Biocontrol of Late Blight Disease (Phytophthora capsici) of Pepper and the Plant Growth Promotion by Paenibacillus ehimensis KWN38

For field application of a bacterial strain used to control Phythophthora capsici, we will need a biologically and economically efficient carrier medium. The known antagonist Paenibacillus ehimensisKWN38 was grown in a grass medium where it showed high antifungal and lytic enzyme activities. To demonstrate the potential of P. ehimensisKWN38 for biocontrol of late blight disease in pepper, pot trials were conducted by treating the 1‐month‐old plants with water (W), a selected grass medium (G3), G plus P. ehimensisKWN38 inoculation (G3P) or synthetic fungicide (F). The shoot dry weight in G3P was higher than that in W and F treatments at 15 days after zoospore infection (DZI). The root dry weight in G3P was also higher than that in W. The root mortality of G3 and W increased over 58 and 80% at 15 DZI, and some plants in those treatments wilted due to the failure of root physiology. The plants in G3P and F survived well because of their better root health conditions. Soil cellulase activity of G3P was consistently higher than that of W and F at earlier observation times (0, 2 and 6 DZI). The root β‐1,3‐glucanase activity of G3P promptly increased to maximum shortly after zoospore infection and reached the maximum value of 51.12 unit g^?1 of fresh weight at 2 DZI. All these results indicate that inoculation of P. ehimensisKWN38 to the root zone of potted pepper plants increases plant growth, root and soil enzyme activities and alleviates the root death caused by infection with P. capsici zoospores.     

Production of volatile organic compounds by an antagonistic strain Paenibacillus polymyxa WR-2 in the presence of root exudates and organic fertilizer and their antifungal activity against Fusarium oxysporum f. sp. niveum

The volatile organic compounds (VOCs) produced by antagonistic microbes have great antifungal potential against soil-borne fungal pathogens. The VOCs produced by Paenibacillus polymyxa strain WR-2 in the presence of root exudates and organic fertilizer were identified and their effects on the growth and spore germination of Fusarium oxysporum f. sp. niveum were evaluated. The VOCs produced by WR-2 inhibited the growth of F. oxysporum by 38%, 36% and 40% in agar medium, sterilized soil and natural soil, respectively. This inhibitory effect was increased to 60%, 58% and 64% with the addition of organic fertilizer in agar medium, sterilized soil and natural soil, respectively. The addition of root exudates did not affect the production of antifungal VOCs by WR-2. The VOCs produced by WR-2 completely inhibited the germination of F. oxysporum spores. Out of 42 identified VOCs, seven VOCs; benzothiazole, benzaldehyde, undecanal, dodecanal, hexadecanal, 2-tridecanone and phenol were found to inhibit the growth of F. oxysporum. The results of these experiments suggest another significance of using organic fertilizer as a carrier material with the biocontrol agents to control soil-borne fungal pathogens.     

Biocontrol of Meloidogyne incognita inciting disease in tomato by using a mixed compost inoculated with Paenibacillus ehimensis RS820

Meloidogyne spp. causes root-knot disease in tomato plants. Biological control of the disease may present economically feasible, agronomically durable and environmentally safe alternative of nematicides. A chitinolytic bacterial strain, Paenibacillus ehimensis RS820, previously isolated from the soil in Korea, produced lytic enzymes in higher amounts and inhibited the growth of phytopathogenic root-knot nematodes. Moreover, the juveniles and eggs of root-knot nematodes induced secretion of lytic enzymes by RS820 including chitinases, gelatinases and collagenases. Furthermore, mixed compost containing increased amounts of chitin and inoculated with RS820 was prepared in the present study. Use of the mixed compost not only reduced the disease caused by root-knot nematodes but also improved the plant growth. The extent of inoculation of the mixed compost with RS820 significantly influenced its ability to control the root-knot disease in tomato. The mixed compost also significantly altered the activity and density of the rhizosphere bacteria. Chitinase and gelatinase producing soil bacteria, as well as their enzyme activities, were significantly influenced by the mixed compost. The mixed compost proposed in the present study may represent a viable alternative to nematicides against the root-knot nematodes in tomato.     

Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177

Summary This study evaluates the potential of Paenibacillus brasilensis strain PB177 to inhibit phytopathogenic fungi commonly causing maize diseases and to colonize maize plants. In vitro assays demonstrated antagonistic activity against the fungal pathogens, Fusarium moniliforme and Diplodia macrospora. The PB177 strain was tagged with the gfp gene, encoding the green fluorescent protein (GFP) and GFP-tagged bacteria were detected attached to maize roots by stereo- and confocal microscopy. The GFP-tagged bacteria were also used to treat maize seeds before challenging the seeds with two phytopathogenic fungi. The results demonstrated that the bacterial cells are mobilized to the maize roots in the presence of the fungal pathogens. The ability of P. brasilensis PB177 to inhibit fungal growth in vitro and its capability of colonization of maize roots in vivo suggest a potential application of this strain as a biological control agent. This is the first report on the successful introduction of the GFP marker gene into a P. brasilensis strain, enabling the direct observation of these promising plant growth promoting bacteria on maize roots in situ.     

Spores of Paenibacillus larvae,Ascosphaera apis,Nosema ceranae and Nosema apis in bee products supervised by the Brazilian Federal Inspection Service

Due to their ecological and economic importance, honey bees have attracted much scientific attention, which has intensified due to the recent population decline of these insects in the several parts of the world. Among the factors related to these patterns, infection by pathogens are the most relevant, mainly because of the easy dissemination of these microorganisms. Although no zoonotic diseases are associated with these insects, the presence of infectious agents in bee products should still be considered because they play a role as disease dispersers, increasing the risk to animal health. Because of the possibility of dispersion of pathogens via bee products, this work aimed to identify the presence of spores of the pathogens Paenibacillus larvae, Ascosphaera apis and Nosema spp. in samples of honey, pollen and royal jelly that are registered with Brazil's Federal Inspection Service (S.I.F.) and commercially available in the state of São Paulo. Of the 41 samples of bee products analyzed, only one showed no contamination by any of these pathogens. N. ceranae and P. larvae had the highest prevalence considering all the samples analyzed (present in 87.80% and 85.37% of the total, respectively), with N. apis present in 26.83% and A. apis present in 73.17% of the samples. These results provide support for the formulation of government regulations for sanitary control of exotic diseases by preventing dispersion of pathogens, including through illegal importation, since local and international trade and the transfer of colonies between regions play important roles in the dispersion of these microorganisms.     

The study of mycolytic properties of aerobic spore-forming bacteria producing extracellular chitinases

The mycolytic activity of 27 strains of antagonistic bacilli belonging to two taxonomic groups (18 strains of Bacillus subtilis and 9 strains of Paenibacillus ehimensis) capable of induced synthesis of chitinolytic enzymes was studied. Most of the B. subtilis strains neither displayed visible mycolytic effects on the phytopathogenic fungus Bipolaris sorokiniana in vitro, nor produced chitinases in the presence of an autoclaved mycelium. On the contrary, P. ehimensis strains grown under conditions favorable for induction of chitinases and other hydrolases exhibited a pronounced lytic effect on B. sorokiniana and actively grew by utilizing mycelium as the sole source of carbon and nitrogen. Comparison of the mycolytic activities of extracellular hydrolases in the studied strains demonstrated low correlation between chitinase production and the ability of the strains to degrade the cell walls of B. sorokiniana. Characterization of enzyme profiles in the studied strains revealed that β-1,3-glucanase was a more significant factor than chitinase for determining the mycolytic potential of bacteria and their ability to utilize the mycelium of phytopathogenic fungi as a growth substrate.     

The prevalence of the honeybee brood pathogens Ascosphaera apis,Paenibacillus larvae and Melissococcus plutonius in Spanish apiaries determined with a new multiplex PCR assay

The microorganisms Ascosphaera apis, Paenibacillus larvae and Melissococcus plutonius are the three most important pathogens that affect honeybee brood. The aim of the present study was to evaluate the prevalence of these pathogens in honeybee colonies and to elucidate their role in the honeybee colony losses in Spain. In order to get it, a multiplex polymerase chain reaction (PCR) assay was developed to simultaneously amplify the16S ribosomal ribonucleic acid (rRNA) gene of P. larvae and M. plutonius, and the 5.8S rRNA gene of A. apis. The multiplex PCR assay provides a quick and specific tool that successfully detected the three infectious pathogens (P. larvae, M. plutonius and A. apis) in brood and adult honeybee samples without the need for microbiological culture. This technique was then used to evaluate the prevalence of these pathogens in Spanish honeybee colonies in 2006 and 2007, revealing our results a low prevalence of these pathogens in most of the geographic areas studied.     

Study on an antimicrobial protein produced by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> JSa-9 isolated from soil

Paenibacillus polymyxa strain JSa-9, a soil isolate showed in vitro inhibitory effects on several pathogens. A protein of about 71.9 kDa, isolated from a culture of JSa-9, exhibited broad-spectrum antibacterial and antifungal activities. The purification procedure consisted of ion-exchange chromatography on DEAE-Sephacel and Sephadex G-100 column chromatography. The purified protein was characterized to be a glycoprotein, which contained 53% of amino acids and 28% of carbohydrate. Its N-terminal sequence was determined as KCATIPVVIKHL and the amino acid composition showed no significant homology with any known antagonistic proteins from P. polymyxa. Because of the excellent antimicrobial activity, this protein may be a good prospect for food preservation and plant disease control.     

Potential Role of Pathogen Signaling in Multitrophic Plant-Microbe Interactions Involved in Disease Protection

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.     

Fungal microbiota isolated from native stingless bee species inhibited pathogens of Apis mellifera

Social bees can establish interactions with microorganisms to keep their colonies free of pathogens and parasites by developing different protection strategies. We explored the fungal microbiota isolated from three species of stingless bees, Tetragonisca fiebrigi, Plebeias sp., and Scaptotrigona jujuyensis, and its potential ability to suppress pathogenic microorganisms of A. mellifera, namely Paenibacillus larvae, Ascosphaera apis and Aspergillus flavus, which were tested and evaluated. Six filamentous fungal strains, Trametes hirsuta, Alternaria alternata, Curvularia spicifera, Skeletocutis sp., Alternaria tenuissima, Monascus spp., as well as the yeast Wickerhamomyces anomalus, were selected for trials and isolated from the heads of foraging bees. The fungal strains were identified by macroscopic and microscopic taxonomic characteristics and by sequencing of the ITS1–5.8S–ITS2 region of ribosomal DNA. All fungal strains inhibited these pathogens of A. mellifera. We also evaluated the effect of the secondary metabolites extracted with and without ethanol. Both metabolites showed antimicrobial properties, and our results suggest that fungi isolated from stingless bees produce bioactive compounds with antibacterial and antifungal effects that could be used to treat Apis mellifera colony diseases and maintain colony health.     

Improvement of the Fungal Biocontrol Agent Trichoderma atroviride To Enhance both Antagonism and Induction of Plant Systemic Disease Resistance

Biocontrol agents generally do not perform well enough under field conditions to compete with chemical fungicides. We determined whether transgenic strain SJ3-4 of Trichoderma atroviride, which expresses the Aspergillus niger glucose oxidase-encoding gene, goxA, under a homologous chitinase (nag1) promoter had increased capabilities as a fungal biocontrol agent. The transgenic strain differed only slightly from the wild-type in sporulation or the growth rate. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted. SJ3-4 had significantly less N-acetylglucosaminidase and endochitinase activities than its nontransformed parent. Glucose oxidase-containing culture filtrates exhibited threefold-greater inhibition of germination of spores of Botrytis cinerea. The transgenic strain also more quickly overgrew and lysed the plant pathogens Rhizoctonia solani and Pythium ultimum. In planta, SJ3-4 had no detectable improved effect against low inoculum levels of these pathogens. Beans planted in heavily infested soil and treated with conidia of the transgenic Trichoderma strain germinated, but beans treated with wild-type spores did not germinate. SJ3-4 also was more effective in inducing systemic resistance in plants. Beans with SJ3-4 root protection were highly resistant to leaf lesions caused by the foliar pathogen B. cinerea. This work demonstrates that heterologous genes driven by pathogen-inducible promoters can increase the biocontrol and systemic resistance-inducing properties of fungal biocontrol agents, such as Trichoderma spp., and that these microbes can be used as vectors to provide plants with useful molecules (e.g., glucose oxidase) that can increase their resistance to pathogens.     

Topographical Mapping of the Rainbow Trout (Oncorhynchus mykiss) Microbiome Reveals a Diverse Bacterial Community with Antifungal Properties in the Skin

The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (Oncorhynchus mykiss). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.     

Paenibacillus terrae AY-38 resistance against Botrytis cinerea in Solanum lycopersicum L. plants through defence hormones regulation

This study aims to investigate the role of Paenibacillus terrae AY-38 to produce bioactive metabolites and to counteract pathogenic infections caused by B. cinerea. The pure culture of P. terrae (AY-38) showed the secretion of significant amount of IAA (indole-3-acetic acid) (109.57?±?3.2?µg?mL^?1). The AY-38 strain also produced siderophore and glucanase, while in the in vitro test, it showed significant antagonism to Botrytis cinerea. In the in vivo plant experiment, the sole application of AY-38 significantly improved plant growth (plant height and leaf area), while in B. cinerea infected plants, AY-38 inoculation not only decreased the disease incidence on leaves and fruits but also reprogramed the plants for higher growth. AY-38 treatments promoted and rescued plant growth by modulating the defence responses of endogenous hormones, such as jasmonic and salicylic acids. Our findings concluded that P. terrae possesses great potential as a possible biocontrol agent against B. cinerea-induced pathogenic infections.     

Penicillium oxalicum directed systemic resistance in tomato against Alternaria alternata

Enhanced yield of tomato has ever been an important issue due to its nutritional value and various dietary consumption forms. But, efficient yield increase can never be achieved without using environmentally safe means e.g. innate resistance. In present study, tomato innate antifungal resistance has been boosted up using Penicillium oxalicum, and then various aspects of resistance modulation have been explored in details. Two tomato varieties of differential antifungal resistance (Dinaar and Red Tara) were treated with six P. oxalicum strains which screened the best inducer strain (Pn 5); which remarkably controlled disease incidence (DI) of Alternaria alternata. Inducer was not only responsible for almost two times production of phenolics, alkaloids and terpenoids in Red Tara, but it also non-significantly triggered same biochemicals in Dinaar. Hemicellulose showed only 40 % increase in variety of least antifungal resistance. During quantification assays of peroxidase (POD), phenyl ammonia lyase and polyphenol oxidase, more or less the same doubling trend was recorded in susceptible variety, while only POD had significant enhancement in resistant variety under the influence of fungal inducer. It was also recorded that inducer not only modulated quantity of enzyme (glucanase), but its isozyme package was also altered. Colorimetric quantifications of lignin, cellulose and pectins proved that biotic inducer strengthened the physical structure of plant cells by increasing these contents from 30 to 120 %. The above investigation collectively comes with the recommendation of an efficient and environmentally safe inducer (P. oxalicum); which, can be used to control fungal pathogens.     

An Interspecies Comparative Analysis of the Predicted Secretomes of the Necrotrophic Plant Pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Phytopathogenic fungi form intimate associations with host plant species and cause disease. To be successful, fungal pathogens communicate with a susceptible host through the secretion of proteinaceous effectors, hydrolytic enzymes and metabolites. Sclerotinia sclerotiorum and Botrytis cinerea are economically important necrotrophic fungal pathogens that cause disease on numerous crop species. Here, a powerful bioinformatics pipeline was used to predict the refined S. sclerotiorum and B. cinerea secretomes, identifying 432 and 499 proteins respectively. Analyses focusing on S. sclerotiorum revealed that 16% of the secretome encoding genes resided in small, sequence heterogeneous, gene clusters that were distributed over 13 of the 16 predicted chromosomes. Functional analyses highlighted the importance of plant cell hydrolysis, oxidation-reduction processes and the redox state to the S. sclerotiorum and B. cinerea secretomes and potentially host infection. Only 8% of the predicted proteins were distinct between the two secretomes. In contrast to S. sclerotiorum, the B. cinerea secretome lacked CFEM- or LysM-containing proteins. The 115 fungal and oomycete genome comparison identified 30 proteins specific to S. sclerotiorum and B. cinerea, plus 11 proteins specific to S. sclerotiorum and 32 proteins specific to B. cinerea. Expressed sequence tag (EST) and proteomic analyses showed that 246 S. sclerotiorum secretome encoding genes had EST support, including 101 which were only expressed in vitro and 49 which were only expressed in planta, whilst 42 predicted proteins were experimentally proven to be secreted. These detailed in silico analyses of two important necrotrophic pathogens will permit informed choices to be made when candidate effector proteins are selected for function analyses in planta.     

Isolation of Paenibacillus sp. and Assessment of its Potential for Enhancing Mineral Weathering

One mineral-solubilizing bacterial strain designated KT was isolated from a soil in Henan Province, China. The full-length 16S rRNA gene sequence showed 95.2 to 96.7% similarity to the bacteria of the genus Paenibacillus, indicating that the strain KT belonged to the genus Paenibacillus. The potential of this strain to release potassium from silicate minerals was investigated using a potassium-bearing rock as the sole source of potassium to support its growth. After inoculation for 7 days, the concentrations of water-soluble Al, Ca and Fe released from the potassium-bearing rock in active bacterial culture were higher than those from the control with autoclaved inoculum, but the concentration of water-soluble K in active bacterial culture was similar to that in the control. The concentrations of HNO₃-extractable Al, Ca, Fe and K from the bacterial culture were also higher than those in the control. These results showed strain KT was able to release potassium from potassium-bearing rock. These results have important implications for extraction of potassium from rocks to support plant growth.     

Partial purification and characterization of 2, 4-diacetylphloroglucinol producing Pseudomonas fluorescens VSMKU3054 against bacterial wilt disease of tomato

We find out the antimicrobial potential of partially purified 2,4-diacetylphloroglucinol (DAPG) against Ralstonia solanacearum and fungal plant pathogens isolated from tomato rhizobacterium Pseudomonas fluorescens VSMKU3054. The present study is mainly focused on the control of wilt disease of tomato by our isolate VSMKU3054 and DAPG. The cell free culture filtrate of P. fluorescens VSMKU3054 was significantly arrested the growth of R. solanacearum and fungal pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum compared to control. The existence of DAPG from the crude metabolites of P. fluorescens VSMKU3054 was confirmed on TLC with Rf value 0.34, which is coincide with that of authentic phloroglucinol. The partially purified DAPG exhibited much higher activity against R. solanacearum at 30 µg/ml than the fungal plant pathogens compared to control. The antimicrobial partially purified compound was identified as DAPG by UV, FT-IR and GC–MS analysis. The percentage of live cells of R. solanacearum when supplemented with DAPG at 30 µg/ml, significantly controlled the living nature of R. solanacearum up to 68% compared to tetracycline and universal control observed under high content screening analysis. The selected isolate P. fluorescens VSMKU3054 and DAPG significantly controlled wilt disease of tomato up to 59.5% and 42.12% on 3rd and 7th days compared to positive and negative control by detached leaf assay. Further, in silico analysis revealed that high interaction of DAPG encoding protease with lectin which is associated with R. solanacearum. Based on our findings, we confirmed that P. fluorescens VSMKU3054 and DAPG could be used a potential bio inoculants for the management of bacterial wilt disease of tomato.     

Isolation and Characterization of a Pseudomonas Strain That Restricts Growth of Various Phytopathogenic Fungi

The characterization of a novel Pseudomonas strain exhibiting antagonism towards many important corn fungal pathogens is presented. This strain was isolated from the caryopses of the grass Tripsacum dactyloides and was identified as Pseudomonas cepacia. The antagonistic activity is due to the production of an antifungal compound. The chromatographic properties of this partially purified compound isolated from growth medium differ from those reported previously for other pseudomonads. The suppression of the growth of economically important phytopathogens by this strain and by the partially purified compound indicates a potential biocontrol agent.     

Enhanced virulence of Beauveria bassiana against Thrips tabaci by the addition of bacterial metabolites derived from Xenorhabdus hominickii

Xenorhabdus and Photorhabdus are two bacterial genera specifically symbiotic to Steinernema and Heterorhabditis, which are the entomopathogenic nematode genera, respectively. These bacteria are well known to produce potent secondary metabolites suppressing insect immune responses. This study aimed to develop a potent microbial insecticide against the onion thrips, Thrips tabaci, using the bacterial metabolites. Among the chemical insecticides that have been used to control the thrips, spinosad was highly effective against both larvae and adults of T. tabaci. Three different entomopathogenic fungi were also effective to kill the thrips. However, the fungal virulence was much less than the control efficacy of the chemical insecticide, spinosad. To enhance the fungal virulence of Beauveria bassiana (Bb), the bacterial culture broth of Xenorhabdus/Photorhabdus was added to suppress the thrips immune defense. Among six different bacterial species, X. hominickii (Xh) produced highly potent metabolites to enhance the fungal virulence. Indeed, four different bacterial metabolites (GameXPeptide, benzylideneacetone, oxindole, and 3-ethoxy-4-methoxyphenol) of the bacteria suppressed the gene expressions of an antimicrobial peptide, lysozyme, which was highly inducible to the fungal infection. To optimize the mixture ratio of fungal and bacterial pathogens, the fungal conidia and bacterial culture broth were freeze-dried and mixed in different ratios. Laboratory and field assays showed that a mixture spray of freeze-dried Xh culture broth (3 g) and Bb conidia (1.17 × 10⁹ conidia) in a liter was effective to control T. tabaci infesting welsh onion.