Quantitative Epidemiology Of Verticillium Dahliae Microsclerotia In Soil

Verticillium dahliae Kleb. is a notorious and widespread soilborne fungal pathogen causing wilt disease on more than 400 plant species. For V. dahliae, the disease cycle begins with dormant microsclerotia, which may survive for more than 10 years in the absence of hosts in soil. Being a monocyclic disease, predicting wilt risk and management decisions to reduce the risk of yield loss need to implement before planting. However, due to the lack of faster quantification techniques for microsclerotia and soil sampling scheme for uncertainty in spatial distribution of microsclerotia, studies of epidemiology and disease predicting were largely limited.The main objective of this study is to access threshold microsclerotial inoculum for cotton Verticillium wilt at the level of individual plants, spatial correction of V. dahliae in soil and cotton plants with wilt symptoms at a range of scales, the efficacy of bio-fumigation-derived products to control V. dahliae microsclerotia, and their temporal effects on soil bacterial and fungal communities. The follow most important results were obtained:1. Developed a new molecular quantification method for quantifying V. dahliae microsclerotia in soil.Combining conventional wet-sieving and SYBR Green I-based real-time qPCR, a new molecular quantification method is developed based on SYBR Green real-time quantitative PCR of wet-sieving samples(wet-sieving qPCR) that is specific, sensitive, and faster than the widely used conventional plating method. This method can detect V. dahliae microsclerotia as low as 0.5 CFU/g of soil. There was a high correlation(r = 0.98) between the estimates of conventional plating analysis and the new wet-sieving qPCR method.2. Threshhold microsclerotial inoculum for cotton Verticillium wilt varied with cultivar,4 and 7 CFU/g of soil for susceptible(JM11) and resistant cultivars(ZHM2).To estimate the inoculum threshold for cotton wilt, 405 soil samples were taken from the rhizosphere of individual plants with or without visual wilt symptoms in experimental and commercial cotton fields. Inoculum density of V. dahliae was estimated using the wet-sieving-qPCR method. The estimated inoculum density, when plotted against the probability being infected and disease category and related to wilt development, closelyfollowed the the logistic model(R2=0.62) and cumulative logit model(R2=0.79) with the exact relationship dependent on the cultivar, respectively. These data suggest that increasing inoculum density led to higher probability being infected and disease category. The estimated by logistics model inoculum threshold varied with cultivar, ranging from 4 and 7 CFU/g of soil for susceptible(JM11) and resistant cultivars(ZHM2), respectively.3. Spatial aggregation of both V. dahliae inoculum and cotton plants with wilt symptoms is not likely to be detected above the scale of 1.0 m for commercial cotton plantations.Spatial patterns of V. dahliae microsclerotia were characterized in three commercial cotton fields through point sampling(based on plant-to-plant distance). Soil samples were assayed for the density of microsclerotia(expressed as CFU g-1 of soil) using wet-sieving qPCR method. The distribution of microsclerotium density was skewed for all testing fields.There were large variations among estimated CFU values within each field. Spatial analyses showed that aggregation of V. dahliae inoculum in soils was usually not detected beyond 1.0m. Similarly, the spatial patterns of wilted cotton plants indicated that spatial aggregation of diseased plants were only observed below the scale of 1.0 m in six commercial cotton plantations. None of the across-row join-count statistics was statistically different from the expected under the assumption of a random pattern. Thus, to estimate the overall inoculum level of a given field for predicting wilt risk prior to planting, a simple random sampling plan with minimum spatial distance of 1 m between any two samples should be able to provide an unbiased estimate.4. Soil biofumigation can significantly reduce V. dahliae microsclerotia. However, these alternatives did not lead to drastic changes in both bacterial and fungal populations.Mini-field-plot experiments were conducted to evaluate the efficacy of individual and combined use of terpene, BioFence? and digestate against V. dahliae; sterile distilled water treatment and untreated control were also included. In addition, we characterised soil bacterial and fungal communities in response to the soil fumigation with microencapsulated terpene,BioFenceTM and chloropicrin in a field trial using DNA metabarcoding techniques. In the field miniplot trials, BioFence?, terpene and digestate significantly reduced wilt inoculum densities. For the combined two or three product treatments, the observed efficacy was significantly less than the expected efficacy on the assumption of Bliss independence. There were no significant differences in all comparisons of pairwise treatments except between digestate and combined three product treatment. Soil fumigation by BioFence? and terpene can reduce V. dahliae microsclerotia by more than 60%. However, these alternatives did not lead to drastic changes in both bacterial and fungal populations. Chloropicrin dramatically altered both bacterial and fungal populations within 4 weeks of application. The effect onbacterial population structure is short-lived and became non-significant 16 weeks after treatment; however, fungal population structure was more persistently affected by chloropicrin.