Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits.     

A thaumatin-like gene in nonclimacteric pepper fruits used as molecular marker in probing disease resistance,ripening, and sugar accumulation

During pepper (Capsicum annuum) fruit ripening, the ripe fruit interaction with the anthracnose fungus, Colletotrichum gloeosporioides, is generally incompatible. However, the unripe fruit can interact compatibly with the fungus. A gene, designated PepTLP (for pepper thaumatin-like protein), was isolated and characterized by using mRNA differential display. The PepTLP gene encodes a protein homologous to other thaumatin-like proteins and contains 16 conserved cysteine residues and the consensus pattern of thaumatin. PepTLP gene expression is developmentally regulated during ripening. The accumulation of PepTLP mRNA and PepTLP protein in the incompatible interaction was higher than that in the compatible one. Furthermore, PepTLP gene expression was stimulated by both jasmonic acid treatment and wounding during ripening, but by wounding only in the unripe fruit. Immunolocalization studies showed that it is localized to the intercellular spaces among cortical cells. The expression of the PepTLP gene upon fungal infection was a rise from the early-breaker fruit. The development of anthracnose became significantly prevented with beginning of fruit ripening, and the sum total of sugar accumulation increased. The results suggest that the PepTLP gene can be used as a molecular marker in probing for disease resistance, ripening, and sugar accumulation in nonclimacteric pepper fruits.     

Ultrastructure at the Host-Parasite Interface of Phytophthora capsici in Roots and Stems of Capsicum annuum

The infecting hyphae of Phytophthora capsici grew intercellularly in infected tissues of roots and stems of pepper (Capsicum annuum). The vascular tissues were not markedly disorganized even when heavily infected. Intercellularly growing hyphae penetrated the host cells by forming haustorium-like bodies. The consistent features of ultrastructural changes in infected tissues of pepper roots and stems were degeneration of cell organelles and dissolution of host cell walls. The cytoplasm detached from the cell wall aggregated abundantly around some haustorium-like bodies or the penetration sites of fungal hyphae. The host cell walls were palely stained, thinned and swollen, possibly being biochemically altered by the action of fungal macerating enzymes. Electron-dense, wall-like material was apposed on the outer wall of xylem vessel contacted by fungal hyphae. The infecting hyphae were also surrounded by granular, dark-staining cytoplasm. Characteristics of host cell responses to the invading P. capsici were the deposition of papilla-like material on host cell walls next to hyphae and the encasement of haustorium-like bodies with wall appositions.     

Inhibition of Pepper mild mottle virus with Commercial Cellulases

Pepper mild mottle virus (PMMoV) is one of the most important pathogens of pepper crops (Capsicum annuum L.) worldwide. We have found that commercial cellulases from Trichoderma reesei and Trichoderma viride strongly inhibit PMMoV infection of plants. When purified PMMoV was mixed with the cellulases, it greatly lost the capacity to induce local lesions on leaves of Nicotiana glutinosa. Pretreatment of sweet pepper leaves (C. annuum L.) with cellulase solutions before PMMoV inoculation greatly reduced the number of infected plants. These effects were superior to the effects of known viral inhibitors such as skim milk or Lentinula edodes culture filtrates. Although the mechanism of inhibition remains unknown, this is the first report of antiviral activity ascribed to commercially available cellulases.     

Susceptibility assessment of bell pepper genotypes to crown and root rot disease

Bell Pepper (Capsicum annuum) is one of the important vegetable crops with valuable food sources, which is used almost around the world. Crown and root rot disease caused by Phytophthora capsici is one of the most important diseases of bell pepper in Iran. The present study was conducted to evaluate the susceptibility of different varieties of bell pepper to crown and root rot disease under glasshouse condition. Fourteen commonly planted genotypes of bell pepper in Iran were evaluated for their susceptibility to infection with the pathogen. For this purpose, disease severity of the chosen genotypes in different growth stages was evaluated. The results indicated that the bell pepper genotypes respond differently to pathogenicity tests. Based on cluster analyses confirmed by the results of SAS analyses, bell pepper cultivars were categorised in five distinct groups.     

Double mutations in eIF4E and eIFiso4E confer recessive resistance to <Emphasis Type="Italic">Chilli veinal mottle virus</Emphasis> in pepper

To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinai mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum ‘Dempsey’ containing an eIF4E mutation (pvr1 ² ) and C. annuum ‘Perennial’ containing an eIFiso4E mutation (pvr6). C. annuum ‘Dempsey’ was susceptible and C. annuum ‘Perennial’ was resistant to ChiVMV. All F₁ plants showed resistance, and F₂ individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F₂ and 329 F₃ plants of 17 families were genotyped with pvr1 ² and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both pvr1 ² and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in eIF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of F2 plants revealed that all plants containing homozygous genotypes of both pvr1 ² and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of eIF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper. These authors contributed equally to this work.     

An SSR-based linkage map of Capsicum annuum

There are five cultivated species of pepper, of which Capsicum annuum is the most widely cultivated as a vegetable or spice and the main experimental material of most pepper breeding programs. However, the number of simple sequence-repeat (SSR) markers known for C. annuum is limited. To develop SSR markers for Capsicum species, we constructed four SSR-enriched libraries from the genomic DNA of C.␣annuum, sequenced 1873 clones, and isolated 626 unique SSR clones. A higher percentage of these SSR markers were taken from dinucleotide motif libraries than from trinucleotide motif libraries. Primer pairs for the 626 SSR clones were synthesized and tested for polymorphisms; 594 amplified products were detected with the expected size. However, only 153 products were polymorphic between the parents of our mapping population. Using 106 highly reproducible pairs from the primer pairs, we constructed a linkage map of C. annuum in an intraspecific doubled haploid population (n=117) that contains nine previously reported SSRs as well as AFLP, CAPS, and RAPD markers and the trait of fruit pungency. The map contains 374 markers, including 106 new SSR markers distributed across all 13 linkage groups, and covers 1042 cM. The polymorphism information content (PIC) of these new SSR markers was calculated using 14 lines of Capsicum species. The average number of alleles per locus was 2.9 and the average PIC value was 0.46, even within C. annuum. The SSR markers developed in this study will be useful for mapping and marker-assisted selection in pepper breeding, and the linkage map provides a reference genetic map for Capsicum species.     

Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N‐vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence‐related genes, such as CASC1, CACHI2 and CABGLU. N‐Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild‐type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never‐ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.     

A New Lignan Amide,Grossamide, from Bell Pepper (Capsicum annuum var. grossurri)

Two new phenolic amides, grossamide (7) and N-cis-feruloyl tyramine (2), have been isolated from the roots of bell pepper (Capsicum annuum var. grossum) together with p-aminobenzaldehyde (1), N-trans-feruloyl tyramine (3), N-trans-p-coumaroyl tyramine (4), N-trans-feruloyl octopamine (5), and N-trans-p-coumaroyl octopamine (6).     

Pathogenesis‐related protein 4b interacts with leucine‐rich repeat protein 1 to suppress PR4b‐triggered cell death and defense response in pepper

To control defense and cell‐death signaling, plants contain an abundance of pathogen recognition receptors such as leucine‐rich repeat (LRR) proteins. Here we show that pepper (Capsicum annuum) LRR1 interacts with the pepper pathogenesis‐related (PR) protein 4b, PR4b, in yeast and in planta. PR4b is synthesized in the endoplasmic reticulum, interacts with LRR1 in the plasma membrane, and is secreted to the apoplast via the plasma membrane. Binding of PR4b to LRR1 requires the chitin‐binding domain of PR4b. Purified PR4b protein inhibits spore germination and mycelial growth of plant fungal pathogens. Transient expression of PR4b triggers hypersensitive cell death. This cell death is compromised by co‐expression of LRR1 as a negative regulator in Nicotiana benthamiana leaves. LRR1/PR4b silencing in pepper and PR4b over‐expression in Arabidopsis thaliana demonstrated that LRR1 and PR4b are necessary for defense responses to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis (Hpa) infection. The mutant of the PR4b Arabidopsis ortholog, pr4, showed enhanced susceptibility to Hpa infection. Together, our results suggest that PR4b functions as a positive modulator of plant cell death and defense responses. However, the activity of PR4b is suppressed by interaction with LRR1.     

Structure of a functional geranylgeranyl pyrophosphate synthase gene from Capsicum annuum

A geranylgeranyl pyrophosphate synthase (GGPPS) gene from Capsicum annuum (bell pepper) was cloned. The nucleotide sequence shows that this gene, like the capsanthin/capsorubin gene but unlike the phytoene synthase gene from C. annuum, is not interrupted by an intron. Southern blot analysis of C. annuum genomic DNA suggests the presence of a single gene highly similar to the cDNA and also of additional related sequences. The present data suggest that this cloned gene is functional.     

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P₀. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P₀ and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK _c 1) gene was increased specifically in the incompatible interaction with TMV-P₀. The expression of CaPK _c 1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK _c 1 responds to several defense-related abiotic stresses in addition to TMV infection. The nucleotide sequence data reported in this paper were submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number DQ114474.     

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant’s resistance to disease.     

In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems withPhytophthora capsici

Summary In situ hybridization and immunogold labeling were performed to examine the temporal and spatial expression pattern of pathogenesis-related protein 1 (CABPR1) mRNA and PR-1 protein in pepper (Capsicum annuum L.) stem tissues infected by virulent and avirulent isolates ofPhytophthora capsici. CABPR1 mRNA accumulation was confirmed in the infected pepper stem tissue by Northern blot analysis and in situ hybridization. Northern blot analysis showed that the temporal expression ofCABPR1 mRNA varied greatly between compatible and incompatible interactions. An earlier expression of theCABPR1 gene, 6 h after inoculation, was observed in the incompatible interaction. In situ hybridization results revealed thatCABPR1 mRNA was expressed in the phloem areas of vascular bundles in infected pepper stem tissues, but especially strongly in the incompatible interaction. PR-1 protein was predominantly found in the intercellular spaces of pepper stem cells in the compatible and incompatible interactions 24 h after inoculation. Strikingly, the immunogold labeling was associated with fibrillar and electron-dense material localized in the intercellular space. Dense labeling of PR-1 protein was also seen at the interface of the pathogen and the host cell wall, whereas few gold particles were detected over the host cytoplasm. However, PR-1 protein was not detected over the fungal cell wall in either interaction.     

Grapevine xylem response to fungi involved in trunk diseases

Grapevine trunk diseases (GTD), caused by a wide range of different fungi, are responsible for decline and productivity losses in vines at all growth stages. Grapevine responses to fungal attack include morphological and physiochemical defence mechanisms in the vascular system to reduce fungal infections. However, the extent to which these responses could control further spread by GTD‐fungi in the xylem vessels is poorly known. This study shows the formation of tyloses inside xylem vessels of diseased grapevines, as well as extracellular ligninolytic activities [lignin peroxidase, manganese peroxidase (MnP) and/or laccase] exhibited by some GTD‐fungi isolated here from symptomatic grapevines. In particular, Botryosphaeriaceae spp. and Phaeoacremonium minimum showed all three lignin‐degrading enzymatic activities. We also examined whether selected vine phenolic compounds, often located in the vascular system in response to fungal infection, could affect the lignin‐degrading activity from those GTD‐fungi as well as fungal colonisation. We found that phenolic compounds appeared to inhibit MnP activity, in addition to reducing fungal growth by causing anomalies in the hyphae morphology. Our results support that affected grapevines can initiate the tylosis formation in order to constrain fungi in the xylem vessels, while highlight the complementary action of the phenolic compounds to inhibit the fungi growth and colonisation. Phenolic compounds are therefore likely to have important role in alternative strategies for preventing trunk diseases.     

Inhibitory Effects of Pepper Mild Mottle Virus Infection by Supernatants of Five Bacterial Cultures in Capsicum annuum L.

Pepper mild mottle virus (PMMoV), one of the most prevalent viruses in chili pepper (Capsicum annuum L.) is a non-enveloped, rod-shaped, single-stranded positive-sense RNA virus classified in the genus Tobamovirus. The supernatants of five bacterial cultures (Pseudomonas putida [PP], Bacillus licheniformis [BLI], P. fluorescens [PF], Serratia marcescens [SER], and B. amyloliquifaciens [BA]) were analyzed to find novel antiviral agents to PMMoV in chili pepper. Foliar spraying with supernatants (1:1, v/v) obtained from Luria-Bertani broth cultures of PP, BLI, PF, SER, and BA inhibited PMMoV infection of chili pepper if applied before the PMMoV inoculation. Double-antibody sandwich enzyme-linked immunosorbent assay showed that treatments of five supernatants resulted in 51–66% reductions in PMMoV accumulation in the treated chili pepper. To identify key compounds in supernatants of PP, BLI, PF, SER, and BA, the supernatants were subjected to gas chromatography-mass spectrometry. The 24 different types of compounds were identified from the supernatants of PP, BLI, PF, SER, and BA. The compounds vary from supernatants of one bacterial culture to another which includes simple compounds—alkanes, ketones, alcohols, and an aromatic ring containing compounds. The compounds triggered the inhibitory effect on PMMoV propagation in chili pepper plants. In conclusion, the cultures could be used to further conduct tissue culture and field trial experiments as potential bio-control agents.     

Biological control of bacterial spot disease and plant growth-promoting effects of lactic acid bacteria on pepper

In our previous studies, we observed the biological control effect of lactic acid bacteria strains (LABs) KLF01, KLC02 and KPD03 against different plant pathogenic bacteria in vitro against Ralstonia solanacearum, and strains KLF01 and KLC02 against Pectobacterium carotovorum under greenhouse and field experiments, respectively. In this study, we observed the efficacy of these bacteria against bacterial spot pathogen (Xanthomonas campestris pv. vesicatoria) and their plant growth-promoting activities in pepper (Capsicum annuum L. var. annuum), under greenhouse and field conditions. LABs significantly (P < 0.05) reduced bacterial spot on pepper plants in comparison to untreated plants in both the greenhouse and the field experiments. The plant growth-promoting effect of LABs on pepper varied; some strains had a significant effect on growth promotion (P < 0.05) compared with untreated plants, while some showed no significant effect in the greenhouse and field experiments. Additionally, LABs were able to colonise roots, produce indole-3-acetic acid (IAA), siderophores and solubilise phosphate. These findings indicate that application of LABs could provide a promising alternative for the management of bacterial spot disease in pepper plants and could therefore be used as a healthy plant growth-promoting agent.     

Construction of an integrated pepper map using RFLP,SSR, CAPS,AFLP, WRKY,rRAMP, and BAC end sequences

Map-based cloning to find genes of interest, markerassisted selection (MAS), and marker-assisted breeding (MAB) all require good genetic maps with high reproducible markers. For map construction as well as chromosome assignment, development of single copy PCR-based markers and map integration process are necessary. In this study, the 132 markers (57 STS from BAC-end sequences, 13 STS from RFLP, and 62 SSR) were newly developed as single copy type PCR-based markers. They were used together with 1830 markers previously developed in our lab to construct an integrated map with the Joinmap 3.0 program. This integrated map contained 169 SSR, 354 RFLP, 23 STS from BAC-end sequences, 6 STS from RFLP, 152 AFLP, 51 WRKY, and 99 rRAMP markers on 12 chromosomes. The integrated map contained four genetic maps of two interspecific (Capsicum annuum ‘TF68’ and C. chinense ‘Habanero’) and two intraspecific (C. annuum ‘CM334’ and C. annuum ‘Chilsungcho’) populations of peppers. This constructed integrated map consisted of 805 markers (map distance of 1858 cM) in interspecific populations and 745 markers (map distance of 1892 cM) in intraspecific populations. The used pepper STS were first developed from end sequences of BAC clones from Capsicum annuum ‘CM334’. This integrated map will provide useful information for construction of future pepper genetic maps and for assignment of linkage groups to pepper chromosomes.