First report of Meloidogyne javanica infecting Bletilla striata in Yunnan, China.

Bletilla striata (Thunb. ex Murray) Rchb. F. (Orchidaceae) is an endangered traditional Chinese medicinal plant and has been traditionally used for hemostasis and detumescence in China (Wang et al. 2022). In March of 2021, during a field survey in Xuanwei city, Yunnan province, China, some B. striata plants with symptoms of plant dwarfing and leaf yellowing were observed. Roots of diseased plants presented numerous galls, typical symptoms of root-knot nematodes (RKNs) infection. The diseased area was approximately 66667 m2, showing a patchy disease distribution pattern. To identify the species of RKNs, females and eggs were isolated from galled tissue, and second-stage juveniles (J2s) were collected from eggs hatched. Nematodes were identified through comprehensive morphological and molecular methods. The perineal pattern of females is round to ovoid with a flat or moderately high dorsal arch and has two conspicuous lateral line striae. Morphological measurements of females (n=20) included body length (L) = 702.9 ± 70.8 (556.2-780.2) μm, body width (BW) = 404.1 ± 48.5 (327.5-470.1) μm, stylet length = 15.5 ± 2.2 (12.3-18.6) μm, distance from base of stylet to dorsal esophageal gland opening (DGO) = 3.7 ± 0.8 (2.1-4.9) μm. The morphometrics of J2s (n=20), L = 438.4 ± 22.6 (354.1-464.8) μm, BW = 17.4 ± 2.0 (12.9-20.8) μm, stylet length = 13.5 ± 0.4 (13.0-14.2) μm, DGO = 3.2 ± 0.6 (2.6-4.7) μm, and hyaline tail terminus = 12.3 ± 1.9 (9.6-15.7) μm. These morphological characteristics were similar to the original descriptions of Meloidogyne javanica (Rammah and Hirschmann 1990). DNA extraction was done 60 times, each from a different single females following the method of Yang et al. (2020). Amplification of ITS1-5.8S-ITS2 region of rDNA and the coxI region of mtDNA was done by using primers 18S/26S (5'-TTGATTACGTCCCTGCCCTTT-3'/5'-TTTCACTCGCCGTTACTAAGG-3') (Vrain et al. 1992) and cox1F/cox1R (5'-TGGTCATCCTGAAGTTTATG-3'/5'-CTACAACATAATAAGTATCATG-3') (Trinh et al. 2019) respectively. The PCR amplification program followed the method described by Yang et al. (2021). The ITS1-5.8S-ITS2 gene sequence (768 bp, GenBank Accession No. OQ091922) showed 99.35-100% identical to the known sequences of M. javanica (GenBank Accession Nos. KX646187, MW672262, KJ739710, KP901063, MK390613). The coxI gene sequence (410 bp, OQ080070) showed 99.75%-100% identical to the known sequences of M. javanica (OP646645, MZ542457, KP202352, KU372169, KU372170). Furthermore, M. javanica species-specific primers Fjav/Rjav (5'-GGTGCGCGATTGAACTGAGC-3'/5'-CAGGCCCTTCAGTGGAACTATAC-3') were used for PCR amplification. An expected fragment of approximately 670 bp was obtained, which was identical to that previously reported for M. javanica (Zijlstra et al. 2000). To verify pathogenicity of this nematode on B. striata, six 1.6-year-old tissue culture seedings of B. striata were maintained in 10-cm-diameter × 9-cm-high plastic pots containing a sterilized mixed soil (humus soil: laterite soil: perlite=3:1:1), and each plant was inoculated with 1000 J2s hatched from eggs of M. javanica. Three non-inoculated B. striata were used as the negative controls. All plants were placed in a greenhouse at approximately 14～26 ℃. After 90 days, the inoculated plants presented symptoms of leaf yellowing, and the roots with root knots identical to those observed in the fields. The root gall rating was 2 according to the 0-5 RKNs rating scale (Anwar and McKenry, 2002) and the reproductive factor (RF= final population/initial population) was 1.6. No symptoms or nematodes were observed on control plants. The nematode was reisolated and identified as M. javanica by morphological and molecular methods as above. To our knowledge, this is the first report of infection of M. javanica on B. striata. The infection of this economically important medicinal plant with M. javanica could pose a great threat to B. striata production in China, and further research will be necessary to develop control strategies.