Pseudomonas syringae pv. actinidiae (Psa), a gram negative bacterium responsible for bacterial canker, is considered to be one of the most devastating global pathogens of kiwifruit (Actinidia deliciosa and A. chinensis), (Vanneste, 2012; Michelotti et al., 2015). Psa biovar 3 is the biovar responsible for causing the recent worldwide outbreak of bacterial canker (Vanneste et al., 2013). Pseudomonas syringae pv. actinidifoliorum (Pfm) was originally thought to be a less virulent form of Psa that caused smaller lesions and localized infection compared with Psa, and was formerly classified as biovar 4, but it has been subsequently classified as a separate pathovar on the basis of differential biochemical, genetic and pathogenicity characteristics (Cunty et al., 2015).

Psa infected vines exhibit leaf spots, cane wilting and dieback, and cankers that are sometimes associated with exudate, and it spreads rapidly in suitable weather conditions, such as high rainfall and cooler temperatures in spring and autumn (Vanneste, 2013; Vanneste et al., 2013). Like most bacterial pathogens, effective control options for Psa are limited, and the mainstays of chemical control are copper pesticides and antibiotics (Reglinski et al., 2013). Disadvantages of using these products include phytotoxicity, heavy metal accumulation in the soil, and development of resistance.

Longer term, more sustainable control measures include breeding for resistance and the use of elicitors in integrated control programs to temporarily induce host defenses. Successful implementation of both resistance breeding and use of elicitors requires a thorough understanding of the mechanisms and genes involved. Breeding for resistance has to be carefully controlled so that other desirable characteristics such as high dry matter, for example, are not lost. Elicitors can induce different phytohormonal defense-signaling pathways (Romanazzi et al., 2016), such as the abscisic acid (ABA) pathway that is involved in generalized stress responses to abiotic and biotic factors (Ton et al., 2009), the salicylic acid (SA) pathway, which tends to be induced by biotrophic pathogens and sap-sucking insects (Loake and Grant, 2007; Erb et al., 2012), and the jasmonic acid (JA) pathway which responds to wounding, chewing insects and invasion by necrotrophic pathogens (Dar et al., 2015). There is often complex cross talk and sometimes antagonism between these pathways, for example, elevation of the SA pathway leading to suppression of the JA pathway and vice versa (Pieterse et al., 2012; Verma et al., 2016). Successful use of an elicitor requires identification of the pathways that are effective in the particular host–pathogen interaction being studied. An understanding of the mode of action of elicitors is equally important because efficacy under field conditions varies with cultivar, timing and frequency of application, and crop phenology (Walters et al., 2011; Bruce, 2014; Reglinski et al., 2014a). In addition, knowledge of the mechanisms involved is needed to minimize the metabolic costs associated with elicitor use, such as reduced fruit production or quality (Walters and Heil, 2007; Cipollini and Heil, 2010). Whilst there are published studies on elicitor-induced responses of different kiwifruit cultivars to Psa, which suggest predominance of the SA-mediated pathway (Petriccione et al., 2013, 2014; Cellini et al., 2014; Reglinski et al., 2014b), there has been no investigation of the molecular and biochemical basis of the variable host response to Psa and the closely related Pfm. Consequently the current study was carried out to address this gap in our knowledge. The study was performed on Actinidia deliciosa ‘Hayward’ because this is the most widely grown cultivar in the world (Nardozza et al., 2013). Specific objectives of this research were to elucidate phytohormone-mediated signaling pathways that play a key role in orchestrating the kiwifruit defense response to Psa and Pfm, and, if possible, to assist identification of molecular markers of plant defense for marker assisted selection (MAS) in breeding programs.

The key finding from this study is that ‘Hayward’ kiwifruit are more resistant to stem inoculation with Pfm than with Psa biovar3, and that the enhanced resistance is associated with a stronger, localized SA-based defense response. Differential responses in hormone concentrations occurred only in the stem tissue close the inoculation site and showed that Pfm inoculation induced a threefold increase in SA relative to Psa inoculation and a smaller 1.6-fold induction of JA. Transcript expression showed that Pfm inoculation of the stem, compared with wounding and Psa inoculation, resulted in the greatest elevation of PR1 and PR6 (SA pathway markers), β-1,3-glucosidase (involved in activation of phytoanticipins and a SA pathway marker), AP2 ERF2 transcription factor (an early defense response marker involved in SA/JA cross talk), and JIH1 (an enzyme degrading the active metabolite JA-Ile in the JA pathway) in stem tissue surrounding the inoculum site. These genes have potential as markers of ‘Hayward’ resistance to Pseudomonas syringae pathovars and, in particular, PR1 and β-1,3-glucosidase appear to be useful marker candidates for distinguishing the host response to Psa and Pfm.

Results from this study indicate that method of inoculation, choice of sampling tissue and timing of collection are important factors to consider when carrying out biochemical and molecular studies. Phytohormone and gene expression measurements showed that significant results were only observed in localized stem tissue that was collected 48 hpi, and not in leaves that were sampled 24 and 48 hpi. However, a systemic response in leaves and in distal stem tissue might have been observed in this study if sample times of greater than 48 hpi had been used. Method of inoculation also needs to be considered. For example, several proteomic studies showed Psa inoculation induced up-regulation of proteins associated with SA-defense in ‘Hayward’ and ‘Hort16A’ leaves 72 hpi (Petriccione et al., 2013, 2014, 2015a), but the inoculation method consisted of spraying all the leaves with Psa, hence a localized response to leaf inoculation was effectively being measured. Cellini et al. (2014) also measured a localized response in leaf tissue, i.e., up-regulation of SA marker genes 24–72 hpi, because their method of inoculation comprised cutting leaf tips with inoculum-dipped scissors. Stem inoculation was utilized in the current study because resistance levels determined by this bioassay method have shown to correlate well with actual levels of resistance observed in the field (Datson et al., 2015; Hoyte et al., 2015; Nardozza et al., 2015). Further research with these molecular markers should include carrying out a time course, because sampling 24–48 dpi may be adequate for determining localized expression of PR proteins that are activated late during a defense response, as indicated by other studies (Cellini et al., 2014; Petriccione et al., 2014), but may be less suitable for capturing earlier expressed localized defenses, or delayed systemic responses.

In both artificial inoculation experiments of ‘Hayward’ plantlets, disease severity, as indicated by lesion length, increased over time and was always significantly greater with Psa (strain #10627) inoculation of the stem than with Pfm (ICMP strain #19098) inoculation. Originally thought to be different biovars of the same pathogen (Vanneste et al., 2013), Pfm was designated as a separate pathovar from Psa due to pathogenic, phenotypic, genetic and phylogenetic differences (Cunty et al., 2015). Pfm is substantially less aggressive than Psa in that it is only able to cause leaf spots and has not been linked with commercial loss of productivity in the New Zealand kiwifruit industry. In contrast, Psa can additionally cause canker and shoot dieback and is considered to be a major kiwifruit pathogen (Vanneste et al., 2013). It is possible that the increased ability of the host to mount a response to Pfm is largely responsible for its lack of virulence. There are several ways to explain this. One is that Pfm lacks a number of critical pathogen factors to suppress the host response that Psa biovar 3 (bv3) possesses. Genome-based comparisons of Psa and Pfm (McCann et al., 2013) clearly indicate a significant series of effectors present in Psa bv3 that are missing from Pfm that could account for this behavior. On the other hand, there could be Pfm factors such as effectors (of which there are several not present in Psa bv3) and/or PAMPs recognized by kiwifruit that turn on a defense response. Typically when a plant recognizes effectors, it initiates resistance gene-based responses, which have characteristics such as hypersensitive responses that are not seen in the response of kiwifruit to Pfm, which makes this scenario less likely. However, it is important to note that resistance responses to effectors are not invariably linked to obvious hypersensitive responses. The most likely explanation, however, is that one or more of the effectors present in Psa bv3, but absent in Pfm, actively suppresses the response mounted by the host against Pfm and this explains the virulence of Psa bv3 and, by contrast, the lack of virulence of Pfm.

Phytohormone measurements suggest predominance of the SA pathway over the JA pathway, in host defense against the pathovars used in this study, with a greater response induced by Pfm than Psa. Phytohormone measurements in leaves were not significant, except for elevated levels of ABA in untreated versus inoculated leaves in Exp 1, but this result was not observed in Exp 2 leaf tissue. Given that no other hormones responded to inoculation treatments in leaf tissue, the Exp 2 result for ABA is more likely to be correct. In contrast, localized induction of SA in stems was up to threefold higher with Pfm inoculation than with Psa (P < 0.001), and a smaller induction (1.6-fold) of JA (P < 0.009) was also observed. P. syringae is able to stimulate both SA and JA pathways (Pieterse et al., 2012), but resistance to many biotrophic bacterial pathogens, which infect aerial parts of plants, including Pseudomonas spp., is mediated via SA-responsive signaling in a number of diverse hosts including rice, tobacco and Arabidopsis (Lee et al., 2013; Yang et al., 2016). In support of these findings, Cellini et al. (2014) found that compounds that stimulated the SA pathway (SA or its synthetic analog, acibenzolar-S-methyl ASM) reduced disease caused by Psa on ‘Hayward,’ while methyl-jasmonate or ethylene increased disease development. These results fit the most simplistic model of antagonistic SA/JA cross talk, where up-regulation of one pathway leads to down-regulation of the other. However, SA/JA cross talk appears much more complex than this, because neutral and synergistic interactions have also been recorded (Schenk et al., 2000; Van Wees et al., 2000; Mur et al., 2006). Mur et al. (2006) showed that the outcome of the SA–JA interaction is dependent of the relative concentration of each hormone. In the current study, SA concentration was elevated by Pfm more than JA, and there was no increase in the bioactive form of jasmonate JA-Ile, suggesting that SA/JA antagonism is occurring. The effect of other phytohormones on SA/JA cross talk also needs to be considered (Pieterse et al., 2012). Wounding significantly reduced ABA levels in the local stem tissue in Exp 2 (P = 0.029), which was most likely associated with movement of all the treated plants (Mock inoc, Psa and Pfm) into a higher humidity environment after inoculation (Okamoto et al., 2009). This suggests that the plant maintains high internal levels of ABA in the stem in response to the presence of P. syringae pathogens, where it may have been acting as a regulator, coordinating stress responses (Bostock et al., 2014; de Zelicourt et al., 2016). Using hormone data alone to draw conclusions must be viewed with caution, as hormone expression is influenced by many factors including tissue type (as shown in the current research), age, external abiotic and biotic factors and circadian rhythms (Zheng et al., 2015). For this reason, the current study also examined gene expression, including mRNA markers of the phytohormone pathways.

Gene expression measurements suggest that host resistance to Pfm is multigenic, localized and predominantly involves the SA rather than the JA pathway, but with a degree of cross talk between SA-dependent and JA/ethylene dependent pathways. Although 16 GoI putative markers were selected to represent as many different pathways and temporal stages in defense as possible, only six genes were significantly affected by inoculation treatment, and exceeded the commonly used bioinformatic cut-off of ±2-fold changes in differential expression (Mosqueira et al., 2012; Meimoun et al., 2014; Damian-Zamacona et al., 2016). Of these 16 genes, 4/5 SA markers were significantly induced (PR1, β-1,3-glucosidase, PR6, WRKY40), and 2/5 of JA markers (AP2 ERF 2, which is known to be involved in SA/JA cross talk, and JIH1 which suppresses the JA pathway). In contrast, RIN4, phenyl propanoid pathway markers (PAL, CHS and CAD) and ABA pathway markers (ABA1 and RD22) all had less than twofold changes in differential expression, which is considered to be the biological equivalent of no change in expression. Expression of PR1, β-1,3-glucosidase, PR6, JIH1 and AP2 ERF2 in stem tissue surrounding the inoculum site was most strongly induced by Pfm inoculation, whilst WRKY40 transcription factor was induced equally in stems by wounding (mock inoculation) and pathovar inoculation. PR1 is the most commonly used marker of the SA pathway (Pieterse et al., 2012). Using the same marker for PR1 as this study, Cellini et al. (2014) showed that PR1 and other SA-pathway markers, PR8 (chitinase) and isochorismate synthase, were more strongly up-regulated in ‘Hayward’ than in ‘Hort16A’ plants treated with the SA-analog, ASM, which correlated with a greater reduction of disease caused by Psa. A recent transcriptome study also showed that pre-treatment of kiwifruit with ASM, leads to a much greater proliferation of defense related sequences associated with the SA pathway upon inoculation with Psa, suggesting that the molecular response is strongly enhanced in ASM-treated plants (Michelotti et al., 2015).

β-1,3-glucosidase in the current study was also most strongly induced by Pfm inoculation. Glucosidases enable the plant to respond immediately to pathogen invasion by converting preformed inert phytoanticipins into their corresponding toxic aglycones by sugar hydrolysis (Osbourn, 1996; Zagrobelny et al., 2004; Morant et al., 2008). In addition to deglycosylation of inactive storage precursors formed via the phenyl propanoid pathway, β-1,3-glucosidases can hydrolyse conjugated plant hormones, thereby altering their bioactivity (Minic, 2008), and enzyme hydrolysis can also produce elicitor compounds. Glucosidases are also considered to be closely related to the PR2 family of enzymes (glucanases) which are often used as markers of the SA-pathway (Thatcher et al., 2005; Minic, 2008). Wurms et al. (2011) showed that β-1,3-glucosidase transcript expression reduced significantly over time (P < 0.001), in concert with increased ripe rot disease incidence caused by Cryptosporiopsis. Other studies have shown that hydrolysis of fruit and leaf phenolic extracts from ‘Hayward’ and ‘Hort16A’ greatly increases the microbial toxicity of these extracts (Wurms et al., 2003; Wurms, 2005). In addition, hydrolysed phenolics of ‘Hort16A’ were more fungitoxic than those from ‘Hayward,’ which correlated positively with greater resistance of ‘Hort16A’ than ‘Hayward’ to Botrytis cinerea and Sclerotinia sclerotiorum infections (Reglinski et al., 2002; Wurms et al., 2003; Wurms, 2005). The same β-1,3-glucosidase transcript that was analyzed in the current study has also been implicated in ‘Hort16A’ defense against scale insects (Hill et al., 2015). In support of these molecular and biochemical findings, a proteomic study of ‘Hayward’ leaf colonization by Psa showed elevated expression of two beta-1,3-glucosidase isoforms 3 days after inoculation compared with a water control (Petriccione et al., 2014).

The strongest induction of JIH (a negative marker of the JA pathway) by Pfm, followed by Psa, in the current study provides further weight to the hypothesis that defense against Pfm and Psa primarily involves the SA pathway. JIH1 degrades the active metabolite JA-Ile, with attenuation of the JA-Ile burst (Woldemariam et al., 2012), possibly allowing the plant to tailor the expression of SA-mediated responses.

Pfm was the strongest inducer of AP2 ERF2 (kiwifruit homolog of AtERF2), particularly in local stem tissue, which suggests some degree of cross talk between SA/JA. Some genes from the AP2/ERF family can be used as markers of ethylene/JA induction, and Van der Does et al. (2013) found that they have an important role to play in JA/SA cross talk. Their data showed that SA can inhibit JA signaling by targeting/negatively affecting GCC-box motifs on JA-responsive promoters of the AP2 ERF transcription factor family, inhibiting accumulation of some members but not others, for example ORA59, but not ERF1. As SA concentrations were higher with Pfm than Psa inoculation, the concentrations of the AP2 ERF2 marker in this study do not appear to be adversely affected by SA. Pré et al. (2008) observed that ERF1, but not AtERF2, was able to activate plant defensin expression (a JA defense response). Other evidence of more complex SA/JA cross-talk occurring in kiwifruit comes from the current study, where WRKY40, which is associated with the SA pathway (Xu et al., 2006; Schoen et al., 2013), was shown to be induced equally strongly by wounding and by Psa/Pfm inoculation, yet wounding is considered to be a JA pathway response (Vasyukova and Ozeretskovskaya, 2009; Wasternack and Song, 2017). In addition, activity of PR6 (a SA pathway marker) was highest with Pfm inoculation, but induction by wounding was almost as strong. Cellini et al. (2014) also found that lipoxygenase 2, a commonly used JA pathway marker (Garcia-Marcos et al., 2013; Wasternack and Hause, 2013), was expressed more highly in ‘Hayward’ than ‘Hort16A’ kiwifruit, which are more resistant to Psa, but expression levels were lower and more transient than that of the SA pathway markers, PR1 and ICS. It is highly unlikely that there is complete antagonism between the SA and JA pathways in response to Psa/Pfm infection in kiwifruit, given that numerous studies in Arabidopsis have shown that activated signaling pathways are not simply linear, but rather form complex networks where considerable cross talk takes place (Thatcher et al., 2005; Pieterse et al., 2012).

One caveat of transcription studies is that correlations between mRNA expression and induced disease resistance alone do not constitute proof of involvement in defense, as mRNA levels may not necessarily always correlate with changes in protein levels (Gygi et al., 1999; Chen et al., 2002). This is further complicated by factors such as post-translational modification which may result in more or less active versions of proteins, or versions that are more or less susceptible to proteolytic degradation. In addition, responses to stresses such as pathogens are likely to have multiple temporal profiles. Response time at the proteomic level is potentially much faster than the transcriptomic response as sometimes all it requires is a post-translational phosphorylation by an existing protein kinase to change protein activity. Bearing in mind these caveats, a proteomic analysis of the localized ‘Hayward’ leaf response to Psa colonization also showed that SA pathway associated PR proteins – PR1, PR2 (glucanases), glucosidases, PR3 (chitinases), PR4 (chitinases), PR5 (thaumatin like proteins), PR9 (peroxidases) and PR17 were the most up-regulated proteins (Petriccione et al., 2014, 2015a). A similar study in ‘Hort16A’ showed significant up-regulation of PR families 1, 2, 3, 4, 5 and 10 (Petriccione et al., 2013), hence proteomic analyses support the gene expression findings of the current study.

To our knowledge, this is the first report of localized versus systemic kiwifruit transcriptomic and phytohormone responses to closely related Pseudomonas syringae pathovars. It suggests that reduced severity of Pfm infection compared with Psa infection is due to a greater ability of kiwifruit to mount defense responses to Pfm. Gaining an understanding of the defense pathways and phytohormones involved in the defense response to Psa can help us to tailor elicitor choice and application to optimize their efficacy. Genes such as PR1 and β-1,3-glucosidase, which proved to be effective markers of resistant phenotype expression in the current study, may be used to screen breeding populations to select for more resistant phenotypes. These strategies are being used to develop an integrated fruit production system that is both more durable and more beneficial to the environment and consumers.

KW – designed experiments, set up trials, carried out all molecular work, including primer design and developing qPCR protocols, data analysis, wrote the manuscript. AH – carried out all molecular work and analysis including qPCR troubleshooting, editorial input into manuscript. AAC – set up trials, carried out disease assessments, molecular work including qPCR troubleshooting, editorial input into manuscript. JB – developed and modified protocols for successful RNA extraction thus overcoming a key technical hurdle, editorial input into manuscript. JP – molecular work including qPCR system optimisation, editorial input into manuscript. JT – set up trials, carried out disease assessments, data analysis, editorial input into manuscript. DJ – carried out all chemical protocols including trouble shooting, editorial input into manuscript. JC – developed phytohormone protocols and carried out all chemical work, editorial input into manuscript. TR – experimental design, input into chemical protocols, identification of most suitable phytohormone markers, data interpretation, major editorial input. MW – statistical analysis and editorial input.

Theauthors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.