Graduate Thesis Or Dissertation
 

Evaluation of assay systems for the determination of susceptibility of the hop (Humulus lupulus) to Verticillium wilt caused by Verticillium dahliae

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  • A rapid and reliable assay is needed to evaluate hop resistance to Verticillium wilt caused by Verticillium dahliae. Assays used in the past are laborious, require long incubation periods, and usually produce mild symptoms which are difficult to evaluate and are often not consistent. A study comparing several methods for evaluating resistance of hops to Verticillium wilt was conducted. Results from isolate pathogenicity / host range tests showed the isolates used in these experiments to differ in their pathogenicity on each host tested. More severe symptoms generally occurred with an increase in inoculum concentration. In the first assay method, cuttings of resistant (R) cultivars Yakima Cluster and Bullion, moderately resistant (MR) Willamette, susceptible (S) Fuggle and Columbia, and three native North American hop types were grown in non sterile field soil artificially infested with microsclerotia of V. dahliae. After seven months, plants of the susceptible cultivars were very stunted and chlorotic. Cultivars reported to be resistant were less stunted. Three native North American hop clones resembled the resistant hop cultivars in visual symptom expression. Although vascular discoloration was observed, Verticillium was not recovered on three isolation media, but an attempt to isolate the fungus by a sterile filter paper method was successful. Analysis of the infested soil by the wet sieve method showed a decreased number of microsclerotia by the end of the trial which may have resulted in the low recovery. In general, root weight increased with increased inoculum concentration among inoculated susceptible cultivars, and decreased for resistant cultivars. Bine dry weight varied significantly among cultivars and in relation to inoculum density, but there were no apparent trends among the cultivars. In the second assay method, roots of the susceptible hop cultivar Hallertauer were inoculated by conidial root dip. Symptoms were mild, with symptomatic plants generally recovering from wilt. Differences in the ability of the isolates to induce symptoms of Verticillium wilt were apparent. There was no significant difference among root or bine dry weight of inoculated versus non-inoculated plants. The proportion of infected plants was significantly different among isolates. Marked symptom expression and recovery of the pathogen were positively correlated. In the third method, standardized conidial suspensions were placed on detached leaves of a number of hop cultivars with different levels of field resistance to Verticillium wilt. Although this method has been used successfully elsewhere, no reaction was seen on the hop leaves in trials repeated numerous times. In a fourth assay method, drops of V. dahliae culture filtrate containing presumed toxin were applied to detached leaves from hop cultivars differing in resistance to Verticillium wilt. Distinctive chlorotic spots were induced on leaves of susceptible cultivars Fuggle and Willamette, while little or no chlorosis was induced on leaves of resistant cultivars Bullion and Yakima Cluster. Boiling the filtrate for 1 min decreased the activity by almost 50%; autoclaving at 15 psi/15 min destroyed the activity. The results of this study suggest that the application of culture filtrates to detached hop leaves is the best assay system of those compared, being both rapid and reliable, and useful to distinguish the relative susceptibility and resistance of hop cultivars to V. dahliae. This method correlated best with field observations on cultivar resistance to Verticillium wilt. Mechanical and physiological responses, however, may be equally important in determining overall resistance. For this reason, initial screening of new plant material or isolates should be done by comparing the results from conidial root dips and tests with the culture filtrate.
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