"Star like" disease of persimmon (Diospyros kaki) fruit caused by Botrytis cinerea in Israel.

Abstract

During May 2016, a corky textured, "star like" symptom, located at the apex on the far side of the fruit was observed on young persimmon fruitlets (Fig. 1), on several persimmon varieties in plantations located along the Mediterranean Sea coast. The lesions caused cosmetic damage, which disqualified the fruit from marketing and can affect as much as 50% of the fruit in the orchard. Symptoms were correlated with the presence of wilting flower parts (petals and stamens) attached to the fruitlet (Fig. 1). Fruitlets with no flower parts attached did not develop the corky star symptom, while almost all fruitlets with an attached wilted flower parts had symptoms underneath the flower parts. Flower parts and fruitlets displaying the phenomenon were sampled (orchard near the town of Zichron Yaccov) and used for fungi isolation. At least ten fruitlets were surface sterilized by immersion in 1% NaOCl for 1 min. Pieces of the infected tissue were then placed on 0.25% potato dextrose agar (PDA) supplemented with 12 µg/mL tetracycline (Sigma, Rehovot, Israel). In addition, the inner parts of at least ten moldy flowers were placed on 0.25% PDA supplemented with tetracycline and incubated at 25°C for 7 days. Two fungi were isolated from the flower parts and the symptomatic fruitlets: Alternaria sp. and Botrytis sp. Koch postulates were conducted by inoculating 10 µl of conidial suspension of each fungus (105 conidia/ml in H2O, single spore originated), on four wounds, made by puncturing a 2 mm deep hole with 21G sterile syringe needle, on the apex of surface sterilized, small, green fruits. Fruits were placed in sealed 2-liter plastic boxes. Symptoms similar to those found on the fruitlets in the orchards were observed on the fruit inoculated with Botrytis sp. (corky like, but not in the shape of stars) about 14 days post inoculation. Botrytis sp. was re-isolated from the symptomatic fruit to fulfill Koch's postulates. Alternaria and water inoculation did not cause any symptoms. The Botrytis sp. colonies, when grown on PDA, grow initially as white colonies becoming gray to brown after about seven days. Elliptical conidia, 8 to 12 μm long and 6 to 10 μm wide, were observed under light microscope. Isolate Pers-1, incubated at 21°C for 21 days, produced blackish, spherical to irregular microsclerotia, ranging from 0.55 to 4 mm (width and length, respectively). For molecular characterization of the Botrytis sp. isolate, Pers-1, fungal genomic DNA was extracted as previously described by Freeman et al. (2013). The internal transcribed spacer (ITS) sequence region of rDNA was amplified using ITS1/ITS4 primers (White et al. 1990), and sequenced. ITS analysis revealed that it belongs to genus Botrytis (MT573470.1 with 99.80% identity). For further confirmation, nuclear protein-coding genes (RPB2 and BT-1, Malkuset et al. 2006 and Glass et al. 1995) were sequenced and found to have 99.87% and 99.80% identity to Botrytis cinerea Pers. Sequences, deposited in GenBank as accessions OQ286390, OQ587946 and OQ409867, respectively. Botrytis was previously reported to cause persimmon fruit scarring and damage of calyces (Rheinländer et al. 2013) and fruit rot during post-harvest (Barkai-Golan. 2001), yet to the best of our knowledge, this is the first report of B. cinerea causing "star like" corky symptoms on persimmon in Israel.