A pioneer nematode effector suppresses plant reactive oxygen species burst by interacting with the class III peroxidase.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease, which can devastate the pine forest ecosystem. Usually, plant cells generate reactive oxygen species (ROS) as a defensive substance or signalling molecules to resist the infection of nematodes. However, little is known about how B. xylophilus effectors mediate the plant ROS metabolism. Here, we identified a pioneer B. xylophilus Prx3-interacting effector 1 (BxPIE1) expressed in the dorsal gland cells and the intestine. Silencing of the BxPIE1 gene resulted in reduced nematode reproduction and a delay in disease progression during parasitic stages, with the upregulation of pathogenesis-related (PR) genes PtPR-3 (class Ⅳ chitinase) and PtPR-9 (peroxidase). The protein-protein interaction assays further demonstrated that BxPIE1 interacts with a Pinus thunbergii class III peroxidase (PtPrx3), which produces H2O2 under biotic stress. The expression of BxPIE1 and PtPrx3 was upregulated during the infection stage. Furthermore, BxPIE1 effectively inhibited H2O2 generating from class III peroxidase and ascorbate can recover the virulence of siBxPIE1-treated B. xylophilus by scavenging H2O2. Taken together, BxPIE1 is an important virulence factor, revealing a novel mechanism utilized by nematodes to suppress plant immunity.

The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Two Novel Bursaphelenchus xylophilus Kunitz Effector Proteins Using Different Infection and Survival Strategies to Suppress Immunity in Pine.

Pine wilt disease, caused by Bursaphelenchus xylophilus, results in tremendous economic loss in conifer production every year. To disturb the host immune responses, plant pathogens secrete a mass of effector proteins that facilitate the infection process. Although several effectors of B. xylophilus have been identified, detailed mechanisms of their functions remain largely unexplored. Here, we reveal two novel B. xylophilus Kunitz effectors, named BxKU1 and BxKU2, using different infection strategies to suppress immunity in Pinus thunbergii. We found that both BxKU1 and BxKU2 could suppress PsXEG1-triggered cell death and were present in the nucleus and cytoplasm in Nicotiana benthamiana. However, they had different three-dimensional structures and various expression patterns in B. xylophilus infection. In situ hybridization experiments showed that BxKU2 was expressed in the esophageal glands and ovaries, whereas BxKU1 was only expressed in the esophageal glands of females. We further confirmed that the morbidity was significantly decreased in P. thunbergii infected with B. xylophilus when BxKU1 and BxKU2 were silenced. The silenced BxKU2I, but not BxKU1, affected the reproduction and feeding rate of B. xylophilus. Moreover, BxKU1 and BxKU2 targeted to different proteins in P. thunbergii, but they all interacted with thaumatin-like protein 4 (TLP4) according to yeast two-hybrid screening. Collectively, our study showed that B. xylophilus could incorporate two Kunitz effectors in a multilayer strategy to counter immune response in P. thunbergii, which could help us better understand the interaction between plant and B. xylophilus.

Optimization of Constitutive Promoters Using a Promoter-Trapping Vector in Burkholderia pyrrocinia JK-SH007.

Selecting suitable promoters to drive gene overexpression can provide significant insight into the development of engineered bacteria. In this study, we analyzed the transcriptome data of Burkholderia pyrrocinia JK-SH007 and identified 54 highly expressed genes. The promoter sequences were located using genome-wide data and scored using the prokaryotic promoter prediction software BPROM to further screen out 18 promoter sequences. We also developed a promoter trap system based on two reporter proteins adapted for promoter optimization in B. pyrrocinia JK-SH007: firefly luciferase encoded by the luciferase gene set (Luc) and trimethoprim (TP)-resistant dihydrofolate reductase (TPr). Ultimately, eight constitutive promoters were successfully inserted into the probe vector and transformed into B. pyrrocinia JK-SH007. The transformants were successfully grown on Tp antibiotic plates, and firefly luciferase expression was determined by measuring the relative light unit (RLU). Five of the promoters (P4, P9, P10, P14, and P19) showed 1.01-2.51-fold higher activity than the control promoter λ phage transcriptional promoter (PRPL). The promoter activity was further validated via qPCR analysis, indicating that promoters P14 and P19 showed stable high transcription levels at all time points. Then, GFP and RFP proteins were overexpressed in JK-SH007. In addition, promoters P14 and P19 were successfully used to drive gene expression in Burkholderia multivorans WS-FJ9 and Escherichia coli S17-1. The two constitutive promoters can be used not only in B. pyrrocinia JK-SH007 itself to gene overexpression but also to expand the scope of application.

Effects of Plant Growth-Promoting Rhizobacteria on the Growth and Soil Microbial Community of Carya illinoinensis.

Plant growth-promoting rhizobacteria (PGPR) are important members of soil microbial communities. In this study, the effects of several PGPR on the growth of Carya illinoinensis plants, the microbial community composition and soil nutrients were investigated by inoculation tests to identify excellent PGPR strains. The experiment showed that after PGPR application, the plant height, ground diameter, and dry weight of C. illinoinensis were significantly increased compared with those of the control group, and Bacillus velezensis YH20 had the most significant effect in promoting growth (p < 0.05). In addition, all the PGPRs used for inoculation promoted plant root growth, and the Brevibacillus reuszeri MPT17 strain had the most significant promoting effect on plant root growth (p < 0.05). The application of PGPRs also affected the nutrient levels in plants and plant rhizosphere soil. For example, compared with the control, the levels of available phosphorus and potassium in rhizosphere soil and the total potassium content in plant roots were significantly increased under Br. reuszeri MPT17 treatment (p < 0.05). The experiment showed that the relative abundance of Mortierella, Dictyophora, and Bacillus in the rhizosphere soil increased significantly after the application of PGPR (p < 0.05). These genera could effectively improve the rate of soil nutrient use, antagonize plant pathogenic bacteria, and promote plant growth. This study provides basic reference data regarding the use of PGPR to improve the microecological environment and promote the growth and development of C. illinoinensis plants.

First Report of Needle Blight on Pinus thunbergii Parl. Caused by Fusarium proliferatum in China.

Pinus thunbergii Parl. (Japanese black pine), an evergreen species, is distributed along the seacoasts of China. In addition, this species has been planted along seacoasts as a windbreak to prevent soil erosion due to its resistance to salt and various environmental stresses. It can also be found in public parks and gardens due to its exquisite appearance and toughness. In August 2020, needle blight symptoms were found on several black pine trees in Sichuan Province, China. Further surveys showed that these symptoms are common. The disease incidence is less than 30% while severity of the disease is high. The tips of old needles first turn grayish green that developed into brown bands ranging from 1 to 2 mm. To determine the pathogen, small needle pieces (3-4 mm2 long) from the margin of fresh lesions were surface-sterilized for 30 s in 75% ethanol, follow by 1% NaOCl for 90 s, then washed three times with sterile water, and then were placed on potato dextrose agar (PDA) with 0.1 mg/mL ampicillin and incubated at 25°C. Pure cultures of 8 isolates were obtained by monosporic isolation, and a representative isolate (SC03) was deposited in the Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University. When cultured on Spezieller Nährstoffarmer Agar medium (Leslie and Summerell, 2006), the SC03 colony was beige-white, cottony from top view and pale orange near the center on the reverse side. The daily growth rate was 11.8 mm/day at 25°C in the dark. Microscopic observations showed hyaline and septate hyphae, slightly curved macroconidia with two to three septa measuring 17.5 - 30 × 3.7 - 7.5 µm (23.2 × 5.7 on average), and aseptate microconidia measuring 7.5 - 12.5 × 2.5 - 5.0 µm, (9.7 × 4.3 on average). The morphological characteristics of conidia and other structures of SC03 matched those of the Fusarium fujikuroi species complex (FFSC) (Abdalla et al. 2000). For accurate identification, translation elongation factor 1-alpha（TEF-1α） , and the second largest RNA polymerase subunit (RPB2) were amplified and sequenced using the primer pairs EF1 and EF2, RPB5f2 and RPB7cr. The sequences were deposited in GenBank [Accession TEF-1α: ON049647, RBP2: ON049648]. A Blast search of GenBank showed that TEF-1α and RPB2 sequences of SC03 matched Fusarium proliferatum (Matsush) Nirenberg at a high level (>99%). Phylogenetic analysis using neighbor joining and concatenated sequences (TEF-1α and RPB2) with MEGA X placed SC03 in F. proliferatum. For the pathogenicity test, a conidial suspension was prepared with a concentration of 2.0 × 107 conidia/ml. The suspension was sprayed onto 3 annual seedlings' needles, and the control was sprayed with sterile water. Inoculated and uninoculated plants were kept in humid chambers in a glasshouse where the average humidity was 60% and the average temperature was 27℃. After 10 days, typical symptoms appeared on inoculated needles, whereas control needles remained symptomless. These symptoms were similar to those observed in field. The fungus, F. proliferatum, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Fusarium proliferatum is a ubiquitous saprophytic fungus on cankers and very rarely reported to cause disease on pine needles. It has been reported to cause leaf blight of Polygonatum cyrtonema (Zhou et al. 2021) and Majesty palm (Polizzi and Vitale 2003). To our knowledge, this is the first report of needle blight on P. thunbergii caused by F. proliferatum in China. The disease represents a threat to producers and more research on the biology and management is needed.

Pathogenicity and Biological Characteristics of Septotinia populiperda Causing Leaf Blotch of Willow.

Salix babylonica is an important landscape tree in China and has been widely planted. In this study, the pathogenicity of Septotinia populiperda causing leaf blotch of Sa. babylonica to four willow species (Sa. matsudana, Sa. chaeomoloides, Sa. matsudana f. tortuosa, and Sa. suchowensis) and Populus tomentosa (Chinese white poplar) was determined. Its sexual stage and biological characteristics were studied. Leaves from four willow species and P. tomentosa were inoculated with mycelial plugs. Typical leaf blotches with sporodochia were produced on all inoculated leaves. Among the isolates studied, some developed conidia but sclerotia were rare. The sclerotia developed apothecia after induction at 4°C for 3 months in an incubator and 2 more months outdoors from January to March. The biological characteristics of S. populiperda showed that mycelium grew better on complete medium than on potato dextrose agar, Czapek's agar, and minimal medium. For mycelial growth, the optimal carbon source was dextrose and the optimal nitrogen source was yeast powder. Conidia germination rate was 59.4% at 24 h. The conidia germinated best in a 4% willow leaf extraction. The optimal temperature for conidia germination was 25°C, and the optimal pH was 4.

Studies on the Requirement of Transthyretin Protein (BxTTR-52) for the Suppression of Host Innate Immunity in Bursaphelenchus xylophilus.

The pinewood nematode, Bursaphelenchus xylophilus, has been determined as one of the world's top ten plant-parasitic nematodes. It causes pine wilt, a progressive disease that affects the economy and ecologically sustainable development in East Asia. B. xylophilus secretes pathogenic proteins into host plant tissues to promote infection. However, little is known about the interaction between B. xylophilus and pines. Previous studies reported transthyretin proteins in some species and their strong correlation with immune evasion, which has also been poorly studied in B. xylophilus. In this study, we cloned and functionally validated the B. xylophilus pathogenic protein BxTTR-52, containing a transthyretin domain. An in situ hybridization assay demonstrated that BxTTR-52 was expressed mainly in the esophageal glands of B. xylophilus. Confocal microscopy revealed that BxTTR-52-RFP localized to the nucleus, cytoplasm, and plasma membrane. BxTTR-52 recombinant proteins produced by Escherichia coli could be suppressed by hydrogen peroxide and antioxidant enzymes in pines. Moreover, silencing BxTTR-52 significantly attenuated the morbidity of Pinus thunbergii infected with B. xylophilus. It also suppressed the expression of pathogenesis-related genes in P. thunbergii. These results suggest that BxTTR-52 suppresses the plant immune response in the host pines and might contribute to the pathogenicity of B. xylophilus in the early infection stages.

A Bursaphelenchus xylophilus Effector, BxSCD3, Suppresses Plant Defense and Contributes to Virulence.

Bursaphelenchus xylophilus is the most economically important species of migratory plant-parasitic nematodes (PPNs) and causes severe damage to forestry in China. The successful infection of B. xylophilus relies on the secretion of a repertoire of effector proteins. The effectors, which suppress the host pine immune response, are key to the facilitation of B. xylophilus parasitism. An exhaustive list of candidate effectors of B. xylophilus was predicted, but not all have been identified and characterized. Here, an effector, named BxSCD3, has been implicated in the suppression of host immunity. BxSCD3 could suppress pathogen-associated molecular patterns (PAMPs) PsXEG1- and INF1-triggered cell death when it was secreted into the intracellular space in Nicotiana benthamiana. BxSCD3 was highly up-regulated in the early infection stages of B. xylophilus. BxSCD3 does not affect B. xylophilus reproduction, either at the mycophagous stage or the phytophagous stage, but it contributes to the virulence of B. xylophilus. Moreover, BxSCD3 significantly influenced the relative expression levels of defense-related (PR) genes PtPR-3 and PtPR-6 in Pinus thunbergii in the early infection stage. These results suggest that BxSCD3 is an important toxic factor and plays a key role in the interaction between B. xylophilus and host pine.

Comparative transcriptomic analysis of candidate effectors to explore the infection and survival strategy of Bursaphelenchus xylophilus during different interaction stages with pine trees.

BACKGROUND: The pine wood nematode (PWN), Bursaphelenchus xylophilus, is a devastating pathogen of many Pinus species in China. The aim of this study was to understand the interactive molecular mechanism of PWN and its host by comparing differentially expressed genes and candidate effectors from three transcriptomes of B. xylophilus at different infection stages. RESULTS: In total, 62, 69 and 46 candidate effectors were identified in three transcriptomes (2.5 h postinfection, 6, 12 and 24 h postinoculation and 6 and 15 d postinfection, respectively). In addition to uncharacterized pioneers, other candidate effectors were involved in the degradation of host tissues, suppression of host defenses, targeting plant signaling pathways, feeding and detoxification, which helped B. xylophilus survive successfully in the host. Seven candidate effectors were identified in both our study and the B. xylophilus transcriptome at 2.5 h postinfection, and one candidate effector was identified in all three transcriptomes. These common candidate effectors were upregulated at infection stages, and one of them suppressed pathogen-associated molecular pattern (PAMP) PsXEG1-triggered cell death in Nicotiana benthamiana. CONCLUSIONS: The results indicated that B. xylophilus secreted various candidate effectors, and some of them continued to function throughout all infection stages. These various candidate effectors were important to B. xylophilus infection and survival, and they functioned in different ways (such as breaking down host cell walls, suppressing host defenses, promoting feeding efficiency, promoting detoxification and playing virulence functions). The present results provide valuable resources for in-depth research on the pathogenesis of B. xylophilus from the perspective of effectors.

First Report of Brown Spot Needle Blight on Pinus thunbergii Parl. Caused by Aureobasidium pullulans in China.

Pinus thunbergii Parl., known as black pine, is widely distributed all over China. This pine variety can prevent soil desertification and promote soil conservation and is excellent for constructing fast-growing forests and shelter belts. The timber of this species can be used for infrastructure construction and furniture production. In August 2020, needle blight symptoms were found on several trees of black pine in Sichuan Province, China. Further surveys showed that these symptoms are common while the disease incidence is less than 30% which indicated the severity of the disease is mild. The tips of old needles first turn grayish green and developed into brown bands ranging from 1 to 2 mm. To determine the pathogen, 20 needle samples with typical symptoms were disinfected with 75% alcohol, and sections of the tissue were cut from joints of diseased and healthy tissues (visually healthy) with a sterilized scalpel, surface sterilized for 45 seconds in 75% alcohol, soaked for 90 seconds in 1.5% NaCIO, rinsed in sterilized water and dried. Small cut tissues were placed on potato dextrose agar (PDA) at 25℃ for 10 days. Pure cultures were obtained by monosporic isolation. The colonies initially appeared white to cream, yeast-like, and later turned to pink and remained at least 10 days. Conidia were hyaline, smooth-walled, single-celled, and ellipsoidal with variable shape and size, 7.5 to 16 × 3.5 to 7 µm (Zalar et al. 2008). DNA was extracted from the mycelium of the isolate by the cetyltriethylammonium bromide (CTAB) method and amplified through polymerase chain reaction (PCR) with the internal transcribed spacer (ITS) region of rDNA and partial ß-tubulin genes of a representative isolate (SC05) were amplified using the ITS1/ITS4 and Bt2a/Bt2b primer pairs, respectively(Wu et al. 2017). The sequences submitted to GenBank (Accession Nos. MW228368 for ITS and MW256762 for ß-tubulin) showed high similarity with BLAST sequences of Aureobasidium pullulans (ITS, KR704881 [100%]; ß-tubulin, MT671934 [99.49%]). For the pathogenicity test, a conidial suspension was prepared with a concentration of 2.0 × 107 conidia/ml. The suspension was sprayed onto 3 annual seedlings' needles, and the control was sprayed with sterile water. Inoculated and non-inoculated plants were kept in humid chambers in a glasshouse. After 10 days, typical symptoms appeared on inoculated needles, whereas control needles remained symptomless. The fungus, A. pullulans, was reisolated from those lesions, confirming Koch's postulates. No symptoms were observed on control plants. Aureobasidium pullulans, a ubiquitous saprophytic fungus on many fruits and very rarely reported to cause disease on pine needles. Only reported invasion of Ozone-injured needles in P. strobus (Costonis and Sinclair 1972) and needles damaged by acid rain in P. sylvestris (Ranta 1990). To our knowledge, this is the first report of brown spot needle blight on P. thunbergii caused by A. pullulans in China. The disease represents a threat to pine manufactures and more research on the pathogenesis and management is needed. .

Effects of Different Culture Conditions on the Biofilm Formation of Bacillus pumilus HR10.

Many studies have focused on the significant role of biofilm formation by Bacillus in the biocontrol process. Bacillus pumilus HR10 is a plant growth-promoting rhizobacterium with multiple biocontrol functions, including promoting growth, controlling pathogens, and assisting in the formation of mycorrhizae. Currently, there is no relevant report on the biofilm formation of B. pumilus HR10 and its influencing factors. B. pumilus HR10 was found to easily form a stable biofilm structure on the surface of media, with awesome swarming ability. The optimum temperature for biofilm formation was 37 °C. B. pumilus HR10 formed most obviously at pH 7.0 and was not extremely sensitive to acidic or alkaline conditions. Most of the polysaccharide components of plant root exudates promoted the biofilm formation by B. pumilus HR10, with glucose having the greatest promoting effect. Low concentrations of Fe2+, Mg2+, Ca2+, K+, and Na+ enhanced biofilm formation. In summary, biofilm formation can improve the tolerance of B. pumilus HR10 to salt and certain heavy metal ion stresses and contribute to its application in different plants and soils with high salinity or heavy metals in the field.

An Effector, BxSapB1, Induces Cell Death and Contributes to Virulence in the Pine Wood Nematode Bursaphelenchus xylophilus.

The pine wood nematode (PWN) Bursaphelenchus xylophilus has caused serious damage to pine forests in China. Effectors secreted by phytonematodes play a role in host infection. We identified and characterized an effector, BxSapB1, based on the B. xylophilus transcriptome at the early stages of infection and the transient expression of proteins in Nicotiana benthamiana. BxSapB1 triggered cell death in N. benthamiana when secreted into the apoplast, and this effect was independent of N. benthamiana brassinosteroid-insensitive 1-associated kinase 1 (NbBAK1) and suppressor of BIR1-1 (NbSOBIR1). The signal peptide of BxSapB1 was proven to be functional in yeast using the yeast signal sequence trap system and BxSapB1 was strongly expressed in the subventral gland cells of B. xylophilus, as revealed by in-situ hybridization. In addition, based on local BLAST analysis, the BxSapB1 showed 100% identity to BUX.s00139.62, which was identified from the B. xylophilus secretome during Pinus thunbergii infection. BxSapB1 was upregulated in a highly virulent strain and downregulated in a weakly virulent strain of PWN at the early stages of infection. RNA interference assays showed that silencing BxSapB1 resulted in decreased expression of pathogenesis-related genes (PtPR-1b, PtPR-3, and PtPR-5) as well as delayed onset of symptoms in P. thunbergii infected by B. xylophilus. The combined data suggest that BxSapB1 can trigger cell death in N. benthamiana and that it contributes to the virulence in B. xylophilus during parasitic interaction.

Major sperm protein BxMSP10 is required for reproduction and egg hatching in Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a disastrous pathogen of pine forests in East Asia and Europe. Despite its decimating effect on pine forests, efficient and environmentally friendly methods available to control the pine wood nematode (PWN) are limited. The most abundant protein in nematode sperm, major sperm proteins (MSPs) have only been discovered in nematodes. In this study, phylogenetic analysis showed that BxMSP10 was highly conserved in the nematode and had a closer phylogenetic relationship with free-living nematodes than with plant-parasitic nematode species. BxMSP10 was specifically expressed in the seminal vesicle of male adults. dsRNA of BxMSP10 significantly decreased reproduction, egg hatching and population maintenance in B. xylophilus. These results indicated that BxMSP10 was a potential candidate for application in the control of B. xylophilus.

Shoot Blight on Chinese Fir (Cunninghamia lanceolata) is Caused by Bipolaris oryzae.

Chinese fir (Cunninghamia lanceolata) is a significant timber species that has been broadly cultivated in southern China. A shoot blight disease on Chinese fir seedlings was discovered in Fujian, China and a fungus was then consistently associated with the symptoms. This fungus was determined to be causing this disease, among others by fulfilling Koch's postulates. Based on morphological characteristics and multilocus phylogenetic analyses with the sequences of the internal transcribed spacer, partial glyceraldehyde-3-phosphate dehydrogenase gene, partial translation elongation factor 1-α gene, and partial 28S large subunit ribosomal RNA gene, the fungus was identified as Bipolaris oryzae. These characteristics and phylogenetic analyses clearly support that this pathogen is different from B. sacchari, which was, until now, considered to be the causal agent of a similar blight on Chinese fir in Guangdong, China. The fungus was also shown to be strongly pathogenic to rice, one of the most susceptible hosts to B. oryzae. Crop rotation involving rice is often carried out with Chinese fir in southern China, a practice that most likely increases the risk of shoot blight on C. lanceolata. To our knowledge, shoot blight caused by B. oryzae is reported for the first time in a gymnosperm species.

Expression Profiling of Autophagy Genes BxATG1 and BxATG8 under Biotic and Abiotic Stresses in Pine Wood Nematode Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchusxylophilus, is the pathogen of pine wilt disease (PWD) and causes huge economic losses in pine forests and shows a 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. Our previous studies showed that autophagy exists in PWN. Therefore, we tested the effects of autophagy inducer rapamycin on PWN and the results revealed that the feeding rate and reproduction were significantly promoted on fungal mats. The gene expression patterns of BxATG1 and BxATG8 under the different stress were determined by quantitative reverse transcription PCR (qRT-PCR). We tested the effects of RNA interference on BxATG1 and BxATG8 in PWN during different periods of infection in Pinus thunbergii. The results revealed that BxATG1 and BxATG8 may play roles in allowing PWN to adapt to changing environmental conditions and the virulence of PWN was influenced by the silence of autophagy-related genes BxATG1 and BxATG8. These results provided fundamental information on the relationship between autophagy and PWN, and on better understanding of gene function of BxATG1 and BxATG8 in PWN.

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 Koch's 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.

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.

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.

Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.