Postharvest Application of Acibenzolar-S-Methyl Activates Salicylic Acid Pathway Genes in Kiwifruit Vines

The plant defence inducer Actigard® (acibenzolar-S-methyl [ASM]) is applied before flowering and after fruit harvest to control bacterial canker in kiwifruit caused by Pseudomonas syringae pv. actinidiae. Pre-flowering application of ASM is known to upregulate defence gene expression; however, the effect of postharvest ASM on defence gene expression in the vine is unknown. In this study, the expression of eight ”defence marker” genes was measured in the leaves of Actinidia chinensis var. chinensis, ”Zesy002,” and Actinidia chinensis var. deliciosa, “Hayward,” vines after postharvest treatment with ASM and/or copper. There were two orchards per cultivar with harvest dates approximately three weeks apart for investigating potential changes in responsiveness to ASM during the harvest period. In all trials, postharvest ASM induced the expression of salicylic-acid-pathway defence genes PR1, PR2, PR5, BAD, DMR6, NIMIN2, and WRKY70. Gene upregulation was the greatest at 1 day and 7 days after treatment and declined to the control level after 3 weeks. In “Zesy002”, the ASM-induced response was greater at the early harvest site than at the late harvest site. This decline was concomitant with leaf yellowing and a reduction in RNA yield. Effects of postharvest ASM on gene expression did not persist into the following spring, nor were vines conditioned to respond more strongly to pre-flowering ASM application.


Introduction
The global pandemic of bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa biovar3) seriously affected kiwifruit production worldwide, resulting in severe economic losses [1,2]. Orchard management of bacterial canker relies on the removal of infected canes to reduce inoculum and the application of copper-based products, antibiotics, resistance inducers, and biological control agents, which operate directly or indirectly against the pathogen [3,4]. In kiwifruit, defence against Psa infection is mediated primarily by the activation of the salicylic acid (SA) defence pathway [5]. Acibenzolar-S-methyl (ASM), a functional analogue of SA [6], is the active ingredient in Actigard ® (Syngenta, Wilmington, ED, USA), a commercial inducer that is used to control Psa in New Zealand kiwifruit orchards. Up to four foliar applications of Actigard (referred to as ASM from hereon) are permitted in New Zealand kiwifruit orchards per season, with sprays on fruiting kiwifruit vines being restricted to the pre-flowering and postharvest periods to limit the risk of chemical residues [7]. Pre-flowering application of ASM in kiwifruit orchards has been shown to induce transcription of SA-pathway defence genes and to reduce Psa leaf necrosis [8]. However, the effects of postharvest ASM application on defence gene expression in the canopy had not been studied. Understanding the vine response to the postharvest application is important because ASM is recommended at this time to protect fruit stalks and leaf scars from infection by the pathogen [9]. Moreover, the harvest period for kiwifruit spans several weeks, and it is not known whether changes in leaf physiology and metabolic activity over this period affect inducibility. This study measured the expression of SA-defence-pathway genes in the leaves of Actinidia chinensis var. chinensis, "Zesy002" and A. chinensis var. deliciosa, "Hayward" after postharvest application with ASM. Gene selection was based on previously published studies [3,8,10] and included transcripts for regulatory proteins (EDS1A, AP2ERF2, NIMIN2, and WRKY70), enzymes involved with hormone biosynthesis and homeostasis (BAD and DMR6), and pathogenesis-related proteins (PR1, PR2, and PR5). The phenomenon described as priming [11] was also studied by measuring the gene expression of young leaves in spring to investigate whether postharvest ASM directly affected gene expression in new leaves and/or conditioned vines for an amplified response to the pre-flowering ASM application.

Year 1 Orchard Trials
In 2019, the expression of defence marker genes in "Hayward" was greater in leaves from ASM + Cu-treated vines than in the Cu controls at both sites ( Table 1). The most strongly induced genes, DMR6, NIMIN2, and WRKY70i, ranged from 2-fold to 11-fold greater in ASM + Cu-treated vines than in the control. At site A (early harvest) the expression of PR1 and PR5 showed a 2-fold increase in ASM + Cu-treated vines compared with the Cu control, whereas at site B (late harvest) PR1, PR2, and PR5 increased by 4-to 5-fold at 6 d after ASM + Cu application. In the following spring, gene expression levels were not statistically different between treatments, indicating that effects of postharvest ASM + Cu on gene expression did not persist. Pre-flowering application of ASM + Cu at site A resulted in the upregulation of the same genes as with the postharvest spray. An accidental overspray of trial plots by contract sprayers at site B meant that no data were obtained. At the early harvest "Zesy002" orchard (site C), there was a 3-to 6-fold increase in the expression of DMR6, NIMIN2, and WRKY70 and a 25-fold increase in the expression of BAD at 1 day after ASM + Cu treatment, compared with the Cu control (Table 2). At 7 days post treatment, DMR6, NIMIN2, and WRKY70i were 5-to 6-fold greater in ASM + Cu-treated vines than in the Cu control, whilst PR1 and PR5 were 3-fold greater. By 19 days after treatment, there was no significant difference in the gene expression level (p > 0.05) between treatments. Following the application of a second postharvest ASM + Cu spray, there was a 10-fold increase in BAD and NIMIN2 expression and a 3-to 7-fold increase in DMR6 and WRKY70i expression after 1 day when compared with the Cu control. After 7 days, DMR6, NIMIN2, WRKY70i, and PR2 were 2-to 5-fold greater in the ASM + Cu-treated vines than in the Cu control. At the late-harvest "Zesy002" (site D), the expression of DMR6, BAD, NIMIN2, and WRKY70i increased by 3-to 11-fold at 1 day after ASM + Cu treatment, compared with the Cu control (Table 2). However, by 7 days post-treatment there was no significant (p > 0.05) difference between treatments. No further data were obtained from this site because of a low RNA yield in the leaves collected at 22 days after Spray 1, and at 1 day and 7 days after Spray 2. These leaves were showing signs of yellowing and senescence ( Figure 1). In "Hayward", severe frost damage after fruit harvest at site E (early harvest) resulted in the experiment at this site being discontinued. At site F (late harvest) the expression of BAD, DMR6, NIMIN2 and WRKY70 were upregulated in ASM + Cu-treated vines at 1 day and/or 7 days after treatment, compared with the control ( Table 2). By 19 days post-application there was no significant difference between treatments. A second postharvest spray was not applied at site F because of insufficient leaf canopy (<10% fill). Table 2. Heat map showing the expression of selected defence-related genes in Actinidia chinensis var. chinensis "Zesy002" vines and A. chinensis var. deliciosa "Hayward" after harvest in 2021. Vines were sprayed with Kocide ® Opti™ (Cu) or with a tank mix containing Kocide Opti and Actigard ® (acibenzolar-S-methyl (ASM) + Cu). Treatments were applied at site C on 29 April and 20 May, at site D on 17 May and 9 June, and at site F on 3 June. Data were log2 transformed for analysis. The numerical values indicate the fold-change relative to the copper control. Fold-changes of 2 or higher (or 0.5 or lower) were all statistically significant (p < 0.05).

Postharvest ASM Has No Direct Effect on Gene Expression in the following Spring
Gene expression levels in young leaves of "Hayward" in 2019 and 2021, and in "Zesy002" in 2021, did not differ statistically (p > 0.05) between those vines treated with ASM + Cu or Cu in the previous autumn (Table 3). This indicates no long-term effect of postharvest ASM on gene expression. Moreover, the postharvest treatment did not affect vine responsiveness to pre-flowering ASM treatment, i.e., no significant (p > 0.05) differences in gene upregulation were observed between the ASM-ASM and Cu-ASM treatments. Thus, postharvest ASM treatment did not prime vine inducibility in the following spring. Following pre-flowering sprays, all marker genes except AP2ERF2 were upregulated in the ASM+Cu-treated vines when compared with the Cu control. Gene upregulation tended to be greater in "Zesy002" than in "Hayward", and greater in "Zesy002" at site D than at site C (Table 3).

Postharvest ASM Has No Direct Effect on Gene Expression in the following Spring
Gene expression levels in young leaves of "Hayward" in 2019 and 2021, and in "Zesy002" in 2021, did not differ statistically (p > 0.05) between those vines treated with ASM + Cu or Cu in the previous autumn (Table 3). This indicates no long-term effect of postharvest ASM on gene expression. Moreover, the postharvest treatment did not affect vine responsiveness to pre-flowering ASM treatment, i.e., no significant (p > 0.05) differences in gene upregulation were observed between the ASM-ASM and Cu-ASM treatments. Thus, postharvest ASM treatment did not prime vine inducibility in the following spring. Following pre-flowering sprays, all marker genes except AP2ERF2 were upregulated in the ASM+Cu-treated vines when compared with the Cu control. Gene upregulation tended to be greater in "Zesy002" than in "Hayward", and greater in "Zesy002" at site D than at site C (Table 3). Table 3. Heat map showing the expression of selected defence-related genes in Actinidia chinensis var. chinensis "Zesy002" and A. chinensis var. deliciosa "Hayward" kiwifruit vines in spring 2021. Vines were sprayed with Kocide ® Opti™ (Cu) or with a tank mix containing Kocide Opti and Actigard ® (acibenzolar-S-methyl (ASM) + Cu). Pre-flowering treatments were applied at site C on 11 October, site D on 13 October, and site E on 3 November. Vines sprayed with Cu after harvest were sprayed with either Cu (Cu/Cu) or ASM + Cu (Cu/ASM + Cu) in spring. Similarly, vines sprayed with ASM + Cu after harvest were sprayed with either Cu (ASM + Cu/Cu) or ASM (ASM + Cu/ASM + Cu) in spring. Data were log2 transformed for analysis. The values indicate the foldchange relative to the copper control (Cu/Cu). Fold-changes of two or higher (or 0.5 or lower) were all statistically significant (p < 0.05).

Discussion
The application of ASM to kiwifruit vines after fruit harvest is recommended to protect fruit stalks and leaf scars from infection by Psa [9], despite poor understanding of defence induction during this period. This study demonstrates for the first time that postharvest ASM induces the upregulation of SA-defence-pathway genes in both "Zesy002" and "Hayward" vines. Vines were sprayed with a tank mix containing ASM + Cu; however, gene upregulation was calculated relative to the copper-treated controls. Hence, it is reasonable to attribute the upregulation to ASM. The most responsive genes, BAD, DMR6, NIMIN2, and WRKY70i, are associated with fine-tuning SA homeostasis (BAD and DMR6) [12,13] and regulating the transcription of pathogenesis-related (PR) proteins (NIMIN2 and WRKY70i) [10]. Specific gene expression patterns varied by orchard and by cultivar, but tended to be greatest at 1 day and 7 days after treatment and were no longer significantly different from control values after 3 weeks. This is consistent with the pattern of gene expression induced by ASM in "Zesy002" and "Hayward" kiwifruit vines during spring [8] and with the transient nature of inducible resistance reported in other plant species [14].
The timing of kiwifruit harvest in commercial orchards is determined by a range of fruit maturity parameters, including soluble solids content, firmness, and flesh colour [15]. The fruit harvest of "Zesy002" is generally several weeks earlier than harvest of "Hayward" vines within the same region [16]. During the postharvest period, an ever-increasing proportion of leaves in the leaf canopy begin to senesce, [17,18] and associated changes in leaf physiology and metabolic activity have the potential to negatively affect defence inducibility [19,20]. In "Hayward" vines in year 1 (2019), the extent of defence gene upregulation by postharvest ASM was similar at both the early-and late-harvest orchards. No comparison between "Hayward" orchards was made in year 2 because of frost damage at one site. However, in "Zesy002", the gene upregulation by ASM was greater at the earlyharvest site than at the late-harvest site, suggesting a decline in vine responsiveness to ASM over time. There was a 3 week gap between fruit harvest at these sites, and an observable difference in canopy quality was observed over this period, with yellowing of the leaves and an associated decline in RNA yield. Leaf yellowing is typically accompanied by a decline in metabolic activity [17,18], and this endorses the kiwifruit industry's recommendations that postharvest ASM should be applied only "if leaves are still green" [9].
There was no direct effect of postharvest ASM treatment on gene expression in "Hayward" and "Zesy002" vines in the spring; nor did the postharvest sprays prime vines for an amplified response to the pre-flowering ASM spray. In general, the most highly upregulated genes by ASM in spring, as at harvest, were BAD, DMR6, NIMIN2, and WRKY70i: the same genes which were the most highly upregulated in the autumn. The upregulation of PR1, PR2, and PR5 tended to occur later than that of the regulatory genes, in accordance with their relative position in the SA defence pathway. Differences in the amplitude of gene upregulation between the two "Zesy002" sites and between cultivars may be a function of leaf maturity because this has been shown to affect the response to pre-flowering ASM application [8].
In orchard experiments on Japanese pear (Pyrus pyrifolia var. culta), postharvest applications of ASM reduced scab lesion development caused by Venturia nashicola on the leaves and shoots of 'Niitaka' trees by over 80% when compared with controls [21]. Furthermore, in the following spring, pseudothecia formation was largely suppressed in fallen leaves from the ASM-treated trees, thus indicating the potential for the postharvest spray to reduce inoculum potential in spring. In the current study, no measurement of Psa leaf necrosis was recorded after harvest because of the difficulty in distinguishing Psa leaf necrosis from other leaf blemishes during this period. However, gene upregulation correlated with Psa control following pre-flowering application of ASM in orchard vines [8], and so gene expression was considered a suitable proxy for ASM efficacy. It is reasonable to propose that gene upregulation after harvest may precede the synthesis of antimicrobial defences that protect leaf scars from new Psa infections and/or suppress existing Psa populations in the vine. Nevertheless, definitive empirical evidence that postharvest ASM protects vines from Psa infection is still lacking.

Orchard Sites
Experiments were conducted in commercial orchards in the Waikato region of New Zealand on the two most commercially important kiwifruit cultivars in New Zealand: Actinidia chinensis var. chinensis "Zesy002" (marketed as Zespri™ SunGold™ Kiwifruit) and A. chinensis var. deliciosa "Hayward" (marketed as Zespri™ Green Kiwifruit). Two orchards per cultivar were selected, with 3-4 weeks separating their respective harvest dates (Table 4).

Treatment and Sampling
After fruit harvest, the vines were treated with Kocide ® Opti™ (Cu) or with a tank mix containing Kocide Opti and Actigard (ASM + Cu). Because the trials were conducted in commercial orchards, the inclusion of untreated controls was not permitted. Copper was applied at a concentration of 90 g/100 L after harvest and at 70 g/100 L for the preflowering application. ASM was applied at 20 g/100 L regardless of season. Treatments were applied at a spray rate of 1000 L/ha using a pressurised handgun. In 2019, there were five single-vine replicates per treatment. In 2021, ten single-vine plots were sprayed per treatment after fruit harvest to accommodate the priming study, in which five of the vines treated with postharvest ASM + Cu were treated with ASM + Cu in spring and five were treated with copper. Similarly, the postharvest copper control plots were treated with either copper or ASM + Cu in the spring (Table 5). At each timepoint, five leaves of similar phenology were collected within a 1.5 m radius of the main trunk of each replicate vine. Care was taken to select unblemished leaves to minimise potential effects of other biotic or abiotic stresses. Six discs (18 mm diameter) were excised from each leaf using a cork borer. They were then pooled by replicate in a plastic vial and snap frozen in liquid nitrogen. The samples were stored at −70 • C until RNA extraction. At orchard F, "Hayward" leaf samples were collected only up to day 19 after the postharvest spray. At this point approximately 10% of the leaf canopy remained, and so no further sprays were applied.

Gene Expression Analysis
Gene expression was determined using the Plexset ® platform from NanoString Technologies Inc. (Seattle, WA, USA), and results were analysed using the nSolver™ 4.0 software (Seattle, WA, USA). Four reference genes and eight target genes were used for the gene expression analysis (Table S1).
All the probes were synthesised by Integrated DNA Technologies Limited (IDT, Singapore). Total RNA was prepared from approximately 100 mg of ground kiwifruit tissue using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Auckland, New Zealand), following the supplier's recommendations. Sample purity and RNA concentrations were determined using a Nanophotometer ® (Implen, CA, USA). RNA samples were sent at −80 • C to the Grafton Clinical Genomics of the School of Medical Science, University of Auckland, for processing.

Statistical Analysis
Relative expression data were log 2 transformed for analysis. For each set of data, the relative expression for each gene was analysed using a linear model with factors for replicate, time, replicate × time, first treatment, second treatment, and the interactions between time and the two treatment factors. Where the effects were significant, the means were compared using least significant differences; comparisons were made within each time. Analysis was performed using Genstat, version 20 (VSNI Ltd., Hemel Hempstead, UK, 2020).

Conclusions
This study confirms that kiwifruit vines are responsive to ASM after fruit harvest. However, there is no evidence that the effect on gene expression persists into the following spring. The amplitude and duration of the postharvest response to ASM depended on the leaf quality at the time of application. Biologically, this makes sense, because defence activation demands actively metabolising, non-senescing tissue; thus, vines will become less responsive to inducers as canopy health declines. Moreover, there is increasing evidence that defence-related phytohormones (SA, jasmonic acid, and ethylene) play differential roles in regulating leaf senescence [22]. Therefore, more detailed investigation of the crossregulatory mechanisms between plant defence and leaf senescence are warranted. Further studies are also required to establish if gene upregulation following postharvest application of ASM correlates with a greater resistance to Psa infection during the postharvest period, and whether this affects Psa symptom expression in the following spring. This remains a significant knowledge gap.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12040833/s1, Table S1. DNA sequence of the capture and reporter probes used to analyse kiwifruit gene expression by PlexSet ® Nanostring.