Antagonistic Endophytic Bacteria Research Articles

Screening of Endophytic Antagonistic Bacteria in Wheat and Evaluation of Biocontrol Potential against Wheat Stripe Rust.

Wheat stripe rust is globally one of the most important diseases affecting wheat. There is an urgent need to develop environmentally safe and durable biological control options to supplement the control that is achieved with breeding and fungicides. In this study, endophytic bacteria were isolated from healthy wheat through the tissue separation method. Antagonistic endophytic bacteria were screened based on the control effect of urediniospore germination and wheat stripe rust (WSR). The taxonomic status of antagonistic strains was determined based on morphological, physiological, and biochemical characteristics and molecular biological identification (16S rDNA and gyrB gene sequence analysis). Finally, the potential growth-promoting effect of different concentrations of antagonists on wheat seedlings and the biological control effect of WSR were studied. A total of 136 strains of endophytic bacteria belonging to 38 genera were isolated. Pseudomonas was the most common bacterial genus, with 29 isolates (21%). The biological control effect of different isolates was assessed using an urediniospore germination assay. The isolate XD29-G1 of Paenibacillus polymyxa had the best performance, with 85% inhibition of spore germination during primary screening. In the deep screening, the control effect of XD29-G1 on wheat stripe rust was 60%. The antagonist XD29-G1 promoted the germination of wheat seeds and the growth of wheat seedlings at a solution dilution of 10-7 cfu/mL. The pot experiment results showed that different dilution concentrations of the strain had different levels of antibacterial activity against WSR, with the concentration of 10-1 cfu/mL having the best control effect and a control efficiency of 61.19%. XD29-G1 has better biological control potential against wheat stripe rust.

Characterization of three novel stem rot pathogens and their antagonistic endophytic bacteria associated with Cistanche deserticola.

Cistanche deserticola is a precious Chinese medicinal material with extremely high health care and medicinal value. In recent years, the frequent occurrence of stem rot has led to reduced or even no harvests of C. deserticola. The unstandardized use of farm chemicals in the prevention and control processes has resulted in excessive chemical residues, threatening the fragile desert ecological environment. Therefore, it is urgent to explore safe and efficient prevention and control technologies. Biocontrol agents, with the advantages of safety and environment-friendliness, would be an important idea. The isolation, screening and identification of pathogens and antagonistic endophytic bacteria are always the primary basis. In this study, three novel pathogens causing C. deserticola stem rot were isolated, identified and pathogenicity tested, namely Fusarium solani CPF1, F. proliferatum CPF2, and F. oxysporum CPF3. For the first time, the endophytic bacteria in C. deserticola were isolated and identified, of which 37 strains were obtained. Through dual culture assay, evaluation experiment and tissue culture verification, a biocontrol candidate strain Bacillus atrophaeus CE6 with outstanding control effect on the stem rot was screened out. In the tissue culture system, CE6 showed excellent control effect against F. solani and F. oxysporum, with the control efficacies reaching 97.2% and 95.8%, respectively, indicating its great potential for application in the production. This study is of great significance for the biocontrol of plant stem rot and improvement of the yield and quality of C. deserticola.

Impact of antagonistic endophytic bacteria on productivity of some economically important legumes.

Understanding the interactions within and between endophytes and their hosts is still obscure. Investigating endophytic bacterial plant growth-promoting (PGP) traits and co-inoculation effects on legumes' performance is a candidate. Endophytic bacteria were isolated from Vicia sativa root nodules. Such endophytes were screened for their PGP traits, hydrolytic enzymes, and antifungal activities. Sterilized Vicia faba and Pisum sativum seedlings were co-inoculated separately with seven different endophytic bacterial combinations before being planted under sterilized conditions. Later on, several growth-related traits were measured. Eleven endophytes (six rhizobia, two non-rhizobia, and three actinomycetes) could be isolated, and all of them were indole-acetic-acid (IAA) producers, while seven isolates could solubilize phosphorus, whereas three, five, five, and four isolates could produce protease, cellulase, amylase, and chitinase, respectively. Besides, some of these isolates possessed powerful antifungal abilities against six soil-borne pathogenic fungi. Co-inoculation of tested plants with endophytic bacterial mixes (Rhizobiamix+Actinomix+non-Rhizobiamix), (Rhizobiamix+Actinomix), or (Rhizobiamix+non-Rhizobiamix) significantly improved the studied growth parameters (shoot, root fresh and dry weights, length and yield traits) compared to controls, whereas co-inoculated plants with (Rhizobiaalone), (non-Rhizobiamix), or (Actinomix) significantly recorded lower growth parameters. Five efficient endophytes were identified: Rhizobium leguminosarum bv. Viciae, Rhizobium pusense, Brevibacterium frigoritolerans, Streptomyces variabilis, and Streptomyces tendae. Such results suggested that these isolates could be utilized as biocontrols and biofertilizers to improve legumes productivity. Also, co-inoculation with different endophytic mixes is better than single inoculation, a strategy that should be commercially exploited.

Identification of Gonatophragmium mori Causing Mulberry Zonate Leaf Spot Disease and Characterization of Their Biological Enemies in Guangxi, China.

Mulberry zonate leaf spot disease (MZLSD) is an important fungal disease of mulberry trees, which seriously affects the productivity and quality of mulberry leaves. MZLSD has been widely reported in sericultural production areas in Guangxi, China, in recent years. In this study, the causal agent of MZLSD was isolated from symptomatic samples and identified as Gonatophragmium mori (Acrospermaceae) based on morphological characterization and molecular analyses using nucleotide sequences of the internal transcribed spacer (ITS) and large subunit ribosomal DNA (LSU rDNA). Pathogenicity tests confirmed that G. mori is the pathogen responsible for MZLSD. Furthermore, we isolated antagonistic endophytic bacteria (AEB) from healthy mulberry leaves. Plate confrontation experiments showed that the lipopeptide crude extracts (LPCE) of three endophytic bacteria can inhibit the growth of G. mori, and the diameter of the antibacterial circle reaches more than 60 mm when their concentration of LPCE is 200 mg/ml. Light microscopy and scanning electron microscopy revealed that LPCE caused drastic changes in mycelial morphology. Fluorescence microscopy and transmission electron microscopy showed that the LPCE-induced apoptosis-like cell death in G. mori hyphae. Finally, based on morphological and molecular features, we identified the three isolates as Bacillus subtilis DS07, B. subtilis DS32, and B. velezensis Q6, respectively. To our knowledge, this is the first time to identify G. mori by combining characterization and molecular analyses, and we provide timely information about the use of biocontrol agents for suppression of G. mori.

The effect of NaCl on growth and volatile metabolites produced by antagonistic endophytic bacteria isolated from Prosopis cineraria

The effect of NaCl on growth and volatile metabolites produced by antagonistic endophytic bacteria isolated from Prosopis cineraria

Bacillus spp. isolated from pepper leaves and their function and inhibition of the fungal plant pathogen Colletotrichum scovillei

BackgroundPepper is a popular ingredient in many Chinese households; however, anthracnose caused by Colletotrichum spp. has greatly decreased pepper production. The genus Bacillus is widely known for its important role in the development and protection of plants from phytopathogenic fungi.ResultsFifty-eight endophytic strains were isolated from pepper leaves and tested for antifungal activity in this study. Specifically, L1-7 and L3-5 displayed growth inhibition rates of 79 and 80% against C. scovillei mycelium, respectively, while 25 of these strains all had growth inhibition rates of greater than 60%. Bacillus amyloliquefaciens and B. velezensis, based on culture and morphological identification with 16S rDNA and gyrB gene sequence analyses, were determined to be the respective species L1-7 and L3-5. Additionally, it was discovered that these two antagonistic endophytic bacteria could fix nitrogen, produce indoleacetic acid (IAA) and have a high salt tolerance. Pot experiments again showed excellent control of the pathogen C. scovillei by L1-7 and L3-5, with 80.64 and 73.39% control, respectively. Therefore, B. amyloliquefaciens (L1-7) and B. velezensis (L3-5) can be applied as biological control agents to protect peppers against C. scovillei-caused anthracnose.ConclusionBacillus amyloliquefaciens (L1-7) and B. velezensis (L3-5) can be applied as biological control agents to protect peppers against C. scovillei-caused anthracnose. Thus, they can serve as promising biocontrol agents and plant growth promoters, and future research on the pertinent bacteria will serve as a useful guide for the creation of microbial resources.

Antagonistic Activity and Potential Mechanisms of Endophytic Bacillus subtilis YL13 in Biocontrol of Camellia oleifera Anthracnose

Anthracnose, caused by the fungus Collectotrichum fructicola (C. fructicola), is a major disease affecting the quality and yield of Camellia oleifera (C. oleifera); it reduces C. oleifera yield by 40%–80%. Bacillus subtilis (B. subtilis) YL13 is an antagonistic endophytic bacteria strain isolated from healthy C. oleifera leaves. This study was aimed at investigating the potential of YL13 for the biocontrol of C. oleifera anthracnose and the possible mechanisms involved. In in vitro assays, YL13 demonstrated remarkable antagonistic activity of C. fructicola. Its cell-free filtrates displayed antagonistic activity, which suggested that the metabolites of YL13 might play important roles. In vivo tests showed that the disease index of YL13-treated plants was obviously reduced under greenhouse conditions. YL13 secretes a variety of bioactive metabolites, including protease, cellulase, and siderophore, which might participate in the resistance to C. fructicola. In addition, C. oleifera treated with the fermentation broth of YL13 demonstrated different defense responses, e.g., accumulation of hydrogen peroxide (H2O2) and activation of the defense-related enzyme peroxidase (POD), which might contribute directly or indirectly to overcome external stresses. The significant biocontrol effect and host defense-induction activity of YL13 suggested that this B. subtilis strain as well as its metabolites have the potential to be exploited as microbial control agents for the efficient management of C. oleifera anthracnose.

Interaction networks reveal highly antagonistic endophytic bacteria in native maize seeds from traditional milpa agroecosystems.

Milpas are traditional Mesoamerican agroecosystems maintained with ancestral practices. Maize landraces are grown in polyculture, creating highly productive and diverse ecosystems. Recent studies suggest that milpas maintain beneficial plant-microbe interactions that are probably absent in modern agroecosystems; however, direct comparisons of the microbiome of plants between traditional and modern agroecosystems are still needed. Here, we studied seed-endophytic bacterial communities from native maize landraces from milpas and hybrid varieties. First, we quantified the abundance of culturable endophytic microbes; next, we assessed pairwise antagonistic interaction networks between bacterial isolates; finally, we compared bacterial community structure by 16S rRNA amplicon sequencing. We found that seeds from native maize landraces harbour a higher endophytic microbial load, including more bacterial strains with antagonistic activity against soil-borne bacteria, and overall harbour more diverse bacterial communities than the hybrid varieties. Noteworthy, most of the seed-endophytic strains with antagonistic activity corresponded to Burkholderia spp. that were only found in native maize seeds, through both culture-dependent and independent strategies. Altogether, our results support that crop modernization alters the functions and structure of plant-associated microbes; we propose native maize from milpas could serve as a model for understanding plant-microbe interactions and the effect of modernization.

Antibacterial Activity of Endophytic Bacteria Against Sugar Beet Root Rot Agent by Volatile Organic Compound Production and Induction of Systemic Resistance.

The volatile organic compounds (VOCs) produced by endophytic bacteria have a significant role in the control of phytopathogens. In this research, the VOCs produced by the endophytic bacteria Streptomyces sp. B86, Pantoea sp. Dez632, Pseudomonas sp. Bt851, and Stenotrophomonas sp. Sh622 isolated from healthy sugar beet (Beta vulgaris) and sea beet (Beta maritima) were evaluated for their effects on the virulence traits of Bacillus pumilus Isf19, the causal agent of harvested sugar beet root rot disease. The gas chromatographymass spectrometry (GC-MS) analysis revealed that B86, Dez632, Bt851, and Sh622 produced 15, 28, 30, and 20 VOCs, respectively, with high quality. All antagonistic endophytic bacteria produced VOCs that significantly reduced soft root symptoms and inhibited the growth of B. pumilus Isf19 at different levels. The VOCs produced by endophytic bacteria significantly reduced swarming, swimming, and twitching motility by B. pumilus Isf19, which are important to pathogenicity. Our results revealed that VOCs produced by Sh622 and Bt851 significantly reduced attachment of B. pumilus Isf19 cells to sugar beetroots, and also all endophytic bacteria tested significantly reduced chemotaxis motility of the pathogen toward root extract. The VOCs produced by Dez632 and Bt851 significantly upregulated the expression levels of defense genes related to soft rot resistance. Induction of PR1 and NBS-LRR2 genes in sugar beetroot slices suggests the involvement of SA and JA pathways, respectively, in the induction of resistance against pathogen attack. Based on our results, the antibacterial VOCs produced by endophytic bacteria investigated in this study can reduce soft rot incidence.

Effects of organic acids on the chemotaxis profiles and biocontrol traits of antagonistic bacterial endophytes against root-rot disease in Panax notoginseng.

Understanding the role of chemotaxis in ecological interactions between plants and microbes in the rhizosphere is necessary to optimize biocontrol strategies targeting plant soil-borne diseases. Therefore, we examined and profiled the antagonistic endophytic bacteria (AEB) population with chemotaxis potential in the medicinal plant Panax notoginseng using a cheA gene-based approach coupled with 16S rRNA sequencing. Phylogenetic analysis of the chemotactic AEB (CAEB) community in P. notoginseng enabled the identification of 56 CAEB strains affiliated with 30 species of Actinobacteria, Firmicutes, and Proteobacteria; Firmicutes, especially Bacillus, were predominant. We then systematically quantified the chemotactic response profiles of CAEB toward five organic acid (OA) attractants: citric acid, fumaric acid (FA), malic acid, oxalic acid, and succinic acid. Further hierarchical cluster analysis revealed that the chemotaxis of CAEB to the same attractant exhibited different patterns among not only genera but also species and even strains of the same species. Following chemotaxis and hierarchical analysis, we selected the strongest chemoattractant, fumaric acid (FA), as the target for evaluating the effects of OAs on the representative CAEB strain Bacillus amyloliquefaciens subsp. plantarum YP1. Application of FA significantly stimulated the chemotaxis ability and growth of YP1, and increased the transcript levels of cheA and biocontrol-related genes in YP1. This is the first study to characterise the diversity of chemotaxis profiles toward OAs in natural bacterial assemblages of P. notoginseng and to highlight how FA promotes the biocontrol-related traits of P. notoginseng-associated CAEB.

Characterisation of Plant Growth-Promoting Endophytic Bacteria from Sugarcane and Their Antagonistic Activity against Fusarium moniliforme.

The use of endophytic bacteria in agriculture provides an effective way of improving crop yield and significantly reducing chemical usage, such as fungicides. This research was conducted to explore endophytic bacteria with plant growth promotion (PGP) and antifungal activities against Fusarium moniliforme AIT01. In this study, we obtained 52 isolates of endophytic bacteria associated with the roots and stems of sugarcane from Nakhon Ratchasima province, Thailand. In vitro antagonistic activity test showed that 14 out of 52 isolates had antagonistic activity against the fungal pathogen F. moniliforme AIT01. These antagonistic endophytic bacteria were identified as belonging to six different species as follows: Nguyenibacter vanlangensis, Acidomonas methanolica, Asaia bogorensis, Tanticharoenia aidae, Burkholderia gladioli and Bacillus altitudinis based on phenotypic characteristics, along with phylogenetic analysis of their 16S rRNA gene sequences. Seven isolates effectively inhibited F. moniliforme AIT01 mycelial growth by up to 40%. The volatile compounds of six isolates reduced the growth of F. moniliforme AIT01 by over 23%. Moreover, riceberry rice seedlings previously treated with B. gladioli CP28 were found to strongly reduce infection with phytopathogen by 80% in comparison to the non-treated control. Furthermore, the isolates also showed relevant PGP features, including ammonia production, zinc and phosphate solubilisation, auxin and siderophore biosynthesis. These results demonstrated that the tested endophytic bacteria could be successfully utilised as a source of PGP and biocontrol agent to manage diseases caused by F. moniliforme.

Screening of Endophytic Bacteria as Biocontrol Agents Against Bacteria Leaf Blight (Xanthomonas oryzae)

Bacterial leaf blight (BLB) is one of major threats in rice production as it can cause 100% yield loss. Concern on the environment and human health has led an attempt to replace existing methods of chemical control and avoid extensive use of bactericides by using endophytic bacteria. The present study was conducted to screen and characterize bacteria isolated from different sources that has potential as antagonistic bacteria against Xanthomonas oryzaepv. oryzae (Xoo), the causal agent of bacterial leaf blight of paddy. Two hundred and thirty-three endophytic bacteria were successfully isolated from roots and leaves from paddy field. Only 17 endophytic bacterial isolates showed positive antagonistic activity indicated by inhibition zone around bacterial colony against Xoo on nutrient agar plate with 2 endophytic isolates (BCA 3 and BCA 12) showed highest inhibitory effect with 35±0.00 mm in diameter. Molecular identification by 16S rRNA amplification successfully identified the antagonistic endophytic bacteria as Pseudomonas fluorescensand Geobacillus thermoparaffinivorans. Findings in this study revealed the biocontrol abilities of isolated endophytes as an excellent option to be used by agriculture sectors to have sustainable environment.

Research Article LC-MS based identification of secondary metabolites from marine antagonistic endophytic bacteria

Halophytes occupy coastal and sub-coastal area of marine environment. They potential candidates for search of novel and new bacterial flora that have immense potential to yield novel therapeutic agents. Six different endophytic bacteria have been isolated from pneumatophores and roots of three halophytes (Salsola imbricata, Avicennia marina and Haplopeplis perfoliata) collected from western coastal area of Jeddah, Saudi Arabia. After testing against five fungal pathogens all were active against oomycetes fungal pathogens, Phytophthora capsici and Pythium ultimum. Molecular identification of the bacteria was done on the basis of 16S rRNA gene sequences which revealed 95.9–99.4% sequence identity to related type strains and were placed in four major genera and two major classes: Actinobacteria (Streptomyces and Nocardioides) and α-Proteobacteria (Inquilinus and Labrezia). Active metabolites of these six bacterial endophytes including EA61, EA83, EA85, EA87, EA97 and EA220 were identified by subjecting to chemical analyses using liquid chromatography-mass spectrometry (LC-MS). LC-MS analyses showed presence of different active compounds in the culture extracts of these isolates. Some of these metabolites are already reported as synthetic molecules and has diverse biological functions as antimicrobial, anti-inflammatory and anthelmintic compounds such as such as Sulfamethoxypyridazine, Sulfamonomethoxine, Sulfamerazine and Dimetridazole, Sulfadiazin

Antagonistic endophytic bacteria associated with nodules of soybean (Glycine max L.) and plant growth-promoting properties.

A total of 276 endophytic bacteria were isolated from the root nodules of soybean (Glycine max L.) grown in 14 sites in Henan Province, China. The inhibitory activity of these bacteria against pathogenic fungus Phytophthora sojae 01 was screened in vitro. Six strains with more than 63% inhibitory activities were further characterized through optical epifluorescence microscopic observation, sequencing, and phylogenetic analysis of 16S rRNA gene, potential plant growth-promoting properties analysis, and plant inoculation assay. On the basis of the phylogeny of 16S rRNA genes, the six endophytic antagonists were identified as belonging to five genera: Enterobacter, Acinetobacter, Pseudomonas, Ochrobactrum, and Bacillus. The strain Acinetobacter calcoaceticus DD161 had the strongest inhibitory activity (71.14%) against the P. sojae 01, which caused morphological abnormal changes of fungal mycelia; such changes include fracture, lysis, formation of a protoplast ball at the end of hyphae, and split ends. Except for Ochrobactrum haematophilum DD234, other antagonistic strains showed the capacity to produce siderophore, indole acetic acid, and nitrogen fixation activity. Regression analysis suggested a significant positive correlation between siderophore production and inhibition ratio against P. sojae 01. This study demonstrated that nodule endophytic bacteria are important resources for searching for inhibitors specific to the fungi and for promoting effects for soybean seedlings.

Endophytes from maize with plant growth promotion and biocontrol activity under drought stress

In the present study, 39 endophytic bacteria were isolated from different crops with main focus on maize roots and seeds. Endophytes were screened for drought stress tolerance, plant growth promoting (PGP) traits and antifungal activity. Out of 39 isolates, 32 could show drought tolerance up to–1.02 matric potential (MPa) and exhibited most of the plant growth promoting traits. But, only five isolates could show antagonistic activity against plant fungal pathogens. Based on the results, 10 promising isolates namely FTR, NFTR, FMZR9, FMZR2, MZ30V92, MRC12, MRC31, MRC33, MRC41 and MRR2 were selected and identified using biochemical and 16S rDNA gene sequencing as Pseudomonas aeruginosa (strains FTR and NFTR), Pseudomonas monteilii (strain FMZR2), Pseudomonas putida (strain FMZR9), Acitenobacter brumalii (strain MZ30V92), Enterobacter asburiae (strain MRC12), Sinorhizobium meliloti (strain MRC31), Pseudomonas thivervalensis (strain MRC33), Pseudomonas fulva (strain MRC41), and Pseudomonas lini (strain MRR2). Further, at–1.02 MPa all the 10 isolates showed PGP traits, and 3 isolates (FTR, NFTR and MRC12) showed antifungal activity. Thus, indicating that drought tolerant plant growth promoting antagonistic endophytic bacteria (PGPAE) helps in plant growth and disease management under drought stress.

Screening and inhibition of antagonistic endophytic bacteria isolated from soybean (Glycine max)nodules against Alternaria longipes.

Using Alternaria longipes as tested phytopathogen, endophytic bacteria isolated from soybean nodules were selected to study antagonistic effects by confrontation and metabolic liquid culture methods. The inhibited hyphae were determined by microscopic observation, and the screened strains were characterized by cell culture, physiological and biochemical tests, 16S rDNA sequencing, phylogenetic analysis and inoculation trials in greenhouse. The results indicated that the seven of the endophytes exerted more than 42% inhibitory effects after the first and the second screening. These strains belonged to genus Bacillus, Pseudomonas, Sinorhizobium and Stenotrophomonas, respectively. Microscopic observation showed that the affected hyphae ends of A. longipes appear deformity of coralline branch, spherical expansions and so on. Antagonistic experiments with metabolites showed that the inhibition of endophytic bacteria against pathogenic fungus played an effective role mainly by bacteria producing extracellular substances. Confrontation tests suggested that endophytic Bacillus rapidly produced biofilm to effectively hinder the growth and extension of pathogen hyphae. Inoculation experiments showed that the disease index of treatment group was significantly lowered compared with the control, suggesting it could be utilized as a biological control resource inhibiting tobacco brown spot.

English

The endophytic bacterial communities in tomato varieties having differing resistance (or susceptibility) to Ralstonia solanacearum were investigated using both cultivation dependent and independent approaches. Both approaches revealed the differences between resistant (Xiahong-1) and susceptible (Baoshi-5) cultivars in terms of diversity and abundance of endophytic bacteria. The amount of the endogenous bacteria in Xiahong-1 at different growth stages was significantly higher than that in Baoshi-5. Furthermore, there were more culturable and antagonistic endophytic bacteria in Xiahong-1 than that in Baoshi-5. Seven endophytic bacterial genetic groups were identified in Xiahong-1 by polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and 16S rDNA sequence, and they were highly similar to Sphingomonas yanoikuya, Pseudomonas pseudoalcaligenes, Serratia marcescens, Bacillus megaterium, Paenibacillus polymyxa, B. pumilus and B. cereus. Four groups were identified in Baoshi-1 which were highly similar to S. yanoikuyae, Pseudomonasfluorescens, Arthrobacter globiformis and Paenibacillus polymyxa. In addition, antagonistic endophytes were identified by 16S rRNA gene analysis, and tested for their abilities to protect tomato plants from infection with R. solanacearum. The relationships between plant resistance and endophytic bacteria diversity are discussed. Key words: Tomato, Ralstonia solanacearum, resistance, endophytic bacteria, diversity,biological control, 16S rRNA gene.

Combining antagonistic endophytic bacteria in different growth stages of cotton for control of Verticillium wilt

Verticillium wilt of cotton is a major limiting factor in cotton production. To investigate the combined control effects of endophytic bacteria in different growth stages of cotton on Verticillium wilt, antagonistic bacteria Paenibacillus xylanilyticus YUPP-1, Paenibacillus polymyxa YUPP-8 and Bacillus subtilis YUPP-2 were isolated from cotton in the seedling, squaring and boll-setting stages, respectively. Assessment of colonization showed that strains YUPP-1, YUPP-8 and YUPP-2 had the highest populations in the seedling, squaring and boll-setting period, respectively. Indoor pot experiment indicated that cotton plants in the combined biocontrol bacteria treatment group were not infected before squaring stage, while infection rates of cotton treated with a single strain in the seedling period were 6.7% (strain YUPP-8), 6.7% (strain YUPP-1) and 13.3% (strain YUPP-2). However, infection rate of the control plants reached 80%. Field experiment conducted during 2010–2011 showed that the combined application of three strains had better effect than individual application. Verticillium wilt morbidity rate and disease index of cotton at the boll-setting period with combined application of three strains in 2010 were 9.4% and 6.5, respectively, while those in the control group were 47.5% and 32.8. The results in 2011 were similar to 2010, with higher disease severity in both treated and control plants. These results illustrated that combined application of endophytic bacteria can achieve good control effects against Verticillium wilt of cotton. This study provides a new strategy for the control of soil-borne diseases.

Biocontrol of verticillium wilt and colonization of cotton plants by an endophytic bacterial isolate

To explore biocontrol potential of 39 DAEB isolates (doubly antagonistic towards both Verticillium dahliae Kleb and Fusarium oxysporum) against verticillium wilt of cotton and to elucidate colonization and category characteristics of an endophytic bacterium with significant biocontrol activity. Thirty-nine antagonistic endophytic bacteria strains were tested for their ability to control verticillium wilt in cotton plants caused by a defoliating pathotype of V. dahliae 107 in cotton under controlled conditions. The biocontrol trial revealed that an endophytic bacterium, designated HA02, showed a significant biocontrol activity to V. dahliae 107. After cotton seedlings were inoculated with a gfp gene-tagged HA02 (HA02-gfp), HA02-gfp populations were higher in the root than in the stem; in addition, the HA02-gfp was distributed in the maturation zone of cotton root. Furthermore, HA02-gfp also colonized seedlings of maize, rape and soybean after the bacteria inoculation. Phylogenetic trees based on 16S rDNA sequences combined with morphological, physiological and identification showed that the bacterium belongs to the Enterobacter genus. Our results showed that only 1 of 39 DAEB isolates demonstrated more efficient biocontrol potential towards V. dahliae 107 in greenhouse and field trials. HA02-gfp mainly colonized cotton in roots. In addition, we quantitatively observed HA02 colonization in other hosts. HA02 belongs to the Enterobacter genus. This is the first study on biocontrol to defoliating pathotype of V. dahliae Kleb by endophytic bacteria. The HA02 showed effective biocontrol to V. dahliae 107 in greenhouse and field trials. Furthermore, we assessed the quantitative and qualitative colonization of HA02 in cotton seedlings. Our study provides basic information to further explore managing strategies to control this critical disease.

Diversity and Characterization of Endophytic Bacteria Associated with Tidal Flat Plants and their Antagonistic Effects on Oomycetous Plant Pathogens

Endophytic bacterial communities of tidal flat plants antagonistic to oomycete plant pathogens were studied by the isolation of 256 root colonizing endophytic bacteria from surface-disinfected root tissues of six plants (Rosa rugosa, Suaeda maritima, Vitex rotundifolia, Carex scabrifolia, Glehnia littoralis and Elymus mollis) growing in a tidal flat area of Namhae Island, Korea. To understand the antagonistic potential, an in vitro antagonistic assay was performed to characterize and identify strains that were antagonistic to the oomycete plant pathogens Phytophthora capsici and Pythium ultimum from the total population. Nine percent of the total number of isolated bacteria exhibited in vitro inhibitory activity against target plant pathogenic oomycetes. Taxonomic and phylogenetic placement of the antagonistic bacteria was investigated by analysis of the 16S rRNA gene sequences. The sequence analysis classified the antagonistic strains into four major classes of the domain bacteria (Firmicutes, α-Proteobacteria, γ-Proteobacteria and Actinomycetes) and 10 different genera. Further production of secondary metabolites, hydrolytic enzymes and plant growth promoting traits were determined for the putative new species of antagonistic endophytic bacteria. These new strains could not be identified as known species of α-Proteobacteria, and so may represent novel bacterial taxa. The unexpected high antagonistic bacterial diversity associated with the tidal flat plants may be indicative of their importance in tidal flat plants as a promising source of novel antimicrobial compounds and biocontrol agents.