First report of Boeremia exigua var. exigua causing branch blight on walnut in China.

Abstract

Walnut (Juglans sinensis L.) is an important economic tree. Its fruit are rich in omega-3 essential fatty acids, which are valuable nutritionally (Cheon et al, 2013). In March 2019, severe branch blight of walnuts (cv. Qingxiang) were observed in two fields in Ganquan Town, Gansu Province, China (N 33°56'/E105°44'). The incidence was about 3% among 10,000 walnuts. Disease symptoms included fusiform or oval black lesions gradually expanded on the branches, blight and dieback of branches, reddish brown dead branch bark with lots of black small spots (pycnidia), and defoliation. To isolate pathogen, 30 diseased tissues collected from different trees were disinfected with 0.1% HgCl solution for 1 min, rinsed in sterilized water 3 times, and placed on potato dextrose agar (PDA) at 25℃ in the dark. About three days later, 4 fungal isolates (3-3, 3-6, 3-9 and H3) with similar morphological characteristics were obtained. Their colonies, with regular margin on OA, 6.1~6.8 cm diam. after 7 days, were loose, greenish olivaceous to olivaceous grey, velvety, floccose to woolly. The conidia (n=60) were 4.77 to 8.84 μm long (mean = 6.88 μm; SD ± 0.91 μm) × 1.71 to 3.89 μm wide (mean = 2.81 μm; SD ± 0.53 μm), cylindrical, ellipsoidal to oblong, hyaline and aseptate. Pycnidia (n=25) were 76.66 ~ 132.86μm diam. (mean = 102.93 μm; SD ± 12.15 μm), variable in shape and size, mostly globose to subglobose. These characteristics were similar to B. exigua var. exigua (Boeremia et al, 2004). Pathogenicity tests of four isolates were performed 3 times on 5 healthy 2-3 years old walnuts (cv. Qingxiang). Plants were wounded by insect needle No.6 and inoculated with 5 mm mycelium block grew on PDA medium or PDA medium as control and kept moist in climatic cabinet (> 85% RH, 25°C). After 3 days, oval black lesions were occurred on branches and gradually expanded, but control was asymptomatic. And original isolates were re-isolated from these diseased shoots. Genomic DNA of four isolates were extracted, and the internal transcribed spacer (ITS), β-tubulin (tub2) and RNA polymerase II second largest subunit (rpb2) gene were amplified and sequenced using the primers ITS1/ITS4, Btub2Fd/Btub4Rd and RPB2-5F2/fRPB2-7CR (White et al, 1990; Woudenberg et al, 2009; Chen et al, 2015), respectively. Sequences were deposited in GenBank (accession no. ITS: MT154621, MT154622, MT154623, MT154624; tub2: MT223481, MT223482, MT223483, MT223484; rpb2: MW448152, MW448153, MW459982, MW459983), and compared with available sequences in NCBI. Results showed that ITS of four isolates have 100% sequence identity to Boeremia spp., tub2 and rpb2 have 100% sequence identity to B. exigua var. exigua (GenBank accession no. MN983734, MN983315) and B. exigua var. linicola (GenBank accession no. MN983785, MT920619). According to host specificity (Boeremia et al, 1976). A 106 conidium/mL spore suspension of four isolates or sterile water were inoculated on stem base of two-month old flax seedlings, placed in climatic cabinet (> 85% RH, 25°C) for moisturing and repeated three times. After two weeks, all inoculated plants still were asymptomatic, indicated that four isolates aren't B. exigua var. linicola. Therefore, they were identified as B. exigua var. exigua based on morphological characteristics, molecular analysis and pathogenicity tests. To our knowledge, this is the first report of B. exigua var. exigua causing walnut branch blight worldwide, which will provide further guidance for prevention and control of walnut branch blight.