| Upland cotton (Gossypium hirsutum L.) is widely cultivated for the important economic value of its fiber. Over 30% area of upland cotton are subject to Verticillium wilt caused by Verticillium dahliae (V. dahliae), and the disease has become the most economically important disease of upland cotton. Traditional methods of breeding for resistance cultivars make these ineffective due to the existence of different strains of V. dahliae. Vascular wilts are particularly notorious because, within the vascular system of host plants, the pathogens cannot be killed by many fungicides. Furthermore, V. dahliae survive as microsclerotia in the soil for many years, facilitating pathogen dispersal through water, farm equipment and personnel. Due to the scarcity of sources of disease resistant host germplasm and fungicides, Verticillium wilt causes significant crop losses.To analyze the relationship between the resistance of cotton cultivars and endophytic bacteria, the composition and antagonistic activity of endophytic bacteria in different resistance cotton were study. The results showed that cotton root endophytic bacteria the composition and diversity are related to the cultivar’s genotype and resistance level. A total of 660 bacterial isolates were screened in vitro for antagonism toward the pathogens Verticillium dahliae Kleb V1070, resulting in 150 active isolates. A high proportion of antagonistic bacteria were found in the resistant cultivars, and significant differences were observed in cultivars with different resistance. Amplified ribosomal DNA restriction analysis (ARDRA) demonstrated that 150 isolates were divided into 19 distinct groups, which indicated a significant genetic diversity among the isolates. Sequencing analysis of the partial 16S rRNA sequence from the representative isolates in each group showed that the most commonly isolated species were Pseudomonas (43.3%), followed by Bacillus (34.7%) and Enterobacter (10%). The potential of the isolates for biocontrol was evaluated by screening for their growth promotion effects and disease control related traits. The greenhouse and field trial revealed that 12 isolates showed a significant biocontrol activity to VI070 and they were isolated from cultivars with high disease resistance. Taken together, the disease resistance of host may be influence by endophytic bacteria through various forms and resistant cultivars may use for selection and characterization of ideal biocontrol agents to V. dahliae in cotton.Non-pathogenic bacteria Pseudomonas spp. can reduce disease in plant tissues through induction of a defence state known as induced systemic resistance (ISR). This resistance is based on multiple bacterial determinants, but nothing is known about the mechanisms underlying bacteria-induced resistance in cotton. Strain 841P-3 inhibited conidial germination and hyphae growth of V. dahliae in vitro; when applied on leaves or to the soil, 841P-3 also inhibited the development of hyphae in a greenhouse, but the effectiveness varied with the length of the interval between the strain treatment and inoculation with the pathogen. In this study, the ability of 841P-3 to induce resistance in cotton against V. dahliae is demonstrated. Bacterial treatment prior to V. dahliae inoculation resulted in less seedling disease development as compared with that in nontreated control plants, in which typical root and leaf symptoms were visible after inoculation with the pathogen. Strain 841 P-3 is an endophyte that can spread in cotton seedlings rapidly and efficiently. Perception of the 841 P-3 also led to activation of some defense enzymes and the terpenoid pathway without challenge with V. dahliae. At the mean time, we found that the PAL, CHI and GLU activity in infection bacterized plant were lies between mock-treated bacterized plant and infection control plants, we speculate that there maybe a functional reciprocal inhibition between the antagonistic bacteria and V. dahliae to induced defence reaction. Perception of the 841 P-3 also led to activation of MAPK and WRKY gene when challenge with V. dahliae. Histological and biochemical analyses conducted on the bacterized plants showed rapid/greater induction of lignification, H2O2 burst, callose accumulation and NO production. And NO paticpated in 841 P-3 induced defence respose.We discovered that both filtrate and methanol extracts of Bacillus amyloliquefaciens 41B-1 inhibited growth of V. dahliae. Using chromatography and mass spectrometry analysis, the antifungal compounds were identified as an iturins and a series of iturins isoforms. The iturins can degrade MS cell wall, led to cell death in plate assays. Iturins can also inhibit germination of V. dahliae MS in soil, prevent the growth of pathogen in plants finally led to symptoms of cotton leaf wilt reduced under greenhouse conditions. Iturins treatment caused cell growth inhibition following ROS cascades and cell swelling, suggesting that oxidative stress and osmotic stress regulate sensitivity of V. dahliae to iturins. Iturins treatment resulted in cell wall degradation and polysaccharide antagonist agents SDS, CW, and CR were all increased V1070 sensitivity to iturins, indicating that iturins treatment induced cell wall stress and the cell wall integrity pathway also contributes to drug resistance in this fungus. Combination of iturins and osmotic stress inducer fludioxonil synergistically inhibited V. dahliae growth. Furthermore, iturins treatment can induced host denfence responses and enhanced PAMP-triggered immunity and at the mean time, led to an efficient reduction of V. dahliae conidia on cotton-roots. These studies provide evidence that iturins mediated multiple defense effects against Verticillium wilts of cotton and provide insight into novel targets for synergistic antifungal drug combinations. |
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