DNA-B Fragments Research Articles

First report of squash leaf curl China virus infecting tomato in China.

Squash leaf curl China virus (SLCCNV) is a species in the genus Begomovirus that possess a bipartite genome. It is transmitted by the whitefly species Bemisia tabaci and infects cucurbit crops in various parts of the Old World (Wu et al., 2020). In 2020, tomato plants with curled, distorted and yellow leaves were found in a greenhouse in Shouguang, Shandong Province, China (Fig. S1). Leaves with these symptoms were collected from 11 plants and the total RNA was extracted with TRIzol reagent (Invitrogen, USA). Five RNA extracts of the highest quality were combined and a small RNA library was generated by the company (BGI-Shenzhen, China). About 22,338,920 clean reads (18-28nt) were acquired and assembled into larger contigs with the software Velvet 1.0.5. These were further compared against nucleotide sequences in the National Center for Biotechnology Information (NCBI) databases with BLASTn searches. Not unexpectedly, there were many assembled contigs that had high identities (90%-100% identities) with known tomato-infecting viruses, including 241 contigs matching tomato chlorosis virus, 26 contigs matching southern tomato virus, and 4 contigs matching tomato yellow leaf curl virus. However, 12 contigs had high identities (90%-100%) with the genomic DNA-A of SLCCNV, while 9 other contigs had high identities (90%-100%) with the genomic DNA-B of SLCCNV. To verify the presence of SLCCNV in tomato plants, two sets of primer pairs were designed according to the specific contigs assembled from derived small interfering RNAs (vsiRNAs). The primer pairs A742-F/A742-R (5'-GTAATACGAGCATCCGCACGGTAG-3'/5'-CGTGGAGGGCGAC AAACAGCTAACG-3') and B539-F/B539-R (5'-GCTACTTTCAAGGACGAAGAAGAGG-3'/5'-CG ACATAGATTTCTGGTCGGTGGGC-3') directed the amplification of 742 bp and 539 bp for DNA-A and DNA-B fragments, respectively, from the total genomic DNA of the 11 tomato samples. The DNA-A and DNA-B of SLCCNV were both detected from all of the tomato samples. After sequencing, the 742 bp PCR products shared 100% nucleotide sequence identity with the DNA-A of SLCCNV isolate GDXW (MW389919), whereas the PCR-amplified 539 bp fragments shared 100% nucleotide sequence identity with the DNA-B of SLCCNV isolate GDXW (MW389920). The full-length of DNA-A and DNA-B components were amplified with back-to-back primers A-F/A-R (Wu et al., 2020) and B-F/B-R (5'-GATAAACACGTCTCATTGCACCGC-3'/5'-GAGACGTGTTTATCAATATGGA CG-3'), respectively. The amplified fragments were further cloned into the PCE2TA/Blunt-Zero vector (Vazyme Biotech Co., China). After sequencing, the complete sequence of DNA-A was 2736 nt in length (MZ682117), while the DNA-B was 2718 nt in length (OK236348). The phylogenetic relationships of the DNA-A and DNA-B components were determined using MEGA 7 based on the full-length sequences of DNA-A and DNA-B, respectively (Kumar et al., 2016). Results showed that the DNA-A formed an independent cluster and was mostly related to the GDHY (MW389917) in the phylogenetic tree constructed using the neighbor-joining (NJ) method, while the DNA-B formed an independent cluster and was mostly related to the SLCCNV isolate BLDG (MW389928) and isolate GDBL (MW389922) (Fig. S2). The nt identities of DNA-A were also calculated with SDT v1.2 by comparison with other begomovirus sequences from the initial BLASTn analysis (Muhire et al., 2014), showing that the virus shared 99.4% sequence identity with SLCCNV isolate GDHY (MW389917). According to the current demarcation threshold for begomoviruses, recommended by the International Committee on Taxonomy of Viruses (ICTV) (91% nt identity) (Brown et al., 2015), this virus identified from tomato is a distinct strain of SLCCNV, designated SLCCNV-SDSG. To the best of our knowledge, this is the first report of a natural infection of SLCCNV on tomato in China. SLCCNV has caused serious problems in cucurbit production in some areas, so it will be important to investigate if tomato plays a role in the disease biology by serving as a reservoir host. The author(s) declare no conflict of interest. Funding: The funding for this research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (QNJJ202131, QNJJ201915, KJCX20200113).

First Report of Tomato Leaf Curl New Delhi Virus Infecting Crossandra infundibuliformis in India

Crossandra infundibuliformis (L.), commonly known as firecracker flower, is a common flowering plant belonging to the family Acanthaceae. It is commonly cultivated in southern India for its attractive flowers. Symptoms of leaf yellowing, leaf mottling, leaf curling, and distortion as well as stunted growth and flower abortion were observed in C. infundibuliformis plants grown in Dharmapuri district (Tamil Nadu, India). In 2011, we observed the first symptom of leaf curl disease, and a partial sequence of tomato leaf curl New Delhi virus (ToLCNDV) was deposited in GenBank (JN410650). Further, in a survey conducted from 2013 to 2015, a total of 30 symptomatic samples suspected of begomovirus infection were collected along with a nonsymptomatic sample. In particular, intact twin icosahedral geminivirus particles (∼18 × 30 nm) were observed by high-resolution transmission electron microscopy in a partially purified extract from symptomatic leaves. Total DNA was extracted and subjected to PCR using coat protein gene-specific degenerate primers (FP/RP) (Riyaz et al. 2013) and replication-associated protein gene-specific degenerate primers ToLCNDVRepFP (5′-ATGAAGTAWGAACAGCCRCAC-3′) and ToLCNDVRepRP (5′-CCATCCGAACATTCAGGGAG-3′). PCR products of the expected sizes of ∼500 and ∼850 bp were obtained from 25 out of 30 plants, and respective PCR products were eluted from gels and sequenced. In sequence analysis, the isolate from C. infundibuliformis (CI1) showed 89 to 92% nucleotide identity with the ToLCNDV isolates reported earlier from India (KM269361, KM269355, and KP868764). Rolling circle amplification (RCA) was performed using a TempliPhi kit (GE Healthcare). RCA products were subjected to PCR amplification employing abutting primers AKKCI1F (5′-ACCTAATGGCAGAATTGTAATTTTG-3′) and AKKCI1R (5′-AATATTATACGAATGGCCGCTTTAG-3′) for DNA-A and BKKCI1F (5′-ACCGAAAGGCCGCGAAAATTTATCCTG-3′) and BKKCI1R (5′-AATATTAGACGAAAGGCCGCTATGA-3′) for DNA-B. The amplified PCR products were ligated into pJET1.2/blunt vector and cloned using a CloneJet PCR cloning kit (Thermo Scientific, U.S.A.). The clones were sequenced by primer walking, and the corresponding sequences assembled to DNA-A and DNA-B fragments were deposited under GenBank accessions MK395548 and MK395549, respectively. DNA-A (2,742 bp) showed 96% sequence identity with the DNA-A component of a ToLCNDV isolate infecting the bitter gourd crop in India (KP868764), whereas DNA-B (2,694 bp) showed 87% identity with the DNA-B component of a ToLCNDV isolate from India infecting chrysanthemum (MG758147). In addition, association of a betasatellite was also confirmed through PCR assay using universal primer pair Beta01/Beta02 (Briddon et al. 2002), followed by sequencing. The nucleotide sequence of the associated betasatellite (1,352 bp) shared 93.7% identity with the betasatellite component of tomato leaf curl New Delhi betasatellite (ToLCuNDB) reported from New Delhi (JX679002) on tomato. The betasatellite sequence was deposited in GenBank (MK040727). This study records the coexistence of bipartite ToLCNDV and ToLCuNDB in C. infundibuliformis with the evidence of their full-length genome for the first time from India.

First Report of Kudzu mosaic virus on Pueraria montana (Kudzu) in China.

Kudzu (Pueraria montana), a weed widely distributed in southern China, is common in the Fuzhou region of Fujian Province, where many plants show yellow vein mosaic disease. In September 2008, four leaf samples from different plants exhibiting yellow vein mosaic symptom were collected in suburban district of Fuzhou (25°15' N, 118°08' E). Whitefly (Bemisia tabaci) infestation was also observed in this region. Total DNA was extracted from all samples using a CTAB method (4). Universal primers (PA/PB) were used to amplify part of the intergenic region and coat protein gene of DNA-A of begomoviruses (1). An amplicon of approximately 500 bp was obtained from all four samples and then sequenced. Comparison of 500-bp fragments (GenBank Accession Nos. FJ539016-18 and FJ539014) revealed the presence of the same virus (98.8 to 99.4%). A pair of back-to-back primers (Yg3FL-F: 5'-GGATCCTTTGTTGAACGCCTTTCC-3'/Yg3FL-R: 5'-GGATCCCACATGTTTAAAGTAAAGC-3') were designed to amplify the full-length DNA-A from the Chinese isolate identified as Yg3. Sequence analysis showed that full-length DNA-A of Yg3 isolate comprised 2,729 nucleotides (GenBank Accession No. FJ539014) and shared the highest nucleotide sequence identity (91.9%) with Kudzu mosaic virus (KuMV, GenBank Accession No. DQ641690) from Vietnam. To further test the association of DNA-B fragments with the four samples from southern China, rolling circle amplification (RCA) was performed (3). When RCA products were digested with Sph I, approximately 2.7 kb was obtained from all samples. Yg3 isolate was chosen to be sequenced. Sequence analysis showed that full-length DNA-B of Yg3 isolate comprised 2,677 nucleotides (GenBank Accession No. FJ539015) and shared the highest nucleotide sequence identity (76.8%) with KuMV DNA-B (GenBank Accession No. DQ641691) from Vietnam. Based on the current convention of begomovirus species demarcation of <89% sequence identity cut-off criterion (2), Yg3 was identified as an isolate of KuMV. To our knowledge, this is the first report of association of KuMV with yellow vein mosaic symptom of kudzu in China.

Bacterial DNA prolongs the survival of inflamed mononuclear cells in haemodialysis patients

Chronic kidney disease (CKD) patients show evidence of chronic inflammation with mononuclear cell activation which is mainly caused by uraemia itself and is exacerbated by haemodialysis. Small fragments of bacterial DNA (DNAb) are ubiquitous contaminants, which are capable of passing through dialyser membranes causing the stimulation of cells of the immune system. The aim of this study was to evaluate whether DNAb contamination may have an effect on apoptosis of activated monocytes from CKD-5 patients. To test the ability of DNAb to stimulate the inflammatory response, mononuclear cells from 10 chronic kidney disease patients who had not begun haemodialysis (ND-CKD-5) and 10 patients undergoing regular dialysis (HD) were cultured in the presence and absence of DNAb. Ten healthy subjects were used as controls. The percentage of IL-1beta cells was higher in HD patients than in ND-CKD-5 (33.9 +/- 3.0% vs 20.0 +/- 2.3%, P < 0.001) and controls (9.4 +/- 2.1%, P < 0.001). The presence of DNAb induced an increase in the percent of cells expressing IL-1beta in controls, ND-CKD5 and HD patients. In addition, the DNAb also increased the release of cytokines in all groups, the effect was more marked in ND-CKD5 and HD than in controls. DNAb only inhibited apoptosis of activated mononuclear cells from, ND-CKD (17.5 +/- 2.8% vs 12.3 +/- 2.6%, P < 0.01) and HD patients (27 +/- 2.5% vs 14.6 +/- 2.9%, P < 0.01). DNAb enhances cytokine production and promotes the survival of inflammatory mononuclear cells from CKD patients. These results strongly suggest that DNAb fragments play an important role in maintaining chronic inflammation in patients on haemodialysis.

Introduction of the New World Squash leaf curl virus to Squash (Cucurbita pepo) in Egypt: A Potential Threat to Important Food Crops

Squash plants showing leaf curling, yellow mottling, and reduced fruit set were observed in fields in Giza, Egypt in spring 2005. These particular symptoms had not been observed previously in zucchini squash plants in Egypt, but were reminiscent of those caused by begomoviruses (Geminiviridae) that are known to occur in the region, including Watermelon chlorotic stunt virus. Squash plants were heavily infested with the whitefly Bemisia tabaci (Genn.), the only known vector of begomoviruses. Total nucleic acids were isolated from symptomatic squash leaves using the cetyltrimethylammoniumbromide method, and extracts were subjected to polymerase chain reaction (PCR) analysis using two sets of PCR primers. One primer set (prAV2644 and prAC1154) was designed to amplify a fragment that contains the entire viral coat protein (Cp), while the second primer set (prBV1855 and prBC656) was designed to amplify the common region (CR) of DNA-B of begomoviruses (1). The expected size fragments were cloned and the sequence was determined for five clones each. Unexpectedly, the Cp and the CR-B fragments shared their highest nucleotide sequence (nt) identity among well-characterized begomoviruses to the bipartite Squash leaf curl virus (SLCV) native to the western United States. A third primer set (prAC344 and prAV1134) (1) was subsequently used to amplify the remainder of the putative SLCV DNA-A. The fragment was cloned and the DNA sequence was determined. Assembly of the overlapping DNA-A fragments resulted in a complete DNA-A component sequence of 2,636 nt, which is identical to the expected size of the SLCV DNA-A component (GenBank Accession No. DQ285019). Comparison with the latter sequence indicated that the Egyptian squash isolate shared 98% nt identity with SLCV. The sequence for the DNA-B fragment (1,162 nt) shared 94% nt identity with SLCV and was deposited in GenBank as Accession No. DQ285020. The high-shared nt identity with SLCV (2) from the United States suggests that this isolate, herein SLCV-EG, has been introduced into Egypt. The relatively low DNA-B nt sequence identity was a not a surprise since this component is normally less conserved even between strains of a single begomoviral species. Introduction of SLCV is not only potentially significant to the domestic production of crop species in the Cucurbitaceae but also for legume crops. SLCV has a broad host range that also includes members of the Fabaceae, which includes species that contribute significant sources of protein for much of Egypt's population. The virus thus far is thought to be present only in Lower Egypt, however, it could feasibly threaten legume and cucurbit crops if it spreads to Upper Egypt. To our knowledge, this is the first begomovirus of New World origin to become established in the Old World.

A New Tomato-Infecting Begomovirus in Barbados.

In September 1998, tomato plants in Barbados exhibited symptoms of severe leaf curling without marginal chlorosis. These symptoms were often associated with an increase in whitefly (Bemisia tabaci) populations. DNA was extracted from leaf tissue from symptomatic tomato plants. Polymerase chain reaction (PCR) was performed with DNA-A degenerate primer pair PAC1v1978/PAV1c715, which amplifies part of the rep gene, the cp gene, and the common region (CR), and with DNA-B primer pair PBC1v2039/PBV1c800, which amplifies part of the bc1 and bv1 genes and the CR (2). The amplified PCR fragments of DNA-A and DNA-B were 1.3 and 1.4 kb, respectively, which are the expected sizes from bipartite, whitefly-transmitted geminiviruses of the Western Hemisphere (2). DNA sequence of the cloned fragments of DNA-A and DNA-B are available as GenBank No. AF213013 and AF213014, respectively. The 181 nucleotides of the CR of DNA-A had a nucleotide identity of 96% with the CR of DNA-B, which indicates that this is a bipartite begomovirus. Pairwise comparisons using DNASTAR (DNASTAR, Madison, WI) of the sequenced part of DNA-A was most similar to Cabbage leaf curl virus (CaLCuV, 69%, U65529) and Squash leaf curl virus extended host range isolate (SqLCV-E, 64%, M38183), and <59% to 13 other bipartite Western Hemisphere geminiviruses and Tomato yellow leaf curl virus from Israel (X15656). Pairwise comparisons of the DNA-B fragment sequence was 59 and 55% similar to CaLCuV (U65530) and SqLCV-E (M38182), respectively. Phylogenetic analysis of DNA-A of the major groups of Western Hemisphere begomoviruses placed the Barbados tomato-infecting geminivirus in the cluster with CaLCuV and SqLCV-E (1), while DNA-B analysis placed it with CaLCuV. The DNA-A amplified fragment was used as a probe at high stringency with the dot blot hybridization assay using the Genius II labeling and detection kit (Boeringer Mannheim) to detect this geminivirus in tomato and several other plant species, which had typical geminiviral symptoms. Strong hybridization signals were obtained for all 23 tomato plants with symptoms, weak signals were observed for two of three muskmelon and two of seven watermelon plants, all with leaf curling symptoms. No hybridization signals were observed for peppers with leaf curling symptoms and two weed species, Macroptilium lathyroides and Rhynchosia minima, with golden mosaic symptoms or with the symptomless plant species used as negative controls. The weak signals observed from watermelon and muskmelon samples indicated the presence of low virus titer or geminiviruses distinct from this tomato virus. The presence of viral DNA in these two plant species was confirmed by PCR with degenerate primers described above. Resulting database searches of sequences in the GenBank revealed that the Barbados tomato virus appears to be a previously unreported virus. This new virus is given the provisional name Tomato leaf curl Barbados virus (ToLCBBV).

First Report of Tomato Mottle Geminivirus Infecting Tomatoes in Yucatan, Mexico

Whitefly-transmitted geminiviruses are a major constraint on tomato production in Mexico (3). In the Yucatan State, these viruses can cause serious losses in late season plantings. As part of an effort to characterize these viruses, leaf samples from four tomato plants showing symptoms of geminivirus infection, such as stunted growth and leaf mottling and deformation, were collected from a single field in the Yucatan State in February, 1996. Geminivirus nucleic acids were detected in leaf samples from all four plants by squash blot hybridization analysis with a general DNA probe for Western Hemisphere whitefly-transmitted geminiviruses (2). Nicotiana benthamiana plants inoculated with sap prepared with leaf tissue from one plant developed stunted growth and leaf mottling and deformation. When graft-transmitted from N. benthamiana to tomato, the geminivirus(es) induced leaf mottling and deformation, which were similar to symptoms in the field-collected tomato plants. The presence of geminivirus DNA in the sap- and graft-inoculated plants was confirmed with the polymerase chain reaction (PCR) and degenerate primers for the DNA-A (PAL1v1978 and PAR1c496) or DNA-B (PBL1v2040 and PCRc1) components of whitefly-transmitted geminiviruses (4). Using PCR and these degenerate primers, approximately 1.1-kb DNA-A and approximately 0.6-kb DNA-B fragments were amplified from DNA extracts prepared from leaves of each of the four Yucatan tomato plants. No DNA fragments were amplified from these extracts with primers for pepper huasteco geminivirus (pAL1c2329 and pAL1v1471, or pBR1c840 and pBL1v1830). To determine the identity of the geminivirus(es) infecting these tomato plants, the PCR-amplified DNA-A and DNA-B fragments from one of the samples were cloned and sequenced. Comparisons made with these sequences revealed two distinct types of DNA-A and DNA-B clones, indicating a mixed infection of at least two bipartite geminiviruses. DNA-A and DNA-B sequences of one set of clones were >97% identical to sequences of tomato mottle geminivirus (ToMoV) from Florida (1). The presence of ToMoV in all four tomato leaf samples was demonstrated by the PCR-mediated amplification of a 0.9-kb DNA-A fragment with ToMoV-specific primers (pAL1v2295 and pAR1c580). The identity of this 0.9-kb DNA fragment was further confirmed based upon its hybridization with a full-length clone of ToMoV DNA-A under high stringency conditions (2). A data base search made with the sequence of the other type of DNA-A clone revealed sequence identities of <70% with various bipartite geminiviruses (e.g., identities of 70% with tomato mottle, 69% with Sida golden mosaic, 67% with bean dwarf mosaic, and 66% with taino tomato mottle and with potato yellow mosaic), which confirmed that a second geminivirus was present in a mixed infection with ToMoV in this tomato leaf sample. To confirm the bipartite nature of this geminivirus, a DNA-B fragment that contained the common region (CR) sequence was amplified from the same sample with PCR and primers PBL1v2040 and PBR1c970 (a degenerate primer that anneals within the BV1 open reading frame; F. M. Zerbini and R. L. Gil-bertson, unpublished data), cloned, and sequenced. The CR sequence of this DNA-B fragment was 96% identical to that of the DNA-A fragment, which establishes the presence of another bipartite geminivirus in this sample. This is the first report of ToMoV in Mexico. These results also suggest that at least two bipartite geminiviruses may infect tomatoes in the Yucatan Peninsula.

Partial Characterization of Two Whitefly-Transmitted Geminiviruses Infecting Tomatoes in Venezuela.

Whitefly-transmitted geminiviruses can cause significant yield losses on tomatoes (Lycopersicon esculentum Mill.) in Venezuela. To identify the geminivirus(es) infecting tomatoes in Venezuela, 20 tomato samples from commercial tomato fields in four states and one weed (Euphorbia heterophylla L.) sample were examined for geminivirus infection. All samples showed symptoms generally associated with geminivirus infection, including golden or yellow mosaic, mottling, crumpling and/or distortion of leaves, and, in some cases, stunted and distorted growth. Through the use of squash blot hybridization analysis and a general probe for Western Hemisphere whitefly-transmitted geminiviruses (4), geminivirus nucleic acids were detected in 19 of 20 tomato samples and the weed sample. No samples were infected with tomato yellow leaf curl virus (TYLCV), based on squash blot hybridization analysis with a TYLCV-specific probe. With polymerase chain reaction (PCR) and degenerate primers for whitefly-transmitted geminiviruses (PAL1v1978 and PAR1c496) (4), an approximately 1.2-kb DNA-A fragment was amplified from the 19 squash blot-positive tomato samples and from the weed sample. No DNA fragment was amplified from any samples when TYLCV-specific primers (PTYC1v2406 and PTYIRc287) (3) were used. PCR-amplified DNA-A fragments from four samples representing four different states [Monagas (5L), Guarico (3M), Aragua (3R), and Portuguesa (2U)] were cloned and sequenced. Partial AC1, AV1, and complete common region (CR) sequences of the 5L, 3M, and 2U DNA-A fragments were 92 to 93, 93, and 95 to 97% identical, respectively, indicating that these were DNA-A clones of the same virus. Furthermore, these sequences were 91 to 92, 92 to 95, and 93 to 95% identical, respectively, to sequences of homologous regions of potato yellow mosaic virus (PYMV), indicating that these tomato-infecting geminiviruses are isolates or strains of PYMV. The partial AC1, AV1, and complete CR sequences of the 3R DNA-A fragment were 79, 95, and 77% identical to those of 5L, 3M, and 2U clones, respectively, suggesting that this is a different geminivirus. These sequences were 75 to 87, 82 to 88, and 73 to 81% identical, respectively, to sequences of homologous regions of other tomato geminiviruses, including tomato golden mosaic from Brazil, tomato mottle from Florida, and tomato leaf crumple from Mexico. The bipartite nature of the geminiviruses that were present in the 5L, 3M, 3R, and 2U samples was suggested by the amplification of a DNA-B fragment with degenerate DNA-B primers (PBL1v2040 and PCRc1) (4). These results suggest at least two distinct bipartite Western Hemisphere whitefly-transmitted geminiviruses are associated with tomato virus diseases in Venezuela, and that one of these (sample 3R) may be an undescribed geminivirus. The sequence of the DNA-A fragment from the weed sample was not closely related to the tomato-infecting geminiviruses and, therefore, this weed was not an alternate host of these viruses. Furthermore, because PYMV has been shown to infect tomatoes and cause yellow mosaic symptoms (1), it would be of interest to determine the relationship of PYMV and tomato yellow mosaic geminivirus (ToYMV), which has been reported infecting tomatoes in Venezuela (2), but has not been characterized on the molecular level.

Pseudorecombination between infectious cloned DNA components of tomato mottle and bean dwarf mosaic geminiviruses

A newly described whitefly-transmitted geminivirus infecting tomato plants in Florida induces yellow mottling symptoms on leaves, and stunted and distorted growth. The DNA-A and DNA-B components were cloned from extracts of field-infected tomato tissue; excised monomers or uncut tandem dimers of these clones were infectious when co-inoculated on to Nicotiana benthamiana by rub-inoculation. Tomato plants inoculated directly with the DNA-A and DNA-B dimers, or indirectly by sap or graft transmission from N. benthamiana plants previously infected with the dimers, developed symptoms similar to those observed in field-infected plants. This tomato geminivirus is different from previously characterized geminiviruses, and has been named tomato mottle geminivirus (ToMoV). DNA sequence comparisons revealed that ToMoV is closely related to bean dwarf mosaic geminivirus (BDMV) and abutilon mosaic geminivirus. Infectious pseudorecombinants were made by exchanging the cloned infectious DNA components of ToMoV and BDMV and inoculating N. benthamiana plants. The presence of the inoculated DNA components in systemically infected plants was confirmed by characterization of DNA-A and DNA-B fragments amplified by the polymerase chain reaction. This is the first report of pseudorecombination between two distinct geminiviruses. The implications of this finding in geminivirus evolution are discussed.