First Report of Fusarium incarnatum-equiseti Species Complex Associated with Boll Rot of Cotton in Pakistan

Cotton (Gossypium hirsutum L.) is grown on over 2.8 million hectares in Pakistan with approximately 12.8 million bales annual production (Pakistan Economic Survey, 2013 to 2014, http://www.finance.gov.pk). During the summers of 2015, 2016, and 2017, symptoms associated with boll rot were regularly observed in cotton-growing areas in the southern Punjab. The initial symptoms on cotton bolls included large, necrotic lesions exhibiting internal decay. As lesions matured, the lesions were obscured with pale pinkish-orange to salmon-colored fungal growth. Samples were collected from fields from five different locations (20 bolls/location). All bolls exhibited the same symptoms, out of which 10 bolls were randomly selected for fungal isolation, morphological identification, and pathogenicity tests. All showed identical morphological characteristics. Symptomatic bolls were surface disinfested with ethanol (70%) for 30 s and sodium hypochlorite (1%) for 5 min and were then washed with sterile water. Small pieces of boll tissue (3 to 4 mm³) were removed from the edge of each lesion and placed on potato dextrose agar (PDA) medium supplemented with 50 mg/liter of streptomycin sulfate. Plates were incubated at 28°C for 7 days with an alternating cycle of light and dark. Colonies grown on PDA had dense, white, aerial mycelium with salmon coloration on the reverse. Morphological characteristics were determined from single-spore cultures grown on carnation leaf agar medium at 25°C for 7 days under a 12-h light/dark cycle. Microconidia were ovoid, hyaline, single celled, nonseptate, measuring 11 to 13 × 3 to 4 μm. Macroconidia were three to five septate, slightly curved, tapered at the apex, measuring 26 to 31 × 3.5 to 5 μm. On the basis of morphological features as observed microscopically, the fungus was believed to be from the genus Fusarium (Leslie and Summerell 2006). DNA was extracted from 7-day-old fungal cultures. Polymerase chain reaction amplification and sequencing of DNA regions including internal transcribed spacer (ITS) using ITS1/ITS4 primers (White et al. 1990) and translation elongation factor 1-alpha (TEF1a) using EF1/ EF2 primers (Geiser et al. 2004) was carried out. Representative consensus sequences of ITS and TEF1a genes were deposited in GenBank with accession numbers MG957236 and MH445456, respectively. A BLAST search of ITS revealed 99% similarity with Fusarium incarnatum (KM519192.1) and F. equiseti (KY523100.1 and MG020431.1). The BLAST result of TEF1a gene showed 100% identity with F. incarnatum (KF993975.1). The pathogen was identified as F. incarnatum (Desm.) Sacc. 1886, which belongs to the F. incarnatum-equiseti species complex on a morphological and molecular basis. To confirm pathogenicity, a 20-µl spore suspension (1 × 10⁵ conidia/ml) of 10 fungal isolates was dispensed to inoculate unopened cotton bolls on plants. Conidial suspensions were applied to stigma scars, wall sutures, and scratch wounds on bracts, calyxes, and bolls by micropipette. The scratch wounds were made by micropipette tip. In total 20 bolls were selected for each fungal isolate from five selected plants, four bolls per plant. In total, 50 plants were inoculated. Control bolls were treated with sterilized distilled water. Inoculated and control bolls were kept in a moist chamber at 26 ± 2°C and observed for the development of disease symptoms after 10 days. To our knowledge, this is the first report from a member of the Fusarium incarnatum-equiseti species complex causing boll rot of cotton in Pakistan.