First report of an important sheat nematode, Hemicycliophora poranga, associated with sugar beet (Beta vulgaris L.) in Vietnam

Plant-parasitic nematodes belonging to the genus Hemicycliophora are commonly known as sheat nematodes that can parasitize various host plants including agricultural crops, fruit and nut trees, and ornamental plants. Strikingly, Hemicycliophora species have been reported from all continents and can induce serious symptoms such as root galls, stubby root, stunted growth, yellowing, and even death of host plants (Chitambar & Subbotin, 2014). Among Hemicycliophora spp., H. poranga (Monteiro & Lordello, 1978) was known as an important pest to agricultural crops and can cause significant damages to many crops such as celery in Argentina (Emilse et al., 2011); Musa sp., cabbage, cowpea, bean, okra, lettuce, and tomato in the USA (Chitambar, 1993); and other 21 crops from different localities listed by Chitambar and Subbotin (2014).

Currently, 135 valid species of the genus Hemicycliophora have been described over the world (Azimi et al., 2020; Chitambar & Subbotin, 2014; Nguyen et al., 2021). In Vietnam, Hemicycliophora spp. have been reported on 12 host plants that were black pepper, banana, cucurbits, grapefruit, orange, rice, tobacco, yardlong bean, bamboo, Malabar cardamom (Nguyen, 1983; Nguyen & Nguyen, 2001; Nguyen et al., 2021). However, only two endemic Hemicycliophora species have been recognized at species level including Hemicycliophora vietnamensis Nguyen and Nguyen (2001) and H. cardamomi Nguyen et al. (2021), therefore, a higher number of Hemicycliophora species is expected in the country. Herein, the first report of H. poranga associated with sugar beet (Beta vulgaris L.) in Vietnam is provided with the support of morphological and molecular characterizations.

Soil samples were collected from the rhizosphere of sugar beet in Lam Dong, Vietnam (GPS coordinates N: 11°56′21′′, E: 108°26′6.5′′) in the winter season. The observation of plant symptoms was recorded and four samples around a host plant were collected at each sampling site (6 sites), after removing the detritus layer to create a bulk sample (collection of 24 soil samples using a core (5 × 25 cm) resulted in 6 bulk samples). Nematodes were extracted from soil samples using the modified Baerman tray method (Whitehead & Hemming, 1965). Permanent slides were made from fixed nematodes following Seinhorst (1959). Subsequently, measurements and pictures were taken using Carl Zeiss Axio Lab.A1 light microscope equipped with an Axiocam ERc5s digital camera.

For molecular analysis, DNA was extracted from a living nematode by cutting and transferring it to a PCR tube with 20 μl of WLB (50 mM KCl;10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45 % NP-40 (Tergitol Sigma); 0.45 % Tween-20). In the next steps, the sample was incubated at −20°C for at least 10 min, followed by adding 1 μl proteinase K (1.2 mg ml−1). Finally, sample was incubated in a PCR machine for 1 h at 65°C and 10 min at 95°C and finished by centrifugation for 1 min at 20 800 g.

D2-D3 expansion segment of 28S rRNA region was amplified using primers D2A (5’-ACAAGTACCGTGGGGAAA GTTG-3’) and D3B (5’-TCGGAAGGAACCAGCTAC TA-3’) (Nunn, 1992) with the following thermal profile: one cycle of 94°C for 4 min, followed by five cycles of 94°C for 30s, 56°C for 30s, 72°C for 2 min, and 45 cycles of 94°C for 30s, 54°C for 30s, 72°C for 1 min and finished at 10°C for 10 min (Nguyen et al., 2021). Successful PCR reactions were purified using the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, Wisconsin, USA) and sequenced commercially by Macrogen (Korea). The obtained forward and reverse sequences were assembled using Geneious R11 (www.geneious.com). Blast search was used to look for closely related sequences from GenBank (Altschul et al., 1997). MUSCLE in Geneious R11 was used to make multiple alignments for all sequences. The Bayesian phylogenetic analysis was carried out using MrBayes 3.2.6 add-in in Geneious R11 following Nguyen et al. (2019). GTR+G+I model was selected following Abadi et al. (2019).

The result of this work has not been published previously and is not under consideration elsewhere.