Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted. Cluster 1 contained Erwinia carotovora subsp. carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp. odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp. atroseptica (subcluster 2a) and Erwinia carotovora subsp. betavasculorum (subcluster 2b) strains. Clusters 3 and 4 contained Erwinia carotovora subsp. wasabiae and E. chrysanthemi strains, respectively. While E. carotovora subsp. carotovora and E. chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E. carotovora subsp. odorifera, E. carotovora subsp. betavasculorum, E. carotovora subsp. atroseptica, and E. carotovora subsp. wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges. The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics. AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.     

Erwinia pel gene homology survey in selected bacteria

A 2.1 kb Bam H1 DNA fragment encoding a pectate lyase (PL) enzyme was isolated from an Erwinia carotovora subsp. atroseptica (Eca) cosmid library. The fragment was labeled with ³²P-CTP and hybridized to total DNA digests from selected bacteria which included plant-invasive as well as plant associative organisms. The pel gene probe hybridized to E. carotovora subsp. carotovora (Ecc) DNA under all conditions tested. Hybridization to DNAs from Agrobacterium tumefaciens and Pseudomonas marginalis was observed at low stringency conditions (45°C). No hybridization was observed between the pel gene probe and six other DNA samples.     

Behavior of bacteriophage P1 inErwinia carotovora subsp.carotovora

Bacteriophage P1KMclr100 was tranferred toErwinia carotovora subsp.carotovora. P1 was stably maintained as detected by hybridization and transfer of kanamycin resistance. Lysogens ofE. carotovora failed to produce any viable P1 phage. Although total DNA from P1 lysogens ofE. carotovora hybridized to³²P-labeled P1 probe, we were not able to detect P1 DNA as an extrachromosomal element. Attempts to use bacteriophage P1 as a vector for transposon Tn5 insertion mutagenesis inE. carotovora were not successful. Our results indicate that lytic replication of P1 DNA does not occur in P1 lysogens ofE. carotovora and that P1 DNA is probably integrated into the bacterial chromosome.Journal paper 10085 from the Purdue Agricultural Experiment Research Station.     

Isolation and Preliminary Characterization of Cryptic Plasmids from Erwinia carotovora

Of the fifty-two Erwinia carotovorastrains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size. Some E. carotovorastrains bore two to five different plasmids. Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins. At the same time, one of the E. carotovorastrains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, the 47.7-kbp pCA 6-2. Three E. carotovorasubsp.carotovorastrains and one E. carotovorasubsp.atrosepticastrain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.     

Biochemical and physiological characterisation of Erwinia species causing tip-over disease of banana

Tip-over disease has become a serious threat to banana plantations in the past decade. The disease is reported to be caused by Erwinia carotovorasubsp. carotovora and Erwinia chrysanthemi. We compared nine Erwinia strains of diseased banana plants from different agroclimatic zones of Karnataka and Andhra Pradesh, Southern India by conventional means. On the basis of morphological, cultural, physiological and biochemical characteristics and pathogenicity tests, the seven isolates I₁ to I₆ and I₈ showed similarities to Erwinia carotovorasubsp. carotovora. Isolate I₉ from Andhra Pradesh expressed characteristics similar to that of Erwinia chrysanthemi and was identified as Erwinia chrysanthemi. The isolate I₇ which showed wider variation, neither confirmed to the characteristics of Erwinia carotovorasubsp. carotovora nor with that of Erwinia chrysanthemi, and possessed characteristics in between the two species. Further we studied the host range of the bacterium causing tip-over disease of banana.     

Occurrence of bacteriophage T4 receptor in Erwinia carotovora

Summary We have tested for the presence of the receptor for the Escherichia coli phage T4 in different isolates of the plant pathogenic enterobacteria Erwinia carotora subsp. carotovora and subsp. atroseptica. Several of the isolates appeared to contain a functional T4 receptor as shown by phage adsorption and phage-induced lysis of the bacteria. Two of the isolates could even sustain lytic growth of T4. In addition, we show that the transducing derivative of T4, T4GT7, can be employed to transfer plasmids from E. coli to E. carotovora thus opening up new possibilities for genetic analysis of Erwinia.     

Derivation of Mutants of Erwinia carotovora subsp. betavasculorum Deficient in Export of Pectolytic Enzymes with Potential for Biological Control of Potato Soft Rot

Erwinia carotovora subsp. betavasculorum Ecb168 produces an antibiotic(s) that suppresses growth of the related bacterium Erwinia carotovora subsp. carotovora in culture and in wounds of potato tubers. Strain Ecb168 also produces and secretes pectolytic enzymes and causes a vascular necrosis and root rot of sugar beet. Genes (out) involved in secretion of pectolytic enzymes by Ecb168 were localized to two HindIII fragments (8.5 and 10.5 kb) of Ecb168 genomic DNA by hybridization to the cloned out region of E. carotovora subsp. carotovora and by complementation of Out^- mutants of E. carotovora subsp. carotovora. Out^- mutants of Ecb168, which did not secrete pectate lyase into the culture medium, were obtained when deletions internal to either HindIII fragment were introduced into the genome of Ecb168 through marker exchange mutagenesis. Out^- mutants of Ecb168 were complemented to the Out⁺ phenotype by introduction of the corresponding cloned HindIII fragment. Out^- mutants of Ecb168 were less virulent than the Out⁺ parental strain on potato tubers. Strain Ecb168 and Out^- derivatives inhibited the growth of E. carotovora subsp. carotovora in culture, indicating that the uncharacterized antibiotic(s) responsible for antagonism was exported through an out-independent mechanism. Strain Ecb168 and Out^- derivatives reduced the establishment of large populations of E. carotovora subsp. carotovora in wounds of potato tubers and suppressed tuber soft rot caused by E. carotovora subsp. carotovora.     

Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in Gram-negative bacteria

The chemical mutagen ethylmethanesulphonate (EMS) has been used to generate mutants of Erwinia carotovora subspecies carotovora which are defective in the secretion of pectinases (Pel) and cellulases (Cel) but unaltered for protease (Prt) secretion. Such mutants, called Out^? still synthesize Pel and Cel but these enzymes accumulate within the periplasm. Cosmid clones carrying wild-type E. carotovra ssp. carotovora DNA, identified by their ability to restore the Out⁺ phenotype when transferred to some Out^? mutants, were classified into six complementation groups using cosmids and cosmid derivatives. Analysis of the nucleotide sequence of a 12.7 kb DNA fragment, encompassing complementing cosmid inserts, revealed a coding capacity for 13 potential open reading frames (ORFs), and these were designated outC-outO. Some of the out gene products were visualized using a T7 gene 10 expression system. The predicted Out proteins are highly similar to components of extracellular enzyme secretion systems from a diverse range of eubacteria including Erwinia chrysanthemi, Klebsiella oxytoca, Aeromonas hydrophila, Pseudomonas aeruginosa and Xanthomonas campestris. Lower levels of similarity exist between Ecc Out proteins and components of macromolecular trafficking systems from Bacillus subtilis, Haemophilus influenzae, Agrobacterium tumefaciens, Yersinia pestis and a protein involved in the morphogenesis of filamentous bacteriophages such as M13.     

Screening Antimicrobial Activities of Basic Protein Fractions from Dry and Germinated Wheat Seeds

Two small cationic peptide fractions (5 kDa) were isolated from dry and germinated seeds of wheat, named WAP and GWAP, respectively. The antifungal and antibacterial activities of the peptides were analyzed using disk diffusion and turbidity measurement assays. The peptides in vitro exhibited effective antifungal activity against four plant pathogenic fungi at minimum concentration of 15 g(protein) cm^–3. Their antimicrobial activity was negatively affected by the presence of 5 mM CaCl₂. The peptides were less effective against Gram-negative bacterium Erwinia carotovora subsp. carotovora, but they demonstrated inhibitory activity against Gram-positive bacterium Clavibacter michiganensis subsp. sepedonicus. The antimicrobial activity of GWAP was more effective than WAP.     

Role of Antibiosis in Competition of Erwinia Strains in Potato Infection Courts

Erwinia carotovora subsp. betavasculorum strains produced a bactericidal antibiotic in vitro that inhibited a wide spectrum of gram-negative and gram-positive bacteria. The optimum temperature for production was 24°C, and the addition of glycerol to culture media enhanced antibiotic production. Antibiotic production by these strains in the infection court of potato was the principal determinant enabling it to gain ascendancy over competing antibiotic-sensitive Erwinia carotovora subsp. carotovora strains. There was a complete correlation between antibiotic production by E. carotovora subsp. betavasculorum in vitro and inhibition of competing E. carotovora subsp. carotovora strains in planta. Inhibition of the latter by the former was apparent after 10 h of incubation in potato tuber wounds. Population densities of sensitive E. carotovora subsp. carotovora strains in mixed potato tuber infections with E. carotovora subsp. betavasculorum were approximately 10⁶-fold lower after 48 h of incubation than in corresponding single sensitive strain infections. E. carotovora subsp. carotovora were not inhibited in tuber infections that were incubated anaerobically. This correlated with the absence of antibiotic production during anaerobic incubation in vitro. Antibiotic-resistant strains of E. carotovora subsp. carotovora were not inhibited in planta or in vitro by E. carotovora subsp. betavasculorum. Moreover, isogenic antibiotic-negative (Ant⁻) mutant E. carotovora subsp. betavasculorum strains were not inhibitory to sensitive E. carotovora subsp. carotovora strains in tuber infections.     

Effects of fluorescent pseudomonads on the potato blackleg syndrome

In four field trials in 1983 and 1984 potato tubers were inoculated by dipping them at planting in a suspension of Erwinia carotovora subsp. atroseptica and then treated with a powder formulation of two strains of fluorescent pseudomonads (B 10 and I 13) isolated from potatoes. lnoculation with E. carotovora increased blackleg and reduced emergence, plant growth, tuber size and weight compared with uninoculated controls. These effects were partially reversed by treatment of tubers with fluorescent pseudomonads which also reduced contamination by E. carotovora and the soft-rot potential of progeny tubers. In some trials a mixture of both pseudomonad isolates delayed the breakdown of the mother tuber although individual treatments did not.     

Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the α-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten “atypical” E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two “atypical” E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Changes in properties of phytopathogenic bacteria effected by plasmid pRD1

Transfer of plasmid pRD1 from Escherichia coli K12J62-1 to phytopathogenic bacteria, Agrobacterium tumefaciens, Xanthomonas beticola and Erwinia carotovora subsp. carotovora caused changes in conjugant properties not determined by the plasmid and the emergence of the properties not present in the parent strains. Clones have been obtained with intermediate properties between donor and recipient, including those with altered or lost virulence. In transconjugants of A. tumefaciens virulence increased. In transconjugants of X. beticola and E. carotovora subsp. carotovora highly virulent as well as avirulent forms have been observed. The loss of virulence in X. beticola correlated with the Nif^* phenotype. Plasmid pRD1 also affected the biochemical properties of the new hosts.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Genetic Regulation of Pathogenicity and Virulence Factors in Bacteria Erwinia carotovora subsp. atroseptica: Identification of Gene kduD

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp. atroseptica. the inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA). Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization. Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined. It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase. The intergene kduI–kduD region in bacteria Erwinia carotovora subsp. atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences. The kduD gene was located at 126.8 min of the Erwinia carotovora subsp. atroseptica genetic map.     

Cloning of the cysB gene of Erwinia carotovora subsp. carotovora, and the identification of its product

Summary The suicide vector pJB4JI was used to generate a range of Tn5-induced mutants of Erwinia carotovora subsp. carotovora (Ecc). One mutant, HC500, was a cysteine auxotroph which had a non-pectolytic, non-cellulolytic, non-proteolytic phenotype when grown under sulphate-limitation. The cysteine lesion of HC500 was shown to be analogous to the cysB mutation of Escherichia coli. The Ecc-cysB ⁺ gene product was identified as a protein of M_r 36000.     

Bacterial soft rot of tomato in plastic greenhouses in Crete

During recent years a new disease has been noticed on tomatoes grown in Polythene greenhouses in Crete. Early symptoms are yellowing of the lower leaves, and a yellow brown discoloration of the pith and stem xylem. As leaves wilt and die there is progressive yellowing towards the top of the plants. A progressive disintegration of the cortical tissues follows which results in a soft rot and a longitudinal splitting of the stem running mainly upwards. Soft rot of the fruits rarely appears. Severely infected plants may wilt and die, but other less affected plants often survive and yield normally. Very vigorous plants grown under humid conditions are more susceptible. Often more than 20% of the plants are infected. Isolations were made from stem (xylem, cortex and pith), from leaf xylem and from fruits of infected tomato plants collected throughout the island from 1979 to 1985. Bacteria of the genus Erwinia and Pseudomonas were consistently isolated. On the basis of physiological and biochemical characters of 49 representative pathogenic isolates, 22 were identified as Erwinia carotovora subsp. carotovora, 10 as Erwinia carotovora subsp. atroseptica, four as Pseudomonas viridiflava and 13 as Pseudomonas fluorescens biotype I. All disease symptoms were reproduced when artificial inoculations were made with the above isolates in the laboratory (20°C and 100% r.h.) on 3–4 week tomato plants and in a commercial greenhouse on 4–5 months tomato plants. Bacteria used for inoculations were reisolated. Results indicated that the disease symptoms as described may be caused by four different bacteria species.