First Record of Ceratocystis fimbriata Causing Quick Wilt of Calotropis gigantea in Pakistan

Calotropis gigantea belongs to the family Asclepiadaceae, native to Asia and commonly known as crown flower. C. gigantea was identified as an important medicinal plant in Asia and also harvested to obtain the fibres from the stem (Ganeshan et al. 2018; Narayanasamy et al. 2020). In April 2021, a quick wilt epidemic of C. gigantea was observed in District Gujranwala (32°05'58.0"N 74°02'38.0"E) Punjab, Pakistan. The wilt symptoms were very severe on mature plants with 60% disease incidence. Affected plants exhibited yellowing and quick wilting of leaves on each branch that eventually spread to the whole plant (Figure 1). Black patches were observed on the stem bark and cross section of infected stem showed black streaks in the xylem. The samples (n=33) were collected from symptomatic plants. Infected tissues were excised into 4-5mm segments, surface disinfested with 1% NaClO and rinsed 3 times with sterilized distilled water. To induce the sporulation of the fungus, the described carrot baiting method of Moller et al., (1968) was used. Single ascoma from carrot baits were picked under a microscope and transferred to malt extract agar (MEA) medium and incubated at 25°C. Purified cultures were obtained by single spore culture method and all the isolates were preserved with 35% glycerol at -80°C. The fungal colony was greyish olive with overripe banana odour. Hyphae were smooth and segmented. The perithecia were globose, black, measuring 190.27 to 257.34 × 186.11 to 243.24 µm, and showed a long black neck (732 to 977.34 µm). Cylindrical conidia (9.46 to 22.97 µm × 2.7 to 5.41 µm), barrel-shaped conidia (6.34 to 7.95× 8.15-10.41 µm) and chlamydospores (7.65-11.38µm×10.17-16.81µm) were observed (Figure 2). The isolates were identified as Ceratocystis fimbriata based on morphology and similar results were reported by Engelbrecht et al. (2005) and Suwandi et al. (2021). Genomic DNA was extracted from all isolates and multi-locus sequence analysis approach was used for molecular identification. The Internal Transcribed Spacers (ITS) region, the Translation Elongation Factor 1-α gene (TEF) and β-tubulin gene (TUB) were amplified using ITS1/4 (White et al. 1990), EF1-728F/ EF1-986R (Carbone et at. 1999) and βt1a/βt1b (Glass and Donaldson, 1995). Based on the BLAST analysis, all isolates were identified as C. fimbriata. The sequences of the representative isolate AK-W17 were submitted to the GenBank, NCBI database with the accession numbers (ITS:MZ711226), (TEF: MZ714595) and (TUB:MZ714596) showing 100% similarity with AF395687(ITS), MG980731(TEF) and AY177227(TUB) accessions of C. fimbriata. Based on Sequences similarity, representative isolate AK-W17 grouped with the isolates which representing C. fimbriata. Pathogenicity test was conducted on healthy C. gigantea seedlings grown in pots. A fungal mycelium plug (4×4mm) from 15 days old culture of AK-W17 was inserted into a slit (5 × 8 mm and made by puncturing the bark surface) in the stem bark, and the artificial wound was covered with wetted tissue paper and secured with Parafilm to maintain humidity, while control treatment was inoculated with only MEA medium plug. The seedlings were incubated under 70% relative humidity (RH) and 28°C in greenhouse. After 24 days, the inoculated seedlings showed 100% wilting identical to the original plant from which they were isolated (Figure 3). The control treatment had no symptoms. The pathogenic fungus was reisolated and identifies as C. fimbriata based on morphological and molecular characterization. C. fimbriata has been also reported to cause wilt disease in Dalbergia sissoo (Harrington et al. 2015) and pomegranate (Alam et al. 2017) in Pakistan. To our knowledge, this is the first report of C. fimbriata to cause C. gigantea quick wilt in Pakistan. C. fimbriata is one of the most aggressive plant pathogens and rapidly spread worldwide, so it is critical to implement appropriate management practices to reduce the fungus attack on plants.