First Report of Aureobasidium pullulans var. pullulans Causing Spot Blight of Pear (Pyrus pyrifolia) in Zhejiang Province of China.

Abstract

Asian pear (Pyrus pyrifolia Nakai cv. cuiguan), is widely grown in Zhejiang province of China. In April 2019, symptoms consisting of small black, round leaf spots and blight flower petals were observed on over 30 % of 'Cuiguan' pear trees in an orchard (ca. 0.8 ha) near Cixi city, Zhejiang province, China. Initially, leaf spots were observed on leaf petioles, which, with time, enlarged and coalesced into necrotic streaks (1-2 cm) along the length of the petioles. Irregularly, reddish brown spots developed on flower petals, which hastened their senescence. Additional symptoms included round, black spots on leaves (2-3 mm in diameter) and necrosis of shoot tips. Symptomatic tissues from petals, petioles and leaves were plated onto potato dextrose agar (PDA). After five days of incubation at 26 °C, slimy fungal colonies (48×48 mm) with pinkish to orange-colored mycelia and with regular annulations were isolated from all tissues. After 10 days, cultured were shiny and dark brown in the center. The color of conidia ranged from hyaline to dark brown. Hyaline conidia were blastic, unicellular, ellipsoidal, smooth, with lengths that ranged from 11.03 to 27.14 (avg. 18.38) μm, and widths that ranged from 3.45 to 8.86 (avg. 6.04) μm (n = 50). Dark brown conidia were 1 to 2 celled, 10.89 to 26.03 (avg. 17.41) μm in length and 4.26 to 12.15 (avg. 6.94) μm in width (n = 50), and a slight septal constriction. Conidiogenous cells were clavate, hyaline, eseptate and top smoothly with 3-11 spores. Indistinct scars remained when the conidia dislodge from the conidiogenous cells. Single spore isolation was used to obtain pure cultures. Mycelia and conidia were scraped from cultures and DNA was extracted using Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech). Amplified PCR products from the internal transcribed spacer (ITS) region ITS1F/ITS4 (White et al. 1990), the partial 28S rDNA (LSU) NL1/NL4 (Boekhout et al. 1995), the β-tubulin (TUB) gene Bt2a/Bt2b (Glass & Donaldson 1995) and the partial elongase gene (ELO) ELO2-F/ELO2-R (Zalar et al. 2008) were sequenced (Tsingke Biotechnology Co., Hangzhou, Zhejiang). A blast search (GenBank Accession No. MT107050, OK485685, OK631951, OK631950) showed 99% Aureobasidium pullulans reference isolate CBS584.75 and EXF-150, which was consistent with the morphological data (Cene et al. 2014). Three-yr-old seedlings from 'Cuiguan' pears were spray with conidial suspension (106 conidia/ml) on the both sides of leaves without wounding. In a greenhouse (26 °C, natural light), six inoculated plants and three noninoculated plants (sprayed with sterile distilled water) enclosed in plastic bags to maintain humidity for 72 h. At 5 days after inoculation, shoot tips blackened and began to wilt. At 15 days after inoculation, symptoms similar to those on the original sample developed on inoculated petioles and leaves, whereas the control plants remained healthy. No symptoms were observed on leaves that were mature at the time of inoculation. Aureobasidium pullulans var. pullulans was reisolated from all inoculated plant. Overall, this disease shortened the life of pear flowers and reduced fruit set. To our knowledge, A. pullulans var. pullulans has not previously been reported as a pathogen of P. pyrifolia.