First report of <i>Graphium euwallaceae</i> causing wilt of sunflower in India

Sunflower (Helianthus annuus, Asteraceae) is globally recognized as a crucial oilseed crop, holding the fourth position in production after palm, soybean, and rapeseed (Blinkov et al. 2022). In February 2024, 23 % sunflower plants exhibited the wilting disease in the fields (36 m 2 ) of sunflower plants at the Vidyasagar University campus, Midnapore, West Bengal, India (22°25'52.1"N 87°18'05.5" E). Affected leaves exhibited brown to dark brown coloured necrotic areas surrounded by a yellow halo, mostly between the veins of the leaves, and it progressed from the margin to the centre or base of the leaves. The affected leaves wilted, curled up at the edges, and eventually perished. Some small pieces of infected tissue (2 × 3 cm) were excised, immersed in 1% sodium hypochlorite (NaOCl) solution for 3 minutes, then rinsed 3 times with Milli-Q water and air-dried. After that, they were placed on potato dextrose agar (PDA) medium and incubated at 25°C for three to four days. Once fungal hyphae emerged, they were transferred to new Petri plates to establish pure cultures. From the pure cultures, one isolate, named HALMP2, was recovered. The colony of this isolate appeared round, flat, and dark olive to black in colour with an entire margin and a rough surface texture. Some aerial hyphae and newly formed peripheral hyphae were off-white in colour. The colony diameter was 1.36 ± 0.17 cm on the 7th day of the culture on PDA medium. The fungi had darkly pigmented synnemata that bore both round and thin conidia at their apex. The thin conidia had a mean length of 2.68 ± 0.10 µm and a breadth of 0.65 ± 0.02 µm. Round conidia were larger, with a mean length of 2.87 ± 0.07 µm and a breadth of 1.76 ± 0.05 µm. By sequencing, internal transcribed spacer (ITS) region (White et al. 1990) and translation elongation factor 1-α (tef1) (Jacobs et al. 2004; Na et al. 2018), it was confirmed that the isolate HALMP2 is Graphium euwallaceae. Sequence analysis of the ITS and tef1-α confirmed that isolate HALMP2 matched with Graphium ewallaceae with identities of 100% with GenBank Accession No. KF540218 and KF534799, respectively. The ITS sequence and tef1-α sequence of HALMP2 were deposited in GenBank, and their GenBank Accession No. is PX270357 and PX227318, respectively. For the pathogenicity test, stem inoculation method was performed. To perform stem inoculation, sunflower plants were taken at an early flowering stage. The epidermis of the stem was carefully peeled at two sites, and 2 cm-diameter agar blocks of the HALMP2 isolate were placed on it and wrapped with parafilm. Control plants underwent the same stem inoculation process, except their agar blocks lacked fungal mycelium (Trivedi et al. 2023). After inoculation, both inoculated and control plants were kept in the shade for 24 hours at 25°C, after which they were exposed to sunlight. On the 4th day, the first symptoms of the disease appeared on the leaves. Brown discoloration was observed on the leaf margin; the leaves were curled upward and wilted. On the 7th day, most of the leaves were affected, brown discoloration developed around the inoculated areas of the stems, and plants were slightly wilted. On the 9th day, the plants were fully wilted and died. But the control plants remain unaffected. Vascular bundles of the inoculated plants exhibited brown discoloration around the inoculated areas, whereas in control plants, vascular bundles remained unchanged. Later, the HALMP2 isolate was reisolated, confirming adherence to Koch’s postulates. A total of five plants were taken for the pathogenicity test, and all plants exhibited wilting symptoms. The disease severity was 72% on the 7th day after pathogen inoculation. The HALMP2 isolate typically developed this disease in the winter season, within the temperature range of 23°C to 30°C, in West Bengal, India. To our best knowledge, this is the first report of Graphium euwallaceae causing wilt disease in sunflower plants. Although the disease outbreak was not evident worldwide, it could become a serious threat to sunflowers in the future, and the risks should not be overlooked.