculated with F. culmorum, compared with the control, often by more than 70%. It performed well under severe disease pressure (F. culmorum at 10⁵ conidia mL^-1) and similarly to a standard fungicide, tebuconazole. Percentages of diseased grains and amounts of DON corresponded well. Small quantities of nivalenol (NIV) occurred in grain samples after treatment with some F. equiseti strains. On wheat inoculated with F. graminearum, F. equisti G2 decreased the percentage diseased grains by 92% and DON by 94%. One strain of F. equiseti (G2) was found to produce small amounts of NIV in pure culture.     

Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death

Filamentous fungal pathogens secrete effectors that modulate host immunity and facilitate infection. Fusarium graminearum is an important plant pathogen responsible for various devastating diseases. However, little is known about the function of effector proteins secreted by F. graminearum. Herein, we identified several effector candidates in the F. graminearum secretome. Among them, the secreted ribonuclease Fg12 was highly upregulated during the early stages of F. graminearum infection in soybean; its deletion compromised the virulence of F. graminearum. Transient expression of Fg12 in Nicotiana benthamiana induced cell death in a light‐dependent manner. Fg12 possessed ribonuclease (RNase) activity, degrading total RNA. The enzymatic activity of Fg12 was required for its cell death‐promoting effects. Importantly, the ability of Fg12 to induce cell death was independent of BAK1/SOBIR1, and treatment of soybean with recombinant Fg12 protein induced resistance to various pathogens, including F. graminearum and Phytophthora sojae. Overall, our results provide evidence that RNase effectors not only contribute to pathogen virulence but also induce plant cell death.     

Multilocus sequence analysis of Fusarium pseudograminearum reveals a single phylogenetic species

Fusarium pseudograminearum causes crown rot of wheat in Australia and most other wheat growing regions, but its evolutionary history is largely unknown. We demonstrate for the first time that F. pseudograminearum is a single phylogenetic species without consistent lineage development across genes. Isolates of F. pseudograminearum, F. graminearum sensu lato, and F. cerealis, were collected from four countries and four single copy, nuclear genes were partially sequenced, aligned with previously published sequences of these and related species, and analysed by maximum parsimony and Bayesian inference. Evolutionary divergence varied between genes, with high phylogenetic incongruence occurring between the gene genealogies. The absence of geographic differentiation between isolates indicates that the introduction of new fungal strains to a region has the potential to introduce new pathogenic and toxigenic genes into the native population through sexual recombination.