NEW Fangled Tactics Towards Cotton Leaf Curl Virus Disease A Review

ABSTRACT CLCuVD is the most ravaging biotic stress to cotton crop and is responsible for the withdrawal of various varieties in cotton cultivation across the globe. CLCuV significantly degrades the quality and productivity of cotton. This notorious viral disease is transmitted by whitefly (Bemisia tabaci Genn)vector. It is challenging to manage this viral disease because of higher recombination rate of viral strains. This virus has broad host range and attacks various host plants like tobacco, tomato, and okra, while new viral strains evolved due to mixed farming practices. CLCuV has been managed by cultural practices and control of insect vector. Various techniques which include netting, use of plant extracts, pathogen-non-pathogen derived approaches, RNAi, CRISPR-cas, biological control and other existing knowledge related to its management is discussed here. Limited research has been conducted to evaluate the efficacy of nanoparticles as an antiviral agent. There is a dire need to explore the mechanisms underlying the role of NPs in the CLCuV- vector interactome. The present manuscript is expected to be fruitful for the further investigation on cutting edge research areas.


Introduction
Cotton crop is cultivated globally for fabric, fuel, foodstuff, and feed (Chakravarthy et al. 2014).Seeds of cotton contain about 23% quality proteins and 21% oil content.The cotton crop occupies 1 st and 2 nd positions for the fiber and oilseed crops, respectively, all over the agricultural world (Gul et al. 2014).The genus Gossypium contains 52 species, among them G. hirsutum and G. barbadense are tetrapolid while G. arboretum and G. herbaceum have diploid (2n) genomes (Khan et al. 2016).Among all these cotton species, Gossypium hirsutum is the most widely cultivated specie which is cultivated in over 60 cotton-growing countries.A number of destructive diseases including Bacterial blight, Angular leaf spot, Fusarium wilt, and Cotton leaf curl virus are known to attack on cotton crop (Leke et al. 2015).Whitefly (Bemisia tabaci) is the insect vector of CLCuV, which transmits this virus in persistent manner.The replication of CLCuV takes place in nuclei of infected cells.Begomoviruses are comprised of small (2.6-2.8 kbp), circular-shaped, and single-stranded DNA i.e. ssDNA genomes (Yadava, Suyal, and Mukherjee 2010).This ssDNA can either be monopartite or bipartite.Countries that are potentially contributing 60% of cotton production globally are India, China, and Pakistan, but these countries carry greater risk for CLCuV (Vyas et al. 2017).The lofty objective of this strive is to review the current state of knowledge including recent investigations for the management of this tremendous pathological malady (CLCuV).The molecular, physiological, and biochemical approaches are discussed in the subsequent sections of the current review.

Constituents of the scientifically significant virus (CLCuV)
The genome of geminiviruses is circular ssDNA with an approximate size of 2.8kb, encapsulated as a twined icosahedron.

Bipartite
Begomoviruses consist of DNA-A along with DNA-B, and these are equally sized (~2.8kb) as shown in Figure 1.These viruses are natively related to New World, whereas fewer are found in Old World.Symptom development requires both components and they share the same region having a repeat and nanomeric sequence that is known as iterons (Argüello-Astorga and Ruiz-Medrano 2001).DNA-B is essentially maintained by this common region 5'-TAATATTAC-3'.Six proteins are encoded by DNA-A which essentially controls replication as well as transmission of the CLCuV (Figure 1).A part of the mono and bipartite type of begomovirus is DNA-A that encodes for various genes two of which lie in the VSS (virion-sense strand) whereas, four genes lie in the VSCS (virion-sense complementary strand).The virion-sense strand encodes the coat protein (CP).CP plays a role in encapsidation, moving in the host, and transmitting through vector (Rahman et al. 2017).Two proteins are known as NSP (nuclear shuttle protein) and MP (movement protein) are encoded by the DNA-B component in the virion-sense strand and in complementary strand, respectively.The vital role is played by these proteins mainly in the movement of virus in the host either within the cell or between cells through plasmodesmata.
The ability to replicate independently exists in alphasatellites, which are made up of ssDNA molecules and do not have any substantial sequence identity with the helper virus.This molecule has a hairpin structure with TAGTATTAC, which constitutes the origin of the virion-strand, and is approximately 1.4 kb in size.Replication associated protein (Rep), which is encoded by these molecules, is one protein that is more genetically related to Rep encoded by nanoviruses -transmitted by aphids.However, both the movement of alphasatellites in plants and their transmission through their vector (whiteflies) depend on helper viruses.Betasatellites are ss DNA molecules (1.4 kb in size) and these molecules are highly conserved in structure, and contain single coding gene (βC1), satellite conserved region (SCR), and A-rich region (Rahman et al. 2017).

Relationship of B. tabaci with CLCuV
Primarily, the whitefly has been noticed as a damaging insect pest in the 1920s in north India and now it has throughout the world except in Antarctica (Chaubey et al. 2015).In the late 1950s, its genetic complexity was determined (Bird 1957).Whitefly is the principle source for transmitting CLCuV.A total of 24 mystifying specie of Bemisia tabaci have been identified through molecular and biological tools.Although study of the begomoviruses (family Geminiviridae) that cause the disease has advanced in recent years, little is known about how the numerous species of whiteflies in the Bemisia tabaci complex transmit these viruses.Four species of whiteflies, two of which are native to Asia and two of which are invasive worldwide, were compared for their ability to transmit the cotton leaf curl Multan virus (CLCuMuV), one of the main begomoviruses associated with cotton leaf curl disease.Only the native Asia II 1 species was able to spread this virus effectively.By quantifying the virus and employing immunofluorescence tests, differential transmission was linked to the varying potential of CLCuMuV to traverse the midgut of diverse whitefly species.The function of coat protein in the transmission of CLCuMuV by whiteflies has been confirmed.It has been investigated that the majority of begomoviruses linked to cotton leaf curl disease may share comparable whitefly transmission traits based on a phylogenetic analysis of the virus coat proteins (Li et al. 2018).
To ascertain the impact of CLCuV on its vector, fecundity and life history traits of a native non-b biotype whitefly were compared 10, 25, and 40 days after post-inoculation in infected and healthy cotton plants.In infected plants, the juvenile stages of whiteflies developed much more quickly, although this did not change with the severity of the symptoms at 25 and 40 DPI (45 and 60 days old plants).Infection with CLCuV boosted the vector's percentage of viable eggs.On CLCuV-infected plants compared to healthy plants, whiteflies drastically reduced the amount of eggs they laid.Plant age had no effect on fertility, and Bemisia tabaci had superior egg viability on younger plants than on older plants.On infected cotton plants, whiteflies lived for a shorter period than on healthy plants (Nogia, Singh, and Meghwal 2014).

Symptoms
Typical symptoms of CLCuV include upward or inward rolling of leaves, leaf enation, thickened, or swelled veins, which lead toward stunted growth of infected plant (Figure 2) (Farooq et al. 2014).

Sick-plot technique
Sick-Plot Technique is easy, cost-effective method usually practiced by phytodoctors at various CRS (cotton research stations) to evaluate the phenotypes of targeted cultivars.In the above method, the disease is spread naturally through a popular and highly susceptible genotype i.e.S-12, where this genotype serves as a spreader and is applied between the rows of the genotypes to be investigated (Rashida, Sultan, and Khan 2005).

Grafting procedure
It is known as the easiest procedure applicable to whitefly transmission, for the reason that symptoms develop just within 14 to 30 days after grafting of plants, depending on the type of variety under testing.In the grafting method, the "scion" can be used as a susceptible source and "rootstock" as resistance for disease transmission.Afterward, the ELISA test is used for confirmation of the presence of the virus (Farooq et al. 2011).

Late sowing
This is the most cost-effective, convenient method which is used for screening of germplasm, segregating populations, and candidate genotypes that can resist CLCuV.The maximum CLCuV attack was reported when the sowing of cotton was delayed to 1 st week of July.The occurrence of CLCuV will be incident within 100 days after sowing.Therefore, the sowing of the candidate genotypes or segregating materials which are to be tested should be delayed to July (Iqbal, Sattar, and Shafiq 2016).

Detection of CLCuV through polymerase chain reaction (PCR)
Amplification of the genome of CLCuV is done either by the use of specific or degenerated primers through PCR.Furthermore, there is also the availability of primers that can detect satellite-like molecules.Many helper viruses along with their recombinants can be amplified through the method of rolling-circle amplification i.e.RCA.Eventually, new assay will be designed with the development of the genome-sequencing tools, so that the vector complex, as well as the whole virus, would be identified and detected in fields and its spread to other crops will be monitored (Saleem et al. 2016).A qPCR assay was developed to detect components of the CLCuD-causing monopartite begomovirus/ betasatellite/alphasatellite complex.The association between symptoms and virus/satellite titer was examined using this.The analysis, unsurprisingly, showed a reasonable correlation between the severity of the symptoms and the virus/satellite titer, with more severe symptoms typically being linked to higher virus/satellite titer (Muhammad et al. 2015).

Immunological assays and Quantum dots as diagnostic tools
For the detection of plant viruses, serological techniques like DIBA (dot immunobinding assay), ISEM (immunosorbent electron microscopy), EBIA (electro-blot immunoassay), and fluorescent antibody technology have been developed.Many researchers have also employed IC-PCR (Immunocapture PCR), which combines serology with PCR, in the identification and characterization of viruses infecting various crops.Over the past 10 years, recombinant antibody (rAb) engineering has emerged as one of the promising methods for plant virus diagnostics (Bhat and Maheshwari 2017).Plant viruses are diagnosed and identified using immunological assays like ELISA (Enzyme-Linked Immunosorbent Assay) and ECIA (Electrochemical Immunoassay) (Zein and Miyatake 2009).Seven plant species were exposed to a stock culture of cotton leaf curl virus Pakistan(CLCuv-PK), which led to symptoms such vein thickening and leaf curling.11 out of 31 monoclonal antibodies produced against the particles of the African cassava mosaic, Indian cassava mosaic, and okra leaf curl viruses were able to recognize it in the TAS-ELISA (Triple Antibody Sandwich ELISA) (Harrison et al. 1997).CLCuV presence in all cotton genotypes was tested by TAS-ELISA.CLCuV concentration in all susceptible cotton genotypes was determined by one-month post-inoculation at ten days interval.Graft inoculation and PCR were used to confirm the indexing of symptomless and ELISA negative genotypes (Shah et al. 2004).A realtime TaqMan PCR combining with magnetic nanoparticles method was developed to detect cotton leaf curl virus.After specificity and sensitivity tests, an MNP fluorescent PCR detection method was established using a TaqMan probe combining with MNPs technique and the coat protein gene as the target gene.Sensitivity by this technique was 525 fg/µl of DNA (Zhang et al. 2013).Small semiconductor nanocrystals known as quantum dots are employed to develop biosensors (Frasco and Chaniotakis 2009).It has been used to detect plant pathogens because it has a unique optical feature that is used in FRET (Algar and Krull 2008).The most intensively explored plant virus is CTV (Citrus Terroreza Virus).The sensor is a prime illustration, which is labeled with a CTV coat protein-derived antibody on the CdTe QD (quantum dot) (Safarnejad et al. 2017).PNRSV (Prunus necrotic ringspot virus) was detected using this technique, and it was discovered that the biosensor and ELISA are quite similar (Jarocka et al. 2013).For the purpose of identifying MCMV, a quartz crystal microbalance immunological sensor based on SAMs was used (Maize chlorotic mottle virus).The biosensor was found to have high sensitivity for related viruses like Wheat streak mosaic virus and an ELISA-like sensitivity with a detection limit of 250 ng/mL (Huang et al. 2014).Later, QD was used to diagnose a number of other viruses, including Potato virus X (PVX), Potato virus M (PVM), Potato virus x, Potato virus A (PVA), and Potato virus Y (PVY) (Drygin et al. 2012).It is the most pressing need to introduce the usage of quantum dot for detection of CLCuV.

Application of gamma rays (irradiation doses)
Genetic variability of the germplasm of cotton is produced by developing tolerant materials that are against CLCuV.Greater genetic variability in different crosses is created by the application of gamma rays (irradiation doses) which range up to 510 GY (Farooq et al. 2014).

Viruliferous whiteflies
The other method through which we can screen the germplasm of cotton against CLCuV is by inoculating the virus through viruliferous whiteflies.Whiteflies carrying the virus are inoculated in a net house either freely or releasing a counted number of these whiteflies on candidate genotypes kept in a polyhouse made of plastic jar periodically (Monga, Chakrabarty, and Kranthi 2011).

Self-Defense Mechanisms (SDM) adopted by cotton plant against CLCuV
Some of the cotton plant's natural defense mechanisms also protect it from the vector.Cuticular waxes make insect attachment to plant surfaces difficult and act as a physical barrier.G. arboreum's cuticular waxes serve as the first line of defense against whiteflies.Other defense strategies may include impeding insect movement or depriving it of food due to the thick waxy layer.Biotechnological strategies against various Begomoviruses, including CLCuV, have been found to be effective in some crops other than cotton.In contrast, G. arboreum's natural defense strategies, such as long trichomes or the presence of inorganic salts with increased wax concentrations, provide a good defense strategy against whiteflies, CLCuV, and other pathogens (Khan et al. 2015).Self-defense mechanisms are adopted by other plants to combat pathogens including viruses.Plants generate the response in their defense when they detect any conserved molecule that is derived from a pathogen.But the effector proteins that are secreted by pathogens help them to suppress the host defense mechanism and get their nutrition (Klein et al. 2015).On the contrary, a significant role is played by resistance "'R'" genes (Tm-1, JAX1, and Scmv1, resistance genes against tomato mosaic virus, RTM1, RTM2, RTM3 genes of Arabidopsis thaliana inhibit systemic transport of potyvirus STV11 of rice against rice stripe virus and Ty-1 and Ty-3 genes of tomato against tomato yellow leaf curl virus) through which the resistance against pathogen is maintained (Figure 3).Whenever a viral infection occurs, these R genes become activated, thus the infected cells are killed by the setup of a localized cell death program, creating a patch that inhibits the infection and its spread.

Management of CLCuV through plant extracts
Insect pests are usually controlled by the application of synthetic pesticides.Furthermore, over the years, the uncontrolled use of synthetic pesticides has led to a number of problems, such as insect resistance and contamination of important planet resources including water, air, and soil.Plant-derived insecticides may therefore be a more environmentally friendly choice than manufactured pesticides (Souto et al. 2021).But alternative strategies are in progress to combat these pests as they have become resistant to synthetic pesticides.Essential oils extracted from plants including petroleum oil repel whiteflies and many other insect pests (Du et al. 2016).Insecticidal activities were noticed in approximately 2000 plant species.These compounds can protect plants against insect pests, bacteria, herbivores, and viruses (Table 2).The poisoning effect is created by oils in interaction with fatty acids of insect bodies thus, destroying their metabolism or respiratory system (Table 2).Extracts of Parthenium hystophorus and Datura alba when applied on cotton, generated resistance against whitefly (Ali et al. 2015).Foliar application of 2-3% mustard oil is also found helpful in controlling the whitefly population.Other plant extracts, including Allium sativum, Eucalyptus globules, Aloe barbadensis, Datura stramonium, and Calotropis Procera were less efficient while comparing to A. indica when, combat with insect vector and pathogen (Table 1).

Salivary secretions
Whitefly These insects are parasitoids.Their larvae live as parasites that eventually kill the host.

E. mundus Salivary secretions
Whitefly E. mundus is an exoparasitoid and it penetrates the host later on, as a 1 st instar that chews into the host nymph from the outside.

Management of CLCuV through netting
Netting provides an efficient hindrance for insects.Various kinds of netting (known as insect-proof netting) are in use to control insect vectors (whiteflies).The method of management of CLCuV through netting is not applicable in tropical areas due to high levels of humidity and elevated temperatures.Nettovering can be efficiently used against whiteflies when combined with pyrethroid and acypermethrin (Shad et al. 2012).

Application of BCAs against cotton leaf curl virus disease
There is no single effective management strategy to completely prevent or reduce the incidence of CLCuV by managing whiteflies.Briefly, biological control is recommended in crop protection after knowing harmful aspects of synthetic pesticides.Pseudomonas aeruginosa (S 1 HL 3 ), Burkholderia sp.(S 1 HL 4 ) and Bacillus sp. were tested against CLCuV in greenhouse conditions (Ramzan et al. 2016).Up till now, various bio-control agents have been used in the world against B. tabaci (the insect vector of CLCuV) which are discussed with their mode of action and secretions/secondary metabolites in Table 02.

DNA markers identification and its contribution to the field of molecular breeding
Valuable traits are being improved via conventional breeding; however, it is a slow progressing process.Furthermore, it is difficult to perform phenotypic selection through these quantitative traits (Zhang et al. 2015).DNA markers are used by breeders on a molecular basis, in the section of valuable traits to prevent phenotypic screening.Multiple genes also termed "quantitative traits" control the major traits including disease resistance, quality, and yield (Bolek et al. 2016).

CRISPR-Cas
Conventional breeding faces significant challenges in keeping up with pathogen evolution and rising food demand, particularly in an era of global climate change.CRISPR/Cas9 is the most effective, durable, and broad-spectrum technique.It has +transformed crop improvement by enabling powerful and precise targeted genome modifications.It paves the way for new methods of genetically improving plant disease resistance and speeds up resistance breeding (Ahmad et al. 2020).A genome editing tool, Clustered Regularly Interspaced Palindromic Repeat CRISPR-associated-protein system, confers resistance through bacteria, to moveable genetic components, archaea, and viruses.CRISPR-Cas technique has captured the focus of researchers belonging to various fields mainly, biologists because it gives a higher specificity level (Iqbal, Sattar, and Shafiq 2016).Adaptive immunity is acquired against the genetic elements of invading pathogens.At present, it is used as a genome editing tool in eukaryotes.During this technique, the nucleic acid (DNA or RNA) of a virus or archaea is chopped by CRISPR spacers.The size of these chopped molecules resembles the RNAi-based sequence, and they are almost 20 nt in length.These are seen in 40% bacterial and 90% archaea genomes.CRISPR-Cas has the potential to overcome several complex organisms by insertion of the guide RNAs and Cas9 protein inside the plant cell (Rojas et al. 2018).As this technique provides adaptability, ease of engineering, and robustness, it can be applied against Geminiviruses (Iqbal, Sattar, and Shafiq 2016).The genomes of cotton leaf curl Kokhran virus strain, Barley yellow dwarf virus, and Merremia mosaic virus have been edited through this approach (Ali et al. 2015).The CRISPR-Cas9 system was successfully tested to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs).We also investigated CLCuV's ability to evade Cas9 endonuclease activity.The use of three gRNAs to target overlapping genes of the most common CLCuVs resulted in virus interference, as evidenced by low virus titer.Furthermore, a multiplex CRISPR-Cas9 construct targeting six CLCuV genes at the same time was found to be more effective at interfering with virus replication than targeting a single region individually.In these research activities, siRNAs are constructed by targeting both regions (coding and non-coding) of viral genomes.
Resistance development was possible in the model plant system using CRISP/Cas9.N. benthamiana demonstrated antiviral tolerance against the CLCuV virus, delaying the onset of symptoms.Modern gene editing approaches like CRISPR/Cas have created new opportunities for manipulating plants to express biomolecules like AMPS, with a high potential for use in obtaining molecules more quickly and efficiently.There is no denying that in a few years, the field of transgenics for AMP production could undergo a revolution attributed to gene editing tools like CRISPR/Cas dos Santos and Cand Franco (2023).

Categories of resistance derived through genetic engineering
Two categories of resistance have been introduced derived through genetic engineering.In PDR (the pathogen-derived resistance) the replication of the virus in the host is blocked by introducing a viral gene (a part or complete).It is considered very efficient against such viruses due to their gene silencing property.There are two categories as it can be either with gene expression or excluding expression of the gene.The protein associated with replication is crucial and plays a significant contribution to replicating the virus in the host plant (Su et al. 2015).It brings the cell into S-phase, synthesizes virus components, and is also involved in the replication of the host cell.Resistance is produced via this protein to combat viral infection.The structure of the gene is modified by applying the siRNA strategy (Khan et al. 2015).The viral components cannot be made by suppressing the Rep-protein structure.It is very vital to introduce genes that can provide antivirus antibodies, coat-binding proteins including DNA-binding proteins in plants to result in resistance potential against the pathogen (CLCuV).
Primarily, the expression of CP gene (viral coat protein) obtained from TMV has been used as a transgenic resistance to combat phytopathogenic viruses.Later on, it was used to control Geminiviruses (Khan et al. 2015).For instance, a protein, namely, Tma12 was recognized in fern for providing resistance against whiteflies.The gene for this protein was inserted in cotton cv.Coker-312 cotton line has expressed an increase in its resistance (>99%) against whitefly.Hence, this protein can produce resistance against whiteflies in the coming time (Shukla et al. 2016).

RNA interference
RNA interference has been introduced on the basis of TGS (Transcriptional Gene Silencing) and PTGS (Post-transcriptional gene silencing).Expression for many valuable traits is improved through RNAi (Meena, Verma, and Kumhar 2017).The function of biologically, physiologically and agronomically valuable traits is studied through this approach.The genes studied through this technology are involved in the development of resistance against stresses (biotic and abiotic), early maturity, improving oil and fiber quality, and embryogenesis fertility.Currently, biotech cotton is manufactured through this technique.The formation of small-RNA (21 to 25 nt) is the main step of RNAi.Small-RNA is formed by the action of the enzymes known as Dicers.The gene silencing at the transcriptional or post-transcriptional level is induced by incorporating the loading strand into RNA-induced silencing complexes (RISCs) having Argonuate proteins.It can be seen in Figure 2. Small RNAs have two main classes: microRNAs and (small-interfering) siRNAs.These are involved in gene silencing.These newly formed siRNAs then bind to their complementary sequence for gene silencing which may be either transcriptional or post-transcriptional. Conversely, the microRNAs use a different mechanism.They either transcribe through their genes or by making structures in fold-back fashion having the ds (double-stranded) portions from introns (Rishishwar and Dasgupta 2019).In the host, and later miRNAs form duplexes, which are then exported to RISCs.RNA Interference was applied to control many phytopathogenic viruses including ACMV and MYMV.Currently, the function of G.hirsutumghr-miR166b is targeting the ATP-synthase gene of (whitefly) B. tabaci is reported and by its overexpression it can also work as a biopesticide thus decreasing B. tabaci population as well viral transmission through this insect (Wamiq and Khan 2018).However, viruses have the ability of encoding suppressor proteins which is the potential of counter defense to escape gene silencing.All viruses encode at least one or more such suppressor proteins for their defense.About four to five suppressor proteins against silencing are also encoded by CLCuV namely C2, C4, V2, and βC1.Moreover, higher suppressor activity is shown by V2, as it does not let the protein formation when siRNA bind to respective mRNA.In CLCuVM, V2 has been known to sequester the dsRNA and inhibit its cleavage through dicers.The protein (AC2/C2) protein can function as PTGS (Posttranscriptional gene silencing) and TGS (Transcriptional Gene Silencing) suppressors.It is reported that this protein acts as a PTGS (Post-transcriptional gene silencing) suppressor in East African cassava mosaic Cameroon virus, Vigna Mungbean yellow mosaic virus (MYMV-Vig), CLCuMV, and Tomato yellow leaf curl virus-China.

Anti-Sense RNA (complementary to the mRNA)
Anti-sense ribonucleic acid (RNA) having its formulation complementary to the mRNA, it forms a duplex with its targeted mRNA and prevents it from translation.This technique is found to be effective against various viruses such as TYLCV including CLCuKoV (Khan et al. 2015).It is also used in transgenic cotton for targeting rep protein, through which the replication mechanism of the virus was inhibited.In another research work, viral AVI was also targeted, which then suppressed the encapsidation, viral movement, and replication in transgenic-cotton plants.Recently, a transgenic cotton (cv.Coker-310) was introduced with insertion of viral gene βC1 under 35S promoter in antisense orientation.Southern blotting was used to confirm the insertion.However, this gene did not show any symptoms of insertion, no symptoms were observed after insertion of this gene in transgenic cotton (Sohrab et al. 2016).

Recent advances using biotechnological tools
Many factors cause limitations of conventional breeding methods, like sudden climatic changes or limited resources.However, plant biotechnology has made possible the control of many diseases including CLCuV via virus cloning and induction of defense strategies thus, improving yields.At present, biotech cotton is broadly marketed, accepted, and cultivated in over 24 M ha areas in the world.At first, the GM crop was commercially used in 1996 and now its use is reached 70% on a global scale (Bakhsh et al. 2015).Biotechnology and genetic engineering has made it possible to incorporate genes, resistance against various diseases in crops of commercial value.Gossypium arboreum is tolerant to CLCuV and various bacterial and fungal diseases.G. arboreum is also used for incorporating and isolating resistant genes into susceptible cultivars through genetic engineering.The Pathogenderived resistance (PDR) technique is also deployed in plants that lack natural resistance against various viral diseases.In the transgenic cotton crop, Agrobacterium tumefaciens (LBA 4404 strain) including interference technology were utilized to fight against CLCuV.However, the comparison of transformed plants with their control groups showed that the overexpression of nucleotides had developed resistance by inhibiting β-satellites and DNA of viral genomes.High transgene expression was observed through Northern blotting in early as well as later stages of growth.

Limitations of new-fangled approaches
In Asian countries like Pakistan, India, Bangladesh, and especially in Afghanistan, growers are reluctant to replace chemical control with more expensive biopesticides and poorly understood biological control agents.Most farmers have a limited understanding of biological control strategies (Falahzadah, Karimi, and Gaugler 2020).Biological control measures have been introduced to a number of plant diseases in order to reduce the risks associated with synthetic chemicals; however, their effectiveness is greatly diminished under natural environmental conditions.Several biocontrol agents do not thrive in newly introduced habitats (Singh et al. 2020).The use of quantum dots for the detection of CLCuV, particularly in developing Asian countries, has received scant interest.The use of nanotechnology in plant disease diagnostics is still in its early stage (Marwal and Gaur 2019).It has been demonstrated that CRISPR/Cas9 can be used to target the CLCuV intervention and generate CLCuD resistance.Due to constitutive gene expression, the integration of the CRISPR/Cas9 cassette may result in unwanted off-target consequences, plant mortality, and restrictions on conducting functional research pertaining to particular developmental or physiological processes (Binyameen et al. 2021).

Future direction
The most pressing need, especially in Pakistan, is to direct research efforts on cutting-edge research fields.It is essential to conduct an in-depth investigation into the development of disease forecasting models at regional basis while taking ecological circumstances into consideration.It has been determined that the loss of varietal resistance to CLCuV has been attributed to the introduction of new pathogenic strains.Pathotyping should be the subject of research because of the diverse and complex nature of pathogen.Further research is a real need to optimize the protocol for administration of potential biocontrol agents and their secondary metabolites as well as to understand the molecular mechanisms underlying cotton leaf curl virus disease resistance.An intense genome project on this pathogen is urgently needed.The advancement of new biotechnology-based techniques such as RNAi and CRISPR/Cas9, which target specific pathways in the virus transmission cycle, can be a foremost stride in limiting virus transmission.A culturable, symbiont-mediated RNAi approach could be a powerful tool for insects to integrate gene function.This technology has already been used in two evolutionarily distinct insect species Rhodnius prolixus and Frankliniella occidentalis.In the future, insect species could be practiced to manipulate bacterial endosymbionts genetically.The endosymbiont-mediated RNAi method is anticipated to be effective in many insect species.Nanotechnology is a rapidly evolving discipline for plant disease management and this nanotechnological approach may prove a fruitful tool to combat this tremendous phytopathological malady.

Conclusion
All the above-mentioned new-fangled tactics that are used to control cotton leaf curl virus disease depend on the conditions.Recommended varieties, agronomic practices, fertilizer, insecticidal control, and biotechnological methods can be employed alone or in combination to fight against this severe disease which is still a challenge even after 20 years of intensive research efforts.Unless a CLCuVD-resistant variety is not introduced, highly tolerant varieties should be grown and losses due to this disease should be minimized by enhancing plant population and intensive inputs in late planting.But the most appropriate and ecofriendly method is the development of resistant varieties.For this purpose, the use of CRISPR/Cas technology is the most suitable and reliable one.

Highlights
• Genome Map of CLCuV • Significant techniques and methods for detection of CLCuV • Self-Defense Mechanisms (SDM) adopted by plants • Application of molecular tools (Innovative Approach) • Categories of resistance derived through genetic engineering

Funding
No additional funding is given to authors for this study.

Figure 1 .
Figure 1.Genome of begomovirus is bipartite or monopartite.The gnome of begomovirus depends on the presence of 1 or 2 components (DNA-A & DNA-B).The virion sense strand of DNA-A encodes pre-coat and coat proteins.The complementary sense strand of DNA-A encodes Rep (Replication associated proteins), TAP (Transcriptional activator protein), REP (Replication enhancer protein) including C4 protein.It is detected that DNA-B encodes NSP (Nuclear shuttle protein) and MP (Movement protein) on the virion sense strand and complementary sense strand respectively.

Figure 2 .
Figure 2. Various typical symptoms of CLCuV on the leaf of cotton plant.

Figure 3 .
Figure 3. Attack of pathogen cause the production of various signaling molecules which plays their significant role in defense.Pathogen produces MAMPs and PAMPs to invade, in response plant produce PRRs (Pattern Recognition Receptors).At this point recognition takes place, (if the Avr protein fits in R gene, means no infection otherwise vice versa).After recognition, plant produces various types of chemical compounds (Phytoalexins, PR Proteins, AMPs, SA (Thianin), Plant defensins) to kill pathogen.This first line of defense is known Pathogen Triggered Immunity (PTI).After first line of defense, pathogen defeated so it send signals to nucleus to produce most powerful Avr gene which give rise to the production of effectors/elicitors.In response of pathogen's elicitors, plant produces receptors.After recognition, plant produce different defense related enzymes like SA. SA spread to the whole plant and develop SAR (Systemic Acquired Resistance).This is known as (ETI) Effector Triggered Immunity.

Table 1 .
Biocontrol agents against CLCuV and whitefly along with their secretions and mode of action.

Table 2 .
Phytoextracts along with their active ingredient and mode of action toward CLCuD.