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A Rapid Microbioassay for Discovery of Antagonistic Bacteria for Phytophthora parasitica var. nicotianae

First, third, sixth, seventh, eighth, ninth, and tenth authors: Guizhou Tobacco Science Institute, Guiyang 550081, P.R. China; second author: Guizhou Institute of Plant Protection, Guiyang 550006, P.R. China; and fourth and fifth authors: College of Agriculture, Yangtze University, Jingzhou 434025, P.R. China. First, second, and third authors contributed equally.

A simple, rapid, small-scale microbioassay for infection of tobacco seedlings by Phytophthora parasitica var. nicotianae was developed here. This assay uses tobacco seedlings cultivated in petri dishes for a standardized method for quantitation of initial zoospore inocula and high-throughput screening of antagonistic bacteria. Zoospore inocula between 10² to 10⁵ spores per petri dish were inoculated on 14-day-old tobacco seedlings for the susceptibility test. The optimum inocula was established to be ten thousand zoospores. One hundred and fifty pure culture bacteria with different pigments, growth rates, and morphologies were isolated from rhizosphere soil of tobacco and screened for protective ability against tobacco black shank. Fifteen bacteria presented high activity against P. parasitica on tobacco seedlings. They were identified by Biolog GEN III MicroPlate and distributed as Bacillus amyloliquefaciens, B. licheniformis, Paenibacillus pabuli, B. atrophaeus, B. subtilis, B. pumilus, and B. endophyticus, respectively. Four antagonists chosen randomly from the 15 bacteria all exhibited the same 100% protective activity in planta as that in the petri dishes. This microassay proved to be a rapid, reproducible, and efficient method for screening of potential biological agents or microorganisms and may be useful for studying mechanisms of infection and control of Phytophthora spp. under hydroponic conditions.