Biological Control of Bacterial Wilt of Bean Using a Bacterial Endophyte, Pantoea agglomerans

Indoor studies were conducted to determine the potential use of Pantoea agglomerans isolate LRC 8311 as a biocontrol agent for control of bacterial wilt of bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens. Soaking seeds of great northern bean cv. US1140 in a suspension of 3 × 10⁸ cfu/ml P. agglomerans resulted in thorough endophytic colonization of the entire bean seedling from root to apical stem after 7 days, regardless of whether the inoculated seeds were hilum injured or not. Colonization of seedlings by P. agglomerans increased seedling height after 10 days, and had no negative effect on seedling emergence. Treatment of hilum‐injured bean seeds of great northern bean cv. US1140 or navy bean cv. Morden003 with a mixture of P. agglomerans + C. flaccumfaciens pv. flaccumfaciens resulted in a high rate of colonization of seedlings by P. agglomerans, reduced frequency of infection by C. flaccumfaciens pv. flaccumfaciens, improved seedling emergence and height, and reduced disease severity, compared with seeds treated with the wilt pathogen alone. Application of P. agglomerans as a soil drench 24 h after planting was also effective in suppressing bacterial wilt in some instances, but was generally not as effective as seed treatment. The study suggests that seed treatment with P. agglomerans may be an effective and practical method for control of bacterial wilt of bean.     

Rhizome development ofPhragmites australis in a reed community

Variations in the height, shoot density, biomasses of above- and below-ground parts and rhizome distributions ofPhragmites australis were investigated along a line-transect in a reed community at Yufutsu Mire, Hokkaido. Relationships of performance of the reed plants to soil conditions and species compositions were also examined. Three types of rhizome development were recognized in reed plants; (1) the central part of the reed community, characterized by well developed rhizomes and dense aerial shoots, (2) the intermediate part, characterized by development of rhizomes along both the peat and surface layers and very dense aerial shoots, and (3) the marginal part, characterized by development of rhizomes only along the peat layer and sparse aerial shoots. Observation showed that rhizomes in the surface layer actively produced aerial shoots, whereas rhizomes in the peat layer contributed to the spreading of their distribution range. With the growth of rhizomes, organic debris originating from dead rhizomes and roots accumulated in the mineral soil to promote organic soil formation. In dense parts of the reed stand, species composition was poor because of the shading and litter accumulation by reed plants. The effects of microtopography and water level on the establishment of reed seedlings were also considered.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

扎龙湿地不同生境芦苇种群根茎构件的年龄结构

芦苇是典型的长根茎型克隆植物,天然种群主要依靠根茎的营养繁殖进行更新。采用单位土体挖掘取样,按实际生活年限划分根茎龄级的方法,对扎龙湿地芦苇种群不同龄级根茎进行调查。结果表明,6月份,4个生境芦苇种群根茎构件均由2—6a的5个龄级组成,7—10月份均由1—6a的6个龄级组成。6—10月份,1a根茎长度比率逐渐增加;除盐碱生境略有增加外,其他3个生境2a、3a比率均小幅减少;4—6a比率逐渐减小,根茎长度比率均以3a最大,依次是2a,4a,1a,5a,最高的6a最小。1—3a根茎生物量比率逐渐增加;4—6a比率逐渐减小,均以3a根茎生物量比率最大,依次是4a,2a,5a,6a,以最低1a最小。不同龄级根茎长度和生物量比率与返青后实际生长时间之间均较好地符合直线函数关系(R~20.91,P0.01;R~20.81,P0.05),6—9月份,根茎长度呈衰退型年龄结构,10月份又转为稳定型结构,整个生长季根茎生物量均为衰退型结构。不同龄级根茎构件在芦苇种群中的地位和作用不同,根茎构件的年龄结构蕴含着种群调节的重要信息。     

Performance evaluation of potent phosphate solubilizing bacteria in potato rhizosphere

Three phosphate solubilizing bacterial isolates identified as Pantoea agglomerans strain P5, Microbacterium laevaniformans strain P7 and Pseudomonas putida strain P13 were assessed for mutual relationships among them, competitiveness with soil microorganisms and associations with plant root using luxAB reporter genes for follow-up studies. Synergism between either P. agglomerans or M. laevaniformans, as acid-producing bacteria, and P. putida, as a strong phosphatase producer, was consistently observed both in liquid culture medium and in root rhizosphere. All laboratory, greenhouse and field experiments proved that these three isolates compete well with naturally occurring soil microorganisms. Consistently, the combinations of either P. agglomerans or M. laevaniformans strains with Pseudomonas putida led to higher biomass and potato tuber in greenhouse and in field trials. It is conceivable that combinations of an acid- and a phosphatase-producing bacterium would allow simultaneous utilization of both inorganic and organic phosphorus compounds preserving the soil structure.     

Detection of virulence and pathogenicity genes in selected phytopathovars

Plant pathogenic organisms are known to infect host cell using various range of secretory proteins. Amongst all other secretion systems, type III secretion system (T3SS) is a key mechanism for bacterial pathogenesis for establishing and maintaining infection into the host. Expression levels of seven genes viz. avrXacE1, avrXacE2, hpaA and hrpG along with bacterial endogenous control lrp (leucine-responsive protein) were studied. The pathogenic organisms selected for the present study includes Enterobacter cloacae, Enterobacter spp., Pantoea ananatis, Xanthomonas campestris pv. Citri, Pantoea agglomerans, Ochrobactrum anthropi and Erwinia chrysanthemi. P. agglomerans and Enterobacter spp. gave high expression of above-mentioned virulence genes compared to Xanthomonas, while E. cloacae and P. ananatis showed similar expression with that of Xanthomonas. The detailed relationship of the expression profiles with respect to the selected organisms is discussed.     

Bacterial Community Composition of Biological Degreasing Systems and Health Risk Assessment for Workers

Biological degreasing system is a new technology based on the degradation capabilities of microorganisms to remove oil, grease, or lubricants from metal parts. No data is available about the potential biological health hazards in such system. Thus, a health risk assessment linked to the bacterial populations present in this new degreasing technology is, therefore, necessary for workers. We performed both cultural and molecular approaches in several biological degreasing systems for various industrial contexts to investigate the composition and dynamics of bacterial populations. These biological degreasing systems did not work with the original bacterial populations. Indeed, they were colonized by a defined and restricted group of bacteria. This group replaced the indigenous bacterial populations known for degrading complex substrates. Klebsiella pneumoniae, Klebsiella oxytoca, Pseudomonas aeruginosa, and Pantoea agglomerans were important members of the microflora found in most of the biological degreasing systems. These bacteria might represent a potential health hazard for workers.     

Acid tolerance response induced in the biocontrol agent Pantoea agglomerans CPA-2 and effect on its survival ability in acidic environments

The aim of this work was to optimize acid stress conditions for induction of acid tolerance response (ATR) in the biocontrol agent Pantoea agglomerans and study the effect of ATR induced on the ability to survive under acidic conditions. Initially, Pantoea agglomerans was grown in mild acidic conditions (pH 6.0, 5.5, 5.0 and 4.0) in order to induce ATR. The highest ATR was induced at initial pH of 5 using malic or citric acid. A first in vitro experiment was carried out. Thus, basal liquid medium at different pHs (3.0, 3.5, 4.0 and non-acidified) were then inoculated with acid-adapted and non-adapted inocula of P. agglomerans and survivals were examined during incubation at 25 or 4 °C. It was found that acid adaptation enhanced the survivals of Pantoea agglomerans CPA-2 cells at pH levels at which the cells were unable to grow (<3.5 and 4.0, at 25 and 4 °C, respectively). In contrast, in pH levels at which the cells were able to grow (pH 4.0 at 25 °C and non-acidified medium at 25 and 4 °C) no-differences were found between adapted and non-adapted cells. In in vivo tests, adapted and non-adapted cells were inoculated in wounds on mandarins and pome fruits. No differences were found between adapted and non-adapted cells and biocontrol efficacy was maintained. The present study demonstrated that exposure of Pantoea agglomerans to mild acidic conditions could induce acid resistance in this biocontrol agent.     

Rhizome age structure of three populations ofPhragmites australis (Cav.) Trin. ex Steud.: Biomass and mineral nutrient concentrations

The rhizome age structure for populations ofPhragmites australis (Cav.) Trin. exSteud. was determined at three sites of the T?eboň Biosphere Reserve (Czech Republic). These sites were classified according to plant species composition and phosphorus availability as oligotrophic (Branná), mesotrophic (Ro?mberk East) and hypertrophic (Ro?mberk West).P.australis stands at these sites were expanding, stabilized and retreating, respectively. Rhizomes were sampled within the terrestrial parts of the reed stands (at a water depth of about 10 cm). The rhizomes were dated according to the position in the branching hierarchy and to supplementary morphological criteria. The analysis of the branching pattern revealed that at the sites studied, the rhizomes typically branch only once a year. The longest life span of rhizomes (six years) was found for the stable reed population at Ro?mberk East. The reduced rhizome life span at Ro?mberk West (four to five years), accompanied by a reduced branching frequency of horizontal rhizomes, was ascribed to a greater incidence of anaerobic conditions associated with the permanent flooding of the hypertrophic organic soil. The maximum life span of the Branná population (four years) probably corresponds to the total age of the reed population. The concentrations of nitrogen, phosphorus, and potassium in the tissue decreased with rhizome age while those of calcium and magnesium increased. The effect of rhizome age on mineral-nutrient concentrations was more pronounced than the effect of site on these concentrations. The mineral nutrient concentrations were not closely associated with reed performance.     

Settlement of the diazotrophic,phytoeffective bacterial strain Pantoea agglomerans on and within winter wheat: An investigation using ELISA and transmission electron microscopy

The aim of this study was to investigate the ability of Pantoea agglomerans, a plant growth-promoting bacterium, to colonize various regions and tissues of the wheat plant (Triticum aestivum L.) by using different inoculation methods and inoculum concentrations. In addition, the enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) were used to determine: (a) the ability of the bacterial cells to grow and survive both on the surface and within internal tissue of the plant and (b) the response of the plant to bacterial infection. After inoculation, cells of the diazotrophic bacterial strain P. agglomerans were found to be located in roots, stems and leaves. Colony development of bacterial cells was only detected within intercellular spaces of the root and on the root surface. However, single bacterial cells were observed in leaves and stems on the surface of the epidermis, in the vicinity to stomatal cells, within intercellular spaces of the mesophyll and within xylem vessels. Inoculated bacterial cells were found to be able to enter host tissues, to multiply in the plant and to maintain a delicate relationship between endophyte and host. The density of bacterial settlement in the plant in all experiments was about 10⁶ to 10⁷ cells per mL root or shoot sap. Establishment was confirmed by a low coefficient of variation of ELISA means at these concentrations.     

Characterization of co-culture of Aeromonas and Pseudomonas bacterial biofilm and spoilage potential on refrigerated grass carp (Ctenopharyngodon idellus)

Aeromonas and Pseudomonas are important bacterial species involved in spoilage of refrigerated freshwater fish. In this study, 10 Aeromonas and seven Pseudomonas bacterial strains were isolated from spoiled grass carp and identified. Twelve of seventeen bacterial strains showed high potential of biofilm formation and 14 of 17 can produce extracellular protease. In order to explore the spoilage capacity of dual-species, the sterile grass carp fillets were inoculated with mono- and dual-species of Aeromonas salmonicida and Pseudomonas azotoformans strains. The results revealed significantly higher levels of the total viable count and total volatile basic nitrogen in dual-species as compared to mono-species from day 6. The higher contents of histamine, cadaverine and serious degradation in muscles tissue were also observed in dual-species after 10 days of storage. Results of in vitro experiments showed that the co-culture of A. salmonicida and P. azotoformans significantly increased the bacterial maximum growth rate, promoted the biofilm formation and improved the spoilage capacity of bacterial strains. This study has revealed that the co-culture of Aeromonas and Pseudomonas bacterial strains accelerated spoilage process of grass carp and increased biofilm formation. It indicates that the mixed-cultures of spoilage micro-organisms pose a huge threat to food industry.     

Biological control of phytopathogenic bacteria Pantoea agglomerans and Erwinia chrysanthemi using 100 essential oils

Plants interact with a wide variety of pathogenic organisms by virtue of their sessile lifestyle. The Pantoea agglomerans and Erwinia chrysanthemi are major plant pathogen amongst them. They are known to cause significant losses in many crop plants. In the present study, bacteria isolated from infected Cajanus cajan and Arachis hypogaea seed are identified by 16S rDNA sequencing as P. agglomerans and E. chrysanthemi, respectively. In vitro antimicrobial properties of 100 essential oils (EOs) were evaluated against P. agglomerans and E. chrysanthemi. The chemical composition of most active EOs was investigated by gas chromatography–mass spectral analysis. The potential properties of these EOs as ecofriendly and economical biocontrol in agriculture is discussed.     

Interactions between plants and associated bacteria in soils contaminated with heavy metals

Interactions were studied between oat (Avena sativa) and two bacterial species, Bacillus subtilis and Pantoea agglomerans, in soils contaminated with heavy metals (HM), cadmium (50 mg/kg), and lead (200 mg/kg). Exposure to HM resulted in decreased (by 30–50%) length, mass, and ratio of shoot to root dimensions. Inoculation with bacteria lead to restoration and further enhancement of plant productivity, raising it above the level achieved via inoculation of oat in uncontaminated soils. It also reduced HM accumulation by plants. Pure cultures of P. agglomerans accumulate HM more intensively than those of B. subtilis (adsorbing activity was studied for both cells and extracellular metabolites). After the introduction of bacteria, lead, and cadmium content in soil decreased four- to fivefold and two- to threefold, respectively. Protection from HM is attributable to reorganizations in the populations of root-associated bacteria: cell number increases in the rhizoplane while decreasing in the rhizosphere.     

Acylcyclohexanediones and biological control agents: combining complementary modes of action to control fire blight

Acylcyclohexanediones and antagonistic bacteria sprayed alone or in combination have been shown to suppress fire blight of apple and pear. Acylcyclohexanediones, such as prohexadione-calcium and trinexapac-ethyl, increase plant resistance and are effective against the shoot blight phase of the disease. Antagonistic bacteria, such as Pantoea agglomerans, compete with the pathogen (Erwinia amylovora) for space and nutrients on stigmas, which prevents blossom blight. Potential synergistic effects of acylcyclohexanediones with P. agglomerans for fire blight suppression were investigated on leaves and flowers of apple and pear. Acylcyclohexanediones modified the composition of apple nectar and stigmatic secretions, which resulted in moderately higher epiphytic populations of P. agglomerans strain P10c. In experiments in apple orchards, the combination of acylcyclohexanediones and P. agglomerans gave the greatest protection against blossom blight and shoot blight. In pear orchards, under natural infection conditions, a similar result was obtained for the 3 of the 4 years of the experiment.     

The mechanics of bacterial cluster formation on plant leaf surfaces as revealed by bioreporter technology

Bacteria that colonize the leaves of terrestrial plants often occur in clusters whose size varies from a few to thousands of cells. For the formation of such bacterial clusters, two non‐mutually exclusive but very different mechanisms may be proposed: aggregation of multiple cells or clonal reproduction of a single cell. Here we assessed the contribution of both mechanisms on the leaves of bean plants that were colonized by the bacterium Pantoea agglomerans. In one approach, we used a mixture of green and red fluorescent P. agglomerans cells to populate bean leaves. We observed that this resulted in clusters made up of only one colour as well as two‐colour clusters, thus providing evidence for both mechanisms. Another P. agglomerans bioreporter, designed to quantify the reproductive success of bacterial colonizers by proxy to the rate at which green fluorescent protein is diluted from dividing cells, revealed that during the first hours on the leaf surface, many bacteria were dividing, but not staying together and forming clusters, which is suggestive of bacterial relocation. Together, these findings support a dynamic model of leaf surface colonization, where both aggregative and reproductive mechanisms take place. The bioreporter‐based approach we employed here should be broadly applicable towards a more quantitative and mechanistic understanding of bacterial colonization of surfaces in general.     

Genotypic comparison ofPantoea agglomeransplant and clinical strains

Background  

Pantoea agglomeransstrains are among the most promising biocontrol agents for a variety of bacterial and fungal plant diseases, particularly fire blight of apple and pear. However, commercial registration ofP. agglomeransbiocontrol products is hampered because this species is currently listed as a biosafety level 2 (BL2) organism due to clinical reports as an opportunistic human pathogen. This study compares plant-origin and clinical strains in a search for discriminating genotypic/phenotypic markers using multi-locus phylogenetic analysis and fluorescent amplified fragment length polymorphisms (fAFLP) fingerprinting.     

Nitrogen and carbohydrate pools of two rhizome types of Phragmites australis (Cav.) Trin. ex Steudel

Carbohydrate and total nitrogen contents as well as free amino acid (FAA) concentrations were determined in distinct types of rhizomes of two genetically homogeneous stands of reed (Phragmites australis (Cav.) Trin. ex Steudel) differing in morphology, productivity and nutrient supply in order to evaluate the storage capacity of vertical rhizomes and expansion rhizomes. The expansion rhizomes possess significantly higher amounts of FAA and of total nitrogen but similar carbohydrate concentrations in comparison to the vertical rhizomes. However, no significant differences were found for total nitrogen, FAA and total carbohydrates between both investigated stands indicating a comparable storage capacity of rhizomes independent of nutrient availability. Only the composition of the FAA pool varied in the alanine/asparagine ratio probably influenced by the oxygen supply of the rhizome/root system.     

Confocal microscopy study to understand Clonostachys rosea and Botrytis cinerea interactions in tomato plants

Clonostachys rosea, a biological control agent for plant diseases, is found in a variety of habitats and colonises and survives in different tissues. This antagonist is effective at controlling grey mould, which is caused by Botrytis cinerea, in different plant species. Despite the existing knowledge regarding the efficiency of C. rosea at biologically controlling grey mould, there are few studies concerning this interaction at the histological level. Therefore, we studied the antagonist–pathogen interactions using confocal microscopy. C. rosea survived in tomato tissues for at least 30 days between 18–30ºC. The antagonist colonised the wounded tomato stems faster and more efficiently than the pathogen. The colonisation of the leaf tissues by C. rosea was slow, and the spore concentration was poor in this experiment. Combined with the pathogen’s direct penetration into the leaves, this slow colonisation could cause the biological control to fail. C. rosea also preyed parasitically upon the pathogen’s hyphae, penetrated the tomato’s leaf tissue through the stomata and colonised the stem’s intercellular spaces. Root colonisation was abundant, with a dense hyphae network forming between epidermal cell junctions. This observation provided evidence that the fungus can penetrate via the roots. This paper will help to better define an application strategy for C. rosea in tomato propagation, with the goal of biological control or growth promotion, because to understand how the antagonist survives and interacts in its habitat will define how and when to apply it.     

Overlapping expression patterns and differential transcript levels of phosphate transporter genes in arbuscular mycorrhizal,P<Subscript>i</Subscript>-fertilised and phytohormone-treated <Emphasis Type="Italic">Medicago truncatula</Emphasis> roots

A microarray carrying 5,648 probes of Medicago truncatula root-expressed genes was screened in order to identify those that are specifically regulated by the arbuscular mycorrhizal (AM) fungus Gigaspora rosea, by P_i fertilisation or by the phytohormones abscisic acid and jasmonic acid. Amongst the identified genes, 21% showed a common induction and 31% a common repression between roots fertilised with P_i or inoculated with the AM fungus G. rosea, while there was no obvious overlap in the expression patterns between mycorrhizal and phytohormone-treated roots. Expression patterns were further studied by comparing the results with published data obtained from roots colonised by the AM fungi Glomus mosseae and Glomus intraradices, but only very few genes were identified as being commonly regulated by all three AM fungi. Analysis of P_i concentrations in plants colonised by either of the three AM fungi revealed that this could be due to the higher P_i levels in plants inoculated by G. rosea compared with the other two fungi, explaining that numerous genes are commonly regulated by the interaction with G. rosea and by phosphate. Differential gene expression in roots inoculated with the three AM fungi was further studied by expression analyses of six genes from the phosphate transporter gene family in M. truncatula. While MtPT4 was induced by all three fungi, the other five genes showed different degrees of repression mirroring the functional differences in phosphate nutrition by G. rosea, G. mosseae and G. intraradices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.