Pathogen-derived Resistance Research Articles

Geminivirus diseases of legumes in India: current status and approaches for management.

India has a large potential for producing a variety of legumes which are proficiently valued for small grower to the highest producers. Plant viruses predominate among the many factors that affect the production of legumes. In tropical and subtropical locations, begomovirus has become a significant productivity barrier for legume production with significant losses. The detection and molecular characterization of various begomoviruses species have been done with regard to phylogenetic analyses, infectivity on host plants, DNA replication, transgenic resistance, promoter analysis, and development of virus-based gene-silencing vectors using several techniques. The molecular detection of begomoviruses involves a variety of techniques, including polymerase chain reaction (PCR), using degenerate primers, reverse transcription PCR (RT-PCR), real time quantitative PCR, rolling-circle amplification PCR (RCA-PCR assay), RCA, and microarray/DNA chip. Begomovirus infections can be prevented by various methods such as by controlling vector populations, use of culture practices, developing virus-free planting materials, developing resistant varieties, following quarantine regulations, and adapting modern methods, including pathogen-derived resistance (PDR), RNA interference (RNAi)-mediated resistance and genome editing approach. This review focuses on current status of geminiviruses infecting various legumes, pathogenesis, genetic flexibility, recombination of begomovirus responsible for the wide host range, modern methods of control, including PDR, RNAi-mediated resistance, small RNA (sRNA)- mediated resistance, Engineered Nucleases, Zinc Finger nucleases, Transcriptional Activator nucleases, CRISPR/Cas9 mediated genome editing and various strategies for management of begomoviruses. The present study entails the view and understanding of different approaches for the begomovirus management which state knowledge about limiting the crop losses.

When an Intruder Comes Home: GM and GE Strategies to Combat Virus Infection in Plants

Viruses are silent enemies that intrude and take control of the plant cell’s machinery for their own multiplication. Infection by viruses and the resulting damage is still a major challenge in the agriculture sector. Plants have the capability to fight back, but the ability of viruses to mutate at a fast rate helps them to evade the host’s response. Therefore, classical approaches for introgressing resistance genes by breeding have obtained limited success in counteracting the virus menace. Genetic modification (GM)-based strategies have been successful in engineering artificial resistance in plants. Several different approaches based on pathogen-derived resistance, antisense constructs, hairpin RNAs, double-stranded RNA, etc., have been used to enhance plants’ resistance to viruses. Recently, genome editing (GE) strategies mainly involving the CRISPR/Cas-mediated modifications are being used for virus control. In this review, we discuss the developments and advancements in GM- and GE-based methods for tackling viral infection in plants.

Natural and Engineered Resistance Mechanisms in Plants against Phytoviruses.

Plant viruses, as obligate intracellular parasites, rely exclusively on host machinery to complete their life cycle. Whether a virus is pathogenic or not depends on the balance between the mechanisms used by both plants and viruses during the intense encounter. Antiviral defence mechanisms in plants can be of two types, i.e., natural resistance and engineered resistance. Innate immunity, RNA silencing, translational repression, autophagy-mediated degradation, and resistance to virus movement are the possible natural defence mechanisms against viruses in plants, whereas engineered resistance includes pathogen-derived resistance along with gene editing technologies. The incorporation of various resistance genes through breeding programmes, along with gene editing tools such as CRISPR/Cas technologies, holds great promise in developing virus-resistant plants. In this review, different resistance mechanisms against viruses in plants along with reported resistance genes in major vegetable crops are discussed.

Evaluation of the Chilli veinal mottle virus CP gene expressing transgenic Nicotiana benthamiana plants for disease resistance against the virus.

Vegetables are an important source of income and high-value crops for small farmers. Chilli (Capsicum spp.) is one of the most economically important vegetables of Pakistan and it is grown throughout the country. It is a rich source of nutrition especially vitamins A, B, C and E along with minerals as folic acid, manganese (Mn), potassium (K) and molybdenum (Mo). Chilli possesses seven times more amount of vitamin C than an orange. Vitamin A, C and beta-carotenoids are strong antioxidants to scavenge the free radicals. Chilli production is restricted due to various biotic factors. Among these viruses, Chilli veinal mottle virus (ChiVMV) is one of the most destructive and menacing agents that inflicts heavy and colossal losses that accounted for 50% yield loss both in quality and quantity. Pathogen-Derived Resistance (PDR) approach is considered one of the effective approaches to manage plant viruses. In this study, ChiVMV was characterized on a molecular level, the coat protein (CP) gene of the virus was stably transformed into Nicotiana benthamiana plants using Agrobacterium tumefaciens. The transgenic plants were challenged with the virus to evaluate the level of resistance of plants against the virus. It was observed that the plants expressing CP gene have partial resistance against the virus in terms of symptoms' development and virus accumulation. Translation of this technique into elite chilli varieties will be resulted to mitigate the ChiVMV in the crop as well as an economic benefit to the farmers.

Transgenic Expression of Synthetic Coat Protein and Synthetic Replication Associated Protein Produces Mild Symptoms and Reduce Begomovirus-Betasatellite Accumulation in Nicotiana benthamiana

Cotton (Gossypium hirsutum L.) is the most essential fiber crop and is widely cultivated within the tropical regions of the world. It has a major impact on socio-economical get-together in Pakistan. Unfortunately, it has been tormented by cotton leaf curl disease (CLCuD) that is produced by distinct species of begomoviruses (Family; Geminiviridae) like Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bu) associated with specific betasatellite—Cotton leaf curl Multan betasatellite (CLCuMuB). Host resistance against CLCuD has not been achieved yet. The present study employed the use of synthetic genes considering the phenomenon of pathogen derived resistance (PDR). The coat protein (CP) and replication associated protein (Rep) genes sequence from CLCuKoV-Bu were codon optimized, synthesized and used to control CLCuKoV-Bu and associated betasatellite related to CLCuD. The Nicotiana benthamiana plants were Agrobacterium mediated transformed with synthetic CP (CPsyn) and synthetic Rep (Repsyn) under the control of Cauliflower mosaic virus 35S promoter. The transgenic plants harboring CPsyn and Repsyn genes were infiltrated with CLCuKoV-Bu alone and CLCuKoV-Bu- CLCuMuB inoculums showed relatively milder symptoms as compared with wild sorts and low virus concentration as checked by southern hybridization. The results confirmed that CPsyn, Repsyn genes may be utilized for the resistance of CLCuKoV-Bu and associated betasatellites related to CLCuD and PDR mechanism may also be developed for the control of CLCuD.

Distinguishing resistances of transgenic sugarcane generated from RNA interference and pathogen‐derived resistance approaches to combating sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) is a causative agent that reduces growth and productivity in sugarcane. Pathogen‐derived resistance (PDR) and RNA interference (RNAi) are the most common approaches to generating resis‐ tance against plant viruses. Two types of transgenic sugarcane have been obtained by PDR and RNAi methods using a gene‐encoding coat protein (CP) of SCMV (SCMVCp). This research aimed to distinguish resistance of the two transgenic sugarcanes in combating SCMV through artificial viral inoculation. The experiment was conducted using transgenic sugar‐ cane lines validated by PCR analysis. Insertion of gene‐encoding CP in the transgenic lines was confirmed by amplification of 702 bp of DNA fragment of SCMVCp. After viral inoculation, mosaic symptoms appeared earlier, at 21 days post inoculation (dpi) in PDR transgenic lines, but was at 26 dpi in RNAi transgenic lines. Symptom observation showed that 77.8% and 50% of the inoculated plants developed mosaic symptoms in PDR and RNAi transgenic lines, respectively. RT‐PCR analysis revealed that the nuclear inclusion protein b (Nib) gene of SCMV was amplified in the symptomatic leaves in plants classified as susceptible lines. Immunoblot analysis confirmed presence of viral CP with a molecular size of 37 kDa in the susceptible lines. Collectively, these results indicated that the RNAi approach targeting the gene for CP effectively produces more resistance against the SCMV infection in transgenic sugarcane compared to the PDR approach.

Yellow Mosaic Disease (YMD) of Mungbean (Vigna radiata (L.) Wilczek): Current Status and Management Opportunities.

Globally, yellow mosaic disease (YMD) remains a major constraint of mungbean production, and management of this deadly disease is still the biggest challenge. Thus, finding ways to manage YMD including development of varieties possessing resistance against mungbean yellow mosaic virus (MYMV) and mungbean yellow mosaic India virus (MYMIV) is a research priority for mungbean crop. Characterization of YMD resistance using various advanced molecular and biochemical approaches during plant–virus interactions has unfolded a comprehensive network of pathogen survival, disease severity, and the response of plants to pathogen attack, including mechanisms of YMD resistance in mungbean. The biggest challenge in YMD management is the effective utilization of an array of information gained so far, in an integrated manner for the development of genotypes having durable resistance against yellow mosaic virus (YMV) infection. In this backdrop, this review summarizes the role of various begomoviruses, its genomic components, and vector whiteflies, including cryptic species in the YMD expression. Also, information about the genetics of YMD in both mungbean and blackgram crops is comprehensively presented, as both the species are crossable, and same viral strains are also found affecting these crops. Also, implications of various management strategies including the use of resistance sources, the primary source of inoculums and vector management, wide-hybridization, mutation breeding, marker-assisted selection (MAS), and pathogen-derived resistance (PDR) are thoroughly discussed. Finally, the prospects of employing various powerful emerging tools like translational genomics, and gene editing using CRISPR/Cas9 are also highlighted to complete the YMD management perspective in mungbean.

Regeneration and protein profile analysis of callus transformant with geminivirus β-satellite fragment

The β-satellite fragment is a single-strand DNA that has a size of 1.4 kb. This fragment plays role in pathogen-derived resistance [PDR]. The genetic transformation of the β-Satellite fragment carried out by the previous researcher is limited by the regeneration of transformant callus. Therefore, this research was aimed to find some factors affecting the regeneration of transformant and non-transformant calli. Furthermore, protein analysis was performed on both calli to identify any specific fragment differentiating both proteins. SDS-PAGE analysis showed 5 and 8 fragments from transformant and transformant respectively. One of the 5 fragments has a size of 13.32 kDa which was predicted as C1 protein. These results proved that the β-Satellite fragment has been successfully inserted and expressed in the transformant calli. Subsequent research is suggested to identify some differential fragments produced by both transformant and non-transformant.

Trichoderma/pathogen/plant interaction in pre-harvest food security

Large losses before crop harvesting are caused by plant pathogens, such as viruses, bacteria, oomycetes, fungi, and nematodes. Among these, fungi are the major cause of losses in agriculture worldwide. Plant pathogens are still controlled through application of agrochemicals, causing human disease and impacting environmental and food security. Biological control provides a safe alternative for the control of fungal plant pathogens, because of the ability of biocontrol agents to establish in the ecosystem. Some Trichoderma spp. are considered potential agents in the control of fungal plant diseases. They can interact directly with roots, increasing plant growth, resistance to diseases, and tolerance to abiotic stress. Furthermore, Trichoderma can directly kill fungal plant pathogens by antibiosis, as well as via mycoparasitism strategies. In this review, we will discuss the interactions between Trichoderma/fungal pathogens/plants during the pre-harvest of crops. In addition, we will highlight how these interactions can influence crop production and food security. Finally, we will describe the future of crop production using antimicrobial peptides, plants carrying pathogen-derived resistance, and plantibodies.

Pectin induced transcriptome of a Rhizoctonia solani strain causing sheath blight disease in rice reveals insights on key genes and RNAi machinery for development of pathogen derived resistance.

RNAi mediated silencing of pectin degrading enzyme of R. solani gives a high level of resistance against sheath blight disease of rice. Rice sheath blight disease caused by Rhizoctonia solani Kuhn (telemorph; Thanatephorus cucumeris) is one of the most devastating fungal diseases which cause severe loss to rice grain production. In the absence of resistant cultivars, the disease is currently managed through fungicides which add to environmental pollution. To explore the potential of utilizing RNA interference (RNAi)-mediated resistance against sheath blight disease, we identified genes encoding proteins and enzymes involved in the RNAi pathway in this fungal pathogen. The RNAi target genes were deciphered by RNAseq analysis of a highly virulent strain of the R. solani grown in pectin medium. Additionally, pectin metabolism associated genes of R. solani were analyzed through transcriptome sequencing of infected rice tissues obtained from six diverse rice cultivars. One of the key candidate gene AG1IA_04727 encoding polygalacturonase (PG), which was observed to be significantly upregulated during infection, was targeted through RNAi to develop disease resistance. Stable expression of PG-RNAi construct in rice showed efficient silencing of AG1IA_04727 and suppression of sheath blight disease. This study highlights important information about the existence of RNAi machinery and key genes of R. solani which can be targeted through RNAi to develop pathogen-derived resistance, thus opening an alternative strategy for developing sheath blight-resistant rice cultivars.

Effect of plating density, amino acid and osmoticum on transformation efficiency of papaya (Carica papaya L.)

Papaya suffers colossal losses due to Papaya Ring Spot Virus (PRSV) disease. PRSV is responsible for about 70% loss in papaya production worldwide. This disease is present all over the world wherever papaya is grown. Transgenic papaya varieties namely SunUp and Rainbow have been developed and commercialized in the USA using Pathogen Derived Resistance (PDR). The present investigation reports a successful gene delivery system in an Indian papaya variety employing PDR. Agro-infected immature young globular embryos of papaya cv. Pusa Delicious were co-cultivated with A. tumefaciens harboring a hairpin loop construct of a truncated PRSV coat protein gene. The Agro-infected explants were plated at a density of 2–3, in the presence of 100 mg L−1 L-cysteine in dark for 72 h. Transformed plants maturation under the influence of 45 mg L−1 PEG produced 4 independent transgenic events which were characterized by PCR and data sequencing.

Genetic Diversity of Groundnut Rosette Disease Causal Agents Towards Its Management: A Review

In this review, the genetic diversity of the three causal agents of Groundnut Rosette Disease (GRD) in Sub-Saharan Africa (SSA) are discussed. Epidemics of GRD viruses in SSA, often reduce groundnut productivity. The etiology of GRD is a complex, involving three agents; Groundnut rosette assistor <i>luteovirus</i> (GRAV), Groundnut rosette <i>umbravirus</i> (GRV) and a Satellite-RNA (Sat-RNA) of GRV. The complex etiology and lack of sensitive and specific diagnostic tools, are major limitations in understanding the epidemiology of GRD viruses, and developing appropriate management strategies for the disease. Nucleotide identity of 97 to 100% among GRAV isolates from different regions in Kenya have been reported. Sat-RNA sequences from Kenya shared nucleotide identity of 95% with Malawian isolate (M24S) and 89% with Nigerian isolate (NG3a). GRAV CP gene was highly conserved (97-99%) regardless of the geographical distance. However, for GRV and Sat-RNA diversity increased with increase in geographical distance. In addition, phylogenetic analysis showed that isolates of GRV (ORF3 and 4) and Sat-RNA clustered together depending on the country of origin. Recent study has unveiled a chlorotic variant of Sat-RNA in Kenya with 97% sequence identity to the Malawian chlorotic isolate (M24S). Pathogen derived resistance (PDR) suitable for each diverse regions where the disease occurs is a promising management strategy which mainly depends in studies to deeply understand the genetic diversity of the three GRD causal agents. Currently, GRAV-CP is the best candidate for PDR.

Incidence of Groundnut Rosette Disease (GRD) and Genetic Diversity of Groundnut Rosette Assistor Virus (GRAV) in Western Kenya

This study determined the incidence of groundnut rosette disease (GRD) and genetic diversity of groundnut rosette assistor virus (GRAV, genus Luteovirus) in western Kenya. The diseases is a major constraint of groundnuts in Sub-Saharan Africa (SSA) causing up to 100% yield losses in severe cases. Among the GRD associated viruses, GRAV plays a crucial role in vector transmission of the other viruses. Therefore understanding the genetics of GRAV across SSA could enhance development of resistance to the disease. In Kenya, groundnuts are mainly grown in western region, however, the yields are poor mainly due to GRD. Information on occurrence and distribution of GRD in western Kenya was not documented and little was known about the characteristics of associated viruses. Two diagnostic surveys were conducted in six counties; Bungoma, Busia, Homabay, Kakamega, Siaya and Vihiga. Symptomatic and asymptomatic groundnut were collected in RNAlater® solution for laboratory analysis. Total RNA was extracted from the leaf samples using RNeasy Mini Kit (Qiagen) according to the manufacturers’ protocol and used for double stranded cDNA synthesis using the SuperScript II kit. The cDNA was column-purified with the DNA Clean & ConcentratorTM-5 – DNA kit. The samples were then processed with the transposon-based chemistry library preparation kit (Nextera XT, Illumina) following manufacturer’s instructions. The fragment sizes structure of the DNA libraries was assessed using the Agilent 2100 Bioanalyzer. The indexed denatured DNA libraries were sequenced (200-bp paired-end sequencing) on the Illumina MiSeq platform (Illumina). Reads quality check was done using FastQC. Trimmed reads were used for de novo assembly and contigs aligned to the viral genomes database using CLC Genomics Workbench 10.1.2. The assembled contigs were subjected to a BLASTn search against the GenBank database. Phylogenetic analyses and comparisons were performed using the MEGA X. Average incidence was 53% and 41% in the short and long rain seasons, respectively. Chlorotic rosette was the dominant symptom followed by Green rosette and Mosaic. The GRAV coat protein (GRAV-CP) gene sequences revealed 97-100% identity with GeneBank isolates showing very slight variations across SSA. The study concludes that GRD incidence is high in western Kenya and that GRAV is highly conserved across SSA. The study recommends an urgent need to curb GRD, possibly through the exploitation of pathogen derived resistance (PDR) with GRAV as the suitable candidate.

Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane.

Sugarcane mosaic virus (SCMV) is a plant pathogenic virus of the family Potyviridae that causes chlorosis, stunting and significantly reduced sugar productivity in sugarcane. Pathogen-derived resistance is a method used to develop SCMV-resistant sugarcane by overexpression of viral DNA. In this study, the gene encoding the coat protein (CP) of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. Nucleotide sequence analysis of amplified cDNA indicated that the 998-bp-long cDNA, termed ScMVCp cDNA, codes for the CP of SCMV from the PS881 isolate. The ScMVCp cDNA was inserted into the binary vector pRI101-ON with two constructs, a full nucleotide sequence (p927) and a sequence coding for N-terminally truncated protein (p702). The constructs were then introduced into sugarcane using Agrobacterium-mediated transformation. Southern blot analysis showed a single hybridized DNA copy inserted into the genome of transgenic sugarcane lines. The inserted genes were expressed at both the RNA transcript and protein levels in the transgenic sugarcane. The highest expression was found in transgenic lines 10, 11 and 13 from the p927 construct. Artificial infection by the virus showed that p927 generated a higher resistance to virus compared with p702. This resistance was passed on to the second generation of transgenic sugarcane with 100 and 20-40% levels of resistance in the p927 and p702 transgenic lines, respectively. This report shows that the full sequence of the CP gene is required to disrupt viral assembly and packaging, thereby generating resistance to SCMV infection.

RNAi-based transgene conferred extreme resistance to the geminivirus causing apical leaf curl disease in potato

Potato apical leaf curl disease is an emerging geminiviral disease in tropics and subtropics. It was reported for the first time in the year 1999 in northern plains of India but quickly spread to almost all potato growing regions of the country largely due to prevalence of warmer weather during early crop growth, thereby favoring whitefly vector. The problem of apical leaf curl disease in India became more severe due to lack of seed indexing for this virus in conventional seed production scheme. Although it accounts for major yield loss, there is no conventional source of resistance available in potato against Tomato Leaf Curl New Delhi Virus-Potato (ToLCNDV-Potato) that causes this disease in potato. In the present study, we have investigated the potential use of RNAi for obtaining resistance against this DNA virus in potato. The replication-associated protein gene (AC1) of the virus was used to obtain pathogen-derived resistance. The AC1 gene was PCR amplified from field-infected potato leaves, cloned and sequenced (JN393309). It showed 93% sequence similarity with the AC1 gene of Tomato Leaf Curl Virus-New Delhi (TOLCV-NDe; DQ169056) virus. Transgenic plants encoding the AC1 gene in three different orientations, viz. sense, antisense and hairpin loop, were raised. Transgenic lines when challenge inoculated with ToLCNDV-Potato showed different levels of resistance for all three constructs. Transgene integration and copy number in selected transgenic lines were determined by qPCR and further confirmed by Southern blot analysis. Though a reduction in viral titer was observed in transgenic lines encoding either antisense or hairpin loop constructs of AC1 gene, the latter transgenics showed most significant results as shown by reduction in the level of symptom expression in glasshouse screening as well as real-time data of in vivo virus concentration. In fact, we obtained a few totally asymptomatic transgenic lines with hairpin loop strategy.

Transformation of Cardamom with the RNA Dependent RNA Polymerase Gene of Cardamom mosaic virus

Cardamom (Elettaria cardamomum Maton) is an important spice crop. It is affected by Cardamom mosaic virus (CdMV). In order to make cardamom plants resistant to CdMV by the pathogen-derived resistance approach, the RNA dependent RNA polymerase gene (NIb) of CdMV in the plant expression vector pAHC17, was introduced into cardamom embryogenic calli along with GFP-BAR by particle bombardment. Transformants were selected on a medium containing bialaphos and the presence of NIb and gfp genes in cardamom plants were confirmed by PCR, Southern hybridization and GFP expression.

The function of small RNAs in plant biotic stress response.

Small RNAs (sRNAs) play essential roles in plants upon biotic stress. Plants utilize RNA silencing machinery to facilitate pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity to defend against pathogen attack or to facilitate defense against insect herbivores. Pathogens, on the other hand, are also able to generate effectors and sRNAs to counter the host immune response. The arms race between plants and pathogens/insect herbivores has triggered the evolution of sRNAs, RNA silencing machinery and pathogen effectors. A great number of studies have been performed to investigate the roles of sRNAs in plant defense, bringing in the opportunity to utilize sRNAs in plant protection. Transgenic plants with pathogen-derived resistance ability or transgenerational defense have been generated, which show promising potential as solutions for pathogen/insect herbivore problems in the field. Here we summarize the recent progress on the function of sRNAs in response to biotic stress, mainly in plant-pathogen/insect herbivore interaction, and the application of sRNAs in disease and insect herbivore control.

Extraction, purification and elicitor activities of polysaccharides from Chrysanthemum indicum

Polysaccharides isolated from Chrysanthemum indicum were studied for their pathogen-derived resistance against Sclerotium rolfsii sacc in Atractylodis maceocephalae koidz. The total sugar content and monosaccharide analysis were determined by phenol–sulfuric acid method and gas chromatography, and infrared spectroscopy performed for simple structure information. The activities of CAT and POD as protective enzymes in A. maceocephalae leaves were evaluated. The purified polysaccharides exhibited strong CAT and POD activities in inoculated with S. rolfsii in A. macrocephala leaves, attained the maximum value 568.3Ug−1min−1 and 604.4Ug−1min−1respectively. Whereas, when compared with the control plants, 20mg/ml purified polysaccharides exhibited the strongest CAT and POD activities. Notably, the treatments of A. macepcephalae seedlings with C. indicum polysaccharides (CIP) decreased disease index development caused by S. rolfsii. The disease index after 10 days was significantly reduced when the seedlings treated with 20mg/ml CIP, 4.41 compared to the control plants 32.00. Given together, these results indicated that purified polysaccharides derived from C. indicum may be useful as a natural inducer.

Characterization of Soybean mosaic virus resistance derived from inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars.

Soybean mosaic virus resistance was significantly improved in multiple soybean cultivars through genetic transformation induced by inverted repeat-SMV- HC - Pro genes based on RNAi and post-transcriptional gene silencing. Here, we demonstrate Soybean mosaic virus (SMV) resistance in transgenic soybean plants. Transformation of five soybean genotypes with a construct containing inverted repeat-SMV-HC-Pro genes-induced high-level SMV resistance. Through leaf-painting assays, polymerase chain reaction (PCR) verification and LibertyLink(®) strip detection, 105 T0 and 1059 T1 plants were confirmed as transgene-positive. Southern blotting confirmed insertion of the T-DNA into the genomic DNA and revealed a low-copy integration pattern. Most T0 plants were fertile and transmitted the exogenous genes to their progenies (ratios of 3:1 or 15:1). In the T1 generation, virus resistance was evaluated visually after inoculation with SMV (strain SC3) and 441 plants were highly resistant (HR). SMV disease rating was classified on a scale with 0=symptomless and 4=mosaic symptoms with severe leaf curl. In the positive T1 plants, the disease rating on average was 1.42 (range 0.45-2.14) versus 3.2 (range 2-4) for the nontransformed plants. With the T2 generation, 75 transgene-positive plants were inoculated with SC3, and 57 HR plants were identified. Virus-induced seed coat mottling was eliminated in the resistant lines. Analysis of SMV levels in the plants was performed using quantitative real-time PCR and double-antibody sandwich enzyme-linked immunosorbent assays; the results revealed no virus or a gradual reduction over time in the viral content, thereby supporting the visual examination results. This is the first report demonstrating pathogen-derived resistance to SMV induced by inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars. Our findings contribute positively to the study of transgenic SMV-resistance using RNA interference.

Recent Research Findings Related to Management Strategies of Begomoviruses

Begomoviruses (family Geminiviridae) cause a number of serious diseases of cultivated crops and they are considered as the major constrains for cultivation of several crops all over the world. Presently human activity and modern day agriculture are one of the key factors in the emergence of begomoviruses in various parts of the world. The available disease management options include the organisation of agricultural practice to reduce disease, the use of cultural control such as sanitation programmes, the control of the vector population through the use of insecticides, and the breeding and growing of resistant crop cultivars. Transgenic resistance against begomoviruses has shown limited success despite the use of a number of strategies. Ability of begomoviruses to evolve rapidly by recombination and mutations is the major limitation to all these strategies. In modern-day biotechnology, focus is required on engineering begomovirus resistance through transgenic approach. Expression of various full length or truncated or defective proteins of the virus has been effective in achieving pathogen-derived resistance. Antisense RNA and RNAi technology have also been used with some success. This review focus on both conventional and nonconventional management strategies methods have been suggested and described over the years for management of begomoviruses diseases in economically important plants.