Boeremia exigua Causes Leaf Spot of Walnut Trees (Juglans regia) in China

Abstract

Walnuts are an important perennial nut crop widely cultivated in China, which are rich in protein, carbohydrate, renieratene, and other beneficial nutrients. China is the largest producer of walnuts in the world, with the largest planting area and output. At the end of April 2020, several unknown necrotic spots on leaves of walnut trees were observed in a Juglans regia field located in Sancha Town, Enshi, China (30°28′N, 109°64′E). Initially, lesions were black, small, sunken, and turning to yellowish-brown, irregular, well surrounded by brown margins. Severely, leaf spots coalesced and resulted in withered and abscised. In order to identify the pathogen, infected leaves were collected. Sections of leaves were aseptically excised from the margins of necrotic spots following surface sterilization and placed on potato dextrose agar (PDA) at 28℃. After 4 days, fungal isolates were obtained and purified by hyphal tip isolation. The isolates looked morphologically similar, producing colonies that appeared hyphae with dark grey, lobed margins, and aerial mycelium with white to light gray. After 15 days of incubation, subglobose, dark brown pycnidia (100-176 μm in wide, 75-95 μm in length) were formed with an orifice in the center, producing conidia. Conidia (3.5 to 9.0 × 1.6 to 4.5 μm) were oval to round, aseptate, occasionally 1-septate. These morphological characteristics lead to the conclusion that the isolates may be identified as Phoma sp. (Boerema et al. 1976). A single isolate was randomly selected and designated for further verification. To confirm the identity, the internal transcribed spacer region (ITS), actin (ACT) and beta-tubulin genes were amplified and sequenced ITS1/ITS4, ACT-512F/ACT-783R, and Bt2a/Bt2b, respectively (White et al. 1990, Groenewald et al. 2013). BLAST analysis of the ITS 505-bp sequence (GenBank accession no. MW282913), actin 269-bp sequence (GenBank accession no. MW201958), and beta-tubulin 347-bp sequence (GenBank accession no. MW273782) showed ≥99% homology with the sequences of B. exigua available in GenBank (GenBank accession no. AB454232, LT158234, and KR010463, respectively). Base on the above results, the strain HTY2 was identified as B. exigua. Pathogenicity was tested. Walnut plants were spray-inoculated with a spore suspension (5 x 105 CFU/mL). Controls were inoculated as described above except that sterile distilled water in the dark at 25 ℃. After seven days, lesions were evident at inoculation points, and equivalent to those observed in field were observed. Control leaves remained symptomless. The pathogenicity test was repeated thrice and the results were the same, fulfilling the Koch’s postulates. The pathogen has been reported on various plants around the world, causing a series of symptoms. Infected plants rarely died, but the presence of lesions decreased their fruit quality and yield. Previous identification of the disease is essential in formulating management strategies.