The Biocontrol Functions of Bacillus velezensis Strain Bv-25 Against Meloidogyne incognita.

Meloidogyne incognita is obligate parasitic nematode with a wide variety of hosts that causes huge economic losses every year. In an effort to identify novel bacterial biocontrols against M. incognita, the nematicidal activity of Bacillus velezensis strain Bv-25 obtained from cucumber rhizosphere soil was measured. Strain Bv-25 could inhibit the egg hatching of M. incognita and had strong nematicidal activity, with the mortality rate of second-stage M. incognita juveniles (J2s) at 100% within 12 h of exposure to Bv-25 fermentation broth. The M. incognita genes ord-1, mpk-1, and flp-18 were suppressed by Bv-25 fumigation treatment after 48 h. Strain Bv-25 could colonize cucumber roots, with 5.94 × 107 colony-forming units/g attached within 24 h, effectively reducing the infection rate with J2s by 98.6%. The bacteria up-regulated the expression levels of cucumber defense response genes pr1, pr3, and lox1 and induced resistance to M. incognita in split-root trials. Potted trials showed that Bv-25 reduced cucumber root knots by 73.8%. The field experiment demonstrated that disease index was reduced by 61.6%, cucumber height increased by 14.4%, and yield increased by 36.5% in Bv-25-treated plants compared with control. To summarize, B. velezensis strain Bv-25 strain has good potential to control root-knot nematodes both when colonizing the plant roots and through its volatile compounds.

Pochonia chlamydosporia Isolate PC-170-Induced Expression of Marker Genes for Defense Pathways in Tomatoes Challenged by Different Pathogens.

Pochonia chlamydosporia is a fungal parasite of nematode eggs. Studies have shown that some strains of Pochonia chlamydosporia can promote plant growth and induce plants' systemic resistance to root-knot nematodes by colonizing in their roots. This study aimed to verify the effect of the PC-170 strain on tomato growth and systemic resistance. Split-root experiments were conducted to observe the systemic resistance induced by PC-170. To explore the defense pathway that was excited due to the colonization by PC-170, we tested the expression of marker genes for defense pathways, and used mutant lines to verify the role of plant defense pathways. Our results showed that PC-170 can colonize roots, and promotes growth. We found a role for jasmonic acid (JA) in modulating tomato colonization by PC-170. PC-170 can activate tomato defense responses to reduce susceptibility to infection by the root-knot nematode Meloidogyne incognita, and induced resistance to some pathogens in tomatoes. The marker genes of the defense pathway were significantly induced after PC-170 colonization. However, salicylic acid (SA)- and jasmonic acid (JA)-dependent defenses in roots were variable with the invasion of different pathogens. Defense pathways play different roles at different points in time. SA- and JA-dependent defense pathways were shown to cross-communicate. Different phytohormones have been involved in tomato plants' responses against different pathogens. Our study confirmed that adaptive JA signaling is necessary to regulate PC-170 colonization and induce systemic resistance in tomatoes.

Biocontrol Efficacy of Bacillus cereus Strain Bc-cm103 Against Meloidogyne incognita.

Root-knot nematodes (Meloidogyne spp.) are soilborne pathogens that infect vegetable crops and cause major economic losses worldwide annually. Therefore, there is an urgent need for novel nematicides or biological control agents to reduce the damage caused by root-knot nematodes. In this study, we tested efficacy of the Bacillus cereus strain Bc-cm103, isolated from the rhizoplane of Cucumis metuliferus, against Meloidogyne incognita. Strain Bc-cm103 fermentation broth caused 100% mortality of the nematode second-stage juveniles within 12 h and decreased the egg hatching rate by 40.06% within 72 h compared with sterile water. Confocal laser-scanning microscopy revealed that strain Bc-cm103 formed a biofilm on cucumber (C. sativus) roots, which protected the roots from the infection of M. incognita. Additionally, strain Bc-cm103 activated the defense-responsive genes PR1, PR2, LOX1, and CTR1 in cucumber. Furthermore, strain Bc-cm103 significantly reduced the appearance of root galls in pot, split-root, and field tests. These results indicated that B. cereus strain Bc-cm103 had a strong suppressive effect on M. incognita and therefore could be used as a potential biocontrol agent against this pathogen.

Volatile Organic Compounds of Bacillus cereus Strain Bc-cm103 Exhibit Fumigation Activity against Meloidogyne incognita.

Bacillus cereus strain Bc-cm103 shows nematicidal activity and, therefore, has been used as a biological control agent to control the root-knot nematode Meloidogyne incognita. However, it remains unknown whether volatile organic compounds (VOCs) produced by B. cereus strain Bc-cm103 are effective in biocontrol against M. incognita. Therefore, in this study, we investigated the activity of Bc-cm103 VOCs against M. incognita. The B. cereus strain Bc-cm103 significantly repelled the second-stage juveniles (J2s) of M. incognita. In vitro evaluation of VOCs produced by the fermentation of Bc-cm103 in a three-compartment Petri dish revealed the mortality rates of M. incognita J2s as 90.8% at 24 h and 97.2% at 48 h. Additionally, evaluation of the ability of Bc-cm103 VOCs to suppress M. incognita infection in a double-layered pot test showed that root galls on cucumber roots decreased by 46.1%. Furthermore, 21 VOCs were identified from strain Bc-cm103 by solid-phase microextraction gas chromatography-mass spectrometry, including alkanes, alkenes, esters, and sulfides. Among them, dimethyl disulfide (30.63%) and S-methyl ester butanethioic acid (30.29%) were reported to have strong nematicidal activity. Together, these results suggest that B. cereus strain Bc-cm103 exhibits fumigation activity against M. incognita.

Degradation of Ferulic Acid by the Endophytic Fungus Colletotrichum gloeosporioides TMTM-13 Associated with Ostrya rehderiana Chun.

Biodegradation of ferulic acid, by an endophytic fungus Colletotrichum gloeosporioides TMTM-13 associated with Ostrya rehderiana Chun, was explored in this study. Ferulic acid was completely degraded by TMTM-13 as its initial concentration was lower than 400 mg L-1. Generally, the initial concentration of ferulic acid and fungal biomass of TMTM-13 kept synchronously growing up as the concentration was lower than 400 mg L-1. Fungal biomass reached a maximum of almost 1.177 g L-1 under concentrations of 400-450 mg L-1. HPLC-MS analysis indicated that ferulic acid ultimately degraded to vanillin, vanillic acid, acetovanillone, and dihydroconiferyl alcohol by TMTM-13. This study was the first report about an endophytic fungus associated with O. rehderiana Chun that has great potential for practical application in ferulic acid contaminated environments.

Transformation of the endophytic fungus Acremonium implicatum with GFP and evaluation of its biocontrol effect against Meloidogyne incognita.

Acremonium implicatum is an endophytic fungus with biocontrol potential against Meloidogyne incognita based on its opportunistic egg-parasitic, hatching inhibition, and toxic properties. To understand its mode of plant endophytism and opportunistic egg parasitism, GFP-tagged A. implicatum was constructed by PEG-mediated protoplast transformation. By laser scanning confocal microscopy (LSCM), we evaluated the endophytism and opportunistic egg parasitism of a stable gfp transformant (Acr-1). Acr-1 could colonize epidermal tissue, cortical tissue, and xylem of roots and form a mutualistic symbiosis with tomato host plants. LSCM of Acr-1 infecting M. incognita eggs revealed that hyphae penetrated the shell and grew inside eggs to form trophic hyphae. A large number of hyphae enveloped parasitized eggs. In addition, the egg shell integrity was destroyed by fungal penetration. The percentage of egg parasitism was 33.8 %. There were no marked differences between the wild type and mutant in nematode second-stage juvenile mortality and egg hatching and in fungal control efficiency in a pot experiment. In conclusion, gfp-transformation did not change the nematicidal activity of A. implicatum and is a tool to examine the mode of plant endophytism and opportunistic egg parasitism of A. implicatum.

Metagenomic analysis of the pinewood nematode microbiome reveals a symbiotic relationship critical for xenobiotics degradation.

Our recent research revealed that pinewood nematode (PWN) possesses few genes encoding enzymes for degrading α-pinene, which is the main compound in pine resin. In this study, we examined the role of PWN microbiome in xenobiotics detoxification by metagenomic and bacteria culture analyses. Functional annotation of metagenomes illustrated that benzoate degradation and its related metabolisms may provide the main metabolic pathways for xenobiotics detoxification in the microbiome, which is obviously different from that in PWN that uses cytochrome P450 metabolism as the main pathway for detoxification. The metabolic pathway of degrading α-pinene is complete in microbiome, but incomplete in PWN genome. Experimental analysis demonstrated that most of tested cultivable bacteria can not only survive the stress of 0.4% α-pinene, but also utilize α-pinene as carbon source for their growth. Our results indicate that PWN and its microbiome have established a potentially mutualistic symbiotic relationship with complementary pathways in detoxification metabolism.

Comparative transcriptomics of two pathogenic pinewood nematodes yields insights into parasitic adaptation to life on pine hosts.

Bursaphelenchus xylophilus and Bursaphelenchus mucronatus are migratory endoparasitic nematodes that live in pine trees. To gain insight into their molecular similarities and differences, transcriptomes of the two nematodes were analysed. A total of 23,765 and 21,782 contigs (>300 bp) were obtained from B. xylophilus and B. mucronatus, respectively. More than 80% of the contigs could map to each other's transcriptome reciprocally. A total of 23,467 and 21,370 Open Reading Frames were predicted, respectively. Besides those known parasitism-related proteins, six new venom allergen-like proteins (VAPs) were found, which were not homologous to known VAPs. Enzymes involved in xenobiotic biodegradation were abundant in the two transcriptomes based on KEGG functional annotation. Metabolism of xenobiotics by cytochrome P450 comprised the main detoxification pathways. The mRNA expression levels of detoxification genes in nematodes living in the host were higher than those in nematodes feeding on fungus. However, there were fewer enzymes involved in the α-pinene degradation. Our results indicate that the two pinewood nematodes have evolved similar molecular mechanisms to adapt to life on pine hosts.

Genome-wide identification of Dicer-like, Argonaute and RNA-dependent RNA polymerase gene families and their expression analyses in response to viral infection and abiotic stresses in Solanum lycopersicum.

Dicer, Argonaute and RNA-dependent RNA polymerase form the core components to trigger RNA silencing. Although tomato (Solanum lycopersicum) is a dicotyledon model plant, no systematic analysis and expression profiling of these genes in tomato has been undertaken previously. In this study, seven Dicer-like (SlDCLs), 15 Argonaute (SlAGOs) and six RNA-dependent RNA polymerase (SlRDRs) genes were identified in tomato. These genes were categorized into four subgroups based on phylogenetic analyses. Comprehensive analyses of gene structure, genomic localization and similarity among these genes were performed. Their expression patterns were investigated by means of expression models in different tissues and organs using online data and semi-quantitative RT-PCR. Many of the candidate genes were up-regulated in response to Tomato yellow leaf curl virus infection and abiotic stresses. The expression models of tandem gene duplications among SlDCL2s indicated the DCL2 family plays an important role in the evolution of tomato.

[Ecological relationships between Bursaphelenchus xylophilus and its companion microorganisms].

Pine wood nematode Bursaphelenchus xylophilus is a notorious invasive species from North America, which can kill a large amount of pine trees and causes economic losses and ecosystem destruction. There is a close relationship and ecological interaction between B. xylophilus and its companion microorganisms. This paper listed the species of companion microorganisms, reviewed their important ecological roles in the propagation and pathogenicity of the nematode, and discussed the pine wilt disease from the viewpoint of microecosystem. The companion fungi can supply food for B. xylophilus, hold the cycle of second infection of the nematode, increase the proportions of dauer juveniles, and benefit the infection and distribution of B. xylophilus. The companion bacteria can enhance the pathogenicity of B. xylophilus, promote the propagation of the nematode, benefit the pinene degradation, and thereby, promote the adaptability of the nematode.

MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing.

BACKGROUND: MicroRNAs (miRNAs) are considered to be very important in regulating the growth, development, behavior and stress response in animals and plants in post-transcriptional gene regulation. Pinewood nematode, Bursaphelenchus xylophilus, is an important invasive plant parasitic nematode in Asia. To have a comprehensive knowledge about miRNAs of the nematode is necessary for further in-depth study on roles of miRNAs in the ecological adaptation of the invasive species. METHODS AND FINDINGS: Five small RNA libraries were constructed and sequenced by Illumina/Solexa deep-sequencing technology. A total of 810 miRNA candidates (49 conserved and 761 novel) were predicted by a computational pipeline, of which 57 miRNAs (20 conserved and 37 novel) encoded by 53 miRNA precursors were identified by experimental methods. Ten novel miRNAs were considered to be species-specific miRNAs of B. xylophilus. Comparison of expression profiles of miRNAs in the five small RNA libraries showed that many miRNAs exhibited obviously different expression levels in the third-stage dispersal juvenile and at a cold-stressed status. Most of the miRNAs exhibited obviously down-regulated expression in the dispersal stage. But differences among the three geographic libraries were not prominent. A total of 979 genes were predicted to be targets of these authentic miRNAs. Among them, seven heat shock protein genes were targeted by 14 miRNAs, and six FMRFamide-like neuropeptides genes were targeted by 17 miRNAs. A real-time quantitative polymerase chain reaction was used to quantify the mRNA expression levels of target genes. CONCLUSIONS: Basing on the fact that a negative correlation existed between the expression profiles of miRNAs and the mRNA expression profiles of their target genes (hsp, flp) by comparing those of the nematodes at a cold stressed status and a normal status, we suggested that miRNAs might participate in ecological adaptation and behavior regulation of the nematode. This is the first description of miRNAs in plant parasitic nematodes. The results provide a useful resource for further in-depth study on molecular regulation and evolution of miRNAs in plant parasitic nematodes.