Abstract: Background and Objective: Oilseed brassica are one of the most exploited agricultural commodities in International trade with diversified use in human and animal consumption besides their potential use in producing green energy in the form of biofuels. Turnip mosaic virus is one of the limiting factors for declining oil content in brassica. The present studies were therefore conducted to confirm the presence of this important virus in brassica through biological and serological assays. Materials and Methods: A total of 518 samples collected from 84 locations spanning across 5 states and 1 union territory from symptomatic plants were collected and assayed in DAS-ELISA using Turnip mosaic virus (TuMV) specific polyclonal antiserum. Biological and serological host range of the virus isolate was established and different varieties/breeding lines of oilseed brassica were screened for developing a resistance panel against TuMV. Results: Turnip mosaic virus incidence ranged between 0.6-8.3% in oilseed brassica and 0.2-17.6% in crucifer vegetables. Turnip mosaic virus was recorded in very high concentration from radish as indicated by the optical density values. Mustard variety Tender Green was established as the best propagative host of Indian radish isolate of Turnip mosaic virus. Out of 32 varieties/breeding lines of oilseed brassica collected from different sources in India, 25 varieties/lines were found to be susceptible to Turnip mosaic virus under glasshouse conditions and DAS-ELISA further confirmed these findings. Conclusion: A radish isolate of Turnip mosaic virus has been identified on the basis of biological and serological assays and results obtained for screening of brassica germplasm against Turnip mosaic virus are expected to help in ascertaining the sources of resistance against this virus.
INTRODUCTION
Mustard rape is a major oil yielding crop throughout the world with India contributing about 11.3% of oilseed brassica and ranking third after Canada and China in its production besides oilseed brassica also occupies first place for edible oil in the country with a share of 23 % in overall production1. Though India is a major producer of oilseed brassica, it still imports edible oils as mustard rape yields have stagnated despite the release of elite cultivars as the crop faces many production challenges particularly from the abiotic stresses like drought, high temperature, salinity besides biotic stresses from bacterial and fungal diseases2 to Turnip mosaic virus3. Plants representing the family Brassicaceae are susceptible to many viruses and Turnip mosaic virus (TuMV), a member of the genus Potyvirus, has the widest host range among all viruses infecting brassica4. The virus is of worldwide occurrence including the temperate and tropical regions of all continents5,6 and it ranks second only to Cucumber mosaic virus for infecting vegetables7.
Biological indexing is probably the best informative and basic method used in plant virology for detecting certain viruses. The utility of biological indexing lies in the fact that it forms the very basis of establishing diagnostic host range of a number of viruses and differentiating between plant viruses in mixed infections8. Subsequent development of enzyme-linked immunosorbent assay (ELISA) as an efficient and rapid detection technique for diagnosis of plant viruses established itself as an imperative and relevant necessity as biological indexing is a time consuming technique. More often than not, it is virtually impossible to diagnose plant viruses on the basis of symptoms on diagnostic hosts as the symptoms often vary depending on the interaction between the plant variety and the strain of the virus9. The symptoms are therefore inconclusive at times and could result from a synergistic effect of mixed infection by two or more viruses. The use of indicator hosts in bioassays is an indispensable tool since original symptoms still play a major role in diagnosis6,10.
A number of laboratory techniques are available to the researchers for the diagnosis of plant viruses. However serology particularly ELISA, has gained popularity over other techniques owing to its high specificity, rapidity and precision11. The strength of ELISA lies in its capability for final identification of viruses decisively and establishing the relationship between different viruses and their strains.
Turnip mosaic virus infects brassica including rapeseed and crucifer vegetables around the globe but a limited knowledge is available on this virus in the states of Himachal Pradesh in India. Mustard is the only edible oilseed crop which holds significant potential towards augmenting the total oilseed production by improving its productivity through hybrid adoption, value addition and disease and pest resistance breeding in an integrated manner1. Screening of brassica germplasm is an important strategy used for identifying sources of resistance to diseases particularly TuMV4. The findings of these studies will help to unravel the dynamics of TuMV in brassica with particular reference to the development of resistance panel against Indian radish isolate of TuMV. The purpose of the present study was to investigate the diversity of TuMV in brassica and crucifer vegetables and uncover the widespread nature of this devastating virus in India.
MATERIALS AND METHODS
Study area: Field surveys were conducted at 84 locations during 2018-2019 in the Indian states of Himachal Pradesh, Punjab, Haryana, Rajasthan and Meghalaya and Union Territory of Jammu and Kashmir.
Surveys: Field surveys were conducted to record the presence and prevalence of Turnip mosaic virus in oilseed brassica and crucifer vegetable crops including mustard, rapeseed, cauliflower, cabbage, radish, turnip, broccoli, kale, knol khol, bok choy, Brussels sprout, lettuce and Chinese cabbage. In each field, ten plants were selected along two diagonals for sampling and a total of 518 plants were marked on the basis of visual symptoms and leaves from infected plants were collected and maintained in the glasshouse for further biological and serological studies. Incidence counts were made during active growing stage of the crop on at least 100 plants by choosing 4-5 locations in the field at random and observations on the number of healthy and diseased plants were recorded. The % disease incidence was calculated by using the following formula12:
Serological detection: Alkaline phosphatase (ALP) based double antibody sandwich ELISA (DAS-ELISA) commercial kits from BIOREBA AG, Switzerland were used for serological detection of TuMV in the collected samples of brassica oilseed crops and crucifer vegetables as per the instructions of the manufacturer. The absorbance value for each sample was read at 405 nm to record the O.D. in MicroScan Plate Reader MS5608A (ECIL, India). Microtiter plates were kept in dark at room temperature in a humid box for 15-60 min till the development of yellow colour. The reaction was stopped by adding 50 μL of 3 M NaOH to each well, if desired. The results obtained in ELISA tests were interpreted and the samples with O.D. values twice the mean values of healthy control samples were considered to be infected with Turnip mosaic virus13.
Maintenance of isolates and host range studies: The collected isolate which tested positive in DAS-ELISA was mechanically sap transmitted to Chenopodium quinoa, C. amranticolor, C. album, Nicotiana tabacum var. White burley, N. glutinosa, Nicandra physalodes, Datura metel and D. festuosa. Young plants with 4-5 leaves were inoculated using 0.01 M phosphate buffer (pH-7.2) and kept under observation in a glasshouse for a period of 4 weeks at 25°C. Prior to the establishment of host range, the isolate was subjected to three successive single local lesion transfers on C. amranticolor. As a standard practice, the isolate was maintained on mustard variety Tender Green (Ed Hume Seeds Inc., Kent, UK), a highly susceptible variety exhibiting typical symptoms of TuMV infection. At least three test plants of each species or cultivar belonging to the family Cruciferae, Chenopodiaceae, Brassicaseae and Solanaceae were inoculated. Four weeks after inoculation with test samples, back inoculations were made to confirm the presence of virus. All inoculated plants including those that did not exhibit any visible symptoms were assayed by DAS-ELISA.
Germplasm screening: Seeds of thirty two different varieties/breeding lines of oilseed brassica procured from different research institutes of India including IARI Regional Station Kullu, research stations of CSKHPKV Palampur, Dr YS Parmar University of Horticulture and Forestry, Solan in Himachal Pradesh, PAU, Ludhiana (Punjab), CCSHAU, Hisar (Haryana) besides commercial varieties from the states of Himachal Pradesh and Rajasthan were inoculated with TuMV isolates maintained under insect-proof glasshouse of the Department of Plant Pathology, Dr Y S Parmar University of Horticulture and Forestry Solan. The inoculated plants were observed for the appearance of visual symptoms and the results were further confirmed by DAS-ELISA.
RESULTS
Incidence of TuMV: Extensive surveys conducted to record the occurrence and distribution of TuMV in brassica oilseed and crucifer vegetable at eighty five locations spread across five major brassica and crucifer vegetables growing states and one Union Territory of India resulted in the collection of 518 samples from symptomatic plants.
| Table 1: | Average incidence of Turnip mosaic virus in different parts of India |
Oilseed brassica was surveyed in all five states and union territory whereas only two states namely Himachal Pradesh and Meghalaya were surveyed for crucifer vegetables as these two states grow crucifer vegetables on commercial scale because of cooler climate. In the present studies, TuMV was found to be prevalent in all the locations surveyed and maximum incidence of TuMV was recorded in Himachal Pradesh with an average disease incidence of 12.5 % across 53 locations surveyed (Table 1). Average incidence of TuMV in Punjab, Haryana, Rajasthan, Jammu and Kashmir and Meghalaya was observed to be 6.72, 5.87, 4.65, 3.29 and 2.18 %, respectively.
The detailed data on visual symptoms and disease incidence at 85 locations surveyed is presented in Table 2. A critical analysis of the variation in symptoms revealed that most of the locations had mosaic, puckering and leaf deformation as the most characteristic symptoms in both oilseed brassica and crucifer vegetable crops though other symptoms such as mottling and crinkling were also observed during surveys. It is evident from the data set out in the Table 2 that a maximum incidence of 8.3% was recorded at Abohar (Punjab) in oilseed brassica whereas minimum incidence of 0.6% was recorded at two locations namely Dharja (Sirmour) and Sharabhai (Kullu). In case of crucifer vegetables, a high incidence of 17.6% was recorded at Shamrod (Solan) and a minimum of 0.3% at Sundernagar (Mandi).
DAS-ELISA assays for TuMV: Leaves from symptomatic plants were subjected to DAS-ELISA test to confirm the presence of TuMV in brassica oilseed and crucifer vegetables. The data on O.D. values recorded at 405 nm presented in Table 3 and it is apparent from the data that out of the 13 hosts tested only radish (Shamrod, Himachal Pradesh) yielded positive results with an O.D. value of 0.886 which was more than twice the O.D. value of negative control (0.226). The data clearly indicates that all other hosts though exhibiting symptoms did not test positive in DAS-ELISA against TuMV.
Biological and serological reaction on indicator hosts: Ten indicator hosts including Mustard var. Tender green were mechanically inoculated with radish isolate of TuMV and the data on symptoms and O.D. values at 405 nm in DAS-ELISA assays is presented in Table 4.
| Table 2: | Incidence of Turnip mosaic virus on oilseed brassica and crucifer vegetable crops |
| Table 3: | DAS-ELISA detection of Turnip mosaic virus in oilseed brassica and crucifer vegetable |
HP: Himachal Pradesh, J and K: Jammu and Kashmir, O.D.: Optical density, A405nm: Absorbance at 405 nm | |
The data reveals that mechanical transmission of TuMV resulted in the development of systemic infection in Mustard var. Tender Green whereas Chenopodium amaranticolor and C. quinoa (Fig. 1) developed local chlorotic lesions 8-12 days post inoculation turning necrotic after 4 weeks of inoculation however, C. album did not develop any symptoms.
| Fig. 1: | Local lesions on Chenopodium amaranticolor and chlorotic spots on Chenopodium quinoa |
| Fig. 2: | Mustard var. Tender green expressing TuMV symptoms |
The remaining indicator hosts representing three genera namely Nicotiana, Datura and Nicandra did not produced any symptom. DAS-ELISA was performed to confirm the findings of biological assays and the data clearly supported these findings as indicated by very high O.D. value of 1.422 against negative control (0.279) and C. amranticolor and C. quinoa also tested positive with O.D. values of 0.930 and 1.008, respectively (Table 4).
Maintenance host: Mustard var. Tender Green was mechanically transmitted for maintaining the radish TuMV isolate and the inoculated plants developed prominent symptoms in the form of mosaic, green vein banding, blistering, puckering and severe leaf deformation under glasshouse conditions (Fig. 2). The symptoms started appearing as diffused mottling 10-12 days after inoculation and developed blisters and puckering 4 weeks after inoculations.
Resistance panel against TuMV: Thirty two entries (varieties/breeding lines) of oilseed brassica were evaluated under insect-proof glasshouse conditions to ascertain their status with regard to resistance or susceptibility against radish isolate of TuMV.
| Fig. 3: | Susceptible reaction of oilseed brassica accessions to TuMV |
| Table 4: | Serological reaction of indicator hosts to Radish isolate of Turnip mosaic virus |
The basic objective of the study was to identify sources of resistance for the development of resistance panel. The results obtained are presented in Table 5. A critical analysis of the data indicates that twenty five out of thirty two accessions exhibited mosaic, mottle, blisters, puckering and leaf deformation (Fig. 3). The findings of mechanical transmission were substantiated by O.D. values recorded in DAS-ELISA assays as all twenty five entries that produced symptoms also tested positive with O.D. values at least two times higher than that of negative control (0.213). Seven accessions testing negative against radish isolate of TuMV can be included in the resistance panel. Out of these seven accessions, breeding line RGN229 had the least O.D. value of 0.112 closely followed by PC-6 with an O.D. value of 0.139. All other entries that did not develop symptoms also recorded very low O.D. values.
| Table 5: | Screening of brassica Germplasm against Turnip mosaic virus using DAS-ELISA |
CSKHPKV: Chaudhary Sarveen Kumar Himachal Pradesh Krishi Vishvavidyalaya, CCSHAU: Chaudhary Charan Singh Haryana Agricultural University, PAU: Punjab Agricultural University | |
DISCUSSION
The prominent symptoms observed on oilseed brassica and crucifer vegetables during surveys conducted for TuMV were severe mosaic, mottling, blister formation, puckering, crinkling and leaf deformation. Turnip mosaic virus is widely known to induce varied symptoms on different hosts including oilseed brassica and crucifers14. Observations of present studies were in conjunction with the findings of many other workers who have also reported different symptoms on many hosts infected with TuMV15-17. Average incidence of TuMV based on visual symptoms ranged between 2.18-12.5% during surveys. Visual symptoms in brassica and crucifer crops have been used by many workers from different parts of the world for recording the incidence of TuMV. Surveys conducted for four viruses including TuMV in 5 natural populations of Brassica oleracea in Dorset, UK found a high TuMV incidence of 43%18. Studies conducted in Iran similar to the present studies reported varying level of incidence of TuMV in brassica and crucifer vegetables19. However, in yet another study in Southern England did not encounter TuMV in wild Brassica rapa ssp. Sylvestris20.
DAS-ELISA assays detected TuMV in radish only and not in any other crucifer vegetable and brassica oilseed crops. These results go in line with the findings of a number of workers who have efficiently used DAS-ELISA for detecting the presence of TuMV in oilseed brassica, radish and lettuce grown in Saudi Arabia, India, Ukraine, UK, Turkey and many other countries21-24. The results of the present studies failed to detect TuMV in crucifer vegetables and brassica which are in contrary to the finding of a study conducted in Turkey that reported the detection of TuMV in Brussels sprout, cabbage and wild mustard besides radish25.
The studies have revealed the C. quinoa and C. amaranticolor are good indicator hosts for biological assays of radish isolate of TuMV under study. Similar results have been reported in C. amaranticolor26 and C. quinoa27 against TuMV. The findings of the present studies are also supported by a study conducted on wild European orchids wherein C. amaranticolor and C. quinoa developed chlorotic spots upon inoculation with TuMV but not on N. tabacum var. White Burley6. Mustard var. Tender Green was observed to be a useful propagative host for maintaining the virus isolate under study and produced typical symptoms of TuMV infection. These observations are in conjunction with a report indicating that Mustard var. Tender Green is a susceptible host to both TuMV UKI and JPNI isolates28.
Efforts to ascertain sources of resistance to TuMV in oilseed brassica resulted in identifying seven breeding lines/varieties that can be used for developing resistance panel. Studies conducted by various research groups have also used oilseed brassica and crucifer vegetable germplasm for screening in quest of sources of resistance against TuMV and have succeeded in identifying the same for use in future breeding programmes14,29-30.
Biological and Serological evidences have confirmed the presence of TuMV isolate in radish and RT-PCR based molecular studies can further substantiate these findings. A wider resistance panel against TuMV can be developed by screening more accessions in the studies.
CONCLUSION
Oilseed brassica and crucifer vegetable crop growing areas in five states and a Union Territory of India resulted in identifying radish isolate of TuMV on the basis of biological and serological assays. Mustard var. Tender Green was found to be the best propagative host of this isolate. Significant findings of the present studies help in characterizing TuMV isolates representing the diversity in India and knowledge of the relative frequency of different pathotypes of the virus in vegetables and oilseed brassica crop develop a panel of resistant germplasm against Indian isolates of TuMV that can be used to screen brassica germplasm.
SIGNIFICANCE STATEMENT
Oilseed brassica and crucifer vegetables though are commercially important crops throughout the world and recent past has witnessed enormous expansion in the area and production under these crops resulting from an ever-increasing demand, Turnip mosaic virus has emerged as a serious threat and hampers commercial cultivation of these crops. Detailed studies on TuMV based on extensive surveys, biological and serological indexing in addition to screening of brassica germplasm conducted in the present study is expected to be of immense use for the further breeding programs which will ultimate help the farming community.
ACKNOWLEDGMENT
The authors sincerely acknowledge Biotechnology and Biological Sciences Research Council (BBSRC), Govt. of UK and Department of Biotechnology, Govt. of India for providing the financial assistance to conduct the research studies under Newton Bhabha PORI Project. The authors also wish to acknowledge Dr Y S Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India for providing infrastructural, laboratory and farm facilities for conducting this research.