ABSTRACT
BACKGROUND: Alternaria alternata is the primary pathogen of potato leaf spot disease, resulting in significant potato yield losses globally. Endophytic microorganism-based biological control, especially using microorganisms from host plants, has emerged as a promising and eco-friendly approach for managing plant diseases. Therefore, this study aimed to isolate, identify and characterize the endophytic fungi from healthy potato leaves which had great antifungal activity to the potato leaf spot pathogen of A. alternata in vitro and in vivo. RESULTS: An endophytic fungal strain SD1-4 was isolated from healthy potato leaves and was identified as Talaromyces muroii through morphological and sequencing analysis. The strain SD1-4 exhibited potent antifungal activity against the potato leaf spot pathogen A. alternata Lill, with a hyphal inhibition rate of 69.19%. Microscopic and scanning electron microscope observations revealed that the strain SD1-4 grew parallel to, coiled around, shrunk and deformed the mycelia of A. alternata Lill. Additionally, the enzyme activities of chitinase and ß-1, 3-glucanase significantly increased in the hyphae of A. alternata Lill when co-cultured with the strain SD1-4, indicating severe impairment of the cell wall function of A. alternata Lill. Furthermore, the mycelial growth and conidial germination of A. alternata Lill were significantly suppressed by the aseptic filtrate of the strain SD1-4, with inhibition rates of 79.00% and 80.67%, respectively. Decrease of leaf spot disease index from 78.36 to 37.03 was also observed in potato plants treated with the strain SD1-4, along with the significantly increased plant growth characters including plant height, root length, fresh weight, dry weight, chlorophyll content and photosynthetic rate of potato seedlings. CONCLUSION: The endophyte fungus of T. muroii SD1-4 isolated from healthy potato leaves in the present study showed high biocontrol potential against potato leaf spot disease caused by A. alternata via direct parasitism or antifungal metabolites, and had positive roles in promoting potato plant growth.
Subject(s)
Alternaria , Endophytes , Plant Diseases , Plant Leaves , Solanum tuberosum , Talaromyces , Alternaria/growth & development , Alternaria/physiology , Plant Diseases/microbiology , Plant Diseases/prevention & control , Solanum tuberosum/microbiology , Talaromyces/genetics , Talaromyces/growth & development , Endophytes/physiology , Endophytes/isolation & purification , Endophytes/genetics , Plant Leaves/microbiology , Hyphae/growth & development , Antibiosis , Chitinases/metabolism , Biological Control Agents , Pest Control, Biological/methodsABSTRACT
To further reveal the interaction mechanism between plants and pathogens, this study used confocal Raman microscopy spectroscopy (CRM) combined with chemometrics to visualize the biopolymers distribution of kiwifruit cell walls at different infection stages at the cellular micro level. Simultaneously, the changes in the content of various monosaccharides in fruit were studied at the molecular level using high-performance liquid chromatography (HPLC). There were significant differences in the composition of various nutrient components in the cell wall structure of kiwifruit at different infection times after infection by Botryosphaeria dothidea. PCA could cluster samples with infection time of 0-9 d into different infection stages, and SVM was used to predict the PCA classification results, the accuracy >96 %. Multivariate curve resolution-alternating least squares (MCR-ALS) helped to identify single substance spectra and concentration signals from mixed spectral signals. The pure substance chemical imaging maps of low methylated pectin (LMP), high methylated pectin (HMP), cellulose, hemicellulose, and lignin were obtained by analyzing the resolved concentration data. The imaging results showed that the lignin content in the kiwifruit cell wall increased significantly to resist pathogens infection after the infection of B. dothidea. With the development of infection, B. dothidea decomposed various substances in the host cell walls, allowing them to penetrate the interior of fruit cells. This caused significant changes in the form, structure, and distribution of various chemicals on the fruit cell walls in time and space. HPLC showed that glucose was the main carbon source and energy substance obtained by pathogens from kiwifruit during infection. The contents of galactose and arabinose, which maintained the structure and function of the fruit cell walls, decreased significantly and the cell wall structure was destroyed in the late stage of pathogens infection. This study provided a new perspective on the cellular structure changes caused by pathogenic infection of fruit and the defense response process of fruit and provided effective references for further research on the mechanisms of host-pathogen interactions in fruit infected by pathogens.
ABSTRACT
BACKGROUND: Phytophthora capsici is a destructive oomycete pathogen, causing huge economic losses for agricultural production. The genus Trichoderma represents one of the most extensively researched categories of biocontrol agents, encompassing a diverse array of effective strains. The commercial biocontrol agent Trichoderma harzianum strain T-22 exhibits pronounced biocontrol effects against many plant pathogens, but its activity against P. capsici is not known. RESULTS: T. harzianum T-22 significantly inhibited the growth of P. capsici mycelia and the culture filtrate of T-22 induced lysis of P. capsici zoospores. Electron microscopic analyses indicated that T-22 significantly modulated the ultrastructural composition of P. capsici, with a severe impact on the cell wall integrity. Dual RNA sequencing revealed multiple biological processes involved in the inhibition during the interaction between these two microorganisms. In particular, a marked upregulation of genes was identified in T. harzianum that are implicated in cell wall degradation or disruption. Concurrently, the presence of T. harzianum appeared to potentiate the susceptibility of P. capsici to cell wall biosynthesis inhibitors such as mandipropamid and dimethomorph. Further investigations showed that mandipropamid and dimethomorph could strongly inhibit the growth and development of P. capsici but had no impact on T. harzianum even at high concentrations, demonstrating the feasibility of combining T. harzianum and these cell wall synthesis inhibitors to combat P. capsici. CONCLUSION: These findings provided enhanced insights into the biocontrol mechanisms against P. capsici with T. harzianum and evidenced compatibility between specific biological and chemical control strategies. © 2024 Society of Chemical Industry.
Subject(s)
Cell Wall , Phytophthora , Cell Wall/metabolism , Phytophthora/physiology , Sequence Analysis, RNA , Plant Diseases/microbiology , Plant Diseases/prevention & control , Hypocreales/physiology , Hypocreales/genetics , AntibiosisABSTRACT
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.
Subject(s)
Actinidia , Allyl Compounds , Ascomycota , Biphenyl Compounds , Phenols , Gene Expression Profiling , Membrane Lipids/pharmacologyABSTRACT
In the original publication [...].
ABSTRACT
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.
Subject(s)
Actinidia , Laccase , Laccase/genetics , Phylogeny , Lignin , Biological Evolution , Actinidia/genetics , Actinidia/microbiology , Pseudomonas syringae/physiology , Plant Diseases/genetics , Plant Diseases/microbiologyABSTRACT
Potato Verticillium wilt, caused by Verticillium dahliae, is a serious soil-borne vascular disease, which restricts the sustainable development of the potato industry, and the pathogenic mechanism of the fungus is complex. Therefore, it is of great significance to explore the important pathogenic factors of V. dahliae to expand the understanding of its pathology. Protein kinase C (PKC) gene is located in the Ca2+ signaling pathway, which is highly conserved in filamentous fungi and involved in the regulation of a variety of biological processes. In the current study, the PKC gene in V. dahliae (VdPKC) was characterized, and its effects on the fungal pathogenicity and tolerance to fungicide stress were further studied. The results showed that the VdPKC positively regulated the growth and development, conidial germination, and production of V. dahliae, which was necessary for the fungus to achieve pathogenicity. It also affected the formation of melanin and microsclerotia and changed the adaptability of V. dahliae to different environmental stresses. In addition, VdPKC altered the tolerance of V. dahliae to different fungicides, which may be a potential target for polyoxin. Therefore, our results strongly suggest that VdPKC gene is necessary for the vegetative growth, stress response, and pathogenicity of V. dahliae.
ABSTRACT
Phytophthora infestans causes late blight on potatoes and tomatoes, which has a significant economic impact on agriculture. The management of late blight has been largely dependent on the application of synthetic fungicides, which is not an ultimate solution for sustainable agriculture and environmental safety. Biocontrol strategies are expected to be alternative methods to the conventional chemicals in controlling plant diseases in the integrated pest management (IPM) programs. Well-studied biocontrol agents against Phytophthora infestans include fungi, oomycetes, bacteria, and compounds produced by these antagonists, in addition to certain bioactive metabolites produced by plants. Laboratory and glasshouse experiments suggest a potential for using biocontrol in practical late blight disease management. However, the transition of biocontrol to field applications is problematic for the moment, due to low and variable efficacies. In this review, we provide a comprehensive summary on these biocontrol strategies and the underlying corresponding mechanisms. To give a more intuitive understanding of the promising biocontrol agents against Phytophthora infestans in agricultural systems, we discuss the utilizations, modes of action and future potentials of these antagonists based on their taxonomic classifications. To achieve a goal of best possible results produced by biocontrol agents, it is suggested to work on field trials, strain modifications, formulations, regulations, and optimizations of application. Combined biocontrol agents having different modes of action or biological adaptation traits may be used to strengthen the biocontrol efficacy. More importantly, biological control agents should be applied in the coordination of other existing and forthcoming methods in the IPM programs. © 2023 Society of Chemical Industry.
Subject(s)
Phytophthora infestans , Solanum lycopersicum , Solanum tuberosum , Solanum tuberosum/microbiology , Plants , Phenotype , Plant Diseases/prevention & control , Plant Diseases/microbiologyABSTRACT
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.
Subject(s)
Actinidia , Laccase , Laccase/genetics , Lignin , Disease Resistance , Metabolome , Gene Expression Profiling , Actinidia/genetics , SulfurABSTRACT
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.
Subject(s)
Actinidia , Bacillus , Pseudomonas syringae , Plant Diseases/prevention & control , Plant Diseases/microbiology , Actinidia/microbiologyABSTRACT
Botryosphaeria dothidea is the source of the deadly kiwifruit disease known as soft rot. In order to explore the role of melatonin in regulating the postharvest quality and disease resistance of kiwifruit at different growth and development stages, in this study, we applied melatonin at different concentrations to kiwifruit at the young fruit, expansion, and late expansion stages to assess its effect on fruit resistance to B. dothidea, minimize soft rot, and maintain postharvest fruit quality. The results showed that melatonin significantly suppressed the mycelial growth of B. dothidea, with 1.0 mmol/L melatonin inhibiting it by up to 50%. However, 0.1-0.3 mmol/L melatonin had the best control over soft rot. Furthermore, spraying MT during kiwifruit growth can successfully increase fruit weight; preserve postharvest fruit firmness; reduce respiration intensity in the early stages of storage; delay the rise in soluble solids, while maintaining a high titratable acid content to ensure suitable solid acid ratio; increase total phenol, flavonoid, chlorophyll, carotenoid, and ascorbic acid contents; and delay the rise in soluble sugar contents in the late stages of storage. These results have a positive effect on maintaining the nutritional composition of kiwifruit. However, the effects on weight loss, dry matter content, and soluble protein content were not significant. In addition, the results of the principal component analysis demonstrated that 0.3 mmol/L MT increased kiwifruit's resistance to soft rot while preserving postharvest fruit quality.
ABSTRACT
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.
Subject(s)
Antifungal Agents , Plant Diseases , Antifungal Agents/pharmacology , Plant Diseases/prevention & control , Plant Diseases/microbiology , AlternariaABSTRACT
BACKGROUND: Kiwifruit rot is an important disease caused by different fungal pathogens, which can lead to huge economic loss in the kiwifruit industry. The aims of this study were to discover an effective botanical compound that significantly inhibits the pathogens causing kiwifruit rot, evaluate its control efficacy against this disease, and reveal the underlying mechanisms. RESULTS: A strain of Fusarium tricinctum (GF-1), isolated from diseased kiwifruit, could cause fruit rot in both Actinidia chinensis var. chinensis and Actinidia chinensis var. deliciosa. Different botanical chemicals were used for antifungal activity test against GF-1 and thymol was the most effective one with a 50% effective concentration (EC50 ) of 30.98 mg L-1 . The minimal inhibitory concentration (MIC) of thymol against GF-1 was 90 mg L-1 . Control efficacy of thymol against kiwifruit rot was evaluated and the results indicated that thymol could effectively decrease the occurrence and spread of kiwifruit rot. The mechanisms underlying the antifungal activity of thymol against F. tricinctum were investigated, and it showed that thymol could significantly damage the ultrastructure, destroy the plasma membrane integrity, and instantaneously increase energy metabolisms of F. tricinctum. Further investigations indicated that thymol could extend shelf life of kiwifruit by increasing their storability. CONCLUSION: Thymol can effectively inhibit F. tricinctum that is one of the causal agents of kiwifruit rot. Multiple modes of action are involved in the antifungal activity. The results of this study indicate that thymol can be a promising botanical fungicide to control kiwifruit rot and provide useful references for thymol application in agriculture system. © 2023 Society of Chemical Industry.
Subject(s)
Actinidia , Thymol , Thymol/pharmacology , Antifungal Agents/pharmacology , Antifungal Agents/metabolism , Fruit/microbiology , Actinidia/chemistryABSTRACT
BACKGROUND: Neonicotinoids are among the most essential chemical insecticides worldwide because of their high activity against many important pests and wide application. However, their application is limited by their toxicity to honeybees. Therefore, the development of a facile route to fabricate efficient and eco-friendly pesticide formulations is of great significance. RESULTS: In this study, clothianidin-loaded zeolitic imidazolate framework-8 (CLO@ZIF-8) nanoparticles were fabricated by a facile one-pot route using zinc nitrate as a Zn2+ source and characterized by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, thermogravimetric analysis, energy-dispersive spectrometry and Fourier transform infrared spectroscopy. Based on the pH response of ZIF-8, a 'burst release effect' was observed for CLO@ZIF-8 at pH 3 and 5 within 12 h, in contrast to the slow and sustainable release at pH 8. CLO@ZIF-8 improved the retention ability of the pesticide liquid and remained 70% control efficacy on Nilaparvata lugens after water rinsed of sprayed CLO@ZIF-8. The pH response of CLO@ZIF-8 allowed it to maintain 43% control efficacy against N. lugens after 10 days of application, which was twice the efficacy of clothianidin solution (SCA). Moreover, CLO@ZIF-8 reduced the acute toxicity to honeybees (Apis mellifera) by ≥120-fold compared with SCA. CONCLUSION: This study provides new insights into the application of ZIF-8 to neonicotinoids and suggests the need for the development of a biocompatible and eco-friendly pesticide formulation. © 2023 Society of Chemical Industry.
Subject(s)
Insecticides , Zeolites , Animals , Bees , Neonicotinoids , Guanidines , Thiazoles , Zeolites/chemistryABSTRACT
Magnolol is a natural compound extracted from the traditional Chinese medicine Magnolia officinalis, which exhibits antimicrobial properties. However, magnolol is insoluble in water and consists of a phenolic hydroxyl group, which is volatile; these factors hinder its application. In this study, a safe and environmentally friendly method to improve the microbial resistance and storability of harvested fruits is developed using the water-soluble carrier carboxymethyl chitosan (CMCS) and magnolol. Magnolol was loaded on CMCS particles to form Magnolol@CMCS antimicrobial particles, a preservation coating agent. Magnolol@CMCS particles effectively solved the problems of water insolubility and agglomeration of magnolol and reduced the size distribution D50 value of magnolol from 0.749 to 0.213 µm. Magnolol@CMCS particles showed greater toxicity against Staphylococcus aureus, Escherichia coli, and Botryosphaeria dothidea than that of magnolol alone, with effective medium concentration (EC50) values of 0.9408, 142.4144, and 8.8028 µg/mL, respectively. Kiwifruit treated with the Magnolol@CMCS solution showed delayed changes in fruit hardness and soluble solid and dry matter contents and significantly higher ascorbic acid (vitamin C) and soluble total sugar contents and sugar:acid ratios compared with that of the control fruit. In addition, no disease spots were observed on fruit treated with the Magnolol@CMCS solution within 7 days after inoculation with B. dothidea. In conclusion, Magnolol@CMCS particles showed antimicrobial activity on harvested fruits, effectively delayed the hardness and nutritional changes of fruits during storage, and improved the storability of kiwifruit.
ABSTRACT
Verticillium dahliae is a soil-borne pathogenic fungus that causes Verticillium wilt in host plants, a particularly serious problem in potato cultivation. Several pathogenicity-related proteins play important roles in the host infection process, hence, identifying such proteins, especially those with unknown functions, will surely aid in understanding the mechanism responsible for the pathogenesis of the fungus. Here, tandem mass tag (TMT) was used to quantitatively analyze the differentially expressed proteins in V. dahliae during the infection of the susceptible potato cultivar "Favorita". Potato seedlings were infected with V. dahliae and incubated for 36 h, after which 181 proteins were found to be significantly upregulated. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses showed that most of these proteins were involved in early growth and cell wall degradation. The hypothetical, secretory protein with an unknown function, VDAG_07742, was significantly upregulated during infection. The functional analysis with knockout and complementation mutants revealed that the associated gene was not involved in mycelial growth, conidial production, or germination; however, the penetration ability and pathogenicity of VDAG_07742 deletion mutants were significantly reduced. Therefore, our results strongly indicate that VDAG_07742 is essential in the early stage of potato infection by V. dahliae.
Subject(s)
Ascomycota , Solanum tuberosum , Verticillium , Solanum tuberosum/microbiology , Virulence/genetics , Proteins , Plant Diseases/microbiologyABSTRACT
The genetic basis underlying loss-of-virulence mutations that arise among natural phytopathogen populations is not well documented. In this study, we examined the virulence of 377 isolates of Pseudomonas syringae pv. actinidiae biovar 3 (Psa3) that were isolated from 76 kiwifruit orchards suffering from bacterial canker disease. Eighty-four nonpathogenic isolates were identified in 40 orchards. A nonpathogenic isolate G166 was found to be defective in hrpL transcription and the downstream type III secretion system (T3SS)-dependent phenotypes. Comparative genomics and complementary expression assay revealed that a single-base "G" insertion in the hrpL promoter blocks gene transcription by reducing promoter activity. The electrophoretic mobility shift assay showed that the genetic variation impairs σ54 /promoter binding during gene transcription under hrp-inducing conditions, resulting in lower expression of hrpL. A PCR-restriction fragment length polymorphism assay was performed to trace the evolutionary history of this mutation, which revealed the independent onset of genetic variations in natural Psa3 populations. We also found that nonpathogenic variants outperformed virulent Psa3 bacteria for both epiphytic and apoplast colonization of kiwifruit leaves in mixed inoculations. Our study highlights a novel mechanism for loss of virulence in Psa3 and provides insight into bacterial adaptive evolution under natural settings.
Subject(s)
Genomics , Pseudomonas syringae , Promoter Regions, Genetic/genetics , Mutation , Bacterial Proteins/genetics , Bacterial Proteins/metabolismABSTRACT
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.
ABSTRACT
'Hongyang' kiwifruit (Actinidia chinensis, cultivar 'Hongyang') black spot disease is caused by the fungal pathogen Didymella glomerata, and is a serious disease, causing considerable losses to the kiwifruit industry during growth of the fruit. Hence, we aimed to identify a potential biocontrol agent against D. glomerata. In this study, bacterial isolates from the rhizosphere soil of kiwifruit were tested for their potential antifungal activity against selected fungal pathogens. Based on a phylogenetic tree constructed using sequences of 16S rDNA and the gyrA gene, BQ-33 with the best antifungal activity was identified as Bacillus mojavensis. We evaluated the antagonistic activity and inhibitory mechanism of BQ-33 against D. glomerata. Confrontation experiments showed that both BQ-33 suspension and the sterile supernatant (SS) produced by BQ-33 possessed excellent broad-spectrum antifungal activity. Furthermore, the SS damaged the cell membrane and cell wall of the mycelia, resulting in the leakage of a large quantity of small ions (Na+, K+), soluble proteins and nucleic acids. Chitinase and ß-1,3-glucanase activities in SS increased in correlation with incubation time and remained at a high level for several days. An in vivo control efficacy assay indicated that 400 mL L-1 of SS completely inhibited kiwifruit black spot disease caused by D. glomerata. Therefore, BQ-33 is a potential biocontrol agent against kiwifruit black spot and plant diseases caused by other fungal pathogens. To our knowledge, this is the first report of the use of a rhizosphere microorganism as a biocontrol agent against kiwifruit black spot disease caused by D. glomerata.
ABSTRACT
Bacterial canker of kiwifruit is a devastating disease caused by Pseudomonas syringae pv. actinidiae (Psa). The type III secretion system (T3SS), which translocates effectors into plant cells to subvert plant immunity and promote extracellular bacterial growth, is required for Psa virulence. Despite that the "HrpR/S-HrpL" cascade that sophisticatedly regulates the expression of T3SS and effectors has been well documented, the transcriptional regulators of hrpR/S remain to be determined. In this study, the OmpR-like transcription factor, previously identified by DNA pull-down assay, was found to be involved in the regulation of hrpR/S genes, and its regulatory mechanisms and other functions in Psa were explored through techniques including gene knockout and overexpression, ChIP-seq, and RNA-seq. The OmpR-like transcription factor had binding sites in the promoter region of the hrpR/S, and the transcriptional level of the hrpR/S increased after the deletion of OmpR-like and decreased upon its overexpression in an OmpR-like deletion background. Additionally, OmpR-like overexpression reduced the strain's capacity to form biofilms and lipopolysaccharides, led to its slow growth in King's B medium, and reduced its swimming ability, although there was no significant effect on its pathogenicity against kiwifruit hosts. Our results indicated that OmpR-like directly and negatively regulates the transcription of hrpR/S and may be involved in the regulation of multiple biological processes in Psa. Our results provide a basis for further understanding the transcriptional regulation mechanism of hrpR/S in Psa.