First Report of Wilt Disease in Curcuma caesia Caused by Fusarium oxysporum in India

Curcuma caesia (black turmeric, kali haldi) is a perennial herb which is native to the Indian subcontinent and Indo-Burma biodiversity hotspot (Sahu et al. 2016; Das et al. 2024). It has dark bluish-black fragrant rhizomes and lanceolate leaves with prominent purple central veins. This plant is integral to traditional systems of medicine in India (Renemma and Reddy 2017) and has potential for pharmaceutical development (Karmakar et al. 2011). During October 2024 (post-monsoon season), diseased leaves of C. caesia were collected from an agricultural field in East Khasi Hills, Meghalaya, India. Observed symptoms included stunted growth, marginal necrosis, vein clearing in young leaves, and wilting of the older basal leaves (Figure 1). For pathogen isolation, infected leaf samples were surface sterilized sequentially in 70% ethanol (one minute), 1% sodium hypochlorite (two minutes), 70% ethanol (30 s), followed by three rinses in sterile distilled water. A 5-mm segment excised from the infected tissue was plated on potato dextrose agar (PDA) and incubated at 29°C for 7 days. The resulting pure cultures produced uniform colonies with abundant, dense, whitish-pink aerial mycelia (Figure 2). Microscopic examination revealed microconidia that were oval to reniform, and aseptate (Figure 3), while macroconidia were faintly falcate to nearly straight, delicate, and 3-septate, and associated with terminal chlamydospores. For molecular identification, genomic DNA was extracted using a manual lysis method. Fresh fungal mycelium was ground using a sterile mortar and pestle and homogenised in lysis buffer (pH 8.0) followed by incubation at 65°C for 1 h. After centrifugation at 12,000 rpm for 15 min at 4°C, the supernatant was extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1). The mixture was centrifuged again at the same speed and timing and the aqueous phase was mixed with half the volume of chloroform. DNA was then precipitated using 3 M sodium acetate (pH 4.5) and 70% ice-cold ethanol. The resulting pellet was air-dried, resuspended in TE buffer, and quantified using a Nanodrop spectrophotometer. For PCR amplification, the internal transcribed spacer (ITS) region was amplified using universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) in a reaction mixture containing 1 × PCR buffer, 1.5 mM MgCl2, 200 µM dNTPs, 0.2 µM of each primer, 0.15 µL Taq polymerase (5 U), and 1 µL template DNA. The amplified product was sequenced by Eurofins Genomics, India. BLASTn analysis confirmed the isolate as Fusarium oxysporum (Figure 4) and the sequence was deposited in GenBank (Accession No. PV169199). Pathogenicity was confirmed by surface-sterilising healthy C. caesia leaves, creating pin-prick wounds, and inoculating them with 10 µL of a spore suspension (1 × 105 spores/mL). Inoculated leaves developed symptoms similar to those observed under natural field conditions within 7 days of incubation at 29°C (Figure 5). Distilled water was used as a negative control, and showed no observable effect on the leaves. The pathogen was consistently re-isolated from the infected tissues and identified based on morphological characteristics, thereby fulfilling Koch's postulates. Fusarium oxysporum is widely recognised as a causal agent of wilt diseases in several economically important crops, including cucumber, tomato and oil palm. Typical symptoms of Fusarium wilt include foliar chlorosis, stunted growth, marginal necrosis, vein clearing in young leaves, drooping of older basal leaves, defoliation and eventual plant death (Sharma and Shukla 2021; Shahid et al. 2023). These symptoms closely correspond with those observed in the present study. Based on morphological characteristics, molecular identification and pathogenicity assays, this study represents the first report of Fusarium oxysporum causing leaf wilt in Curcuma caesia in India. The first author sincerely thanks the Department of Botany, North-Eastern Hill University, Shillong, Meghalaya, India, for providing the necessary facilities to carry out this research. The author also gratefully acknowledges the UGC Non-NET Fellowship for the financial support received during the course of this study.