First Report of Leaf Spot Caused by <i>Lasiodiplodia theobromae</i> on Bitter Gourd in India

Bitter gourd (Momordica charantia L.), a member of the Cucurbitaceae family, is an economically important vegetable crop cultivated in India, valued for its nutritional and medicinal properties (Mallikarjuna et al. 2023). In May 2024, leaf spot disease symptoms were observed on bitter gourd in Dakshin Charilam (23.630 N, 91.320E), Sepahijala, Tripura, India with disease incidence of leaf spot ranged from 6% to 11% (n = 100 plants) in a 1-ha field. Symptoms included small, water-soaked, angular, gray or straw-colored spots with a yellow halo. As the disease progressed, spots became dry and dropped leaving irregularly shaped holes in the leaves. Ten symptomatic diseased leaves were randomly collected from 10 individual plants and cut into small pieces (5.0 mm2) at the junction between diseased and healthy tissue using a sterilized scalpel. The pieces were disinfected with 1% sodium hypochlorite (NaOCl) solution for 2 min and subsequently samples were washed thoroughly with sterilized distilled water three times. Disinfected tissue was transferred to Petriplates containing potato dextrose agar (PDA) and incubated at 25±2°C. Out of ten symptomatic leaves, eight isolates with similar morphological characterizations were isolated and purified. Two isolates (Bitter G-3 and Bitter G-3′) were selected for further morphological and molecular identification. After 7 days at 25±2ºC, colonies on PDA were fast growing, dark greyish black, with hyaline to olivaceous grey, septate, smooth-walled, 3–6.5 (x̄ = 4) μm wide, sporulating hyaline form. Conidia were aseptate, ovoid to ellipsoid (6.2 ± 1.01 × 14.5 ± 0.96 μm; n = 20). Genomic DNA was extracted from 7 days old freshly harvested mycelia using a fungus genomic DNA extraction kit (Biomiga, U.S.A.) and the partial internal transcribed spacer (ITS) region and translation elongation factor 1-alpha (tef1-α) gene were amplified, sequenced with primer pairs ITS4 / ITS5 (White et al. 1990) and EF1- 983F/EF1-2218R (Rehner and Buckley 2005), respectively, and submitted to GenBank (GenBank accession nos. PV248196, PV962840 for ITS and PV274565, PV976015 for tef1). BLASTn searches with the obtained sequences in GenBank revealed 99.79 % identity [466/467 bp; 100% Query Cover; and 0 gaps] with L. theobromae CBS 164.96 (Type; Sequence ID: NR_111174.1) based on ITS gene region and 100 % identity [314/314 bp; 100% Query Cover; and 0 gaps] with L. theobromae isolate CUZ044 (Sequence ID: PV441873.1) based on tef1 gene. A phylogenetic tree was constructed by MEGA11.0 using the Neighbor-Joining method (Saitou and Nei, 1987). Based on morphology and molecular analysis, the fungus was identified as L. theobromae. For pathogenicity test, twelve healthy leaves of three healthy bitter gourd plants were inoculated with a 5-mm-diameter mycelial disc and an additional three healthy plants inoculated with sterile PDA discs as control (He et al. 2021). After 5 days of inoculation, L. theobromae inoculated leaves of bitter gourd exhibited leaf spot symptoms similar to those observed in the field, while the control plants had no symptoms. The pathogen was reisolated (isolate Tri BG-3) from the symptomatic leaves in three successive trials, and identified as L. theobromae, thus fulfilling Koch’s postulates. DNA sequence for ITS and tef1-α gene were submitted to GenBank (GenBank accession nos. PV962841 for ITS and PV976016 for tef1). To the best of our knowledge, this is the first report of L. theobromae causing leaf spot in bitter gourd in India and it is crucial to monitor the occurrence of this pathogen in other bitter gourd growing areas as a potential threat to bitter gourd production.