First Report of Fusarium fujikuroi Causing Black Stem Rot of Zanthoxylum bungeanum in China.

Abstract

Chinese prickly ash (Zanthoxylum bungeanum Maxim.), native to China, is an important tree species for soil and water conservation, barren mountain afforestation, and garden greening. Its fruit is commonly used for seasoning and medicine. In August 2016, black stem rot of Z. bungeanum was first observed in Hanyuan County, Ya'an City. In June 2019, the symptoms were observed on > 60% of 10,000 plants in Hanyuan County. At its early stage, the bark was wet and rotten, slightly concave, and accompanied by gummosis. The lesions were dark brown and long oval, peeling off the rotten bark covered with white hyphae. At the later stage, the lesions shrunk and cracked, with many orange-red particles (conidia) and dense black particles (ascospores). Larger lesions often caused large-scale bark necrosis. After the lesions girdled the trunk, the plants rapidly died. A total of 36 isolates were isolated from 320 infested tissue fragments (5 × 5 mm) that were surface sterilized for 60 s in 3% sodium hypochlorite, and 60 s in 75% ethanol, rinsed three times in sterilized water, placed onto potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml), and incubated in the dark at 25°C. Among them, 28 exhibited morphological characteristics described as Fusarium fujikuroi Nirenberg. On PDA, the fungus produced white to grey orange, circular colonies. On carnation leaf agar (CLA), the microconidia were club shaped with a flattened base and 0 to 1 septum, 5 to 15 × 2 to 4 μm in size, whereas macroconidia were relatively slender, medium length with no significant curvature, and had a tapered apical cell and a poorly developed, notched basal cell, 3 to 5 septa, 20 to 50 × 3 to 5 μm in size. For molecular identification, DNA was extracted from a representative isolate using a fungus genomic DNA extraction kit (Solarbio, Beijing). PCRs were performed with primers EF1f/EF2r for translation elongation factor 1α (EF-1α) region, and primers 5f2/7cr for RNA polymerase II genes (RPB2) region. PCRs were amplified and their products (GenBank accession nos. MT448248 and MT448247) were sequenced and blasted, showing 99 to 100% sequence homology with known F. fujikuroi isolates (GenBank accession nos. MN102101.1 and MN193888.1). To conduct pathogenicity test, twigs of 42 three-year-old potted Z. bungeanum plants were superficially wounded with a needle on the cortex, and each twig was inoculated by dropping a 100 µl conidial suspension (1×106 conidia/ml) of the fungus onto its wound surface. The inoculated areas were bandaged with gauze moistened with sterilized water. The wounded wigs that were inoculated with sterilized water served as the controls. Treated plants were maintained in a greenhouse with temperature ranging from 22 to 29°C and RH from 70% to 80%. One month later, 100% of inoculated plants showed the symptoms similar to those observed in the field, with the size of lesions ranging from 0.5 to 1.0 cm2. The fungus was re-isolated from the branch cortexes and confirmed morphologically and molecularly. To our knowledge, this is the first report of F. fujikuroi as a causal agent of black stem rot disease on Z. bungeanum in China. These results will help correctly identify this disease and develop proper strategies to manage the disease.