Volatiles of bacterial antagonists inhibit mycelial growth of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

Bacterial antagonists are bacteria that negatively affect the growth of other organisms. Many antagonists inhibit the growth of fungi by various mechanisms, e.g., secretion of lytic enzymes, siderophores and antibiotics. Such inhibition of fungal growth may indirectly support plant growth. Here, we demonstrate that small organic volatile compounds (VOCs) emitted from bacterial antagonists negatively influence the mycelial growth of the soil-borne phytopathogenic fungus Rhizoctonia solani Kühn. Strong inhibitions (99–80%) under the test conditions were observed with Stenotrophomonas maltophilia R3089, Serratia plymuthica HRO-C48, Stenotrophomonas rhizophila P69, Serratia odorifera 4Rx13, Pseudomonas trivialis 3Re2-7, S. plymuthica 3Re4-18 and Bacillus subtilis B2g. Pseudomonas fluorescens L13-6-12 and Burkholderia cepacia 1S18 achieved 30% growth reduction. The VOC profiles of these antagonists, obtained through headspace collection and analysis on GC-MS, show different compositions and complexities ranging from 1 to almost 30 compounds. Most volatiles are species-specific, but overlapping volatile patterns were found for Serratia spp. and Pseudomonas spp. Many of the bacterial VOCs could not be identified for lack of match with mass-spectra of volatiles in the databases.     

Optimal conditions for chitinase production by<Emphasis Type="Italic">Serratia marcescens</Emphasis>

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.     

Seasonal variation in allelopathic potential of<Emphasis Type="Italic">Artemisia princeps</Emphasis> var.<Emphasis Type="Italic">orientalis</Emphasis> on plants and microbes

We investigated seasonal variations in allelopathic potential ofArtemisia princeps var.orientalis. Aqueous and meth-anol extracts and volatile substances were prepared in the laboratory from samples collected monthly (April through October). Their impacts were then assessed on the germination and seedling growth ofLactuca sativa andAchyranthes japonica. The allelopathic potential varied with the time of sample collection and the concentration tested. For example, germination ofL. sativa was not inhibited by the aqueous extract but seedling growth (shoots and roots) was, with its seasonal effect being significant. ForA. japonica, seed germination was not inhibited at lower concentrations (except for August samples). However, at higher concentrations and in certain months (especially July), germination was more negatively affected. The degree of seedling growth inhibition also differed by month and by extract concentration, with roots being impacted more than shoots. Volatile substances also had a time-dependent influence on the germination and seedling elongation ofA. japonica. In a separate experiment, the ethyl-acetate and water fractions of a crude methanol extract were prepared monthly fromA. princeps var.orientalis. Here, we examined their antimicrobial activities against three gram-positive bacteria (Bacillus cereus, Bacillus subtilis, andStaphylococcus aureus), two gramnegative bacteria (Escherichia coli andPseudomonas fluorescens), and one lactic acid bacterium,Lactobacillus plantar urn. The ethyl-acetate fraction that was sampled in September was remarkably potent againstB. cereus andB. subtilis, whereas the water fraction collected in August and September showed great antimicrobial activity against the grampositive and -negative bacteria. In contrast,L. plantarum was not inhibited by the water fraction, regardless of the sampling month. Likewise, the ethyl-acetate and water fractions collected in April and October had the lowest levels of antimicrobial activity.     

The Attractive Serratia plymuthica BMA1 Strain With High Rock Phosphate-Solubilizing Activity and Its Effect on the Growth and Phosphorus Uptake by Vicia faba L. Plants

Abstract

The present work aims to investigate the attractive ability of the newly isolated bacterium Serratia plymuthica BMA1, to release phosphorus (P) from rock phosphate (RP) and also to assess its beneficial effect in promoting the growth of Vicia faba. This strain exhibited the highest RP-solubilization activity ever reported, with 450?mg l^?1 of soluble P at 30?°C. At 10 and 20?°C, its RP-solubilization was estimated at 100 and 200?mg l^?1, respectively. HPLC analysis revealed that RP-solubilizing activity was associated with the release of gluconic acid. The hydroxyapatite (HA) solubilization activity concomitantly occurred with the secretion of gluconic acid and lactic acid. Under greenhouse conditions, application of BMA1 strain as an inoculant in presence of RP as the sole P source, considerably increased phosphorus uptake by V. faba L. and upgraded its overall growth in terms of dry weight and length by 76% and 39%, respectively. This growth promoting effect was accompanied by a substantial increase in chlorophyll contents. Additionally, phosphorus levels within roots and shoots of S. plymuthica BMA1-treated plants were approximately three times greater, compared to the non-inoculated control plants. When HA was used instead of RP, bacterization with BMA1 strain also enhanced the plant growth parameters and P contents, but significantly less than RP. These findings revealed that S. plymuthica BMA1 could be a potential candidate to improve the agronomic effectiveness of RP, toward a clean P-nutrition through the formulation of bio-phosphate fertilizers for plant growth promotion.     

Improvement of seed bio-priming of oilseed rape (Brassica napus ssp. oleifera) with Serratia plymuthica and Pseudomonas chlororaphis

Seed bio-priming of oilseed rape (Brassica napus) with the antagonistic rhizobacteria Serratia plymuthica and Pseudomonas chlororaphis was improved. With the imbibition of water, bacteria are transported into the seed where they survive better. To obtain a minimum bacterial density in the seed of log₁₀ 5 colony-forming-units (CFUs) seed^?1, the bacterial density in the bio-priming suspension should be >log₁₀ 9 CFUs mL^–1 for S. plymuthica and >log₁₀ 8 CFUs mL^–1 for P. chlororaphis. Priming duration was reduced from 12 to 2 h for S. plymuthica and 4 h for P. chlororaphis. Among other priming solutions tested, the addition of MgSO₄ best supported establishment in the seeds and also improved germination. The optimal bio-priming temperature for S. plymuthica is 28°C and for P. chlororaphis 22°C. Survival of the bacteria inside the seeds was moderately improved by storage at low temperature but considerably prolonged by storage under anaerobic conditions. P. chlororaphis survived significantly longer than S. plymuthica.     

Involvement of the global regulators GrrS,RpoS, and SplIR in formation of biofilms in <Emphasis Type="Italic">Serratia plymuthica</Emphasis>

Most bacteria exist in the natural environment as biofilms, multicellular communities attached to various surfaces. Biofilms have a characteristic architecture and are enclosed in the exopolymer matrix. Bacterial cells in biofilms are extremely resistant to antibacterial factors. It was shown in this work that the GrrA/GrrS system of global regulators of gene expression and the sigma S subunit of RNA polymerase (RpoS) play a significant role in positive regulation of biofilm formation in the rhizospheric bacterium Serratia plymuthica IC1270. Inactivation of grrS and rpoS genes resulted in an up to six-to-sevenfold and four-to-fivefold reduction in biofilm formation, respectively. Mutation in the grrS gene decreased the capacity of the bacterium for swarming motility. The splIR Quorum Sensing (QS) system was shown to negatively influence the biofilm formation. Transfer of the recombinant plasmid containing cloned genes splI/splR of S. plymuthica HRO-C48 into S. plymuthica IC1270 cells led to a twofold decrease of their ability to form biofilms. Inactivation of the splI gene coding for the synthase of N-acyl-homoserine lactones in S. plymuthica HRO-C48 resulted in a 2–2.5-fold increase in the level of biofilm formation, whereas the transfer of plasmid carrying the cloned splI/splR genes into these mutant cells restored the biofilm formation to the normal level. The results obtained demonstrate that the formation of biofilms in S. plymuthica is positively regulated by the GrrA/GrrS and RpoS global regulators and is negatively regulated by the SplIR QS system.     

Antimicrobial activities ofStreptomyces pulcher,S. canescens andS. citreofluorescens against fungal and bacterial pathogens of tomatoin vitro

Thirty-seven actinomycete species isolated from fertile cultivated soils in Egypt were screened for the production of antimicrobial compounds against a variety of test organisms. Most of the isolates exhibited antimicrobial activities against Gram-positive, Gram-negative, and acid-fast bacteria, yeasts and filamentous fungi, with special attention to fungal and bacterial pathogens of tomato. On starch-nitrate agar, 14 strains were active againstFusarium oxysporum f.sp.lycopersici (the cause ofFusarium wilt), 18 againstVerticillium albo-atrum (the cause ofVerticillium wilt), and 18 againstAlternaria solani (the cause of early blight). In liquid media, 14 isolates antagonizedPseudomonas solanacearum (the cause of bacterial wilt) and 20 antagonizedClavibacter michiganensis ssp.michiganensis (the cause of bacterial canker). The most active antagonists of the pathogenic microorganisms studied were found to beStreptomyces pulcher, S. canescens (syn.S. albidoflavus) andS. citreofluorescens (syn.S. anulatus). The antagonistic activities ofS. pulcher andS. citreofluorescens against pathogenic bacteria in liquid media under shaking conditions. The optimum culture conditions were determined.     

Field performance of a weed-suppressing Serratia plymuthica strain applied with conventional spraying equipment

In previous glasshouse experiments, the soilbacterium Serratia plymuthica, strainA153, showed strong growth-suppressingactivities against a range of broad-leavedweeds after foliar spraying. In field tests ofthis strain in spring wheat, spring barley andpotatoes, variable effects were achieved on arange of weeds including Chenopodiumalbum, Stellaria media, Polygonumconvolvulus and Galeopsis speciosa. Atone site, good suppression of C. albumwas observed when the strain was applied in atank mix with another bacterial isolate or withreduced doses of a herbicide. Effects on weedsappeared to be independent of the applicationvolume (1000, 600, 500 l ha^–1), but weedswere in some cases more strongly suppressed athigher bacterial doses. Barley yields weresomewhat reduced by the bacterial application,but wheat yields were less affected. AlthoughS. plymuthica suppressed certain weedswhen applied in the field in a simple aqueousformulation and with conventional sprayingequipment, the level of weed suppression wasunsatisfactory from a practical standpoint.     

Inhibition ofHelminthosporium oryzae andAlternaria solani byStreptomyces longisporus (Krasil'nikov) Waksman

Summary Streptomyces longisporus, isolated from field soil in association with wheat stubble, showed antagonism againstHelminthosporium oryzae andAlternaria solani in vitro as well as in soil (autoclaved). The organism or its filtrate showed formation of a distinct zone of inhibition in culture against the pathogens. Infected potato seed pieces or rice grains dipped in the culture filtrate prevented or reduced disease incidence to different degrees depending on the period of treatment. Infestation of soil (autoclaved) withS. longisporus and the pathogens in various combinations also reduced disease incidence. Among such combinations, infestation withS. longisporus followed by the pathogens proved to be the most efficient.     

Antimicrobial activity of naphthoquinones from Fusaria

Twenty-two naphthoquinone compounds isolated or derived synthetically from culture extracts ofFusarium solani andF. oxysporum were examined for antimicrobial activity. Fifteen exhibited antibiotic activity againstStaphylococcus aureus, and 12 were active againstStreptococcus pyogenes, but none were active at the highest rate of 128 g/ml againstEscherichia coli, Klebsiella pneumoniae, Salmonella typhi, Proteus vulgaris, Serratia marcescens, orPseudomonas aeruginosa. Of 8 plant pathogenic bacteria tested against 11 naphthoquinones,Corynebacterium poinsettiae was inhibited by 6 compounds, andPseudomonas viridiflava was weakly inhibited by one. Only one of a group of 6 fluorescent soil pseudomonads was inhibited by one naphthoquinone. Antifungal activity of 10 compounds against 8 fungal plant pathogens was limited to inhibition ofPhytophthora parasitica by one naphthopyran.South Atlantic, Agricultural Research Service, U.S. Department of Agriculture. Mention of a trademark or proprietary product is for identification only and does not imply a warranty or guarantee of the product by the U.S. Department of Agriculture over other products which may also be suitable.     

Microbial Diversity Inside Pumpkins: Microhabitat-Specific Communities Display a High Antagonistic Potential Against Phytopathogens

Recent and substantial yield losses of Styrian oil pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) are primarily caused by the ascomycetous fungus Didymella bryoniae but bacterial pathogens are frequently involved as well. The diversity of endophytic microbial communities from seeds (spermosphere), roots (endorhiza), flowers (anthosphere), and fruits (carposphere) of three different pumpkin cultivars was studied to develop a biocontrol strategy. A multiphasic approach combining molecular, microscopic, and cultivation techniques was applied to select a consortium of endophytes for biocontrol. Specific community structures for Pseudomonas and Bacillus, two important plant-associated genera, were found for each microenvironment by fingerprinting of 16S ribosomal RNA genes. All microenvironments were dominated by bacteria; fungi were less abundant. Of the 2,320 microbial isolates analyzed in dual culture assays, 165 (7%) were tested positively for in vitro antagonism against D. bryoniae. Out of these, 43 isolates inhibited the growth of bacterial pumpkin pathogens (Pectobacterium carotovorum, Pseudomonas viridiflava, Xanthomonas cucurbitae); here only bacteria were selected. Microenvironment-specific antagonists were found, and the spermosphere and anthosphere were revealed as underexplored reservoirs for antagonists. In the latter, a potential role of pollen grains as bacterial vectors between flowers was recognized. Six broad spectrum antagonists selected according to their activity, genotypic diversity, and occurrence were evaluated under greenhouse conditions. Disease severity on pumpkins of D. bryoniae was significantly reduced by Pseudomonas chlororaphis treatment and by a combined treatment of strains (Lysobacter gummosus, P. chlororaphis, Paenibacillus polymyxa, and Serratia plymuthica). This result provides a promising prospect to biologically control pumpkin diseases.     

Draft Genome Sequence of the Biocontrol Strain Serratia plymuthica A30, Isolated from Rotting Potato Tuber Tissue

Serratia plymuthica A30 is a Gram-negative bacterium expressing antagonistic activity toward blackleg- and soft rot-causing Dickeya sp. biovar 3 (“Dickeya solani”). Here, we present the draft genome sequence of strain A30, which has been isolated from rotten potato tuber tissue.     

Impact of formulation procedures on the effect of the biocontrol agent Serratia plymuthica HRO-C48 on Verticillium wilt in oilseed rape

Verticillium wilt is an important disease in oilseed rape with an increasing importance worldwide. Currently, there are no methods available to suppress the pathogen. A biological protection strategy on the basis of the plant-beneficial bacterium Serratia plymuthica HRO-C48 to control Verticillium dahliae in oilseed rape was developed. Three different techniques to apply the biocontrol agent to seeds, namely pelleting, film coating and bio-priming, were evaluated considering the influence on the control activity, cell stability during storage and practical feasibility. Neither the treatment nor the inoculum density was found to influence the abundances of HRO-C48 in the rhizosphere after 30 days. Serratia treatment using bio-priming and pelleting resulted in a statistically significant biocontrol in comparison to the non-bacterized controls. Additionally, survival of HRO-C48 differed between treatments, and was the highest using bio-priming at 20°C, and pelleting at 4°C. In conclusion, the procedure of bio-priming, which was developed in line with this study, resulted in a stable and efficient formulation of S. plymuthica on rape seed. This technology opens a possibility to develop a commercial Serratia formulation to protect oilseed against V. dahliae.     

Quantitative and specific detection of the biocontrol agent, Serratia plymuthica, in plant extracts using a real-time TaqMan? assay

A Serratia plymuthica-specific TaqMan? assay was designed based on the consensus nucleotide sequence from the 3??- end of the luxS gene present in all S. plymuthica strains tested. The specificity of the assay was demonstrated by testing 21 Serratia spp. strains and 30 isolates belonging to various species that can potentially coexist with S. plymuthica in the same environment. Positive reactions in the TaqMan? assay were observed only for S. plymuthica isolates and not for other bacteria. The TaqMan? assay could detect down to 1.95 ng of S. plymuthica DNA, down to 5 bacterial cells per reaction (100?cfu ml^?1) in vitro, down to 50 bacterial cells per reaction (1,000?cfu ml^?1) in spiked potato root extracts and down to 5 bacterial cells per reaction (100?cfu ml^?1) in spiked potato haulm extracts. We used this assay to quantify S. plymuthica A30 cells in potato and tomato haulms and roots grown from S. plymuthica A30-inoculated potato seed tubers and tomato seeds. The results were comparable with the spread-plating of plant extracts on a newly developed S. plymuthica A30 selective medium (CVTR2Arif). The TaqMan? assay can be used to quantify S. plymuthica isolates in different ecosystems and in complex substrates.     

Mechanisms of biocontrol of soil-borne plant pathogens by Rhizobacteria

Bacterial antagonism, responsible for biological control, may operate by antiobiosis, competition or parasitism. Parasitism relies on lytic enzymes for the degradation of cell walls of pathogenic fungi. Serratia marcescens was found to be an efficient biocontrol agent of Sclerotium rolfsii and Rhizoctonia solani under greenhouse conditions. Populations of 10⁵ or 10⁶ colony forming units g^-1 soil were the most effective. Drench and drip application of S. marcescens suspension were more effective in controlling S. rolfsii than spraying, mixing in soil or seed coating. The highest population density of the bacteria in the rhizosphere was found on the proximal portion of the root, decreasing significantly until the tips, where it increased again. The isolated Serratia, found to possess chitinolytic activity, was able to release N-acetyl D-glucosamine from cell walls of S. rolfsii. The gene coding for chitinase was cloned into Escherichia coli and the enzyme was uniquely excreted from the bacterium into its growth medium. When S. rolfsii was sprayed by partially purified chitinase produced by the cloned gene, rapid and extensive bursting of the hyphal tips was observed. This chitinase preparation was effective in reducing disease incidence caused by S. rolfsii in beans and R. solani in cotton, under greenhouse conditions. A similar effect was obtained when a viable E. coli cell, containing the plasmid with the chitinase gene (pLCHIA), was applied. It appears that genetic engineering of the lytic enzymes, such as chitinase which play an important role in plant disease control, may improve the efficacy of biocontrol agents.     

Biocontrol of Fusarium sambucinum,dry rot of potato,by Serratia plymuthica 5–6

Serratia grimesii 4–9 and Serratia plymuthica 5–6, isolated from the rhizosphere of pea, Pisum sativum (L), were evaluated for their potential to suppress growth of Fusarium sambucinum in vitro and to reduce Fusarium dry rot in stored potatoes (Solanum tuberosum L). In vitro studies indicated that these bacterial isolates suppressed growth of F. sambucinum by 60% or more at both 15 and 25°C. In a potato tuber slice assay the number of infection sites in potato slices exposed to F. sambucinum and treated with S. grimesii 4–9 and S. plymuthica 5–6 was reduced by 96 and 97%, respectively, at 15°C. The diameter (mm) of the infection sites was reduced 91 and 96%, respectively, when compared to slices treated with F. sambucinum alone. Studies with Fusarium-infected whole potato tubers also showed significant reduction in dry rot formation following treatment with the bacterial isolates or the fungicide thiabendazole. When applied simultaneously with the pathogen, S. grimesii 4–9 and S. plymuthica 5–6 suppressed development of Fusarium dry rot by 60 and 77%, respectively, at 15°C and by 63 and 84%, respectively, at 25°C compared to tubers inoculated with the pathogen alone. Thiabendazole suppressed development of Fusarium dry rot by 66 and 81% at 15 and 25°C, respectively, compared to tubers inoculated with the pathogen alone. These studies demonstrate the potential of soil bacteria as biofungicides for managing post-harvest crop diseases. Due to the potential risks to human health associated with S. grimesii 4–9, S. plymuthica 5–6 is recommended for further study for biofungicide development.     

Acetylene reduction and indoleacetic acid production by Azospirillum isolates from Cactaceous plants

Azospirillum isolates were obtained from rhizosphere soil and roots of three cactaceae species growing under arid conditions. All Azospirillum isolates from rhizosphere and roots ofStenocereus pruinosus andStenocereus stellatus were identified asA. brasilense; isolates of surface-sterilized roots fromOpuntia ficus-indica were bothA. brasilense andA. lipoferum. Azospirilla per g of fresh root in the three species ranged from 70×10³ to 11×10³. The most active strains in terms of C₂H₂ reduction (25–49.6 nmol/h·ml) and indoleacetic acid (IAA) production (36.5–77 μg/ml) were those identified asA. brasilense and isolated from Stenocereus roots.A. lipoferum isolated from Opuntia roots produced low amounts of IAA (6.5–17.5 μg/ml) and low C₂H₂-reduction activity (17.8–21.2 nmol/h·ml).     

Microbial mineralization of organic phosphate in soil

Summary Phosphate-dissolving microorganisms were isolated from non-rhizosphere and rhizosphere of plants. These isolates included bacteria, fungi and actinomycetes. In broth cultures, Gram-negative short rod,Bacillus andStreptomyces species were found to be more active in solubilizing phosphate thanAspergillus, Penicillium, Proteus, Serratia, Pseudomonas andMicrococcus spp. The sterile soils mixed with isolated pure culture showed slower mineralization of organic phosphate than that of non-sterile soil samples at all incubation periods. Maximum amount of phosphate mineralization by isolated microorganisms were obtained at the 60th and the 75th day of incubation in sterile and non-sterile soils respectively. The mixed cultures were most effective in mineralizing organic phosphate and individuallyBacillus sp. could be ranked next to mixed cultures. Species ofPseudomonas andMicrococcus were almost the same as that of the control under both sterile and non-sterile conditions.     

Effect of inoculation ofAzospirillum spp. on nitrogen accumulation by field-grown wheat

Summary Two experiments were performed to examine the effects of inoculation of field grown wheat with various Azospirillum strains. In the first experiment the soil was sterilized with methyl bromide to reduce the Azospirillum population and¹⁵N labelled fertilizer was added to all treatments. Two strains ofAzospirillum brasilense isolated from surface sterilized wheat roots and theA. brasilense type strain Sp7 all produced similar increases in grain yield and N content. From the¹⁵N and acetylene reduction data it was apparent that these increases were not due to N₂ fixation. In the second experiment performed in the same (unsterilized) soil, twoA. brasilense strains (Sp245, Sp246) and oneA. amazonense strain (Am YTr), all isolated from wheat roots, produced responses of dry matter and N content while the response to the strain Sp7 was much smaller. These data confirm earlier results which indicate that if natural Azospirillum populations in the soil are high (the normal situation under Brazilian conditions), strains which are isolated from wheat roots are better able to produce inoculation responses than strains isolated from other sources. The inoculation of a nitrate reductase negative mutant of the strain Sp245 produced only a very small inoculation response in wheat. This suggests that the much greater inoculation response of the original strain was not due to N₂ fixation but to an increased nitrate assimilation due to the nitrate reductase activity of the bacteria in the roots. Consultant Inter-American Institute for Cooperation in Agriculture IICA/EMBRAPA World Bank Project.     

An endophytic bacterium isolated from roots of the halophyte <Emphasis Type="Italic">Prosopis strombulifera</Emphasis> produces ABA,IAA, gibberellins A<Subscript>1</Subscript> and A<Subscript>3</Subscript> and jasmonic acid in chemically-defined culture medium

This paper informs the characterization by 16SrDNA partial sequence analysis of an endophytic diazotrophic bacterium isolated from roots of the halophyte shrub Prosopis strombulifera. The bacterium produced ABA, IAA, GA₁, GA₃ and jasmonic acid in chemically-defined culture medium as assessed by GC-EIMS. The results emphasize the role of phytohormones produced by endophytic bacteria in the association host-beneficial microorganisms, especially under conditions of adverse environments.