Morphological, Metabolomic and Genomic Evidences on Drought Stress Protective Functioning of the Endophyte Bacillus safensis Ni7.

The metabolomic and genomic characterization of an endophytic Bacillus safensis Ni7 was carried out in this study. This strain has previously been isolated from the xerophytic plant Nerium indicum L. and reported to enhance the drought tolerance in Capsicum annuum L. seedlings. The effects of drought stress on the morphology, biofilm production, and metabolite production of B. safensis Ni7 are analyzed in the current study. From the results obtained, the organism was found to have multiple strategies such as aggregation and clumping, robust biofilm production, and increased production of surfactin homologues under the drought induced condition when compared to non-stressed condition. Further the whole genome sequencing (WGS) based analysis has demonstrated B. safensis Ni7 to have a genome size of 3,671,999 bp, N50 value of 3,527,239, and a mean G+C content of 41.58%. Interestingly the organism was observed to have the presence of various stress-responsive genes (13, 20U, 16U,160, 39, 17M, 18, 26, and ctc) and genes responsible for surfactin production (srfAA, srfAB, srfAC, and srfAD), biofilm production (epsD, epsE, epsF, epsG, epsH, epsI, epsK, epsL, epsM, epsN, and pel), chemotaxis (cheB_1, cheB_2, cheB_3, cheW_1, cheW_2 cheR, cheD, cheC, cheA, cheY, cheV, and cheB_4), flagella synthesis (flgG_1, flgG_2, flgG_3, flgC, and flgB) as supportive to the drought tolerance. Besides these, the genes responsible for plant growth promotion (PGP), including the genes for nitrogen (nasA, nasB, nasC, nasD, and nasE) and sulfur assimilation (cysL_1&L_2, cysI) and genes for phosphate solubilization (phoA, phoP_1& phoP_2, and phoR) could also be predicted. Along with the same, the genes for catalase, superoxide dismutase, protein homeostasis, cellular fitness, osmoprotectants production, and protein folding could also be predicted from its WGS data. Further pan-genome analysis with plant associated B. safensis strains available in the public databases revealed B. safensis Ni7 to have the presence of a total of 5391 gene clusters. Among these, 3207 genes were identified as core genes, 954 as shell genes and 1230 as cloud genes. This variation in gene content could be taken as an indication of evolution of strains of Bacillus safensis as per specific conditions and hence in the case of B. safensis Ni7 its role in habitat adaptation of plant is well expected. This diversity in endophytic bacterial genes may attribute its role to support the plant system to cope up with stress conditions. Overall, the study provides genomic evidence on Bacillus safensis Ni7 as a stress alleviating microbial partner in plants.

In vitro and in silico investigation of effects of antimicrobial peptides from Solanaceae plants against rice sheath blight pathogen Rhizoctinia solani.

Rhizoctonia solani, the causative agent of sheath blight disease in rice, poses a significant threat to agricultural productivity. Traditional management approaches involving chemical fungicides have been effective but come with detrimental consequences for the ecosystem. This study aimed to investigate sustainable alternatives in the form of antifungal peptides derived from Solanaceous plant species as potential agents against R. solani. Peptide extracts were obtained using an optimized antimicrobial peptide (AMP) extraction method and desalted using the solid-phase extraction technique. The antifungal potential of peptide-rich extracts from Solanum tuberosum and Capsicum annum was assessed through in vitro tests employing the agar well diffusion method. Furthermore, peptide-protein docking analysis was performed on HPEPDOCK and HDOCK server; and molecular dynamics simulations (MDS) of 100 ns period were performed using the Gromacs 2020.4. The results demonstrated significant inhibition zones for both extracts at concentrations of 100 mg/mL. Additionally, the extracts of Solanum tuberosum and Capsicum annum had minimum inhibitory concentrations of 50 mg/mL and 25 mg/mL, respectively with minimum fungicidal concentrations of 25 mg/mL. Insights into the potential mechanisms of key peptides inhibiting R. solani targets were gleaned from in-silico studies. Notably, certain AMPs exhibited favorable free energy of binding against pathogenicity-related targets, including histone demethylase, sortin nexin, and squalene synthase, in protein-peptide docking simulations. Extended molecular dynamics simulations lasting 100 ns and MM-PBSA calculations were performed on select protein-peptide complexes. AMP10 displayed the most favorable binding free energy against all target proteins, with AMP3, AMP12b, AMP6, and AMP15 also exhibiting promising results against specific targets of R. solani. These findings underscore the potential of peptide extracts from S. tuberosum and C. annum as effective antifungal agents against rice sheath blight caused by R. solani.

Identification and mechanism characterization of Streptomyces griseoaurantiacus XQ-29 with biocontrol ability against pepper southern blight caused by Sclerotium rolfsii.

Pepper southern blight, caused by Sclerotium rolfsii, is a devastating soil-borne disease resulting in significant loss to pepper, Capsicum annuum L. production. Here, we isolated an antagonistic bacterial strain XQ-29 with antifungal activity against S. rolfsii from rhizospheric soil of pepper. Combining the morphological and biochemical characteristics with the 16S rDNA sequencing, XQ-29 was identified as Streptomyces griseoaurantiacus. It exhibited an inhibition of 96.83% against S. rolfsii and displayed significant inhibitory effects on Botrytis cinerea, Phytophthora capsica and Rhizoctonia solani. Furthermore, XQ-29 significantly reduced the pepper southern blight by 100% and 70.42% during seedling and growth stages, respectively. The antifungal mechanism involved altering the mycelial morphology, disrupting cell wall and membrane integrity, accompanied by accumulation of reactive oxygen species and lipid peroxidation in S. rolfsii mycelia. Furthermore, XQ-29 promoted growth and stimulated resistance of pepper plants by increasing defense-related enzyme activities and upregulating defense-related genes. Correspondingly, XQ-29 harbors numerous functional biosynthesis gene clusters in its genome, including those for siderophores and melanin production. The metabolic constituents present in the ethyl acetate extracts, which exhibited an EC50 value of 85.48 ± 1.62 µg/mL, were identified using LC-MS. Overall, XQ-29 demonstrates significant potential as a biocontrol agent against southern blight disease.

Pepper immunity against Ralstonia solanacearum is positively regulated by CaWRKY3 through modulation of different WRKY transcription factors.

BACKGROUND: Several WRKY transcription factors (TFs), including CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40 are known to govern the resistance of pepper (Capsicum annuum L.) plants to Ralstonia solanacearum infestation (RSI) and other abiotic stresses. However, the molecular mechanisms underlying these processes remain elusive. METHODS: This study functionally described CaWRKY3 for its role in pepper immunity against RSI. The roles of phytohormones in mediating the expression levels of CaWRKY3 were investigated by subjecting pepper plants to 1 mM salicylic acid (SA), 100 µM methyl jasmonate (MeJA), and 100 µM ethylene (ETH) at 4-leaf stage. A virus-induced gene silencing (VIGS) approach based on the Tobacco Rattle Virus (TRV) was used to silence CaWRKY3 in pepper, and transiently over-expressed to infer its role against RSI. RESULTS: Phytohormones and RSI increased CaWRKY3 transcription. The transcriptions of defense-associated marker genes, including CaNPR1, CaPR1, CaDEF1, and CaHIR1 were decreased in VIGS experiment, which made pepper less resistant to RSI. Significant hypersensitive (HR)-like cell death, H2O2 buildup, and transcriptional up-regulation of immunological marker genes were noticed in pepper when CaWRKY3 was transiently overexpressed. Transcriptional activity of CaWRKY3 was increased with overexpression of CaWRKY6, CaWRKY22, CaWRKY27, and CaWRKY40, and vice versa. In contrast, Pseudomonas syringae pv tomato DC3000 (Pst DC3000) was easily repelled by the innate immune system of transgenic Arabidopsis thaliana that overexpressed CaWRKY3. The transcriptions of defense-related marker genes like AtPR1, AtPR2, and AtNPR1 were increased in CaWRKY3-overexpressing transgenic A. thaliana plants. CONCLUSION: It is concluded that CaWRKY3 favorably regulates phytohormone-mediated synergistic signaling, which controls cell death in plant and immunity of pepper plant against bacterial infections.

Isolation and evaluation of probiotics from traditional Chinese foods for aflatoxin B1 detoxification: Geotrichum candidum XG1 (yeast) and mechanistic insights.

Identifying aflatoxin-detoxifying probiotics remains a significant challenge in mitigating the risks associated with aflatoxin contamination in crops. Biological detoxification is a popular technique that reduces mycotoxin hazards and garners consumer acceptance. Through multiple rounds of screening and validation tests, Geotrichum candidum XG1 demonstrated the ability to degrade aflatoxin B1 (AFB1) by 99-100%, exceeding the capabilities of mere adsorption mechanisms. Notably, the degradation efficiency was demonstrably influenced by the presence of copper and iron ions in the liquid medium, suggesting a potential role for proteases in the degradation process. Subsequent validation experiments with red pepper revealed an 83% reduction in AFB1 levels following fermentation with G. candidum XG1. Furthermore, mass spectrometry analysis confirmed the disruption of the AFB1 furan ring structure, leading to a subsequent reduction in its toxicity. Collectively, these findings establish G. candidum XG1 as a promising candidate for effective aflatoxin degradation, with potential applications within the food industry.

Differential effects of plant-beneficial fungi on the attraction of the egg parasitoid Trissolcus basalis in response to Nezara viridula egg deposition.

There is increasing evidence that plant-associated microorganisms play important roles in defending plants against insect herbivores through both direct and indirect mechanisms. While previous research has shown that these microbes can modify the behaviour and performance of insect herbivores and their natural enemies, little is known about their effect on egg parasitoids which utilize oviposition-induced plant volatiles to locate their hosts. In this study, we investigated how root inoculation of sweet pepper (Capsicum annuum) with the plant-beneficial fungi Beauveria bassiana ARSEF 3097 or Trichoderma harzianum T22 influences the olfactory behaviour of the egg parasitoid Trissolcus basalis following egg deposition by its host Nezara viridula. Olfactometer assays showed that inoculation by T. harzianum significantly enhanced the attraction of the egg parasitoid, while B. bassiana had the opposite effect. However, no variation was observed in the chemical composition of plant volatiles. Additionally, fitness-related traits of the parasitoids (wasp body size) were not altered by any of the two fungi, suggesting that fungal inoculation did not indirectly affect host quality. Altogether, our results indicate that plant inoculation with T. harzianum T22 can be used to enhance attraction of egg parasitoids, which could be a promising strategy in manipulating early plant responses against pest species and improving sustainable crop protection. From a more fundamental point of view, our findings highlight the importance of taking into account the role of microorganisms when studying the intricate interactions between plants, herbivores and their associated egg parasitoids.

Effect of ethylene production by four pathogenic fungi on the postharvest diseases of green pepper (Capsicum annuum L.).

Ethylene produced by plants generally induces ripening and promotes decay, whereas the effect of ethylene produced by pathogens on plant diseases remains unclear. In this study, four ethylene-producing fungi including Alternaria alternata (A. alternata, Aa), Fusarium verticilliodes (F. verticillioides, Fv), Fusarium fujikuroi 1 (F. fujikuroi 1, Ff-1) and Fusarium fujikuroi 2 (F. fujikuroi 2, Ff-2) were severally inoculated in potato dextrose broth (PDB) media and postharvest green peppers, the ethylene production rates, disease indexes and chlorophyll fluorescence parameters were determined. The results showed that Ff-2 and Fv in the PDB media had the highest and almost the same ethylene production rates. After inoculation with green peppers, Ff-2 treated group still exhibited the highest ethylene production rate, whereas Aa treated group had a weak promotion effect on ethylene production. Moreover, the ethylene production rate of green peppers with mechanical injury was twice that without mechanical injury, and the ethylene production rates of green peppers treated with Aa, Ff-1, Ff-2 and Fv were 1.2, 2.6, 3.8 and 2.8 folds than those of green peppers without treatment, respectively. These results indicated that pathogen infection stimulated the synthesis of ethylene in green peppers. Correlation analysis indicated that the degreening of Fusarium-infected green pepper was significantly positively correlated with the ethylene production rate of green pepper, whereas the disease spot of Aa-infected green pepper had a significant positive correlations with the ethylene production rate of green peppers. Chlorophyll fluorescence results showed that the green peppers already suffered from severe disease after being infected with fungi for 4 days, and Fusarium infection caused early and serious stress, while the harm caused by A. alternata was relatively mild at the early stage. Our results clearly showed that α-keto-γ-methylthiobutyric acid (KMBA)-mediated ethylene synthesis was the major ethylene synthesis pathway in the four postharvest pathogenic fungi. All the results obtained suggested that ethylene might be the main infection factor of Fusarium spp. in green peppers. For pathogenic fungi, stimulating green peppers to produce high level of ethylene played a key role in the degreening of green peppers.

Paenarthrobacter aromaticivorans sp. nov., a paraoxon-degrading bacterium isolated from red pepper cultivation soil.

A bacterial strain designated MMS21-TAE1-1T, capable of degrading paraoxon, was isolated from red pepper soil (36° 25' 26.0″ N, 126° 25' 47.0″ E) and subjected to polyphasic taxonomic characterisation. MMS21-TAE1-1T was an aerobic, non-motile and Gram-stain-positive bacterium. MMS21-TAE1-1T showed growth at 10-37 °C (optimum, 30 °C), at pH 4-10 (optimum, pH 7) and in the presence of 0-6 % NaCl (optimum, 0 %). On the basis of the results of 16S rRNA gene sequence analysis, MMS21-TAE1-1T could be assigned to the genus Paenarthrobacter and shared the highest sequence similarities with Paenarthrobacter aurescens NBRC 12136T (99.72 %), then with Paenarthrobacter nitroguajacolicus G2-1T (99.65 %) and Paenarthrobacter ilicis DSM 20138T (99.17 %). However, the results of genome-based comparison using orthologous average nucleotide identity (orthoANI) and digital DNA-DNA hybridisation indicated that MMS21-TAE1-1T could be readily distinguished from all species of the genus with validly published names. The predominant menaquinone of MMS21-TAE1-1T was MK-9(H2). The diagnostic polar lipids were diphosphatidylglycerol and phosphatidylinositol, and unidentified glycolipids were also present. The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0. The chemotaxonomic properties of MMS21-TAE1-1T were generally consistent with those of members of the genus Paenarthrobacter. The genome of MMS21-TAE1-1T contained genes related to degradation of aromatic compounds. It is evident from the results of this study that strain MMS21-TAE1-1T merits recognition as representing a novel species of the genus Paenarthrobacter, for which the name Paenarthrobacter aromaticivorans sp. nov. is proposed. The type strain is MMS21-TAE1-1T (=KCTC 49652T = LMG 32368T).

Insights into the genetic architecture of Phytophthora capsici root rot resistance in chile pepper (Capsicum spp.) from multi-locus genome-wide association study.

BACKGROUND: Phytophthora root rot, a major constraint in chile pepper production worldwide, is caused by the soil-borne oomycete, Phytophthora capsici. This study aimed to detect significant regions in the Capsicum genome linked to Phytophthora root rot resistance using a panel consisting of 157 Capsicum spp. genotypes. Multi-locus genome wide association study (GWAS) was conducted using single nucleotide polymorphism (SNP) markers derived from genotyping-by-sequencing (GBS). Individual plants were separately inoculated with P. capsici isolates, 'PWB-185', 'PWB-186', and '6347', at the 4-8 leaf stage and were scored for disease symptoms up to 14-days post-inoculation. Disease scores were used to calculate disease parameters including disease severity index percentage, percent of resistant plants, area under disease progress curve, and estimated marginal means for each genotype. RESULTS: Most of the genotypes displayed root rot symptoms, whereas five accessions were completely resistant to all the isolates and displayed no symptoms of infection. A total of 55,117 SNP markers derived from GBS were used to perform multi-locus GWAS which identified 330 significant SNP markers associated with disease resistance. Of these, 56 SNP markers distributed across all the 12 chromosomes were common across the isolates, indicating association with more durable resistance. Candidate genes including nucleotide-binding site leucine-rich repeat (NBS-LRR), systemic acquired resistance (SAR8.2), and receptor-like kinase (RLKs), were identified within 0.5 Mb of the associated markers. CONCLUSIONS: Results will be used to improve resistance to Phytophthora root rot in chile pepper by the development of Kompetitive allele-specific markers (KASP®) for marker validation, genomewide selection, and marker-assisted breeding.

Revealing core functional microorganisms in the fermentation process of Qicaipaojiao (Capsicum annuum L.) based on microbial metabolic network.

Paojiao, a typical Chinese traditional fermented pepper, is favored by consumers for its unique flavor profile. Microorganisms, organic acids, amino acids, and volatile compounds are the primary constituents influencing the development of paojiao's flavor. To elucidate the key flavor compounds and core microorganisms of Qicaipaojiao (QCJ), this study conducted a comprehensive analysis of the changes in taste substances (organic acids and amino acids) and volatile flavor compounds during QCJ fermentation. Key flavor substances in QCJ were identified using threshold aroma value and odor activity value and the core microorganisms of QCJ were determined based on the correlation between dominant microorganisms and the key flavor substances. During QCJ fermentation, 16 key taste substances (12 free amino acids and 4 organic acids) and 12 key aroma substances were identified. The fermentation process involved 10 bacteria and 7 fungal genera, including Lactiplantibacillus, Leuconostoc, Klebsiella, Pichia, Wickerhamomyces, and Candida. Correlation analysis revealed that the core functional microorganisms encompassed representatives from 8 genera, including 5 bacterial genera (Lactiplantibacillus, Weissella, Leuconostoc, Klebsiella, and Kluyvera) and 3 fungal genera (Rhodotorula, Phallus, and Pichia). These core functional microorganisms exhibited significant correlations with approximately 70 % of the key flavor substances (P < 0.05). This study contributes to an enhanced understanding of flavor formation mechanisms and offers valuable insight into flavor quality control in food fermentation processes.

CaWRKY01-10 and CaWRKY08-4 Confer Pepper's Resistance to Phytophthora capsici Infection by Directly Activating a Cluster of Defense-Related Genes.

Phytophthora blight of pepper, which is caused by the notorious oomycete pathogen Phytophthora capsici, is a serious disease in global pepper production regions. Our previous study had identified two WRKY transcription factors (TFs), CaWRKY01-10 and CaWRKY08-4, which are prominent modulators in the resistant pepper line CM334 against P. capsici infection. However, their functional mechanisms and underlying signaling networks remain unknown. Herein, we determined that CaWRKY01-10 and CaWRKY08-4 are localized in plant nuclei. Transient overexpression assays indicated that both CaWRKY01-10 and CaWRKY08-4 act as positive regulators in pepper resistance to P. capsici. Besides, the stable overexpression of CaWRKY01-10 and CaWRKY08-4 in transgenic Nicotiana benthamiana plants also significantly enhanced the resistance to P. capsici. Using comprehensive approaches including RNA-seq, CUT&RUN-qPCR, and dual-luciferase reporter assays, we revealed that overexpression of CaWRKY01-10 and CaWRKY08-4 can activate the expressions of the same four Capsicum annuum defense-related genes (one PR1, two PR4, and one pathogen-related gene) by directly binding to their promoters. However, we did not observe protein-protein interactions and transcriptional amplification/inhibition effects of their shared target genes when coexpressing these two WRKY TFs. In conclusion, these data suggest that both of the resistant line specific upregulated WRKY TFs (CaWRKY01-10 and CaWRKY08-4) can confer pepper's resistance to P. capsici infection by directly activating a cluster of defense-related genes and are potentially useful for genetic improvement against Phytophthora blight of pepper and other crops.

In vitro, in planta, and comparative genomic analyses of Pseudomonas syringae pv. syringae strains of pepper (Capsicum annuum var. annuum).

Pseudomonas syringae pv. syringae (Pss) is an emerging phytopathogen that causes Pseudomonas leaf spot (PLS) disease in pepper plants. Pss can cause serious economic damage to pepper production, yet very little is known about the virulence factors carried by Pss that cause disease in pepper seedlings. In this study, Pss strains isolated from pepper plants showing PLS symptoms in Ohio between 2013 and 2021 (n = 16) showed varying degrees of virulence (Pss populations and disease symptoms on leaves) on 6-week-old pepper seedlings. In vitro studies assessing growth in nutrient-limited conditions, biofilm production, and motility also showed varying degrees of virulence, but in vitro and in planta variation in virulence between Pss strains did not correlate. Comparative whole-genome sequencing studies identified notable virulence genes including 30 biofilm genes, 87 motility genes, and 106 secretion system genes. Additionally, a total of 27 antimicrobial resistance genes were found. A multivariate correlation analysis and Scoary analysis based on variation in gene content (n = 812 variable genes) and single nucleotide polymorphisms within virulence genes identified no significant correlations with disease severity, likely due to our limited sample size. In summary, our study explored the virulence and antimicrobial gene content of Pss in pepper seedlings as a first step toward understanding the virulence and pathogenicity of Pss in pepper seedlings. Further studies with additional pepper Pss strains will facilitate defining genes in Pss that correlate with its virulence in pepper seedlings, which can facilitate the development of effective measures to control Pss in pepper and other related P. syringae pathovars. IMPORTANCE: Pseudomonas leaf spot (PLS) caused by Pseudomonas syringae pv. syringae (Pss) causes significant losses to the pepper industry. Highly virulent Pss strains under optimal environmental conditions (cool-moderate temperatures, high moisture) can cause severe necrotic lesions on pepper leaves that consequently can decrease pepper yield if the disease persists. Hence, it is important to understand the virulence mechanisms of Pss to be able to effectively control PLS in peppers. In our study, in vitro, in planta, and whole-genome sequence analyses were conducted to better understand the virulence and pathogenicity characteristics of Pss strains in peppers. Our findings fill a knowledge gap regarding potential virulence and pathogenicity characteristics of Pss in peppers, including virulence and antimicrobial gene content. Our study helps pave a path to further identify the role of specific virulence genes in causing disease in peppers, which can have implications in developing strategies to effectively control PLS in peppers.

Evaluation of sterols as markers of fungal spoilage in red pepper powder.

Red pepper powder (RPP) made from ground dried red pepper (Capsicum annuum L.) is prone to adulteration with fungal-spoiled RPP to gain unfair profits in Korea. This study aimed to investigate the effects of fungal infection on the ergosterol and phytosterol content of RPP and evaluate the potential of the sterol content as a marker for identifying fungal-spoiled RPP. Ergosterol was detected only in fungal-spoiled RPP and not in unspoiled RPP [

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.

Evolution of microbial community and the volatilome of fresh-cut chili pepper during storage under different temperature conditions: Correlation of microbiota and volatile organic compounds.

The effect of temperature conditions on the evolution of microbial communities and volatile organic compounds (VOCs) in fresh-cut chili peppers during storage was investigated. Results showed that Proteobacteria and Actinobacteriota were the dominant phyla in fresh-cut chili peppers. During storage, bacterial communities changed more dramatically than fungi. Different temperature conditions significantly affected the shift of bacteria at the genus level. At the beginning of storage, Rhodococcus, Pantoea, and Pseudomonas dominated the bacteria. However, on day 8, Pantoea and Enterobacter became the predominant genera at 5 °C and high temperatures (10, 15 °C, dynamic temperature), respectively. No significant variability in bacterial species was observed between different batches. Additionally, 140 VOCs were determined in fresh-cut chili peppers. Twenty-two VOCs were screened and could be recommended as potential spoilage markers. Based on Spearman's correlation analysis results, Enterobacter and Enterococcus were the most positive microorganisms correlated with spoilage markers.

Microstructural and physicochemical quality maintenance in green bell pepper infected with Botrytis cinerea and treated with thyme essential oil combined with carnauba wax.

Bell pepper presents rapid weight loss and is highly susceptible to gray mold caused by the fungus Botrytis cinerea. The most employed method to control this disease is the application of synthetic fungicides such as thiabendazole (TBZ); however, its continued use causes resistance in fungi as well as environmental problems. For these reasons, natural alternatives arise as a more striking option. Currently, bell pepper fruits are coated with carnauba wax (CW) to prevent weight loss and improve appearance. Moreover, CW can be used as a carrier to incorporate essential oils, and previous studies have shown that thyme essential oil (TEO) is highly effective against B. cinerea. Therefore, this study aimed to evaluate the effect of CW combined with TEO on the development of gray mold and maintenance of microestructural and postharvest quality in bell pepper stored at 13°C. The minimal inhibitory concentration of TEO was 0.5%. TEO and TBZ provoked the leakage of intracellular components. TEO and CW + TEO treatments were equally effective to inhibit the development of gray mold. On the quality parameters, firmness and weight loss were ameliorated with CW and CW + TEO treatments; whereas lightness increased in these treatments. The structural analysis showed that CW + TEO treatment maintained the cell structure reducing the apparition of deformities. The results suggest that CW + TEO treatment could be used as a natural and effective antifungal retarding the appearance of gray mold and maintaining the postharvest quality of bell pepper. PRACTICAL APPLICATION: CW and TEO are classified as generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). This combination can be employed on the bell pepper packaging system to extend shelf life and oppose gray mold developments. Bell pepper fruits are normally coated with lipid-base coatings such as CW before commercialization; therefore, TEO addition would represent a small investment without any changes on the packaging system infrastructure.

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.

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.

Defensin-like peptides from Capsicum chinense induce increased ROS, loss of mitochondrial functionality, and reduced growth of the fungus Colletotrichum scovillei.

In the present study, we identified and characterized two defensin-like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed-phase in a high-performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse-phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1-Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 µg mL-1, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 µg mL-1 and 200 µg mL-1, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1-Fraction indicated the presence of two defensin-like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1-Fraction was also found to inhibit the activity of insect α-amylases. In summary, the F1-Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin-like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.