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Distribution and diversity of bacterial endophytes from four Pinus species and their efficacy as biocontrol agents for devastating pine wood nematodes.

ABSTRACT: In this study, we isolated a total of 238 culturable putative bacterial endophytes from four Pinus species (Pinus densiflora, P. koraiensis, P. rigida, and P. thunbergii) across 18 sampling sites in Korea. The samples were cultured in de Man Rogosa Sharpe and humic acid-vitamin agar media. These selective media were used to isolate lactic acid bacteria and Actinobacteria, respectively. Analysis using 16S ribosomal DNA sequencing grouped the isolated putative bacterial endophytes into 107 operational taxonomic units (OTUs) belonging to 48 genera. Gamma-proteobacteria were the most abundant bacteria in each sampling site and three tissues (needle, stem and root). The highest OTU richness and diversity indices were observed in the roots, followed by stem and needle tissues. Total metabolites extracted from three isolates (two isolates of Escherichia coli and Serratia marcescens) showed significant nematicidal activity against the pine wood nematode (Bursaphelenchus xylophilus). Our findings demonstrated the potential use of bacterial endophytes from pine trees as alternative biocontrol agents against pine wood nematodes.

SUBMITTER: Liu Y

PROVIDER: S-EPMC6713764 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Distribution and diversity of bacterial endophytes from four Pinus species and their efficacy as biocontrol agents for devastating pine wood nematodes.

Liu Yunran Y Ponpandian Lakshmi Narayanan LN Kim Hoki H Jeon Junhyun J Hwang Buyng Su BS Lee Sun Keun SK Park Soo-Chul SC Bae Hanhong H

Scientific reports 20190828 1

In this study, we isolated a total of 238 culturable putative bacterial endophytes from four Pinus species (Pinus densiflora, P. koraiensis, P. rigida, and P. thunbergii) across 18 sampling sites in Korea. The samples were cultured in de Man Rogosa Sharpe and humic acid-vitamin agar media. These selective media were used to isolate lactic acid bacteria and Actinobacteria, respectively. Analysis using 16S ribosomal DNA sequencing grouped the isolated putative bacterial endophytes into 107 operati ...[more]

PMID: 31462658

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Project description:Pine wilt disease is a dangerous pine disease globally. We used Masson pine (Pinus massoniana) clones, selected through traditional breeding and testing for 20 years, to study the molecular mechanism of their high resistance to pine wood nematodes (PWN,Bursaphelenchus xylophilus). Nine strains of seedlings of genetically stable Masson pine screened from different families with high resistance to PWN were used. The same number of sensitive clones were used as susceptible controls. Total proteins were extracted for tandem mass tag (TMT) quantitative proteomic analysis. The key proteins were verified by parallel reaction monitoring (PRM). A threshold of upregulation greater than 1.3-fold or downregulation greater than 0.3-fold was considered significant in highly resistant strains versus sensitive strains. A total of 3491 proteins were identified from the seedling tissues, among which 2783 proteins contained quantitative information. A total of 42 proteins were upregulated and 96 proteins were downregulated in the resistant strains. Functional enrichment analysis found significant differences in the proteins with pectin esterase activity or peroxidase activity. The proteins participating in salicylic acid metabolism, antioxidant stress reaction, polysaccharide degradation, glucose acid ester sheath lipid biosynthesis, and the sugar glycosaminoglycan degradation pathway were also changed significantly. The PRM results showed that pectin acetyl esterase, carbonic anhydrase, peroxidase, and chitinase were significantly downregulated, while aspartic protease was significantly upregulated, which was consistent with the proteomic data. These results suggest that Masson pine can degrade nematode-related proteins by increasing protease to inhibit their infestation, and can enhance the resistance of Masson pine to PWN by downregulating carbon metabolism to limit the carbon available to PWN or for involvement in cell wall components or tissue softening. Most of the downregulated proteins are supposed to act as an alternative mechanism for latter enhancement after pathogen attacks. The highly resistant Masson pine, very likely, harbors multiple pathways, both passive and active, to defend against PWN infestation.

Project description:The pinewood nematode (PWN) Bursaphelenchus xylophilus is a forestry quarantine pest and causes an extremely dangerous forest disease that is spreading worldwide. Due to the complex pathogenic factors of pine wood nematode disease, the pathogenesis is still unknown. B. xylophilus ultimately invades a host and causes death. However, little is known about the defence-regulating process of host pine after infection by B. xylophilus at the molecular level. Therefore, we wanted to understand how Pinus massoniana regulates its response to invasion by B. xylophilus. P. massoniana were artificially inoculated with B. xylophilus solution, while those without B. xylophilus solution were used as controls. P. massoniana inoculated with B. xylophilus solution for 0 h, 6 h, 24 h, and 120 h was subjected to high-throughput sequencing to obtain transcriptome data. At various time points (0 h, 6 h, 24 h, 120 h), gene transcription was measured in P. massoniana inoculated with PWN. At different time points, P. massoniana gene transcription differed significantly, with a response to early invasion by PWN. According to Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, P. massoniana response to PWN invasion involves a wide range of genes, including plant hormone signal transformation, flavonoid biosynthesis, amino sugar and nucleoside sugar metabolism, and MAPK signalling pathways. Among them, inoculation for 120 hours had the greatest impact on differential genes. Subsequently, weighted gene coexpression network analysis (WGCNA) was used to analyse transcriptional regulation of P. massoniana after PWN infection. The results showed that the core gene module of P. massoniana responding to PWN was "MEmagenta", enriched in oxidative phosphorylation, amino sugar and nucleotide sugar metabolism, and the MAPK signalling pathway. MYB family transcription factors with the highest number of changes between infected and healthy pine trees accounted for 20.4% of the total differentially expressed transcription factors. To conclude, this study contributes to our understanding of the molecular mechanism of initial PWN infection of P. massoniana. Moreover, it provides some important background information on PWN pathogenic mechanisms.

Project description:BackgroundPine wilt disease is caused by the pine wood nematode, Bursaphelenchus xylophilus, which threatens pine forests and forest ecosystems worldwide and causes serious economic losses. In the 40 years since the pathogen was identified, the physiological changes occurring as the disease progresses have been characterized using anatomical and biochemical methods, and resistant trees have been selected via breeding programs. However, no studies have assessed the molecular genetics, e.g. transcriptional changes, associated with infection-induced physiological changes in resistant or susceptible trees.ResultsWe constructed seven subtractive suppression hybridization (SSH) cDNA libraries using time-course sampling of trees inoculated with pine wood nematode at 1, 3, or 7 days post-inoculation (dpi) in susceptible trees and at 1, 3, 7, or 14 dpi in resistant trees. A total of 3,299 sequences was obtained from these cDNA libraries, including from 138 to 315 non-redundant sequences in susceptible SSH libraries and from 351 to 435 in resistant SSH libraries. Using Gene Ontology hierarchy, those non-redundant sequences were classified into 15 subcategories of the biological process Gene Ontology category and 17 subcategories of the molecular function category. The transcriptional components revealed by the Gene Ontology classification clearly differed between resistant and susceptible libraries. Some transcripts were discriminative: expression of antimicrobial peptide and putative pathogenesis-related genes (e.g., PR-1b, 2, 3, 4, 5, 6) was much higher in susceptible trees than in resistant trees at every time point, whereas expression of PR-9, PR-10, and cell wall-related genes (e.g., for hydroxyproline-rich glycoprotein precursor and extensin) was higher in resistant trees than in susceptible trees at 7 and 14 dpi.ConclusionsFollowing inoculation with pine wood nematode, there were marked differences between resistant and susceptible trees in transcript diversity and the timing and level of transcripts expressed in common; in particular, expression of stress response and defense genes differed. This study provided new insight into the differences in the physiological changes between resistant and susceptible trees that have been observed in anatomical and biochemical studies.

Dataset Information

Distribution and diversity of bacterial endophytes from four Pinus species and their efficacy as biocontrol agents for devastating pine wood nematodes.

Publications

Distribution and diversity of bacterial endophytes from four Pinus species and their efficacy as biocontrol agents for devastating pine wood nematodes.

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