向日葵黄萎病病原菌轮枝菌的多重DPO-PCR检测方法

为建立可同时快速检测引起向日葵黄萎病害的2种检疫性病原菌大丽轮枝菌Verticillium dahliae Kleb.和黑白轮枝菌V.albo-atrum Reinke et Berthold的方法,根据2种病原菌的β-tubulin基因分别设计特异性DPO引物,建立多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅大丽轮枝菌和黑白轮枝菌可分别扩增出225 bp与151 bp的特异性条带,其它向日葵病害的7种病原菌及阴性对照均无目的条带;反应体系中引物终浓度为0.2μmol/L、退火温度为60℃时,30个扩增循环的检测灵敏度均可达0.05 ng菌丝DNA量;在45~65℃退火温度范围内均可高效扩增靶基因片段,表明该方法退火温度范围宽。所建立的检测方法能够准确、高效地检测引起向日葵黄萎病的大丽轮枝菌和黑白轮枝菌,可用于向日葵种子带菌筛查及田间病害诊断检测。     

Relationship between the virulence of hop isolates of Verticillium albo-atrum and their in vitro secretion of cell-wall degrading enzymes

The in vitro production of extracellular polygalacturonase, pectin lyase, and cellulase by hop isolates of Verticillium albo-atrum was studied. A total of 24 isolates belonging to the two pathotypes commonly described as fluctuating or progressive, according to the symptoms produced in certain susceptible hop cultivars, were grown in liquid shake cultures. The media used contained glucose, pectin or acetone-extracted hop tissue as a carbon source. The production of all three enzymes was significantly correlated with pathotype when the medium contained hop tissue. Pectin lyase production was also correlated with pathotype when pectin was used as the carbon source. The influence of carbon source on enzyme output and possible reasons for some exceptions to the correlations observed are discussed.     

茄子黄萎病的发生及病菌生长影响因子

江苏扬州等地棚室栽培条件下,茄子黄萎病一般始见于现蕾期,5月上、中旬进入座果期开始大量发生,5月中下旬至6月上旬达发病高峰;病田土壤2年未栽作物仍能造成茄植株较高发病率.病叶混入土壤引起茄植株发病率最高,而病根混入土壤未见发病.分离自茄子、辣椒和棉花的黄萎病菌菌株在供试的玉米粉、燕麦、理查、马铃薯等培养基上均生长良好,茄子、辣椒黄萎病菌在理查培养基上出现明显的紫红色,棉花黄萎病菌则未出现;三种病菌在pH4～9的培养基上均能生长;棉叶、茄叶、辣椒叶的汁液能刺激茄黄萎病菌分生孢子的萌发,其中以茄叶汁液最明显;病菌经55℃水浴处理20 min,各菌株均未见生长.     

Accumulation of phytoalexins in susceptible and resistant near-isogenic lines of tomato infected with Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici

The time course of accumulation of two phytoalexins, the terpenoid rishitin and the polyacetylene cis-tetradeca-6-ene-1,3-diyne-5,8-diol, was determined in near-isogenic susceptible and resistant tomato lines inoculated with either Verticillium albo-atrum or Fusarium oxysporum f.sp. lycopersici.Cultivars containing the Ve gene for verticillium wilt resistance accumulated phytoalexins at a rate similar to that in susceptible plants following stem inoculation with V. albo-atrum. Higher amounts of phytoalexins were isolated from susceptible than from resistant plants at 11 days after inoculation. Inoculum concentrations of 10⁵, 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ had no differential effect on phytoalexin accumulation at 3 days after inoculation. Also, no differences were observed between fungal growth in susceptible and resistant cultivars during that period.A cultivar containing the I-1 gene for fusarium wilt resistance contained more rishitin than did susceptible plants at 2 and 3 days after inoculation with 10⁷ conidia of F. oxysporum f.sp. lycopersici ml⁻¹, but at 7 and 11 days after inoculation more rishitin had accumulated in the susceptible plants.No difference was observed between the rate of accumulation of phytoalexin in stem segments from resistant and susceptible plants inoculated by vacuum-infiltration.To estimate the concentration of phytoalexins in the xylem fluid, sap was expressed from vascular tissue and amounts of phytoalexins were determined in the sap and in the expressed tissue. Less than 5% of the phytoalexins present in stem segments was recovered from the sap, indicating that their concentration in the xylem fluid may be relatively low.The role of phytoalexins in resistance to verticillium and fusarium wilt is discussed.     

新疆棉花黄萎病的发生现状及其病原菌的分子鉴定与ISSR分析

为掌握新疆主要植棉区棉花黄萎病的发生现状及其病原菌大丽轮枝菌Verticillium dahliae的落叶型菌系分布以及遗传变异情况,于2015年对26个新疆主要植棉区棉花黄萎病的发生情况进行了随机调查,统计新疆大丽轮枝菌的培养性状,利用大丽轮枝菌落叶型特异引物D1/D2、INTD2F/INTD2R与非落叶型特异性引物ND1/ND2、INTNDF/INTNDR对新疆大丽轮枝菌菌系进行互补鉴定,并对部分菌系的遗传变异进行简单序列重复区间(inter simple sequence repeat,ISSR)分析。结果表明:2015年新疆棉花黄萎病发病田比例为54.0%,其中病情指数在10.0以上的发病田与2013年持平,而病情指数在20.0以上的严重发病田比例为10.8%,比2013年增加3.8个百分点;新疆大丽轮枝菌的培养性状以菌核型为主,比例为70.1%,菌丝型与中间型比例分别为13.4%和16.5%;新疆大丽轮枝菌落叶型菌系比例为53.2%,26株菌株的来源地全部检出落叶型菌系;聚类分析结果显示,当遗传相似系数为0.66时,新疆大丽轮枝菌落叶型与非落叶型菌系聚为2个谱系,菌系地理来源、培养性状与大丽轮枝菌的遗传分化无明显相关性。     

Tolerance to a non-host isolate of Verticillium dahliae in tomato

Tolerance to Verticillium spp. is a condition in which a host plant develops few symptoms despite substantial colonization by the pathogen. In the present paper we have shown that Craigella tomatoes are tolerant to a non-host isolate of V. dahliae, Dvd E6. Symptom expression was used to quantify disease and quantitative PCR to assess the amount of fungus in the stems. The classical incompatible and compatible interactions between Craigella resistant or Craigella susceptible near isolines and V. dahliae, race 1 were used for comparative purposes. Additional experiments using cytological assessment and quantitative PCR showed that in the tolerant interactions one plant defence response, vascular coating, was deployed as effectively as in resistant plants, limiting pathogen distribution. However, a second defence response, which causes the cyclical elimination of fungus from the stem in the classical interactions either does not occur or is substantially delayed in tolerant plants. Thus, the Verticillium population remains stable and substantial throughout the studied time course.     

大丽轮枝菌侵染抗感棉种的组织学过程观察

 棉花黄萎病是一种极难防治的土传性真菌病害,研究病原菌侵染棉花的组织学过程对致病机理解析和抗病资源利用具有重要意义。本研究利用绿色荧光蛋白标记的大丽轮枝菌系统研究了其对抗病棉种海岛棉7124和三裂棉、感病棉种军棉1号和戴维逊棉的侵染过程。结果表明,大丽轮枝菌对抗/感棉种的初始侵染没有明显差异,接菌5 h后,分生孢子均能吸附在感病和抗病棉种的根表面。但侵染过程存在显著差异,侵染感病棉种中病原菌3~5 d到达皮层,5~7 d达到维管束,随后迅速扩展繁殖,侵染14 d后即完成系统侵染,并开始产生黄萎病症状;而病原菌侵染抗病棉种,5~7 d才侵入皮层,7~10 d到达维管束,14 d后仍无法扩展,病原菌的定殖与发展受到限制,无法形成系统侵染,较少形成黄萎病症状。本研究通过绿色荧光蛋白标记大丽轮枝菌对抗/感棉种的侵染过程研究,为大丽轮枝菌致病机理研究和抗性资源利用提供了强有力的理论依据。     

The use of Verticillium dahliae and Diplodia scrobiculata to induce resistance in Pinus halepensis against Diplodia pinea infection

The pathogenic fungi Verticillium alboatrum and Diplodia scrobiculata were assayed for biological control of Diplodia pinea on Aleppo pine (Pinus halepensis) in Catalonia (north-eastern Spain). Young shoots were pre-treated with inoculations of either V. dahliae or D. scrobiculata, by placing colonized agar plugs on wounds made by removing a single needle fascicle. An inoculation with D. pinea was performed 15 days later. Two months after the shoot inoculations, the canker length on the stems was measured and the percentage of shoot dieback calculated. Verticillium dahliae and D. scrobiculata were found to significantly reduce the canker length of D. pinea (P < 0.05) when compared with positive controls. Diplodia pinea was slightly more sensitive to V. dahliae than to D. scrobiculata, but no significant differences (P > 0.05) were observed in the mean canker length between the two treatments. Trees pre-inoculated with V. dahliae resulted in 31.12% shoot dieback, while those pre-inoculated with D. scrobiculata resulted in 32.18% shoot dieback, compared with positive controls (42.85%).     

三重PCR检测茄子青枯病菌、黄萎病菌和白绢病菌

为快速、准确对田间茄子发病植株和土壤中3种土传病害的病原菌青枯病菌Ralstonia solanacearum、黄萎病菌Verticillium dahliae和白绢病菌Sclerotium rolfsii进行检测,筛选这3种病菌的特异性引物,建立这3种病菌的三重PCR检测体系,对其退火温度、引物浓度、10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶进行优化,对优化后体系的特异性和灵敏度进行检测,并利用优化体系对田间采集的病害样品和土壤样品进行检测。结果表明,在优化后的50μL三重PCR检测体系中,RS-1-F/RS-3-R、dllz1/dllz2和SRITSF/SRITSR引物的最佳浓度分别为0.16、0.16和0.28μmol/L,10×PCR Buffer(Mg2+plus)、dNTP和Taq DNA聚合酶体积分别为6、5和1μL,检测体系的最佳退火温度为54℃。按照优化后的反应条件能分别扩增出青枯病菌、黄萎病菌和白绢病菌3种病菌的特异条带,分别为716、350和500 bp,其他对照病菌无条带。该体系对病菌DNA检测灵敏度达到0.1 ng/μL。该...     

利用木霉菌防治棉花黄萎病

在对3个木霉菌株的剂型、施用方法和复配可能性研究的基础上，进行了温室盆栽和大田防治棉花黄萎病的试验。结果表明：生防菌麦麸制剂和PD制剂之间的防病效果差别不大；生防菌之间复配不能增强防病作用；三种生防菌制剂及其与无病土营养钵育苗相结合的处理对棉花黄萎病防病效果明显，尤以T3-4与营养钵配合使用效果最佳，并且其防治效果都以第一次发病高峰优于第二次发病高峰。     

青枯菌GMI1000效应蛋白RipQ对致病力的作用研究

青枯菌通过Ⅲ型分泌系统向寄主植物细胞分泌100多种效应蛋白,对寄主植物的抗感病性产生影响。青枯菌效应蛋白RipQ启动子区存在典型的HrpB识别序列PIP box (5′-TTCGG-N15-TTCGC-3′),但其功能尚未明确。本研究分析了RipQ在青枯菌4种演化型菌株中的分布情况。以青枯菌GMI1000为出发菌,构建ripQ缺失突变体和过表达菌株,研究效应蛋白RipQ在青枯菌-番茄植物互作中的功能。结果显示,ripQ广泛分布于除演化型IV的不同青枯菌类群中。与野生型菌株相比,ripQ突变体在番茄上的致病力有一定程度的增强,而ripQ过表达菌株的致病力显著降低。突变体和过表达菌株在培养基中的生长与野生型没有区别,但过表达菌株在番茄体内的繁殖能力下降。RipQ过表达菌株侵染番茄后hrpB、hrpG和epsA基因表达量显著下调,且能够诱导番茄叶片H₂O₂的大量累积,过敏性坏死反应标志基因hin1和水杨酸信号通路标志基因PR1a的诱导表达。另外,在番茄上瞬时表达ripQ也可以观察到H₂O₂积累及叶片细胞坏...     

Inhibitory and stimulatory effects of essential oils and individual monoterpenoids on growth and sporulation of four soil-borne fungal isolates of Aspergillus terreus, Fusarium oxysporum, Penicillium expansum, and Verticillium dahliae

The effect of essential oils and individual monoterpenoids on soil-borne fungi, in pure and mixed cultures, in growth media and in the soil environment, was investigated. Essential oils were extracted from lavender (Lavandula stoechas), oregano (Origanum vulgare subsp. hirtum), sage (Salvia fruticosa) and spearmint (Mentha spicata). The monoterpenoids tested were fenchone, carvacrol, 1,8-cineole, carvone, α-pinene and terpinen-4-ol. Their effect was examined on growth and sporulation of Aspergillus terreus, Fusarium oxysporum, Penicillium expansum and Verticillium dahliae isolated from an organic cultivation of tomato. All tested essential oils and individual monoterpenoids inhibited mycelial growth in all fungi and conidial production in most fungi. The strongest inhibitory activity on mycelial growth was exhibited by oregano and spearmint oils and by carvacrol and carvone, respectively their main constituents. The inhibitory activity was clearly fungistatic in A. terreus and F. oxysporum but fungicidal in V. dahliae. On sporulation, clearly stimulatory effects were observed alongside inhibitory ones. Conidial production was always promoted by α-pinene in P. expansum and by sage oil in F. oxysporum. At certain dosages it was promoted by cineole and carvone in F. oxysporum, and by lavender oil in A. terreus and V. dahliae. Experiments with carvone and carvacrol against mixed fungal cultures in a soil environment showed that V. dahliae was the most sensitive and A. terreus the most tolerant of the four fungi. Our results demonstrate strong but divergent effects and selectivity of action of the lower terpenoids on fungal strains that can become serious pests of tomato. Of special importance is the complete inhibition of growth and conidial production of V. dahliae, a pathogen otherwise very resistant to chemical control.     

新疆棉花黄萎病菌的培养特性及致病力分化

新疆是我国最主要的棉花生产基地,产量占全国的85％左右.由大丽轮枝菌引起的黄萎病严重阻碍了新疆棉花产业的可持续发展.因此,本研究针对新疆棉区采集分离的140个菌株进行研究,结果发现不同地域菌株的生长速度和产孢量差异显著,新疆棉区以菌核型、落叶型菌株为主;与此同时,建立了一种苗期棉花黄萎病抗性快速鉴定新方法——育苗块定量...     

江苏省大丽轮枝菌的培养、遗传及致病特性分析

由大丽轮枝菌引起的黄萎病是我国棉花和茄子的主要病害之一,对棉花和茄子的生产造成巨大损失。为了研究大丽轮枝菌的群体遗传变异以及对棉花和茄子的交互致病性,本文对分离自江苏省的63个棉花黄萎菌和10个茄子黄萎菌进行了培养、遗传和致病特性分析。根据菌株在PDA培养基上生长时形成微菌核的多少来划分培养类型,结果菌核型占83.6%,成为主要的培养类型。用PCR技术检测菌株的致病类型、交配型以及是否具有无毒基因Ave1,结果落叶型菌株占86.3%,为优势种群,但是10株茄子黄萎菌100%都是非落叶型菌株;供试的所有江苏菌株交配型都是MAT1-2型,并且都没有无毒基因Ave1。选择江苏省的6个棉花黄萎菌和4个茄子黄萎菌在室内苗期接种棉花和茄子,进行交互致病性测定,结果这10个菌株都可以侵染棉花和茄子,而且来源于不同寄主作物的菌系之间致病力分化明显,表现在不同菌株对同一寄主的致病力不同,同一菌株对不同寄主的致病力也不同。研究结果为深入研究大丽轮枝菌群体遗传结构和制定黄萎病防治措施提供了理论支持。     

大丽轮枝菌效应蛋白VdSRP2的功能分析

为明确效应蛋白VdSRP2在大丽轮枝菌Verticillium dahliae中的生物学功能,从大丽轮枝菌落叶型菌株V592中克隆VdSRP2基因并进行生物信息学分析,利用酵母转化酶分泌系统对其信号肽活性进行测定,采用实时荧光定量PCR(real-time fluorescence quantitative PCR,qPCR)技术分析VdSRP2基因在大丽轮枝菌中的表达模式,并以V592菌株为材料获得VdSRP2基因的敲除突变体和过表达体菌株,通过表型分析和致病性测定确定VdSRP2基因的生物学功能。结果显示,VdSRP2基因编码232个氨基酸,含有5个半胱氨酸残基,N-端信号肽具有分泌活性,为真菌的典型效应蛋白;VdSRP2基因主要在大丽轮枝菌菌丝和微菌核中表达,其中经棉花根系诱导培养24 h时表达量最高;与野生型菌株V592相比,VdSRP2基因敲除导致大丽轮枝菌的产孢量和孢子萌发率显著下降,不能形成微菌核,对棉花的致病力明显减弱;但VdSRP2基因敲除不影响大丽轮枝菌的穿透能力;VdSRP2基因在本氏烟和棉花叶片瞬时表达不会诱导细胞死亡。表明VdSRP2是大丽轮枝菌微菌核形成必需...     

棉秆生物炭对大丽轮枝菌生长及毒素作用的影响

棉花黄萎病难以防治的根源在于大丽轮枝菌Verticillium dahliae Kleb在土壤中形成的微菌核能抵抗不良环境,并在土壤中长期存活,将棉秆加工成生物炭施入棉田,可克服棉秆直接还田的缺点,从源头上防止黄萎病菌进入土壤。为探索棉秆生物炭还田对棉花黄萎病原菌的影响机理,采用液态摇床培养及平皿固态培养法研究生物炭对大丽轮枝菌菌丝生长及微菌核萌发的影响;通过平皿内棉花和甜瓜种子发芽试验研究生物炭对大丽轮枝菌毒素的减毒作用。结果表明:液态培养条件下,棉秆生物炭对大丽轮枝菌菌丝生长抑制作用不明显,在固态培养下对菌丝体生长有一定促进作用;生物炭能减慢微菌核的萌发速率,但对最终萌发率无影响;液态培养中加入棉秆生物碳对大丽轮枝菌毒素有较强的减毒作用。在摇床培养中期,加入2.0 g·L-1生物炭处理与对照棉种在培养12天时的发芽率分别为53.3%与33.3%(P0.05);前期、中期及后期加入0.5 g·L-1生物炭处理的棉花幼苗总长度分别为对照的4.9、2.1倍及3.2倍;加入棉秆生物碳处理甜瓜种子发芽率、胚根长度、胚轴长度及总苗长较对照均有不同程度增加。表明棉杆生物炭对棉花黄萎病菌大丽轮枝菌菌丝生长、微菌核萌发的抑制作用较弱,对大丽轮枝菌毒素危害有较强的减毒作用。     

脂肽类抗生素fengycin对大丽轮枝菌孢子萌发和微菌核形成的影响

 枯草芽胞杆菌NCD-2菌株能够产生脂肽类抗生素fengycin和surfactin。本研究分别比较了NCD-2野生型菌株、丧失fengycin合成的突变子MF和丧失surfactin合成的突变子MS对大丽轮枝菌的抑菌活性。结果表明,NCD-2菌株和突变子MS周围能形成清晰的透明圈,而突变子MF周围透明圈模糊,说明fengycin对大丽轮枝菌具有抑菌活性。不同浓度的脂肽提取物对大丽轮枝菌孢子萌发的抑制试验表明,NCD-2野生型菌株的脂肽提取物能抑制大丽轮枝菌孢子萌发,在0.1 mg·mL^-1浓度下对孢子萌发的抑制率为46.30%,且随着脂肽浓度的提高抑制效果更加明显;同野生型菌株相比,突变子MF脂肽提取物显著降低了对大丽轮枝菌孢子萌发的抑制效果,在0.1 mg·mL^-1浓度下对孢子萌发的抑制率仅为18.48%,而突变子MS的脂肽提取物对大丽轮枝菌孢子萌发抑制效果与野生型菌株没有显著差异。通过测试脂肽提取物对微菌核形成的影响,发现野生型菌株和突变子MS脂肽提取物均能够显著抑制微菌核的形成,而突变子MF几乎丧失了对微菌核形成的抑制作用。RT-qPCR结果证明,野生型菌株和突变子MS脂肽提取物能抑制微菌核形成相关基因的表达,而突变子MF丧失了对微菌核形成相关基因表达的抑制作用。以上结果证明,fengycin在NCD-2菌株抑制大丽轮枝菌孢子萌发和微菌核形成中发挥主要作用。     

脂肽类抗生素fengycin对大丽轮枝菌孢子萌发和微菌核形成的影响

枯草芽胞杆菌NCD-2菌株能够产生脂肽类抗生素fengycin和surfactin。本研究分别比较了NCD-2野生型菌株、丧失fengycin合成的突变子MF和丧失surfactin合成的突变子MS对大丽轮枝菌的抑菌活性。结果表明,NCD-2菌株和突变子MS周围能形成清晰的透明圈,而突变子MF周围透明圈模糊,说明fengycin对大丽轮枝菌具有抑菌活性。不同浓度的脂肽提取物对大丽轮枝菌孢子萌发的抑制试验表明,NCD-2野生型菌株的脂肽提取物能抑制大丽轮枝菌孢子萌发,在0.1 mg·mL^-1浓度下对孢子萌发的抑制率为46.30%,且随着脂肽浓度的提高抑制效果更加明显;同野生型菌株相比,突变子MF脂肽提取物显著降低了对大丽轮枝菌孢子萌发的抑制效果,在0.1 mg·mL^-1浓度下对孢子萌发的抑制率仅为18.48%,而突变子MS的脂肽提取物对大丽轮枝菌孢子萌发抑制效果与野生型菌株没有显著差异。通过测试脂肽提取物对微菌核形成的影响,发现野生型菌株和突变子MS脂肽提取物均能够显著抑制微菌核的形成,而突变子MF几乎丧失了对微菌核形成的抑制作用。RT-qPC...