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Current IssueSpecial IssueEditorial BoardOnline First ArticlesArchive

ABSTRACT

Background: Okra enation leaf curl virus (OELCV) belongs to the genus Begomovirus and family Geminiviridae. This virus adversely affects the potential and productivity of okra crops and in present scenario it is an emerging disease. The present study was conducted to elucidate the virus responsible for the disease.

Methods: The virus transmission study was done with whitefly in which acquisition access period (AAP) and inoculation access period (IAP) was recorded. Detection was through transmission electron microscopy (TEM) and molecular characterization from infected leaf sample.

Result: First symptom was appeared after 30 DAS as twisting of petiole. Infected plants showed pronounced vein thickening, cup shaped leaves, small pinhead-sized enations on underside of the leaves. The disease symptom in plants initiated with 33.33 per cent at 1 hour of acquisition access period (AAP) and 2 hours of inoculation access period (IAP) of the whitefly and increased progressively up to cent per cent in 24 hrs. Incubation period decreased with increase in AAP and IAP. In TEM geminate particles were detected. Viral DNA isolated from the infected leaf was subjected to PCR and the amplified products were confirmed to be OELCV and submitted to the NCBI GenBank (Accession No. OR270161, Accession No. OR270162).

INTRODUCTION

Okra [Abelmoschus esculentus (L.) Moench] is an essential plant belonging to the family Malvaceae. Although it is believed that okra originated from tropical Africa, it is now cultivated all around the world. Okra generally takes between 60 and 65 days to mature (Kumar et al., 2013). Okra seeds are also used as a substitute for coffee. It is also a potential source of natural fibers suitable for textile, paper and other engineering applications (Gupta and Patra, 2021).
       
Okra is cultivated over a period of 2 million hectares across the world producing around 10 million tons of pods every year. India produces the highest percentage of okra in the world. India accounts for about 60 per cent of total okra production in the world. Due to its annual production of about 6 million tonnes, it also plays an important role in exporting vegetable to different countries around the globe. Among the states, West Bengal accounts for the highest production of okra in India. It produces about 15 per cent of the okra produced in India. Bihar, Gujarat and Odisha are some other okra-producing states (Australian Government Department of Agriculture, 2021).
       
Several biotic and abiotic factors play an important role in affecting the quality as well as production of okra yield. Biotic factors that affect okra crop are different types of viruses, fungi, bacteria and nematodes. Among these factors viral infections such as Okra enation leaf curl virus (OELCV) and Okra yellow vein mosaic virus (OYVMV) affect the performance of okra crop (Kucharek, 2004).
       
Among the viral diseases, okra enation leaf curl disease is a major issue in okra production which falls under the genus Begomovirus belonging to the family Geminiviridae (Venkataravanappa et al., 2013). The vector involved in transmitting the pathogen is the whitefly species known as Bemisia tabaci (Venkataravanappa et al., 2015). Bemisia tabaci (Gennadius) is a polyphagous pest, found in tropical, subtropical and temperate regions. The overall impact of whitefly has been very devastating with yield losses ranging from 20 to 100 per cent, causing significant crop losses by sucking sap from the plants and also through the transmission of viral diseases particularly caused by the genus Begomovirus (Singh and Chandi, 2019; Swathi et al., 2023). The first case reported concerning OELCD is in Bangalore, Karnataka, India during the early 1980s (Singh, 1996). Several symptoms are shown by the plant when infected with OELCD such as the presence of tiny enations that resemble pinheads on the lower surface of the leaf, stem twisting and tough leaves. The stage when the infection occurs affects the losses in yields. The yield losses are at 93.8, 83.68 and 49.38 percent when the infection occurs at 20, 35 and 50 days after germination, respectively. In contrast, plants infected within five and ten days after germination fail to bear fruits resulting in one hundred per cent yield loss (Singh, 1996).  
       
Based on the importance of crops and their negative impacts due to Okra enation leaf curl virus, the current study was carried out to identify the virus behind the infection with the following objectives.
• To study symptomatology and transmission of okra enation leaf curl disease.
• To detect and characterize Okra enation leaf curl virus through molecular techniques.

MATERIALS AND METHODS

Symptomatology and transmission of okra enation leaf curl disease
 
Symptomatology
 
Characteristic symptoms of okra enation leaf curl disease were recorded on plants naturally grown in the field and transmitted by whitefly from its inception to full development. Time taken for appearance of full symptoms was determined.
 
Transmission of Okra enation leaf curl virus
 
Whitefly transmission experiments were performed in pots placed in insect-proof cages maintained inside the glasshouse of Anand Agricultural University, Anand, Gujarat. Virus culture used in this experiment was prepared using infected okra plants. Stock colony of whiteflies was maintained using brinjal seedlings raised in pots. Insects were collected from field in the early hours of the morning using aspirator. Three okra seedlings were tested with whiteflies in each test. Acquisition access period (AAP) was determined by starving the insect populations for 2 hrs prior to feeding on the infected plant for various periods of AAP, which were 15 min, 30 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs and 24 hrs. Similarly, IAP was also determined in the same way. One plant was kept per pot and three such pots were used in each test. To kill whiteflies after requisite time, the plants were sprayed with systemic insecticide.
       
Observations on number of plants infected, transmission percentage were recorded.
       
Per cent transmission was calculated as per the following formula (Meena et al., 2002).

 
Detection and characterization of Okra enation leaf curl virus through molecular techniques
 
Transmission electron microscopy of Okra enation leaf curl virus
 
Transmission electron microscopy (TEM) of Okra enation leaf curl virus was done from infected leaf at sophisticated instrumentation centre for applied research and testing (SICART), Sardar Patel Centre for Science andTechnology, Charutar Vidya Mandal, Vallabh Vidyanagar, Anand, Gujarat. Crude sap preparation was made from OELCV-infected leaf samples by grinding them in 20 mM tris buffer, pH 7.8 with a mortar and pestle. Then crude sap was observed under the transmission electron microscope (Electron Source: -W emitter and LaB, Objective lens: -S-TWIN, Line Resolution: 2.0 nm).
 
Detection and identification of Okra enation leaf curl virus through PCR
 
The leaves of the healthy and plants infected with OELCV were collected and used for DNA isolation. Genomic DNA was isolated from the fresh, tender leaves through Cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). DNA concentration was quantified by measuring absorbance at 230, 260 and 280 nm through NanoDrop Spectrophotometer.
       
The DNA was amplified through PCR using Begomovirus universal CP gene along with self-designed OELCV specific primer. PCR was performed for both healthy leaves and those leaves infected with OELCV. The sequence obtained after PCR amplification of viral DNA by Begomovirus universal primer was used to develop gene-specific primer for OELCV using Primer3 Software. Further, the specificity of the primer was analyzed by amplifying it with the help of Mungbean yellow mosaic virus, Xanthomonas sp. and Chaetomium sp. The detail information of the primer used in the study has been shown in Table 1.

Table 1: Primer details.


       
Thermal cycling was performed to screen the primers for detecting the appropriate genes of virus infection in all samples. The components of PCR reaction include 5.00 μl Dream Taq green PCR Master mix (MBI, Fermentas AG), 0.50 μl of forward primer (10 pM/μl), 0.50 μl of reverse primer (10 pM/μl), 3.00 μl of Nuclease free water, 1.00 μl DNA per reaction.
       
Thermal cycling process for amplification was programmed in the following manner: Step-1: 94°C for 4 minutes (Initial Denaturation), followed by 30 cycles of Step-2: 94°C for 30 seconds (Denaturation after every cycle), Step-3: 56°C (Universal Primer) and 56°C (Specific Primer) for 45 seconds (Annealing), Step-4: 72°C for 1 minute (Extension of annealing primer) and then Step-5: 72°C for 10 minutes (Final extension). Approximately 10 µl of amplified DNA samples from each PCR tube along with 2 µl of loading dye were loaded on a 1.8 percent agarose gel electrophoresis at 80 volts for 1 hour. The gel was examined in a transilluminator under UV light and the picture was captured by using the Alpha Ease FC 4.0.0 gel documentation system.
       
Sequencing of amplified products was done at Eurofins Genomics India Pvt. Ltd., Bangalore, Karnataka. The sequences were analyzed through similarity search using BLASTN program that is available on NCBI database, USA (Altschul et al., 1997). The multiple sequence alignment and pairwise alignment was done using BioEdit version 5.09. A phylogenetic tree was constructed based on similarities using NCBI program.

RESULTS AND DISCUSSION

Symptomatology and transmission of okra enation leaf curl disease
 
Symptomatology
 
The symptoms of the disease were studied under natural field condition. First disease symptom was appeared after 30 days of sowing in field condition as “S” shaped bending or twisting of petiole which is a characteristic symptom. Severely infected plants included pronounced thickening of veins, leaves curled into a cup shape, small pinhead-sized enations on underside of the leaves and plants became smaller with deformed internodes, stunted growth. There were no peculiar symptoms on fruit but the infected plant had limited number of fruits or no fruit setting at all. As the infection progresses, the leaves become thick and leathery (Fig 1). The disease incidence of 47.78 per cent was recorded in the GAO 7 variety under field condition.

Fig 1: Symptoms of the okra plants infected with Okra enation leaf curl virus.


 
Transmission of Okra enation leaf curl virus
 
The minimum AAP and IAP needed for the virus was found to be 1 hour and 2 hour respectively. The results indicate that as the AAP and IAP increased, there was a corresponding increase in OELCV transmission. The maximum AAP and IAP were 24 hours and it resulted in a cent per cent transmission rate of the virus. The relative effectiveness of different AAP and IAP of OELCV by whiteflies is depicted Fig 2A and Fig 2B. The symptom expression of the plants is clearly depicted in the Fig 3. Additionally, as the AAP and IAP increased, the transmission percentage of OELCV also increased (Table 2 and 3).

Fig 2: Disease occurrence at different acquisition and inoculation access periods during transmission study of OELCV.



Fig 3: Symptom of OELCV infected plant in transmission study.



Table 2: Influence of different acquisition access periods on transmission of OELCV by whitefly.



Table 3: Influence of different inoculation access periods on transmission of OELCV by whitefly.


 
Transmission electron microscopy of Okra enation leaf curl virus
 
In TEM, geminate particles of OELCV were detected from the sap of infected okra leaves (Fig 4).

Fig 4: Transmission electron microscopy of Okra enation leaf curl virus.


 
Detection and characterization of Okra enation leaf curl virus through molecular techniques
 
Detection and Identification of Okra enation leaf curl virus through PCR
 
The integrity of isolated DNA samples was confirmed on 0.8% agarose gel using gel electrophoresis technique (Fig 5).  A PCR reaction at annealing temperature of 54°C and 56°C for the Begomovirus universal primer and self-designed OELCV specific primers respectively gave very specific band of amplicons which was observed in 1.8% agarose gel. The primer pair CpDeng541F and CpDeng540R yielded amplicon of ~500 bp fragment size (Fig 6) and the specific primer pair oelcF and oelcR yielded ~355 bp amplicon of virus genomic DNA (Fig 7A). It showed negative results when amplified with healthy okra leaves DNA.

Fig 5: DNA extraction by CTAB method from okra leaves (Lane 1:3: DNA of OELCV extracted from infected okra leaves, Lane 4: DNA extracted from healthy leaves).



Fig 6: PCR amplification of OELCV from infected leaf by using Begomovirus universal primer.



Fig 7: PCR amplification of Okra enation leaf curl virus sample by using OELCV specific primer and specificity check of self-designed OELCV specific primer.


       
The specificity of OELCV specific primer was checked through PCR technique by amplifying the primer pair with the genomic DNA of Mungbean yellow mosaic virus, Xanthomonas sp. And Chaetomium sp. in which it showed negative reaction. Hence, it proved that the self-designed primer is specific to the OELCV (Fig 7B).
 
Nucleotide sequence analysis
 
The sequences were aligned and assessed in the NCBI nucleotide BLAST to determine the similarity of newly sequenced samples to the GenBank databases and they were matched at diverse global similarity levels. The viral sequences from infected leaves were all confirmed to be Okra enation leaf curl virus and submitted to the NCBI GenBank. Percentage of similarity of the virus sequences ranged from 100.00-95.21 per cent. Sequence obtained from universal primer corresponds to V2 and V1 gene of OELCV which codes for precoat and coat protein of the virus respectively. Similarly, the sequence obtained from the OELCV specific primer resembles with AV2 and AV1 gene which also codes for precoat and coat protein of the virus respectively. Phylogenetic tree was constructed for both the sequences which represented similarities with different isolates of Okra enation leaf curl virus (Fig 8 and Fig 9). Most closely related virus has been depicted in Table 4.

Fig 8: Phylogenetic tree of Okra enation leaf curl virus AAU1 (NCBI gene bank version no. OR270161.1).



Fig 9: Phylogenetic tree of Okra enation leaf curl virus Anand (NCBI gene bank version no. OR270162.1).



Table 4: Sequence analysis of Okra enation leaf curl virus.


       
Okra enation leaf curl disease was reported for the first time in Karnataka, India, where it caused symptoms such as small pinhead-sized enations that formed on the lower surface of the leaves. Enation refers to a condition where there is an abnormal growth of projection-like structures in plant tissues (Singh, 1986). This disease has been previously reported in different parts of India by different authors (Tiendrébéogo et al., 2010; Serfraz et al., 2015; Yadav et al., 2018 and Kumar et al., 2019). This disease is a new threat to okra cultivation (Yadav et al., 2018). There are previous studies on the transmission of Okra yellow vein mosaic virus, which is another begomovirus affecting okras, by different researchers (Capoor and Varma, 1950; Sanwal et al., 2014; Sheikh et al., 2013 and Venkataravanappa et al., 2017). However, there are not many studies that have been carried out on the transmission of Okra enation leaf curl virus through whiteflies. Okra leaf curl virus (OLCV) obtained from okra plants was analyzed using electron microscopy, where it was found that the virus contains geminate particles with a size of 20 × 28 nm. Which proves that the virus belongs to Geminiviridae family (Ghanem, 2003). An alphasatellite DNA in association with OELCV has been identified in India. The alphasatellite DNA that has been characterized in the study consists of a total of 1,350 nucleotides. Its genome structure is the same as in alphasatellites. In addition to the fact that this alphasatellite DNA exhibits the highest nucleotide sequence identity (79.7 per cent) with the symptomless alphasatellite in Hollyhock yellow vein virus (HoYVSLA), this high nucleotide sequence identity suggests an evolutionary relationship between the two. Sohrab et al., (2013) investigated leaf curl and yellow vein mosaic diseases of okra. The scientists performed nucleotide sequencing of the DNA-β molecule and the coat protein gene of the virus. According to their results, the highest identity of the nucleotide sequence of the complete DNA-β is 91.7 per cent in isolates of BYVMV (NC_003405) and its lowest identity is 48.5 per cent in isolates of OKLCV (NC_004093) and OKLCV (GQ245761). The above-mentioned numbers indicate the level of identity between the DNA sequences. As for the coat protein gene, it demonstrates the highest level of similarity (99%) with isolates of BYVMV-AJ278861, AF465619 and FN645917. However, the lowest identity of 92 per cent was recorded for the isolates of BYVMV (FJ561298) and MYMBV (EU360303). The results obtained indicate that the viral disease of okra plants under consideration has close relationship with BYVMV in terms of similarity of nucleotide sequences of both the complete DNA-β and coat protein gene. Based on the sequences obtained through amplification of the complete genome of OELCV using PCR method, OELCV has a similar origin with most other Begomoviruses (Venkataravanappa et al., 2015). Moreover, specific primers to amplify the coat protein gene of BYVMV and OELCV have been conducted, in which the isolates show nucleotide similarities ranging from 99 to 100 per cent to OELCV. Therefore, the virus associated with BYVMD has a close relationship with OELCV, implying that the two viruses might be linked together (Chinju, 2017). In the current study, it has been found out that Okra enation leaf curl virus is the most threatening virus to okra growing areas in Gujarat. In Gujarat, an incidence rate of 47.78 per cent has been reported (Mishra et al., 2024). Some of the symptoms identified include petiole twisting, veins thickening, upward curling of leaves and small pinhead sized enations on under surface of leaves. The transmission dynamics of OELCV by whiteflies align with findings reported for soybean yellow mosaic virus (Swathi et al., 2023). Consistent with the established vector efficiency in other legumes, increasing acquisition and inoculation access periods significantly elevate transmission rates. Furthermore, transmission efficiency is positively influenced by pre-acquisition starvation, which enhances the feeding propensity and subsequent viral uptake, a phenomenon observed in various Begomovirus-vector systems. Transmission electron microscopy confirmed the existence of geminate OELCV in sap of infected leaves. Amplification of ~500 bp and ~355 bp fragments of genes using two universal and OELCV specific primers indicated 100 percent similarities with previously reported isolates of OELCV.

CONCLUSION

The study confirms that Okra Enation Leaf Curl Virus (OELCuV) is a serious and emerging threat to okra cultivation in Gujarat. The disease is characterized by distinct symptoms such as twisting of the petiole, vein thickening, upward curling of leaves and the formation of small pinhead-sized enations on the underside of leaves, which significantly affect plant growth and yield.
       
Efficient transmission of the virus by whiteflies was observed, particularly when longer acquisition and inoculation access periods were allowed, highlighting the critical role of the vector in disease spread. Transmission electron microscopy further validated the presence of characteristic geminate virus particles in infected leaf sap, confirming its association with Begomoviruses.
       
Molecular analysis through PCR amplification produced fragments of approximately 500 bp using universal primers and 355 bp using OELCuV-specific primers, providing strong confirmation of the virus identity.
       
Overall, the combined symptomatological, vector transmission, electron microscopy and molecular evidence conclusively establish the presence and active spread of OELCuV in okra-growing regions of Gujarat, emphasizing the urgent need for effective management strategies to control whitefly populations and limit disease dissemination.

ACKNOWLEDGEMENT

The authors are grateful to the Department of Plant Pathology, Anand Agricultural University, Anand, Gujarat, India for providing all the resources in this research work.
 
Statements and declaration
 
Authors contribution
 
Sonal Mishra carried out the research work and prepared the manuscript. Dr. Puja Pandey and Dr. Ramjibhai G. Parmar provided the conception or design of the work and finalized the manuscript with necessary corrections.
 
Data availability statement
 
Data from which results are drawn is available within the manuscript in table format under results section.
 
Research involving human and/or animals
 
This research does not involve use of any kind of animal and human being.
 
Informed consent
 
There is no need of any informed consent for this manuscript as it does not involve any concerning images of vulnerable people or images in sensitive contexts. 

CONFLICT OF INTEREST

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

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    ABSTRACT

    Background: Okra enation leaf curl virus (OELCV) belongs to the genus Begomovirus and family Geminiviridae. This virus adversely affects the potential and productivity of okra crops and in present scenario it is an emerging disease. The present study was conducted to elucidate the virus responsible for the disease.

    Methods: The virus transmission study was done with whitefly in which acquisition access period (AAP) and inoculation access period (IAP) was recorded. Detection was through transmission electron microscopy (TEM) and molecular characterization from infected leaf sample.

    Result: First symptom was appeared after 30 DAS as twisting of petiole. Infected plants showed pronounced vein thickening, cup shaped leaves, small pinhead-sized enations on underside of the leaves. The disease symptom in plants initiated with 33.33 per cent at 1 hour of acquisition access period (AAP) and 2 hours of inoculation access period (IAP) of the whitefly and increased progressively up to cent per cent in 24 hrs. Incubation period decreased with increase in AAP and IAP. In TEM geminate particles were detected. Viral DNA isolated from the infected leaf was subjected to PCR and the amplified products were confirmed to be OELCV and submitted to the NCBI GenBank (Accession No. OR270161, Accession No. OR270162).

    INTRODUCTION

    Okra [Abelmoschus esculentus (L.) Moench] is an essential plant belonging to the family Malvaceae. Although it is believed that okra originated from tropical Africa, it is now cultivated all around the world. Okra generally takes between 60 and 65 days to mature (Kumar et al., 2013). Okra seeds are also used as a substitute for coffee. It is also a potential source of natural fibers suitable for textile, paper and other engineering applications (Gupta and Patra, 2021).
           
    Okra is cultivated over a period of 2 million hectares across the world producing around 10 million tons of pods every year. India produces the highest percentage of okra in the world. India accounts for about 60 per cent of total okra production in the world. Due to its annual production of about 6 million tonnes, it also plays an important role in exporting vegetable to different countries around the globe. Among the states, West Bengal accounts for the highest production of okra in India. It produces about 15 per cent of the okra produced in India. Bihar, Gujarat and Odisha are some other okra-producing states (Australian Government Department of Agriculture, 2021).
           
    Several biotic and abiotic factors play an important role in affecting the quality as well as production of okra yield. Biotic factors that affect okra crop are different types of viruses, fungi, bacteria and nematodes. Among these factors viral infections such as Okra enation leaf curl virus (OELCV) and Okra yellow vein mosaic virus (OYVMV) affect the performance of okra crop (Kucharek, 2004).
           
    Among the viral diseases, okra enation leaf curl disease is a major issue in okra production which falls under the genus Begomovirus belonging to the family Geminiviridae (Venkataravanappa et al., 2013). The vector involved in transmitting the pathogen is the whitefly species known as Bemisia tabaci (Venkataravanappa et al., 2015). Bemisia tabaci (Gennadius) is a polyphagous pest, found in tropical, subtropical and temperate regions. The overall impact of whitefly has been very devastating with yield losses ranging from 20 to 100 per cent, causing significant crop losses by sucking sap from the plants and also through the transmission of viral diseases particularly caused by the genus Begomovirus (Singh and Chandi, 2019; Swathi et al., 2023). The first case reported concerning OELCD is in Bangalore, Karnataka, India during the early 1980s (Singh, 1996). Several symptoms are shown by the plant when infected with OELCD such as the presence of tiny enations that resemble pinheads on the lower surface of the leaf, stem twisting and tough leaves. The stage when the infection occurs affects the losses in yields. The yield losses are at 93.8, 83.68 and 49.38 percent when the infection occurs at 20, 35 and 50 days after germination, respectively. In contrast, plants infected within five and ten days after germination fail to bear fruits resulting in one hundred per cent yield loss (Singh, 1996).  
           
    Based on the importance of crops and their negative impacts due to Okra enation leaf curl virus, the current study was carried out to identify the virus behind the infection with the following objectives.
    • To study symptomatology and transmission of okra enation leaf curl disease.
    • To detect and characterize Okra enation leaf curl virus through molecular techniques.

    MATERIALS AND METHODS

    Symptomatology and transmission of okra enation leaf curl disease
     
    Symptomatology
     
    Characteristic symptoms of okra enation leaf curl disease were recorded on plants naturally grown in the field and transmitted by whitefly from its inception to full development. Time taken for appearance of full symptoms was determined.
     
    Transmission of Okra enation leaf curl virus
     
    Whitefly transmission experiments were performed in pots placed in insect-proof cages maintained inside the glasshouse of Anand Agricultural University, Anand, Gujarat. Virus culture used in this experiment was prepared using infected okra plants. Stock colony of whiteflies was maintained using brinjal seedlings raised in pots. Insects were collected from field in the early hours of the morning using aspirator. Three okra seedlings were tested with whiteflies in each test. Acquisition access period (AAP) was determined by starving the insect populations for 2 hrs prior to feeding on the infected plant for various periods of AAP, which were 15 min, 30 min, 1 hr, 2 hrs, 4 hrs, 6 hrs, 8 hrs and 24 hrs. Similarly, IAP was also determined in the same way. One plant was kept per pot and three such pots were used in each test. To kill whiteflies after requisite time, the plants were sprayed with systemic insecticide.
           
    Observations on number of plants infected, transmission percentage were recorded.
           
    Per cent transmission was calculated as per the following formula (Meena et al., 2002).

     
    Detection and characterization of Okra enation leaf curl virus through molecular techniques
     
    Transmission electron microscopy of Okra enation leaf curl virus
     
    Transmission electron microscopy (TEM) of Okra enation leaf curl virus was done from infected leaf at sophisticated instrumentation centre for applied research and testing (SICART), Sardar Patel Centre for Science andTechnology, Charutar Vidya Mandal, Vallabh Vidyanagar, Anand, Gujarat. Crude sap preparation was made from OELCV-infected leaf samples by grinding them in 20 mM tris buffer, pH 7.8 with a mortar and pestle. Then crude sap was observed under the transmission electron microscope (Electron Source: -W emitter and LaB, Objective lens: -S-TWIN, Line Resolution: 2.0 nm).
     
    Detection and identification of Okra enation leaf curl virus through PCR
     
    The leaves of the healthy and plants infected with OELCV were collected and used for DNA isolation. Genomic DNA was isolated from the fresh, tender leaves through Cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle, 1987). DNA concentration was quantified by measuring absorbance at 230, 260 and 280 nm through NanoDrop Spectrophotometer.
           
    The DNA was amplified through PCR using Begomovirus universal CP gene along with self-designed OELCV specific primer. PCR was performed for both healthy leaves and those leaves infected with OELCV. The sequence obtained after PCR amplification of viral DNA by Begomovirus universal primer was used to develop gene-specific primer for OELCV using Primer3 Software. Further, the specificity of the primer was analyzed by amplifying it with the help of Mungbean yellow mosaic virus, Xanthomonas sp. and Chaetomium sp. The detail information of the primer used in the study has been shown in Table 1.

    Table 1: Primer details.


           
    Thermal cycling was performed to screen the primers for detecting the appropriate genes of virus infection in all samples. The components of PCR reaction include 5.00 μl Dream Taq green PCR Master mix (MBI, Fermentas AG), 0.50 μl of forward primer (10 pM/μl), 0.50 μl of reverse primer (10 pM/μl), 3.00 μl of Nuclease free water, 1.00 μl DNA per reaction.
           
    Thermal cycling process for amplification was programmed in the following manner: Step-1: 94°C for 4 minutes (Initial Denaturation), followed by 30 cycles of Step-2: 94°C for 30 seconds (Denaturation after every cycle), Step-3: 56°C (Universal Primer) and 56°C (Specific Primer) for 45 seconds (Annealing), Step-4: 72°C for 1 minute (Extension of annealing primer) and then Step-5: 72°C for 10 minutes (Final extension). Approximately 10 µl of amplified DNA samples from each PCR tube along with 2 µl of loading dye were loaded on a 1.8 percent agarose gel electrophoresis at 80 volts for 1 hour. The gel was examined in a transilluminator under UV light and the picture was captured by using the Alpha Ease FC 4.0.0 gel documentation system.
           
    Sequencing of amplified products was done at Eurofins Genomics India Pvt. Ltd., Bangalore, Karnataka. The sequences were analyzed through similarity search using BLASTN program that is available on NCBI database, USA (Altschul et al., 1997). The multiple sequence alignment and pairwise alignment was done using BioEdit version 5.09. A phylogenetic tree was constructed based on similarities using NCBI program.

    RESULTS AND DISCUSSION

    Symptomatology and transmission of okra enation leaf curl disease
     
    Symptomatology
     
    The symptoms of the disease were studied under natural field condition. First disease symptom was appeared after 30 days of sowing in field condition as “S” shaped bending or twisting of petiole which is a characteristic symptom. Severely infected plants included pronounced thickening of veins, leaves curled into a cup shape, small pinhead-sized enations on underside of the leaves and plants became smaller with deformed internodes, stunted growth. There were no peculiar symptoms on fruit but the infected plant had limited number of fruits or no fruit setting at all. As the infection progresses, the leaves become thick and leathery (Fig 1). The disease incidence of 47.78 per cent was recorded in the GAO 7 variety under field condition.

    Fig 1: Symptoms of the okra plants infected with Okra enation leaf curl virus.


     
    Transmission of Okra enation leaf curl virus
     
    The minimum AAP and IAP needed for the virus was found to be 1 hour and 2 hour respectively. The results indicate that as the AAP and IAP increased, there was a corresponding increase in OELCV transmission. The maximum AAP and IAP were 24 hours and it resulted in a cent per cent transmission rate of the virus. The relative effectiveness of different AAP and IAP of OELCV by whiteflies is depicted Fig 2A and Fig 2B. The symptom expression of the plants is clearly depicted in the Fig 3. Additionally, as the AAP and IAP increased, the transmission percentage of OELCV also increased (Table 2 and 3).

    Fig 2: Disease occurrence at different acquisition and inoculation access periods during transmission study of OELCV.



    Fig 3: Symptom of OELCV infected plant in transmission study.



    Table 2: Influence of different acquisition access periods on transmission of OELCV by whitefly.



    Table 3: Influence of different inoculation access periods on transmission of OELCV by whitefly.


     
    Transmission electron microscopy of Okra enation leaf curl virus
     
    In TEM, geminate particles of OELCV were detected from the sap of infected okra leaves (Fig 4).

    Fig 4: Transmission electron microscopy of Okra enation leaf curl virus.


     
    Detection and characterization of Okra enation leaf curl virus through molecular techniques
     
    Detection and Identification of Okra enation leaf curl virus through PCR
     
    The integrity of isolated DNA samples was confirmed on 0.8% agarose gel using gel electrophoresis technique (Fig 5).  A PCR reaction at annealing temperature of 54°C and 56°C for the Begomovirus universal primer and self-designed OELCV specific primers respectively gave very specific band of amplicons which was observed in 1.8% agarose gel. The primer pair CpDeng541F and CpDeng540R yielded amplicon of ~500 bp fragment size (Fig 6) and the specific primer pair oelcF and oelcR yielded ~355 bp amplicon of virus genomic DNA (Fig 7A). It showed negative results when amplified with healthy okra leaves DNA.

    Fig 5: DNA extraction by CTAB method from okra leaves (Lane 1:3: DNA of OELCV extracted from infected okra leaves, Lane 4: DNA extracted from healthy leaves).



    Fig 6: PCR amplification of OELCV from infected leaf by using Begomovirus universal primer.



    Fig 7: PCR amplification of Okra enation leaf curl virus sample by using OELCV specific primer and specificity check of self-designed OELCV specific primer.


           
    The specificity of OELCV specific primer was checked through PCR technique by amplifying the primer pair with the genomic DNA of Mungbean yellow mosaic virus, Xanthomonas sp. And Chaetomium sp. in which it showed negative reaction. Hence, it proved that the self-designed primer is specific to the OELCV (Fig 7B).
     
    Nucleotide sequence analysis
     
    The sequences were aligned and assessed in the NCBI nucleotide BLAST to determine the similarity of newly sequenced samples to the GenBank databases and they were matched at diverse global similarity levels. The viral sequences from infected leaves were all confirmed to be Okra enation leaf curl virus and submitted to the NCBI GenBank. Percentage of similarity of the virus sequences ranged from 100.00-95.21 per cent. Sequence obtained from universal primer corresponds to V2 and V1 gene of OELCV which codes for precoat and coat protein of the virus respectively. Similarly, the sequence obtained from the OELCV specific primer resembles with AV2 and AV1 gene which also codes for precoat and coat protein of the virus respectively. Phylogenetic tree was constructed for both the sequences which represented similarities with different isolates of Okra enation leaf curl virus (Fig 8 and Fig 9). Most closely related virus has been depicted in Table 4.

    Fig 8: Phylogenetic tree of Okra enation leaf curl virus AAU1 (NCBI gene bank version no. OR270161.1).



    Fig 9: Phylogenetic tree of Okra enation leaf curl virus Anand (NCBI gene bank version no. OR270162.1).



    Table 4: Sequence analysis of Okra enation leaf curl virus.


           
    Okra enation leaf curl disease was reported for the first time in Karnataka, India, where it caused symptoms such as small pinhead-sized enations that formed on the lower surface of the leaves. Enation refers to a condition where there is an abnormal growth of projection-like structures in plant tissues (Singh, 1986). This disease has been previously reported in different parts of India by different authors (Tiendrébéogo et al., 2010; Serfraz et al., 2015; Yadav et al., 2018 and Kumar et al., 2019). This disease is a new threat to okra cultivation (Yadav et al., 2018). There are previous studies on the transmission of Okra yellow vein mosaic virus, which is another begomovirus affecting okras, by different researchers (Capoor and Varma, 1950; Sanwal et al., 2014; Sheikh et al., 2013 and Venkataravanappa et al., 2017). However, there are not many studies that have been carried out on the transmission of Okra enation leaf curl virus through whiteflies. Okra leaf curl virus (OLCV) obtained from okra plants was analyzed using electron microscopy, where it was found that the virus contains geminate particles with a size of 20 × 28 nm. Which proves that the virus belongs to Geminiviridae family (Ghanem, 2003). An alphasatellite DNA in association with OELCV has been identified in India. The alphasatellite DNA that has been characterized in the study consists of a total of 1,350 nucleotides. Its genome structure is the same as in alphasatellites. In addition to the fact that this alphasatellite DNA exhibits the highest nucleotide sequence identity (79.7 per cent) with the symptomless alphasatellite in Hollyhock yellow vein virus (HoYVSLA), this high nucleotide sequence identity suggests an evolutionary relationship between the two. Sohrab et al., (2013) investigated leaf curl and yellow vein mosaic diseases of okra. The scientists performed nucleotide sequencing of the DNA-β molecule and the coat protein gene of the virus. According to their results, the highest identity of the nucleotide sequence of the complete DNA-β is 91.7 per cent in isolates of BYVMV (NC_003405) and its lowest identity is 48.5 per cent in isolates of OKLCV (NC_004093) and OKLCV (GQ245761). The above-mentioned numbers indicate the level of identity between the DNA sequences. As for the coat protein gene, it demonstrates the highest level of similarity (99%) with isolates of BYVMV-AJ278861, AF465619 and FN645917. However, the lowest identity of 92 per cent was recorded for the isolates of BYVMV (FJ561298) and MYMBV (EU360303). The results obtained indicate that the viral disease of okra plants under consideration has close relationship with BYVMV in terms of similarity of nucleotide sequences of both the complete DNA-β and coat protein gene. Based on the sequences obtained through amplification of the complete genome of OELCV using PCR method, OELCV has a similar origin with most other Begomoviruses (Venkataravanappa et al., 2015). Moreover, specific primers to amplify the coat protein gene of BYVMV and OELCV have been conducted, in which the isolates show nucleotide similarities ranging from 99 to 100 per cent to OELCV. Therefore, the virus associated with BYVMD has a close relationship with OELCV, implying that the two viruses might be linked together (Chinju, 2017). In the current study, it has been found out that Okra enation leaf curl virus is the most threatening virus to okra growing areas in Gujarat. In Gujarat, an incidence rate of 47.78 per cent has been reported (Mishra et al., 2024). Some of the symptoms identified include petiole twisting, veins thickening, upward curling of leaves and small pinhead sized enations on under surface of leaves. The transmission dynamics of OELCV by whiteflies align with findings reported for soybean yellow mosaic virus (Swathi et al., 2023). Consistent with the established vector efficiency in other legumes, increasing acquisition and inoculation access periods significantly elevate transmission rates. Furthermore, transmission efficiency is positively influenced by pre-acquisition starvation, which enhances the feeding propensity and subsequent viral uptake, a phenomenon observed in various Begomovirus-vector systems. Transmission electron microscopy confirmed the existence of geminate OELCV in sap of infected leaves. Amplification of ~500 bp and ~355 bp fragments of genes using two universal and OELCV specific primers indicated 100 percent similarities with previously reported isolates of OELCV.

    CONCLUSION

    The study confirms that Okra Enation Leaf Curl Virus (OELCuV) is a serious and emerging threat to okra cultivation in Gujarat. The disease is characterized by distinct symptoms such as twisting of the petiole, vein thickening, upward curling of leaves and the formation of small pinhead-sized enations on the underside of leaves, which significantly affect plant growth and yield.
           
    Efficient transmission of the virus by whiteflies was observed, particularly when longer acquisition and inoculation access periods were allowed, highlighting the critical role of the vector in disease spread. Transmission electron microscopy further validated the presence of characteristic geminate virus particles in infected leaf sap, confirming its association with Begomoviruses.
           
    Molecular analysis through PCR amplification produced fragments of approximately 500 bp using universal primers and 355 bp using OELCuV-specific primers, providing strong confirmation of the virus identity.
           
    Overall, the combined symptomatological, vector transmission, electron microscopy and molecular evidence conclusively establish the presence and active spread of OELCuV in okra-growing regions of Gujarat, emphasizing the urgent need for effective management strategies to control whitefly populations and limit disease dissemination.

    ACKNOWLEDGEMENT

    The authors are grateful to the Department of Plant Pathology, Anand Agricultural University, Anand, Gujarat, India for providing all the resources in this research work.
     
    Statements and declaration
     
    Authors contribution
     
    Sonal Mishra carried out the research work and prepared the manuscript. Dr. Puja Pandey and Dr. Ramjibhai G. Parmar provided the conception or design of the work and finalized the manuscript with necessary corrections.
     
    Data availability statement
     
    Data from which results are drawn is available within the manuscript in table format under results section.
     
    Research involving human and/or animals
     
    This research does not involve use of any kind of animal and human being.
     
    Informed consent
     
    There is no need of any informed consent for this manuscript as it does not involve any concerning images of vulnerable people or images in sensitive contexts. 

    CONFLICT OF INTEREST

    All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

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