Harlapur Sharanappa Ishwarappa, Ilger Krishnanand, Salakinkop S R, Talekar Sidramappa Channappa, Kachapur R M, Balol Gurupada, Patil Sanjay B, Tippannavar P S
University of Agricultural Sciences Dharwad, Plant Pathology, KELGERI ROAD, Research Complex, UAS, Dharwad, Dharwad,India, DHARWAD, KARNATAKA, India, 580008.
University of Agricultural Sciences Dharwad, UNIVERSITY OF AGRICULTURAL SCIENCES,DHARWAD, AICRP on Maize, Research Complex, UAS, Dharwad, Research Complex, UAS, Dharwad, Dharwad,India, Dharwad, University of Agricultural Sciences, Dharwad, India, 580005;
Plant Dis. 2022 Sep 15. doi: 10.1094/PDIS-08-22-1764-PDN.
Maize is a widely grown cereal crop in India and ranks third to wheat and rice in production (https://iimr.icar.gov.in). During a field survey in Kharif season in 2018, foliar chlorosis at the base and middle of leaves, and twisted top symptoms were observed in 40-50 days old maize plants in Belagavi district, Karnataka, India. Again during Kharif season in 2021, similar symptoms were observed on commercial maize hybrids and sugarcane at Agricultural Research Station, Sankeshwar Karnataka. The symptoms resembled Pokkah boeng disease of sugarcane (Vishwakarma et al. 2013). Symptomatic sugarcane and maize leaves were sampled, surface sterilized with 1.0% sodium hypochlorite, and 70% ethanol, and transferred on Potato dextrose agar, incubated for 10 days at 27±1°C. Fungal growth initiated with white mycelium later turned to pinkish-white with hyaline spores. The morphological features and sporulation patterns of maize and sugarcane samples were similar (e-Xtra 1). Microconidia were formed in long chains and clusters with oval to club-shaped, 0-septate, monophialide-borne microspores. DNA from representative pure culture isolates was extracted using the CTAB protocol (Doyle and Doyle, 1990). The ITS region of r-DNA was amplified with ITS1/ITS4 primers and sequenced. BLAST analyses of sequences of maize and sugarcane culture isolates at NCBI database revealed 100% homology with Fusarium verticillioides MK264336 (Lin et al., 2016). PCR amplification with Fusarium verticilliodes specific primers VER1/VER2 (Mule et al., 2004) confirmed the organism. CBS-KNAW Fungal Biodiversity Centre's Fusarium MLST database also revealed over 98.89% homology with Fusarium verticilliodes (NRRL 46612). The fungal isolates were named Fusarium verticilliodes maize isolate SNK 01 (ON110289) and Fusarium verticilliodes sugarcane isolate SNK 01 (ON564879), and their sequences were deposited in the GenBank. To test pathogenicity, artificial inoculation using maize isolate SNK 01 and cross-inoculation of sugarcane isolate SNK 01 were done on ten maize plants by spraying a conidial suspension (2×106 conidia ml-1) on nonwounded leaves. The plants sprayed with sterile water were used as control. After ten days, typical Pokkah boeng symptoms were observed in the plants inoculated with both maize and sugarcane isolates. Diseased leaves turned pale yellowish-green with small brown spots and a chlorotic appearance, further, these developed into stripes (e-Xtra 2). Wrinkling of leaves was noticed followed by splitting and rotting. No symptoms were noticed in the water-treated control. The pathogens re-isolated from diseased plants inoculated with maize and sugarcane isolates were similar morphologically and identical to the original isolates, fulfilling Koch's postulates. Hitherto, Fusarium verticilliodes was known to cause post-flowering stalk rot in maize. However, this is the first report of Pokkah boeng disease on maize in India caused by F. verticillioides. Considering the economic value of the maize crop, this identification can help develop appropriate disease management strategies to control the disease. References Lee, S. B., et al. 1988. A rapid, high yield mini-prep method for isolation of total genomic DNA from fungi. Fungal Genet. Newsl. 35:23-24. Lin, Z., et al. 2016. Deciphering transcriptomic response of Fusarium verticillioides in relation to nitrogen availability and the development of sugarcane Pokkah boeng disease. Sci. Rep. 6, 29692. Mule, G., et al. 2004. A Species-Specific PCR assay based on the Calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. European J. Plant Path. 110:495-502 Vishwakarma, S.K., et al. 2013. Pokkah Boeng: an emerging disease of sugarcane. J. Plant Pathol. Microb. 4(3):170. https://iimr.icar.gov.in. Director's desk, ICAR-Indian Institute of Maize Research. (Accessed September 8, 2022).
玉米是印度广泛种植的谷类作物，产量仅次于小麦和水稻，位列第三（https://iimr.icar.gov.in）。2018年雨季实地调查期间，在印度卡纳塔克邦贝拉尔加维区40 - 50日龄的玉米植株上，观察到叶片基部和中部出现叶部黄化以及顶部扭曲症状。2021年雨季期间，在卡纳塔克邦桑凯什瓦尔农业研究站的商业玉米杂交种和甘蔗上也观察到了类似症状。这些症状与甘蔗的梢腐病相似（Vishwakarma等人，2013年）。对出现症状的甘蔗和玉米叶片进行采样，用1.0%次氯酸钠和70%乙醇进行表面消毒，然后转移到马铃薯葡萄糖琼脂培养基上，在27±1°C下培养10天。真菌生长初期为白色菌丝体，随后变为粉白色，并带有透明孢子。玉米和甘蔗样本的形态特征和产孢模式相似（补充材料1）。分生孢子形成长链和簇状，呈椭圆形至棍棒状，0隔膜，单瓶梗着生的小孢子。使用CTAB方法（Doyle和Doyle，1990年）从代表性的纯培养分离物中提取DNA。用ITS1/ITS4引物扩增r - DNA的ITS区域并进行测序。在NCBI数据库中对玉米和甘蔗培养分离物的序列进行BLAST分析，结果显示与轮枝镰孢菌MK264336（Lin等人，2016年）具有100%的同源性。用轮枝镰孢菌特异性引物VER1/VER2（Mule等人，2004年）进行PCR扩增，证实了该生物体。CBS - KNAW真菌生物多样性中心的镰刀菌多位点序列分型数据库也显示与轮枝镰孢菌（NRRL 46612）的同源性超过98.89%。这些真菌分离物被命名为轮枝镰孢菌玉米分离株SNK 01（ON110289）和轮枝镰孢菌甘蔗分离株SNK 01（ON564879），它们的序列已存入GenBank。为了测试致病性，通过在10株玉米植株的未受伤叶片上喷洒分生孢子悬浮液（2×106个分生孢子/毫升），使用玉米分离株SNK 01进行人工接种，并对甘蔗分离株SNK 01进行交叉接种。喷洒无菌水的植株用作对照。10天后，在接种了玉米和甘蔗分离株的植株上观察到典型的梢腐病症状。患病叶片变为淡黄绿色，带有小褐色斑点并呈现黄化外观，进而发展成条纹（补充材料2）。注意到叶片出现皱纹，随后出现开裂和腐烂。水处理对照未出现症状。从接种了玉米和甘蔗分离株的患病植株上重新分离出的病原体在形态上相似，且与原始分离株相同，符合柯赫氏法则。此前，已知轮枝镰孢菌会导致玉米开花后茎腐病。然而，这是印度首次报道由轮枝镰孢菌引起的玉米梢腐病。考虑到玉米作物的经济价值，这一鉴定有助于制定适当的病害管理策略来控制该病。参考文献：Lee, S. B.,等人，1988年。一种从真菌中分离总基因组DNA的快速、高产小量制备方法。真菌遗传学通讯。35:23 - 24。Lin, Z.,等人，2016年。解析轮枝镰孢菌与氮素有效性及甘蔗梢腐病发生相关的转录组反应。科学报告。第6卷，29692。Mule, G.,等人，2004年。基于钙调蛋白部分基因的种特异性PCR检测方法用于鉴定轮枝镰孢菌、层出镰孢菌和亚粘团镰孢菌。欧洲植物病理学杂志。110:495 - 502。Vishwakarma, S.K.,等人，2013年。梢腐病：甘蔗的一种新出现病害。植物病理学与微生物学杂志。4(3):170。https://iimr.icar.gov.in。ICAR - 印度玉米研究所所长办公室。（2022年9月8日访问）
