同步检测棉黄萎病菌、枯萎病菌和炭疽病菌三重PCR方法的建立

棉黄萎病、枯萎病和炭疽病是对棉花生产和产垣威胁最大、危害最重的病害,为此建立了同步检测进境棉籽中携带上述3种病害的三重PCR办法。结果表明:单一PCR检测棉黄萎痫菌、枯萎病菌和炭疽病菌的最低DNA量分别为56.988 pg、2.633 pg和32.325 pg.同步检测3种真菌混合物最低DNA量为1.061 ng。建立的三重PCR方法特异性强、灵敏度高,节省了检测时间和成本,可在我国检验检疫口岸和农林生产部门推广应用。

三株木霉生防菌的生物学特性研究

对三株棉黄萎病菌的木霉生防菌的生物学特性研究表明 :其生长最适温度为 2 5～ 30℃ ;最适pH值为 4～5 ;最适光照条件为半黑暗半光照 ;孢子萌发对营养环境要求不严格 ,但在营养丰富的条件下萌发得更好。

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Antagonistic Endophytic Bacteria Isolated from Different Growth Stages of Cotton for the Control of Cotton Verticillium wilt

[Objective] This study aimed to investigate the combined control effects of endophytic bacteria at different growth stages against cotton Verticfllium wilt and pro- vide a new strategy for the biocontrol of other soil-borne diseases. [Method] Endophytic bacteria with high resistance against Verticillium wilt were isolated from seedling, squaring and boll-setting cotton vascular, respectively. Their 16S rDNA se- quences were detected for comparative analysis. Three biocontrol strains were se- lected and identified, whose colonization roles in cotton plants were explored. The control efficiency was determined with indoor and field experiments. [Result] Accord- ing to the 16S rDNA sequence homology, the three strains were identified as Paeni- bacillus polyrnyxa YUPP-8, Paenibacillus xylanilyticus YUPP-1 and Bacillus subtilis YUPP-2, respectively. Results of colonization assessment showed that three strains all could be successfully colonized in cotton vascular. However, application amount had a positive effect on the number of colonized biocontrol bacteria in cotton, strain YUPP-8 had the largest number of colonized biocontrol bacteria in seedling period, strain YUPP-1 had the largest number of colonized biocontrol bacteria in squaring period, and strain YUPP-2 had the largest number of colonized biocontrol bacteria in boll-setting period. Indoor pot experiment showed that cotton plants in combined bio- control bacteria treatment group were not infected in flowing period, while Verticillium wilt morbidity rate of cotton treated with single strain in seedling period were 6.7% （YUPP-8）, 6.7% （YUPP-1） and 13.3% （YUPP-2）; however, Verticillium wilt morbidity rate wilt of the control reached 80%. Field experiment conducted during 2010-2011 showed that the combined application of three strains had better effect than separate application; specifically, Verticillium wilt morbidity rate and disease index of cotton in boll-setting period with combined application of three strains in 2010 were 9.4% and 6.5, respectively, while those in control group were 47.5% and 32.8; results in 2011 were similar to 2010, with higher disease severity. These results indicate that com- bined application of endophytic bacteria at different growth stages has great applica- tion potential in control of cotton Verticillium wilt. [Conclusion] This study preliminarily overcomes the defects in the application of biocontrol bacteria and provided reference for the prevention and treatment of other soil-borne diseases.

Expression of pathogenesis-related genes in cotton roots in response to Verticillium dahliae PAMP molecules

Verticillium wilt disease becomes a major threat to many economically important crops. It is unclear whether and how plant immunity takes place during cotton-Verticillium interaction due to the lack of marker genes. Taking advantage of cotton （Gossypium hirsutum） genome, we discovered pathogenesis-related （PR） gene families, which have been widely used as markers of immune responses in plants. To profile the expression of G. hirsutum PR genes in the process of plant immunity, we treated cotton roots with two immunogenic peptides, fig22 and nlp20 known as pathogen-associated molecular patterns, as well as three Verticillium dahliae-derived peptides, nlp20vd2, nlp23vd3, and nlp23vd4 which are highly identical to nlp20. Quantitative real-time PCR results revealed that 14 G. hirsutum PR gene （GhPR） families were induced or suppressed independently in response to fig22, nip20, nlp20va2, nlp23vd3, and nlp23vd4. Most GhPR genes are expressed highest at 3 h post incubation of immunogenic peptides. Compared to fig22 and nlp20, nlp20vd2 is more effective to trigger up-regulated expression of GhPR genes. Notably, both nlp23vd3 and nlp23vd4 are able to induce GhPR gene up-regulation, although they do not induce necrosis on cotton leaves. Thus, our results provide marker genes and new immunogenic peptides for further investigation of cotton-V, dahliae interaction.

Overexpression of GhPFN2 enhances protection against Verticillium dahliae invasion in cotton

Growing evidence indicates that actin cytoskeleton is involved in plant innate immune responses, but the functional mechanism remains largely unknown. Here, we investigated the behavior of a cotton profilin gene （GhPFN2） in response to Verticillium dahliae invasion, and evaluated its contribution to plant defense against this soil-borne fungal pathogen. GhPFN2 expression was up-regulated when cotton root was inoculated with V. dahliae, and the actin architecture was reorganized in the infected root cells, with a clear increase in the density of filamentous actin and the extent of actin btmdling. Compared to the wild type, GhPFN2-overexpressing cotton plants showed enhanced protection against V. dahliae infection and the actin cytoskeleton organization in root epidermal cells was clearly altered, which phenocopied that of the wild-type （WT） root cells challenged with V. dahliae. These results provide a solid line of evidence important for defense against V. dahliae infection. showing that actin cytoskeleton reorganization involving GhPFN2 is