Site-Specific Septoria Leaf Blotch Risk Assessment in Winter Wheat Using Weather-Radar Rainfall Estimates.

The Septoria leaf blotch prediction model PROCULTURE was used to assess the impact on simulated infection rates when using rainfall estimated by radar instead of rain gauge measurements. When comparing infection events simulated by PROCULTURE using radar- and gauge-derived data, the simulated probability of detection (POD) of infection events was high (0.83 on average), and the simulated false alarm ratio (FAR) of infection events was not negligible (0.24 on average). For most stations, simulation-observed FAR decreased to 0 and simulation-observed POD increased (0.85 on average) when the model outputs for both datasets were compared against visual observations of disease symptoms. An analysis of 148 infection events over 3 years at four locations showed no significant difference in the number of infection events of simulations using either dataset, indicating that, for a given location, radar estimates were as reliable as rain gauges for predicting infection events. Radar also provided better estimates of rainfall occurrence over a continuous space than weather station networks. The high spatial resolution provides radar with an important advantage that could significantly improve existing warning systems.

Genetic diversity of the mating type and toxin production genes in Pyrenophora tritici-repentis.

Pyrenophora tritici-repentis, the causal agent of tan spot on wheat, is a homothallic loculoascomycete with a complex race structure. The objectives of this study were to confirm the homothallic nature of the pathogen, characterize mating type diversity and toxin production genes in a global collection of strains, and analyze how these traits are associated between each other and with existing races. The pseudothecia production capacity, race identification, mating type locus (MAT), internal transcribed spacer, and glyceraldehyde-3-phosphate dehydrogenase regions were analyzed in a selection of 88 strains originating from Europe, North and South America, North Africa, and Central and South Asia. Some (60%) strains produced pseudothecia containing ascospores, independent of their origin. Race identification obtained using the multiplex polymerase chain reaction targeting host-selective toxin (HST) genes was consistent, overall, with the results based on the inoculation of a set of differential wheat cultivars and confirmed the predominance of race 1/2 strains ( approximately 83%). However, discrepancies in race identification, differences from the reference tester strains, and atypical ToxA profiles suggest the presence of new races and HSTs. The MAT1-1 and MAT1-2 coding regions are consecutively arranged in a single individual, suggesting putative heterothallic origin of P. tritici-repentis. Upstream from the MAT is an open reading frame of unknown function (ORF1) containing a MAT-specific degenerate carboxy-terminus. The phylogenetic analysis of the MAT locus reveals two distinct groups, unlinked to geographical origin or ToxA profile. Group I, the best-represented group, is associated with typical tan spot lesions caused by races 1, 2, 3, and 5 on wheat. It is more homogenous than group II encompassing race 4 strains, as well as isolates associated primarily with small spot lesions on wheat leaves or other hosts. Group II could contain several distinct taxa.

Assessing the Accuracy of Simulation Model for Septoria Leaf Blotch Disease Progress on Winter Wheat.

A mechanistic model, PROCULTURE, for assessing the development of each of the last five leaf layers and the progress of Septoria leaf blotch, caused by Septoria tritici (teleomorph Mycosphaerella graminicola), has been applied on susceptible and weakly susceptible winter wheat (Triticum aestivum) cultivars in two locations (Everlange and Reuland) in Luxembourg over a 3-year period (2000 to 2002). A double performance assessment of PROCULTURE was conducted in this study. First, the capability of PROCULTURE to correctly simulate S. tritici incidence was checked. Second, the model's ability to accurately estimate disease severity was assessed on the basis of the difference between simulated and observed levels of disease development at each leaf layer. The model accurately predicted disease occurrence in the 2000 and 2002 seasons, on susceptible and semi-susceptible cultivars, with a probability of detection (POD) exceeding 0.90. However, in 2001, even though the POD never fell below 0.90, the false alarm ratio (FAR) was too high to consider the simulations satisfactory. Concerning the evaluation of disease severity modeling, statistical tests revealed accurate simulations performed by PROCULTURE for susceptible cultivars in 2000 and 2002. By contrast, for weakly susceptible cultivars, the model overestimated disease severity, especially for the upper leaves, for the same period.

Barley yellow mosaic virus is overcoming RYM4 resistance in Belgium.

Barley yellow mosaic virus (BaYMV) is the causal agent of a soil-borne systemic mosaic disease on barley. It has been reported in Belgium since the 1980s. The control of this disease is managed almost exclusively through the use of resistant varieties. The resistance of most commercial barley cultivars grown in Europe is conferred mainly by a single recessive gene, rym4. This monogenic resistance provides immunity against BaYMV pathotype 1 and has been mapped on barley chromosome 3HL and shown to be caused by mutations in the translation initiation factor eIF4E. Another pathotype, BaYMV pathotype 2, which appeared in the late 1980s (in Belgium, in the early 1990s), is able to overcome the rym4-controlled resistance. Until recently, this pathotype remained confined to specific locations. During a systematic survey in 2003, mosaic symptoms were observed only on susceptible barley cultivars collected in Belgian fields. BaYMV was detected by ELISA and RT-PCR on the susceptible cultivars and only by RT-PCR on the resistant cultivars. In 2004, mosaic symptoms were observed on susceptible and resistant cultivars. BaYMV was detected by ELISA and RT-PCR on both cultivars. In addition to developing RT-PCR methods for detecting and identifying BaYMV and Barley mild mosaic virus (BaMMV), an RT-PCR targeting the VPg/NIa viral protein part of the genome, known to discriminate the two BaYMV pathotypes, was set up to accurately identify the pathotype(s) now present in Belgium. The sequences from the generated amplicons revealed the single nucleotide substitution resulting in an amino acid change from lysine to asparagine specific to BaYMV pathotype 2. The possible reasons for the change in the BaYMV pathotype situation in Belgium, such as climatic change or a progressive build-up of soil inoculum potential, will be discussed, as well as the use of eIF4E-based resistance.

A certain but non-exclusive association between Polymyxa graminis special forms and cereals.

Polymyxo graminis, a ubiquitous plasmodiophorid obligate root endoparasite, is recognized as the vector of about 15 viruses on cereals and groundnut in temperate and tropical areas. Within the species, five special forms have been distinguished on the basis of specific ribotypes. Three of them occur in tropical areas: P. graminis f.sp. colombiana on rice, P. graminis f.sp. subtropicalis on cereals cropped in the tropics such as maize, pearl millet and sorghum but also on barley and/or wheat, and P. graminis f.sp. tropicalis mainly on maize, pearl millet and sorghum. Their particular host ranges distinguish them significantly from P. graminis f.sp. temperata and P. graminis f.sp. tepida found in temperate areas on barley and wheat. In order to assess whether these special forms commonly infect these cereals, barley and wheat plants were grown under controlled conditions on two soils from Belgium and France and both infested by P. graminis f.sp. temperata and P. graminis f.sp. tepida. The infection of each cereal species by each form was quantified by real-time quantitative PCR with specific primers and Taqman probes. The infection of P. graminis f.sp. temperata was significantly higher on barley than on wheat, whereas the quantities of P. graminis f.sp. tepida on wheat were higher than on barley. These results show that the distinction between these special forms, based on the ribotype, reflects differences in ecological features.

Identification and Quantification of Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida on Barley and Wheat.

Polymyxa graminis f. sp. temperata and P. graminis f. sp. tepida are distinguished on the basis of their specific ribosomal DNA sequences. In order to evaluate whether or not host specialization is associated with the special form, the occurrence of infection of both forms on barley and wheat was studied. P. graminis inocula were obtained from soils collected in Belgium and France. Their ribotypes were characterized using molecular tools specific to P. graminis f. sp. temperata or P. graminis f. sp. tepida such as restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rDNA, nested and multiplex PCR. Both special forms were found in each country and coexisted in some soils. The host specificity of P. graminis special forms for barley and wheat was studied from two soils collected at Gembloux (Belgium) and Chambon-sur-Cisse (France), each infested by bymo- and furoviruses. P. graminis f. sp. temperata is more frequent on barley and P. graminis f. sp. tepida on wheat. Furthermore, the quantification of each form on barley and wheat by two separated real-time quantitative PCR assays confirms the observations on the vector specialization. These results suggest a certain but not exclusive host specificity of P. graminis special forms.

Widespread Occurrence of Wheat spindle streak mosaic virus in Belgium.

In order to assess the occurrence of Wheat spindle streak mosaic virus (WSSMV) in Belgium, a reverse-transcription polymerase chain reaction (RT-PCR) was developed, targeting WSSMV isolates from Canada, France, Germany, Italy, and the United States. The primers also were designed for virus quantification by real-time RT-PCR with SYBR-Green. No cross-reaction with soilborne cereal viruses such as Barley mild mosaic virus, Barley yellow mosaic virus, Soilborne cereal mosaic virus, and Soil-borne wheat mosaic virus was observed. The RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection of WSSMV than enzymelinked immunosorbent assay. The incidence of WSSMV in Belgium was evaluated using a bioassay with wheat cvs. Cezanne and Savannah and rye cv. Halo, grown in 104 Belgian soils. The presence of WSSMV was detected from plants grown in 32% of the soils. The RT-PCR methods developed here, combined with large sampling, allowed WSSMV to be detected for the first time in Belgium. The real-time quantitative RT-PCR was developed as a tool for evaluating the resistance to WSSMV by quantifying the virus concentration in wheat cultivars.

Individual and combined effects of dosages of azoxystrobin and epoxiconazole in wheat.

The effects of single fungicide applications on Mycosphaerella graminicola (septoria leaf blotch) control and winter wheat yield were evaluated in field trials conducted in central Belgium between 2000 and 2004. Individual applications of 25, 50, 75 and 100% of the manufacturer's recommended dose rates of azoxystrobin and epoxiconazole, and all the combinations of these treatments, were made at GS 39 in 2001 to 2004 and at GS 59 in 2000. Disease assessments were made at growth stage 75, some 7-8 weeks after the last applications. Between 2000 and 2003, no significant difference was observed for disease control between the products when applied alone. With regard to the dose responses, the differences between the recommended dose rates and the 50% reduced dosages were not important. In 2004, azoxystrobin was less effective than epoxiconazole. This was probably the result of strobilurin-resistant isolates of M. graminicola reaching an occurrence of 32% before fungicide application. The combination of different dosages of azoxystrobin and epoxiconazole revealed that there was very little synergy between these products when applied in a single application. The combinations of these products were better than individual applications only when high dosages of both compounds were used.

First detection of resistance to QoI fungicides in Mycosphaerella graminicola on winter wheat in Belgium.

A total of 740 Mycosphaerella graminicola strains were isolated between 2000 and 2002 from winter wheat F1 or F2 leaves showing Septoria leaf blotch lesions (SLB) collected mainly at the soft dough stage in fungicide trials, analysing at 12 locations in Belgium the possibilities and risks associated with the use of epoxiconazole and azoxystrobin at various doses, mixtures and application dates. Fungicide sensitivity tests were performed in microtitre plates on potato dextrose broth amended with various concentrations of azoxystrobin. A wide range of sensitivity to azoxystrobin was observed, with EC50 values ranging for 735 strains between 0.002 to 0.7 microg/ml, the highest frequency gradually shifting from EC50 classes 0.01 and 0.02 microg/ml azoxystrobin in 2000 to EC50 classes 0.02 and 0.04 microg/ml in 2002. No clear selection effect of particular fungicide use strategies was observed. Among the 382 strains isolated in 2002, five originating from 2 locations, showed azoxystrobin EC50 values >1 microg/ml. On medium amended with 100 microg/ml salicylhydroxamic acid (SHAM), 58% of the 2002 strains were strongly inhibited, which affected adequate azoxystrobin ED50 determination. This suggests widespread occurrence of M. graminicola strains relying in vitro on the alternative respiration pathway. In the presence of SHAM, strains 339 and 880 showed azoxystrobin EC50 values of 3 and >30 microg/ml, respectively. This high level of resistance to a QoI fungicide was confirmed by analysing mycelium growth inhibition on PDA. Cross-resistance to trifloxystrobin and kresoxim-methyl was demonstrated. Greenhouse assays on wheat plants revealed that control of QoI resistant strains by azoxystrobin is decreased, compared to control of sensitive ones. This highlights the risk of resistance to QoI fungicides also in M. graminicola populations, although up to now no decrease in field performance was noticed. It is recommended to delay build up of QoI resistance by an integrated approach, combining optimised fungicide use with the choice of SLB resistant cultivars and the application of farming practices promoting stubbles break down and so the reduction of the teleomorph stage.

Lessons from the year 2001 Mycosphaerella graminicola epidemic on winter wheat in Belgium.

Infection by Mycosphaerella graminicola (anamorph Septoria tritici) was monitored between April and July 2001 on F6 to flag leaf in 11 farmers' fields or fungicide trials. Data were analysed by mean of the decision support system "Proculture" which links an automatic weather station of the PAMESEB network to a particular field, simulates plant development with adjustment by one phenological observation during the stem elongation and analyses superposition of emerged leaves and infection events (http://www.fymy.ucl.ac.be/proculture). Several climatic events favourable for the infection and dissemination of M. graminicola occurred between October 2000 and March 2001 and allowed build up of a large amount of inoculum on the lower leaves at the end of the winter. The start of stem elongation was associated with frequent rainy periods during April, causing early infection of F5, F4 and up to F3 in some precocious fields. Dry weather with only a few local showers during most of May and June slowed down spread of infection to the upper leaves, leading to absence of M. graminicola infection of the flag leaf in 9 out of the 11 fields. Yield increase by a single fungicide spray ranged from 800 to 2200 kg/ha. A second treatment was cost effective in none of the fields. The interest and limitation of the decision support system for understanding M. graminicola epidemic and for guiding decision on spray timing are discussed.

Characterization of fluorescent and nonfluorescent peptide siderophores produced by Pseudomonas syringae strains and their potential use in strain identification.

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in beta-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.

Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses.

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.

Modulation of plant plasma membrane H+-ATPase by phytotoxic lipodepsipeptides produced by the plant pathogen Pseudomonas fuscovaginae.

Pseudomonas fuscovaginae produces the lipodepsipeptides syringotoxin, fuscopeptin A and fuscopeptin B concurrently. These phytotoxins inhibit acidification of the external medium by fusicoccin-treated rice leaf sheath discs. When tested in vitro on H+-ATPase of rice shoot plasma membranes, syringotoxin and its structural analogue syringomycin, produced by P. syringae pv. syringae, displayed a double effect. At low concentrations they stimulated the ATPase activity of native right-side-out membrane vesicles in a detergent-like manner. At higher concentrations, however, this stimulation was reversed. With membranes treated with the detergent Brij 58, inhibition of ATPase activity was observed at low concentrations of the nonapeptides. The latter effect required the presence of an intact lactone ring formed by the nonapeptide head of these molecules. In contrast, fuscopeptins A and B inhibited enzyme activity regardless of the orientation of the vesicles. These observations were confirmed using plasma membranes from a yeast strain whose own H+-ATPase had been replaced by a single plant H+-ATPase isoform, PMA2, from Nicotiana plumbaginifolia. The kinetics of inhibition induced by the most active compound fuscopeptin B, showed a non-competitive pattern, with a Ki of about 1 microM. The combination of syringotoxin (or syringomycin) with the more hydrophobic fuscopeptins, in amounts with little or no effect, resulted in strong inhibition of the enzyme activity of rice membranes, suggesting a synergistic effect for the two types of toxins.

Xanthomonas translucens from Small Grains: Diversity and Phytopathological Relevance.

ABSTRACT Sixty-eight presumptive Xanthomonas translucens strains isolated from 15 small grains or grass species were studied by pathogenicity tests on barley, bread wheat, oat, and bromegrass species, and also by AFLP, analysis of fatty acid methyl esters (FAME), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of protein extracts. The X. translucens strains were divided into three pathogenicity types based on differences observed on barley and bread wheat. Two unspeciated strains producing atypical symptoms formed a fourth pathogenicity type. Pathogenicity on oat and bromegrass species varied within these types. Clusterings observed by AFLP analysis and, to a lesser extent, by FAME analysis were consistent with these pathogenicity groupings. The current results, as well as those of previous restriction fragment length polymorphism analyses of the same strains, support the recent reclassification of X. translucens pv. translucens and X. translucens pv. hordei as true synonyms. X. translucens pv. cerealis, X. translucens pv. translucens, and X. translucens pv. undulosa cluster in different groups by AFLP and FAME analyses. Even though distinction by simple biochemical tests is not clear-cut, the data indicate that the pathovars cerealis, translucens, and undulosa correspond to true biological entities.

Structure of fuscopeptins, phytotoxic metabolites of Pseudomonas fuscovaginae.

The structure of the fuscopeptins, bioactive lipodepsipeptides produced in culture by the gramineae pathogen Pseudomonas fuscovaginae, has been determined. The combined use of FAB mass spectroscopy NMR spectroscopy and chemical and enzymatic procedures allowed one to define a peptide moiety corresponding to Z-Dhb-D-Pro-L-Leu-D-Ala-D-Ala-D-Ala-D-Ala-D-Val-Gly-D-Ala-D-Val-D-Ala-D- Val-Z-Dhb-Da-Thr-L-Ala-L-Dab-D-Dab-L-Phe with the terminal carboxyl group closing a macrocyclic ring on the hydroxyl group of the allothreonine residue. The N-terminus is in turn acylated by 3-hydroxyoctanoate in fuscopeptin A and 3-hydroxydecanoate in fuscopeptin B. Some preliminary data on the biological activity of fuscopeptins are also reported.

Genetic Diversity among Xanthomonas campestris Strains Pathogenic for Small Grains.

A collection of 51 Xanthomonas campestris strains from throughout the world was studied to detect and assess genetic diversity among pathogens of small grains. Isolates from barley, bread wheat, bromegrass, canary grass, cassava, maize, orchard grass, rice, rough-stalked meadow grass, rye, timothy, and triticale were analyzed by pathogenicity tests on bread wheat cv. Alondra and barley cv. Corona, indirect immunofluorescence, and restriction fragment length polymorphism (RFLP). Three probes were used for the RFLP analysis. They were an acetylaminofluorene-labelled 16S+23S rRNA probe from Escherichia coli and two (sup32)P-labelled restriction fragments from either plasmidic (pBSF2) or chromosomal (pBS8) DNA of X. campestris pv. manihotis. Strains clustered in 9 and 20 groups with the rRNA probe and the pBSF2 DNA probe, respectively. Strains of X. campestris pv. graminis, X. campestris pv. phleipratensis, and X. campestris pv. poae are shown to be related but are also distinguishable by RFLP patterns, serology, and pathogenicity on bread wheat. Strains pathogenic only for barley and not for wheat grouped together. Another group is temporarily designated deviant X. campestris pv. undulosa. These South American isolates from bread wheat did not react by indirect immunofluorescence and produced atypical lesions in pathogenicity tests. The results stress the need to perform pathogenicity tests before strains are named at the pathovar level. The importance of the different probes used for epidemiological studies or phylogenetic studies of closely related strains is underlined.

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta).

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.