Cucumber Rhizosphere Research Articles

Responses of Soil Microbial Communities in the Rhizosphere of Cucumber (Cucumis sativus L.) to Exogenously Applied p-Hydroxybenzoic Acid

Changes in soil biological properties have been implicated as one of the causes of soil sickness, a phenomenon that occurs in continuous monocropping systems. However, the causes for these changes are not yet clear. The aim of this work was to elucidate the role of p-hydroxybenzoic acid (PHBA), an autotoxin of cucumber (Cucumis sativus L.), in changing soil microbial communities. p-Hydroxybenzoic acid was applied to soil every other day for 10 days in cucumber pot assays. Then, the structures and sizes of bacterial and fungal communities, dehydrogenase activity, and microbial carbon biomass (MCB) were assessed in the rhizosphere soil. Structures and sizes of rhizosphere bacterial and fungal communities were analyzed by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and real-time PCR, respectively. p-Hydroxybenzoic acid inhibited cucumber seedling growth and stimulated rhizosphere dehydrogenase activity, MBC content, and bacterial and fungal community sizes. Rhizosphere bacterial and fungal communities responded differently to exogenously applied PHBA. The PHBA decreased the Shannon-Wiener index for the rhizosphere bacterial community but increased that for the rhizosphere fungal community. In addition, the response of the rhizosphere fungal community structure to PHBA acid was concentration dependent, but was not for the rhizosphere bacterial community structure. Our results indicate that PHBA plays a significant role in the chemical interactions between cucumber and soil microorganisms and could account for the changes in soil microbial communities in the continuously monocropped cucumber system.

Study of the siderophore‐producing Trichoderma asperellum Q1 on cucumber growth promotion under salt stress

Trichoderma spp. are versatile beneficial fungi which can stimulate growth and plant resistance to biotic and abiotic stresses. In this study, the potential of Trichoderma isolate in promoting the cucumber growth under salt stress and its possible mechanisms were investigated. Strain Q1 was isolated from the rhizosphere of cucumber in greenhouse in China and identified as Trichoderma asperellum based on its morphological features and the molecular phylogenetic analyses. It exhibited some plant growth-promoting attributes of phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, auxin and siderophore production. In pot trials, applying strain Q1 to cucumber plant had significantly promoted seedlings growth and alleviated the growth suppression induced by salt stress as confirmed by the changes in growth phenotype and several biochemical and physiological parameters. In solution culture experiments, the growth of cucumber seedlings was increased and the percentage of wilted cucumber seedlings was decreased in the treatment of siderophore-containing culture filtrate (SCF) of strain Q1 with insoluble Fe(3+) under salt stress. These results indicated that T. asperellum Q1 has a real potential to enhance cucumber growth by inducing physiological protection under saline stress, and its siderophores showed sign of alleviating negative effect of salinity and available iron deficiency.

Effects of amendments of ferulic acid on soil microbial communities in the rhizosphere of cucumber (Cucumis sativus L.)

Phenolic compounds are secondary metabolites often implicated in allelopathic interactions. However, how these compounds would affect soil microbial communities is not yet fully understood. In this experiment, effects of amendments of ferulic acid on soil dehydrogenase activity, microbial biomass carbon (MBC) content, and bacterial and fungal community structures and abundances in the rhizosphere of cucumber were evaluated. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and real-time PCR, on bacterial 16S rRNA genes or fungal ITS regions, were used to analyze bacterial and fungal community structures and sizes, respectively. Results showed that amendments of ferulic acid inhibited cucumber seedling growth, and stimulated soil dehydrogenase activity, MBC content, and bacterial and fungal community sizes. Amendments of ferulic acid also changed rhizosphere bacterial and fungal community structures. Bacterial and fungal communities responded differently to the ferulic acid amendment: numbers of visible bands and diversity indices of microbial community were lower in ferulic acid-amended soils than in the unamended soil for bacterial community, while were higher for fungal community. Overall, our results suggested that ferulic acid might account for the autotoxicity of cucumber through changing soil microbial communities.

Effect of Biocontrol Agent Pseudomonas fluorescens 2P24 on Soil Fungal Community in Cucumber Rhizosphere Using T-RFLP and DGGE

Fungi and fungal community play important roles in the soil ecosystem, and the diversity of fungal community could act as natural antagonists of various plant pathogens. Biological control is a promising method to protect plants as chemical pesticides may cause environment pollution. Pseudomonas fluorescens 2P24 had strong inhibitory on Rastonia solanacearum, Fusarium oxysporum and Rhizoctonia solani, etc., and was isolated from the wheat rhizosphere take-all decline soils in Shandong province, China. However, its potential effect on soil fungal community was still unknown. In this study, the gfp-labeled P. fluorescens 2P24 was inoculated into cucumber rhizosphere, and the survival of 2P24 was monitored weekly. The amount decreased from 108 to 105 CFU/g dry soils. The effect of 2P24 on soil fungal community in cucumber rhizosphere was investigated using T-RFLP and DGGE. In T-RFLP analysis, principle component analysis showed that the soil fungal community was greatly influenced at first, digested with restriction enzyme Hinf I and Taq I. However, there was little difference as digested by different enzymes. DGGE results demonstrated that the soil fungal community was greatly shocked at the beginning, but it recovered slowly with the decline of P. fluorescens 2P24. Four weeks later, there was little difference between the treatment and control. Generally speaking, the effect of P. fluorescens 2P24 on soil fungal community in cucumber rhizosphere was just transient.

Genetic diversity among crown and root rot isolates of Rhizoctonia solani isolated from cucurbits using PCR-based techniques

Twenty three (n=23) isolates of Rhizoctonia solani were obtained from root, crown and rhizosphere of cucumber, pumpkin, watermelon and melon plants. Investigation of the anastomosis groups (AGs) showed that all of the isolates belonged to AG4. Twenty three of R. solani AG4 were measured for disease severity according to their geographical origins and host plants. The pathogenicity test revealed that the disease severity varied from 8.3 to 91.6%, when all isolates were inoculated on their corresponding hosts. The disease severity indicated that R. solani isolates were classified into five groups. Genetic diversity was performed among isolates of R. solani AG4 by combination of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) techniques. Amplicon size of six RAPD primers out of fifteen and four ISSR primers out of ten, ranged from 0.5 to 3 Kb in isolates. All isolates were divided into seven groups at >30% similarity level. The results exhibited a high genetic variability in the R. solani AG4 population, without any correlation between genetic diversity, the host plants and geographical regions. Key words: Cucurbits, genetic diversity, random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), pathogenicity, Rhizoctonia solani.

Bacillus subtilis SQR 9 can control Fusarium wilt in cucumber by colonizing plant roots

Fusarium wilt is one of the major constraints on cucumber production worldwide. Several strategies have been used to control the causative pathogen, Fusarium oxysporum f. sp. cucumerinum J. H. Owen, including soil solarization, fungicide seed treatment and biological control. In this study, F. oxysporum f. sp. cucumerinum was successfully controlled by a newly isolated strain, Bacillus subtilis SQR 9, in vitro and in vivo. Greenhouse experiments were carried out to evaluate the effect of inoculation and solid fermentation of organic fertilizer with B. subtilis SQR 9, hereby defined as bio-organic fertilizer (BIO), on the control of Fusarium wilt. In comparison with the control, the wilt incidence was significantly reduced (49–61% reduction) by application of BIO. The rhizosphere population of F. oxysporum f. sp. cucumerinum, as detected both by selective plating and realtime PCR, was significantly lower in BIO-treated plants than the control. The localization of bacterial cells, pattern of colonization and survival of B. subtilis SQR 9 in the rhizsosphere of cucumber, was examined by fluorescent microscopy and explored following recovery of the green fluorescent protein (gfp)-labeled SQR 9 with the new gfp-marked shuttle vector pHAPII through selective plating. The preferential sites of the labeled strain were the differentiation and elongation zone, root hair and the lateral root junctions. The population of the strain was 106 cfu/g root in rhizoplane. These results indicate that the strain was able to survive well in the rhizosphere of cucumber, suppressed growth of F. oxysporum in the rhizosphere of cucumber and protected the host from the pathogen.

Screening of Rhizobacteria for Biological Control of Cucumber Root and Crown Rot Caused by Phytophthora drechsleri

Antagonistic rhizobacteria, more specifically fluorescent pseudomonads and certain species of Bacillus, are known as biocontrol agents of fungal root diseases of agronomic crops. In this study, 144 bacteria were isolated from cucumber rhizosphere and screened as potential biological control agents against Phytophthora drechsleri, the causal agent of cucumber root rot, in vitro condition. Non-volatile compounds of 23 isolates showed noticeable inhibition zone (> 30%) against P. drechsleri, whereas volatile compounds of 7 isolates could prevent more than 30% of the mycelial growth of the fungus. All promising isolates, except of Pseudomonas flourescens V69, promoted significantly plant growth under in vitro condition. P. flourescens CV69 and V11 exhibited the highest colonization on the root. Results of the greenhouse studies showed that a reduction in disease incidence by use of some strains, and particularly use of strains CV6 and V11 as a soil treatment, exhibited a reduction in disease incidence so that suppressed disease by 85.71 and 69.39% respectively. Pseudomonas flourescens CV6 significantly suppressed disease in comparison to Ridomil fungicide. The use of mixture bacterial strains in the soil inoculated by the fungus resulting in falling down the most of the plants which didn't show significant difference with infected control soils without bacteria.

Improved bacterial community diversity and cucumber yields in a rotation with kidney bean–celery–cucumber

Using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR–DGGE) methods, we investigated the effects of seven different crop rotation modes on cucumber rhizosphere soil bacterial communities. The bacterial diversity indexes in three growth periods showed that the diversity and evenness indexes of the kidney bean–celery–cucumber rotation were higher than those of the following rotations: tomato–kidney bean–cucumber, tomato–celery–cucumber, kidney bean–tomato–cucumber, cucumber–kidney bean–cucumber, cucumber–celery–cucumber, cucumber–tomato–cucumber, and cucumber–cucumber–cucumber. DGGE profiles of various rhizosphere soils showed six major bacterial populations: Proteobacteria, Sphingobacteria, Flavobacteria, Actinobacteria, Clostridia, and Acidobacteria. The yield of cucumber in the kidney bean–celery–cucumber rotation was higher than in other crop rotations (p<0.05). Cucumber yield was strongly and positively correlated with soil microorganism diversity index (p<0.01), and was also positively correlated with evenness index (p<0.05). Our results suggest that the kidney bean–celery–cucumber rotation is suitable for cucumber cropping.

Root exudates as an important factor of formation of nanomolecular structures of the rhizosphere

The content and qualitative composition of organic acids and sugars in the rhizosphere of tomato, cucumber, and wheat is investigated. Preservation of their high content in the rhizosphere of vegetable crops with inoculation of the plants with rhizobacteria is shown. The possible influence of this effect on the formation of nanomolecular structures of the rhizosphere is discussed.

Assessment of the Effects of Genetically Modified Pseudomonas spp. Expressing Chitinase on the Soil Microbial Community in the Cucumber Rhizosphere

In our previous study, a genetically-modified (GM) rhizosphere bacterium, Pseudomonas putida strain 101-97 harboring the chitinase-expression vector pKAC9-p07, showed over-production of chitinase and suppressed the damping-off of cucumber seedlings caused by the plant-pathogenic fungus Rhizoctonia solani. To assess the effects of the GM bacterium on other soil microorganisms, we monitored the microbial population in the cucumber rhizosphere after inoculation with the GM bacterium by culture-dependent and -independent methods. In the culture-dependent analysis, total bacterial and fungal populations in the cucumber rhizosphere did not differ after inoculation with either the GM bacterium or its parental strain, P. putida 101R. In the cultureindependent analysis, we determined changes in the microbial communities by PCR-denaturing gradient gel electrophoresis (DGGE). The DGGE profiles of bacterial 16S rDNA genes differed significantly depending on whether they had been inoculated with the GM bacterium or its parental strain, whereas the profiles of fungal 18S rDNA genes remained similar.

Effects of summer catch crop, residue management, soil temperature and water on the succeeding cucumber rhizosphere nitrogen mineralization in intensive production systems

Nitrogen nutrient management is crucially important in shallow-rooted vegetable production systems characterized by high input and high environmental risk. To investigate the effects of summer catch crop (sweet corn, common bean, garland chrysanthemum and edible amaranth), residue management, and soil temperature and water on the succeeding cucumber rhizosphere nitrogen mineralization in intensive production systems, we determined the rates of net nitrogen mineralization and nitrification in a 4-year field experiment on greenhouse cucumber double-cropping systems. Summer catch crop and its residue significantly increased the succeeding cucumber rhizosphere mineral nitrogen contents, when compared to conventional practices. In general, summer catch crop and its residue significantly increased the rates of both net nitrogen mineralization and net nitrogen nitrification at 4 or 40°C, and increased the rates of net nitrogen immobilization (negative mineralization) and net nitrogen nitrification at 15 or 28°C, in succeeding cucumber rhizosphere after four-year treatment. Soil temperature and water had more influence than catch crops and residue management on N mineralization. The effect of carbon on nitrogen mineralization was more pronounced than that of nitrogen, and the effect of microbial carbon on the different forms of inorganic N was more pronounced than that of organic carbon. When the effects of soil temperature and water content were eliminated, cumulative net nitrogen mineralization and nitrification in catch crop and residue management plots were 296–784 and 57–84% higher, respectively, than conventional practices plots. Catch crops and residue management influenced change of ammonium-N more significantly than that of nitrate-N. Additionally, there were complex relationships between fruit yield and soil N mineralization in catch crop- and residue management-induced systems.

Soil microbial community structure in cucumber rhizosphere of different resistance cultivars to fusarium wilt

Cucumber fusarium wilt is a common soil-borne disease. We hypothesize that there is a relationship between the severity of disease and soil microbial ecology. In this work, culturable microbial populations, lipid fatty acid and community-level physiological profiles (CLPP) from rhizosphere soils of four different cucumber cultivars were investigated. Comparatively higher actinomycetes, mycorrhizal colonization and higher ratios of bacteria to fungi were found in the two resistant cultivars compared with the two susceptible cultivars. CLPP analysis showed that catabolic diversity indices were higher in the presence of two resistant cultivars. Phospholipid fatty acid (PLFA) profiles suggested that fungal (18:2omega6,9c) PLFA was enriched in the rhizosphere soils of the two susceptible cultivars, but some bacterial (16:0 and 15:0a) PLFAs were found in a lower relative abundance in these soils. The neutral lipid fatty acid 16:1omega5, which is an indicator of arbuscular mycorrhizal fungi, was enriched in the rhizosphere soils of the two resistant cultivars. All the three methods suggested that plant genotype had a significant impact on the soil microbial community composition and activity, and the differences in the rhizosphere microbial community may result in the differences in the resistance to fusarium wilt.

Bacterial diversity in cucumber ( Cucumis sativus) rhizosphere in response to salinity, soil pH, and boron

Soil salinity is a major factor relating microbial communities to environmental stress in the microbial selection process as stress can reduce bacterial diversity. In the San Joaquin Valley (SJV) of California, the problem of increasing salinity and consequently, decreasing crop productivity, due to reuse of saline drainage water are major concerns. An experiment was conducted in a closed, recirculating volumetric lysimeter system (VLS) consisting of 24 experimental plant growth units to determine the interactive effects of salinity, boron and pH on rhizosphere and non-rhizosphere microbial composition of cucumber ( Cucumis sativus L. cv. Seminis Turbo hybrid). Plants in the VLS were irrigated from individual reservoirs containing a modified half-strength Hoagland's nutrient solution combined with salinity, boron (B), and pH treatments. The results indicated that salinity and pH were the most influential factors affecting the growth of plants and the effect of boron on the plant was more severe under slightly acidic conditions. Total bacterial DNA was extracted from rhizosphere and non-rhizosphere samples, and a 236-bp DNA fragment in the V3 region of the small subunit ribosomal RNA genes of eubacteria was amplified. The 16S rRNA and the products were subjected to denaturing gradient gel electrophoresis (DGGE) and sequencing. Analyses of bacterial diversity showed that the effects of salinity, boron, and pH were more severe on the rhizosphere bacterial population during the first week of growing cucumber, with decreasing impacts with plant growth. However, there was no salinity–B–pH interaction effects on plant biomass, but the effects were seen in the number of heterotrophic bacteria in the rhizosphere and on species richness and diversity during week seven of the study. These suggest that the effects of salinity–B–pH interactions may influence microorganisms first before plants and may pose long term effects on soil quality.

Effects of rotation and interplanting on soil bacterial communities and cucumber yield

We studied the effects of rotation and interplanting on soil bacterial communities and crop yields using cucumber as the main vegetable. Onion and garlic were used as interplanting species, and wheat, soybean, villose (more commonly, villous) vetch, clover, and alfalfa were used as rotation plants. T-RFLP techniques were used to show the effects of rotation and interplanting systems on diversity index and richness index of cucumber rhizosphere soil bacterial communities. The results showed that both rotation and interplanting systems increased the richness index and diversity index of soil bacterial community structures, except where alfalfa was used as the rotation plant. The diversity index of the cucumber rhizosphere bacterial community structure was highest in the wheat rotation cropping system, and the richness index of soil bacteria was highest in wheat and clover rotations and in the onion interplanting system. Our results show that rotation and interplanting systems beneficially altered community structures of dominant soil bacteria, and increased cucumber yield and soil bacterial diversity. The best cultivation system to increase cucumber yield utilized onion as an interplanting species and wheat as a rotation plant.

Effect of cinnamic acid on soil microbial characteristics in the cucumber rhizosphere

In order to elucidate the effect of allelochemicals on soil microbial characteristics in the cucumber rhizosphere, the soil microbial biomass and respiration, community functional diversity and RAPD marker diversity as affected by exogenous cinnamic acid were studied. Exogenous cinnamic acid increased soil microbial respiration and the metabolic quotient, but decreased soil microbial biomass-C. Soil microbial community functional diversity and genetic diversity (as indicated by RAPD markers) were also significantly altered by exogenous cinnamic acid. These results suggest that allelochemicals can change soil microbial genetic diversity, biological activity and microbial metabolic activity, which alter soil microbial ecology and accordingly affect the growth of cucumber with accumulation in the soil of allelochemicals.

Interactions between the arbuscular mycorrhizal fungus Glomus mosseae and plant growth-promoting fungi and their significance for enhancing plant growth and suppressing damping-off of cucumber ( Cucumis sativus L.)

This study investigated the interactions between the arbuscular mycorrhizal fungus (AMF) Glomus mosseae and the plant growth-promoting fungi (PGPF) Penicillium simplicissimum GP17-2 and Trichoderma harzianum GT3-2 in relation to their colonization of roots and the rhizosphere of cucumber ( Cucumis sativus L.), and their effect on plant growth and suppression of the damping-off pathogen Rhizoctonia solani. Combined inoculation of T. harzianum with G. mosseae increased the percentage of AMF root colonization, but the presence of P. simplicissimum had no significant effect on mycorrhizal formation. The existence of G. mosseae decreased the population development of T. harzianum in and/or around the roots, however, growth of P. simplicissimum was not affected. Both PGPF isolates were effective in increasing the plant shoot and root dry weight, G. mosseae, however, was not. Nevertheless, co-inoculation of G. mosseae with T. harzianum synergistically enhanced plant growth. A comparable additive effect on plant growth was not observed when P. simplicissimum was combined with G. mosseae. Treating plants with the PGPF either simultaneously with the pathogen or prior to pathogen infection suppressed damping-off disease. G. mosseae was significantly effective in reducing disease severity when inoculated prior to the pathogen but not when inoculated simultaneously with the pathogen. Interestingly, the levels of disease protection achieved by the single inoculation of P. simplicissimum, T. harzianum or G. mosseae were significantly increased by combined inoculation of each PGPF with G. mosseae.

Interactions between the arbuscular mycorrhizal fungus Glomus intraradices and the plant growth promoting rhizobacteria Paenibacillus polymyxa and P. macerans in the mycorrhizosphere of Cucumis sativus

Interactions between the arbuscular mycorrhizal (AM) fungus Glomus intraradices and bacteria from the genus Paenibacillus ( P. macerans and P. polymyxa) were examined in a greenhouse pot experiment with Cucumis sativus with and without organic matter amendment (wheat bran). P. polymyxa markedly suppressed AM fungus root colonization irrespective of wheat bran amendment, whereas P. macerans only suppressed AM fungus root colonization in combination with wheat bran amendment. Dual inoculation with P. macerans and G. intraradices in combination with wheat bran amendment also caused severe plant growth suppression. Inoculation with G. intraradices was associated with increased levels of dehydrogenase activity and available P in the growth substrate suggesting that mycorrhiza formation accelerated the decomposition of organic matter resulting in mobilization of phosphorus. Inoculation with both Paenibacillus species increased all measured microbial fatty acid biomarkers in the cucumber rhizosphere, except for the AM fungus biomarker 16:1ω5, which was reduced, though not significantly. Similarly, inoculation with G. intraradices increased all measured microbial fatty acid biomarkers in the cucumber rhizosphere, except for the Gram-positive bacteria biomarker 15:0 anteiso, which was overall decreased by G. intraradices inoculation. In combination with wheat bran amendment G. intraradices inoculation caused a 39% reduction in the amount of 15:0 anteiso in the treatment with P. polymyxa, suggesting that G. intraradices suppressed P. polymyxa in this treatment. In conclusion, plant growth promoting species of Paenibacillus may have suppressive effects of AM fungi and plant growth, especially in combination with organic matter amendment. The use of an inert plant growth media in the present study allowed us to study rhizosphere microbial interactions in a relative simple substrate with limited interference from other soil biota. However, the results obtained in the present work mainly show potential interactions and should not be directly extrapolated to a soil situation.

Differential effects of Paenibacillus spp. on cucumber mycorrhizas

The effects of 17 Paenibacillus strains on root colonization by Glomus intraradices or Glomus mosseae and plant growth parameters (shoot and root weight) of mycorrhizal cucumber plants were examined. The Paenibacillus strains were originally isolated from mycorrhizal (G. intraradices) and non-mycorrhizal cucumber rhizosphere and/or hyphosphere, except for strain EJP73, which originated from a Pinus sylvestris-Lactarius rufus ectomycorrhiza. Root colonization of cucumber plants by G. intraradices or G. mosseae was unaffected by all seven strains of Paenibacillus polymyxa, but was decreased or increased by four strains of Paenibacillus macerans and strain EJP73 of Paenibacillus sp. Overall, shoot dry weight of cucumber grown in symbioses with either G intraradices or G. mosseae was unaffected by inoculation with all of the Paenibacillus strains, except for strain MB02-429 of P. macerans, which increased the shoot dry weight in the cucumber-G. mosseae symbiosis. On the other hand, several Paenibacillus strains caused altered root growth. Three strains of P. polymyxa and four strains of P. macerans increased the root fresh weight of the cucumber–G. intraradices symbiosis, whereas three strains of P. polymyxa and one strain of P. macerans as well as Paenibacillus sp. EJP73, decreased the root fresh weight of the cucumber–G. mosseae symbiosis. In conclusion, our results show that bacteria from several species of Paenibacillus differentially affect cucumber mycorrhizas.

Detection of Plant-Modulated Alterations in Antifungal Gene Expression in Pseudomonas fluorescens CHA0 on Roots by Flow Cytometry

The biocontrol activity of the root-colonizing Pseudomonas fluorescens strain CHA0 is largely determined by the production of antifungal metabolites, especially 2,4-diacetylphloroglucinol. The expression of these metabolites depends on abiotic and biotic environmental factors, in particular, elements present in the rhizosphere. In this study, we have developed a new method for the in situ analysis of antifungal gene expression using flow cytometry combined with green fluorescent protein (GFP)-based reporter fusions to the phlA and prnA genes essential for the production of the antifungal compounds 2,4-diacetylphloroglucinol and pyrrolnitrin, respectively, in strain CHA0. Expression of phlA-gfp and prnA-gfp in CHA0 cells harvested from the rhizosphere of a set of plant species as well as from the roots of healthy, leaf pathogen-attacked, and physically stressed plants were analyzed using a FACSCalibur. After subtraction of background fluorescence emitted by plant-derived particles and CHA0 cells not carrying the gfp reporters, the average gene expression per bacterial cell could be calculated. Levels of phlA and prnA expression varied significantly in the rhizospheres of different plant species. Physical stress and leaf pathogen infection lowered phlA expression levels in the rhizosphere of cucumber. Our results demonstrate that the newly developed approach is suitable to monitor differences in levels of antifungal gene expression in response to various plant-derived factors. An advantage of the method is that it allows quantification of bacterial gene expression in rhizosphere populations at a single-cell level. To our best knowledge, this is the first study using flow cytometry for the in situ analysis of biocontrol gene expression in a plant-beneficial bacterium in the rhizosphere.

Laboratory Culture Media-Dependent Biocontrol Ability of Burkholderia gladioli strain B543

Cultivation of a biocontrol agent on a certain medium often results in reduced biocontrol efficacy and alters physiological state. In our previous study, Burkholderia gladioli strain B543 with long-term subculture on tryptic soy agar resulted in significantly reduced biocontrol ability against cucumber damping-off caused by P. ultimum. Therefore, we investigated the influence of laboratory culturing media on biocontrol activity and physiological state of Burkholderia gladioli strain B543 by using long-term repeated culture on a certain medium. When isolate B543 were successionally cultured on King`s B agar (KBA), tryptic soy agar, nutrient agar (NA), or soil extract agar more than 20 times, the isolate cultured on KBA or NA showed a significantly enhanced biocontrol efficacy and higher population density in the rhizosphere of cucumber compared to that of the others. However, the isolates cultured on KBA more than 20 times showed the lowest production of protease, siderophore, or antifungal substance(s), measured by skim milk agar, Chrome-Azurol-S agar, and potato dextrose agar amended with 10% of the culture filtrate, respectively. Our results suggest that adaptation to proper culturing medium can alter biocontrol ability and physiological state, and we must consider laboratory media in optimizing the use of biocontrol agents.