Understanding the mechanism of action of protease inhibitors in controlling the growth of the Candida Genus: potential candidates for development of new antifungal molecules.

There is a growing imperative for research into alternative compounds for the treatment of the fungal infections. Thus, many studies have focused on the analysis of antifungal proteins and peptides from different plant sources. Among these molecules are protease inhibitors (PIs). Previously, PIs present in the peptide-rich fractions called PEF1, PEF2 and PEF3 were identified from Capsicum chinense seeds, which have strong activity against phytopathogenic fungi. The aim of this study was to evaluate the mechanism of action and antimicrobial activity of PIs from PEF2 and PEF3 on the growth of yeasts of the genus Candida. In this work, analyses of their antimicrobial activity and cell viability were carried out. Subsequently, the mechanism of action by which the PIs cause the death of the yeasts was evaluated. Cytotoxicity was assessed in vitro by erythrocytes lysis and in vivo in Galleria mellonella larvae. PEF2 and PEF3 caused 100% of the growth inhibition of C. tropicalis and C. buinensis. For C. albicans inhibition was approximately 60% for both fractions. The PEF2 and PEF3 caused a reduction in mitochondrial functionality of 54% and 46% for C. albicans, 26% and 30% for C. tropicalis, and 71% and 68% for C. buinensis, respectively. These fractions induced morphological alterations, led to membrane permeabilization, elevated ROS levels, and resulted in necrotic cell death in C. tropicalis, whilst demonstrating low toxicity toward host cells. From the results obtained here, we intend to contribute to the understanding of the action of PIs in the control of fungal diseases of medical importance.

Characterization and Antioxidant and Antibacterial Activities of Carboxymethylated Tamarind Seed Polysaccharide Composite Films Incorporated with ε-Polylysine and Their Application in Fresh-Cut Green Bell Pepper Preservation.

Traditional petroleum-based food-packaging materials have poor permeability, limited active packaging properties, and difficulty in biodegradation, limiting their application. We developed a carboxymethylated tamarind seed polysaccharide composite film incorporated with ε-polylysine (CTPε) for better application in fresh-cut agricultural products. The CTPε films exhibit excellent water vapor barrier properties, but the mechanical properties are slightly reduced. Fourier transform infrared spectroscopy and X-ray diffraction spectra indicate the formation of hydrogen bonds between ε-PL and CTP, leading to their internal reorganization and dense network structure. With the increase of ε-PL concentration, composite films showed notable inhibition of postharvest pathogenic fungi and bacteria, a significant enhancement of 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activity, and gradual improvement of wettability performance. Cytotoxicity experiments confirmed the favorable biocompatibility when ε-PL was added at 0.3% (CTPε2). In fresh-cut bell pepper preservation experiments, the CTPε2 coating effectively delayed weight loss and malondialdehyde increase preserved the hardness, color, and nutrients of fresh-cut peppers and prolonged the shelf life of the fresh-cut peppers, as compared with the control group. Therefore, CTPε composite films are expected to be a valuable packaging material for extending the shelf life of freshly cut agricultural products.

Exploiting the role of plant growth promoting rhizobacteria in reducing heavy metal toxicity of pepper (Capsicum annuum L.).

Microorganisms are cost-effective and eco-friendly alternative methods for removing heavy metals (HM) from contaminated agricultural soils. Therefore, this study aims to identify and characterize HM-tolerant (HMT) plant growth-promoting rhizobacteria (PGPR) isolated from industry-contaminated soils to determine their impact as bioremediators on HM-stressed pepper plants. Four isolates [Pseudomonas azotoformans (Pa), Serratia rubidaea (Sr), Paenibacillus pabuli (Pp) and Bacillus velezensis (Bv)] were identified based on their remarkable levels of HM tolerance in vitro. Field studies were conducted to evaluate the growth promotion and tolerance to HM toxicity of pepper plants grown in HM-polluted soils. Plants exposed to HM stress showed improved growth, physio-biochemistry, and antioxidant defense system components when treated with any of the individual isolates, in contrast to the control group that did not receive PGPR. The combined treatment of the tested HMT PGPR was, however, relatively superior to other treatments. Compared to no or single PGPR treatment, the consortia (Pa+Sr+Pp+Bv) increased the photosynthetic pigment contents, relative water content, and membrane stability index but lowered the electrolyte leakage and contents of malondialdehyde and hydrogen peroxide by suppressing the (non) enzymatic antioxidants in plant tissues. In pepper, Cd, Cu, Pb, and Ni contents decreased by 88.0-88.5, 63.8-66.5, 66.2-67.0, and 90.2-90.9% in leaves, and 87.2-88.1, 69.4-70.0%, 80.0-81.3, and 92.3%% in fruits, respectively. Thus, these PGPR are highly effective at immobilizing HM and reducing translocation in planta. These findings indicate that the application of HMT PGPR could be a promising "bioremediation" strategy to enhance growth and productivity of crops cultivated in soils contaminated with HM for sustainable agricultural practices.

Zinc priming enhances Capsicum annuum immunity against infection by Botrytis cinerea- From the whole plant to the molecular level.

Micronutrient manipulation can enhance crop resilience against pathogens, but the mechanisms are mostly unknown. We tested whether priming Capsicum annuum plants with zinc (5 µM Zn) or manganese (3 µM Mn) for six weeks increases their immunity against the generalist necrotroph Botrytis cinerea compared to deficient (0.1 µM Zn, 0.02 µM Mn) and control conditions (1 µM Zn, 0.6 µM Mn). Zinc priming reduced the pathogen biomass and lesion area and preserved CO2 assimilation and stomatal conductance. Zinc mobilization at the infection site, visualized by micro-X-ray fluorescence, was accompanied by increased Zn protein binding obtained by size exclusion HPLC-ICP/MS. A common metabolic response to fungal infection in Zn- and Mn-primed plants was an accumulation of corchorifatty acid F, a signaling compound, and the antifungal compound acetophenone. In vitro tests showed that the binding of Zn2+ increased, while Mn2+ binding decreased acetophenone toxicity against B. cinerea at concentrations far below the toxicity thresholds of both metals in unbound (aquo complex) form. The metal-specific response to fungal infection included the accumulation of phenolics and amino acids (Mn), and the ligand isocitrate (Zn). The results highlight the importance of Zn for pepper immunity through direct involvement in immunity-related proteins and low molecular weight Zn-complexes, while Mn priming was inefficient.

A Putative Apoplastic Effector of Clavibacter capsici, ChpGCc as Hypersensitive Response and Virulence (Hrv) Protein in Plants.

Clavibacter bacteria use secreted apoplastic effectors, such as putative serine proteases, for virulence in host plants and for hypersensitive response (HR) induction in nonhost plants. Previously, we have shown that Clavibacter capsici ChpGCc is important for the necrosis development in pepper (Capsicum annuum) leaves. Here, we determine the function of ChpGCc, along with three paralogous proteins, for HR induction in the apoplastic space of a nonhost plant, Nicotiana tabacum. The full-length and signal peptide-deleted (ΔSP) mature forms of all proteins fused with the tobacco PR1b signal sequence were generated. The full-length and ΔSP forms of ChpGCc and only the ΔSP forms of ChpECc and Pat-1Cc, but none of the ChpCCc, triggered HR. Based on the predicted protein structures, ChpGCc carries amino acids for a catalytic triad and a disulfide bridge in positions like Pat-1Cm. Substituting these amino acids of ChpGCc with alanine abolished or reduced HR-inducing activity. To determine whether these residues are important for necrosis development in pepper, alanine-substituted chpGCc genes were transformed into the C. capsici PF008ΔpCM1 strain, which lacks the intact chpGCc gene. The strain with any variants failed to restore the necrosis-causing ability. These results suggest that ChpGCc has a dual function as a virulence factor in host plants and an HR elicitor in nonhost plants. Based on our findings and previous results, we propose Clavibacter apoplastic effectors, such as ChpGCc, Pat-1Cm, Chp-7Cs, and ChpGCm, as hypersensitive response and virulence (Hrv) proteins that display phenotypic similarities to the hypersensitive response and pathogenicity (Hrp) proteins found in gram-negative bacteria. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Harnessing CRISPR/Cas9 for Enhanced Disease Resistance in Hot Peppers: A Comparative Study on CaMLO2-Gene-Editing Efficiency across Six Cultivars.

The Capsicum annuum Mildew Locus O (CaMLO2) gene is vital for plant defense responses against fungal pathogens like powdery mildew, a significant threat to greenhouse pepper crops. Recent advancements in genome editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, have unlocked unprecedented opportunities for modifying disease-resistant genes and improving crop characteristics. However, the application of CRISPR technology in pepper cultivars has been limited, and the regeneration process remains challenging. This study addresses these limitations by investigating the feasibility of using the validated CaMLO2 genetic scissors system in six commercial hot pepper cultivars. We assessed the gene-editing efficiency of the previously reported high-efficiency Cas9/CaMLO2single-guide RNA (sgRNA)1-ribonucleoprotein (RNP) and the low-efficiency Cas9/CaMLO2sgRNA2-RNP systems by extending their application from the bell pepper 'Dempsey' and the hot pepper 'CM334' to six commercial hot pepper cultivars. Across the six cultivars, CaMLO2sgRNA1 demonstrated an editing efficiency ranging from 6.3 to 17.7%, whereas CaMLO2sgRNA2 exhibited no editing efficiency, highlighting the superior efficacy of sgRNA1. These findings indicate the potential of utilizing the verified Cas9/CaMLO2sgRNA1-RNP system to achieve efficient gene editing at the CaMLO2 locus in different Capsicum annuum cultivars regardless of their cultivar genotypes. This study provides an efficacious genome-editing tool for developing improved pepper cultivars with CaMLO2-mediated enhanced disease resistance.

Genome-wide association study of resistance to anthracnose in pepper (Capsicum chinense) germplasm.

BACKGROUND: Anthracnose is a fungal disease caused by Colletotrichum spp. that has a significant impact on worldwide pepper production. Colletotrichum scovillei is the most common pathogenic anthracnose-causing species in the Republic of Korea. RESULTS: The resistances of 197 pepper (Capsicum chinense) accessions deposited in Korea's National Agrobiodiversity Center were evaluated for their response against the virulent pathogens Colletotrichum acutatum isolate 'KSCa-1' and C. scovillei isolate 'Hana') in the field and in vitro methods for three consecutive years (2018 to 2020). The severity of the disease was recorded and compared between inoculation methods. Six phenotypically resistant pepper accessions were selected based on three years of disease data. All of the selected resistant pepper accessions outperformed the control resistant pepper in terms of resistance (PI 594,137). A genome-wide association study (GWAS) was carried out to identify single nucleotide polymorphisms (SNPs) associated with anthracnose resistance. An association analysis was performed using 53,518 SNPs and the disease score of the 2020 field and in vitro experiment results. Both field and in vitro experiments revealed 25 and 32 significantly associated SNPs, respectively. These SNPs were found on all chromosomes except Ch06 and Ch07 in the field experiment, whereas in the in vitro experiment they were found on all chromosomes except Ch04 and Ch11. CONCLUSION: In this study, six resistant C. chinense accessions were selected. Additionally, in this study, significantly associated SNPs were found in a gene that codes for a protein kinase receptor, such as serine/threonine-protein kinase, and other genes that are known to be involved in disease resistance. This may strengthen the role of these genes in the development of anthracnose resistance in Capsicum spp. As a result, the SNPs discovered to be strongly linked in this study can be used to identify a potential marker for selecting pepper material resistant to anthracnose, which will assist in the development of resistant varieties.

Discovery of Novel Small Molecule Growth Inhibitors to Manage Pseudomonas Leaf Spot Disease on Peppers (Capsicum sp.).

Pseudomonas leaf spot (PLS) disease in peppers caused by Pseudomonas syringae pv. syringae (Pss) is an emerging seedborne phytopathogen. Pss infection can severely reduce the marketable yield of peppers in favorable environmental conditions and cause significant economic losses. The intensive use of copper-sulfate and streptomycin-sulfate to control PLS and other bacterial diseases is associated with antimicrobial-resistant Pss strains, making these control methods less effective. So, there is an urgent need to develop novel antimicrobials effective against Pss in peppers. Several studies, including those done in our laboratory, have shown that small molecule (SM) antimicrobials are ideal candidates as they can be effective against multidrug resistant bacteria. Therefore, our study aims to identify novel SM growth inhibitors of Pss, assess their safety, and evaluate their efficacy on Pss-infected pepper seeds and seedlings. Using high-throughput screening, we identified 10 SMs (PC1 to PC10) that inhibited the growth of Pss strains at 200 µM or lower concentrations. These SMs were effective against both copper- and streptomycin-resistant as well as biofilm-embedded Pss. These SMs were effective against other plant pathogens (n = 22) at low concentrations (<200 µM) and had no impact on beneficial phytobacteria (n = 12). Furthermore, these SMs showed better or equivalent antimicrobial activity against Pss in infested pepper seeds and inoculated seedlings compared with copper-sulfate (200 µM) and streptomycin (200 µg/ml). Additionally, none of the SMs were toxic to pepper tissues (seeds, seedlings, or fruits), human Caco-2 cells, and pollinator honeybees at 200 µM. Overall, the SMs identified in this study are promising alternative antimicrobials for managing PLS in pepper production.

Biocontrol Capabilities of Bacillus subtilis E11 against Aspergillus flavus In Vitro and for Dried Red Chili (Capsicum annuum L.).

As a condiment with extensive nutritional value, chili is easy to be contaminated by Aspergillus flavus (A. flavus) during field, transportation, and storage. This study aimed to solve the contamination of dried red chili caused by A. flavus by inhibiting the growth of A. flavus and detoxifying aflatoxin B1 (AFB1). In this study, Bacillus subtilis E11 (B. subtilis) screened from 63 candidate antagonistic bacteria exhibited the strongest antifungal ability, which could not only inhibit 64.27% of A. flavus but could also remove 81.34% of AFB1 at 24 h. Notably, scanning electron microscopy (SEM) showed that B. subtilis E11 cells could resist a higher concentration of AFB1, and the fermentation supernatant of B. subtilis E11 could deform the mycelia of A. flavus. After 10 days of coculture with B. subtilis E11 on dried red chili inoculated with A. flavus, the mycelia of A. flavus were almost completely inhibited, and the yield of AFB1 was significantly reduced. Our study first concentrated on the use of B. subtilis as a biocontrol agent for dried red chili, which could not only enrich the resources of microbial strains for controlling A. flavus but also could provide theoretical guidance to prolong the shelf life of dried red chili.

In vivo and In vitro evaluation of the antifungal activity of the PGPR Bacillus amyloliquefaciens RaSh1 (MZ945930) against Alternaria alternata with growth promotion influences on Capsicum annuum L. plants.

Alternaria alternata that threatens pepper production and causes major economic harm is responsible for the leaf spot/blight disease. Chemical fungicides have been widely employed; unfortunately, fungicidal resistance is a current concern. Therefore, finding new environmentally friendly biocontrol agents is a future challenge. One of these friendly solutions is the use of bacterial endophytes that have been identified as a source of bioactive compounds. The current study investigates the in vivo and in vitro fungicidal potential of Bacillus amyloliquefaciens RaSh1 (MZ945930) against pathogenic A. alternata. In vitro, the results revealed that RaSh1 exhibited strong antagonistic activity against A. alternata. In addition to this, we inoculated pepper (Capsicum annuum L.) plants with B. amyloliquefaciens RaSh1 and infected them with A. alternata. As a result of A. alternata infection, which generated the highest leaf spot disease incidence (DI), the plant's growth indices and physio-biochemical characteristics significantly decreased, according to our findings. Our results also showed the abnormal and deformed cell structure using light and electron microscopy of A. alternata-infected leaves compared with other treatments. However, DI was greatly reduced with B. amyloliquefaciens RaSh1 application (40%) compared to pepper plants infected with A. alternata (80%), and this led to the largest increases in all identified physio-biochemical parameters, including the activity of the defense-related enzymes. Moreover, inoculation of pepper plants with B. amyloliquefaciens RaSh1 decreased electrolyte leakage by 19.53% and MDA content by 38.60% as compared to A. alternata infected ones. Our results show that the endophyte B. amyloliquefaciens RaSh1 has excellent potential as a biocontrol agent and positively affects pepper plant growth.

Genome-wide comprehensive analysis of miRNAs and their target genes expressed in resistant and susceptible Capsicum annuum genotypes during Phytophthora capsici infection.

Despite extensive works on miRNA's role during plant-oomycete interaction, its role in Capsicum annuum-Phytophthora capsici pathosystem is not fully explored. Therefore, the present study was designed to identify known and novel miRNAs along with their target genes in two contrasting chili peppers genotypes, i.e., GojamMecha_9086 (resistant) and Dabat_80045 (susceptible) under P. capsici infection associated with modulating the defense response during disease pathogenesis. The result demonstrated 79 known miRNAs corresponding to 24 miRNAs families and 477 novel miRNAs along with 22,895 potential targets, including 30 defense-related target genes against P. capsici infection. The expression analysis of 29 known and 157 novel miRNAs in resistant and 30 known and 177 novel miRNAs in susceptible genotypes revealed differential accumulation patterns. qRT-PCR analysis of 8 defense-related miRNAs representing 4 novels (Pz-novel-miR428-1, Pz-novel-miR160-1, Pz-novel-miR1028-1, Pz-novel-miR204-1) and 4 known miRNAs (Pz-known-miR803-1, Pz-known-miR2059-1, Pz-known-miR2560-1, Pz-known-miR1872-1) revealed differential accumulation pattern in both resistant and susceptible genotypes. Additionally, validation of eight target genes of miRNAs using regional amplification quantitative RT-PCR (RA-PCR), a superior technique to 5'-RNA Ligase-Mediated-rapid amplification of cDNA ends (5' RLM-RACE), revealed expression of six target genes positively correlated with their corresponding miRNAs in RC versus RI leaf, while five target genes observed an inverse correlation with their corresponding miRNAs in SC versus SI leaf, suggesting their key role during disease response. The Pz-known-miR1872-PODs pair showed perfect inverse relation in all four samples. The significant findings of the current study provide comprehensive genome-wide information about the repertoire of miRNAs and their target genes expressed in resistant and susceptible chili pepper genotypes, which can serve as a valuable resource for better understanding the post-transcriptional regulatory mechanism during C. annuum-P. capsici pathosystem.

Inhibition of Serine Protease, α-Amylase and Growth of Phytopathogenic Fungi by Antimicrobial Peptides from Capsicum chinense Fruits.

Plant fungal diseases cause major problems for the global economy. Antimicrobial peptides have aroused great interest in the control of phytopathogens, as they are natural molecules and have a broad spectrum of inhibitory activity. Herein, we have tried to identify and characterize antimicrobial peptides present in fruits of Capsicum chinense and to evaluate their enzymatic and antifungal activities. The retained fraction obtained in the anion exchange chromatography with strong antifungal activity was subjected to molecular exclusion chromatography and obtained four fractions named G1, G2, G3, and G4. The 6.0-kDa protein band of G2 showed similarity with protease inhibitors type II, and it was able to inhibit 100% of trypsin and α-amylase activities. The protein band with approximately 6.5 kDa of G3 showed similarity with sequences of protease inhibitors from genus Capsicum and showed growth inhibition of 48% for Colletotrichum lindemuthianum, 49% for Fusarium lateritium, and 51% for F. solani and F. oxysporum. Additionally, G3 causes morphological changes, membrane permeabilization, and ROS increase in F. oxysporum cells. The 9-kDa protein band of G4 fraction was similar to a nsLTP type 1, and a protein band of 6.5 kDa was similar to a nsLTP type 2. The G4 fraction was able to inhibit 100% of the activities of glycosidases tested and showed growth inhibition of 35 and 50% of F. oxysporum and C. lindemuthianum, respectively. C. chinense fruits have peptides with antifungal activity and enzyme inhibition with biotechnological potential.

Role of the cAMP signaling pathway in the dissemination and development on pepper fruit anthracnose disease caused by Colletotrichum scovillei.

The ascomycete fungus Colletotrichum scovillei causes severe anthracnose disease on the fruit of sweet pepper and chili pepper (Capsicum annuum L.) worldwide. Understanding the biology of C. scovillei would improve the management of fruit anthracnose diseases. The cyclic adenosine monophosphate (cAMP) signaling pathway regulates diverse cellular and physiological processes in several foliar fungal pathogens. We investigated the roles of the cAMP signaling pathway in C. scovillei using pharmaceutical and genetic approaches. Exogenous cAMP was found to increase conidiation, appressorium formation, and anthracnose disease development in C. scovillei. CsAc1, CsCap1, and CsPdeH, which regulate the intracellular cAMP level, were deleted by homology-dependent gene replacement. Expectedly, the intracellular cAMP level was significantly decreased in ΔCsac1 and ΔCscap1 but increased in ΔCspdeh. All three deletion mutants exhibited serious defects in multiple fungal developments and pathogenicity, suggesting regulation of the intracellular cAMP level is important for C. scovillei. Notably, exogenous cAMP recovered the defect of ΔCsac1 in appressorium development, but not penetration, which was further recovered by adding CaCl2. This result suggests that CsAc1 is associated with both the cAMP and Ca2+ signaling pathways in C. scovillei. ΔCscap1 produced morphologically abnormal conidia with reduced tolerance to thermal stress. ΔCspdeh was completely defective in conidiation in C. scovillei, unlike other foliar pathogens. Taken together, these results demonstrate the importance of cAMP signaling in anthracnose disease caused by C. scovillei.

Microbial transformation of capsaicin by several human intestinal fungi and their inhibitory effects against lysine-specific demethylase 1.

Capsaicin widely exists in the Capsicum genus (e.g., hot peppers) and is commonly used as a food additive or medicinal material. In this work, microbial transformation of capsaicin was performed based on the three cultivated human intestinal fungi. Fourteen metabolites were obtained, and their chemical structures were elucidated by spectroscopic data analysis, including 13 compounds with undescribed structures. Hydroxylation, lactylation, succinylation, citric acylation, and acetylation were observed for these microbial metabolites derived from capsaicin, which indicated diverse catalytic characteristics of human intestinal fungi. In an in vitro bioassay, four metabolites and capsaicin inhibited the activity of lysine-specific demethylase 1 (LSD1) with a more than 70% inhibitory rate at 10 µM. In particular, 9,5'-dihydroxycapsaicin displayed the strongest inhibitory effect with an IC50 of 1.52 µM. Therefore, capsaicin analogs displayed potential application as LSD1 inhibitors against the invasion and migration of cancer cells.

Dynamic Fluctuation and Niche Differentiation of Fungal Pathogens Infecting Bell Pepper Plants.

The plant microbiome is shaped by plant development and microbial interaction. Fungal pathogens infecting bell pepper plants may fluctuate across the growing seasons. Dynamic fluctuation of the microbiome and fungal pathogens in bell pepper plants is poorly understood, and the origin of fungal pathogens causing fruit rot and leaf wilt has been barely investigated. In this study, we used amplicon sequencing (i.e., 16S rRNA and internal transcribed spacer [ITS] sequencing) to explore the compositional variations of the microbiome in bell pepper plants and studied the fluctuation of fungal pathogens across the growing seasons. Co-occurrence network analysis was applied to track the origin and dissemination route of fungal pathogens that infected bell pepper plants. ITS and 16S rRNA sequencing analyses demonstrated that fungal pathogens infecting fruits and leaves probably belonged to the Penicillium, Cladosporium, Fusarium, and unclassified_Sclerotiniaceae genera rather than one specific genus. The dominant fungal pathogens were different, along with the development of bell pepper plants. Both plant development and fungal pathogens shaped microbial communities in bell pepper plants across the growing seasons. Fungal pathogens decreased species richness and diversity of fungal communities in fungus-infected fruit and leaf tissues but not the uninfected stem tissues. Bacterial metabolic functions of xenobiotics increased in fungus-infected leaves at a mature developmental stage. Competitive interaction was present between fungal and bacterial communities in leaves. Co-occurrence network analysis revealed that the origins of fungal pathogens included the greenhouse, packing house, and storage room. Niche differentiation of microbes was discovered among these locations. IMPORTANCE Bell peppers are widely consumed worldwide. Fungal pathogen infections of bell peppers lead to enormous economic loss. To control fungal pathogens and increase economic benefit, it is essential to investigate the shifting patterns of the microbiome and fungal pathogens in bell pepper plants across the growing seasons. In this study, bell pepper plant diseases observed in fruits and leaves were caused by different fungal pathogens. Fungal pathogens originated from the greenhouse, packing house, and storage room, and niche differentiation existed among microbes. This study improves the understanding of dynamic fluctuation and source of fungal pathogens infecting bell pepper plants in the farming system. It also facilitates precise management of fungal pathogens in the greenhouse.

Bacterial communities in home-made Doushen with and without chili pepper.

Doushen is a traditional Chinese fermented soybean product prepared with sterilizated soybeans under open conditions. However, little is known on the bacterial community and their influence on the flavor of Doushen. In the present study, Doushen samples with and without chili pepper were collected to reveal the bacterial community and assess a correlation between bacterial community and VFCs in the two kinds of Doushen samples. We identified four phyla and 97 bacterial genera in the two kinds of Doushen samples. In addition, a total of 17 significantly different OTUs were detected by LEfSe (Line Discriminant Analysis (LDA) Effect Size). Results of Principal coordinates analysis (PCoA), unweighted pair-group method (UPGMA) and functional and phenotypes prediction showed that bacterial communities in the two kinds of Doushen were significantly different. Spearman correlation analysis showed that all the dominant genera, except Clostridium Sensu stricto 1, were significantly correlated with the characteristic VFCs. This study provides a theoretical basis for improving the flavour quality of traditional homemade Doushen.

CsPOM1, a DYRK Family Kinase, Plays Diverse Roles in Fungal Development, Virulence, and Stress Tolerance in the Anthracnose Pathogen Colletotrichum scovillei.

Colletotrichum scovillei is the major anthracnose fungus of sweet pepper and chili pepper (Capsicum annuum L.), causing significant losses in the yield and quality of the pepper fruits. Molecular mechanisms governing development and pathogenicity have been widely studied in many foliar fungal pathogens, but the information on fruit diseases is still limited. In this study, we determined the functional roles of the dual-specificity tyrosine phosphorylation-regulated kinase CsPOM1 in C. scovillei. Knockout mutant for CsPOM1 gene was obtained via homology-dependent gene replacement. The ΔCspom1 mutant exhibited a reduction in vegetative growth on osmotic stress, surface hydrophobicity, and conidiation compared with wild-type. Conidia of the ΔCspom1 mutant were already two-celled before inoculation on an induction surface, indicating that CsPOM1 negatively regulates conidial cell division. The ΔCspom1 mutant, similar to wild-type, formed appressoria on the plant surface, but was significantly reduced on hydrophobic coverslips, probably due to a defect in the recognition of surface hydrophobicity. Treatment of conidia with cutin monomers restored appressorium formation on hydrophobic coverslips in the ΔCspom1 mutant. On pepper fruits, the ΔCspom1 mutant exhibited delayed penetration and invasive growth, leading to significantly reduced virulence. Collectively, the results showed that CsPOM1 is important for stress tolerance, conidiation, surface hydrophobicity, appressorium formation, and virulence in C. scovillei.

Identification and Mapping of bs8, a Novel Locus Conferring Resistance to Bacterial Spot Caused by Xanthomonas gardneri.

Although cultivars possessing recessive resistance alleles provide effective control of bacterial spot of pepper (Capsicum annuum), the deployed resistance gene, bs5, is ineffective against Xanthomonas gardneri, one of the pathogenic species. Resistance against X. gardneri was identified in C. annuum accession PI 163192, and this study sought to characterize this novel resistance and to map the resistance gene(s) to the pepper genome. We crossed PI 163192 with the susceptible cultivar Early Calwonder (ECW) to develop resistant near-isogenic lines (NILs) of ECW, designated ECW80R. The novel resistance in ECW80R was determined to be quantitative, recessively inherited, and non-hypersensitive-response causing, and inhibits lesion expansion and chlorosis. Presence of the resistance in NILs decreased the in planta bacterial population by ninefold compared with ECW. Bulked segregant analysis of resistant and susceptible individuals from an F2 population using whole genome single nucleotide polymorphisms identified a major resistance locus within an approximate 6-Mbp interval on the subtelomeric region of chromosome 11. We developed markers spanning this region and used these to genotype backcross F2 populations, which further delimited the resistance locus within a 2.3-Mbp interval. The novel resistance locus has been designated bs8. ECW80R and the linked markers developed in this study should prove useful for breeders seeking to advance this resistance into commercially relevant germplasm and for pyramiding bs8 with other resistance alleles such as bs5 and bs6. The allele bs8 will help prolong the durability of bacterial spot resistance in pepper and improve resistance to multiple species of Xanthomonas.

The Xanthomonas type-III effector XopS stabilizes CaWRKY40a to regulate defense responses and stomatal immunity in pepper (Capsicum annuum).

As a critical part of plant immunity, cells that are attacked by pathogens undergo rapid transcriptional reprogramming to minimize virulence. Many bacterial phytopathogens use type III effector (T3E) proteins to interfere with plant defense responses, including this transcriptional reprogramming. Here, we show that Xanthomonas outer protein S (XopS), a T3E of Xanthomonas campestris pv. vesicatoria (Xcv), interacts with and inhibits proteasomal degradation of WRKY40, a transcriptional regulator of defense gene expression. Virus-induced gene silencing of WRKY40 in pepper (Capsicum annuum) enhanced plant tolerance to Xcv infection, indicating that WRKY40 represses immunity. Stabilization of WRKY40 by XopS reduces the expression of its targets, which include salicylic acid-responsive genes and the jasmonic acid signaling repressor JAZ8. Xcv bacteria lacking XopS display significantly reduced virulence when surface inoculated onto susceptible pepper leaves. XopS delivery by Xcv, as well as ectopic expression of XopS in Arabidopsis thaliana or Nicotiana benthamiana, prevented stomatal closure in response to bacteria and biotic elicitors. Silencing WRKY40 in pepper or N. benthamiana abolished XopS's ability to prevent stomatal closure. This suggests that XopS interferes with both preinvasion and apoplastic defense by manipulating WRKY40 stability and downstream gene expression, eventually altering phytohormone crosstalk to promote pathogen proliferation.

Deciphering the influence of Bacillus subtilis strain Ydj3 colonization on the vitamin C contents and rhizosphere microbiomes of sweet peppers.

Bacillus subtilis strain Ydj3 was applied to sweet peppers to understand the influence of this bacterium on the growth, fruit quality, and rhizosphere microbial composition of sweet pepper. The promotion of seed germination was observed for sweet pepper seeds treated with the Ydj3 strain, indicating that Ydj3 promoted seed germination and daily germination speed (131.5 ± 10.8 seeds/day) compared with the control (73.8 ± 2.5 seeds/day). Strain Ydj3 displayed chemotaxis toward root exudates from sweet pepper and could colonize the roots, which enhanced root hair growth. Following the one-per-month application of strain Ydj3 to sweet pepper grown in a commercial greenhouse, the yield, fruit weight, and vitamin C content significantly increased compared with those of the control. Additionally, the composition of the rhizosphere bacterial community of sweet pepper changed considerably, with the Bacillus genus becoming the most dominant bacterial genus in the treated group. These results suggested that B. subtilis Ydj3 promotes seed germination and enhances fruit quality, particularly the vitamin C content, of sweet pepper. These effects may be partly attributed to the B. subtilis Ydj3 colonization of sweet pepper roots due to Ydj3 chemotaxis toward root exudates, resulting in the modulation of the rhizosphere bacterial community.