Multiplex PCR for Identification and Detection of Cassava Mosaic Begomoviruses in Togo

Cassava mosaic disease (CMD) caused by Cassava Mosaic Begomoviruses (CMBs) is one of the most devastating crop diseases and a major constraint for cassava production. In order to ensure surveillance for epidemic prevention, low-cost diagnostic tools are appropriate for large-scale testing of cassava viruses. Multiplex PCR diagnosis is one approach that can reduce diagnostic costs and delays. A multiplex PCR approach was developed for simultaneous detection of African cassava mosaic virus (ACMV), East African Cassava Mosaic Virus and East African cassava mosaic Cameroon virus (EACMV/CM) in Togo CMD-infected cassava leaves. Three primers pairs were used to target their respective viruses in a single tube PCR. Multiplex PCR detected ACMV, EACMV and EACMV/CM in plant DNA extracts prepared from cassava leaves infected with CMB. The primers amplified 783 bp specific to ACMV, 650 bp specific to EACMV and 560 bp specific to EACMCV/CM in both uniplex and multiplex formats. Multiplex PCR is an excellent tool for the effective control of cassava diseases. .

Begomoviruses伴随分子的研究进展

根据国内外有关植物双生病毒伴随分子的报道,总结了begomoviruses伴随分子DNAβ、DNA1和缺陷型小分子DNA的基因组结构特点、基因功能、与辅助病毒和寄主植物的互作以及应用等方面的研究现况,指出存在问题并加以讨论,最后分析了植物病毒伴随分子的研究前景。

Special Issue Introduction-The Whitefly Bemisia tabaci Species Complex and Begomoviruses:Research Progress and Future Prospects

The whitefly Bemisia tabaci species complex and the begomoviruses they transmit are devastating agricultural pests worldwide.With the rapid and further inva-sions by some members of the whitefly species complex into new regions,their impacts on human society are likely to increase in the years to come.

Back to Basics:Are Begomoviruses Whitefly Pathogens?

Begomoviruses and whiteflies have interacted for geological times.An assumed long-lasting virus-vector intimate relationship of this magnitude implies that the partners have developed co-evolutionary mechanisms that insure on one hand the survival and the efficient transmission of the virus,and on the other hand the safeguard of the insect host from possible deleterious effects of the virus.Several studies have indicated that viruses belonging to the Tomato yellow leaf curl virus（TYLCVs）family from China,Israel and Italy are reminiscent of insect pathogens.TYLCVs like all begomoviruses are transmitted in a circulative manner by the whitefly Bemisia tabaci.The survival of the virus in the haemolymph of B.tabaci is ensured by a GroEL homologue produced by a whitefly secondary endosymbiont.Following acquisition and transfer to non-host plants,the virus may remain associated with the insect for its entire 4-5 wk-long adult life.During this period,the ability of the insects to inoculate plants steadily decreased,but did not disappear.The long-term presence of TYLCVs in B.tabaci was associated with a decrease in the insect longevity and fertility.Viral DNA was transmitted to progeny,but seldom infectivity.TYLCV transcripts were found associated with the insects,raising the possibility of replication and expression in the vector.TYLCVs may spread amidst whiteflies during copulation.Functional genomics tools such as microarrays,deep sequencing,quantitative PCR and gene silencing allow revisiting the proposition that TYLCVs have retained,or acquired,some characteristics of an insect pathogen.

East African Cassava Mosaic Virus and East African Cassava Mosaic Cameroon Virus: Two Species Emerging in Togo

Cassava mosaic disease (CMD) caused by the whitefly-transmitted Begomoviruses (family Geminiviridae) is a major threat to production of cassava (Manihot esculenta Crantz) in Togo. Survey was conducted in 2020 in the 5 agroecological zones of Togo to assess the status of East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCMV) and its distribution. Polymerase chain reaction (PCR) and Sanger sequencing were used for the detection of cassava mosaic Begomoviruses (CMBs) in the sampled leaves. The incidence of EACMV was 47.93% (278/580) and varied between 41.30% (Zone V) and 62.29% (Zone IV) across the agroecological zone but no significant difference was observed. The EACMCMV incidence was 13.67% (38/278) and varied significantly (p ≤ 0.001) through the agroecological zone. Phylogenetic analysis of the viral isolates showed that they are closely related to those from Nigeria, Ghana, and Ivory coast. Nucleotide sequence analysis of CP revealed an overall genetic diversity (π) of around 3.4%. These results showed that EACMV was the predominant virus and that EACMCMV incidence could be more widespread in Togo.

A WW Domain-Containing Protein Forms Immune Nuclear Bodies against Begomoviruses

The bipartite begomoviruses (Geminiviridae family), which are DNA viruses that replicate in the nucleus of infected cells, encode the nuclear shuttle protein (NSP) to facilitate the translocation of viral DNA from the nucleus to the cytoplasm via nuclear pores. This intracellular trafficking of NSP-DNA complexes is accessorized by the NSP-interacting guanosine triphosphatase (NIG) at the cytosolic side. Here, we report the nuclear redistribution of NIG by AtWWPI, a WW domain-containing protein that forms immune nuclear bodies (NBs) against begomoviruses. We demonstrated that AtWWPI relocates NIG from the cytoplasm to the nucleus where it is confined to AtWWP1-NBs, suggesting that the NIG-AtWWP1 interaction may interfere with the NIG pro-viral function associated with its cytosolic localization. Consistent with this assumpti on joss AtWWPI functi on cuased plants more susceptible to begomovirus infection, whereas overexpression of AWWP1 enhaneed plant resistance to begomovirus. Furthermore, we found that a mutant versi on of AtWWPI defective for NB formatio n was noIonger capable of interacti ng with and relocating NIG to the nucleus and lost its immune function against begomovirus. The antiviral function of AtWWP1-NBs, however, could be antagonized by viral infection that induced either the disruption or a decrease in the number of AtWWP1-NBs. Collectively, these results led us to propose that AtWWPI organizes nuclear structures into nuclear foci, which provide intrinsic immunity against begomovirus infection.

Molecular Identification and Characterization of a Begomovirus Associated with Okra Enation Leaf Curl Disease in Mali

Okra is one of the most widespread vegetable crops in the world, particularly in West Africa. However, several factors influence okra crops as biotic and abiotic factors. Among the diseases affecting its culture, okra leaf curl disease is a major threat. This study aims to assess begomoviruses from okra plants with symptoms like leaves curl by molecular approach. A total of thirteen serologically positive samples were tested by PCR assay, and one sample was sequenced among them. The begomovirus was found in six isolates, Bhendi yellow vein mosaic virus (BYVMV) in three isolates, and Okra enation leaf curl virus (OELCuV) in three isolates, respectively. The begomovirus isolate sequences shared 90% identity with the Cotton leaf curl Gezira virus. Mixed infections between these viruses were found. Thus, these results highlight the need to monitor the spread of these disease-causing viruses for okra crops in Mali. In addition, they can also lead to a considerable loss in okra fields in Koulikoro, which is an agricultural region by excellence.

侵染朱槿的木尔坦棉花曲叶病毒及其卫星DNA全基因组结构特征

从广州朱槿上分离到病毒分离物G6,全序列测定结果表明,G6DNA-A全长为2737个核苷酸。序列比较显示,G6DNA-A与木尔坦棉花曲叶病毒(CLCuMV)各分离物的同源率均大于89%,其中与CLCuMV-[62]的同源率最高(96.1%),与拉贾斯坦棉花曲叶病毒(CLCuRV)的同源率87.1%～89.8%,而与其他菜豆金色花叶病毒属病毒同源率均在87%以下。DNA-A系统进化关系分析显示,G6与CLCuMV各分离物的亲缘关系最近,聚在一起形成一个分支,而与其他几种双生病毒的亲缘关系相对较远。利用DNAβ特异引物β01和β02,从G6中扩增到卫星DNA分子(DNAβ)。序列分析结果表明,G6DNAβ全长1346个核苷酸,推导其互补链上编码一个ORF(C1)。序列比较结果表明,G6DNAβ与CLCuMV DNAβ的同源率最高(92.1%),与CLCuRV DNAβ的同源率为88.7%,而与其他已报道的DNAβ的同源率均在80%以下。DNAβ系统进化关系分析显示,G6DNAβ与CLCuMV DNAβ形成一个独立的分支,再与CLCuRV及MYVV-[Y47]的DNAβ形成一个较大分支。从上述研究结果可以得出,侵染广东朱槿的病毒分离物G6应该是CLCuMV一个分离物。

侵染广西烟草的中国番茄黄化曲叶病毒及其伴随的卫星DNA分子的基因组特征

从中国广西靖西的烟草病株上分离到病毒分离物G102和G103,用双生病毒特异性引物均扩增出约500bp的片段,两者序列同源性达99%。对G102基因组DNA-A全序列测定表明,其全长为2728个核苷酸,与中国番茄黄化曲叶病毒(TYLCCNV)同源性最高,达96.5%。进一步研究发现,G102和G103都伴随有长为1342个核苷酸的卫星DNA分子(DNAβ),这两个DNAβ分子的全序列与TYLCCNV的DNAβ同源性最高,分别为92.9%和93.4%。这是首次明确广西分离的TYLCCNV也伴随有卫星分子。

Bemisia tabaci Phylogenetic Groups in India and the Relative Transmission Efficacy of Tomato leaf curl Bangalore virus by an Indigenous and an Exotic Population

Bemisia tabaci adults from various host-plant species were collected from 31 regions across India.266 B.tabaci samples were first screened by RAPD-PCR to examine molecular variability and to select individuals with different fingerprints.Host-plant and region of collection were then used to select 25 individuals for PCR amplification and sequencing of their partial mitochondrial cytochrome oxidase subunit one（mtCOI）genes.Pairwise comparisons with mtCOI consensus sequences showed that the majority of these samples had 〈3.5% sequence divergence from groups currently termed Asia I,Asia II-5,Asia II-7,and Asia II-8.The biotype-B B.tabaci from India clustered into the Middle East-Asia Minor 1 group.A new group of B.tabaci from Coimbatore,collected from pumpkin,was related most closely to the Asia I group（6.2% sequence divergence from the consensus Asia I sequence）.To increase our understanding of the epidemiology of tomato leaf curl disease（ToLCD）and the key B.tabaci genetic groups involved in virus spread,the indigenous Asia I and the exotic biotype-B population from South India were used to carry out transmission experiments using Tomato leaf curl Bangalore virus（ToLCBV）.The acquisition access periods（AAP）,inoculation access periods（IAP）,latent periods（LP）,and ToLCBV transmission efficiencies of the two populations were compared and the biotype-B had the more efficient transmission characteristics.These results are discussed in relation to recent changes in the epidemiology of tomato leaf curl disease in South India.

Genetic Diversity of Tomato(Solanum lycopersicum L.)Begomovirus in Togo

Geminiviruses, in particular the members of the genus Begomovirus , are considered to be a major phytosanitary problem for tomato crops production in the world. They are responsible for yield losses of up to 20% to 100%. Regrettably, Togo is not spared from this situation. This work aims to show the genetic diversity of the begomoviruses affecting tomato crops production in Togo and their relationship with other begomoviruses. To achieve these objectives, 307 samples of tomato leaves and wild plant species with typical virus symptoms were collected in the Maritime, Plateaus, Central, Kara and Savannah regions and submitted to PCR analysis. The results revealed the presence of begomovirus in 25.40% of the analyzed samples. The PCR products obtained were submitted to direct sequencing. Phylogenetic analysis of sequences of DNA-A different regions of begomovirus identified in this work with that of other begomoviruses showed a nucleotide identity of 96% respectively for Tomato leaf curl Togo virus-Fontem, Tomato Leaf Curl Togo Virus , Ageratum leaf curl Cameroon Alphasatellite;98% respectively to Tomato leaf curl Nigeria virus , Ageratum leaf curl Cameroon virus , Tomato leaf curl Cameroon virus-Fontem, Ageratum leaf curl Cameroon virus and 99% respectively to Tomato leaf curl Kumasi virus , Pepper yellow vein Mali virus Bazegahot and Pepper yellow vein Mali virus-Ouaga. These results suggest a high degree of genetic diversity of tomato begomoviruses identified in Togo.

Homopteran Vector Biomarkers for Efficient Circulative Plant Virus Transmission are Conserved in Multiple Aphid Species and the Whitefly Bemisia tabaci

Plant viruses in the families Luteoviridae and Geminiviridae are phloem restricted and are transmitted in a persistent,circulative manner by homopteran insects.Using fluorescence 2-D difference gel electrophoresis to compare the proteomes of F2 genotypes of Schizaphis graminum segregating for virus transmission ability,we recently discovered a panel of protein biomarkers that predict vector competency.Here we used aphid and whitefly nucleotide and expressed sequence tag database mining to test whether these biomarkers are conserved in other homopteran insects.S.graminum gene homologs that shared a high degree of predicted amino acid identity were discovered in two other aphid species and in the whitefly Bemisia tabaci.Selected reaction monitoring mass spectrometry was used to validate the expression of these biomarkers proteins in multiple aphid vector species.The conservation of these proteins in multiple insect taxa that transmit plant viruses along the circulative transmission pathway creates the opportunity to use these biomarkers to rapidly identify insect populations that are the most efficient vectors and allow them to be targeted for control prior to the spread of virus within a crop.

Socio-Economic and Scientific Impact Created by Whitefly-Transmitted,Plant-Virus Disease Resistant Tomato Varieties in Southern India

Research carried out to assess the impact of open-pollinated Tomato leaf curl virus（ToLCV）-resistant tomatoes and hybrids on the livelihoods of resource-poor farmers in Southern India is described and discussed.Three high-yielding ToLCV-resistant tomato varieties were developed initially using conventional breeding and screening techniques involving inoculation by ToLCV-viruliferous whitefly,Bemisia tabaci.In 2003 and 2004,respectively,these varieties were released officially by the Karnataka State Seed Committee and the Indian Ministry of Agriculture through notification in the Gazette of India.From 2003 to 2005,eleven seed companies bought breeder seed of the ToLCV-resistant varieties and used them to begin breeding F1 hybrids from them.Socio-economic studies carried out to assess the benefits obtained from growing the ToLCV-resistant varieties found that farmers could gain up to 10 times the profit by growing the ToLCV-resistant varieties compared to the pre-existing ToLCV-susceptible varieties.Adoption of ToLCV-resistant tomatoes was also associated with reduced pesticide use.Extra income from tomato sales was prioritised by farmers to pay for children＇s education,better nutrition and medicines.In a joint effort with the commercial seed sector in India,a promotional field day was organised in 2007.As well as the three ToLCV-resistant varieties,62 ToLCV-resistant hybrid tomatoes were exhibited during a farmer-field day by 17 commercial seed companies and several public institutes.Tomatoes with ToLCV-resistance are now grown widely in South India and seeds of the three open-pollinated varieties have been distributed to more than 12 countries.In 2007,a conservative estimate of the financial-benefit to cost of the research ratio was already more than 837：1.

Cryptic species composition and genetic diversity within Bemisia tabaci complex in soybean in India revealed by mtCOI DNA sequence

Bemisia tabaci is a cryptic species complex, causing signiifcant loss on many agricultural y important crops worldwide. Knowledge on species composition and diversity within B. tabaci complex is critical for evolving sustainable pest management strategies. Here we investigate the whitelfy species complex in soybean in major soybean growing states of India. The mitochondrial cytochrome oxidase gene subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian methods indicated the existence of three cryptic species namely Asia I, Asia II 1, and Asia II 7. Al the haplotypes detected in the study could be assigned to these three cryptic species fol owing the species demarcation criteria of 3.5%divergence threshold. Of these, Asia II 1 was found to be predominant with wide spread distribution across the surveyed regions from cool temperate zones to hot and humid tropical plains. On the contrary, cryptic species Asia II 7 showed localized distribu-tion. The Asia II 1 exhibited the highest haplotype diversity and Asia I showed high level of nucleotide diversity. There was a signiifcantly high genetic differentiation among these three cryptic species. The MEAM 1, a dreadful invasive species was not detected in the specimens tested in the current study. The diversity and distribution of three cryptic species is discussed in the light of current knowledge on distribution of whitelfy species in India and yel ow mosaic disease observed during sampling survey.

Occurrence of Cassava Mosaic Begomovirus New Species and Ageratum Leaf Curl Cameroon Virus on Pepper(Capsicum annuum L.)in Togo

Cassava mosaic disease caused by the whitefly-transmitted begomoviruses (family Geminiviridae) is a major threat to cassava (Manihot esculenta Crantz) production, which can be intercropped with other plants such as pepper (Capsicum annuum L.). The aim of this study is to identify cassava begomoviruses on other crops in cassava intercropping systems. Thus, foliar samples showing typical symptoms of virus diseases in cassava intercropping systems were collected from pepper and submitted to PCR analysis and direct sequencing. Three begomovirus species ACMV, EACMV and ALCCMV were identified and characterized in samples. Isolates of these species shared respectively 90% - 93%, 74% and 80% nucleotide identities with begomoviruses. These findings show that cassava begomoviruses can infect other crops and will help in understanding the epidemiology related to whitefly-transmitted begomoviruses in cassava intercropping systems.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo

Tomato leaf curl New Delhi virus(ToLCNDV)is a member of the genus Begomovirus,and causes devastating disease in the world.In recent years,ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture.In this study,we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China.We constructed a full-length infectious cDNA clone of ToLCNDV,which could induce systemic infection with typical symptoms in Nicotiana benthamiana,Cit-rullus melo,and Citrullus lanatus plants through agrobacterium-mediated inoculation.Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible.We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV.Collectively,the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions,which might provide new insights into breeding resistance germplasm in crops.

Tobacco curly shoot virus isolated in Yunnan is a distinct species of Begomovirus

Virus isolate Y1 was obtained from tobacco showing curly shoot symptoms in Baoshan, Yunnan Province. Whitefly transmission test and virion morphology observa-tion showed that it is a begomovirus. In reactions with 14 monoclonal antibodies raised against begomoviruses, Y1 was readily differentiated from begomoviruses reported in China, Pakistan and India. The complete nucleotide sequence of DNA-A was determined, it contains 2746 nucleotides, with two ORFs in virion-sense DNA and four ORFs in comple-mentary-sense DNA. Comparisons with total DNA-A, inter-genie region and deduced amino acid sequences of individual ORFs showed that Y1 is a distinct Begomovirus species, for which the name Tobacco curly shoot virus (TCSV) is pro-posed. The total DNA-A of TCSV is most closely related to that of Tomato leaf curl virus from India (85% sequence identity). In contrast, the deduced coat protein of TCSV is most like that of Cotton leaf curl virus 72b isolate from Paki-stan (98% amino acid sequence identity).

Viral infection of tobacco plants improves performance of Bemisia tabaci but more so for an invasive than for an indigenous biotype of the whitefly

The ecological effects of plant-virus-vector interactions on invasion of alien plant viral vectors have been rarely investigated. We examined the transmission of Tomato yellow leaf cur/China virus （TYLCCNV） by the invasive Q biotype and the indigenous ZHJ2 biotype of the whitefly Bemisia tabaci, a plant viral vector, as well as the influence of TYLCCNV-infection of plants on the performance of the two whitefly biotypes. Both whitefly biotypes were able to acquire viruses from infected plants and retained them in their bodies, but were unable to transmit them to either tobacco or tomato plants. However, when the Q biotype fed on tobacco plants infected with TYLCCNV, its fecundity and longevity were increased by 7- and 1-fold, respectively, compared to those of the Q biotype fed on uninfected tobacco plants. When the ZHJ2 biotype fed on virus-infected plants, its fecundity and longevity were increased by only 2- and 0.5-fold, respectively. These data show that the Q biotype acquired higher beneficial effects from TYLCCNV-infection of tobacco plants than the ZHJ2 biotype. Thus, the Q biotype whitefly may have advantages in its invasion and displacement of the indigenous ZHJ2 biotype.

Identification of a novel DNA molecule associated with Tobacco leaf curl virus

A circular DNA molecule, designated as DNAp, was identified in tobacco plants infected with Tobacco leaf curl virus (TLCV) isolates Y5 and Y8 by PCR using primers based on the conserved region of the two reported DNAp sequences of whitefly-transmitted geminiviruses (WTGs). The complete nucleotide sequences of DNAβ of Y5 and Y8 (TLCV DNAβ) were determined. Y5 DNAβ comprises 1333 nucleotides encoding 8 predicted ORFs with 4 ORFs in virion-sense DNA and 4 ORFs in complementary-sense DNA; Y8 DNAP consists of 1338 nucleotides encoding 7 predicted ORFs with 4 ORFs in virion-sense DNA and 3 ORFs in complementary-sense DNA. TLCV DNAβhas little sequence homology to DNA-A of TLCV, except that it shares conserved TAATATTAC loop sequence with TLCV DNA-A. Sequence comparison showed that Y5 DNAβ shared 85% sequence homology with Y8 DNAβ, and both Y5 DNAβ and Y8 DNAβ had relatively low sequence identity (51%-65%) with the reported DNAβ molecules associated with Agera-tum yellow vein virus and Cotton leaf

Gene expression profiling of the whitefly （Bemisia tabaci） Middle East- Asia Minor 1 feeding on healthy and Tomato yellow leaf curl China virus-infected tobacco

Begomoviruses are exclusively transmitted by whitefly （Bemisia tabaci） in a circulative, non-propagative manner. The influences of begomoviruses on whitefly vector are complex with both direct and indirect effects. However, the molecular mechanisms underlying these effects are poorly understood and the transcriptional profiles of whitefly on healthy and virus-infected plants have not yet been studied. Using suppression subtractive hybridization, we investigated the differentially expressed genes in whitefly Middle East - Asia Minor 1 feeding on healthy and Tomato yellow leaf curl China virus （TYLCCNV） infected tobacco. From the forward cDNA library, 124 differentially expressed expression sequence tags （ESTs） were obtained which represent up-regulated genes in the whiteflies feeding on the infected tobacco. From the reverse library, 112 ESTs were isolated which represent down-regulated genes. Among the up-regulated genes, we identified several genes that are probably involved in direct interaction between whitefly and TYLCCN~, including a 26/29-kDa proteinase that appears to participate in the elimination of foreign proteins, heparan sulfate proteoglycan which mediates the entry of several viruses into host cells and two genes of Rickettsia-a secondary endosymbiotic bacterium of whitefly. In addition, we identified a number of genes involved in metabolism, transcription and translation which might be the result of indirect effects of TYLCCNV on the whitefly via host plants. Collectively, our results suggest that TYLCCNV-infected tobacco changes the gene expression profiles of whitefly via both direct and indirect interactions. This study revealed a number of genes involved in whitefly-TYLCCNV-tobacco interactions and provided useful information for future study on this complex system.