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.     

小麦响应禾谷镰刀菌侵染的转录组学研究进展

由禾谷镰刀菌引起的小麦赤霉病直接为害作物穗部,不仅严重影响小麦产量,还可因为毒素污染问题威胁人畜健康。近年来对小麦与禾谷镰刀菌互作的转录组学研究带来了很多新见解,概述了小麦响应禾谷镰刀菌侵染的转录组学研究进展,主要比较了不同抗性品种、不同器官、不同籽粒发育时期的小麦穗部在禾谷镰刀菌侵染时的基因表达特征,总结了赤霉病感染时小麦的激素响应、信号传导、转录调控和防卫相关基因的表达规律,以期促进研究者对小麦响应禾谷镰刀菌侵染规律的理解。     

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。     

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.     

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。     

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.     

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。     

禾谷镰孢菌蛋白激酶FgCdc15功能的研究

细胞分裂是真核细胞生长发育的重要环节。本研究利用生物信息学的方法,通过使用酵母菌Cdc15蛋白激酶序列比对,发现禾谷镰孢菌中只存在一个Cdc15蛋白激酶。基因缺失的功能研究表明FgCDC15（FGSG_10381）基因敲除后,突变体菌落生长速度减慢,对小麦和玉米无致病力。FgCDC15基因敲除突变体产生的分生孢子外观形态正常,但隔膜数量变少,同时发现该基因在分生孢子阶段表达量最高。在有性生殖阶段,FgCDC15基因敲除突变体可产生极少量的子囊壳,但不产生子囊和子囊孢子。本文研究表明,禾谷镰孢菌蛋白激酶FgCdc15可能参与了有性和无性阶段的细胞分裂和生长,同时影响禾谷镰刀菌的致病毒力。     

Isolation of 4-acetyl-benzoxazolin-2-one (4-ABOA) and diferuloylputrescine from an extract of gibberella ear rot-resistant corn that blocks mycotoxin biosynthesis, and the insect toxicity of 4-ABOA and related compounds

Bioassay-directed fractionation of an extract of a gibberella ear rot-resistant corn hybrid resulted in the isolation of a fraction that inhibited mycotoxin production, but not growth, of Fusarium culmorum. The fraction contained diferuloylputrescine and a new allelochemical 4-acetyl-benzoxazolin-2-one (4-ABOA). This report describes the isolation of the bioactive fraction and tests of the effect of 4-ABOA on 3-acetyl deoxynivalenol (3ADON) and aflatoxin production. In addition, data are reported on the effects of 4-ABOA and related compounds on a number of insect species and some preliminary studies on the mechanism of insect toxicity. The effect of 4-ABOA on F. culmorum and Apergillus flavus was similar. Production of toxin was reduced without a concomitant reduction of growth. A 50% inhibition of 3ADON production occurred at ca. 4 μM and 0.12 mM for alfatoxin. 4-ABOA was slightly toxic to Spodoptera frugiperda.     

小麦抗赤霉病利器——他山之石

赤霉病是我国乃至世界小麦(Triticum aestivum)产区的重要病害, 给农业生产和人畜健康造成重大威胁。分离鉴定优质抗病基因、培育抗病品种, 是控制我国麦区赤霉病的重要手段。最近, 山东农业大学孔令让团队完成了二倍体长穗偃麦草(Thinopyrum elongatum)基因组的组装, 并在此基础上通过精细定位和图位克隆分离得到来自长穗偃麦草的抗赤霉病基因Fhb7。他们发现Fhb7编码1个谷胱甘肽转移酶, 对禾谷镰孢菌(Fusarium graminearum)分泌的包括呕吐毒素等在内的多种毒素具有解毒作用, 是1个广谱持久抗病基因。他们还发现Fhb7很可能最初源于内生真菌, 经过基因水平转移进入到偃麦草基因组中。此外, Fhb7不影响其它农艺性状, 且其抗性不受小麦遗传背景影响。这一系列工作揭示了作物抗病演化中的全新机制, 对小麦抗赤霉病育种以及更好地利用长穗偃麦草的丰富基因资源都具有重要意义。     

尖孢镰刀菌FoHeli转座元件的结构特征及功能验证

Helitron是一种广泛存在于真核生物中的可移动遗传元件。与其他转座子不同,自主Helitron元件可编码具有复制引发（Rep）和解旋酶（Hel）结构域的转座酶,并通过滚环复制的方式在基因组中进行扩张。本研究对9种尖孢镰刀菌中的自主Helitron元件进行系统分析,结果表明尖孢镰刀菌中存在两类自主Helitron元件FoHeli1与FoHeli2。其中FoHeli1成员间序列高度相似,并具有明晰的边界特征：3’端为保守的“TATTTT”序列,其上游可形成稳定的发夹结构,且发夹上游可与5’端形成12bp的反向互补结构。基于上述分析结果,从尖孢镰刀菌Fo4287菌株中克隆获得完整的FoHeli1元件,并通过构建双元转座系统及PEG介导的原生质体转化,证明尖孢镰刀菌中的FoHeli元件可在禾谷镰刀菌PH-1菌株的基因组中发生跳转。     

蛋白激酶FgBud32参与禾谷镰刀菌的生长发育、致病和胁迫应答

由禾谷镰刀菌引起的小麦赤霉病是一种毁灭性的小麦真菌病害,在世界范围内造成小麦产量和质量的巨大损失。实验室前期在禾谷镰刀菌中共鉴定到116个蛋白激酶,其中FgBUD32基因的缺失会造成营养生长和有性生殖方面的严重缺陷,但其在禾谷镰刀菌中的详细功能尚未报道。本研究通过系统比较Fgbud32突变体与野生型PH-1及互补菌株的表型差异,对FgBud32在禾谷镰刀菌中的生物学功能进行了解析。研究结果显示Fgbud32突变体在多个表型方面存在缺陷,与野生型菌株以及互补菌株相比,其生长速率急剧下降,菌丝弯曲且分支减少;分生孢子的产量显著降低,形态变短,隔膜减少,萌发率降低且萌发速率延迟;在有性生殖时期不能产生子囊壳或子囊壳前体;对小麦穗和胚芽鞘的致病力以及DON毒素的合成能力均显著下降。进一步胁迫试验表明,FgBUD32基因的缺失导致禾谷镰刀菌对氧化胁迫(H₂O₂)以及DNA损伤胁迫(羟基脲和甲磺甲酯)的敏感性增加。此外,我们还发现FgBud32在细胞核和细胞质中均有定位,且在一定时期或条件下会从细胞质向细胞核内聚集。综上所述,FgBUD32基因参与了禾谷镰刀菌的营养生长、极性生长、无性/有性生殖、DON毒素合成、致病以及对氧化胁迫和DNA损伤胁迫的应答等多种生命活动,但其具体的作用机制还有待深入研究。     

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。     

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.     

小麦赤霉病新抗源的发掘与抗性位点的检测分析

小麦赤霉病是由禾谷镰刀菌引起的世界性重要病害,发掘优异的抗性种质资源、培育抗病品种是持续防治赤霉病最经济且环境友好的措施。为发掘新的赤霉病抗源,本研究于2017—2021年在弥雾保湿大棚中,采用单花滴注法对642份小麦种质资源的赤霉病抗扩展性进行鉴定,同时利用已知抗赤霉病基因/位点Fhb1~Fhb7的分子标记对筛选出的抗性种质基因型进行分析。结果表明,不同年份间赤霉病病小穗率的相关性均达到极显著水平。筛选到3年及以上赤霉病抗性优于扬麦158的种质81份,主要来自长江中下游麦区,其中33份种质连续4年抗性优于扬麦158;筛选到3年及以上抗性与苏麦3号相当的种质9份,分别为望水白、Grandin、浩麦1号、剑子麦、魁小麦、农林26、软秆洋麦、苏麦2号和武农6号,其中剑子麦、软秆洋麦、苏麦2号和Grandin连续4年抗性与苏麦3号相当。对抗性种质携带的抗赤霉病基因/位点进行分析发现,浩麦1号、冀师7225-28、南农13Y110、石优17和武农6号不携带任何已知抗赤霉病基因/QTL,为小麦抗赤霉病研究和品种培育提供了新的种质资源和理论依据。     

Growth inhibition of the cereal root pathogens Rhizoctonia solani AG8, R. oryzae and Gaeumannomyces graminis var. tritici by a recombinant 42-kDa endochitinase from Trichoderma harzianum

The objective of this study was to determine the effectiveness of a 42-kDa endochitinase coded by the ThEn42 gene from Trichoderma harzianum as a potential source of transgenic resistance to Rhizoctonia root rot of barley caused by Rhizoctonia solani AG8 and/or R. oryzae. The gene cThEn42 was codon optimized (GC content increased from 53.3 to 65.1%) and then synthesized to produce the modified cThEn42GC in Pichia pastoris for in vitro tests. Two expression vectors were constructed: one with the fungal signal peptide and the fungal activation peptide [FSP-FAP-cThEn(GC)] and the other with barley chitinase 26 signal peptide followed by the fungal signal and activation peptides [SP(HVChi26)-FSP-FAP-cThEn(GC)]. N-terminal sequencing showed that, of two proteins secreted into liquid medium, FSP was cleaved off faithfully in one protein and both FSP and FAP were cleaved from the other protein. Purified endochitinase provided strong in vitro inhibition of both R. solani AG8 and R. oryzae. The enzyme had an intermediate inhibitory activity against Gaeumannomyces graminis var. tritici, and no inhibitory activity against Fusarium graminearum, F. pseudograminearum, and F. culmorum.     

Secondary Metabolites Produced by a Strain of Fusarium oxysporum Used for Striga Control in West Africa

The production of fusaric acid and other toxins by a strain of Fusarium oxysporum used for control of the weed Striga hermonthica (Del.) Benth. was investigated. Culturing of the strain under optimal conditions for toxin production produced small amounts of fusaric acid and dehydrofusaric acid but no other toxins reported to be produced by some strains of F. oxysporum. Culturing of the fungus under conditions similar to those that would be used in the field produced no detectable toxins of concern for human health.     

苹果连作生防细菌B6对平邑甜茶幼苗生物量及连作土壤环境的影响

以苹果连作障碍病原真菌层出镰刀菌、串珠镰刀菌、尖孢镰刀菌和腐皮镰刀菌为靶标菌,通过平板对峙法对分离自苹果根际土壤的细菌进行反复筛选比较,对筛选出的拮抗效果最优的菌株进行形态学、生理生化特征和16S rDNA序列分析鉴定,并于盆栽条件下探讨其菌肥对平邑甜茶幼苗生长及连作土壤环境的影响.结果表明: 菌株B6对上述4种病原真菌的抑菌率最高,分别达到71.8%、70.1%、72.6%、91.5%.经鉴定,菌株B6为甲基营养型芽孢杆菌.盆栽试验表明,与连作处理(CK₁)相比,B6菌肥处理(T)可以不同程度地促进平邑甜茶幼苗生物量的增加,其中地径、鲜质量和干质量分别显著增加18.3%、51.2%;显著提高连作土壤中可培养细菌和放线菌数量,使真菌数量下降为连作土壤的37.7%,促使土壤类型向细菌型转化;显著提高连作土壤中的蔗糖酶、磷酸酶、脲酶和过氧化氢酶的活性,增长率分别为37.3%、24.0%、42.9%、49.4%.表明B6菌肥可以优化苹果连作障碍土壤中可培养微生物群落结构,提高土壤酶活性,增加平邑甜茶幼苗生物量.