Modern approaches for breeding high quality apples with durable resistance to scab, powdery mildew and fire blight

New methods to allow for more precise selection of tree and fruit characters in breeding programmes were developed in recent years. Marker-assisted selection (MAS) is common practice in the ACW apple breeding programme at Wädenswil. Genetic markers can reduce the number of plants and the time required for evaluation, thus new varieties become commercially available sooner. How can this molecular selection method reasonably be applied in an apple breeding programme? Application of phenotypic and molecular selection techniques in the ACW apple breeding programme and results are presented

QTL mapping of fire blight resistance in apple

Fire blight caused by the bacterium Erwinia amylovora is a severe threat to apple and pear orchards worldwide. Apple varieties exhibit a wide range of relative susceptibility/tolerance to fire blight. Although, no monogenic resistance against fire blight has been identified yet, recent evidence indicates the existence of quantitative resistance. Potential sources of fire blight resistance include several wild Malus species and some apple cultivars. F1 progenies of ‘Fiesta'בDiscovery' were inoculated with the Swiss strain Ea 610 and studied under controlled conditions to identify quantitative trait loci (QTLs) for fire blight resistance. Disease was evaluated at four time points after inoculation. Shoot lesion length and the area under disease progress curve (AUDPC) values were used for QTL analysis. One significant (LOD score of 7.5-8.1, p<0.001) QTL was identified on the linkage group 7 of ‘Fiesta' (F7). The F7 QTL explained about 37.5-38.6% of the phenotypic variatio

Broad spectrum microarray for fingerprint-based bacterial species identification

BACKGROUND: Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. RESULTS: A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. CONCLUSIONS: These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups

Erwinia amylovora in the genomics era : from genomes to pathogen virulence, regulation, and disease control strategies

The publication of the first Erwinia amylovora genome has greatly accelerated and advanced our understanding of the fire blight organism. With the availability of multiple genomes, it quickly became clear that chromosomal diversity is relatively small, and that most of the pan-genome variance is attributable to plasmids. In addition to gaining a more detailed view of the known virulence factors, genomics has enabled new breakthrough studies of virulence regulation mechanisms. Furthermore, several niche adaptation and ecological fitness factors, though not directly influencing virulence, have been studied in greater detail, providing novel insights into the physiology and ecology of the bacterium. Additionally, application of genome data has yielded improved diagnostics and enabled population studies at different geographic scales

Selection of a biocontrol agent based on a potential mechanism of action: degradation of nicotinic acid, a growth factor essential for Erwinia amylovora

This work describes a medium-based screening method for selecting microbial biocontrol agents against Erwinia amylovora based on the degradation of a specific growth factor. Erwinia amylovora, the causal agent of the devastating fire blight disease, requires nicotinic acid or nicotinamide as an essential growth factor. Potential biocontrol agents are either selected for antimicrobial production in plate or directly on immature pears or apple blossoms. In this work, we have attempted to streamline the selection of a new potential biocontrol agent with a lower risk of non-target effects by isolation based on the ability to degrade nicotinic acid in vitro, using therefore few plant materials. A total of 735 bacteria and 1237 yeast were isolated from apple blossoms and pre-screened for nicotinic acid-degradation. Pseudomonas rhizosphaerae strain JAN was able to degrade both nicotinic acid and nicotinamide. Mutants deficient in this ability were constructed. JAN, but not the mutants, controlled E. amylovora on pear slices. On detached apple blossoms, JAN colonized apple hypanthia and strongly suppressed E. amylovora growth. Under greenhouse conditions, JAN was more effective in controlling blossom blight than P. fluorescens A506, a commercial biocontrol agent of fire blight unable to degrade nicotinic acid and nicotinamide.[Int Microbiol 2010; 13(4):195-206

Phenotypic comparison of clinical and plant-beneficial strains of Pantoea agglomerans

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity

Phenotypic comparison of clinical and plant-beneficial strains of Pantoea agglomerans

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity. [Int Microbiol 2014; 17(2):81-90]Keywords: Pantoea agglomerans &middot; Erwinia amylovora &middot; Meloidogyne javanica &middot; Penicillium expansum &middot; Caenorhabditis elegans &middot; biocontrol &middot; biosafety &middot; toxicity &middot; hemolytic activity &middot; Ames tes

Signaling between bacterial and fungal biocontrol agents in a strain mixture

The use of bacterial and fungal strain mixtures is a promising way to improve efficacy of biocontrol treatments. Certain Pseudomonas and Trichoderma strains belong to the most common studied biocontrol agents. One key factor for the biocontrol efficacy of several P. fluorescens strains is the synthesis of 2,4-diacetylphloroglucinol (DAPG). Production of chitinases, such as the ECH42 endochitinase and the NAG1 N-acetyl-β-d-glucosaminidase, is a primary mechanism of action for T. atroviride. We examined the molecular interactions between the DAPG-producing P. fluorescens strains CHA0 and Q2-87 and chitinase-producing T. atroviride P1. Interactions were monitored using the reporter gene constructs, phlA'-'lacZ translational fusion in P. fluorescens CHA0 and ech42-goxA or nag1-goxA fusions in T. atroviride P1. We found that DAPG enhanced nag1, but not ech42 expression, whereas an unidentified substance from P. fluorescens CHA0 repressed expression of both Trichoderma chitinases. Addition of T. atroviride P1 culture filtrates to growing cultures of P. fluorescens enhanced phlA expression transiently during growth. These results indicate that negative and positive effects on expression of key biocontrol genes may occur while mixing antagonist

Role of the type VI secretion systems during disease interactions of Erwinia amylovora with its plant host

Background: Type VI secretion systems (T6SS) are widespread among Gram-negative bacteria and have a potential role as essential virulence factors or to maintain symbiotic interactions. Three T6SS gene clusters were identified in the genome of E. amylovora CFBP 1430, of which T6SS-1 and T6SS-3 represent complete T6SS machineries, while T6SS-2 is reduced in its gene content. Results: To assess the contribution of T6SSs to virulence and potential transcriptomic changes of E. amylovora CFBP 1430, single and double mutants in two structural genes were generated for T6SS-1 and T6SS-3. Plant assays showed that mutants in T6SS-3 were slightly more virulent in apple shoots while inducing less disease symptoms on apple flowers, indicating that T6SSs have only a minor effect on virulence of E. amylovora CFBP 1430. The mutations led under in vitro conditions to the differential expression of type III secretion systems, iron acquisition, chemotaxis, flagellar, and fimbrial genes. Comparison of the in planta and in vitro transcriptome data sets revealed a common differential expression of three processes and a set of chemotaxis and motility genes. Additional experiments proved that T6SS mutants are impaired in their motility. Conclusion: These results suggest that the deletion of T6SSs alters metabolic and motility processes. Nevertheless, the difference in lesion development in apple shoots and flower necrosis of T6SS mutants was indicative that T6SSs influences the disease progression and the establishment of the pathogen on host plants