Singh Dinesh, Sharma Sapna, Das Amrita, Thakuria Dwipendra, Sahoo Lingaraj, Kharbikar Lalit Laxman, Samal Swarnmala, Chand Gireesh, Bahadur Amar, Shakywar Ramesh Chandra
Indian Agricultural Research Institute, Division of Plant Pathology, Division of Plant Pathology, IARI, Pusa, New Delhi- 110012, Delhi, Delhi, India, 110012.
Indian Agricultural research Institute, Division of Plant Pathology, Division of Plant Pathology, IARI, Pusa, New Delhi- 110012, Delhi, Delhi, India, 110012;
Plant Dis. 2024 Jul 19. doi: 10.1094/PDIS-06-23-1156-PDN.
Khasi mandarin (Citrus reticulata Blanco) is the most economically important crop among the citrus growing region in the north-eastern India (Singh et al. 2016). An extensive survey was conducted to identify the causal agent of citrus root rot and gummosis in north eastern states (Meghalaya, Tripura, Manipur, Arunachal Pradesh, Sikkim, Nagaland and Assam) of India during October 2021-23. The gummosis disease incidence ranged from 5 to 95 % in 10 to 25 years old Khasi mandarin plants showing relatively more chronic symptoms on mature trees. Yellowing and dropping of leaves, twigs die back, gum oozing from infected bark and loss of feeder roots were the typical symptoms of the disease. Infected bark tissue and young lemon leaf baits in rhizosphere soil were plated on corn meal agar medium supplemented with pimaricin (10 µg/ml), ampicillin (250 µg/ml), rifamycin (10 µg/ml) and 300µg/ml carbendazim and incubated at 26℃. Fifty isolates were purified and maintained on Carrot agar medium. These isolates showed similar cultural and morphological characteristics. Two representative isolates from Arunachal Pradesh (AP21 and AP26) were selected for further experiments and deposited to Indian Type culture collection (ITCC), New Delhi with accession no. 9156 and 9157 respectively. The colonies were fast growing, showing rosette pattern along with whitish blooming mycelium appearance with no visual sporulation at the surface. The hyphae were coenocytic with initially right-angled branching. Sporangia were globose or sub globose and papillated. Oogonia were smooth and globose (16.29-21.09 µm) in diameter. Antheridia were irregular, cylindrical and broadly attached to oogonia. Empty sporangia were also observed. Multilocus phylogenetic analysis using internal transcribed spacer region (Das et al. 2011), β tubulin (Blair et al. 2008) and Cytochrome oxidase II gene (Noireung et al. 2020) showed that these isolates formed a stable clade with Phytopythium vexans (CBS119.80) sequence retrieved from NCBI database. BLAST analysis showed that ITS sequence of AP21 (OQ372986) and AP26 (OQ381083) had >99 % identity with P. vexans isolate NS-3 (ON533631). Further, BLAST analysis of β tubulin (AP21 OQ446053, AP26 OR405377) and Cox II gene (AP21 OQ473414, AP26 OR552422) sequences showed that our Indian isolates showed >99 % similarity with P. vexans voucher strain CBS119.80. To fulfil Koch's postulates, Khasi mandarin (Citrus reticulata) seedlings were inoculated by adding 100 ml zoospore suspension of P. vexans (1x105 spores/ml) in sterilized soil (Thao et al. 2020). The experiment was carried out in triplicate. Yellowing of leaves and leaf drop were observed 7 days post inoculation while 30 days post inoculation, treated plants started showing symptoms of root rot, including mild root decay. No symptoms were observed in control treatment. The pathogen was reisolated from symptomatic roots and confirmed through colony and sporangium morphology. Recently, it was reported that P. vexans is associated with apple and pear decline in the Saiss plain of Morocco (Jabiri et al. 2021), root rot on mandarin in Thailand (Noireung et al. 2020) and on Durian in Vietnam (Thao et al. 2020). As per our knowledge, this is the first report of P. vexans causing root rot and gummosis in Khasi mandarin from north eastern states of India. This finding is significantly important for the development of a successful disease management strategy in India.
卡西蜜柑（Citrus reticulata Blanco）是印度东北部柑橘种植区经济上最重要的作物（Singh等人，2016年）。2021年10月至2023年期间，在印度东北部各邦（梅加拉亚邦、特里普拉邦、曼尼普尔邦、阿鲁纳恰尔邦、锡金邦、那加兰邦和阿萨姆邦）进行了广泛调查，以确定柑橘根腐病和流胶病的病原体。在10至25年树龄的卡西蜜柑植株上，流胶病发病率为5%至95%，在成年树上表现出相对更慢性的症状。叶片发黄和脱落、嫩枝枯死、受感染树皮渗出树胶以及须根丧失是该病的典型症状。将感染的树皮组织和根际土壤中的幼柠檬叶诱饵接种在添加了匹马霉素（10 µg/ml）、氨苄青霉素（250 µg/ml）、利福霉素（10 µg/ml）和300 µg/ml多菌灵的玉米粉琼脂培养基上，并在26℃下培养。纯化了50个分离株，并保存在胡萝卜琼脂培养基上。这些分离株表现出相似的培养和形态特征。从阿鲁纳恰尔邦选取了两个代表性分离株（AP21和AP26）进行进一步实验，并分别以登录号9156和9157保藏于新德里的印度模式培养物保藏中心（ITCC）。菌落生长迅速，呈莲座状，表面有白色菌丝体，无可见孢子形成。菌丝多核，最初呈直角分支。孢子囊球形或近球形，有乳头状突起。藏卵器光滑，球形（直径16.29 - 21.09 µm）。雄器不规则，圆柱形，广泛附着于藏卵器。还观察到空孢子囊。使用内转录间隔区（Das等人，2011年）、β微管蛋白（Blair等人，2008年）和细胞色素氧化酶II基因（Noireung等人，2020年）进行的多位点系统发育分析表明，这些分离株与从NCBI数据库检索到的Phytopythium vexans（CBS119.80）序列形成了一个稳定的分支。BLAST分析表明，AP21（OQ372986）和AP26（OQ381083）的ITS序列与P. vexans分离株NS - 3（ON533631）的同源性>99%。此外，β微管蛋白（AP21 OQ446053，AP26 OR405377）和Cox II基因（AP21 OQ473414，AP26 OR552422）序列的BLAST分析表明，我们的印度分离株与P. vexans模式菌株CBS119.80的相似性>99%。为了满足科赫法则，在灭菌土壤中添加100 ml P. vexans游动孢子悬浮液（1×105个孢子/ml）对接种卡西蜜柑（Citrus reticulata）幼苗（Thao等人，2020年）。实验重复三次。接种后7天观察到叶片发黄和落叶，而接种后30天，处理过的植株开始出现根腐症状，包括轻度根腐烂。对照处理未观察到症状。从有症状的根中重新分离出病原体，并通过菌落和孢子囊形态进行了确认。最近，有报道称P. vexans与摩洛哥塞伊斯平原的苹果和梨衰退有关（Jabiri等人，2021年）、泰国蜜柑根腐病（Noireung等人，2020年）以及越南榴莲根腐病（Thao等人，2020年）。据我们所知，这是P. vexans引起印度东北部各邦卡西蜜柑根腐病和流胶病的首次报道。这一发现对印度成功制定病害管理策略具有重要意义。
