First report of leaf spot caused by Nigrospora hainanensis on Oxalis corymbosa in China
First report of leaf spot caused by Nigrospora hainanensis on Oxalis corymbosa in China
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国内首例由海南黑孢菌引起的酢浆草叶斑病
DOI：
10.1094/pdis-10-21-2149-pdn
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发表时间：
--
期刊：
影响因子：
4.5
通讯作者：
Yan Hui Wang
中科院分区：
文献类型：
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作者：
Tao Zheng;Lin Zhao;Meng Ge Huang;Jian Xin Deng;Yan Hui Wang
紫叶酢浆草，世纪中期作为观赏植物引入中国，是一种重要的药用和食用多年生草本植物（Zhou et al. 2021）。原产于南美洲，它是一种入侵性的，广泛分布于农业农场，花园和草坪的杂草，特别是在中国广西省的甘蔗田。O.甘蔗田的伞房花序一般大于70%，有时达100%。2021年3月，一株O.在广西南宁市甘蔗田发现了一株甘蔗伞房花序。早期症状表现为小而黄的圆形斑点。斑点变得不规则，通常在中心显示淡棕色坏死，深棕色坏死，边缘清楚。严重感染的叶子枯萎死亡。为了分离病原体，将患病叶组织碎片（4× 4 mm）在75%乙醇中浸泡10 s，然后在2%NaOCl中浸泡1 min，并在无菌水中冲洗3次。将它们转移到马铃薯葡萄糖琼脂（PDA）培养基中并在25 ℃下培养.通过从菌落收集菌丝尖端获得纯培养物。殖民地特征彼此相似：最初为絮状和白色，在90 mm培养皿中完全覆盖在PDA上7天后变为棕色、深棕色或黑色。分生孢子测定根据Wang等人在合成贫营养琼脂（SNA）培养基上进行。（2017年）。分生孢子大量分散在SNA上，SNA由分生孢子梗产生，通常由菌丝产生的分生孢子细胞减少。产孢细胞为单胚层、透明、球形或壶状，直径6 ~ 8.5（~ 12.5）× 5 ~ 7.5（~ 9）μm（n= 50）。分生孢子单生，光滑，黑色，球形或椭圆形，直径（11 ~ 13 ~ 16.5）×（8 ~ 10 ~ 15.5）μm（n= 100）。未观察到刚毛。根据形态特征鉴定该真菌为黑孢菌属（Nigrospora sp.）。选择代表性菌株（FSC-3）进行基因组DNA提取。分别使用引物对ITS 1/ITS 4（白色et al. 1990）、EF 1 - 728 F和EF 2（Carbone and Kohn 1999; Crous et al. 2013）以及Bt 2a和Bt 2b（Glass and唐纳森1995）扩增rDNA转录间隔区（ITS）、部分翻译延伸因子（TEF 1）和β-微管蛋白片段（TUB）的序列。将它们保藏在NCBI GenBank（登录号：0 K 083685 [541 bp]、0 K184809 [481 bp]和0 K 086377 [421 bp]）。BLASTn分析表明，该菌株的ITS、TEF 1和TUB基因序列与模式菌株（CGMCC 3. 18129）的海南黑孢菌（Nigrospora hainanensis）（GenBank登录号Nos. NR 153480、KY 019415和KY 019464）。在RAxML v.7.2中使用ITS、TEF 1和TUB的串联基因序列进行最大似然分析。8（Stamatakis 2006），实施GTRCAT模型，具有1，000个自举重复。系统发育分析结果表明，FSC-3为N. hainanensis的形态学特征也证实了这一点。hainanensis（Wang et al. 2017）.对活O.伞房花序叶（每种试验三株植物），由生长3周的栽培根产生。将5 μl分生孢子悬浮液（105个分生孢子/ml）滴在叶片上（每片叶片两个位点），在25 ℃和90 - 100%相对湿度下在单独的容器中培养。对照组用无菌蒸馏水处理。24 h后出现淡褐色小斑点，并长成较大的褐色斑点。症状与田间症状相似，而对照植物保持健康。致病性试验重复两次，结果相似。从接种的叶片中重新分离出病原体。
Oxalis corymbosa DC., introduced to China as an ornamental plant in the mid-19th century, is an important medicinal and edible perennial herb (Zhou et al. 2021). Native to South America, it is an invasive, widely distributed weed found in agricultural farms, gardens, and lawns, especially in the sugarcane fields of Guangxi Province, China. The coverage of O. corymbosa in sugarcane fields is normally> 70%, and sometimes 100%. In March 2021, a leaf spot disease of O. corymbosa from sugarcane fields was encountered in Nanning City, Guangxi. Early symptoms appeared as small, yellowish, round spots. The spots became irregular, usually exhibiting pale brown necrosis in the center with dark brown, necrotic, and well-defined margins. Severely infected leaves became blighted and died. To isolate the pathogen, diseased leaf tissue fragments (4× 4 mm) were soaked in 75% ethanol for 10 s followed by 2% NaOCl for 1 min and rinsed in sterile water three times. They were transferred to potato dextrose agar (PDA) medium and cultured at 25 C. Pure cultures were obtained by collecting hyphal tips from colonies. The colony features were similar to each other: floccose and white at first, becoming brown, dark brown, or black after 7 days fully covered on PDA in 90-mm Petri dishes. Conidial determination was conducted on synthetic nutrient-poor agar (SNA) medium according to Wang et al.(2017). Conidia were 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, and 6 to 8.5 (to 12.5)× 5 to 7.5 (to 9) μm (n= 50). Conidia were solitary, smooth, black, spherical or ellipsoidal, and (11 to) 13 to 16.5×(8 to) 10 to 15.5 μm (n= 100). Setae were not observed. The fungus was identified as Nigrospora sp. based on the morphology. A representative strain (FSC-3) was selected for genomic DNA extraction. Sequences of the transcribed spacer region of rDNA (ITS), partial translation elongation factor (TEF1), and beta-tubulin fragment (TUB) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F and EF2 (Carbone and Kohn 1999; Crous et al. 2013), and Bt2a and Bt2b (Glass and Donaldson 1995), respectively. They were deposited in NCBI GenBank (accession nos. OK083685 [541 bp], OK184809 [481 bp], and OK086377 [421 bp]). BLASTn analysis showed that the ITS, TEF1, and TUB gene sequences shared 99 to 100% identity with the type strain (CGMCC3. 18129) of Nigrospora hainanensis (GenBank accession nos. NR153480, KY019415, and KY019464, respectively). A maximum likelihood analysis using the 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 FSC-3 was N. hainanensis, which was also confirmed by a morphological comparison with N. hainanensis (Wang et al. 2017). Pathogenicity was tested on living O. corymbosa leaves (three plants per test) arising from cultivated roots grown for 3 weeks. It was conducted by dropping 5 μl of conidial suspension (105 conidia/ml) on leaves (two sites per leaf) and incubating them in separate containers at 25 C with 90 to 100% relative humidity. Controls were treated with sterile distilled water. Pale brown small spots appeared after 24 h and grew into larger brown spots. Symptoms were similar to field ones, while the control plants remained healthy. The pathogenicity test was repeated twice with similar results. Reisolation of the pathogen from inoculated leaves was determined …