Honokiol inhibits Botryosphaeria dothidea, the causal pathogen of kiwifruit soft rot, by targeting membrane lipid biosynthesis.

BACKGROUND: Kiwifruit soft rot is mainly caused by Botryosphaeria dothidea, representing a considerable threat to kiwifruit industry. This investigation assessed the inhibitory consequences and mechanisms of honokiol against B. dothidea, evaluating the inhibitory effects and underlying mechanism. RESULTS: A strain of B.dothidea (XFCT-2) was isolated from infected soft rot kiwifruit. The findings indicate that honokiol hindered the mycelial growth, conidial germination, and pathogenicity of B. dothidea in a dose-dependent manner, both in vitro and in vivo. Furthermore, ultrastructural examinations showed that honokiol impaired the integrity of B. dothidea, leading to an elevation in cell membrane permeability, engendering a multitude of intracellular substance extravasations and hampering energy metabolism. Transcriptome analysis exhibited that honokiol-regulated genes were related to membrane lipid biosynthesis, comprising ACC1, FAS2, Arp2, gk, Cesle, and Etnk1. These findings indicate that honokiol impedes B. dothidea by obstructing lipid biosynthesis within the cell membrane and compromising its integrity, halting the growth of the mycelia, which could potentially cause cellular demise. CONCLUSION: This investigation illustrates how honokiol functions as an eco-friendly approach to prevent the occurrence of soft rot in kiwifruits. © 2023 Society of Chemical Industry.

Correction: Chen et al. Identification of the Causal Agent of Brown Leaf Spot on Kiwifruit and Its Sensitivity to Different Active Ingredients of Biological Fungicides. Pathogens 2022, 11, 673.

In the original publication [...].

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

BACKGROUND: Kiwifruit 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. RESULTS: We 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. CONCLUSIONS: These results are valuable for further analysis of the resistance function and molecular mechanism of laccases in kiwifruit.

Metabolome and Transcriptome Analysis of Sulfur-Induced Kiwifruit Stem Laccase Gene Involved in Syringyl Lignin Synthesis against Bacterial Canker.

Kiwifruit canker is caused by Pseudomonas syringae pv. actinidiae and is one of the most destructive diseases of kiwifruit worldwide. Sulfur can improve the deposit of lignin in kiwifruit stems and induce disease resistance, but the action mechanism at the molecular level remains unclear. This omics-based study revealed that sulfur-induced S lignin synthesis contributes to disease resistance. Histological staining verified sulfur-enhanced total lignin deposition in kiwifruit stems. High-performance liquid chromatography and confocal Raman microscopy showed that sulfur-activated S lignin was mainly deposited in the cell corner. Metabolome and transcriptome analysis revealed that the levels of phenylpropanoid pathway S lignin precursors sinapic acid and sinapyl alcohol were significantly increased and 16 laccase genes were upregulated. Sulfur-induced resistance defense promoted elevated laccase activity by activating the laccase genes, participating in sinapic acid and sinapyl alcohol substance synthesis, and ultimately polymerizing S lignin at cell corner against kiwifruit canker disease.

Assessment of the Biocontrol Potential of Bacillus velezensis WL-23 against Kiwifruit Canker Caused by Pseudomonas syringae pv. actinidiae.

Kiwifruit canker disease, caused by Pseudomonas syringae pv. actinidiae (Psa), is the main threat to kiwifruit production worldwide. Currently, there is no safe and effective disease prevention method; therefore, biological control technologies are being explored for Psa. In this study, Bacillus velezensis WL-23 was isolated from the leaf microbial community of kiwifruit and used to control kiwifruit cankers. Indoor confrontation experiments showed that both WL-23 and its aseptic filtrate had excellent inhibitory activity against the main fungal and bacterial pathogens of kiwifruit. Changes in OD600, relative conductivity, alkaline proteinase, and nucleic acid content were recorded during Psa growth after treatment with the aseptic filtrate, showing that Psa proliferation was inhibited and the integrity of the cell membrane was destroyed; this was further verified using scanning electron microscopy and transmission electron microscopy. In vivo, WL-23 promoted plant growth, increased plant antioxidant enzyme activity, and reduced canker incidence. Therefore, WL-23 is expected to become a biological control agent due to its great potential to contribute to sustainable agriculture.

Antifungal activity and mechanism of tetramycin against Alternaria alternata, the soft rot causing fungi in kiwifruit.

Kiwifruit rot caused by the fungus Alternaria alternata occurs in many countries, leading to considerable losses during kiwifruit production. In this study, we evaluated the antifungal activity and mechanism of tetramycin against kiwifruit soft rot caused by Alternaria alternata. Tetramycin exerted antifungal effects through the suppression of mycelial growth, conidial germination, and the pathogenicity of A. alternata. Scanning electron microscopic observations revealed that tetramycin destroyed the mycelial structure, causing the mycelia to twist, shrink, and even break. Furthermore, transmission electron microscopy revealed that tetramycin caused severe plasmolysis and a decrease in cell inclusions, and the cell wall appeared thinner with blurred boundaries. In addition, tetramycin destroyed cell membrane integrity, resulting in the leakage of cellular components such as nucleic acids and proteins in mycelial suspensions. Moreover, tetramycin also caused cell wall lysis by enhancing the activities of chitinase and ß-1,3-glucanase and inducing the overexpression of related chitinase gene (Chit) and ß-1,3-glucanase gene (ß-1,3-glu) in A. alternata. In field trials, tetramycin not only decreased the incidence of kiwifruit rot but also create a beneficial living space for kiwifruit growth. Overall, this study indicated that the application of tetramycin could serve as an alternative measure for the management of kiwifruit rot.

Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation.

Soft rot causes significant economic losses in the kiwifruit industry. This study isolated strain CTXW 7-6-2 from healthy kiwifruit tissue; this was a gram-positive bacterium that produced the red pigment pulcherrimin. The phylogenetic tree based on 16S ribosomal RNA, gyrA, rpoB, and purH gene sequences identified CTXW 7-6-2 as a strain of Bacillus subtilis. CTXW 7-6-2 inhibited hyphal growth of pathogenic fungi that cause kiwifruit soft rot, namely, Botryosphaeria dothidea, Phomopsis sp., and Alternaria alternata, by 81.76, 69.80, and 32.03%, respectively. CTXW 7-6-2 caused the hyphal surface to become swollen and deformed. Volatile compounds (VOC) produced by the strain inhibited the growth of A. alternata and Phomopsis sp. by 65.74 and 54.78%, respectively. Whole-genome sequencing revealed that CTXW 7-6-2 possessed a single circular chromosome of 4,221,676 bp that contained 4,428 protein-coding genes, with a guanine and cytosine (GC) content of 43.41%. Gene functions were annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, Carbohydrate-Active enZYmes, and Rapid Annotations using Subsystem Technology databases, revealing non-ribosomal pathways associated with antifungal mechanisms, biofilm formation, chemotactic motility, VOC 3-hydroxy-2-butanone, cell wall-associated enzymes, and synthesis of various secondary metabolites. antiSMASH analysis predicted that CTXW 7-6-2 can produce the active substances bacillaene, bacillibactin, subtilosin A, bacilysin, and luminmide and has four gene clusters of unknown function. Quantitative real-time PCR (qRT-PCR) analysis verified that yvmC and cypX, key genes involved in the production of pulcherrimin, were highly expressed in CTXW 7-6-2. This study elucidates the mechanism by which B. subtilis strain CTXW 7-6-2 inhibits pathogenic fungi that cause kiwifruit soft rot, suggesting the benefit of further studying its antifungal active substances.

Identification of the Causal Agent of Brown Leaf Spot on Kiwifruit and Its Sensitivity to Different Active Ingredients of Biological Fungicides.

Kiwifruit (Actinidia chinensis) is an important commercial crop in China, and the occurrence of diseases may cause significant economic loss in its production. In the present study, a new pathogen that causes brown leaf spot disease on kiwifruit was reported. The fungus was isolated from an infected sample and identified as Fusarium graminearum based on morphological and molecular evaluation. Koch's postulates were confirmed when the pathogen was re-isolated from plants with artificially induced symptoms and identified as F. graminearum. Based on the biological characteristics of the pathogen, it was determined that: its optimal growth temperature was 25 °C; optimal pH was 7; most suitable carbon source was soluble starch; most suitable nitrogen source was yeast powder; and best photoperiod was 12 h light/12 h dark. Further investigations were conducted by determining 50% effective concentrations (EC50) of several active ingredients of biological fungicides against F. graminearum. The results showed that among the studied fungicides, tetramycin and honokiol had the highest antifungal activity against this pathogen. Our findings provide a scientific basis for the prevention and treatment of brown leaf spot disease on kiwifruit.