Identification of Pathogenicity Defective Mutants from a T-DNA Insertion Mutant Library of Verticillium dahliae

[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes. [Method] In total, 294 T-DNA insertion mutants of V. dahliae were tested for their virulence using a cotton infection assay. Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant. DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR. [Result] Based on the virulence assay, the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991. The Southern blot assay revealed that only one mutant contained two T-DNA insertions, while the remaining eight mutants harbored a single T-DNA insert. An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991. The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17. Furthermore, the pathogenicity-related genes were cloned from strain Vd991. [Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V. dahliae on a genome-wide scale. This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V. dahliae.     

Effect of Pathogen Synergistic Pathogenicity on Severity of Verticillium Wilt of Cotton

Verticillium dahliae is a soil-borne and vascular pathogen causing Verticillium wilt of cotton, which is one of the most severe diseases affecting cotton production in China. Recently, a soil-inhabiting opportunistic pathogen was isolated and identified as Scopulariopsis gossypii; it could infect the host through wounds and increase disease severity in combination with V. dahliae. S. gossypii was able to form conidia in a nutritionally poor substrate and had similar biological characteristics to other vascular pathogens, while ultrastructure observation also confirmed that it was present in vascular tissue of cotton plants and produced conidia. Nematode activity in soil could cause wounds on the surface of cotton roots resulting in infection by the pathogens. On the basis of our analysis of the biological characteristics and infection process of S. gossypii and its interactions with other pathogens in cotton plants, we propose establishing a research program for multipathogen-host interactions in the cotton rhizosphere, which will enable detailed investigation of the synergistic pathogenesis of S. gossypii and V. dahliae.     

两种杀菌剂对棉花黄萎病的防治效果比较

棉花黄萎病对棉花产量造成极大影响,目前仍无很好的解决办法。因此,需要筛选最佳化学药剂,为棉花黄萎病的防治提供科学依据。本文通过室内抑菌试验、田间药剂防治和产量测定研究两种低毒杀菌剂80%代森锰锌可湿性粉剂和0.3%四霉素水剂对棉花黄萎病的防治效果。室内毒力结果测定显示,0.3%四霉素水剂浓度在0.6g/L时,抑菌效果最好,抑菌率可达98.44%;田间喷施药剂显示,在推荐剂量下,0.3%四霉素水剂防效为39.8%,显著高于80%代森锰锌可湿性粉剂,试验结果与室内毒力测定一致;0.3%四霉素水剂施药区棉花产量比对照区高30.28%,比示范区低10.78%,80%代森锰锌可湿性粉剂仅比对照区高4.06%。四霉素能有效减缓棉花黄萎病蔓延,减少病害对棉花产量造成的损失,在化学防治棉花黄萎病时,可推荐使用0.3%四霉素水剂为防治药剂。     

不同培养基继代培养蜡蚧轮枝菌对产孢量和烟粉虱毒力的影响

为了探究不同培养基继代培养的蜡蚧轮枝菌对烟粉虱的作用效果,通过继代培养方法测定JMC-01、JMC-02菌株的产孢量、对烟粉虱的毒力及其几丁质酶活性。结果表明,JMC-01、JMC-02菌株在添加蝉蜕和烟粉虱的PDA培养基中连续培养5代的产孢量、对烟粉虱的毒力、几丁质酶活性均高于无添加的PDA培养基,且JMC-01菌株的各项参数均高于JMC-02菌株。JMC-01菌株在添加蝉蜕的PDA培养基中T_1和T_3代的产孢量最大,均为1.279×10~7个/mL,T_3代对烟粉虱的毒力最大,为88.33%,T_5代的几丁质酶活最高,为0.268 3 U/mL;在添加烟粉虱的PDA培养基中,JMC-01菌株的最大产孢量、对烟粉虱的最高毒力和最高几丁质酶活分别出现在S_2、S_4和S_5代,分别为1.270×10~7个/mL、88.33%和0.325 4 U/mL。回归分析结果显示,JMC-01、JMC-02菌株几丁质酶活性与烟粉虱校正死亡率呈显著的正相关,且拟合优度较好。本试验表明JMC-01、JMC-02菌株均对烟粉虱具有较强的毒力,添加蝉蜕和烟粉虱的培养基可以维持菌株生长特性、增加产孢量、延缓菌株退化、增强菌株的几丁质酶活性,为烟粉虱的生物防治提供了新的菌种资源。     

西兰花植株残体还田对棉花黄萎病的防治效果及其安全性评价

本研究探讨了温室和田间试验条件下西兰花植株残体还田对棉花黄萎病的防治效果以及西兰花残体对棉花生长的安全性，以期为深入研究西兰花植株残体对棉花黄萎病的生态防治机制提供依据。将粉碎的西兰花植株残体与培养基质分别按1%、3%、5%、7%和14%处理混匀，在（28±2）℃条件下分别腐解7和30 d后种植棉花，测定其对棉花出苗率、株高的影响以及对黄萎病的防治效果。研究结果表明，当腐解时间为7 d，西兰花残体处理比例为1%和3%时，棉花出苗率与不添加西兰花残体的空白对照处理不存在显著差异，但对株高具有显著促生长作用，增幅分别为19.46%、42.41%和36.96%；西兰花残体添加比例为5%、7%和14%时，对出苗具有不同程度的抑制作用。当腐解时间为30 d时，上述各比例西兰花残体处理后的棉花出苗率和株高与空白对照之间差异不显著。西兰花残体处理显著降低了棉花黄萎病的发病率和病情指数，其21 d后的防治效果达到72.55%。田间试验表明，西兰花残体在棉花全生育期内对黄萎病的防治效果为43.06%。综合分析表明，西兰花残体的施入，促进了棉花生长，降低了黄萎病的发病率和病情指数，且在发病初期的防治效果优于后期。研究结果为深入研究西兰花残体防治棉花黄萎病的生态机制提供理论依据和技术指导，最终为棉花黄萎病的绿色防控和化学农药的减量提供了新途径和思路。     

历山国家级自然保护区虫生真菌资源及杀虫活性研究

对地处山西东南部的历山国家级自然保护区的虫生真菌种类资源及其杀虫活性作了初步的研究。共分离鉴定真菌3目13属19种，2种为山西特有种，其中的丝孢目真菌为该地区的优势种群。选取其中的多形白僵菌和印度轮枝菌，分别对林业害虫杨二尾舟蛾、杨梢叶甲幼虫进行杀虫活性试验，多形白僵菌的平均杀虫效果高于印度轮枝菌16．4％。     

球孢白僵菌×蜡蚧轮枝孢菌(BV制剂)对松墨天牛幼虫寄生试验初报

用球孢白僵菌(Beauveriabassiana)、蜡蚧轮枝孢菌(Verticilliumlecanii)及2种菌混合物配制成0 5×109～5 0×109孢子/mL,在室内和野外对松墨天牛幼虫进行了防治试验。室内试验结果表明,球孢白僵菌和蜡蚧轮枝孢菌对松墨天牛幼虫均有较强的致病性,2种菌混合使用致病作用倍增。球孢白僵菌对松墨天牛的寄生率为5%～32%,平均20%;蜡蚧轮枝菌的寄生率为2 9%～25%,平均14 9%;2种菌混合物寄生率为12%～76%,平均47 9%。野外试验表明,球孢白僵菌防治效果为50 43%,蜡蚧轮枝孢菌防治效果为38 66%,2菌混合物防治效果为69 69%。     

进境美国苜蓿草中苜蓿黄萎病菌的检疫鉴定

从一批进境的美国苜蓿草样品中分离得到了一株与苜蓿黄萎病菌(Verticillium albo-atrum)相似的分离物M1。该分离物原始菌落为白色,边缘规则呈圆形,菌丝较致密,气生菌丝较少,苜蓿组织块不被菌丝覆盖,菌落生长速度为小于2.5 mm/d;M1在PDA上进行纯培养,前6d内,菌落为白色圆形,容易产生分子孢子轮枝状分生孢子梗,7d后菌落中央表面因产生休眠菌丝开始变成黑褐色至黑色,20d后菌落的表面和背面大部分均变黑色,仍不产生微菌核和厚垣孢子。M1的DNA用V.albo-atrum特异引物Vaa1/Vaa2进行检测,PCR扩增后得到预期330 bp的产物片段,产物序列与V.albo-atrum相应序列的相似性为100%。该分离物接种苜蓿草根部,15d后引起苜蓿黄萎病的典型症状。根据分离物的形态特征、PCR检测结果、PCR产物序列分析,以及致病性测定结果,将进境美国苜蓿草样品中的分离物M1鉴定为苜蓿黄萎病菌。     

厚壁轮枝菌摇瓶液体发酵培养的研究

厚壁轮枝菌对根结线虫虫卵有很强的寄生力,是线虫的生防菌,本文对其最适培养条件进行了研究.每12 h对其产孢情况进行跟踪,探讨了不同C、N源,初始pH值,无机盐对其产孢量的影响,确定了适宜的培养条件及发酵终止时间.结果显示,28 ℃下,在160 r·min-1 摇床上,以500 mL三角瓶200 mL的装量进行液体培养,筛选出的最佳复合培养基配方为:葡萄糖20 g·L-1,玉米粉20 g·L-1, 黄豆粉20 g·L-1,KH2PO4 0.5 g·L-1 ,MgSO4·7 H2O 0.5 g·L-1.最佳pH值范围5.0～7.0,最适发酵终止时间120～144 h.     

Biological control agents (BCAs) of verticillium wilt: influence of application rates and delivery method on plant protection,triggering of host defence mechanisms and rhizosphere populations of BCAs

Verticillium dahliae causes severe yield reductions in a variety of important annual crops worldwide. Control of verticillium wilt has relied on soil fumigation; however, the use of the main soil fumigant, methyl bromide, has been banned in the European Union since 2010, creating a demand for novel crop protectants. As such, the use of biocontrol agents (BCAs) is an appealing management strategy. Prerequisites for the development of a successful BCA are an understanding of the modes of action of the antagonist, its ecological fitness and an efficient and economically feasible delivery system. Therefore, two BCAs (Paenibacillus alvei K165 or the nonpathogenic Fusarium oxysporum F2) and two release strategies (seed coating or amendment of the transplant soil plug) were assessed against verticillium wilt of aubergine (eggplant). Mixing the transplant soil plug with K165 or F2, at a rate of 10 and 20% (v/v), respectively, reduced verticillium wilt symptom development. Furthermore, a positive correlation was revealed between the release strategy and the BCA rhizosphere population. Correlation analysis also showed that disease severity was negatively correlated to the rhizosphere size of the BCA population. In addition, qPCR analysis showed that both BCAs induced the expression of the pathogenesis‐related (PR) proteins PR1 and PR4 in the stem of aubergines before and after inoculation with V. dahliae in a manner that suggests a link with the rhizosphere size of the BCA population.