A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.     

Identification of Bacillus Strains for Biological Control of Catfish Pathogens

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10⁷ CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.     

Symbiont Interactions in a Tripartite Mutualism: Exploring the Presence and Impact of Antagonism between Two Fungus-Growing Ant Mutualists

Mutualistic associations are shaped by the interplay of cooperation and conflict among the partners involved, and it is becoming increasingly clear that within many mutualisms multiple partners simultaneously engage in beneficial interactions. Consequently, a more complete understanding of the dynamics within multipartite mutualism communities is essential for understanding the origin, specificity, and stability of mutualisms. Fungus-growing ants cultivate fungi for food and maintain antibiotic-producing Pseudonocardia actinobacteria on their cuticle that help defend the cultivar fungus from specialized parasites. Within both ant-fungus and ant-bacterium mutualisms, mixing of genetically distinct strains can lead to antagonistic interactions (i.e., competitive conflict), which may prevent the ants from rearing multiple strains of either of the mutualistic symbionts within individual colonies. The success of different ant-cultivar-bacterium combinations could ultimately be governed by antagonistic interactions between the two mutualists, either as inhibition of the cultivar by Pseudonocardia or vice versa. Here we explore cultivar-Pseudonocardia antagonism by evaluating in vitro interactions between strains of the two mutualists, and find frequent antagonistic interactions both from cultivars towards Pseudonocardia and vice versa. To test whether such in vitro antagonistic interactions affect ant colonies in vivo, we performed sub-colony experiments using species of Acromyrmex leaf-cutting ants. We created novel ant-fungus-bacterium pairings in which there was antagonism from one, both, or neither of the ants'' microbial mutualists, and evaluated the effect of directional antagonism on cultivar biomass and Pseudonocardia abundance on the cuticle of workers within sub-colonies. Despite the presence of frequent in vitro growth suppression between cultivars and Pseudonocardia, antagonism from Pseudonocardia towards the cultivar did not reduce sub-colony fungus garden biomass, nor did cultivar antagonism towards Pseudonocardia reduce bacteria abundance on the cuticle of sub-colony workers. Our findings suggest that inter-mutualist antagonism does not limit what combinations of cultivar and Pseudonocardia strains Acromyrmex fungus-growing ants can maintain within nests.     

Effect of the combination of bio-organic fertiliser with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease,crop production and banana rhizosphere culturable microflora

Soil amended with organic amendments has been suggested to be a strategy for managing the Fusarium wilt disease which severely hindered the banana production. The effects of four fertilisation regimes, including chemical fertiliser, manure composts and bio-organic fertiliser (BIO) containing Bacillus amyloliquefaciens NJN-6 for 2-year continuous application on the banana Fusarium wilt disease incidence, crop yield and rhizosphere culturable microbial community were investigated. To explore the soil microflora, plate counting method, in vitro screening method for antagonism, eco-physiological index and culture-dependent denaturing gradient gel electrophoresis method (CD DGGE) were used. The highest banana yield, culturable bacteria, actinobacteria and Bacillus populations, culturable bacteria to fungi (B/F) value, antagonistic Bacillus ratio and lowest Fusarium wilt disease incidence were observed in the BIO treatment. Based on CD DGGE results, the BIO application significantly altered the soil bacteria structure and showed highest richness and diversity. The phylogenetic analysis of the selected bands showed that the most abundant phyla were Proteobacteria and Bacteroidetes and BIO application enriched the genera Comamonas, Chitinophaga, the species Bacillus flexus and uncultured Bacillus. All the results showed that 2-year continuous application of BIO containing B. amyloliquefaciens NJN-6 more effectively controlled Fusarium wilt disease and improved fruit yields under field conditions and modulated banana rhizosphere microflora.     

Isolation and characterization of plant growth promoting endophytic diazotrophic bacteria from Korean rice cultivars

We have isolated 576 endophytic bacteria from the leaves, stems, and roots of 10 rice cultivars and identified 12 of them as diazotrophic bacteria using a specific primer set of nif gene. Through 16S rDNA sequence analysis, nifH genes were confirmed in the two species of Penibacillus, three species of Microbacterium, three Bacillus species, and four species of Klebsiella. Rice seeds treated with these plant growth-promoting bacteria (PGPB) showed improved plant growth, increased height and dry weight and antagonistic effects against fungal pathogens. In addition, auxin and siderophore producing ability, and phosphate solubilizing activity were studied for the possible mechanisms of plant growth promotion. Among 12 isolates tested, 10 strains have shown higher auxin producing activity, 6 isolates were confirmed as strains with high siderophore producing activity while 4 isolates turned out to have high phosphate-solubilizing activity. These results strongly suggest that the endophytic diazotrophic bacteria characterized in this study could be successfully used to promote plant growth and inducing fungal resistance in plants.     

Biocontrol bacteria selected by a direct plant protection strategy against avocado white root rot show antagonism as a prevalent trait

Aim: This study was undertaken to study bacterial strains obtained directly for their efficient direct control of the avocado white root rot, thus avoiding prescreening by any other possible mechanism of biocontrol which could bias the selection. Methods and Results: A collection of 330 bacterial isolates was obtained from the roots and soil of healthy avocado trees. One hundred and forty‐three representative bacterial isolates were tested in an avocado/Rosellinia test system, resulting in 22 presumptive protective strains, all of them identified mainly as Pseudomonas and Bacillus species. These 22 candidate strains were screened in a more accurate biocontrol trial, confirming protection of some strains (4 out of the 22). Analyses of the potential bacterial traits involved in the biocontrol activity suggest that different traits could act jointly in the final biocontrol response, but any of these traits were neither sufficient nor generalized for all the active bacteria. All the protective strains selected were antagonistic against some fungal root pathogens. Conclusions: Diverse bacteria with biocontrol activity could be obtained by a direct plant protection strategy of selection. All the biocontrol strains finally selected in this work were antagonistic, showing that antagonism is a prevalent trait in the biocontrol bacteria selected by a direct plant protection strategy. Significance and Impact of the Study: This is the first report on the isolation of biocontrol bacterial strains using direct plant protection strategy in the system avocado/Rosellinia. Characterization of selected biocontrol bacterial strains obtained by a direct plant protection strategy showed that antagonism is a prevalent trait in the selected strains in this experimental system. This suggests that antagonism could be used as useful strategy to select biocontrol strains.     

Diversity and antifungal activity of endophytic bacteria associated with <Emphasis Type="Italic">Panax ginseng</Emphasis> seedlings

Ginseng (Panax ginseng C.A. Meyer) is a medicinal crop that requires a long culture time before it is ready to harvest, thus generating high economic and environmental costs. Symbiotic bacteria that live within the plant provide the host plant with many advantages in terms of metabolism and disease resistance. Here, we isolated endophytic bacteria from various tissues of P. ginseng seedlings using a culture-dependent method and we compared their tissue distribution. In addition, their antimicrobial activity against two fungal pathogens was investigated. Based on 16S rRNA sequencing, we identified 21 bacterial strains from ginseng seedlings. Leaves and rhizomes showed higher bacterial species diversity than root bodies and tails. While Bacillus strains were detected in all tissues, Xanthomonas and Micrococcaceae strains were specifically isolated from rhizome and leaf tissues, respectively. Fourteen bacterial strains showed antimicrobial activities against Cylindrocarpon destructans and/or Botrytis cinerea, with different activities. Among them, two strains (PgKB29 and PgKB35) showed strong antimicrobial activities against both fungi. Taken together, these results provide a better understanding of endophytic bacteria in P. ginseng seedlings and suggest the possibility of biological control of fungal pathogens using endophytic bacteria.     

Phylogenetic diversity of bacterial endophytes of Panax notoginseng with antagonistic characteristics towards pathogens of root-rot disease complex

Endophytes play an important role in protection of host plants from infection by phytopathogens. Endophytic bacteria were isolated from five different parts (root, stem, petiole, leaf and seed) of Panax notoginseng and evaluated for antagonistic activity against Fusarium oxysporum, Ralstonia sp. and Meloidogyne hapla, three major pathogens associated with root-rot disease complex of P. notoginseng. From 1000 endophytic bacterial strains evaluated in vitro, 104 strains exhibited antagonistic properties against at least one of these three pathogens. Phylogenetic analyses of their 16S rRNA gene sequences showed that these 104 antagonistic bacteria belong to four clusters: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. Members of the Firmicutes, in particular the Bacillus spp., were predominant in all analyzed tissues. The root was the main reservoir for antagonistic bacteria. Of the 104 antagonists, 51 strains showed antagonistic activities to one pathogen only, while 43 and 10 displayed the activities towards two and all three pathogens, respectively. The most dominant species in all tissues were Bacillus amyloliquefaciens subsp. plantarum and Bacillus methylotrophicus, which were represented by eight strains with broad antagonistic spectrum to the all three test pathogens of root-rot disease complex of P. notoginseng.     

Isolation and Characterization of Antagonistic Bacillus Strains Capable to Degrade Ethylenethiourea

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.     

Effect of different west african species and strains ofArthrobotrys nematophagous fungi onMeloidogyne species

Different species ofArthrobotrys nematophagous fungi and several strains ofA. oligospora have been studied for their antagonistic effects against nematodes of the genusMeloidogyne, important pests of vegetables. All fungi trappedM. mayaguensis andM. incognita juvenilesin vitro but had no effect on the juveniles ofM. javanica. In pot experiments withM. mayaguensis, all fungi reduced the nematode populations and stimulated the growth of tomato seedlings. In a field trial, a strain of A.oligospora, isolated in Senegal and incorporated into compost blocks, was efficient in increasing the tomato seedling growth. The introduction of nematophagous fungi in compost blocks as a biological biocontrol technique against phytophagous nematodes adapted for developing countries is discussed.     

Genome shuffling enhances biocontrol abilities of Streptomyces strains against two potato pathogens

Aims: To employ the genome shuffling technique for improving the phenotype of a biocontrol control agent of the genus Streptomyces. Methods and Results: Two rounds of genome shuffling (GS) were carried out with Streptomyces melanosporofaciens EF‐76, a geldanamycin producer. Six fusants that showed optimized in vitro antagonistic activity against Streptomyces scabies or Phytophthora infestans, two important pathogens of potato crops, were selected. All selected fusants retained the capacity to produce geldanamycin, but none overproduced this antibiotic. The higher antagonism ability appeared to result from a diversification of secreted metabolites. Seven or eight metabolites were detected in the HPLC profiles of parental strains, whereas 12–15 were detected in fusant strains. Biocontrol assays revealed that four of six fusants protected tubers more efficiently than parental strains. Conclusions: GS emerged as an elegant and rapid tool to optimize the antagonistic ability of Streptomyces strains. Optimization of the in vitro antagonistic activity against plant pathogens appears to be an effective approach to select for improved biocontrol agents. The enhanced phenotype did not depend on an overproduction of a specific antibiotic but rather on the secretion of a wider variety of secondary metabolites. Significance and Impact of the Study: Improved capacities of a biocontrol agent compensate for the lack of efficient chemical control of potato scab.     

Isolation and characterization of Bacillus sp. strain BC01 from soil displaying potent antagonistic activity against plant and fish pathogenic fungi and bacteria

Fungal and bacterial pathogens infect a diverse range of hosts including various plant and animal species. Fungal and bacterial diseases, especially of plants and aquatic animals, such as fish, lead to significant damage to crops and aquaculture, respectively, worldwide. The present study was conducted to isolate and characterize potent Bacillus strains with significant antagonistic activity against the major plant and fish pathogenic fungi and bacteria. We randomly collected 22 isolates of Bacillus from the soil, rhizosphere, and sediment from different parts of Bangladesh. Initial characterization, based on in vitro antagonistic activity on the culture plate, resulted in the selection of four gram-positive Bacillus sp. isolates. Among these, the isolate BC01, obtained from soil demonstrated the highest broad-spectrum anti-bacterial and anti-fungal activities. We confirmed the genus of BC01 to be Bacillus by morphological and biochemical tests as well as using molecular data analysis tools, including the study of 16s rDNA, phylogenetic relationship, and evolutionary divergence scores. The isolate significantly inhibited the mycelial growth of the plant pathogen, Penicillium digitatum and fish pathogen, Aphanomyces invadans in vitro. The anti-bacterial effect of the isolate was also evaluated against Pseudomonas spp. and Xanthomonas spp., the two deadliest plant pathogens, and Aeromonas veronii, Pseudomonas fluorescens, and Streptococcus iniae, three major fish pathogens that are primarily responsible for global aquaculture loss. The results of the present study could pave the way for developing potent drugs to combat microbial infection of plants and fish.     

Antagonistic bacteria of composted agro-industrial residues exhibit antibiosis against soil-borne fungal plant pathogens and protection of tomato plants from Fusarium oxysporum f.sp. radicis-lycopersici

Rhizospheric and root-associated/endophytic (RAE) bacteria were isolated from tomato plants grown in three suppressive compost-based plant growth media derived from the olive mill, winery and Agaricus bisporus production agro-industries. Forty-four (35 rhizospheric and 9 RAE) out of 329 bacterial strains showed in vitro antagonistic activity against at least one of the soil-borne fungal pathogens, Fusarium oxysporum f.sp. radicis-lycopersici (FORL), F. oxysporum f.sp. raphani, Phytophthora cinnamomi, P. nicotianae and Rhizoctonia solani. The high percentage of total isolates showing antagonistic properties (13%) and their common chitinase and β-glucanase activities indicate that the cell wall constituents of yeasts and macrofungi that proliferate in these compost media may have become a substrate that favours the establishment of antagonistic bacteria to soil-borne fungal pathogens. The selected bacterial strains were further evaluated for their suppressiveness to tomato crown and root rot disease caused by FORL. A total of six rhizospheric isolates, related to known members of the genera Bacillus, Lysinibacillus, Enterobacter and Serratia and one RAE associated with Alcaligenes faecalis subsp. were selected, showing statistically significant decrease of plant disease incidence. Inhibitory effects of extracellular products of the most effective rhizospheric biocontrol agent, Enterobacter sp. AR1.22, but not of the RAE Alcaligenes sp. AE1.16 were observed on the growth pattern of FORL. Furthermore, application of cell-free culture extracts, produced by Enterobacter sp. AR1.22, to tomato roots led to plant protection against FORL, indicating a mode of biological control action through antibiosis.     

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria.In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism.Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied.The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.     

Evaluation of indigenous bacterial strains for biocontrol of the frogeye leaf spot of soya bean caused by Cercospora sojina

Aims: Assessment of biological control of Cercospora sojina, causal agent of frogeye leaf spot (FLS) of soya bean, using three indigenous bacterial strains, BNM297 (Pseudomonas fluorescens), BNM340 and BNM122 (Bacillus amyloliquefaciens). Methods and Results: From cultures of each bacterial strain, cell suspensions and cell‐free supernatants were obtained and assayed to determine their antifungal activity against C. sojina. Both mycelial growth and spore germination in vitro were more strongly inhibited by bacterial cell suspensions than by cell‐free supernatants. The Bacillus strains BNM122 and BNM340 inhibited the fungal growth to a similar degree (I～52–53%), while cells from P. fluorescens BNM297 caused a lesser reduction (I～32–34%) in the fungus colony diameter. The foliar application of the two Bacillus strains on soya bean seedlings, under greenhouse conditions, significantly reduced the disease severity with respect to control soya bean seedlings and those sprayed with BNM297. This last bacterial strain was not effective in controlling FLS in vivo. Conclusions: Our data demonstrate that the application of antagonistic bacteria may be a promising and environmentally friendly alternative to control the FLS of soya bean. Significance and Impact of the Study: To our knowledge, this is the first report of biological control of C. sojina by using native Bacillus strains.     

双歧醋对常见病原菌的生物拮抗作用研究

目的研究双歧醋对常见病原菌的生物拮抗作用及影响因素。方法采用纸片扩散法(K-B法)、琼脂打孔法和试管稀释法(M IC法)测定双歧醋及其处理物(加热、不同pH)对常见病原菌的拮抗作用,并讨论pH、加热对拮抗作用的影响,同时用某市售醋作为对照。结果双歧醋及其处理物均具有一定的抑菌作用。结论双歧醋对常见病原菌有一定的拮抗作用,抑菌作用强于市售醋。     

Enhancing compost quality by using whey-grown biosurfactant-producing bacteria as inocula

Wild screening of bacterial strains from compost materials was performed. Several biosurfactant-producer strains were isolated and then cultured in whey as a low-cost medium for biosurfactant production. Two strains, identified as Bacillus sp. and Streptomyces sp., were the best biosurfactant producers and were selected for determination of compost quality enhancing. The effect of cell biomass, cell-free supernatant, and a consortium of these two strains on compost quality were determined and specific parameters of compost were analyzed. The results showed that using these bacteria (or supernatants) in compost processing have slight stimulatory effect on bacterial population (8.08 log₁₀ CFU/g), surface tension reduction (to 42.6 mN/m at 24 h), and heavy metal bioremediation (>50 % in most treatments), speeding up the decomposition rate of organic matter (42.3 % OM at the end of experiment), accelerating the stabilization process by reduction of NH ₄ ⁺ to NO ₃ ^? ratio (reduced from 0.2 to 0.026), decreasing the biotoxicity (tested by seed germination and root length of germinated seed), and also reduction of pathogens (reduced from 2100 to 120 MPN/g in fecal coliform).     

神农架林区和自然保护区链霉菌拮抗性的研究(Ⅱ)

用琼脂移块法对由神农架林区和自然保护区土壤中分离并鉴定了的３６种５７株链霉菌进行拮抗性试验,结果显示３６种５５株链霉菌对细菌或真菌有拮抗性,或对细菌和真菌都有拮抗性,并发现这些链霉菌以较高的百分率拮抗丝状真菌和酵母菌。     

大连海域沉积物中可培养海洋链霉菌活性及其多样性

以大肠杆菌、金黄色葡萄球菌和尖孢镰刀枯萎病菌作为测试靶目标,采用9种分离培养基从大连海域13个不同采样点的海洋沉积物样品中分离到165株海洋链霉菌。从165株海洋放线菌中筛选到对金黄色葡萄球菌具有抑制活性的菌株85株,占总菌株数的51.5%;对大肠杆菌具有抑制活性的菌株27株,占总菌株数的16.4%;对尖孢镰刀枯萎病菌具有抑制活性的菌株仅有6株,占总菌株数的3.6%。因此,海洋链霉菌的活性更多地表现为对细菌的抗性,尤其对革兰氏阳性细菌具有更高的抑制活性。对其中具有抑制活性或形态独特的菌株进行了16S r DNA序列分析,并构建系统发育树,显示活性海洋链霉菌具有丰富的种类多样性和广谱抗菌活性。同种海洋链霉菌与土壤链霉菌活性比较结果也表明,海洋链霉菌多表现抗革兰氏阳性细菌活性。