Hudie Shao

Bursaphelenchus mucronatus is the sibling species of B. xylophilus, which causes pine wilt disease. Sex pheromone-mediated mating behavior underlies the development of B. xylophilus populations. The study of the molecular mechanism of sex pheromone receptor genes is vital for the control of B. xylophilus. The pivotal role of the 5-HT receptor ser-1 in Caenorhabditis elegans’s mating has been demonstrated, but there is little known in B. mucronatus and B. xylophilus. In our study, the molecular features and biological functions of Bmu-ser-1 are explored. qPCR results showed that Bmu-ser-1 was expressed at all ages, especially at J2 and J4, with significantly high expression. Notably, the expression levels in males were significantly higher than that in females. The results of in situ hybridization suggest that the Bmu-ser-1 gene was expressed at the site of the digestive system during the embryonic stage, the whole body during the J2 stage, and mainly at the end of the germinal primordium during the J3 stage, as well as centrally in the vulva of females and in the gonads and tail of males during the J4 and adult stages. The RNAi results indicate a significant decrease in hatchability and stagnation at the J1 stage after interference. Treated J2 had reduced motility and stunted growth. Males after interference showed mismatch and females showed spawning difficulties. Sexual arousal experiments further validated Bmu-ser-1 as a receptor gene for males receiving female sex pheromones. Collectively, our findings strongly suggest that the Bmu-ser-1 gene has a classical role, the control of nematode growth; and a novel role involved in mating. The study on the molecular mechanisms of growth and reproduction in B. mucronatus could provide a reference for understanding the population expansion and disease epidemics of B. xylophilus.

Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus. The result shows that cyclobutrifluram is the most effective agent, with an LC₅₀ value of 0.1078 mg·L⁻¹. At an LC₂₀ dose, it significantly reduced the population size to 10.40 × 10³ ± 737.56—approximately 1/23 that of the control group. This notable impact may stem from the agent’s ability to diminish egg-laying and hatching rates, as well as to impede the nematodes’ development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc-dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

The distribution range of root-knot nematode Meloidogyne graminicola is rapidly expanding, posing a severe threat to rice production. In this study, the sequences of cytochrome oxidase subunit I (COI) genes of rice M. graminicola populations from all reported provinces in China were amplified and sequenced by PCR. The distribution pattern and phylogenetic tree showed that all 54 M. graminicola populations in China have distinct geographical distribution characteristics; specifically, cluster 1 (southern China), cluster 2 (central south and southwest China), and cluster 3 (central and eastern China). The high haplotype diversity (Hd = 0.646) and low nucleotide diversity (π = 0.00682), combined with the negative value of Tajima’s D (−1.252) and Fu’s Fs (−3.06764), suggested that all nematode populations were expanding. The existence of high genetic differentiation (Fst = 0.5933) and low gene flow (Nm = 0.3333) indicated that there was a block of gene exchange between most populations. Mutation accumulation with population expansion might be directly responsible for the high genetic differentiation; therefore, the tested nematode population showed high within-group genetic variation (96.30%). The haplotype Hap8 was located at the bottom of the network topology, with the widest distribution and the highest frequency (59.26%), indicating that it was the ancestral haplotype. The populations in cluster 3 were newly invasive according to the lowest frequency of occurrence of Hap8, the highest number of endemic haplotypes, and the highest total haplotype frequency (60%). In contrast, cluster 1 having the highest genetic diversity (Hd = 0.772, π = 0.01127) indicated that it was the most primitive. Interestingly, the highest gene flow (Nm > 1), lowest genetic differentiation (Fst ≤ 0.33), and closest genetic distance (0.000) only occurred between the Guangdong/Hainan population and others, which suggested that there might be channels for gene exchange between them and that long-distance dispersal occurred. This suggestion is further confirmed by the weak correlation between genetic distance and geographical distance. Based on these data, a hypothesis can be drawn that M. graminicola populations in China were spreading from south to north, specifically from Guangdong and Hainan Provinces to other regions. Natural selection (including anthropogenic) and genetic drift were the main drivers of their evolution. Coincidentally, this hypothesis was consistent with the gradual warming trend and the chronological order of reporting these populations. The main factors influencing current M. graminicola population expansion and distribution patterns might be geography, climate, long-distance seedling transport, interregional operations of agricultural machinery, and rotation mode. It reminds human beings of the necessity to be vigilant about preventing nematode disease according to local conditions all year round.

The cereal cyst nematodes Heterodera avenae and Heterodera filipjevi are recognized as cyst nematodes that infect cereal crops and cause severe economic losses worldwide. Rapid, visual detection of cyst nematodes is essential for more effective control of this pest. In this study, recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (formerly known as cpf1) was developed for the rapid detection of H. avenae and H. filipjevi from infested field samples. The RPA reaction was performed at a wide range of temperatures from 35 to 42°C within 15 min. There was no cross-reactivity between H. avenae, H. filipjevi, and the common closely related plant-parasitic nematodes, indicating the high specificity of this assay. The detection limit of RPA-Cas12a was as low as 10⁻⁴ single second-stage juvenile (J2), 10⁻⁵ single cyst, and 0.001 ng of genomic DNA, which is 10 times greater than that of RPA-lateral flow dipstick (LFD) detection. The RPA-Cas12a assay was able to detect 10⁻¹ single J2 of H. avenae and H. filipjevi in 10 g of soil. In addition, the RPA-LFD assay and RPA-Cas12a assays could both quickly detect H. avenae and H. filipjevi from naturally infested soil, and the entire detection process could be completed within 1 h. These results indicated that the RPA-Cas12a assay developed herein is a simple, rapid, specific, sensitive, and visual method that can be easily adapted for the quick detection of H. avenae and H. filipjevi in infested fields.

Globodera rostochiensis is an important quarantine pest, it causes serious potato yield losses annually. Reliable and rapid molecular detection of G. rostochiensis is pivotal to effective early disease diagnosis and managements. Herein, recombinase polymerase amplification integrated with lateral flow assays method (RPA-LFA) was developed to target the internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) of the golden cyst nematode (G. rostochiensis), which allowed for the rapid diagnosis and detection of this nematode from crude extracts of cysts and juveniles within 30 min. Sensitivity test results showed that 10⁻¹ single juvenile and 10⁻³ single cyst can be reliably detected. Moreover, the RPA-LFA method can directly diagnose and detect G. rostochiensis from infested field soil. This is the first RPA-LFA method for diagnosis G. rostochiensis, it is a fast, accurate, and sensitive detection method and can be developed for detection of G. rostochiensis in fields and laboratories lacking large instrument and equipment.

Cyst nematodes are one of the most important pathogens worldwide. Most cyst nematode species have been reported recently in China. From 2016 to 2020, an extensive survey of cyst nematodes was conducted in the Bashang region of Hebei Province. A total of 158 soil samples were collected, and cyst-forming nematodes were isolated from five soil samples. Morphological and molecular characterization showed that four of the cyst-forming nematode populations were Heterodera glycines (SCN), named populations of ZM, KM, CB and FN, respectively. These SCN populations were collected from Zhangbei County, Kangbao County, Chabei Management Area of Zhangjiakou and Fengning Manchu Autonomous County of Chengde, respectively, where the corresponding cyst densities were 57, 41, 103 and 31 cysts/200 cc soil. Furthermore, the populations of ZM, KM and CB were identified as race 4, whereas the FN population was identified as race 3. The cyst-forming nematode population was collected from Zhangbei County of Zhangjiakou, which was confirmed to be Heterodera schachtii (SBCN), named population ZZ, and the cyst density was 94 cysts/200 cc soil. It is a new disease of Chinese cabbage caused by SBCN based on Koch’s postulates. Fourteen cultivars from five plant families were evaluated as hosts for SBCN. Chinese cabbage (cv. Linglonghuang012) and cabbage (cv. Chunwang) were suitable hosts, while celery (cv. Yuhuang), potato (cv. Helan 15) and eggplant (cv. Junlang) were nonhosts. The obtained results regarding the occurrence, distribution, races of SCN and hosts of SBCN in the Bashang region in this study provide a reference for SCN and SBCN management.

There is little information about nematode pests associated with yam in China. Between 2020 and 2021, surveys of yam fields were conducted to investigate the abundance and prevalence of plant-parasitic nematodes in major yam growing areas. A total of 110 bulk soil samples from the yam rhizosphere and 48 yam tubers were collected from seven counties in Jiangxi and Shandong provinces. Standard protocols were used to extract nematodes from soil and tubers and identified at the genus level. In this study, 16 species and 13 nematode genera were recorded. The five most prominent species on the yam rhizosphere according to mean population densities were Pratylenchus coffeae (291/individuals), Meloidogyne (262/individuals), Rotylenchulus reniformis (225/individuals), Merlinius (224/individuals), and Helicotylenchus dihystera (171/individuals). In the tubers, the three most prominent species were Pratylenchus coffeae (415/individuals), Meloidogyne (331/individuals), and Rotylenchulus reniformis (115/individuals). These species were verified with appropriate molecular analysis. The high prevalence of the ectoparasite (Merlinius spp.) on the rhizosphere of yam also revealed that Merlinius spp. May be more important to yam than previously thought. Morphological and molecular analyses further confirmed the identity of the species as Merlinius brevidens and were characterized for the first time on yam in China. Minor morphometrical differences (slightly longer body and stylet) were observed in Chinese populations of M. brevidens compared to the original description. Additionally, this study reveals that M. brevidens isolated from China showed a higher nucleotide sequence in the ITS region compared to M. brevidens populations from India. This finding provides baseline information on the nematode pest occurrence on yam in China and calls for effective management.