Fusarium graminearum sensu stricto和F.asiaticum在玉米秸秆和水稻秸秆上子囊壳形成的比较

为探究麦田前茬作物玉米秸秆和水稻秸秆上中国小麦赤霉病菌的两个优势种Fusarium graminearum sensu stricto(F.graminearum)和F.asiaticum子囊壳形成和发育成熟过程的差异,分别选择3株F.graminearum菌株(SE81,3-ADON型;LcA-2,15-ADON型;HX5-1,NIV型)和2株F.asiaticum菌株(M4A,3-ADON型;M31-2,NIV型),用其孢子悬浮液接种玉米秸秆和水稻秸秆,放置于花泥和地表两种环境条件下,比较接种不同菌株秸秆上子囊壳的形成和发育成熟情况及5个菌株对‘郑麦366’(高感小麦赤霉病品种)的致病力。结果表明:花泥环境下产生子囊壳的速度要快于地表,以玉米秸秆为载体子囊壳产生速度比水稻秸秆快且多数密度大,3个F.graminearum菌株(SE81、LcA-2和HX5-1)在同种秸秆相同环境下比2个F.asiaticum菌株(M4A和M31-2)产生子囊壳的速度快且子囊壳密度大。5个菌株在不同环境的不同秸秆上产生的子囊壳的成熟度均无规律,接种‘郑麦366’后其病情指数间有极显著差异(P<0.01),在花泥和地表的玉米秸秆和水稻秸秆上3次调查的子囊壳密度与‘郑麦366’的病情指数呈显著正相关,不同环境条件下的子囊壳产生速度(AUPGC)与‘郑麦366’的病情指数呈极显著正相关。本研究将为阐明F.graminearum和F.asiaticum在中国小麦赤霉病上表现区域性地理分布的原因提供参考依据。

FgGyp8 as a putative FgRab1 GAP is required for growth and pathogenesis by regulating FgSnc1-mediated secretory vesicles fusion in Fusarium graminearum

Fusarium graminearum is an important plant pathogenic fungus that causes disease and yield reduction in many cereal crops, such as wheat and barley. Gyp8 stimulates GTP hydrolysis on Ypt1 in yeast. However, the functions of Gyp8 in plant pathogenic fungi are still unknown. In this study, we investigated the roles of Fg Gyp8 in F. graminearum by genetic and pathological analyses. Through gene knockout and phenotypic analyses, we found that Fg Gyp8 is required for vegetative growth in F. graminearum. The conidiation, conidial size and number of septa per conidium of ΔFggyp8 mutant are significantly reduced when compared to the wild type PH-1. Furthermore, Fg Gyp8 is crucial for pathogenicity on wheat coleoptiles and wheat heads. Fg Gyp8 contains a conserved TBC domain. Domain deletion analysis showed that the TBC domain, C-and N-terminal regions of Fg Gyp8 are all important for its biological functions in F. graminearum. Moreover, we showed that Fg Gyp8 catalyzes the hydrolysis of the GTP on Fg Rab1 to GDP in vitro, indicating that Fg Gyp8 is a GTPase-activating protein(GAP) for Fg Rab1. In addition, we demonstrated that Fg Gyp8 is required for Fg Snc1-mediated fusion of secretory vesicles with the plasma membrane in F. graminearum. Finally, we showed that Fg Gyp8 has functional redundancy with another Fg Rab1 GAP, Fg Gyp1, in F. graminearum. Taken together, we conclude that Fg Gyp8 is required for vegetative growth, conidiogenesis, pathogenicity and acts as a GAP for Fg Rab1 in F. graminearum.

Mitochondrial dynamics caused by QoIs and SDHIs fungicides depended on FgDnm1 in Fusarium graminearum

Fusarium head blight(FHB) caused by Fusarium graminearum is a devastating fungal disease on small grain cereal crops,because it reduces yield and quality and causes the mycotoxin contamination to the grain.Dynamins and dynamin-related proteins(DRPs) are large GTPase superfamily members,which are typically involved in the budding and division of vesicles in eukaryotic cells,but their roles in Fusarium spp.remain unexplored.Here,we found that FgDnm1,a DRP and homolog to Dnm1 in Saccharomyces cerevisiae,contributes to the normal fungal growth,sexual reproduction and sensitivity to fungicides.In addition,we found FgDnm1 co-localizes with mitochondria and is involved in toxisome formation and deoxynivalenol(DON) production.Several quinone outside inhibitors(QoIs) and succinate dehydrogenase inhibitors(SDHIs) cause fragmentated morphology of mitochondria.Importantly,the deletion of FgDnm1displays filamentous mitochondria and blocks the mitochondrial fragmentation induced by QoIs and SDHIs.Taken together,our studies uncover the effect of mitochondrial dynamics in fungal normal growth and how such events link to fungicides sensitivity and toxisome formation.Thus,we concluded that altered mitochondrial morphology induced by QoIs and SDHIs depends on FgDnm1.

ZmMYC7 directly regulates ZmERF147 to increase maize resistance to Fusarium graminearum

The jasmonic acid(JA)signaling pathway is involved in plant growth,development,and response to abiotic or biotic stresses.MYC2,a bHLH transcription factor,is a regulatory hub in the pathway.The function of ZmMYC7,a putative MYC2 ortholog,in jasmonate-signaled defense responses of maize has not been reported.In this study,we found that ZmMYC7 possesses JID,TAD,bHLH and Zip domains and essential characteristics of transcription factors:a nuclear location and transactivation activity.The ZmMYC7mutants showed markedly increased sensitivity to Fusarium graminearum and Setosphaeria turcica.The expression levels of the defense-associated genes ZmPR1,ZmPR2,ZmPR3,ZmPR5,ZmPR6,and ZmPR7 in response to F.graminearum infection were downregulated in ZmMYC7 mutants,while ZmPR4 and ZmPR10 were up-regulated.ZmMYC7 interacted with members of the ZmJAZ family,including ZmJAZ8,ZmJAZ11,and ZmJAZ12.ZmMYC7 physically interacted with G-box cis-elements in the ZmERF147 promoter in vitro and transcriptional activation of ZmERF147 by ZmMYC7 was inhibited by ZmJAZ11 and ZmJAZ12.ZmERF147 mutants were more susceptible to F.graminearum infection than inbred line B73with concomitant down-regulation of all defense-associated ZmPRs except ZmPR4.These findings indicate that ZmMYC7 functions in maize resistance to F.graminearum and sheds light on maize defense responses to pathogenic fungi via the JA signaling pathway.

禾谷镰刀菌(Fusarium graminearum)毒素对小麦幼胚愈伤组织诱导和分化的影响

为了给普通小麦抗赤霉细胞工程育种中抗毒素细胞系选择技术体系的建立和方法的选择提供理论依据,研究了离体培养条件下禾谷镰刀菌毒素胁迫对小麦幼胚愈伤组织诱导和分化的影响。结果表明,禾谷镰刀菌毒素对小麦幼胚胚性愈伤组织的诱导具有低浓度促进和高浓度抑制、毒害作用,这种毒害作用降低了胚性愈伤组织诱导率和胚性愈伤组织质量,显著影响胚性愈伤组织再生频率。同时禾谷镰刀菌毒素胁迫降低了幼胚愈伤组织产量,并随胁迫浓度的升高,愈伤组织产量迅速下降。禾谷镰刀菌毒素对小麦幼胚培养特性的影响,表现出基因型间的差异,对感病基因型的毒害作用远大于抗病品种。

禾谷镰刀菌(Fusarium graminearum)的酯酶电泳分析及其在分类上的意义

对不同地理来源的41个禾谷镰刀菌菌株酯酶同工酶的聚丙烯酰胺凝胶电泳分析,基本明确了代表禾谷镰刀菌种的标志性酯酶带,根据标志性酶带的变化特点,将供试菌株的酯酶同工酶谱划分为4种类型,其中第一类型占总菌株的80.5％,是禾谷镰刀菌的主体型,其它3种类型的比例均在8％以下。结果对应用酯酶同工酶分析禾谷镰刀菌种的鉴定提供了依据,并使利用生化手段于分类迈向实用化阶段。

禾谷镰刀菌(Fusarium graminearum)几丁质酶基因特征分析

禾谷镰刀菌是引发麦类赤霉病的优势种,对禾谷镰刀菌基因组中几丁质酶基因的研究可以帮助理解其致病机理。通过对禾谷镰刀菌基因组数据库的搜索分析,发现了16个ORFs编码的假定几丁质酶,它们都属于糖水解酶18家族。通过与它们的糖水解酶家族比较,对这些几丁质酶进行了系统的命名,按pI值从小到大进行编号为Chi18-1～Chi18-16,研究分析这些几丁质酶蛋白的信号肽、活性位点和亚细胞定位,进行了多序列比对,并建立了系统发生树,为禾谷镰刀菌中的几丁质酶在其致病性和相关生物防治方面的研究奠定了基础。

Transcriptomic responses in resistant and susceptible maize infected with Fusarium graminearum

Gibberella ear rot(GER)caused by Fusarium graminearum(teleomorph Gibberella zeae)is a common maize disease that not only severely reduces grain yield but also contaminates maize grain with mycotoxins.We investigated the molecular mechanism underlying the host defense responses against pathogen infection using comparative transcriptomic analysis.We injected F.graminearum spore suspensions into plants of resistant(IBM-81)and a susceptible(IBM-85)maize inbred line after pollination and performed RNA-seq 48,72,and 96 h after inoculation.Respectively 487 and 410 differentially expressed genes(DEGs)were induced in the resistant and susceptible lines across three time points,indicating that a stronger defense response was activated in the resistant than in the susceptible line.Among them,198 genes commonly induced in the two lines were subjected to pathway analysis,revealing that most of the DEGs were closely associated with defense and a wide range of metabolic activities.DEGs associated with pathogenesis-related protein 1(PR1)and regulation of salicylic acid were significantly enriched during F.graminearum infection,suggesting that these DEGs play dominant roles in maize resistance to GER.Our results provide a resource for future gene discovery and facilitate elucidation of the complex defense mechanisms involved in resistance to GER.

FgHAT2 is involved in regulating vegetative growth, conidiation, DNA damage repair, DON production and virulence in Fusarium graminearum

Histone lysine acetylation is catalyzed by acetyltransferases(HATs), which is important in regulating gene expression and physiological function in eukaryotic cells. HATs can be classified into two main types: A-and B-type HATs. Recently, in Fusarium graminearum, it has been reported that A-type HATs are involved in hyphal development, conidiation, sexual reproduction and virulence. However, the biological roles of B-type HATs are unknown. Here we report the identification and characterization of two B-type HATs(FgHat1 and FgHat2) in F. graminearum. Targeted deletion of FgHAT1 did not result in any detectable phenotypes. However, ΔFghat2 mutants were severely defective in vegetative growth, conidia production and morphogenesis, deoxynivalenol(DON) biosynthesis and virulence. Interestingly, deletion of Fg HAT2 resulted in significantly increased sensitivity to the DNA-damaging agent methyl methanesulfonate(MMS). Furthermore, double deletion mutants(ΔFghat1ΔFghat2) displayed similar phenotypes to the ΔFghat2 mutants. Taken together, we conclude that FgHat2 but not FgHat1 plays essential roles in regulating morphogenesis, DNA damage repair, DON production and virulence in F. graminearum.

Sensitivity of Fusarium graminearum to Carbendazim and Prochloraz

[ Objective] The paper was to understand the resistance of Fusarium graminearum to carbendazim and prochloraz in the main wheat producing areas in Hubei Province. [ Method] Totally 350 F. graminearum strains isolated in 2014 were determined to clear their sensitivity to carbendazim and prochloraz. [ Result] ALl isolates were sensitive to carbendazim and procidoraz in Hubei Province, and carbendazim was still valuable in controlling Fusarium head blight. The control effect of prochloraz was better than that of carbendazim, and the combination of prochloraz and carbendazim at the proportion of 1：7 received the best control effect. [ Conclusion ] Combination of chemical agents will occupy a more and more important position in future prevention and control against Fusarium head blight.

The transcription factor FgNsf1 regulates fungal development,virulence and stress responses in Fusarium graminearum

Nutrient and stress factor 1(Nsf1),a transcription factor containing the classical Cys2-His2(C_(2)H_(2))zinc finger motif,is expressed under non-fermentable carbon conditions and in response to salt stress in Saccharomyces cerevisiae.However,the role of Nsf1 in filamentous fungi is not well understood.In this study,the orthologue of Nsf1 was investigated in Fusarium graminearum(named FgNsf1),a causal agent of Fusarium head blight(FHB).The functions of FgNsf1 were evaluated by constructing a FgNSF1 deletion mutant,designated asΔFgNsf1,and its functional complementation mutantΔFgNsf1-C.Gene deletion experiments showed that the mycelial growth rate,asexual and sexual reproduction ofΔFgNsf1 were significantly reduced,but the pigment production ofΔFgNsf1 was remarkably increased compared with the PH-1 andΔFgNsf1-C.In addition,the tolerance ofΔFgNsf1 to osmotic pressures,cell wall-damaging agents and oxidative stress increased significantly.Sensitivity tests to different fungicides revealed thatΔFgNsf1 exhibited increased sensitivity to carbendazim(MBC)and tebuconazole,and enhanced tolerance to fludioxonil and iprodione than PH-1 andΔFgNsf1-C.The virulence ofΔFgNsf1 to wheat coleoptiles and flowering wheat heads were dramatically decreased,which was consistent with the decrease in the yield of deoxynivalenol(DON).All of these defects were restored by target gene complementation.These results indicated that FgNsf1 plays a crucial role in vegetative growth,asexual and sexual reproduction,stress responses,fungicide sensitivity,and full virulence in F.graminearum.

小麦赤霉病菌(Fusarium graminearum)与拮抗细菌拮抗作用初探

对自广东、福建、云南、黑龙江、江苏和上海6个省市材料中筛选到的14个细菌菌株与小麦赤霉病菌(F.graminearum)的拮抗作用进行了初步研究,结果表明菌株可分为3种类型:1型,细菌对赤霉病菌分生孢子几乎没有抑制作用(分生孢子萌发率仍在90％以上)芽管正常;2型,分生孢子萌发率高于90％,但芽管顶端膨大成球形;3型,分生孢子萌发率低于5％或不萌发。平板拮抗试验表明:1型、2型的拮抗圈较小。边缘不清;3型的拮抗圈大,边缘清晰而光滑。细菌间拮抗试验表明,拮抗菌间存在着相互拮抗现象,且只发生于不同拮抗类型的拮抗细菌间。另外,每一种拮抗细菌拮抗谱宽窄和受到其它细菌拮抗的数量也有很大不同,这为拮抗菌的选择和混配提供了理论依据。

Cloning and characterization of a novel barley gene,HvORG4,induced by Fusarium graminearum infection

Barley Fusarium head blight(FHB),caused by species of the Fusarium fungus,is a devastating disease that is reemerging worldwide in recent years.In this study,a novel gene,HvORG4,was cloned from barley by using cDNA library and suppression subtractive hybridization(SSH) library strategies.The SSH library and cDNA library were constructed from the Chinese barley cultivar Jing02-461(resistance to FHB) infected by Fusarium graminearum isolate Huanggang-1.For the SSH analysis,more than 120 differentially expressed cDNAs were identified and sequenced.One of them showed high homology to the AtORG4 gene and was used as a probe to screen the cDNA library of Jing02-461.Six positive clones were identified and one of them contained a full-length cDNA,which was named HvORG4.Sequence analysis showed that HvORG4 encoded a deduced basic protein of 197 amino acids.Northern blotting analysis showed that HvORG4 was constitutively expressed in root and stalk,not in leaf or spike,and strongly induced in barley spikelets in response to infection with F.graminearum isolate Huanggang-1.Its homology and expression profile suggest that the HvORG4 might function as a transcription factor,playing an important role in signal transduction pathway for defense against FHB in barley.

Fusarium graminearum sensu stricto和F.asiaticum3种分子鉴定方法的比较

Fusarium graminearum sensu stricto和F.asiaticum是中国主要的禾谷镰刀菌复合种(FGSC)成员,为了提高这2个种的鉴定效率,选取48个引起小麦赤霉病和玉米茎腐病的FGSC菌株,比较了系统发育谱系分析、PCR-RFLP(聚合酶链式反应-限制性片段长度多态性)和多重PCR 3种分子鉴定方法。系统发育谱系分析首先扩增菌株的EF-1α和Tri101两个基因的部分序列,测序之后利用MEGA 6.06软件构建系统发育树,结果表明,有11个菌株与F.asiaticum聚在一起,37个菌株与F.graminearum sensu stricto聚为一枝,此法繁琐、复杂、成本高,但鉴定结果准确。PCR-RFLP基于组蛋白H3基因特殊的酶切位点,设计引物H3d StyⅠ/H3R1,通过PCR和酶切反应进行鉴定,电泳检测发现,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,该法工作量大且对操作过程要求高。多重PCR基于CYP51A基因设计特异引物Fa F/FaR和Fg F/FgR进行鉴定,电泳检测结果表明,有11个菌株为F.asiaticum,37个菌株为F.graminearum sensu stricto,此法操作简单、效率高。以上3种分子鉴定方法结果一致,但多重PCR方法具有快速、准确、高效的特点。

Soil application of Trichoderma asperellum GDFS1009 granules promotes growth and resistance to Fusarium graminearum in maize

Of diseases affecting maize(Zea mays), Fusarium graminearum is one of the most common pathogenic fungi that cause stalk rot. In the present study, the Trichoderma asperellum GDFS1009 strain was shown to be an effective biocontrol agent against stalk rot. In a confrontation culture test, Trichoderma strain displayed an approximately 60% inhibition rate on the mycelial growth of F. graminearum. In pot trials, the application of 2 g/pot of T. asperellum GDFS1009 granules had the best control effect on stalk rot at the seedling stage(up to 53.7%), while the average plant height and fresh weight were also significantly improved. Additionally when fertilizer was added at 8 g/pot, the application of 3 g/pot of Trichoderma granules had the best control effect on maize stalk rot(40.95%). In field trials, when inoculating F. graminearum alone, the disease index for inoculating was 62.45, but only 31.43 after treatment with T. asperellum GDFS1009 granules, suggesting a control efficiency of 49.67%. Furthermore, in a naturally F. graminearum-infected field, Trichoderma granules, when applied for 3 consecutive years, showed significant control of stalk rot and increased yields.

Inhibition Effect and Synergistic Effect of Prochloraz and Tebuconazole and Their Mixture on Fusarium graminearum

Wheat scab caused by Fusarium graminearum is increasingly severe in the middle and lower reaches of the Yangtze River region. With long-term use of single chemical pesticide,the drug resistance of F. graminearum is growing day by day. In order to reveal the inhibition effect and synergistic effect of prochloraz and tebuconazole and their mixture on F. graminearum,new compound agents for controlling wheat scab were developed. The inhibition effect and synergistic effect of prochloraz and tebuconazole mixed at seven different ratios on F. graminearum were studied. The results showed that the efficiency of the mixture was the best when the proportion of prochloraz and tebuconazole was 1:7; the EC50 was 0.145 36 and the synergistic coefficient was 1.853 9. The results provide a theoretical basis for development of new compound agents and effective control of wheat scab.

落葵薯内生真菌Fusarium graminearum TSQ-2化学成分研究

目的研究落葵薯内生真菌禾谷镰刀菌(Fusarium graminearum)TSQ-2的化学成分。方法利用大孔树脂、硅胶、Sephadex LH-20、Prep-HPLC等色谱方法对禾谷镰刀菌固体发酵物乙酸乙酯部位化学成分进行分离纯化,根据理化性质及现代波谱技术对单体化合物进行结构鉴定。初步评价化合物1~5对两种人类肿瘤细胞(SMMC-7721、HepG2)的体外细胞增殖抑制活性和对两种致病菌(MRSA、E.coil)的抗菌活性。结果从禾谷镰刀菌固体发酵物乙酸乙酯部位分离得到5个化合物,分别鉴定为zearalenone(1)、clavatustide C(2)、W493 C(3)、4,4,24-trimethyl cholesta-8,14,24(28)-trien-2α,3β,11α,12β,tetrol 12-acetate(4)和glycerolmonopalmitate(5)。活性实验结果显示化合物4对SMMC-7721、HepG2细胞增殖具有抑制活性(IC50值分别为23.1和24.2μmol·^(L-1))。结论共分离得到5个化合物,其中化合物2、5为首次从该属真菌中分离得到。

玉米茎腐病病原菌Fusarium graminearum拮抗真菌的筛选与鉴定

针对吉林省玉米茎腐病的主要优势致病菌禾谷镰孢菌进行拮抗菌株的筛选和鉴定,为玉米茎腐病生防菌剂的研制奠定基础。从300份土壤中分离出685个菌株,选取生长势较好的212个菌株,分别与禾谷镰孢菌进行平板对峙试验,筛选出拮抗效果较好的拮抗菌株5株。将这5株菌进行抑菌试验,其中M-43号菌株抑菌率最高,为75.71%,选取M-43号真菌菌株进行后续研究。基于形态学显微观察和ITS序列分析,鉴定M-43号菌株为Talaromyces pinophilus,该种菌为吉林省新纪录种。

Herbicide 2,4-dichlorophenoxyacetic acid interferes with MAP kinase signaling in Fusarium graminearum and is inhibitory to fungal growth and pathogenesis

Plant hormones are important for regulating growth,development,and plant-pathogen interactions.Some of them are inhibitory to growth of fungal pathogens but the underlying mechanism is not clear.In this study,we found that hyphal growth of Fusarium graminearum was significantly reduced by high concentrations of IAA and its metabolically stable analogue 2,4-dichlorophenoxyacetic acid(2,4-D).Besides inhibitory effects on growth rate,treatments with 2,4-D also caused significant reduction in conidiation,conidium germination,and germ tube growth.Treatments with 2,4-D had no obvious effect on sexual reproduction but significantly reduced TRI gene expression,toxisome formation,and DON production.More importantly,treatments with 2,4-D were inhibitory to infection structure formation and pathogenesis at concentrations higher than 100μM.The presence of 1000μM 2,4-D almost completely inhibited plant infection and invasive growth.In F.graminearum,2,4-D induced ROS accumulation and FgHog1 activation but reduced the phosphorylation level of Gpmk1 MAP kinase.Metabolomics analysis showed that the accumulation of a number of metabolites such as glycerol and arabitol was increased by 2,4-D treatment in the wild type but not in the Fghog1 mutant.Transformants expressing the dominant active FgPBS2^(S451D T455D) allele were less sensitive to 2,4-D and had elevated levels of intracellular glycerol and arabitol induced by 2,4-D in PH-1.Taken together,our results showed that treatments with 2,4-D interfere with two important MAP kinase pathways and are inhibitory to hyphal growth,DON biosynthesis,and plant infection in F.graminearum.