First Report of Aphelenchoides besseyi Causing White Tip Disease of Rice in Heilongjiang Province of China

Abstract

Aphelenchoides besseyi is considered one of the most damaging plant parasitic nematodes to rice and can cause white tip disease. It was first discovered in Kyushu, Japan in 1915 and introduced into China from Japan in the 1940s. The disease is mainly found in rice-planting areas in South China (Zhu et al. 2016; Wang et al. 2017; Xu et al. 2019), causing yield losses of 30%-50% in irrigated rice (Wang et al. 2014). In summer 2021, rice plants displaying distortion and discoloration on new leaves, white tips on flag leaves, erect and shorter panicles, and fewer or less filled spikelets were observed in the rice field in Acheng district, Harbin city, Heilongjiang Province, China (13°32′33″S, 58°48′51″W). Plant samples with the typical symptoms of ‘white tip’ in the flag leaves were collected and transported to the laboratory for further pathogen identification. Nematodes were extracted from the leaves and panicles of diseased plants using Baermann funnel method, and the population density of nematode was an average of 57 individuals per sample (total 28 diseased samples). The main morphological characteristics of females (n = 20) and males (n = 15) were individually determined using a microscope (LEICA, DM2500). Female body was slender, and straight to ventrally curved when heat-relaxed, with a rounded cephalic region, the lateral field with four incisures, the excretory pore close to the anterior edge of the nerve ring, a narrow post-vulval sac and less than 1/3 the distance from the vulva to the anus, and a conoid tail with star-shaped mucro. Female measurements: body length = 724.6 (593.3 to 867.5) μm, stylet = 11.7 (10.9 to 12.5) μm, tail length = 39.4 (35.8 to 44.4) μm, a = 45.2 (38.6 to 51.0), b = 10.1 (9.4 to 10.7), c = 18.47 (14.7 to 21.4), and V = 71.1 (65.0 to 79.6). The body of male usually showed in a ‘J’ shape when relaxed, spicule length between 17.1 and 19.4 μm, and a conoid tail, with terminal mucro bearing two to four pointed processes. Male measurements: body length = 546.3 (521.7 to 587.6) μm, stylet = 11.9 (10.7 to 12.6) μm, tail length = 32.54 (29.53 to 34.35) μm, a = 37.7 (35.7 to 40.4), b = 8.7 (8.5 to 9.1), c = 17.8 (15.8 to19.4). The key morphological characteristics were coincident with the description of A. besseyi by Christie (1942), Ou et al. (2014) and Chen et al. (2020). DNA was extracted from six fresh nematodes, and the partial 18S-rRNA was amplified with a pair of primers 1813F/2646R (5’-CTGCGTGAGAGGTGAAAT-3’; 5’-GCTACCTTGTTACGACTTTT-3’) (Holterman et al. 2006). PCR products of expected size were purified and sequenced. The newly obtained sequence (accession no. ON629604) was submitted to GenBank and displayed more than 99.5% identity with that of A. besseyi isolates (KT943536, KT454963, JQ957877). Additionally, the isolated nematodes were further confirmed using the species-specific primers Abess_11F/Abess_11R (5’-GTATTCAATCCCGCGACACT-3’; 5’-CATCCTGTTCGGGCATAGTT-3’), and the PCR amplification generated a 570 bp fragment which was specific to the 28S rDNA region of A. besseyi reported previously (Noronha et al. 2020). The pathogenicity test was confirmed in a pot assay. Approximately 2000 mixed-stage A. besseyi (juveniles, males and females) were obtained from the field-diseased rice plants, and ten-day-old rice seedlings (Japonica rice variety, Keyou 1) (n = 5) were inoculated with 400 nematodes per seedling and maintained in a greenhouse. During early growth stages (fifty days after inoculation), some young leaves of infected plants showed the chlorotic tips. Ninety days after inoculation, similar field symptoms (white tip on leaves) were observed on the aerial tissues of the inoculated plants, and an average of 237 A. besseyi were re-isolated from each plant. To the best of our knowledge, this is the first report of A. besseyi on rice plant in Heilongjiang Province of Northeast China. This finding indicates that A. besseyi could become a severe threat to rice production in Heilongjiang Province and contributes to the further inspection and prevention of white tip disease in rice-growing areas of Heilongjiang Province.