Levansucrase (EC 2.4.1.10), an exoenzyme of Pseudomonas syringae pv. phaseolicola, was purified to homogeneity from the cell supernatant by chromatography on TMAE-Fraktogel and butyl-Fraktogel. The enzyme has molecular masses of 45 kDa under denaturing conditions and 68 kDa during gel filtration of the native form. In isoelectric focusing, active bands appeared at pH 3.55 and 3.6. Maximum sucrose cleaving activities were measured at pH 5.8 to 6.6 and 60؇C. The enzyme was highly tolerant to denaturing agents, proteases, and repeated freezing and thawing. The molecular weight of the produced levan depended on temperature, salinity, and sucrose concentration. The enzyme had levan-degrading activity and did not accept raffinose as a substrate. Comparison of the N-terminal amino acid sequence with the predicted amino acid sequence of levansucrases from Erwinia amylovora and Zymomonas mobilis showed 88 and 69% similarity, respectively, in amino acids 5 to 20. No similarity could be detected to levansucrases of gram-positive bacteria in the first 20 amino acids. By comparison of all levansucrases which have been sequenced to date, the enzyme seems to be conserved in the gram-negative bacteria. The rheological behavior of the product levan prompted a new assessment of the enzyme's role in pathogenesis. Depending on formation conditions, levan solutions exclude other polymer solutions. This behavior supports the presumption that the levansucrase is important in the early phase of infection by creating a separating layer between bacteria and plant cell wall to prevent the pathogen from recognition.

Azole fungicides play a prominent role for reliable plant disease management. However, quantitative azole resistance has been shown to develop in fungal pathogens, including Fusarium graminearum, the causal agent of Fusarium head blight (FHB). Due to widespread application of azole fungicides, resistance may accumulate to higher degrees in fungal field populations over time. Although azole fungicides are prominent components in FHB control, little effort has been made to investigate azole resistance in F. graminearum. We allowed F. graminearum strain NRRL 13383 to adapt to an azole fungicide in vitro, applying a strongly growth-reducing but sublethal dose of tebuconazole. Two morphologically distinguishable azole-resistant phenotypes were recovered that differed with regard to levels of fitness, fungicide resistance, virulence, and mycotoxin production. Isolates of the adapted "phenotype 1" exhibited azole-specific cross-resistance, whereas "phenotype 2" isolates displayed the phenomenon of multidrug resistance because the sensitivity to amine fungicides was also affected. Assessment of individual infected spikelets for mycotoxin contents by high-performance liquid chromatography mass spectrometry and for Fusarium DNA by quantitative polymerase chain reaction indicated that some of the adapted isolates produced significantly higher levels of nivalenol per fungal biomass than the NRRL 13383 strain.

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BackgroundIn soils with a low phosphorus (P) supply, sugar beet is known to intake more P than other species such as maize, wheat, or groundnut. We hypothesized that organic compounds exuded by sugar beet roots solubilize soil P and that this exudation is stimulated by P starvation.ResultsRoot exudates were collected from plants grown in hydroponics under low- and high-P availability. Exudate components were separated by HPLC, ionized by electrospray, and detected by mass spectrometry in the range of mass-to-charge ratio (m/z) from 100 to 1000. Eight mass spectrometric signals were enhanced at least 5-fold by low P availability at all harvest times. Among these signals, negative ions with an m/z of 137 and 147 were shown to originate from salicylic acid and citramalic acid. The ability of both compounds to mobilize soil P was demonstrated by incubation of pure substances with Oxisol soil fertilized with calcium phosphate.ConclusionsRoot exudates of sugar beet contain salicylic acid and citramalic acid, the latter of which has rarely been detected in plants so far. Both metabolites solubilize soil P and their exudation by roots is stimulated by P deficiency. These results provide the first assignment of a biological function to citramalic acid of plant origin.

SummaryTryptophan is a precursor for many biologically active secondary metabolites. We have investigated the origin of indole pigments first described in the pityriasis versicolor-associated fungus Malassezia furfur. Some of the identified indole pigments have properties potentially explaining characteristics of the disease. As M. furfur is not amenable to genetic manipulation, we used Ustilago maydis to investigate the pathway leading to pigment production from tryptophan. We show by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance analysis that the compounds produced by U. maydis include those putatively involved in the etiology of pityriasis versicolor. Using a reverse genetics approach, we demonstrate that the tryptophan aminotransferase Tam1 catalyses pigment biosynthesis by conversion of tryptophan into indolepyruvate. A forward genetics approach led to the identification of mutants incapable of producing the pigments. These mutants were affected in the sir1 gene, presumably encoding a sulphite reductase. In vitro experiments with purified Tam1 showed that 2-oxo 4-methylthio butanoate serves as a substrate linking tryptophan deamination to sulphur metabolism. We provide the first direct evidence that these indole pigments form spontaneously from indolepyruvate and tryptophan without any enzymatic activity. This suggests that compounds with a proposed function in M. furfur-associated disease consist of indolepyruvate-derived spontaneously generated metabolic by-products.

A survey for the natural occurrence of Fusarium mycotoxins in maize for human consumption in four south-western states of Nigeria using High Performance Liquid Chromatography coupled with Mass Spectroscopy (HPLC/MS) showed that 93.4% of the samples were contaminated with zearalenone (ZON), alpha- and beta-zearalenols (alpha- and beta-ZOL), fumonisin B(1) (FB(1)) or enniatins (ENNs). The fractions of contaminated samples were 73% for FB(1) (mean:117 microg kg(-1), range:10-760 microg kg(-1)); 57% for ZON (mean:49 microg kg(-1), range:115-779 microg kg(-1)) and 13% for alpha-ZOL (mean: 63.6 microg kg(-1), range:32-181 microg kg(-1)), while ENNs A1, B and B(1) were present in 3, 7 and 3% of the samples respectively. There was no beta-ZOL present above the quantification limits of 50 microg kg(-1). Only the FB(1) content was significantly different at the 95% confidence level among the four states. The Fusarium species most frequently isolated from maize seeds were F. verticillioides (70%), followed by F. sporotrichioides (42%), F. graminearum (30%), F. pallidoroseum (15%), F. compactum (12%), F. proliferatum (12%), F. equiseti (9%), F. acuminatum (8%) and F. subglutinans (4%). This is the first report of the occurrence of alpha-zearalenol and enniatins in Nigerian maize.

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Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae,Erwinia amylovora, and other bacterial species. Thelsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene inEscherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3.0-kbPstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to theE. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring thelsc gene. Expression of lsc was found to be dependent on the vector-based P lac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.