Fungal Pathogen Botrytis Cinerea Research Articles

Citral-loaded chitosan/carboxymethyl cellulose copolymer hydrogel microspheres with improved antimicrobial effects for plant protection

The extensive use of chemical pesticides in agricultural production has caused great damage to the soil and ecological environment. Citral, a monoterpene aldehyde, has been shown to inhibit the growth of a variety of pathogenic fungi by affecting mitochondrial structure. However, the high volatility and chemical instability of citral may restrict its applications in the agricultural industries. In this study, a concise and facile method for the preparation of modified copolymer chitosan/carboxymethyl cellulose (CS/CMC) hydrogels microspheres loaded with citral was developed to increase and stabilize the bioavailability of this natural bioactive substance. Polyelectrolyte composite scaffold hydrogel microspheres were synthesized by polycationic chitosan (CS) and polyanionic carboxymethyl (CMC). Citral was embedded into the microspheres by coupling the carbonyl group of citral with the amino group of CS to form a Schiff base structure. The effects of three parameters including CS/CMC weight ratio, concentration of CMC and citral on the loading ratio were investigated and optimal loading of 68% was achieved based on single-factor experiments. X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM) were employed to confirm and characterize the structure and surface morphology of the microspheres. Both the XRD and FTIR spectra indicated that the microspheres contain -C=N- covalent bonds between CS and citral. The hydrogel microspheres with incorporated citral exhibited effective and improved in vitro antibacterial effects against E. coli, S. aureus and B. subtilis than non-loaded CS microspheres. Moreover, CS/CMC-citral-MPs showed a good antifungal effect in vivo in reducing disease incidence caused by the plant pathogenic fungus Botrytis cinerea in Solanum lycopersicum. Different from the previous applications of CS and citral in the preservation of picked fruits, citral was embedded into CS/CMC microspheres to achieve improved plant protection against Botrytis cinerea. The microspheres are a promising green antimicrobial agent against plant pathogens in crop protection and other fields.

RcMYB84 and RcMYB123 mediate jasmonate-induced defense responses against Botrytis cinerea in rose (Rosa chinensis).

Jasmonates (JAs) are important for pathogen resistance in many plants, but the role of these phytohormones in fungal pathogen resistance in rose is unclear. Here, we determined that exogenous application of methyl jasmonate increased resistance to the important fungal pathogen Botrytis cinerea in Rosa chinensis 'Old blush', whereas silencing the JA biosynthetic pathway gene Allene Oxide Synthase (AOS) and JA co-receptor gene CORONATINE INSENSITIVE 1 (COI1) suppressed this response. Transcriptome profiling identified various MYB transcription factor genes that responded to both JA and B. cinerea treatment. Silencing Ri-RcMYB84/Ri-RcMYB123 increased the susceptibility of rose plants to B. cinerea and inhibited the protective effects of JA treatment, confirming the crucial roles of these genes in JA-induced responses to B. cinerea. JAZ1, a key repressor of JA signaling, directly interacts with RcMYB84 and RcMYB123 to deplete their free pools. The JAZ1-RcMYB84 complex binds to the RcMYB123 promoter via the CAACTG motifs to block its transcription. Upon JA treatment, the expression of RcMYB123 is de-repressed, and free forms of RcMYB84 and RcMYB123 are released due to JAZ1 degradation, thereby activating the defense responses of plants to B. cinerea. These findings shed light on the molecular mechanisms underlying JA-induced pathogen resistance in roses.

Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

A novel endophytic strain of Lactobacillus plantarum CM-3 with antagonistic activity against Botrytis cinerea on strawberry fruit

The fungal pathogen Botrytis cinerea causes grey mold, a severely damaging disease of strawberry. Lactic acid bacteria (LAB) are known for their biopreservative activity. Meanwhile, LAB dominate in plant organs over time, and are of pivotal importance for plant defense. The goal of the present study was to isolate endophytic LAB and to evaluate for their biocontrol activities against B. cinerea. Of the tested endophytic bacteria isolates, Lactobacillus plantarum CM-3 demonstrated the highest potential for in vitro and in vivo control of B. cinerea. The in vitro results showed that L. plantarum CM-3 reduced the mycelial growth of B. cinerea on Potato Dextrose Agar (PDA) between 55.27% and 79.80%. Spore germination of the pathogen was greatly inhibited in the presence of living cell suspensions. In in vivo biocontrol assays, the decay incidence and lesion diameter of grey mold decay of strawberries treated by L. plantarum CM-3 cells were significantly (P < 0.05) reduced compared with the control fruit. At concentrations of 1 × 108 and 1 × 109 CFU mL−1, the incidence of gray mold was reduced by 48.23% and 75.00% respectively, compared to the control, after storage at 20 °C for 6 d. Moreover, rapid colonization of L. plantarum CM-3 in wounds was observed both in the presence or absence of B. cinerea at 20 °C during the storage period. The concentrations of the antagonist had significant effects on biocontrol effectiveness both in vitro and in vivo. However, cell-free filtrates of the strain failed to provide any protection against B. cinerea. Findings of the present study suggested that L. plantarum CM-3 could be a promising biocontrol agent for application during the post-harvest storage and marketing of horticultural products. This study reports the first assessment of endophytic LAB isolated from strawberry fruit and selection of isolates for the biocontrol of B. cinerea.

Leaf resistance traits influence endophytic fungi colonization and community composition in a South American temperate rainforest

Abstract Despite the ubiquitous presence of fungal endophytes in woody plants, mechanisms underlying variation in foliar fungal endophyte communities are poorly understood. Given that endophytes in woody plants are predominantly horizontally transmitted, fungal endophyte colonization of foliar tissues is likely to be influenced by plant resistance traits. Here, we evaluated the association between leaf resistance traits and colonization and community composition of horizontally transmitted endophytes (HTE) in 10 dominant trees species in a temperate rainforest in Southern Chile. Because resistance traits might restrict colonization of HTE, it was hypothesized that host trees with similar leaf resistance traits would show similar HTE communities. We measured leaf traits with reported roles in plant resistance against pathogens and/or herbivores: cell wall polysaccharides, leaf toughness, flavonoids, anthocyanins, terpenoids and chitinases. We also determined variation in fungal read counts, number of operational taxonomic units (OTUs), diversity and community structure across tree species. Foliar fungal endophyte communities were characterized using Illumina MiSeq sequencing of tagged amplicons of the ITS1 region. Fungal read counts were significantly different among host trees, while number of OTUs and diversity were similar among them. Multivariate analyses showed that community composition of endophytic fungi significantly differed among hosts, and that variation in cell wall, flavonoids, anthocyanins and terpenoids was associated with differences in HTE communities. We found that host trees sharing leaf resistance traits harbour similar HTE communities. Fungal read counts decreased with increasing content of cell wall polysaccharides, while endophyte diversity decreased with increasing anthocyanin levels. Fungal read counts, in contrast, were positively associated with terpenoids. Additionally, culture‐based data indicated that some HTE strains inhibited the growth of the common fungal pathogen Botrytis cinerea. Synthesis. Overall, we found that leaf resistance traits may influence fungal endophyte colonization and community composition in tree species, likely through deterrent effects of structural and chemical traits. We suggest that endophytic fungi that overcome resistance traits might play a role in plant protection.

Tomato Plants Treated with Systemin Peptide Show Enhanced Levels of Direct and Indirect Defense Associated with Increased Expression of Defense-Related Genes.

Plant defense peptides represent an important class of compounds active against pathogens and insects. These molecules controlling immune barriers can potentially be used as novel tools for plant protection, which mimic natural defense mechanisms against invaders. The constitutive expression in tomato plants of the precursor of the defense peptide systemin was previously demonstrated to increase tolerance against moth larvae and aphids and to hamper the colonization by phytopathogenic fungi, through the expression of a wealth of defense-related genes. In this work we studied the impact of the exogenous supply of systemin to tomato plants on pests to evaluate the use of the peptide as a tool for crop protection in non-transgenic approaches. By combining gene expression studies and bioassays with different pests we demonstrate that the exogenous supply of systemin to tomato plants enhances both direct and indirect defense barriers. Experimental plants, exposed to this peptide by foliar spotting or root uptake through hydroponic culture, impaired larval growth and development of the noctuid moth Spodoptera littoralis, even across generations, reduced the leaf colonization by the fungal pathogen Botrytis cinerea and were more attractive towards natural herbivore antagonists. The induction of these defense responses was found to be associated with molecular and biochemical changes under control of the systemin signalling cascade. Our results indicate that the direct delivery of systemin, likely characterized by a null effect on non-target organisms, represents an interesting tool for the sustainable protection of tomato plants.

NADPH Oxidase Is Crucial for the Cellular Redox Homeostasis in Fungal Pathogen Botrytis cinerea.

During interactions, both plants and pathogens produce reactive oxygen species (ROS). Plants generate ROS for defense induction, while pathogens synthesize ROS for growth, sporulation, and virulence. NADPH oxidase (NOX) complex in the plasma membrane represents a main protein complex for ROS production in pathogens. Although NOX plays a crucial role in pathogenicity of pathogens, the underlying molecular mechanisms of NOX, especially the proteins regulated by NOX, remain largely unknown. Here, we applied an iodoacetyl tandem mass tag-based redox proteomic assay to investigate the protein redox dynamics in deletion mutant of bcnoxR, which encodes a regulatory subunit of NOX in the fungal pathogen Botrytis cinerea. In total, 214 unique peptidyl cysteine (Cys) thiols from 168 proteins were identified and quantified in both the wild type and ∆bcnoxR mutant. The Cys thiols in the ∆bcnoxR mutant were generally more oxidized than those in the wild type, suggesting that BcNoxR is essential for maintaining the equilibrium of the redox state in B. cinerea. Site-specific thiol oxidation analysis indicated that 142 peptides containing the oxidized thiols changed abundance significantly in the ∆bcnoxR mutant. Proteins containing these differential peptides are classified into various functional categories. Functional analysis revealed that one of these proteins, 6-phosphate dehydrogenase, played roles in oxidative stress response and pathogenesis of B. cinerea. These results provide insight into the potential target proteins and the ROS signal transduction pathway regulated by NOX.

Deletion of the Nonribosomal Peptide Synthetase Gene nps1 in the Fungus Clonostachys rosea Attenuates Antagonism and Biocontrol of Plant Pathogenic Fusarium and Nematodes.

Secondary metabolites produced by biological control agents may influence the outcome of their interactions with plant pathogenic microorganisms and plants. In the present study, we investigated the role of the nonribosomal peptide synthetase gene nps1 expressed by the biocontrol fungus Clonostachys rosea. A gene expression analysis showed that nps1 was induced during confrontations with the plant pathogenic fungus Botrytis cinerea. Gene deletion strains of nps1 displayed increased growth rates and conidiation. However, the nematicidal activity of culture filtrates from C. rosea Δnps1 strains was significantly weaker than that from wild-type filtrates (P ≤ 0.001); after 24 h of incubation with culture filtrates from nps1 deletion strains, only 13 to 33% of a mixed community of nematodes were dead compared with 42% of nematodes incubated with wild-type culture filtrates. The Δnps1 strains also showed reduced biocontrol efficacy during pot experiments, thus failing to protect wheat seedlings from foot rot disease caused by the plant pathogenic fungus Fusarium graminearum. Furthermore, C. rosea Δnps1 strains were not able to reduce populations of plant-parasitic nematodes in soil or in roots of wheat as efficiently as the wild-type strain. Both C. rosea wild-type and Δnps1 strains increased the dry shoot weight and shoot length of wheat by 20 and 13%, respectively. We showed that NPS1, a putative nonribosomal peptide synthetase encoded by nps1, is a biocontrol factor, presumably by producing a hitherto unknown nonribosomal peptide compound with antifungal and nematicidal properties that contributes to the biocontrol properties of C. rosea.

Cell death regulation but not abscisic acid signaling is required for enhanced immunity to Botrytis in Arabidopsis cuticle-permeable mutants.

Prevailing evidence indicates that abscisic acid (ABA) negatively influences immunity to the fungal pathogen Botrytis cinerea in most but not all cases. ABA is required for cuticle biosynthesis, and cuticle permeability enhances immunity to Botrytis via unknown mechanisms. This complex web of responses obscures the role of ABA in Botrytis immunity. Here, we addressed the relationships between ABA sensitivity, cuticle permeability, and Botrytis immunity in the Arabidopsis thaliana ABA-hypersensitive mutants protein phosphatase2c quadruple mutant (pp2c-q) and enhanced response to aba1 (era1-2). Neither pp2c-q nor era1-2 exhibited phenotypes predicted by the known roles of ABA; conversely, era1-2 had a permeable cuticle and was Botrytis resistant. We employed RNA-seq analysis in cuticle-permeable mutants of differing ABA sensitivities and identified a core set of constitutively activated genes involved in Botrytis immunity and susceptibility to biotrophs, independent of ABA signaling. Furthermore, botrytis susceptible1 (bos1), a mutant with deregulated cell death and enhanced ABA sensitivity, suppressed the Botrytis immunity of cuticle permeable mutants, and this effect was linearly correlated with the extent of spread of wound-induced cell death in bos1. Overall, our data demonstrate that Botrytis immunity conferred by cuticle permeability can be genetically uncoupled from PP2C-regulated ABA sensitivity, but requires negative regulation of a parallel ABA-dependent cell-death pathway.

Spermine Is a Potent Plant Defense Activator Against Gray Mold Disease on Solanum lycopersicum, Phaseolus vulgaris, and Arabidopsis thaliana.

Polyamines (PAs) are ubiquitous aliphatic amines that play important roles in growth, development, and environmental stress responses in plants. In this study, we report that exogenous application of spermine (Spm) is effective in the induction of resistance to gray mold disease, which is caused by the necrotrophic fungal pathogen Botrytis cinerea, on tomato (Solanum lycopersicum), bean (Phaseolus vulgaris), and Arabidopsis thaliana. High throughput transcriptome analysis revealed a priming role for the Spm molecule in the genus Arabidopsis, resulting in strong upregulation of several important defense-associated genes, particularly those involved in systemic-acquired resistance. Microscopic analysis confirmed that Spm application potentiates endogenous defense responses in tomato leaves through the generation of reactive oxygen species and the hypersensitive response, which effectively contained B. cinerea growth within the inoculated area. Moreover, co-application of Spm and salicylic acid resulted in a synergistic effect against the pathogen, leading to higher levels of resistance than those induced by separate applications of the two compounds. The Spm plus salicylic acid treatment also reduced infection in systemic nontreated leaves of tomato plants. Our findings suggest that Spm, particularly when applied in combination with salicylic acid, functions as a potent plant defense activator that leads to effective local and systemic resistance against B. cinerea.

Glucose oxidase as a control agent against the fungal pathogen Botrytis cinerea in postharvest strawberry

Botrytis cinerea is an economically devastating necrotrophic fungus with many host species, and the fungicides commonly used to control it cause serious food and environmental safety problems. It is therefore urgent to develop alternative control measures for these pathogen fungicides. The glucose oxidase enzyme (GOX) is an oxido-reductase that catalyzes the oxidation of glucose to hydrogen peroxide (H2O2) and gluconic acid. GOX has been widely used as a food additive because of its safety and oxidation properties, but little is known about its antifungal effects against B. cinerea, and there have been no reports of its application for pathogen control of postharvest fruit. Here, we examined the antimicrobial activity of GOX against B. cinerea spores and developed a GOX-glucose (10 g/L) system that can significantly restrict fungal growth on postharvest strawberry fruit. A variety of optical and electron microscopy investigations revealed that GOX inhibits B. cinerea growth by altering the morphology of mycelial membranes, among other deleterious effects. Biochemical analyses supported the observation that GOX-mediated growth inhibitory effects result from its competition of carbon and enzymatic production of H2O2 and from the acidifying influence of gluconic acid. Our demonstration of this GOX-glucose system as a control agent to protect postharvest fruit opens the door for the application of this technology to other crops and for further investigation of the mechanisms that can interrupt B. cinerea growth.

Biocontrol of Botrytis cinerea by chitin-based cultures of Paenibacillus elgii HOA73

The chitinase-producing bacterium, Paenibacillus elgii HOA73, is a biocontrol agent that limits the damage caused to plants by microbial pathogens, insects, and nematodes. However, the mechanisms involved in the biocontrol of plant diseases by HOA73 have not been determined. The objective of this study was to elucidate the role of extracellular chitinase obtained from isolate HOA73 in the control of the fungal pathogen Botrytis cinerea, the causative agent of gray mold in tomato. The HOA73 strain grew efficiently in a chitin-containing broth and produced chitin oligomers through chitinase activity; protease, lipase, and Fe-chelating siderophores were also secreted by the bacterium. Cultures containing intact bacteria inhibited B. cinerea conidia germination to a greater extent than did the bacterial cells alone or the cell-free culture supernatant. The antifungal activity increased with culture age and was heat-sensitive because of chitinase-mediated production of long-chain chitin oligomers. The biocontrol efficacy of undiluted bacterial cultures against gray mold in tomato was comparable to that of a standard fungicide. This study demonstrated that P. elgii HOA73 bacterial cultures grown on chitin-based minimal medium may be an effective formulation for the integrated control of gray mold.

Different Antifungal Activity of Anabaena sp., Ecklonia sp., and Jania sp. against Botrytis cinerea.

Water extracts and polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. were tested for their activity against the fungal plant pathogen Botrytis cinerea. Water extracts at 2.5, 5.0, and 10.0 mg/mL inhibited B. cinerea growth in vitro. Antifungal activity of polysaccharides obtained by N-cetylpyridinium bromide precipitation in water extracts was evaluated in vitro and in vitro at 0.5, 2.0, and 3.5 mg/mL. These concentrations were tested against fungal colony growth, spore germination, colony forming units (CFUs), CFU growth, and on strawberry fruits against B. cinerea infection with pre- and post-harvest application. In in vitro experiments, polysaccharides from Anabaena sp. and from Ecklonia sp. inhibited B. cinerea colony growth, CFUs, and CFU growth, while those extracted from Jania sp. reduced only the pathogen spore germination. In in vitro experiments, all concentrations of polysaccharides from Anabaena sp., Ecklonia sp., and Jania sp. reduced both the strawberry fruits infected area and the pathogen sporulation in the pre-harvest treatment, suggesting that they might be good candidates as preventive products in crop protection.

Occurrence and management of fungicide resistance in Botrytis cinerea on tomato from greenhouses in Hebei, China

AbstractGrey mould, caused by the fungal pathogen Botrytis cinerea, is one of the most devastating tomato diseases, and the control of this disease is mainly by the application of chemicals. In this study, 512 isolates of B. cinerea were collected from tomato grown in greenhouses at 10 locations in 10 cities of Hebei Province from 2011 to 2016 and tested for their sensitivities to carbendazim (Car), diethofencarb (Die), iprodione (Ipr) and pyrimethanil (Pyr). Of these tested isolates, 95.7%, 95.2%, 31.6% and 89.4% were resistant to Car, Die, Ipr and Pyr, respectively. There were nine fungicide‐resistant phenotypes in the tested isolates. CarRPyrRDieRIPRS and CarRPyrRDieRIPRR were the most common phenotypes, accounting for 59.6%, and 31.1% of the tested isolates, respectively. The field trials showed that the control efficacies (CE) of carbendazim + diethofencarb (WP, 25% + 25%), pyrimethanil (EC, 40%) and iprodione (WP, 50%) at the recommended doses were 22.75%–29.23%, 58.44%–64.19% and 61.02%–65.17%, respectively, significantly lower than those of boscalid (WG, 50%) and pyrisoxazole (EC, 25%). The resistance management trial conducted from 2015 to 2017 indicated that the CE of tomato grey mould in the experimental fields was higher than 90% and the sensitivity to carbendazim, diethofencarb and pyrimethanil of B. cinerea isolates from the experimental fields increased on a yearly basis. These results showed that the frequency of resistance to Car, Die, Ipr and Pyr was high, and these four fungicides could not effectively control tomato grey mould. Tomato grey mould could be controlled by using biopesticides and newly synthesized fungicides with different modes of action. Our findings would be useful in designing and implementing fungicide resistance management spray programmes for the control of tomato grey mould.

Grey mould of strawberry, a devastating disease caused by the ubiquitous necrotrophic fungal pathogen Botrytis cinerea.

SummaryThe fungal pathogen Botrytis cinerea causes grey mould, a commercially damaging disease of strawberry. This pathogen affects fruit in the field, storage, transport and market. The presence of grey mould is the most common reason for fruit rejection by growers, shippers and consumers, leading to significant economic losses. Here, we review the biology and epidemiology of the pathogen, mechanisms of infection and the genetics of host plant resistance. The development of grey mould is affected by environmental and genetic factors; however, little is known about how B. cinerea and strawberry interact at the molecular level. Despite intensive efforts, breeding strawberry for resistance to grey mould has not been successful, and the mechanisms underlying tolerance to B. cinerea are poorly understood and under‐investigated. Current control strategies against grey mould include pre‐ and postharvest fungicides, yet they are generally ineffective and expensive. In this review, we examine available research on horticultural management, chemical and biological control of the pathogen in the field and postharvest storage, and discuss their relevance for integrative disease management. Additionally, we identify and propose approaches for increasing resistance to B. cinerea in strawberry by tapping into natural genetic variation and manipulating host factors via genetic engineering and genome editing.

Calcium chloride treatment modifies cell wall metabolism and activates defense responses in strawberry fruit (Fragaria × ananassa, Duch).

Fruit dips in calcium ions solutions have been shown as an effective treatment to extend strawberries (Fragaria × ananassa, Duch) quality during storage. In the present work, strawberry fruit were treated with 10g L-1 calcium chloride solution and treatment effects on cell wall enzymes activities and the expression of encoding genes, as well as enzymes involved in fruit defense responses were investigated. Calcium treatment enhanced pectin methylesterase activity while inhibited those corresponding to pectin hydrolases as polygalacturonase and β-galactosidase. The expression of key genes for strawberry pectin metabolism was up-regulated (for FaPME1) and down-regulated (for FaPG1, FaPLB, FaPLC, FaβGal1 and FaAra1) by calcium dips. In agreement, a higher firmness level and ionically-bound pectins (IBPs) amount were detected in calcium-treated fruit compared with controls. The in vitro and in vivo growth rate of fungal pathogen Botrytis cinerea was limited by calcium treatment. Moreover, the activities of polyphenol oxidases, chitinases, peroxidases and β-1,3-glucanases were enhanced by calcium ion dips. News insights concerning the biochemical and molecular basis of cell wall preservation and resistance to fungal pathogens on calcium-treated strawberries are provided. © 2019 Society of Chemical Industry.

Bryophyte extracts suppress growth of the plant pathogenic fungus Botrytis cinerea

In this study, the extracts of three selected bryophyte species are shown to have inhibitory effects on grey mould disease (Botrytis cinerea). Methanol extracts of one leafy liverwort (Porella platyphylla) and two mosses, one aquatic (Cinclidotus fontinaloides) and one terrestrial (Anomodon viticulosus), were applied in vitro to Botrytis cinerea, after which tests showed suppression of fungal development.

Proteomic analysis of the inhibitory effect of oligochitosan on the fungal pathogen, Botrytis cinerea.

The fungal pathogen Botrytis cinerea infects a broad range of horticultural plants worldwide, resulting in significant economic losses. A derivative of chitosan, oligochitosan, has been reported to be an eco-friendly alternative to synthetic fungicides. Oligochitosan can greatly inhibit B. cinerea spore germination and induce protein carbonylation. To further investigate the molecular mechanism underlying the inhibitory effect, a comparative proteome analysis was conducted of oligochitosan-treated versus non-treated B. cinerea spores. The cellular proteins were obtained from B. cinerea spore samples and subjected to two-dimensional gel electrophoresis. In total, 21 differentially expressed proteins (DEPs) were identified. Three DEPs were up-regulated in the oligochitosan-treated versus the untreated spores, including scytalone dehydratase and a serine carboxypeptidase III precursor. By contrast, seven DEPs, including Hsp 88 and cell division cycle protein 48, were down-regulated by oligochitosan treatment. Notably, 10 DEPs, including phosphatidylserine decarboxylase proenzyme and ATP-dependent molecular chaperone HSC82, were only detected in the control spores, whereas one DEP, a non-annotated predicted protein, was only detected in the oligochitosan-treated spores. Oligochitosan may affect the spore germination of B. cinerea by impairing protein function. These findings have practical implications with respect to the use of oligochitosan for controlling fungal pathogens. © 2018 Society of Chemical Industry.

Design of a bacterial speck resistant tomato by CRISPR/Cas9-mediated editing of SlJAZ2.

SummaryDue to their different lifestyles, effective defence against biotrophic pathogens normally leads to increased susceptibility to necrotrophs, and vice versa. Solving this trade‐off is a major challenge for obtaining broad‐spectrum resistance in crops and requires uncoupling the antagonism between the jasmonate (JA) and salicylate (SA) defence pathways. Pseudomonas syringae pv. tomato (Pto) DC3000, the causal agent of tomato bacterial speck disease, produces coronatine (COR) that stimulates stomata opening and facilitates bacterial leaf colonization. In Arabidopsis, stomata response to COR requires the COR co‐receptor AtJAZ2, and dominant AtJAZ2Δjas repressors resistant to proteasomal degradation prevent stomatal opening by COR. Here, we report the generation of a tomato variety resistant to the bacterial speck disease caused by Pto DC3000 without compromising resistance to necrotrophs. We identified the functional ortholog of AtJAZ2 in tomato, found that preferentially accumulates in stomata and proved that SlJAZ2 is a major co‐receptor of COR in stomatal guard cells. SlJAZ2 was edited using CRISPR/Cas9 to generate dominant JAZ2 repressors lacking the C‐terminal Jas domain (SlJAZ2Δjas). SlJAZ2Δjas prevented stomatal reopening by COR and provided resistance to Pto DC3000. Water transpiration rate and resistance to the necrotrophic fungal pathogen Botrytis cinerea, causal agent of the tomato gray mold, remained unaltered in Sljaz2Δjas plants. Our results solve the defence trade‐off in a crop, by spatially uncoupling the SA‐JA hormonal antagonism at the stomata, entry gates of specific microbes such as Pto DC3000. Moreover, our results also constitute a novel CRISPR/Cas‐based strategy for crop protection that could be readily implemented in the field.

RNA sequencing-based transcriptome analysis of mature strawberry fruit infected by necrotrophic fungal pathogen Botrytis cinerea

Botrytis cinerea, the causal agent of gray mold disease, is regarded as the most impactful postharvest fungal pathogen, as it causes serious losses in fresh fruits, ornamentals, and vegetables worldwide. To understand the molecular defense mechanisms of the affected plants, we analyzed transcriptome using RNA sequencing (RNA-seq) of mature strawberry fruit that were infected by B. cinerea at 12, 24, and 48 h. Overall, 18 cDNA libraries were produced generating 923,482,642,150 base pair reads. A total of 1459 differentially expressed genes (DEGs) were identified (≥2.0-fold change and FDR ≤ 0.05), among which only 26 were down-regulated. The majority of changes in gene expression occurred 24 h after inoculation, before significant lesion development, suggesting that this is an important stage during B. cinerea infection. Further analysis revealed that the DEGs were predominately involved in pathogen recognition (pattern recognition receptors), signaling transduction (MAPK cascades, CDPKs), defense response (transcription factors, secondary metabolism biosynthesis, pathogenis-related), as well as different sorts of transporter encoding genes. These data provided a global insight into mature strawberry fruit-B. cinerea interaction and might be used for fruit postharvest pathogens and diseases management.