First report on the seed association of <i>Lasiodiplodia theobromae</i> on Cotton in India and its rapid detection

Cotton (Gossypium spp.) is a major fiber and oilseed crop of global economic importance, belonging to the family Malvaceae. Cotton lint is the most important natural fibre, providing the raw material for the global textile industry (Smith and Cothren 1999). India has the largest area under cotton cultivation (11.36 million ha), with a production of 5.09 million tonnes and a productivity of 448 kg ha⁻¹ (ICAC 2025). However, cotton production is affected by several diseases, seedborne diseases being one of the major constraints limiting the yield. With the aim to investigate the presence of seedborne pathogens in cotton, different seed samples were collected and surface sterilized with 1% sodium hypochlorite and incubated in potato dextrose agar medium at 27±2°C for 5 days. Isolation and examination of seedborne pathogens led to the identification of Lasiodiplodia theobromae associated with G. hirsutum samples which was purified by hyphal tip cutting method (Fig. 1A). In PDA medium, the isolate was fast growing, initially white, turning dark grey to black with dense, fluffy, aerial and septate mycelium (Fig. 1B). The pathogen produced intercalary hyphal cells which became enlarged and thickened, measuring 7.20 to 7.73 µm × 6.51 to 7.22 µm (Fig. 1C). The pathogenicity of L. theobromae (isolate SK2) was evaluated by seed inoculation using mycelial suspension prepared from a 7-day-old culture grown in potato dextrose broth for 30min, wherein L. theobromae resulted in reduction in germination (by 40%) against untreated control (100%) (16 seeds). Further, germinated seedlings of LRA 5166 variety showed yellowing of cotyledonary leaves, later wilting and drying of the seedlings (Fig. 2). The pathogen was re-isolated from the stem of diseased plants and colonies were identical to the isolate (SK2). The fungal DNA was extracted from mycelium using the CTAB method (Murray and Thompson 1980). The Internal transcribed spacer (ITS) region (ITS1 and ITS4) (White et al. 1990) and β-tubulin gene (TUB2) (Glass and Donaldson 1995) of the pathogen were amplified and sequenced (Sanger sequencing). The sequence identity for both the regions was compared with the reference sequences available in NCBI database (Fig. 3) wherein, sequence of ITS (GenBank Acc. no. PV300409) showed 98.28 % similarity with reference strain L. theobromae MML4305 (GenBank Acc. no. MH049696) and that of β-tubulin (GenBank Acc. no. PX552539) showed 99.34 % similarity with L. theobromae isolate Bot-2017-LT6 (GenBank Acc. no. MW574066). Further, to enable the rapid detection of L. theobromae from seeds, primers were designed to amplify the L. theobromae-specific markers identified in this study, targeting the ITS region (LasITSF: 5’-TCTCCCACCCTTTGTGAACG-3’/ LasITSR: 5’-ACTACGCTTGAGGGCTGAAC-3’) and TUB2 gene (LasBTR: 5’-ATGGCTCCGGTGTGTAAGTG-3’/ LasBTR: 5’-GACCCTTGGCCCAGTTGTTA-3’). These L. theobromae specific markers produced amplicons of 362bp (Fig. 4A) and 322bp (Fig. 4B), respectively. Both the designed primers, along with specificity to L. theobromae, also showed very high sensitivity of 1pg and 0.1ng respectively (Fig. 5). The primers were further validated using DNA from L. theobromae inoculated seeds. Previously, Lasiodiplodia sp. has been reported to cause twig and stem blight in cotton (Naz 2021). To our knowledge, this is the first report of the seed association of L. theobromae with cotton seeds in India along with the development of a rapid detection tool.