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Pinus, a conifer, dominates the world’s forest ecosystems. But it is seriously infected with pine wood nematode (PWN). Transcription factors (TFs) are key regulators in regulating plant resistance. However, the molecular mechanism of TFs remains thus far unresolved in P. thunbergii inoculated with Bursaphelenchus xylophilus. Here, we used RNA-seq technology to identify differentially expressed TFs in resistant and susceptible pines. The results show that a total of 186 differentially expressed transcription factors (DETFs), including 99 upregulated and 87 downregulated genes were identified. Gene ontology (GO) enrichment showed that the highly enriched differentially expressed TFs were responsible for secondary biosynthetic processes. According to KEGG pathway analysis, the differentially expressed TFs were related to chaperones and folding catalysts, phenylpropanoid biosynthesis, and protein processing in the endoplasmic reticulum. Many TFs such as NAC, LBD, MYB, bHLH, and WRKY were determined to be quite abundant in the DETFs. Moreover, the NAC transcription factor PtNAC9 was upregulated in PWN-resistant and susceptible P. thunbergii and especially distinctly upregulated in resistant pines. By purifying recombinant PtNAC9 protein in vitro, we found that overexpression of PtNAC9 at the early stage of B. xylophilus infection could reduce the degree of disease. We also demonstrated the content of salicylic acid (SA) and the related genes were increased in the PtNAC9 protein-treated plants. These results could be helpful in enhancing our understanding of the resistance mechanism underlying different resistant pine.

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 β-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.

Persistence in the soil is a function of seed physiology, particularly non-germination and inherent lifespan. However, for seeds with mechanical dormancy, non-germination is also a function of the composition and activity of the soil microbiota. We attempted to screen out microorganisms in the soil that can specifically and rapidly decompose the hard fruit pericarps of Tilia miqueliana Maxim., a unique native tree species in China. Using the classical replica plating method, more than 100 different culturable microorganisms that could rapidly erode the pericarp were collected from the surface of pericarps under different culture conditions. At the same time, we successfully extended the concept of metagenomics and applied it to the identification of mixed artificial cultures. The decomposition process of the pericarps in soil was also simulated artificially. The physical and chemical data suggested a potential mechanism of microbial scarification and cracking in pericarp, whilst the embryos inside the eroded fruits retained good viability. Our discoveries could pave the way for the removal of physical and mechanical obstacles that prevent hard coat seeds from germinating. We anticipate that the use of this technology will improve the germination of other hard coat seeds. More research is needed to investigate the impacts on other seeds. The findings of this research can inform the design of experiments on the seed ecology of persistence.

Previous research has indicated that Pseudomonas aurantiaca ST-TJ4 possesses a notable antagonistic impact on Phytophthora cinnamomi and holds promising potential for biocontrol. In this study, a combination of a single-factor experiment, a Plackett–Burman design and a response surface approach was employed to investigate the optimal formula of ST-TJ4 fermentation medium. Furthermore, the stability of ST-TJ4 fermentation filtrate and its biocontrol effect on Ph. cinnamomi in vivo were also evaluated. The results revealed that the optimal culture conditions for ST-TJ4 involved the use of 20.59 g/L of glucose and 18.76 g/L of yeast extract powder. Following optimization, the fermentation filtrate of ST-TJ4 exhibited an inhibition rate of 76.5%, representing a 15% increase compared to previous levels. Additionally, phzA, phzB, phzD, phzE, phzF and phzO genes involved in the synthesis of phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) were also upregulated. The ST-TJ4 fermentation filtrate demonstrated strong alkali resistance, weak acid resistance and favorable temperature and UV light stability. Furthermore, in vitro inoculation experiments confirmed that optimizing the fermentation medium reduced Ps. cinnamomi’s ability to infect the leaves of Rhododendron pulchrum.

Keypoints Fermentation medium with high antagonistic activity of HR10 strain was determined. Antagonistic crude protein of HR10 strain purified by Ion-Exchange Chromatography. Composition of the antagonist protein of HR10 strain was analyzed using LC-MS-MS.

Abstract Background Bursaphelenchus xylophilus is the causal agent of pine wilt disease (PWD) that has caused enormous ecological and economic losses in China. The mechanism in the interaction between nematodes and pine remains unclear. Plant parasitic nematodes (PPNs) secrete effectors into host plant tissues. However, it is poorly studied that role of effector in the infection of pine wood nematode (PWN). Results We cloned, characterized and functionally validated the B. xylophilus effector BxML1, containing an MD-2-related lipid-recognition (ML) domain. This protein inhibits immune responses triggered by the molecular pattern BxCDP1 of B. xylophilus. An insitu hybridization assay demonstrated that BxML1 was expressed mainly in the dorsal glands and intestine of B. xylophilus. Subcellular localization analysis showed the presence of BxML1 in the cytoplasm and nucleus. Furthermore, number of B. xylophilus and morbidity of pine were significantly reduced in Pinus thunbergii infected with B. xylophilus when BxML was silenced. Using yeast two-hybrid (Y2H) and coimmunoprecipitation (CoIP) assays, we found that the BxML1 interacts with cyclophilin protein PtCyP1 in P. thunbergii. Conclusions This study illustrated that BxML1 plays a critical role in the B. xylophilus–plant interaction and virulence of B. xylophilus.

Volatile organic compounds (VOCs) produced by microorganisms are considered promising environmental-safety fumigants for controlling soil-borne diseases. Verticillium dahliae, a notorious fungal pathogen, causes economically important wilt diseases in agriculture and forestry industries. Here, we determined the antifungal activity of VOCs produced by Trichoderma koningiopsis T2. The VOCs from T. koningiopsis T2 were trapped by solid-phase microextraction (SPME) and tentatively identified through gas chromatography–mass spectrometry (GC/MS). The microsclerotia formation, cell wall-degrading enzymes and melanin synthesis of V. dahliae exposed to the VOC mixtures and selected single standards were examined. The results showed that the VOCs produced by strain T2 significantly inhibited the growth of V. dahliae mycelium and reduced the severity of Verticillium wilt in tobacco and cotton. Six individual compounds were identified in the volatilome of T. koningiopsis T2, and the dominant compounds were 3-octanone, 3-methyl-1-butanol, butanoic acid ethyl ester and 2-hexyl-furan. The VOCs of strain T2 exert a significant inhibitory effect on microsclerotia formation and decreased the activities of pectin lyase and endo-β-1,4-glucanase in V. dahliae. VOCs also downregulated the VdT3HR, VdT4HR, and VdSCD genes related to melanin synthesis by 29. 41-, 10. 49-, and 3.11-fold, respectively. Therefore, T. koningiopsis T2 has potential as a promising biofumigant for the biocontrol of Verticillium wilt disease.

Plant growth-promoting rhizobacteria are important for improving plant iron nutrition, but the interactions among inoculants, host plants and soil microorganisms have not been greatly explored. Rahnella aquatilis JZ-GX1 was applied to treat the increasingly serious iron deficiency chlorosis in Cinnamomum camphora, and the resulting improvement in chlorosis was determined by assessing the contents of chlorophyll, active iron, Fe2+ and antioxidant enzymes in leaves, the effects on the soil microbial community and the metabolism in the rhizosphere by high-throughput sequencing techniques and liquid chromatography–mass spectrometry (LC–MS). The results showed that inoculation with JZ-GX1 significantly increased the chlorophyll content of C. camphora, which promoted the redistribution of active iron in roots and leaves, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and thus reduced membrane damage in iron-deficient C. camphora caused by reactive oxygen species. According to genome prediction and ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) analysis, the JZ-GX1 strain could secrete desferrioxamine (DFO), and the concentration of DFO in C. camphora rhizosphere was 21-fold higher than that in uninoculated soil. The exogenous application of DFO increased the SPAD and Fe2+ contents in leaves. In addition, the inoculant affected the fungal community structure and composition in the C. camphora rhizosphere soil and increased the abundances of specific taxa, such as Glomus, Mortierella, Trichoderma, and Penicillium. Therefore, R. aquatilis JZ-GX1 application promoted iron absorption in C. camphora trees by secreting DFO and alleviated iron deficiency chlorosis through interactions with the local fungal community.

The migratory plant-parasitic nematode Bursaphelenchus xylophilus is the pathogen of the pine wilt disease (PWD), causing serious damage to pine forests in China. During the process of plant resistance to multiple pathogens, plant immunity plays a key role. In this current study, the pathogen-associated molecular pattern (PAMP) BxCDP1 in B. xylophilus has been identified, but the host target protein of BxCDP1 and its key amino acid region inducing the plant immunity have yet to be elucidated. We found that BxCDP1 could trigger superoxide production, H2O2 production, and callose deposits. A RING-H2 finger protein 1 (RHF1) of Pinus thunbergii was screened and characterized as a target protein of BxCDP1 by yeast two-hybrid and co-immunoprecipitation (Co-IP). Moreover, two peptides (namely M9 and M16) proved to be key regions of BxCDP1 to induce PAMP-triggered immunity (PTI) in Nicotiana benthamiana, which also induced the expression of pathogenesis-related (PR) genes (PtPR-3, PtPR-4, and PtPR-5) in P. thunbergii and enhanced the resistance of the host to B. xylophilus. These results indicate that BxCDP1 plays a critical role in the interaction between B. xylophilus and P. thunbergii, and both peptides M9 and M16 have the potential to be developed and utilized as immune inducers of pines against B. xylophilus in future.

Salt stress is an environmental stress that severely limits plant growth, development and productivity. The use of symbiotic relationships with beneficial microorganisms provides an efficient, cost-effective and environmentally friendly preventative method. The plant growth-promoting rhizobacteria (PGPR) Rahnella aquatilis JZ-GX1 is a moderately salinophilic strain with good probiotic properties, although its ability to improve woody plant salt tolerance has not been reported. In this study, the effect of JZ-GX1 on Cinnamomum camphora under different salt concentrations (0, 50 and 100 mM NaCl) was investigated to reveal the mechanism by which JZ-GX1 improves salt tolerance in C. camphora. The results showed that JZ-GX1 promoted plant growth and root development. The relative electrolyte leakage (REL) and malondialdehyde (MDA) production of inoculated C. camphora plants were reduced by 37.38% and 21.90%, respectively, and the superoxide dismutase (SOD) activity in the leaves was enhanced by 321.57% under a 100 mM NaCl treatment. It was observed by transmission electron microscopy that under 100 mM salt concentration conditions, the inoculated C. camphora leaf cells showed a significant reduction in plasma membrane–cell wall separation and intact chloroplast structures, with tightly packed thylakoids. Importantly, inoculation reduced Na+ accumulation and promoted K+ accumulation in the seedlings, and these changes were consistent with the upregulated expression of the K+ channel SKOR and the vesicular membrane (Na+, K+)/H+ reverse cotransporter NHX1 in the plant roots. This study revealed the mechanism of the Rahnella aquatilis JZ-GX1 enhancing salt tolerance of C. camphora.

Abstract 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.

There are complex interactions between mycorrhizal helper bacteria (MHBs) and ectomycorrhizal (ECM) fungi, with MHBs promoting mycorrhizal synthesis and ECM fungi regulating plant rhizobacterial colonization, diversity, and function. In this study, to investigate whether the ECM fungus Hymenochaete sp. Rl affects the survival and colonization of the MHB strain Bacillus pumilus HR10 in the rhizosphere, the biomass of B. pumilus HR10 was measured in the rhizosphere and mycorrhizosphere. In addition, extracts of Hymenochaete sp. Rl and Pinus thunbergii were evaluated for their effect on B. pumilus HR10 colonization (growth, sporulation, biofilm formation, extracellular polysaccharide and extracellular protein contents, flagellar motility, and expression of colonization-related genes). The results showed that inoculation of Hymenochaete sp. Rl significantly increased the biomass of B. pumilus HR10 in the rhizosphere; however, while extracts of Hymenochaete sp. Rl and P. thunbergii did not affect the biomass or spore formation of HR10, they did affect its biofilm formation, extracellular polysaccharide and extracellular protein production, and flagellar motility. Furthermore, the addition of symbiont extracts affected the expression of chemotaxis-related genes in HR10. When the extracts were added separately, the expression of srf genes in HR10 increased; when the extracts were added simultaneously, the expression of the flagellin gene fliG in HR10 increased, but there was no significant effect on the expression of srf genes, consistent with the results on biofilm production. Thus, Hymenochaete sp. Rl and P. thunbergii roots had a positive effect on colonization by B. pumilus HR10 at the rhizosphere level through their secretions.

Genomic information for bacteria within the genus Rahnella remains limited. Rahnella sp. JZ-GX1 was previously isolated from the Pinus massoniana rhizosphere in China and shows potential as a plant growth-promoting (PGP) bacterium. In the present work, we combined the GridION Nanopore ONT and Illumina sequencing platforms to obtain the complete genome sequence of strain JZ-GX1, and the application effects of the strain in natural field environment was assessed. The whole genome of Rahnella sp. JZ-GX1 comprised a single circular chromosome (5,472,828 bp, G + C content of 53.53%) with 4,483 protein-coding sequences, 22 rRNAs, and 77 tRNAs. Based on whole genome phylogenetic and average nucleotide identity (ANI) analysis, the JZ-GX1 strain was reidentified as R. victoriana. Genes related to indole-3-acetic acid (IAA), phosphorus solubilization, nitrogen fixation, siderophores, acetoin, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, gamma-aminobutyric acid (GABA) production, spermidine and volatile organic compounds (VOCs) biosynthesis were present in the genome of strain JZ-GX1. In addition, these functions were also confirmed by in vitro experiments. Importantly, compared to uninoculated control plants, Pyrus serotina, Malus spectabilis, Populus euramericana (Dode) Guinier cv. “San Martino” (I-72 poplar) and Pinus elliottii plants inoculated with strain JZ-GX1 showed increased heights and ground diameters. These findings improve our understanding of R. victoriana JZ-GX1 as a potential biofertilizer in agriculture.

The MYB-CC family is a subtype within the MYB superfamily. This family contains an MYB domain and a predicted coiled-coil (CC) domain. Several MYB-CC transcription factors are involved in the plant’s adaptability to low phosphate (Pi) stress. We identified 30, 34, and 55 MYB-CC genes in Brassica rapa, Brassica oleracea, and Brassica napus, respectively. The MYB-CC genes were divided into nine groups based on phylogenetic analysis. The analysis of the chromosome distribution and gene structure revealed that most MYB-CC genes retained the same relative position on the chromosomes and had similar gene structures during allotetraploidy. Evolutionary analysis showed that the ancestral whole-genome triplication (WGT) and the recent allopolyploidy are critical for the expansion of the MYB-CC gene family. The expression patterns of MYB-CC genes were found to be diverse in different tissues of the three Brassica species. Furthermore, the gene expression analysis under low Pi stress revealed that MYB-CC genes may be related to low Pi stress responses. These results may increase our understanding of MYB-CC gene family diversification and provide the basis for further analysis of the specific functions of MYB-CC genes in Brassica species.

Abstract As important plant growth-promoting rhizobacteria, phosphate-solubilizing bacteria (PSB) fix nitrogen, dissolve potassium, promote growth, improve the soil micro-environment, and enhance soil fertility. A high-efficiency PSB strain from the pine tree rhizosphere, Burkholderia multivorans WS-FJ9, was screened in our laboratory. In this study, using a Bio Screener fully automatic microbial growth curve meter to determine the growth of the WS-FJ9 strain in phosphate-removing medium, the growth and mineral phosphate solubilization of WS-FJ9 were measured by Mo-Sb colorimetry and organophosphate-solubilization plate assays. Second-generation sequencing technology was used to obtain genomic information and to analyze possible phosphate decomposition genes. The related expression levels of these genes under different soluble phosphate levels were determined by quantitative real-time PCR. The results showed that WS-FJ9 had strong adaptability and capacity for mineral phosphate solubilization at low soluble phosphate levels, which is characterized by its low soluble phosphate induction and high soluble phosphate inhibition. The amount of solubilized mineral phosphate could exceed 140 mg/L. The total length of the WS-FJ9 genome was 7,497,552 bp after splicing, and the GC content was 67.37%. Eight phosphate-related genes were selected to determine their expression patterns at different soluble phosphate levels. Among them, AP-2, GspE and GspF were only related to organic phosphate, HlyB was only related to inorganic phosphate, and PhoR, PhoA, AP-1 and AP-3 were related to both. The WS-FJ9 strain utilizes multiple pathways for mineral phosphate solubilization, and the solubilization processes of different phosphate sources are interrelated and independent, indicating that the WS-FJ9 strain can adapt to different phosphate source environments and has good potential for future applications.

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.

Salt stress is one of the major abiotic stresses that affects plant growth and development. The use of plant growth-promoting rhizobacteria to mitigcate salt stress damage in plants is an important way to promote crop growth under salt stress conditions. Rahnella aquatilis JZ-GX1 is a plant growth-promoting rhizobacterial strain, but it is not clear whether it can improve the salt tolerance of plants, and in particular, the role of volatile substances in plant salt tolerance is unknown. We investigated the effects of volatile organic compounds (VOCs) from JZ-GX1 on the growth performance, osmotic substances, ionic balance and antioxidant enzyme activities of acacia seedlings treated with 0 and 100mm NaCl and explored the VOCs associated with the JZ-GX1 strain. The results showed that compared to untreated seedlings, seedlings exposed to plant growth-promoting rhizobacterium JZ-GX1 via direct contact with plant roots under salt stress conditions exhibited increases in fresh weight, lateral root number and primary root length equal to approximately 155.1, 95.4, and 71.3%, respectively. Robinia pseudoacacia seedlings exposed to VOCs of the JZ-GX1 strain showed increases in biomass, soil and plant analyser development values and lateral root numbers equal to 132.1, 101.6, and 166.7%, respectively. Additionally, decreases in malondialdehyde, superoxide anion (O2−) and hydrogen peroxide (H2O2) contents and increases in proline contents and superoxide dismutase, peroxidase and glutathione reductase activities were observed in acacia leaves. Importantly, the sodium-potassium ratios in the roots, stems, and leaves of acacia exposed to VOCs of the JZ-GX1 strain were significantly lower than those in the control samples, and this change in ion homeostasis was consistent with the upregulated expression of the (Na+, K+)/H+ reverse cotransporter RpNHX1 in plant roots. Through GC-MS and creatine chromatography, we also found that 2,3-butanediol in the volatile gases of the JZ-GX1 strain was one of the important signaling substances for improving the salt tolerance of plants. The results showed that R. aquatilis JZ-GX1 can promote the growth and yield of R. pseudoacacia under normal and salt stress conditions. JZ-GX1 VOCs have good potential as protectants for improving the salt tolerance of plants, opening a window of opportunity for their application in salinized soils.

Iron deficiency causes chlorosis and growth inhibition in Cinnamomum camphora, an important landscaping tree species. Siderophores produced by plant growth-promoting rhizobacteria have been widely reported to play an indispensable role in plant iron nutrition. However, little to date has been determined about how microbial siderophores promote plant iron absorption. In this study, multidisciplinary approaches, including physiological, biochemical and transcriptome methods, were used to investigate the role of deferoxamine (DFO) in regulating Fe availability in C. camphora seedlings. Our results showed that DFO supplementation significantly increased the Fe2+ content, SPAD value and ferric-chelate reductase (FCR) activity in plants, suggesting its beneficial effect under Fe deficiency. This DFO-driven amelioration of Fe deficiency was further supported by the improvement of photosynthesis. Intriguingly, DFO treatment activated the metabolic pathway of glutathione (GSH) synthesis, and exogenous spraying reduced glutathione and also alleviated chlorosis in C. camphora. In addition, the expression of some Fe acquisition and transport-related genes, including CcbHLH, CcFRO6, CcIRT2, CcNramp5, CcOPT3 and CcVIT4, was significantly upregulated by DFO treatment. Collectively, our data demonstrated an effective, economical and feasible organic iron-complexing agent for iron-deficient camphor trees and provided new insights into the mechanism by which siderophores promote iron absorption in plants.

Iron deficiency in soil has crucially restricted agricultural and forestry production. Volatile organic compounds (VOCs) produced by beneficial microorganisms have been proven to play an important role in inducing abiotic stress tolerance in plants. We investigated the effects of VOCs released by the rhizobacterium Rahnella aquatilis JZ-GX1 on the growth and root parameters of Arabidopsis thaliana under iron deficiency. The effect of the rhizobacterial VOCs on the gene expression in iron uptake and hormone signaling pathways were detected by RT-qPCR. Finally, the VOCs of the JZ-GX1 strain that could promote plant growth under iron deficiency stress were screened. The results showed that the JZ-GX1 strain could induce A. thaliana tolerance to iron deficiency stress by promoting the development of lateral roots and root hairs and increasing the activities of H+ ATPase and Fe3+ reductase. In addition, the AHA2, FRO2, and IRT1 genes of A. thaliana exposed to JZ-GX1-emitted VOCs were upregulated 25-, 1. 81-, and 1.35-fold, respectively, and expression of the abscisic acid (ABA) synthesis gene NCED3 was upregulated on both the 3rd and 5th days. Organic compounds were analyzed in the headspace of JZ-GX1 cultures, 2-undecanone and 3-methyl-1-butanol were found to promote Medicago sativa and A. thaliana growth under iron-limited conditions. These results demonstrated that the VOCs of R. aquatilis JZ-GX1 have good potential in promoting iron absorption in plants.

Salinity is one of the strongest abiotic factors in nature and has harmful effects on plants and microorganisms. In recent years, the degree of soil salinization has become an increasingly serious problem, and the use of plant growth-promoting rhizobacteria has become an option to improve the stress resistance of plants. In the present study, the salt tolerance mechanism of the rhizosphere bacterium Rahnella aquatilis JZ-GX1 was investigated through scanning electron microscopy observations and analysis of growth characteristics, compatible solutes, ion distribution and gene expression. In addition, the effect of JZ-GX1 on plant germination and seedling growth was preliminarily assessed through germination experiments. R. aquatilis JZ-GX1 was tolerant to 0–9% NaCl and grew well at 3%. Strain JZ-GX1 promotes salt tolerance by stimulating the production of exopolysaccharides, and can secrete 60.6983 mg/L of exopolysaccharides under the high salt concentration of 9%. Furthermore, the accumulation of the compatible solute trehalose in cells as the NaCl concentration increased was shown to be the primary mechanism of resistance to high salt concentrations in JZ-GX1. Strain JZ-GX1 could still produce indole-3-acetic acid (IAA) and siderophores and dissolve inorganic phosphorus under salt stress, characteristics that promote the ability of plants to resist salt stress. When the salt concentration was 100 mmol/L, strain JZ-GX1 significantly improved the germination rate, germination potential, fresh weight, primary root length and stem length of tomato seeds by 10.52, 125.56, 50.00, 218.18, and 144.64%, respectively. Therefore, R. aquatilis JZ-GX1 is a moderately halophilic bacterium with good growth-promoting function that has potential for future development as a microbial agent and use in saline-alkali land resources.

Determining the mechanisms of pine wilt disease (PWD) resistance in Pinus is a popular research topic, but information on volatile organic substances (VOS) and their role in PWD is lacking. Whether the difference in VOS among Pinus thunbergii parl. that have different levels of resistance with pine wood nematodes (PWNs) is the reason for the differing resistance needs to be studied. In this study, resistant P. thunbergii introduced from Japan and susceptible P. thunbergii native to China were used to investigate the effects of different lines inoculated with PWN. We determined the expression levels of the terpene synthesis-related genes geranylgeranyl diphosphate synthase (GGPPS), 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (HMDH1), two kinds of alpha-farnesene synthase (PT) genes. The types and the relative percentage content of terpenoids in the pine needles were also determined by gas chromatography coupled with mass spectrometry (GC-MS). Results show that the growth, population size and migration of PWNs were significantly inhibited. The expression of terpene synthesis genes in the resistant P. thunbergii was higher than that in the susceptible one. The analysis of terpenoids revealed a total of 41 terpenoids, of which resistant P. thunbergii contained 39 and susceptible P. thunbergii only 28; 14 terpenoids were specific to resistant P. thunbergii, in which 8 of the terpenoids were constitutive terpenes and 6 were inducible terpenes. There were 3 terpenes unique to the susceptible P. thunbergii, and only 1 inducible terpene. Our results showed that the reduction in the expression of disease symptom and suppression of PWNs in resistant P. thunbergii was likely related to differences in the types and content of resistance-related substances in the trees. This study does not specifically connect elevated compounds in resistant P. thunbergii to resistance to PWN and assays should be conducted to establish direct effects of terpenoids on pinewood nematode activity and reproduction.

Verticillium dahliae is one of the most destructive fungal pathogens, causing substantial economic losses in agriculture and forestry. The use of plant growth-promoting rhizobacteria (PGPR) is an effective and environmentally friendly strategy for controlling diseases caused by V. dahliae. In this study, 90 mm in diameter Petri plates were used to test the effect of volatile organic compounds (VOCs) produced by different concentrations of Pseudomonasaurantiaca ST-TJ4 cells suspension on V. dahliae mycelia radial growth and biomass. The mycelial morphology was observed by using scanning electron microscopy. The conidia germination and microsclerotia formation of V. dahliae were evaluated. The VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The VOCs produced by strain ST-TJ4 significantly inhibited the growth of mycelium of V. dahliae. The morphology of the hyphae was rough and wrinkled when exposed to VOCs. The VOCs of strain ST-TJ4 have a significant inhibitory effect on V. dahliae conidia germination and microsclerotia formation. At the same time, the VOCs also reduce the expression of genes related to melanin synthesis in V. dahliae. In particular, the expression of the hydrophobin gene (VDAG-02273) was down-regulated the most, about 67-fold. The VOCs effectively alleviate the severity of cotton root disease. In the volatile profile of strain ST-TJ4, 2-undecanone and 1-nonanol assayed in the range 10–200 µL per plate revealed a significant inhibitory effect on V. dahliae mycelial radial growth. These compounds may be useful to devise new control strategies for control of Verticillium wilt disease caused by V. dahliae.

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.

Fungal esophagitis is a common infectious disease, although the pathogenic clinical characteristics remain incompletely clear, especially in South China. The goal of this study was to investigate the pathogenic clinical characteristics of fungal esophagitis and the efficacy of different therapeutic strategies at a tertiary hospital in South China. A retrospective study was conducted from January 2007 to December 2017. Data from 113,390 patients who were treated in the endoscopic unit were retrieved and analyzed. To further understand the pathogen and risk factors for fungal esophagitis, we performed a case-control analysis of 101 patients and 202 controls. Of the 113,390 patients, 932 (0.82%) were positive. The annual detection rate ranged from 0.345% to 1.106%, showing an initially increasing and subsequently decreasing trend. The patients’ median age was 49 years (range from 8 to 85), and most were men (615/932, 65.99%). Candida albicans was found in samples collected from 36 patients, without any drug-resistant strains. Age (P=0.018), malignancy (OR=4.031, 95% CI: 1.562~10.407), cigarette smoking (OR=3.017, 95% CI: 1.645~5.533), and the use of antibiotics (OR=2.178, 95% CI: 1.078~4.400) or immunosuppressants (OR=6.525, 95% CI: 1.089~39.105) were independently associated with esophageal candidiasis. Fluconazole had a better curative effect than nystatin (OR=4.047, 95% CI: 1.282~12.772) or simple observation (OR=8.91, 95% CI: 2.61~30.49). In conclusion, fungal esophagitis primarily affects men and elderly individuals; it develops in the setting of malignancy, smoking, and certain previous medication use. Candida albicans is the most common pathogen and is sensitive to antifungal agents. Fluconazole has a good therapeutic effect.

Rhizosphere fungi have the beneficial functions of promoting plant growth and protecting plants from pests and pathogens. In our preliminary study, rhizosphere fungus JP-NJ4 was obtained from the soil rhizosphere of Pinus massoniana and selected for further analyses to confirm its functions of phosphate solubilization and plant growth promotion. In order to comprehensively investigate the function of this strain, it is necessary to ascertain its taxonomic position. With the help of genealogical concordance phylogenetic species recognition (GCPSR) using five genes/regions (ITS, BenA, CaM, RPB1, and RPB2) as well as macro-morphological and micro-morphological characters, we accurately determined the classification status of strain JP-NJ4. The concatenated phylogenies of five (or four) gene regions and single gene phylogenetic trees (ITS, BenA, CaM, RPB1, and RPB2 genes) all show that strain JP-NJ4 clustered together with Talaromyces brevis and Talaromyces liani, but differ markedly in the genetic distance (in BenA gene) from type strain and multiple collections of T. brevis and T. liani. The morphology of JP-NJ4 largely matches the characteristics of genes Talaromyces, and the rich and specific morphological information provided by its colonies was different from that of T. brevis and T. liani. In addition, strain JP-NJ4 could produce reduced conidiophores consisting of solitary phialides. From molecular and phenotypic data, strain JP-NJ4 was identified as a putative novel Talaromyces fungal species, designated T. nanjingensis.

The use of volatile organic compounds (VOCs) produced by microorganisms for the biological control of plant diseases has attracted much attention in recent years. In this study, the antifungal activity and identity of VOCs produced by Rahnella aquatilis JZ-GX1 isolated from the rhizosphere soil of pine were determined and analyzed. The effect of the VOCs on the mycelial growth of Colletotrichum gloeosporioides, the pathogen of Liriodendron chinense × tulipifera black spot, was determined by a joined-petri dish fumigation method. An in vitro leaf inoculation method was used to determine the fumigation effect of the VOCs on Liriodendron black spot. VOCs with antifungal activity were collected by headspace solid-phase microextraction (SPME), and their components were analyzed by gas chromatography-mass spectrometry (GC-MS). The results showed that the VOCs secreted by JZ-GX1 inhibited the mycelial growth of the tested pathogen. The VOCs destroyed the morphology of the mycelium, significantly increased the permeability of the cell membrane and downregulated the expression of pathogenicity-related genes during mycelial infection, thus inhibiting the expansion of anthracnose disease spots in leaves. In the volatile compound profile, 3-methyl-1-butanol and 2-phenylethyl methyl ether significantly inhibited the mycelial growth and spore germination of C. gloeosporioides. This work provides a new strategy for the research and application of microorganisms and bioactive compounds to control plant anthracnose.

Objective: To establish a complete technical solution for the radiation biological dose estimation, to enable prediction of individuals’ response to radiotherapy (RT), and to control treatment dose for reduced irradiation injury and promote repair; and to evaluate the risk of radiation-induced late effects for patients undergoing external photon beam RT and provide the reliable dose–response relationships. Methods: Select 49 tumor patients using 60 Co and linear accelerator for radiation therapy; initial radiation dose was 250 cGy. Chromosome aberration and blood count were analyzed before radiation therapy and 2 hours after the first day of RT. Results: Two hours after the first day of RT, peripheral blood cell count of lymphocytes of patients with cancer was significantly decreased ( P < .01). The frequency of chromosome aberration was higher ( P < .01). Conclusion: High-dose radiation of the radiation therapy makes significant injuries to peripheral blood lymphocytes.

Microbes in soil can degrade insoluble inorganic and organic phosphorus, which are components of the soil phosphorus cycle and play an important role in plant growth. Pinus massoniana is a pioneer tree species used for afforestation in southern China and grows in poor, acidic soil. A shortage of available phosphorus in soil limits the growth of P. massoniana. To alleviate this situation, it is necessary to improve soil fertility. A fungal strain (JP-NJ2) with the ability to solubilize phosphate was isolated from the P. massoniana rhizosphere. The ability of JP-NJ2 to solubilize inorganic and organic phosphorus and promote the growth of P. massoniana was evaluated. It showed that JP-NJ2 could grow in NBRIP inorganic phosphate (AlPO4, FePO4·4H2O, and Ca3[PO4]2) fermentation broths, with the highest phosphorus concentration (1.93 mg/ml) and phosphate-solubilizing rate (43.7%) for AlPO4 and in Monkina organic phosphate fermentation broth with a phosphorus concentration of 0.153 mg/ml. The phosphate-solubilizing capability in inorganic and organic fermentation broths was negatively correlated with pH. JP-NJ2-produced acids at a total concentration of 4.7 g/l, which included gluconic (2.3 g/l), oxalic (1.1 g/l), lactic (0.7 g/l) and malonic (0.5 g/l) acids. It prioritized extracellular acidic phosphatase and combined with phytase to solubilize organic phosphates. The fungal suspension and extracellular metabolites from phosphate-solubilizing fungi promoted the shoot length of P. massoniana seedlings by 97.7% and 59.5%, respectively, while increasing the root crown diameter by 46.8% and 27.7%. JP-NJ2 was identified as Penicillium guanacastense based on its morphology and phylogenetic analyses of five genes/regions (ITS, ben A, cmd, cox1 and tef). This is the first report on P. guanacastense isolated from pine tree rhizosphere soil in China and its high phosphate-solubilizing capability, which promoted the growth of P. massoniana. P. guanacastense JP-NJ2 has potential use as a biological fertilizer in forestry and farming.

Phytase plays an important role in crop seed germination and plant growth. In order to fully understand the plant growth-promoting mechanism by Rahnella aquatilis JZ-GX1, the effect of this strain on germination of maize seeds was determined in vitro, and the colonization of maize root by R. aquatilis JZ-GX1 was observed by scanning electron microscope. Different inoculum concentrations and Phytate-related soil properties were applied to investigate the effect of R. aquatilis JZ-GX1 on the growth of maize seedlings. The results showed that R. aquatilis JZ-GX1 could effectively secrete indole acetic acid and had significantly promoted seed germination and root length of maize. A large number of R. aquatilis JZ-GX1 cells colonized on the root surface, root hair and the root interior of maize. When the inoculation concentration was 107 cfu/mL and the insoluble organophosphorus compound phytate existed in the soil, the net photosynthetic rate, chlorophyll content, phytase activity secreted by roots, total phosphorus concentration and biomass accumulation of maize seedlings were the highest. In contrast, no significant effect of inoculation was found when the total P content was low or when inorganic P was sufficient in the soil. R. aquatilis JZ-GX1 promotes the growth of maize directly by secreting IAA and indirectly by secreting phytase. This work provides beneficial information for the development and application of R. aquatilis JZ-GX1 as a microbial fertilizer in the future.

The alternative sigma factor D is known to be involved in at least three biological processes in Bacilli: flagellin synthesis, methyl-accepting chemotaxis and autolysin synthesis. Although many Bacillus genes have been identified as SigD regulon, the list may be not be complete. With microarray-based systemic screening, we found a set of genes downregulated in the sigD knockout mutant of the plant growth-promoting rhizobacterium B. amyloliquefaciens subsp. plantarum FZB42. Eight genes (appA, blsA, dhaS, spoVG, yqgA, RBAM_004640, RBAM_018080 and ytk) were further confirmed by quantitative PCR and/or northern blot to be controlled by SigD at the transcriptional level. These genes are hitherto not reported to be controlled by SigD. Among them, four genes are of unknown function and two genes (RBAM_004640 and RBAM_018080), absent in the model strain B. subtilis 168, are unique to B. amyloliquefaciens stains. The eight genes are involved in sporulation, biofilm formation, metabolite transport and several other functions. These findings extend our knowledge of the regulatory network governed by SigD in Bacillus and will further help to decipher the roles of the genes.

Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD). The disease has caused significant economic losses and damage to forests. However, the pathogenic mechanism of B. xylophilus remains unclear. Cystatins are involved in various biological processes where they regulate normal proteolysis and also play a role in pathogenicity, but their functions in B. xylophilus are unknown. Therefore, we cloned the full-length cDNA of a cystatin gene of B. xylophilus (Bx-cpi-1) by rapid-amplification of cDNA ends and analyzed its characteristics with bioinformatic methods. In situ mRNA hybridization analyses showed that transcripts of Bx-cpi-1 were abundantly expressed in the reproductive organs of B. xylophilus. The expression of Bx-cpi-1 was investigated using qPCR. Bx-cpi-1 was expressed during each of the different developmental stages of B. xylophilus. The highest gene expression was at the egg stage. After infection of Pinus massoniana, the expression of Bx-cpi-1 increased. The functions of Bx-cpi-1 were verified by RNA interference. The feeding rate, reproduction and pathogenicity of B. xylophilus all decreased as a result of silencing of the Bx-cpi-1 gene. These results revealed that Bx-cpi-1 may be a variant of a type II cystatin gene which is involved in the development and pathogenic process of B. xylophilus.

The data presented in this article are related to the publication entitled “Malonylome analysis of rhizobacterium Bacillus amyloliquefaciens FZB42 reveals involvement of lysine malonylation in polyketide synthesis and plant–bacteria interactions”(doi:10.1016/j.jprot.2016.11.022) (B. Fan, Y. Li, L. Li et al.) [1]. This article presented the raw information of all malonyllysine sites identified by LC-MS/MS in the Bacillus amyloliquefaciens FZB42. Further, the functional features and conservation of the malonylated peptide/proteins were analyzed and made publicly available to enable critical or extended analyses.

Stenotrophomonas maltophilia influences the reproduction, pathogenicity, and gene expression of aseptic Bursaphelenchus xylophilus after inoculation of aseptic Pinus massoniana. Pine wilt disease is a destructive pine forest disease caused by B. xylophilus, and its pathogenesis is unclear. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. S. maltophilia is one of the most dominant bacteria in B. xylophilus, and its effect is ambiguous. This study aims to explore the role of S. maltophilia in pine wilt disease. The reproduction and virulence of aseptic B. xylophilus and B. xylophilus containing S. maltophilia were examined by inoculating aseptic P. massoniana seedlings. The gene expressions of two nematode treatments were identified by transcriptome sequencing. The reproduction and virulence of B. xylophilus containing S. maltophilia were stronger than that of aseptic nematodes. There were 4240 differentially expressed genes between aseptic B. xylophilus and B. xylophilus containing S. maltophilia after inoculation of aseptic P. massoniana, including 1147 upregulated genes and 2763 downregulated genes. These differentially expressed genes were significantly enriched in some immune-related gene ontology (GO) categories, such as membrane, transporter activity, metabolic processes, and many immune-related pathways, such as the wnt, rap1, PI3K-Akt, cAMP, cGMP-PKG, MAPK, ECM-receptor interaction, and calcium signaling pathways. The polyubiquitin-rich gene, leucine-rich repeat serine/threonine-protein kinase gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, acetyl-CoA carboxylase gene, and heat shock protein genes were the key genes associated with immune resistance. Moreover, there were four cell wall hydrolase genes, thirty-six detoxification- and pathogenesis-related protein genes, one effector gene and ten cathepsin L-like cysteine proteinase genes that were differentially expressed. After inoculation of the host pine, S. maltophilia could affect the virulence and reproduction of B. xylophilus by regulating the expression of parasitic, immune, and pathogenicity genes of B. xylophilus.

The biocontrol process mediated by plant growth-promoting rhizobacteria (PGPR) relies on multiple mechanisms. Biofilm formation plays an important role in the ability of PGPR to control plant diseases. Bacillus pumilus HR10, one such PGPR, promotes the growth of Pinus thunbergii. This study showed that the wild-type strain B. pumilus HR10 produces a stable and mature biofilm in vitro. Biofilm-deficient mutants of B. pumilus HR10 with different phenotypes were screened by mutagenesis. The contents of extracellular polysaccharides (EPS) and proteins produced by the mutant strains were significantly reduced, and the biofilms of the mutants were weakened to varying degrees. The swarming abilities of the wild-type and mutant strains were positively correlated with biofilm formation. A colonization assay demonstrated that B. pumilus HR10 could colonize the roots of Pinus massoniana seedlings in a large population and persist, while biofilm-deficient mutants showed weak colonization ability. Furthermore, a biocontrol assay showed that biocontrol efficacy of the mutants was reduced to a certain degree. We determined the inhibitory activity of B. pumilus HR10 and its ability to induce systemic resistance against Rhizoctonia solani of plants. The synthesis of lipopeptide antibiotics is probably involved in biofilm formation by B. pumilus HR10. These observations not only provide a reference for further research about the coordinated action between biofilm formation and the multiple biocontrol mechanisms of B. pumilus HR10 but also improve the understanding of the regulatory pathway of biofilm formation by B. pumilus HR10.

The pine wood nematode (PWN) Bursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD), a devastating forest disease. PWN-associated bacteria may play a role in PWD. However, little is known about the endo-bacteria in PWN. We analyzed the diversity of endo-bacteria in nine isolates of PWNs from Pinus massoniana Lamb. in nine epidemic areas from three Chinese provinces by high-throughput sequencing of 16S rDNA and isolated and identified culturable endo-bacteria through construction of a 16S rDNA phylogenetic tree and Biolog microbial identification. We also examined the effects of endo-bacteria on PWN fecundity, antioxidant capacity, and virulence using sterile nematodes as a control. While the dominant endo-bacteria in PWNs from different regions exhibited no significant difference in the classification levels of class and genus, their proportions differed. Pseudomonas and Stenotrophomonas were highly abundant in all PWN isolates. A total of 15 endo-bacterial strains were successfully isolated and identified as six species: Stenotrophomonas maltophilia, Pseudomonas fluorescens, Kocuria palustris, Microbacterium testaceum, Rhizobium radiobacter, and Leifsonia aquatica. We also found that P. fluorescens significantly increased the egg production of PWN, and that both P. fluorescens and S. maltophilia enhanced the mobility of PWN under oxidative stress and reduced the content of reactive oxygen species by increasing antioxidant enzyme activity in PWN. These strains also accelerated the development of PWD, and P. fluorescens had a more beneficial effect on PWN than S. maltophilia. Diversity exists among the endo-bacteria in PWNs from different regions, and some endo-bacteria can promote PWN infestation by enhancing the fecundity and antioxidant capacity of the nematode. Our study contributes to clarifying the interaction between endo-bacteria and PWN.

Plant growth-promoting rhizobacteria (PGPR) can potentially be used as an alternative strategy to control plant diseases. In this study, strain ST–TJ4 isolated from the rhizosphere soil of a healthy poplar was found to have a strong antifungal activity against 11 phytopathogenic fungi in agriculture and forestry. Strain ST–TJ4 was identified as Pseudomonas sp. based on 16S rRNA-encoding gene sequences. The bacterium can produce siderophores, cellulase, and protease, and has genes involved in the synthesis of phenazine, 1–phenazinecarboxylic acid, pyrrolnitrin, and hydrogen cyanide. Additionally, the volatile compounds released by strain ST–TJ4 can inhibit the mycelial growth of plant pathogenic fungi more than diffusible substances can. Based on volatile compound profiles of strain ST–TJ4 obtained from headspace collection and GC–MS/MS analysis, 1-undecene was identified. In summary, the results suggested that Pseudomonas sp. ST–TJ4 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.

Phosphate-solubilizing fungi (PSF) generally enhance available phosphorus (P) released from soil, which contributes to plants' P requirement, especially in P-limiting regions. In this study, two PSF, TalA-JX04 and AspN-JX16, were isolated from the rhizosphere soil of moso bamboo (Phyllostachys edulis) widely distributed in P-deficient areas in China and identified as Talaromyces aurantiacus and Aspergillus neoniger, respectively. The two PSF were cultured in potato dextrose liquid medium with six types of initial pH values ranging from 6.5 to 1.5 to assess acid resistance. Both PSF were incubated in Pikovskaya's liquid media with different pH values containing five recalcitrant P sources, including Ca3(PO4)2, FePO4, CaHPO4, AlPO4, and C6H6Ca6O24P6, to estimate their P-solubilizing capacity. No significant differences were found in the biomass of both fungi grown in media with different initial pH, indicating that these fungi could grow well under acid stress. The P-solubilizing capacity of TalA-JX04 was highest in medium containing CaHPO4, followed by Ca3(PO4)2, FePO4, C6H6Ca6O24P6, and AlPO4 in six types of initial pH treatments, while the recalcitrant P-solubilizing capacity of AspN-JX16 varied with initial pH. Meanwhile, the P-solubilizing capacity of AspN-JX16 was much higher than TalA-JX04. The pH of fermentation broth was negatively correlated with P-solubilizing capacity (p

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.

AIM: To study curative effect of Ranibizumab with Ex-PRESS drainage device for neovascular glaucoma. METHODS: From January 2012 to December 2014 in our hospital, 51 cases(62 eyes)with neovascular glaucoma were enrolled as the research objects. Under the unified enrolling criteria and measuring methods, according to the actual clinical situation and combined with the doctor's judgment, the patients were divided into control group with 23 cases(28 eyes)and observation group with 28 cases(34 eyes). The patients in observation group received the injection of ranibizumab combined the Ex-PRESS drainage device implantation. The patients in control group were only given the Ex-PRESS drainage device implantation. The vision, intraocular pressure(IOP)before and after treatments were compared as well as the outcomes and incidences of adverse reactions of the two groups.RESULTS: Before treatments, IOP of the two groups did not have statistically significant different(P>0.05). After treatments, IOP of the two groups were significantly lower, and the differences were statistically significant(PPP>0.05). After treatments, the visual acuity of the control group improved in 2 cases(3 eyes), didn't change in 18 cases(22 eyes), and declined in 3 cases(3 eyes). While the visual acuity of observation group improved in 12 cases(12 eyes), didn't change in 15 cases(21 eyes), and declined in 1 case(1 eye). The distribution of the visual acuity of the two groups was significantly different(PPP>0.05). CONCLUSION: For patients with neovascular glaucoma, the injection of ranibizumab combined with the Ex-PRESS the drainage device implantation can effectively reduce IOP, improve visual function in patients with impaired visual function or delay the recession of visual function, with outstanding curative effect, high security features and is worthy of clinical application.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen responsible for pine wilt disease (PWD), a devastating forest disease with a pathogenic mechanism that remains unclear. Autophagy plays a crucial role in physiological and pathological processes in eukaryotes, but its regulatory mechanism and significance in PWN are unknown. Therefore, we cloned and characterized three autophagy genes, BxATG5, BxATG9, and BxATG16, in PWN. BxATG9 and BxATG16 were efficiently silenced through RNA interference, and we found that BxATG16 positively regulated the expression of BxATG5. Silencing BxATG9 and BxATG16 severely inhibited feeding and reproduction in PWN, indicating that autophagy is essential for these processes. We then examined the expression patterns of these three autophagy genes in PWN under the stresses of α-pinene and H2O2, the main defense substances of pine trees, and during the development of PWD using quantitative reverse transcription polymerase chain reaction. The expression levels of BxATG5, BxATG9, and BxATG16 all significantly increased after nematodes were stressed with α-pinene and H2O2 and inoculated into pine trees, suggesting that autophagy plays an important role in the defense and pathogenesis of PWN. In this study, the molecular characteristics and functions of the autophagy genes BxATG5, BxATG9, and BxATG16 in PWN were elucidated.

Bursaphelenchus xylophilus, the causal agent of pine wilt disease, is a destructive threat to pine forests. The role of bacteria associated with B. xylophilus in pine wilt disease has attracted widespread attention. This study investigated variation in bacterial communities and the virulence of surface-sterilized B. xylophilus from different Pinus spp. The predominant culturable bacteria of nematodes from different pines were Stenotrophomonas and Pseudomonas. Biolog EcoPlate analysis showed that metabolic diversity of bacteria in B. xylophilus from P. massoniana was the highest, followed by P. thunbergii and P. densiflora. High-throughput sequencing analysis indicated that bacterial diversity and community structure in nematodes from the different pine species varied, and the dominant bacteria were Stenotrophomonas and Elizabethkingia. The virulence determination of B. xylophilus showed that the nematodes from P. massoniana had the greatest virulence, followed by the nematodes from P. thunbergii and P. densiflora. After the nematodes were inoculated onto P. thunbergii, the relative abundance of the predominant bacteria changed greatly, and some new bacterial species emerged. Meanwhile, the virulence of all the nematode isolates increased after passage through P. thunbergii. These inferred that some bacteria associated with B. xylophilus isolated from different pine species might be helpful to adjust the PWN’s parasitic adaptability.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

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.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, 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.

Bursaphelenchus xylophilus is the pathogen of pine wilt disease. Bursaphelenchus mucronatus is similar to B. xylophilus in morphology. Both species share a common niche, but they are quite different in pathogenicity. Presently, the role of bacteria in pine wilt disease development has been widely speculated. The diversity of bacteria associated with B. xylophilus and B. mucronatus with different virulence remains unclear. In this study, virulence of four B. xylophilus and four B. mucronatus strains were evaluated by inoculating Pinus thunbergii. High-throughput sequencing targeted 16S rDNA of different virulence nematode strains was carried out. The associated bacterial community structures of the eight strains were analyzed. The results showed that 634,051 high-quality sequences were obtained from the eight nematode strains. The number of OTUs of bacteria associated with B. mucronatus was generally greater than those of B. xylophilus. The richness of the community of bacteria associated with high virulent B. xylophilus ZL1 and AmA3 was higher than moderately virulent B. xylophilus AA3, HE2, and all B. mucronatus strains. While the diversity of bacteria associated with B. mucronatus was higher than B. xylophilus. Stenotrophomonas, Pseudomonadaceae_Unclassified or Rhizobiaceae_Unclassified were predominant in the nematode strains with different virulence. Oxalobacteraceae and Achromobacter were found more abundant in the low virulent B. xylophilus and non-virulent B. mucronatus strains.

Bacillus amyloliquefaciens subsp. plantarum FZB42 is a representative of Gram-positive plant-growth-promoting rhizobacteria (PGPR) that inhabit plant root environments. In order to better understand the molecular mechanisms of bacteria-plant symbiosis, we have systematically analyzed the primary transcriptome of strain FZB42 grown under rhizosphere-mimicking conditions using differential RNA sequencing (dRNA-seq). Our analysis revealed 4,877 transcription start sites for protein-coding genes, identified genes differentially expressed under different growth conditions, and corrected many previously mis-annotated genes. We also identified a large number of riboswitches and cis-encoded antisense RNAs, as well as trans-encoded small noncoding RNAs that may play important roles in the gene regulation of Bacillus. Overall, our analyses provided a landscape of Bacillus primary transcriptome and improved the knowledge of rhizobacteria-host interactions.

Pine wilt disease (PWD) caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, is responsible for devastating epidemics in pine trees in Asia and Europe. Recent studies showed that bacteria carried by the PWN might be involved in PWD. However, the molecular mechanism of the interaction between bacteria and the PWN remained unclear. Now that the whole genome of B. xylophilus (Bursaphelenchus xylophilus) is published, transcriptome analysis is a unique method to study the role played by bacteria in PWN. In this study, the transcriptome of aseptic B. xylophilus, B. xylophilus treated with endobacterium (Stenotrophomonas maltophilia NSPmBx03) and fungus B. xylophilus were sequenced. We found that 61 genes were up-regulated and 830 were down-regulated in B. xylophilus after treatment with the endobacterium; 178 genes were up-regulated and 1122 were down-regulated in fungus B. xylophilus compared with aseptic B. xylophilus. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to study the significantly changed biological functions and pathways for these differentially expressed genes. Many pathogenesis-related genes, including glutathinone S-transferase, pectate lyase, ATP-binding cassette transporter and cytochrome P450, were up-regulated after B. xylophilus were treated with the endobacterium. In addition, we found that bacteria enhanced the virulence of PWN. These findings indicate that endobacteria might play an important role in the development and virulence of PWN and will improve our understanding of the regulatory mechanisms involved in the interaction between bacteria and the PWN.

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.

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.