Extensin and Phenylalanine Ammonia-Lyase Gene Expression Altered in Potato Tubers in Response to Wounding, Hypoxia, and Erwinia carotovora Infection

Potato (Solanum tuberosum L.) tubers are susceptible to infection by Erwinia carotovora, causal agent of bacterial soft rot, when wounded and subjected to wet, hypoxic environments. The expression of two putative plant defense genes, extensin and phenylalanine ammonia-lyase (PAL), was examined by monitoring their respective mRNA levels and cell wall hydroxyproline levels in tuber tissues under various conditions leading to susceptibility or resistance and after inoculation with E. carotovora in order to assess the possible roles of these genes and their products in this plant-pathogen interaction. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels accumulated markedly in response to wounding and subsequent aerobic incubation. Extensin and PAL mRNA levels as well as cell wall hydroxyproline levels decreased in response to wounding and subsequent anaerobic incubation; these changes were correlated with high susceptibility of tuber tissue to E. carotovora infection. Inoculation of wound sites with E. carotovora caused some additional accumulation of the wound-regulated extensin and PAL mRNAs under certain aerobic conditions, but never under anaerobic conditions.     

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.     

Characterization of Monoclonal Antibodies Specific for Erwinia carotovora subsp. atroseptica and Comparison of Serological Methods for Its Sensitive Detection on Potato Tubers

Seven monoclonal antibodies (MAbs) to Erwinia carotovora subsp. atroseptica have been produced. One, called 4G4, reacted with high specificity for serogroup I of E. carotovora subsp. atroseptica, the most common serogroup on potato tubers in different serological assays. Eighty-six strains belonging to different E. carotovora subsp. atroseptica serogroups were assayed. Some strains of serogroup XXII also reacted positively. No cross-reactions were observed against other species of plant pathogenic bacteria or 162 saprophytic bacteria from potato tubers. Only one strain of E. chrysanthemi from potato cross-reacted. A comparison of several serological techniques to detect E. carotovora subsp. atroseptica on potato tubers was performed with MAb 4G4 or polyclonal antibodies. The organism was extracted directly from potato peels of artificially inoculated tubers by soaking or selective enrichment under anaerobiosis in a medium with polypectate. MAb 4G4 was able to detect specifically 240 E. carotovora subsp. atroseptica cells per ml by indirect immunofluorescence and immunofluorescence colony staining and after soaking by ELISA-DAS (double-antibody sandwich enzyme-linked immunosorbent assay) after enrichment. The same amount of cells was detected by using immunolectrotransfer with polyclonal antibodies, and E. carotovora subsp. atroseptica and subsp. carotovora were distinguished by the latter technique. ELISA-DAS using MAb 4G4 with an enrichment step also efficiently detected E. carotovora subsp. atroseptica in naturally infected tubers and plants.     

Zur Rolle des Pektins bei der Ausprägung sorten- und jahresbedingter Unterschiede der Naßfäuleanfälligkeit von Kartoffelknollen

The influence of soil moisture and mean day temperature of July to September on pectin structure and soft rot resistance of potato tubers was investigated. Tuber samples of middle early and middle late potato cultivars respectively were tested from 1976 to 1979. Cultivar characteristics as well as growth conditions effect the pectin structure. The tuber tissue of soft rot resistant cultivars contains relative high amounts of esterified pectin. The pectin esterification was promoted by drought during vegetation periods (1976, 1978). A late rise of metabolic activity caused by rainfall at the end of the vegetation period (1978) increased the amount of soluble pectin in the cell wall. This lack of pectin stabilization facilitates tissue maceration by soft rot bacteria. Increased pectin solubility was also caused by unfavorable storing conditions of potato tubers. A low degree of esterification and high solubility of the cell wall pectin affect the relative resistance of potato tubers against soft rot caused by Erwinia carotovora var. atro-septica (van Hall) Dye adversely.     

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.     

Infection of potato tubers with soft rot bacteria

Stolons attached to developing potato tubers were inoculated with the soft rot bacterium Erwinia carotovora var. atroseptica. Almost all the stolons rotted, but soft rots developed in less than 10% of new tubers; the bacterium was isolated later from these tubers. No rots developed in the other tubers but the bacterium was later isolated from about half of them. It could not be isolated from tubers attached to inoculated stolons where the rot on them did not extend to the tuber or from tubers attached to stolons that were not inoculated though many of these rotted. The bacterium was reisolated from almost all arrested lesions in tubers inoculated 8 month earlier with E. carotovora var. atroseptica. Blackleg did not develop from plants grown fom these tubers under various soil conditions. It did develop in a large proportion of plants from tubers inoculated shortly before planting and grown in cool, wet soil. Less than 1% blackleg developed in plants grown from tubers from plants with blackleg or from plants immediately adjacent. The presence of pectolytic bacteria and E. caratovora var. atroseptica in seed and new tubers was investigated during June, July and August. Although E. caratovora var. atroseptica was obtained from c. 40% tubers, only c. 0·3% of c. 8400 plants developed blackleg. The bacterium was isolated from only three of 160 new tubers sampled during the summer.     

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.     

Transformation of Erwinia carotovora ssp. atroseptica with the Green Fluorescent Protein Gene from Aequorea victoria

Erwinia carotovora ssp. atroseptica has been transfortned with the green fluorescent protein (GFP) gene from Aequorea victoria. Transformants had very high levels of GFP expression but some plasmid instability was apparent when bacteria were not grown on atnpicillin. Confocal microscopy showed variation between cells in the level of expression of GFP. Transfonnants retained their pathogenicity on potatoes, rotting tubers and causing typical blackleg symptoms when inoculated into stems, but the level of fluorescence was not suitable for observing bacterial movement. In contrast, E. coli producing GFP could be used to follow bacterial movement within vascular tissue of potato leaves.     

Sites of contamination and numbers of Erwinia carotovora present in stored seed potato stocks in Scotland

Forty-eight bulk and crate stored commercial seed potato stocks from the east of Scotland were examined for contamination by Erwinia carotovora in 1967 and 1968. Both E. carotovora var. carotovora and E. carotovora var. atroseptica were present, the latter, the blackleg pathogen, being detected four times more frequently. Most contamination was located on the tuber surface and in the lenticels; it was rare in the vascular ring and the organism was never detected in the cortex. The level of contamination of lenticels was only slightly affected after winter storage, which, however, substantially reduced surface contamination. More than 10³ cells were detected on the surface of tubers in over half the stocks and in half the stocks each lenticel sampled contained more than 10² bacteria.     

Genotypic differences in the uptake and distribution of radiocaesium in plants

The ability of endophytic bacteria to influence Erwinia carotovora var. atroseptica (Eca) growth and disease development was examined in potatoes. Bacterial populations isolated from within the tubers of five potato (Solanum tuberosum L.) cultivars (Kennebec, Butte, Green Mountain, Russet Burbank and Sebago) showed antibiosis toward Eca in an in vitro assay. Sebago was host to the highest percentage of bacterial isolates inhibiting Eca growth in vitro (49.5%), followed by Green Mountain (33.3%), Kennebec (29.3%), Russet Burbank (12.9%) and Butte (1.8%). Of these, Curtobacterium luteum was the most common species. Few endophytic bacteria from Butte were inhibitory to Erwinia; all were from Pantoea agglomerans. Significantly higher populations of Erwinia-inhibiting bacteria were recovered from Kennebec (1.89 × 10⁶ cfu fresh weight tuber tissue) as compared to the other cultivars; the lowest populations were recovered from Butte (0.01 × 10⁶ cfu per g fresh weight tuber tissue). Published levels of cultivar disease resistance to blackleg did not correspond to actual bacterial soft rot development (induced by Eca) in an in vivo (tuber) assay. However, bacterial soft rot development was negatively correlated with the density of tuber populations of endophytic bacteria found able to inhibit Eca growth in vitro (R=−0.879, p=0.05).     

Needle in a haystack: Antibacterial activity-guided fractionation of a potato wound tissue extract

Erwinia carotovora is a major cause of potato tuber infection, which results in disastrous failures of this important food crop. There is currently no effective antibiotic treatment against E. carotovora. Recently we reported antibacterial assays of wound tissue extracts from four potato cultivars that exhibit a gradient of russeting character, finding the highest potency against this pathogen for a polar extract from the tissue formed immediately after wounding by an Atlantic cultivar. In the current investigation, antibacterial activity-guided fractions of this extract were analyzed by liquid chromatography-mass spectrometry (LC-MS) utilizing a quadrupole-time-of-flight (QTOF) mass spectrometer. The most active chemical compounds identified against E. carotovora were: 6-O-nonyl glucitol, Lyratol C, n-[2-(4-Hydroxyphenyl)] ethyldecanamide, α-chaconine and α-solanine. Interactions among the three compounds, ferulic acid, feruloyl putrescine, and α-chaconine, representing metabolite classes upregulated during initial stages of wound healing, were also evaluated, offering possible explanations for the burst in antibacterial activity after tuber wounding and a chemical rationale for the temporal resistance phenomenon.     

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 g/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.Abbreviations AP acidic carboxyl terminal polypeptide - Eca Erwinia carotovora ssp. atroseptica - Ecc E. carotovora ssp. carotovora - Ech E. chrysanthemi - IF intercellular fluid - SP signal peptide - TPNI (tpnI) tachyplesin I     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

Effect of certain cultural practices on susceptibility of potato tubers to soft rot disease caused by Erwinia carotovora pv. carotovora

Erwinia soft rot causes destructive and serious damage to many vegetable crops including potato in the field, transit and storage periods. The effect of certain cultural practices on the susceptibility of potato tubers to soft rot bacteria was studied and the results of this work can be summarised in the following: potato tubers harvested on 1 May first exhibited the highest disease incidence compared with those harvested on 15 May or 30 May. Harvesting on 15 June resulted in the lowest disease infection. The application of high levels of nitrogen fertiliser as urea (46.5%), ammonium nitrate (31%) and ammonium sulphate (20.5%) resulted in an increase of the susceptibility of potato tubers to bacterial soft rot disease. In contrast, the addition of phosphorous as superphosphate (15.5%) fertiliser caused the reverse effect. The addition of potassium as potassium sulphate (48%) alone at any of the tested levels showed no effect. The susceptibility of potato tubers to bacterial soft rotting disease was increased by increasing storage periods at 4°C for 1, 2, 3 and 4 months. Spraying copper sulphate exhibited the highest decrease in soft rot incidence disease followed by manganese, zinc and iron. However, spraying of boron increased susceptibility to the disease. Potato tubers obtained from plants sprayed with copper and stored for different periods showed the lowest susceptibility to disease incidence. Tuber sprayed with zinc, iron, manganese and finally boron came next.     

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.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

The extent and survival of contamination of potato stocks in Scotland by Erwinia carotovora var. carotovora and E. carotovora var.atroseptica*

The following results were obtained when fifty-seven bulk and crate-stored commercial seed potato stocks from the East of Scotland were examined in 1966-8 for contamination by pectolytic Erwinia spp. (1) Most tubers of all the cultivars and stocks examined, irrespective of whether they were obtained from blackleg-infected or blackleg-free crops, were contaminated with E. carotovora; (2) some 80% of the Erwinia isolates obtained were identified as var. atroseptica, the rest being var. carotovora; (3) the organisms survived in and on tubers for 6–7 months of bulk storage over the winter and up to planting time the following spring; (4) contrary to what is generally thought, the high incidence of contamination of all stocks, while suggesting that the seed itself is the major source of E. carotovora for the growing crop, emphasizes that other factors affect manifestation of blackleg in the field and soft rot in store.