The effect of nitrogen and carbon sources on growth of the biocontrol agent Pantoea agglomerans strain CPA-2

AIM: The effect of several nitrogen and carbon sources on the growth of Pantoea agglomerans (strain CPA-2) was studied for the first time. METHODS AND RESULTS: Synthetic nitrogen and carbon sources were tested to obtain a suitable medium. Synthetic yeast extract provided maximum growth and disaccharides such as sucrose, lactose and trehalose improved this growth significantly from 3.2 x 10(9) to 5.5 x 10(9) cfu ml-1. CONCLUSION: Pantoea agglomerans can be produced in a combination of nitrogen sources such as yeast extract with carbohydrates such as sucrose in shake flask and a laboratory fermenter (5 l). SIGNIFICANCE AND IMPACT OF THE STUDY: Results suggest good production of this biocontrol agent on a laboratory scale and the potential of scaling up the process.     

Production of the biocontrol agent Pantoea agglomerans strain CPA-2 using commercial products and by-products

The aim of this paper was to find the nitrogen and carbon sources that provide maximum biomass production of strain CPA-2 of the biocontrol agent Pantoea agglomerans and minimum cost of media, whilst maintaining biocontrol efficacy. To reduce the cost of media, commercial products and by-products were tested. P. agglomerans can be produced using a combination of nitrogen sources such as yeast extract (5 g l(-1)) and dry beer yeast (10 g l(-1)) with inexpensive carbohydrates such as sucrose (10 g l(-1)) and molasses (20 g l(-1)), respectively, maintaining the efficacy of the biocontrol agent against Penicillium digitatum and P. italicum on oranges. The results obtained in this study could be used to provide a reliable basis for a scale-up of this fermentation process to an industrial level.     

The structure of the O-specific polysaccharide of the lipopolysaccharide from Pantoea agglomerans strain FL1

A neutral O-specific polysaccharide consisting of d-rhamnose was obtained by mild acid hydrolysis of the lipopolysaccharide of the plant pathogenic bacterium Pantoea agglomerans strain FL1, a common epiphyte of many plant species, and associated with Pseudomonas savastanoi pv. savastanoi in young and apparently intact olive knots. By means of compositional and methylation analyses, and NMR spectroscopy, the chemical repeating unit of the polymer was identified as a linear tetrasaccharide of the structure:     

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.     

Effect of package and storage conditions on viability and efficacy of the freeze-dried biocontrol agent Pantoea agglomerans strain CPA-2

AIMS: To reduce concentrations of protective and rehydrating media and to evaluate the effect of storage temperature, packaging and atmosphere conditions on the stability of freeze-dried Pantoea agglomerans cells. Efficacy against Penicillium digitatum of freeze-dried cells in orange fruits was also evaluated. METHODS AND RESULTS: Several concentrations of protective and rehydration media were tested to reduce processing costs. Freeze-dried cells were packed in glass vials or plastic bags under vacuum or nitrogen conditions at 4 and 25 degrees C. After 1 and 3 months, efficacy of freeze-dried P. agglomerans against P. digitatum was tested. CONCLUSIONS: The results indicate that it is possible to reduce the concentration of non-fat skimmed milk as a rehydration medium from 10% to 1%, maintaining viabilities of 100%. Moreover, freeze-dried cells could be stored in glass vials or in high barrier plastic bags at 4 degrees C for 3 months while maintaining high viabilities and efficacy against P. digitatum. SIGNIFICANCE AND IMPACT OF THE STUDY: The major obstacle in the commercialization of biocontrol products is the development of a shelf-stable formulated product that retains biocontrol activity at a level similar to that of fresh cells. This study suggests that it is possible to maintain viability and efficacy of freeze-dried P. agglomerans cells for at least 3 months.     

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.     

一株西瓜枯萎病生防菌的鉴定与田间防效

[背景]西瓜枯萎病是由尖孢镰孢菌西瓜专化型(Fusarium oxysproum f.sp.niveum)引起的土传病害,是西瓜最为严重的病害之一.利用拮抗微生物抑制病原菌繁殖,降低其危害程度,已成为西瓜枯萎病防控领域的研究热点.[目的]筛选对西瓜枯萎病病原菌具有拮抗作用的菌株,并探究其抑菌作用,为有效防控西瓜枯萎病提...     

黄瓜枯萎病生防菌HD-087产抗菌物质条件的 优化及抑菌作用初探

【目的】优化比基尼链霉菌HD-087摇瓶发酵条件,提高菌株发酵液对黄瓜枯萎病菌HU-M的抑制率,并通过拮抗试验初步评价发酵液的抑菌作用效果。【方法】采用单因素筛选及正交试验对HD-087的发酵培养基和发酵条件进行优化。经发酵液处理后,光学显微镜观察HU-M菌丝形态和孢子萌发抑制率,测定HU-M菌丝电导率。【结果】改进的发酵培养基配方为:淀粉1.00%、黄豆粉0.80%、酵母粉0.12%、CaCO30.40%。对发酵条件的研究表明:pH为6.8,180 r/min、28°C条件下,250 mL三角瓶装液量为40 mL,接种1 mL种龄为2 d的种子,发酵5 d为最佳培养条件。抑菌结果表明,HD-087产抗菌物质能造成病原菌HU-M菌丝细胞质渗漏,菌丝畸形,分生孢子萌发受抑制,5倍发酵稀释液孢子抑制率达72.1%;除此之外还能引起菌丝电解质渗漏,造成菌丝细胞膜受损。【结论】优化后的摇瓶发酵条件能提高生防菌HD-087发酵液抑菌效果,并且发酵液可破坏细胞膜明显抑制病原菌HU-M生长,具有较大开发应用潜力。     

Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying

The effect of initial cell density, protective agents and rehydration media on the viability of biocontrol agent Pantoea agglomerans CPA-2 when subjected to freeze-drying was studied. Several additives were tested as protective agents against freeze-drying injury. Maximum viability of the bacterial cells was obtained with disaccharides (survival levels > 60%). Freeze-dried samples were rehydrated with several media; the highest percentage viability was obtained with 10% non-fat skim milk (100%+). The effect of initial bacterial load on the final recovery was dependent on protectant but not on rehydration media. Sucrose was an effective protectant when a high initial concentration (10(10) cfu ml(-1) was used; the opposite occurred with non-fat skim milk. The use of 10(10) cfu ml(-1) as an initial concentration, sucrose as a protectant and non-fat skim milk as a rehydration medium enabled 100% of P. agglomerans viability to be conserved after freeze-drying. Results suggest the possibility of achieving a good formulation system for the studied biocontrol agent with a high number of viable cells to be used toward pathogens, which is desirable for the industrial development of the product.     

Effects of a lipopolysaccharide from Pantoea agglomerans on the cocaine-induced place preference

A lipopolysaccharide from Pantoea agglomerans (LPSp) was purified, and its effect on the cocaine-induced place preference was examined in rats. Cocaine (4 mg/kg, i.p.) produced a significant place preference. Administration of LPSp (5 – 1000 μg/kg, i.p.) alone resulted in neither preference nor aversion for either the drug- or saline-associated place. However, pretreatment with LPSp (500 and 1000 μg/kg, i.p.) abolished the place preference that had been induced by cocaine. Furthermore, treatment with LPSp (500 μg/kg, i.p.) abolished cocaine (20 mg/kg, i.p.)-induced locomotor enhancement in mice. These results suggest that while LPSp itself may possess neither reinforcing nor locomotor enhancing effects, it blocks both the reinforcing and the locomotor enhancing effects of cocaine. Therefore, LPSp might be useful in pharmacotherapy for prevention of recurrent cocaine abuse.     

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses"

Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."     

一株内生砖红镰刀菌促进烟草生长和增强青枯病抗性

课题组前期报道了一株对马铃薯具有促生防病作用的内生砖红镰刀菌Fusarium lateritium (FL617)。为拓展该菌株的应用范围,本研究以同为茄科作物的烟草为研究对象,探究了砖红镰刀菌对其生长和抗病的影响。结果表明,与对照组相比,处理组叶表面积、主根数、叶片数、叶绿素a和叶绿素b含量分别提高了5.0、3.9、1.4、1.3和1.3倍;该结果表明砖红镰刀菌对烟草具有促生作用。生测结果表明,砖红镰刀菌增强了烟草对青枯病的抗病性,其青枯病病情指数下降约30%。植物激素合成相关基因表达模式分析,发现处理组植物激素合成相关基因表达显著上调(1.6-39.9倍);用青枯病菌Ralstonia solanacearum感染寄主植物后分析其水杨酸(SA)、茉莉酸(JA)和R基因信号相关基因的转录模式,发现与对照组相比,处理组SA、JA相关基因均显著上调(1.2-8.3倍),仅有一个R基因显著下调(50%)。进一步用GFP标记的菌株进行荧光定殖观察,发现植物根系周围簇生着带有绿色荧光信号的真菌菌丝,表明砖红镰刀菌可以定殖于烟草根系。综上所述,推测砖红镰刀菌F. lateritium能够通过定殖于烟草根系介导植物激素、免疫防御相关基因的表达从而影响植株的生长发育和抗病性。     

5'-Deoxyisopentenyladenosine and other cytokinins in culture filtrates of the bacterium Pantoea agglomerans

A Pantoea agglomerans isolate from barley seeds, selected for inducing growth promotion in tomato test plants, was found to excrete several hormones of the cytokinin class into its culture medium. In addition to isopentenyladenine, isopentenyladenosine and the 2-methylthiol derivates of these, an unknown compound with affinity to anticytokinin antibodies was also isolated. Mass spectroscopy indicated the structure of this to be a deoxyisopentenyladenosine. The structure of 9-(5'-deoxy- β - d -ribofuranosyl)-6-(3-methyl-2-butenylamino)purine was verified after synthesis of standards and analysis with GC–MS. The synthesized 5'-deoxyisopentenyl-adenosine showed activity in the Amaranthus bioassay, specific for cytokinins. To our knowledge this is the first report of a naturally occurring cytokinin containing 5'-deoxyribose.     

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.     

Effect of leaf surface waxes on leaf colonization by Pantoea agglomerans and Clavibacter michiganensis

To evaluate the influence of leaf cuticular waxes on bacterial colonization of leaves, bacterial colonization patterns were examined on four glossy maize (Zea mays L.) mutants that were altered in their cuticular wax biosynthesis. Mutant gl3 was indistinguishable from the wild-type maize in its ability to foster colonization by the two bacterial species, Pantoea agglomerans and Clavibacter michiganensis subsp. nebraskensis. In contrast, the other three mutants supported the development of populations that significantly differed in size from those on the wild type. Mutant gl5 gl20 supported smaller populations of P. agglomerans, but not C. michiganensis, while mutant gl1 supported larger populations of C. michiganensis but not P. agglomerans. Mutant gl4 supported larger populations of both bacterial species. The exceptional ability of mutant gl4 to support bacterial colonization was hypothesized to result from the lower density of the crystalline waxes on gl4 than on the wild type, because a reduced crystal density could promote capillary water movement and water trapping among the wax crystals. This hypothesis was supported by the demonstration that the mechanical introduction of gaps among the wax crystals of the wild-type leaves resulted in the establishment of larger P. agglomerans populations on the altered leaves. These results provide the first direct evidence that leaf surface waxes affect bacterial leaf colonization at various stages of colonization and in a bacterial species-dependent manner.     

Evaluation of neem seed powder for Fusarium wilt and Meloidogyne control on tomato

Fusarium wilt and root-knot are important diseases of tomato. The use of chemical is becoming less appealing because of the health implications. Also, the chemicals required are often not within the reach of farmers in most of the developing part of the world. This research is aimed at finding an alternative mode of control. Tomato variety Roma VF inoculated with Meloidogyne and Fusarium were treated with 2 g/kg soil neem seed powder in the screenhouse and 2 Mg ha ^{? 1} in the field. An untreated and Furadan treated plot in the field served as control. Neem seed powder significantly reduced the disease severity of Fusarium and root-knot in both screenhouse and field. Results suggest the possible use of neem seed powder for control of the root-knot nematodes - Fusarium wilt disease complex.     

苦豆子根瘤内生细菌分离及其对棉花枯萎病的生物防治效果测定

对从塔里木盆地苦豆子中分离得到的内生细菌进行皿内涂布拮抗实验、对峙培养法拮抗实验、胞外分泌物拮抗性测定和盆栽控病实验等研究,结果表明塔里木盆地苦豆子根瘤中存在大量的棉花枯萎病菌拮抗性内生细菌。皿内涂布法筛选结果表明60株苦豆子根瘤内生细菌中有48株相对抑菌率超过50%。对峙培养法对48株拮抗性内生细菌进一步筛选的结果表明,对棉花枯萎病菌抑菌距离超过20 mm的菌株有40株。40株拮抗性内生细菌胞外分泌物对棉花枯萎病菌的抑菌距离超过5 mm的有26株。KDRE12和KDRE41对棉花枯萎病的盆栽控病平均防效分别为67.11%和72.65%,具有应用潜力。     

防治苦瓜枯萎病的拮抗放线菌分离筛选及鉴定

以苦瓜枯萎病菌为靶标菌,通过对峙培养试验和发酵滤液抑菌试验对分离自苦瓜根际土壤的放线菌进行筛选。候选菌株0250具有广谱抗真菌活性,根据培养特征、生理生化特性以及与同源性相近的菌株进行平均核苷酸一致性分析,被鉴定为Streptomyces rhizosphaericus,并评估了该菌株在温室和田间对苦瓜的促生长和防治枯萎病效果。结果表明: 链霉菌菌株0250对苦瓜枯萎病菌的平板抑制率为69.2%,对17种植物病原真菌的平板抑制率达64.3%~85.6%;该菌株的菌悬液处理能促进盆栽和田间苦瓜植株根、茎生长发育,提升产量,对苦瓜枯萎病的防病效果分别为66.9%和61.5%。预先用菌株0250菌悬液处理土壤再接种病原菌,对土壤尖镰孢菌数量抑制率达62.1%,显著提高了苦瓜幼苗苯丙氨酸解氨酶、过氧化物酶和β-1,3-葡聚糖酶活性以及根系活力。总之,菌株0250是一株对苦瓜枯萎病具有巨大生防潜力的放线菌资源。     

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector

A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.