Jian-Ren Ye

Role of human-mediated dispersal in the spread of the pinewood nematode in China.

Background Intensification of world trade is responsible for an increase in the number of alien species introductions. Human-mediated dispersal promotes not only introductions but also expansion of the species distribution via long-distance dispersal. Thus, understanding the role of anthropogenic pathways in the spread of invading species has become one of the most important challenges nowadays. Methodology/Principal Findings We analysed the invasion pattern of the pinewood nematode in China based on invasion data from 1982 to 2005 and monitoring data on 7 locations over 15 years. Short distance spread mediated by long-horned beetles was estimated at 7.5 km per year. Infested sites located further away represented more than 90% of observations and the mean long distance spread was estimated at 111–339 km. Railways, river ports, and lakes had significant effects on the spread pattern. Human population density levels explained 87% of the variation in the invasion probability (P<0.05). Since 2001, the number of new records of the nematode was multiplied by a factor of 5 and the spread distance by a factor of 2. We combined a diffusion model to describe the short distance spread with a stochastic, individual based model to describe the long distance jumps. This combined model generated an error of only 13% when used to predict the presence of the nematode. Under two climate scenarios (stable climate or moderate warming), projections of the invasion probability suggest that this pest could expand its distribution 40–55% by 2025. Conclusions/Significance This study provides evidence that human-induced dispersal plays a fundamental role in the spread of the pinewood nematode, and appropriate control measures should be taken to stop or slow its expansion. This model can be applied to Europe, where the nematode had been introduced later, and is currently expanding its distribution. Similar models could also be derived for other species that could be accidentally transported by humans.

Specific and functional diversity of endophytic bacteria from pine wood nematode Bursaphelenchus xylophilus with different virulence.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is one of the most devastating diseases of Pinus spp. The PWN was therefore listed as one of the most dangerous forest pests in China meriting quarantine. Virulence of the PWN is closely linked with the spread of PWD. However, main factors responsible for the virulence of PWNs are still unclear. Recently epiphytic bacteria carried by PWNs have drawn much attention. But little is known about the relationship between endophytic bacteria and virulence of B. xylophilus. In this research, virulence of ten strains of B. xylophilus from different geographical areas in six provinces of China and four pine species were tested with 2-year-old seedlings of Pinus thunbergii. Endophytic bacteria were isolated from PWNs with different virulence to investigate the relationship between the bacteria and PWN virulence. Meanwhile, the carbon metabolism of endophytic bacteria from highly and low virulent B. xylophilus was analyzed using Biolog plates (ECO). The results indicated that ten strains of PWNs showed a wide range of virulence. Simultaneously, endophytic bacteria were isolated from 90% of the B. xylophilus strains. The dominant endophytic bacteria in the nematodes were identified as species of Stenotrophomonas, Achromobacter, Ewingella, Leifsonia, Rhizobium, and Pseudomonas using molecular and biochemical methods. Moreover, S. maltophilia, and A. xylosoxidans subsp. xylosoxidans were the predominant strains. Most of the strains (80%) from P. massoniana contained either S. maltophilia, A. xylosoxidans, or both species. There was a difference between the abilities of the endophytic bacteria to utilize carbon sources. Endophytic bacteria from highly virulent B. xylophilus had a relatively high utilization rate of carbohydrate and carboxylic acids, while bacteria from low virulent B. xylophilus made better use of amino acids. In conclusion, endophytic bacteria widely exist in B. xylophilus from different pines and areas; and B. xylophilus strains with different virulence possessed various endophytic bacteria and diverse carbon metabolism which suggested that the endophytic bacteria species and carbon metabolism might be related with the B. xylophilus virulence.

Pathogenicity of aseptic Bursaphelenchus xylophilus.

Pine wilt is a disease of pine (Pinus spp.) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus. However, the pathogenic mechanism of pine wilt disease (PWD) remains unclear. Although the PWN was thought to be the only pathogenic agent associated with this disease, a potential role for bacterial symbionts in the disease process was recently proposed. Studies have indicated that aseptic PWNs do not cause PWD in aseptic pine trees, while PWNs associated with bacteria cause wilting symptoms. To investigate the pathogenicity of the PWN and its associated bacteria, 3-month-old microcuttings derived from certain clones of Pinus densiflora Siebold & Zucc. produced in vitro were inoculated under aseptic conditions with aseptic PWNs, non-aseptic PWNs and bacteria isolated from the nematodes. Six-month-old aseptic P. densiflora microcuttings and 7-month-old P. massoniana seedlings were also inoculated under aseptic conditions with aseptic PWNs and non-aseptic PWNs. The results showed that the aseptic microcuttings and seedlings inoculated with aseptic PWNs or non-aseptic PWNs wilted, while those inoculated with bacterial isolates did not wilt. Nematodes were recovered from wilted microcuttings and seedlings inoculated with aseptic PWNs and non-aseptic PWNs, and the asepsis of nematodes recovered from aseptic PWN-inoculated microcuttings and seedlings was reconfirmed by culturing them in NB liquid medium at 30°C for more than 7 days. Taken together, the results indicate that the asepsis of PWN did not cause the loss of pathogenicity.

Specifically expressed genes of the nematode Bursaphelenchus xylophilus involved with early interactions with pine trees.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD.

Deep sequencing analyses of pine wood nematode Bursaphelenchus xylophilus microRNAs reveal distinct miRNA expression patterns during the pathological process of pine wilt disease.

Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs and novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Functional analysis revealed that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes. In addition, another set of atypical miRNAs, termed mirtrons, was also identified in this study. Thus, our research has provided detailed characterization of B. xylophilus miRNA expression patterns during the pathological process of pine wilt disease. These findings would contribute to more in-depth understanding of this devastating plant disease.

Colletotrichum gloeosporioides sensu stricto Is a Pathogen of Leaf Anthracnose on Evergreen Spindle Tree (Euonymus japonicus)

The genus Colletotrichum is considered the eighth most important group of plant-pathogenic fungi in the world due to its scientific and economic importance. Colletotrichum spp. cause anthracnose disease in a wide range of economically important plants. Euonymus japonicus Thunb. (Celastraceae) is a broad-leaved evergreen tree that is widely planted in the parks and landscapes of China. An anthracnose occurs on E. japonicus in China but there has been a disagreement on the identity of the fungal pathogen. In this study, the fungal isolate HYCG2-3 was determined by Kochs postulates to be a pathogen on E. japonicus. Based on the morphological and molecular methods, isolate HYCG2-3 was identified as Colletotrichum gloeosporioides sensu stricto within the C. gloeosporioides species complex.

Deciphering the Molecular Variations of Pine Wood Nematode Bursaphelenchus xylophilus with Different Virulence

Bursaphelenchus xylophilus is the causative agent of pine wilt disease which has caused huge economic losses in many countries. It has been reported that two forms of pine wood nematodes existed in its native region, i.e., with strong virulence and weak virulence. However, little is known about the molecular differences between the two forms. To better understand their molecular variations, transcriptome and genome sequences of three strongly virulent and one weakly virulent strains were analyzed. We found 238 transcripts and 84 exons which showed notable changes between the two virulent forms. Functional analyses of both differentially expressed transcripts and exons indicated that different virulence strains showed dissimilar nematode growth, reproduction, and oxidoreductase activities. In addition, we also detected a small number of exon-skipping events in B. xylophilus. Meanwhile, 117 SNPs were identified as potential genetic markers in distinguishing the two forms. Four of them were further proved to have undergone allele specific expressions and possibly interrupted the target site of evolutionary conserved B. xylophilus miR-47. These particular SNPs were experimentally verified by including eight additional strains to ensure the validity of our sequencing results. These results could help researchers to better diagnose nematode species with different virulence and facilitate the control of pine wilt disease.

Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes, BxATG1 and BxATG8.

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.

The effect of endobacteria on the development and virulence of the pine wood nematode, Bursaphelenchus xylophilus

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is a destructive forest disease. Recent studies showed that bacteria associated with PWN might play important roles in PWD. In this study, the effects of endobacteria isolated from PWN on the development and virulence of PWN were evaluated. The results revealed that endobacteria isolated from high virulence PWN could partially promote the development of the nematode, while endobacteria from PWN with low virulence could inhibit the development of nematode. Both bacteria-free and non-sterilised nematodes were able to infect microcuttings of Pinus densiflora. The endobacterial isolates, Stenotrophomonas maltophiliaNSBx.14 and Achromobacter xylosoxidansssp. xylosoxidansNSBx.22, enhanced the virulence of low virulence PWN but reduced the virulence of high virulence PWN. The results suggest the pathogenicity of PWN is not affected by loss of bacteria and endobacteria might play a role in the development and virulence of PWN.

Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD.