Abstract
Pseudomonas syringae pv. actinidiae (Psa) has caused severe damage to kiwifruit plantations worldwide. It is still crucial to find compounds that are able to control the disease. In this work we developed a time and space-saving leaf disk assay using highly susceptible yellow-fleshed cultivars. The assay enabled 29 molecules/compounds to be screened in relation to their capacity to condition the development of necrosis on leaf disks by treating them before the inoculation. A necrosis index was thereby created. Fosetyl-aluminum, DL-β-Amino-n-butyric acid (BABA) and saccharin (BIT) reduced symptoms significantly, and Fosetyl-aluminum was the most effective. Maxim®, triclopyr and Methyl jasmonate enhanced the necrosis index significantly. S-Methyl 1,2,3-benzothiadiazole-7-carbothioate (BTH) also enhanced symptom severity, though there was no statistical support confirming this. Salicylic acid did not condition symptom development. In vitro investigations into the direct effect of the compounds on Psa highlighted that Fosetyl-aluminum and the polyamines, spermidine and cadaverine, inhibited growth and also had a biocidal effect in a dose-dependent fashion. Salicylic acid and spermine also strongly reduced bacterial growth. Long-term trials with potted plants in the greenhouse confirmed the protective effect of Fosetyl-aluminum and clearly showed that BTH enhances the susceptibility of kiwifruit to Psa. Fosetyl-aluminum significantly decreased symptom expression in kiwifruit plantations (cv. Hayward) subjected to natural Psa infection. Monitoring Psa growth dynamics in the leaf disks by time-point-based Real-Time PCR, showed that treatment with Fosetyl-aluminum restricts bacterial multiplication within the host plant.
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Acknowledgements
This research was supported by: i) a Mipaaf (the Italian Ministry of Agricultural and Forestry Policies) project, “INTERACT/ARDICA, Interventi di coordinamento ed implementazione alle azioni di ricerca, lotta e difesa al cancro batterico dell’Actinidia”; ii) a Latium Region PSR (Programma di Sviluppo Rurale) project, “PRO.ACTI.BA.RE, Protezione dell’actinidia dal cancro batterico (Pseudomonas syringae pv. actinidiae) mediante l’uso di induttori di resistenza”.
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Online Resource 1
Details on the preparation of compounds to be tested for the anti-bacterial activity and for their effect on the infection of Pseudomonas syringae pv. actinidiae (Psa) in the leaf disk assays (PDF 110 kb)
Online Resource 2
Symptom scoring for the inference of the disease severity index of the greenhouse trials (McKinney 1923) and data on the outcome of the trials. A. Classes of symptoms observed on potted plants treated with Fosetyl-aluminum, BTH and water and inoculated with Pseudomonas syringae pv. actinidiae (Psa), and the attributed scores. The score derived from the combination of different classes was obtained from the sum of the values of each class. Class combinations including F are valued as F. B. Score assigned to each plant within each treatment and trial (A, B1 and B2) based on symptom class ranking. (PDF 68 kb)
Online Resource 3
Optical density (OD) of Pseudomonas syringae pv. actinidiae (Psa) after growing in NSB supplemented with compounds/molecules, which were also used in the leaf disk assay. All measurements were made with a spectrophotometer at 660 nm after 20–22 h incubation, when non- compound-supplemented Psa cultures (called Control 1, C1) reached approximately the value of 1.0. A: OD measurements for each compound/molecule at different concentration. The comparison is with C1. Control 2 (C2) is the absorbance of non-inoculated NSB. Different letters indicate significant differences between groups (p < 0.01). The vertical bars indicate standard deviations of the means. Asterisks indicate the bactericidal concentrations. B: compounds/molecules and concentrations that caused a growth reduction ≥40% compared with C1. Differences are expressed as growth percentage compared with C1 (= 100%) and as percentage growth reduction (PDF 161 kb)
Online Resource 4
Example of non-inoculated leaf disks used as the control in order to assess any wound, time and compound-related senescence/phytotoxicity. The figure includes the non-treated leaf disks (NT) and leaf disks treated twice with BTH, following the protocol assay. Ten leaf disks were treated with BTH suspensions at different concentrations, and the experiment was repeated three times. We show close-ups of five disks for each concentration. The image was taken 20 days after BTH treatments. Belen was used as the source of the leaf disks (PDF 268 kb)
Online Resource 5
Confirmation of the outcome of the leaf disk assay on Actinidia chinensis var. deliciosa cv. Hayward: significant decrease in the necrosis index of leaf disks treated with Fosetyl-aluminum and BABA and subsequently inoculated with Pseudomonas syringae pv. actinidiae (Psa). The comparison is with the control non-treated and inoculated with Psa (NT-Psa). Necrosis indexes marked by different letters are significantly different from each other [p < 0.01 (Fosetyl-aluminum), p < 0.05 (BABA)]. The numbers indicate the independent statistical analyses conducted separately on data from two temporally-distinct recordings: the first in grey, the second black. The vertical bars indicate the standard error of the means (PDF 34 kb)
Online Resource 6
The IAE response (Increase and Anticipation of symptom Expression) obtained by treating the leaf disks with triclopyr and MeJA at various concentrations and then inoculating with Pseudomonas syringae pv. actinidiae (Psa). NT-Psa = non-treated and inoculated with Psa; NT = non-treated and non-inoculated (PDF 318 kb)
Online Resource 7
Examples of compounds/molecules whose treatment on leaf disks (cv. Belen) did not influence the outcome of Pseudomonas syringae pv. actinidiae (Psa) inoculation in terms of the necrosis index. The comparison is with the control non-treated and inoculated with Psa (NT-Psa). Necrosis index values marked by different letters are significantly different from each other (p < 0.05). The numbers indicate the independent statistical analyses conducted separately on data from two temporally-distinct recordings: the first in grey, the second black. The vertical bars indicate the standard error of the means (PDF 37.4 kb)
Online Resource 8
Compounds/molecules whose treatment on leaf disk (cv. Belen) did not statistically differ from the control non-treated and inoculated with Pseudomonas syringae pv. actinidiae (Psa) (NT-Psa) in terms of necrosis index, similarly to those depicted in Online resource 7. Unlike the effects reported in Online resource 7, the mean values of the necrosis index in this figure were repeatedly and evidently lower or higher than the NT-Psa in all the experiments. This prompts a hypothesis on a fine tuning by these compounds in the direction of resistance/susceptibility induction. Necrosis indexes marked by the different letters are significantly different from each other (p < 0.05). The numbers indicate the independent statistical analyses conducted separately on data from two temporally-distinct recordings: the first in grey, the second black. The vertical bars indicate the standard error of the means. Concentrations of BTH expressed in mM and g/l refer to the active principle and the commercial product (Bion) respectively (PDF 34.3 kb)
Online Resource 9
Healthy-looking Fosetyl-aluminum-treated/Psa-inoculated plants (a) compared with BTH-treated/Psa-inoculated plants (b), which were dead or severely stunted (B2 trial, during spring/summer 2016) (PDF 175 kb)
Online Resource 10
Rainfall (gray line) and average (green), maximum (red) and minimum (azure) temperature during the field trial. The trial was conducted from 9 April to 8 July (PDF 91 kb)
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Brunetti, A., Pucci, N., Modesti, V. et al. In vitro and in planta screening of compounds for the control of Pseudomonas syringae pv. actinidiae in Actinidia chinensis var. chinensis. Eur J Plant Pathol 158, 829–848 (2020). https://doi.org/10.1007/s10658-020-02119-1
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DOI: https://doi.org/10.1007/s10658-020-02119-1