The effects of several factors on the estimation of Verticillium dahliae in soil by the wet-sieving method were studied. The following factors were important for maximising recovery: the removal of soil particles of < 20 nm size from suspensions before plating; the medium used for plating; the amount of sieved soil inoculated to plates; the length of time of incubating plates; and the method of incubating soil on plates. There was no short-term effect of air-drying soil before analysis. Using sodium hexametaphosphate to aid soil dispersion, treating soi) suspensions with sodium hypochlorite. or making suspensions from different amounts of soil had no consistent effect on recovery. Excessive sample-to-sample variability in the results of some experiments was attributed to non-random distribution of fungus propagules in soil, or to the difficulty of standardizing the sieving part of the analysis, or both. Wet-sieving was generally more effective in detecting I'. dahliae and gave higher counts than a sucroseflotation method. Bait methods with eggplants or antirrhinums proved ineffective for estimating V. dahliae in naturally infested soil.

The incidence of wilt was recorded in runner and fruiting crops of 13 strawberry cultivars at 72 locations in southern England in 1989 and 1990, and soil samples from the sites were analysed for Verticillium dahliae. Linear regressions of wilt incidence on inoculum concentration in soil for runner crops of the susceptible cv. Elsanta in both years were significant whilst that for runner crops of the susceptible cv. Hapil in 1989 approached significance; the regression for cv. Elsanta fruiting crops in 1990 was not significant. The inclusion of sand content of soil in the regression model improved the fit for the cv. Hapil data but not for the cv. Elsanta data; neither clay nor silt content of soil significantly improved the fit of the models for any data set. There were insufficient data in either year for regression analysis for other cultivars, but the levels of wilt generally corresponded with the degree of soil infestation and broadly reflected known field resistance. The data were used to estimate an inoculum concentration which corresponds to 5% wilt incidence (IC5) for cv. Elsanta. It is suggested that this could be used as a yardstick for determining the risk of unacceptable levels of wilt in susceptible cultivars on the basis of pre‐planting soil analysis.  For the 44 sites where the cropping history over the 15 years prior to soil analysis was available there was no clear association between any crop and soil infestation levels at or above the IC5. However, V.  dahliae was more common at sites with a history of vegetatively propagated crops than at sites which had only supported crops grown from true seed.

Two isolates (CVd‐WHw and CVn‐WHg) recovered from Verticillium‐wilt‐symptomatic cotton grown in Hubei Province of China were identified based on their morphology, growth characteristics in culture, specific amplification and identification of internal transcribed spacer (ITS) rDNA sequence. According to the morphological characteristics, specific PCR amplification and ITS sequences, CVd‐WHw was identified as V. dahliae and CVn‐WHg as Gibellulopsis nigrescens. In bioassays, the two isolates had significantly lower pathogenicity to cotton plant than V. dahliae isolate CVd‐AYb. Cotton pre‐inoculated with isolate CVn‐WHg or CVd‐WHw exhibited reduced disease indices of Verticillium wilt compared with those inoculated with CVd‐AYb alone. Cotton co‐inoculated with CVn‐WHg or CVd‐WHw and CVd‐AYb provided increased protection from subsequent CVd‐AYb inoculation. These results suggest that the two isolates have the potential to be developed as biocontrol agents for the control of Verticillium wilt in cotton. To our knowledge, this is the first report of a cross‐protection phenomenon using Gibellulopsis nigrescens against Verticillium wilt caused by V. dahliae on cotton.

Quantification of Verticillium dahliae microsclerotia is an important component of wilt management on a range of crops. Estimation of microsclerotia by dry or wet sieving and plating of soil samples on semiselective medium is a commonly used technique but this method is resource-intensive. We developed a new molecular quantification method based on Synergy Brands (SYBR) Green real-time quantitative polymerase chain reaction of wet-sieving samples (wet-sieving qPCR). This method can detect V. dahliae microsclerotia as low as 0.5 CFU g(-1) of soil. There was a high correlation (r=0.98) between the estimates of conventional plating analysis and the new wet-sieving qPCR method for 40 soil samples. To estimate the inoculum threshold for cotton wilt, >400 soil samples were taken from the rhizosphere of individual plants with or without visual wilt symptoms in experimental and commercial cotton fields at the boll-forming stage. Wilt inoculum was estimated using the wet-sieving qPCR method and related to wilt development. The estimated inoculum threshold varied with cultivar, ranging from 4.0 and 7.0 CFU g(-1) of soil for susceptible and resistant cultivars, respectively. In addition, there was an overall relationship of wilt incidence with inoculum density across 31 commercial fields where a single composite soil sample was taken at each field, with an estimated inoculum threshold of 11 CFU g(-1) of soil. These results suggest that wilt risk can be predicted from the estimated soil inoculum density using the new wet-sieving qPCR method. We recommend the use of 4.0 and 7.0 CFU g(-1) as an inoculum threshold on susceptible and resistant cultivars, respectively, in practical risk prediction schemes.