Yan L Y, Kang Y P, Lei Y, Huang J Q, Wan L Y, Liao B S
Oil Crops Research Institute (OCRI) of Chinese Academy of Agricultural Sciences (CAAS)/Key Lab of Oil Crop Biology of the Ministry of Agriculture, Hubei, Wuhan 430062, China.
Plant Dis. 2014 Jan;98(1):156. doi: 10.1094/PDIS-05-13-0476-PDN.
Peanut, Arachis hypogaea L., is an important source of vegetable oil and protein in China with a planting area of 5 million ha and annual production of 16.2 million metric tons. In September of 2012, typical symptoms of Sclerotinia blight were first observed on peanut in Shuangcheng, Heilongjiang Province, China. Approximately 50% of the plants in a field were infected. Symptoms began as a chlorotic wilt on the foliage and developed into necrosis of basal stems and developed very quickly in the field. In advanced stages of the disease, stems and branches became bleached and eventually died. White, fluffy mycelium and black irregular sclerotia (3.5 to 5.4 mm diameter) were observed on the infected stems. Infected branches and pegs were shredded, and most pods dropped on the soil during harvest. To isolate the causal agent of the disease, sclerotia were collected from the field. Twenty sclerotia were surface disinfected in 1% NaOCl for 3 min, rinsed three times with sterile water, placed on potato sucrose agar (PSA) with 100 μg/ml streptomycin, and then incubated at 22°C in the dark for 10 days. Fungal DNA was extracted from mycelia with a TIANGEN DNAsecure Plant Kit (Beijing) and amplified by PCR with the universal fungal primer ITS1 and ITS4. PCR products of five replicates were sequenced and subjected to an NCBI BLAST search. The BLAST search revealed that our sequences (GenBank Accession No. KC935388) had 100% identity with reported sequences of Sclerotinia sclerotiorum. The isolates were identified as S. sclerotiorum (Lib.) de Bary based on mycelia, sclerotia, and rDNA sequence analysis. To conduct pathogenicity tests, nine potted peanut plants (1 month old) were each inoculated with a 5-mm-diameter disk of colonized PSA by placing the inoculum on the base of the stem. Plants were inoculated with a plug of non-colonized PSA as controls. All inoculated plants were covered with a plastic bag for 5 days to maintain high humidity and incubated at 22 to 24°C in the growth chamber with a 12-h photoperiod. After 2 days, the inoculated plants showed water-soaked brown symptoms on the stem base and whole plants wilted after 5 days under high moisture conditions. The symptoms were identical to those observed on peanut plants in Shuangcheng, Heilongjiang Province, whereas the control plants remained symptom-free. Re-isolation of the fungus from the inoculated plants confirmed that the causal agent was S. sclerotiorum. S. sclerotiorum has been reported on peanut in the United States and Argentina (1,2,3). To our knowledge, this is the first report of S. sclerotiorum as a pathogen causing Sclerotinia blight on peanut in northeastern China. References: (1) A. Marinelli et al. Int. J. Pest Manage. 44:251, 1998. (2) J. E. Woodward et al. Plant Dis. 90:111, 2006. (3) J. E. Woodward et al. Plant Dis. 92:1468, 2008.
花生,落花生(Arachis hypogaea L.),是中国植物油和蛋白质的重要来源,种植面积达500万公顷,年产量为1620万吨。2012年9月,在中国黑龙江省双城市的花生上首次观察到菌核病的典型症状。一块田地里约50%的植株受到感染。症状始于叶片的褪绿萎蔫,发展为基部茎干坏死,且在田间发展迅速。在病害后期,茎干和枝条变白,最终死亡。在受感染的茎干上观察到白色、蓬松的菌丝体和黑色不规则菌核(直径3.5至5.4毫米)。受感染的枝条和果针被撕裂,收获时大多数荚果掉落在土壤上。为分离该病害的病原菌,从田间采集菌核。将20个菌核在1%次氯酸钠中表面消毒3分钟,用无菌水冲洗三次,置于添加100μg/ml链霉素的马铃薯蔗糖琼脂(PSA)上,然后在22°C黑暗条件下培养10天。用天根植物基因组DNA提取试剂盒(北京)从菌丝体中提取真菌DNA,并使用通用真菌引物ITS1和ITS4通过PCR进行扩增。对五个重复的PCR产物进行测序,并在NCBI上进行BLAST搜索。BLAST搜索显示,我们的序列(GenBank登录号KC935388)与已报道的核盘菌序列具有100%的同一性。基于菌丝体、菌核和rDNA序列分析,这些分离物被鉴定为核盘菌(Sclerotinia sclerotiorum (Lib.) de Bary)。为进行致病性测试,将九个盆栽花生植株(1月龄)通过在茎基部放置一块直径5毫米的已在PSA上定殖的菌盘进行接种。用一块未在PSA上定殖的菌盘接种植株作为对照。所有接种的植株用塑料袋覆盖5天以保持高湿度,并在生长室中12小时光周期、22至24°C条件下培养。2天后,接种的植株在茎基部出现水渍状褐色症状,并在高湿度条件下5天后整株萎蔫。这些症状与在黑龙江省双城市花生植株上观察到的症状相同,而对照植株未出现症状。从接种的植株上再次分离出真菌,证实病原菌为核盘菌。在美国和阿根廷的花生上已报道过核盘菌(参考文献(1)(2)(3))。据我们所知,这是核盘菌作为引起中国东北地区花生菌核病的病原菌的首次报道。参考文献:(1) A. Marinelli等人,《国际害虫管理杂志》44:251,1998年。(2) J. E. Woodward等人,《植物病害》90:111,2006年。(3) J. E. Woodward等人,《植物病害》92:1468,2008年。