Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

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

AIMS: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.     

Isolation by genomic subtraction of DNA probes specific for Erwinia carotovora subsp. atroseptica.

Erwinia carotovora subsp. atroseptica is a pathogen of potatoes in Europe because of its ability to induce blackleg symptoms early in the growing season. However, E. carotovora subsp. carotovora is not able to produce such severe symptoms under the same conditions. On the basis of the technique described by Straus and Ausubel (Proc. Natl. Acad. Sci. USA 87:1889-1893, 1990), we isolated DNA sequences of E. carotovora subsp. atroseptica 86.20 that were absent from the genomic DNA of E. carotovora subsp. carotovora CH26. Six DNA fragments ranging from ca. 180 to 400 bp were isolated, cloned, and sequenced. Each fragment was further hybridized with 130 microorganisms including 87 E. carotovora strains. One probe was specific for typical E. carotovora subsp. atroseptica strains, two probes hybridized with all E. carotovora subsp. atroseptica strains and with a few E. carotovora subsp. carotovora strains, and two probes recognized only a subset of E. carotovora subsp. atroseptica strains. The last probe was absent from the genomic DNA of E. carotovora subsp. carotovora CH26 but was present in the genomes of many strains, including those of other species and genera. This probe is homologous to the putP gene of Escherichia coli, which encodes a proline carrier. Further use of the probes is discussed.     

Involvement of N-acylhomoserine lactones throughout plant infection by Erwinia carotovora subsp. atroseptica (Pectobacterium atrosepticum)

Erwinia carotovora subsp. atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage. The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step. Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL). The role of HSL throughout plant infection was analyzed. To this purpose, HSL produced by a specific E. carotovora subsp. atroseptica wild-type strain, which was particularly virulent on potato, were identified. A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated. The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development. Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated. Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants. Therefore, the use of QS quenching strategies for biological control in E. carotovora subsp. atroseptica cannot prevent initial infection and multiplication of this pathogen.     

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 alpha-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.     

Rapid detection and identification of Erwinia carotovora subsp. atroseptica by a conjugated Staphylococcus aureus slide agglutination test

A. MCLEOD AND M.C.M. PEROMBELON. 1992. A conjugated Staphylococcus aureus slide agglutination test was used to detect and identify the potato blackleg pathogen, Erwinia carotovora subsp. atroseptica. Agglutination was obtained with > 10⁸ cfu/ml of the homologous strain with a polyclonal antiserum (171) against E.c. atroseptica serogroup I which is the predominant E.c. atroseptica serogroup on potatoes in Scotland. The titre of antiserum 171 against live cells of E.c. atroseptica groups I and XXII was 2000 whereas that of other serogroups was considerably less; only 1 and 4 out of 22 serogroups of E. carotovora subsp. carotovora reacted at 1:1500 and 1:1000 antiserum dilutions, respectively and one of the three less common other E.c. atroseptica serogroups reacted at 1:1000. When tested against 24 different bacterial species including E. chrysanthemi and saprophytic bacteria present in potato tuber rots, negative results were obtained with 1:1000 antiserum dilution. The titre against heat-treated (1 h, 70°C) cells of E.c. atroseptica serogroups I and XXII was1700–2000 whereas it was < 10 against other bacteria including E.c. carotovora. Detection of E.c. atroseptica serogroups I and XXII in diseased potato tissues was achieved directly by the slide agglutination test, but lower antiserum dilutions (1:700–1000) were needed. Still lower antiserum dilutions were needed with heat-treated test material for E.c. atroseptica identification.     

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.     

Rhizosphere bacteria affected by transgenic potatoes with antibacterial activities compared with the effects of soil, wild-type potatoes, vegetation stage and pathogen exposure

A greenhouse experiment was performed to analyze a potential effect of genetically modified potatoes expressing antibacterial compounds (attacin/cecropin, T4 lysozyme) and their nearly isogenic, nontransformed parental wild types on rhizosphere bacterial communities. To compare plant transformation-related variations with commonly accepted impacts caused by altered environmental conditions, potatoes were cultivated under different environmental conditions, for example using contrasting soil types. Further, plants were challenged with the blackleg pathogen Erwinia carotovora ssp. atroseptica. Rhizosphere soil samples were obtained at the stem elongation and early flowering stages. The activities of various extracellular rhizosphere enzymes involved in the C-, P- and N-nutrient cycles were determined as the rates of fluorescence of enzymatically hydrolyzed substrates containing the highly fluorescent compounds 4-methylumbelliferone or 7-amino-4-methyl coumarin. The structural diversity of the bacterial communities was assessed by 16S rRNA-based terminal restriction fragment length polymorphism analysis, and 16S rRNA gene clone libraries were established for the flowering conventional and T4 lysozyme-expressing Desirée lines grown on the chernozem soil, each line treated with and without E. carotovora ssp. atroseptica. Both genetic transformation events induced a differentiation in the activity rates and structures of associated bacterial communities. In general, T4 lysozyme had a stronger effect than attacin/cecropin. In comparison with the other factors, the impact of the genetic modification was only transient and minor, or comparable to the dominant variations caused by soil type, plant genotype, vegetation stage and pathogen exposure.     

Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp. atroseptica: identification of kduD gene

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-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 kdul-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.     

Inhibition of fungal and bacterial plant pathogens by synthetic peptides: in vitro growth inhibition, interaction between peptides and inhibition of disease progression

Four synthetic cationic peptides, pep6, pep7, pep11 and pep20, were tested alone and in combinations for their antimicrobial activities against economically important plant pathogenic fungi (Phytophthora infestans and Alternaria solani) and bacteria (Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica). In in vitro studies, P. infestans and A. solani were inhibited by all four peptides, while E. carotovora subsp. carotovora and E. carotovora subsp. atroseptica were inhibited only by pep11 and pep20. All peptides completely inhibited P. infestans and A. solani on potato leaves and P. infestans on tubers at concentrations comparable to the in vitro IC50 (effective concentration for 50% growth inhibition) values, suggesting that these peptides are more potent in preventing infection than in inhibiting hyphal growth in vitro. Microscopic observations of P. infestans and A. solani when treated with these peptides revealed hyphal anomalies. In tuber-infectivity assays, pep11 and pep20 reduced bacterial softrot symptoms by 50% at 2.0 to 2.30 microM and by 100% at 20 microM. In assays involving two-way combinations of these peptides, growth inhibitions of fungi and bacteria by the combinations were no more than the sum of growth inhibitions by each peptide when used alone, indicating that they act additively. pep11 and pep20 are not phytotoxic to potato plants at 200 microM. With strong and broad-spectrum antimicrobial activities of pep11 and pep20 against fungi and bacteria, and with no antagonistic activities, the expression of these peptides in transgenic potato plants could lead to enhanced disease resistance against these pathogens.     

Soft rot Erwinia bacteria in surface and underground waters in southern Scotland and in Colorado, United States

An anaerobic liquid enrichment method followed by plating on a selective medium revealed that the soft rot coliform bacterium Erwinia carotovora subsp. carotovora was generally present in water from drains, ditches, streams, rivers and lakes (including reservoirs) in southern Scotland and in Colorado, United States, in mountainous, upland and arable areas through the year. Many sites were remote from susceptible or diseased crops. Erwinia carotovora subsp. atroseptica was isolated much less frequently and no Erwinia bacteria were isolated from underground waters. Erwinia bacteria were also found in rain-water in Scotland, in winter snow from mountain passes in Colorado, and in sea water from the west and east coasts of Scotland and from the coasts of Oregon, California, Texas, Louisiana and Florida. The significance of the occurrence of these bacteria in water is discussed in relation to the control of blackleg and soft rot diseases of potato by production of Erwinia -free stocks.     

Survival of soft rot coliforms, Erwinia carotovora subsp. carotovora and E. carotovora subsp. atroseptica in soil in Scotland

An enrichment method was used to monitor Erwinia carotovora in soil or the rhizosphere of different crops and weeds in 17 fields with different cropping histories on three farms. The bacteria were detected in all fields not cropped with potatoes, although not consistently, and the mean annual frequency of detection was generally low (< 10%). Fields in which potatoes were grown were extensively contaminated after harvest in September but contamination declined over the winter to very low levels by early summer in the following year. Contamination level tended to rise in some fields without potatoes regardless of their cropping history but for only a short time during autumn and winter. The bacteria were no more frequent in rhizosphere soil of any of the weeds or crops examined, with the exception of brassicas, than in bare soil. In fields where more than 16 months had elapsed since cropping with potatoes, 91% of erwinia isolates obtained were E. carotovora subsp. carotovora , the remainder being E. carotovora subsp. atroseptica. The bacteria were shortlived in soil and in the rhizospheres of inoculated field and pot grown crop and weed plants. Longevity was greater in dry (10% moisture) than in wet (21% moisture) soil and decreased as temperatures rose, particularly above 25°C. Survival was best in association with brassica plants, moderate on grasses and cereals, and least on potatoes and weeds. E.c. carotovora survived better than E.c. atroseptica. Because survival of the bacteria in soil is apparently restricted, their presence in fields could be attributed to recurrent introductions from different sources.     

Blackleg potential of potato seed: determination of tuber contamination by Erwinia carotovora subsp. Atroseptica by immunofluorescence colony staining and stock and tuber sampling

Two methods to determine numbers of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, in tuber peel extract were compared; (1) growth and cavity formation on crystal violet pectate (CVP) medium (Pérombelon, Lumb & Hyman, 1987) and (2) immunofluorescent colony (IFC) staining (Van Vuurde & Roozen, 1990) using an antiserum against the bacterium conjugated with fluorescein isothiocyanate. Detection, identification and quantification of the bacterium based on the differential effect of temperature on growth in the CVP method were severely restricted and in some cases could not be done at low peel extract dilutions containing > 106 saprophytic bacteria ml“1 and > 103 cells ml-1 of E. carotovora subsp. carotovora. In contrast, although recovery was c. 50% relative to growth of E.c. atroseptica alone on nutrient agar, numbers of the bacteria could be determined by the IFC method regardless of numbers of saprophytic bacteria and E.c. carotovora present. Moreover, the tedium of counting colonies on a UV microscope could be avoided by automation using an imaging system on photograph film negatives of the microscope fields. Readily accessible tubers from the top layer of one tonne boxes in commercial stores were c. 10 times less contaminated than those from the middle of the boxes. For the two methods of peel extract preparation examined, the estimated sample size needed with an allowable error of log1010 E.c. atroseptica cells ml“1 extract with 95% confidence, was c. five tubers per box and 14 boxes for extract prepared from individual tubers and c. three lots of 10 tubers per box and 10 boxes for extract from 10 pooled tubers. A blackleg potential index for seed stocks was proposed based on the summation of the weighted number of individually tested tubers in different classes of contamination level.     

Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.)

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 degrees C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

pULB113, an RP4::mini-Mu plasmid, mediates chromosomal mobilization and R-prime formation in Erwinia amylovora, Erwinia chrysanthemi, and subspecies of Erwinia carotovora.

The RP4::mini-Mu plasmid pULB113, transferred from Escherichia coli strain MXR, was stable and transfer proficient in Erwinia amylovora strain EA303, E. carotovora subsp. atroseptica strain ECA12, E. carotovora subsp. carotovora strain ECC193, and E. chrysanthemi strain EC183. The plasmid mobilized an array of Erwinia sp. chromosomal markers (E. amylovora: his+,ilv+,rbs+,ser+,thr+;E. chrysanthemi:arg+,his+,ilv+,leu+; E. carotovora subsp. atroseptica: arg+,gua+,leu+,lys+,pur+,trp+; E. carotovora subsp. carotovora: arg+,gua+,leu+,lys+,out+[export of enzymes],pur+,trp+), suggesting random interactions of the plasmid with the chromosomes. In E. carotovora subsp. carotovora, pULB113-mediated two-factor crosses revealed linkage between three auxotrophic markers and the out loci. The export of pectate lyase, polygalacturonase, and cellulase and the maceration of potato tuber tissue occurred with Out+, but not Out-, strains of E. carotovora subsp. carotovora, indicating the importance of enzyme export in plant tissue maceration. Erwinia sp. donors harboring pULB113 complemented mutations in various biosynthetic and catabolic genes (arg, gal, his, leu, met, pro, pur, thy) in Escherichia coli recA strains. Escherichia coli transconjugants harbored pULB113 primes as indicated by the cotransfer of Erwinia genes and pULB113 markers and a change in plasmid mass. Moreover, the PstI and SmaI cleavage patterns of selected pULB113 primes were different from those of pULB113. pULB113 primes carried DNA insertions ranging from 3 to about 160 kilobases. These findings indicate that pULB113 is useful for in vivo gene cloning and genetic analysis of various enterobacterial phytopathogens.     

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.     

Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities

To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach. The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA. The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities. However, endophytic populations correlated to a certain extent with plant growth performance. Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes. A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations. In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris. The proportion of isolates with antagonistic activity was highest against Streptomyces sp. (43%) followed by those against Xanthomonas sp. (29%). As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists. The antifungal activity of the isolates was generally low. The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high. Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.     

Response of endophytic bacterial communities in banana tissue culture plantlets to Fusarium wilt pathogen infection

Endophytic bacteria reside within plant hosts without having pathogenic effects, and various endophytes have been found to functionally benefit plant disease suppressive ability. In this study, the influence of banana plant stress on the endophytic bacterial communities, which was achieved by infection with the wilt pathogen Fusarium oxysporum f. sp. cubense, was examined by cultivation-independent denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA directly amplified from plant tissue DNA. Community analysis clearly demonstrated increased bacterial diversity in pathogen-infected plantlets compared to that in control plantlets. By sequencing, bands most similar to species of Bacillus and Pseudomonas showed high density in the pathogen-treated pattern. In vitro screening of the isolates for antagonistic activity against Fusarium wilt pathogen acquired three strains of endophytic bacteria which were found to match those species that obviously increased in the pathogen infection process; moreover, the most inhibitive strain could also interiorly colonize plantlets and perform antagonism. The evidence obtained from this work showed that antagonistic endophytic bacteria could be induced by the appearance of a host fungal pathogen and further be an ideal biological control agent to use in banana Fusarium wilt disease protection.