Pectolytic Erwinia Research Articles

Detection of bacterial soft-rot of crown imperial caused by Pectobacterium carotovorum subsp. carotovorum using specific PCR primers.

Pectobacterium is one of the major destructive causal agent in most crop plants throughout the world. During a survey in spring of 2005 in the rangeland of Kermanshah and Isfahan, provinces of Iran, samples of bulbs and stems of crown imperial with brown spot and soft rot were collected. Eight strains of pectolytic Erwinia were isolated and purified from these samples. Phenotypic tests indicated that the strains were gram-negative, facultative anaerobic, rod shaped, motile with peritrichous flagella. They were oxidase negative, catalase positive and also able to macerate potato slices. Pathogenicity of all the strains were confirmed on corn, philodendron and crown imperial by inoculation of these crops with a bacterial suspension and reisolation of the strain from symptomatic tissues. A pair of specific PCR primers was used to detect these bacterial strains. The primer set (EXPCCF/EXPCCR) amplified a single fragment of the expected size (0.55 kb) from genomic DNA of all strains used in this study. In nested PCR, the primer set (INPCCR/INPCCF) amplified the expected single fragment (0.4 kb) from the PCR product of first PCR amplification. On the basis of the biochemical and phenotypic characteristics and PCR amplification by the specific PCR primers, these strains were identified as Pectobacterium carotovorum subsp. carotovorum. This is the first report of occurrence of crown imperial bacterial soft-rot in Iran.

Maritime Regional Meeting, 2004

Epidemiology and management of fire blight in red raspberry. P.G. Braun and P.D. Hildebrand. Atlantic Food and Horticulture Research Centre, Agriculture and Agri- Food Canada, 32 Main Street, Kentville, NS B4N 1J5, Canada. Investigations on the potential use of reflectance spectrophotometry for detection of virus-infected potato plants. M.B. Crane, R.H Coffin, W. Hardy, M. Singh, and H.A. MacEwen. Gentec Ltd, P.O. Box 579, Kensington, PE C0B 1M0, Canada; (R.H.C., W.H., H.A.M.) Cavendish Farms, New Annan, Kensington, PE C0B 1M0, Canada; and (M.S.) Agriculture Certification Services, 1030 Lincoln Road, Fredericton, NB E3B 8B7, Canada. Detection and differentiation of pectolytic erwinias in potato by multiplex PCR. S.H. De Boer and J.M. D’Aubin. Centre for Animal and Plant Health, Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada. Assessing the in vitro sensitivity of Alternaria solani and Alternaria alternata isolates from Prince Edward Island to azoxystrobin. W. MacDonald, R.D. Peters, R.H. Coffin, and C. Lacroix. Department of Biology, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE C1A 4P3, Canada; (R.D.P.) Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (R.H.C.) Cavendish Farms, New Annan, Kensington, PE C0B 1M0, Canada. Potato virus A (PVA) coat protein transgene suppresses PVA-induced hypersensitive response in potato cultivar ‚Shepody’. X. Nie, R.P. Singh, J. Zeng, and T. Molen. Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, NB E3B 4Z7, Canada. Managing nitrogen for control of pests and diseases. D.G. Patriquin. Department of Biology, Life Science Centre, Dalhousie University, 1355 Oxford Street, Halifax, NS B3H 4J1, Canada. Recent investigations regarding the soilborne pathogen Phytophthora erythroseptica and the management of potato pink rot. R.D. Peters, A.V. Sturz, H.W. (Bud) Platt, and W.J. Arsenault. Crops and Livestock Research Centre, Agriculture and Agri-Food Canada, 440 University Avenue, Charlottetown, PE C1A 4N6, Canada; and (A.V.S.) Plant Health Research and Diagnostics, Prince Edward Island Department of Agriculture and Forestry, P.O. Box 1600, Charlottetown, PE C1A 7N3, Canada. Comparison of PCR primers for the detection of bacterial ring rot. M. Taghavi, S.H. De Boer, and L.J. Ward. College of Agriculture, Shiraz University, Shiraz, Iran; and (S.H.D.B., L.J.W.) Centre for Animal and Plant Health, Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada. Ultra-sensitive methods for PCR detection of potato wart in soil. L.J. Ward, S.H. De Boer, and C.A. Lévesque. Centre for Animal and Plant Health, Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada; and (C.A.L.) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada. Identification of a Canadian alfalfa mosaic virus potato isolate and its detection in potato by RT-PCR and RFLP. H. Xu and J. Nie. Centre for Animal and Plant Health, Canadian Food Inspection Agency, 93 Mount Edward Road, Charlottetown, PE C1A 5T1, Canada.

Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil

To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia. Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E. carotovora subspecies and E. chrysanthemi. Pathogenicity and maceration ability of the Brazilian strains were greater than those of E. carotovora subsp. atroseptica, the causal agent of potato blackleg in temperate zones. Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E. carotovora subsp. atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia. Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E. carotovora and from E. chrysanthemi. A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR. The bacterium that causes the blackleg disease of potato in Brazil differs from E. carotovora subsp. atroseptica, the blackleg pathogen in temperate zones. It also differs from other subspecies of E. carotovora and from E. chrysanthemi and warrants status as a new subspecies, which would be appropriately named E. carotovora subsp. brasiliensis. The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions. The Brazilian strain is more virulent than E. carotovora subsp. atroseptica, the usual causal agent of potato blackleg.

Characterization of Pectolytic Erwinias as Highly Sophisticated Pathogens of plants

Erwinia carotovora and Erwinia chrysanthemi are the two most important soft rotting bacteria of commercially-grown plants. They are genetically diverse as is evident from polymorphisms in the pel and recA genes as well as in rrn, the ribsomal gene cluster. Subpopulations grouped into biovars, pathovars, or subspecies associated with various hosts and in different geographic regions suggest specialization in host preference and/or survival in diverse environments. Previous characterization of the pectolytic erwinias as opportunistic pathogens is being replaced by a realization that this group of bacteria exhibits a sophisticated repertoire of pathogenicity and virulence genes and regulators. The presence of an entire hrp gene cluster and associated type III secretion system, and global regulators which regulate virulence determinants such as exoenzyme production and motility, attest to a highly specialized pathogen. The fact that production of extracellular plant cell wall-degrading enzymes are coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone in a population density-dependent manner may explain the occurrence of pectolytic erwinia in asymptomatic plant tissues. Transgenic plants expressing bacterial quorum-sensing signal molecules modulate this sensory system and exhibit resistance to soft rot infection. The pectolytic erwinias, being significant plant pathogens that are neither of quarantine concern nor a human health hazard while readily isolated from field sources, make an ideal model for investigating the genetic basis of plant pathogenesis and environmental fitness.

An Oligonucleotide Array for the Identification and Differentiation of Bacteria Pathogenic on Potato

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.

Incidence of pectolytic erwinias associated with blackleg of potato in Rio Grande do Sul

Erwinia carotovora subsp. atroseptica (Eca), E. carotovora subsp. carotovora (Ecc) and E. chrysanthemi (Ech) may cause potato (Solanum tuberosum) blackleg. To determine the occurrence of these pathogens in the conditions found in the State of Rio Grande do Sul (RS), potato plants showing blackleg symptoms were harvested from 22 fields in nine counties in Serra do Nordeste, Planalto, Depressão Central, and Grandes Lagoas, from September to December of 1999 (Spring-Summer season). Green pepper (Capsicum annuum) fruits were used as a host to enrich for pectolytic erwinia from potato stems with blackleg symptoms. Bacteria were subsequently isolated on non-selective medium. Isolates that were Gram-negative, facultatively anaerobic, and pitted crystal-violet-pectate medium were tested for biochemical traits to identify the species and subspecies. Four hundred strains were identified as either Eca, Ecc or Ech. Although the three erwinias were found in RS potato fields, only three strains of Ech were found in one field. Frequencies of Eca and Ecc were 55 and 42%, respectively. Eight strains could not be assigned based on the biochemical characterization.

The Study of Erwinia carotovora Bacteriocins with the Aid of Nalidixic Acid–Resistant Bacterial Indicator Cells

A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs). The approach lies in the bacteriocinogeny of pectolytic erwinia being studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs. The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E. carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics. Preliminary studies using this approach showed that there is no correlation between the occurrence of MCTVs in particular E. carotovora strains and the habitat of the host plants from which these strains were isolated. There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.

Defective lysogeny in Erwinia carotovora

The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles. E. carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid. The tails were 128-192 nm in length and 13-21 nm in diameter. Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm. The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain. It was shown that cells of the same species may contain several different types of phage tails and heads. The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E. carotovora J2 was studied in more detail. The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.

Description of a new disease on Erythrina sp. in Martinique (French West Indies) and preliminary characterization of the causal agent as a novel Erwinia species

In spring 1995, symptoms of partial defoliation were observed on Erythrina indica var. fastigiata trees, commonly used as windbreaks in banana plantations in Martinique (French West Indies). Browning of the bark surface was consistently observed at the base of defoliated branches. Bacteria were isolated as nearly pure cultures from typical necrotic lesions on the bark. Results of Gram stain, staining of flagella and biochemical tests indicated that all isolates belonged to the Enterobacteriaceae, and to the genus Erwinia. Numerical analysis of the results of 48 biochemical properties showed that Erwinia isolates from diseased erythrina trees constituted a homogenous phenotypic cluster, and that, although weakly pectolytic, these isolates were closely related to pectolytic Erwinia species: E. carotovora ssp. betavasculorum and ssp. wasabiae, and E. cacticida. However, they could be clearly distinguished by different biochemical characteristics from type strains of known plant pathogenic species of Erwinia, Pantoea and Enterobacter. Under greenhouse conditions, the isolates were pathogenic to Erythrina indica var. fastigiata cuttings, but not to banana and pineapple, which are major crops in Martinique.

Rapid Detection of Pectolytic Erwinia sp. in Aglaonema sp.

The purpose of this research was to develop a rapid microplate assay for detecting and presumptively identifying pathogenic pectolytic Erwinia sp. in ornamental propagative stock and to readily distinguish them from nonpathogenic bacteria associated with Aglaonema. The assay was developed by modifying an existing crystal violet-sodium polypectate (CVP) medium to visualize the depolymerization of pectate by addition of bromcresol purple (BCP), then acidifying with a dilute hydrochloric acid (HCl) solution. The assay was sufficiently sensitive to detect latent (symptomless) infections in small sections of leaf or stem tissue. Based on inoculum titration assays, 40 to 600 colony-forming units (CFU) of Erwinia chrysanthemi (Burkholder et al.) were required to produce symptoms in Aglaonema stems and leaves, respectively. The microplate assay was able to detect the pathogen at low levels (40 to 60 CFU) in tissue segments of ≈1 cm2. In tests of bacterial strains isolated from 211 samples from Aglaonema `Silver Queen', `Emerald Beauty', and `San Luis' grown in Hawaii, only the pectolytic and pathogenic Erwinia sp. reacted positively in the microplate assay. Other bacteria associated with Aglaonema, including Pseudomonas paucimobilis (Holmes), P. vesicularis (Galarneault and Leifson), and nonpectolytic Erwinia sp., were not detected by the assay. Pectolytic strains of Ralstonia (Pseudomonas) solanacearum (Smith) were differentiated from pectolytic Erwinia sp. by the yellow color that developed in wells of the latter strains after acidification of the medium with dilute HCl. The test is visual and can be performed with minimal equipment and cost.

Bandstab: a PCR-based alternative to cloning PCR products.

Bandstab: a PCR-based alternative to cloning PCR products.

Components of partial resistance to potato blackleg caused by pectolytic Erwinia carotovora subsp. atroseptica and E. chrysanthemi

The various phases of the infection process in potato blackleg, caused by pectolytic Erwinia spp., were interpreted as components of partial resistance and studied in detail. Mother tubers of glasshouse‐grown plants of two potato cultivars were inoculated with antibiotic resistant marker strains of Erwinia carotovora subsp. atroseptica (Eca) or E. chrysanthemi (Ech). Stem tissue samples of these plants were collected 3, 7, 14 and 21 days after inoculation and subsequently analysed for the presence of the bacteria. As soon as 3 days after inoculation, Erwinia bacteria were detected in the stem tissue of the plants. However, blackleg developed only when the rotting mother tuber tissue reached the sites where the stems were attached to the mother tuber. An inoculation method was developed for determining cultivar differences in the incidence and rate at which rot in tuber cylinders proceeds to sprouts on top of such cylinders. A set of 12 cultivars was screened for this putative component of resistance which was called ‘stem base resistance’. Significant differences for stem base resistance were found among the cultivars. These differences were clearer when inoculation was carried out with Ech in contrast to Eca. Glasshouse‐grown plants of the same set of cultivars were also screened for resistance of the above ground part of the stem. Cultivars differed significantly for this so‐called ‘stem tissue resistance’, but results of some cultivars were not consistent among years of testing. Multiple regression analysis revealed that stem base resistance, stem tissue resistance, tuber tissue resistance and resistance of mother tubers in the field account for 63% (Eca) and 75% (Ech) of the variance found after screening for resistance to blackleg in the field. It was concluded that if results of this study are extrapolated to any set of potato clones, selection for resistance in breeding programmes under laboratory or glasshouse conditions, would be most efficient when directed to clonal differences for stem base resistance.

Erwinia soft rot resistance of potato cultivars expressing antimicrobial peptide tachyplesin I

Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1×106 colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 μg/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35S promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atroseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.

Factors Affecting Potato Soft‐rot Resistance to Pectolytic Erwinia Species in a Tuber‐slice Assay

AbstractPotato‐tuber slices and small tubers of 12 cultivars were inoculated with isolates of Erwinia carotovora subsp. atroseptica (Eca), E. c. subsp. carotovora (Ecc) and E. chrysanthemi (Ech). Cultivar resistance was expressed as the mean diameter of the rotted‐tissue area. Grouping of the cultivars into classes of relatively high, intermediate or low resistance, was different after incubation in air in comparison to incubation under anaerobic conditions, and depended on the (sub‐)species of bacteria used. When compared to the effect of inoculation with different (sub‐)species, grouping of the cultivars was less affected by isolates within (sub‐)species. Cultivar grouping was hardly affected by site of inoculation, being either in the medullary tissue or in the cortex. In general, the results of experiments with either field‐grown seed tubers or glasshouse‐grown small tubers from 2 years were reproducible, except when tuber tissue was inoculated with Ech and incubated, aerobically, in which case cultivars only showed relatively small differences for resistance. Correlation coefficients between the results from slice and small‐tuber screenings were 0.71 and 0.64 for Eca and Ecc, respectively. This implies, some potential for applying the method in the early stages of potato‐breeding programmes.The results of this study show that soft‐rot resistance of clones to different pectolytic Erwinia spp. can be determined efficiently and reproducibly if the oxygen concentration during incubation is standardized.

Selective Medium for Isolation of PectolyticErwiniasp.

Miller-Schroth medium modified by replacement of most of the agar with sodium polypectate appeared superior to the crystal violet polypectate medium for the selective isolation of pectolytic Erwinia sp. NaOH and MOPS (3-[N-morpholino]propanesulfonic acid) were added to raise the pH and buffer the medium, respectively. Pectolytic erwinias appeared as pink to orange colonies located in deep pits. Nonpectolytic erwinias formed pink-orange colonies with no pits (.)

Taxonomy and Pathogenicity of Erwinia cacticida sp. nov.

A total of 108 pectolytic, soft-rotting Erwinia strains were collected from 11 types of cacti growing in Arizona, Texas, northern Mexico, and Australia between 1958 and 1989. Four strains were collected from soils beneath or close to naturally rotting saguaro cacti. Collectively, these strains caused soft rots of saguaro, organ pipe, and senita cacti, Opuntia (cactus) fruits and pads, tomato fruits, and potato slices, but only occasionally caused soft rots of slices of carrot roots. A numerical cluster analysis showed that 98 of the 112 strains formed a uniform group (cluster 1A) that was distinguished from other pectolytic erwinias by an API 20E code of 1205131, by negative reactions in API 50CHE tests for L-arabinose, myo-inositol, D-cellobiose, melibiose, and D-raffinose, and, in supplemental tests, by positive reactions for malonate and growth at 43 degrees C. The average levels of DNA relatedness of 22 cluster 1A strains to the proposed type strain (strain 1-12) as determined by the hydroxyapatite method were 88% in 60 degrees C reactions (with 1% divergence within related sequences) and 87% in 75 degrees C reactions. The levels of relatedness to the type strains of other Erwinia spp. were less than or equal to 38% in 75 degrees C reactions. Cluster 1A strains also had a characteristic cellular fatty acid profile containing cyclo-(11,12)-nonadecanoic acid (C19:0 Cyclo C11-12) and missing tridecanoic acid (C13:0), heptadecanoic acid (C17:0), and cis-9-heptadecenoic acid (C17:1 CIS 9), which separated them from other pectolytic erwinias. Collectively, these data indicate that the members of cluster 1A are members of a new species, which we name Erwinia cacticida. Three cactus strains in cluster 1B appear to represent a second new species that is closely related to E. cacticida; these strains are designated E. cacticida-like pending the availability of additional strains for testing. The remaining cactus strains (in cluster 4) have the physiological, DNA, and fatty acid profiles of Erwinia carotovora.

An improved formulation and method of preparation of crystal violet pectate medium for detection of pectolytic erwinia

An improved procedure for the preparation of CVP medium is described on which pectolytic erwinia rapidly develop deep, localised pits. The medium can be prepared quickly and easily and forms a smooth, firm gel suitable for streaking.

Quantitative assessment of tuber contamination by pectolytic erwinia and its possible use in the prediction and control of blackleg

A Most Probable Number Technique based on the use of carbohydrate-free McConkey broth and crystal violet pectate agar for the quantification of tuber-borne pectolytic erwinia has been developed. The population of the organisms on naturally contaminated tubers has been shown to be log-normally distributed. The possibility of using tuber contamination and standards for tuber quality for the prediction and control of blackleg is explored.

Serological and biochemical variation among potato strains of Erwinia carotovora subsp. atroseptica and their taxonomic relationship to other E. carotovora strains

The serological and biochemical characteristics of 32 Erwinia carotovora subsp. atroseptica strains from potato were compared with 48 other pectolytic Erwinia strains. Biochemical characteristics were examined by the API 20E and API 50CHE systems. Numerical analysis using the Euclidean distance coefficients and clustering by the unweighted average pair group method indicated that these E. carotovora subsp. atroseptica strains formed a distinct cluster (subphenon A1) that could be differentiated from other E. carotovora strains. Three non‐potato strains also belonged to this group; two of these were from tomato and the other from Chinese cabbage. Named E. carotovora subsp. atroseptica strains from other hosts clustered into other phenons. Sixty‐three per cent of subphenon A1 strains tested in this study typed into serogroup I. One potato strain in another phenon also typed into this serogroup. The subphenon A1 strains that did not type into serogroup I typed into serogroups XVIII, XX, or XXII. Many of these strains, however, expressed several different O antigens which were also expressed by E. carotovora strains in other phenons.

Control of potato blackleg by tuber pasteurisation: the determination of time-temperature combinations for the inactivation of pectolytic erwinia

Pasteurisation of potato tubers by immersion in hot water is being examined as a method of blackleg control. Studies on the in vitro heat resistance of representative pectolytic erwinia have enabled prediction curves for the selection of suitable times and temperatures of pasteurisation to be prepared. High levels of contamination on some tubers may require heat treatment that will reduce the erwinia population by more than 80% to ensure that only innocuous levels remain.