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Fusarium basal rot is a worldwide disease problem in onions, and causes substantial losses in onion production, both during the growing season and in the storage. To minimize the post-harvest losses, a protocol for screening of latent infections with pathogenic Fusarium oxysporum strains from harvested onions was developed. This protocol is based on a dual PCR test with primers specific for the fungal species and new SIX3 primers specific for the onion-pathogenic F. oxysporum strains. A pooled sample containing pieces from 50 harvested symptomless onions was prepared for the dual PCR using microwave disruption of the filamentous Fusarium fungi and Whatman FTATM filter paper matrix technology, or as a reference protocol, by extracting DNA with a commercial kit. The two sample preparation protocols gave consistent results with the tested onion samples. Detection limit of the dual PCR protocol was 100 pg of F. oxysporum DNA, in a mixture with onion DNA, when the FTA card was applied. The new protocol reported here is simple and sensitive enough for routine testing, enabling the detection of latent infections in harvest lots even at the infection levels under 10%. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium basal rot causes serious problems in onion production. To minimize post-harvest losses, a simple protocol based on FTATM technology and a dual PCR test with Fusarium oxysporum species-specific and pathogenicity-specific primers was developed. By testing pooled onion samples using this method, latent infections with F. oxysporum can be screened from a representative sample of the harvest. This screening method could be a useful tool to manage the post-harvest losses caused by latent infections with F. oxysporum and, with modification of the PCR protocol, with other Fusarium species pathogenic to onion.

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'Candidatus Liberibacter solanacearum' (CLso) haplotype C is associated with disease in carrots and transmitted by the carrot psyllid Trioza apicalis. To identify possible other sources and vectors of this pathogen in Finland, samples were taken of wild plants within and near the carrot fields, the psyllids feeding on these plants, parsnips growing next to carrots, and carrot seeds. For analyzing the genotype of the CLso-positive samples, a multilocus sequence typing (MLST) scheme was developed. CLso haplotype C was detected in 11% of the T. anthrisci samples, in 35% of the Anthriscus sylvestris plants with discoloration, and in parsnips showing leaf discoloration. MLST revealed that the CLso in T. anthrisci and most A. sylvestris plants represent different strains than the bacteria found in T. apicalis and the cultivated plants. CLso haplotype D was detected in 2 of the 34 carrot seed lots tested, but was not detected in the plants grown from these seeds. Phylogenetic analysis by unweighted-pair group method with arithmetic means clustering suggested that haplotype D is more closely related to haplotype A than to C. A novel, sixth haplotype of CLso, most closely related to A and D, was found in the psyllid T. urticae and stinging nettle (Urtica dioica, Urticaceae), and named haplotype U.

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In the present study, we have analysed the ability of Streptococcus pyogenes [Group A streptococcus (GAS)] to activate the NACHT-domain-, leucine-rich repeat- and PYD-containing protein 3 (NALP3) inflammasome complex in human monocyte-derived macrophages and the molecules and signalling pathways involved in GAS-induced inflammatory responses. We focused upon analysing the impact of dynamin-dependent endocytosis and the role of major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in the immune responses induced by GAS. These virulence factors are involved in immune evasion by forming pores in host cell membranes, and aid the bacteria to escape from the endosome-lysosome pathway. We analysed cytokine gene expression in human primary macrophages after stimulation with live or inactivated wild-type GAS as well as with live SLO and SLS defective bacteria. Interleukin (IL)-1ß, IL-10, tumour necrosis factor (TNF)-α and chemokine (C-X-C motif) ligand (CXCL)-10 cytokines were produced after bacterial stimulation in a dose-dependent manner and no differences in cytokine levels were seen between live, inactivated or mutant bacteria. These data suggest that streptolysins or other secreted bacterial products are not required for the inflammatory responses induced by GAS. Our data indicate that inhibition of dynamin-dependent endocytosis in macrophages attenuates the induction of IL-1ß, TNF-α, interferon (IFN)-ß and CXCL-10 mRNAs. We also observed that pro-IL-1ß protein was expressed and efficiently cleaved into mature-IL-1ß via inflammasome activation after bacterial stimulation. Furthermore, we demonstrate that multiple signalling pathways are involved in GAS-stimulated inflammatory responses in human macrophages.

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In the present study we have characterized T helper type 2 (Th2) [interleukin (IL)-10]/Th1 (IL-12) cytokine expression balance in human primary macrophages stimulated with multiple non-pathogenic Gram-positive bacteria used in the food industry and as probiotic substances. Bacteria representing Lactobacillus, Bifidobacterium, Lactococcus, Leuconostoc, Propionibacterium and Streptococcus species induced anti-inflammatory IL-10 production, although quantitative differences between the bacteria were observed. S. thermophilus was able to induce IL-12 production, while the production of IL-12 induced by other bacteria remained at a low level. The highest anti-inflammatory potential was seen with bifidobacteria, as evidenced by high IL-10/IL-12 induction ratios. All studied non-pathogenic bacteria were able to stimulate the expression of suppressor of cytokine signalling (SOCS) 3 that controls the expression of proinflammatory cytokine genes. Lactobacillus and Streptococcus species induced SOCS3 mRNA expression directly in the absence of protein synthesis and indirectly via bacteria-induced IL-10 production, as demonstrated by experiments with cycloheximide (CHX) and anti-IL-10 antibodies, respectively. The mitogen-activated protein kinase (MAPK) p38 signalling pathway played a key role in bacteria-induced SOCS3 gene expression. Enhanced IL-10 production and SOCS3 gene expression induced by live non-pathogenic Lactobacillus and Streptococcus is also likely to contribute to their immunoregulatory effects in vivo.

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The nucleotide sequence of the 3' terminal 3105 nucleotides (nt) of RNA2 of blackcurrant reversion associated virus (BRAV), the first mite-transmitted member of the nepovirus group, has been determined. The sequence contains an open reading frame of 1744 nt in the virus-sense strand, a 3' untranslated region of 1360 nt and a 3' poly(A) tail. Analysis of the amino-terminal residues of purified coat protein (CP) suggests that the CP gene is located between nts 1361 and 2959 (from the 3' terminus) in the RNA2, and that Asp/Ser is the proteolytic cleavage site of CP in the RNA2 encoded polyprotein. The predicted translation product from the CP gene is a polypeptide of 533 amino acids with a calculated Mr of 57 561. The amino acid sequence of BRAV CP showed highest similarity to blueberry leaf mottle virus (BLMV), and tomato ringspot virus (ToRSV), two members of the proposed sub-group three of nepoviruses possessing large RNA2 components. Nucleic and amino acid sequence comparisons between BRAV CP and the CPs of other nepoviruses indicate that specific conserved nepovirus CP domains occur in the BRAV CP thus confirming that BRAV is a member of the subgroup three of nepoviruses. reserved.

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ABSTRACT Black currant reversion is a virus-like disease whose causal agent has not been identified. In rooted cuttings of a black currant plant affected with the severe form of the disease, pronounced chlorotic line patterns and ringspots developed in newly emerging leaves. From such symptom-bearing leaves, a virus was mechanically transmitted with difficulty to Chenopodium quinoa and, from this host, to other herbaceous test plants. The virus was purified and partially characterized, and the purified viri-ons were used for antiserum production. Virus particles were isometric, approximately 27 nm in diameter, and sedimented as two nucleoprotein components. They contained a protein species with a molecular mass of 55 kDa, which was readily degraded into a 54-kDa protein and two major RNA components of about 6,700 and 7,700 nucleotides (nt), each with a poly(A) tail. Most of these properties are shared by nepoviruses, but the virus was serologically unrelated to 14 nepoviruses or putative nepovi-ruses tested. However, the deduced sequence of 1,260 nt at the 3' end of one of the viral RNA species was distinct from any known viral sequence, except that it contained short regions of homology to the 3' terminal sequences of RNAs of seven other nepoviruses and two comovi-ruses. To detect this virus in Ribes plants, primers were designed from the known sequence to amplify a 210-nt region of the cDNA of the virus RNA using an immunocapture reverse transcriptase polymerase chain reaction (IC-RT-PCR) protocol. Using this assay for the virus, we associated its presence with two recognized forms of black currant reversion disease occurring in Finland, Scotland, or New Zealand. We also detected the virus in vector gall mites from reverted plants and in black currant plants on which such vector mites had fed. However, the virus was not detected by IC-RT-PCR in known healthy Ribes plants; in Ribes plants free from reversion, but affected by three other distinct virus-like diseases of Ribes; or in plants infected with arabis mosaic, strawberry latent ringspot, or raspberry ringspot nepoviruses. These data suggest that this virus may be the causal agent of reversion disease, and it is tentatively called black currant reversion associated virus.