البرايمرات الخاصة بمجموعة Tobamovirus كشفت عن الإصابة المشتركة بفايروس موزائيك التبغ وفايروس تبرقش الفلفل الطفيف على الطماطه في العراق

During the summer of 2015, leaf deformations and chlorotic mosaic symptoms on leaves and unusual necrotic lesions on leaves, stems and fruits were observed on pepper (Capsicum annuum) plants grown in Almeria (Spain). Symptomatic leaf and fruit samples from eight collected plants, carrying L3 and L4 resistance against tobamovirus, were analyzed by ELISA specific for potyviruses, Cucumber mosaic virus and each of the following possible pepper-infecting viruses: Paprika mild mottle virus, Tobacco mosaic virus, Tomato mosaic virus, Tobacco mild green mosaic virus, and Pepper mild mottle virus (PMMoV). The eight plants tested negative for all viruses except for PMMoV. Mechanically inoculated extracts from two PMMoV-positive, singly infected symptomatic pepper plants systemically infected C. annuum cv Mazo (Unigenia), but not C. chinense PI159236, C. chacoense PI260429, four commercial L4, and two L3 resistant pepper plants, which exhibited local necrosis symptoms. Total RNAs from two plants inoculated with single infections were extracted, analyzed by reverse transcription PCR using primers designed to specifically amplify the coat protein gene (CP) of PMMoV (Antignus et al., 2008) and directly sequenced. Two independent samples yielded identical nt sequences (GenBank accession No. KT962848) and BLAST analysis showed the highest deduced aa identities of 99.4% with P1,2 isolates (M81413, AB000709, EF434393) of PMMoV. However, the predicted aa sequence possessed an Ala→Thr mutation at residue 66 of the CP. To our knowledge, although PMMoV has been shown to have variants that fail to infect L3 and L4 resistant pepper plants, based on unique CP coding sequences, this is the first report of a new P1,2 pathotype in Spain.

In the pathogen infection and host defence equilibrium, plant viruses have evolved to efficiently replicate their genomes, to resist the attack from host defence responses and to avoid causing severe negative effect on growth and metabolism of the hosts. In this study, we generated chimeric tobacco mosaic virus (TMV) variants, in which the coat protein (CP) sequences were substituted with that of cucumber green mottle mosaic virus (CGMMV) or pepper mild mottle virus (PMMoV) to address the role of these in virus infection and host symptomology. The results showed that the chimeric viruses (TMV-CGCP or TMV-PMCP) induce stunting and necrotic symptoms in tobacco plants. We analyzed the transcriptomic changes in tobacco plants after infection of TMV and its chimeras using a high-throughput RNA sequencing approach and found that infection of the chimeric TMV induced significant up-regulation of host defence responsive genes together with salicylic (SA) or abscisic acid (ABA) responsive genes, but down-regulation of auxin (Aux) responsive genes. We further confirmed the increase in the levels of SA and ABA, together with the reduced levels of Aux after infection of chimeric TMV in tobacco plants. These data suggest novel roles of tobamovirus CP in induction of host symptoms and defence responses.

The study described here concerns the proteins, synthesized as a result of tobacco mosaic virus (TMV) multiplication in tobacco protoplasts and in cowpea protoplasts. The identification of proteins involved in the TMV infection, for instance in the virus RNA replication, helps to elucidate the infection process in the plant cell. Not only virus coded proteins, but possibly also host coded proteins may play a part in the TMV multiplication.Research on proteins encoded by the TMV RNA, carried out in cell-free protein synthesizing systems, has revealed that five polypeptides are synthesized under the direction of TMV (subgenomic) mRNAs (see table 1.2., chapter L). Whether the polypeptides, synthesized invitro with TMV RNA as messenger, are of functional significance for the TMV infection may only be determined by means of investigating TMV infected leaves and protoplasts.The TMV multiplication runs synchronously in all protoplasts that are infected. Therefore, proteins synthesized in small amounts upon infection, may be thus detected.The search for proteins sythesized in protoplasts as a result of TMV infection has long been hindered by the fact that various factors in the cultivation of the tobacco plants may adversely influence the quality of the protoplasts. The cultivation of the tobacco plants: Nicotiana Tabacum cv. L. Samsun, Samsun NN and Xanthi nc, could be standardized however, as described in chapter 2. When the tobacco plants were cultivated in this way, at least 50 % of the tobacco protoplasts could be infected with TMV and 70 % or more of the protoplasts survived the subsequent incubation period of 36 hours. This could be achieved every time the protoplasts were isolated. The intensity and quality of the light, the way of watering, the age of the tobacco plants and of the leaf, from which the protoplasts are isolated, among others, appeared to affect the quality of the protoplasts (chapter 3.).The proteins, synthesized upon TMV infection, have to be distinguished among a great variety of host proteins. For this reason it is important to determine the incorporation of radioactive amino acids into protein synthesized as a result of TMV multiplication, in comparison with the incorporation into host proteins that are formed independently from the virus infection. Therefore the specific activity of TMV coat protein (cpm/mg protein) and of the proteins of the 27,000 x g supernatant fraction, synthesized in infected tobacco protoplasts were compared. It appeared that the specific activity of TMV coat protein was at least four times higher than of the proteins in the 27,000 x g supernatant (chapter 4.).The proteins synthesized as a result of TMV multiplication were studied not only in tobacco protoplasts, but also in protoplasts from the primary leaves of cowpea ( Vigna unguiculata (L.) Walp. var. 'Blackeye Early Ramshorn'). The method used for the infection of tobacco protoplasts with TMV was not suitable for the infection of cowpea protoplasts with TMV. Best results were obtained when both protoplasts and virus were incubated in the presence of poly-D-lysine, for 7.5 min. before infection. The protoplasts were pre-incubated in 0.1 M potassium phosphate buffer (pH 5.4) at 0°C, at a concentration of 4 x 10 5 protoplasts/mI and 0.75 μg poly-D-lysine/ml. TMV was pre-incubated in the same buffer at room temperature at a concentration of 2 μg TMV/mI and 2 μg poly-D-lysine/ml. During infection the cowpea protoplasts were incubated together with TMV and poly-D-lysine in a concentration of 2 x 10 5 protoplasts/ml, 1 μg TMV/ml and 1 μg poly-D-lysine/ml, for 7.5 min, in the buffer mentioned above at 0°C. In this way 50 to 70 % of the cowpea protoplasts could be infected with TMV.The course of TMV synthesis in cowpea protoplasts was comparable with that in tobacco protoplasts. The TMV multiplication in cowpea protoplasts was preceeded, however, by a period of 16 hours, during which the increase of TMV is slight, while the TMV multiplication in tobacco protoplasts was preceeded by a lag period of 8 hours. A possible explanation is that a much smaller amount of TMV particles penetrates into cowpea protoplasts during inoculation and/or starts to multiply than is the case in tobacco protoplasts (chapter 5.).The proteins of TMV infected and mock-infected protoplasts were analysed therupon by means of SDS-polyacrylamide slabgel electrophoresis and the polypeptide patterns were visualized by autoradiography.Ten polypeptides were distinguished, which are synthesized as a result of TMV multiplication in polypeptide patterns of proteins from infected tobacco protoplasts. The molecular weights were estimated to be 260,000, 240,000, 170,000, 116,500, 96,000, 90,000, 82,000, 72,000, 30,000 and 17,500 (coat protein). Polypeptides of similar molecular weight were absent or were present to much less extent in polypeptide patterns of proteins from mock-infected tobacco protoplasts. Many polypeptides were observed for reason that the detection capacity was improved by means of subcellular fractionation of the protoplast homogenates.The polypeptides of molecular weight 170,000, 116,500, 72,000 and coat protein were present in the 31,000 x g supernatant fraction and the pellet fractions as well. The polypeptide of molecular weight of 30,000 was present exclusively in the pellet fractions. The other polypeptides were observed exclusively in polypeptide patterns of protein of the 31,000 x g supernatant fraction (see table 6. l., chapter 6.).Eight polypeptides were observed, which were synthesized as a result of TMV multiplication in cowpea protoplasts. The molecular weights of the polypeptides were approximately 150,000, 116,500, 86,000, 72,000, 17,500 (coat protein), 16,000,14,000 and 10,000. Polypeptides of similar molecular weight were absent or present on a far less extent in polypeptide patterns of proteins from mockinfected cowpea protoplasts.The polypeptides of molecular weight 116,500, 72,000 and coat protein were present in the 3 1,000 xg pellet and 3 1,000 xg supernatant. The other polyeptides were present exclusively in the 3 1,000 xg supernatant (table 7. l., chapter 7.).It was assumed that the TMV coded polypeptides are similar in different hosts and, on the other hand, that the host polypeptides, synthesized upon TMV infection differ from host to host. When the TMV specific polypeptides, synthesized in infected tobacco protoplasts were compared with the specific polypeptides synthesized in TMV infected cowpea protoplasts, it appeared that only the polypeptides of molecular weight 116,500, 72,000 and coat protein are of similar size in both hosts (table 7.2., chapter 7). This is an indication that not only the polypeptide of 116,500 daltons and coat protein are TMV coded polypeptides, but that also the polypeptide of 72,000 daltons is encoded in the TMV RNA. It has not been reported that a polypeptide of this size is observed when TMV RNAs are translated in cell-free protein synthesizing systems.A polypeptide of 170,000 daltons is synthesized in vitro under the direction of the TMV RNA. It appeared that the polypeptide synthesized in TMV infected tobacco leaves, has a slightly less electrophoretic mobility than the product of 170,000 daltons synthesized in vitro from TMV RNA as messenger. A polypeptide of similar electrophoretic mobility was present to a lesser extent in mockinfected tobacco protoplasts. Furthermore, a polypeptide of 170,000 daltons was not observed in TMV infected cowpea protoplasts. For these reasons it is likely, that the polypeptide of 170,000 daltons, synthesized in TMV infected tobacco protoplasts, is encoded in the genome of tobacco or is encoded in the TMV RNA, but then the polypeptide has no functional significance in the TMV multiplication process.Further the polypeptide of 30,000 was observed only in TMV infected tobacco protoplasts, whereas a polypeptide of similar molecular weight was shown to be synthesized in vitro from a TMV subgenomic mRNA. The polypeptide of 30,000 daltons was detected exclusively in the polypeptide patterns of protein from the pellet fractions of TMV infected tobacco protoplasts. Polypeptide patterns of protein from corresponding fractions of cowpea protoplasts had a predominant, grey background. Due to this the polypeptide of 30,000 daltons may not be distinguished in TMV infected cowpea protoplasts, whereas the polypeptide of 30,000 daltons synthesized in TMV infected tobacco protoplasts can in fact be a polypeptide coded by TMV RNA. The other polypeptides synthesized in infected tobacco protoplasts or cowpea protoplasts as a result of TMV multiplication are presumably synthesized under the genome of tobacco or cowpea respectively.Finally, it was attempted to examine in what way the polypeptides of 116,500 and 72,000 are involved in the TMV infection process. Both polypeptides were shown to be present in the 31,000 x g pellet of TMV infected tobacco and cowpea protoplasts. It was studied whether virus specific polypeptides of similar molecular weight can be observed in RNA-dependent RNA polymerase preparations isolated from the 31,000 x g pellet fraction of cowpea leaves infected with the cowpea strain of TMV (C-TMV). The RNA-dependent RNA polymerase preparations were isolated by extraction of the 31,000 x g pellet fraction and were further purified by means of subsequent DEAE-BioGel column chromatography and glycerol gradient centrifugation. The purification procedure used was the same procedure as described for the isolation of RNA-dependent RNA polymerase from cowpea leaves infected with cowpea mosiac virus (CPMV).Four specific polypeptides of molecular weight of 98,000, 90,000, 72,000 and 46,000 were distinguished in RNA-dependent RNA polymerase preparations from C-TMV infected cowpea leaves, after glycerol gradient purifications. A polypeptide of molecular weight 116,500 was not observed. Polypeptides of molecular weights 72,000 and 46,000 were not found and those of molecular weights 98,000 and 90,000 were distinguished to a less extent in polypeptide patterns of preparations isolated in exactly the same way from mock-inoculated cowpea leaves.RNA-dependent RNA polymerase activity was also observed in preparations isolated from mock-inoculated cowpea leaves. The specific activity (cpm/mg protein) of the preparation from mock-inoculated leaves was one sixth of the specific activity of the RNA-dependent RNA polymerase preparations from CTMV infected cowpea leaves. The RNA-dependent RNA polymerase activity in C-TMV infected cowpea leaves might therefore be attributed to the increase of one or several polypeptides, present already before inoculation. Since it was thought that the polypeptide of 72,000 daltons is a TMV coded polypeptide, it was examined which specific polypeptides are present in RNA-dependent RNA polymerase preparations isolated in a similar way from CPMV infected cowpea leaves. It appeared, that in addition to CPMV specific polypeptides, the polypeptides of molecular weight 98,000 and 90,000 were also observed in RNAdependent RNA polymerase preparations from CPMV infected leaves. The polypeptides of 72,000 and 46,000 daltons were distinguished only in preparations isolated from C-TMV infected cowpea leaves. These results suggest that the polypeptide of 72,000 daltons in involved is the synthesis of TMV RNA (chapter 8.).

The efficiency of forced-air distribution systems in new residential construction was investigated to determine current conditions and identify improvements. Previous research was reviewed, survey data analyzed to assess current construction practices, and relevant codes and standards were examined. The decision-making process of HVAC design and installation was explored through focus groups with builders and contractors. Performance analysis of duct systems at 22 data collection sites measured duct leakage, house infiltration, and thermal performance. A best practice was identified to help builders move ductwork from attic spaces to interior walls when practical and economical.

Paris polyphylla var. yunnanensis is a perennial herb in the family Trilliacea. The plants have immense medicinal and economic importance (Chen et al., 2021). Large-scale artificial planting has led to the emergence of various viral diseases in Paris polyphylla var. yunnanensis, including paris virus 1 (ParV1), paris mosaic necrosis virus (PMNV), paris polyphylla virus X, and pepper mild mottle virus (PMMoV) (Chen et al., 2021; Chen et al., 2022). However, tobacco mosaic virus (TMV) had not been reported as a pathogen on this host. In September 2021, symptoms of leaf shrinking, withering and mottling, and the plants demonstrating dwarfing first observed on Paris polyphylla var. yunnanensis in Qujing Province, Yunnan, China (Suppl Figure 1A). Leaves with these characteristic symptoms were collected from 20 plants. Virus particles in the samples were observed by transmission electron microscopy (TEM) using negative staining (Zhang et al., 2016). These samples revealed the presence of rod-shaped virions, which were approximately 300 nm long with a diameter of approximately 18nm (Suppl Figure 1B). Based on particle morphology these were identified as a putative Tobamovirus. To further identify the exact virus, total RNA was obtained using an RNA-easy Isolation Reagent (TaKaRaBiotech, Dalian, China), cDNA synthesis was performed and RT-PCR assays allowed to amplify a fragment of the CP gene of TMV using specific primers (Suppl table 1). A 480 bp fragment (Suppl Figure 1C) was obtained and cloned into the pMD-18T vector (TaKaRa Biotech, Dalian, China) and sequenced. BLASTn- analysis revealed that the 20 amplicons were identical and shared coat sequence (100%) identity with the TMV isolates Mile-1 (acc. no. MK584554.1) and the diseased P. polyphylla was infected with TMV. The sequence was deposited in the GenBank database with the accession number OM366238 (CP). The sap from infected plants was used as inoculum for transmission of TMV to 10 healthy Nicotiana glutinosa and N. tabacum K326, respectively. 15 days post-inoculation, obvious symptoms of necrosis and chlortisis for viral infection were observed on inoculated and systemic leaves. The systemic leaves of 20 from two species plants were collected, and tested positive for TMV by RT-PCR with the specific primers (Suppl table 1). The sequences of the movement protein (MP) gene (807 bp, OM3662406) and RNA-dependent RNA polymerase (RdRp) gene (3351 bp, OM366242) of TMV were obtained by RT-PCR assays using MP-and RdRp-specific primers (Suppl Table 1). A disease incidence survey was conducted by our team in three Paris polyphylla var. yunnanensis fields in Qujing province and we observed a symptom incidence of 60% across all three fields. To confirm that the symptoms corresponded to TMV infection, leaf samples from 20 plants were collected from per field and all plants tested positive for TMV using RT-PCR assays. To the best of our knowledge, this is the first report of TMV infection in P. polyphylla var. yunnanensis in China. This report, in combination with another recent report of new viruses (Paris mitovirus 1, Paris virus 2) that infects the plants (Chen et al., 2022), points toward a need to intensively monitor the viruses in fields to protect the P. polyphylla var. yunnanensis industry.

Pepper (Capsicum annuum L.) is one of the major vegetable crops in Jordan. During the winter growing seasons of 2015 and 2016, virus symptoms including stunting of young plants, puckering, and yellow mottling of leaves in sweet pepper plants grown under plastic houses in Jordan Valley were observed. The most obvious symptoms were the misshapen fruits that affected the market value of the crop, which resembled recent descriptions of tomato brown rugose fruit virus (ToBRFV) on tomato fruits (Salem et al. 2016). Symptoms on mechanically inoculated indicator hosts including Chenopodium quinoa, Datura stramonium, D. metel, Nicotiana glutinosa, and N. tabacum, and serological testing using double-antibody sandwich ELISA (antiserum A128 of the PLAVIT collection at IPSP-CNR, Italy) suggested the presence of a tobamovirus. Total RNA was extracted from fruits and leaves of 39 symptomatic pepper plants, using an SV-Total RNA Extraction kit (Promega, U.S.A.). Samples were tested by reverse transcription polymerase chain reaction (RT-PCR) for the most common tobamoviruses infecting pepper, including tomato mosaic virus, tobacco mosaic virus, tobacco mild green mosaic virus (TMGMV), and pepper mild mottle virus (Takeuchi et al. 2005). In addition, generic primers for detection of tobamoviruses were also used (Dovas et al. 2004). In RT-PCR, amplicons of the expected product size (400 and 728 bp) using the generic tobamovirus and TMGMV-specific primers, respectively, in all symptomatic plant samples were obtained, but such amplicons were not obtained from healthy plant extracts or water negative controls. RT-PCR products of the RdRp (400 bp) and CP (728 bp) partial regions were purified and ligated into pGEM T-Easy Vector (Promega), and two clones for each PCR product were sequenced and deposited in NCBI GenBank (accession nos. MK816313 to 16). BLASTN analysis showed that these nucleotide sequences had 96 to 99% identity to TMGMV genome sequences (JX534224 and MH730962) in NCBI GenBank. Furthermore, total RNA was extracted with TRIzol reagent (Invitrogen, U.S.A.) according to the manufacturer’s specifications. After ribosomal RNA depletion, the cDNA library was constructed using a TruSeq RNA Sample Prep kit (Illumina, U.S.A.) and sequenced by Illumina HiSeq X-ten platform (Biomarker, China). Raw sequencing data were analyzed using CLC Genomics Workbench 9.5 (Qiagen, Denmark). After raw reads were processed, a total of 80,818,734 paired-end reads of 150 bp were obtained, generating 266,412 contigs (>200 nt) with de novo assembly by CLC Genomics Workbench 9.5. BLASTN analysis of the assembled contigs revealed the presence of two virus-derived contigs: TMGMV (6,414 nt, MK648158) and ToBRFV (6,388 nt, MK648157), which represented a nearly full-length genome. To confirm the ToBRFV identity, all samples were tested by RT-PCR using two pairs of primers: ToBRFV F1 (5′-GTATTTTTGTTTTACAACATATACCAAC-3′) and ToBRFV R1 (5′-AGTGCGAATGTGATTTAAAACTGTGAA-3′), and ToBRFV F7 (5′-GGAAGAAGTCCCGATGTCTGTAAGGCTT-3′) and ToBRFV R7 (5′-GATGCAGGTGCAGAGGACCATTGTAAAC-3′), designed on ToBRFV genome (KT383474; Salem et al. 2016). Twenty-two out of 39 tested samples originated specific amplicons (1,300 and 697 bp for RdRp and CP, respectively). RT-PCR products of two samples were purified and sent for direct sequencing, and results were deposited in NCBI GenBank (MK834288, MK834289, MK834294, and MK834295), revealing nucleotide identity of 99 to 100% to ToBRFV for both RdRp and CP (KT383474 and KX619418). To our knowledge, this is the first report of TMGMV and ToBRFV infecting pepper in Jordan. The occurrence of these tobamoviruses that are transmitted through seeds, in Jordan Valley, the main area for pepper production, may represent a potential threat to other susceptible vegetables and requires careful monitoring to avoid future outbreaks and significant yield losses.

We conducted a survey on pepper virus diseases in 31 regions in Korea from November 2001 to December 2004. Using electron microscopy, test plant reaction, rapid immuno-filter paper assay (RIPA), reverse transcription-polymerase chain reaction (RT-PCR) and/or analysis of viral nucleotide sequences, we found a number of viruses from 1,056 samples that we collected. These included Cucumber mosaic virus (CMV), Pepper mottle virus (PepMoV), Pepper mild mottle virus (PMMoV), Broad bean wilt virus 2 (BBWV2), Tobacco mild green mosaic virus (TMGMV), and Tomato spotted wilt virus (TSWV). Of the samples analyzed, <TEX>$343(32.5\%)$</TEX> were infected with CMV, <TEX>$209(19.8\%)$</TEX> with PepMoV, <TEX>$141(13.4\%)$</TEX> with PMMoV, <TEX>$12(1.1\%)$</TEX> with BBWV2, <TEX>$40(3.8\%)$</TEX> with TMGMV, <TEX>$5(0.5\%)$</TEX> with TSWV, <TEX>$153(14.5\%)$</TEX> with CMV and PepMoV, <TEX>$54 (5.1\%)$</TEX> with CMV and PMMoV, <TEX>$31(2.9\%)$</TEX> with PepMoV and PMMoV, <TEX>$3(0.3\%)$</TEX> with CMV and BBWV2, <TEX>$1(0.1\%)$</TEX> with CMV, PepMoV and BBWV2, <TEX>$8(0.8\%)$</TEX> with CMV, PepMoV and PMMoV, and <TEX>$30 (2.8\%)$</TEX> samples were infected with viruses which were not identified. CMV was the most predominant virus in all inspected fields and the number of the samples infected with PMMoV was relatively low as compared PepMoV infection level in pepper. TMGMV was only found in the southern part of Korea, while TSWV was isolated in Anyang and Yesan. However, we did not encounter in this survey the Alfalfa mosaic virus (AMV), Potato virus Y (PVY), Tobacco mosaic virus (TMV), and Pepper vein chlorosis virus (PVCV).

We investigated the roles of different concentrations of chemical synthetic spherical silver nanoparticles (AgNPs) in protecting pepper seedlings of the Mecca region, which were naturally and artificially infected by the pepper mild mottle virus (PMMoV). The virus shows many infection symptoms, including pepper leaf deformation with filiform leaves and severe mosaic symptoms. Our study focused on the antiviral activity of different concentrations of spherical nanoparticles in controlling PMMoV infecting pepper seedlings. PMMoV identification was confirmed via DAS-ELISA using the following antiserum: PMMoV, cucumber mosaic virus (CMV), tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), potato virus Y (PVY), and tomato spotted wilt virus (TSWV). The presence of PMMoV was confirmed using electron microscopy and reverse transcription polymerase chain reaction (RT-PCR). We evaluated the effects of exogenously applied different concentrations of AgNPs on CMV infection rate, infection severity, virus concentration, and the concentrations of photosynthetic pigments chlorophyll a, chlorophyll b, carotenoid content, phenolic compounds, and protein components in virus-infected plant cells that were treated with three different concentration of nanoparticles (200, 300, and 400 µg/L) compared to the positive and negative control.

N' resistance is intrinsically broken by tobacco mosaic virus but is still effective against pepper mild mottle virus (PMMoV), including those breaking L resistance in peppers. To evaluate the durability of N' resistance to PMMoV, we performed random mutagenesis of the coat protein (CP) gene of PMMoV. We isolated 11 CP mutants with two to six amino acid changes that escaped the N'-mediated resistance response in Nicotiana sylvestris. Some mutants and their derivatives, which had minimal mutations to escape N'-mediated resistance, exhibited reduced accumulation in inoculated leaves and loss of systemic infectivity in a susceptible pepper (Capsicum annuum) cultivar, as determined by RT-PCR analysis. Although the mutant CPs also escaped recognition by L3 and L4 resistance proteins from pepper in transient expression assays, the loss of systemic infectivity suggests that the mutants are unlikely to overcome L-mediated resistance. In Nicotiana benthamiana, a highly susceptible systemic host of PMMoV, ELISA and RT-qPCR indicated that the mutants consistently infected the host systemically, albeit with attenuated virulence and reduced virus accumulation, especially in younger leaves. The results collectively suggest that the reduced virus accumulation enabled the mutant PMMoV to escape N'-mediated resistance, and as a trade-off, compromised its virulence. The results also suggest that PMMoV CP modulates the systemic symptoms.

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

The biological, serological, and molecular characteristics of a newly isolated L4 resistance-breaking isolate of Pepper mild mottle virus (PMMoV) were studied. The new pathotype of PMMoV is closely related to the Israeli pathotypes P1,2 and P1,2,3 of the virus; however, the mosaic symptoms caused by this new pathotype on pepper plants with an L4 genotype were more severe than the mild mosaic symptoms caused by other common pathotypes of the virus in susceptible plants. The predicted amino acid sequence of the putative coat protein (CP) of the newly described pathotype has two amino acid mismatches when compared with the CP of pathotype P1,2, leucine to glutamine at position 47, and alanine to glycine at position 87. The CP of the new pathotype has one amino acid mismatch when compared with P1,2,3, having alanine instead of glycine at position 87. Based on its biological characteristics, the new pathotype was designated P1,2,3,4 of PMMoV-Is. A method is described for the differentiation among the three PMMoV pathotypes using restriction cleavage analysis of reverse-transcription polymerase chain reaction products made from virus-infected plants. An additional unique MnlI site in the CP gene of the newly isolated P1,2,3,4 allows its distinction from the other two isolates, while BglI cleaved only products of the P1,2 pathotype.

Pepper (Capsicum annuum L.), an important crop in Hun- gary, is frequently infected by Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV) and, recently, also by Pepper mild mottle virus (PMMoV) and Tomato spotted wilt virus (TSWV). In the spring of 2015, mild mosaic symptoms were observed on cv. Ho F1. Symptoms on the fruits were more obvious, consisting of reduction in size, mottling and color changes, brown necrotic streaks and spots. To identify the putative pathogen(s), symptomatic fruits were collected and used for host range determination. Symptoms on mechani- cally inoculated Nicotiana tabacum cv. Samsun, N. tabacum cv. Xanthi-nc, Nicotiana benthamiana, Capsicum annuum cv. Albaregia (L+ gene), C. annuum cv. Feherozon (L1) and C. annuum cv. Brendon F1 (L3) suggested the presence of a tobamovirus belonging to pathotype 0 (Boukema, 1980). Moreover, total RNA was extracted from a symptomatic pepper fruit with the RNeasy plant mini kit (Qiagen, Ger- many) and used in RT-PCR using universal tobamovirus primers for the coat protein gene (Kalman et al., 2001). A products of the expected size (700 bp) was obtained, cloned into pGEM-T Easy Vector (Promega, USA) and sequenced (Biomi, Hungary). The sequence was deposited in GenBank under the accession No. KT374283. Blast analysis showed a 99% identity at nucleotide level with a Spanish isolate (P04/17, accession No. FN594859) of Tobacco mild green mosaic virus (TMGMV). To our knowledge, this is the first report of TMGMV on pepper in Hungary.

Datura stramonium L. produces tropane alkaloids, and the hyoscyamine is dominant among them. Hyoscyamine is produced by hairy root cultures in vitro derived from native plants or plants with the genetically modified biosynthetic pathway for hyoscyamine. A common procedure is extraction from cultivated plants. Elicitors for increased production can be used in both cases. Live viruses are not well known for use as elicitors, therefore, D. stramonium plants grown in soil were artificially infected with the tobamoviruses Pepper mild mottle virus (PMMoV), Tomato mosaic virus (ToMV), and Tobacco mosaic virus (TMV). Differences in the content of hyoscyamine were between capsules and roots of infected and non-infected plants. Elicitation increased content of hyoscyamine in capsules 1.23-2.34 times, compared to the control. The most effective viruses were PMMoV and ToMV (isolate PV143), which increased content to above 19 mg/g of fresh weight of a capsule. The effect of each virus elicitor was expressed also in hyoscyamine content in roots. Elicited plants contained 5.41-16.54 times more hyoscyamine in roots compared to non-elicited plants. The most effective elicitor was ToMV SL-1, which raised production above 20 mg/g fresh weight of roots. It has been shown that tobamoviruses can be used as biotic elicitors.

Key West nightshade (Solanum bahamense) is a perennial solanaceous weed found in the extreme southern portion of Florida. It can be propagated by seed and cuttings and is absent from the noxious weed lists of all U.S. states. Its susceptibility to five viruses common to Florida was evaluated by mechanical inoculation of leaves with Tomato spotted wilt virus (TSWV), Tobacco mosaic virus (TMV), Pepper mild mottle virus (PMMoV), Cucumber mosaic virus (CMV), and a putative tobamovirus recently isolated from hibiscus in Florida (HV). TSWV induced chlorotic rings on inoculated leaves and mosaic and malformation of uninoculated leaves. CMV induced necrotic local lesions on inoculated leaves. No symptoms were observed following inoculation with TMV, PMMoV, or HV. TSWV, TMV, and PMMoV systemically infected S. bahamense as determined by the use of enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction, viral-associated double-stranded RNA analysis, and/or indicator hosts. Active growth of infected plants continued for 7 months following inoculation, making S. bahamense suitable for long-term maintenance of viruses in planta. We suggest that S. bahamense may be a useful host for virus culture collections and for studies involving large numbers of virus isolates where fresh, infected tissue is continuously required.

This study was conducted to evaluate the applicability of crAssphage, pepper mild mottle virus (PMMoV), and tobacco mosaic virus (TMV) as indicators of the reduction of human enteric viruses during wastewater treatment. Thirty-nine samples were collected from three steps at a wastewater treatment plant (raw sewage, secondary-treated sewage, and final effluent) monthly for a 13-month period. In addition to the three indicator viruses, eight human enteric viruses [human adenoviruses, JC and BK polyomaviruses, Aichi virus 1 (AiV-1), enteroviruses, and noroviruses of genogroups I, II, and IV] were tested by quantitative PCR. Indicator viruses were consistently detected in the tested samples, except for a few final effluents for crAssphage and TMV. The mean concentrations of crAssphage were significantly higher than those of most tested viruses. The concentrations of crAssphage in raw sewage were positively correlated with the concentrations of all tested human enteric viruses (p <0.05), suggesting the applicability of crAssphage as a suitable indicator to estimate the concentrations of human enteric viruses in raw sewage. The reduction ratios of AiV-1 (1.8 ± 0.7 log10) were the lowest among the tested viruses, followed by TMV (2.0 ± 0.3 log10) and PMMoV (2.0 ± 0.4 log10). Our findings suggested that the use of not only AiV-1 and PMMoV but also TMV as indicators of reductions in viral levels can be applicable during wastewater treatment.