Characterization of Pectobacterium carotovorum subsp. carotovorum Causing Watermelon Soft Rot Disease in Iran

The aim of this study was characterized Pectobacterium carotovorum subsp. carotovorum (Pcc) the causal pathogen of watermelon soft rot disease in Iran. Of fifty bacterial isolates with white grey and convex colonies on nutrient agar obtained from symptomatic watermelon, ten isolates were selected for further tests. Pathogenicity tests results showed that all test isolates developed typical water‐soak symptoms after 2 days and signs of soft rot began 4 days after inoculation on watermelon fruits. Based on the phenotypic properties, the isolates were identified as Pectobacterium carotovorum subsp. carotovorum. The 16S rDNA sequences of isolates were 99% similar to the corresponding 16S rDNA sequence of the reference Pcc isolate. BOX and ERIC‐PCR analysis indicated that genetic diversity was present among the isolated Pcc isolates did not relate to the geographic location isolated from. To the best of our knowledge, this is the first study of biochemical and genotypic characterization of Pcc isolates the causal agents of soft rot disease on watermelon, in Iran.     

胡萝卜软腐果胶杆菌鞭毛钩基因flgK 的功能

【目的】为了研究鞭毛钩基因flgK在胡萝卜软腐果胶杆菌胡萝卜亚种(Pectobacterium carotovorum subsp.carotovorum,P.c.c)的功能。【方法】本研究采用两亲同源交换法构建了基因缺失突变体ΔflgKpcc并构建了互补菌株ΔflgKpcc-KH,测定突变体及其互补菌株的菌体形态、运动性、致病因子、致病性等表型。【结果】与野生菌株PccS1相比,ΔflgKpcc鞭毛缺失,菌体易沉降,在0.3%半固体培养基上运动能力明显降低,生长速率无明显变化,但是纤维素酶和蛋白酶的活性、生物膜形成能力明显下降,对感病寄主的致病力显著减弱。基因互补可以使上述突变表型恢复。【结论】实验表明,鞭毛基因flgK突变导致了菌体的运动性降低、病原菌毒性相关的酶活力下降,从而导致致病力下降。     

胡萝卜软腐果胶杆菌CpxP蛋白纯化及抑菌功能鉴定

胡萝卜软腐果胶杆菌是世界十大植物病原菌之一,主要侵染十字花科的经济作物和观赏花卉。文中从胡萝卜软腐果胶杆菌的基因组中克隆1个抗菌基因cpxP(GeneID:29704421),将其构建在原核表达质粒pET-15b上,并转化至大肠杆菌Escherichia coli BL21 (DE3)进行表达,经纯化后进行稳定性和抑菌实验。结果显示,IPTG的诱导终浓度为1mmol/L,实现了蛋白的高效外源表达,纯化后电泳无杂蛋白残留,且该蛋白具有良好的热稳定性和pH稳定性。CpxP蛋白抑菌试验结果显示其对胡萝卜切片的抑菌率可达到44.89%,对马铃薯切片的抑菌率可达到59.41%。为进一步解释其抑菌机理,研究该蛋白的空间结构可为软腐病的防治和新型蛋白农药靶点研究提供新思路。     

Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot on Paprika in Korea

During summer 2011 in South Korea, severe fruit rot of paprika was observed, causing severe economic losses in paprika production. Symptoms of fruit and pedicel decay were consistent with symptoms caused by Pectobacterium carotovorum subsp. brasiliense (Pcb) as recently described in Brazil, the United States, Israel and South Africa. Physiological analysis and pathogenicity test of strains isolated from paprika fruit revealed that the pathogen was the bacterium Pcb. Sequencing and phylogenetic analysis of the 16S rDNA and partial 16S–23S rDNA intergenic spacer region confirmed that the isolates were Pcb. This is the first report of Pcb in Korea, which has a significant economic impact on Korean paprika production.     

Efficacy of potent antagonistic yeast Wickerhamiella versatilis against soft rot disease of potato caused by Pectobacterium carotovorum subsp. carotovorum

Abstract

In this study, an antagonistic yeast isolate, Wickerhamiella versatilis was considered as a promising biocontrol agent against Pectobacterium carotovorum subsp. Carotovorum (Pcc) the causal agent of soft rot disease of potato. Antagonistic yeast inhibited the growth of Pcc in vitro, and reducing the soft rot severity of infected potato tubers (cv. Diamant) under greenhouse conditions. Consequently, cellulase and pectinase hydrolytic activities in infected potato tubers with yeast?+?Pcc were decreased compared with infected tubers with Pcc. The histological characterization of treated potato tubers with antagonistic yeast W. versatilis using scanning electron microscope showed the accumulation of extracellular substances that may induce plant resistant and protects potato tubers from hydrolysis and damages. This study may introduce the possibility of using the antagonistic yeast isolate, as a biocontrol agent against soft rot of potato tubers.     

Bacteriophages Isolated in China for the Control of Pectobacterium carotovorum Causing Potato Soft Rot in Kenya

Soft rot is an economically significant disease in potato and one of the major threats to sustainable potato production. This study aimed at isolating lytic bacteriophages and evaluating methods for and the efficacy of applying phages to control potato soft rot caused by Pectobacterium carotovorum. Eleven bacteriophages isolated from soil and water samples collected in Wuhan, China, were used to infect P. carotovorum host strains isolated from potato tubers showing soft rot symptoms in Nakuru county, Kenya. The efficacy of the phages in controlling soft rot disease was evaluated by applying individual phage strains or a phage cocktail on potato slices and tubers at different time points before or after inoculation with a P. carotovorum strain. The phages could lyse 20 strains of P. carotovorum, but not Pseudomonas fluorescens control strains. Among the 11 phages, Pectobacterium phage Wc5 r, interestingly showed cross-activity against Pectobacterium atrosepticum and two phage-resistant P. carotovorum strains. Potato slice assays showed that the phage concentration and timing of application are crucial factors for effective soft rot control. Phage cocktail applied at a concentration of 1 9 109 plaque-forming units per milliliter before or within an hour after bacterial inoculation on potato slices, resulted in C 90%reduction of soft rot symptoms. This study provides a basis for the development and application of phages to reduce the impact of potato soft rot disease.     

Combining flow cytometry and gfp reporter gene for quantitative evaluation of Pectpbacterium carotovorum ssp. carotovorum in Ornithogalum dubium plantlets

Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.     

Characterisation of Pectobacterium wasabiae and Pectobacterium carotovorum subsp. carotovorum isolates from diseased potato plants in Finland

To identify bacteria causing soft rot and blackleg in potato in Finland, pectinolytic enterobacteria were isolated from diseased potato stems and tubers. In addition to isolates identified as Pectobacterium atrosepticum and Dickeya sp., many of the isolated strains were identified as Pectobacterium carotovorum subsp. carotovorum. Phylogenetic analysis and biochemical tests indicated that one of the isolates from potato stems resembled Pectobacterium wasabiae. Furthermore, two blackleg‐causing P. carotovorum strains recently isolated in Europe clustered with P. wasabiae, suggesting that at least some of these isolates were originally misidentified. All the other Finnish P. carotovorum isolates resembled the subsp. carotovorum type strain in biochemical tests but could be clustered into two distinct groups in the phylogenetic analysis. One of the groups mainly contained strains isolated from diseased tubers, whereas the other mainly included isolates from potato stems. In contrast to the tuber isolates, the stem isolates lacked genes in Type III secretion genes, were not able to elicit a hypersensitive response in tobacco leaves and produced only small amounts of autoinducers in the stationary phase in vitro. P. wasabiae isolate was able to cause similar amount of blackleg‐like symptoms as P. atrosepticum in a field experiment with vacuum‐infiltrated tubers, whereas both P. atrosepticum and P. carotovorum isolates reduced emergence and delayed growth more than P. wasabiae. Our findings confirm the presence of P. wasabiae in Finland and show that the Finnish P. carotovorum subsp. carotovorum isolates can be divided into two groups with specific characteristics and possibly also different ecologies.     

Batch and fed-batch bioreactor studies for the enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428

The effect of dissolved oxygen (DO) level and pH (controlled/uncontrolled) was first studied to enhance the production of novel glutaminase-free L-asparaginase by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor. The optimum level of DO was found to be 20%. The production of L-asparaginase was found to be maximum when pH of the medium was maintained at 8.5 after 12?h of fermentation. Under these conditions, P. carotovorum produced 17.97?U/mL of L-asparaginase corresponding to the productivity of 1497.50?U/L/h. The production of L-asparaginase was studied in fed-batch bioreactor by feeding L-asparagine (essential substrate for production) and/or glucose (carbon source for growth) at the end of the reaction period of 12?h. The initial medium containing both L-asparagine and glucose in the batch mode and L-asparagine in the feeding stream was found to be the best combination for enhanced production of glutaminase-free L-asparaginase. Under this condition, the L-asparaginase production was increased to 38.8?U/mL, which corresponded to a productivity of 1615.8?U/L/h. The production and productivity were increased by 115.8% and 7.9%, respectively, both of which are higher than those obtained in the batch bioreactor experiments.     

Genome Sequence of Pectobacterium carotovorum subsp. carotovorum Strain PCC21, a Pathogen Causing Soft Rot in Chinese Cabbage

Pectobacterium carotovorum is a plant-pathogenic enterobacterium responsible for soft rot in various commercially important plants. Here we report the complete genome sequence and automatic annotation of strain PCC21.     

Ectopically expressed sweet pepper ferredoxin PFLP enhances disease resistance to Pectobacterium carotovorum subsp. carotovorum affected by harpin and protease‐mediated hypersensitive response in Arabidopsis

Plant ferredoxin‐like protein (PFLP) is a photosynthesis‐type ferredoxin (Fd) found in sweet pepper. It contains an iron–sulphur cluster that receives and delivers electrons between enzymes involved in many fundamental metabolic processes. It has been demonstrated that transgenic plants overexpressing PFLP show a high resistance to many bacterial pathogens, although the mechanism remains unclear. In this investigation, the PFLP gene was transferred into Arabidopsis and its defective derivatives, such as npr1 (nonexpresser of pathogenesis‐related gene 1) and eds1 (enhanced disease susceptibility 1) mutants and NAHG‐transgenic plants. These transgenic plants were then infected with the soft‐rot bacterial pathogen Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovora ssp. carotovora, ECC) to investigate the mechanism behind PFLP‐mediated resistance. The results revealed that, instead of showing soft‐rot symptoms, ECC activated hypersensitive response (HR)‐associated events, such as the accumulation of hydrogen peroxide (H₂O₂), electrical conductivity leakage and expression of the HR marker genes (ATHSR2 and ATHSR3) in PFLP‐transgenic Arabidopsis. This PFLP‐mediated resistance could be abolished by inhibitors, such as diphenylene iodonium (DPI), 1‐l ‐trans‐epoxysuccinyl‐leucylamido‐(4‐guanidino)‐butane (E64) and benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylketone (z‐VAD‐fmk), but not by myriocin and fumonisin. The PFLP‐transgenic plants were resistant to ECC, but not to its harpin mutant strain ECCAC5082. In the npr1 mutant and NAHG‐transgenic Arabidopsis, but not in the eds1 mutant, overexpression of the PFLP gene increased resistance to ECC. Based on these results, we suggest that transgenic Arabidopsis contains high levels of ectopic PFLP; this may lead to the recognition of the harpin and to the activation of the HR and other resistance mechanisms, and is dependent on the protease‐mediated pathway.     

The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

The evaluation of novel chromogenic substrates for the detection of lipolytic activity in clinical isolates of Staphylococcus aureus and MRSA from two European study groups

Pinellia ternata , a traditional Chinese herb that has been used in China for over 1000 years, is susceptible to a soft rot disease, which may cause major loss of yield. The use of bacteria as potential antagonists against Pectobacterium carotovorum SXR1, the causal agent of the disease on P. ternata , was evaluated. Altogether, 1107 candidate bacteria were isolated from the rhizosphere and surface-sterilized plants of P. ternata . In Petri dish tests, 55 isolates inhibited the growth of strain SXR1, and 21 of these reduced the disease development on P. ternata slices by over 50%. Four selected antagonists significantly reduced the disease incidence on tissue culture seedlings, and also prevented the disease on the transplants. Agonist P-Y2-2 yielded a good prevention level of 81.9%. The four antagonists rapidly colonized the tissue culture seedlings and transplants, whereas greater populations of the antagonists (10⁷–10⁹ CFU g⁻¹ fresh tissues) were observed in the seedlings and in the preinoculated transplants than in those inoculated during transplanting. The use of pathogen-free tissue culture seedlings pre-inoculated with antagonist may provide a strategy for production of P. ternata plantlets resistant to soft rot disease. This is the first report on the efficacy of biocontrol agents against pathogens on P. ternata .     

Inactivation of the pgsA Gene Affects Biosynthesis and Secretion of Alkaline Phosphatase in Escherichia coli

Inactivation of pgsA, which is responsible for biosynthesis of anionic phospholipid phosphatidyl-glycerol (PG), was shown to affect biosynthesis and secretion of alkaline phosphatase (PhoA) in Escherichia coli. A decrease in PG, but not in total anionic phospholipids, correlated with reduction of PhoA secretion, suggesting the role of PG in this process. A dramatic decrease in PG (from 18 to 3, but not 8, percent of the total phospholipids) inhibited not only secretion, but also synthesis of PhoA. In addition, pgsA inactivation expedited repression of PhoA synthesis by exogenous orthophosphate.     

Disruption of the ste22 Gene Encoding a Glycosyltransferase and Its Function in Biosynthesis of Ebosin in Streptomyces sp. 139

Streptomyces sp.139 produces an exopolysaccharide (EPS) designated Ebosin with remarkable anti-rheumatic arthritis activity in vivo. The ste (Streptomyces eps) gene cluster required for Ebosin biosynthesis has been identified. According to similarities with other proteins in the database, ste22 shows high homology with glycosyltransferases originated from different microorganisms. In this study, the ste22 gene was disrupted by double crossover via homologous recombination. The EPS produced by the mutant strain Streptomyces sp.139 (ste22⁻) has a different monosaccharide composition profile in comparison with that of Ebosin. This derivative of Ebosin retained the original antagonistic activity of IL-1R in vitro but lost the bioactivities of anti-inflammation and pain relief in vivo.