First report of Leaf Spot Disease Caused by Exserohilum rostratum on Bottle Gourd in India

Abstract

Bottle gourd (Lagenaria siceraria) is a medicinal and nutritious domesticated cucurbit vegetable having annual production of 2.37 million metric tons with a pantropical distribution in India. In August 2017, typical symptoms of leaf spot disease were observed on 3-month-old bottle gourd leaves sampled from open fields in Karnal, India, of sporadic level of about 10% severity. During monsoon season, initial symptoms appeared mostly on young leaves with grayish white to brown lesions surrounded by a yellowish halo, measuring 0.5 to 1.7 × 0.4 to 0.8 cm. These lesions expanded gradually from circular to cylindrical, coalescing and covering almost the entire leaf and ultimately leading to blighting of foliage and defoliation. Symptomatic advancing lesions leaf bits of 4 to 6 mm were surface sterilized with 0.1% HgCl2 for 45 s, rinsed three times in sterile distilled water, placed onto potato dextrose agar (PDA) medium, and incubated at 27°C for 7 to 10 days. The emerging fungal colonies on plates were circular, gray to brown, with abundant cottony aerial mycelium. Conidia were straight to slightly curved, ellipsoidal to narrowly obclavate or rostrate, brown, 5 to 14 septate, with basal septum darker and thicker than other septa, 22.4 to 154.4 × 8 to 14.7 μm, with a distinctly protruding basal hilum. Conidiophores were single, cylindrical, dark brown, geniculate, and 4 to 6 μm thick with a swollen basal cell. Further, it was identified by Indian Type Culture Collection, New Delhi (no. 10.643.17) as E. rostratum. For molecular identification, genomic DNA of isolate Ex-1 was extracted from a single conidial culture, and the internal transcribed spacer regions of rDNA (ITS1-5.8S-ITS4) were amplified and sequenced with universal primers ITS1 and ITS4 (Lin et al. 2011) and deposited in GenBank (MG711857). BLASTn analysis revealed 100% identity with E. rostratum isolates (GenBank accession nos. JN711432.1, FJ949084.1, and KU204880.1). A partial sequence of glyceraldehyde-3-phosphate dehydrogenase-like (Gpd) gene of Ex-1 was also amplified using primers GPD1 and GPD2 (Berbee et al. 1999; Liu et al. 2016), sequenced, and deposited in GenBank (MG918126). BLASTn analysis revealed 99% sequence similarity with an E. rostratum isolate (KU935741.1). Based on morphological characteristics and molecular analysis, the isolate was identified as E. rostratum (Drechs.) Leonard & Suggs (Ahmadpour et al. 2013; Lin et al. 2011). A pathogenicity experiment was conducted using five bottle gourd plants (three leaves inoculated in each plant). Newly matured leaves were inoculated by double spray at a 24-h interval with isolate Ex-1 conidial suspension (105 conidia/ml). Leaves sprayed with sterile water served as a negative control. Right after inoculation, plants were covered with plastic bags to maintain high relative humidity and incubated at ≈30°C with a natural photoperiod. Seven days later, all pathogen-inoculated plants showed symptoms identical to those observed in the field, whereas control plants remained asymptomatic. Koch’s postulates were proved with the reisolation of the fungus from leaves. E. rostratum has been reported as the causal agent of leaf spots on Zea mays, Ananas comosus, Musa paradisiaca, and Hevea brasiliensis in China (Liu et al. 2016). To our knowledge, this is the first report of E. rostratum causing leaf spot disease in bottle gourd in India. This disease occurred in quite a large bottle gourd area, and identification of pathogens was essential for developing integrated pest management strategies.