Four endophytic fungi were isolated from the medicinal plant, Catharanthus roseus, and were identified as Diaporthe spp. with partial translation elongation factor 1-alpha (TEF1), beta-tubulin (TUB), histone H3 (HIS), calmodulin (CAL) genes, and rDNA internal transcribed spacer (ITS) region (TEF1-TUB-HIS--CAL-ITS) multigene phylogeny suggested for species delimitation in the Diaporthe genus. Each fungus produces a unique mixture of volatile organic compounds (VOCs) with an abundant mixture of terpenoids analyzed by headspace solid-phase microextraction (SPME) fiber-GC/MS. These tentatively-detected terpenes included α-muurolene, β-phellandrene, γ-terpinene, and α-thujene, as well as other minor terpenoids, including caryophyllene, patchoulene, cedrene, 2-carene, and thujone. The volatile metabolites of each isolate showed antifungal properties against a wide range of plant pathogenic test fungi and oomycetes, including Alternaria alternata, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium graminearum, and Phytophthora cinnamomi. The growth inhibition of the pathogens varied between 10% and 60% within 72 h of exposure. To our knowledge, the endophytic Diaporthe-like strains are first reported from Catharanthus roseus. VOCs produced by each strain of the endophytic Diaporthe fungi were unique components with dominant monoterpenes comparing to known Diaporthe fungal VOCs. A discussion is presented on the inhibitive bioactivities of secondary metabolites among endophytic Diaporthe fungi and this medicinal plant.
A moderately halophilic bacterium, designated strain 9-2(T), was isolated from saline and alkaline soil collected in Lindian county, Heilongjiang province, China. The strain was observed to be strictly aerobic, Gram-negative, rod-shaped, oxidase-positive, catalase-positive and motile. It was found to require NaCl for growth and to grow at NaCl concentrations of 0.5-14 % (w/v) (optimum, 7-10 %, w/v), at temperatures of 10-45 °C (optimum 25-30 °C) and at pH 5.0-10.0 (optimum pH 8.0). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 9-2(T) is a member of the genus Halomonas and is closely related to Halomonas desiderata DSM 9502(T) (96.68 %), Halomonas campaniensis DSM 1293(T) (96.46 %), Halomonas ventosae DSM 15911(T) (96.27 %) and Halomonas kenyensis DSM 17331(T) (96.27 %). The DNA-DNA hybridization value was 38.9 ± 0.66 % between the novel isolate 9-2(T) and H. desiderata DSM 9502(T). The predominant ubiquinones were identified as Q9 (75.1 %) and Q8 (24.9 %). The major fatty acids were identified as C16:0 (22.0 %), Summed feature 8 (C18:1 ω6c/C18:1 ω7c, 19.6 %), Summed feature 3 (C16:1 ω6c/C16:1 ω7c, 12.6 %), C12:0 3-OH (12.0 %) and C10:0 (11.7 %). The DNA G+C content was determined to be 69.7 mol%. On the basis of the evidence presented in this study, strain 9-2(T) is considered to represent a novel species of the genus Halomonas, for which the name Halomonas heilongjiangensis sp. nov. is proposed. The type strain is 9-2(T) (=DSM 26881(T) = CGMCC 1.12467(T)).
Two alkaliphilic and halotolerant Gram-stain positive, rod-shaped and endospore-forming bacteria, designated strains 12-3(T) and 12-4, were isolated from saline and alkaline soils collected in Lindian county, Heilongjiang province, China. Both strains were observed to grow well at a wide range of temperature and pH values, 10-45 °C and pH 8-12, with optimal growth at 37 °C and pH 9.0, respectively. Growth of the two strains was found to occur at total salt concentrations of 0-12 % (w/v), with an optimum at 4 % (w/v). The G+C contents of the genomic DNA of strains 12-3(T) and 12-4 were determined to be 42.7 and 42.4 mol%, respectively, and the major cellular fatty acids were identified as anteiso-C15:0 and anteiso-C17:0. In isolate 12-3(T), meso-diaminopimelic acid was found to be the diagnostic diamino acid of the cell wall peptidoglycan; diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol were identified as the major cellular polar lipids; and menaquinone-7 was identified as the predominant isoprenoid quinone. Strains 12-3(T) and 12-4 share very close 16S rRNA gene sequence similarity (99.74 %) and their DNA-DNA relatedness was 95.3 ± 0.63 %, meaning that the two strains can be considered to belong to the same species. 16S rRNA gene sequence-based phylogenetic analysis revealed strains 12-3(T) and 12-4 exhibit high similarities to Bacillus pseudofirmus DSM 8715(T) (98.7 %), Bacillus marmarensis DSM 21297(T) (97.2 %) and Bacillus nanhaiisediminis CGMCC 1.10116(T) (97.1 and 97.0 %, respectively). DNA-DNA hybridization values between isolate 12-3(T) and the type strains of closely related Bacillus species were below 30 %. On the basis of the polyphasic evidence presented, strains 12-3(T) and 12-4 are considered to represent a novel species of the genus Bacillus, for which the name Bacillus lindianensis sp. nov. is proposed. The type strain is 12-3(T) (DSM 26864(T) = CGMCC 1.12717(T)).
Pine wilt disease is a malady caused by a complex interaction of various factors such as pine wood nematodes, host plants, vector insects, associated fungi and bacteria, human economic and logistics activities, and environmental factors. The use of microorganisms to biologically control pine wilt disease is a potentially environmentally friendly means for the prevention and control of the disease. In this study, we carried out a systematic review of the progress in research on the biocontrol of pine wilt disease, by focusing on the pathogenic pine wood nematode, its vector beetle, and the host pine tree species. Then, we discuss the implementation prospects and research trends associated with the biocontrol of pine wood disease. This study provides reference information for the understanding and application of various biocontrol microorganisms in the prevention and control of pine wood disease and for the establishment of an environmentally friendly prevention and control strategy.
SummaryNatural volatiles released by the fungus, Annulohypoxylon sp. FPYF3050, were evaluated against the pine wood nematode (PWN), Bursaphelenchus xylophilus. Our results showed that volatile organic compounds (VOCs) caused 64.1 and 58.4% mortality of second-stage juveniles (J2) and mixed-stages (eggs, J2, third- and fourth-stage juveniles, and adults) of populations of PWN, but no inhibitive effects were detected on nematode eggs in the experiment. Analysis of the gases within the Petri plate by gas chromatography-mass spectrometry (GC-MS) showed a yield of an unique volatile with dominant 1,8-cineole in 77.4% relative area (RA) after 72 h treatment of nematodes with Annulohypoxylon sp. FPYF3050 and Botrytis cinerea. The commercial 1,8-cineole at concentrations of 2, 5, 10 and 15 μl ml−1 was applied to examine nematicidal activity. The results showed that 1,8-cineole had a 40-100% inhibition on the nematode eggs during 48 h treatment, more than 82.9% mortality of J2 after 24 h, 48 h and 96 h, and 18.7-91.9% mortality of the mixed-stage population, depending on the period after exposure. This result indicates that 1,8-cineole in the volatile gas emissions of Annulohypoxylon sp. FPYF3050 may play a crucial inhibitory effect on the pine wood nematode The nematicidal volatile gas from fungi may provide a useful biocontrol agent for controlling B. xylophilus.
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