Sympatric occurrence of sibling Phytophthora species associated with foot rot disease of black pepper in India.

Foot rot disease caused by Phytophthora capsici is a serious threat to black pepper cultivation in India and globally. High diversity exists among the Phytophthora isolates of black pepper and hence detailed investigations of their morphology and phylogenetic taxonomy were carried out in the present study. In order to resolve the diversity, 182 isolates of Phytophthora, collected from different black pepper-growing tracts of South India during 1998-2013 and maintained in the National Repository of Phytophthora at ICAR-Indian Institute of Spices Research, Kozhikode, were subjected to morphological, molecular and phylogenetic characterization. Morphologically all the isolates were long pedicellate with umbellate/simple sympodial sporangiophores and papillate sporangia with l/b ranging from 1.63 to 2.55 µm. Maximum temperature for the growth was ~ 34 °C. Chlamydospores were observed in "tropicalis" group, whereas they were absent in "capsici" group. Initial molecular studies using internal transcribed spacer (ITS) marker gene showed two clear cut lineages-"capsici-like" and "tropicalis-like" groups among them. Representative isolates from each group were subjected to host differential test, multilocus sequence typing (MLST) and phylogeny studies. MLST analysis of seven nuclear genes (60S ribosomal protein L10, beta-tubulin, elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA and TigA gene fusion protein) clearly delineated black pepper Phytophthora isolates into two distinct species-P. capsici and P. tropicalis. On comparing with type strains from ATCC, it was found that the type strains of P. capsici and P. tropicalis differed from black pepper isolates in their infectivity on black pepper. The high degree of genetic polymorphism observed in black pepper Phytophthora isolates is an indication of the selection pressure they are subjected to in the complex habitat which ultimately may lead to speciation. So based on the extensive analysis, it is unambiguously proved that the foot rot disease of black pepper in India is predominantly caused by two species of Phytophthora, viz. P. capsici and P. tropicalis. Presence of multiple species of Phytophthora in the black pepper agro-ecosystem warrants a revisit to the control strategy being adopted for managing this serious disease. The silent molecular evolution taking place in such an ecological niche needs to be critically studied for the sustainable management of foot rot disease.

Determination of pepper microbial contamination for low energy e-beam irradiation.

Electrons with energies of 300 keV or lower have the potential to decontaminate the surfaces of various types of food products with minimal loss of quality. The aim of the present work was to determine the thickness of the layer inhabited by microorganisms. The food samples tested were black and white pepper irradiated with 200 keV, 230 keV, 300 keV and 9 MeV beams of electron energy. To determine the depth from the surface which can be inhabited by microorganisms two approaches were tested. The methods used were based on the application of different microbiological recovery techniques and the microbial effectiveness of the irradiation process depending on the energy of the electron beam. It was observed that the layer which microorganisms may contaminate differed for the tested samples it was estimated as being below 100 µm thick for white pepper and about 200 µm for black pepper. The penetration ability was significant in experiments performed, and as a result the electron beam at the lowest levels tested (200 and 230 keV) was found to be insufficient to effectively decontaminate the black pepper samples. The beam of energy 300 keV was found to have a similar microbial inactivation effect as the high energy electron beam (9 MeV).

Efficacy of bioagents against Pythium deliense Meurs associated with yellowing of black pepper.

Yellowing and wilting of black pepper vines is a serious concern in many black pepper growing tracts where Pythium deliense was recently emerged as a pathogen from the rhizosphere of affected vines, which is proved to be pathogenic by Koch's postulates. As a measure to manage the symptoms, bioagents were evaluated against infection by P. deliense. Among the seven bioagents tested, Trichoderma harzianum and Streptomyces albulus showed 100% inhibition in vitro followed by one Streptomyces sp. and S. rimosus (75.33%). The potential ones were further evaluated under the hydroponic system in vivo by challenge inoculation. No root infection was noticed with T. harzianum and S. albulus inoculation, instead, the inoculated plants showed root regeneration. This suggests the efficiency of these bioagents on plant growth promotion as well as on disease suppression. Biochemical analysis of the hydroponic medium showed an increase in membrane conductivity in all the treatments except in T. harzianum. The release of phenolic compounds into the medium was lowest with T. harzianum indicating the prevention of pathogen invasion. In planta evaluation under greenhouse condition and field evaluation also showed the protective effect of T. harzianum and S. albulus with a reduction in the intensity of yellowing to an extent of 73.1% and 71.2%, respectively. The study revealed that T. harzianum and the actinomycete S. albulus had the potential to prevent the root rot caused by P. deliense.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella in ground black pepper at different water activities.

Thermal inactivation kinetics of Salmonella in low moisture foods are necessary for developing proper thermal processing parameters for pasteurization. The effect of water activity on thermal inactivation kinetics of Salmonella and Enterococcus faecium NRRL B-2354 in ground black pepper has not been studied previously. Identification of a suitable surrogate assists in conducting in-plant process validations. Ground black pepper was inoculated with a 5-serotype Salmonella cocktail or E. faecium NRRL B-2354, equilibrated to water activities of 0.25, 0.45 or 0.65 in a humidity-controlled chamber, and isothermally treated at different temperatures. The survivor data were used for fitting the log-linear models to obtain the D and z-values of Salmonella and E. faecium in ground black pepper. Modified Bigelow models were developed to evaluate the effects of temperature and water activity on the thermal inactivation kinetics of Salmonella and E. faecium. Water activity and temperature showed significant negative effects on the thermal resistance of Salmonella and E. faecium in ground black pepper. For example, significantly higher D values of Salmonella were observed at water activity of 0.45 (D70°C = 20.5 min and D75°C = 7.8 min) compared to water activity of 0.65 (D70°C = 3.9 min and D75°C = 2.0 min). D-values of E. faecium were significantly higher than those of Salmonella at all three water activities, indicating that E. faecium is a suitable surrogate for Salmonella in thermal processing validation.

A quasimetagenomics method for concerted detection and subtyping of Salmonella enterica and E. coli O157:H7 from romaine lettuce.

Quasimetagenomics refers to the sequencing of a modified food microbiome to facilitate combined detection and subtyping of targeted pathogens in a single workflow. Through quasimetagenomic sequencing, pathogens are detected and subtyped in a shortened time frame compared to traditional culture enrichment and whole genome sequencing-based analyses. While this method was previously used to detect and subtype Salmonella enterica from chicken, iceberg lettuce, and black pepper, it has not been applied to investigate multiple pathogens in one workflow. A quasimetagenomic method to concertedly detect and subtype Salmonella enterica and Escherichia coli O157:H7 from artificially contaminated romaine lettuce in a single workflow was developed. All quasimetagenomic samples with initial target pathogen inoculum levels of ~1 CFU/g were detected and serotyped after co-enrichment of the two pathogens for 12 h. Single nucleotide polymorphism typing was achievable for some initial pathogen inoculum levels as low as ~0.1 CFU/g. Our results suggest that this method can be used for concerted detection and subtyping of multiple bacterial pathogens from romaine lettuce even at low contamination levels.

Gaseous antimicrobial treatments to control foodborne pathogens on almond kernels and whole black peppercorns.

Gaseous treatments with ClO2 and O3 on low-moisture foods (LMFs) have been reported for their efficient bacterial reduction without affecting the external quality of food. However, these studies were conducted on a small scale, which limits their application to LMF industries. We aimed to evaluate the effectiveness of gaseous antimicrobial intervention with ClO2 or O3 to reduce foodborne pathogens (Shiga toxin-producing Escherichia coli, serovars of Salmonella enterica, and Listeria monocytogenes) inoculated on almonds and peppercorns maintained under various conditions. Almonds were treated for over 4 or 6 h. Peppercorns were treated for over 2.5 or 4 h. Gaseous O3 treatment was used for 6 h on almonds and 2 or 4 h on peppercorns. Additionally, the effects of relative humidity (RH) during the treatment of peppercorns and post-treatment heating on almonds were evaluated. Heating at 65 °C post-ClO2 treatment yielded the highest bacterial log reduction of 4.6 CFU/g on almonds, while 80% RH resulted in 3.7-log bacterial reduction on peppercorns. Gaseous O3 resulted in maximum log reductions of 1.3 and 2.5 CFU/g on almonds and peppercorns, respectively. No visual damage was observed. In conclusion, ClO2 was more efficient than O3 and the treatment can be incorporated into industrial practices.

Plant growth-promoting bacteria as potential bio-inoculants and biocontrol agents to promote black pepper plant cultivation.

Black pepper production in Malaysia was restricted by various diseases. Hazardous chemical products appear to be the best solution to control diseases in black pepper cultivation. However, persistence of chemical residues in peppercorns could affect the quality of exports and consumptions. Application of fertilizers is crucial to sustain pepper growth and high yield. But, continuous use of chemical fertilizers could affect the soil ecosystem and eventually restrict nutrient uptake by pepper roots. Therefore, we propose biological approaches as an alternative solution instead of chemical products to sustain pepper cultivation in Malaysia. In this study, we have isolated a total of seven indigenous rhizobacteria antagonistic to soil-borne Fusarium solani, the causal fungus of slow decline, the most serious debilitating disease of black pepper in Malaysia. The isolated bacteria were identified as Bacillus subtilis, Bacillus siamensis, Brevibacillus gelatini, Pseudomonas geniculata, Pseudomonas beteli, Burkholderia ubonensis and Burkholderia territorii. These bacteria were effective in production of antifungal siderophore with the amount of 53.4 %-73.5 % per 0.5 mL of cell-free supernatants. The bacteria also produced appreciable amount of chitinase with chitinolytic index was ranged from 1.19 to 1.76. The bacteria have shown phosphate solubilizing index within 1.61 to 2.01. They were also efficient in ACC deaminase (0.52 mM-0.62 mM) and ammonia (60.3 mM-75.3 mM) production. The isolated antagonists were efficacious in stimulation of black pepper plant growth and root development through IAA (10.5 µg/mL-42.6 µg/mL) secretion. In conclusion, the isolated rhizobacteria are potent to be developed not only as biocontrol agents to minimize the utilization of hazardous chemicals in black pepper disease management, but also developed as bio-fertilizers to improve black pepper plant growth due to their capabilities in plant growth-promotion.

Transcriptomic analysis of pepper plants provides insights into host responses to Fusarium solani infestation.

Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation.

Correlação estatística entre parâmetros microscópicos e microbiológicos em pimenta-do-reino moída (Piper nigrum L) comercializada em Minas Gerais / Statistical correlation between microscopic and microbiological parameters in ground black pepper (Pipper nigrum L) sold in Minas Gerais

A ausência de Boas Práticas de Fabricação durante a colheita, processamento e manuseio da pimenta-do-reino pode acarretar a contaminação do produto com sujidades microscópicas e microrganismos. Foram analisadas 227 amostras de pimenta-do-reino, comercializadas em Minas Gerais, coletadas entre 2008 e 2018, quanto à presença de fragmentos de pelo de roedor e de insetos, coliformes a 45°C ou Escherichia coli e Salmonella spp. Para verificar se havia correlação entre os contaminantes, foi empregado método estatístico de regressão linear múltipla. As análises microscópicas evidenciaram presença de fragmentos de pelo de roedor e de insetos em 26,0% e 30,5% das amostras, respectivamente, em valores superiores ao limite tolerado pela RDC 14/2014. Quanto às análises microbiológicas, 10% das amostras apresentaram coliformes a 45°C ou E. coli acima dos limites tolerados pela RDC 12/2001 e em 8,8% das amostras foi detectada presença de Salmonella spp. A avaliação estatística mostrou que houve correlação entre presença de fragmentos de insetos e de pelos de roedor e a contaminação por Salmonella spp. em pimenta-do-reino. Os resultados demonstraram a importância das análises microscópica e microbiológica simultaneamente para detecção dos contaminantes presentes bem como das possíveis relações existentes entre eles e a melhor compreensão dos fatores que favorecem as contaminações.

The absence of Good Manufacturing Practices during harvesting, processing and handling of black pepper can lead to contamination of the product with microscopic dirt and microrganisms. Two hundred and twenty seven black pepper samples, commercialized in Minas Gerais, collected between 2008 and 2018, were analyzed for the presence of rodent and insect fragments, coliforms at 45°C or Escherichia coli and Salmonella spp. To verify whether there was a correlation between the contaminants, a statistical method of multiple linear regression was used. Microscopic analysis showed the presence of rodent and insect fragments in 26.0% and 30.5% of the samples, respectively, in values above the limit tolerated by RDC14/2014. As for the microbiological analysis, 10% of the samples presented coliforms at 45°C or E.coli above the limits tolerated by the RDC 12/2001 and in 8.8% of the samples Salmonella spp. were detected. The statistical evaluation showed that there was a relationship between the presence of insect fragments and rodent hair and contamination by Salmonella spp. in black pepper. The results demonstrated the importance of simultaneous microscopic and microbiological analysis to detect the contaminants present, as well as the possible relationships between them and better understanding of the factors that favor contamination.

Efficacy of pulsed-ultraviolet light for inactivation of Salmonella spp on black peppercorns.

Efficacy of pulsed ultraviolet (PUV) to inactivate Salmonella pure culture and on inoculated black peppercorns was evaluated. Black peppercorns inoculated with Salmonella were subjected to PUV treatment (0.28 J/cm2 /pulse) using two different sample holders, on a traditional flat surface or on a wave-shaped surface to increase surface exposure of peppercorns to PUV through light reflection. The temperature change on black peppercorns surface during treatment was recorded, and the effect of cooling period during PUV treatment was studied. PUV treatment of two pulses reduced Salmonella population by more than 6 log CFU/mL in phosphate-buffered saline. Continuous PUV treatment (80 pulses on each side) using a wave-shaped surface was able to reduce Salmonella by 1.9 log CFU/g; same treatment using flat surface reduced Salmonella by less than 1.5 log CFU/g. The temperature on peppercorns surface increased to 65 °C after 80 pulses continuous PUV treatment. Adding 280 s cooling time after every 20 pulses reduced the temperature from 65 to 40 °C and achieved similar Salmonella inactivation (P > 0.05) as the continuous PUV treatment. Results from this study showcase the effectiveness of PUV treatment for reducing Salmonella level on black peppercorns surface and provided insights on the potential implementation of PUV treatment at the industrial level. PRACTICAL APPLICATION: Results from this study showcased the effectiveness of PUV treatment for reducing Salmonella level on black peppercorns surface and provided insights on the potential implementation of PUV treatment at the industrial level.

Genetic diversity and biocontrol efficacy of indigenous Trichoderma isolates against Fusarium wilt of pepper.

Trichoderma species are recognized as biocontrol agents with great potential in inhibiting fungal pathogens that cause significant crop losses. In this study, 15 Trichoderma isolates were collected from various Egyptian locations. Internal transcribed spacer sequencing revealed four different Trichoderma species; Trichoderma harzianum, Trichoderma asperellum, Trichoderma longibrachiatum, and Trichoderma viride. The antagonistic activity of Trichoderma isolates against Fusarium oxysporum f. sp. capsici was evaluated in vitro. The effect of Trichoderma isolates on pepper growth plants in the presence of F. oxysporum was studied in planta. The inhibition of pathogen mycelial growth in vitro ranged between 35.71% and 85.75%. The isolates Ta3 and Tl had the highest antagonistic ability in vitro against F. oxysporum f. sp. capsici. However, Th7 and Th6 of T. harzianum isolates showed the highest values of disease severity reduction under greenhouse conditions. The genetic diversity of the Trichoderma isolates (Ta1, Ta2, Th1, Th2, Th3, Th4, Th5, and Tv) was investigated on the basis of ISSR and SCoT markers. SCoT primers generated a total of 28 bands, out of which 14 (50%) were polymorphic. ISSR primers gave 32 bands, and 11 of these bands (34.37%) were polymorphic.

Survival kinetics of Listeria monocytogenes on chickpeas, sesame seeds, pine nuts, and black pepper as affected by relative humidity storage conditions.

Nuts and seeds have been increasingly associated with recalls due to contamination with Listeria monocytogenes. Storage of these food commodities occurs at various relative humidity (RH) conditions for months or years. The objective of this study was to assess L. monocytogenes survival on four commodities representing dried legumes, seeds, and spices categories: chickpeas, sesame seeds, pine nuts, and black pepper kernels. Inoculated products at 10 log CFU/g were stored for 180 days (6 months) at 25°C and different relative humidity (RH) levels: 25% (low), 45% (ambient), and 75% (high). After 180 days at 25% RH, L. monocytogenes populations decreased to 2.67-6.59 log CFU/g; the highest survival of the pathogen was observed on pine nuts and sesame seeds with decay rates of -0.014± 0.001 log CFU/g per d. Significantly greater population reductions on all products were observed during storage at 45 and 75% RH. At 45% RH, L. monocytogenes levels decreased to 1.90-6.36 log CFU/g. On chickpeas and black pepper stored at 75% RH, the pathogen population decreased to below the limit of enumeration (1 log CFU/g) yet were still detected via enrichments. The lowest survival of L. monocytogenes occurred at 75% RH on black pepper with a decay rate of -0.058±0.003 log CFU/g per d. Overall, regardless of RH level, the ability of the products to support survival of the pathogen may be expressed in the following order: pine nuts > sesame seeds > chickpeas > black pepper. The results of this study can aid in understanding how L. monocytogenes survives on dried legumes, seeds, and spices, and the data can contribute to the risk assessment of this pathogen.

The CaAP2/ERF064 Regulates Dual Functions in Pepper: Plant Cell Death and Resistance to Phytophthora capsici.

Phytophthora blight is one of the most destructive diseases of pepper (Capsicum annuum L.) globally. The APETALA2/Ethylene Responsive Factors (AP2/ERF) genes play a crucial role in plant response to biotic stresses but, to date, have not been studied in the context of Phytophthora resistance in pepper. Here, we documented potential roles for the pepper CaAP2/ERF064 gene in inducing cell death and conferring resistance to Phytophthora capsici (P. capsici) infection. Results revealed that the N-terminal, AP2 domain, and C-terminal of CaAP2/ERF064 protein is responsible for triggering cell death in Nicotiana benthamiana (N. benthamiana). Moreover, the transcription of CaAP2/ERF064 in plant is synergistically regulated by the Methyl-Jasmonate (MeJA) and ethephon (ET) signaling pathway. CaAP2/ERF064 was found to regulate the expression of CaBPR1, which is a pathogenesis-related (PR) gene of pepper. Furthermore, the silencing of CaAP2/ERF064 compromised the pepper plant resistance to P.capsici by reducing the transcript level of defense-related genes CaBPR1, CaPO2, and CaSAR82, while the ectopic expression of CaAP2/ERF064 in N. benthamiana plant elevated the expression level of NbPR1b and enhanced resistance to P.capsici. These results suggest that CaAP2/ERF064 could positively regulate the defense response against P. capsici by modulating the transcription of PR genes in the plant.

Veiculação de bactérias de importância alimentar em especiarias comercializadas em feira livre

O objetivo desse trabalho foi analisar a qualidade microbiológica das especiariascanela, orégano e pimenta do reino comercializadas na feira livre de Cruz das Almas no Recôncavo da Bahia, por meio da quantificação de coliformes a 45°C, Staphylococcus spp., Staphylococcus coagulase positiva, Bacillus spp., Escherichia coli, Bacillus cereus e Salmonella spp. A pimenta do reino diferiu estatísticamente (p>0,05) apresentando maior contagem quando comparado com a canela e o orégano. A prevalência de S. coagulase positiva foi de 60%, 57% e 47% para pimenta do reino moída, orégano e canela, respectivamente. E. coli e Salmonella estiveram presentes na pimenta do reino em 94% e 20%, respectivamente. A contagem de coliformes a 45ºC na pimenta foi maior (3,4 log NMP. g-1) em relação a canela (0,5 log NMP.g-1) e orégano (ausência). A pimenta do reino moída apresentou alta carga microbiana com patógenos de importância alimentar como Salmonella spp., Staphylococcus coagulase positiva e B. cereus.

Radiofrequency pasteurization process for inactivation of Salmonella spp. and Enterococcus faecium NRRL B-2354 on ground black pepper.

Salmonella persistence in ground black pepper has caused several foodborne outbreaks and created public concern about the safety of low water activity (aw) foods. In this study, radiofrequency (RF) processing was evaluated for pasteurization of ground black pepper. Stability and homogeneity tests were done for both Salmonella spp. and E. faecium during moisture equilibration before RF heating to evaluate the inoculation method. Moisture content of samples were conditioned such that the final moisture content after RF heating reached the optimal storage moisture. RF heating was shown to provide more than 5.98 log CFU/g reduction for Salmonella spp. and the reduction of 3.89 log CFU/g for E. faecium with a 130 s of treatment time. The higher thermal resistance of E. faecium indicated its suitability as surrogate for Salmonella spp. during RF heating of ground black pepper. Piperine, total phenolics, volatile compounds, and antioxidant activity were assessed as quality parameters for ground black pepper. The results demonstrated that the RF processing provided effective inactivation of Salmonella spp. with insignificant (p > 0.05) quality deterioration.

Secondary metabolite profiles of small-spored Alternaria support the new phylogenetic organization of the genus.

The group of the small-spored Alternaria species is particularly relevant in foods due to its high frequency and wide distribution in different crops. These species are responsible for the accumulation of mycotoxins and bioactive secondary metabolites in food. The taxonomy of the genus has been recently revised with particular attention on them; several morphospecies within this group cannot be segregated by phylogenetic methods, and the most recent classifications proposed to elevate several phylogenetic species-groups to the taxonomic status of section. The purpose of the present study was to compare the new taxonomic revisions in Alternaria with secondary metabolite profiles with special focus on sections Alternaria and Infectoriae and food safety. A total of 360 small-spored Alternaria isolates from Argentinean food crops (tomato fruit, pepper fruit, blueberry, apple, wheat grain, walnut, pear, and plum) was morphologically identified to species-group according to Simmons (2007), and their secondary metabolite profile was determined. The isolates belonged to A. infectoria sp.-grp. (19), A. tenuissima sp.-grp. (262), A. arborescens sp.-grp. (40), and A. alternata sp.-grp. (7); 32 isolates, presenting characteristics overlapping between the last three groups, were classified as Alternaria sp. A high chemical diversity was observed; 78 different metabolites were detected, 31 of them of known chemical structure. The isolates from A. infectoria sp.-grp. (=Alternaria section Infectoriae) presented a specific secondary metabolite profile, different from the other species-groups. Infectopyrones, novae-zelandins and phomapyrones were the most frequent metabolites produced by section Infectoriae. Altertoxin-I and alterperylenol were the only compounds that these isolates produced in common with members of section Alternaria. None of the well-known Alternaria toxins, considered relevant in foods, namely alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN) or altenuene (ALT), were produced by isolates of this section. On the other hand, strains from section Alternaria (A. tenuissima, A. arborescens, and A. alternata sp.-grps.) shared a common metabolite profile, indistinguishable from each other. AOH, AME, ALT, TEN, and TeA were the most frequently mycotoxins produced, together with pyrenochaetic acid A and altechromone A. Alternaria section Alternaria represents a substantial risk in food, since their members in all types of crops are able to produce the toxic metabolites.

Radio-Frequency Processing for Inactivation of Salmonella enterica and Enterococcus faecium NRRL B-2354 in Black Peppercorn.

Several Salmonella outbreaks linked to black pepper call for effective inactivation processes, because current decontamination methods result in quality deterioration. Radio-frequency (RF) heating provides a rapid heating rate and volumetric heating, resulting in a shorter come-up time. This allows for choosing a high-temperature and short-time combination to achieve the desired inactivation with minimal quality deterioration. The objectives of this study were to evaluate RF heating for inactivation of Salmonella enterica and Enterococcus faecium in black peppercorn and evaluate quality changes of RF-treated black peppercorn. Black peppercorns were inoculated with a five-strain cocktail of Salmonella or E. faecium to attain initial population levels of 6.8 and 7.3 log CFU/g, respectively, and were then adjusted to a moisture content of 12.7% (wet basis) and a water activity of 0.60 at room temperature. A stability test was performed to quantify the microbial reduction during inoculation and equilibration before RF heating inactivation. During RF heating, the cold spot was determined to be at the center on the top surface of the treated sample. In addition to inoculating the entire sample, an inoculated packed sample was placed at the cold spot of the tray. An RF heating time of 2.5 min provided a 5.31- and 5.26-log CFU/g reduction in the entire sample contained in the tray for Salmonella and E. faecium, respectively. Color parameters (L*, a*, b*), piperine content, total phenolics, scavenging activity, and most of the volatile compounds of 2.5-min RF-treated samples were not significantly different from those of the control samples. These data suggest that RF heating is a promising thermal inactivation treatment for Salmonella without significant quality deterioration, and E. faecium seems to be a suitable surrogate for Salmonella to validate the efficacy of RF heating of black peppercorn.

Trichoderma harzianum MTCC 5179 impacts the population and functional dynamics of microbial community in the rhizosphere of black pepper (Piper nigrum L )

Abstract Employing Illumina Hiseq whole genome metagenome sequencing approach, we studied the impact of Trichoderma harzianum on altering the microbial community and its functional dynamics in the rhizhosphere soil of black pepper (Piper nigrum L.). The metagenomic datasets from the rhizosphere with (treatment) and without (control) T. harzianum inoculation were annotated using dual approach, i.e., stand alone and MG-RAST. The probiotic application of T. harzianum in the rhizhosphere soil of black pepper impacted the population dynamics of rhizosphere bacteria, archae, eukaryote as reflected through the selective recruitment of bacteria [Acidobacteriaceae bacterium (p = 1.24e-12), Candidatus koribacter versatilis (p = 2.66e-10)] and fungi [(Fusarium oxysporum (p = 0.013), Talaromyces stipitatus (p = 0.219) and Pestalotiopsis fici (p = 0.443)] in terms of abundance in population and bacterial chemotaxis (p = 0.012), iron metabolism (p = 2.97e-5) with the reduction in abundance for pathogenicity islands (p = 7.30e-3), phages and prophages (p = 7.30e-3) with regard to functional abundance. Interestingly, it was found that the enriched functional metagenomic signatures on phytoremediation such as benzoate transport and degradation (p = 2.34e-4), and degradation of heterocyclic aromatic compounds (p = 3.59e-13) in the treatment influenced the rhizosphere micro ecosystem favoring growth and health of pepper plant. The population dynamics and functional richness of rhizosphere ecosystem in black pepper influenced by the treatment with T. harzianum provides the ecological importance of T. harzianum in the cultivation of black pepper.

Genome-wide identification of the auxin/indole-3-acetic acid (Aux/IAA) gene family in pepper, its characterisation, and comprehensive expression profiling under environmental and phytohormones stress.

Auxin is an essential phytohormone that plays a crucial role in the growth and development of plants in stressful environments. Here, we analysed the auxin/indole-3-acetic acid (Aux/IAA) gene family, which produces auxin in pepper, and succeeded in identifying 27 putative members containing four conserved domains (I. II. III and IV) in their protein sequences. Sequence analysis, chromosomal mapping and motif prediction of all identified CaAux/IAA genes were performed. It was observed that these genes contained four conserved motifs divided into nine different groups and distributed across nine chromosomes in pepper plants. RNA-seq analysis revealed the organ specific expression of many CaAux/IAA genes. However, the majority of genes were expressed with high expression levels in the early stages of fruit development. However, the maximum expression level of the CA03g34540 gene was observed in the breaker stage. Moreover, thirteen CaAux/IAA genes were labelled as early responsive genes to various phytohormone and abiotic stresses. Furthermore, RNA-seq analysis in response to pathogen inoculation (PepMoV, TMV strains P0/P1, and Phytophthora capsici) showed distinct expression profiles of all identified genes, suggesting the diverse expression nature of genes under these stress conditions. Overall, this study provides insight into the dynamic response of CaAux/IAA genes under environmental and phytohormones stress conditions, providing bases to further explore the importance of these genes through mutant/transgenic analysis in pepper.

Cold atmospheric pressure ambient air plasma inhibition of pathogenic bacteria on the surface of black pepper.

In present study, the inhibition effect of low temperature plasma on Bacillus subtilis, Escherichia coli, Salmonella Enteritidis and B. subtilis endospores inoculated on the surface of black peppercorns was studied. Plasma was generated by Diffuse Coplanar Surface Barrier Discharge (DCSBD) at atmospheric pressure in ambient air. Plasma treatment time of 300 s led to log10 CFU/g reduction of B. subtilis from 7.36 to 2.30 and B. subtilis endospores from 4.42 to 2.39. Plasma treatment reduced the number of E. coli and Salmonella Enteritidis to below detection level (1.0 log10 CFU/g) from initial populations of 7.45 log10 CFU/g and 7.60 log10 CFU/g, respectively. The inactivation kinetics was explained by Weibull model. Decimal reduction times (D-values) for B. subtilis, E. coli, Salmonella Enteritidis, and B. subtilis endospores were determined as 43 s, 47 s, 58 s, and 142 s, respectively. The surface morphology observed by Scanning Electron Microscopy showed no significant changes after the plasma treatment. The influence of plasma on chemical bonds on the surface and inside the peppercorns was studied by Attenuated Total Reflectance - Fourier Transform Infrared Spectroscopy.