First Report of Round Spot Disease of Zanthoxylum armatum DC Caused by Fusarium fujikuroi in China.

Abstract

Zanthoxylum armatum DC, a deciduous tree in Rutaceae, has significant economic value as an important food condiment, spice, and medicine (Cao et al. 2019). Recently, an unknown round leaf spot disease has been found on Z. armatum in Meishan and surroundings areas of Sichuan province. The disease mainly affected the leaves, mostly on seedlings, with incidence of approximately 50%. Isolate HJYB-4 was isolated from typical diseased leaves and purified on potato dextrose agar (PDA). The isolate produced floccose white, magenta, or grey aerial mycelium. On the reverse side of the culture, the colony had the pigment of pale gray or magenta, with concentric rings of dark red and pale brown in the center. Morphological characteristics were recorded using a pure culture grown on PDA and Synthetic low nutrient agar (SNA). The hyphae of the isolate were colorless and septate. There were two types of conidia on SNA, microconidia, and macroconidia. Macroconidia were long and slender with parallel dorso-ventral sides, usually 3 to 5 septate, 21.89 to 47.21 × 3.33 to 4.73 μm. Microconidia were oval, melon-shaped, ovate, 0-1 septate, mostly no septate, 5.35 to 11.22 × 2.24 to 3.79 μm. A few pyriform microconidia and chlamydospores were observed. DNA of the isolate was extracted using the Column Fungal DNAout 2.0 (Tiandz Inc., Irvine, Beijing, China). PCR was performed using the following primers, ITS1/ITS4, TEF1/TEF2, TUBT1/TUBT2, fRPB2-5f2/fRPB2-7cr, LR0R/LR5, and NL1/NL4 to amplify the loci of the representative isolate in the ribosomal internal transcribed spacers (ITS), translation elongation factor EF-1α (TEF1), β-tubulin 2 (TUB2), RNA polymerase II largest-subunit (RPB2), the Large subunit (LSU) region of rDNA, 26S rDNA D1/D2 domain. The products were sequenced and Blasted. Blast analysisf the ITS, TFE, TUB2, RPB2, LSU, and NL amplicon revealed more than 99% of sequence identify with Fusarium fujikuroi. These sequences were submitted to GenBank and the GenBank accession numbers were as follows MT864359 (ITS), MT864358 (LSU), MT877222 (NL), MT902141 (RPB2), MT902140 (TEF), and MT902139 (TUB2). The phylogenetic tree was inferred from the combined datasets (TEF, TUB, and PRB2) from members of the F. fujikuroi species complex analyzed in this study (Jayawardena et al. 2019). The Phylogenetic tree revealed isolate HJYB-4 matched F. fujikuroi with a clade credibility value of 100%. According to the morphological characteristics and multi-gene phylogenetic analysis, the isolate was identified as F. fujikuroi. To complete pathogenicity tests, healthy leaves were needle-wound inoculated with mycelial plugs. The leaves that were inoculated with PDA-only plugs served as the control. After 3 days incubation at 26±2°C and 100% relative humidity, brown lesions that developed from inoculated leaves were similar to those in the field. No symptoms developed in the controlled leaves. Typical fungal cultures consistently isolated from symptomatic leaves, indicating that the fungus was responsible for the development of the disease. F. fujikuroi has been reported to cause root rot on Reineckia carnea (Sun et al. 2018), black rot on Macleaya cordata (Yu et al. 2019), and a wilt disease on sugarcane (Bao et al. 2020). This is the first report of F. fujikuroi attacking leaf of Z. armatum in China. The identification of this disease could provide the basis for the prevention and control of the disease at the seedling stage of Z. armatum.