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

Recent Developments in Flavin-Based Catalysis:Enzymatic Sulfoxidation

The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.</p

Constraints on the evolution of azole resistance in plant pathogenic fungi

The durability of azole fungicides in controlling agriculturally important pathogenic fungi is unique amongst modern single site fungicides. Today, azoles are still relied on to control pathogens of many crops including cereals, fruits and vegetables, canola and soybeans. Significantly, this widespread use continues despite many reports of azole-resistant fungal strains. In this review, recent reports of azole resistance and the mechanisms associated with resistant phenotypes are discussed. The example of the complex evolution of the azole target sterol 14-demethylase (CYP51) enzyme in modern European populations of the wheat pathogen Mycosphaerella graminicola is used to describe the quantitative and epistatic effects on fungicide sensitivity and enzyme function of target site mutations, and to explore the hypothesis that constraints on CYP51 evolution have ensured the longevity of azoles. In addition, the threats posed by alternative resistance mechanisms causing cross-resistance to all azoles or even unrelated fungicides are discussed, and postulations are made on how using new genomic technologies to gain a greater understanding of azole resistance evolution should enhance the ability to control azole-resistant strains of plant pathogenic fungi in the future

A novel gamma-N-methylaminobutyrate demethylating oxidase involved in catabolism of the tobacco alkaloid nicotine by Arthrobacter nicotinovorans pAO1

Nicotine catabolism, linked in Arthrobacter nicotinovorans to the presence of the megaplasmid pAO1, leads to the formation of gamma-N-methylaminobutyrate from the pyrrolidine ring of the alkaloid. Until now the metabolic fate of gamma-N-methylaminobutyrate has been unknown. pAO1 carries a cluster of ORFs with similarity to sarcosine and dimethylglycine dehydrogenases and oxidases, to the bifunctional enzyme methylenetetrahydrofolate dehydrogenase/cyclohydrolase and to formyltetrahydrofolate deformylase. We cloned and expressed the gene carrying the sarcosine dehydrogenase-like ORF and showed, by enzyme activity, spectrophotometric methods and identification of the reaction product as gamma-aminobutyrate, that the predicted 89 395 Da flavoprotein is a demethylating gamma-N-methylaminobutyrate oxidase. Site-directed mutagenesis identified His67 as the site of covalent attachment of FAD and confirmed Trp66 as essential for FAD binding, for enzyme activity and for the spectral properties of the wild-type enzyme. A K-m of 140 mum and a k(cat) of 800 s(-1) was determined when gamma-N-methylaminobutyrate was used as the substrate. Sarcosine was also turned over by the enzyme, but at a rate 200-fold slower than gamma-N-methylaminobutyrate. This novel enzyme activity revealed that the first step in channelling the gamma-N-methylaminobutyrate generated from nicotine into the cell metabolism proceeds by its oxidative demethylation

Lack of an Intron in Cytochrome b and Overexpression of Sterol 14 alpha-Demethylase Indicate a Potential Risk for QoI and DMI Resistance Development in Neophysopella spp. on Grapes

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14 alpha-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 mu g/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 mu g/ml for N. tropicalis and 0.46 mu g/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 mu g/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp

The Multi-Fungicide Resistance Status of Aspergillus fumigatus Populations in Arable Soils and the Wider European Environment

The evolution and spread of pan-azole resistance alleles in clinical and environmental isolates of Aspergillus fumigatus is a global human health concern. The identification of hotspots for azole resistance development in the wider environment can inform optimal measures to counteract further spread by minimizing exposure to azole fungicides and reducing inoculum build-up and pathogen dispersal. We investigated the fungicide sensitivity status of soil populations sampled from arable crops and the wider environment and compared these with urban airborne populations. Low levels of azole resistance were observed for isolates carrying the CYP51A variant F46Y/M172V/E427K, all belonging to a cluster of related cell surface protein (CSP) types which included t07, t08, t13, t15, t19, and t02B, a new allele. High levels of resistance were found in soil isolates carrying CYP51A variants TR34/L98H and TR46/Y121F/T289A, all belonging to CSP types t01, t02, t04B, or t11. TR46/Y121F/M172V/T289A/G448S (CSP t01) and TR46/Y121F/T289A/S363P/I364V/G448S (CSP t01), a new haplotype associated with high levels of resistance, were isolated from Dutch urban air samples, indicating azole resistance evolution is ongoing. Based on low numbers of pan-azole resistant isolates and lack of new genotypes in soils of fungicide-treated commercial and experimental wheat crops, we consider arable crop production as a coldspot for azole resistance development, in contrast to previously reported flower bulb waste heaps. This study also shows that, in addition to azole resistance, several lineages of A. fumigatus carrying TR-based CYP51A variants have also developed acquired resistance to methyl benzimidazole carbamate, quinone outside inhibitor and succinate dehydrogenase (Sdh) inhibitor fungicides through target-site alterations in the corresponding fungicide target proteins; beta-tubulin (F200Y), cytochrome b (G143A), and Sdh subunit B (H270Y and H270R), respectively. Molecular typing showed that several multi-fungicide resistant strains found in agricultural soils in this study were clonal as identical isolates have been found earlier in the environment and/or in patients. Further research on the spread of different fungicide-resistant alleles from the wider environment to patients and vice versa can inform optimal practices to tackle the further spread of antifungal resistance in A. fumigatus populations and to safeguard the efficacy of azoles for future treatment of invasive aspergillosis

Mesozoic-Cenozoic crustaceans preserved within echinoid tests and bivalve shells

Associations of crustaceans with echinoids (Echinodermata) and bivalves (Mollusca) are not uncommon in modern oceans. Here we record the occurrence of anomurans, brachyurans and isopods within echinoid tests and bivalve shells from the Middle Jurassic of France, the Upper Jurassic of the Czech Republic, the Eocene of Croatia and the Miocene of Austria. Additionally a new genus and species of fossil cirolanid isopod from the Middle Jurassic of France is described. The present examples are interpreted as crustacean sheltering, probably for safe and undisturbed moulting (ecdysis), within a vacant host test or shell. However, accidental association (washed in) or even food remains cannot be ruled out entirelyWeb of Science90361160

A phylogenetically distinct lineage of Pyrenopeziza brassicae associated with chlorotic leaf spot of Brassicaceae in North America

Light leaf spot, caused by the ascomycete Pyrenopeziza brassicae Sutton & Rawlinson, is an established disease of Brassicaceae in the United Kingdom (UK), continental Europe, and Oceania (OC, including New Zealand and Australia). The disease was reported in North America (NA) for the first time in 2014 on Brassica spp. in the Willamette Valley of western Oregon, followed by detection in Brassica juncea cover crops and on B. rapa weeds in northwestern Washington in 2016. Preliminary DNA sequence data and field observations suggest that isolates of the pathogen present in NA might be distinct from those in the UK, continental Europe, and OC. Comparisons of isolates from these regions genetically (multilocus sequence analysis, MAT gene sequences, and rep-PCR DNA fingerprinting), pathogenically (B. rapa inoculation studies), biologically (sexual compatibility), and morphologically (colony and conidial morphology) demonstrated two genetically distinct evolutionary lineages. Lineage 1 comprised isolates from the UK, continental Europe, and OC, and included the P. brassicae type specimen. Lineage 2 contained the NA isolates associated with recent disease outbreaks in the Pacific Northwest region of the USA. Symptoms caused by isolates of the two lineages on B. rapa and B. juncea differed, so ‘chlorotic leaf spot’ is proposed for the disease caused by lineage 2 isolates of P. brassicae. Isolates of the two lineages differed in genetic diversity as well as sensitivity to the fungicides carbendazim and prothioconazole

Epidemiological studies of pan-azole resistant Aspergillus fumigatus populations sampled during tulip cultivation show clonal expansion with acquisition of multi-fungicide resistance as potential driver

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungi-cides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), have recently been reported. These fungicide groups are not used in medicine but can play an important role in further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR34/L98H and TR46/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have an advantage in environments where residues of multiple fungicides belonging to different modes of action are presen