Biocontrol potential of endophytic bacterium Bacillus altitudinis GS-16 against tea anthracnose caused by Colletotrichum gloeosporioides.

Background: As one of the main pathogens causing tea anthracnose disease, Colletotrichum gloeosporioides has brought immeasurable impact on the sustainable development of agriculture. Given the adverse effects of chemical pesticides to the environment and human health, biological control has been a focus of the research on this pathogen. Bacillus altitudinis GS-16, which was isolated from healthy tea leaves, had exhibited strong antagonistic activity against tea anthracnose disease. Methods: The antifungal mechanism of the endophytic bacterium GS-16 against C. gloeosporioides 1-F was determined by dual-culture assays, pot experiments, cell membrane permeability, cellular contents, cell metabolism, and the activities of the key defense enzymes. Results: We investigated the possible mechanism of strain GS-16 inhibiting 1-F. In vitro, the dual-culture assays revealed that strain GS-16 had significant antagonistic activity (92.03%) against 1-F and broad-spectrum antifungal activity in all tested plant pathogens. In pot experiments, the disease index decreased to 6.12 after treatment with GS-16, indicating that strain GS-16 had a good biocontrol effect against tea anthracnose disease (89.06%). When the PE extract of GS-16 treated mycelial of 1-F, the mycelial appeared deformities, distortions, and swelling by SEM observations. Besides that, compared with the negative control, the contents of nucleic acids, protein, and total soluble sugar of GS-16 group were increased significantly, indicating that the PE extract of GS-16 could cause damage to integrity of 1-F. We also found that GS-16 obviously destroyed cellular metabolism and the normal synthesis of cellular contents. Additionally, treatment with GS-16 induced plant resistance by increasing the activities of the key defense enzymes PPO, SOD, CAT, PAL, and POD. Conclusions: We concluded that GS-16 could damage cell permeability and integrity, destroy the normal synthesis of cellular contents, and induce plant resistance, which contributed to its antagonistic activity. These findings indicated that strain GS-16 could be used as an efficient microorganism for tea anthracnose disease caused by C. gloeosporioides.

Herbaspirillum camelliae sp. nov., a novel endophytic bacterium isolated from Camellia sinensis L.

Bacterial strain WT00CT is an endophytic bacterium that was isolated from the tea plant (Camellia sinensis L.). The phylogenetic analysis of 16S rRNA genes demonstrated that strain WT00CT was a member of the genus Herbaspirillum. This strain is microaerobic, gram-negative and non-pigmented, and its cells are rod shaped, with a polar flagellum. It grew optimally at 34-37 °C, pH 5.0-8.0 and 0-1.5% NaCl (w/v). The G + C content of its genomic DNA was 62.36 mol%. C16:0, iso-C15:0, iso-C17:0, anteiso-C15:0 and anteiso-C17:0 were major fatty acids. The strain WT00CT contained six polar lipids, namely DPG (diphosphatidylglycerol), PE (phosphatidylethanolamine), PG (phosphatidylglycerol), PC (phosphatidylcholine), GL (glycolipid) and APL (aminophospholipids), and its respiratory quinone was Q8. The strain WT00CT had a genome size of 6.08 Mb with a total ORF of 5,537, in which one gene cluster (36 genes) encoding a type IV secretion system was absent in other members of the Herbaspirillum genus. ANI values of genomic comparison between the strain WT00CT and other Herbaspirillum species were 75-96%. Based on the phylogenetic, chemotaxonomic and phenotypic data presented here, the strain WT00CT represents a novel species in the Herbaspirillum genus, for which the name Herbaspirillum camelliae sp. nov. is proposed. The type strain of H. camelliae sp. nov. is WT00CT (AB 2018017 T and KCTC 62527 T).

Characterization and Initial Application of Endophytic Bacillus safensis Strain ZY16 for Improving Phytoremediation of Oil-Contaminated Saline Soils.

Hydrocarbon-degrading and plant-growth-promoting bacterial endophytes have proven useful for facilitating the phytoremediation of petroleum-contaminated soils with high salinity. In this study, we identified Bacillus safensis strain ZY16 as an endophytic bacterium that can degrade hydrocarbons, produce biosurfactants, tolerate salt, and promote plant growth. The strain was isolated from the root of Chloris virgata Sw., a halotolerant plant collected from the Yellow River Delta. ZY16 survived in Luria-Bertani (LB) broth with 0-16% (w/v) sodium chloride (NaCl) and grew well in LB broth supplemented with 0-8% NaCl, indicating its high salt tolerance. The endophytic strain ZY16 effectively degraded C12-C32 n-alkanes of diesel oil effectively, as well as common polycyclic aromatic hydrocarbons under hypersaline conditions. For example, in mineral salts (MS) liquid medium supplemented with 6% NaCl, ZY16 degraded n-undecane, n-hexadecane, n-octacosane, naphthalene, phenanthrene, and pyrene, with degradation percentages of 94.5, 98.2, 64.8, 72.1, 59.4, and 27.6%, respectively. In addition, ZY16 produced biosurfactant, as confirmed by the oil spreading technique, surface tension detection, and emulsification of para-xylene and paraffin. The biosurfactant production ability of ZY16 under hypersaline conditions was also determined. Moreover, ZY16 showed plant-growth-promoting attributes, such as siderophore and indole-3-acetic acid production, as well as phosphate solubilization. To assess the enhanced phytoremediation of saline soils polluted by hydrocarbons and the plant-growth-promotion ability of ZY16, a pot trial with and without inoculation of the endophyte was designed and performed. Inoculated and non-inoculated plantlets of C. virgata Sw. were grown in oil-polluted saline soil, with oil and salt contents of 10462 mg/kg and 0.51%, respectively. After 120 days of growth, significant enhancement of both the aerial and underground biomass of ZY16-inoculated plants was observed. The soil total petroleum hydrocarbon degradation percentage (a metric of phytoremediation) after incubation with ZY16 was 63.2%, representing an elevation of 25.7% over phytoremediation without ZY16 inoculation. Our study should promote the application of endophytic B. safensis ZY16 in phytoremediation by extending our understanding of the mutualistic interactions between endophytes and their host plants.

Toxoflavin Produced by Burkholderia gladioli from Lycoris aurea Is a New Broad-Spectrum Fungicide.

Fungal infections not only cause extensive agricultural damage but also result in serious diseases in the immunodeficient populations of human beings. Moreover, the increasing emergence of drug resistance has led to a decrease in the efficacy of current antifungals. Thus, screening of new antifungal agents is imperative in the fight against antifungal drug resistance. In this study, we show that an endophytic bacterium, Burkholderia gladioli HDXY-02, isolated from the medicinal plant Lycoris aurea, showed broad-spectrum antifungal activity against plant and human fungal pathogens. An antifungal ability assay indicated that the bioactive component was produced from strain HDXY-02 having an extracellular secreted component with a molecular weight lower than 1,000 Da. In addition, we found that this new antifungal could be produced effectively by liquid fermentation of HDXY-02. Furthermore, the purified component contributing to the antifungal activity was identified to be toxoflavin, a yellow compound possessing a pyrimido[5,4-e][1,2,4]triazine ring. In vitro bioactivity studies demonstrated that purified toxoflavin from B. gladioli HDXY-02 cultures had a significant antifungal activity against the human fungal pathogen Aspergillus fumigatus, resulting in abolished germination of conidia. More importantly, the growth inhibition by toxoflavin was observed in both wild-type and drug-resistant mutants (cyp51A and non-cyp51A) of A. fumigatus Finally, an optimized protocol for the large-scale production of toxoflavin (1,533 mg/liter) has been developed. Taken together, our findings provide a promising biosynthetic resource for producing a new antifungal reagent, toxoflavin, from isolates of the endophytic bacterium B. gladioliIMPORTANCE Human fungal infections are a growing problem associated with increased morbidity and mortality. Moreover, a growing number of antifungal-resistant fungal isolates have been reported over the past decade. Thus, the need for novel antifungal agents is imperative. In this study, we show that an endophytic bacterium, Burkholderia gladioli, isolated from the medicinal plant Lycoris aurea, is able to abundantly secrete a compound, toxoflavin, which has a strong fungicidal activity not only against plant fungal pathogens but also against human fungal pathogens Aspergillus fumigatus and Candida albicans, Cryptococcus neoformans, and the model filamentous fungus Aspergillus nidulans More importantly, toxoflavin also displays an efficacious inhibitory effect against azole antifungal-resistant mutants of A. fumigatus Consequently, our findings provide a promising approach to abundantly produce toxoflavin, which has novel broad-spectrum antifungal activity, especially against those currently problematic drug-resistant isolates.

Biotransformation of ginsenoside Rb1 to Gyp-XVII and minor ginsenoside Rg3 by endophytic bacterium Flavobacterium sp. GE 32 isolated from Panax ginseng.

The rare ginsenoside Rg3 is attracting more attention because of its good physiological activity and urgent need. There are many pathways to obtain ginsenoside Rg3, including chemical and biological methods. Among these, the conversion of the protopanaxadiol-type ginsenosides by microbial hydrolysis is a trend due to its high efficiency and mild conditions. For effectively extracting from the other panaxadiol saponins, the conversion process for ginsenoside Rg3 was investigated using ß-glycosidase-producing endophytic fungus in Panax ginseng in this study. The metabolic pathways are as follows: ginsenoside Rb1 â†’ Gyp-XVII and ginsenoside Rb1 â†’ ginsenoside Rd â†’ ginsenoside Rg3. Phylogenetic analysis of 16S rDNA gene sequence, showed that GE 32 strain belonged to Flavobacterium species. These results suggest that the process of rare ginsenoside Rg3 production by endophytic bacteria GE 32 is efficient for the industrial production and application. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on cultivable ß-glycosidase-producing endophytic bacteria from Panax ginseng. Flavobacterium sp. GE32 could convert major ginsenoside Rb1 into Gyp-XVII and minor ginsenoside Rg3. Strain GE 32 has potential to be applied on the preparation for minor ginsenoside Rg3 in pharmaceutical industry.

Characterization and antifungal activity against Pestalotiopsis of a fusaricidin-type compound produced by Paenibacillus polymyxa Y-1.

Dendrobium nobile (D. nobile) is a valuable Chinese herbal medicine. The discovery of microbial resources from has provided a wealth of raw materials. Stalk rot, which is caused by Pestalotiopsis, is one of the most serious diseases of D nobile and has resulted in serious losses in production. However, an effective method for the prevention and control of stalk rot remains lacking. In this study, we aimed to identify a biocontrol strain against Pestalotiopsis. We isolated Paenibacillus polymyxa Y-1, an endophytic bacterium, from the stem of D. nobile. Three pairs of active metabolites isolated from this bacterium were identified as fusaricidin compounds. We then investigated the mechanism of fusaricidin compounds on Pestalotiopsis via proteomics. Proteomics data showed that the compounds mainly inhibit energy generation in the respiratory chain and amino acid biosynthesis of Pestalotiopsis.

Volatiles released by endophytic Pseudomonas fluorescens promoting the growth and volatile oil accumulation in Atractylodes lancea.

Atractylodes lancea is a well-known, but endangered, Chinese medicinal plant whose volatile oils are its main active components. As the volatile oil content in cultivated A. lancea is much lower than that in the wild herb, the application of microbes or related elicitors to promote growth and volatile oil accumulation in the cultivated herb is an important area of research. This study demonstrates that the endophytic bacterium Pseudomonas fluorescens ALEB7B isolated from the geo-authentic A. lancea can release several nitrogenous volatiles, such as formamide and N,N-dimethyl-formamide, which significantly promote the growth of non-infected A. lancea. Moreover, the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A. lancea via activating plant defense responses. Notably, the bacterial nitrogenous volatiles cannot be detected in the A. lancea - Pseudomonas fluorescens symbiont while the benzaldehyde can be detected, indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant. This study firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact, extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interaction.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.