NIb Protein Research Articles

Turnip mosaic virus NIb weakens the function of eukaryotic translation initiation factor 6 facilitating viral infection in Nicotiana benthamiana.

Viruses rely completely on host translational machinery to produce the proteins encoded by their genes. Controlling translation initiation is important for gaining translational advantage in conflicts between the host and virus. The eukaryotic translation initiation factor 4E (eIF4E) has been reported to be hijacked by potyviruses for virus multiplication. The role of translation regulation in defence and anti-defence between plants and viruses is not well understood. We report that the transcript level of eIF6 was markedly increased in turnip mosaic virus (TuMV)-infected Nicotiana benthamiana. TuMV infection was impaired by overexpression of N. benthamiana eIF6 (NbeIF6) either transiently expressed in leaves or stably expressed in transgenic plants. Polysome profile assays showed that overexpression of NbeIF6 caused the accumulation of 40S and 60S ribosomal subunits, the reduction of polysomes, and also compromised TuMV UTR-mediated translation, indicating a defence role for upregulated NbeIF6 during TuMV infection. However, the polysome profile in TuMV-infected leaves was not identical to that in leaves overexpressing NbeIF6. Further analysis showed that TuMV NIb protein, the RNA-dependent RNA polymerase, interacted with NbeIF6 and interfered with its effect on the ribosomal subunits, suggesting that NIb might have a counterdefence role. The results propose a possible regulatory mechanism at the translation level during plant-virus interaction.

P3 and NIa-Pro of Turnip Mosaic Virus Are Independent Elicitors of Superinfection Exclusion.

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus.

A Novel Isolate of Bean Common Mosaic Virus Isolated from Crownvetch (Securigera varia L. Lassen)

Bean common mosaic virus from the genus Potyvirus has a wide range of hosts and a very negative impact on cultivated crops from the genus Phaseolus. The risk of viral infection of economically important crops increases even if the carriers of the virus are related plant species growing on agroecological interfaces. Such plant species have emerged as new hosts for BCMV, usually harboring novel genetic variants of the virus. A novel genetic variant of BCMV was isolated from a symptomatic crownvetch plant, where the presence of this virus was confirmed via Western blot analysis and via amino acid identities in peptide fragments of CI, HC-pro, and CP proteins using the nanoLC-ESI-Q-TOF. The novel BCMV SVK isolate differed from the most genetically similar one in 0.91% of nucleotides and 1.55% of amino acids. The highest number of amino acid substitutions (8.8% of amino acids) was in the P1 protein, followed by CP (2.44% of amino acids). Minor substitutions were in Hc-pro, CI, and Nib proteins. The symptomatic crownvetch plant was confirmed as a new host and carrier of the novel BCMV isolate.

First Report of Cucurbit Chlorotic Yellows Virus Infecting Pumpkin in India.

Pumpkin (Cucurbita moschata), a member of the family Cucurbitaceae, is widely cultivated throughout the world including India. During August 2020 to January 2021, stunted pumpkin plants (cv. Pusa Vishwas), showing chlorotic patches, mosaic, and vein banding on leaves (e-Xtra Fig.1), were observed in the experimental fields of the Indian Agricultural Research Institute (IARI), New Delhi, India. Leaf-dip electron microscopy (EM) of the symptomatic plants (12 out of 37 samples) revealed the association of long flexuous virus particles measuring 650-950nm×10-12nm, suggestive of the presence of either crinivirus or potyvirus or both. Subsequently, a reverse transcription-polymerase chain reaction (RT-PCR) was performed on RNA extracted from the samples that had long flexuous virus particles using generic primers for criniviruses i.e. CriniPol-F: GCY CCS AGR GTK AAT GA and CriniPol-R: ACC TTG RGA YTT RTC AAA targeting partial RNA-dependent RNA polymerase coding region (Martin et al. 2003) and specific primers for papaya ringspot virus (PRSV) targeting a part of 3' NIb and full coat protein (CP) gene (Basavaraj et al., 2019) separately. All tested samples were positive for both crinivirus and PRSV as expected size amplicons were obtained, accounting for about 32% prevalence. As PRSV is a well-studied virus infecting cucurbits, further work was not carried on this virus and only the RT-PCR amplicon indicative of crinivirus (~515 bp) was cloned into the pGEM-T easy cloning vector (Promega, Madison, WI) and sequenced for further confirmation of the virus presence. The obtained sequence (GenBank accession No MZ318672) shared up to 90% nucleotide and 100% amino acid sequence identity with the corresponding genomic region of a cucurbit chlorotic yellows virus (CCYV) isolate from Greece (LT841297). To confirm the identity of the crinivirus species present in the same pumpkin sample, the CP gene (753bp) was amplified and sequenced using CCYV CP gene-specific primers CP-F (5'-ATG GAG AAG ACY GAC AAT AAA CAA AAT GAT GA-3') and CP-R (5'-TTA TTT ACT ACA ACC TCC CGG TGC CAA C-3') (modified from Kheireddine et al. 2020). Sequence analysis using the BioEdit tool (version 2.0) revealed that the crinivirus present in pumpkin (KC577202) shared 95 to 100% nucleotide (and 98 to 100% amino acid) sequence identity with the corresponding gene sequences of CCYV isolates originating from cucurbitaceous hosts from diverse locations. The presence of CCYV was further validated by a whitefly transmission-based bioassay followed by RT-PCR confirmation. The bioassay was performed by the whitefly species Bemisia tabaci (biotype Asia II7) using the acquisition access period and inoculation access period of 24 hours each. Six whitefly individuals per plant were used for inoculating ten pumpkin plants (cv. Pusa Vishwas) at the first true leaf stage grown in pots containing soilrite as the medium in insect-proof cages. All ten plants inoculated using whiteflies exhibited chlorosis and stunting symptoms 12-15 days post-inoculation (e-Xtra Fig.2) and were found positive for CCYV in RT-PCR assay performed using CCYV CP gene-specific primers. Though CCYV had been reported worldwide (Tzanetakis et al. 2013), its occurrence had not been reported from India. Results of the present study confirm the infection of pumpkin plants by CCYV and constitute the first report of its presence in India. Further, there is a need to investigate the extent of its spread and impact of this virus on the production of cucurbitaceous crops in the country.

Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection.

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

Genome-wide identification and analysis of the regulation wheat DnaJ family genes following wheat yellow mosaic virus infection

The co-chaperone DnaJ plays an important role in protein folding and regulation of various physiological activities, and participates in several pathological processes. DnaJ has been extensively studied in many species including humans, drosophila, mushrooms, tomatoes, and Arabidopsis. However, few studies have examined the role of DnaJ in wheat (Triticum aestivum), and the interaction mechanism between TaDnaJs and plant viruses. Here, we identified 236 TaDnaJs and performed a comprehensive genome-wide analysis of conserved domains, gene structure and protein motifs, chromosomal positions and duplication relationships, and cis-acting elements. We grouped these TaDnaJs according to their domains, and randomly selected six genes from the groups for tissue-specific analysis, and expression profiles analysis under hormone stress, and 17 genes for plant virus infection stress. In qRT-PCR, we found that among the 17 TaDnaJ genes tested, 16 genes were up-regulated after wheat yellow mosaic virus (WYMV) infection, indicating that the TaDnaJ family is involved in plant defense response. Subsequent yeast two-hybrid assays verified the WYMV NIa, NIb and 7KD proteins interacted with TaDJC (TraesCS7A02G506000), which had the most significant changes in gene expression levels after WYMV infection. Insights into the molecular mechanisms of TaDnaJ-mediated stress tolerance and sensitivity could inform different strategies designed to improve crop resistance to abiotic and biotic stress. This study provides a basis for future investigation of the TaDnaJ family and plant defense mechanisms.

Geography-Driven Evolution of Potato Virus A Revealed by Genetic Diversity Analysis of the Complete Genome.

Potato virus A (PVA), a member of the genus Potyvirus, is an important potato pathogen that causes 30%–40% yield reduction to global potato production. Knowledge on the genetic structure and the evolutionary forces shaping the structure of this pathogen is limited but vital in developing effective management strategies. In this study, we investigated the population structure and molecular evolution of PVA by analyzing novel complete genomic sequences from Chinese isolates combined with available sequences from Europe, South America, Oceania, and North America. High nucleotide diversity was discovered among the populations studied. Pairwise FST values between geographical populations of PVA ranged from 0.22 to 0.46, indicating a significant spatial structure for this pathogen. Although purifying selection was detected at the majority of polymorphic sites, significant positive selection was identified in the P1, NIa, and NIb proteins, pointing to adaptive evolution of PVA. Further phylogeny–trait association analysis showed that the clustering of PVA isolates was significantly correlated with geographic regions, suggesting that geography-driven adaptation may be an important determinant of PVA diversification.

Nuclear exportin 1 facilitates turnip mosaic virus infection by exporting the sumoylated viral replicase and by repressing plant immunity.

Exportin 1/XPO1 is an important nuclear export receptor that binds directly to cargo proteins and translocates the cargo proteins to the cytoplasm. To understand XPO1 protein functions during potyvirus infections, we investigated the nuclear export of the NIb protein encoding the RNA-dependent RNA polymerase (RdRp) of turnip mosaic virus (TuMV). Previously, we found that NIb is transported to the nucleus after translation and sumoylated by the sumoylation (small ubiquitin-like modifier) pathway to support viral infection. Here, we report that XPO1 interacts with NIb to facilitate translocation from the nucleus to the viral replication complexes (VRCs) that accumulate in the perinuclear regions of TuMV-infected cells. XPO1 contains two NIb-binding domains that recognize and interact with NIb in the nucleus and in the perinuclear regions, respectively, which facilitates TuMV replication. Moreover, XPO1 is involved in nuclear export of the sumoylated NIb and host factors tagged with SUMO3 that is essential for suppression of plant immunity in the nucleus. Deficiencies of XPO1 in Arabidopsis and Nicotiana benthamiana plants inhibit TuMV replication and infection. These data demonstrate that XPO1 functions as a host factor in TuMV infection by regulating NIb nucleocytoplasmic transport and plant immunity.

Presence of Potyvirus in the Norte de Santander department and molecular characterization of NIb protein in Colombian SCMV isolates

Potyviruses are the largest genus of plant viruses that cause significant losses over a wide range of crops. In this paper, the presence of potyvirus in different plant crops in the provinces of Ocaña and Pamplona located in the north and south of the Department of Norte de Santander (Colombia) was evaluated with RT-PCR analysis using universal oligonucleotides specific to the region that encodes the NIB protein. The results indicate the presence of several potyvirus in Pamplona in economically important crops such as corn (Zea mays), tomato (Solanum lycopersicum), potato (Solanum tuberosum) and zucchini (Cucurbita pepo). In Ocaña, potyvirus was found in bean (Phaseolus vulgaris), corn and pumpkin (Cucurbita maxima). In corn, one of the most important crops, the presence of the Sugarcane mosaic virus (SCMV) was confirmed with nucleotide sequencing. This is the first report of this virus in the department. The presence of several potyviruses in different crops in Norte de Santander indicate an alarming phytosanitary condition that must be addressed with priority to establish detection and control systems that maximize production, ensure agricultural sustainability, and propose certification schemes and improvement programs to reduce economic losses.

The Potyviruses: An Evolutionary Synthesis Is Emerging.

In this review, encouraged by the dictum of Theodosius Dobzhansky that “Nothing in biology makes sense except in the light of evolution”, we outline the likely evolutionary pathways that have resulted in the observed similarities and differences of the extant molecules, biology, distribution, etc. of the potyvirids and, especially, its largest genus, the potyviruses. The potyvirids are a family of plant-infecting RNA-genome viruses. They had a single polyphyletic origin, and all share at least three of their genes (i.e., the helicase region of their CI protein, the RdRp region of their NIb protein and their coat protein) with other viruses which are otherwise unrelated. Potyvirids fall into 11 genera of which the potyviruses, the largest, include more than 150 distinct viruses found worldwide. The first potyvirus probably originated 15,000–30,000 years ago, in a Eurasian grass host, by acquiring crucial changes to its coat protein and HC-Pro protein, which enabled it to be transmitted by migrating host-seeking aphids. All potyviruses are aphid-borne and, in nature, infect discreet sets of monocotyledonous or eudicotyledonous angiosperms. All potyvirus genomes are under negative selection; the HC-Pro, CP, Nia, and NIb genes are most strongly selected, and the PIPO gene least, but there are overriding virus specific differences; for example, all turnip mosaic virus genes are more strongly conserved than those of potato virus Y. Estimates of dN/dS (ω) indicate whether potyvirus populations have been evolving as one or more subpopulations and could be used to help define species boundaries. Recombinants are common in many potyvirus populations (20%–64% in five examined), but recombination seems to be an uncommon speciation mechanism as, of 149 distinct potyviruses, only two were clear recombinants. Human activities, especially trade and farming, have fostered and spread both potyviruses and their aphid vectors throughout the world, especially over the past five centuries. The world distribution of potyviruses, especially those found on islands, indicates that potyviruses may be more frequently or effectively transmitted by seed than experimental tests suggest. Only two meta-genomic potyviruses have been recorded from animal samples, and both are probably contaminants.

Genomic sequence variability of an Italian Zucchini yellow mosaic virus isolate

Zucchini yellow mosaic virus (ZYMV) was first reported in Italy, but no Italian isolates have been sequenced so far. A ZYMV isolate, designated Z104, was isolated from a zucchini plant grown in the open field in Piedmont, Italy, and showing severe disease symptoms. We report the full-length genome sequence of Z104 as determined by Sanger sequencing and small (s)RNA deep sequencing. The latter technique was useful to confirm the isolate sequence, that was deposited in the NCBI database with the accession number MK956829 and to investigate the sequence variability of Z104 isolate. The genome of 9611 nucleotides (nt) consisted of an open reading frame of 9243 nt encoding a polyprotein of 3080 amino acids (349.88 kDa), flanked by the 5′- and 3′-untranslated regions, and an overlapping ORF of 74 amino acids, the P3N-PIPO. As in previously sequenced ZYMV isolates, Z104 polyprotein consists of 10 putative functional proteins. Several domains in the HC-Pro and P3 proteins related to the transmissibility by aphids and virus aggressiveness are conserved in Z104. Symptoms induced both in the field and laboratory by Z104 together with its molecular features indicate that it is an aggressive strain. Deep sequencing of sRNAs revealed a particularly high variability in the genome sequence at eight nucleotide positions, three of which resulted in missense mutations in the P3, 6K1, and NIb proteins. This may be indicative of the natural ZYMV genome variability within the host. The phylogenetic analysis of the whole genome or the sole coat protein of several ZYMV isolates did not provide evidence for a close relationship of Z104 with other isolates from European countries. To the best of our knowledge, this is the first-reported complete genome of a ZYMV isolate from Italy.

Variable 3\u2019polyadenylation of Wheat yellow mosaic virus and its novel effects on translation and replication

BackgroundPolyadenylation influences many aspects of mRNA as well as viral RNA. variable polyadenylation at the 3' end have been reported in RNA viruses. It is interesting to identify the characteristic and potential role of 3' polyadenylation of Wheat yellow mosaic virus (WYMV), which has been reported to contain two genomic RNAs with 3' poly(A) tails and caused severe disease on wheat in East Asia region.Methods3' RACE was used to identify sequences of the 3′ end in WYMV RNAs from naturally infected wheat by WYMV. In vitro translation assay was performed to analyze effect of UTRs of WYMV with or without 3’polyadenylation on translation. In vitro replication mediated by WYMV NIb protein were performed to evaluate effect of variable polyadenylation on replication.ResultsVariable polyadenylation in WYMV RNAs was identified via 3′ RACE. WYMV RNAs in naturally infected wheat in China simultaneously present with regions of long, short, or no adenylation at the 3' ends. The effects of variable polyadenylation on translation and replication of WYMV RNAs were evaluated. 5’UTR and 3’UTR of WYMV RNA1 or RNA2 synergistically enhanced the translation of the firefly luciferase (Fluc) gene in in vitro WGE system, whereas additional adenylates had an oppositive effect on this enhancement on translation mediated by UTRs of WYMV. Additional adenylates remarkably inhibited the synthesis of complementary strand from viral genome RNA during the in vitro replication mediated by WYMV NIb protein.Conclusions3' end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3' polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3' polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.

Ribosome profiles and riboproteomes of healthy and Potato virus A- and Agrobacterium-infected Nicotiana benthamiana plants.

Summary Nicotiana benthamiana is an important model plant for plant–microbe interaction studies. Here, we compared ribosome profiles and riboproteomes of healthy and infected N. benthamiana plants. We affinity purified ribosomes from transgenic leaves expressing a FLAG‐tagged ribosomal large subunit protein RPL18B of Arabidopsis thaliana. Purifications were prepared from healthy plants and plants that had been infiltrated with Agrobacterium tumefaciens carrying infectious cDNA of Potato virus A (PVA) or firefly luciferase gene, referred to here as PVA‐ or Agrobacterium‐infected plants, respectively. Plants encode a number of paralogous ribosomal proteins (r‐proteins). The N. benthamiana riboproteome revealed approximately 6600 r‐protein hits representing 424 distinct r‐proteins that were members of 71 of the expected 81 r‐protein families. Data are available via ProteomeXchange with identifier PXD011602. The data indicated that N. benthamiana ribosomes are heterogeneous in their r‐protein composition. In PVA‐infected plants, the number of identified r‐protein paralogues was lower than in Agrobacterium‐infected or healthy plants. A. tumefaciens proteins did not associate with ribosomes, whereas ribosomes from PVA‐infected plants co‐purified with viral cylindrical inclusion protein and helper component proteinase, reinforcing their possible role in protein synthesis during virus infection. In addition, viral NIa protease‐VPg, RNA polymerase NIb and coat protein were occasionally detected. Infection did not affect the proportions of ribosomal subunits or the monosome to polysome ratio, suggesting that no overall alteration in translational activity took place on infection with these pathogens. The riboproteomic data of healthy and pathogen‐infected N. benthamiana will be useful for studies on the specific use of r‐protein paralogues to control translation in infected plants.

Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication.

Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb.

First Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in Pennsylvania

HomePlant DiseaseVol. 100, No. 4First Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in Pennsylvania PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of White clover mosaic virus and Turnip mosaic virus Mixed Infection on Garlic Mustard in PennsylvaniaK. Zhao, P. Margaria, and C. RosaK. ZhaoSearch for more papers by this author, P. MargariaSearch for more papers by this author, and C. RosaSearch for more papers by this authorAffiliationsAuthors and Affiliations K. Zhao P. Margaria C. Rosa , Pennsylvania State University, University Park, PA 16802. Published Online:10 Feb 2016https://doi.org/10.1094/PDIS-09-15-1083-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In spring 2015, 12 weedy garlic mustard (Alliaria petiolata) plants with mosaic symptoms were found in a garden in State College, PA. The same symptoms are seen on many weedy garlic mustards in the area each spring, as they were in previous years at this location. Incidence was ∼10% in the garden, with 2 of ∼20 groups of 4 to 6 plants infected. Quality of RNA extracted from garlic mustard was poor, so symptomatic leaves were used for mechanical transmission to Nicotiana benthamiana. Seven days after mechanical inoculation, plants showed necrotic spots and leaf deformation on the systemic leaves. Double-stranded RNA was extracted from symptomatic N. benthamiana leaves and electrophoresis was performed on 6% polyacrylamide gel to detect dsRNA, produced in plants infected with RNA viruses (Valverde et al. 1990). A band of high molecular weight confirmed the presence of dsRNA. Total RNA was then extracted from the N. benthamiana plants using Spectrum Plant Total RNA Kit (Sigma-Aldrich). A tobravirus or a potyvirus were suspected as possible causal agents, based on literature search for the potyvirus and on the presence a tobravirus infected ornamental plant in the same garden. Two pairs of degenerate primers (Chen et al. 2001; Jones et al. 2008) were used separately in one tube reverse-transcription PCR (RT-PCR) and 835-bp and 654-bp amplicons were cloned into pGEM-T Easy Vector (Promega) and sequenced by Sanger sequencing. The sequences were deposited as GenBank Accession Nos. KT799530 and KT799528, respectively. BLAST results showed that the first sequence had 96% nucleotide identity with the RNA dependent RNA polymerase (RdRp) of a previously reported White clover mosaic virus (WClMV) sequence (AB669182). The second sequence had 95% nucleotide identity to a Turnip mosaic virus (TuMV) sequence across the NIb and coat protein genes (AB252117). Two newly designed primer pairs W_1F (5′-CAGAACGAGCCATCCGCAGAA), W_1500R (5′-TCAGTGCAGTCAGCATTATGAG) and TuMV_F (5′-TCTTGGACGAAGCATGGAGC), TuMV_R (5′-TCGGACTGCCTCTTTGCCTG) were used to further confirm the presence in N. benthamiana of WClMV and TuMV, respectively. A 1,457-bp amplicon was obtained by using W_1F and W_1500R and sequenced. This sequence (KT799531) had 95 to 96% nucleotide identity with several WClMV RdRp sequences (AB056720, AB669182, and X16636). A 577-bp amplicon was obtained by using the primer pair specific to TuMV sequence. The sequenced amplicon (KT788529) showed 97% nucleotide identity to a TuMV sequence spanning the P3 and 6K1 protein (AB701705). Due to the poor quality of RNA we could not test the original or other garlic mustard plants for the presence of the two viruses, but all weeds had uniform symptoms appearance and severity. To our knowledge, White clover mosaic virus and Turnip mosaic virus have not been previously reported in garlic mustard plants in Pennsylvania. Other potyviruses including TuMV have been reported in garlic mustard and Brassicaceae while WClMV to our knowledge was never reported in Brassicaceae or in garlic mustard before. TuMV is a member of the Potyviridae family, is transmitted by aphids in a nonpersistent manner, and causes a variety of symptoms in many Brassica spp.(Tomlinson and Ward 1978) The presence on garlic mustard of a strain of TuMV very similar to a strain already reported in Europe on arugula (Eruca or Brassica vesicaria subsp. sativa) could indicate that this virus can be transmitted between cultivated and not cultivated Brassicaceae and that wild Brassicaceae can potentially serve as virus reservoir (Lockhart 2012). The mode of transmission of WClMV is not known and reports of its transmission by contact or by dodder (Cuscuta spp.) (probably under experimental conditions) have been controversial; therefore, it is hard to speculate if this virus can move from crops to natural environment and vice versa. This is the first report of infection of garlic mustard with TuMV and WClMV, which have the potential to reduce the fitness of this plant species that is considered invasive in North America.

GENETIC VARIABILITY OF THE REPLICASE (NIB) GENE OF PAPAYA RINGSPOT VIRUS IN NORTHERN INDIA INDICATES COMMON ANCESTRY WITH ISOLATES FROM CHINA AND TAIWAN

The purpose of the present study was to investigate the genetic variability and phylogeny of papaya ringspot virus (PRSV) on the basis of NIb (replicase) gene. The NIb and coat protein (CP) regions of about 2.5 kb of seven PRSV isolates from North India were cloned and sequenced. The North Indian isolates R2R and R5R have higher and lower sequence similarity with other PRSV isolates, respectively. Sequence comparison of NIb gene of 42 PRSV isolates, revealed that the average evolutionary divergence of all sequence pairs were 10.6% and 11.2% at amino acid and nucleotide level, respectively. Transitions were more frequent than transversions in the analyzed sequences. Maximum likelihood phylogenetic tree of combined NIb and CP regions indicated the common ancestry of North Indian isolates with Chinese and Taiwanese isolates. One potential recombination breakpoint in isolate R5R, together with its higher sequence identity and common ancestry with Chinese PRSV isolates, suggest the possible import of the former from the Chinese subcontinent.

Changes in some biochemical qualities during drying of pulp pre-conditioned and fermented cocoa (Theobroma cacao) beans.

Fermentation and drying are critical to the development of flavour precursors that generate into distinctive chocolate flavour notes during industrial manufacture. These processes also lead to reduction in acidity and free fatty acids of nibs, which dictates the levels of bitterness and colour development in chocolates. This study investigated changes in nib acidity, flavour precursors (sugars concentration and proteins) and free fatty acids during drying of pulp pre-conditioned and fermented cocoa beans using a 4 x 3 full factorial experimental design with pod storage (0, 3, 7 and 10 days) and drying time (0, 3 and 6 days) as the principal factors. Non-volatile (titratable) acidity, pH, sugars (reducing, non-reducing and total sugars), changes in protein content and free fatty acids of the beans were studied using standard analytical methods. Increasing pod storage consistently increased pH of the fermented nibs at the end of drying with consequential decrease in titratable acidity. The pH increased from 4.92 for the freshly harvested pods to 6.00 for pods stored for 10 days at the end of the drying process. Similarly, pH of the fermented beans increased with increasing drying time for all pod storage treatment except for pods stored for 10 days. The pH of fermented beans whose pods were stored for 3 and 7 days were 5.26 and 5.56 respectively after drying for 7 days. Protein, reducing sugars, non-reducing sugars and total sugars decreased significantly (p<0.05) with increasing duration of drying at all pod storage periods. Pod storage and drying significantly (p<0.05) increased the free fatty acids content of the fermented nibs. The FFAs of the dried beans increased from 0.47% for the unstored (freshly harvested) pods to 0.55% for pods stored for 3 and 7 days and 0.58% for pods stored for 10 days. However, FFAs content of all the dried fermented beans were below the acceptable limits of 1.75% oleic acid equivalent in cocoa butter at all pod storage periods. Storage of cocoa pod between 3–7 days with 7 days of drying (after 6 days fermentation) led to considerable reductions in nib acidity, reducing sugars, nonreducing, total sugars and proteins and acceptable FFA levels.

First Report of Leek yellow stripe virus in Allium sativum in Western India.

Garlic (Allium sativum L.) is an important bulbous spice crop in India as well as other parts of world. Garlic is well known for its medicinal properties. Degeneration due to viral infections is one of the important constraints in exploiting its yield potential. Leek yellow stripe virus (LYSV), genus Potyvirus, family Potyviridae, is a prominent virus known to infect garlic worldwide (4). During July 2013, potyvirus-like symptoms such as mosaic, streaking, stunting, mottling of leaves were observed on garlic cv. G-41 and landrace Ranibennur local, collected from Karnataka, India, and maintained at the Directorate of Onion and Garlic Research, Rajgurunagar, Pune, India. The incidence of symptomatic plants was estimated at 70% for Ranibennur local and 68% for cv. G-41. The symptomatic leaves were sampled diagonally from the field. Twenty symptomatic plants per cultivar with each sample was composited from young, middle, and lower (basal) leaves of the plant. These samples were tested by double-antibody sandwich (DAS)-ELISA for LYSV using commercially available kit (Agdia Inc., Elkhart, IN). ELISA-positive plants were further subjected to molecular studies. Total RNA from the infected leaf samples were extracted by RNeasy Plant Mini kit (Qiagen GmbH, Hilden, Germany) and assayed by reverse transcription (RT)-PCR using primer pair LYSV-F 2 (5'-GCACCATACAGTGAATTGAG-3') (1), LYSV-R (5'-GCCTCGCGCGCTCTAA-3') (3) to amplify 874 bases of partial Nib and partial coat protein gene. The amplified product of 874 bp derived from A. sativum isolate was purified (QIAquick PCR Purification Kit, Qiagen) and cloned using vector pDrive (Qiagen). The recombinant clones were sequenced and submitted in NCBI database (GenBank Accession No. KF850539). The sequence analysis performed on CLC Main Workbench Version 6.8.4 gave confirmation of LYSV. Further, phylogenetic analysis of the 874-nt sequence revealed the highest nucleotide identity (80 to 82%) with LYSV isolates (DQ925453, JN127339, AB005611, and JX429965). To the best of our knowledge, this is the first report of natural infection of garlic by LYSV in western India. LYSV is known to cause direct losses in garlic and other related Allium spp. Up to 54% reduction in bulb weight was observed due to single infection of this virus (2). Hence, our first report about this virus has significant impact on garlic production scenario, if this virus found to be widespread in the country. For this, additional surveys and genotype screenings are needed to obtain a better understanding of the potential impact of LYSV on garlic production in India.

First Report of Wild tomato mosaic virus Infecting Tobacco (Nicotiana tabacum) in China

Wild tomato mosaic virus (WTMV), a potyvirus, has been reported in Laichau, Vietnam, infecting Solanum torvum (wild tomato) in 2008 (3), and Kanchanaburi, Thailand, infecting Capsicum spp. in 2013 (KF250353). In mid-May 2013, Nicotiana tabacum showing yellowing, mosaic, and/or ringspot symptoms were found in natural tobacco fields of Nanxiong, Guangdong Province, China. Total RNA was extracted from symptomatic leaves and reverse transcribed with M4T (5'-GTTTTCCCAGTCACGAC (T)15-3') as the 3' anchoring primer (1). The cDNA was used as template in a PCR assay using primers M4: 5'-GTTTTCCCAGTCACGAC-3' and Sprimer: 5'-GGXAAYAAYAGYGGXCAZCC-3', which amplifies a region comprising part of the NIb protein gene, the entire coat protein (CP) gene and the 3' nontranslated region (UTR) of a potyvirus (1). A ~1,700-bp product was amplified from the cDNA derived from three of the five diseased plants. The product (KF639967) showed 87% and 84% nucleotide sequence identities with those of WTMV isolates KAN and Laichau, respectively. The CP deduced from the sequence of the product shared 87% and 86% nucleotide and 94% and 93% amino acid sequence identities with those of WTMV isolates KAN and Laichau, respectively. The 3'-UTR of the putative virus shared 93% and 92% nucleotide sequence identities to those of WTMV isolates KAN and Laichau, respectively. Thus, according to the molecular criteria for potyvirus species demarcation (2), the virus we identified should be an isolate of WTMV (isolate GD1). One of the diseased samples was homogenized in 0.1 mol/liter phosphate buffer (pH 7.0) and used to inoculate the potyvirus to healthy, two to four leaf-stage Capsicum annuum L., N. tabacum, and N. benthamiana. The inoculated, as well as mock-treated plants, which were inoculated only with phosphate buffer, were grown in soil under 12 h day/12 h night at 25°C. All inoculated N. tabacum and N. benthamiana plants developed yellowing and mosaic symptoms by 14 days post inoculation (dpi). For N. benthamiana, the symptom became very severe by 21 dpi and some diseased plants died prematurely. About 10% of inoculated C. annuum L. developed very mild veinal chlorosis 18 dpi. Cloning and sequencing experiments showed that all the symptomatic plants tested were WTMV positive, but Cucumber mosaic virus, Tobacco mosaic virus, and Tobacco etch virus negative. To our knowledge, this is the first report of WTMV in China. Also, it is the first report that WTMV infects Nicotiana spp. Although further experiments are needed to definitively attribute the disease observed in the field to WTMV, our results indicate that WTMV, which forms a monophyletic clade with a number of other potyviruses infecting Solanaceae species in phylogenetic analysis, is widely distributed, or is spreading in Southeast Asia. It may pose a threat to Solanaceae species cultivation in this region.

Changes in Nib Acidity, Proteolysis and Sugar Concentration as Influenced by Pod Storage and Roasting Conditions of Fermented Cocoa (Theobroma cacao) Beans

Changes in nib acidity, protein and sugar concentration during roasting of pulp pre-conditioned and fermented cocoa (Theobroma cacao) beans were investigated. A 4 × 4 full factorial design with the principal experimental factors as pod storage (0, 3, 7 and 10 d) and roasting time (0, 15, 30 and 45 min) were used. The roasted samples were evaluated for pH, titratable acidity, protein content and sugars concentrations using standard methods. Increasing pod storage caused consistent increases in pH with concomitant decreases in titratable acidity, whereas increasing roasting time caused only marginal and insignificant changes in pH but significantly decreased the titratable acidity. The protein content decreased significantly (P < 0.05) with increasing pod storage and roasting time. Reducing sugars increased marginally with increasing pod storage treatments whiles increasing roasting time significantly (P < 0.05) decreased the reducing sugars of the beans for all pod storage. The non-reducing sugar and total sugar content of the beans decreased significantly from 3.493 mg/g to 2.641 mg/g and from 9.284 mg/g to 8.891 mg/g, respectively, for pods stored from 0 to 10 days while roasting time caused slight decreases in non-reducing sugars with a considerable decrease in total sugars. Pod storage up to seven days decreased considerable the nib acidity (non-volatile acids), non-reducing sugars and total sugars while roasting up to 45 min at 120 °C caused dramatic decreases in the nib acidity and reducing sugars with only marginal decreases in non-reducing sugars and total sugars.