Bacterial Canker Of Kiwifruit Research Articles

Synthesis of organic-inorganic hybrid nanocomposites modified by catalase-like catalytic sites for the controlling of kiwifruit bacterial canker.

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. Actinidiae (Psa), is one of the most important diseases in kiwifruit, creating huge economic losses to kiwifruit-growing countries around the world. Metal-based nanomaterials offer a promising alternative strategy to combat plant diseases induced by bacterial infection. However, it is still challenging to design highly active nanomaterials for controlling kiwifruit bacterial canker. Here, a novel multifunctional nanocomposite (ZnO@PDA-Mn) is designed that integrates the antibacterial activity of zinc oxide nanoparticles (ZnO NPs) with the plant reactive oxygen species scavenging ability of catalase (CAT) enzyme-like active sites through introducing manganese modified polydopamine (PDA) coating. The results reveal that ZnO@PDA-Mn nanocomposites can efficiently catalyze the conversion of H2O2 to O2 and H2O to achieve excellent CAT-like activity. In vitro experiments demonstrate that ZnO@PDA-Mn nanocomposites maintain the antibacterial activity of ZnO NPs and induce significant damage to bacterial cell membranes. Importantly, ZnO@PDA-Mn nanocomposites display outstanding curative and protective efficiencies of 47.7% and 53.8% at a dose of 200 μg mL-1 against Psa in vivo, which are superior to those of zinc thiozole (20.6% and 8.8%) and ZnO (38.7% and 33.8%). The nanocomposites offer improved in vivo control efficacy through direct bactericidal effects and decreasing oxidative damage in plants induced by bacterial infection. Our research underscores the potential of nanocomposites containing CAT-like active sites in plant protection, offering a promising strategy for sustainable disease management in agriculture.

Comparative genomic analyses provide insight into the pathogenicity of three Pseudomonas syringae pv. actinidiae strains from Anhui Province, China

BackgroundPseudomonas syringae pv. actinidiae (Psa) is an important bacterial plant pathogen that causes severe damage to the kiwifruit industry worldwide. Three Psa strains were recently obtained from different kiwifruit orchards in Anhui Province, China. The present study mainly focused on the variations in virulence and genome characteristics of these strains based on the pathogenicity assays and comparative genomic analyses.ResultsThree strains were identified as biovar 3 (Psa3), along with strain QSY6 showing higher virulence than JZY2 and YXH1 in pathogenicity assays. The whole genome assembly revealed that each of the three strains had a circular chromosome and a complete plasmid. The chromosome sizes ranged from 6.5 to 6.6 Mb with a GC content of approximately 58.39 to 58.46%, and a predicted number of protein-coding sequences ranging from 5,884 to 6,019. The three strains clustered tightly with 8 Psa3 reference strains in terms of average nucleotide identity (ANI), whole-genome-based phylogenetic analysis, and pangenome analysis, while they were evolutionarily distinct from other biovars (Psa1 and Psa5). Variations were observed in the repertoire of effectors of the type III secretion system among all 15 strains. Moreover, synteny analysis of the three sequenced strains revealed eight genomic regions containing 308 genes exclusively present in the highly virulent strain QSY6. Further investigation of these genes showed that 16 virulence-related genes highlight several key factors, such as effector delivery systems (type III secretion systems) and adherence (type IV pilus), which might be crucial for the virulence of QSY6.ConclusionThree Psa strains were identified and showed variant virulence in kiwifruit plant. Complete genome sequences and comparative genomic analyses further provided a theoretical basis for the potential pathogenic factors responsible for kiwifruit bacterial canker.

Evaluation of cold plasma activated water composite reagent on kiwifruit plants with bacterial canker and the external qualities of kiwifruit after spray treatment

This study aimed to investigate the efficacy of cold plasma activated water (PAW) and its complex solution (PKC) in conjunction with kasugamycin copper oxychloride wettable powder (KC) against kiwifruit bacterial canker (KBC) caused by Pseudomonas syringae pv. actinidiae (PSA). Through morphological identification and 16S rDNA sequence analysis, Pantoea agglomerans was identified as the pathogen responsible for KBC in the 'Bairui' orchard. Results indicated that PKC exhibited the highest effectiveness in controlling KBC, particularly on mildly (II) infested plants, with a remarkable reduction of 63.64 % in PKC II disease index and a control efficiency of 38.64 %. Additionally, the application of PKC significantly increased fruit yield, improved fruit weight, and enhanced fruit morphology, resulting in a more uniform and refined fruit shape. Analysis of fruit skin conformational parameters revealed that KBC-affected fruit exhibited reduced hardness and brittleness, increased toughness, and thinner skin. These findings are pivotal for developing effective strategies to further prevent and manage KBC outbreaks. In future studies, further investigations could focus on elucidating the mechanism of action of PAW in complex solution with copper-based pesticides and the potential mechanism of inactivation of PSA.

E3 Ubiquitin Ligase PUB23 in Kiwifruit Interacts with Trihelix Transcription Factor GT1 and Negatively Regulates Immune Responses against Pseudomonas syringae pv. actinidiae.

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) is the most serious disease threatening kiwifruit production. Our previous study found genes encoding the U-box containing proteins were significantly regulated by Psa infection. Here, we report a U-box type E3 ubiquitin ligase PUB23 in kiwifruit which acts as a negative regulator of immune responses against Psa. PUB23 was found to physically interact with GT1, a trihelix transcription factor, in vitro and in vivo. The expression of GT1 was up-regulated in PUB23-silenced plants, indicating that interacting with PUB23 may directly or indirectly suppress GT1 expression. The silencing of PUB23 led to enhanced immune responses of PAMP-triggered immunity (PTI), including a higher expression level of defense marker genes PR1 and RIN4, and increased accumulation of hydrogen peroxide and superoxide anion. Our results reveal a negative role PUB23 plays in kiwifruit immune responses against Psa and may regulate gene expression by interacting with GT1.

Streptothricin-F Inhibition of FtsZ Function: A Promising Approach for Controlling Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is a significant pathogenic bacterium affecting the kiwifruit industry. This study investigated the target sites of streptothricin-F (ST-F), produced by Streptomyces lavendulae gCLA4. The inhibition of ST-F on Psa was examined by the microscopic structural differences of Psa before and after treatment with ST-F, as well as the interaction between ST-F and cell division-related proteins. The results revealed filamentation of Psa after ST-F treatment, and fluorescence microscopy showed that ST-F inhibited the formation of the Z-ring composed of FtsZ protein. In vitro experiments and molecular docking demonstrated that ST-F can bind to FtsZ with a binding energy of 0.4 μM and inhibit FtsZ's GTP-dependent polymerization reaction. In addition, ST-F does not exert inhibitory effects on cell division in Psa strains overexpressing ftsZ. In conclusion, FtsZ is one of the target sites for ST-F inhibition of Psa, highlighting its potential as a therapeutic target for controlling Psa-induced kiwifruit bacterial canker.

Scientific and technological advances in the development of sustainable disease management tools: a case study on kiwifruit bacterial canker.

Plant disease outbreaks are increasing in a world facing climate change and globalized markets, representing a serious threat to food security. Kiwifruit Bacterial Canker (KBC), caused by the bacterium Pseudomonas syringae pv. actinidiae (Psa), was selected as a case study for being an example of a pandemic disease that severely impacted crop production, leading to huge economic losses, and for the effort that has been made to control this disease. This review provides an in-depth and critical analysis on the scientific progress made for developing alternative tools for sustainable KBC management. Their status in terms of technological maturity is discussed and a set of opportunities and threats are also presented. The gradual replacement of susceptible kiwifruit cultivars, with more tolerant ones, significantly reduced KBC incidence and was a major milestone for Psa containment - which highlights the importance of plant breeding. Nonetheless, this is a very laborious process. Moreover, the potential threat of Psa evolving to more virulent biovars, or resistant lineages to existing control methods, strengthens the need of keep on exploring effective and more environmentally friendly tools for KBC management. Currently, plant elicitors and beneficial fungi and bacteria are already being used in the field with some degree of success. Precision agriculture technologies, for improving early disease detection and preventing pathogen dispersal, are also being developed and optimized. These include hyperspectral technologies and forecast models for Psa risk assessment, with the latter being slightly more advanced in terms of technological maturity. Additionally, plant protection products based on innovative formulations with molecules with antibacterial activity against Psa (e.g., essential oils, phages and antimicrobial peptides) have been validated primarily in laboratory trials and with few compounds already reaching field application. The lessons learned with this pandemic disease, and the acquired scientific and technological knowledge, can be of importance for sustainably managing other plant diseases and handling future pandemic outbreaks.

Genome-wide identification and expression patterns of the laccase gene family in response to kiwifruit bacterial canker infection

BackgroundKiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a destructive disease worldwide. Resistance genes that respond to Psa infection urgently need to be identified for controlling this disease. Laccase is mainly involved in the synthesis of lignin in the plant cell wall and plays a prominent role in plant growth and resistance to pathogen infection. However, the role of laccase in kiwifruit has not been reported, and whether laccase is pivotal in the response to Psa infection remains unclear.ResultsWe conducted a bioinformatics analysis to identify 55 laccase genes (AcLAC1–AcLAC55) in the kiwifruit genome. These genes were classified into five cluster groups (I–V) based on phylogenetic analysis, with cluster groups I and II having the highest number of members. Analysis of the exon–intron structure revealed that the number of exons varied from 1 to 8, with an average of 5 introns. Our evolutionary analysis indicated that fragment duplication played a key role in the expansion of kiwifruit laccase genes. Furthermore, evolutionary pressure analysis suggested that AcLAC genes were under purifying selection. We also performed a cis-acting element analysis and found that AcLAC genes contained multiple hormone (337) and stress signal (36) elements in their promoter regions. Additionally, we investigated the expression pattern of laccase genes in kiwifruit stems and leaves infected with Psa. Our findings revealed that laccase gene expression levels in the stems were higher than those in the leaves 5 days after inoculation with Psa. Notably, AcLAC2, AcLAC4, AcLAC17, AcLAC18, AcLAC26, and AcLAC42 showed significantly higher expression levels (p < 0.001) compared to the non-inoculated control (0 d), suggesting their potential role in resisting Psa infection. Moreover, our prediction indicated that 21 kiwifruit laccase genes are regulated by miRNA397, they could potentially act as negative regulators of lignin biosynthesis.ConclusionsThese results are valuable for further analysis of the resistance function and molecular mechanism of laccases in kiwifruit.

Two transcription factors, AcREM14 and AcC3H1, enhance the resistance of kiwifruit Actinidiachinensis var. chinensis to Pseudomonas syringae pv. actinidiae.

Kiwifruit bacterial canker is a global disease caused by Pseudomonas syringae pv. actinidiae (Psa), which poses a major threat to kiwifruit production worldwide. Despite the economic importance of Actinidia chinensis var. chinensis, only a few resistant varieties have been identified to date. In this study, we screened 44 kiwifruit F1 hybrid lines derived from a cross between two A. chinensis var. chinensis lines and identified two offspring with distinct resistance to Psa: resistant offspring RH12 and susceptible offspring SH14. To identify traits associated with resistance, we performed a comparative transcriptomic analysis of these two lines. We identified several highly differentially expressed genes (DEGs) associated with flavonoid synthesis, pathogen interactions, and hormone signaling pathways, which play essential roles in disease resistance. Additionally, using weighted gene co-expression network analysis, we identified six core transcription factors. Moreover, qRT-PCR results demonstrated the high expression of AcC3H1 and AcREM14 in Psa-induced highly resistant hybrid lines. Ultimately, Overexpression of AcC3H1 and AcREM14 in kiwifruit enhanced disease resistance, and this was associated with upregulation of enzymatic activity and gene expression in the salicylic acid (SA) signaling pathway. Our study elucidates a molecular mechanism underlying disease resistance in kiwifruit and contributes to the advancement of research on kiwifruit breeding.

Adapting the inoculation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from branch microbiome.

Pseudomonas syringae pv. actinidiae (Psa), the bacterium that causes kiwifruit bacterial canker, is a common field occurrence that is difficult to control globally. Currently, exploring the resources for efficient biocontrol bacteria is a hot spot in the field. The common strategy for isolating biocontrol bacteria is to directly isolate biocontrol bacteria that can secrete diffusible antibacterial substances, most of which are members of Bacillus, Pseudomonas and Streptomycetaceae,from disease samples or soil. Here, we report a new approach by adapting the typical isolation methods of kiwifruit canker disease to identify efficient biocontrol bacteria from the branch microbiome. Using this unique approach, we isolated a group of kiwifruit biocontrol agents (KBAs) from the branch microbiome of Psa-resistant varieties. Thirteen of these showed no antagonistic activity invitro, which depends on the secretion of antibacterial compounds. However, they exhibited antibacterial activity via cell-to-cell contacts mimicked by co-culture on agar plates. Through biocontrol tests on plants, two isolates, KBA13 and KBA19, demonstrated their effectiveness by protecting kiwifruit branches from Psa infection. Using KBA19, identified as Pantoea endophytica, as a representative, we found that this bacterium uses the type VI secretion system (T6SS) as the main contact-dependent antibacterial weapon that acts via translocating toxic effector proteins into Psa cells to induce cell death, and that this capacity expressed by KBA19 is common to various Psa strains from different countries. Our findings highlight a new strategy to identify efficient biocontrol agents that use the T6SS to function in an antibacterial metabolite-independent manner to control wood diseases.

Identification and genetic characterization of Pseudomonas syringae pv. actinidiae from kiwifruit in Sichuan, China.

Pseudomonas syringae pv. actinidiae (Psa) causes kiwifruit bacterial canker and poses a major threat to the kiwifruit industry. This study aimed to investigate the genetic characteristics of the Psa population from kiwifruit in Sichuan, China. Sixty-seven isolates obtained from diseased plants were characterized using morphological features, multiplex-PCR and multilocus sequence analysis (MLSA). The isolates exhibited the typical colony morphology of Psa. Multiplex PCR amplification identified every isolate as Psa biovar 3. MLSA analysis of the three housekeeping genes gapA, gyrB, and pfk, revealed that the reference strains of the five described biovars were clearly distinguished by a combined phylogenetic tree, and all the tested isolates clustered with the reference strains of Psa biovar 3. Through a phylogenetic tree constructed from a single gene, it was found that pkf gene alone could distinguish biovar 3 from the other biovars. Furthermore, all Psa isolates analyzed by BOX-A1R-based repetitive extragenic palindromic (BOX)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR clustered into four groups. The clustering results of BOX- and ERIC-PCR indicated that group III had the largest number of isolates, accounting for 56.72% and 61.19% of all sixty-seven isolates, respectively, and the two characterization methods were similar and complementary. The results of this study revealed that the genomes of Psa isolates from Sichuan had rich genetic diversity, but no obvious correlation was found between clustering and geographical region. This research provides novel methodologies for rapidly detecting kiwifruit bacterial canker pathogen and a molecular differentiation at genetic level of Psa biovars diversity in China.

Satureja montana and Mentha pulegium essential oils’ antimicrobial properties against Pseudomonas syringae pv. actinidiae and elicitor potential through the modulation of kiwifruit hormonal defenses

Pseudomonas syringae pv. actinidiae (Psa) is responsible for the kiwifruit bacterial canker, the most severe disease of Actinidia spp. The use in agriculture of antibiotics and cooper-based compounds is increasingly being restricted, demanding for new sustainable alternatives to current agrochemicals. We aimed to characterize the anti-Psa potential of essential oils (EOs) of Mentha pulegium and Satureja montana and investigate if they elicit the plant-host hormonal defenses. The EOs were characterized through gas-chromatography with flame ionization detector (GC-FID) and mass spectrometry (MS). Pulegone (78.6%) and carvacrol (43.5%) were the major constituents of M. pulegium and S. montana EO, respectively. Only S. montana EO showed relevant anti-Psa activity in vitro. To evaluate if the EOs also elicited host defenses, in vitro shoots were treated with 2 mg shoot−1 of EO-solution and subsequently inoculated with Psa three days later. Shoots were analyzed 10 min, three days (and 10 min after Psa-inoculation), four and ten days after EO application. The up/down regulation of RNA-transcripts for hormone biosynthesis, Psa biofilm production and virulence genes were quantified by real-time quantitative PCR (RT-qPCR). Phytohormones were quantified by High-Performance Liquid Chromatography (HPLC). S. montana EO showed the most promising results as a defense elicitor, increasing 6-benzylaminopurine (BAP) by 131.07% and reducing indole-3-acetic acid (IAA) levels by 49.19%. Decreases of salicylic acid (SA), and gibberellic acid 3 (GA3) levels by 32.55% and 33.09% respectively and an increase of abscisic acid (ABA) by 85.03%, in M. pulegium EO-treated shoots, revealed some protective post-infection effect. This is the most comprehensive research on the Psa’s impact on phytohormones. It also unveils the protective influence of prior EO exposure, clarifying the plant hormonal response to subsequent infections. The results reinforce the hypothesis that carvacrol-rich S. montana EO can be a suitable disease control agent against Psa infection. Its dual action against pathogens and elicitation of host plant defenses make it a promising candidate for incorporation into environmentally friendly disease management approaches. Nonetheless, to fully leverage these promising results, further research is imperative to elucidate the EO mode of action and evaluate the long-term efficacy of this approach.

Paenibacillus polymyxa YLC1: A promising antagonistic strain for biocontrol of Pseudomonas syringae pv. actinidiae, causing kiwifruit bacterial canker.

Kiwifruit bacterial canker (KBC) caused by Pseudomonas syringae pv. actinidiae (Psa) is the main limiting factor in the kiwifruit industry. This study aimed to identify bacterial strains with antagonistic activity against Psa, analyze antagonistically active substances, and provide a new basis for the biological control of KBC. A total of 142 microorganisms were isolated from the rhizosphere soil of asymptomatic kiwifruit. Among them, an antagonistic bacterial strain was identified as Paenibacillus polymyxa YLC1 by 16S rRNA sequencing. KBC control by strain YLC1 (85.4%) was comparable to copper hydroxide treatment (81.8%) under laboratory conditions and field testing. Active substances of strain YLC1 were identified by genetic sequence analysis using antiSMASH. The six biosynthetic active compound gene clusters that were identified as encoding ester peptide synthesis, such as polymyxins. An active fraction was purified and identified as polymyxin B1 using chromatography, hydrogen nuclear magnetic resonance (NMR), and liquid chromatography-mass spectrometry. In addition, polymyxin B1 was also found significantly to suppressed the expression of T3SS-related genes but did not affect the growth of Psa at low concentrations. In this study, a biocontrol strain P. polymyxa YLC1 obtained from kiwifruit rhizosphere soil exhibited excellent control effects on KBC in vitro and in field tests. Its active compound was identified as polymyxin B1, which inhibits a variety of pathogenic bacteria. We conclude that P. polymyxa YLC1 is a biocontrol strain with excellent prospects for development and application. This article is protected by copyright. All rights reserved.

Recombinase polymerase amplification-lateral flow (RPA-LF) assay for rapid visual detection of Pseudomonas syringae pv. actinidiae in kiwifruit

Efficient detection of the causal pathogen Pseudomonas syringae pv. actinidiae (Psa) is vital to prevent and control Kiwifruit Bacterial Canker. In this present study, a novel isothermal amplification assay was developed for detecting Psa. The assay was highly specific for Psa, and could be completed in two steps within 35 min. Firstly, a set of RPA specific primers, which was designed based on the conserved region of avrD1 gene of Psa, was used to amplify a specific fragment of the avrD1 gene of Psa in a 30 min recombinase polymerase amplification (RPA) reaction. Secondly, lateral flow (LF) dipstick was used to detect and visualize the RPA amplicons of Psa within 5 min. An optimized detection system for Psa was established, with probe concentration of 0.10 μM, primer concentration of 0.42 μM, incubation time of 30 min and 5 min, and reaction temperature of 27–39 °C. The optimized RPA-LF method displayed high sensitivity to Psa, with 103 times higher sensitivity than that of conventional PCR, and could detect as low as 7.61 × 10−6 μg/μL genomic DNA of Psa in a 12.5 μL reaction system. Results in this study indicated that this rapid, specific, and sensitive RPA-LF assay has potential application prospects in detecting and monitoring of Kiwifruit Bacterial Canker, especially under limited time and resources.

Discovery and structure-activity relationship studies of novel tetrahydro-β-carboline derivatives as apoptosis initiators for treating bacterial infections

Developing and excavating new agrochemicals with highly active and safe is an important tactic for protecting crop health and food safety. In this paper, to discover the new bactericide candidates, we designed, prepared a new type of 1,2,3,4-tetrahydro-β-carboline (THC) derivatives and evaluated the in vitro and in vivo bioactivities against the Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac), and Pseudomonas syringae pv. actinidiae (Psa). The in vitro bioassay results exhibited that most title molecules possessed good activity toward the three plant pathogenic bacteria, the compound A17 showed the most active against Xoo and Xac with EC50 values of 7.27 and 4.89 mg mL–1 respectively, and compound A8 exhibited the best inhibitory activity against Psa with EC50 value of 4.87 mg mL–1. Pot experiments showed that compound A17 exhibited excellent in vivo antibacterial activities to manage rice bacterial leaf blight and citrus bacterial canker, with protective efficiencies of 52.67 and 79.79% at 200 mg mL–1, respectively. Meanwhile, compound A8 showed good control efficiency (84.31%) against kiwifruit bacterial canker at 200 mg mL–1. Antibacterial mechanism suggested that these compounds could interfere with the balance of the redox system, damage the cell membrane, and induce the apoptosis of Xoo cells. Taken together, our study revealed that tetrahydro-β-carboline derivatives could be a promising candidate model for novel broad-spectrum bactericides.

Design, Synthesis and Bioactivity Evaluation of Novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles Containing an Imidazole Fragment as Antibacterial Agents.

Imidazole alkaloids, a common class of five-membered aromatic heterocyclic compounds, exist widely in plants, animals and marine organisms. Because of imidazole's extensive and excellent biological and pharmacological activities, it has always been a topic of major interest for researchers and has been widely used as an active moiety in search of bioactive molecules. To find more efficient antibacterial compounds, a series of novel imidazole-fragment-decorated 2-(pyrazol-4-yl)-1,3,4-oxadiazoles were designed and synthesized based on our previous works via the active substructure splicing principle, and their bioactivities were systematically evaluated both in vitro and in vivo. The bioassays showed that some of the target compounds displayed excellent in vitro antibacterial activity toward three virulent phytopathogenic bacteria, including Xanthomonas oryzae pv. oryzae (Xoo), Xanthomonas axonopodis pv. citri (Xac) and Pseudomonas syringae pv. actinidiae (Psa), affording the lowest EC50 values of 7.40 (7c), 5.44 (9a) and 12.85 (9a) μg/mL, respectively. Meanwhile, compound 7c possessed good in vivo protective and curative activities to manage rice bacterial leaf blight at 200 μg/mL, with control efficacies of 47.34% and 41.18%, respectively. Furthermore, compound 9a showed commendable in vivo protective and curative activities to manage kiwifruit bacterial canker at 200 μg/mL, with control efficacies of 46.05% and 32.89%, respectively, which were much better than those of the commercial bactericide TC (31.58% and 17.11%, respectively). In addition, the antibacterial mechanism suggested that these new types of title compounds could negatively impact the cell membranes of phytopathogenic bacteria cells and cause the leakage of the intracellular component, thereby leading to the killing of bacteria. All these findings confirm that novel 2-(pyrazol-4-yl)-1,3,4-oxadiazoles containing an imidazole fragment are promising lead compounds for discovering new bactericidal agents.

DCas9-BE3 and dCas12a-BE3 Systems Mediated Base Editing in Kiwifruit Canker Causal Agent Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker of kiwifruit with heavy economic losses. However, little is known about the pathogenic genes of Psa. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas-mediated genome editing technology has dramatically facilitated the characterization of gene function in various organisms. However, CRISPR genome editing could not be efficiently employed in Psa due to lacking homologous recombination repair. The base editor (BE) system, which depends on CRISPR/Cas, directly induces single nucleoside C to T without homology recombination repair. Here, we used dCas9-BE3 and dCas12a-BE3 systems to create substitutions of C to T and to convert CAG/CAA/CGA codons to stop codons (TAG/TAA/TGA) in Psa. The dCas9-BE3 system-induced single C-to-T conversion frequency of 3 to 10 base positions ranged from 0% to 100%, with a mean of 77%. The dCas12a-BE3 system-induced single C-to-T conversion frequency of 8 to 14 base positions in the spacer region ranged from 0% to 100%, with a mean of 76%. In addition, a relatively saturated Psa gene knockout system covering more than 95% of genes was developed based on dCas9-BE3 and dCas12a-BE3, which could knock out two or three genes at the same time in the Psa genome. We also found that hopF2 and hopAO2 were involved in the Psa virulence of kiwifruit. The HopF2 effector can potentially interact with proteins such as RIN, MKK5, and BAK1, while the HopAO2 effector can potentially interact with the EFR protein to reduce the host's immune response. In conclusion, for the first time, we established a PSA.AH.01 gene knockout library that may promote research on elucidating the gene function and pathogenesis of Psa.

Contribution of Sucrose Metabolism in Phloem to Kiwifruit Bacterial Canker Resistance.

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (Psa), is a catastrophic disease affecting kiwifruit worldwide. As no effective cure has been developed, planting Psa-resistant cultivars is the best way to avoid bacterial canker in kiwifruit cultivation. However, the differences in the mechanism of resistance between cultivars is poorly understood. In the present study, five local kiwifruit cultivars were used for Psa resistance evaluation and classified into different resistance categories, tolerant (T), susceptible (S), and highly susceptible (HS), based on their various symptoms of lesions on the cane. Susceptible and highly susceptible varieties had a higher sucrose concentration, and a greater decrease in sucrose content was observed after Psa inoculation in phloem than in tolerant varieties. Three invertase activities and their corresponding gene expressions were detected in the phloem with lesions and showed the same trends as the variations in sucrose concentration. Meanwhile, after Psa inoculation, enzyme activities involved in antioxidant defense responses, such as PAL, POD, and CAT, were also altered in the phloem of the lesion position. With no differences among cultivars, PAL and POD activities in phloem first increased and then decreased after Psa inoculation. However, great differences in CAT activities were observed between T and S/HS categories. Our results demonstrate that sucrose content was negatively correlated with the disease resistance of different cultivars and that the increase in immune response enzymes is likely caused by increased sucrose metabolism in the phloem.

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.

In vitro evaluation of Actinidia chinensis cultivars for their resistance to Pseudomonas syringae pv. actinidiae

Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has been the most destructive disease to kiwifruit plants since 2010, and so far there has been no way to completely solve it. Breeding and planting Psa-resistant cultivars is considered one of the most effective disease-control strategies, but the resistance of most existing kiwifruit cultivars to Psa is poorly understood. At present, almost all commercially important cultivars are Actinidia chinensis species, which are generally considered to be susceptible to Psa. In the present study, 62 cultivars of A. chinensis were evaluated on the resistance to Psa through an in vitro assay. In order to find early index for these cultivars’ resistance to Psa, activities of the defense-related enzymes including peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were also analyzed after inoculation of Psa. The results showed that there were a more-than-expected number of A. chinensis cultivars (about 38.71%) classified as Psa-resistant in comparison to Hayward which has been recognized as a Psa-tolerant cultivar. In particular, some red- and yellow-fleshed cultivars were first reported with strong tolerance to Psa. Further study found that these cultivars have an obvious tendency of increasing resistance with ploidy level increasing. A positive correlation was found between the POD and CAT activity to the resistance of the test cultivars after 10 days of inoculation, these two enzyme activities could be used as indicators for assessing kiwifruit resistance to Psa to some extent. Our findings greatly increase the understanding of the resistance of A. chinensis cultivars to Psa and lay a foundation for the use of existing kiwifruit cultivars and the breeding of improved Psa-resistant cultivars.

A report of Pectobacterium carotovorum subsp. actinidiae causing kiwifruit bacterial canker in GuangXi, China.

A bacterial pathogen strain was isolated from susceptible tissue of Hongyang variety kiwifruit in Zhongfeng Town, Ziyuan County, Guilin City, Guangxi, China. Due to the relatively single variety of kiwifruit in Guangxi, the control technology of fruit farmers is backward, and the climate is humid, which is suitable for the growth of pathogenic bacteria, resulting in frequent occurrence of diseases. In this study, the pathogen strain was identified on the basis of morphological, physiological and biochemical tests, 16S rRNA , PCR detection with specific primers, and Biolog analysis. The results showed that a tobacco allergic reaction could be induced by inoculation with the pathogenic bacteria, Additionally, brown necrotic plaques appeared on kiwifruit leaves, necrotic phloem lesions appeared, and wounds on kiwifruit branches turned brown. The characteristics identified by morphological, physiological, biochemical and Biolog identification were similar to those caused by Pectobacterium sp. Through 16S rRNA sequence analysis and PCR identification with specific primers, bands with a size corresponding to target bands, indicating that the pathogen was Pectobacterium carotovorum subsp. actinidiae . This is the first report of kiwifruit canker disease caused by Pectobacterium carotovorum subsp. actinidiae in Guangxi, China. In addition, through this study, a preliminary understanding of the pathogen has been obtained, which will lay the foundation for the prevention and control of the disease in the future.