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Chapter Title Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases Copyright Year 2016 Copyright Holder Springer India Corresponding Author Family Name Awasthi Q1 Particle Given Name L.P. Suffix Division Department of Plant Pathology Organization/University N.D. University of Agriculture and Technology Street Kumarganj City Faizabad Postcode 224229 Country India Email [email protected] Author Family Name Singh Particle Given Name S.P. Suffix Author Family Name Verma Particle Given Name H.N. Suffix Division Organization/University Jaipur National University City Jaipur Country India Abstract Plants, animals, and other microorganisms are provided, in their genetic makeup, with a certain range of antimicrobial compounds. With respect to viruses, a few plants show resistance to their infection. This resistance, in many cases, has been associated with the protective chemicals within the plant cells which are known for their antifungal or antimicrobial property and reported to be proteinaceous in nature. Many higher plants have developed a variety of defense systems to combat pathogen attack which is essential for their survival. Some of these plants possess endogenous proteins that act as virus inhibitors. They are generally basic proteins with molecular weight ranging from 24 to 32 kDa and effective against a wide range of plant viruses. The viral inhibitors are well studied in Phytolacca americana, Dianthus caryophyllus, Mirabilis jalapa, Bougainvillea spectabilis, and Celosia cristata. These viral inhibitors are most effective when mixed with the virus inoculum or when they are applied one day before or shortly after mechanical inoculation.

AUTHOR QUERIES

Q1 Please confirm the author affiliation. 1 Role of Defensive Antiviral 2 Proteins from Higher Plants 12 3 in the Management of Viral 4 Diseases

[AU1]5 L.P. Awasthi, S.P. Singh, and H.N. Verma

6 12.1 Introduction applied one day before or shortly after mechani- 28 cal inoculation. 29 [AU2]7 Plants, animals, and other microorganisms are 8 provided, in their genetic makeup, with a certain 9 range of antimicrobial compounds. With respect 12.2 History 30 10 to viruses, a few plants show resistance to their 11 infection. This resistance, in many cases, has Duggar and Armstrong (1925) reported for the 31 12 been associated with the protective chemicals first time that the crude sap extract of Pokeweed 32 13 within the plant cells which are known for their (Phytolacca decandra L.) markedly inhibited the 33 14 antifungal or antimicrobial property and reported infectivity of tobacco mosaic virus (TMV). 34 15 to be proteinaceous in nature. Many higher plants Kuntz and Walker (1947) made first attempt to 35 16 have developed a variety of defense systems to investigate the nature and property of the spinach 36 17 combat pathogen attack which is essential for extract. A variety of plants belonging to different 37 18 their survival. Some of these plants possess taxonomic families were subsequently used for 38 19 endogenous proteins that act as virus inhibitors. viral disease management. Loebenstein and Ross 39 20 They are generally basic proteins with molecular (1963) demonstrated formation of virus interfer- 40 21 weight ranging from 24 to 32 kDa and effective ing substances in sap extracted from resistant 41 22 against a wide range of plant viruses. The viral apical uninoculated halves of Datura leaves, 42 23 inhibitors are well studied in Phytolacca ameri- whose basal halves had been inoculated ten days 43 24 cana, Dianthus caryophyllus, Mirabilis jalapa, earlier with TMV. The sap from resistant halves 44 25 Bougainvillea spectabilis, and Celosia cristata. of leaves when mixed with virus reduced the 45 26 These viral inhibitors are most effective when infectivity of TMV, as compared to control sap. 46 27 mixed with the virus inoculum or when they are Verma et al. (1979a, b, c) and Verma and Awasthi 47 (1979a, b, c) conducted experiments with antivi- 48 ral substance of plant origin and found consider- 49 L.P. Awasthi (*) able reduction in infection of viruses. Awasthi 50 Department of Plant Pathology, N. D. University of and Mukherjee (1980) found protection of potato 51 Agriculture and Technology, virus infection by extract from some medicinal 52 Kumarganj, Faizabad 224229, India e-mail: [email protected] plants. The control of viral diseases of some 53 cucurbitaceous crops was also reported by the 54 S.P. Singh same group (Verma et al. 1980). Awasthi et al. 55 H.N. Verma (1984) observed that pre-inoculation sprays of 56 Jaipur National University, Jaipur, India Boerhaavia diffusa root extract were effective 57

© Springer India 2016 L.P. Awasthi (ed.), Recent Advances in the Diagnosis and Management of Plant Diseases, DOI 10.1007/978-81-322-2571-3_12 L.P. Awasthi et al.

58 against tobacco mosaic virus in tobacco and diffusa. Surendran et al. (1999) observed the anti- 107 59 tomato, cucumber mosaic virus in cucumber, viral activity of plant extracts (Azadirachta 108 60 Cucumber green mottle mosaic virus in melon, indica, Clerodendrum infortunatum, Ocimum 109 61 sunn hemp rosette virus in Crotalaria juncea, and sanctum, and Vitex negundo) against Brinjal 110 62 Gomphrena globosa. Verma et al. (1985) sug- mosaic virus on local lesion host Datura stramo- 111 63 gested possible control of natural infection of nium. The pre-inoculation sprays of 10 % leaf 112 64 Mung bean yellow mosaic virus (MYMV) in extract or oil formulations of A. indica were 113 65 mung bean and urdbean by plant extracts. found effective in reducing the virus infection 114 66 Zaidi et al. (1988) reported inhibitory effect of under field conditions. Singh 2002( ) and Singh 115 67 neem extract (A. indica) against Spinach mosaic and Awasthi (2002) reported that aqueous root 116 68 virus in Chenopodium amaranticolor. Verma extract of B. diffusa effectively reduced mung 117 69 et al. (1994) observed the efficacy of leaf extracts bean yellow mosaic and bean common mosaic 118 70 of different species of Clerodendrum, when virus disease in mung bean and urdbean along 119 71 applied to leaves of several hypersensitive hosts. with increased grain yield in field conditions. 120 72 The aqueous leaf extract prevented the infection Later, Awasthi and Kumar (2003a, b), Kumar and 121 73 of viruses by increasing the resistance of the host Awasthi (2003a, b) revealed that weekly sprays 122 74 plants towards subsequent virus infection. Verma of aqueous root extract of B. diffusa significantly 123 75 and Varsha (1995) used Clerodendrum aculea- prevented infection, multiplication, and spread of 124 76 tum alone and with certain proteinaceous modi- Cucumber mosaic virus, Bottle gourd mosaic 125 77 fiers (CA-M) against sunn hemp rosette virus virus, Cucumber green mottle mosaic virus, and 126 78 (SHRV) in Crotalaria juncea and observed that Pumpkin mosaic virus in cucurbitaceous crops. 127 79 in CA-M (with papain) sprayed plants, disease Kumar and Awasthi (2008) were able to prevent 128 80 incidence was much lower when treated plants infection and spread of cucumber mosaic disease 129 81 were challenged with SHRV 6 days after the in cucumber through plant proteins. Singh and 130 82 treatment. Verma et al. (1996) purified a non-­ Awasthi (2009) tested various medicinal plants 131 83 phytotoxic systemic resistance inducer from C. for the management of yellow mosaic disease of 132 84 aculeatum leaves. A water-soluble basic protein mung bean (Vigna radiata) Yadav et al. (2009). 133 85 of mol. wt. 34 kDA present in Clerodendrum Awasthi and Yadav (2009) worked on the man- 134 86 aculeatum (Ca-SRI) when applied prior to virus agement of viral diseases of tomato by seed treat- 135 87 inoculation reduced more than 90 % of local ment and foliar sprays of Boerhaavia diffusa root 136 88 lesions in N. ghtinosa by TMV. extract and Clerodendrum aculeatum leaf extract. 137 89 Bharathi (1999) reported that extract of 90 Mirabilis jalapa completely inhibited Cucumber 91 mosaic virus in brinjal (Solanum melongena L.), 12.3 Virus Inhibitors and Their 138 92 while the inhibition of CMV by the plant extract Characteristics 139 93 of Prosopis chinensis, Bougainvillea spectabilis, [AU3] 94 and Eucalyptus citriodora was 83 %, 75 %, and Antiviral resistance-inducing proteins act on any 140 95 58 %, respectively. In pre-inoculation treatments step of virus synthesis, i.e., from the uncoating of 141 96 with M. jalapa, the percent infection of CMV on viral proteins to the appearance of symptoms. 142 97 brinjal ranged from 0 to 56 % (Awasthi and Rizvi Proteins inhibit virus infection or multiplication 143 98 1999). They also found that infection of Tomato when applied before or after virus multiplication. 144 99 yellow leaf curl virus, a vector-borne virus, was Virus inhibitory property of virus inhibitors 145 100 checked significantly by the application ofB. dif- depends on their concentration and time of appli- 146 101 fusa root extract. Jayashree et al. (1999) studied cation. Functions of proteins are also affected by 147 102 the efficacy of 10 plant extracts against Pumpkin temperature and pH. For example, virus inhibitor 148 103 yellow vein mosaic virus in pumpkin and in Boerhaavia diffusa roots was inactivated at 149 104 observed maximum inhibition of virus transmis- 95 °C and pH 4 but not at pH 10 (Verma and 150 105 sion by insect vector Bemisia tabaci by Awasthi 1979c). 151 106 Bougainvillea spectabilis extract followed by B. 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

152 Types of virus inhibitors: On the basis of mode an infectious preparation of TMV can be recov- 196 153 of action, the virus inhibitors may be grouped ered after centrifugation at 59, 000 g for 60 min. 197 154 into two types: Crude AVF preparation retained the activity for 198 several months when stored at 4–10 °C, and for 199 155 (A) Inhibitors of virus infection several days at room temperature. It was further 200 156 (B) Inhibitors of virus multiplication suggested that AVF acts as an antimetabolite to 201 the biosynthesis of virus nucleic acid, or it blocks 202 some sites essential for virus multiplication. 203 [AU5] Verma et al. (1979a, b, c) and Verma and 204 157 12.4 Inhibitors of Virus Infection Awasthi (1979a, b, c) conducted experiments 205 with antivirus substance of plant origin and found 206 158 Occurrence of highly potent inhibitors of virus considerable reduction in infection by the viruses. 207 159 infection has been reported from different plants. Later on, Awasthi and Mukherjee (1980) found 208 160 A number of reviews have adequately listed vari- protection of potato virus infection by extract 209 161 ous plants showing virus inhibitory activity from some medicinal plants. The control of viral 210 162 (Bawden 1954; Ragetli 1975; Verma 1982; diseases of some cucurbitaceous crops was also 211 163 Awasthi and Singh 2009). Duggar and Armstrong reported by the same group (Verma et al. 1980). 212 [AU6]164 (1925) observed that when the extract from poke- Awasthi et al. (1984) observed that pre-­ 213 165 weed was mixed with infective sap of TMV, there inoculation sprays of Boerhaavia diffusa root 214 166 was complete inhibition of the virus. They also extract were effective against wide range of 215 167 found that extracts of Datura stramonium and viruses in different susceptible hosts. Verma et al. 216 168 Pelargonium sp. also inhibited viral infectivity (1985) suggested possible control of natural 217 169 when mixed with the virus. Kuntz and Walker infection of Mung bean yellow mosaic virus 218 170 (1947) made first attempt to investigate the nature (MYMV) in mung bean and urdbean by plant 219 171 and property of the spinach extract. A variety of extracts. The infection, on these crops, by 220 172 plants belonging to different taxonomic families MYMV was suppressed by aqueous, partially 221 173 were used for viral disease management. clarified leaf extract of Clerodendrum fragrans, 222 174 Inhibitors present in extracts of a particular plant Aerva sanguinolenta, and root extract of B. dif- 223 175 species are effective only when host species were fusa. The treatments were administered as foliar 224 176 inoculated with virus along with inhibitors. sprays after 3–4 days from the seedling stage. 225 [AU4]177 Loebenstein and Ross(1963) demonstrated the The extract from C. fragrans reduced the virus 226 178 formation of virus interfering substance(s) in sap infection, delayed the appearance of disease 227 179 extracted from resistant apical uninoculated symptoms, and promoted flowering and conse- 228 180 halves of Datura leaves, whose basal halves had quent fruiting. The treatment also increased the 229 181 been inoculated ten days earlier with TMV. The nodulation and yield. 230 182 sap from resistant halves of leaves when mixed Prevention of Oat sterile dwarf virus infection 231 183 with virus reduced the infectivity of TMV, as and suppression of disease symptoms were 232 184 compared to control sap. Subsequently, observed by some phytochemicals (Awasthi et al. 233 185 Loebenstein and Ross (1963) studied the charac- 1989). Verma and Verma (1993) revealed that 234 186 teristics of the induced interfering substances or leaf extract of C. aculeatum along with soil 235 187 agents from resistant leaves of Datura that inter- amendment with dry leaf powder showed two-­ 236 188 fered with infection by TMV. The agent was a fold increase in nodulation and grain yield with 237 189 protein, with a molecular weight considerably 50 % reduction in disease incidence caused by 238 190 less than those characteristic of viruses. The Mung bean yellow mosaic virus. Verma and 239 191 inhibitory activity was lost on heating at 78 °C Singh (1994) reported that C. aculeatum may be 240 192 for 50 min and by aging for 5 days at 3 °C. The a possible prophylactic agent against natural viral 241 193 agent was non-dialyzable and partially sedi- infection in mung bean plants. The plants grown 242 194 mented at 93,000 g. It did not inactivate virus in pots and kept in the field were protected against 243 195 in vitro, since from a mixture of interfering agent, natural viral infection by spraying with leaf 244 L.P. Awasthi et al.

245 extract of C. aculeatum, together with soil Singh (2002) and Singh and Awasthi (2002) [AU11]293 246 amendment with dry leaf powder or fresh extract. reported that aqueous root extract of B. diffusa 294 247 Unsprayed plants showed severe disease symp- effectively reduced mung bean yellow mosaic 295 248 toms, while treated plants showed only mild and bean common mosaic virus disease in mung 296 249 symptoms. Soil treatment with dry leaf pow- bean and urdbean along with increased grain 297 250 der + sprays with fresh leaf extract were effective yield in field conditions. Later, Awasthi and 298 251 in increasing the yield as well as in reducing dis- Kumar (2003a, b), Kumar and Awasthi (2003a, 299 252 ease incidence and severity. b) revealed that weekly sprays of aqueous root 300 253 Verma et al. (1994) observed the efficacy of extract of B. diffusa significantly prevented infec- 301[AU7] 254 leaf extracts of different species of Clerodendrum, tion, multiplication, and spread of Cucumber 302 255 when applied to leaves of several hypersensitive mosaic virus, Bottle gourd mosaic virus, 303 256 hosts. The extracts prevented the infection of Cucumber green mottle mosaic virus, and 304 257 viruses by increasing the resistance of the host Pumpkin mosaic virus in cucurbitaceous crops. 305 258 plants. The numbers of local lesions produced on Singh et al. (2004a, b), Singh and Awasthi [AU12]306 259 treated leaves were much lower as compared to (2004) and Singh et al. (2005) reported the pre- [AU13]307 260 untreated leaves. The decrease in lesion number by vention of yellow mosaic disease of mung bean 308 261 different species of Clerodendrum was variable. and urdbean by clarified aqueous root extract of 309 262 Verma and Varsha (1995) used Clerodendrum B. diffusa. Six sprays of B. diffusa root extract 310[AU8] 263 aculeatum alone and with certain proteinaceous (10 %) reduced 80–90 % disease incidence and 311 264 modifiers (CA-M) against sunn hemp rosette increased nodulation, plant height, primary and 312 265 virus (SHRV) in Crotalaria juncea and observed secondary branches, pod formation, and grain 313 266 that in CA-M (modifies – papain) sprayed plants, yield. Awasthi and Singh (2006) reported that the 314 267 disease incidence was very low when treated most effective treatment was seed treatment with 315 268 plants were challenged with SHRV 6 days after B. diffusa root extract + three foliar sprays, which 316 269 the treatment. Verma et al. (1996) purified a non-­ exhibited 70 % reduction in disease incidence. 317 270 phytotoxic systemic resistance inducer from C. Inhibitory effect of the extract of A. indica was 318 271 aculeatum leaves. The purified basic protein reported against Spinach mosaic virus in 319 272 (CA-SRIP) having a molecular weight of 34 kDa Chenopodium amaranticolor (Zaidi et al. 1988). 320 273 completely prevented virus infection in N. gluti- Spraying with neem leaf extract on upper surface 321 274 nosa, when sprayed prior to virus inoculation. of the test plant was effective up to 4 h and the 322 275 The prevention of Tomato yellow leaf curl efficacy decreased gradually with increase in 323[AU9] 276 vector-borne virus was checked significantly by time interval between treatment and inoculation 324 277 the application of B. diffusa root extract (Awasthi (Sangar and Dhingra 1982). Aqueous neem 325 278 and Rizvi 1999). Jayashree et al. (1999) studied extract was more active when mixed with virus 326 279 the efficacy of 10 plant extracts against Pumpkin inoculum of Spinach mosaic virus. An aqueous 327 280 yellow vein mosaic virus in pumpkin and showed extract of neem leaf also inhibited ring mosaic of 328 281 maximum inhibition of virus transmission by pea caused by tomato spotted wilt virus (TSWV) 329 282 Bemisia tabaci, by Bougainvillea spectabilis under laboratory conditions (Ganapathy and 330 283 extract followed by B. diffusa. Narayanaswamy 1990). Singh et al. (1988) 331 284 Surendran et al. (1999) observed antiviral reported that leaf and bark extract of neem inhib- 332[AU10] 285 activity of plant extracts (Azadirachta indica, ited the infection of C. amaranticolor by Cowpea 333 286 Clerodendrum infortunatum, Ocimum sanctum, mosaic comovirus. Louis Vimi and Balakrishan 334 287 and Vitex negundo) against Brinjal mosaic virus (1995) reported that five medicinal plants, viz., 335 288 on local lesion host Datura stramonium. The pre-­ Basella alba, Glycyrrhiza glabra, Phyllanthus 336 289 inoculation sprays of 10 % leaf extract or oil for- fraternus, Plumbago rosea, and Thespesia popul- 337 290 mulations of A. indica were found effective in nea, decreased Pumpkin mosaic virus infection in 338 291 reducing the number of local lesions and also in systemic hosts. 339 292 preventing virus infection under field conditions. Bharathi (1999) reported that extract of 340[AU14] Mirabilis jalapa completely inhibited Cucumber 341 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

342 mosaic virus in brinjal (Solanum melongena L.) 12.5 Purification of Virus 388 343 while the inhibition of CMV by the plant extract Inhibitors Resistance 389 344 of Prosopis chilensis, Bougainvillea spectabilis, Inducers from Plants 390 345 and Eucalyptus citriodora was 83 %, 75 %, and 346 58 %, respectively. In pre-inoculation treat- Purification of antiviral agents from different 391 347 ments with M. jalapa the percent infection of plants involved different purification techniques, 392 348 CMV on brinjal ranged from 0 to 56 % as com- depending on the nature of the compound present 393 349 pared to control. in the crude extract of plants. For purification of 394 [AU15]350 Kumar et al. (1997) reported that the leaf antiviral substances (mostly polysaccharides, pro- 395 351 extract of Clerodendrum aculeatum significantly teins, or glycoproteins), the protocol adopted by 396 352 reduced infection of a mosaic disease in various workers varied, however. In general, fresh 397 353 Amorphophallus campanulatus. Efforts were or dried leaves/roots from outdoor plants have 398 354 made by Singh and Awasthi (2008) to manage been used. The leaves/roots were ground in suit- 399 355 ring spot disease of papaya through antiviral able buffer; the juice was expressed through 400 356 agents of plant origin along with milk protein. cheese cloth and then centrifuged at low speed 401 357 Similarly, Kumar and Awasthi (2008) were able (5000–7000 rpm for 15–30 min). The supernatant 402 358 to prevent infection and spread of cucumber fluid was clarified either by high-speed centrifu- 403 359 mosaic disease in cucumber through plant pro- gation, heat treatment, or organic solvents. 404 [AU16]360 teins. Recently, Singh and Awasthi (2009) tested Afterwards, polysaccharide inhibitors were pre- 405 361 various medicinal plants for the management of cipitated with ethanol and proteinaceous or glyco- 406 362 yellow mosaic disease of mung bean (Vigna radi- proteinaceous inhibitors with different saturations 407 363 ata). Yadav et al. (2009) and Awasthi and Yadav of ammonium sulfate (40–100 %). The precipi- 408 364 (2009) worked on the management of viral dis- tate was dissolved in low ionic strength buffer and 409 365 eases of tomato by seed treatment and foliar dialyzed. Subsequently, the solution was passed 410 366 sprays of Boerhaavia diffusa root extract and through a DEAE-cellulose column or Sephadex 411 367 Clerodendrum aculeatum leaf extract. Awasthi G-25 column to remove pigmented material. 412 368 and Singh (2008) reported a possible mechanism First inhibitor purified and characterized was 413 369 of action for the inhibition of the plant viruses by from carnation plant by Ragetli and Weintraub 414 370 an antiviral glycoprotein isolated from B. diffusa (1962). The scheme for purification of the inhibitor 415 371 roots. Baranwal et al. (2002) purified antiviral involved steps like low-speed centrifugation, dialy- 416 372 protein from Celosia cristata. The protein inhib- sis through semipermeable membrane, DEAE treat- 417 373 ited the lesion formation by TMV, sunn hemp ment, and exclusion chromatography over Sephadex 418 374 rosette virus, and Potato virus X (PVX) in a few G-75 column. The DEAE treatment completely 419 375 hypersensitive hosts. eliminated all RNase activity. The characteristic 420 376 A large number of plants have been tested for features of the inhibitor from carnation were: 421 377 their antiviral activity using different host-virus 378 combinations. The investigations have revealed • The inhibitor was proteinaceous and con- 422 379 that they were not identical in chemical compo- tained 16 amino acids. 423 380 sition and behavior. Marked fluctuations of the • Its molecular weight was 14, 000 Da. 424 381 inhibitor contained in many plants occurred dur- • The protein showed positive charges up to 425 382 ing different seasons and various stages of plant pH 7.8. 426 383 growth. Although substances that interfere with • At a concentration of 0.6 μg inhibitor/ml, 427 384 the virus infection have been reported to occur 100 % inhibition of TMV was observed. 428 385 in several plants, even so, only a few of the • The inhibitor was inactivated at a temperature 429 386 inhibitory substances have been isolated and of 80 °C. 430 387 characterized. Verma and Awasthi (1979a, b, c) isolated a 431 strong and highly potent inhibitor of virus from 432 L.P. Awasthi et al.

433 roots of B. diffusa. The partial purification by before and after virus inoculation. It was also 478 434 organic solvent, protein precipitants and believed that the inhibition took place as a result 479 435 Sephadex gel filtration, revealed that the inhibitor of competition between virus and inhibitor. 480 436 was a glycoprotein and had molecular weight of Presumably, infection in such cases was pre- 481 437 16–18 kDa. The purified preparation contained vented either by blocking entrance of the virus to 482 438 70–80 % protein and carbohydrate. a susceptible region of the host plant or tying up 483 439 Leaf extract from Capsicum applied to the some constituents within cell required for virus 484 440 under surface of bean (Phaseolus vulgaris) leaves multiplication. 485 441 inhibited alfalfa mosaic virus (AMV) infection on Francki (1964) stated that loss of infectivity of 486 442 the upper surface. Inhibitors from plant extract do Cucumber mosaic virus (CMV) on exposure to 487 443 not irreversibly inactivate viruses, because the orig- cucumber leaf extracts could be due to the aggre- 488 444 inal virus regains its infectivity when the mixture is gation of virus particles and the formation of a 489 445 diluted or ultracentrifuged (Fischer and Nienhaus complex between some host materials and virus 490 446 1973). Baranwal et al. (2002) used ammonium sul- particles, thus preventing infection. Infection of 491 447 fate to sediment Celosia cristata antiviral protein Gomphrena globosa with Potato virus X (PVX) 492 448 (25 kD) (CCP25). The sediment was dissolved in was inhibited by leaf extract from all potato vari- 493 449 buffer, dialyzed and subjected to DEAE-cellulose eties that are tolerant, hypersensitive, or immune 494 450 column chromatography. Infectivity of the viruses to the virus. There was no indication that the 495 451 was completely lost by leaf extract of Pelargonium inhibitors from different resistant types of potato 496 452 hortorum, Chenopodium album, and C. amaranti- differed in their effectiveness (Mooker and Kim [AU17]497 453 color. The Pelargonium juice was resistant to heat- 1962). Therefore, degree of host resistance has 498 454 ing at 100 °C for 10 min. no direct relationship to the inhibitory capacity of 499 the extract. Inhibitors introduced within host tis- 500 sues probably produce some stimulatory effect, 501 455 12.6 Characteristics of Virus which translocates through cells to the upper epi- 502 456 Inhibitors Resistance dermis. The chemical nature of some of the virus 503 457 Inducers inhibitors present in healthy plants has been elu- 504 cidated. The well-known inhibitor from poke- 505 458 Kassanis and Kleczkowski (1948) for the first weed (Phytolacca americana) is a basic protein 506 459 time purified virus inhibitors from pokeweed, consisting of about 116 amino acid residues and 507 460 Phytolacca americana (esculenta). It was found possessing a molecular weight of 13, 000 Da 508 461 to contain 8–12 % carbohydrate and 14–15 % (Wyatt and Shepherd 1969). McKeen (1956) and 509 462 nitrogen. They suggested that the inhibitor was Rao and Raychaudhuri (1965) attempted to 510 463 probably a glycoprotein. It was basic in nature investigate the nature of inhibitors present in the 511 464 and combined reversibly with purified TMV, like extracts of cucumber, tobacco, and Datura. They 512 465 pancreatic ribonuclease – another inhibitor of suggested that the inhibitory substances in the 513 466 plant viruses. Since carbohydrate was constantly extracts were proteinaceous in nature. 514 467 associated with the protein, the inhibitor was pre- Ragetli (1957) working on a potent inhibitor 515 468 sumed to be a glycoprotein. However, Benda from carnation (D. caryophyllus) made a detailed 516 469 (1956) later on found two types of substances in attempt to study the mode of action of the inhibi- 517 470 the sap of New Zealand spinach (Tetragonia tor. The inhibitor was effective when applied to 518 471 expansa). A relatively stable protein was an leaf surface simultaneously with virus or prior to 519 472 inhibitor and the other a soluble oxalate salt was it. When it was administered after virus inocula- 520 473 an augmenter and increased the number of local tion, marked interference with the infection pro- 521 474 lesions. Subsequently, it was found that inhibi- cess was not observed beyond 15–30 min. Virus 522 475 tors from a several other plants also had no effect and inhibitors could be separated easily by cen- 523 476 on viruses but their action was on the host plants. trifugation in vitro. Thus, it was concluded that 524 477 This was shown by applying the inhibitory sap the inhibitor acted at the time, the virus came in 525 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

526 contact with the plant, presumably by binding the many species retained their full inhibitory action 571 527 receptor site and preventing the infection. after storage for several weeks at or near 4 °C. 572 4. Effect of chemicals: The various inhibitors 573 present in the crude extracts of plants were 574 528 12.7 Physical Properties mostly insoluble in organic solvents such as 575 529 of the Virus Inhibitory Plant petroleum ether, chloroform, benzene, and 576 530 Proteins diethyl ether. In several plants, the inhibitors 577 contained in crude extract could be precipi- 578 tated with 90–95 % ethanol with only slight 579 531 1. Dilution: The inhibitory property of the plant loss in activity, whereas after precipitation 580 532 extracts was reduced greatly by dilution. Ten-­ with 10 % TCA activity was generally lost. 581 533 fold dilutions of plant extracts with distilled 5. Effect of dialysis: Mostly, the inhibitors con- 582 534 water in most of the cases removed the inhibi- tained in crude extracts of plants were non-­ 583 535 tion or decreased it remarkably. The juice from dialyzable, indicating thereby that they had a 584 536 Dianthus barbatus, D. caryophyllus, Boerhaavia molecular weight of more than 10000 Da. This 585 537 diffusa, etc. appears to be very powerful, since is in sharp contrast to most antifungal and anti- 586 538 their action is still apparent at dilution of 1:2000 microbial substances occurring in plants 587 539 or more (Verma and Awasthi 1979b). whose molecular weights are typically lower. 588 540 2. Effect of heat: The activity of inhibitors pres- 6. Effect of various enzymes: The effect of 589 541 ent in different plants was found to be greatly enzymes such as trypsin, chymotrypsin, 590 542 influenced by heating the crude extract. papain, pronase, and RNase has been tested 591 543 Heating plant extracts for 10 min at 60–70 °C on a few plant extracts. The results following 592 544 removed inhibitory activity partially or com- treatment with enzymes varied with different 593 545 pletely in many cases but was generally less plant extracts. In some cases, activity was 594 546 efficient than dilution. On the basis of thermal abolished by incubation in the presence of 595 547 inactivation of inhibitors present in saps of those enzymes, whereas in other cases it was 596 548 different plants, inhibitors have been classi- not destroyed. Normally, the proteinaceous 597 549 fied into two types: substances were influenced by proteolytic 598 550 (a) Inhibitory activity lost after 10 min at enzymes, whereas glycoproteinaceous sub- 599 551 60–80 °C. For example, Amaranthus, stances remained unaffected. 600 552 Basella, Cuscuta, Datura stramonium, 7. Effect of pH: The stability of inhibitors in 601 553 Hablitzia, Beta sp., etc. crude extract was greatly influenced by 602 554 (b) Inhibitory activity lost after 10 min at pH. Mostly, the inhibitors remained active 603 555 80–100 °C. For example, A. retroflexus, between pH 5 and 7. However, in some cases, 604 556 Chenopodium amaranticolor, C. quinoa, C. such as Acacia arabica, Basella alba, 605 557 album, Atriplex, Pelargonium, Salsola, etc. Clerodendrum aculeatum, Datura metel, and 606 558 Heating had not much effect on the inhibitory Syzygium cumunis inhibitors were stable 607 559 activity of extracts or juices from Amaranthus between pH 4 and 10. The presence of mer- 608 560 mangostanus, Boerhaavia diffusa, captoethanol in the solution helped to increase 609 561 Clerodendrum aculeatum, C. indicum, C. the activity of the inhibitory extracts. The 610 562 phlomoides, C. inerme, etc. activity was considerably decreased, however, 611 563 3. Longevity in vitro: The inhibitors containing after treatment with SDS or 6 M urea. 612 564 preparations of crude sap could be stored at 8. Effect of high-speed centrifugation: Inhibitors 613 565 room temperature for different periods before in plant extracts generally did not sediment on 614 566 losing activity. For most, inhibitory activity was ultracentrifugation up to 40, 000 rpm or 120, 615 567 lost after a week of storage; some extracts 000 g for 2 hours. Activity following ultracen- 616 568 retained activity up to one month. The most sta- trifugation always remained in the supernatant 617 569 ble inhibitors were generally those which pos- and was unaffected biologically by 618 570 sessed high thermal stability. Plant extracts of ultracentrifugation. 619 L.P. Awasthi et al.

620t1.1 Various plants and the nature and characteristics of the inhibitors present in them t1.2 Characteristics of the purified t1.3 Name inhibitors Action References t1.4 Abutilon striatum Polysaccharide Inhibits Abutilon mosaic Flores et al. (1967) t1.5 virus and tobacco virus t1.6 infection, forms an unstable t1.7 complex, and aggregation of t1.8 virus particles was observed t1.9 Boerhaavia diffusa Glycoprotein MW 20,000; Inhibits the infectivity of Verma and Awasthi t1.10 carbohydrate 8–13 %; protein many plant viruses (1979a, b, c) t1.11 70–80 % Induces systemic resistance t1.12 reversible by actinomycin D t1.13 Provokes formation of t1.14 antiviral agent which t1.15 inactivates virus in vitro t1.16 Brassica oleracea Polysaccharide MW 23,000 Alters the susceptibility of Varma (1973) t1.17 host by changing the cell t1.18 wall permeability t1.19 Chenopodium amaranticolor Basic protein MW 29,000 Inhibits infectivity of many Singh et al. (1988) t1.20 plant viruses t1.21 Clerodendrum aculeatum Basic protein MW 32,000; Induces systemic resistance, Verma et al. (1991) t1.22 resistant to proteases reversible by actinomycin t1.23 D. Inhibits infectivity of t1.24 many plant viruses t1.25 Dianthus caryophyllus Glycoprotein; dianthin Induces systemic resistance Ragetli and t1.26 30 MW 29,500; dianthin and inhibits infectivity of 17 Weintraub (1962) t1.27 32 MW 31,700 plant viruses, including t1.28 TMV RNA t1.29 Mirabilis jalapa Basic protein MW 24,200 Inhibits infectivity and Habuka et al. t1.30 MAP has compressed mechanical transmission of (1990) t1.31 structure which confers many plant viruses t1.32 resistance to proteases t1.33 Inhibitory activity of MAP is t1.34 substantially increased (22 t1.35 times) by elimination of t1.36 disulfide bonds with genetic t1.37 engineering t1.38 Phytolacca americana Basic protein MW 29,000; pl Inhibits infectivity of many Wyatt and t1.39 8.1 plant viruses Shepherd (1969) t1.40 PAP Basic protein MW 30,000; pl Like TMV, CMV, WMV, t1.41 8.3. Contains greater and Sugarcane mosaic virus t1.42 proportion of basic amino acid t1.43 residues as compared to PAP t1.44 PAP-11 Contains higher concentration Ribosome-inactivating t1.45 of tyrosine protein (RIP) t1.46 PAP-11 Does not cross react with t1.47 anti-Pap antibodies t1.48 PAP-s t1.49 Spinacia oleracea Basic protein MW 29, 000; pl Inhibits infectivity of many Kuntz and Walker t1.50 10.3. Serologically related to plant viruses (1947) t1.51 inhibitory proteins occurring t1.52 in Phytolacca dianthus and t1.53 Chenopodium amaranticolor t1.54 Yucca recurvifolia Basic protein MW 23, 000; pl Inhibits infectivity of many Okuyama et al. t1.55 9.4. Exhibits amino acid plant viruses (1978) 621t1.56 composition similar to PAP 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

12.8 Detailed Studies on Virus roots have been widely used for the treatment of 622665 Inhibitors from dyspepsia, jaundice, enlargement of spleen, 623666 abdominal pain, abdominal tumors, and cancers 667 12.8.1 Boerhaavia diffusa (Kirtikar and Basu 1956). 624668

The plant was named in honor of Herman As Medicine in the Ayurvedic System The 625669 Boerhaave, a famous Dutch physician of the roots and leaves with flowers have been found to 626670 eighteenth century (Chopra 1969). Boerhaavia, a be highly potent in Ayurvedic medicine; different 627671 herbaceous plant, belongs to the Nyctaginaceae parts of this plant were reported to have various 628672 (four o’clock) family. Order Thymilae, group medicinal properties (CSIR 1988). 629673 [AU18] Dicotyledons, and phylum Angiosperms (Rendle 630 1925). Six species are found in India: B. diffusa, Pharmacological and Clinical Properties 631674 B. chinensis, B. erecta, B. repens, B. rependa, Pharmacological studies have demonstrated that 632675 and B. rubicunda (Chopra 1969; CSIR 1988). Punarnava possesses punarnavoside, which 633676 The whole plant or its specific parts (leaves, exhibits a wide range of properties – diuretic, anti- 634677 stem, and roots) are known to have medicinal inflammatory, antifibrinolytic, anticonvulsant, 635678 properties and have a long history of use by antibacterial, anti-stress agent, antihepatotoxic, 636679 indigenous and tribal people in India. It has many antiasthmatic, antiscabies, and anti-urethritis. 637680 ethnobotanical uses (the leaves are used as vege- 638 table; the root juice is used to cure asthma, uri- Antiviral Activity of Boerhaavia diffusa The 639681 nary disorders, leucorrhea, rheumatism, and roots of B. diffusa are a rich source of a basic pro- 640682 encephalitis) and is medicinally used in the tradi- tein, which is used for inducing systemic resis- 641683 tional Ayurvedic system. Besides, B. diffusa tance in many susceptible crops against commonly 642684 shows potent antiviral efficacy of this plant occurring viruses (Verma and Awasthi 1979a, b, 643685 against phytopathogenic viruses. Antiviral agent c, 1980; Verma et al. 1979a, b, c; Awasthi et al. 644686 isolated from this plant was found to be a glyco- 1984, 1985, 1989). Maximum antiviral activity, 645687 protein with a molecular weight of 16–20 kDa in each case, was recorded with the aqueous 646688 (Verma and Awasthi 1979a, b, c). extract of dried root powder applied before virus 647689 inoculation. The active principle was purified and 690 12.8.1.1 Chemical Composition isolated (Verma et al. 1979a, b, c). This protein or 648691 of Boerhaavia diffusa antiviral agent was active against tobacco mosaic 649692 The Boerhaavia diffusa plant contains a large virus in Nicotiana glutinosa, Datura metel, 650693 number of compounds such as flavonoids, alka- Chenopodium amaranticolor, and Nicotiana 651694 loids, steroids, triterpenoids, lipids, lignins, car- tabacum (Ky58 White Burley and NP31); sunn 652695 bohydrates, proteins, glycoproteins, punarnavin, hemp rosette virus in Cyamopsis tetragonoloba, 653696 and punaravoside (Agrawal and Dutt 1936; Basu Vigna unguiculata, and Crotalaria juncea; 654697 et al. 1947; Surange and Pendse 1972; Ahmad Gomphrena mosaic virus in Chenopodium ama- 655698 and Hossain 1968; Jain and Khanna 1989). A ranticolor, Vigna unguiculata, and Gomphrena 656699 glycoprotein having a molecular weight of globosa when applied a few hours (2–24 h) before 657700 16 kDa was isolated and studied in detail for its inoculation by the respective inoculum of viruses 658701 biological activity (Mishra and Tiwari 1971; (Verma and Awasthi 1979a, b, c; Awasthi et al. 659702 Verma et al. 1979a, b, c). 1984). The antiviral agent was a basic glycopro- 660703 tein (70–80 % protein and 8–13 % carbohydrates) 704 12.8.1.2 Biological Activity with a molecular weight of 16–20 kDa as deter- 661705 mined by gel filtration chromatography (Verma 706 As Medicine in the Traditional System B. dif- et al. 1979a, b, c). After application of systemic 662707 fusa plants have been widely used by indigenous resistance-inducing protein, the susceptible 663708 tribes in the traditional system of medicine. The healthy hosts produced a virus inhibitory agent 664709 L.P. Awasthi et al.

710 (VIA). The VIA showed the characteristics of basic protein. The glycoprotein occurring in B. 715 711 protein, and upon incubation with the viruses, diffusa roots functions as a signal molecule and is 716 712 reduced their infectivity of both in vitro and of great interest as it has a role in stimulating the 717 713 in vivo. The biophysical characteristics of induced defense systems of plants against viruses (Verma 718 714 VIA were also studied and it was found to be a and Awasthi 1980; Awasthi et al. 1987). 719

720t2.1 Prevention and management of viral diseases of crops in fields by Boerhaavia diffusa inhibitor/resistance inducer t2.2 Disease Increase in t2.3 Virus Crop protection (%) yield (%) Reference t2.4 Potato virus X Potato 68 22 Awasthi and Mukherjee (1980) t2.5 Tomato leaf curl virus Tomato 71 24 Awasthi et al. (1984), Awasthi t2.6 and Rizvi (1999) t2.7 Complex infection of tomato Tomato 75 16 Awasthi et al. (1985) t2.8 Mosaic and Cucumber green Cucumber 62 9 Awasthi et al. (1985) t2.9 mottle mosaic virus t2.10 Tomato mosaic virus Tomato 78 12 Awasthi et al. (1985) t2.11 Brinjal mosaic virus Brinjal 64 9 Awasthi et al. (1985) t2.12 Oat sterile dwarf virus Oats 42 NA Kempiak et al.(1991) t2.13 Tomato yellow mosaic virus Tomato 68 29 Awasthi and Rizvi (1998) t2.14 Bean common mosaic virus Black gram 42 28 Singh and Awasthi (2002)

721t2.15 Bottle gourd mosaic virus Bottle gourd 68 42 Kumar and Awasthi (2003a, b)

722 12.8.2 Clerodendrum aculeatum 12.8.2.1 Phytochemistry 742 Clerodendrum is reported in various indigenous 743 723 The genus Clerodendrum L. [Family Lamiaceae systems of medicine throughout the world for the 744 724 (Verbenaceae)] is very widely distributed in trop- treatment of various diseases. Efforts have been 745 725 ical and subtropical regions of the world and made by various researchers to isolate and iden- 746 726 comprises of small trees, shrubs, and herbs. The tify biologically active principle and other major 747 727 first description of the genus was given by chemical constituents from various species of the 748 728 Linnaeus in 1753, with identification ofC. infor- genus. Research reports on the genus show that [AU19]749 729 tunatum. After a decade later, in 1763, Adanson the major class of chemical constituents present 750 730 changed the Latin name “Clerodendrum” to its are steroids in various Clerodendron species 751 731 Greek form “Clerodendron.” Clerodendrum is a such as C. inerme, C. phlomidis, C. infortuna- 752 732 very large and diverse genus and till now 580 tum, C. paniculatum, C. cyrtophyllum, C. fra- 753 733 species of the genus have been identified and are grans, C. splendens, and C. campbellii (Bolger 754 734 widely distributed in Asia, Australia, Africa, and et al. 1970; Abdul-Alim 1971; Joshi et al. 1979; 755 735 America. Sinha et al. 1980, 1982; Singh and Singhi 1981; 756 736 Some of the major chemical constituents Singh and Prakash 1983; Singh and Singh 1983; 757 737 of Clerodendrum genus: Hispudilin, Pinto and Nes 1985; Akihisa et al. 1989; Atta-Ur- 758 738 −O-ethylclerodendricin, Iridiod diglucoside, Rehman et al. 1997; Goswami et al. 1996; Yang 759 739 Colebrin, Clerodermic acid, Jionoside D, et al. 2000, 2002; Kanchanapoom et al. 2001, 760 740 Uncinatone, Apigenin, Clerostero, Serratagenic 2005; Gao et al. 2003; Pandey et al. 2003; Lee 761 741 acid, and Scutellarin. et al. 2006). 762 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

763t3.1 A few species of genus Clerodendrum and their distribu- antitumor, antidiabetic, antihyperlipidemic, 792 764t3.2 tion in the world larvicidal, and antidiarrheal activities. 793 t3.3 Scientific name Synonym Distribution 4. Antiphytoviral activity. An endogenous agent 794 t3.4 C. inerme Gaertn. India, Sri that occurs in Clerodendrum aculeatum 795 t3.5 Clerodendrum Lanka, leaves induced a very high degree of systemic 796 t3.6 aculeatum Southeast Asian t3.7 Countries resistance (CA-SRI) against virus infection in 797 t3.8 C. phlomoidis C. multiforum India plants when lower leaves were treated with 798 t3.9 Linn. f. Burm Clerodendrum aculeatum leaf extract. The 799 t3.10 C. serratum Spreng India induction of systemic resistance by CA leaf 800 t3.11 C. siphonanthus C. indicum India extract was very fast, was reversed by actino- 801 t3.12 R. Br. (Linn) Kuntze mycin D, and was associated with the devel- 802 t3.13 C. colebrookianum Tropical regions opment of a virus inhibitory agent (VIA) in 803 t3.14 of Asia the extract-treated healthy susceptible plants. 804 t3.15 C. myricoide India The VIA was present both in treated and non-­ 805 t3.16 C. commersonii China 806 t3.17 Spreng treated leaves of plants treated with C. acu- t3.18 C. bungei Steud Japan leatum leaf extract. Such endogenously 807 occurring substances from plants, which can 808 765t3.19 C. glabrum E. Mey Southern Africa function as signal molecules, are of particular 809 interest and deserve greater emphasis, because 810 they are not antiviral themselves but they act 811 766 12.8.2.2 Biological Activity by inducing the hosts to produce VIA(s). 812

767 1. Anti-inflammatory activity – Inflammation is a 768 very complex pathophysiological process 769 involving a variety of biomolecules responsi- 12.8.3 Phytolacca americana 813 770 ble for causing it, such as leucocytes, macro- 771 phages, mast cells, platelets, and lymphocytes American Pokeweed (Phytolacca americana) is 814 772 by releasing eicosanoids and nitric oxide. Pro-­ a large herbaceous perennial plant. It is also 815 773 inflammatory cytokines such as TNF-α and known as American nightshade, cancer jalap, 816 774 IL-1β are also responsible for various inflam- coakum, garget, inkberry, pigeonberry, pocan 817 775 matory conditions. bush, pokeroot, pokeweed, redweed, scoke, red 818 776 2. Antimicrobial activity. Anti-infective com- ink plant, and chui xu shang lu (in Chinese medi- 819 777 pounds from natural resources are of great cine). Broadly distributed in fields and waste 820 778 interest as the existing drugs are getting less places. Phytolacca americana was the first plant 821 779 effective due to increased tolerance of micro- species shown to contain an inhibitor (Duggar 822 780 organisms. Essential oil obtained from leaves and Armstrong 1925). The inhibitor in Phytolacca 823 781 of the plant showed antifungal activity against sap is probably the most potent. 824 782 variety of fungal species such as Alternaria 783 species, Aspergillus species, Cladosporium Chemical Composition The plant has been 825 784 herbarum, Cunnimghamella echinulata, reported to contain triterpenes, saponins, 826 785 Helminthosporium sacchari, Microsporum Phytolaccoside A,B,C,D,E,F,G (esculentoside 827 786 gypseum, Mucor mucedo, Penicillium digita- E), phytolaccagenin, jaligonic acid, esculentic 828 787 tum, and Rhizopus nigricans (Sharma and acid, 3-oxo-30-carbomethoxy-23-norolean-12-en- 829 788 Singh 1979). 28-oic acid, phytolaccagenic acid, oleanolic acid, 830 789 3. Other biological activities of Clerodendrum phytolaccatoxin, canthomicrol, astragalin, pro- 831 790 genus. Other major biological activities tein PAP-R, mitogen (a series of glycoproteins), 832 791 reported for this genus are antihypertensive, caryophyllene. 833 L.P. Awasthi et al.

834 Antiviral Property Phytolacca species are of (leaves and roots) are known to have medicinal 877 835 economic and medicinal interest. The medicinal properties. Mirabilis jalapa contains a ribosome-­ 878 836 value is attributed to the antibacterial, antifungal, inactivating protein (RIP), called Mirabilis anti- 879 837 and antiviral activities. These activities mainly viral protein (MAP); the protein was tested 880 838 depend on the phytochemical constituent charac- against infection by Potato virus X, Potato virus 881 839 teristic of this genus (Abdel-Mogib et al. 2002). Y, Potato leafroll virus, and Potato spindle tuber 882 viroid. Root extracts of M. jalapa sprayed on test 883 840 Anticancer The anticancer effects appear to plants 24 h before virus or viroid inoculation 884 841 work primarily based upon antitumor and anti-­ inhibited infection by almost 100 %, as corrobo- 885 842 inflammatory properties, along with immune rated by infectivity assays and the nucleic acid 886 843 stimulant functions. Anticancer, antileukemic, or spot hybridization test. Antiviral activity of MAP 887 844 antitumor constituents include: ascorbic acid, extracts was observed against mechanically 888 845 astragalin, beta-carotene, caryophyllene, isoquer- transmitted viruses but not against aphid trans- 889 846 citrin, oleanolic acid, riboflavin, tannin, and mitted viruses. Purified MAP showed the same 890 847 thiamine. antiviral effect as the crude extract. MAP was 891 purified to homogeneity and was found to be 892 848 Anti-Inflammatory Constituents include sapo- lysine rich and basic (pI 9.8), with a molecular 893 849 nins, alpha-spinasterol, ascorbic acid, calcium weight close to 24.2 kDa. Purified MAP has been 894 850 oxalate, caryophyllene, isoquercitrin, jialigonic shown to inhibit the mechanical transmission of 895 851 acid, and oleanolic acid in the roots and berries. tomato mosaic virus (TMV) in tobacco, tomato, 896 and pepper plants and cucumber green mottle 897 852 Action Crude extract acts as an antiphytoviral mosaic virus in cucumber plants. Moreover, 898 853 agent, against different plant viruses like tobacco MAP was also shown to inhibit protein synthesis 899 854 necrosis virus (TNV), tobacco mosaic virus in Escherichia coli as well as in eukaryotes and to 900 855 (TMV), and tomato spotted wilt virus (TSWV). possess repellent properties against aphids and 901 856 When it was applied onto Phaseolus vulgaris, white flies. Kataoka et al. showed that MAP was 902 857 Datura stramonium, and Chenopodium amaran- compartmentalized in M. jalapa vacuoles, 903 858 ticolor as pre-inoculation spray (in vivo), it sequestering its ribosome-inactivating activity 904 859 reduced the infectivity of above viruses up to away from its own ribosomes. 905 860 90 %. However, when the extract was mixed with 861 the virus inoculum (in vitro), it inhibited the local Inhibitory Activity of M. jalapa Extracts 906 862 lesion development by 100 % after one hour of against PVX and PVY M. jalapa root extracts 907 863 mixing with TNV, and three hours for both TMV were applied to the leaves of G. globosa, an indi- 908 864 and TSWV (Allam et al. 1979; Verma and cator plant which reacts hypersensitively to 909 865 Baranwal 1983; Barakat 1988; Hansen 1989; PVXCP infection. Results show that the root and 910 866 Takanami et al. 1990; Othman et al. 1991; Meyer leaf extracts diluted 1:5 (vol/vol) in sterile water 911 867 et al. 1995; Yordanova et al. 1996; El–Dougdoug were strongly inhibitory to PVX infection, 912 868 1997; Shoman 2002). because almost 100 % inhibition was observed. 913 The inhibitory activity of MAP was not affected 914 by dilution even extracts diluted with tap water 915 869 12.8.4 Mirabilis jalapa gave an inhibitory effect. Similar effects were 916 found by using leaf or root tissues. Purified MAP 917 870 Mirabilis jalapa belongs to family Nyctaginaceae. showed high antiviral activity. 918 871 The whole plant or its specific parts (leaves and 872 roots) are known to have medicinal properties. M. 873 jalapa hails from tropical South America, but has 12.8.5 Tagetes minuta L. 919 874 become natural throughout tropical and warm 875 temperate regions and in cooler temperate Tagetes minuta also known as Mexican marigold, 920 876 regions. The whole plant or its specific parts mint marigold, wild marigold, or stinking roger. 921[AU20] 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

922 The volatile oils of plants have been recognized 12.8.6 Bougainvillea spectabilis 969 923 since antiquity to possess biological activity and 924 a number of plant fractions and pure isolates have Bougainvillea spectabilis belongs to family 970 925 been mentioned as containing substances which Nyctaginaceae; it also contain an endogenous 971 926 interfere with or inhibit infection of viruses. virus inhibitor which confers resistance to 972 927 Tagetes minuta oil and its components act as Tospovirus, tobacco mosaic virus (TMV), cucum- 973 928 potent antiviral agent. ber mosaic virus (CMV), and cowpea aphid-­ 974 borne mosaic virus (CAMV) in their respective 975 929 Compounds Present in Tagetes minuta Z-β-­ susceptible hosts. The viral inhibitor in 976 930 ocimene and dihydrotagetone present in Tagetes Bougainvillea spectabilis is very potent, stable, 977 931 minuta oil have been found to inhibit carnation and is 28 kDa basic protein (BAP). The partial 978 932 ring spot (CaRSV) and carnation vein mottle cDNA encoding the Bougainvillea antiviral pro- 979 933 viruses (CaVMV) (Matthews 1991). The freshly tein was synthesized from the leaf of 980 934 distilled Tagetes minuta oil contains ocimene 55 % Bougainvillea spectabilis, cloned, and sequenced. 981 935 and dihydrotagetone 33 % (Singh et al. 1992). The Homology with other antiviral proteins was 982 936 whole oil of Tagetes minuta and its pure compo- studied. 983 937 nents, i.e., ocimene and dihydrotagetone were 938 tested individually with virus cultures of CaVMV Sequence Homology Homology search was 984 [AU21]939 and CaRSV on Chenopodium amaranticolor. performed using NCBI BLAST program. The 985 putative region was chosen among the three open 986 940 12.8.5.1 Screening of Antiviral Activity reading frames for further homology studies. 987 941 Activity of volatile oils was tested against carna- Alignment of the peptide sequences with known 988 942 tion ringspot and carnation vein mottle virus in antiviral/ribosome-inactivating protein sequences 989 943 different dilutions. Most of the tests were per- revealed weak homology of BAP-cDNA 990 944 formed by using 0.5 % and 2.5 % concentration sequence with the reported AVP/RIP sequences, 991 945 of essential oils as beyond this concentration viz., Mirabilis antiviral protein, Pokeweed antivi- 992 946 phytotoxic effect appeared on Chenopodium ral protein, and Clerodendrum aculeatum 993 947 amaranticolor leaves, at higher concentrations. AVP. The results of the sequence homology anal- 994 948 The 0.5 and 2.5 % concentration of essential oils ysis infer that the cDNA may be specific to 995 949 was mixed with crude sap containing each virus Bougainvillea spectabilis. 996 950 and incubated at room temperature for 24 h. After 951 incubation, sap containing virus was inoculated 952 individually on bioassay host Chenopodium 12.8.7 Dianthus caryophyllus L. 997 953 amaranticolor after adding Celite (as abrasive) to (carnation) 998 954 monitor the inhibitory effect. 955 Tagetes minuta plant grows wild in the hilly Dianthin 30 and dianthin 32, two proteins iso- 999 956 areas like Himachal Pradesh, Jammu and lated from the leaves of Dianthus caryophyllus 1000 957 Kashmir, Uttar Pradesh, and North Eastern States (carnation), were purified to homogeneity by 1001 958 of India and cultivated as commercial Tagetes oil chromatography on nitrocellulose. The molecu- 1002 959 crop, hence easily available in bulk quality. The lar weight of dianthin 30 is 29,500 and that of 1003 960 oil and pure isolates are natural products and dianthin 32 is 31,700. Both dianthins are glyco- 1004 961 hence no threat to environment. Application of proteins containing mannose. 1005 962 oil and pure isolates ensure quick and efficient 963 recovery from viral infections. It also helps in the Antiviral Activity Tobacco mosaic virus was 1006 964 plant virus management. Since Tagetes crop mixed with the substances to be tested or with an 1007 965 grows wild and can be distilled in rich pockets/ equal volume of water as a control. Inoculum, 1008 966 places with prototype distillation unit, hence the containing 600 grit Carborundum as an abrasive, 1009 967 oil will be a cheap, eco-friendly, and easily was rubbed on to leaves of the local lesion 1010 968 ­available antiviral natural product. host Nicotiana glutinosa in a glasshouse at 1011 L.P. Awasthi et al.

1012 20–240C. Each treatment was replicated 10 times ferent location on the phenolic ring. Although, 1056 1013 and randomized on whole leaves of the test among the essential oil constituents, phenolic 1057 1014 plants. Lesions were counted after 3 days of compounds with hydroxyl groups were previ- 1058 1015 infection. Dianthin 30 and dianthin 32, mixed ously described as antimicrobial agents and 1059 1016 with tobacco mosaic virus before infection, pre- antiphytoviral agents. When the oil was applied 1060 1017 vented local lesions in the leaves of Nicotiana onto N. glutinosa plants as a pre-inoculation 1061 1018 glutinosa by more than 50 % at concentrations of spray, the number of local lesions was signifi- 1062 1019 0.5 and 1 ug/ml, respectively (Stevens et al. cantly inhibited. Crude extract and the essential 1063 1020 1981). Both dianthins markedly decreased the oil of Plectranthus tenuiflorus also showed inhib- 1064 1021 production of lesions by tobacco mosaic virus, itory effect against tobacco necrosis virus, 1065 1022 and this presumably account for the antiviral tobacco mosaic virus, and tomato spotted wilt 1066 1023 properties of carnation leaf extract (Van Kammen virus. Monoterpenes were responsible for the 1067 1024 et al. 1961; Ragetli and Weintraub 1962) and antiviral activity of the oil and may show syner- 1068 1025 have been compared with interferon (Fantes and gism in their antiviral effect. When the oil was 1069 1026 O’Neill 1964). applied on local hosts simultaneously with the 1070 infecting virus, the number of local lesions was 1071 reduced by TMV infection and CMV infection. 1072 1027 12.8.8 Satureja montana L. ssp. When applied individually, thymol and carvacrol 1073 1028 Variegate reduced the number of local lesions on both 1074 CMV- and TMV-infected plants of Chenopodium 1075 1029 It belongs to the Lamiaceae and is a very impor- amaranticolor. 1076 1030 tant source of essential oils and other biologically 1031 active molecules. Essential oils are variable mix- 1032 tures, principally of terpenoids and specifically of 12.9 Conclusions 1077 1033 monoterpenes and sesquiterpenes, although 1034 diterpenes may also be present. Monoterpenes It has been demonstrated that interferon-like 1078 1035 are detected in every essential oil comprising native inhibitors of plant virus infection occur in 1079 1036 from as little as 1 % to more than 95 % of the oil a few plants, growing wild in nature or grown for 1080 1037 and are usually present as main constituents in oil ornamental purposes, which prevented virus 1081 1038 fractions of Satureja plants. They play an impor- infection in healthy susceptible hosts prior to 1082 1039 tant role in the resistance against diseases and virus infection. The endogenous virus inhibitors 1083 1040 insects. Essential oils and their components having strong antiviral property lack virus speci- 1084 1041 exhibit antiviral, antimycotic, antioxygenic, anti- ficity and had an association with DNA-­ 1085 1042 parasitic, and insecticidal properties. The phenol dependent protein synthesis. They are pH and 1086 1043 components with hydroxyl groups were found to heat stable, like interferon found in vertebrate 1087 1044 posses the major antimicrobial activity. system. The endogenous virus inhibitors them- 1088 1045 Carvacrols had anti-inflammatory activity and selves have no direct effect on the virus. Their 1089 1046 limonenes showed antiviral activity. treatment on plants results in the production of 1090 the actual virus inhibitory substances like PR 1091 1047 12.8.8.1 S. montana Essential Oil proteins which later on circulate in the whole 1092 1048 and Its Major Components plant system to cause systemic resistance against 1093 1049 Thymol and carvacrol affected the development viruses. 1094 1050 of local lesions caused by tobacco mosaic virus Possibilities of using biological proteins in the 1095 1051 and cucumber mosaic virus. Both phenolic treatment of plant virus diseases under field dis- 1096 1052 ­compounds are biologically active – thymol has eases are undergoing serious evaluations. 1097 1053 antiseptic and carvacrol possesses antifungal Although the present work may not be of great 1098 1054 properties. Thymol and carvacrol are structurally commercial importance just now, its achievement 1099 1055 very similar, having the hydroxyl group at a dif- itself is vital. The knowledge gained will spawn 1100 12 Role of Defensive Antiviral Proteins from Higher Plants in the Management of Viral Diseases

1101 more effective virus disease control methods. Awasthi LP, Kumar P (2003b) Protection of some cucur- 1153 bitaceous crops against natural infection of virus 1154 1102 The intention has been to combine the features of through Boerhaavia diffusa. Ind Phytopathol 56:317 1155 1103 inducer yielding plants as well as other biological Awasthi LP, Mukherjee K (1980) Protection of potato 1156 1104 agents with the virus protective agricultural virus X infection by plant extracts. Biol Plant 1157 1105 plants. The use of natural resources from plant 22:205–206 1158 Awasthi LP, Rizvi SMA (1998) Prevention of infection by 1159 1106 species in the treatment of plant viral diseases has a vector borne virus of Tomato by Boerhaavia diffusa 1160 1107 not been extensively explored and may provide glycoprotein. In: National conference on integrated 1161 1108 some new information about antiphytoviral activ- pest management. 23–35, February, 1998. Sultan 1162 1109 ity of plants. Qaboos University. 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1330 Pinto WJ, Nes WR (1985) 24β-ethylsterols, n-alkanes Singh S, Awasthi LP, Khan MN (2005) Management of 1389 1331 and n-alkanols of Clerodendrum splendens. yellow mosaic disease of mungbean and urdbean 1390 1332 Phytochemistry 24:1095–1097 through aqueous root extract of Boerhaavia diffusa. 1391 1333 Ragetli HWJ (1957) Behavior and nature of a virus inhibi- New Bot XXXII:55–62 1392 1334 tor occurring in D. caryophyllus. Tijdschr Planteziekten Singh S, Awasthi LP, Verma HN (2004b) Prevention and 1393 1335 63:245–344 control of yellow mosaic disease of mungbean by 1394 1336 Ragetli HWJ (1975) The mode of action of natural plant application of aqueous root extract of Boerhaavia dif- 1395 1337 virus inhibitors. Curr Adv Plant Sci 19:321–334 fusa. 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Campridge University Press, T (1982) Acteoside from the flowers ofClerodendron 1405 1347 London infortunatum. Indian J Chem 22B:97–98 1406 1348 Sangar RBS, Dhingra MK (1982) Potato virus inhibitor Sinha NK, Pandey VB, Shah AH, Dasgupta B (1980) 1407 1349 from Neem leaf extract. J Indian Potato Assoc Chemical constituents of the flowers ofClerodendron 1408 1350 9:143–149 infortunatum. Indian J Pharm Sci 42:21 1409 1351 Sharma SK, Singh VP (1979) The antifungal activity of Stevens WA, Spurdon C, Onyon LJ, Stirpe F (1981) Effect 1410 1352 some essential oils. Indian Drug Pharm Ind 14:3–6 of inhibitors of protein synthesis from plants on 1411 1353 Shoman SA (2002) Role of salicylic acid in plant resis- tobacco mosaic virus infection. Experientia 37:28–29 1412 1354 tance to tobacco necrosis and tobacco mosaic viruses Surange SR, Pendse GS (1972) Pharmacognostic study of 1413 1355 infection. Az J Microbiol 58:178–191 roots of Boerhaavia diffusa Willd. (punarnava). J Res 1414 1356 Singh AK, Singh M, Singh AK (1988) Antiviral activity India Med 7:1 1415 1357 and physical proportion of the extract of Azadirachta Surendran M, Shanmugam V, Rajagopalan B, Ramanian 1416 1358 indica. Indian J Virol 4:76–81 N (1999) Efficacy of botanicals on Brinjal mosaic 1417 1359 Singh B, Sood RP, Singh V (1992) Chemical composition virus. Plant Dis Res 14(1):63–66 1418 1360 of Tagetes minuta L. oil from Himachal Pradesh Takanami Y, Kuwata S, Ideda T, Kubo S (1990) 1419 1361 (India). J Ess Oil Res 4:525–526 Purification and characterization of the antiplant viral 1420 1362 Singh P, Singhi CL (1981) Chemical investigation of protein from Mirabilis Jalapa L. Ann Phytopath Soc 1421 1363 Clerodendron fragrans. J Indian Chem Soc Jpn 56:488–494 1422 1364 58:626–627 Van Kammen A, Noordam D, Thung T (1961) The mech- 1423 1365 Singh R, Prakash L (1983) Chemical examination of anism of inhibition of infection with tobacco mosaic 1424 1366 stems of Clerodendron inerme (L) Gaertn. virus by an inhibitor from carnation sap. Virology 1425 1367 (Verbenaceae). Pharmazie 38:565 14:100–108 1426 1368 Singh S (2002) Studies on management of yellow mosaic Varma JP (1973) Isolation and characterization of a virus 1427 1369 disease of mungbean (Vigna mungo (L.) Hepper) inhibitor from cabbage (Brassica oleracea var.wirs- 1428 1370 through botanicals. M.Sc. (Ag.) thesis, N.D. University ing) leaves. Indian Phytopathol 26:713–722 1429 1371 of Agriculture & Technology, Kumarganj, Faizabad Verma A (1988) The economic impact of filamentous 1430 1372 (U.P.), pp. 1–100 plant viruses: the Indian subcontinent. In: Milne RG 1431 1373 Singh S, Awasthi LP (2002) Prevention of infection and (ed) The viruses. Plenum Press, New York, 1432 1374 spread of Bean common mosaic virus disease of pp 371–378 1433 1375 mungbean and urdbean through botanicals. Indian J Verma A, Singh RB (1994) Clerodendrum aculeatum a 1434 1376 Plant Pathol 11(1 & 2):63–65 possible prophylactic agent against natural viral infec- 1435 1377 Singh S, Awasthi LP (2004) Prevention of infection and tion in mungbean. Ann Plant Prot Sci 2(2):60–63 1436 1378 spread of mungbean yellow mosaic virus (MYMV) on Verma A, Verma HN (1993) Management of viral disease 1437 1379 urdbean (Vigna mungo) through Boerhaavia diffusa of mungbean by Clerodendrum leaf extracts. Indian J 1438 1380 root extract. Indian J Plant Pathol 22(1&2):50–55 Plant Pathol 11(1 & 2):63–65 1439 1381 Singh S, Awasthi LP (2008) Management of ring spot dis- Verma HN (1982) Inhibitor of plant viruses from higher 1440 1382 ease of papaya (Carica papaya L.) through antiviral plants. In: Singh BP, Raychoudhury SP (eds) Current 1441 1383 agents of plant origin along with milk protein. Indian J trends in plant virology. Today and Tomorrow’s 1442 1384 Virol 19(1):106–107 Printers and Publishers, New Delhi, pp 151–159 1443 1385 Singh S, Awasthi LP, Verma HN (2004a) Prevention and Verma HN, Awasthi LP (1979a) Prevention of virus infec- 1444 1386 control of yellow mosaic disease of mungbean through tion and multiplication by leaf extract of Euphorbia 1445 1387 aqueous root extract of Boerhaavia diffusa. Indian hirta and the properties of the virus inhibitor. New Bot 1446 1388 Phytopathol 57:303–307 6:49–59 1447 L.P. Awasthi et al.

1448 Verma HN, Awasthi LP (1979b) Further studies on a mosaic Verma HN, Chowdhury B, Rastogi P (1994) Antiviral 1484 1449 virus of Gomphrena globosa. Phytopath Z 95:178–182 activity in leaf extracts of different Clerodendrum spe- 1485 1450 Verma HN, Awasthi LP (1979c) Antiviral activity of cies. Z Pflanzenk Pflanzenschuz 91(1):34–41 1486 1451 Boerhaavia diffusa root extract and the physical prop- Verma HN, Rastogi P, Prasad V, Srivastava A (1985) 1487 1452 erties of the virus inhibitor. Can J Bot 57:926–932 Possible control of natural virus infection on Vigna 1488 1453 Verma HN, Awasthi LP (1980) Occurrence of a highly radiatus and Vigna mungo by plant extracts. Ind J 1489 1454 antiviral agent in plants treated with Boerhaavia dif- Plant Pathol 3:21–24 1490 1455 fusa inhibitor. Can J Bot 58:2141–2144 Verma HN, Srivastava S, Varsha, Kumar D (1996) 1491 1456 Verma HN, Baranwal VK (1983) Antiviral activity and the Induction of systemic resistance in plants against 1492 1457 physical properties of the leaf extract of Chenopodium viruses by a basic protein from Clerodendrum aculea- 1493 1458 ambrosoides L. Proc Indian Acad Sci (Plant Sci) tum leaves. Phytopathology 86:485–492 1494 1459 92:461–465 Verma HN, Varsha, Baranwal VK (1995) Agricultural role 1495 1460 Verma HN, Varsha (1995) Prevention of natural occur- of endogenous antiviral substances of plant origin. In: 1496 1461 rence of tobacco leaf curl disease by primed Chessin M, De Borde D, Zipf A (eds) Antiviral pro- 1497 1462 Clerodendrum aculeatum leaf extracts. In: Verma JP, teins in higher plants. CRC Press, Boca Raton, 1498 1463 Verma A, Kumar D (eds) Detection of plant pathogens pp 23–37 1499 1464 and their management. Angkor Publishers (P) Ltd, Wyatt SD, Shepherd RJ (1969) Isolation and characteriza- 1500 1465 New Delhi, pp 202–206 tion of a virus inhibitor from Phytolacca americana. 1501 1466 Verma HN, Awasthi LP, Mukerjee K (1979a) Prevention Phytopathology 69:1787–1794 1502 1467 of virus infection and multiplication by extracts from Yadav CP, Awasthi LP, Singh S (2009) Management of 1503 1468 medicinal plants. Phytopathol Z 96:71–76 viral diseases of tomato through biopesticides: an eco- 1504 1469 Verma HN, Awasthi LP, Mukerjee K (1979b) Induction of friendly approach. Indian J Virol 20(1):42 1505 1470 systemic resistance by antiviral plant extracts in Yang H, Hou A-J, Mei S-X, Sun H-D, Che C-T (2002) 1506 1471 ­non-­hypersensitive hosts. Zeitschrift Pflanzenk Constituents of Clerodendrum bungei. J Asian Nat 1507 1472 Pflanzenschutz 86:735–740 Prod Res 4:165–169 1508 1473 Verma HN, Awasthi LP, Saxena KC (1979c) Isolation of Yang H, Jiang B, Hou A-J, Lin Z-W, Sun H-D (2000) 1509 1474 the virus inhibitor from the root extract of Boerhaavia Colebroside A, a new diglucoside of fatty acid ester of 1510 1475 diffusa inducing systemic resistance in plants. Can J glycerin from Clerodendrum colebrookianum. J Asian 1511 1476 Bot 57:1214–1217 Nat Prod Res 2:177–185 1512 1477 Verma HN, Varsha, Srivastava S (1991) Antiviral agents Yordanova A, Korparov NE, Stomenova, Starcheva M 1513 1478 from plants for control of viral diseases, Abstracts: (1996) Antiphytoviral activity of 1–morpholinomethyl 1514 1479 international conference on virology in the tropics. tetrahydro 2–Pyrimidinone (DDB). Plant Pathol 1515 1480 Lucknow, India, p 250 45:547–551 1516 1481 Verma HN, Awasthi LP, Kumar V, Chaudhary B, Rastogi Zaidi ZB, Gupta VP, Samad A, Naqvi QA (1988) 1517 1482 P, Duvedi SD (1980) Control of plant virus diseases by Inhibition of spinach mosaic virus by extracts of some 1518 1483 extract from higher plants. J Indian Bot Soc 59:30 medicinal plants. Curr Sci 57(3):151–152 1519 Author Queries Chapter No.: 12 0002561477

Queries Details Required Author’s Response AU1 Please confirm the author affiliation. AU2 Please confirm the identified head level. AU3 Please check if “chinensis” should be changed to “chilensis.” AU4 Please check the sentence “Loebenstein and Ross(1963) demonstrated the formation..... TMV, as compared to control sap” as these seem to be a repetition of the previous sentence.

AU5 Please check the sentence “Verma et al. (1979a, b, c) and Verma and Awasthi (1979a, b, c) conducted ...... also reported by the same group (Verma et al. 1980).” as these seem to be a repetition of the previous sentence.

AU6 Please check the sentence “Awasthi et al. (1984) observed that pre-inoculation...... mung bean and urdbean by plant extracts” as these seem to be a repetition of the previous sentence.

AU7 Please check the sentence “Verma et al. (1994) observed the efficacy ..... resistance of the host plants” as these seem to be a repetition of the previous sentence. AU8 Please check the sentences “Verma and Varsha (1995) used Clerodendrum aculeatum alone..... , when sprayed prior to virus inoculation” as these seem to be a repetition of the previous sentence. AU9 Please check the sentence “The prevention of Tomato yellow leaf curl vector-borne virus...... extract followed by B. diffusa” as these seem to be a repetition of the previous sentence.

AU10 Please check the sentences “Surendran et al. (1999) observed antiviral activity..... preventing virus infection under field conditions” as these seem to be a repetition of the previous sentence. AU11 Please check “Singh (2002) and Singh and Awasthi (2002) reported that aqueous … and Pumpkin mosaic virus in cucurbitaceous crops” as these seem to be a repetition of the previous sentence.

AU12 Please fix “a” or “b” for the references Singh et al. (2004), Verma and Awasthi (1979), Verma et al. (1979), Kumar and Awasthi (2003). AU13 Please check the sentence “Singh et al. (2004a, b), Singh and Awasthi (2004) and..... clarified aqueous root extract of B. diffusa” as these seem to be a repetition of the previous sentence. AU14 Please check the sentence “Bharathi (1999) reported that extract of Mirabilis jalapa.... ranged from 0 to 56 % as compared to control” as these seem to be a repetition of the previous sentence.

AU15 Kumar et al. (2004), Rao and Raychaudhury (1956) have been changed to Kumar et al. (1997), Rao and Raychaudhuri (1965) respectively as per the reference list. Please check if okay. AU16 Please check the sentences “Recently, Singh and Awasthi (2009) tested various medicinal plants...... root extract and Clerodendrum aculeatum leaf extract” as these seem to be a repetition of the previous sentence. AU17 Please provide details of Mooker and Kim (1962), Singh and Singh (1983) in the reference list. AU18 Please check sentence starting “Order Thymilae, group …” for completeness. AU19 Please check if edit to sentence starting “Research reports on…” is okay. AU20 Please check sentence starting “The whole plant…” as this seems to be a repetition of the previous. AU21 Please expand “NCBI BLAST.”

AU22 Please provide in-text citation for the references Batista et al. (1995), Verma (1988), Verma et al. (1995). AU23 Please confirm the inserted volume number for the reference Fantes and O’Neill, (1964). AU24 Please provide page range for the reference Vimi and Balakrishan (1996).

AU25 Please provide article title for the reference Okuyama et al. (1978).