The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH.     

THE COLIFORM BACTERIA ASSOCIATED WITH POTATO BLACK-LEG AND OTHER SOFT ROTS

Twelve glasshouse trials using twenty-five isolates of coliform soft-rot bacteria obtained from various countries showed that all the isolates produced typical black-leg of potato at temperatures of 76° F. (24.5° C.) or over, while some of them could also produce the disease at temperatures below 66° F. (19° C.).
Thus the isolates fall into two main groups of which the high-temperature group originates mostly from tropical or subtropical countries or from plants grown in heated glasshouses, whereas the low-temperature group is indigenous in the north temperate regions. The high-temperature group comprises Pectobacterium carotovorum (Erwinia carotovora), P. carotovorum var. chrysanthemi (E. chrysanthemi) and P. carotovorum var. aroideae (E. aroideae). The low-temperature group consists of one variety, P. carotovorum var. atrosepticum (E. atroseptica).
This temperature relation provides a possible means of discovering the origin of the black-leg pathogen in crops grown from Scottish seed in certain tropical or subtropical countries.
As a result of this investigation it is considered that all soft-rot coliform bacteria are varieties of a single species, P. carotovorum (Jones) Waldee.     

Pectobacterium carotovorum elicits plant cell death with DspE/F but the P. carotovorum DspE does not suppress callose or induce expression of plant genes early in plant-microbe interactions

The broad-host-range bacterial soft rot pathogen Pectobacterium carotovorum causes a DspE/F-dependent plant cell death on Nicotiana benthamiana within 24 h postinoculation (hpi) followed by leaf maceration within 48 hpi. P. carotovorum strains with mutations in type III secretion system (T3SS) regulatory and structural genes, including the dspE/F operon, did not cause hypersensitive response (HR)-like cell death and or leaf maceration. A strain with a mutation in the type II secretion system caused HR-like plant cell death but no maceration. P. carotovorum was unable to impede callose deposition in N. benthamiana leaves, suggesting that P. carotovorum does not suppress this basal immunity function. Within 24 hpi, there was callose deposition along leaf veins and examination showed that the pathogen cells were localized along the veins. To further examine HR-like plant cell death induced by P. carotovorum, gene expression profiles in N. benthamiana leaves inoculated with wild-type and mutant P. carotovorum and Pseudomonas syringae strains were compared. The N. benthamiana gene expression profile of leaves infiltrated with Pectobacterium carotovorum was similar to leaves infiltrated with a Pseudomonas syringae T3SS mutant. These data support a model where Pectobacterium carotovorum uses the T3SS to induce plant cell death in order to promote leaf maceration rather than to suppress plant immunity.     

THE EFFECTS OF STREPTOMYCIN AND TERRAMYCIN, SINGLY AND IN COMBINATION, ON THE LEAF BLIGHT DISEASE OF MAIZE CAUSED BY BACTERIUM CAROTOVORUUM F. ZEAE SABET

The growth in vitro of Bacterium carotovorum f. zeae Sabet, the cause of the root and stalk rot and leaf blight disease of maize, was inhibited by dihydrostreptiomycin sulphate and by Terramycin (oxytetracycline). Joint applications showed a synergistic action, and the bacterium was most susceptible to a 1:1 mixture of the two antibiotics. This mixture is effective in checking the development of resistant strains which develop rapidly after treatment with streptomycin alone.
Roots of maize seedlings in water cultures, and leaves of older plants readily absorb both antibiotics. High water content of soil favoured absorption; sunlight had no effect. Absorption from leaf applications first increased with higher concentration and frequency of spraying, but after eight daily applications of streptomycin toxic symptoms appeared and there was evidence that the antibiotic was inactivated.
Antibiotic treatment of maize leaves shortly before inoculation with B. carotovorum f. zeae reduced the incidence and severity of leaf blight. The therapeutic effect extends at first both downward and upward but later only upward. Treatment of the leaves did not induce resistance to infection in the stems and roots.     

CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum

Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum.     

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.     

Soft rot Erwinia bacteria in the rhizosphere of weeds and crop plants in Colorado, United States and Scotland

The soft rot coliform bacteria Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica were isolated by an enrichment method from the rhizosphere of many weed species and crop plants, collected in commercial potato fields either currently in potatoes or in a different crop as part of the rotation. Erwinia carotovora was isolated from 24 plant species in Colorado and 47 species in Scotland. Weeds contaminated with E. carotovora were found in fields growing other crops in which potatoes had not been grown for 1–2 and sometimes much longer. Weeds collected from virgin land in Colorado were not contaminated with E. carotovora but in Scotland virgin soils containing weed roots yielded E. carotovora subsp. carotovora . In general, the numbers of contaminated weeds rose from nil or low levels in spring and early summer to considerably higher levels during mid-season, and fell to progressively lower levels later. Erwinia carotovora subsp. carotovora was the predominant organism recovered from the rhizosphere, but E. carotovora subsp. atroseptica was less common, especially in Scotland, and its incidence varied in different seasons depending on factors such as temperature and moisture conditions. The bacteria could apparently persist in the root zone for an extended period of time and may be a source of inoculum to contaminate soft rot erwinia-free seed potato stocks; the origin of the bacteria was uncertain.     

Variation in antibiosis ability, against potato pathogens, of bacterial communities recovered from the endo- and exoroots of potato crops produced under conventional versus minimum tillage systems

The culturable component of bacterial communities found in the endoroot and associated exoroot (root zone soil) was examined in potatoes (Solanum tuberosum L.) grown under either conventional or minimum tillage systems. Bacterial species--abundance relationships were determined and in vitro antibiosis ability investigated to discover whether tillage practice or bacteria source (endo- or exoroot) influenced bacterial community structure and functional versatility. Antibiosis abilities against Phytophthora erythroseptica Pethyb. (causal agent of pink rot of potatoes), Streptomyces scabies (Thaxt.) Waksm. and Henrici) (causal agent of potato common scab), and Fusarium oxysporum Schlecht. Emend. Snyder and Hansen (causal agent of fusarium potato wilt) were selected as indicators of functional versatility. Bacterial community species richness and diversity indices were significantly greater (P = 0.001) in the exoroot than in the endoroot. While both endo- and exoroot communities possessed antibiosis ability against the phytopathogens tested, a significantly greater proportion (P = 0.0001) of the endoroot population demonstrated antibiosis ability than its exoroot counterpart against P. erythroseptica and F. oxysporum. Tillage regime had no significant influence on species-abundance relationships in the endo- or exoroot but did influence the relative antibiosis ability of bacteria in in vitro challenges against S. scabies, where bacteria sourced from minimum tillage systems were more likely to have antibiosis ability (P = 0.0151). We postulate that the difference in the frequency of isolates with antibiosis ability among endoroot versus exoroot populations points to the adaptation of endophytic bacterial communities that favour plant host defence against pathogens that attack the host systemically.     

Identification of a DNA restriction-modification system in Pectobacterium carotovorum strains isolated from Poland

AIMS: Polish isolates of pectinolytic bacteria from the species Pectobacterium carotovorum were screened for the presence of a DNA restriction-modification (R-M) system. METHODS AND RESULTS: Eighty-nine strains of P. carotovorum were isolated from infected potato plants. Sixty-six strains belonged to P. carotovorum ssp. atrosepticum and 23 to P. carotovorum ssp. carotovorum. The presence of restriction enzyme Pca17AI, which is an isoschizomer of EcoRII endonuclease, was observed in all isolates of P. c. atrosepticum but not in P. c. carotovorum. The biochemical properties, PCR amplification, and sequences of the Pca17AI restriction endonuclease and methyltransferase genes were compared with the prototype EcoRII R-M system genes. Only when DNA isolated from cells of P. c. atrosepticum was used as a template, amplification of a 680 bp homologous to the gene coding EcoRII endonuclease. CONCLUSIONS: Endonuclease Pca17AI, having a relatively low temperature optimum, was identified. PCR amplification revealed that the nucleotide sequence of genes for EcoRII and Pca17AI R-M are different. Dcm methylation was observed in all strains of Pectobacterium and other Erwinia species tested. The sequence of a DNA fragment coding Dcm methylase in P. carotovorum was different from that of Escherichia coli. SIGNIFICANCE AND IMPACT OF THE STUDY: Pca17AI is the first psychrophilic isoschizomer of EcoRII endonuclease. The presence of specific Dcm methylation in chromosomal DNA isolated from P. carotovorum is described for the first time. A 680 bp PCR product, unique for P. c. atrosepticum strains, could serve as a molecular marker for detection of these bacteria in environmental samples.     

Complete Genome Sequence of Phytopathogenic Pectobacterium carotovorum subsp. carotovorum Bacteriophage PP1

Pectobacterium carotovorum subsp. carotovorum is a phytopathogen causing soft rot disease on diverse plant species. To control this plant pathogen, P. carotovorum subsp. carotovorum-targeting bacteriophage PP1 was isolated and its genome was completely sequenced to develop a novel biocontrol agent. Interestingly, the 44,400-bp genome sequence does not encode any gene involved in the formation of lysogen, suggesting that this phage may be very useful as a biocontrol agent because it does not make lysogen after host infection. This is the first report on the complete genome sequence of the P. carotovorum subsp. carotovorum-targeting bacteriophage, and it will enhance our understanding of the interaction between phytopathogens and their targeting bacteriophages.     

Antimicrobial effects of sanitizers against planktonic and sessile Listeria monocytogenes cells according to the growth phase

An entomopathogenic bacterium, Xenorhabdus nematophila, is known to have potent antibiotic activities to maintain monoxenic condition in its insect host for effective pathogenesis and ultimately for optimal development of its nematode symbiont, Steinernema carpocapsae. In this study we assess its antibacterial activity against plant-pathogenic bacteria and identify its unknown antibiotics. The bacterial culture broth had significant antibacterial activity that increased with development of the bacteria and reached its maximum at the stationary growth phase. The antibiotic activities were significant against five plant-pathogenic bacterial strains: Agrobacterium vitis, Pectobacterium carotovorum subsp. atrosepticum, P. carotovorum subsp. carotovorum, Pseudomonas syringae pv. tabaci, and Ralstonia solanacearum. The antibacterial factors were extracted with butanol and fractionated using column chromatography with the eluents of different hydrophobic intensities. Two active antibacterial subfractions were purified, and the higher active fraction was further fractionated and identified as a single compound of benzylideneacetone (trans-4-phenyl-3-buten-2-one). With heat stability, the synthetic compound showed equivalent antibiotic activity and spectrum to the purified compound. This study reports a new antibiotic compound synthesized by X. nematophila, which is a monoterpenoid compound and active against some Gram-negative bacteria.     

Seasonal variations in the activity in soil of Phytophthora cactorum, P. syringae and P. citricola in relation to collar rot disease of apple

Phytophthora species were isolated from infested orchard soils using apple fruit as bait at intervals during the periods July 1956 to August 1957, January 1957 to June 1958, and January 1969 to June 1970. When baited soils were kept out of doors P. cactorum and P. citricola were isolated only from April or May to October, when mean temperatures exceeded 8 and 10 °C respectively; P. syringae was isolated in all months except June, July and August.
The results did not suggest that the incidence of these species was particularly associated with apple as a host plant, but the periods of activity of P. cactorum and P. syringae in soils coincided closely with the periods when apple trees were susceptible to infection by either pathogen. With collar rot disease caused by P. cactorum it was considered that, the time of commencement of activity of the pathogen in the soil, together with the availability of water, might be critical in determining the severity of disease outbreaks.     

Detection and characterization of bacteria from the potato rhizosphere degrading N-acyl-homoserine lactone

Quorum sensing plays a role in the regulation of soft rot diseases caused by the plant pathogenic bacterium Pectobacterium carotovorum subsp. carotovorum. The signal molecules involved in quorum sensing in P. carotovorum subsp. carotovorum belong to the group of N-acyl homoserine lactones (AHLs). In our study, we screened bacteria isolated from the potato rhizosphere for the ability to degrade AHLs produced by P. carotovorum subsp. carotovorum. Six isolates able to degrade AHLs were selected for further studies. According to 16S rDNA sequence analysis and fatty acid methyl ester profiling, the isolates belonged to the genera Ochrobactrum, Rhodococcus, Pseudomonas, Bacillus, and Delftia. For the genera Ochrobactrum and Delftia, for the first time AHL-degrading isolates were found. Data presented in this study revealed for the first time that Ochrobactrum sp. strain A44 showed the capacity to inactivate various synthetic AHL molecules; the substituted AHLs were inactivated with a lower efficiency than the unsubstituted AHLs. Compared with the other isolates, A44 was very effective in the degradation of AHLs produced by P. carotovorum subsp. carotovorum. It was verified by polymerase chain reaction, DNA-DNA hybridization, and a lactone ring reconstruction assay that Ochrobactrum sp. strain A44 did not possess AHL lactonase activity. AHL degradation in Ochrobactrum sp. strain A44 occurred intracellularly; it was not found in the culture supernatant. AHL-degrading activity of A44 was thermo sensitive. Experiments in planta revealed that Ochrobactrum sp. strain A44 significantly inhibited the maceration of potato tuber tissue. Since A44 did not produce antibiotics, the attenuation of the decay might be due to the quenching of quorum- sensing-regulated production of pectinolytic enzymes. The strain can potentially serve to control P. carotovorum subsp. carotovorum in potato.     

Isolation and properties of psychrotrophic and psychrophilic, pectolytic strains of Clostridium

Fourteen strains of pectolytic clostridia have been isolated that were capable of growth at 5–10°C in 7 d; two strains were psychrophiles and failed to grow at 20°C in 14 d and the remainder were psychrotrophs. The bacteria formed pectate lyase enzymes and were capable of degrading potato tissue; they are therefore a potential cause of soft rot of potatoes stored at low temperatures. Doubling times for representative strains were 15–19 h at 10°C and 21–53 h at 5°C. Twelve strains were classified with Group I Clostridium species and two strains with Group II. In the case of one strain the mature spores were not released from the sporangium. Electron microscopy of ultrathin sections of this strain showed the presence of disorganized lamellar structures associated with the spore coat.     

Pupal polymorphism in the butterfly Danaus chrysippus (L.): environmental, seasonal and genetic influences

The pupae of the tropical butterfly Danaus chrysippus are either green or pink the switch being operated by a ‘greening’ hormone produced in the larval head. Both environmental and genetic cues are involved in controlling the endocrine mechanism. The environmental factors identified are of two distinct kinds: proximate factors influence pupal colour after the larva has selected its pupation site, whereas ultimate factors are effective at an earlier stage, either prompting choice of pupation site by the larva or priming pupation physiology in a particular direction. Genetic factors preadapt the larva to form a pupa which will be cryptic in the normal or average conditions, climatic or biogeographical, anticipated in its environment. The proximate factors demonstrated are background colour, darkness, light quality (wavelength) and humidity. There is some evidence that substrate texture may also be relevant. Ultimate factors are temperature, humidity and species of larval foodplant. Two closely linked gene loci which govern the phenotype of adult morphs and races either have a pleiotropic effect on pupa colour or are closely linked with other genes which do so. Moreover, the two loci interact epistatically with respect to their pupation effects. Factors producing predominantly green pupae are plant substrates, yellow background, darkness, yellow light, high humidity, high temperature, the b allele at the B locus when homozygous and, on non-plant substrates, the C allele at the C locus. High frequencies of pink pupae result on non-plant substrates, red backgrounds, in blue light, low humidity, low temperatures and in B- and cc genotypes. The C locus alleles, C and c, interact epistatically with the B alleles, their effect on choice of pupation site being determined by linkage phase. Of the two foodplants tested, Calotropis produced a high frequency of green pupae and Tylophora of pinks. The seasonal cycling of rainfall, temperature, availability or condition of foodplant, and gene frequencies are all correlated with oscillations in the frequencies of green and pink pupae. Though genotype influences pupa colour, all genotypes are capable of forming pupae of both colours. The variation can therefore be attributed to an environmental polyphenism superimposed upon a genetic polymorphism. The hormone producing green pupae emanates from the head during the prepupal period. Denied hormonal influence, the pupa is pink. Pupal colour is judged to be aposematic at close range and cryptic at distance.     

A baiting technique for the isolation of bacteria producing starch-hydrolysing enzymes

By baiting litter soils with raw potatoes, species of bacilli producing thermostable amylases and raw starch-degrading amylase were selectively isolated.     

Pseudomonas syringae pv. coriandricola, Incitant of Bacterial Umbel Blight and Seed Decay of Coriander (Coriandrum sativum L.) in Germany

Since 1987 a devastating disease has occurred in coriander in Germany, characterized by dark-brown discoloration of blossoms and umbels. water-soaked and brown spots on leaves and stems, seed decay and willing. Infected tissue always contained large quantities of Gramnegative, rod-shaped, motile bacteria with few polar flagella. Tests for LOPAT reactions showed the bacteria to be positive for levan-production and tobacco hypersensitivity reaction but negative for oxidase reaction, rot of potato slices and arginine dihydrolase. The bacteria failed to produce fluorescent pigment on King's medium B but revealed a blue fluorescence after growing in a liquid medium without Fe, According to further standard nutritional, biochemical and physiological tests the coriander pathogen belongs to Pseudomonas group la. i.e. Pseudomonas syringae . Also, the fatty acid composition revealed a very close similarity to Pseuodomonas syringae . On Biolog plates the coriander strains showed a uniform metabolic pattern and could clearly be distinguished from other Pseudomonas syringae pathovars.
Typical hosts of Pseudomonas syringae pv. syringae were not infected by the coriander pathogen. Also, most tested umbelliferae species reacted resistant towards the pathogen. Typical disease symptoms, such as persistent water-soaked lesions, were incited only a Coriandrum saticum Animi majus and Levisticum offieinale . The studies revealed that the pathogen described is a separate pathovar of Pseudomonas syringae not included in the approved list of P. syringae pathovars. The name Pseudomonas syringae pv. coriandricola is proposed. Strain GSPB 1965 has been deposited in the NCPPB as pathotype strain (no. 3781).     

Diversity of polyamine patterns in soft rot pathogens and other plant-associated members of the Enterobacteriaceae

Polyamine profiles of 91 pectolytic and other plant-associated strains from 30 taxa of the Enterobacteriaceae were obtained by gradient high performance liquid chromatography (HPLC). Pectobacterium carotovorum, basonym Erwinia carotovora, contained a high amount of putrescine and less diaminopropane. Diaminopropane was absent in Pectobacterium chrysanthemi, basonym E. chrysanthemi, whereas cadaverine was present in addition to the major compound putrescine. This chemotaxonomic difference reflects the deepest phylogenetic branching point within the recently emended genus Pectobacterium which lies between the two species P. carotovorum and P. chrysanthemi. Both important soft rot pathogens are easily distinguishable from each other and from the type species of the genus Erwinia as diaminopropane is the only major polyamine compound in E. amylovora. Chemotaxonomic heterogeneity is also emerging with respect to DYE's Amylovora group proposed in an early phytopathological concept.     

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 .     

Bacteria Are Omnipresent on Phanerochaete chrysosporium Burdsall

Bacteria have been isolated from 10 different strains of Phanerochaete chrysosporium, a white rot fungus which degrades lignocellulosic materials. The investigations showed that one or more bacterial species were always associated with the fungus. Various attempts to eliminate the bacteria on the fungus were unsuccessful. Three different bacterial species were isolated and identified. One of these was Agrobacterium radiobacter, while another may represent a new taxon close to the genus Burkholderia. A third strain remains unidentified but is most probably a member of rRNA superfamily IV or the Woese (alpha) group. Besides P. chrysosporium, 23 other white rot fungi and 9 brown rot fungi were also investigated. None of these was associated with bacteria. The physiological significance of the association between the fungus and the bacteria remains to be elucidated.