Background: The pine wood nematodeBursaphelenchus xylophilusis a destructive pest ofPinustrees worldwide and lacks effective control measures. Screening for nematotoxic proteins has been undertaken to develop new strategies for nematode control.Results: The results of the present study provided initial insights into the responses ofB. xylophilusexposed to a nematotoxic cytolytic-like protein (CytCo) based on transcriptome profiling. A large set of differentially expressed genes (DEGs = 1265) was found to be related to nematode development, reproduction, metabolism, motion, and immune system. In response to the toxic protein,B. xylophilusupregulated DEGs encoding cuticle collagens, transporters, and cytochrome P450. In addition, many DEGs related to cell death, lipid metabolism, major sperm proteins, proteinases/peptidases, phosphatases, kinases, virulence factors, and transthyretin-like proteins were downregulated. Gene Ontology enrichment analysis showed that the CytCo treatment substantially affected DEGs involved in muscle contraction, lipid localization, and the mitogen-activated protein kinase cascade. The pathway richness of the Kyoto Encyclopedia of Genes and Genomes showed that the DEGs were concentrated in lysosomes and involved in fatty acid degradation. Weighted co-expression network analysis indicated that the hub genes affected by CytCo were associated with the nematode cuticular collagen.Conclusions: These results showed that CytCo toxin interferes with gene expression to exert multiple nematotoxic effects, thereby providing insights into its potential use in pine wood nematode control.

Introduction: Pakchoi is an important leafy vegetable in China. Due to industrialization and urbanization, pakchoi has been cultivated in newly reclaimed mountainous lands in Zhejiang Province, China in recent years. However, immature soil is not suitable for plant growth and needs to be modified by the application of different organic fertilizer or microbial fertilizer based plant-growth-promoting microbe. In 2021, a high efficient plant-growth-promoting fungi (PGPF; Aspergillus brunneoviolaceus HZ23) was obtained from newly reclaimed land of Zhejiang Province, China. In order to valuate microbial fertilizer based A. brunneoviolaceus HZ23 (MF-HZ23) on pakchoi growth in immature soil, we investigated the effect of MF-HZ23 on soil properties, rhizosphere bacterial community structure, and metabolites of pakchoi rhizosphere soil samples.Methods: The field experiment (four treatments, MF-HZ23, MF-ZH23 + CCF, CCF and the control) was completely randomly designed and carried out on newly reclaimed land in Yangqingmiao Village of Fuyang district, Hangzhou City, Zhejiang Province, China. In order to evaluate the influence of microbial fertilizer based A. brunneoviolaceus HZ23 on pakchoi in the newly reclaimed land, the number of pakchoi leaves, total fresh and dry weight of the seedlings was counted. In addition, the soil properties, including the pH, OMC, total N, AHN, available P, the genome sequencing, and metabolomics assay were also detected.Results: The results revealed a significant difference between MF-HZ23 and the control in soil properties, bacterial community structure, and metabolites. Indeed, compared with the control, MF-HZ23 caused 30.66, 71.43, 47.31, 135.84, and 2099.90% increase in the soil pH, organic matter contents (OMC), total nitrogen (N), alkaline hydrolysis nitrogen (AHN), and available phosphorus (P), respectively. Meanwhile, MF-HZ23 caused 50.78, 317.47, and 34.40% increase in the relative abundance of Proteobacteria, Bacteroidota, and Verrucomicrobiota and 75.55, 23.27, 69.25, 45.88, 53.42, and 72.44% reduction in the relative abundance of Acidobacteriota, Actinobacteriota, Chloroflexi, Planctomycetota, Patescibacteria, and WPS-2, respectively, compared with the control based on 16S amplicon sequencing of soil bacteria. Furthermore, redundancy discriminant analysis (RDA) of bacterial communities and soil properties indicated that the main variables of bacterial communities included available P, AHN, pH, OMC, and total N. In addition, non-targeted metabolomics techniques (UHPLC–MS analysis) revealed that MF-HZ23 resulted in a great change in the kinds of metabolites in the rhizosphere soil. Indeed, in MF-HZ23 and the control group, there were six differentially expressed metabolites (DEMs) belong to organoheterocyclic compounds, organic acids and derivatives, organic nitrogen compounds, and these six DEMs were significantly positively correlated with 23 genus of bacteria, which showed complicated interactions between bacteria and DEMs in pakchoi rhizosphere soil.Conclutions: Overall, the results of this study revealed significant modification in physical, chemical, and biological properties of pakchoi soil. Microbial fertilizer based PGPF A. brunneoviolaceus HZ23 (MF-HZ23) can be used as a good amendment for newly reclaimed land.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5′-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 µg/mL) in the soaking buffer for 24 h in the dark at 25 °C. The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

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.

Tetrastigma hemsleyanum Diels & Gilg (TDG) has been recently planted in reclaimed lands in Zhejiang Province, China, to increase reclaimed land use. Winter cold stress seriously limits the growth and development of TDG and has become the bottleneck limiting the TDG planting industry. To investigate the defense mechanisms of TDG toward winter cold stress when grown on reclaimed land, a combined analysis of soil bacterial communities, metabolites, and physicochemical properties was conducted in this study. Significant differences were observed in the composition of soil bacterial communities, metabolites, and properties in soils of a cold-tolerant variety (A201201) compared with a cold-intolerant variety (B201810). The fresh weight (75.8% of tubers) and dry weight (73.6%) of A201201 were significantly higher than those of B201810. The 16S rRNA gene amplicon sequencing of soil bacteria showed that Gp5 (25.3%), Gemmatimonas (19.6%), Subdivision3 (16.7%), Lacibacterium (11.9%), Gp4 (11.8%), Gp3 (10.4%), Gp6 (7.0%), and WPS-1 (1.2%) were less common, while Chryseolinea (10.6%) were more common in A201201 soils than B201810 soils. Furthermore, linear discriminant analysis of effect size identified 35 bacterial biomarker taxa for both treatments. Co-occurrence network analyses also showed that the structures of the bacterial communities were more complex and stable in A201201 soils compared to B201810 soils. In addition, ultra-high-performance liquid chromatography coupled to mass spectrometry analysis indicated the presence of significantly different metabolites in the two soil treatments, with 10 differentially expressed metabolites (DEMs) (8 significantly upregulated by 9.2%–391.3% and 2 significantly downregulated by 25.1%–73.4%) that belonged to lipids and lipid-like molecules, organic acids and derivatives, and benzenoids. The levels of those DEMs were significantly correlated with the relative abundances of nine bacterial genera. Also, redundancy discriminant analysis revealed that the main factors affecting changes in the bacterial community composition were available potassium (AK), microbial biomass nitrogen (MBN), microbial biomass carbon (MBC), alkaline hydrolysis nitrogen (AHN), total nitrogen (TN), available phosphorus (AP), and soil organic matter (SOM). The main factors affecting changes in the metabolite profiles were AK, MBC, MBN, AHN, pH, SOM, TN, and AP. Overall, this study provides new insights into the TDG defense mechanisms involved in winter cold stress responses when grown on reclaimed land and practical guidelines for achieving optimal TDG production. IMPORTANCE: China has been undergoing rapid urbanization, and land reclamation is regarded as a viable option to balance occupation and compensation. In general, the quality of reclaimed land cannot meet plant or even cultivation requirements due to poor soil fertility and high gravel content. However, Tetrastigma hemsleyanum Diels & Gilg (TDG), extensively used in Chinese herbal medicine, can grow well in stony soils with few nutrients. So, to increase reclaimed land use, TDG has been cultivated on reclaimed lands in Zhejiang Province, China, recently. However, the artificial cultivation of TDG is often limited by winter cold stress. The aim of this study was to find out how TDG on reclaimed land deal with winter cold stress by looking at the bacterial communities, metabolites, and physicochemical properties of the soil, thereby guiding production in practice.

To improve the conductivity of nematocidal emamectin benzoate (EB) in pine trees (Pinus massoniana Lamb.), EB-inclusive nanocapsules (EB-NCs) were developed as trunk injections and spraying agents. Microscopy revealed that the EB-NCs were spherical in shape (100–200 nm in diameter) and micro-agglomerated with an obvious core-shell structure. The EB-NCs formulation maintained high toxicity in Bursaphelenchus xylophilus pine wood nematodes (LC₅₀ = 0.44 mg L⁻¹). Moreover, the formulation had better EB slow-release and photostability. In addition, the EB-NCs formulation was effectively absorbed and transported in the pine trees via either trunk injection or spraying. In terms of trunk injection, the average residues of EB in the lower, middle, and upper branches of P. massoniana 30 days after application in summer were 8.11, 16.42, and 6.98 mg L⁻¹. In terms of spraying treatment, the EB-NC formulation was successfully conducted from the cortical tissue to the cambium and the EB fluorescence intensity inside of the branches was better than that of the 2% EB micro-emulsion. The EB-NC formulation has suitable conductivity in pine trees; hence, this study provides a potential agent for the control of pine wilt disease in the field under all weather conditions.