河南省小麦全蚀病菌的分离及变种类型鉴定

 从河南省10个地市采集的小麦病根样品中分离得到82株病原分离株, 通过形态特征观察、回接致病性测定、以及分子生物学的方法对所分离菌株进行鉴定。其中47个菌株菌丝分支处都形成典型的“∧”状;子囊壳埋生或半埋生, 子囊棍棒状, 子囊孢子线形, 稍弯曲, 无色, 具有5~10个分隔, 大小为68~94 μm×2~4 μm;对82个分离株rDNA-ITS区进行PCR扩增和序列测定, BLAST序列比对结果表明, 47株菌株与小麦全蚀病菌的ITS序列同源性达97%～99%, 据此确定47株菌株为小麦全蚀病菌。应用小麦全蚀病菌4个变种的特异性引物进行PCR扩增都得到禾顶囊壳小麦变种(870 bp)的特异性片段, 鉴定47株菌株均为禾顶囊壳小麦变种 (Gaeumannomyces graminis var.tritici)。     

河南省小麦全蚀病菌的鉴定、进化分析及标志基因筛选

为了解河南省郑州市中牟市小麦全蚀病菌的变种类型及进化情况,筛选全蚀病菌侵染小麦的分子标记,采用形态学观察、科赫氏法则验证、ITS序列分析和系统进化树构建等方法对分离的小麦全蚀病菌进行鉴定,并对其侵染后小麦病原相关蛋白(pathogen-related protein,PR)基因的表达进行实时定量PCR分析。结果表明,显微形态观察初步确定分离的小麦全蚀病菌为禾顶囊壳Gaeumannomyces graminis(Sacc.)v. Arx.Olivier,经科赫氏法则验证、ITS序列比对及系统进化树分析进一步证明该病菌均为禾顶囊壳。本研究分离的小麦全蚀病菌与地域相距较远的英国、美国的小麦全蚀病菌间的同源性比与来自中国陕西省的小麦全蚀病菌的同源性更高。采用禾顶囊壳4个变种的特异性引物进行PCR扩增,所有分离病菌均扩增出了小麦变种的特异性条带,证实分离菌株为禾顶囊壳小麦变种G. graminis var. tritici。实时定量PCR分析发现,病原相关蛋白基因PR4a、PR4b、PR2、PR10受到小麦全蚀病菌侵染后表达量均显著上调,在侵染后第5～6天达到最高峰,表明这些基因可作为小麦全蚀病菌侵染小麦的标志性基因。     

利用插入突变分离小麦全蚀病菌致病性突变体

 利用限制性内切酶介导的整合(restriction enzyme-mediated integration,REMI)技术转化禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici,Ggt),以分离致病性突变体。将含有潮霉素磷酸转移酶基因的质粒pUC ATPH线性化后转化Ggt W-1菌株,从获得的1501个转化体中筛选出1个对小麦品种内乡13完全丧失致病能力的突变体,与野生型出发菌株相比,该突变体生长缓慢。对此突变体进行Southern杂交分析,显示出该突变体是外源质粒单位点串联重复插入的结果。     

水稻全蚀菌生物学研究

水稻全蚀病是辽宁省近年发现的新病害,其病原菌为禾顶囊壳菌水稻变种。病原菌菌落初期白色绒毛状,后期菌落变灰褐色至黑褐色,气生菌丝较多;在5 ～32 ℃范围内均能生长,最适生长温度25～28 ℃;对pH 值适应范围较广,最适pH 为7-5 ;对水稻致病力很强,主要危害水稻。     

禾顶囊壳小麦变种对小麦种子根的侵染过程

 光镜和电镜观察表明,禾顶囊壳小麦变种(Gaeumannomyces graminis var.tritici,小麦全蚀病菌)对小麦种子根的侵染过程可分为侵入前、侵入表皮层、进入皮层和进入中柱等4个连续阶段。麦根接菌后在15℃下培养,48 h后侵入表皮层细胞,60 h后进入皮层,120 h后进入中柱。病原菌主要以侵染菌丝直接侵入表皮层,表皮细胞间隙和根毛基细胞是主要侵入部位,少数由附着枝侵入。菌丝穿透细胞壁有明显的酶解作用特征,菌丝先端前方胞壁上还产生电子密物质。皮层细胞是病原菌定殖和发展的主要场所,病原菌还能离解胞间层,形成胞外空间,特别有利于菌丝和菌丝束的扩展。在侵入位点的寄主细胞壁和质膜之间,形成多种形状的木质管,其数量与侵入菌丝的数目相对应,但木质管不能阻止菌丝进入细胞。菌丝进入中柱后,可阻塞导管和筛管。小麦细胞发生退行性病变,尤以细胞壁膨大崩坏和早期质壁分离最明显,细胞间隙还产生性质不明的黄色物质。     

小麦全蚀病菌拮抗细菌的筛选及生防效果

为筛选小麦全蚀病菌Gaeumannomyces tritici的拮抗细菌,从健康小麦根际土壤中分离和筛选拮抗细菌,采用对峙培养法和室内盆栽试验测定拮抗细菌对小麦全蚀病菌的抑制活性及生防效果,通过形态学特征观察、生理生化特性测定和保守序列分析确定其分类地位。结果表明,从小麦根际土壤中分离并筛选获得2株具有较好拮抗效果的菌株XJ-3和XJ-4,对小麦全蚀病菌菌丝生长的抑制率分别为64.31%和65.25%。基于菌株XJ-3和XJ-4的形态学特征、生理生化特性和gyrB基因系统进化分析结果,将其均鉴定为贝莱斯芽胞杆菌Bacillus velezensis。菌株XJ-3和XJ-4的发酵液中均含有一类具抗真菌活性、耐热性的非蛋白类次生代谢产物;经菌株XJ-3和XJ-4发酵液处理小麦幼苗后,株高较对照分别显著增加了6.48%和13.12%,干重较对照分别增加了1.30%和14.29%,其中菌株XJ-4的促生效果相对较好;对小麦全蚀病的盆栽防效分别达到51.29%和52.46%。表明筛选得到的贝莱斯芽胞杆菌XJ-3和XJ-4可用于小麦全蚀病的生物防治,具有开发为生防制剂的潜力。     

我国北方玉米全蚀病菌变种类型研究

 988~1992年先后于我国北方10省(自治区)22市、县采集到玉米全蚀病罹病根茬,经分离培养获得21个致病力较强的菌株。根据各菌株的培养性状、形态特征、生理特性及致病性等可分为两类:一类M₁、M₂、M₃a、M₃b、M₃e、M₃f、M₃g、M₄a、M₄c、M₄d、M₅a、M₅b、M₆、M₇a、M₇b、M₈a、M₈b、M₉18个菌株属禾顶囊壳菌玉米变种[Gaeumannomyces graminis(Sacc.) Arx et Olivier vat.maydis Yao,wang et Zhu];另一类M₃c、M₃d、M₄b菌株属禾顶囊壳菌水稻变种[Gaeumannomyces graminis(Sacc.) Arx.et Olivier var.graminis Trans.]在玉米上的一个生理小种。变种内在培养性状、致病力、瓶梗孢子产生数量等方面存在着生理型差别和某些生理分化现象。     

荧光93菌体和代谢物对全蚀病菌和小麦生长影响的初步研究

对荧光假单胞菌工程菌剂“荧光93”菌体和代谢物的作用特性进行了比较。结果表明:该菌剂能保护小麦根系免遭全蚀病危害,对植株生长有促进作用。菌体的抑菌能力强于代谢物,浸种处理防效分别为44.8％和27.8％;代谢物具有更强的刺激植株生长作用,在无病条件下,代谢物灌根处理鲜重增加34.5％,菌体处理鲜重增加7.5％。培养基成份对代谢物的作用有一定影响:以马铃薯葡萄糖培养产生的代谢物,抑菌效果好;营养肉汁酵母粉培养产生的代谢物,促生作用好。据此推断,荧光93菌剂代谢产物中含有抑制全蚀病菌和刺激植株生长的物质。     

小麦全蚀病的发生、诊断和防治

1 小麦全蚀病的分布和发生情况 小麦全蚀病是世界各地普遍发生的重要病害,已有46个国家和地区发生为害.我国自1931年前后在浙江发现此病,目前已蔓延至我国绝大部分省(自治区,直辖市);包括山东、山西、内蒙、宁夏、甘肃、青海、陕西、黑龙江、新疆、西藏、云南、贵州、四川、浙江、江苏、河北、河南、安徽、福建、辽宁、上海、湖北等22个省(自治区、直辖市).     

Effects of different cultivated or weed grasses, grown as pure stands or in combination with wheat, on take-all and its suppression in subsequent wheat crops

Grass species were grown in plots, as pure stands or mixed with wheat, after a sequence of wheat crops in which take-all ( Gaeumannomyces graminis var. tritici ) had developed. Annual brome grasses maintained take-all inoculum in the soil as well as wheat (grown as a continuous sequence), and much better than cultivated species with a perennial habit. Take-all developed more in wheat grown after Anisantha sterilis (barren brome) or Bromus secalinus (rye brome), with or without wheat, than in continuous grass-free wheat in the same year, where take-all decline was apparently occurring. It was equally or more severe, however, in wheat grown after Lolium perenne (rye-grass) or Festuca arundinacea (tall fescue), despite these species having left the least inoculum in the soil. It was most severe in plots where these two grasses had been grown as mixtures with wheat. It is postulated that the presence of these grasses inhibited the development of take-all-suppressive microbiota that had developed in the grass-free wheat crops. The effects of the grasses appeared to be temporary, as amounts of take-all in a second subsequent winter wheat test crop were similar after all treatments. These results have important implications for take-all risk in wheat and, perhaps, other cereal crops grown after grass weed-infested cereals or after set-aside or similar 1-year covers containing weeds or sown grasses, especially in combination with cereal volunteers. They also indicate that grasses might be used experimentally in wheat crop sequences for investigating the mechanisms of suppression of, and conduciveness to, take-all.     

利用小麦内生细菌防治小麦全蚀病的初步研究

植物内生细菌是指能定殖在健康植物组织内,并与植物建立了和谐关系的一类细菌。内生细菌对植物的益生作用主要表现为促进植物生长、抑制植物病原物、增加植物的抗逆性和他感作用等几个方面。小麦全蚀病（wheat take-all）作为一种世界毁灭性病害,目前,由于缺乏抗病品种和有效的化学防治药剂,所以利用微生物之间的拮抗作用来控制小麦全蚀病危害具有广阔的应用前景。本研究通过从小麦里分离出内生细菌,从中筛选出对小麦全蚀病菌具有拮抗作用的菌株,在研究其拮抗机制和定殖作用基础上,对其防治小麦全蚀病的作用进行了初步研究。     

Size, shape and intensity of aggregation of take-all disease during natural epidemics in second wheat crops

Point pattern analysis (fitting of the beta-binomial distribution and binary form of power law) was used to describe the spatial pattern of natural take-all epidemics (caused by Gaeumannomyces graminis var. tritici ) on a second consecutive crop of winter wheat in plots under different cropping practices that could have an impact on the quantity and spatial distribution of primary inoculum, and on the spread of the disease. The spatial pattern of take-all was aggregated in 48% of the datasets when disease incidence was assessed at the plant level and in 83% when it was assessed at the root level. Clusters of diseased roots were in general less than 1 m in diameter for crown roots and 1–1·5 m for seminal roots; when present, clusters of diseased plants were 2–2·5 m in diameter. Anisotropy of the spatial pattern was detected and could be linked to soil cultivation. Clusters did not increase in size over the cropping season, but increased spatial heterogeneity of the disease level was observed, corresponding to local disease amplification within clusters. The relative influences of autonomous spread and inoculum dispersal on the size and shape of clusters are discussed.     

小麦全蚀病生防细菌的筛选和鉴定

为获得对小麦全蚀病菌有良好拮抗效果的生防菌株,分别从河南省商丘市及驻马店市小麦全蚀病发生田块中采集小麦根际土样,采用稀释平板涂布法共分离到1051株细菌,通过与全蚀病菌G1037菌株进行平板对峙筛选,最终获得9株具有明显拮抗效果且生长状况良好的菌株。16S rDNA序列比对及生理生化性状分析结果表明：菌株P155、P154、P16及P147为荧光假单胞菌Pseudomonas fluorescens,菌株P188和P97为恶臭假单胞菌Pseudomonas putida,菌株LY3为产酶溶杆菌Lysobacter enzymogenes,菌株S38为嗜根寡养单胞菌Stenotrophomonas rhizophila,菌株B20为洋葱伯克霍尔德Burkholderia cepacia。产抗生素相关基因的检测结果发现,菌株P147含吩嗪和硝吡咯菌素合成基因,菌株B20含硝吡咯菌素合成基因。除B20、LY3、S38和P147外,其余菌株均可产生嗜铁素。9株细菌都产蛋白酶。除P97、B20和S38外,其余菌株均可产脂肽类物质。盆栽试验结果表明,9株生防菌对小麦全蚀病都具有较好的防治效果,菌株P155和P154的防治效果最好,相对防效分别为67.11%和63.82%,略高于3%的苯醚甲环唑种衣剂的防治效果。研究结果表明这些细菌具有作为小麦全蚀病生防菌的潜力。     

小麦全蚀病菌不同致病力菌株的致病特点

利用小麦种子根接种小麦全蚀病菌Gaeumannomyces graminis var．tritici，研究了不同致病力菌株的致病特点。结果表明，弱致病菌株可侵染小麦，但罹病过程缓慢，接种第5天仅在皮层观察到少量菌丝体，13天有少量菌丝进入中柱，中柱组织在菌丝侵入前褐变，未出现导管堵塞现象，也不能导致典型的黑根症状。强致病菌株接种第2天可侵入皮层，8天即进入中柱，并在寄主组织内产生大量菌丝体，致使寄主皮层组织和中柱细胞大量褐变和坏死，以及导管堵塞。     

小麦全蚀病菌重组酶聚合酶检测方法的建立及应用

由禾顶囊壳小麦变种Gaeumannomyces graminis var.tritici引起的小麦全蚀病是危害极大的小麦土传真菌病害。本研究以小麦全蚀病菌β-tubulin为靶标基因设计重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)引物Gg-RPA-F/Gg-RPA-R和RPA探针Gg-LF-Probe,结合侧流层析试纸条,建立了小麦全蚀病菌RPA快速检测方法,该方法在38℃恒温条件下30 min内完成可视化检测,摆脱PCR仪等仪器设备的限制。小麦全蚀病菌RPA快速检测方法具有较高的检测特异性,其检测极限为10 pg/μL,与普通PCR一致。此外,小麦全蚀病菌RPA快速检测方法可从土壤中快速检测到小麦全蚀病菌,具有较强的实用性。因此,本研究建立的小麦全蚀病菌RPA快速检测方法具备简便高效、实用性强的特点,为小麦全蚀病菌的快速检测和病害早期诊断提供新技术。     

Sensitivity to silthiofam, tebuconazole and difenoconazole of Gaeumannomyces graminis var. tritici isolates from China

BACKGROUND: Wheat take‐all caused by Gaeumannomyces graminis var. tritici (Ggt) has become an emerging threat to wheat production in the last few years. Silthiofam is very effective against Ggt, and recently it has been widely used for the control of take‐all in China. However, farmers have noted a decline in control efficacy with this compound in some wheat fields, suggesting that the pathogen may have developed resistance to silthiofam. RESULTS: Of the 66 Ggt isolates collected from different locations in China, 27 were resistant to silthiofam. There was no cross‐resistance between silthiofam and tecuconazole or difenoconazole. The effectiveness of silthiofam in controlling take‐all was compromised on wheat inoculated with silthiofam‐resistant isolates. Based on the DNA fingerprinting generated by microsatellite PCR, two predominant genetic clusters were found among these isolates and were clearly associated with the sensitivity to silthiofam. CONCLUSION: Silthiofam has a high risk in the development of resistance in Ggt. Tebuconazole and difenoconazole show great potential for control of take‐all on wheat. Results from this study provide useful information for take‐all control and the management of fungicide resistance. Copyright © 2012 Society of Chemical Industry     

新型吡唑-3-甲酸酯类化合物HNNK-1对小麦全蚀病菌的毒力及防治效果

为了研究新型吡唑-3-甲酸酯类化合物HNNK-1对小麦全蚀病的防治效果,采用菌丝生长速率法测定了其对小麦全蚀病菌的毒力;并通过拌种处理在温室和田间测定了其对小麦全蚀病的防治效果;同时观察了HNNK-1拌种对小麦生长的影响。菌丝生长速率法测定结果表明,HNNK-1对小麦全蚀病菌的EC₅₀为0.28 mg/L,低于硅噻菌胺的EC₅₀(0.58 mg/L);盆栽试验结果表明,用50 g/L HNNK-1 SC拌种(药种质量比1∶3 000)后对小麦全蚀病的防效为60.10%;对小麦株高、根长等生长指标无显著影响。田间药效试验表明,用50 g/L HNNK-1 SC拌种(药种质量比1∶3 000)对小麦全蚀病的防效为73.13%(开封)和71.15%(周口)。HNNK-1可以有效控制田间小麦全蚀病发生和危害。     

小麦白粉病菌分生孢子田间传播的初步研究

2015年度采用定容式孢子捕捉器对田间空气中小麦白粉病菌分生孢子传播的初步研究表明,在病害发生初期病菌分生孢子捕捉量比较低,但随着菌源中心病害的逐渐加重,病菌分生孢子在距菌源中心20 m和40 m远处捕捉量随之增大。线性弧度相关分析结果发现,距菌源中心北向20 m和40 m处的孢子捕捉量与风向存在显著正相关性;距菌源中心不同距离处的孢子捕捉量之间均存在极显著正相关性,且在相同方向上,距菌源中心20 m处孢子捕捉量显著高于40 m处孢子捕捉量。