Erwinia amylovoraAuxotrophic Mutant Exometabolomics and Virulence on Apples

ABSTRACTThe Gram-negative bacteriumErwinia amylovoracauses fire blight disease of apples and pears. While the virulence systems ofE. amylovorahave been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophicE. amylovoramutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-typeE. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenicE. amylovoraauxotrophs could have utility as fire blight biocontrol agents.IMPORTANCEThis study has revealed the availability of a range of host metabolites toE. amylovoracells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterialde novosynthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophicE. amylovoramutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulentE. amylovoraon flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenicE. amylovoraauxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

Mutation of an Arginine Biosynthesis Gene Causes Reduced Pathogenicity in Fusarium oxysporum f. sp. melonis

Restriction enzyme-mediated integration (REMI) mutagenesis was used to tag genes required for pathogenicity of Fusarium oxysporum f. sp. melonis. Of the 1,129 REMI transformants tested, 13 showed reduced pathogenicity on susceptible melon cultivars. One of the mutants, FMMP95–1, was an arginine auxotroph. Structural analysis of the tagged site in FMMP95-1 identified a gene, designated ARG1, which possibly encodes argininosuccinate lyase, catalyzing the last step for arginine biosynthesis. Complementation of FMMP95–1 with the ARG1 gene caused a recovery in pathogenicity, indicating that arginine auxotrophic mutation causes reduced pathogenicity in this pathogen.