Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera

Stewart Hall

Research output: ThesisDoctoral Thesis

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Abstract

Vitis vinifera is one of the world’s most economically important fruit crops. Recently, extensive yield losses in wine grape production, caused by bacterial inflorescence rot (BIR), have been reported in some cool climate Australian vineyards. This disease is caused by the bacterium Pseudomonas syringae pv. syringae (P. s. syringae). Symptoms on grapevine caused by P. s. syringae include the production of leaf spots with chlorotic haloes, necrotic lesions on petioles and shoots, and necrosis of inflorescences.

The aims of the current study were to i) comprehensively evaluate the relatedness and distribution of P. s. syringae isolates from Australian cool climate vineyards, ii) characterise isolates from grapevine, using traditional biochemical techniques, including toxin production and host range, iii) determine whether phenotypic and genotypic data are related to the production of bacterial inflorescence rot or pathogenicity in grapevine using analysis of molecular variance, and iv) determine the grapevine host defence response to P. s. syringae infection to better understand how the pathogen may be
manipulating host responses.

Putative P. s. syringae isolates from infected grapevines within a range of Australian vineyards were identified using LOPAT and RNA polymerase β-subunit (rpoB) gene sequencing for pathovar allocation. The isolates were then characterised by a combination of multi-locus sequence typing (MLST) and biochemical tests. Additionally, the production of syringomycin and syringopeptin was assessed, along with genotyping for these toxins including identification of the syringolin A biosynthesis gene (sylC).

Plant defence responses to pathogenic and non-pathogenic P. s. syringae were
investigated on potted Chardonnay grapevines. Callose deposition was observed by aniline blue staining under epifluorescence microscopy and quantified using high intensity pixels from digital photographs. Relative expression of defence gene targets for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and stilbene synthase were monitored by semi-quantitative PCR (qPCR) from reverse transcribed RNA.

This study identified eight vineyards in six Australian viticultural regions affected by P. s. syringae, with symptoms of BIR and/or leaf spot. Bacterial isolates from these vineyards were grouped by MLST data into two well supported P. s. syringae clades, each containing a mixture of pathogenic and non-pathogenic grapevine isolates. Pathogenic P. s. syringae isolates were also obtained from grapevine sucker shoots, suggesting that sucker shoots may allow ‘overwintering’ of the pathogen.

Pathogenicity was associated with tyrosinase negative phenotype whereas those from healthy and non-BIR vineyards were tyrosinase positive. The pathogenicity of P. s. syringae was also found to be associated with syringolin A genotypes (sylC). Although both pathogenic and non-pathogenic P. s. syringae isolates were able to induce callose deposition in grapevine leaves, the effect was less for pathogenic P. s. syringae. Semi-quantitative PCR showed that inoculation of grapevine leaves by pathogenic P. s. syringae caused increases in the activity of the SA and JA/ET mediated pathways in potted Chardonnay.

The current study has demonstrated that, in cool climate Australian vineyards,
genetically distinct strain groups of P. s. syringae can be isolated from grapevines affected by BIR. Phenotypic and genotypic characterisation suggests that P. s. syringae isolates that produce syringolin A but lack tyrosinase activity are associated with this disease. Finally, the defence gene studies provide insight into the grapevine defence responses to pathogenic P. s. syringae, which may open up knowledge for effective targeted treatment and effective disease management in affected regions.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
Supervisors/Advisors
  • Weckert, Melanie, Co-Supervisor
  • Dry, Ian, Co-Supervisor, External person
  • Blanchard, Christopher, Co-Supervisor
Award date01 Nov 2015
Place of PublicationAustralia
Publisher
Publication statusPublished - 2016

Fingerprint

Pseudomonas syringae
vineyards
Vitis vinifera
plant pathogens
inflorescences
leaf spot
pathogenicity
callose
adventitious shoots
jasmonic acid
climate
salicylic acid
signs and symptoms (plants)
shoots
ethylene
quantitative polymerase chain reaction
toxins
genes
Pseudomonas syringae pv. syringae
lesions (plant)

Cite this

Hall, Stewart. / Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera. Australia : Charles Sturt University, 2016. 261 p.
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title = "Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera",
abstract = "Vitis vinifera is one of the world’s most economically important fruit crops. Recently, extensive yield losses in wine grape production, caused by bacterial inflorescence rot (BIR), have been reported in some cool climate Australian vineyards. This disease is caused by the bacterium Pseudomonas syringae pv. syringae (P. s. syringae). Symptoms on grapevine caused by P. s. syringae include the production of leaf spots with chlorotic haloes, necrotic lesions on petioles and shoots, and necrosis of inflorescences.The aims of the current study were to i) comprehensively evaluate the relatedness and distribution of P. s. syringae isolates from Australian cool climate vineyards, ii) characterise isolates from grapevine, using traditional biochemical techniques, including toxin production and host range, iii) determine whether phenotypic and genotypic data are related to the production of bacterial inflorescence rot or pathogenicity in grapevine using analysis of molecular variance, and iv) determine the grapevine host defence response to P. s. syringae infection to better understand how the pathogen may bemanipulating host responses.Putative P. s. syringae isolates from infected grapevines within a range of Australian vineyards were identified using LOPAT and RNA polymerase β-subunit (rpoB) gene sequencing for pathovar allocation. The isolates were then characterised by a combination of multi-locus sequence typing (MLST) and biochemical tests. Additionally, the production of syringomycin and syringopeptin was assessed, along with genotyping for these toxins including identification of the syringolin A biosynthesis gene (sylC).Plant defence responses to pathogenic and non-pathogenic P. s. syringae wereinvestigated on potted Chardonnay grapevines. Callose deposition was observed by aniline blue staining under epifluorescence microscopy and quantified using high intensity pixels from digital photographs. Relative expression of defence gene targets for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and stilbene synthase were monitored by semi-quantitative PCR (qPCR) from reverse transcribed RNA.This study identified eight vineyards in six Australian viticultural regions affected by P. s. syringae, with symptoms of BIR and/or leaf spot. Bacterial isolates from these vineyards were grouped by MLST data into two well supported P. s. syringae clades, each containing a mixture of pathogenic and non-pathogenic grapevine isolates. Pathogenic P. s. syringae isolates were also obtained from grapevine sucker shoots, suggesting that sucker shoots may allow ‘overwintering’ of the pathogen.Pathogenicity was associated with tyrosinase negative phenotype whereas those from healthy and non-BIR vineyards were tyrosinase positive. The pathogenicity of P. s. syringae was also found to be associated with syringolin A genotypes (sylC). Although both pathogenic and non-pathogenic P. s. syringae isolates were able to induce callose deposition in grapevine leaves, the effect was less for pathogenic P. s. syringae. Semi-quantitative PCR showed that inoculation of grapevine leaves by pathogenic P. s. syringae caused increases in the activity of the SA and JA/ET mediated pathways in potted Chardonnay. The current study has demonstrated that, in cool climate Australian vineyards,genetically distinct strain groups of P. s. syringae can be isolated from grapevines affected by BIR. Phenotypic and genotypic characterisation suggests that P. s. syringae isolates that produce syringolin A but lack tyrosinase activity are associated with this disease. Finally, the defence gene studies provide insight into the grapevine defence responses to pathogenic P. s. syringae, which may open up knowledge for effective targeted treatment and effective disease management in affected regions.",
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year = "2016",
language = "English",
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address = "Australia",
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Hall, S 2016, 'Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera', Doctor of Philosophy, Charles Sturt University, Australia.

Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera. / Hall, Stewart.

Australia : Charles Sturt University, 2016. 261 p.

Research output: ThesisDoctoral Thesis

TY - THES

T1 - Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera

AU - Hall, Stewart

PY - 2016

Y1 - 2016

N2 - Vitis vinifera is one of the world’s most economically important fruit crops. Recently, extensive yield losses in wine grape production, caused by bacterial inflorescence rot (BIR), have been reported in some cool climate Australian vineyards. This disease is caused by the bacterium Pseudomonas syringae pv. syringae (P. s. syringae). Symptoms on grapevine caused by P. s. syringae include the production of leaf spots with chlorotic haloes, necrotic lesions on petioles and shoots, and necrosis of inflorescences.The aims of the current study were to i) comprehensively evaluate the relatedness and distribution of P. s. syringae isolates from Australian cool climate vineyards, ii) characterise isolates from grapevine, using traditional biochemical techniques, including toxin production and host range, iii) determine whether phenotypic and genotypic data are related to the production of bacterial inflorescence rot or pathogenicity in grapevine using analysis of molecular variance, and iv) determine the grapevine host defence response to P. s. syringae infection to better understand how the pathogen may bemanipulating host responses.Putative P. s. syringae isolates from infected grapevines within a range of Australian vineyards were identified using LOPAT and RNA polymerase β-subunit (rpoB) gene sequencing for pathovar allocation. The isolates were then characterised by a combination of multi-locus sequence typing (MLST) and biochemical tests. Additionally, the production of syringomycin and syringopeptin was assessed, along with genotyping for these toxins including identification of the syringolin A biosynthesis gene (sylC).Plant defence responses to pathogenic and non-pathogenic P. s. syringae wereinvestigated on potted Chardonnay grapevines. Callose deposition was observed by aniline blue staining under epifluorescence microscopy and quantified using high intensity pixels from digital photographs. Relative expression of defence gene targets for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and stilbene synthase were monitored by semi-quantitative PCR (qPCR) from reverse transcribed RNA.This study identified eight vineyards in six Australian viticultural regions affected by P. s. syringae, with symptoms of BIR and/or leaf spot. Bacterial isolates from these vineyards were grouped by MLST data into two well supported P. s. syringae clades, each containing a mixture of pathogenic and non-pathogenic grapevine isolates. Pathogenic P. s. syringae isolates were also obtained from grapevine sucker shoots, suggesting that sucker shoots may allow ‘overwintering’ of the pathogen.Pathogenicity was associated with tyrosinase negative phenotype whereas those from healthy and non-BIR vineyards were tyrosinase positive. The pathogenicity of P. s. syringae was also found to be associated with syringolin A genotypes (sylC). Although both pathogenic and non-pathogenic P. s. syringae isolates were able to induce callose deposition in grapevine leaves, the effect was less for pathogenic P. s. syringae. Semi-quantitative PCR showed that inoculation of grapevine leaves by pathogenic P. s. syringae caused increases in the activity of the SA and JA/ET mediated pathways in potted Chardonnay. The current study has demonstrated that, in cool climate Australian vineyards,genetically distinct strain groups of P. s. syringae can be isolated from grapevines affected by BIR. Phenotypic and genotypic characterisation suggests that P. s. syringae isolates that produce syringolin A but lack tyrosinase activity are associated with this disease. Finally, the defence gene studies provide insight into the grapevine defence responses to pathogenic P. s. syringae, which may open up knowledge for effective targeted treatment and effective disease management in affected regions.

AB - Vitis vinifera is one of the world’s most economically important fruit crops. Recently, extensive yield losses in wine grape production, caused by bacterial inflorescence rot (BIR), have been reported in some cool climate Australian vineyards. This disease is caused by the bacterium Pseudomonas syringae pv. syringae (P. s. syringae). Symptoms on grapevine caused by P. s. syringae include the production of leaf spots with chlorotic haloes, necrotic lesions on petioles and shoots, and necrosis of inflorescences.The aims of the current study were to i) comprehensively evaluate the relatedness and distribution of P. s. syringae isolates from Australian cool climate vineyards, ii) characterise isolates from grapevine, using traditional biochemical techniques, including toxin production and host range, iii) determine whether phenotypic and genotypic data are related to the production of bacterial inflorescence rot or pathogenicity in grapevine using analysis of molecular variance, and iv) determine the grapevine host defence response to P. s. syringae infection to better understand how the pathogen may bemanipulating host responses.Putative P. s. syringae isolates from infected grapevines within a range of Australian vineyards were identified using LOPAT and RNA polymerase β-subunit (rpoB) gene sequencing for pathovar allocation. The isolates were then characterised by a combination of multi-locus sequence typing (MLST) and biochemical tests. Additionally, the production of syringomycin and syringopeptin was assessed, along with genotyping for these toxins including identification of the syringolin A biosynthesis gene (sylC).Plant defence responses to pathogenic and non-pathogenic P. s. syringae wereinvestigated on potted Chardonnay grapevines. Callose deposition was observed by aniline blue staining under epifluorescence microscopy and quantified using high intensity pixels from digital photographs. Relative expression of defence gene targets for salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and stilbene synthase were monitored by semi-quantitative PCR (qPCR) from reverse transcribed RNA.This study identified eight vineyards in six Australian viticultural regions affected by P. s. syringae, with symptoms of BIR and/or leaf spot. Bacterial isolates from these vineyards were grouped by MLST data into two well supported P. s. syringae clades, each containing a mixture of pathogenic and non-pathogenic grapevine isolates. Pathogenic P. s. syringae isolates were also obtained from grapevine sucker shoots, suggesting that sucker shoots may allow ‘overwintering’ of the pathogen.Pathogenicity was associated with tyrosinase negative phenotype whereas those from healthy and non-BIR vineyards were tyrosinase positive. The pathogenicity of P. s. syringae was also found to be associated with syringolin A genotypes (sylC). Although both pathogenic and non-pathogenic P. s. syringae isolates were able to induce callose deposition in grapevine leaves, the effect was less for pathogenic P. s. syringae. Semi-quantitative PCR showed that inoculation of grapevine leaves by pathogenic P. s. syringae caused increases in the activity of the SA and JA/ET mediated pathways in potted Chardonnay. The current study has demonstrated that, in cool climate Australian vineyards,genetically distinct strain groups of P. s. syringae can be isolated from grapevines affected by BIR. Phenotypic and genotypic characterisation suggests that P. s. syringae isolates that produce syringolin A but lack tyrosinase activity are associated with this disease. Finally, the defence gene studies provide insight into the grapevine defence responses to pathogenic P. s. syringae, which may open up knowledge for effective targeted treatment and effective disease management in affected regions.

M3 - Doctoral Thesis

PB - Charles Sturt University

CY - Australia

ER -

Hall S. Effects of the Plant Pathogen Pseudomonas syringae pathovar syringea on Vitis vinifera. Australia: Charles Sturt University, 2016. 261 p.