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.     

Anaerobic Nitrate Respiration by Erwinia carotovora subsp. atroseptica during Potato Tuber Invasion

The in planta induction of anaerobic nitrate respiration by Erwinia carotovora subsp. atroseptica in relation to the in situ oxygen status in soft rotting potato tubers has been investigated. In vitro experiments have shown that nitrate was required for the induction of respiratory nitrate reductase activity in E. carotovora. In addition, oxygen was found to repress this activity. Expression of respiratory nitrate reductase was found in E. carotovora cells extracted from soft rotting potato tuber tissue. However, the rate of nitrite production in these cells was approximately 70-fold lower than the rate recorded in fully induced anaerobic cultures. Oxygen measurements in soft rotting potato tubers indicated that the invading bacteria encounter the lowest oxygen concentration at the interphase between healthy and macerated tissue. Consequently, growth of bacteria present in this specific zone will be stimulated by nitrate which is present in sufficient amounts in tuber tissue. A high nitrate content of the tuber will most likely facilitate the proliferation of E. carotovora in the tuber tissue.     

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.     

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.     

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.     

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.     

Potato tuber lenticels: susceptibility to infection by Erwinia carotovora var. atroseptica and Phytophthora infestans

In laboratory tests, proliferation increased the susceptibility of lenticels on mature tubers to infection by Erwinia carotovora var. atroseptica. No differences in susceptibility to E. c. atroseptica and Phytophthora infestans were detected on tubers lifted at different stages of growth from soil kept wet or dry for 3 wk prior to each sampling. As tubers aged, fewer lenticels became infected by either pathogen or were penetrated by fluorescein dye, presumably because cells had been suberized and cork barriers formed.     

A method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica)

In experiments to develop a method for assessing the field susceptibility of potato cultivars to blackleg (Erwinia carotovora subsp. atroseptica) seed tubers were stab-inoculated near the stolon (attachment end), with a suspension of the bacterium, or with water, before planting. Disease symptoms were recorded in three years (1980–1982) and plant growth and yield in 1982. Estima and Maris Bard were the most susceptible cultivars with many plants failing to emerge and most of those that did showing disease symptoms. Pentland Crown was the most resistant: few plants failed to emerge and few showed blackleg. Nevertheless compared with water-inoculated plants bacterial inoculation of the seed tubers of this cultivar caused loss of yield and differences in tuber size distribution. Cara, Wilja and King Edward showed intermediate reactions.     

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.     

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     

Effects of treating seed potatoes with trace elements on sprouting and microbial spoilage during storage under tropical conditions

Attempts were made to extend the storage life of seed potatoes by treating them with trace elements as antimicrobial and antisprouting agents. In tests to measure the growth of Fusarium oxysporum, and Erwinia carotovora pv. carotovora it was found to be decreased more by iron, copper and zinc than by boron, manganese and molybdenum. Dipping seed tubers in aqueous solutions of trace element salts decreased both sprouting and microbial spoilage during storage at ambient conditions. Field testing at the end of storage proved that treating seed potatoes with ferrous sulphate did not decrease the yield of tubers or increase their iron content. It is suggested that trace elements may be used to extend the storage life of seed potato tubers, in tropical conditions.     

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 effect of Erwinia carotovora subsp. atroseptica (blackleg) on potato plants. I. Growth and yield of different cultivars

The effect of planting seed tubers inoculated either near the stolon attachment (heel end), among the eyes at the apex (rose end) or mid-way along the tuber with Erwinia carotovora subsp. atroseptica, was assessed in terms of growth of the plant, disease symptoms and yield. Invariably rose-end inoculation had the greatest and heel-end the least effect in decreasing yield when compared with uninoculated plants. Cultivars Majestic and King Edward were the most susceptible, Pentland Crown showed some resistance to invasion of stems (blackleg) although plant vigour, expressed in terms of plant height and stem number was affected and Maris Piper was the most resistant.     

Screening of cassava (Manihot esculenta Crantz) accessions against Phytophthora palmivora induced tuber rot of cassava

Cassava (Manihot esculenta Crantz), is an important tropical tuber crop with global importance and plays a significant role in the food, nutritional and livelihood security of around 500 million people. In India, the low productivity of cassava attributes to the soil borne disease, particularly tuber rot caused by Phytophthora palmivora (Butl.) which is destructive and the attack is spreading in alarming rate in all the cassava growing regions causing heavy yield loss of more than 50%. Introduction of disease resistant varieties may alleviate the problem to a certain extent. This paper describes the screening procedures and findings on the disease resistant variety of cassava accession against tuber rot. Variety Sree Padmanabha imparted high resistance against tuber rot, while Sree Sahya was moderately resistant and all other accessions studied were found to be susceptible in in vitro and in field trials. In screening studies, a reproducible positive correlation was obtained between attached tubers in live plant with detached tubers which showed that detached tuber part can be used for the prediction of resistance in attached live plants of cassava for cultivar resistance. The procedure described here could be used as a simple, rapid and efficient method for screening of cassava accessions against tuber rot of cassava.     

The Influence of the Inoculum Source of Rhizoctonia solani on Development of Black Scurf on Potato

Rhizoctonia solani, the causal agent of stem canker and black scurf on potato, survives as sclerotia on tubers, in soil and in plant residues. The objective of the present study was to evaluate the importance of inoculum source on disease development. Disease‐free minitubers and seed tubers contaminated with low levels of R. solani were planted in fumigated or artificially inoculated growth mixture in greenhouse experiments. Black scurf incidence and severity were significantly higher when the inoculum was present in both seed tubers and soil, compared with either of them separately. The severity of disease symptoms on the subterranean parts of the plant also were significantly higher in plots where both seed tubers and soil were contaminated, compared with plots where the inoculum source was either the seed tubers or the soil. Thus, both major sources of inoculum, seed tubers and soil, are important in disease development. However, when both sources are present, black scurf incidence and severity are increased, leading to economical damage to tuber yield and quality. Additional results from field trials support these findings. Disease incidence and severity on daughter tubers were correlated with levels of contamination in seed tubers and soil. When seed tubers and soil were heavily infested, the levels of black scurf incidence and severity on daughter tubers were very high; when seed tuber and soil infestation were very low, black scurf incidence and severity on progeny were also lower. Disease levels were reduced by in‐furrow fungicide treatment, but were less effective when the initial levels of the fungus on the seed tubers and in the soil were high.     

Control of contamination of potatoes with air-borne Erwinia carotovora by foliar application of copper oxychloride

In field experiments in 1983 and 1984, micropropagated plants of cv. Désirée planted in soil tested and found free of erwinias became contaminated with airborne erwinias, mostly E. carotovora pv. carotovora, in September when rain was more abundant, but not earlier. Control of contamination of progeny tubers harvested in September and October was achieved by spraying plants with copper oxychloride (‘Cuprokylt’; 5 g litre^-1) twice weekly from June or July but not from August nor by weekly or fortnightly treatments from June. Bactericidal activity of copper in the effective spray regimes was not expressed on the leaves or in the soil and contamination was similar in the sprayed and unsprayed treatments. In contrast, multiplication of erwinias in senescent leaves on the soil surface (leaf debris) was inhibited in the sprayed treatment but not in the unsprayed treatment and population numbers of c. 10³ cells g^-1 fresh weight were detected. It is suggested that fewer erwinias would be washed from leaf debris by rain in the sprayed than in the unsprayed treatment to contaminate the progeny tubers.     

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.