First report of Helicotylenchus dihystera parasitizing Lady Tankerville orchids (Phaius tankervilleae) in Vietnam.

ABSTRACT

Lady Tankerville, a rare orchid species (Phaius tankervilleae (Banks ex L'Hér.) Blume) in Vietnam, not only enhances the aesthetic value of the surroundings with its enchanting blooms but also holds high economic value (Aver'janov & Averyanova, 2003). Investigating the necrosis symptom on the root system and declined Lady Tankerville in the market in Hai Duong province in Vietnam from 11/2020 to 12/2021, we discovered a substantial infestation (24/24 plants were infected) of a spiral nematode, Helicotylenchus sp. The nematode population was extracted from the rhizosphere soil, roots, and stems of a single orchid using the modified Baermann tray technique (Whitehead & Hemming, 1965), for thorough characterization. The average nematode density was measured at 525±85 (350-670) nematodes/250 cm3 of soil, 202±56 (198-264) nematodes/15 g of roots, and 80±15 (72-95) nematodes/15 g of stem. Two hundred nematodes from the same population were inoculated into another healthy orchid for preservation and further infection tests. This species was morphologically identified as Helicotylenchus dihystera according to the key of Fotedar and Kaul (1985) and the description of Sher (1966). Morphometric measurements of the females (n = 12) are as follows body length = 632-725 (681 ± 32) µm; a = 24-34 (28 ± 3); b = 5.1-7.2 (6.0 ± 0.6); b' = 4.3-6.4 (5.3 ± 0.6); c = 33-46 (39 ± 5); c' = 1.1-1.4 (1.2 ± 0.1); V = 62-78 (65 ± 4)%; O = 30 -49 (39 ± 6); stylet length = 21-26 (24 ± 2) µm; DGO = 9.2-12.4 (10.8 ± 1.1) µm. To validate morphological observations, molecular analyses of the ITS (Vrain et al., 1992) and the D2-D3 of 28S rRNA (Subbotin et al., 2006) were conducted. The ITS and D2-D3 sequences from this study (accession number PP060444 and PP033748) exhibited the highest similarity of 99.8% and 100% to the sequence of H. dihystera in GenBank (KM506885 and MW023215), respectively. The infection test took place in a greenhouse at 28 ± 2℃. Three-month-old Phaius tankervilleae (n=8) were individually grown in 15 x 15 cm deep pots filled with sterilized sand and inoculated with 500 gravid females of H. dihystera (Rashid & Azad, 2013). Two noninoculated plants served as controls. After 60 days of inoculation, symptoms such as sunspots and root necrosis, observed in the market, appeared in the tested plants (Fig. S1). The presence and diagnosis of H. dihystera infestation in soil, roots, and stems across growth stages of orchids indicates the nematode to be an obligate parasite. The nematodes penetrate the roots, causing characteristic necrotic lesions initially yellow, then turn reddish-brown to black, along with brown root flecks in discolored tissues. Heavy infestations post-flowering led to extensive necrosis, distortion, and decay of the roots. The average reproduction factor (final population/initial population) of H. dihystera in this study was 22.2. Control plants remained symptom-free. Notably, 100% of tested plants were infected, highlighting the severe impact of H. dihystera. The nematodes were successfully reisolated and identified as H. dihystera through molecular analyses (accession number PP060615 (ITS) and PP033748 (D2-D3)), reaffirming its identity. In addition to 32 host plants in Vietnam (Nguyen et al., 2023), our study reveals a strain of H. dihystera parasitizing Lady Tankerville orchids with a relatively high reproduction factor and infection rate. This marks the first reported instance of H. dihystera parasitizing Lady Tankerville orchids in Vietnam, emphasizing the need for proactive measures to protect this plant.