Colonization pattern of the biocontrol strain Pseudomonas chlororaphis MA 342 on barley seeds visualized by using green fluorescent protein.

Pseudomonas chlororaphis MA 342 is a potent biocontrol agent that can be used against several seed-borne diseases of cereal crops, including net blotch of barley caused by the fungus Drechslera teres. In this study, strain MA 342 was tagged with the gfp gene (encoding the green fluorescent protein) in order to study the fate of cells after seed inoculation. The gfp-tagged strain, MA 342G2, had the same biocontrol efficacy as the wild type when it was applied at high cell concentrations to seeds but was less effective at lower cell concentrations. By comparing cell counts determined by microscopy to the number of CFU, we found that the number of culturable cells was significantly lower than the total number of bacteria on seeds which were inoculated and dried for 20 h. Confocal microscopy and epifluorescence stereomicroscopy were used to determine the pattern of MA 342G2 colonization and cell aggregation on barley seeds. Immediately after inoculation of seeds, bacteria were found mainly under the seed glume, and there was no particular aggregation pattern. However, after the seeds were sown, irregularly distributed areas of bacterial aggregation were found, which reflected epiphytic colonization of glume cells. There was a trend towards bacterial aggregation near the embryo but never within the embryo. Bacterial aggregates were regularly found in the groove of each seed formed by the base of the coleoptile and the scutellum. Based on these results, we suggest that MA 342 colocalizes with the pathogen D. teres, which facilitates the action of the fungistatic compound(s) produced by this strain.     

Root Colonization by Pseudomonas sp. DSMZ 13134 and Impact on the Indigenous Rhizosphere Bacterial Community of Barley

Over the last few decades, the ability of rhizosphere bacteria to promote plant growth has been considered to be of scientific, ecological, and economic interest. The properties and mechanisms of interaction of these root-colonizing bacteria have been extensively investigated, and plant protection agents that are based on these bacterial strains have been developed for agricultural applications. In the present study, the root colonization of barley by Pseudomonas sp. DSMZ 13134, that is contained in the commercially available plant protection agent Proradix®, was examined using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. In the first phase of root colonization, the inoculant strain competed successfully with seed and soil-borne bacteria (including Pseudomonads) for the colonization of the rhizoplane. Pseudomonas sp. DSMZ 13134 could be detected in all parts of the roots, although it did not belong to the dominant members of the root-associated bacterial community. Gfp-tagged cells were localized particularly in the root hair zone, and high cell densities were apparent on the root hair surface. To investigate the impact of the application of Proradix® on the structure of the dominant root-associated bacterial community of barley, T-RFLP analyses were performed. Only a transient community effect was found until 3 weeks post-application.     

Development and evaluation of SCAR markers for a Pseudomonas brassicacearum strain used in biological control of snow mould

Biological control microorganisms have long been promoted as an alternative to conventional pesticides. Before registration of a microbial biocontrol product for commercial sale, it must be evaluated as regards potential spread and persistence after release. In this study, strainspecific sequence-characterized amplified region (SCAR) markers were developed to monitor the biocontrol candidate strain Pseudomonas brassicacearum MA250, which is effective against snow mould (Microdochium nivale). One SCAR marker, OPA2-73, was used in quantitative real-time PCR (Q-PCR) on samples from a climate chamber experiment in which winter wheat seeds were treated with the bacterium or a chemical control agent, or left untreated. The results showed that MA250 persisted for up to 3 weeks after sowing on the kernel residues and also colonized the roots of treated seedlings. Total MA250 cell numbers on biocontrol treated seedlings after three weeks were approximately 10⁶ cells, compared with the original inoculum of 10⁶–10⁷ cells per seed. Corresponding cell numbers of MA250 on chemically treated and untreated seedlings were below the detection limit. This study shows that SCAR marker OPA2-73 is a specific and sensitive tool for monitoring the biocontrol microorganism MA250 in environmental samples.     

Contrasting colonization and plant growth promoting capacity between wild type and a gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar

This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619::gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.     

Colonization pattern of plant root and leaf surfaces visualized by use of green-fluorescent-marked strain of <Emphasis Type="Italic">Methylobacterium suomiense</Emphasis> and its persistence in rhizosphere

The localization of bacterial cell, pattern of colonization, and survival of Methylobacterium suomiense CBMB120 in the rhizosphere of rice and tomato plants were followed by confocal laser scanning, scanning electron microscopy, and selective plating. M. suomiense CBMB120 was tagged with green fluorescent protein (gfp), and inoculation was carried out through seed source. The results clearly showed that the gfp marker is stably inherited and is expressed in planta allowing for easy visualization of M. suomiense CBMB120. The colonization differed in rice and tomato—intercellular colonization of surface-sterilized root sections was visible in tomato but not in rice. In both rice and tomato, the cells were visible in the substomatal chambers of leaves. Furthermore, the strain was able to compete with the indigenous microorganisms and persist in the rhizosphere of tomato and rice, assessed through dilution plating on selective media. The detailed ultra-structural study on the rhizosphere colonization by Methylobacterium put forth conclusively that M. suomiense CBMB120 colonize the roots and leaf surfaces of the plants studied and is transmitted to the aerial plant parts from the seed source.     

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.     

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

Genes Involved in Cyclic Lipopeptide Production Are Important for Seed and Straw Colonization by Pseudomonas sp. Strain DSS73

Survival in natural bulk soil and colonization of sugar beet seeds and barley straw residues were determined for Pseudomonas sp. strain DSS73 and Tn5 mutants in amsY (encoding a peptide synthetase involved in production of the cyclic lipopeptide amphisin) and gacS (encoding the sensory kinase of the two-component GacA/GacS regulatory system). No differences in survival or growth in response to carbon amendment (citrate) were observed in bulk soil. However, both mutants were impaired in their colonization of sugar beet seeds and barley straw residues by an inoculum established in the bulk soil. The two mutants had comparable colonization phenotypes, suggesting that amphisin production is more important for colonization than other gacS-controlled traits.     

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.     

Screening of high toxic Metarhizium strain against Plutella xylostella and its marking with green fluorescent protein

Entomopathogenic fungus is proposed to be one of the best biocontrol agents against the destructive insect pest Plutella xylostella. In this study, we tested the virulence of 11 Metarhizium strain isolates against P. xylostella using a leaf dipping method, and found one strain, named 609, which had displayed the highest pathogenicity. Bioassay results showed that the accumulated corrected mortality rate was 86.7 % on the eighth day after inoculation with a spore concentration 1 × 10⁸ conidia/mL, and that the time to 50 % lethality was 5.7-day. The strain was identified as Metarhizium anisopliae var. acridum by internal transcribed spacer (ITS) region sequencing. A green fluorescent protein (GFP) marker containing vector, camben-gfp, was constructed and delivered into strain 609 by Agrobacterium tumefaciens-mediated transformation. Six positive clones expressing GFP were selected and tested for toxicity against P. xylostella, all of which displayed the same toxicity as the parental wild type strain. The survival rate of transformant T1 was investigated by monitoring GFP levels at 4-day intervals after inoculation into soil. We found that the concentration of Metarhizium spores decreased sharply from 1 × 10⁷ conidia/g to 1 × 10⁶ conidia/g in the first 5 days after inoculation. The decreasing trend then stabilized and the spore count declined to approximately 1 × 10⁴–10⁵ conidia/g after 1 month. The results of this study indicate that the expression of gfp gene in strain 609 does not alter the virulence capability of Metarhizium. This strain will therefore be useful for the control of P. xylostella and as a tool to study molecular biology properties and monitor colonization of M. anisopliae in the field.     

In Vivo Study of Trichoderma-Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.     

Role of pfkA and General Carbohydrate Catabolism in Seed Colonization by Enterobacter cloacae

Enterobacter cloacae A-11 is a transposon mutant of strain 501R3 that was deficient in cucumber spermosphere colonization and in the utilization of certain carbohydrates (D. P. Roberts, C. J. Sheets, and J. S. Hartung, Can. J. Microbiol. 38:1128–1134, 1992). In vitro growth of strain A-11 was reduced or deficient on most carbohydrates that supported growth of strain 501R3 but was unaffected on fructose, glycerol, and all amino acids and organic acids tested. Colonization by strain A-11 was significantly reduced (P ≤ 0.05) for cucumber and radish seeds compared to that of strain 501R3, but colonization of pea, soybean, sunflower, and sweet corn seeds was not reduced. Pea seeds released several orders of magnitude more total carbohydrates and amino acids than cucumber and radish seeds and approximately 4,000-fold more fructose. Fructose was the only carbohydrate detected in the seed exudates which supported wild-type levels of in vitro growth of strain A-11. Soybean, sunflower, and sweet corn seeds also released significantly greater amounts of fructose and total carbohydrates and amino acids than cucumber or radish seeds. The exogenous addition of fructose to cucumber and radish seeds at quantities similar to the total quantity of carbohydrates released from pea seeds over 96 h increased the populations of strain A-11 to levels comparable to those of strain 501R3 in sterile sand. Molecular characterization of strain A-11 indicated that the mini-Tn5 kanamycin transposon was inserted in a region of the genome with significant homology to pfkA, which encodes phosphofructo kinase. A comparison of strain A-11 with Escherichia coli DF456, a known pfkA mutant, indicated that the nutritional loss phenotypes were identical. Furthermore, the pfkA homolog cloned from E. cloacae 501R3 complemented the nutritional loss phenotypes of both E. coli DF456 and E. cloacae A-11 and restored colonization by strain A-11 to near wild-type levels. These genetic and biochemical restoration experiments provide strong evidence that the quantities of reduced carbon sources found in seed exudates and the ability of microbes to use these compounds play important roles in the colonization of the spermosphere.     

Barley 4H QTL confers NFNB resistance to a global set of <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> isolates

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres Drechs., is prevalent in barley-growing regions worldwide. A population of 132 recombinant inbred lines (RILs) developed from a cross of the barley varieties ‘Falcon’ and ‘Azhul’ were used to evaluate resistance to NFNB due to their differential reactions to isolates of P. teres f. teres from Australia, Canada, Japan, and the USA. Falcon is a six-rowed, hulless feed barley harboring resistance to NFNB, while Azhul is a six-rowed, hulless food barley with high levels of susceptibility to many P. teres f. teres isolates. Seedling disease resistance data were collected on seedlings of parents, RILs, and checks in a growth chamber. The population was genotyped using Illumina’s GoldenGate assay, and quantitative trait loci (QTL) were detected on chromosomes 2H, 3H, 4H, and 6H. We identified a single genetic region on barley chromosome 4H that provided varying levels of resistance to all P. teres f. teres isolates evaluated.     

Colonization of Vitis vinifera by a Green Fluorescence Protein-Labeled, gfp-Marked Strain of Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis of Grapevine

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.     

Fate and behaviour of a seed-applied Pseudomonas brassicacearum strain in a winter wheat field trial,as determined by analysis with SCAR markers

The fate and behaviour of the seed-applied biocontrol strain Pseudomonas brassicacearum MA250 in a field trial with winter wheat was determined using sequence-characterised amplified region (SCAR) markers. Samples of belowground plant parts from healthy and withered (due to snow mould infection) seedlings were collected approximately one and seven months after sowing, which was performed in early autumn. DNA was extracted from roots and remaining parts of seeds with adhering soil, and the abundance of the strain was determined in quantitative real-time PCR (qPCR) assays. The results show that the introduced strain persisted over the whole trial-period of seven months. On termination of the trial (after seven months) the belowground plant parts of each plant housed 10⁶–10⁷ cells, substantially less than the original approximately 10⁹ cells inoculated onto the seed. In healthy seedlings, there was a shift in cell numbers from seeds to roots between the samplings, suggesting colonisation of the roots during this time. The results show that with sufficient attention given to analytical control measures and the possibility of resident background populations, SCAR markers in combination with qPCR provide valuable information regarding the fate and behaviour of biocontrol micro-organisms under field conditions.     

Improvement of biological control capacity of Paenibacillus polymyxa E681 by seed pelleting on sesame

Sesame is an important vegetable crop for the production of oil in Korea. The main obstacle of sesame cultivation is the occurrence of damping-off diseases and wilt caused by a complex of soil-borne pathogens in fields cultivated for two or more successive years. To protect sesame seedlings against these diseases, Paenibacillus polymyxa E681, a plant growth-promoting rhizobacterium (PGPR) previously shown to suppress disease incidence and promote growth on cucumber and pepper in the greenhouse and field experiments, was evaluated for its capacity for biological control and growth promotion in vitro and in situ. Seed treatment with strain E681 alone did not show consistent protection. Therefore, seed pelleting with strain E681 was attempted to increase the seed size and improve the stability and effectiveness of biocontrol capacity by strain E681. Through screening of pelleting materials, a combination of clay and vermiculite was selected for further experiments to enhance seed germination and root colonization of strain E681 on sesame. In greenhouse trials, formulations of strain E681 reduced disease incidence in disease-conducive soil. In the field, pelleting of sesame seeds with strain E681 significantly reduced pre- and post-emergence damping-off compared to the non-treated or pelleting alone controls; pelleting also promoted the plant growth and the grain yield. Furthermore, the efficacy of strain E681 for biological control and plant growth promotion was improved by sesame seed pelleting compared to the treatment with strain E681 alone. Hence, the application of strain E681 via seed pelleting offers potential to overcome some of the problems associated with successive years of sesame cultivation.     

Genetic analysis of net form net blotch resistance in barley lines CIho 5791 and Tifang against a global collection of <Emphasis Type="Italic">P. teres</Emphasis> f. <Emphasis Type="Italic">teres</Emphasis> isolates

Key message

A CIho 5791 × Tifang recombinant inbred mapping population was developed and used to identify major dominant resistance genes on barley chromosomes 6H and 3H in CI5791 and on 3H in Tifang.

Abstract

The barley line CIho 5791 confers high levels of resistance to Pyrenophora teres f. teres, causal agent of net form net blotch (NFNB), with few documented isolates overcoming this resistance. Tifang barley also harbors resistance to P. teres f. teres which was previously shown to localize to barley chromosome 3H. A CIho 5791 × Tifang F₆ recombinant inbred line (RIL) population was developed using single seed descent. The Illumina iSelect SNP platform was used to identify 2562 single nucleotide polymorphism (SNP) markers across the barley genome, resulting in seven linkage maps, one for each barley chromosome. The CIho 5791 × Tifang RIL population was evaluated for NFNB resistance using nine P. teres f. teres isolates collected globally. Tifang was resistant to four of the isolates tested whereas CIho 5791 was highly resistant to all nine isolates. QTL analysis indicated that the CIho 5791 resistance mapped to chromosome 6H whereas the Tifang resistance mapped to chromosome 3H. Additionally, CIho 5791 also harbored resistance to two Japanese isolates that mapped to a 3H region similar to that of Tifang. SNP markers and RILs harboring both 3H and 6H resistance will be useful in resistance breeding against NFNB.

     

Association mapping utilizing diverse barley lines reveals net form net blotch seedling resistance/susceptibility loci

Key message

A diverse collection of barley lines was phenotyped with three North American Pyrenophora teres f. teres isolates and association analyses detected 78 significant marker-trait associations at 16 genomic loci.

Abstract

Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the causal agent of the economically important foliar disease net form net blotch (NFNB) of barley. The deployment of effective and durable resistance against P. teres f. teres has been hindered by the complexity of quantitative resistance and susceptibility. Several bi-parental mapping populations have been used to identify QTL associated with NFNB disease on all seven barley chromosomes. Here, we report the first genome-wide association study (GWAS) to detect marker-trait associations for resistance or susceptibility to P. teres f. teres. Geographically diverse barley genotypes from a world barley core collection (957) were genotyped with the Illumina barley iSelect chip and phenotyped with three P. teres f. teres isolates collected in two geographical regions of the USA (15A, 6A and LDNH04Ptt19). The best of nine regression models tested were identified for each isolate and used for association analysis resulting in the identification of 78 significant marker-trait associations (MTA; ?log10p value?>3.0). The MTA identified corresponded to 16 unique genomic loci as determined by analysis of local linkage disequilibrium between markers that did not meet a correlation threshold of R ²?≥?0.1, indicating that the markers represented distinct loci. Five loci identified represent novel QTL and were designated QRptts-3HL, QRptts-4HS, QRptts-5HL.1, QRptts-5HL.2, and QRptts-7HL.1. In addition, 55 of the barley lines examined exhibited a high level of resistance to all three isolates and the SNP markers identified will provide useful genetic resources for barley breeding programs.

     

Quantification of Pyrenophora teres in infected barley leaves using real-time PCR

Net blotch is a barley foliar disease caused by two forms of Pyrenophora teres: Pyrenophora teres f. teres (PTT) and Pyrenophora teres f. maculata (PTM). To monitor and quantify their occurrence during the growing season, diagnostic system based on real-time PCR was developed. TaqMan MGB (Minor Groove Binder) primers and probes were designed that showed high specificity for each of the two forms of P. teres. As a host plant internal standard, TaqMan MGB primers and probe based on RacB gene sequence were designed. The method was optimised on pure fungal DNA and on plasmid standard dilutions. Quantification was accomplished by comparing Ct values of unknown samples with those obtained from plasmid standard dilutions. The assay detects down to five gene copies per reaction. It is able to produce reliable quantitative data over a range of six orders of magnitude. The developed assay was used to differentiate and quantify both forms of P. teres in infected barley leaves. Correlation R² = 0.52 was obtained between the Ct values and size of symptoms areas in early stage of infection. Application of the TaqMan MGB technology to leaf samples collected in 20 barley varieties in the region Kromeriz during the growing season of 2003 and 2004 revealed that P. teres f. teres predominated in these 2 years. The developed method is an important tool to quantify and monitor the dynamics of the two forms of P. teres during the growing season.     

Genetic Control of Virulence of <Emphasis Type="Italic">Pyrenophora teres</Emphasis> Drechs., the Causative Agent of Net Blotch in Barley

The genetic control of virulence was studied in four isolates of the fungus Pyrenophora teres f. teres, originating from various geographic regions in experiments with nine barley accessions, possessing known resistance genes. Experiments were performed with the ascospore progeny of two crosses. The results of segregation for virulence in the progeny of direct crosses were confirmed by analysis of backcrosses and sib crosses. One to four genes for avirulence toward various barley genotypes were found in the isolates under study. It is suggested that dominant suppressor genes are involved in the genetic control of avirulence toward four barley genotypes.