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

BACKGROUND: Pseudomonas 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. RESULTS: Three 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. CONCLUSION: Three 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.

An efficient genetic transformation system mediated by Rhizobium rhizogenes in fruit trees based on the transgenic hairy root to shoot conversion.

Genetic transformation is a critical tool for gene editing and genetic improvement of plants. Although many model plants and crops can be genetically manipulated, genetic transformation systems for fruit trees are either lacking or perform poorly. We used Rhizobium rhizogenes to transfer the target gene into the hairy roots of Malus domestica and Actinidia chinensis. Transgenic roots were generated within 3 weeks, with a transgenic efficiency of 78.8%. Root to shoot conversion of transgenic hairy roots was achieved within 11 weeks, with a regeneration efficiency of 3.3%. Finally, the regulatory genes involved in stem cell activity were used to improve shoot regeneration efficiency. MdWOX5 exhibited the most significant effects, as it led to an improved regeneration efficiency of 20.6% and a reduced regeneration time of 9 weeks. Phenotypes of the overexpression of RUBY system mediated red roots and overexpression of MdRGF5 mediated longer root hairs were observed within 3 weeks, suggesting that the method can be used to quickly screen genes that influence root phenotype scores through root performance, such as root colour, root hair, and lateral root. Obtaining whole plants of the RUBY system and MdRGF5 overexpression lines highlights the convenience of this technology for studying gene functions in whole plants. Overall, we developed an optimized method to improve the transformation efficiency and stability of transformants in fruit trees.

Antibacterial synergy between a phage endolysin and citric acid against the Gram-negative kiwifruit pathogen Pseudomonas syringae pv. actinidiae.

Horticultural diseases caused by bacterial pathogens provide an obstacle to crop production globally. Management of the infection of kiwifruit by the Gram-negative phytopathogen Pseudomonas syringae pv. actinidiae (Psa) currently includes copper and antibiotics. However, the emergence of bacterial resistance and a changing regulatory landscape are providing the impetus to develop environmentally sustainable antimicrobials. One potential strategy is the use of bacteriophage endolysins, which degrade peptidoglycan during normal phage replication, causing cell lysis and the release of new viral progeny. Exogenous use of endolysins as antimicrobials is impaired by the outer membrane of Gram-negative bacteria that provides an impermeable barrier and prevents endolysins from accessing their target peptidoglycan. Here, we describe the synergy between citric acid and a phage endolysin, which results in a reduction of viable Psa below detection. We show that citric acid drives the destabilization of the outer membrane via acidification and sequestration of divalent cations from the lipopolysaccharide, which is followed by the degradation of the peptidoglycan by the endolysin. Scanning electron microscopy revealed clear morphological differences, indicating cell lysis following the endolysin-citric acid treatment. These results show the potential for citric acid-endolysin combinations as a possible antimicrobial approach in agricultural applications. IMPORTANCE: The phytopathogen Pseudomonas syringae pv. actinidiae (Psa) causes major impacts to kiwifruit horticulture, and the current control strategies are heavily reliant on copper and antibiotics. The environmental impact and increasing resistance to these agrichemicals are driving interest in alternative antimicrobials including bacteriophage-derived therapies. In this study, we characterize the endolysin from the Otagovirus Psa374 which infects Psa. When combined with citric acid, this endolysin displays an impressive antibacterial synergy to reduce viable Psa below the limit of detection. The use of citric acid as a synergistic agent with endolysins has not been extensively studied and has never been evaluated against a plant pathogen. We determined that the synergy involved a combination of the chelation activity of citric acid, acidic pH, and the specific activity of the ΦPsa374 endolysin. Our study highlights an exciting opportunity for alternative antimicrobials in agriculture.

Microbial community diversity analysis of kiwifruit pollen and identification of potential pathogens.

The kiwifruit industry typically uses commercial pollen for artificial pollination. However, during the collection of male flowers and pollen production, pollen can be easily contaminated by pathogenic bacteria that cause diseases such as canker and flower rot. Consequently, it is crucial to understand the structure of the pollen microbial community. This study employed Illumina high-throughput sequencing technology to analyze the fungal and bacterial composition in pollen samples from various regions in Shaanxi Province. Concurrently, potential pathogenic strains were isolated using traditional microbial isolation and cultivation techniques, and their molecular identification was performed through 16S rDNA sequence analysis. A tieback test was conducted on healthy branches to verify the pathogenicity of the strains. The results revealed a rich diversity of fungi and bacteria in kiwifruit pollen. At the phylum level, pollen fungi were mainly distributed in Ascomycota, and bacteria were mainly distributed in Proteobacteria and Firmicutes. The dominant fungal genera were Mycosphaerella, Aspergillus, and Cladosporium; the dominant bacterial genera were Weissella, Pantoea, Enterobacter, and Pseudomonas, respectively. Additionally, both Erwinia persicina and Pseudomonas fluorescens, isolated from pollen, exhibited high pathogenicity toward healthy kiwifruit branches. These findings contribute to a deeper understanding of the microbial diversity in commercial kiwifruit pollen used for mass pollination.

Simultaneous inoculation of non-Saccharomyces yeast and lactic acid bacteria for aromatic kiwifruit wine production.

To further explore strain potential and develop an aromatic kiwifruit wine fermentation technique, the feasibility of simultaneous inoculation by non-Saccharomyces yeast and lactic acid bacteria was investigated. Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum, which have robust ß-glucosidase activity as well as good acid and ethanol tolerance, were inoculated for simultaneous fermentation with Zygosaccharomyces rouxii and Meyerozyma guilliermondii, respectively. Subsequently, the chemical compositions and sensory characteristics of the wines were comprehensively evaluated. The results showed that the majority of the simultaneous protocols effectively improved the quality of kiwifruit wines, increasing the content of polyphenols and volatile compounds, thereby enhancing sensory acceptability compared to the fermentation protocols inoculated with non-Saccharomyces yeast individually. Particularly, the collaboration between Lacp. plantarum and Z. rouxii significantly increased the diversity and content of esters, alcohols, and ketones, intensifying floral and seeded fruit odors, and achieving the highest overall acceptability. This study highlights the potential significance of simultaneous inoculation in kiwifruit wine production.

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.

A dual-branch selective attention capsule network for classifying kiwifruit soft rot with hyperspectral images.

Kiwifruit soft rot is highly contagious and causes serious economic loss. Therefore, early detection and elimination of soft rot are important for postharvest treatment and storage of kiwifruit. This study aims to accurately detect kiwifruit soft rot based on hyperspectral images by using a deep learning approach for image classification. A dual-branch selective attention capsule network (DBSACaps) was proposed to improve the classification accuracy. The network uses two branches to separately extract the spectral and spatial features so as to reduce their mutual interference, followed by fusion of the two features through the attention mechanism. Capsule network was used instead of convolutional neural networks to extract the features and complete the classification. Compared with existing methods, the proposed method exhibited the best classification performance on the kiwifruit soft rot dataset, with an overall accuracy of 97.08% and a 97.83% accuracy for soft rot. Our results confirm that potential soft rot of kiwifruit can be detected using hyperspectral images, which may contribute to the construction of smart agriculture.

Kiwifruit resistance to gray mold is enhanced by yeast-induced modulation of the endophytic microbiome.

The influence of endophytic microbial community on plant growth and disease resistance is of considerable importance. Prior research indicates that pre-treatment of kiwifruit with the biocontrol yeast Debaryomyces hansenii suppresses gray mold disease induced by Botrytis cinerea. However, the specific underlying mechanisms remain unclear. In this study, Metagenomic sequencing was utilized to analyze the composition of the endophytic microbiome of kiwifruit under three distinct conditions: the healthy state, kiwifruit inoculated with B. cinerea, and kiwifruit treated with D. hansenii prior to inoculation with B. cinerea. Results revealed a dominance of Proteobacteria in all treatment groups, accompanied by a notable increase in the relative abundance of Actinobacteria and Firmicutes. Ascomycota emerged as the major dominant group within the fungal community. Treatment with D. hansenii induced significant alterations in microbial community diversity, specifically enhancing the relative abundance of yeast and exerting an inhibitory effect on B. cinerea. The introduction of D. hansenii also enriched genes associated with energy metabolism and signal transduction, positively influencing the overall structure and function of the microbial community. Our findings highlight the potential of D. hansenii to modulate microbial dynamics, inhibit pathogenic organisms, and positively influence functional attributes of the microbial community.

Kiwifruit-Agaricus blazei intercropping effectively improved yield productivity, nutrient uptake, and rhizospheric bacterial community.

Intercropping systems have garnered attention as a sustainable agricultural approach for efficient land use, increased ecological diversity in farmland, and enhanced crop yields. This study examined the effect of intercropping on the kiwifruit rhizosphere to gain a deeper understanding of the relationships between cover plants and kiwifruit in this sustainable agricultural system. Soil physicochemical properties and bacterial communities were analyzed using the Kiwifruit-Agaricus blazei intercropping System. Moreover, a combined analysis of 16S rRNA gene sequencing and metabolomic sequencing was used to identify differential microbes and metabolites in the rhizosphere. Intercropping led to an increase in soil physicochemical and enzyme activity, as well as re-shaping the bacterial community and increasing microbial diversity. Proteobacteria, Bacteroidota, Myxococcota, and Patescibacteria were the most abundant and diverse phyla in the intercropping system. Expression analysis further revealed that the bacterial genera BIrii41, Acidibacter, and Altererythrobacter were significantly upregulated in the intercropping system. Moreover, 358 differential metabolites (DMs) were identified between the monocropping and intercropping cultivation patterns, with fatty acyls, carboxylic acids and derivatives, and organooxygen compounds being significantly upregulated in the intercropping system. The KEGG metabolic pathways further revealed considerable enrichment of DMs in ABC transporters, histidine metabolism, and pyrimidine metabolism. This study identified a significant correlation between 95 bacterial genera and 79 soil metabolites, and an interactive network was constructed to explore the relationships between these differential microbes and metabolites in the rhizosphere. This study demonstrated that Kiwifruit-Agaricus blazei intercropping can be an effective, labor-saving, economic, and sustainable practice for reshaping bacterial communities and promoting the accumulation and metabolism of beneficial microorganisms in the rhizosphere.

Genome sequencing and CAZymes repertoire analysis of Diaporthe eres P3-1W causing postharvest fruit rot of 'Hongyang' kiwifruit in China.

Postharvest rot caused by various fungal pathogens is a damaging disease affecting kiwifruit production and quality, resulting in significant annual economic losses. This study focused on isolating the strain P3-1W, identified as Diaporthe eres, as the causal agent of 'Hongyang' postharvest rot disease in China. The investigation highlighted cell wall degrading enzymes (CWDEs) as crucial pathogenic factors. Specially, the enzymatic activities of cellulase, ß-galactosidase, polygalacturonase, and pectin methylesterases peaked significantly on the second day after infection of D. eres P3-1W. To gain a comprehensive understanding of these CWDEs, the genome of this strain was sequenced using PacBio and Illumina sequencing technologies. The analysis revealed that the genome of D. eres P3-1W spans 58,489,835 bp, with an N50 of 5,939,879 bp and a GC content of 50.7%. A total of 15,407 total protein-coding genes (PCGs) were predicted and functionally annotated. Notably, 857 carbohydrate-active enzymes (CAZymes) were identified in D. eres P3-1W, with 521 CWDEs consisting of 374 glycoside hydrolases (GHs), 108 carbohydrate esterase (CEs) and 91 polysaccharide lyases (PLs). Additionally, 221 auxiliary activities (AAs), 91 glycosyltransferases (GTs), and 108 carbohydrate binding modules (CBMs) were detected. These findings offer valuable insights into the CAZymes of D. eres P3-1W.

Functional characterization of AcWRKY94 in response to Pseudomonas syringae pv. actinidiae in kiwifruit.

WRKY transcription factors are essential for coping with various biotic stresses. Pseudomonas syringae pv. actinidiae (Psa)-induced kiwifruit canker is a major problem restricting kiwifruit yield. Nevertheless, it's unclear how the kiwifruit WRKY genes respond to Psa. Through genome-wide identification, 112 WRKY members were found in 'Hongyang' genome in this work. Promoter analysis revealed that there were many cis-acting elements associated with stress responses in the AcWRKY gene's promoter region. According to transcriptomic analysis, 90 of the AcWRKY genes were differently expressed following Psa, salicylic acid (SA), or methyl jasmonate (MeJA) treatments. Almost all group III WRKYs were responsive to at least one of these treatments, with tissue-specific expression patterns. Quantitative RT-PCR study provided more evidence that Psa and SA treatments significantly induced the expression of the group III WRKY gene AcWRKY94, whereas MeJA treatment repressed it. AcWRKY94 was a transcriptionally active protein localized in the nucleus. Transient overexpression of AcWRKY94 in the leaves of 'Hongyang' enhanced the resistance of kiwifruit to Psa. Overexpression of AcWRKY94 in kiwifruit callus remarkably promoted the expression of PR and JAZ genes associated with SA and JA signals, respectively. These data imply that AcWRKY94 controls the signaling pathway dependent on SA and JA, thereby enhancing resistance to Psa. Taken together, this study establishes the basis for functional research on WRKY genes and provides important information for elucidating the resistance mechanism of kiwifruit canker disease.

Hydrogen sulfide enhances kiwifruit resistance to soft rot by regulating jasmonic acid signaling pathway.

As the third active gas signal molecule in plants, hydrogen sulfide (H2S) plays important roles in physiological metabolisms and biological process of fruits and vegetables during postharvest storage. In the present study, the effects of H2S on enhancing resistance against soft rot caused by Botryosphaeria dothidea and the involvement of jasmonic acid (JA) signaling pathway in kiwifruit during the storage were investigated. The results showed that 20 µL L-1 H2S fumigation restrained the disease incidence of B. dothidea-inoculated kiwifruit during storage, and delayed the decrease of firmness and the increase of soluble solids (SSC) content. H2S treatment increased the transcription levels of genes related to JA biosynthesis (AcLOX3, AcAOS, AcAOC2, and AcOPR) and signaling pathway (AcCOI1, AcJAZ5, AcMYC2, and AcERF1), as well as the JA accumulation. Meanwhile, H2S promoted the expression of defense-related genes (AcPPO, AcSOD, AcGLU, AcCHI, AcAPX, and AcCAT). Correlation analysis revealed that JA content was positively correlated with the expression levels of JA biosynthesis and defense-related genes. Overall, the results indicated that H2S could promote the increase of endogenous JA content and expression of defense-related genes by regulating the transcription levels of JA pathway-related genes, which contributed to the inhibition on the soft rot occurrence of kiwifruit.