Fusarium solani and Lasiodiplodia pseudotheobromae, fungal pathogens causing stem rot disease on durian trees (Durio zibethinus) in Eastern Thailand

Abstract

Durian (Durio zibethinus) is known as the king of fruit in Southeast Asia (Bampenrat et al., 2020), and is one of the most economically important export fruits in Thailand (Charoensumran et al., 2021). However, a major problem affecting durian plantations in Thailand is stem rot disease. Symptoms of the infected trees are bark and trunk canker, twig blight, yellow leaves, and wilting. Thirty symptomatic bark and wood samples were collected in November 2020 from different durian orchards in Rayong and Chanthaburi Provinces in the Eastern Thailand (Fig. 1). The samples were cut into 1×1 cm fragments, surface sterilised with 0.5% sodium hypochlorite solution, and washed three times in sterilised water similar to the method used by Chantarasiri et al. (2021). The sterilised samples were plated on dichloran Rose Bengal chloramphenicol (DRBC) agar as a selective medium and potato dextrose agar (PDA) as a relatively rich medium. The agar plates were incubated at 30°C for seven days under a 12-hour light/dark photoperiod. The isolated fungal colonies and hyphal tips were cut and placed on the PDA for culture purification. All fungal isolates were primarily categorised according to their colony and conidial morphology. The first fungal group had a white and cottony colony, producing slightly curved macroconidia with two-four septa (Fig. 2). The second group also had a white and woolly colony but these colonies grew rapidly, turned dark grey after seven days, and did not produce conidia on PDA (Fig. 3). DNA of representative fungal isolates was extracted using the GF Fungus DNA Extraction Kit (Vivantis, Malaysia) and PCR amplified with ITS1/ITS4 primers (White et al., 1990). The resulting PCR products were sequenced by Macrogen Inc. (South Korea) and deposited in the GenBank with Accession Nos. MZ461923, MZ461933, MZ493230, and MZ493231, respectively. Sequence alignment using BLASTn showed that the first fungal group was related to the Fusarium solani strain GP (MN658459.1) with 100% coverage and 95.81-98.99% identity. The second group was related to the Lasiodiplodia pseudotheobromae isolate KEGA_11 (MK922068.1) with 99–100% coverage and 96.64-98.94% identity. Phylogenetic analysis using SeaView version 5.0.2 software confirmed the sequence alignment results (Figure 4). One hundred milliliters of each fungus cultured in potato dextrose broth was inoculated to the trunk of durian seedlings and placed in a glasshouse at ambient temperature for seven days. The same volume of sterilised water was inoculated for the controls. The experiment was performed in triplicate. White mycelia and brown lesions were visually observed in the inoculated seedlings, whereas the controls remained healthy. The fungi were re-isolated and identified by PCR as F. solani and L. pseudotheobromae, thus fulfilling Koch's postulates. This report provides evidenced that F. solani and L. pseudotheobromae are the fungal pathogens causing stem rot disease on durian trees. Previous reports described these fungi causing stem rot in hackberry and mango (Khanzada et al., 2004; Liang et al., 2020). To date, Phytophthora sp. has been attributed as the cause of stem rot disease on durian trees in Thailand. Misidentification of the stem rot fungal pathogens could result in the use of inappropriate methods to control the disease. The authors would like to thank the orchardists in Rayong and Chanthaburi Provinces for kindly providing the samples used in this study.