Conductimetric detection of Pseudomonas syringae pathover pisi in pea seeds and soft rot Erwinia spp. on potato tubers

Pea bacterial blight and potato blackleg are diseases caused by Pseudomonas syringae pv. pisi ( Psp ) and soft rot Erwinia spp., respectively. The primary source of inoculum for these bacteria is contaminated plant propagation material, i.e. pea seeds and potato tubers. One of the best ways to control the diseases is the use of healthy planting material. To check the health status of this material, sensitive and specific methods are needed to detect the bacteria.In Chapter 2 the use of a conductimetric assay to detect Psp in pea seed extracts is described. The conductance medium used was based on Special Peptone Yeast Extract broth (SPYE) with the addition of the selective agents boric acid, cycloheximide, cephalexin and cefuroxime to restrict the growth of other micro- organisms. Conductimetric assays, immunofluorescence cell staining (IF) and an enzyme-linked immunosorbent assay (ELISA) for detecting Psp in pea seed extracts were compared with dilution plating by two extraction methods, viz. 6 h soaking of pea seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Psp with conductimetric, IF and dilution plating assays in the extracts of the ground and 2 h-soaked pea samples was less sensitive than the detection in the extracts of the 6 h-soaked pea samples. The detection thresholds of these assays varied per seed lot between 0 and 10 4cfu ml -1for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 10 4and 10 6cfu ml -1. Detection times recorded in conductimetric assays correlated well with the number of Psp added to seed extracts at 27 as well as at 17 °C. Due to the presence of saprophytic Pseudomonas spp., which were able to overgrow Psp and to generate conductance responses, conductimetric detection in SPYEC was not useful for routine testing.In Chapter 3 a medium based on L-asparagine conversion (AM) was found more suitable for conductimetric detection of Psp than SPYE, because higher and more specific conductance responses were obtained for Pseudomonas spp., Psp included. However, AM supplemented with the same selective agents as in SPYEC (AMC) was still not sufficiently selective for a direct conductimetric detection of Psp in pea seed extracts, mainly due to the presence of interfering conductance responses caused by Pseudomonas fluorescens and Pseudomonas putida. Although the medium selectivity could not be improved further by addition of other selective agents, AMC was shown to be useful in an enrichment procedure. In comparison with SPYEC, higher yields of Psp were obtained after enrichment of seed extracts in AMC. With IF an initial concentration of less than 10 3Psp cells ml -1could be detected in naturally contaminated seed extracts after 48 h enrichment in AMC at 27 °C. However, although serological detection of Psp in seed extracts after enrichment was sensitive, false negative and false positive reactions, due to the presence of unusual serotypes of Psp and cross reacting Pseudomonas syringae pv. syringae ( Pss ), respectively, can still be obtained in serology. Consequently, for an accurate detection the presence of Psp in enriched seed extracts, found positive with serology, has to be confirmed with other specific tests. Suitable techniques are pathogenicity testing and the polymerase chain reaction (PCR), provided that specific primers are available for the latter technique to exclude false positive reactions in serology due to the presence of cross-reacting Pss .To develop a specific conductimetric assay for detection of potato pathogenic Erwinia spp., Erwinia carotovora subsp. atroseptica ( Eca ), Erwinia carotovora subsp. carotovora ( Ecc ), Erwiniachrysanthemi (Ech) and a set of potatoassociated saprophytes were tested for their ability to generate conductance responses in various media (Chapter 4). In SPYE, all bacteria tested, including the genera Bacillus , Enterobacter, Flavobacterium, Klebsiella, Pseudomonas and Xanthomonas , generated conductance responses, while in minimal medium supplemented with glucose and trimethylamine N-oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses. Additionally, in minimal medium supplemented with L- asparagine, only Pseudomonas and Erwinia spp. were able to generate large conductance responses rapidly, whereas with polypectate as sole carbon source only Erwinia spp. produced distinct conductance responses.The high conductance responses of Erwinia spp. in pectate media were due to the release of large amounts of saturated and unsaturated oligogalacturonates during depolymerization of pectate by a combined action of extracellular polygalacturonases (PGs) and pectate lyases (PLs) (Chapter 5). Other highly pectolytic bacteria, such as Klebsiella and an unidentified saprophyte, could only generate weak conductance responses in pectate media, due to PL activity.Because of its specificity, minimal medium with polypectate as sole carbon source (PM) was most suitable for conductimetric detection of Erwinia spp. in potato peel extracts. Due to bacterial conversion of asparagine/aspartic acid present in potato peel extract itself, generating an interfering conductance response in PM, only small samples or 10-fold dilutions of potato peel extracts could be tested conductimetrically (Chapter 4). The detection threshold for Eca in inoculated potato peel extracts was ca 10 4cells ml -1, when samples were considered positive on the basis of a response at 20 °C. within 48 h, while ca 10 5cells of Ech ml -1were detected at 25 °C. within 36 h (Chapters 4 and 6). Samples with a positive conductance response had to be confirmed with other techniques, such as ELISA or PCR, for presence of Eca and Ech, since Ecc was also able to generate a conductance response. The conductimetric detection of contamination levels of Eca higher than 10 4cells ml -1 peel extract was specific and efficient, because most of the seed lots tested were negative in conductimetry, meaning that an additional test to check the presence of Eca was superfluous (Chapter 6). Consequently, large-scale certification of seed lots for contamination levels of Eca higher than 10 4cells ml -1peel extract to control blackleg can be done with automated conductance measurements as a primary screening, after which PCR can be used to verify the positive samples. For Ech, the conductimetric detection was less specific and sensitive, and unefficient, due to the presence of low contamination levels of Ech and high numbers of Ecc after enrichment, which interfered with the test (Chapter 6). Further research is needed to improve the sensitivity and specificity of the conductimetric assays for Erwinia spp., which for example might be achieved by the use of an immunomagnetic separation procedure or a selective pre-enrichment step before applying conductimetry.Immunofluorescence colony staining (IFC), for both Eca and Ech (Chapter 6) and enrichment combined with IF or PCR, for Eca (Chapters 6 and 7), were suitable to detect and quantify lower numbers of bacteria, viz. 10-10 4cells ml -1in potato peel extracts. With regard to serology, false positive and false negative reactions were observed (Chapters 6 and 7). However, since the chance of false negative reactions caused by unusual serotypes of Ech and Eca is negligible, only false positive reactions in serology are considered as a major problem for laboratory testing. To exclude false positive reactions in WC or in IF using enrichment, verification with PCR was applied succesfully (Chapters 6 and 7). If required, false negative serological reactions of enriched peel extracts can be excluded by testing all samples with PCR. The latter test protocol, although being laborious and expensive, might be very useful in small-scale blackleg indexing of valuable young clonal material from the top of the selection system in order to eradicate the disease, because of the specificity and the extremely low detection threshold of 10 cells ml -1potato peel extract.