Transmission Of Plant Viruses Research Articles

Identification and Characterization of Three Novel Solemo-like Viruses in the White-Backed Planthopper, Sogatella furcifera

Agricultural insects play a crucial role in transmitting plant viruses and host a considerable number of insect-specific viruses (ISVs). Among these insects, the white-backed planthoppers (WBPH; Sogatella furcifera, Hemiptera: Delphacidae) are noteworthy rice pests and are responsible for disseminating the southern rice black-streaked dwarf virus (SRBSDV), a significant rice virus. In this study, we analyzed WBPH transcriptome data from public sources and identified three novel viruses. These newly discovered viruses belong to the plant-associated viral family Solemoviridae and were tentatively named Sogatella furcifera solemo-like virus 1-3 (SFSolV1-3). Among them, SFSolV1 exhibited a prevalent existence in different laboratory populations, and its complete genome sequence was obtained using rapid amplification of cDNA ends (RACE) approaches. To investigate the antiviral RNA interference (RNAi) response in WBPH, we conducted an analysis of virus-derived small interfering RNAs (vsiRNAs). The vsiRNAs of SFSolV1 and -2 exhibited typical patterns associated with the host’s siRNA-mediated antiviral immunity, with a preference for 21- and 22-nt vsiRNAs derived equally from both the sense and antisense genomic strands. Furthermore, we examined SFSolV1 infection and distribution in WBPH, revealing a significantly higher viral load of SFSolV1 in nymphs’ hemolymph compared to other tissues. Additionally, in adult insects, SFSolV1 exhibited higher abundance in male adults than in female adults.

Effects of natural Ionones and derived novel analogues with simplified structures on behavioral responses of whiteflies.

Whiteflies are major pests in agriculture, causing damage to crops and transmitting plant viruses. Using Volatile Organic Compounds (VOCs) as semiochemicals offers a sustainable approach for combating whiteflies. One such group of compounds, represented by β-ionone, has been found to possess repellent/attractant properties. To further explore the behavioral effects of these compounds on whiteflies, we selected five natural ionone compounds and synthesized six novel analogues to examine the impact of structural variations on whitefly behavior. Our results demonstrated that β-ionone and its analogues have a significant impact on the behavior of whiteflies. Among them, 0.01% pseudo ionone solution exhibited an attractant effect on whiteflies. Notably, the application of 1% β-ionone and 0.1% β-ionol solution demonstrated a notable repellent effect and oviposition deterrent effect on whitefly. We also found that the novel ionone analogue (±)1A exhibited a strong repellent effect. Both β-ionol and compound (±)1A possess high logP values and low saturation vapor pressures, which contribute to enhanced lipophilicity, making them more likely to penetrate insect antennae and prolong their presence in the air. The newly discovered ionone analogue (±)1A and β-ionol exhibit improved repellent effects, while pseudo ionone shows an attractant effect. These three compounds hold promising potential for development as novel biological control agents. Our work highlights the efficacy of VOCs as a protection method against whiteflies. These findings indicate that our new technology for a 'push-pull' control method of B. tabaci can offer a novel tool for integrated pest management (IPM). © 2024 Society of Chemical Industry.

Plant virus transmission during seed development and implications to plant defense system.

Most plants produce large amounts of seeds to disperse their progeny in the environment. Plant viruses have evolved to avoid plant resistance mechanisms and use seeds for their dispersal. The presence of plant pathogenic viruses in seeds and suppression of plant host defenses is a major worldwide concern for producers and seed companies because undetected viruses in the seed can represent a significant threat to yield in many economically important crops. The vertical transmission of plant viruses occurs directly through the embryo or indirectly by getting in pollen grains or ovules. Infection of plant viruses during the early development of the seed embryo can result in morphological or genetic changes that cause poor seed quality and, more importantly, low yields due to the partial or ubiquitous presence of the virus at the earliest stages of seedling development. Understanding transmission of plant viruses and the ability to avoid plant defense mechanisms during seed embryo development will help identify primary inoculum sources, reduce virus spread, decrease severity of negative effects on plant health and productivity, and facilitate the future of plant disease management during seed development in many crops. In this article, we provide an overview of the current knowledge and understanding of plant virus transmission during seed embryo development, including the context of host-virus interaction.

Vector transmission of parsley yellow leaf curl virus by the leafhopper Austroagallia sinuata.

Parsley yellow leaf curl virus (PYLCV) is a new member of the family Geminiviridae that has not yet been assigned to an established genus due to limited information about its biological properties. In this study, the ability of Austroagallia leafhoppers, which are commonly found on vegetable farms in Kerman province (Iran), to transmit this virus was studied. After a two-day acquisition access period, Austroagallia sp. successfully transmitted the virus from PYLCV-infected parsley to healthy seedlings. On the basis of male genitalia morphology, the species of leafhopper was identified as A. sinuata. This is the first report of a transmission of plant virus by a member of the genus Austroagallia.

Substrate-borne vibrations produced during the interaction with natural enemies alter aphids probing behavior

AbstractThe “ecology of fear”, i.e., physiological and behavioral alterations displayed by pests in response to predation risk, has recently been proposed as a sustainable alternative to chemicals for pest control. However, the development of such a strategy requires a detailed understanding of the signals and cues underlying the pest-antagonist interaction and eliciting the prey behavioral alteration. Here, we characterized the substrate-borne vibrations produced during the interaction between the green peach aphid Myzus persicae and its antagonists, the parasitoid wasp Aphidius colemani and the ladybug Adalia bipunctata. Thereafter, coupling the electrical penetration graph (EPG) with a stimulus controller, we evaluated whether the playback of the vibrations, alone and in combination with the alarm pheromone, impacted aphid probing behavior and interaction with the host plant. Aphids responded to vibrations exhibiting longer non-probing, shorter intracellular probes, i.e. the behavior through which the insect evaluates host plant quality, delay in accessing the phloem vessels and decrease of the frequency of phloem salivation events. In contrast, on plants treated with the alarm pheromone, insects displayed longer intracellular probes. We hypothesize that the alarm pheromone, signaling a distant threat, might induce a careful evaluation of the host plant in order to decide the magnitude of the reaction. On the other hand, vibrations might indicate a closely approaching threat pushing the aphid to rush the host evaluation process and the whole feeding process. The possible repercussion of the behavioral alterations observed on the dynamics of aphid-borne plant virus transmission is also discussed.

Transcriptome Analysis Reveals a Diverse Range of Novel Viruses in Australian Sugarcane Soldier Fly (Inopus flavus) Larvae.

In Australia, Soldier flies (Inopus spp.) are economically significant pests of sugarcane that currently lack a viable management strategy. Despite various research efforts, the mechanisms underlying the damage caused by soldier fly larvae remain poorly understood. Our study aims to explore whether this damage is associated with the transmission of plant viruses during larval feeding. We also explore the larval transcriptome to identify any entomopathogenic viruses with the potential to be used as biocontrol agents in future pest management programs. Seven novel virus sequences are identified and characterised using de novo assembly of RNA-Seq data obtained from salivary glands of larvae. The novel virus sequences belong to different virus families and are tentatively named SF-associated anphevirus (SFaAV), SF-associated orthomyxo-like virus (SFaOV), SF-associated narna-like virus (SFaNV), SF-associated partiti-like virus (SFaPV), SF-associated toti-like virus (SFaTV-1 and SFaTV-2) and SF-associated densovirus (SFaDV). These newly identified viruses are more likely insect-associated viruses, as phylogenetic analyses show that they cluster with other insect-specific viruses. Small RNA analysis indicates prominent peaks at both 21 nt and 26-29 nt, suggesting the activation of host siRNA and piwiRNA pathways. Our study helps to improve understanding of the virome of soldier flies and could identify insect viruses for deployment in novel pest management strategies.

A chromosome-level genome for the flower thrips Frankliniella intonsa

The flower thrips Frankliniella intonsa (Thysanoptera: Thripidae) is a common insect found in flowers of many plants. Sometimes, F. intonsa causes damage to crops through direct feeding and transmission of plant viruses. Here, we assembled a chromosomal level genome of F. intonsa using the Illumina, Oxford Nanopore (ONT), and Hi-C technologies. The assembled genome had a size of 209.09 Mb, with a contig N50 of 997 bp, scaffold N50 of 13.415 Mb, and BUSCO completeness of 92.5%. The assembled contigs were anchored on 15 chromosomes. A set of 14,109 protein-coding genes were annotated in the genome with a BUSCO completeness of 95.0%. The genome contained 491 non-coding RNA and 0.57% of interspersed repeats. This high-quality genome provides a valuable resource for understanding the ecology, genetics, and evolution of F. intonsa, as well as for controlling thrips pests.

Management of Bemisia tabaci on vegetable crops using entomopathogens

The sweetpotato whitefly, Bemisia tabaci Gennadius, is a devastating insect pest on vegetable crops around the world. This pest causes significant damage by directly feeding on vegetables, excreting honeydew, inducing plant physiological disorders, and transmitting plant viruses. The combined direct and indirect damage to vegetable crops has led to substantial economic losses. The heavy reliance on chemical insecticides for whitefly control has led to considerable resistance to many insecticides in whitefly populations. Therefore, integrated pest management (IPM) that includes entomopathogens is considered as a sustainable and effective approach against B. tabaci. This review focuses on biological control of B. tabaci using entomopathogens, including entomopathogenic fungi (EPF), entomopathogenic nematodes (EPN), and entomopathogenic bacteria (EPB). Among them, EPF is the mostly explored group of entomopathogens for B. tabaci control. In particular, Beauveria bassiana (Balsamo-Crivelli) Vuillemin, Akanthomyces lecanii (Zimm.) Spatafora, Kepler and B. Shrestha, Cordyceps javanica (Frieder. & Bally) Kepler, B. Shrestha & Spatafora, and C. fumosorosea (Wize) Kepler, B. Shrestha & Spatafora are the most commonly used EPF species for whitefly control. The control efficacy of EPN against B. tabaci varies with strains/species and concentrations of the EPN, whitefly nymphal instars, host plants, and environmental conditions. Most research on evaluating the virulence of various EPB isolates against whiteflies has been conducted in the laboratory. Entomopathogens offer a promising strategy for whitefly management in vegetables. This review discusses research updates on the three entomopathogen groups for whitefly control, barriers in broad adoption by vegetable growers, and future directions.

The plant virus transmissions database.

Plant viruses are transmitted mechanically or by vegetative propagation, and by vectors such as arthropods, fungi, nematodes, or parasitic plants. Sources to access available information regarding plant virus transmissions are scattered and require extensive literature searches. Here, a recently created plant virus transmission database is described. This was developed to provide access to the modes of transmission and vectors of over 1600 plant viruses. The database was compiled using over 3500 publication records spanning the last 100 years. The information is publicly accessible via https://library.wur.nl/WebQuery/virus and fully searchable by virus name, taxonomic position, mode of transmission or vector.

Genome-wide identification of cuticle protein superfamily in Frankliniella occidentalis provide insight into the control of both insect vectors and plant virus.

The structural cuticle proteins (CPs) play important roles in the development and fitness of insects. However, knowledge about CP gene superfamily is limited in virus-transmitting insect vectors, although its importance on transmission of plant virus has been gradually emphasized. In this study, the genome-wide identification of CP superfamily was conducted in western flower thrips Frankliniella occidentalis that is the globally invasive pest and plant virus vector pest. The pest transmits notorious tomato spotted wilt virus (TSWV) around the world, causing large damage to a wide array of plants. One hundred and twenty-eightF. occidentalis CP genes (FoCPs) were annotated in this study and they were classified into 10 distinct families, including 68 CPRs, 16 CPAP1s, 6 CPAP3s, 2 CPCFCs, 10 Tweedles, 4 CPFs, 16 CPLCPs, and 6 CPGs. The comprehensive analysis was performed including phylogenetic relationship, gene location and gene expression profiles during different development stages of F. occidentalis. Transcriptome analysis revealed more than 30% FoCPs were upregulated at least 1.5-fold when F. occidentalis was infected by TSWV, indicating their potential involvement in TSWV interactions. Our study provided an overview of F. occidentalis CP superfamily. The study gave a better understand of CP's role in development and virus transmission, which provided clues for reducing viral damages through silencing CP genes in insect vectors.

Change in the Physiological Aspects of Soybean Caused by Infestation by Bemisia tabaci MEAM1

Whiteflies cause significant crop losses through direct sap feeding, inducing plant physiological disorders and promoting the growth of sooty mold. Moreover, whiteflies can indirectly harm plants by transmitting plant viruses, particularly begomoviruses and criniviruses, resulting in severe viral disease epidemics. This study aimed to evaluate the physiological characteristics of susceptible and resistant soybean cultivars to B. tabaci. The experiments were conducted in a greenhouse. Eleven soybean cultivars were selected and infested with 100 adults of B. tabaci at the V3 stage. The evaluation of photosynthetic parameters, such as photosynthetic rate, leaf transpiration, stomatal conductance, and internal CO2 concentration, revealed that B. tabaci infestation influenced gas exchange in soybean plants. The photosynthetic rate was higher in cultivars AS3810 and M8349 during the V6 stage. Infestations caused alterations in photosynthetic parameters, suggesting increased energy demand to maintain photosynthetic activity. However, the response to infestation varied among the different cultivars, indicating varying levels of resistance and tolerance to the whitefly’s damage. Furthermore, the infestation had a more notable impact during the vegetative phenological stage. In summary, infestation by B. tabaci has a discernible impact on the physiology of soybean plants, resulting in alterations in gas exchange parameters and water use efficiency. The reaction to infestation exhibited variations among different soybean cultivars, indicating potential differences in resistance to the pest. This study underscores the significance of assessing the physiological consequences of whitefly infestations on soybean crops.

Comprehensive Assessment of Reference Gene Expression within the Whitefly Dialeurodes citri Using RT-qPCR.

The citrus whitefly, Dialeurodes citri, is a destructive pest that infests citrus plants. It is a major vector in transmitting plant viruses such as citrus yellow vein clearing virus (CYVCV), which has caused severe economic losses worldwide, and therefore efficient control of this pest is economically important. However, the scope of genetic studies primarily focused on D. citri is restricted, something that has potentially limited further study of efficient control options. To explore the functionalities of D. citri target genes, screening for specific reference genes using RT-qPCR under different experimental conditions is essential for the furtherance of biological studies concerning D. citri. The eight candidate reference genes were evaluated by dedicated algorithms (geNorm, Normfinder, BestKeeper and ΔCt method) under five specific experimental conditions (developmental stage, sex, tissue, population and temperature). In addition, the RefFinder software, a comprehensive evaluation platform integrating all of the above algorithms, ranked the expression stability of eight candidate reference genes. The results showed that the best reference genes under different experimental settings were V-ATP-A and RPS18 at different developmental stages; α-tubulin, 18S and V-ATP-A in both sexes; EF1A and α-tubulin in different tissues; Actin and Argk under different populations; and RPS18 and RPL13 in different temperatures. The validation of selected reference genes was further identified using heat shock protein (Hsp) 70 as a reporter gene. Our study, for the first time, provides a detailed compilation of internal reference genes for D. citri that are suitable for RT-qPCR analysis, which is robust groundwork for comprehensive investigation of the functional target genes of D. citri.

D-Limonene Affects the Feeding Behavior and the Acquisition and Transmission of Tomato Yellow Leaf Curl Virus by Bemisia tabaci.

Bemisia tabaci (Gennadius) is an important invasive pest transmitting plant viruses that are maintained through a plant-insect-plant cycle. Tomato yellow leaf curl virus (TYLCV) can be transmitted in a persistent manner by B. tabaci, which causes great losses to global agricultural production. From an environmentally friendly, sustainable, and efficient point of view, in this study, we explored the function of d-limonene in reducing the acquisition and transmission of TYLCV by B. tabaci as a repellent volatile. D-limonene increased the duration of non-feeding waves and reduced the duration of phloem feeding in non-viruliferous and viruliferous whiteflies by the Electrical Penetration Graph technique (EPG). Additionally, after treatment with d-limonene, the acquisition and transmission rate of TYLCV was reduced. Furthermore, BtabOBP3 was determined as the molecular target for recognizing d-limonene by real-time quantitative PCR (RT-qPCR), fluorescence competitive binding assays, and molecular docking. These results confirmed that d-limonene is an important functional volatile which showed a potential contribution against viral infections with potential implications for developing effective TYLCV control strategies.

Dissecting Diagnostic and Management Strategies for Plant Viral Diseases: What Next?

Recent advancements in molecular biology have revolutionized plant disease diagnosis and management. This review focuses on disease diagnosis through serological techniques, isothermal amplification methods, CRISPR-based approaches, and management strategies using RNA-based methods. Exploring high-throughput sequencing and RNA interference (RNAi) technologies like host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), this review delves into their potential. Despite the precision offered by RNAi in pest and pathogen management, challenges such as off-target effects and efficient dsRNA delivery persist. This review discusses the significance of these strategies in preventing aphid-mediated plant virus transmission, emphasizing the crucial role of meticulous dsRNA design for effective viral RNA targeting while minimizing harm to plant RNA. Despite acknowledged challenges, including off-target effects and delivery issues, this review underscores the transformative potential of RNA-based strategies in agriculture. Envisaging reduced pesticide dependency and enhanced productivity, these strategies stand as key players in the future of sustainable agriculture.

Fusion dsRNA designs incorporating multiple target sequences can enhance the aphid control capacity of an RNAi-based strategy.

RNA interference (RNAi) is the sequence-dependent suppression of gene expression by double-stranded RNA (dsRNA). This is a promising strategy for the control of insect pests because dsRNA can be rationally designed to maximize efficacy and biosafety, the latter by using sequences that are found in target pests but are safe for non-target insects. However, this has yet to be optimized in aphids, destructive sap-sucking pests that also transmit plant viruses. We used the green peach aphid (Myzus persicae) as a case study to optimize the efficiency of RNAi by applying a novel fusion dsRNA design. Comparative transcriptomics revealed a number of genes that are induced in feeding aphids, and eight candidate genes were chosen as RNAi targets. To improve RNAi efficiency, our fusion dsRNA design approach combined optimal gene fragments (highly conserved in several aphid species but with less homology in beneficial insects such as the predator ladybeetle Propylea japonica) from three candidate genes. We compared this RNAi-based biological control approach with conventional chemical control using imidacloprid. We found that the fusion dsRNA strategy inhibited the aphid population to a significantly greater extent than single-target RNAi and did not affect ladybeetle fitness, allowing an additive effect between RNAi and natural predation, whereas imidacloprid was harmful to aphids and ladybeetles. Our fusion dsRNA design approach enhances the ability of RNAi to control aphids without harming natural predators. © 2024 Society of Chemical Industry.

High-throughput molecular gut content analysis of aphids identifies plants relevant for potato virus Y epidemiology.

Aphids are phloem-feeding insects that reduce crop productivity due to feeding and transmission of plant viruses. When aphids disperse across the landscape to colonize new host plants, they will often probe on a wide variety of nonhost plants before settling on a host suitable for feeding and reproduction. There is limited understanding of the diversity of plants that aphids probe on within a landscape, and characterizing this diversity can help us better understand host use patterns of aphids. Here, we usedgut content analysis (GCA) to identify plant genera that were probed by aphid vectors of potato virus Y (PVY). Aphids were trapped weekly near potato fields during the growing seasons of 2020 and 2021 in San Luis Valley in Colorado. High-throughput sequencing of plant barcoding genes, trnF and ITS2, from 200 individual alate (i.e., winged) aphids representing nine vector species of PVY was performed using the PacBio sequencing platform, and sequences were identified to genus using NCBI BLASTn. We found that 34.7% of aphids probed upon presumed PVY host plants and that two of the most frequently detected plant genera, Solanum and Brassica, represent important crops and weeds within the study region. We found that 75% of aphids frequently probed upon PVY nonhosts including many species that are outside of their reported host ranges. Additionally, 19% of aphids probed upon more than one plant species. This study provides the first evidence from high-throughput molecular GCA of aphids and reveals host use patterns that are relevant for PVY epidemiology.

How does vector diversity influence the transmission efficiency of yellow dwarf virus? Perspectives from a review

AbstractCereals are some of the most important global crops that contribute directly and indirectly to the production of food for human consumption. Cereal aphids can cause significant damage to wheat, barley and oats, particularly via the transmission of plant viruses that cause devastating plant diseases, such as yellow dwarf disease. High levels of yellow dwarf disease can result in yield losses of around 20%, rising to 80% if infection is severe. Yellow dwarf disease is caused by multiple viruses, including viruses within the families Tombusviridae and Solemoviridae. These include yellow dwarf virus species within the genus Luteovirus (Barley yellow dwarf virus) and Polerovirus (Cereal yellow dwarf virus, Wheat yellow dwarf virus, Maize yellow dwarf virus). Some yellow dwarf virus species are primarily vectored by one aphid species whereas others can be transmitted by multiple vectors. Biological diversity within a given vector species (e.g., genotype, biotype) can influence virus transmission efficiency. However, it is unclear what biological factors drive this variation within a given vector species. Understanding how biological variation in vector populations influences virus transmission efficiency can help to identify biological traits that underpin successful transmission in competent vector populations. Here, the available literature on yellow dwarf virus transmission efficiency is synthesized and significant variation in yellow dwarf virus transmission efficiency is detected between different populations for several vector species. Three biological mechanisms that potentially underpin this variation are proposed.

Genetic Diversity and DNA Barcoding of Thrips in Bangladesh.

Thrips are economically important pests, and some species transmit plant viruses that are widely distributed and can damage vegetables and cash crops. Although few studies on thrips species have been conducted in Bangladesh, the variation and genetic diversity of thrips species remain unknown. In this study, we collected thrips samples from 16 geographical locations throughout the country and determined the nucleotide sequences of the mitochondrial cytochrome c oxidase subunit 1 (mtCOI) gene in 207 thrips individuals. Phylogenetic analysis revealed ten genera (Thrips, Haplothrips, Megalothrips, Scirtothrips, Frankliniella, Dendrothripoides, Astrothrips, Microcephalothrips, Ayyaria, and Bathrips) and 19 species of thrips to inhabit Bangladesh. Among these, ten species had not been previously reported in Bangladesh. Intraspecific genetic variation was diverse for each species. Notably, Thrips palmi was the most genetically diverse species, containing 14 haplotypes. The Mantel test revealed no correlation between genetic and geographical distances. This study revealed that thrips species are expanding their host ranges and geographical distributions, which provides valuable insights into monitoring the diversity of and control strategies for these pests.

Leafhopper salivary vitellogenin mediates virus transmission to plant phloem

Salivary effectors of piercing-sucking insects can suppress plant defense to promote insect feeding, but it remains largely elusive how they facilitate plant virus transmission. Leafhopper Nephotettix cincticeps transmits important rice reovirus via virus-packaging exosomes released from salivary glands and then entering the rice phloem. Here, we report that intact salivary vitellogenin of N. cincticeps (NcVg) is associated with the GTPase Rab5 of N. cincticeps (NcRab5) for release from salivary glands. In virus-infected salivary glands, NcVg is upregulated and packaged into exosomes mediated by virus-induced NcRab5, subsequently entering the rice phloem. The released NcVg inherently suppresses H2O2 burst of rice plants by interacting with rice glutathione S-transferase F12, an enzyme catalyzing glutathione-dependent oxidation, thus facilitating leafhoppers feeding. When leafhoppers transmit virus, virus-upregulated NcVg thus promotes leafhoppers feeding and enhances viral transmission. Taken together, the findings provide evidence that viruses exploit insect exosomes to deliver virus-hijacked effectors for efficient transmission.

Phylo-geographic analysis of whitefly on the basis of mitochondrial cytochrome oxidase 1 gene

Abstract Bemisia tabaci is a species complex that causes damage to its broad range of plant hosts through serious feeding. It transmits plant viruses of different groups to important agricultural crops. Some important cash crops of Pakistan are sugar cane, rice, tobacco and seed oil. It shows high genetic variability and is differentiated as races or biotypes. Biotypes are, biotype Q, biotype B, biotype B2, biotype M, biotype L, biotype A, biotype H, biotype C, biotype K, biotype N, biotype R, biotype E, biotype P, biotype J, biotype S, biotype AN. Although the current report based on the Bayesian study of mitochondrial cytohrome oxidase gene1 (CO1) DNA sequences has classified the different populations of whiteflies into twelve genetic groups which are Mediterranean, Sub-Saharan Africa silverleafing, Indian Ocean, Asia II, Asia I, Australia, New World, Italy, China, Sub-Saharan Africa non-silverleafing, Mediterranean/Asia Minor/Africa and Uganda sweet potato. Begomoviruses is largest group of viruses transmitted by B. tabaci and cause major diseases of crops such as tomato and chili leaf curl disease, cassava mosaic disease; yellow mosaic disease of legumes and cotton leaf curl disease. The main objective of current study is to inculpate knowledge regarding genetic diversity of whitefly in cotton fields across Pakistan via analysis of partial DNA sequence of mitochondrial gene Cytochrom Oxidase I (mtCO1).