First report of Fusarium incarnatum associated with fruit rot disease of drumstick (Moringa oleifera L.) in India.

Moringa oleifera Lam. (Moringaceae) is one of the important leafy vegetable trees widely spread from India to Africa and widely used as food and medicine. During field investigation (July, 2021) of drumstick fields in Hiriyur (13°95'79" N; 76°64'45" E), Karnataka, fruits of drumstick plants showed a characteristic rot disease with an incidence ranging from 10-12% in an area of 5 hectares surveyed. Initially water soaked lesions turned to small necrotic lesions and later coalesced to form larger areas covered with white mycelial growth leading to softening and later mummification of fruits. Infected fruits were collected (n=5) and infected fruit parts (margins of healthy and infected tissues) were cut into small pieces, surface sterilized with sodium hypochlorite (2%, v/v) and blotter dried after three sterile water washes. An associated fungal species was isolated on PDA medium amended with Streptomycin (40 mg/L) and incubated at 28 ºC for 1 week. The fungal isolate grown on PDA had dense, white, aerial mycelium with light brown coloration on the reverse side of the agar medium. Morphological characteristics of conidia were determined for single-spore cultures grown on water agar media. Microconidia were single-celled, hyaline, non septate, ovoid, and 8.4 - 9.8 × 1.8 - 2.94 μm (n=20). Macroconidia were three- to five-septate, slightly curved, tapered at the apex, and 24.4 - 28 × 2 - 4 μm (n=20). Based on morphological characters' pathogen was identified as Fusarium sp. (Leslie and Summerell, 2006). Further, three representative isolate (SMG, MYS1 & MYS2) were subjected for molecular identification. The genomic DNA was extracted following CTAB method and ITS-rDNA was amplified using ITS1/ITS4 primers (White et al., 1990) and translation elongation factor (tef-1α) gene was amplified using EF1/EF2 primers (O'Donnell et al., 2009) respectively. ITS-rDNA sequence shared 100% sequence similarity (650bp / 650bp) with reference sequence F. incarnatum (ON226997) followed by 100% sequence identity to F. equiseti (KT277307 & MT953927). But, tef-1α gene sequence analysis in nBLAST showed that the sequence shared 100% (123/123bp) identity with F. incarnatum (F. incarnatum NEAU-TG1 MH920853; M2JP3 OP312673; WEH ON456146). Combined phylogenetic analysis revealed that the isolate shared a common clade with reference sequence of F. incarnatum in the Fusarium-incarnatum-equiseti species complex thus confirming the identity of the isolated pathogen as F. incarnatum (Rob. ex Desm.) Sacc. 1886. The sequences obtained in the present study are deposited in GenBank database (ITS: OP508729, OQ159019, OQ159020 and tef-1α: OP477394, OQ176254, OQ176255). To prove Koch's postulates, pathogenicity test was carried out by inoculating healthy drumstick fruits cv. Bhagya (n=10) with spore suspension (105 conidia/ml). Control fruits (n=5) were sprayed with sterile water. The experiments were conducted in triplicates and repeated twice. Inoculated and control fruits were kept in a moist chamber at 26 ± 2°C for 2 days and observed at regular intervals. Development of disease symptoms were recorded on 52 out of 60 inoculated fruits which were identical with symptoms seen in the field and all control fruits remained symptomless. Identity was confirmed after re-isolation by morphology and culture studies. To the best of our knowledge, this is the first report of F. incarnatum causing a fruit rot of drumstick in India. This disease affected the cost of drumstick production and contributed to the decline in production in India.