The Apple Fruitlet Model System for Fire Blight Disease

Klee, Sara M.; Sinn, Judith P.; McNellis, Timothy W.

doi:10.1007/978-1-4939-9458-8_17

Cited by 11 publications

(13 citation statements)

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“…Virulence in apple fruitlets. Detached immature apple (Malus pumila) fruits (apple fruitlets) represent a convenient and inexpensive fire blight infection model system (39), and we used this system to assay virulence for the large number of auxotrophic mutants identified in our genetic screen. The entire series of mutants was inoculated onto apple fruitlets, and disease severity was measured after 1 week using a disease severity rating scale (39).…”

Section: Resultsmentioning

confidence: 99%

“…Detached immature apple (Malus pumila) fruits (apple fruitlets) represent a convenient and inexpensive fire blight infection model system (39), and we used this system to assay virulence for the large number of auxotrophic mutants identified in our genetic screen. The entire series of mutants was inoculated onto apple fruitlets, and disease severity was measured after 1 week using a disease severity rating scale (39). Mutant disease severity was scored as a ratio of disease severity relative to wild-type E. amylovora, with a value of 1 being equal to disease severity caused by the wild type and ratios less than 1 indicating less disease severity than that generated by wild type.…”

Section: Resultsmentioning

confidence: 99%

“…Virulence in apple tree shoots. While immature apple fruit serve as a highthroughput model system for fire blight infection (39), bacteria naturally colonize flowers or open wounds on trees, only sometimes reaching fruit later in severe infections (1). Eight auxotrophic mutants, including three with virulence similar to the wild type on apple fruitlets (cysI3, hisA, and ppc mutants) and five that were less virulent than the wild type on apple fruitlets (ilvA, leuB, metE, purL, and trpB mutants), were selected for inoculation onto apple shoots by shoot tip wounding.…”

Section: Resultsmentioning

confidence: 99%

“…Virulence assays. The virulence of each auxotrophic mutant was tested using immature 'Gala' apples, as described in detail elsewhere (39). Each disease screening tray allowed testing of eight mutants, as well as wild-type and mock inoculations.…”

Section: Methodsmentioning

confidence: 99%

“…Within a tray, each strain was inoculated into five separate immature apple halves by dipping a sterile toothpick into bacterial culture on an LB plate and inserting the toothpick into a wound in the skin of the fruitlet. After 7 days, the disease severity of each fruitlet was scored from 1 to 8 according to the following rating scale: 1, no symptoms; 2, 1 to 10% of fruit is water soaked; 3, Ͼ10% water soaking and/or Ͻ10% necrosis; 4, 10 to 30% necrotic/oozing; 5, Ͼ30 to 50% necrotic/oozing; 6, Ͼ50 to 70% necrotic/oozing; 7, Ͼ70 to 90% necrotic/oozing; and 8, Ͼ90% necrotic/oozing (39). Each mutant was tested at least twice in two different trays.…”

Section: Methodsmentioning

confidence: 99%

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Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples

Klee

Sinn

Finley

et al. 2019

Appl Environ Microbiol

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The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have 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 auxotrophic E. amylovora mutants, 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-type E. 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 nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents. IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on 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 nonpathogenic E. amylovora auxotrophs 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.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%