Polypectate Medium Research Articles

First Report of Pectobacterium carotovorum subsp. brasiliense Causing Soft Rot and Blackleg of Potatoes in Kenya.

During the 2012/2013 growing season, potato tubers and stems showing rotting tissue and black discoloration, respectively, were obtained for analysis from Nyandarua and Mau Narok areas of Kenya, where potatoes are widely grown. During this period, more than 50% of the farms across Kenya reported cases of soft rot and blackleg diseases. Soft rot and blackleg diseases account for as much as 1/4 of the annual potato losses in Kenya. Bacteria from infected potato tuber and stem samples were isolated on nutrient agar then transferred to crystal violet polypectate medium (CVP) according to established standard procedures (3). All pit-forming (n = 48) strains were purified on nutrient agar and stored in 30% glycerol at -80°C for further use. All strains grew at 28°C and 37°C. PCR with pel gene specific primers (Y1/Y2) produced a 434-bp product and confirmed that all 48 strains have the gene sequence coding for pectate lyase specific for Pectobacterium spp. (1). Primers (Br1f/L1r) identified 1/3 of these strains as Pectobacterium carotovorum subsp. brasiliense based on their characteristic 322 bp (2). The other Pectobacterium spp. are currently undergoing further characterization. To further identify these pectolytic strains, a multi locus sequence typing (MLST) approach was employed (4). To this end, partial nucleotide sequences of the housekeeping genes mdh and gapA (accession nos. KF72004 to KF72009) showed 92% similarity to the Pcb1692 reference strain in GenBank. These results were in agreement with those obtained by species-specific primers. Phylogenetic analysis of the 679-bp concatenated partial gene sequences grouped strains collected in this study together with Pectobacterium subsp. brasiliense strains identified in other parts of the world with a 98% bootstrap support value. Three randomly selected Kenyan strains and Pcb1692 reference strain were inoculated into potato tubers in our research laboratory by making 1-cm holes into the tubers using a sterile pipette tip and thereafter injecting 10 μl (at 1.0 × 106 cfu/ml) into the tuber for pathogenicity assays. A negative control of 10 mM MgSO4 was included and all the inoculated holes sealed with petroleum jelly to avoid contamination. This experiment consisted of five potato tubers per strain in three independent assays. All three representative strains induced water soaked soft symptoms similar to the symptoms previously observed on infected potato tubers. Furthermore, when bacterial suspensions of 1.0 × 106 cfu/ml isolated strains and the Pcb1692 reference strain were inoculated onto potato stems maintained at 28°C, blackleg and wilting of the stems occurred within a period of 3 to 21 days. No symptoms were observed in potato tubers or stems inoculated with the negative control (MgSO4). PCR with Br1f/L1r primers confirmed that the re-isolated bacteria were P. carotovorum subsp. brasiliense. To our knowledge, this is the first occurrence of P. carotovorum subsp. brasiliense on potatoes in Kenya.

Characterization of Erwinia Populations from Nursery Retention Ponds and Lakes Infecting Ornamental Plants in Florida.

Water shortages in Florida are occurring due to intense utilization of the aquifer system by municipalities and agriculture, and because of continued deficits in annual rainfall. Water districts therefore, are, recommending the use of recycled irrigation water, stormwater runoff, reclaimed municipal sewage water, and lakes for agricultural use. With recycled water, however, there is potential for both introducing and concentrating plant pathogens. In Florida, Erwinia soft-rot bacteria (synonym Pectobacterium) cause extensive crop losses in ornamental plant production. To determine Erwinia spp. population levels, samples were taken monthly for 1 year from four hypereutropic lakes and eight nursery retention ponds. Seventy-seven Erwinia strains were collected by both direct plating and by an enrichment process. With the direct plating method, 0 to 29 CFU/ml were detected on sodium polypectate medium. Significantly higher populations of Erwinia were detected in retention ponds of nurseries that were actively reutilizing their water. Erwinia strains were identified to species by fatty acid analysis and biochemical tests. Strains were further characterized by repetitive element-polymerase chain reaction (rep-PCR) and compared with 120 strains of Erwinia collected from ornamentals over a 17-year period in Florida. Using rep-PCR, most strains were clustered into two heterogeneous populations of E. chrysanthemi and E. carotovora subsp. carotovora in a 1:2 and a 1:4 ratio for isolates from ornamentals and from water, respectively. Within each population of E. chrysanthemi and E. carotovora, genetically different subpopulations could be identified that contained high percentages of Erwinia strains from water sources. Even though genetic differences exist, 99% of the strains from water sources were found to be pathogenic on dieffenbachia. Without water treatment of irrigation and stormwater runoff, there is a potential for both introducing and concentrating Erwinia populations within these water sources.

Pectate lyase isozymes and the pathogenicity of soft rotting strains of Erwinia for melon and cucumber

Two strains of an Erwinia sp. obtained from soft rotting melon (Cucumis melo) plants in Egypt were found to be highly pathogenic on melon but non-pathogenic on the related crop cucumber (C. sativus). Typical isolates of E. carotovora ssp. carotovora (Ecc) were pathogenic on both species while isolates of the related ssp. atroseptica were pathogenic in neither. Diagnostic tests showed that the two strains had characteristics which were not wholly typical of Ecc nor of the related species E. chrysanthemi (Echr) but indicated affinities with both. Thus. both strains produced indole, which is typical of Echr but not Ecc, but fermented lactose and trehalose, which are characteristics of Ecc but not Echr. They also produced basic pectate lyases only on a polypectate medium, a characteristic which is more typical of Ecc than of Echr. One of the strains was tested for its ability to grow in cucumber cotyledons and although the generation time and final population achieved were less than half of those in melon, substantial growth did occur. Thus, although not a soft rotting pathogen of cucumber, it is clearly a parasite of this species. Culture supernates of both isolates were capable of soft rotting cucumber tissue and so the failure to cause a soft rot was not due to the resistance of the tissues to the enzymes produced by the two strains but could be due to the failure of cucumber tissue to induce the production of the appropriate enzymes. An analysis of pectate lyase production by one of the strains showed that it produced basic isozymes only when grown on a polypectate medium or on autoclaved cucumber tissue but it produced an acidic isozyme in addition to basic isozymes when grown on autoclaved melon tissue and when colonising melon seedlings. It is suggested that the failure of the two melon strains to cause a soft rot in cucumber seedlings is due to the failure of the tissue to provide the appropriate environment for the induction of the set of lyase isozymes required for tissue maceration.

Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp. and other pectolytic bacteria isolated from potato tubers.

Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests. For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography. The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium. In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp. rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL. The responses of Erwinia spp. were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity. Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity. The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger. Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions.

Detection of soft rot Erwinia spp. on seed potatoes: conductimetry in comparison with dilution plating, PCR and serological assays

Automated conductance measurements in polypectate medium were used for the detection of pathogenic soft rot Erwinia spp. in potato peel extracts. The detection threshold for Erwinia carotovora subsp. atroseptica (Eca) in inoculated peel extracts was ca. 104 colony forming units (cfu) ml-1 when samples were considered positive on the basis of a response within 48 h at 20 °C. Detection of E. chrysanthemi (Ech) was less sensitive, only 105 cfu ml-1 peel extract were detected within 36 h at 25 °C. The linear correlation between detection times in conductimetry and inoculum levels of Eca and Ech in peel extracts was used for a quantitative estimation of Eca and Ech in naturally contaminated peel extracts. Samples giving a positive conductimetric response had to be confirmed with an enzyme-linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR) for the presence of Eca and Ech, because E. carotovora subsp. carotovora (Ecc) also generated a conductance response. Conductimetry was sensitive and efficient for detection of contamination levels of Eca higher than 104 cfu ml-1 peel extract. For Ech, conductimetric detection was less sensitive and inefficient due to low contamination levels of Ech and the presence of high numbers of Ecc in many samples after enrichment, which interfered with the test. Immunofluorescence cell staining (IF) combined with enrichment and immunofluorescence colony staining (IFC) were suited to detect and quantify low numbers of Eca and Ech at less than 104 cfu ml-1 in peel extracts. However, since false positive and negative reactions in serology were observed, the use of PCR after enrichment, or in combination with IFC to confirm positive results, was required for accurate detection.

Effects of aluminium in acid streams on growth and sporulation of aquatic hyphomycetes

We investigated, by field and laboratory experiments, the effects of aluminium in an acid stream (pH 5.0) on the growth and sporulation of aquatic hyphomycete fungi which degrade organic litter. The stream water had monomeric aluminium (Al m) concentrations of 9.1–13.4 μ m — fifty times higher than a nearby circumneutral stream. Alder leaves submersed in the stream accumulated Al, most of which was tightly bound. Growth rates of four species of aquatic hyphomycetes were altered by inclusion of Al m in the culture medium. On a polypectate substrate, and on low-phosphate medium with glucose, growth rates increased significantly. On a low-nutrient substrate of homogenized alder leaves, growth rates were inhibited by aluminium. The pattern of mycelial growth was found to be different on a polypectate medium including Al m, compared with a control without aluminium. There was a significant increase in hyphal radial growth and a decrease in the hyphal growth unit. The effect resembled the growth of a starved fungal colony. Treatment with Al m decreased pectinase production by the four fungal species tested. The capacity of these species to sporulate was reduced by flooding culture plates with Al m solution. These deleterious metabolic effects were most severe in isolates taken from circumneutral streams and less marked, though significant, in species originating from acid streams.

Comparison of Methods for Detection of Erwinia carotovora ssp. atroseptica in Progeny Tubers Derived from Inoculated Tubers of Solanum tuberosum L.

AbstractSeveral techniques for detection of Erwinia carotovora ssp. atroseptica (Eca) on potato tubers were used to study the transmission of Eca from inoculated mother tubers into progeny tubers for 12 different potato cultivars. The sensitivity of the polymerase chain reaction (PCR) for a direct detection of Eca in crude peel extracts was only 107‐108 cells/ml, due to inhibition of PCR amplification by potato tuber‐derived compounds. With dilution plating and ELLSA, 105 and 107 cells/ml were detected, respectively, Immunofluorescence cell staining (IF), with a detection threshold of 104‐105 cells/ml, was shown to be most sensitive for direct detection, and transmission of Eca was found for six potato cultivars, whereas with the other techniques tested no transmission was found. After 48 h enrichment of peel extract in polypectate medium at 20 C, IF and PCR detected an initial inoculum concentration of 10 Eca cells/ml, whereas 102, 104 and 104 cells/ml were detected in dilution plating, ELISA and conductimetry, respectively. With dilution plating, PCR and IF, performed after enrichment, transmission of Eca was found for, respectively, four, six and seven out of 12 potato cultivars tested. No transmission was found using ELISA and conductimetry, due to their lower sensitivity, Erwinia carotovora ssp, carotovora (Ecc) was only detected in the progeny tubers of control plants. No relation was found between the transmission of Eca to progeny tubers and blackleg incidence in the field.

Selective Medium for Isolation of PectolyticErwiniasp.

Miller-Schroth medium modified by replacement of most of the agar with sodium polypectate appeared superior to the crystal violet polypectate medium for the selective isolation of pectolytic Erwinia sp. NaOH and MOPS (3-[N-morpholino]propanesulfonic acid) were added to raise the pH and buffer the medium, respectively. Pectolytic erwinias appeared as pink to orange colonies located in deep pits. Nonpectolytic erwinias formed pink-orange colonies with no pits (.)

Cellular and environmental factors affecting the synthesis of polygalacturonate lyase byBacillus subtilis

Cellular and environmental factors affecting the synthesis of polygalacturonate lyase in batch and chemostat cultures ofBacillus subtilis were investigated. The lyase was produced constitutively during growth on a wide range of carbon sources in a defined minimal medium and in medium containing complex organic carbon and nitrogen sources. The highest activity was obtained during batch growth in minimal medium containing glucose and ammonium sulphate. Over 99% of the activity was present extracellularly in the supernatant medium at all stages of the batch growth cycle. Two distinct differential rates of synthesis were observed during exponential growth. The lyase was unable to attack pectin rapidly unless pectin methyl-esterase was also present. Pectin was a poor substrate for growth and polygalacturonate lyase induction because the organism did not produce pectin methyl-esterase. In continuous-flow chemostat cultures with glucose medium, polygalacturonate lyase activity declined to a very low level owing to the selection of non-productive mutant strains. Loss of activity did not occur when polypectate was the carbon source. Steady-state specific polygalacturonate lyase activity in polypectate medium was relatively independent of dilution rate in the range 0.04 to 0.36/h. When polypectate was supplied in excess of the growth requirement lyase activity was 5 times higher than during polypectate-limited growth.