A Genome-Wide Analysis of Pathogenesis-Related Protein-1 (PR-1) Genes from Piper nigrum Reveals Its Critical Role during Phytophthora capsici Infection.

Black pepper (Piper nigrum L.) is a prominent spice that is an indispensable ingredient in cuisine and traditional medicine. Phytophthora capsici, the causative agent of footrot disease, causes a drastic constraint in P. nigrum cultivation and productivity. To counterattack various biotic and abiotic stresses, plants employ a broad array of mechanisms that includes the accumulation of pathogenesis-related (PR) proteins. Through a genome-wide survey, eleven PR-1 genes that belong to a CAP superfamily protein with a caveolin-binding motif (CBM) and a CAP-derived peptide (CAPE) were identified from P. nigrum. Despite the critical functional domains, PnPR-1 homologs differ in their signal peptide motifs and core amino acid composition in the functional protein domains. The conserved motifs of PnPR-1 proteins were identified using MEME. Most of the PnPR-1 proteins were basic in nature. Secondary and 3D structure analyses of the PnPR-1 proteins were also predicted, which may be linked to a functional role in P. nigrum. The GO and KEGG functional annotations predicted their function in the defense responses of plant-pathogen interactions. Furthermore, a transcriptome-assisted FPKM analysis revealed PnPR-1 genes mapped to the P. nigrum-P. capsici interaction pathway. An altered expression pattern was detected for PnPR-1 transcripts among which a significant upregulation was noted for basic PnPR-1 genes such as CL10113.C1 and Unigene17664. The drastic variation in the transcript levels of CL10113.C1 was further validated through qRT-PCR and it showed a significant upregulation in infected leaf samples compared with the control. A subsequent analysis revealed the structural details, phylogenetic relationships, conserved sequence motifs and critical cis-regulatory elements of PnPR-1 genes. This is the first genome-wide study that identified the role of PR-1 genes during P. nigrum-P. capsici interactions. The detailed in silico experimental analysis revealed the vital role of PnPR-1 genes in regulating the first layer of defense towards a P. capsici infection in Panniyur-1 plants.

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.

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.

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.

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

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.

Regulation of Black Pepper Inflorescence Quantity by Shading at Different Growth Stages.

Black pepper is a perennial plant that can bloom throughout the year. It is generally expected that pepper inflorescence quantity could be minimized at the nonfull-bloom stage. The objective of this study was to find an appropriate shading measure that could inhibit blooming at other growing stages except the full-bloom stage and did not cause any reduction in pepper yield and quality. In this study, pepper trees were shaded up to 15%, 30%, 60% and 75%, respectively, and the inflorescence quantity, photosynthetic characteristics, pepper yield and quality traits were investigated at every growing stage. The results showed that the effect of shading on pepper yield decreased as time progressed. Shading treatment did not alter the composition of piperine and volatile oil, but reduced the moisture content. Based on the correlation between photosynthetic parameter and inflorescence number, the appropriate shading intensities for regulating inflorescence quantity at different phenological stages were determined. Moreover, it was found that the regulation of inflorescence quantity could be achieved by controlling leaf temperature during recovery to filling period. This research outcome also will give us some guidelines to develop other management strategies that control leaf temperature and regulate inflorescence quantity to consequently improve pepper yield.

Protocols for Improvement of Black Pepper (Piper nigrum L.) Utilizing Biotechnological Tools.

Black pepper, Piper nigrum L., the "King of spices" is the most widely used spice growing in the South-Western region of India. The humid tropical evergreen forest bordering the Malabar Coast (Western Ghats is one of the hot spot areas of plant bio-diversity on earth) is its center of origin and diversity. However, the crop faces constraints like rampant fungal and viral diseases, lack of disease free planting material, hence biotechnological tools can be utilized to address these problems and strides have been made successfully. The standardization of micropropagation, somatic embryogenesis, in vitro conservation, protoplast isolation, and genetic transformation protocols are described here. The protocols could be utilized to achieve similar goals in the related species of Piper too.

Integrated plant nutrient system - with special emphasis on mineral nutriton and biofertilizers for Black pepper and cardamom - A review.

Integrated Plant Nutrition System (IPNS) as a concept and farm management strategy embraces and transcends from single season crop fertilization efforts to planning and management of plant nutrients in crop rotations and farming systems on a long-term basis for enhanced productivity, profitability and sustainability. It is estimated that about two-thirds of the required increase in crop production in developing countries will have to come from yield increases from lands already under cultivation. IPNS enhances soil productivity through a balanced use of soil nutrients, chemical fertilizers, combined with organic sources of plant nutrients, including bio-inoculants and nutrient transfer through agro-forestry systems and has adaptation to farming systems in both irrigated and rainfed agriculture. Horticultural crops, mainly plantation crops, management practices include application of fertilizers and pesticides which become inevitable due to the depletion of soil organic matter and incidence of pests and diseases. The extensive use of chemical fertilizers in these crops deteriorated soil health that in turn affected the productivity. To revitalize soil health and to enhance productivity, it is inexorable to enrich the soil using microorganisms. The lacunae observed here is the lack of exploitation of indigenous microbes having the potential to fix atmospheric nitrogen (N) and to solubilize Phosphorus (P) and Potassium (K). The concept of biofertilizer application appears to be technically simple and financially feasible, but the task of developing biofertilizers with efficient strains in appropriate combinations in a consortia mode is not easier. More than developing consortia, a suitable delivery system to discharge the microbial inoculants warranted much effort. This review focuses on the integrated plant nutrition system incorporating biofertilizer with special emphasis on developing and formulating biofertilizer consortium.

The Effect of Long-Term Continuous Cropping of Black Pepper on Soil Bacterial Communities as Determined by 454 Pyrosequencing.

In the present study, 3 replanted black pepper orchards with continuously cropping histories for 10, 21, and 55 years in tropical China, were selected for investigating the effect of monoculture on soil physiochemical properties, enzyme activities, bacterial abundance, and bacterial community structures. Results showed long-term continuous cropping led to a significant decline in soil pH, organic matter contents, enzymatic activities, and resulted in a decrease in soil bacterial abundance. 454 pyrosequencing analysis of 16S rRNA genes revealed that the Acidobacteria and Proteobacteria were the main phyla in the replanted black pepper orchard soils, comprising up to 73.82% of the total sequences; the relative abundances of Bacteroidetes and Firmicutes phyla decreased with long-term continuous cropping; and at genus level, the Pseudomonas abundance significantly depleted after 21 years continuous cropping. In addition, bacterial diversity significantly decreased after 55 years black pepper continuous cropping; obvious variations for community structures across the 3 time-scale replanted black pepper orchards were observed, suggesting monoculture duration was the major determinant for bacterial community structure. Overall, continuous cropping during black pepper cultivation led to a significant decline in soil pH, organic matter contents, enzymatic activities, resulted a decrease in soil bacterial abundance, and altered soil microbial community membership and structure, which in turn resulted in black pepper poor growth in the continuous cropping system.

Effect of meadowsweet flower extract-pullulan coatings on rhizopus rot development and postharvest quality of cold-stored red peppers.

The study involved an examination of the antifungal activity on red peppers of pullulan coating (P) and pullulan coating containing either water-ethanol (P + eEMF) or ethanol extract of meadowsweet flowers (P + eEMF). Pullulan was obtained from a culture of Aureobasidium pullulans B-1 mutant. Both non-inoculated peppers and those artificially inoculated with Rhizopus arrhizus were coated and incubated at 24 °C for 5 days. The intensity of the decay caused by Rhizopus arrhizus in the peppers with P and P + eEMF coatings was nearly 3-fold lower, and in the case of P + weEMF 5-fold lower, than that observed in the control peppers. Additionally, the P + weEMF coating decreased, almost two-fold the severity of pepper decay compared to other samples. The influence of coating of pepper postharvest quality was examined after 30 days of storage at 6 °C and 70%-75% RH. All coatings formed a thin and well-attached additional layer of an intensified gloss. During storage, color, total soluble solid content and weight loss of coated peppers were subject to lower changes in comparison with uncoated ones. The results indicate the possibility of the application of pullulan coatings containing MFEs as an alternative to the chemical fungicides used to combat pepper postharvest diseases.

Landscape heterogeneity shapes host-parasite interactions and results in apparent plant-virus codivergence.

Knowledge on how landscape heterogeneity shapes host-parasite interactions is central to understand the emergence, dynamics and evolution of infectious diseases. However, this is an underexplored subject, particularly for plant-virus systems. Here, we analyse how landscape heterogeneity influences the prevalence, spatial genetic structure, and temporal dynamics of Pepper golden mosaic and Pepper huasteco yellow vein begomoviruses infecting populations of the wild pepper Capsicum annuum glabriusculum (chiltepin) in Mexico. Environmental heterogeneity occurred at different nested spatial scales (host populations within biogeographical provinces), with levels of human management varying among host population within a province. Results indicate that landscape heterogeneity affects the epidemiology and genetic structure of chiltepin-infecting begomoviruses in a scale-specific manner, probably related to conditions favouring the viruses' whitefly vector and its dispersion. Increased levels of human management of the host populations were associated with higher virus prevalence and erased the spatial genetic structure of the virus populations. Also, environmental heterogeneity similarly shaped the spatial genetic structures of host and viruses. This resulted in the congruence between host and virus phylogenies, which does not seem to be due to host-virus co-evolution. Thus, results provide evidence of the key role of landscape heterogeneity in determining plant-virus interactions.

Propagação vegetativa de espécies silvestres do gênero Piper, com potencial para uso como porta enxertos em pimenta-do-reino (Piper nigrun) / Vegetative propagation of wild species of the genus Piper, with potential for use as rootstocks in black pepper (Piper nigrun)

O estabelecimento de espécies usadas como porta-enxerto, importantes na olericultura, podem ser multiplicadas por meio da estaquia com auxílio de fitorreguladores. Objetivou-se com este trabalho avaliar a influência do AIB no estabelecimento de espécies nativas do gênero Piper com potencial de uso como porta-enxerto de Piper nigrum. Estacas de P. Arboreum, P. amplum e P. sp foram submetidas a diferentes dosagens de AIB (0, 2000, 4000, 6000 e 8000 mg.L-1) e mantidas em estufa agrícola com irrigação intermitente com temperatura e umidade controlados. O delineamento experimental utilizado foi inteiramente casualizado em esquema fatorial 3x5 (3 espécies de Piper x 5 dosagens de AIB) e composto por 3 repetições por tratamento, sendo cada parcela composta por 10 estacas. A avaliação foi realizada aos 45 dias de transplantio e as variáveis analisadas foram: índice de sobrevivência, número médio de folhas, número médio de brotos e comprimento médio do maior broto. A espécie Piper arboreum apresentou bons resultados referentes ao crescimento da parte aérea independentemente da concentração de AIB empregada.

The establishment of species used as rootstock, which are important in horticulture crop scan may be multiplied, with the aid of growth regulators, by cutting. The objective of this study was to evaluate the influence of IBA on the establishment of native species of the genus Piper with potential use as rootstock of Piper nigrum. Cuttings of P. arboreum, P. amplum and P. sp were treated with different concentrations of IBA (0 ,2000, 4000, 6000 and 8000mg.L-1) and maintained in a greenhouse with intermittent irrigation, with the temperature and humidity controlled. The experimental design was completely randomized in a factorial 3x5 plan (3 Piper species x5 doses of IBA) and comprising of three repetitions per treatment, each plot consisting of 10 cuttings. The evaluation was performed at 45 days of transplanting and the analyzed variables were: survival rate, the number of leaves, the number of shoots and the length of the largest shoot. The Piper arboretum species showed the best growth results for the aerial part independent of the IBA concentration used.

Chlorophyll breakdown during pepper fruit ripening in the chlorophyll retainer mutation is impaired at the homolog of the senescence-inducible stay-green gene.

The pepper chlorophyll retainer (cl) mutation is characterized by inhibition of chlorophyll degradation during fruit ripening. Ripe fruit of cl pepper containing chlorophyll and red carotenoids is brown, while ripe fruit containing chlorophyll and yellow carotenoids is green. In addition to the inhibitory effect during fruit ripening caused by cl, we show that chlorophyll degradation is inhibited during natural and dark-induced leaf senescence. Therefore, the cl mutation has the characteristics of the stay-green (sgr) mutants described in many other species. Upon the recent discovery of the SGR gene in various plant species, we isolated pepper SGR (CaSGR) and found that it genetically cosegregates with cl in a BC1 mapping population. Furthermore, sequencing the wild-type and mutant alleles revealed an amino-acid substitution of tryptophan (aromatic amino acid) to arginine (basic amino acid) at position 114 in the protein sequence. The single-nucleotide polymorphism (SNP) that differentiates the wild-type and mutant alleles was exploited to develop a PCR marker useful for marker-assisted selection. Expression of CaSGR as measured by semiquantitative RT-PCR was mostly induced upon fruit ripening and to a lesser extent upon leaf senescence. Taking together, our genetic, sequence and expression data all indicate that CaSGR is a candidate for controlling the cl mutation in pepper.

Diversity and activity of biosurfactant-producing Pseudomonas in the rhizosphere of black pepper in Vietnam.

AIMS: Phytophthora capsici is a major pathogen of black pepper and zoospores play an important role in the infection process. Fluorescent pseudomonads that produce biosurfactants with zoosporicidal activities were isolated from the black pepper rhizosphere in Vietnam, and their genotypic diversity and potential to control Phy. capsici root rot was determined. METHODS: Biosurfactant-producing pseudomonads were genotypically and biochemically characterized by BOX-polymerase chain reaction (PCR), 16S-rDNA sequencing, reverse-phase-high-performance liquid chromatography and liquid chromatography-mass spectrometry analyses. RESULTS: Biosurfactant-producing fluorescent pseudomonads make up c. 1.3% of the culturable Pseudomonas population in the rhizosphere of black pepper. Although BOX-PCR revealed substantial genotypic diversity, the isolates were shown to produce the same biosurfactants and were all identified as Pseudomonas putida. When applied to black pepper stem cuttings, several of the biosurfactant-producing strains provided significant disease control. In absence of the disease, several of the bacterial strains promoted shoot and root growth of black pepper stem cuttings. CONCLUSIONS: Biosurfactant-producing pseudomonads indigenous to the rhizosphere of black pepper plants are genotypically diverse and provide a novel resource for the control of Phy. capsici root rot and growth promotion of black pepper stem cuttings. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide a strong basis for further development of supplementary strategies with antagonistic bacteria to control foot and root rot of black pepper and to promote plant growth.

[Allelopathy of decomposing pepper stalk on pepper growth].

With decomposing pepper stalk as test material, this paper studied its allelopathy on the growth of pepper plants. The results showed that after 60 days of decomposition, the decomposed pepper stalk could decrease the plant height, stem diameter, dry weights of above-and underground biomass, leaf area, and chlorophyll content of pepper plants by 0.0374 - 0.0646, 0.0020 - 0.0097, 0.0050 - 0.0355 and 0.0916 - 0.3584, 0.0016 - 0.0251, and 0.0043 - 0.0242 respectively. These inhibitory effects were enhanced after 120 days of decomposition, but the difference with CK was not significant. The root vigor and its SOD, POD and CAT activities of pepper plants were decreased, while the MDA content and relative conductivity were increased with the increasing concentration of decomposed pepper stalk and with the prolong of treating time. The allelopathic effects of decomposed pepper stalk on the physiological indices of pepper root activity ranged from 0.0163 to 0.6507, which was significantly higher than that of plant growth index.