A simplified slice method for assessing tuber susceptibility of potato cultivars to Erwinia carotovora subsp atroseptica

The susceptibility of 24 potato cultivars to soft rot caused by Erwinia carotovora subsp. atroseptica was assessed in a simplified form of a tuber slice test described previously. Freshly made wounds on tuber slices were inoculated with drops (0–02 ml) containing 10⁸, 10⁷ or 10⁶ cells/ml and incubated aerobically for 3 days at 15° or 20°C. At 20°C differences between cultivars were clear at all concentrations but at 15°C less so at the lowest concentration unless incubation time was extended. The rank order of cultivar susceptibility was similar to that found in previous seasons.     

Contamination of potatoes by Erwinia carotovora during grading

Considerable tuber contamination by soft rot erwinias in rotting tubers can occur when grading potato stocks. Erwinia carotovora pv. carotovora , from a single rotting tuber, contaminated c. 100 kg of potatoes during mechanical grading, c. 50% being contaminated with 10⁴–10⁵ bacteria per tuber. Survival of the bacteria during storage was related to the degree of damage sustained by the tubers during grading which in turn depended on the model of oscillating riddle grader used and the cultivar of potato. Chemical disinfection of the grader immediately before use and of tubers immediately after grading reduced contamination.     

Detection of Erwinia carotovora subsp. atroseptica and Erwinia chrysanthemi in potato tubers using polymerase chain reaction

Soft rot erwiniae are a group of notorious plant pathogens for which currently available detection methods are inadequate. Based on the polymerase chain reaction, specific and sensitive detection of Erwinia carotovora subsp. atroseptica and E. chrysanthemi in potato tubers has been achieved. The composition of the PCR primers used in two specific detection systems is based on identification of the consensus of sequences of metalloprotease-coding genes present in soft rot erwiniae. Bacterial DNA was extracted from the potato tuber matrix by differential centrifugation in order to avoid interference of potato-derived compounds with the performance of the PCR assay. The PCR assay jjerformed with the E. carotovora subsp. atroseptica specific primer set was found to be capable of distinguishing E. carotovora subsp. atroseptica from all other Erwinia species and the closely related subspecies E. carotovora subsp. carotovora. With the E. chrysanthemi specific primer set, agarose gel electrophoresis is required for unequivocal differentiation between E. chrysanthemi and other erwiniae. Combined with the efficient extraction procedure, the assay allowed specific detection of less than 10³ culturable erwiniae per tuber. The specificity and sensitivity of the assay were not reduced in the presence of a 100-fold excess of DNA from both related and unrelated bacteria. This PCR-based method for detection of erwiniae in potato tubers provides a relatively fast and sensitive alternative to routinely applied serological methods.     

Microbiological, immunological and molecular methods suitable for commercial detection and quantification of the blackleg pathogen, Erwinia carotovora subsp. atroseptica, on seed potato tubers: a review

Contamination of seed potato tubers by Erwinia carotovora subsp. atroseptica is widespread with the bacteria usually sited superficially in lenticels and suberized wounds. As seed contamination level is related to blackleg incidence, seed health is best assessed by determining the number of cells of E. c. atroseptica per mL of tuber-peel extract. The relative specificity, sensitivity and ease of use of four recently developed microbiological, immunological and molecular methods to detect and/or quantify tuber contamination are discussed in relation to the testing of commercial seed stock. Sensitivities of all four methods are at or below the threshold level for blackleg development (< 10³ cells mL^-1), but there are differences regarding their specificity and ease of use. Three of them allow enumeration of most live cells of the bacterium, using specific monoclonal and polyclonal antibodies against the predominant serogroup I: (a) immunomagnetic separation of E. c. atroseptica before viable count on a selective-diagnostic growth medium, crystal violet pectate, (b) immunofluorescence staining and counting of colonies in pour-plate medium in tissue culture plates and (c) enrichment of the bacterium in peel-extract dilutions directly in microtitre plates prior to DAS-ELISA. In the fourth method, both live and dead cells are detected, but not quantified, by PCR amplification of target sequences using specific primers for E. c. atroseptica regardless of serogroup.     

Characterisation of Erwinia carotovora subspecies and detection of Erwinia carotovora subsp. atroseptica in potato plants, soil and water extracts with PCR-based methods

A PCR-RFLP test based on a pectate-lyase encoding gene permits the detection of several Erwinia carotovora subspecies, but requires complete DNA extraction. This paper reports on the suitability of a simplified PCR-RFLP protocol to characterise E. carotovora strains and on the performance of PCR, using the same primers, to detect the atroseptica subspecies in substrates of epidemiological significance. A collection of 140 strains from various hosts and geographical origins was characterised for biochemical traits and PCR-RFLPs. PCR performed on boiled bacterial suspensions yielded an amplification product of 434 bp in 109 of the 140 strains. None of the E. carotovora subsp. betavasculorum strains was amplified, even after complete DNA extraction. RFLPs of the PCR product yielded 24 groups, 3 of which were new. Twenty one groups were specific to one subspecies. Several strains biochemically similar to E. carotovora subsp. atroseptica, but growing at 37 °C, showed PCR-RFLP profiles characteristic of E. carotovora subsp. carotovora. Phenetic and cladistic analyses gave three main domains, not strictly related to hosts or geographical origins. The atroseptica (RFLP groups 1 and 2) and wasabiae (group 21) subspecies constituted one of the domains, despite clustering distantly from one another. Host specialisation and molecular homogeneity suggest a clonal structure within these subspecies. Conversely, E. carotovora subsp. odorifera, despite its limited host range and geographical distribution, and E. carotovora subsp. carotovora showed great molecular diversity, spreading respectively across five and 19 RFLP groups. These two subspecies shared RFLP groups 4, 5 and 6. The tree nodes in the phenograms showed a low robustness when bootstrapping the data matrix. PCR coupled with a 48h enrichment step in a polypectate-rich medium improved detection thresholds of E. carotovora subsp. atroseptica (1.5.10²- 1.5.10³ bacteria/ml in leaves, stems, and tuber peel extracts to 4.10⁷ bacteria/ml in wash water) relative to either immunomagnetic separation coupled with PCR or DAS-ELISA (2.10⁵ in plant samples to 2.10⁷ bacteria/ml in wash water).     

Potato diseases caused by soft rot erwinias: an overview of pathogenesis

Three soft rot erwinias, Erwinia carotovora ssp. carotovora , E. carotovora ssp. atroseptica and E. chrysanthemi are associated with potatoes causing tuber soft rot and blackleg (stem rot). Latent infection of tubers and stems is widespread. As opportunistic pathogens, the bacteria tend to cause disease when potato resistance is impaired. Pathogenesis or disease development in potato tubers and stems is discussed in terms of the interaction between pathogen, host and environment, microbial competition and recent findings on the molecular basis of pathogenicity. Emphasis is placed on the role of free water and anaerobiosis in weakening tuber resistance and in providing nutrient for erwinias to multiply. Blackleg symptoms are expressed when erwinias predominate in rotting mother tubers, invade the stems and multiply in xylem vessels under favourable weather conditions. Soft rot erwinias tend to out-compete other bacteria in tuber rots because of their ability to produce larger quantities of a wider range of cell wall-degrading enzymes. However, despite extensive studies on their induction, regulation and secretion, little is known about the precise role of the different enzymes in pathogenesis. The putative role of quorum-sensing regulation of these enzymes in disease development is evaluated. The role certain pathogenicity-related characters, including motility, adhesion, siderophores, detoxifying systems and the hrp gene complex, common to most bacteria including symbionts and saprophytes, could play in latent and active infections is also discussed.     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

Incidence of bacteria inhibitory to Erwinia amylovora from blossoms in New Zealand apple orchards

Populations of total bacteria and Erwinia herbicola were monitored on apple blossom in selected orchards at two different geographical regions in New Zealand (Canterbury and Hawke's Bay). In all four orchards surveyed E. herbicola populations remained negligible (less than 50cfu/blossom) throughout flowering, increasing rapidly at petal drop to reach levels of 1 × 10³ cfu/blossom (Hawke's Bay) to 1 × 10⁵ cfu/blossom (Canterbury). Total bacterial populations increased 100-fold at petal drop in both locations. Pseudomonas populations were predominant in Canterbury throughout all flowering stages and in Hawkes Bay after early flowering. When screened for inhibitory activity to E. amylovora , 26% of all isolates from Canterbury showed some inhibition in vitro , but none of the isolates from Hawke's Bay showed inhibition of E. amylovora in vitro. Two Canterbury isolates with strong in vitro inhibitory activity also inhibited E. amylovora in immature pear fruit. One isolate was identified as E. herbicola and one isolate as Pseudomonas fluorescens.     

Quantification of viable cells of Clavibacter michiganensis subsp. michiganensis using a DNA binding dye and a real-time PCR assay

Viable cells of Clavibacter michiganensis subsp. michiganensis (CMM), the causal agent of bacterial canker of tomato, were discriminated from the dead cells by quantitative real-time polymerase chain reaction (PCR), after the bacterial solution was treated with the DNA binding dye ethidium monoazide (EMA). The primers and TaqMan probe, based on the 16S-23S rDNA spacer sequences, were highly specific for CMM at the subspecies level. The detection limit of the direct real-time PCR was 10³ colony forming units per mL (cfu mL⁻¹) in samples and with an apparent sensitivity of 2 cfu of target cells in PCR reaction solution. Application of this method allows for selective quantification of viable cells of CMM and facilitates monitoring the pathogen in tomato seeds.     

An oligonucleotide array for the identification and differentiation of bacteria pathogenic on potato

ABSTRACT Oligonucleotides, 16 to 24 bases long, were selected from the 3' end of the 16S gene and the 16S-23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.     

Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Effect of formulation on the rhizosphere competence and biocontrol ability of Trichoderma atroviride C52

The rhizosphere competence of the biological control agent Trichoderma atroviride isolate C52 was studied on onion roots both in the glasshouse and in the field when introduced into soil in a range of formulations. Proliferation of T. atroviride in the rhizosphere was formulation-dependent. A pellet formulation maintained the fungal concentration at 10⁵ cfu per g soil, whereas solid-substrate and seed-coating formulations gave concentrations of 10⁴ and 10¹ cfu per g soil, respectively. To facilitate rhizosphere-competence studies, a UP-PCR band profile generated with primer L45 for isolate C52 was used to enable conclusive identification of T. atroviride C52 when recovered from soil. When isolate C52 was introduced into Sclerotium cepivorum -infested soil as both pellet and solid-substrate formulations, there was no statistically significant difference in the disease control between these treatments, but the pellet treatment doubled the percentage of healthy plants compared with the control treatment.     

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.     

Interaction of a bioherbicide and glyphosate for controlling hemp sesbania in glyphosate-resistant soybean

The bioherbicidal fungus, Colletotrichum truncatum (Schwein.) Andrus & Moore, was tested at different inoculum concentrations alone and in combination with, prior to or following treatment with different rates of glyphosate ( N -[phosphonomethyl]glycine) (Roundup Ultra) for the control of hemp sesbania ( Sesbania exaltata [Raf.] Rydb. ex A.W. Hill) in Roundup Ready soybean field plots. Colletotrichum truncatum and glyphosate were applied in all pair-wise combinations of 0, 1.25, 2.5, 5.0, and 10.0 × 10⁶ spores mL⁻¹ (i.e. 3.125, 6.25, 12.5, and 25 × 10¹¹ spores ha⁻¹), and 0.15, 0.30, 0.60, and 1.2 kg ha⁻¹, respectively. Weed control and disease incidence were enhanced at the two lowest fungal and herbicidal rates when the fungal spores were applied after glyphosate treatment. The application of the fungus in combination with or prior to glyphosate application at 0.30 kg ha⁻¹ resulted in reduced disease incidence and weed control regardless of the inoculum's concentration. At the highest glyphosate rates, the weeds were controlled by the herbicide alone. These results suggest that it might be possible to utilize additive or synergistic herbicide and pathogen interactions to enhance hemp sesbania control.     

A detection method for Pseudomonas solanacearum in symptomless potato tubers and some data on its sensitivity and specificity

A detection method is described for latent infections of Pseudomonas solanacearum race 3 in potato. This method is based on extraction of 200 heel ends of potato, followed by screening with an indirect antibody stain (IFAS) and (in IFAS-positive cases) a pathogenicity test on tomato (PT). Using the method in some sensitivity and specificity tests with more than 600 samples it appears that: (a) its detection level is 10⁴ cells ml^-1 in IFAS and 10²— 10⁴ cells ml^-1 in PT; (b) per cent recovery is 0.1–10% (10% at lower contamination levels); (c) false-positive samples due to cross-reacting bacteria in IFAS were limited to only 2–3%, using two polyclonal antisera against whole cells of P. solanacearum. The merits of the method in comparison with others (ELISA, selective media) are discussed.     

Biochemical and molecular diversity among Erwinia isolates from potato in Algeria

The variability within a collection of 100 isolates of Erwinia collected from various potato cultivars and locations in Algeria was studied using physiological, biochemical and molecular tests. The comparison of their biochemical characteristics with those of the type isolates CFBP 1526 ( E. carotovora ssp. atroseptica ), CFBP 2046 ( E. carotovora ssp. carotovora ) and CFBP 2048 ( E. chrysanthemi ) indicated that all the isolates collected in Algeria belonged to the species E . carotovora . They included 40 typical E. carotovora ssp. carotovora and 14 E. carotovora ssp. atroseptica ; the remaining 46 isolates could not be classified as E. carotovora ssp. atroseptica or ssp. carotovora , even though they were true Erwinia. Amplification of total genomic DNA with the primers Y1 and Y2, specific for E. carotovora , yielded an amplified fragment of the expected size in 99 isolates. The primers Y45 and Y46 specifically amplified a 439-bp DNA fragment in all E. carotovora ssp. atroseptica isolates tested, but not in isolates of the other E. carotovora subspecies or in atypical isolates, as expected from the characteristics of these primers . The digestion patterns of the 99 amplified products with the restriction enzymes Alu I, Hae II, Hpa II and Sau3A I yielded 12 RFLP groups, three of which were undescribed. The 14 isolates of E. carotovora ssp. atroseptica shared a single restriction pattern (RFLP group 1), while the typical isolates of E. carotovora ssp. carotovora and the atypical isolates composed the remaining groups (3, 4, 8–10, 12, 14, 22 and 25–27), reflecting the heterogeneity among these isolates.     

Factors influencing infection of eucalypts by Cylindrocladium pteridis

The pattern of Cylindrocladium pteridis adhesion, germination and penetration in eucalypt leaves was assessed using scanning electron microscopy. The effects of inoculum concentration, leaf wetness period, plant age and branch position of cylindrocladium leaf blight and defoliation severity were assessed in greenhouse studies using two Eucalyptus grandis × E. urophylla hybrid clones. Penetration occurred through stomata, and there was no difference in the number of penetrations between young and old leaves. Percentage leaf area with lesions and defoliation increased with the increase in inoculum concentration (1 × 10² to 10⁵ conidia mL⁻¹), duration of leaf wetness period (6 to 48 h) and plant age (60 to 180 days). Branch position in plants also significantly affected the percentage leaf area with lesions and defoliation, the latter variable being significantly higher at the stem base. The highest values of lesion area were also observed on leaves at the stem base in both clones. The Pearson correlation between defoliation and leaf area with lesions was significant in all experiments ( r  > 0·9) indicating a high association between these two variables.     

Effects of avirulent bacteriocin-producing strains of Pseudomonas solanacearum on the control of bacterial wilt of tobacco

Root systems of tobacco dipped in suspensions containing 2 × 10⁹ colony forming units (CFU)/ml of avirulent bacteriocin-producing strains (ABPS) of Pseudomonas solanacearum and assayed immediately after planting in steam-sterilized soil had 8 × 10⁶ CFU/root system of ABPS. The bacterial population declined to an average of 5·3 × 10⁵ CFU/root system after 30 days. Roots of seedlings dipped in bacterial suspensions of ABPS were more effectively protected against wilt caused by P. solanacearum than those dipped in suspensions of an avirulent nonbacteriocin-producing strain (ANBPS). Lipopolysaccharide (LPS) isolated from one ABPS (121) inhibited the attachment of bacteria on roots by 70% but had no effect on the reduction of wilt, whereas bacterial cells significantly reduced the disease severity as compared to LPS or water treatment. In steam-sterilized soil containing a 1:1 mixture (5 × 10⁵ CFU/g of oven-dried soil) of ABPS 121 or 237 and the virulent strain K-60, ABPS 121 reduced multiplication of the virulent strain in soil and in the rhizosphere of seedlings. When roots of seedlings were dipped in a suspension of 2 × 10⁹ CFU/ml of ABPS before planting, root colonization by the virulent strain added to steam-sterilized soil at 2 × 10⁶ CFU/g of oven-dried soil was significantly reduced. When roots were dipped in a suspension of ABPS and assayed 20 days after planting, 98% of the bacterial population was found in the original zone of inoculation and only 2% was detected in new growths of the root system. Plants which were grown in soil infested with ABPS 121 or K-60 had both strains present at variable populations along all sections of roots.     

Effects of the safeners CGA 154281, oxabetrinil and fenclorim on uptake and degradation of metolachlor in corn (Zea mays L.) seedlings

Summary. The influence of three crop safeners on the uptake and degradation of ¹⁴C-metolachlor was investigated in two corn varieties. Following application of herbicide and safener together to seedling shoots the concentrations of non-metabolized ¹⁴C-metolachlor in the tissues was found to be lower in the tolerant variety LG 9 than in the susceptible variety 211A. The difference between varieties was due to differences in both uptake and degradation of ¹⁴C-metolachlor.
Following shoot application most of the radioactivity was retained in the coleoptile and the mesocotyl. Two hours after application 95% of the herbicide had been degraded in coleoptiles and mesocotyls, whereas approximately 20% of non-metabolized ¹⁴C-metolachlor was present in the enclosed developing shoot leaves. In both corn varieties the safener CGA 154281 caused a substantial lowering of tissue levels of parent ¹⁴C-metolachlor. This was primarily due to an enhanced degradation. Glutalhione- S -transfer-ase (GST) enzyme activity in shoot tissues was found to be enhanced in both varieties by CGA 154281. Oxabetrinil and fenclorim were less effective than CGA 154281 both in reducing tissue levels of non-metabolized ¹⁴C-metolachlor and in enhancing GST activity in either variety.     

Biocontrol of seedborne Botrytis cinerea in chickpea with Gliocladium roseum

An isolate of Gliocladium roseum proved highly antagonistic to Botrytis cinerea . Sporulation of B. cinerea on chickpea seed naturally infected or inoculated with B. cinerea was suppressed by seed treatment with conidial suspensions of G. roseum at 10⁷ and 10⁸ conidia/mL, respectively. Establishment of healthy seedlings in punnets (small trays) 5 weeks after sowing with inoculated seed was increased from 29.2% to 59.7% by treatment with G. roseum at 3×10⁷ conidia/mL, and from 1.4% to 69.4% with G. roseum at 3×10⁸ conidia/mL, the latter being equivalent to disease control by Thiram. There was no significant effect of Rhizobium on disease suppression by G. roseum , and treatment with G. roseum at 10⁸ conidia/mL did not reduce nodulation. Amendment with culture filtrates of G. roseum did not affect the growth rate of B. cinerea on potato dextrose agar, indicating that constitutive production of an antibiotic is not involved in biocontrol. A selective medium was developed to enumerate propagules of G. roseum on seed recovered from soil. There was no significant change in the population of G. roseum on seed after incubation for 4 weeks in soil to which the isolate of G. roseum was indigenous.