First report of Meloidogyne enterolobii infecting mulberry cv. Chiang Mai 80 (Morus alba) in Thailand

White mulberry (Morus alba) is a perennial crop of global significance, prized for its fruit and for food processing. Mulberry leaves play a crucial role as a protein source in the silkworm (Bombyx mori)-rearing industry in Thailand (Sawangloke et al., 2023) and in other countries. In December 2021, root gall symptoms, linked to root-knot nematode (RKN) infestation, were noted in an orchard of white mulberry (cv. Chiang Mai 80) in the Pak Chong district, Nakhon Ratchasima province, Thailand. Of particular interest was the absence of conspicuous above-ground symptoms, such as leaf abscission, yellowing, or growth stunting (Fig. 1), aligning with observations reported by Jindapunnapat et al. (2023) in RKN-infected rain trees. RKN-infected roots were collected and cleaned under running tap water until free of soil debris. Subsequently, a single egg mass was manually isolated and introduced onto a two-week-old okra seedling grown in 14-cm-diameter pot filled with 400 g sterilised soil. The plants were grown in a greenhouse for two months prior to nematode identification and pathogenicity assessments. To diagnose the underlying cause of the disease, nematodes were carefully extracted from the roots of the okra plant. A morphological examination of female perineal patterns and second-stage juveniles was conducted. The perineal patterns exhibited an oval shape with moderate to high elevation, a squarish dorsal arch, coarse and smooth striae in the ventral arch, and an absence of lateral lines (Fig. 2). The morphological measurements of second-stage juveniles (n = 20) were as follows: body length (mean ± standard deviation) 449.1 ± 14.7 µm, width 15.2 ± 1.7 µm, stylet length 12.6 ± 0.8 µm, tail length 56.6 ± 7.5 µm, tail diameter at anus 10.6 ± 0.9 µm, and hyaline tail terminus 18.4 ± 2.5 µm. These observed characteristics closely aligned with those described for M. enterolobii, as detailed by Jindapunnapat et al. (2023). For molecular identification, two primer sets were employed: SSU18A/SSU26R (5′-AAAGATTAAAGCCATGCATG-3′/5′-CATTCTT GGCAAATGCTTTCG-3′) (Mayer et al., 2007) targeting the 18S rRNA gene, and C2F3/1108 (5′-GTCAATGTTCAGAAATTTGTGG-3′/5′-TACCTTTGACCAATCACGCT-3′) (Powers & Harris, 1993) targeting the cytochrome oxidase subunit II (COII) - 16S rRNA region. PCR yielded amplicons of c. 900 and 700 bp, respectively. Subsequent BLAST searches revealed 99% identity to several sequences of M. enterolobii (GenBank Accession Nos. OK076893, ON496981, MN269940, and MW167109). The sequences were deposited in GenBank, PP779709- PP779711 for the 18S rRNA and PP798378-PP798380 for the COII - 16S rRNA. Additionally, PCR assays utilising species-specific primers MK7F/MK7R (5′-GATCAGAGGCGGGCGCATTGCGA-3′/5′-CGAACTCGCTCGAACTCGAC-3′) were conducted to confirm the species identity, following the protocol outlined by Tigano et al. (2010). A distinct band of approximately 520 bp was obtained, corroborating the identification of M. enterolobii as the causative agent of root-knot disease in mulberry trees in Thailand. To demonstrate Koch's postulates, 20-cm-long cuttings of mulberry cv. Chiang Mai 80 were inserted into a 20-cm-diameter pots containing 1 kg of sterilised soil, one cutting per pot. The cuttings were maintained in a greenhouse for three months to facilitate root development before being inoculated with five thousand M. enterolobii second-stage juveniles. The experiment was conducted with three replicates, each with or without RKN inoculation. Three months post-inoculation, mulberry plants exhibiting root galls resembling those observed in the orchard were observed, whereas no galls were evident in the control plants (Fig. 3). The nematodes were subsequently re-isolated and identified, confirming the presence of M. enterolobii in the mulberry roots. To the best of our knowledge, this study represents the first report of M. enterolobii infecting mulberry in Thailand. However, further investigation is required to elucidate the potential impacts of this nematode on mulberry in the region. The authors express their gratitude to Rai Nai Ying farm for generously providing the samples and valuable assistance during field surveys.