*4.7. Biocontrol Activity of KOF112 against Downy Mildew*

An in vivo bioassay using grape leaf disks was performed as described previously, with minor modifications [51]. Briefly, Koshu leaves were collected from potted seedlings. Five leaf disks each having a diameter of 13 mm were cut out from the leaves using a cork polisher and placed upside down on moistened filter paper in square Petri dishes (140 mm × 100 mm). KOF112 was incubated in SCD medium overnight and adjusted to 1 × <sup>10</sup>8, <sup>1</sup> × 107, 1 × <sup>10</sup>6, and 1 × <sup>10</sup><sup>5</sup> cfu/mL with SCD medium and sterile water (all KOF112 solutions contained 10% SCD medium). Ten microliters of KOF112 solution was dropped onto four locations on the abaxial surface of the leaf disk. Ten percent SCD medium and *Agrobacterium* sp. isolate CHB3 (1 × 108 cfu/mL, containing 10% SCD) were used as control. The leaf disks were dried at room temperature in a flow cabinet for 3 h.

Inoculation of *P. viticola* zoosporangia was performed as described previously with minor modifications [6]. Briefly, field-isolated *P. viticola* was maintained on leaves of potted Koshu seedlings in a growth chamber under light irradiation (11.8 Wm−2/16 h/d) at 21 ◦C. Fresh zoosporangia of *P. viticola* were washed off with sterile water from the symptoms on the Koshu leaves. Ten microliters of the zoosporangium suspension (1 × 104 zoosporangia/mL) was dropped onto the same locations as the pretreated KOF112 solutions on the leaf disks. The Petri dishes containing the leaf disks were placed in a plastic box containing moistened paper towel. The box was incubated in the dark for 24 h and then in an incubator (21 ◦C, 11.8 Wm−2/16 h/d). Downy mildew symptoms on each disk were assessed 7 days post *P. viticola* inoculation. Disease severity was scored by evaluating the symptom on each disk as described previously [51]: 0, no symptoms; 1, white symptom occupies up to 1/6 of the disk; 2, white symptom occupies up to 1/3 of the disk; 3, white symptom occupies up to half of the disk; 4, white symptom occupies up to two-thirds of the disk; 5, white symptom occupies more than two-thirds of the disk. Two independent experiments were performed with five leaf disks.
