Colonization of grape leaves by endophytic Bacillus subtilis JL4 and its control of grape downy mildew

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In order to understand the colonization pattern of Bacillus subtilis JL4 both on and inside grape leaves, and its control of grape downy mildew, a shuttle vector pGFP78, carrying the GFP gene, was transformed into B. subtilis JL4, and a GFP-labelled transformant designated as JL4-gfp was obtained successfully. The stability of the marker and antagonistic activity to Plasmopara viticola of JL4-gfp were tested. JL4-gfp was spray inoculated on grape leaves in a vine yard and colonization of the leaves was investigated by dilution plating on selective medium. Leaves treated with JL4-gfp were collected and taken to the laboratory for inoculation of a sporangial suspension of P. viticola, to determine its control effect on grape downy mildew. The green fluorescence of the marked strain was stable for at least 10 subcultures, and JL4-gfp maintained wild type antagonistic activity against P. viticola. JL4-gfp was recovered from the grape leaves by dilution plating on medium supplemented with antibiotics. Numbers recovered from the leaf surface of grape leaves were 3.6×105, 2.7×105 and 3.1×103 CFU·g-1 at 0, 3 and 7 days after inoculation, and the population density inside the leaf tissue reached a maximum of 9.6×104 CFU·g-1 at 3 days after inoculation, but could not be recovered after 14 days. The efficiency of downy mildew control by the marked strain was more than 88.0% at 3 days after inoculation, but no significant control effect was observed after 7 days. Our results suggested that there was a positive correlation between the JL4-gfp population density and control efficiency of grape downy mildew, and a threshold colonization level at 105 CFU·g-1 was a prerequisite for this Bacillus strain to present efficient control effects.