Alternaria alternata Causing Brown Spot Disease on Walnut in Xinjiang, China

Walnut is one of the Xinjiang's characteristic dried fruits and the main source of income for farmers in walnut growing areas. In September 2019, Juglans regia leaves with brown spots were observed in a 10 hm2 orchard in Hotan area, the diseased leaf rate reached more than 25%. The leaf lesions were suborbicular to irregular, black-brown, 3 to 8 mm in diameter, with distinct dark borders. Colonies were isolated from 10 diseased leaves collected from two trees in the orchard. Leaf sections (4 × 4 mm) from diseased leaves were surface disinfested with 75% ethyl alcohol for 30 s and 2% NaClO for 3 min, washed with sterile water three times and then plated on potato dextrose agar (PDA) and incubated at 27℃ with a 12h/12h light/dark photoperiod for 4 days. A total of 7 fungal isolates were obtained by single-spore isolation. All the colonies were dark olivaceous on the PDA plates, with loose, cottony mycelium. On potato carrot agar (PCA), all fungal isolates produced conidial chains with numerous secondary chains. The conidia were ellipsoid or obpyriform with 0-3 longitudinal septa and 2-4 transverse septa, measuring 20.6 to 35.8 × 6.8 to 11.2 μm (25.5 ± 0.4 × 8.7 ± 0.2 μm, n=50). The morphological characteristics of the seven fungal isolates were consistent with the A. alternata descriptions of Simmons (2007). DNA was extracted from 50 mg of mycelia for the representative isolate HLP17-7. The internal transcribed spacer (ITS) region was PCR amplified using the universal primers ITS1 / ITS4 (White et al.1990), the partial coding sequence of endopolygalacturonase (endoPG) and the partial region of the histone 3 (H3) were amplified using primers PG2b / PG3 (Andrew et al. 2009) and H3-1a / H3-1b (Glass and Donaldson 1995) respectively. The products were sequenced and deposited in GenBank database under the accession numbers MW514319 [ITS], ON806938 endoPG, MW489301 [H3]. ITS, endoPG and H3 sequences had 99.81% (1/535 nt difference), 99.78% (1/448 nt difference) and 100% (0/417 nt difference) homology with homologous sequences of A. alternata strains (KP124306 [ITS], KP124006 [endoPG], MK085979 [H3]), respectively. During the early autumn, pathogenicity tests were carried out on the healthy mature leaves of seven-year-old Juglans regia plants in the field. Thirty leaves (five leaves per plant) were wounded with a sterile needle and then sprayed with a spore suspension prepared from 10-day-old PDA culture. Five wounded leaves per plant were sprayed with sterile water as control. All the treated leaves were covered with clear plastic bags for 3 days, and the experiment was replicated three times. On the 8th day after inoculation, brown spots appeared on the inoculated leaves, but no spots were observed in the control. Morphological observation and gene sequencing confirmed that the original fungal pathogen was re-isolated from the inoculated leaves. No colony was isolated from the control leaves. The pathogen causing the brown spot was identified as A. alternata based on morphological features and sequence analysis. A. alternata has been reported previously in Sichuan (Yang et al., 2017) causing brown spot in walnut. Xinjiang is dry with little rain and abundant sunshine, so there are few diseases on walnuts. However, the occurrence of brown spot disease has alarmed fruit farmers, walnuts are still at the risk of A. alternata infections even in dry environment with little rain. To our knowledge, this is the first report of A. alternata causing brown spot in walnut in Xinjiang, China. References: Andrew, M., et al. 2009. Mycologia. 101:95 Glass, M. L., and Donaldson, G. C. 1995. Appl. Environ. Microbiol. 61:1323. Simmons, E. G. 2007. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands. White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA. Yang, L., et al. 2017. Forest Research. 30(6):1004-1008.