First Report of Leaf Spot Caused by Nigrospora hainanensis on Oxalis corymbosa in China.

Abstract

Oxalis corymbosa DC. introduced into China as an ornamental plant in the mid-19th century is commonly known as an important medicinal and edible perennial herb (Zhou et al. 2021). The plant native to South America is also an invasive and widely distributed weed found in agricultural farms, gardens, and lawns, especially in sugarcane fields of Guangxi province, China. The coverage rate of O. corymbosa in sugarcane fields was normally more than 70%, sometimes up to 100%. In March of 2021, a leaf spot disease of O. corymbosa from sugarcane fields was encountered in Nanning city of Guangxi province, China. Early symptoms appeared as small yellowish round spots. The spots turned to be irregularly, usually exhibiting pale brown necrosis in the center with dark brown necrotic well-defined margins. Severely infected leaves turned to be blighted, then dead. To isolate the pathogen, diseased leave tissue fragments (4 mm × 4 mm) were soaked in 75% ethanol for 10 s followed by 2% sodium hypochlorite for 1 min, and rinsed by sterile water for three times. They were transferred to potato dextrose agar (PDA) medium cultured at 25 °C. Pure cultures were obtained by collected hypha tip from upcoming colonies. The colony features were similar to each other, floccose, white at first, becoming brown, dark brown or black on PDA after 7 days fully covered the 90 mm petri-dishes. Conidial determination were conducted on synthetic nutrient-poor agar medium (SNA) according to Wang et al. (2017). Conidia abundantly dispersed on SNA arising from conidiophores, which normally reduced to conidiogenous cells generated from hyphae. The conidiogenous cells were monoblastic, hyaline, globose or ampulliform, 6-8.5 (-12.5) × 5-7.5 (-9) μm in size (n=50). Conidia were solitary, smooth, black, sphaerical or ellipsoidal, (11-) 13-16.5 × (8-) 10-15.5 μm in size (n=100). Setae were not observed during the observation. The fungus was identified as Nigrospora sp. based on the morphology. One of the representative strains (FSC-3) was selected for genomic DNA extraction. The sequences of transcribed spacer region of rDNA (ITS), the partial translation elongation factor (TEF1), and the Beta-tubulin fragment (TUB) were respectively amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F and EF2 (Carbone & Kohn 1999, Crous et al. 2013) and Bt2a and Bt2b (Glass & Donaldson 1995), deposited in the NCBI GenBank with accession numbers of OK083685 (541 bp), OK184809 (481 bp) and OK086377 (421 bp). BLASTn analysis showed that those ITS, TEF1 and TUB gene sequences shared 99%-100% identity with the type strain (CGMCC3.18129) of Nigrospora hainanensis (GenBank accession nos. NR153480, KY019415, KY019464, respectively). In addition, a maximum likelihood analysis using concatenated gene sequences of ITS, TEF1 and TUB was performed in RAxML v.7.2.8 (Stamatakis 2006) implementing the model of GTRCAT with 1,000 bootstrap replicates. The phylogenetic results indicated that the strain FSC-3 was N. hainanensis, which also confirmed after a morphological comparison with N. hainanensis (Wang et al. 2017). Pathogenicity was tested on living Oxalis corymbosa leaves (3 plants for each test) arising from cultivated roots grown for three weeks. It was conducted by dropping 5 μL conidial suspensions (105 conidia / mL) on the living leaves (two sites per leave) incubated in separate containers at 25 °C with 90-100% relative humidity after inoculation. Controls were treated with sterile distilled water. Pale brown small spots came up after 24 h, and then extended to brown larger spots. Symptoms after inoculation were similar to field ones, while the control plants remained healthy. The pathogenicity test was repeated twice with the similar results. Re-isolation of the pathogen from the inoculated leaves was determined based on morphology and sequence analysis to fulfill Koch's postulates. Nigrospora hainanensis had been found from diseased root and leaf tissues of sugarcane in Liuzhou city, Guangxi province (Raza et al. 2019). The results indicated that O. corymbosa was another host in sugarcane fields in Guangxi, China. To our knowledge, this is the first report of Nigrospora hainanensis causing leaf spot on Oxalis corymbosa in China.