SciFed Journal of Immunology

Studies on the Production of Virus Inhibitory Agent (VIA) in Different osts Treated with Partially Purified Phytoprotein Isolated from Leaves of Clerodendrum Aculeatum

Research Article

Received on: June 18, 2017

Accepted on: November 20, 2017

Published on: December 06, 2017

Awasthi LP

*Corresponding author: Awasthi LP, Amity Center for Research and Innovation, Varanasi-221007, India. E-mail: lpawasthi14@gmail.com; Tel: +91-9415718904

Abstract

          The synthesis of virus inhibitory agent (VIA), could be induced, in treated as well as nonrated leaves of hypersensitive and systemic host plants, by the application of partially purified phytoprotein isolated from the leaves of Clerodendrum aculeatum plants (CAP). Exvivo inactivation of virus using LS-VIA (Lagenaria siceraria -Virus Inhibitory Agent), CS-VIA (Cucumis sativus -Virus Inhibitory Agent) and CJ-VIA (Crotolaria juncea-Virus Inhibitory Agent) revealed that each of the VIA sample isolated either from of LS- VIA, CS-VIA or CJVIA posses strong ability of ex vivo inactivation of viruses.
           It was revealed that the effectiveness of VIAs isolated from different sources was host dependent. VIA isolated from Lagenaria siceraria plants, pre-treated with CAP (LS-VIA) gave best response when assayed after incubation with SHRV. The VIA at a concentration of 1:10 was found to be optimum which could not enhance its effect on the ex vivo inactivation of Virus.
Keywords
             Phytoprotein; Virus Inhibitory Agent; Hypersensitive; Systemic; Inactivation

FullText

Introduction
        Viral diseases are of immense importance considering the extensive damage and severe losses they cause to crops. Because of their peculiar nature and characteristic association with hosts and vectors, no therapeutic method to completely control them has been found successful [1]. The worldwide losses caused by viral diseases are estimated at about US $220 billions per year [2]. However, certain preventive measures can be of great help in avoiding virus diseases [3, 4].
        Many higher plants have the ability to resist pathogen attack including virus infection [5, 6, 7, 8]. Some of the plants are known to contain endogenous proteins that act as antiviral agents [9, 10. 11]. There is no indication, however, that all the plants contain same type of inhibitor or that the antiviral mechanism is the same in all cases. Although, attempts have been made to explain the mechanism of antiviral action of plant products [12] no mechanism could fully and satisfactorily explain the phenomenon of virus inaction. In most of the cases, the proposed mechanism for the antiviral action of the widely occurring proteinacious inhibitors seems to be the result of the inhibitor ribosome-specific-N glycosides activity in-vitro [13, 14].
        Endogenously occurring substances, in a few higher exotic plants have been reported to induce systemic resistance in susceptible hosts against virus infections [15, 16, 17]. Such plant extracts have been used for protecting economically important crops against virus infections [311, 18, 19]. The leaves of C. aculeatum and roots of B. diffusa have been shown to contain potent endogenous virus inhibitory phytoproteins called as CA-SRIP and BD-SRIP, respectively [17, 20]. These phytoproteins ,referred as systemic resistance inducing protein (SRIP), could induce the production of potent virus inhibitory agents (VIAs), when applied on to the leaves of different susceptible healthy host, and confer strong systemic resistance in such plants against a number of plant viruses [7 , 1420, 21, 22, 23, 24, 25, 26, 27, 28].
        The present investigations were carried out to study the production of Virus Inhibitory Agent (VIA), in different hosts, treated with partially purified phytoprotein, isolated from the leaves of Clerodendrum aculeatum plants.
Materials and Methods
       Procedure for raising of test plants, maintenance of virus culture, preparation of virus inoculum and the induction of systemic resistance were the same as described earlier [29, 30].
Preparation of Virus Inhibitor and the Isolation and Partial Purification of Phytoprotein
       The leaves of C. aculeatum were harvested and ground in freshly prepared 0.2 M phosphate buffer (PB) of pH 6.6 containing 0.1 % β mercaptoethanol in the ratio of 1:2. It was then squeezed through double-layered muslin cloth. The extracted sap was centrifuged at 8,000 g for 10 minutes to remove the cell debris. A saturated solution of ammonium sulphate was added to the supernatant with continuous stirring and then left overnight at 4OC. The mixture was centrifuged at 8,000 g for 15 minutes and the precipitate in the form of thick pellets was collected. It was then suspended in a small amount of buffer [20g fresh weight/ml of 0.2M PB (pH 6.6)] and then dialyzed, in a dialysis bag, against running water for overnight, to obtain total phytoprotein fraction. The dialyzed phytoprotein fraction was either diluted as per requirement or was ̊concentrated through freeze-drying. Lyophilized phytoprotein sample, stored at 200C  was dissolved in sodium acetate buffer and .further purified by elution through a Sephadex G- 25 (Pharmacia Fine chemicals, Uppsala, Sweden) column, following the procedure as described Earlier [31]. The eluted fractions, showing antiviral activity, referred as 'Partially purified phytoprotein' were used for VIA induction in susceptible host plants, viz. Lagenaria siceraria, Cucumis sativus, Crotolaria juncea and Cyamopsis tetragonoloba plants.
Induction of Systemic Resistance and its Purification
        The systemic resistance inducing partially purified C. aculeatum phytoprotein (CAP), was applied on Cyamopsis tetragonoloba plants. The leaves from Cyamopsis tetragonoloba plants, sprayed 24 hours earlier with CAP, and also the leaves from untreated control plants (treated with distilled water) were harvested separately, weighed, washed well with distilled water and frozen immediately. After 24 hours frozen leaves were homogenized in 0.2 M sodium acetate buffer, pH 5.2 containing 0.1% β mercaptoethanol. The homogenates thus obtained were squeezed through two fold of muslin cloth and the solutions in each case were centrifuged at 10,000g for 15 minutes. The pellets were discarded and supernatant was precipitated with a saturated solution (60 % w/v) of ammonium sulphate. Following overnight of precipitation, the sample was centrifuged at 5,000g for 15 minutes and the pellet obtained was dissolved in minimum volume of 0.02M sodium acetate buffer, pH 5.2 containing 0.01% β mercaptoethanol. The dissolved pellet was cleared by centrifugation at 8,000g for 15 minutes and clear supernatant was collected. The supernatant thus obtained was assayed for VIA activity in the samples against SHRV. The samples were incubated with SHRV inoculum, in equal ratio for 4 hours and then assayed for antiviral efficacy on the leaves of hypersensitive host.
Production of Virus Inhibitory Agent (VIA)
        Partially purified phytoprotein isolated from the leaves of C. aculeatum was sprayed with the help of a small atomizer on vigorously growing 4-6 young and healthy seedlings of Lagenaria siceraria, Cucumis sativus, and Crotolaria juncea. The leaves of each plant treated 24 hours earlier with phytoprotein from C.aculeatum were harvested separately, weighed, washed and homogenized using standard protein purification protocol, and centrifuged at 10,000g for 15 minutes. Supernatants, in each case, collected separately were screened for their antiviral state and designated as LS-VIA (Lagendaria siceraria -Virus Inhibitory Agent), CS-VIA (Cucumis sativus -Virus Inhibitory Agent) and CJ-VIA (Crotalaria juncea-Virus Inhibitory Agent).
Each of the VIA sample isolated either from of LS- VIA, CS-VIA or CJ-VIA posses an ability of ex vivo inactivation of viruses. The samples of LS- VIA, CS-VIA and CJ-VIA were incubated, with an equal amount of virus inoculum (V/V), for 4 hours at room temperature, then assayed on to the leaves of Cyamopsis tetagaonoloba plants. An equal number of identical plants of same age group, height and vigor, treated with distilled water, served as control. Leaves of all the plants (treated and control), treated 24 hours earlier, either with VIA or distilled water alone were challenge inoculated with Sun hemp rosette virus. Local lesions appeared, on the leaves of treated and control plants, in the form of necrotic spots, four to six days following virus inoculation were counted separately. Biological activity, in terms of percent reduction in local lesions production, by Sun hemp rosette virus (SHRV) challenge inoculation, on the leaves of Cyamopsis tetragonoloba plants was calculated for the degree of the induction of systemic resistance. The percent reduction in local lesion production / percent decrease in virus infectivity was calculated, separately for treated site and remote site (non-treated site) in comparison to control/ untreated plants, by the following formula.
               Where: C=Average number of local lesions produced on control plants and
                T = Average number of local lesions produced on treated plants
The percent reduction corresponds to antiviral activity; hence it is supposed to be due to systemic antiviral resistance induction and concomitant VIA induction as well. Data obtained were analyzed statistically for the significance of results [32].
Results
Resistance Induction by Partially Purified CA Protein
      The partially purified CA phytoprotein, sprayed on to the leaves of Cyanosis tetagaonoloba plants, revealed a tremendous reduction in the number of local lesions produced by SHRV on the leaves of Cyanosis tetagaonoloba plants treated earlier with VIA . The percent reduction in lesion number was drastically reduced by about 92%. The reduction of virus infection to this extent was most probably due to the induction of virus inhibitory agent (VIA) in the test host plants (Plate -1).
Ex-vivo Inactivation of Virus using VIA
      Results presented on the ex-vivo inactivation of virus using VIA from different source hosts and the screening of antiviral efficacy of LS-VIA (Lagenaria siceraria -Virus Inhibitory Agent), CS-VIA (Cucumis sativus -Virus Inhibitory Agent) and CJ-VIA (Crotolaria juncea-Virus Inhibitory Agent) have clearly indicated that each of the VIA sample isolated either from of LSVIA, CS-VIA or CJ-VIA posses an ability of ex- vivo inactivation of viruses. Data, on comparison with DW-SHRV control plants, clearly indicated significant reduction of about 92%, 85% and 76%.
                                                
Effect of partially purified phytoprotein isolated from the leaves of Clerodendrum aculeatum (CA) on the production of Lagenaria siceraria virus inhibitory agent (LS-VIA) in Cyamopsis tetragonoloba
(a) Control distilled water (DW)
(b) LS -VIA (1:1)
(c) LS ? VIA (1:5)
(d) LS ? VIA (1:10)
(e) LS ? VIA (1:20)
Graph 1: Ex-vivo inactivation of SHRV using LS-VIA, CS-VIA and CJ-VIA on Cyamopsis tetragonoloba
                         
Graph 2: Ex-vivo inactivation of SHRV using LS-VIA, CS-VIA and CJ-VIA on Cyamopsis tetragonoloba
                         
Table 1: Ex-vivo inactivation of SHRV using LS-VIA on Cyamopsis tetragonoloba
                      
+ SEM = Standard Error of Mean
LS ? VIA = Lagenaria siceraria -Virus Inhibitory Agent
L1, L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 50ml distilled water [virus inoculum (1:50)]
Table 2: Ex-vivo inactivation of SHRV using LS-VIA on Cyamopsis tetragonoloba
                       
+ SEM = Standard Error of Mean
LS ? VIA = Lagenaria siceraria -Virus Inhibitory Agent
L1, L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 100ml distilled water [virus inoculum (1:100)]
Table 3: Ex-vivo inactivation of SHRV using CS-VIA on Cyamopsis tetragonoloba
                       
+ SEM = Standard Error of Mean
CS-VIA = Cucumis sativus -Virus Inhibitory Agent
L1, L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 50 ml distilled water [virus inoculum (1:50)]
Table 4: Ex-vivo inactivation of SHRV on using CS-VIA on Cyamopsis tetragonoloba
                       
+ SEM = Standard Error of Mean
CS-VIA = Cucumis sativus -Virus Inhibitory Agent
L1, L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 100 ml distilled water [virus inoculum (1:100)]
Table 5: Ex-vivo inactivation of SHRV using CJ-VIA on Cyamopsis tetragonoloba
                       
+ SEM = Standard Error of Mean
CJ-VIA = Crotolaria juncea -Virus Inhibitory Agent L1,
L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 50 ml distilled water [virus inoculum (1:50)]
Table 6: Ex-vivo inactivation of SHRV using CJ-VIA on Cyamopsis tetragonoloba
                       
+ SEM = Standard Error of Mean
CJ-VIA = Crotolaria juncea -Virus Inhibitory Agent L1,
L2, L3 and L4 represent the number of leaves treated
1.0g of SHRV infected leaf tissue in 50 ml distilled water [virus inoculum (1:100)]
Discussion
        Virus Inhibitory agent (VIA) was induced in host plants, following the treatment with phytoprotein, isolated from extract of a non-host plant, endogenously produced in them [33, 34]. Such phytoproteins mediated induced systemic resistance showed partial resemblance with SAR.VIA, which is an inducible gene product likewise AVF, IVR and PR-proteins [35, 36, 37]. VIA induction in host plants following application of endogenously produced plant protein extracts, are capable of exvivo virus inactivation [38, 39, 40]. Efforts were made to screen the production of VIA in different hosts [41, 42]. The sources used for VIA production were bottle gourd (Lagenaria siceraria), cucumber (Cucumis sativus) and sun hemp (Crotolaria juncea) plants [29]. These antiviral proteins may induce the host for the production of virus inhibitory agents. Probably some protein (VIA) diffuses to surrounding tissues and other plant parts [17]. The VIA have been isolated from leaves of plants treated with antiviral agents and they have been shown conclusively to inactivate the viruses in vitro [43, 44, 45, 46, 47]. The release of resistance seems to be an activation of a pre-existing system and hence is easily stimulated. It is probably able to move from one leaf to another through the vascular system of the plant [48, 49, 50, 51].
        Verma and Awasthi [43] reported that the synthesis of VIA is inhibited, if Actinomycin D was applied soon after phytoprotein treatment. The VIA synthesized was neither virus specific nor host specific [1516, 52]. Extracts containing VIA when incubated with the viruses reduced their infectivity [53]. VIAs from a few hosts have been characterized [20]. The VIA synthesized in the leaves of N.glutinosa treated with B.diffusa root extract reduced infectivity of TMV on N.glutinosa, Datura stramonium and D. metel [38]. It was, however, less effective in inhibiting TRSV and GMV on C. amaranticolor. The VIA production was maximum after 24 hours of treatment with the extract. The VIA, synthesized in C. tetragonoloba plants ,following treatment with B. spectabilis leaf extract , was proteinacious in nature and could prevent the infection of tobamoviruses in seven hypersensitive hosts [44].
These experiments resulted in the conclusion that VIA produced in different host plants bottle gourd (Lagenaria siceraria), cucumber (Cucumis sativus), sunnhemp (Crotolaria juncea) and (Guar) Cyamopsis tetragonoloba, can serve as an effective control measures for the management treatment of viral diseases of economically important plants.

References

  1. Verma HN, Varsha, Baranwal VK (1995a) Endogenous virus inhibitors from plants: Their physical and biological properties. In: Antiviral Proteins in Higher Plants CRC Press Boca Raton FI 1-21.
  2. Carr JP, Loebenstein GD (2010) Natural and Engineered Resistance to Plant Viruses. Part B. Advances in Virus Research 76: 269.
  3. Awasthi LP, Chaudhury B, Verma HN (1984) Prevention of plant virus diseases by Boerhaavia diffusa inhibitor. In Jour Trop Plant Dis 2: 41-44.
  4. Pandey AK, Awasthi LP, Pandey VP, et al. (2014) Bio-efficacy of root extract of Boerhaavia diffusa on yellow disease of ginger. AIJRFANS 5: 79-80.
  5. Awasthi LP, Pathak SP, Gautam NC, et al. (1985) Control of virus diseases of vegetable crops by glycoprotein isolated from Boerhaavia diffusa. Ind Jour Plant Pathol 3: 59-63.
  6. Kempiac G, Schuster G, Awasthi LP, et al. (1991) Attempts to reduce damage caused by oat sterile dwarf virus in oats using virazole, 2,4-Dioxohexahydrotriazine,Boerhaavia inhibitor and Alkane-monosuphonate. Acta Phytopath Et Entomol Hungarica 26: 219-226.
  7. Awasthi LP, Verma HN (2006) Boerhaavia diffusa-A wild herb with potent Biological and Antimicrobial properties. Asian Agri History 10: 55-68.
  8. Awasthi LP, Singh Samir Pratap (2015) Clerodendrum-A Novel Herb having Broad Spectrum Antimicrobial Properties Asian Agri History 19: 33-44.
  9. Verma HN, Sivastava A, Gupta RK (1998a) Seasonal variation in systemic resistance inducing basic protein isolated from leaves of Clerodendrum aculeatum. Ind Plant Pathol 16: 9-13.
  10. Verma HN, Baranwal VK, Srivastava S (1998b) Antiviral substances of plant origin. In Plant virus disease control, edited by American Phytopathol. Society (APS) Press St Paul Minnesota 154-162.
  11. Verma HN, Baranwal VK (1999) Antiviral phytoproteins as biocontrol agents for efficient management of plant viruse diseases. In: Biocontrol potential and their exploitation in Crop Pest and Disease Management (Eds RL Rajak and Rajeev K Upathyay) Aditya Book Pvt Ltd New Delhi.
  12. Hansen AJ (1989) Antiviral chemicals for plant disease control. Plant Science. 8: 45-88.
  13. Batelli MG, Stirpe F (1995) Ribosome inactivating proteins from plants. In: Antiviral Proteins in Higher Plants CRC Press USA.
  14. Awasthi LP, Verma HN, Kluge S (2016) A possible mechanism of action for the Inhibition of plant viruses by an antiviral glycoprotein isolated from Boerhaavia diffusa roots. J Virol Antivir Res 5: 1-8.
  15. Verma HN, Awasthi LP (1979a) Prevention of virus infection and multiplication by leaf extract of Euphorbia hirta and the properties of the virus inhibitor. New Botanist 6: 49-59.
  16. Verma HN, Awasthi LP (1979b) Antiviral activity of Boerhaavia diffusa root extract and the physical properties of the virus inhibitor Can J Bot 57: 926-932.
  17. Verma HN, Srivastava S, Varsha, et al. (1996) Induction of systemic resistance in plants against viruses by a basic protein from Clerodendrum aculeatum leaves. Phytopathol 86: 485-492.
  18. Chaubey, AN, Awasthi LP, Singh SP (2014) Eco-friendly management of viral diseases of potato. International Research Journal of Life Sciences 2: 8-12.
  19. Chaubey AN, Mishra RS, Awasthi LP (2017) Ecofriendly management of leaf curl disease of chilli through botanical biopesticides. SF J Virol 1: 1-7.
  20. Awasthi LP, Singh SP, Verma, HN, et al. (2013) Further Studies on the Antiviral Agent(s) Isolated from Host Plants Pre-treated with Boerhaavia diffusa Glycoprotein. Virol Mycol 3: 124.
  21. Gupta RK, Srivastava A, Verma HN (2004) Callus culture and organogenesis in Boerhaavia diffusa: A potent antiviral protein containing plant. Physiol Mol Bio Plant 10: 263.
  22. Srivastava A, Gupta RK, Verma HN (2004) Micro propagation of Clerodendurum aculeatum through adventitious shoot induction and production of consistent amount of virus resistance inducing protein. Indian J Experimental Biol 42: 1200-1207.
  23. Singh S, Awasthi LP (2006) Protection of mungbean and urdbean crops against vector borne Mung bean yellow mosaic virus through botanicals. Indian J Virology 17: 112-113.
  24. Singh AK, Najam A, Hussain MM, et al. (2009) Effect of phytoproteins isolated from roots of Boerhaavia diffusa and leaves of Clerodendrum aculeatum on the activity of Semliki forest Virus in albino mice model. Indian J of Virology 20: 78-82.
  25. Verma HN, Srivastava S, Varsha, Kumar D (1996) Induction of systemic resistance in plants against viruses by a basic protein from Clerodendrum aculeatum leaves. Phytopathol 86: 485-492.
  26. Verma HN, Sivastava A, Gupta RK (1998a) Seasonal variation in systemic resistance inducing basic protein isolated from leaves of Clerodendrum aculeatum. Ind Plant Pathol 16: 9-13.
  27. Verma HN, Baranwal VK, Srivastava S (1998b) Antiviral substances of plant origin. In Plant virus disease control. Kozenzava (American Phytopathol. Society Press St Paul, Minnesota) 154-162.
  28. Verma HN, Baranwal VK (1999) Antiviral phytoproteins as biocontrol agents for efficient management of plant viruse diseases. In: Biocontrol potential and their exploitation in Crop Pest and Disease Management (Eds RL Rajak and Rajeev K Upathyay) Aditya Book Pvt Ltd New Delhi.
  29. Najam A, Awasthi LP, Verma HN, et al. (2017a) Effect of bioenhancers on the antiviral resistance inducing activity of phytoproteins, isolated from roots of Boerhaavia diffusa and leaves of Clerodendrum aculeatum plants. SF J Virol 1: 1-19.
  30. Najam A, Awasthi LP, Verma HN (2017b) Management of viral diseases of crops through phytoproteins isolated from Boerhaavia diffusa and Clerodendrum aculeatum plants alongwith bioenhancers. Virology: Current Research.
  31. Verma HN, Awasthi LP (1979a) Prevention of virus infection and multiplication by leaf extract of Euphorbia hirta and the properties of the virus inhibitor. New Botanist 6: 49-59.
  32. Snedecor GW (1961) Statistical Methods, Allied Pacific Private Limited Bombay.
  33. Awasthi LP (1981) The purification and nature of an antiviral protein from Cuscuta reflexa plants. Arch Virol 70: 215-223.
  34. Awasthi LP (1982) Characteristics and mode of action of a virus inhibitor from Cascuta reflexa plants. Zentralblatt Mikrobiol 137: 509-518.
  35. Awasthi LP, Singh Shyam (2009a) Management of ring spot disease of papaya through plant products. Indian Phytopath 62: 369-375.
  36. Awasthi LP, Singh Shyam (2009b) Response of Papaya (Carica papaya L) Cultivars against Viral Diseases under Field Conditions. Journal of Plant Disease Sciences 4: 72-75.
  37. Sharma NK, Awasthi LP (2017) Molecular characterization of antiviral Proteins, isolated from host Plants, pretreated with antiviral glycoprotein, isolated from roots of Boerhaavia diffusa plants. J Hum Virol Retrovirol 5: 1-5.
  38. Awasthi LP, Mukerjee K (1980) Protection of Potato virus X infection by plant extracts. Biologia Plantarum 22: 205-209.
  39. Awasthi LP, Menzel G (1986) Effect of root extract from Boerhaavia diffusa L.containing an antiviral principle upon plaque formation of RNA Bacteriophages. Zentralblat Mikrobiol 141: 415-419.
  40. Awasthi LP, Singh Shyam, Sharma NK, et al. (2011) Induction of systemic resistance through antiviral agents of plant origin against papaya ring spot virus disease in papaya (Carica papaya L) International Journal of Sustainable Agriculture 3: 54-57.
  41. Awasthi LP, Singh Samir Pratap, Tripathi D (2014a) Eco-friendly Management of the Viral Diseases of Chilli (Capsicum annum L) Research & Review J Agr Sci Tech 3: 11-16.
  42. Awasthi LP, Singh SP, Chaube AN, et al. (2014b) Molecular characterization of potato viruses through RT-PCR and Electron-microscopy. Carib j SciTech 2: 405-410.
  43. Verma, HN, Awasthi LP (1980) Occurrence of a antiviral agent in plants treated with Boerhaavia diffusa inhibitor. Can J Bot 58: 2141-2144.
  44. Verma HN, Khan MMAA, Dwivedi SD (1985) Biological properties of highly antiviral agents present in Pseudoranthemum atropurpureum and Bougainvillea spectabilis extract. Indian J Plant Pathol 3: 13-20.
  45. Verma HN, Varsha Baranwal VK (1995a) Endogenous virus inhibitors from plants: Their physical and biological properties. In: Antiviral Proteins in Higher Plants M Chessin D De Borde and A Zipf Eds CRC Press BocaRaton FI 1-21.
  46. Verma HN, Varsha Baranwal VK (1995b) Agricultural role in endogenous antiviral substance of plant origin. In: Antiviral Proteins in Higher Plants M Chessin D De Borde and A Zipf Eds CRC Press BocaRaton FI 23-37.
  47. Verma HN, Srivastava S, Varsha Kumar D (1996) Induction of systemic resistance in plants against viruses by a basic protein from Clerodendrum aculeatum leaves. Phytopathol 86: 485-492.
  48. Kumar P, Awasthi LP (2007) Management of mosaic disease in bottle gourd through botanicals Indian J Virology 18: 83-88.
  49. Kumar Pardeep, Awasthi LP (2009) Prevention of Infection and Spread of Viral Diseases in Cucumber (Cucvmis Sativs L.) Through Botanicals. Journal of Plant Disease Sciences 4: 25-32.
  50. Singh SK, Awasthi LP, Singh S, et al. (2011) Protection of mungbean and urdbean crops against vector borne mungbean yellow mosaic virus through botanicals Curr Bot 2: 08-11.
  51. Singh AK, Ahmad Najam Pant AB, Awasthi LP Verma, et al. (2012) Modulatory effect of phytoproteins isolated from roots of Boerhaavia diffusa and leaves of Clerodendrum aculeatum on neoplastic growth of human breast cancer cell line. Open Access Scientific Reports 1: 1-6.
  52. Verma HN, Awasthi LP, Mukerjee K (1979c) Induction of systemic resistance by antiviral plant extracts in non-hypersensitive hosts. Zeitschrift Pflanzenk. Pflanzenschutz 86: 735-740.
  53. Yadav CP, Awasthi LP (2009) Response of tomato cultivars against tomato leaf curl visus (TLCV) under natural field conditions. International Journal of Plant Protection 2: 234-236.

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