Effect of crop rotational position and nitrogen supply on root development and yield formation of winter wheat.

The lower yield of wheat grown after wheat (second wheat) compared with the first wheat after a break crop is frequently attributed to fungal disease occurrence, but has also been found without visible disease infection; thus, other factors might be responsible for the lower yield of the second wheat. The aims of this study were to analyze the effects of growing wheat as first and second wheat after oilseed rape, as well as monoculture in a long-term field experiment over three years on (i) aboveground biomass formation, root development and nutrient acquisition during the growing season, (ii) take-all occurrence, and (iii) grain yield and yield components. Subsoil root length density of winter wheat was significantly higher after oilseed rape as pre-crop than after wheat, which was independent of take-all occurrence. Differences in wheat aboveground biomass occurred at early growth stages and were persistent until harvest. Grain yield loss correlated well with take-all disease severity in a wet year but yield differences among crop rotational positions occurred also in a dry year without visible fungal infection. Thus, an effect of the crop rotational position of wheat beyond take-all disease pressure can be assumed. Overall, wheat root length density might be the key to understand wheat biomass formation and grain yield in different crop rotational positions.

Evaluation of the AID carbapenemase line probe assay for rapid detection and identification of carbapenemase genes in Gram-negative bacilli.

Objectives: This study evaluated the AID carbapenemase line probe assay (LPA) for the detection and identification of carbapenem resistance genes in Enterobacteriaceae and other Gram-negative bacilli (GNB) using bacterial cultures and DNA extracts directly from patient urine samples. Methods: The AID carbapenemase LPA detects 13 different carbapenemase genes. Test probe accuracy was verified for using clinical Enterobacteriaceae isolates harbouring bla KPC , bla VIM , bla NDM , bla GIM , bla AIM , bla SPM , bla IMP and bla OXA-48 and a well-characterized set of Escherichia coli DH5α strains transformed with the vector plasmid pUC57- kan harbouring bla BIC , bla SIM , bla DIM , bla IMI-3 , bla IMI-1 and bla NMC-A . Sensitivity and specificity was determined by testing 151 clinical GNB strains previously characterized for the production of carbapenemase activity and carbapenemase genes. Direct detection of carbapenemase genes using the LPA was determined using 299 clinical urine specimens. Analytical sensitivity for detection in urine was determined by testing serial dilutions of bla KPC and bla NDM in clinical Klebsiella pneumoniae strains. Results: All carbapenemase gene probes showed 100% accuracy without cross-reactions. Sensitivity and specificity of the LPA using clinical isolates was 100% for each. Analytical sensitivity for detection of bla KPC and bla NDM in urine was 10 1 -10 2 cfu. The LPA detected carbapenemase genes in 20 urines, which were confirmed in 12 samples by conventional multiplex PCR. Remarkably, 0 of the 20 urines grew carbapenemase-suspicious GNB applying EUCAST recommendations. Conclusions: The AID carbapenemase LPA is an accurate, sensitive and easy-to-use test for the detection and identification of carbapenemase genes, which can readily be implemented in any diagnostic laboratory.

Development and Validation of LNA-Based Quantitative Real-Time PCR Assays for Detection and Identification of the Root-Knot Nematode Meloidogyne enterolobii in Complex DNA Backgrounds.

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

Development of species-, strain- and antibiotic biosynthesis-specific quantitative PCR assays for Pantoea agglomerans as tools for biocontrol monitoring.

Pantoea agglomerans is a cosmopolitan plant epiphytic bacterium that includes some of the most effective biological antagonists against the fire blight pathogen Erwinia amylovora, a major threat to pome fruit production worldwide. Strain E325 is commercially available as Bloomtime Biological™ in the USA and Canada. New quantitative PCR (qPCR) assays were developed for species- and strain -specific detection in the environment, and for detection of indigenous strains carrying the biocontrol antibacterial peptide biosynthesis gene paaA. The qPCR assays were highly specific, efficient and sensitive, detecting fewer than three cells per reaction or 700 colony forming units per flower, respectively. The qPCR assays were tested on field samples, giving first indications to the incidence of P. agglomerans E325 related strains, total P. agglomerans and pantocin A producing bacteria in commercial orchards. These assays will facilitate monitoring the environmental behavior of biocontrol P. agglomerans after orchard application for disease protection, proprietary strain-tracking, and streamlined screening for discovery of new biocontrol strains.

Lipopolysaccharide biosynthesis genes discriminate between Rubus- and Spiraeoideae-infective genotypes of Erwinia amylovora.

Comparative genomic analysis revealed differences in the lipopolysaccharide (LPS) biosynthesis gene cluster between the Rubus-infecting strain ATCC BAA-2158 and the Spiraeoideae-infecting strain CFBP 1430 of Erwinia amylovora. These differences corroborate rpoB-based phylogenetic clustering of E. amylovora into four different groups and enable the discrimination of Spiraeoideae- and Rubus-infecting strains. The structure of the differences between the two groups supports the hypothesis that adaptation to Rubus spp. took place after species separation of E. amylovora and E. pyrifoliae that contrasts with a recently proposed scenario, based on CRISPR data, in which the shift to domesticated apple would have caused an evolutionary bottleneck in the Spiraeoideae-infecting strains of E. amylovora which would be a much earlier event. In the core region of the LPS biosynthetic gene cluster, Spiraeoideae-infecting strains encode three glycosyltransferases and an LPS ligase (Spiraeoideae-type waaL), whereas Rubus-infecting strains encode two glycosyltransferases and a different LPS ligase (Rubus-type waaL). These coding domains share little to no homology at the amino acid level between Rubus- and Spiraeoideae-infecting strains, and this genotypic difference was confirmed by polymerase chain reaction analysis of the associated DNA region in 31 Rubus- and Spiraeoideae-infecting strains. The LPS biosynthesis gene cluster may thus be used as a molecular marker to distinguish between Rubus- and Spiraeoideae-infecting strains of E. amylovora using primers designed in this study.

Involvement of Lsp, a member of the LraI-lipoprotein family in Streptococcus pyogenes, in eukaryotic cell adhesion and internalization.

Three open reading frames (ORFs) were identified by a genome walking strategy in the genomes of serotype M49 group A streptococcal (GAS) strains CS101 and 591. These ORFs were located between the mga core regulon and the dipeptide permease operon. The deduced amino acid (aa) sequences contained signature sequences indicative of a lipoprotein (306 aa), an intracellular protein (823 aa), and a secreted peptide (66 aa), respectively. ORF1 (named Lsp for lipoprotein of Streptococcus pyogenes) and ORF2 exhibited a high degree of homology to the lmb/ORF2 genes of S. agalactiae (B. Spellerberg et al., Infect. Immun. 67:871-878, 1999). The three ORFs were found to be present in each of the 27 GAS serotype strains tested. Transcription analysis revealed a polycistronic lsp/ORF2 and a monocistronic ORF3 message that were detected primarily at the transition from exponential to stationary growth phase. lsp and ORF2 mutants, ORF2- and ORF3-luciferase reporter fusions, and antiserum against recombinant Lsp were produced to examine the biological role of these genes. Although high Zn(2+) and Cu(2+) ion concentrations decreased lsp operon expression, Lsp did not transport divalent cations as described for other LraI-type operons. The lsp mutant had reduced fibronectin binding. Although no direct binding of Lsp to fibronectin could be demonstrated, the lsp mutant showed decreased transcription of prtF2 encoding the fibronectin-binding protein F2. Both the lsp and ORF2 mutants showed decreased laminin binding. Adherence to and internalization into A549 epithelial cells of both mutants was reduced without a detectable effect on eukaryotic cell viability. The transcription of a number of virulence factors was altered in the lsp mutants and ORF2 mutants. The changes in laminin binding and eukaryotic cell internalization could be explained by changes in transcription of speB (cysteine protease) and/or the global regulators mga, csrRS, and nra.

Group A streptococcal RofA-type global regulators exhibit a strain-specific genomic presence and regulation pattern.

RofA-like protein (RALP) type regulators have been shown to exist in different forms in group A streptococci (GAS) and to regulate the expression of important bacterial adhesins. This study shows that the vast majority of strains from different GAS M serotypes carried a rofA virulence regulator gene in their genome and that this gene could be detected in combination with other RALP genes and RALP-dependent adhesin genes in a strain-specific manner. The gene encoding the Nra regulator was predominantly found in opacity factor (OF)-negative serotypes. When analysing a rofA mutant in a serotype M2 strain, the strain specificity was also found in the positive and negative regulatory functions of RALP genes as well as in the type and number of virulence genes and functions controlled by the RALP genes. Of 17 virulence-associated genes tested, only one, the putative streptolysin S gene, was observed to be derepressed in RALP mutants of three different GAS serotype strains. This strain-specific variability of RALP regulon sizes is associated with different patterns of host cell attachment and internalization. In addition, RofA2 was shown to control expression of the ribosomal protein gene rpsL. As a consequence, it was demonstrated for the first time in streptococci that aminoglycoside resistance mediated by rpsL expression is apparently controlled by a virulence gene regulator.

Integrated Control of Rhizoctonia Crown and Root Rot of Sugar Beet with Fungicides and Antagonistic Bacteria.

Rhizoctonia crown and root rot, caused by the fungus Rhizoctonia solani AG 2-2, is one of the most damaging sugar beet diseases worldwide and causes significant economic losses in more than 25% of the sugar beet production area in the United States. We report on field trials in the years 1996 to 1999 testing both experimental fungicides and antagonistic Bacillus sp. for their potential to reduce disease severity and increase sugar yield in trials inoculated with R. solani AG 2-2. Fungicides were applied as in-furrow sprays at planting or as band sprays directed at the crown at the four-leaf stage, or four- plus eight-leaf stage, while bacteria were applied at the four-leaf stage only. The fungicides azoxystrobin and tebuconazole reduced crown and root rot disease by 50 to 90% over 3 years when used at rates of 76 to 304 g a.i./ha and 250 g a.i./ha, respectively. The disease index at harvest was reduced and the root and sugar yield increased with azoxystrobin compared with tebuconazole. The combination of azoxystrobin applied at 76 g a.i./ha and the Bacillus isolate MSU-127 resulted in best disease reduction and greatest root and sucrose yield increase.