Thangavelu Raman, Muthukathan Gopi, Pushpakanth Periaswamy, Murugan Loganathan, Edwin Raj Esack, N Marimuthu, M Prabakaran, Uma Subbaraya
ICAR-National Research Centre for Banana, Plant Pathology, ICAR-National, Tiruchirapalli, India, 620102.
4/115, 16th cross, shanmuganagar UKTIndia;
Plant Dis. 2020 Dec 2. doi: 10.1094/PDIS-09-20-2052-PDN.
Fusarium wilt caused by Fusarium oxysporum f.sp. cubense (Foc) is the most devastating disease affecting commercial and subsistence cultivation of banana (Musa spp.) worldwide. Generally, the Cavendish bananas are resistant to Foc race 1 that destroyed cv. 'Gros Michel' (AAA) and susceptible to tropical race 4 (TR4), which is causing severe epidemics in different banana-growing countries including India (Thangavelu et al. 2019). In 2019, a roving survey was conducted in major banana growing states of India such as Bihar, Uttar Pradesh, Gujarat and Tamil Nadu to assess the incidence of Fusarium wilt disease in Cavendish bananas and also to characterize the pathogens by different methods including Vegetative Compatibility Grouping (VCG) and molecular methods. The Fusarium wilt incidence in cv. Grand Naine (Cavendish group-AAA) was 6-65% in Bihar, 30-45% in Uttar Pradesh, 5-15% in Gujarat and 15- 21% in Tamil Nadu. For characterization, a total of 61 samples from the Fusarium wilt infected Cavendish bananas were collected and single spore culture of Foc was obtained. The morphological characterization revealed the presence of one to two oval- to kidney-shaped cells in false heads and sickle-shaped macroconidia and a foot-shaped basal cell. The pathogenicity was demonstrated by adopting randomized block design with five replications on cv. Grand Naine. The Koch's postulate was successfully completed by re-isolation of the inoculated Foc pathogen and characterization by PCR method. The VCG analysis carried out using nit-M testers of all known VCGs indicated the presence of VCG 0125 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Tamil Nadu (Cumbum of Theni district), VCG 01220 from the Foc samples collected from cv. Grand Naine grown in Uttar Pradesh (Siswabazar of Maharakanj district) and Gujarat (Kamrej of Surat district,) and VCG 01213/16 from Foc samples collected from Uttar Pradesh (Siswabazar of Maharakanj district) and Bihar (Falka village of Katihar district) . The molecular confirmation of these VCGs 0125, and 01220 (Foc R1) isolates was carried out by PCR method using the primer set SIX6b_210_F and SIX6b_210_R (Carvalhais et al. 2019) for Foc R1, primer sets Foc TR4-F & Foc TR4 -R (Dita et al. 2010) for Foc TR4 and primer set Foc-1/Foc -2 (Lin et al. 2009) for Race 4. The results showed that only the primer set for Foc R1 has generated the expected amplicon size of 210 bp in the Foc isolates of VCG 0125 and 01220. Besides, the sequencing of Translation Elongation Factor (TEF) 1-α gene and BLAST searches in Genbank for the representative Foc isolates of VCG 0125 (Genbank no. MW 286800) showed 99.84% similarity to Foc R1 (KX365393.1) and Foc isolates of VCG 01220 (Genbank no. MW 286803) showed 99.69% similarity to Foc R1 (KX365413.1). Further, a phylogenetic analysis performed using the TEF1-α gene sequences showed that the Foc race 1 isolates (VCGs 0125 and 01220) from India were grouped with known Foc race 1 isolates from Tanzania and Australia. Based on the experimental results the study has confirmed the presence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India. As these VCGs are most widely distributed and do not found to infect Cavendish bananas so far (Mostert et al. 2017), this report is very important from the quarantine and management perspectives. To the best of our knowledge, this is the first report of the occurrence of VCGs 0125 and 01220 of Foc Race 1 in cv. Grand Naine in India.
由尖孢镰刀菌古巴专化型(Foc)引起的枯萎病是影响全球香蕉(Musa spp.)商业种植和自给种植的最具毁灭性的病害。一般来说,卡文迪什香蕉对摧毁了“大麦克”品种(AAA)的Foc 1号生理小种具有抗性,而对热带4号生理小种(TR4)敏感,TR4正在包括印度在内的不同香蕉种植国家引发严重疫情(Thangavelu等人,2019年)。2019年,在印度主要香蕉种植邦,如比哈尔邦、北方邦、古吉拉特邦和泰米尔纳德邦进行了一次巡回调查,以评估卡文迪什香蕉中枯萎病的发病率,并通过包括营养体亲和群分组(VCG)和分子方法在内的不同方法对病原菌进行鉴定。在比哈尔邦,“大奈”品种(卡文迪什组-AAA)的枯萎病发病率为6-65%,北方邦为30-45%,古吉拉特邦为5-15%,泰米尔纳德邦为15-21%。为了进行鉴定,从感染枯萎病的卡文迪什香蕉中总共收集了61个样本,并获得了Foc的单孢培养物。形态学鉴定显示,在假头状体中存在一到两个椭圆形至肾形细胞,有镰刀形大分生孢子和足形基部细胞。通过在“大奈”品种上采用随机区组设计并重复五次来证明致病性。通过对接种的Foc病原菌进行再分离并用PCR方法进行鉴定,成功完成了科赫法则。使用所有已知VCG的nit-M测试菌株进行的VCG分析表明,从北方邦(马哈拉坎杰区的西斯瓦巴扎尔)和泰米尔纳德邦(特尼区的坎布姆)种植的“大奈”品种收集的Foc样本中存在VCG 0125,从北方邦(马哈拉坎杰区的西斯瓦巴扎尔)和古吉拉特邦(苏拉特区的坎雷杰)种植的“大奈”品种收集的Foc样本中存在VCG 01220,从北方邦(马哈拉坎杰区的西斯瓦巴扎尔)和比哈尔邦(卡蒂哈尔区的法尔卡村)收集的Foc样本中存在VCG 01213/16。使用引物对SIX6b_210_F和SIX6b_210_R(Carvalhais等人,2019年)通过PCR方法对这些VCG 0125和01220(Foc R1)分离株进行分子确认,使用引物对Foc TR4-F和Foc TR4 -R(Dita等人,2010年)对Foc TR4进行确认,使用引物对Foc-1/Foc -2(Lin等人,2009年)对4号生理小种进行确认。结果表明,只有用于Foc R1的引物对在VCG 0125和01220的Foc分离株中产生了预期的210 bp扩增子大小。此外,对翻译延伸因子(TEF)1-α基因进行测序,并在Genbank中对VCG 0125(Genbank编号MW 286800)的代表性Foc分离株进行BLAST搜索,结果显示与Foc R1(KX365393.1)的相似性为99.84%,对VCG 01220(Genbank编号MW 286803)的Foc分离株进行搜索,结果显示与Foc R1(KX365413.1)的相似性为99.69%。此外,使用TEF1-α基因序列进行的系统发育分析表明,来自印度的Foc 1号生理小种分离株(VCGs 0125和01220)与来自坦桑尼亚和澳大利亚的已知Foc 1号生理小种分离株归为一组。基于实验结果,该研究证实了印度“大奈”品种中存在Foc 1号生理小种的VCGs 0125和01220。由于这些VCGs分布最广泛,且迄今为止未发现感染卡文迪什香蕉(Mostert等人,2017年),本报告从检疫和管理角度来看非常重要。据我们所知,这是印度“大奈”品种中首次报道Foc 1号生理小种的VCGs 0125和01220的发生情况。