Soft Rot Erwinia Research Articles

Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI of 10.0 and the minor one had a pI of 6.7. A single PL with a pI of greater than or equal to 10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae. PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitation and anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees. The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and 35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwinia spoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pI appeared to be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rot bacteria.

Screening of seedlings of wildSolanum species for resistance to bacterial stem rot caused by soft rot Erwinias

A standard effective screening procedure for evaluating resistance of young seedlings ofSolanum species to bacterial stem rot caused byErwinia species was developed. Susceptibility to stem rot caused byErwinia carotovora subsp.carotovora varied greatly among 500 differentSolanum accessions; generally accessions resistant toE. carotovora subsp.carotovora were also resistant toE. carotovora subsp.atroseptica andE. chrysanthemi. Most of the lines ofSolarium berthaultii, S. bulbocastanum, S. chacoense, S. stoloniferum, S. tarijense, andS. tuberosum subsp.andigenum were very resistant to stem rot.

A Note on the Limitations of Identifying Soft Rot Erwinias by Temperature Tolerances and Sensitivity to Erythromycin on a Pectate Medium

AbstractForty‐five erwinia strains from 14 different hosts have been tested lor growth on a crystal violet pectate medium with or without 70 μ/ml erythromycin at 27, 33.5 and 37°C. On the basis of positive data in the literature it was tried to identify the strains as Erwnia carotovora subsp. atroseptica, E. carotovora subsp. carotovora or E. chrysanthemi using this method.It appeared that relatively few strains reacted differentially, apparently due to strain variation (all strains were identified also by biochemical and physiological tests). The conclusion is that for a reliable identification of field isolates of soft rot crwinias other (biochemical, physiological and serological) methods have to be applied.

Sero-groups ofErwinia carotovoraassociated with water, soil, tuber, and stems of potato plants in Western Australia

Abstract Soft-rot erwinias were isolated from irrigation water, soil, tubers, and stems from spring (1986) and summer (1987) crops of potato at a field in Manjimup, Western Australia. Most of the isolates from all environments in both seasons belonged to Erwinia carotovora pv. carotovora (Ecc), with the exception of those from stems of the summer crop which were predominantly (68%) Erwinia carotovora pv. atroseptica (Eca) and belonged to sero-group SGI. In spring, SGIII was recorded from all environments tested, while SGI was found only in stems and tubers. SGVI was recorded from stems (8%) and soil (15%) only in summer and in water (6.4%) only in spring. In spring, SGV, which was the predominant (28.2%) sero-group isolated from stems, was also found in water, soil, and tubers. In summer, of the SGs isolated from stems, SGIII occurred in soil and tubers and SGVI in soil. Although SGXXIX was the most common in water in both seasons, it was isolated from stems only in summer. In a pathogenicity test, repres...

Assessing potato cultivars for resistance to tuber soft rot (Erwinia carotovora subsp.atroseptica) at four test centres in the UK

Tuber slices of 15 cultivars were inoculated withErwinia carotovora subsp.atroseptica on two occasions at four test centres and the results compared. Correlations in mean lesion diameter between inoculum concentrations, between the same test on different occasions, between centres and between cultivars in different tests were mostly significant in spite of small differences in procedure at each centre. Satisfactory agreement was reached between centres, particularly concerning the most resistant (Cara, Pentland Hawk) and most susceptible (Record, Wilja, Pentland Crown, King Edward) of the cultivars tested. The implications of the findings are discussed in relation to results published elsewhere and to other methods of inoculation.

Irrigation Water as a Source of Inoculum of Soft Rot Erwinias for Aerial Stem Rot of Potatoes

Seed pieces from a single seed lot of potato cultivar Russet Burbank were planted at two production areas in Oregon and Colorado in 1985 and 1986. Seed tubers, field soil, irrigation water, symptomless leaflets, and stems with aerial stem rot symptoms were assayed for the presence of soft rot erwinias. More than 95 and 90% of all strains recovered from Oregon and Colorado sources, respectively, were Erwinia carotovora subsp. carotovora. A total of 3.556 strains of E. carotovora were tested serologically against 21 different antisera by Ouchterlony agar double diffusion. In Oregon, seven different serogroups we detected in irrigation water first during the season, then on leaflets, and later in diseased stems. In Colorado, five different serogroups were detected in irrigation water before or at the same time they were detected on potato leaflets. However, only one plant developed aerial stem rot symptoms. In Oregon, 39% of the serogroups, identified were common to irrigation water, leaflets, and diseased stems, whereas in Colorado 50% of the serogroups identified were common to irrigation water and leaflets. Contaminated water sources are a potentiel source of inoculum for aerial stem rot in Oregon

Ｅｒｗｉｎｉａ属細菌のペクチン質分解酵素 軟腐性Ｅｒｗｉｎｉａ属細菌の病原性とペクチン質分解酵素

Ｅｒｗｉｎｉａ属細菌のペクチン質分解酵素 軟腐性Ｅｒｗｉｎｉａ属細菌の病原性とペクチン質分解酵素

Molecular Genetics of Pathogenesis by Soft-Rot Erwinias

The soft-rot erwinias are enterobacteria able to cause diseases, a symptom of which is the maceration of host tissues, on a wide range of plants. In the past, this group of pathogens was grouped into a single species, Erwinia caroto­ vora, divided into subspecies (129). Now, the group is split into two main species, Erwinia chrysanthemi and E. carotovora, the latter being divided into the three subspecies, carotovora, atroseptica, and betavasculorum (89). Two other minor pathogens with restricted host range are included in the soft-rot group: Erwinia cypripedii and Erwinia rhapontici (108). The general characteristics and the ecology of these bacteria have been previously re­ viewed (107, 108, 129). The soft-rot erwinias can infect plants in culture as well as in storage. Besides soft rot itself, other symptoms may be observed on the growing plant. For instance, E. chrysanthemi induces a wilt on carnation. E. carotovora subsp. atroseptica, E. carotovora subsp. carotovora, and E. chrysanthemi can cause blackleg on potato, which has leaf wilting as a symptom (107, 108). Infection of chrysanthemu?l by E. chrysanthemi generally induces a canker on the stem, starting from the crown (25). In plants that reproduce vegetative­ ly, the infection can be systemic and, when conditions for development of the disease are not met, latent. Host specificity, although not as strict as that observed in plant-pathogenic pseudomonads and xanthomonads (78, 130), seems to occur in E. chrysanthemi strains. It has been proposed that E. chrysanthemi be divided into five pathotypes (51, 52). Soft-rot erwinias produce several extracellular enzymes that can attack components of the plant

Pathogenicity of soft rot erwinias to potato plants in Scotland and Israel

The pathogenicity of Erwinia carotovora pv. carotovora, E. carotovora pv. atroseptica and E. chrysanthemi to potato plants was examined in experimental and commercial crops in Scotland and Israel in 1983 and 1984. Symptoms caused by the three erwinias tended to be different depending on when they were expressed during the different growing seasons. Field symptoms were grouped into four types based on the origin and extent of stem rot and of leaf desiccation. Most were reproduced on inoculated potted plants held under different combinations of relative humidities (ca 70 and ca 100%) and temperatures (17° and 30°C). In plots planted with seed inoculated with E. c. atroseptica alone or together with E. c. carotovora or E. chrysanthemi (in Israel only), stem rot symptoms developed throughout the growing season in Scotland (Invergowrie) but only before May in February‐sown crops and after November in September‐sown crops in Israel (Negev). In contrast, E. chrysanthemi alone or together with one or the other erwinias in Israel caused extensive leaf desiccation with no stem rot only in May and before December in the spring‐and autumn‐sown growing seasons, respectively. None of 13 E. c. carotovora strains inoculated into erwinia‐free tubers was pathogenic in the spring growing season in Israel. More than one erwinia type was frequently present in both healthy and diseased stems, usually the same bacteria which were inoculated in ihe seed tubers. Isolations made from diseased stems showed that the temporal patterns of detection frequency of E. c. atroseptica and E. chrysanthemi in stem rots and in plants with leaf desiccation symptoms were similar to those for expression of the two symptom types. Erwinia carotovora pv. carotovora alone was never found in diseased stems although it could be isolated together with the other erwinias in both Scotland and Israel. Similar results were obtained when diseased stems from commercial potato fields were examined, except that E. c. carotovora was the sole erwinia present in aerial stem rots. The patterns observed in the pathogenicity of three erwinias were tentatively explained in terms of temperature changes during the growing season, the transition points being in the region of 20°C for maximum soil temperature at tuber depth or 25°C for maximum air temperature above which E. c. atroseptica and below which E. chrysanthemi were not apparently pathogenic.

Use of TnphoA to enrich for extracellular enzyme mutants of Erwinia carotovora subspecies carotovora.

Soft rot Erwinia species secrete a range of enzymes into the extracellular environment. Therefore, the genetically amenable Erwinia system is a useful model for the study of protein secretion by Gram-negative bacteria. We have used a lambda-sensitive derivative of Erwinia carotovora subspecies carotovora (Ecc) and the transposon TnphoA, to isolate a range of extracellular enzyme mutants. The use of TnphoA provides an enrichment for extracellular enzyme mutants over other transposon-based systems. In these mutants, the alkaline phosphatase activity of the hybrid protein is found in the periplasm, and is under the control of the Ecc promoters. Three TnphoA-induced extracellular enzyme mutants were studied in detail. One proved to be an enzyme structural gene mutant, whilst the other two appeared to be secretory mutants.

Erwinia carotovora subsp. wasabiae subsp. nov. Isolated from Diseased Rhizomes and Fibrous Roots of Japanese Horseradish (Eutrema wasabi Maxim.)

A gram-negative bacterium with peritrichous flagella isolated from internally discolored rhizomes of Japanese horseradish, Eutrema wasabi Maxim. (Japanese common name, wasabi), was compared with other soft rot Erwinia spp. The wasabi bacterium induced soft rot on slices of various vegetables, such as wasabi rhizomes, potato tubers, and carrot and radish roots, on midribs of chinese cabbage and intact wasabi, and on tomato and tobacco plants, but not on rice, corn, or chrysanthemum. Although this bacterium was more similar to Erwinia carotovora than to Erwinia chrysanthemi in physiological and biochemical properties, it differed from E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and E. carotovora subsp. betavasculorum in being negative in the o-nitrophenyl-β-d-galactopyranoside test, curd formation and peptonization of litmus milk, growth in yeast extract-peptone broth containing 0.075% KCN or 5% NaCI, growth at 36°C, and fermentation of lactose, raffinose, and melibiose. This bacterium also differed in having a 24-h lag in galactose fermentation, rapid fermentation of trehalose, and different serological properties. The guanine-plus-cytosine content of deoxyribonucleic acid was 51.4 to 51.7 mol%. Such significant differences warranted the designation of the wasabi bacterium as a new subspecies of E. carotovora, and the name Erwinia carotovora subsp. wasabiae subsp. nov. is proposed. Strain SR91 (=ATCC 43316, and PDDCC 9121) is designated the type strain.

A rapid method to identify and quantify soft rot erwinias on seed potato tubers1

Potato tubers are usually contaminated by more than one species or pathovar of soft rot erwinia and, because blackleg incidence is related to the contamination level of seed tubers, the disease potential of seed stocks may be assessed by determining seed‐tuber contamination level. A method is described for identifying and quantifying directly from tubers the three soft rot erwinias commonly associated with potatoes. Replicate lots of 10–15 tubers are peeled by dry abrasion in a commercial potato peeler and the peel weight determined by weighing the tubers before and after peeling. Sap is expressed from the peel, an antioxidant (0.075% dithiothreitol) added, and the sap is dilution‐plated on a diagnostic selective medium (crystal violet pectate [CVP]). After incubating for 24 h at 20°C, the plates are velvet‐replicated onto fresh plates of CVP with or without 35 μg ml‐1 erythromycin and incubated for 48 h at 27°C and 24 h at 33.5 or 37°C. Soft‐rot erwinias typically form deep cup‐like cavities on CVP and they can be identified and enumerated according to the pattern of cavity formation. Cavities are formed by Erwinia carotovora pv. atroseptica only at 27°C, by E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C, whereas E. chrysanthemi forms cavities at all temperatures but fails to grow in the presence of erythromycin. Contamination levels can be expressed as the number of different erwinias per tuber or per g peel.

Reducing the potential for bacterial soft rot in potato tubers by chemical treatments and drying

The potential for bacterial soft rot caused byErwinia carotovora in freshly inoculated potato tubers was reduced up to 99% by immersion for 5 min in solutions of sodium hypochlorite (chlorine bleach) containing up to 10,000 ppm chlorine. Reductions up to 93% were achieved using a combination treatment of immersion in 1% citric acid for 5 min followed by airdrying. Immersion treatment alone in 1% aqueous solutions of citric, acetic, ascorbic, or malonic acid also significantly reduced the soft rot potential. In contrast, no reductions accompanied treatment with solutions of potassium or calcium acetate. All treatments including the combination immersion/airdrying treatment were much less effective if tubers had been infiltrated initially with soft rotErwinia or had numerous mechanical injuries. If tubers had not been infiltrated with the causal organism, immersing them in 1% citric acid reduced the potential nearly as much as a similar treatment with 1000 ppm chlorine. A 30-sec immersion in the latter was less effective than a similar treatment with 500 ppm a.i. CGA 78039, an experimental bactericide. However, air-drying, coupled with provisions for keeping tuber surfaces free from moisture, remains the most effective means of reducing losses to bacterial soft rot.

Contamination of potatoes by Erwinia carotovora during grading

Considerable tuber contamination by soft rot erwinias in rotting tubers can occur when grading potato stocks. Erwinia carotovora pv. carotovora, from a single rotting tuber, contaminated c. 100 kg of potatoes during mechanical grading, c. 50% being contaminated with 104–105 bacteria per tuber. Survival of the bacteria during storage was related to the degree of damage sustained by the tubers during grading which in turn depended on the model of oscillating riddle grader used and the cultivar of potato. Chemical disinfection of the grader immediately before use and of tubers immediately after grading reduced contamination.

A rapid method for identifying and quantifying soft rot erwinias directly from plant material based on their temperature tolerances and sensitivity to erythromycin

A method is described for identifying and quantifying three soft rot erwinias directly from plant tissue and from other sources that is particularly useful in epidemiological studies. Colonies of these bacteria form characteristic deep cavities on selective‐diagnostic crystal violet pectate (CVP) medium. Bacteria from individual presumptive erwinia colonies on CVP plates spot inoculated on plates of CVP medium with or without erythromycin (35 μg/ml) added and incubated at 27, 33°5 and 37°C can be identified according to the pattern of cavity formation. Erwinia carotovora pv. atroseptica forms the characteristic cavities only at 27°C and E. carotovora pv. carotovora at 27 and 33.5°C but not at 37°C on CVP with or without erythromycin. Erwinia chrysanthemi forms cavities at all temperatures and can also be identified by failure to grow at 27°C on CVP with erythromycin. Similarly, erwinias in mixed populations can be quantified by dilution plating on CVP with or without erythromycin and incubating at the different temperatures. Using this method, ca 80% of 183 erwinia strains in a culture collection were correctly identified, the precision increasing to over 95% when recently isolated erwinia strains were examined.

Taxonomic study on soft rot bacteria isolated from diseased rhizomes and roots of wasabi (Eutrema wasabi Maxim.).

Various kinds of soft rot bacteria were isolated from rhizomes of wasabi or Japanese horse radish (Eutrema wasabi Maxim.) with the internal black rot syndrome as well as from fibrous roots showing black stele symptoms. These bacteria included Erwinia spp. and Pseudomonas spp. Based on the bacteriological characteristics, Erwinia soft rot bacteria were divided into 4 groups. The strains of group 1 were identified as E. rhapontici which failed to produce pink diffusible pigment on any kinds of media used in the study. The strains of group 4 were identified as E. carotovora subsp. carotovora. The strains of group 3 were also identical with subsp. carotovora although they differed from the former in a few phenotypic characters. The strains of group 2 differed from subsp. carotovora in properties such as delayed acid production from lactose, absence of β-galactosidase, inability to utilize melibiose and raffinose, absence of growth in KCN broth, in 5% NaCl broth and at 36 C. This group should be classified as a new taxon in the genus Erwinia. Pseudomonas soft rot bacteria were divided into 5 groups by the bacteriological tests. The strains of groups 1, 2, and 5 were identified as P. marginalis (=biovar II of P. fluorescens) based on their overall similarity of phenotypic characteristics. The strains of group 4 were identified as P. viridiflava although they differed from the description of the bacterium in the literature in some minor characters. The strains of group 3 were also identical to P. viridiflava in the characteristics of the LOPAT test. However, the physiological and biochemical properties were significantly different from those of any of the soft rot pseudomonads hitherto described. Therefore, the taxonomic attributes of this group remain to be determined by more detailed comparative studies in future.

Pretreatment of Pectic wastewater from orange canning by soft-rot Erwinia corotovora

Erwinia carotovora FERM P-7576, a soft-rot pathogen screened for biochemical pulping, was used to dispose of pectic wastewater discharged from orange canning factories as a pretreatment for an activated sludge process. Twelve-hour bacterial pretreatment made it possible to reduce COD and total uronic acids by 10% and 84%, respectively, with supplementation of some nutrients (N, P, and K) and keeping DO constant. Enzymatic pretreatment using crude enzyme from this strain degraded almost all the pectic substances in this waste to the level of tetramers and pentamers within 24 h. These pretreatment was found to provide more rapid decomposition of the chemically very resistant pectic substances than other chemical, physical, or biological methods.

Presence of DNA damaging agents in plants as the possible inducers of pectin lyases of soft-rot Erwinia.

When the strains of E. chrysanthemi and E. carotovora subsp. carotovora were inoculated onto potato or radish disks, the activity of pectin lyase reached the same order as that of pectate lyase in some combination of the pathogen and plant. When time-course samples were taken after the addition of the extract of radish into the culture of E. chrysanthemi (EC183), the specific activities of both pectate lyase and pectin lyase were consistently elevated. The specific activities of both enzymes were also elevated, when E. chrysanthemi (ALE8292P) was grown in the medium containing the ethanol extract from 12 different plants. Thus, some compound responsible for the induction of these enzymes seem to be commonly present in plants. Ten out of these 12 ethanol extracts from plants inhibited the growth of rec- strain more severely than that of rec+ strain of Bacillus subtilis (rec assay). Therefore, DNA damaging agents also seem to be commonly present in plants. Since the induction of pectin lyase in soft-rot Erwinia has been shown to occur only by DNA-damaging agents, these DNA damaging agents found in plants may be responsible for the induction of pectin lyase at the infection site.

Inoculation of potato tubers withErwinia carotovora during simulated commercial washing and fluming practices

The potential for bacterial soft rot in potato tubers was affected by laboratory simulation of some components of fluming and washing practices at commercial packinghouses. The potential was expressed as disease severity (average percentage surface decayed) after a standard 4-day incubation at 20°C in a mist chamber. Increased disease resulted when there was an increase in the following factors: a) duration of immersion of tubers in water; b) population of bacteria suspended in water; c) hydrostatic pressure on submerged tubers, and d) concentration of a surfactant (Triton X-100) in the suspensions. A sample of tubers from each tuber lot was uniformly moistened with tap water and then incubated to determine a base-level disease severity. Disease severity in Russet Burbank tubers from commercial storage increased from the base-level of 0.8% to 76% when tubers were immersed in 5 × 106 colony-forming units (cfu)/ml ofErwinia carotovora pv.carotovora (Ecc) for 64 min prior to incubation. Disease severity was 64% when tubers were immersed in a similar suspension for only 0.1 min, but with a hydrostatic pressure of 230 cm of water. Severity values fourto fivefold above control levels (from immersion in water) resulted from an immersion for 5 min in 5 × 103 cfu/ml with hydrostatic pressures of 5, 180, or 370 cm of water. A 20-fold increase in severity occurred when Red LaSoda tubers were immersed briefly (less than 5 s) in 5 × 106 cfu/ml. Rinsing these tubers with tap water after inoculation, but prior to incubation, did not affect the subsequent development of decay. Thus, the high potential for bacterial soft rot in tubers that have been flumed or washed by water concentrated with soft rotErwinia cannot be reduced by washes or rinses with clean water.

Reduced Severity of Erwinia Soft Rot in Potato Tubers with Increased Calcium Content

Reduced Severity of Erwinia Soft Rot in Potato Tubers with Increased Calcium Content