First report of Colletotrichum siamense causing Fruit Drop in Kinnow mandarin (Citrus nobilis x Citrus deliciosa) in Punjab, India

Kinnow, a hybrid developed by crossing two cultivars of mandarin i.e. King (Citrus nobilis) x Willow leaf (Citrus deliciosa) accounts for its high economic productivity and possess high processing quality along with great aromatic flavour. Kinnow mandarin cultivation occupies 4,73,000 ha of land in India with an annual production of 62,65,000 MT (Anonymous, 2021) and Punjab is the leading Kinnow producing state in India. Fruit drop is a major problem in Kinnow mandarin causing serious damage in the quality and yield of the fruit. During 2020-21, the symptoms of fruit drop were observed in Kinnow orchards of Punjab as; drying of twigs from the tip downwards, brown discoloured circular areas on stem-end of the fruit, rotting, and dropping of the fruit. To identify the casual agent, the infected fruit tissue bits (2 mm) was surface sterilized with 1% mercuric chloride solution for 30-40 s and given three washings of distilled water and placed on whatman filter paper-1 to soak all the excessive moisture. The bits was placed on Potato Dextrose Agar (PDA) plates and incubated at 25 +/- 1 degrees C. The colony colour ranged from creamy white to light gray with orange conidial mass in the centre. Based on various morphological characteristics i.e., conidial shape, size, spore production, presence or absence of fruiting body and size of fruiting body the fungus was identified as Colletotrichum siamense. For molecular confirmation ITS region, actin (ACT) and beta-tubulin2 (TUB2) genes were amplified using primers ITS-6/ITS-4, ACT-512F/ ACT-783R and T1/Bt2b, respectively. PCR amplicons for three loci (ITS, ACT and TUB2) were sequenced and sequences were deposited in GenBank as accession numbers OQ857285, OQ983464 and OQ983465 for isolate no. C-4 and OQ888798, OQ983466 and OQ983467 for isolate no. C-19, respectively. The BLAST analysis and multigene phylogenetic analysis showed that the isolates collected in present study were phylogenetically similar to the C. siamense. Thus, based on morphological, phylogenetic analysis and pathogenicity assays, the causal agent was identified as Colletotrichum siamense. This is the first report of C. siamense causing fruit drop of kinnow mandarin in Punjab, India.