Pine wilt disease was first discovered in Dongtang town, Liaoning Province, China, in 2017. However, no record of Monochamus alteratus existed in Fengcheng, where M. saltuarius is an indigenous insect, and no experimental evidence has thus far indicated that M. saltuarius can transport the Bursaphelenchus xylophilus in China. In this study, we investigated whether M. saltuarius is a vector of B. xylophilus in China. On the sixth day after eclosion, beetles began to transmit nematodes into the twigs. The transmission period of nematodes is known to be able to last for 48 days after beetle emergence. In laboratory experiments, M. saltuarius fed and transmitted B. xylophilus not only on pines but also on other non-Pinus conifers. The non-Pinus conifers preferred by M. saltuarius for feeding are Picea pungens, Picea asperata, and Abies fabri. The experimental results show that M. saltuarius functions as a vector of B. xylophilus in northeast China.

Quantitative reverse transcription polymerase chain reaction (RT-qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization under different experimental conditions. Bursaphelenchus mucronatus, a pine-parasitic nematode varying in virulence, is widely distributed in natural pine forests throughout the northern hemisphere, but has not been investigated with respect to the identification of reference genes suitable for the normalization of RT-qPCR data. In the present study, eight candidate reference genes were analyzed in B. mucronatus under different habitat conditions and at different developmental stages. The expression stability of these genes was assessed by geNorm, NormFinder, BestKeeper, delta Cq, and RefFinder algorithms. In general, our results identified encoding beta-tubulin as the most stable gene. Moreover, pairwise analysis showed that three reference genes were sufficient to normalize the gene expression data under each set of conditions, with genes encoding beta-tubulin, 18S ribosomal RNA and ubiquitin-conjugating enzyme being the most suitable reference genes for different habitat conditions, whereas genes encoding beta-tubulin, histone, and 18S ribosomal RNA exhibited the most stable expression at different developmental stages. Validation of the selected reference genes was performed by profiling the expression of the fatty acid- and retinol-binding protein gene in different habitats, and by profiling the expression of the arginine kinase gene at different developmental stages. This first systematic analysis for the selection of suitable reference genes for RT-qPCR in B. mucronatus will facilitate future functional analyses and deep mining of genetic resources in this nematode.

Cyclocarya paliurus is an extremely valuable and multifunctional tree species whose leaves have traditionally been used in used in medicine or as a medicinal tea in China. In recent years, anthracnose has been frequently observed on young leaves of C. paliurus in several nurseries located in Jiangsu Province, resulting in great yield and quality losses. To date, no information is available about the prevalence of C. paliurus anthracnose in China. The main purpose of the present study was to characterize the etiology of C. paliurus anthracnose. Phylogenetic analysis of the eight-loci concatenated dataset revealed that all 44 single-spore Colletotrichum isolates belonged to three species in the Colletotrichum gloeosporioides species complex, namely, Colletotrichum aenigma, Colletotrichum fructicola, and C. gloeosporioides sensu stricto. Phenotypic features, including the colony appearance and the morphology of conidia, appressoria, and ascospores, were consistent with the phylogenetic grouping. Virulence tests validated that the three Colletotrichum species could cause typical symptoms of anthracnose on C. paliurus leaves, similar to those observed in the field. The optimum mycelial growth temperature ranged from 25 to 30°C for all representative isolates, while C. gloeosporioides s. s. isolates exhibited greater tolerance to high temperature (40°C). Fungicide sensitivity assays indicated that all three Colletotrichum species were sensitive to tetramycin, which may be a potential alternative for the management of C. paliurus anthracnose. To our knowledge, this study provides the first report of C. aenigma, C. fructicola, and C. gloeosporioides s. s. causing C. paliurus anthracnose in China as well as in the world.

BACKGROUND: Bursaphelenchus xylophilus, the pinewood nematode, kills millions of pine trees worldwide every year, and causes enormous economic and ecological losses. Despite extensive research on population variation, there is little understanding of the population-wide variation spectrum in China.RESULTS: We sequenced an inbred B. xylophilus strain using Pacbio+Illumina+Bionano+Hi-C and generated a chromosomelevel assembly (AH1) with six chromosomes of 77.1 Mb (chromosome N50: 12 Mb). The AH1 assembly shows very high continuity and completeness, and contains novel genes with potentially important functions compared with previous assemblies. Subsequently, we sequenced 181 strains from China and the USA and found ∼7.8 million single nucleotide polymorphisms (SNPs). Analysis shows that the B. xylophilus population in China can be divided into geographically bounded subpopulations with severe cross-infection and potential migrations. In addition, distribution of B. xylophilus is dominated by temperature zones while geographically associated SNPs are mainly located on adaptation related GPCR gene families, suggesting the nematode has been evolving to adapt to different temperatures. A machine-learning based epidemic tracking method has been established to predict their geographical origins, which can be applied to any other species.CONCLUSION: Our study provides the community with the first high-quality chromosome-level assembly which includes a comprehensive catalogue of genetic variations. It provides insights into population structure and effective tracking method for this invasive species, which facilitates future studies to address a variety of applied, genomic and evolutionary questions in B. xylophilus as well as related species.

Summary Previous risk prediction studies have mostly shown that suitable areas for pine wood nematode, Bursaphelenchus xylophilus (PWN), in China are mainly concentrated in eastern and southern China, with pest reaching no further northern than the Yangtze River basin. The Chinese pine (Pinus tabuliformis, Chinese pine or Chinese red pine) is the main, native, afforestation species in areas to the north of the Yangtze River basin. To our knowledge, there is no report on PWN infecting Chinese pines in the north of the Yangtze. In this study, we used Baermann funnel method and several PCR primers to detect the presence of PWN in Chinese pine forest fields in 2010 in Shaanxi, China, which is far to the north of the Yangtze River and a cause for concern. Pinus koraiensis (Korean pine) may also be at risk and should also be monitored to prevent invasion by PWN.

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Phosphate-solubilizing bacteria (PSB) have beneficial effects on plant health and soil composition. To date, studies of PSB in soil have largely been performed under field or greenhouse conditions. However, less is known about the impact of introducing indigenous PSB in the field, including their effects on the local microbial community. In this study, we conducted greenhouse and field experiments to explore the effects of the addition of indigenous PSB on the growth of Chenmou elm (Ulmus chenmoui) and on the diversity and composition of the bacterial community in the soil. We obtained four bacterial isolates with the highest phosphate-solubilizing activity: UC_1 (Pseudomonas sp.), UC_M (Klebsiella sp.), UC_J (Burkholderia sp.), and UC_3 (Chryseobacterium sp.). Sequencing on the Illumina MiSeq platform showed that the inoculated PSB did not become the dominant strains in the U. chenmoui rhizosphere. However, the soil bacterial community structure was altered by the addition of these PSB. The relative abundance of Chloroflexi decreased significantly in response to PSB application in all treatment groups, whereas the populations of several bacteria, including Proteobacteria and Bacteroidetes, increased. Network analysis indicated that Chloroflexi was the most strongly negatively correlated with Proteobacteria, whereas Proteobacteria was strongly positively correlated with Bacteroidetes. Our findings indicate that inoculation with PSB (UC_1, UC_M, UC_J, and UC_3) can improve the growth of U. chenmoui and regulate its rhizosphere microbial community. Therefore, inoculation with these bacterial strains could promote the efficient cultivation and production of high-quality plant materials.

Summary The internal transcribed spacer (ITS) regions of rDNA have been routinely employed for identification and phylogenetic analysis of many nematode species. In this study, the intra‐ and interspecies ITS genetic diversity of Bursaphelenchus xylophilus and Bursaphelenchus mucronatus was evaluated. Ninety‐one isolates of the two nematode species collected from 14 Chinese provinces, Japan and Korea were used for ITS‐PCR and sequencing. An unweighted pair group cluster analysis dendrogram clustered them as two B. mucronatus and one B. xylophilus independent clades. Principal component analysis showed the phylogenetic relationship of the two nematode species more clearly; B. mucronatus isolates were separated into more than four groups, whereas B. xylophilus isolates still clustered into a group. The results of the Mantel test indicated the correlation of genetic distance matrices and geographic distance matrices was significant for both nematode species. The genetic differentiation coefficient (Gst) and gene flow (Nm) of B. mucronatus were 0.341 and 1.091, respectively, suggesting the importance of landscape heterogeneity and considerable obstacles for genetic exchange among B. mucronatus isolates in China. However, Gst and Nm of B. xylophilus were 0.188 and 2.151, respectively, very different compared to B. mucronatus. This could be owing to the short‐term introduction of B. xylophilus into China and a rapid spread through anthropogenic pathways. Our work adds to the understanding of the genetic diversity and genetic relationship of the two pine‐parasitic nematode species, and will aid in controlling them in the future.