First report of Nigrospora sphaerica associated with leaf spot disease of Crossandra infundibuliformis in India

Crossandra (Crossandra infundubuliformis (L.) Nees.) is one of the main floriculture crops in Karnataka. In 2020 (March-June), a characteristic leaf spot disease of unknown etiology with an incidence ranging from 10–12% (~30 ha area evaluated) was observed in Southern Karnataka (Mysore, Mandya). Initially, the symptoms developed as small specks (3 to 8 mm), characterized by circular to irregular shapes in the beginning and coalesced to form larger lesions. Ten samples were collected in polybags followed by the isolation of associated fungal pathogen on potato dextrose agar (PDA) medium amended with Chloramphenicol (60 mg/L). Briefly, small pieces of infected leaves were cut into small pieces and surface sterilized with 2% sodium hypochlorite (NaOCl) solution, rinsed three times with sterile distilled water (SDW), blot dried, then inoculated onto PDA medium, and incubated at room temperature (27 ± 2°C) for 3 – 5 days. Fungal colonies developed from the segments and were subcultured through hyphal tipping to fresh PDA plates to get pure cultures. A total of 12 pure cultures were obtained. Mycelia were initially white and eventually turned gray. The conidia were black, single-celled, smooth, spherical to subspherical, 9 to 18 μm in diameter (n=50), and borne singly on a hyaline vesicle at the tip of each conidiophore. The identity was initially established based on the cultural features and conidial morphology as Nigrospora sp. (Deepika et al., 2021). To confirm the identity of fungal isolates based on molecular sequence analysis was performed for two representative isolates (CIT1 & CIT2). ITS-rDNA, tub2 & EF-1α gene were amplified using primers ITS1/ITS4, T1/T22 & EF1-728F/986R (White et al., 1990; O’Donnel and Cigelnik, 1997; Carbone and Kohn, 1999), then purified and sequenced. The BLASTn analysis of ITS, tub2 and EF-1α gene showed 99-100% similarity with reference sequences from the GenBank database to Nigrospora sphaerica (ITS: 520bp, KX985935 – LC7312; MH854878 – CBS:166.26; tub2: 357bp, MZ032030 - WYR007, 350bp, KY019606 – LC7298, KY019522 – LC4278, KY019520 – LC4274; EF-1α: 472bp, KY019397 – LC7294, KY019331 – LC4241; MN864137 - HN-BH-3) and the sequences were deposited in GenBank (ITS: OL672271 & OL672272; tub2: OL782120 & OL782121; EF-1α: ON051604 & ON051605) (Wang et al., 2017). The associated fungal pathogen was identified as N. sphaerica (Sacc.) Mason (Chen et al. 2018; Deepika et al., 2021) based on the cultural, morphological, microscopic, and molecular characteristics. Further, pathogenicity tests were conducted on healthy plants (Crossandra cv. Arka; n=30) grown under greenhouse conditions (28±2 °C; 80% RH). Inoculations were made with conidial suspension (18 days old N. sphaerica isolate CIT1, 106 conidia/ml) prepared in SDW, and healthy plants sprayed with SDW (n=10) served as controls. All the plants were covered with polyethylene bags for 24–48 hr and observations were made at regular intervals. Typical necrotic lesions developed on 16 plants after 12 days after inoculation but no symptoms were observed on the control plants. The associated pathogen was re-isolated from diseased leaves and confirmed their identity based on morphology and cultural characteristics. Earlier, N. sphaerica was associated with various tree species as an endophyte, and recently several reports have appeared to cause disease on various crop plants (Deepika et al., 2021). However, there are no previous reports on the association of N. sphaerica causing leaf spot disease on C. infundibuliformis from India. Early diagnosis of this leaf spot disease will help the floriculturist adopt suitable management practices to avoid significant economic loss.