Kiwifruit Resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. actinidiae and Defence Induction by Acibenzolar-S-methyl and Methyl Jasmonate Are Cultivar Dependent.

Pathogen susceptibility and defence gene inducibility were compared between the Actinidia arguta cultivar 'Hortgem Tahi' and the two cultivars of A. chinensis 'Hayward' and 'Zesy002'. Plants were treated with acibenzolar-s-methyl (ASM) or methyl jasmonate (MeJA) one week before inoculation with Pseudomonas syringae pv. actinidiae (Psa biovar3) or Sclerotinia sclerotiorum, or secondary induction with chitosan+glucan (Ch-Glu) as a potential pathogen proxy. Defence expression was evaluated by measuring the expression of 18 putative defence genes. 'Hortgem Tahi' was highly susceptible to sclerotinia and very resistant to Psa, whereas 'Zesy002' was highly resistant to both, and 'Hayward' was moderately susceptible to both. Gene expression in 'Hayward' and 'Zesy002' was alike but differed significantly from 'Hortgem Tahi' which had higher basal levels of PR1-i, PR5-i, JIH1, NPR3 and WRKY70 but lower expression of RD22 and PR2-i. Treatment with ASM caused upregulation of NIMIN2, PR1-i, WRKY70, DMR6 and PR5-i in all cultivars and induced resistance to Psa in 'Zesy002' and 'Hayward' but decreased resistance to sclerotinia in 'Zesy002'. MeJA application caused upregulation of LOX2 and downregulation of NIMIN2, DMR6 and PR2-i but did not affect disease susceptibility. The Ch-Glu inducer induced PR-gene families in each cultivar, highlighting its possible effectiveness as an alternative to actual pathogen inoculation. The significance of variations in fundamental and inducible gene expression among the cultivars is explored.

Host-specific signal perception by PsaR2 LuxR solo induces Pseudomonas syringae pv. actinidiae virulence traits.

Plant-associated bacteria, including pathogens, recognise host-derived signals to activate specific responses. The genome of Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of bacterial canker of kiwifruit, encodes for three putative LuxR-like receptors. Proteins of this family are usually involved in the quorum sensing system, through the perception of autoinducers (AHLs) produced by a cognate LuxI. However, Psa does not produce AHLs according to the lack of LuxI-encoding gene. It has been proposed that the so-called LuxR solos may be involved in the perception of environmental stimuli. We thus hypothesised that Psa LuxR-like receptors could be involved in host-derived signal sensing. Psa virulence traits, i.e., biofilm formation, motility and endophytic colonisation, were stimulated by growing the pathogen in host plant extracts, but not in non-host plant extracts or rich medium. Moreover, the phenotypic analyses of Psa mutant strains lacking the LuxR solo-encoding genes, demonstrated that PsaR2 plays a major role in host recognition and induction of virulence responses. The heterologous expression of PsaR2, followed by affinity chromatography and fraction activity assessment, confirmed the specific recognition of plant-derived components by this sensor. Overall, these data provide a deeper understanding of the regulation of Psa virulence through interkingdom communication, which represents a interesting target for the development of tolerant/resistant genotypes or innovative control strategies.

Effector loss drives adaptation of Pseudomonas syringae pv. actinidiae biovar 3 to Actinidia arguta.

A pandemic isolate of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) has devastated kiwifruit orchards growing cultivars of Actinidia chinensis. In contrast, A. arguta (kiwiberry) is not a host of Psa3. Resistance is mediated via effector-triggered immunity, as demonstrated by induction of the hypersensitive response in infected A. arguta leaves, observed by microscopy and quantified by ion-leakage assays. Isolates of Psa3 that cause disease in A. arguta have been isolated and analyzed, revealing a 51 kb deletion in the exchangeable effector locus (EEL). This natural EEL-mutant isolate and strains with synthetic knockouts of the EEL were more virulent in A. arguta plantlets than wild-type Psa3. Screening of a complete library of Psa3 effector knockout strains identified increased growth in planta for knockouts of four effectors-AvrRpm1a, HopF1c, HopZ5a, and the EEL effector HopAW1a -suggesting a resistance response in A. arguta. Hypersensitive response (HR) assays indicate that three of these effectors trigger a host species-specific HR. A Psa3 strain with all four effectors knocked out escaped host recognition, but a cumulative increase in bacterial pathogenicity and virulence was not observed. These avirulence effectors can be used in turn to identify the first cognate resistance genes in Actinidia for breeding durable resistance into future kiwifruit cultivars.

Pseudomonas syringae pv. actinidiae: Ecology, Infection Dynamics and Disease Epidemiology.

Since 2008, the kiwifruit industry has been devastated by a pandemic outbreak of Pseudomonas syringae pv. actinidiae (Psa), the causal agent of bacterial canker. This disease has become the most significant limiting factor in kiwifruit production. Psa colonizes different organs of the host plant, causing a specific symptomatology on each of them. In addition, the systemic invasion of the plant may quickly lead to plant death. Despite the massive risk that this disease poses to the kiwifruit industry, studies focusing on Psa ecology have been sporadic, and a comprehensive description of the disease epidemiology is still missing. Optimal environmental conditions for infection, dispersal and survival in the environment, or the mechanisms of penetration and colonization of host tissues have not been fully elucidated yet. The present work aims to provide a synthesis of the current knowledge, and a deeper understanding of the epidemiology of kiwifruit bacterial canker based on new experimental data. The pathogen may survive in the environment or overwinter in dormant tissues and be dispersed by wind or rain. Psa was observed in association with several plant structures (stomata, trichomes, lenticels) and wounds, which could represent entry points for apoplast infection. Environmental conditions also affect the bacterial colonization, with lower optimum values of temperature and humidity for epiphytic than for endophytic growth, and disease incidence requiring a combination of mild temperature and leaf wetness. By providing information on Psa ecology, these data sets may contribute to plan efficient control strategies for kiwifruit bacterial canker.

N-Acyl Homoserine Lactones and Lux Solos Regulate Social Behaviour and Virulence of Pseudomonas syringae pv. actinidiae.

The phyllosphere is a complex environment where microbes communicate through signalling molecules in a system, generally known as quorum sensing (QS). One of the most common QS systems in Gram-negative proteobacteria is based on the production of N-acyl homoserine lactones (AHLs) by a LuxI synthase and their perception by a LuxR sensor. Pseudomonas syringae pv. actinidiae (Psa), the aetiological agent of the bacterial canker of kiwifruit, colonises plant phyllosphere before penetrating via wounds and natural openings. Since Psa genome encodes three LuxR solos without a cognate LuxI, this bacterium may perceive diffusible signals, but it cannot produce AHLs, displaying a non-canonical QS system. The elucidation of the mechanisms underlying the perception of environmental cues in the phyllosphere by this pathogen and their influence on the onset of pathogenesis are of crucial importance for a long-lasting and sustainable management of the bacterial canker of kiwifruit. Here, we report the ability of Psa to sense its own population density and the presence of surrounding bacteria. Moreover, we show that Psa can perceive AHLs, indicating that AHL-producing neighbouring bacteria may regulate Psa virulence in the host. Our results suggest that the ecological environment is important in determining Psa fitness and pathogenic potential. This opens new perspectives in the use of more advanced biochemical and microbiological tools for the control of bacterial canker of kiwifruit.

Multiple quantitative trait loci contribute to resistance to bacterial canker incited by Pseudomonas syringae pv. actinidiae in kiwifruit (Actinidia chinensis).

Pseudomonas syringae pv. actinidiae (Psa) biovar 3, a virulent, canker-inducing pathogen is an economic threat to the kiwifruit (Actinidia spp.) industry worldwide. The commercially grown diploid (2×) A. chinensis var. chinensis is more susceptible to Psa than tetraploid and hexaploid kiwifruit. However information on the genetic loci modulating Psa resistance in kiwifruit is not available. Here we report mapping of quantitative trait loci (QTLs) regulating resistance to Psa in a diploid kiwifruit population, derived from a cross between an elite Psa-susceptible 'Hort16A' and a resistant male breeding parent P1. Using high-density genetic maps and intensive phenotyping, we identified a single QTL for Psa resistance on Linkage Group (LG) 27 of 'Hort16A' revealing 16-19% phenotypic variance and candidate alleles for susceptibility and resistance at this loci. In addition, six minor QTLs were identified in P1 on distinct LGs, exerting 4-9% variance. Resistance in the F1 population is improved by additive effects from 'Hort16A' and P1 QTLs providing evidence that divergent genetic pathways interact to combat the virulent Psa strain. Two different bioassays further identified new QTLs for tissue-specific responses to Psa. The genetic marker at LG27 QTL was further verified for association with Psa resistance in diploid Actinidia chinensis populations. Transcriptome analysis of Psa-resistant and susceptible genotypes in field revealed hallmarks of basal defense and provided candidate RNA-biomarkers for screening for Psa resistance in greenhouse conditions.

Integrated Use of Aureobasidium pullulans Strain CG163 and Acibenzolar-S-Methyl for Management of Bacterial Canker in Kiwifruit.

An isolate of Aureobasidium pullulans (strain = CG163) and the plant defence elicitor acibenzolar-S-methyl (ASM) were investigated for their ability to control leaf spot in kiwifruit caused by Pseudomonas syringae pv. actinidiae biovar 3 (Psa). Clonal Actinidia chinensis var. deliciosa plantlets ('Hayward') were treated with ASM, CG163 or ASM + CG163 at seven and one day before inoculation with Psa. ASM (0.2 g/L) was applied either as a root or foliar treatments and CG163 was applied as a foliar spray containing 2 × 107 CFU/mL. Leaf spot incidence was significantly reduced by all treatments compared with the control. The combination of ASM + CG163 had greater efficacy (75%) than either ASM (55%) or CG163 (40%) alone. Moreover, treatment efficacy correlated positively with the expression of defence-related genes: pathogenesis-related protein 1 (PR1), ß-1,3-glucosidase, Glucan endo 1,3-ß-glucosidase (Gluc_PrimerH) and Class IV chitinase (ClassIV_Chit), with greater gene upregulation in plants treated with ASM + CG163 than by the individual treatments. Pathogen population studies indicated that CG163 had significant suppressive activity against epiphytic populations of Psa. Endophytic populations were reduced by ASM + CG163 but not by the individual treatments, and by 96-144 h after inoculation were significantly lower than the control. Together these data suggest that ASM + CG163 have complementary modes of action that contribute to greater control of leaf spotting than either treatment alone.

Comparative transcriptome analysis of the interaction between Actinidia chinensis var. chinensis and Pseudomonas syringae pv. actinidiae in absence and presence of acibenzolar-S-methyl.

BACKGROUND: Since 2007, bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has become a pandemic disease leading to important economic losses in every country where kiwifruit is widely cultivated. Options for controlling this disease are very limited and rely primarily on the use of bactericidal compounds, such as copper, and resistance inducers. Among the latter, the most widely studied is acibenzolar-S-methyl. To elucidate the early molecular reaction of kiwifruit plants (Actinidia chinensis var. chinensis) to Psa infection and acibenzolar-S-methyl treatment, a RNA seq analysis was performed at different phases of the infection process, from the epiphytic phase to the endophytic invasion on acibenzolar-S-methyl treated and on non-treated plants. The infection process was monitored in vivo by confocal laser scanning microscopy. RESULTS: De novo assembly of kiwifruit transcriptome revealed a total of 39,607 transcripts, of which 3360 were differentially expressed during the infection process, primarily 3 h post inoculation. The study revealed the coordinated changes of important gene functional categories such as signaling, hormonal balance and transcriptional regulation. Among the transcription factor families, AP2/ERF, MYB, Myc, bHLH, GATA, NAC, WRKY and GRAS were found differentially expressed in response to Psa infection and acibenzolar-S-methyl treatment. Finally, in plants treated with acibenzolar-S-methyl, a number of gene functions related to plant resistance, such as PR proteins, were modulated, suggesting the set-up of a more effective defense response against the pathogen. Weighted-gene coexpression network analysis confirmed these results. CONCLUSIONS: Our work provides an in-depth description of the plant molecular reactions to Psa, it highlights the metabolic pathway related to acibenzolar-S-methyl-induced resistance and it contributes to the development of effective control strategies in open field.

The Scientific, Economic, and Social Impacts of the New Zealand Outbreak of Bacterial Canker of Kiwifruit (Pseudomonas syringae pv. actinidiae).

The introduction of Pseudomonas syringae pv. actinidiae (Psa) severely damaged the New Zealand kiwifruit industry, which in 2010 was based on only two cultivars. Despite an extraordinarily quick and strong response by industry, government, and scientists to minimize the economic and social impacts, the economic consequences of this outbreak were severe. Although our understanding of Psa epidemiology and control methods increased substantively over the past six years, the kiwifruit industry largely recovered because of the introduction of a less-susceptible yellow-fleshed cultivar. The New Zealand population of Psa is clonal but has evolved rapidly since its introduction by exchanging mobile genetic elements, including integrative conjugative elements (ICEs), with the local bacterial populations. In some cases, this has led to copper resistance. It is currently believed that the center of origin of the pathogen is Japan or Korea, but biovar 3, which is responsible for the global outbreak, originated in China.

Genomic analysis of the Kiwifruit pathogen Pseudomonas syringae pv. actinidiae provides insight into the origins of an emergent plant disease.

The origins of crop diseases are linked to domestication of plants. Most crops were domesticated centuries--even millennia--ago, thus limiting opportunity to understand the concomitant emergence of disease. Kiwifruit (Actinidia spp.) is an exception: domestication began in the 1930s with outbreaks of canker disease caused by P. syringae pv. actinidiae (Psa) first recorded in the 1980s. Based on SNP analyses of two circularized and 34 draft genomes, we show that Psa is comprised of distinct clades exhibiting negligible within-clade diversity, consistent with disease arising by independent samplings from a source population. Three clades correspond to their geographical source of isolation; a fourth, encompassing the Psa-V lineage responsible for the 2008 outbreak, is now globally distributed. Psa has an overall clonal population structure, however, genomes carry a marked signature of within-pathovar recombination. SNP analysis of Psa-V reveals hundreds of polymorphisms; however, most reside within PPHGI-1-like conjugative elements whose evolution is unlinked to the core genome. Removal of SNPs due to recombination yields an uninformative (star-like) phylogeny consistent with diversification of Psa-V from a single clone within the last ten years. Growth assays provide evidence of cultivar specificity, with rapid systemic movement of Psa-V in Actinidia chinensis. Genomic comparisons show a dynamic genome with evidence of positive selection on type III effectors and other candidate virulence genes. Each clade has highly varied complements of accessory genes encoding effectors and toxins with evidence of gain and loss via multiple genetic routes. Genes with orthologs in vascular pathogens were found exclusively within Psa-V. Our analyses capture a pathogen in the early stages of emergence from a predicted source population associated with wild Actinidia species. In addition to candidate genes as targets for resistance breeding programs, our findings highlight the importance of the source population as a reservoir of new disease.