Plant Growth-Promoting Rhizobacteria Promote Growth of Seedlings, Regulate Soil Microbial Community, and Alleviate Damping-Off Disease Caused by Rhizoctonia solani on Pinus sylvestris var. mongolica.

As the excessive use of chemical fertilizers harms organisms and adversely affects the soil environment, the replacement of chemical fertilizers with biological fertilizers has attracted widespread attention as an environmental protection strategy. In this study, the effects of rhizosphere bacteria inoculation on growth of Pinus sylvestris var. mongolica seedlings, soil parameters, soil microbial community structure, and the biocontrol of damping-off were studied by pot experiments. The results showed that all three rhizosphere bacteria (Pseudomonas chlororaphis, Pseudomonas extremaustralis, and Acinetobacter lwoffii A07) tested exhibited growth-promoting properties, such as the production of indole-3-acetic acid, hydrolase, siderophores, and hydrogen cyanide; nitrogen fixation; and phosphorus solubilization. The application of the three bacteria increased plant biomass, root structure, and nutrient content and also increased soil nutrient content and enzyme activity. Bacterial inoculation promoted the growth of beneficial bacteria and antagonistic bacteria by adjusting the physicochemical properties of the soil, thereby improving the bacterial community structure. Among the soil features, available nitrogen, total nitrogen, available potassium, and urease activity were the main influencing factors. In addition, it was also found that bacterial inoculation significantly increased the activities of plant superoxide dismutase, catalase, peroxidase, and other defense enzymes; enhanced plant disease resistance; effectively inhibited damping-off; and promoted plant growth. In summary, the application of three rhizosphere bacteria systematically affected the interaction between plants, soil parameters, and soil microbial communities. These results provide a basis for understanding how rhizosphere bacteria promote the growth of P. sylvestris var. mongolica, thereby offering a promising sustainable alternative to chemical fertilizers.

Diversity and structure of the microbial community in rhizosphere soil of Fritillaria ussuriensis at different health levels.

Fritillaria wilt is a kind of soil-borne disease that causes a large reduction in the yield of Fritillaria ussuriensis. The diversity and structure of the soil microbial community are important factors affecting the health of Fritillaria ussuriensis. The analysis of the microbial community in the diseased and healthy soils provided a theoretical basis for revealing the pathological mechanism and prevention of Fritillaria wilt disease. In the present study, we sequenced the soil microorganisms from healthy (H), pathology (P) and blank (B) soil samples by Illumina MiSeq. Determined the soil physicochemical properties respectively, analyzed the soil microbial diversity and structure, and constructed single factor co-correlation networks among microbial genera. The results showed that Ascomycota (48.36%), Mortierellomycota (23.06%), Basidiomycota (19.00%), Proteobacteria (31.74%), and Acidobacteria (20.95%) were dominant in the soil. The diversity of healthy soil was significantly greater than that of diseased soil samples (P and B) (P < 0.05). The populations of Fusarium and Humicola significantly increased in the diseased soil sample (P and B) (P < 0.05). RB41 (4.74%) and Arthrobacter (3.30%) were the most abundant genera in the healthy soil. Total nitrogen (TN), available nitrogen (AN), total potassium (TK), available potassium (AK), and inorganic salt (salt) were significantly correlated with soil microbial communities (P < 0.05). The relationship between fungi and the plant was mostly positive, whereas bacteria showed the opposite trend. In conclusion, the diversity and structure of the soil microbial community were closely related to the health level of Fritillaria ussuriensis. Fusarium and Humicola affect the severity of Fritillaria wilt disease, while RB41 and Arthrobacter are the important indicators for maintaining the health of Fritillaria ussuriensis. Moreover, environmental factors greatly affect the abundance and formation of soil microbial community. The interactions in microbial communities also influence the healthy growth of Fritillaria ussuriensis.

Inhibitory Mechanism of Trichoderma virens ZT05 on Rhizoctonia solani.

Trichoderma is a filamentous fungus that is widely distributed in nature. As a biological control agent of agricultural pests, Trichoderma species have been widely studied in recent years. This study aimed to understand the inhibitory mechanism of Trichoderma virens ZT05 on Rhizoctonia solani through the side-by-side culture of T. virens ZT05 and R. solani. To this end, we investigated the effect of volatile and nonvolatile metabolites of T. virens ZT05 on the mycelium growth and enzyme activity of R. solani and analyzed transcriptome data collected from side-by-side culture. T. virens ZT05 has a significant antagonistic effect against R. solani. The mycelium of T. virens ZT05 spirally wraps around and penetrates the mycelium of R. solani and inhibits the growth of R. solani. The volatile and nonvolatile metabolites of T. virens ZT05 have significant inhibitory effects on the growth of R. solani. The nonvolatile metabolites of T. virens ZT05 significantly affect the mycelium proteins of R. solani, including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), selenium-dependent glutathione peroxidase (GSH-Px), soluble proteins, and malondialdehyde (MDA). Twenty genes associated with hyperparasitism, including extracellular proteases, oligopeptide transporters, G-protein coupled receptors (GPCRs), chitinases, glucanases, and proteases were found to be upregulated during the antagonistic process between T. virens ZT05 and R. solani. Thirty genes related to antibiosis function, including tetracycline resistance proteins, reductases, the heat shock response, the oxidative stress response, ATP-binding cassette (ABC) efflux transporters, and multidrug resistance transporters, were found to be upregulated during the side-by-side culture of T. virens ZT05 and R. solani. T. virens ZT05 has a significant inhibitory effect on R. solani, and its mechanism of action is associated with hyperparasitism and antibiosis.

Effects of Dark Septate Endophytes Strain A024 on Damping-off Biocontrol, Plant Growth and the Rhizosphere Soil Enviroment of Pinus sylvestris var. mongolica Annual Seedlings.

Dark septate endophytes (DSEs) exert a vital role in promoting plant growth, improving mineral absorption, biological disease control, and enhancing plant stress resistance. The effects of dark septate endophyte strain, Phialocephala bamuru A024 on damping-off biocontrol, plant development, nutrients within the rhizosphere soil, as well as bacterial communities in the annual seedlings of P. sylvestris var. Mongolica were studied. According to our findings, following P. bamuru A024 inoculation, the damping-off disease morbidity decreased significantly compared with control, some physiological indices such as ß-1,3-glucanase, chitinase enzyme activity as well as a soluble protein and proline content in P. sylvestris var. mongolica were elevated under R. solani stress. After inoculation with P. bamuru A024, the biomass in seedlings, nutrients in soil, root structure index, together with activities of soil enzymes were remarkably up-regulated relative to control (p < 0.05). As suggested by the results of high-throughput sequencing, the microbial structure in the rhizosphere soil of the P. sylvestris var. mongolica showed significant differences (p < 0.05) after P. bamuru A024 inoculation compared to control treatment and the rhizosphere soil bacterial community structure after DSE A024 inoculation was positively correlated to the main soil nutrition indices.

Expression of Pleurotus ostreatus Laccase Gene in Pichia pastoris and Its Degradation of Corn Stover Lignin.

Pleurotus ostreatus is a species of white-rot fungi that effectively degrades lignin. In this study, we aimed to efficiently express the lac-2 gene of Pleurotus ostreatus in the Pichia pastoris X33 yeast strain. The enzymatic properties of recombinant yeast were determined, and its ability to degrade corn stover lignin was determined. The results showed the optimum pH values of recombinant laccase for 2,2'-Azinobis-3-ethylbenzothiazoline-6-sulfonic acid, 2,6-dimethoxyphenol, and 2-methoxyphenol were 3.0, 3.0, and 3.5, respectively. The optimum reaction temperature was 50 °C, and it had good thermal stability and acid and alkali resistance. The degradation rate of lignin in corn stover by recombinant laccase was 18.36%, and the native Pleurotus ostreatus degradation rate was 14.05%, the difference between them is significant (p < 0.05). This experiment lays a foundation for the study of the degradation mechanism of lignin by laccase.

The anti-phytoalexin gene Bx-cathepsin W supports the survival of Bursaphelenchus xylophilus under Pinus massoniana phytoalexin stress.

BACKGROUND: Pine trees challenged by Bursaphelenchus xylophilus invasion produce phytoalexins to combat this nematode. Nevertheless, the phytoalexins of Asian pine trees are ineffective against B. xylophilus. The anti-phytoalexin genes of B. xylophilus disable almost all Asian pine phytoalexins, which has allowed B. xylophilus to devastate pine forests in eastern Asia over the last four decades. However, to date, the factors that stimulate anti-phytoalexin gene expression and the mechanisms by which these genes act are not well understood. RESULTS: Here, we described anti-phytoalexin genes in B. xylophilus using transcriptomic and bioinformatics analyses. The genes that were induced by both Pinus massoniana and carvone and had similarly elevated expression trends were considered anti-phytoalexin genes. Altogether, 187 anti-phytoalexin genes were identified, including 4 cathepsin genes. KEGG pathway enrichment indicated that those cathepsins were related to the Lysosome pathway. Since cathepsins help to maintain metabolic homeostasis by participating in the degradation of heterophagic and autophagic material, the lysosomal cathepsin gene Bx-cathepsin W was cloned and characterized. The results of the RNAi assessment indicated that the knockdown of Bx-cathepsin W reduced the survival rates of B. xylophilus under carvone or P. massoniana stress. The correlation between Bx-cathepsin W and the susceptibility of pines showed that Bx-cathepsin W might help improve the anti-phytotoxin ability of B. xylophilus. CONCLUSIONS: The results indicated that the anti-phytoalexin gene Bx-cathepsin W supported the survival of B. xylophilus under P. massoniana phytoalexin stress. The cDNA library sequencing, differentially expressed gene identification, and WGCNA algorithm analysis provided insight at a systemic level into the gene regulation of B. xylophilus in response to the immune reaction of P. massoniana. These results will lead to a better understanding of the function of nematode defenses in host innate immunity.

Isolation, Purification, and Structural Identification of an Antifungal Compound from a Trichoderma Strain.

Trichoderma strain T-33 has been demonstrated to have inhibitory effect on the fungus species Cytospora chrysosperma. Here, an active antifungal compound was obtained from Trichoderma strain T-33 extract via combined separation technologies, including organic solvent extraction, liquid chromatography, and thin-layer chromatography. The purified compound was further characterized by advanced analytical technologies to elucidate its chemical structure. Results indicated that the active antifungal compound in Trichoderma strain T-33 extract is 2,5- cyclohexadiene-1,4-dione-2,6-bis (1,1-dimethylethyl).

[Antifungal sctivities and stability of extracts from culture liquid of Hypoxylon perforatum to Sphaeropsis sapinea].

OBJECTIVE: Our research objective is to obtain the active substances from culture liquid of Hypoxylon perforatum with inhibitory effect on Sphaeropsis sapinea growth and germination. METHODS: Water and ester were used for extracting active substances from culture liquid of Hypoxylon perforatum, either by extracting directly from cultural liquid, or extracting under ultrasonic. Growth-inhibiting rate and germination-inhibiting rate were used as the index of antifungal activity. RESULTS: Five extracts from culture liquid of Hypoxylon perforatum showed antifungal activities to Sphaeropsis sapinea growth and germination. CONCLUSION: Ethyl acetate-extract from culture liquid of Hypoxylon perforatum has a highly antifungal activity to Sphaeropsis sapinea, high stability in the natural environment. So it has a highly development of the value and application prospect. The mainly inhibiting active substance is methyl p-methoxy cinnamate, which as medicine intermediate was used in cosmetic to be UV-protection absorbefacient. Diisobutyl phthalate and Dibutyl phthalate are all the common plastic-enhancer for PolyvinylChloride (PVC). It is very important in medicinal industry and chemistry industry to discovering the natural presence of the three compounds.

[Effect of ectomycorrhizal fungi on the seedlings growth of Korea spruce].

To enhance the effects of ectomycorrhizal fungi to promote the growth of Korea spruce seedlings, based on previous research, the combinations of different ectomycorrhizal fungi strains were screened by dual culture method. 3 years transplanting seedlings of Korea spruce were inoculated by different combinations of ectomycorrhizal fungi strains using lister inoculating method in the field, and those were inoculated by different single strains were designed as control respectively. Thus the effects of different combinations and different single fungus strains to promote the growth of Korea spruces were studied. The results showed that all single strains and combinations in this experiment can promote the growth of Korea spruce seedlings. The growth characteristics of seedlings were observed 100 days after inoculation. The growth promoting effect of strain L15 was the best in all combinations and single strains. Comparing with control, the average height of seedlings inoculated by strain L15 was increased 30.88%, the average collar diameter of these was increased 15.29%. The growth promoting effect of combinations L15/025 or L15/009 were better than strain 009 or 025. Comparing with control, the leaf chlorophyll contents of seedling inoculated by strain 010 and combination L15/025 were increased significantly, the contents of chlorophyll a were increased 59.15% and 54.61% respectively, and the contents of chlorophyll b were increased 76.34% and 67.78% respectively. Except seedling inoculated by strain 010, the activities of hydrogen peroxidase of other treated seedlings were lower than one of control. The root activities of all treated seedlings were lower than one of control. In conclusion, inoculation by the mixture of high-effect strain and other single strain weakens the effect of high-effect strain to promote the growth of Korea spruce seedlings, the activity of hydrogen peroxidase and the root activity of seedling are not correlated with its biomass.

[Effect of ectomycorrhizae on the growth of Picea koraiensis seedlings].

Basidioscarps of Agaricales in different Picea koraiensis forest plantations were collected during August-October, 2000. 36 isolaters of species of Agaricales were obtained by isolating and culturing to the basidioscarps. Through indoor inoculation test on seedlings of Picea koraiensis, 6 ectomycorrhizae fungi cultures were obtained from 36 isolaters. The inoculation results show that the period for ectomycorrhizae inoculation to 1-year seedlings of Picea koraiensis should be about 30 days after seedlings emerging, the suitable temperature for ectomycorrhizae forming is about 20 degrees C. 6 ectomycorrhizae strains all have growth-promoting effect to the seedlings of Picea koraiensis. The contents of chlorophyll a of the seedlings inoculated strains of Agaricus silvaticus, 031 and L15 were significantly higher than other strains and control. The contents of chlorophyll b in the seedlings inoculated strains 009, 004, Agaricus silvaticus and L15 were significantly higher than other strains and control. The weights of seedlings which inoculated strains 009, 025, 031, Agaricus silvaticus and L15 were significantly different to control, the weight of seedlings inoculated strains of Agaricus silvaticus and L15 are 19.23% and 23.08% more than control; The heights of the seedlings inoculated 6 strains all have significant difference to control, the weight of seedlings inoculated strains of Agaricus silvaticus and L15 are 17.83% and 16.37% more than control. The results of outdoor inoculation show that the seedlings inoculated Agaricus silvaticus grow best on height, 9.25% more than control after inoculated 70 days; the seedlings inoculated strain L15 grow best on collar diameter, 9.92% more than control after inoculated 70 days; the lateral root numbers of seedlings inoculated strain 009 is largest, 51.91% more than control after inoculated 70 days; the main roots of seedlings inoculated strain 009 are longest, 3.36% more than control after inoculated 70 days; the ectomycorrhizae fungi infestation percentage of seedlings inoculated strain 031 is highest, 13.90% more than control after inoculated 70 days; the photosynthesis of seedlings inoculated strain 031 is more powerful, 20.02% more than control after inoculated 70 days. To sum up, strain 009, strain 031 and strain L15 should be fine strains to seedlings of Picea koraiensis.