The Virome of Piper nigrum: Identification, Genomic Characterization, Prevalence, and Transmission of Three New Viruses of Black Pepper in China.

Viral diseases are one of the main categories of diseases that cause substantial yield losses in black pepper. Disease symptoms in black pepper are generally complex and are often caused by both known and undescribed viruses. To identify and clarify the etiology of viral diseases in black pepper in Hainan, China, we conducted high-throughput sequencing (HTS) by targeting purified double-stranded RNA (dsRNA) and ribosomal RNA depleted total RNA (rRNA-depleted totRNA). Analysis of the data revealed the presence of one known virus, piper yellow mottle virus (PYMoV), and three newly identified viruses: black pepper virus F (BPVF) in the genus Fabavirus, black pepper virus E (BPVE) in the genus Enamovirus, and black pepper virus B (BPVB) in the genus Badnavirus. The dominant viruses in P. nigrum sampled in Hainan are PYMoV, with an incidence of 100%, followed by BPVF (84%, 133 of 158) and BPVB (66%, 105 of 158). Mechanical inoculation of sap extracts from source plants containing PYMoV, BPVF, and BPVB gave negative results on both herbaceous and woody host plants 60 days postinoculation (dpi). BPVF and PYMoV were successfully transmitted to virus-free seedlings of black pepper through bark grafting, while BPVB was experimentally undetectable up to 150 dpi. Seed transmission experiments showed that no target viruses were present in all 59 germinated seedlings. This study provides information on diagnosis, prevalence, and transmission of black-pepper-associated viruses.

Transcriptome profiling of pepper leaves by RNA-Seq during an incompatible and a compatible pepper-tobamovirus interaction.

Upon virus infections, the rapid and comprehensive transcriptional reprogramming in host plant cells is critical to ward off virus attack. To uncover genes and defense pathways that are associated with virus resistance, we carried out the transcriptome-wide Illumina RNA-Seq analysis of pepper leaves harboring the L3 resistance gene at 4, 8, 24 and 48 h post-inoculation (hpi) with two tobamoviruses. Obuda pepper virus (ObPV) inoculation led to hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV) inoculation resulted in a systemic infection without visible symptoms (compatible interaction). ObPV induced robust changes in the pepper transcriptome, whereas PMMoV showed much weaker effects. ObPV markedly suppressed genes related to photosynthesis, carbon fixation and photorespiration. On the other hand, genes associated with energy producing pathways, immune receptors, signaling cascades, transcription factors, pathogenesis-related proteins, enzymes of terpenoid biosynthesis and ethylene metabolism as well as glutathione S-transferases were markedly activated by ObPV. Genes related to photosynthesis and carbon fixation were slightly suppressed also by PMMoV. However, PMMoV did not influence significantly the disease signaling and defense pathways. RNA-Seq results were validated by real-time qPCR for ten pepper genes. Our findings provide a deeper insight into defense mechanisms underlying tobamovirus resistance in pepper.

Complete nucleotide sequences of M and L RNAs from a new pepper-infecting tospovirus, Pepper chlorotic spot virus.

Pepper chlorotic spot virus (PCSV), newly found in Taiwan, was identified as a new tospovirus based on the molecular characterization of its S RNA. In this study, the complete M and L RNA sequences of PCSV were determined. The M RNA has 4795 nucleotides (nts), encoding the NSm protein of 311 aa (34.5 kDa) in the viral (v) strand and the glycoprotein precursor (Gn/Gc) of 1122 aa (127.6 kDa) in the viral complementary (vc) strand. The L RNA has 8859 nts, encoding the RNA-dependent RNA polymerase (RdRp) of 2873 aa (330.8 kDa) in the vc strand. Analyses of the NSm, Gn/Gc and RdRp of PCSV revealed that PCSV is phylogenetically clustered within the watermelon silver mottle virus-related clade. Based on the whole genome sequence, PCSV is closely related to Tomato necrotic ringspot virus and should be classified as a new tospovirus species.

Influence of temperature on symptom expression, detection of host factors in virus infected Piper nigrum L.

Expression of symptoms in black pepper plants (Piper nigrum) infected with Piper yellow mottle virus (PYMoV) vary depending on the season, being high during summer months. Here, we explored the influence of temperature on symptom expression in PYMoV infected P. nigrum. Our controlled environment study revealed increase in virus titer, total proteins, IAA and reducing sugars when exposed to temperature stress. There was change in the 2-D separated protein before and after exposure. The 2-D proteomics LC-MS identified host and viral proteins suggesting virus-host interaction during symptom expression. The analysis as well as detection of host biochemical compounds may help in understanding the detailed mechanisms underlying the viral replication and damage to the crop, and thereby plan management strategies.

The complete genome sequence of Piper yellow mottle virus (PYMoV).

This study reports the first complete genome sequence of Piper yellow mottle virus (PYMoV, KC808712) identified in black pepper. The genome is 7,622 nucleotides long, possessing four open reading frames (ORFs). ORF1, ORF2 and ORF4 of PYMoV are reported as hypothetical proteins of unknown function with a predicted molecular mass of 15.7, 17.1 and 17.9 kDa, respectively. ORF3 of PYMoV encodes a polyprotein of 218.6 kDa and consists of a viral movement protein (MP), trimeric dUTPase, zinc finger, retropepsin, RT-LTR, and RNAse H. Detailed PYMoV genome analysis confirmed that it is a member of the family Caulimoviridae, genus Badnavirus. Fragments of two additional novel sequences resembling those found in members of the family Caulimoviridae were also identified in the black pepper sample, and the viruses from which they were derived were tentatively named Piper DNA virus 1 and 2.

Rapid detection of Piper yellow mottle virus and Cucumber mosaic virus infecting black pepper (Piper nigrum) by loop-mediated isothermal amplification (LAMP).

The loop-mediated isothermal amplification (LAMP) assay for Piper yellow mottle virus and the reverse transcription (RT) LAMP assay for Cucumber mosaic virus each consisted of a set of five primers designed against the conserved sequences in the viral genome. Both RNA and DNA isolated from black pepper were used as a template for the assay. The results were assessed visually by checking turbidity, green fluorescence and pellet formation in the reaction tube and also by gel electrophoresis. The assay successfully detected both viruses in infected plants whereas no cross-reactions were recorded with healthy plants. Optimum conditions for successful amplification were determined in terms of the concentrations of magnesium sulphate and betaine, temperature, and duration. The detection limit for both LAMP and RT-LAMP was up to 100 times that for conventional PCR and up to one-hundredth of that for real-time PCR. The optimal conditions arrived at were validated by testing field samples of infected vines of three species from different regions.

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.

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR.

AIMS: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. METHODS AND RESULTS: A multiplex RT-PCR method consisting of one-tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT-PCR detected up to 10(-6) dilution of total RNA extracted from infected leaves. Multiplex RT-PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. CONCLUSIONS: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.

Geminivirus mixed infection on pepper plants: synergistic interaction between PHYVV and PepGMV.

BACKGROUND: PHYVV and PepGMV are plant viruses reported in Mexico and Southern US as causal agents of an important pepper disease known as "rizado amarillo". Mixed infections with PHYVV and PepGMV have been reported in several hosts over a wide geographic area. Previous work suggested that these viruses might interact at the replication and/or movement level in a complex manner. The aim of present report was to study some aspects of a synergistic interaction between PHYVV and PepGMV in pepper plants. These include analyses of symptom severity, viral DNA concentration and tissue localization of both viruses in single and mixed infections. RESULTS: Mixed infections with PepGMV and PHYVV induced symptoms more severe than those observed in single viral infections. Whereas plants infected with either virus (single infection) presented a remission stage with a corresponding decrease in viral DNA levels, double-infected plants did not present symptom remission and both viral DNA concentrations dramatically increased. In situ hybridization experiments revealed that both viruses are restricted to the vascular tissue. Interestingly, the amount of viral DNA detected was higher in plants inoculated with PepGMV than that observed in PHYVV-infected plants. During mixed infections, the location of both viruses remained similar to the one observed in single infections, although the number of infected cells increases. Infections with the tripartite mixture PHYVV (A+B) + PepGMV A produced a similar synergistic infection to the one observed after inoculation with both full viruses. On the contrary, tripartite mixture PepGMV (A+B) + PHYVV A did not produce a synergistic interaction. In an attempt to study the contribution of individual genes to the synergism, several mutants of PHYVV or PepGMV were inoculated in combination with the corresponding wild type, second virus (wt PepGMV or wt PHYVV). All combinations tested resulted in synergistic infections, with exception of the TrAP mutant of PepGMV (PepGMV TrAP-) + PHYVV. CONCLUSION: In this report, we have demonstrated that synergistic interaction between PHYVV and PepGMV during a mixed infection is mainly due to an increased DNA concentration of both viruses, without any noticeable effect on the localization of either virus on infected plant tissue. Our results have shown that the viral component A from PepGMV is important for synergism during PHYVV-PepGMV mixed infections.

Evidence that the nonstructural protein of Tomato spotted wilt virus is the avirulence determinant in the interaction with resistant pepper carrying the TSW gene.

All known pepper cultivars resistant to Tomato spotted wilt virus (TSWV) possess a single dominant resistance gene, Tsw. Recently, naturally occurring resistance-breaking (RB) TSWV strains have been identified, causing major concerns. We used a collection of such strains to identify the specific genetic determinant that allows the virus to overcome the Tsw gene in Capsicum spp. A reverse genetic approach is still not feasible for TSWV; therefore, we analyzed reassortants between wild-type (WT) and RB strains. Our results confirmed that the S RNA, which encodes both the nucleocapsid protein (N) and a nonstructural protein (NSs), carries the genetic determinant responsible for Tsw resistance breakdown. We then used full-length S RNA segments or the proteins they encode to compare the sequences of WT and related RB strains, and obtained indirect evidence that the NSs protein is the avirulence factor in question. Transient expression of NSs protein from WT and RB strains showed that they both can equally suppress post-transcriptional gene silencing (PTGS). Moreover, biological characterization of two RB strains carrying deletions in the NSs protein showed that NSs is important in maintaining TSWV infection in newly emerging leaves over time, preventing recovery. Analysis of another RB strain phenotype allowed us to conclude that local necrotic response is not sufficient for resistance in Capsicum spp. carrying the Tsw gene.

Sodium sulphite enhances RNA isolation and sensitivity of Cucumber mosaic virus detection by RT-PCR in black pepper.

Isolation of intact high quality RNA suitable for RT-PCR from black pepper is greatly hindered by the presence of polyphenols and polysaccharides. These compounds adversely affect the sensitivity of virus detection by RT-PCR. The present study evaluated the effect of sodium sulphite in enhancing RNA yield and quality in a modified acid guanidium thiocyanate-phenol-chloroform (AGPC) protocol. The results were compared with the standard AGPC method and RNeasy Plant Mini Kit (Qiagen) for detection of Cucumber mosaic virus through RT-PCR. The addition of sodium sulphite in the extraction buffer increased the sensitivity of virus detection. Higher sensitivity of detection (than obtained from the kit) was seen when sodium sulphite was used at 0.5%. Similar levels of sensitivity were also observed for the detection of Cucumber mosaic virus from Piper longum.