The First Report of Juglans regia Leaf Spot Caused by Fusarium proliferatum in China.

Abstract

Juglans regia is planted widely in China because of its significant economic value as a woody oil species. In August 2019, leaf spots were discovered in a walnut orchard in Tang County, Baoding City, Hebei Province. In the early stages of the disease, irregular brown spots appeared on the edges of leaves, causing them to shrivel; in a later stage, the disease spread inward along the edges to the whole leaves, which then withered. The diseased leaves were collected from the walnut orchard. Select 3-4 mm2 of diseased and healthy junction for each diseased leaf, and the surfaces were sterilized with 75% ethanol for 30 s and 0.1% HgCl2. The leaves were incubated on potato dextrose agar (PDA) at 28 ℃ in darkness, each diseased leaf was repeated 10 times, and purified after 6 days. The diameters of the suspected Fusarium isolates reached 8.3 cm, and the aerial mycelia were white to cream and wool-like, later turning violet and dark purple with age. On PDA, the macroconidia exhibited 3-5 septa and were straight or slightly curved, with a size of 21.6-47.4×2.5-4.6 µm (average: 31.9×3.5 µm, n=50). The morphological features of these fungi were noted to be in line with those of Fusarium proliferatum (Leslie and Summerell, 2006; Zhao et al., 2019). For molecular analysis, DNA was extracted from the mycelium and used the specific primers to amplify the translation elongation factor EF-1α, and RNA polymerase II largest-subunit (RPB2) further. The amplicons were sequenced and deposited in GenBank under accession numbers MZ891582, and MZ891583. BLAST analysis revealed that all these sequences showed more than 99% sequence identification with F. proliferatum. We used Fusarium MLST for the identification of this strain and the results showed that EF-1α and RPB2 genes have more than 99% similarity with the database F. proliferatum data, with 100% sequence duplication(Deposited of EF-1α in GenBank under accession numbers: MH582346, and MH582347; Deposited of RPB2 in GenBank under accession numbers: MH582193, MH582151, MH582196, and MH582172). We created a multigenic tree using Fusarium MLST. Based on the results of the morphology tests and the molecular sequences, the isolates from the diseased leaves were identified as F. proliferatum. To confirm the pathogenicity, healthy walnut leaves of tree planted in pot were inoculated using 50 µL of conidia suspension (1.0×107 CFU) from a pure clone, and those inoculated using only distilled water were taken as controls. All treatments were repeated three times and placed under 28°C, RH 100% for 5 days. The inoculated plants showed the same symptoms as those observed in the original diseased plants, while the control plants were asymptomatic. The same fungus was isolated again from the inoculated leaves of tree planted in pot. The re-isolated pathogen from diseased plants was identical to the purified strain used for inoculation, confirmed Koch's postulates. According to the above results, F. proliferatum was identified as the pathogen behind the Juglans regia leaf spots, and it is clearly different from other pathogens, such as Colletotrichum gloeosporioides. It is reported that F. proliferatum can lead to necrotic leaf lesions and the bulb rot of stored onions (Beck et al., 2020), as well as light brown discoloration and dryness on palm leaves and branches (Alananbeh et al., 2021). At the same time, F. proliferatum has been found to have the highest colonization ability on jujube leaves and is becoming a serious pathogen on jujube (Saleh et al., 2016). To our knowledge, this is the first report of walnut leaf spots caused by F. proliferatum in China.