STUDIES ON THE INHIBITION OF TOBACCO MOSAIC VIRUS

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STUDIES ON THE INHIBITION OF TOBACCO MOSAIC VIRUS STUDIES ON THE INHIBITION OF TOBACCO MOSAIC VIRUS

INFECTION BY PLANT JUICES, GROWTH REGULATORS, AND INFECTION BY PLANT JUICES, GROWTH REGULATORS, AND

SYSTEMIC INSECTICIDES SYSTEMIC INSECTICIDES

VICTOR WEERARATNE

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W E E R A R A T N E , V ic to r , 1 9 2 9 - STUDIES ON THE INHIBITION O F TOBACCO MOSAIC VIRUS IN FECTIO N B Y P L A N T JU ICES, GROWTH REGULATO RS, AND SYSTEM IC IN SECTICIDES.

U n iv e r s ity o f N ew H a m p sh ire , P h .D ., 1961 B otan y

University M icrofilm s, Inc., A nn Arbor, M ichigan

Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

STUDIES ON THE INHIBITION OP TOBACCO MOSAIC VIRUS INFECTION BY PLANT JUICES, GROWTH REGULATORS, AND SYSTEMICINSECTICIDES

BYVICTOR WEERARATNE

B, S., University of Ceylon, 19^6

A DISSERTATION Submitted to the University of New Hampshire

In Partial Fulfillment of The Requirements for the Degree of

Doctor of Philosophy

Graduate School Department of Botany

May, 1961


This dissertation has been examined and approved,

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'SUHtX!

/f^/Dare


ACKNOWLEDGMENT

The writer wishes to express his deep gratitude to Dr. Avery E. Rich for his valuable assistance in directing, planning and oritiolzing the investigation and the preparation of the dissertation, to Albion R, Hodgdon for his taxonomic assistance, to Dr. R. A. Kilpatrick for his advice and suggestions in preparation of the dissertation and photographs, to Dr. Mathias G, Richards for his assistance in preparation of the dissertation, to Miss Gall Grégoire for her aid in preparation of the manuscript and to all other members of the Department of Botany, University of New Han^shire for their aid both direct and indirect. The writer wishes to express his deep appreciation to Dr, Theodore G, Metcalf and the other members of the Department of Bacteriology for their advice and permission to use the facilities of the laboratory. The writer is Indebted to Dr. Ceaare Slbilia for his assistance in planning the first phase of the investigation and permission to use the facilities of the Stazion© Fatologia Vegetal©, Rome, Italy, to Dr, Osvaldo Lovisolo for his valuable guidance in the development of the first phase of the investigation and to all other members of the Stazlone Fatologia Vegetal©, Rome, Italy for their aid in experimental work. The writer wishes to thank Dr. Jussepp© lÆiaina of the University of Rome for his taxonomic assistance.


TABLE OP CONTENTSPag©

LIST OF TABLES.................................. illLIST OP ILLUSTRATIONS.......................... v

I. INTRODUCTION............ 1II. REVIEW OF LITERATURE .............. 3III. MATERIALS AND METHODS. .................... 2$

1. Viruses and Their Sources. . . . . . . . . . . 252. Local Lesion Hosts .......................... 263. Sources of Teat Inhibitors . . . . . ......... 26A. Plant Juices . . . . . .................. 26B. Growth Regulators. ..................... 2?C. Systemic Insecticides......................30Ij.. Method of Assaying Virus Inhibitory Activity . 30A. Plant Juices and Growth Regulators . . . . 30B. Systemic Insecticides. .................. 32C. Recording and Analysis of Data.............32IV. PROCEDURE AND RESULTS.............................. 35

1. Test Inhibitors......... ................... 35A. Angiosperms............................... 38B. Gymnosperms. . . . ............ . . . . . 42C. Perns.......................... „. .. . . 43D. Marine Alga© . . . . . . . . . . . . . . . 43E. Growth Regulators. . . . . . . . . . . . . 4?P. Systemic Insecticides. . . . . . . . . . . 492. Properties of Inhibitors in Agropyron repenaand Coconut Meat Extract........... 5lA. Preapplication and Postapplication ofInhibitor to Virus Inoculation . . . . . . 5lB. Effect of Aging of Inhibitor ^ Vitro. , . 61C. Effect of Dilution and the Concentrationof the Inhibitor ..................... 6?D. Effect of Heating of the Inhibitor . . . . 73V. . DISCUSSION AND CONCLUSIONS........................ 79VI. SUMMARY........................................... 91

BIBLIOGRAPHY, . .................... 93

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Table 1,

Table 2.

Table 3.

Table 4*

Table 5*

Table 6«

Table 7.

Table 8.

Table 9.

Table 10.

Table 11.

Table 12.

Table 13.

Table 14»

LIST OP TABLESPage

Number of species of different groups of plants tested for inhibition of virus infection. . . . . . . ...................... 36Angiosperms tested for the inhibition of tobacco mosaic virus . . . .................. 37Families of dicotyledons tested for inhibition of TMV Infection . . . . . . . . . . . . . . . 38Inhibition of TMV Infection by juices of angiosperms excluding Gramineae. . . . . . . . 39Inhibition of TMV Infection by various species of Gramineae..................................44Inhibition of TMV infection by grmnosperms, ferns, and marine algae................. . . 4&Effect of growth regulators on the inhibition of TMV infection............................. 48Inhibition of TMV infection by systemic insecticides applied at various time intervals before virus inoculation............................. 50Effect of time interval between application of Agropyron repens sap and TMV inoculation on TnElDiÊTôh""ôfTGT^ infection............... 54Effect of time interval between TMV inoculation and application of Agropyron repens juice on inhibition of TMV infection......... 55Effect of time interval between application of coconut meat extract and TMV inoculation on inhibition of TMV infection............... 58Effect of time interval between TMV inoculation and application of coconut meat extract on inhibition of TMV infection . . . . 59Effect of aging of juice of Agropyron repens on Inhibition of TMV 63Effect of aging of coconut meat extract on inhibition of TMV................... 65

ill


LIST OF TABLES, CONTINUEDPage

Table 15. Effect of dilution of the juice ofAgropyron repens on inhibition of TMV infection, ................................69Table 16. Effect of dilution of coconut meat extracton the inhibition of TMV infection . . . . . 71Table 17, Effect of heating the juice of Agropyronrepena on the inhibition of TMV...............75Table I8. Effect of heating the coconut meat extracton the inhibition of T M V .............. 77

iv


LIST OP ILLUSTRATIONS

Figure 1,

Figure 2,

Figure 3*

Figure 4»

Figure 5.

Figure 6.

Figure 7.

Figure 8.

Figure 9.

Figure 10,

Pag©The separation of coconut meat extract into three layers by centrifugation at 3500 rpm........................... 29The appearance of local lesions produced on the leaf of Nicotians glutlnoaa 7 days after tobacco mosaic virus inoculation.(The lesions are produced on the controlleft half of the leaf while no lesions are produced on the right half of the leaf treated with an effective inhibitor . . . 34Inhibitory action of Agropyron repens sap on TMV infection when appliedat various intervals before suid after TMV inoculation............... 56Inhibitory action of coconut meat extract on TMV infection when applied at various intervals before and after TMV inoculation......... 60Effect of aging of juice of Agropyron repena on inhibition of TMV . . . . . . . 64Effect of aging of coconut meat extract on inhibition of TMV, . . . . . . . . . . 66Effect of dilution of the juice of Agropyron repena on inhibition of TMV infection................... 70Effect of dilution of coconut meat extract on inhibition of TMV infection........... 72Effect of heating juice of Agropyron repens on inhibition of TMV. . . . . . . . . . . 76Effect of heating of coconut meat extract on inhibition of TMV. . . . . . . . . . . 7 8


SECTION I

INTRODUCTION

The discovery of viruses dates back to the end of the last century. However, some diseases now known to be caused by viruses were known for thousands of years. The Chinese description of pestilence in 10th century B.C. apparently refers to smallpox. Yellow fever, known for centuries in Africa as a scourge of ships, probably was responsible for the legends of cursed ships such as those of the Ancient Mariner and the Plying Dutchman.

Plant viruses such as potato leaf roll can be traced to records hundreds of years back. The ornamentally appreciated color variegation known as tulip break in tulip, caused by a virus, has been a prized condition of the plant since the l6th century, A.D,

Plant viruses can cause systemic infection and persist in the vegetative parts of the plant for long periods. They can withstand unfavorable climatic conditions under which most other pathogens would perish.Once perennial plants are infected by a virus, the infection is indefinitely carried and the progeny of the vegetatively propagated plants are very often found to be infected. Therefore, the losses due to virus diseases are not limited to one season or one generation of plants, but may continue as long as the virus infected plants are


propagated. Hence the economic importance of plant virus diseases can not be underestimated. However the control measures for plant virus diseases are few in number.

Heat therapy is a method of plant virus disease control. The practical application of this method Is limited partly due to the Injurious effect Of heat to the host tissue and partly due to the difficulty of large scale application of it to growing plants without any injurious effect to neighbouring healthy plants.

Chemotherapy has been studied widely. There are reports of virus inhibitory activity in plant extracts, inorganic and organic chemical compounds, and some physical conditions such as electric current and pressure. However, we are still in search of an ideal virus inhibitor,

The purpose of this dissertation is to summarize the work done by others and to report results of a search by the writer for inhibitors of tobacco mosaic virus infection and multiplication in plant extracts, growth regulators and insecticides.


SEOTIOH II

REVIEW OP LITERATURE

New virus diseases are constantly being discovered. However, the known control measures to combat these diseases remain comparatively few. One© a plant has contracted a virus disease, the methods of eradicating the disease are even more limited. Frequently the destruction of the diseased plant is advocated as the only effective control measure.

Heat therapy has been reported by several workers as a method of plant virus control. Kunkel (1935) was the first to report that a plant virus disease can be cured by heat. He inactivated the peach yellows virus by keeping the small trees at 35^0 for two weeks or more. Bawden (1950), discussing the reports of several workers, stated that sugar cane cuttings infected with sereh disease, usually produced healthy plants after immersion in water at 52°C for periods up to one hour. The virus is thus inactivated probably as a result of Its protein being denatured by heat. Viruses such as tobacco mosaic (TMV) and sugar-beet curly top can withstand temperatures as high as 70°C (Smith, 1957). Heat therapy has strict limitations in its application to diseased plants because of the injurious effect of heat to the protein part of the protoplasm of the host cells. Hence a search was made for


other methods of plant virus control.Plant Juices. Allard (1914» 19l8) reported that

sap from the mosaic-infected pokeweed plant, Phytolacca decandra L,, failed to produce the disease in tobacco or other solanaceous plants such as tomato, petunia, Physalls, Datura and Solantun. Also, he failed to produce the disease in healthy pokeweed plants by mechanical inoculation with sap from mosaic-diseased pokeweed plants. He gave no explanation for this observed phenomenon. Clinton (1914) failed to Infect P. deoandra with juice of tobacco plants infected with Calico (the term used by him to describe the mosaic disease of tobacco).

Duggar and Armstrong (1925) tested the juices of pokeweed, jimson weed (Datura Stramonium L. ), geranium (Pelargonium sp.), cotton and squash, with Irish potato tubers, sweet potatoes and apples of the variety Ben Davis. They reported that the juice of pokeweed effectively inactivated the agency of tobacco mosaic in a relatively short time. They stated that although the juice had no adverse effect on the growth of Aspergillus nlger Thom & Raper or Bacterium prodigiosum Lehm & Meum. and so is not "germicidal" in the customary sense, when it is added to TMV healthy tobacco plants are not infected. The juice of D, Stramonium had a similar effect on TMV but was leas effective than that of pokeweed. Kassanis and Kleczkowski (1948) ultimately Isolated the Inhibitor from Fbytolacca esoulenta Van Houtte and identified it as a glycoprotein


containing 14 - 15^ nitrogen and 8 - 12^ carbohydrate.Doolittle and Walker (1925), using aphids as

vectors, were able to transfer cucumber mosaic virus from infected pokeweed to healthy cucumber plants, but they failed to infect the cucumber plants by mechanical inoculation with the juice from diseased pokeweed plants.Grant (1934) reported that although Beta vulgaris E., B, vulgaris var. cicla. and Spinacia oleracea L. were susceptible to TMV, the extracts from healthy plants of these species greatly reduced the InfectIvlty of the virus in juice extracted from infected tobacco plants. The inhibitory property of these juices was found to be greatly reduced by dilution and heat.

Kuntz and Walker (1947) reported that extracts of the leaves of spinach, garden beet, sugar beet, and chard, fidien mixed in equal parts with infectious juice of tobacco containing TMV and the infectious juice of cabbage containing cabbage mosaic virus (Turnip virus I), completely or almost completely inhibited the Infectivlty of those juices. liAen the spinach extract was mixed In equal parts with plant juices containing tobacco ring spot virus, necrotic potato ring spot virus and cucumber mosaic virus, the infeotlvity of those juices was also completely or nearly completely inhibited. The action of spinach extract was Instantaneous and did not Increase with time. The extract retained Its Infeotlvity for at least 15 months at room temperature.


The addition of Juices from healthy leaves of Nicotiana rustlea L. or Hlcotlana Tabacum L, to inoculum prepared from tobacco streak virus-infected N. rustica reduced the number of local lesions produced by the inoculum, according to Diachun (1948). Crushing the leaves of infected plants reduced the number of local lesions as compared with the inoculum from leaves not so thoroughly crushed.

T he juices of Pberidlum aqlllnum (L,) Kuhn and Scolonendrlum vulgare Smith (Pteridophyta), Chenonodiumalbum E,, Ghenopodium Bonus-Henrlcus L., Heuchera micrantha L., Saxifrage crasaifolia L., Astilbe .japonica Morren &Dene«, Artemisia vulgaris L., Sambuaus nigra L., Gonvallaria roàjalis L., and Fragaria veaoa JL. reduced TMV Infection (Manil, 1949a, 1949b).

Bawden and Kleczkowski (1945) found that aqueous extracts from all the parts of strawberry plants except fruits reduced infactivity of TMV. They concluded that specific antisera could not be produced against extracts of virus-infected strawberry plants, possibly because of the properties of the host. They were unable to extract soluble proteins from the leaves, runners or roots. All extracts contained tannin which precipitated serum proteins, The presence of virus inhibitors in raspberry leaves is reported by Cadman (1959), and Bawden and Kleezkowski (1945)® According to Cadman, the Inhibitor is lost on dialysis, precipitated by nicotine or added


protein, unaffected by boiling or freezing, and la reportedas probably a phenolic tannin agent. % e degree to which the tannin substance from raspberry inhibits virus infection depends on the virus and not on the species of the test plant, it combines irreversibly with some viruses but with others the combination is reversible by dilution or increase of pH, He was able to demonstrate the presence of protein in raspberry leaves by removing the tannin with nicotine.He concluded that neither the raspberry tannin nor tannic acid alters the susceptibility of the host plant to virus infection. His conclusion was based on the fact that the virus and the inhibitor combine in a fixed ratio, Thorn- berry (1935)» working with TMV and bean plants (He used Scotia bean plants while Cadman used French bean plants.), reported that tannic acid inhibits infection of TMV when applied to the test plant before the inoculation of the virus, thus showing that tannic acid alters the susceptibility of the host plant. The results are in conflict with the conclusions drawn by 0adman.

Van der Want (1951), reported that sap of healthy carnation leaves was able to inhibit tobacco necrosis virus and rattle virus. Weintraub and Gilpatrick (1952) inoculated four seedling clones of Dianthua barbatus L. with tobacco ring spot virus and found that one clone was not susceptible. They reported that the difficulty of transmitting the virus from D. barbatus to tobacco was caused by an inhibitor in the healthy leaves of D. barbatus.


8

The inhibitor had no effect in transmitting the virus toD. barbatus but in cucumber. Datura Stramonium. and snapdragon, it caused either resistance to infection or delay of symptom expression. The inhibtor also reduced the infactivity of TMV, cucumber mosaic and tobacco etch virus.Ho definite conclusions were reached as to whether the inhibitor acted on the host or the virus.

The expressed juice from Capsicum frutescens L. contains a substance that markedly reduced local lesions and primary lesions by certain viruses on cowpea, Ghenopodium hybridum L., and to a lesser extent on H. Tabacum and Physalls peruviana L. (McKeen, 1956), McKeen also stated that the decrease in number of lesions by pepper juice is independent of the virus concerned and depends only on the species to which the inoculation is made. Pepper juice inhibits the production of local lesions when it is applied to virus Inoculum or to the upper or lower leaf surface before Inoculation; but if applied to the leaf surface a few minutes after inoculation, the local lesion production is not appreciably decreased. The application of the inhibitor at some distance from the point of virus inoculation also inhibited lesions formation. The inhibitive component of pepper juice was thermolablle; it resisted aging and drying ^ vitro, and did not pass through a cellopheme membrane, thus indicating that it is of protein nature.The protein component of the pepper juice, precipitated either by 95^ ethanol or by ammonium sulphate, was inhlb-


itively active but less so than the raw juice.Hooker and Kim (1959) demonstrated by local lesion

infection of Gomphrena globosa E. the presence of inhibitors of potato virus X (PVX) in leaves of potato varieties. The infection was unimpaired when leaves were first inoculated and the leaf extract was placed on the surface 1 minute after inoculation. Mien the Inhibitor was spread on the surface of the leaves before inoculation infection was reduced to 35^ of the control. However, they postulated that the inhibitor affects the virus more directly than it does the host plant. The application of the inhibitor prior to virus inoculation reduced infection considerably and hence the writer believes that their results indicate that the inhibitor affects the host plant more than the virus.

Blaszczak ejb al, (1959) reported that juices from 26 species or varieties of plants inhibited infection of brown spot strain of potato X (PVX). The Infectivlty was wholly suppressed by juices from Pelargonium hortorum Ballev; G. album, C. amarantlcolor L. and two varieties of C. frutesoens.--Inhibition was distinct with juices of D. Stramonium. D. metel L., Solanum Integrlfollum Poir,8. tuberosum L., Spinacia oleracea L., Phaseolus vulgaris L., Trifolium pratense L. and Vicia faba L. Tenfold dilution of juices with distilled water in most cases removed the Inhibition or decreased It markedly. The moderate dilution of the juices of D. metel, G. amarantlcolor and


10

Gapalciun frutesoens increased the infectivlty of each teat juice. Juice of P. hortorum did not lose its inhibitory activity when heated 10 minutes at 100°G. The inhibition was still complete when the juice of P. hortorum was sprayed on assay plants and inoculated 20 days later. The juices of £. album, S. oleracea and 0. frutescens sprayed on the leaves were still inhibitory when Inoculation was delayed 6-7 days. Juices of many species retained their inhibitory action after storage for several weeks.

8111 (1951), and Sill and Walker (1952) have shown that parts of the cucumber plant except the corolla contain a stable inhibitor that interferes with the infectionof cowpea by cucumber mosaic virus. Inhibitory activitywas evident 20 days after extraction. On dilution the

-2 “ 3inhibitor lost its potency between 10 and 10 . Amongother cucurbits tested watermelon was the only one having a comparable inhibitor. Johnson (194D has reported that extracts of citrus and tomato fruit inactivated TMV.

Inhibition of TIfV by extracts of rice is reported by Jones e^ al. (1959). The inhibitor was reported to reduce Infection idien the virus was inoculated 1-3 days after application of the inhibitor. The inhibitor in rice was considered to be protein in nature.

Fungi and Bacteria. The search for virus inhibitorshas not been limited to higher plants. Several workers have tested fungi and bacteria for possible virus Inhibitors.In search for an answer to the observed fact that when TMV


11

la added to the soil its infactivity is lost, Mulvania (1926) incubated the virus with cultures of bacteria and found that some species make TMV noninfectious Wiereas others do not.

Johnson and Hoggan (1937) Incubated many different fungi and bacteria and found that almost all fungi and some bacteria decreased the infectivity of the viruses.These results were interpreted as an indication that microorganisms destroyed the virus. However, this interpretation was amended by Johnson (1938» 1941 ) 1^ light of the results he obtained with Aspergillus niger and Aerobacter aerogenes (Kruse) Beijerinck. These organisms produced some material which when added to TMV solution, immediately decreased infectivity.

Yeast is reported to inhibit TMV infection by Takahashi (1942, 1946)• He stated that the inhibitor is hydrolysed by boiling with S% HCl and yields 85^ of its weight as reducing sugars measured as glucose.

Gupta and Price (1950) tested filtrates of 49 species of fungi grown on cornraeal-beef-dextrose broth and found that 40 species decreased the infection of the southern bean mosaic, tobacco mosaic and tobacco necrosis viruses. Growth products of Triohothecium roseum Link and Neurosnora sitophila Shear & Dodge reduced Infection by more than 90^. The filtrate of T. roseum was effective in inhibiting infection when applied to host plant leaves before or up to 30 minutes after inoculation with the virus. The substance


12

responsible for virus inhibition in T. roseum was thought to be made up of large non-proteinaceoua particles because it did not diffuse throu^ cellophane and it withstood boiling. Bawden and Freeman (1952) proved that there were 2 heat-stable inhibitors in T. roseum, one tricothecin, an antifungal substance, and the other a polysaccharide.

Neutralization of tobacco necrosis virus by certain complexes derived from a fiItrate of Bacillus subtilis Eisenberg culture was reported by Ramon et al. (1948)»

Utech and Johnson (1950) tested substances obtained from 94 species of bacteria and fungi for their ability to inhibit TMV, Basidiomycetes were the most uniform of all the groups tested and all species tested inhibited TMV to a marked degree. Boletinue pictus Pk., Amanita phalloïdes (Bull.) Fr. and Pleurotus ostreatua (Jacq.)Fr. yielded over 90^ inhibition. Of the Ascomycetes tested Morchella sp,, Rhodotorula sp, and Pénicillium splculisporum Lehman, produced marked Inhibition. Most Phycomycetes tested yielded over 90/ Inhibition.

Bobyr (1959b, 1959c) reported inhibitory effects of cultures of filtrates of Aspergillus niger, Pestalozzia truncata Lev, Btachybotrys altarnans Bon., Tricboderma llgnoruBi (Tode) Harz, and T. roseum on TMV. He also reported inhibitory effects of the antibiotics imanine, arenarin, gramicidins, microcide and a new antibiotic, I604, on TMV, The inactivation was partly or completely reversible at relatively low concentrations.


13

The Inhibition of southern bean mosaic virus and TMV by noformicin, an antibiotic, produced by a species of Nocardia is reported by Gray (1955)» The application of noformicin to roots of Pinto been plants growing in sand reduced the number of local lesions produced by the viruses. The virus Infection is reported to be inhibited when the antibiotic is applied to leaves, starting 1 hour or 1 day after inoculation as well as when sprays were applied several days before inoculation.

A complete prevention of local lesion formation by bean mosaic virus and TMV by cytovirin, isolated from a culture filtrate of an unidentified species of Strepto- myces was reported by Gray (1957»» 1957h) and Wittman (1958)» Cytovirin is claimed to be effective in inhibiting virus infection both before and after virus inoculation.

Inorganic and Organic Chemical Compounds. Extensive work has been done in the field of inorganic and organic chemicals in search of virus inhibitors of infection and multiplication, however the space in this dissertation does not permit an exhaustive review of the literature available in this field. It would be opportune to mention some of the important literature available.

Of inorganic compounds that inhibit virus infection mercuric chloride has been extensively studied (Stanley, 1935» Went, 1937» Manil, 1938; Johnson, 1941» Kassanis Kleczkowski, 1944 aod Beat and Samuel, 1936).

Nitric acid is reported to inhibit TMV and southern


14celery mosaic virus (Johnson, 1941» Wellman, 1934)» Iodine is reported to be effective against both plant and animal viruses (Johnson, 1941I Kaiser, 1 9 3 9 ).

Pukishi (1930) and Bobyr (1959a) reported that several alkaloids inhibit virus infection. Bobyr found that 23 of the 61 alkaloids tested inhibited the necrotic reaction caused by TMV. None of them inhibited the necrotic reaction when used 24 hours after infection.Effect of mustard gas on virus inactivation has been stnidied by Harriot (1949) and Pong (1955)* Harriot stnidled the action of mustard gas on animal, plant and bacterial viruses, He reported that viruses containing DMA were inactivated faster than those containing HNA.

Inhibition of TMV and carnation mosaic by zinc sulphate is reported by Rijkov and Smirnova (1949) and Rumley and Thomas (1 9 5 1 ). However, Johnson (1 9 4 D did not obtain any inhibition of TMV with zinc sulphate. The positive results obtained by former research workers suggest that zinc sulphate behaves as an inhibitor of virus increase, because they treated the host already Infected with the virus. Weintraub e^ al. (1952) reported zinc chloride and zinc sulphate inhibited TMV.

Shepherd (1959) reported that there was less virus in boron-deficient plants. However this difference disappeared after a long period of concentration of the virus. He also reported that magnesium deficiency limited virus multiplication.


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Inhibition of TMV and Influenza virus by formaldehyde is reported by several workers (Ross and Stanley,19381 Johnson, 194-1; Lauffer and Wieatley, 1949).

Inhibitory effect of halogens on virus infection has been reported by Johnson (1941) and I^nsen ert al. (1949)»

Inhibitory effect of potassium salicylate on TMV and tomato spotted wilt is reported by Beat (1945)® Several workers have reported the inhibition of both plant and animal viruses by methylene blue (Perdrau and Todd, 1933» Blrkeland, 1934» Samuel, 1936; Best, 1939).

Takahashi (1948) »»d Norris (1953) reported inactivation of TMV and PVX, respectively, by malachite green.

Thiouracil greatly retarded the rate of TMV multiplication according to Conanoner and Mercer (1951» 1952).They demonstrated that the inhibitory effect of thiouracil on virus multiplication could be counteracted by an excess of uracil. Bawden and Kassanis (1954) confirmed that thiouracil can inhibit multiplication of TMV, and that uracil but not adenine, cytosine, or guanine, con counteract the effect of thiouracil. Virus multiplication decreased when tobacco leaves were sprayed daily with solutions of thiouracil, but less so than when leaves were floated in such solutions. Nichols (1953) found that TMV was produced more rapidly and extensively when inoculated tobacco leaves were floated in water and kept in the light than when they were in the dark; by contrast when floated in thiouracil solution more virus was produced in the dark


16

than In the light. The results obtained by Schlegel (1954) by floating discs of tobacco leaves in a solution of thiouracil agreed with the results obtained by Nichols. Bawden and Kassanis (1954) showed that thiouracil did not act specifically against TMV but that it did impede the multiplication in tobacco of potato virus X and Y, henbane mosaic and tobacco necrosis virus.

By floating TMV-infected tobacco leaves in various organic solutions, Schlegel (1954) demonstrated that Merck antibiotic MK 61, diazouracil, L-isoleuolne, L-taurine and benzaldehyde thiosemicarbazone inhibited TMV infection appreciably in the light. In the dark or low light environment, the most effective inhibitors were Merck antibiotic MK 61, diazouracil and alpha-piconilic acid. Employing the same method as Schlegel, Schneider (1953) showed that TMV infection was inhibited by 8-azaguanine, 8-azaadenine, benzimidazole, 6-nitrobenzimidazole, 2,6-diaminopurine, 2-methyltbioadenine and 2-idoadenine, while uric acid and adenine stimulated TMV infection, and 2-6-diamlnopurine delayed symjptom appearance when sprayed on leaves.

The inhibitory effect of urea was demonstrated by Stanley and Lauffer (1939 ) and Bawden and Pirie (1940)-

According to Stanley & Lauffer (1939) inactivation was accompanied by separation of virus into nucleic acid and protein.

The inhibition of TMV by dinitrophenol, aniline, phenylhydrazine, hydrozylamine and rivanol was reported


17

by Ryzhkov et al. (1946). It is concluded, that the action of the inhibitor is to inactivate a specific aldehyde necessary as a substrate for the virus. However, their findings that urea did not inhibit TMV are in conflict with the findings of several other workers. By placing nucleic acid in perforations of potato tubers infected with potato leaf roll vimia, Hippel (1949, 1950) demonstrated that nucleic acid inhibits virus infection.

Caldwell (1933) and Stanley (1934^) were able to prove that trypsin inhibited TMV. It is of interest to note that Stanley concluded that the effect of trypsin on the virus was not due to proteolytic activity of trypsin. He found the proteolytic activity of trypsin was limited to a narrow range of hydrogen ion concentration, yet the inhibitory activity of trypsin on TMV was spread over a wider range of hydrogen ion concentration. He was unable to demonstrate that the virus combines with the inhibitor. Hence he concluded that the inhibitory effect of trypsin was chiefly due to the action of trypsin on the host. On the other hand Stanley (1934^), working with TMV and pepsin, established that the inactivation of virus by pepsin was due to proteolytic action. In contrast to trypsin, he reported that pepsin inactivated the virus only under conditions favorable for proteolytic activity. The rate of inactivation of the virus varied directly with the concentration of the pepsin. Here is one of the earliest hints to the possible existence of 2 kinds of


18

viîTus inhibitors, one that affects the host and the other that affects the virus in such a way that it is completely inactivated by bringing about a chemical or physical change in the virus. The action of pepsin on THV has become a subject of controversy. Some workers claim it has an inhibitory effect, but more claim it has no inhibitory effect on TMV. Ross and Vinson (1937) claimed that after incubating the TMV with pepsin for many days at pH 3.0 and 37°Gf the virus lost its Infectivity. Lojkin and Vinson (1931), Caldwell (1933), Chester (1936), Ekukkm and Pirie (1937), and Eleozkowski (1944) found no effect on either infectivity or serological activity. The failure of lojkin and Vinson to obtain TMV infection is not difficult to explain. Apparently they conducted the experiment at a pH where pepsin is inactive as a proteolytic enzyme. They stated that this lack of action may have been due to unfavorable pH, since the virus without the addition of the enzyme, was inactivated to a considerable extent at the optimum reaction for pepsin. Caldwell carried out his experiment at 2^°C and he incubated the virus - pepsin mixture for only 24 hours. The period of incubation and the temperature at which he carried out the experiments are different from those of Stanley, who obtained positive inhibition. Further, there is no mention at what pH Caldwell conducted his experiment. In view of these discrep- anoies, it is not surprizing that Caldwell did not obtain virus inhibition with pepsin. Chester conducted his exper-


19

iments under conditions similar to those of Stanley,However, it is not difficult to find an explanation for the negative results he obtained. He incubated antiserum- virus precipitate with pepsin. At least part of the pepsin if not all was used in destroying the antibodies in the precipitate. He used nearly the same concentration of pepsin as Stanley in his eêriments, however Stanley incubated virus and pepsin only. It is reasonable to assume that practically all of the pepsin present in the antiserum- virus precipitate was used in destroying antibodies, and very little or none was left to act on the virus released.An additional experiment with pepsin added to the virus released after destruction of the antibodies could have established beyond doubt whether pepsin had any effect on the virus. It is of interest to note that Chester did not obtain complete recovery of the virus in all of the tests which he conducted. In 13 out of 15 tests, the recovery of virus after incubation with pepsin occurred.One of the 2 tests where he failed to recover virus was the only test in which virus was diluted tenfold more than the others. Stanley stated that the rate of inactivation of the virus by pepsin was proportional to the concentration of pepsin. Hence, it is quite conceivable that in the test where the virus was in diluted form, there was sufficient pepsin left over to act on the virus, which was In diluted form. Further, his report that the amount of infective virus recovered from the mixtures


20

after incubation with pepsin was only 9.4^ of the maximum theoretical yield, tends to support the above interpretation. However, no explanation can be given for the negative results obtained by Eleczkowski.

Lojkin and Vinson (1931) reported that erpsin and papain inhibit TMV infection. Loring (1942) showed that weight by weight, ribonuclease was about 100 times more effective than trypsin in decreasing the infectivity of TMV.

lauffer and Robinson (1949) reported that glyceroland aniline decreased infectivity of TMV. Caldwell (1936) reported that casein, albumen and peptone inhibited TMV. Hydrogen in the presence of platinized platinum arrested the normal aerobic inactivation of tomato spotted wilt (Beat, 1939). Stoddard (1942, 1947) reported that quinhydrone completely inactivated the X disease of peach, while 8-hydroxyquinoline sulphate, hydroquinone, p-nitro- phenol. Calcium 8-hydroxyquinolate, urea, sodium thio- sulphate and some of their derivatives inactivated the virus to a lesser degree. The virus content of the sap of tobacco infected with potato X and potato Y viruses is reported to decrease by spraying with 2- methyl - 4 - chlorophenoxyacetic acid and 2-4-dichlorophenoxy acetic acid (2-4”h) (Zd-nusaset et al. » 1948)- However, Nour-Eldin (1953) reported that 2-4-D stimulated TMV infection in the dark, while in the light there was neither stimulation nor inhibition. Ryzhkov and Sukhov (1944) stated that


21 .

ascorbic acid, cysteine and thiamin inhibited TMV.Naphthalene acetic acid at the concentration of

10 decreases the virus concentration approximately to one third of the control in tobacco tissue culture (Kutsky and Rawlins, 1950).

Miscellaneous. The ability of normal rabbit serum to inhibit TMV Infection is reported by several workers. (Mulvania, 1926; Purdy, 1929; Matsumuto, 1930; Silber- schmldt, 1933; Chester, 1934; Spooner and Bawden, 1935; Eassenls, 1943)* Chester (1934) stated that specific neutralizing action of homologous antisera differs quantitatively from the unspecific one of normal sera. He further stated that antisera produce their full effect only if they are incubated with the virus in vitro, whereas normal serum produces its full effect immediately when it is added to a virus. The neutralization of TMV by antisera follows the law of multiple proportion, whereas the neutralization of the normeil serum does not, but a constant amount of the serum decreases the infection by a constant percentage regardless of the concentration of the virus in the Inoculum. He concluded that the nonspecific inhibition occurs because the resistance of the host plant is increased. The specific inhibition occurs because the infectivity of the virus is directly affected. Chester (1936) reported that all protein fractions of normal serum could neutralize infectivity, but that pseudoglobulin was more effective than albumin or euglobulin.


22

Johnson (1941) found that horse blood and horse blood serum Inactivated TMV, He stated that serum inactivation is immediate. The blood of earth worm and fish are reported to inhibit infection of various plant viruses(Smith, 1941).

Johnson (1941)» reported that cow's milk inhibitedvirus infection to a remarkable degree. The inactivation was instantaneous and practically complete on full concentration of virus treated with an equal part of milk.It was effective at dilutions of 1:10 but somewhat lesseffective at 1:100, Pasteurisation, sterilization, or boiling did not destroy the inhibiting property appreciably. Crowley (1958) reported that skim milk inhibited TMV, Skimmed milk and whey were equally as effective as whole milk. Hagborg and Glelack (I960) reported that whey inhibited infection of stripe mosaic of barley. Lucas and Hare (1959) and Hare and Ducas (I960) stated that lactal- bumen, alpha-lactoglobulin and betalactoglobulin were weak inhibitors, whereas casein and crude whey fraction of milk were much stronger inhibitors of TMV. Bovine blood, gamma- globulin, fibrinogen and albumen were weak inhibitors but alpha- globulin and beta- globulin were stronger.

Gendron (1950) stated that coconut milk and extract of copra inhibited the multiplication of TMV and PVX.He concluded that coconut milk and extract of copra affected the host plant. Extracts of insects have been


23

reported as having an inhibitory effect on 6 different viruses (Black, 1939). Saith (194D found that extracts of caterpillars inhibited infection of TMV. He states that there is no relationship between the ability of the Insect to transmit viruses and the occurrence of the inhibitor. Loss of virulence of tomato spotted wilt was greatly accelerated by stirring and bubbling air (Bald and Samuel, 1934)» Tomato aucuba mosaic and ordinary mosaic virus (TMV) in living tissue were Inactivated by passage of direct electric current (Bolas, 1934)»

Inactivation of TMV by high-frequency sound waves is reported by Stanley (1934&)» Pressure of about 130,000 lbs, inactivated TMV (Giddings et al., 1929).

McKinney (1935) found that the virus of common mosaic of tobacco (TMV) is shown to act as an Immunizing agent against yellow mosaic virus, suppressing the development of the latter. Hagaich (1958) reported that a tobacco-mosaic-type virus from tomato, itself noninfectious to Pinto beans, reduced the number of local lesions produced on beans by TMV.

Inactivation of viruses by heat has been reported by several workers. Eunkel (1935) found that keeping diseased peach yellows trees at 35°G for 2 weeks or more leads to recovery of the poach trees. Heat treatment also frees peach trees from phony disease (Hutchins and La Rue, 1939) and yellow-red disease (Hildebrand, 1941)« Potato tubers were freed from leaf-rollby keeping them


24

at 37®C for 10 to 20 days (Kaasànla, 1949).


25

SECTION III

MATERIALS AND METHODS

1. Viruses and Their Sources

The cultures of all viruses used in the experimentswere maintained in systeroically infected host plants ininsect-proof cages in the greenhouse. The tobacco mosaicvirus (TMV), supplied by Osvaldo Lovisolo of Italy, was

a/maintained in Nicotians tabaoum L. cv.-' White Burley. Infected plants were kept in insect-proof cages.

The experiments were conducted throughout the year in the greenhouse maintained at a temperature between 20° and 30®C. lÆienever a source of virus was required for a test, leaves were removed from infected plants the day before inoculation and stored overnight in the refrigerator at 4®C.

Virus Extraction. The leaf tissue of the infected plants was macerated in a mortar, and the crude infectious juice was extracted from the pulp by squeezing through 2 layers of cheese cloth. The crude juice was centrifuged at 3500 rpm and the supemate was retained for the test.

a/ Cultivar


26

2. Local Lesion Hosts The inhibitory activity of the various plant juices

and chemicals was tested on a host plant (N. glutinoaa L.) which produced local lesions (Holmes,1929) with the TMV.

N. glutinosa plants were grown in pasteurized soil in pots in the greenhouse. Plants having 8-10 well-formed leaves were selected for the test, A week priorto the test, the growing tips and the lower, smaller leaves were removed leaving 6-7 leaves on each plant. By this procedure the physiological characters of the leaves were rendered more nearly uniform (Samuel al., 1935» and Takahashi, 1956). A day prior to the test all except 5 of the leaves were removed. The plants were then kept in a dark chamber for 24 hours to increase the sensitivity of the leaves to virus infection (Samuel ej al., 1935).

3. Sources of Test InhibitorsThe samples for testing of virus inhibitory activity

were selected from plant juices, growth regulators and systemic insecticides. However, the major part of theresearch program was devoted to the testing of virus inhibitory activity of plant juices.

A. Plant Juices. The plants were obtained either from naturally occurring vegetation or from cultivated plants. Plant collections were made from the greenhouse of the department of Botany, University of Rome, Italy, from the Botanical Gardens of the University of Rome (Orto Botanico), from the Horticulture Farm, University of New


27

Hampshire, Durham, New Hampshire and Strafford County Farm, Dover, New Hampshire. Plant collections were also made from the natural vegetation in the vicinity of the University of New Hampshire, Durham, New Hampshire.

The leaves were collected a day before the teat and stored in the refrigerator at in polyethylene bags. The day following, the leaves were removed from the refrigerator and immediately chopped into small pieces. They were then macerated in a mortar or a meat grinder and the crude juice was extracted from the pulp by squeezing through 2 layers of cheese cloth. The extraction of the crude juice from the leaves was also made by using a hydraulic press at a pressure of l500 lbs. The crude juice was centrifuged at 3500 rpm and the supernate was retained for the test,

B. Growth Regulators. Three commercially availablegrowth regulators tested were indoleacetic acid (lAA),^

2/indolebutyrio acid ( I B A ) a n d chlorophenoxyacetic acid3/(P-GPxAA), all of which were obtained from Eastern Organic

Chemical, Distillation Product Industries, Rochester 3,New York. The naturally occurring growth regulator tested was coconut meat extract. The commercially available growthregulators at the concentration of 100 ppm were used as

1/ 3-indoleacetlc acid G^Hj^NHCH : GH2C00H.J __________J

2/ 3-indolebutyrlc acid C^Hj^NHOH : G(CH2)^G00H.3/ p-Ghlorophenoxyacetic acid.


28

the teat solution® (Avery and Johnson, 1947)* The coconut meat extract was made from the coconuts obtained from the local market*

The milk from the coconuts was drained, and it was retained for the preliminary test. The coconut meat was homogenized with distilled water added (1 ml/5 g meat). A meat grinder and a Waring Blendor were used for the homogenization (Mauney ejb al., 1952). The homogenate was squeezed through 2 layers of cheese cloth. A preliminary test carried out with the crude meat extract inhibited TMV infection considerably. However, the inhibition of virus infection was accompanied by phytotoxicity to test plants. There was sufficient evidence available to attribute the phytotoxic affect to the fatty portion of the extract. In order to remove the fatty portion, the crude extract was centrifuged at 3500 rpm. On centrifugation the extract was separated into 3 layers (Fig. 1}, the upper solid layer (fat), the middle liquid layer and the lower solid layer. The contents of the tubes were decanted and filtered through 2 layers of cheese cloth, thereby separating the liquid portion from the solids. The liquid portion was retained for the test.


29

Fig* 1* The separation of coconut meat extract Into three layers by centrifugation at 3500 rpm.


30

G* Systeailo Ihsectlcldea, Three systemic insec-y 5/ 6/ticides, Systox, Dimethoate'^ €ind OMPA were tested for

virus inhibition activity. One ml of Systox was dissolved in 100 ml of pure acetone while 1 ml portions of Dimethoate and OMPA were dissolved in 100 ml portions of distilled water. The resulting solutions were tested for virus inhibition.

4. Method of Assaying Virus Inhibitory ActivityA. Plant Juices and Growth Regulators. The experi-

mental procedure used for the testing of the virus inhibitoryactivity of plant extracts and growth regulators was the same. N. glutinoaa was used as the local lesion host to test the inhibition of TMV infection. Holmes* half-leaf method was employed in these experiments. In the first series of the experiments the pH of the plant extracts was recorded. However, in the latter series of experiments this procedure was abandoned, as no correlation was found to exist between the pH and the inhibitory activity of

y Systox. Synonyms. Demeton, 0.0-Diethyl 0-2- (ethylthio) ethyl phosphorodlthioate.( GgHÔ ) gPS.OGHgCHg;. SCH2CH3

y Dimethoate. Synonyms. American Gynamide 12880.0,0-Dimethyl 8- methylcarbamoylmethyl phosphorodithioate.

6/ OMPA. Synonyms. Sobradan. Pestox 3, Bis(dimethylamino) phosphonoua anhydride. (Me2N)2P0.0.P0(NMe2)2 where Me - Gh^


31

the plant extracts. In the first series of experiments 1 volume of sap containing the virus was added to 9 volumes of the inhibitor (Grant, 1934)» The 2 portions were thoroughly mixed by shaking the mixture in a teat tube for 2-3 minutes. For the standard control, 1 volume of sap containing the virus was added to 9 volumes of distilled water and the 2 portions were thoroughly mixed as before» In the latter series of experiments, 1 volume of sap containing Ihe virus was added to an equal volume of inhibitor and the 2 portions were thoroughly mixed (Euntz and Walker, 1947)* For the standard control 1 volume of sap containing the virus was added to an equal volume of distilled water and the 2 portions were mixed thorou^ly. The pH of the test solution and of the control solution was adjusted to pH 7 by the addition of a buffer solution.

Three N. glutinosa plants, each having 5 leaves were used for a single treatment. Finely ground carbo- rundum was lightly and evenly dusted'over the entire leaf surface before the inoculation. The left half of each leaf was inoculated with the control mixture, and the right half was inoculated with the test mixture. The inoculation was carried out with a camel-hair brush (Takahashi, 1956). During inoculation the leaf was supported on a paper pad, tdiich absorbed any excess inoculum dripping from the leaf. The brush was dipped into the solution and the leaf was inoculated twice over, from the


32

midrib toward the margin. Uniform pressure and movement were used during the inoculation to avoid leaf injury.The paper pad was discarded at the end of each half-leaf inoculation and a new pad was used for the next half leaf. This procedure was adopted to avoid the contamination of one side of the leaf with the solution from the other side.

B. Systemic Insecticides. Each systemic insecticide was assayed on 3 N. glutinosa plants, having 5 leaves each. The plants were placed on a rotating table and were uniformly sprayed 7 times with the test insecticide. A de Vilbiss atomizer No. 152, was used to spray the insecticides. Three N, glutinoaa plants of the same age having the same number of leaves were used as a control. At intervals after spraying, the leaves were inoculated with TMV, using the procedure described above.At the same time the leaves of the control plants were inoculated with the same stock sap containing the virus.Equal volumes of sap containing the virus were contained in 2 separate beakers. Two separate brushes were utilized to inoculate the treated plants and the control plants, thus avoiding any contamination of the control plants.

C. Recording and Analysis of Data. On N, glutinosa, local lesions appeared within 48-72 hours after inoculation (Holmes, 1929). The local lesion counts were taken 5-8days after inoculation. In assaying TMV inhibition by plant juices and growth regulators, the number of local


33

lesions was counted on the treated and on the control halves of the leaves separately (Pig. 2), The average number of local lesions per half leaf was calculated from the data. The percentage inhibition was calculated from the number of local lesions per treated half leaf compared to the number of local lesions per half leaf for the controls.

In the analysis of TMV inhibition by systemic insecticides an entire N. glutinosa leaf was taken as the unit. The total number of local lesions on the 15 treated and the l5 control leaves was counted. The average number of local lesions per treated and control leaf was calculated as given in Example 1.

example 1Calculation of Percentage Inhibition

No. lesions on Av. No. lesions 15 leaves per half leafTreated 50 3.36Control 311 20.6?Percentage Inhibition = 100 - ( 3.36 ^ |

The detailed study of inhibitors was confined only to some of those Inhibitors that resulted in more thaninhibition of virus infection in several trials.


34

Fig. 2. The appearance of local lesions produced on the leaf of yiootlana glutinoaa 7 days after tobacco mosaic virus inoculation. (The lesions are produced on the control left half of the leaf while no lesions are produced on the right half of the leaf treated with an effective inhibitor.}


35

SECTION IV

PROCEDURE AND RESUIITS

1, Teat Inhibitors

Fifty two species of plants representing anglosperms gyninosperms, ferns, and marine algae were included in the investigation (Table 1). The selection of various groups and species of plants was based on the following factors :(a) a wide distribution of different families of angiosperme;(b) availability of plants; (c) facility of extraction of the juice; and (d) the work done by Grant (Grant, 1934) on host range and behavior of tobacco mosaic virus. He was unable to infect any monocotyledonous plants although be inoculated 16 species in 5 families.

The families tested, and the numbers of genera and species in each family are shown in Table 2,


3è

Table 1.- Number of species of different groups of plants tested for inhibition of virus infection.

Name of the group of plants Number of species testeda/Angiosperras-' 45

Gymnosperras 1Ferns 2Marine algae 4

Total 52

a/ Three cultivars (varieties) each of Sorgum vulgare and Secale cereale were tested. In this text the terms cultivar (variety) are used according to the recommendations of International Code of Nomenclature for cultivated plants. Hegnum Vegetabile Vol. 10. February, 1958* Utrecht, Netherlands.


37

Table 2.- Anglosperms tested for the mosaic virus.

inhibition of tobacco

Family name Number of generatested

Number of species tested

Azioaceae 1 1Amaryllidaceae 1 1Araceae 1 1Asclepladaceae 1 1Broraellaceae 2 2Caryophyllaceae 1 1Composltae 1 1Euph orb i ac e ae Graminoae

119

Lablatae 2 2Idllaceae 3 3Orchldacea© 1 1Palmae 3 3Rutaceae 1 1

Total 38 44

a/ Three cultivars (varieties) each of Sorgum vulgare and Secale cereale were tested.


38

A. Angioaperima Dicotyledons.-Eight speciea of dicotyledons rep

resenting 8 genera and 7 families were tested for the inhibition of TMV infection (Table 3). The juices of Mesembryanthemuro ap. (Aizoaceae), Dianthua Caryophyllus L. (Caryophyllaceae) and Euphorbia Charaolas Host. (Euphor- biaceae), inhibited TMV infection by more than 90^. Hilomis fruticosa Steber. (I^biatae), Inhibited TMV infection by 87 (Table 4)» However, the percentage inhibition obtained by Salvia aclarea L, the other member of the same family, was considerably less. The species tested from the families Asclepladaceae, Composltae and Rutaceae resulted in rather low TMV inhibition.

Table 3.-Families of dicotyledons tested for inhibition ofTMV infection

Family name

AsclepladaceaeAizoaceaeCaryophyllaceaeComposltaeEuphorbiaceaeLabiataeRutaceae

Number of genera tested

21

Number of species tested1111121

Total 8 8


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Monocotyledons.-Thirty seven species of Monocotyledons representing 5 families were tested for the inhibition of TMV infection. Ninety per cent of the species tested inhibited TMV infection by 60^ or more; 70^ inhibited TMV infection by 70g or more, and 30^ inhibited TMV infection by 80^ or more.

All 3 species Aloe suocotrina Schult., Sanseviera zylanica Roxb, and Scilia ap. of the family Eiliaeeae tested, inhibited TMV by 80^ or more. The only species tested in the family Araceae inhibited TMV infection by 8l . The percentage inhibition of TMV infection obtained from the 4 members of the family Palma© ranged from 52-71. The species of Amaryllidacae, Bromeliaceae and Orchldaceae tested did not produce sufficient inhibition of TMV infection to warrant further investigation.

Gramineae. Species of gramineae tested for TMV inhibition.-Grant (1934) inoculated 390 plants representing 12 species of grasses with TMV. H© obtained no infection In any of the plants Included In his study. To ascertain whether there is any inverse correlation between suscep- tibllity of plants to virus infection and their content of inhibitors, a number of species of grasses were included in this investigation. The major part of the work on plant juices was devoted to testing of juices of grasses. In this investigation 24 species representing 19 genera of grasses were included. The genera of grasses included were, Avena, Tritlcum, Hordeum« Secale, Sorgum, Bambusa,


42

Echlnochloa, Satari a». Panicum. Dlgitarla, Agropyron, Poa, Phleum. Daotylia, Bromus, Spartina. Loliuin, Pfaalarla and Festuoa» The results are summarized in Table 5*

Fifty per cent of the species tested inhibited TMV infection by 60^ or more; 30^ inhibited TMV infection by 70^ or more ; and 20^ of the species tested inhibited TMV infection by 8o^ or more. The genera Avena, Agropyron, Festuca, Spartina, Secale and Bambusa inhibited TMV infection by 80^ or more. However, several repetitions of the experiments with Secale did not confirm the earlier results of 100^ inhibition. All 3 subsequent repetitions produced 60“70^ virus inhibition. Several repetitions of the experiments with A. repens consistently gave TMV inhibitions of over 90^, thus warranting the detailed investigationof the juice of this species. The experiments with the genera Avena, Festuca. Spartina, and Bambusa were conducted only once and hence cannot be reported as confirmatory.

It is interesting to note that the experiments conducted with 4 cultivars (varieties) of Sorgum produced leas then 10^ inhibition in all oases (Table 5).

B. Gymnosperms One species of Qymnosperm Pinus aylveatris L.

collected from the garden of the Stazione Patologia Vegetal©, Rome, Italy was tested for inhibition of TMV infection.

JË/rhe results revealed that inhibitory activity of A. repens varied from one clone of A. repens to another.


U3

P. aylveatrla, the only species of Gymmoaperm tested, inhibited TMV infection by 97^ (Table 6),

C, PernsTwo species of ferns collected from the botanical

gardens of the University of Rome, Italy were tested for TMV infection. However, they did not produce sufficient inhibition of TMV infection to warrant further investigation (Table 6).

D. Marine AlgaePour species of marine algae were tested for the

inhibition of TMV infection (Table 6). All Ij. algae species were collected from the beach of Santamarinelli, Italy in December» 1957» The collected algae were stored at

in a refrigerator for hours. Before extraction of the juice, the saline water adhering to the algae was removed by squeezing through cheesecloth.

The results obtained with marine algae are of special Interest. All species tested Inhibited TMV Infection by 80^ or more, and 2 of them inhibited TMV infection by 90^ or more (Table 6),


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47

E. Growth RegulatorsThree commercially available growth regulators,

indoleacetic acid, indolebutyric acid, and p-cblorophenoxy- acetic acid, were tested for inhibition of TMV infection at the concentrations of 100 ppm. Inhibition obtained with indoleacetic acid and indolebutyric acid was very low, and p-chlorophenoxyacetio acid increased TMV infection by 3*5^* The three commercially available growth regulators had no substantial effect on the inhibition of TMV infection at the concentrations used in these studies (Table 7).

Coconut meat extract. The naturally occurring growth regulator, coconut meat extract, inhibited TMV infection by 97^ in the first trial. Repetition of the experiment gave equally high percentage inhibitions in all trials. Hence, coconut meat extract was selected for detailed investigation. The coconut milk, however, inhibited TMV infection by a relatively low percentage. Therefore, no further work was done with coconut milk. The results revealed that the inhibitory activity of coconut meat extract varied from one coconut to another (Table 7).


48

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49

F. Systemic InsecticidesThree systemic insecticides, Diraethoate, Syatox and

OMPA were tested for inhibition of TMV infection. The experiments were designed to test the effect on virus infection at various intervals, after the test plants had been sprayed with the systemic insecticides. In the Initial experiments the effect on virus infection was tested at Intervals of 12 and 2i| hours after the host plants had been sprayed with the systemic insecticides.A separate experiment was set up to test the effect at each time Interval, The experiments with Diraethoate were extended to test the effect on virus infection at intervals of 1|8 and 72 hours after the spraying of the test plants with the systemic insecticide. This extension of time Interval was necessary because the 2i{.-hour interval after spraying with the systemic insecticide gave significantly more inhibition of TMV infection than the 12-hour interval.

With OMPA the effect on TMV infection was tested at intervals of 21 and hours after spraying the test plants with the systemic insecticide.

None of the systemic insecticides tested proved to be of value in inhibition of TMV infection. It is of interest to note that Diraethoate increased TMV infection by at least 70% when the sprayed plants were inoculated at intervals of 12, 14.8, and 72 hours after Diraethoate was applied. TMV Infection was increased by 77% and 26^ respectively, when the plants were inoculated 12 and 2i|. hours


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21

after spraying with Systox. OMPA inhibited TMV Infection by a very small percentage {Table 8).

2. Properties of Inhibitors in Agropyron repena and Coconut Meat Extract

The repetition of the experiments with coconut meat extract and juice of jA. repens gave confirmatory results for the presence of virus inhibitors in both substances.The results of several experiments showed that coconut meat extract a n d the juice of A , repens inhibited T M V

infection an average of 92^ and 93^* respectively. Hence experiments^were designed to study the effect of preapplication and postapplication of inhibitor in relation to virus inoculation, the effect of aging ^ vitro, the dilution end point and the thermal inactivation point,

A, Preapplication and Postapplication of Inhibitor toVirus Inoculation

Preapplication of inhibitor to virus Inoculation.- Experiments were designed to teat the effect of virus inoculation at intervals of 3, 6, 12, 2i|, W , 96, and 192 hours after the application of the inhibitor. For each treatment, the inhibitor was applied to 15 half leaves of N. glutinoaa on 3 plants. After a given time Interval following application of the inhibitor, the virus was -inoculated to both the treated half leaves and the untreated half leaves. The application of the inhibitor and the7. In these experiments pH of the mixtures was not altered.


22

virus inoculation were both carried, out with a camel hair brush. The stock virus solution was divided into 2 equal parts. One part was used to inoculate the treated half leaves with 1 brush and the other part was used to inoculate the untreated half leaves with another brush. By this procedure any contamination of the control half leaves by the inhibitor was avoided.

Postapplication of inhibitor to virus inoculation,- The preliminary experiments were designed to test the effect of inhibitor application at intervals of 3, 6, and 12 hours after the inoculation of the virus. As the local lesions appear on N. glutinosa about 2ij. hours after TMV inoculation the experiments were designed to test the effect of post inhibitor application only up to 12 hours after virus inoculation. However, there were plans to extend the experiment to test the effect of post inhibitor application to virus inoculation at longer intervals, in the event the post inhibitor application at 12 hours produced substantial virus inhibition.

Preapplication and postapplication of juice of A . repens to TMV inoculation.-The TÎ4V inoculated at 3 and 6 hours after the application of the A. repena inhibitor inhibited Infection by a slightly higher degree than that of a 1:1 virus-inhibitor mixture. However, the effect of inhibitor on inhibition of TMV infection began to decline 12 hours after the application of the inhibitor. This decline in inhibitory activity was neither rapid nor appre-


53

clable. The percentage inhibition obtained 144 hours after the application of the inhibitor was 77%» which was only an Q% reduction in inhibitory activity from that of a 1:1 virus-inhibitor mixture (Table 9 and Pig. 3)*

In the application of inhibitor following virus inoculation, the inhibitor applied 12 hours after the TMV inoculation inhibited TMV infection by only 12^. The results (Table 10 and Pig. 3) show that the application of inhibitor following TMV inoculation had little or no effect on inhibition of TMV infection.


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57

Ereappllcatlon and postapplication of coconut meat extract to TMV Inoculation,-The TMV inoculation at 3» & and 12 hours after the application of the coconut meat extract inhibited infection by almost the same percentage as that of the 1:1 virus-inhibitor mixture. TMV inoculated 192 hours (8 days) after the application of the inhibitor, inhibited infection by 82^, which is only a 12^ reduction of inhibition from that of the 1:1 fresh virus-inhibitor mixtures (Table 11 and Fig. 4)»

The postapplication of the inhibitor following TMV inoculation had very little effect in inhibiting TMV infection. The inhibitor applied 12 hours after the TMV inoculation inhibited infection by only Z2% (Table 12 and Pig. 4).


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61

B, Effect of Aging of Inhibitors In Vitro Experiments were designed to study the effect of aging

in vitro of both the juice of A, repens and the coconut meat extract on their virus inhibitory activity. The freshly extracted inhibitor was divided into 2 equal portions of 50 ml each in stoppered 2$0 ml Erlenmeyer flasks. One portion was maintained at in the refrigerator, while the other portion was maintained at room temperature (approximately 22®G), The effect of aging on the inhibitory activity was tested at intervals of 8, 16, 32 and 64 days.One volume of the aged inhibitor was mixed with an equal volume of the TMV-infected tobacco juice. The mixture was inoculated to a half leaf of N. glutinosa. For a control,1 volume of the same stock, TMV infected tobacco juice was mixed with an equal volume of distilled water and this mixture was inoculated on the opposite half of the N. glutinosa leaf. Fifteen leaves' on 3 plants were inoculated for each test. At time of extraction the inhibitor was tested for the fresh inhibitory action in the same manner. Comparison was made between the freshly extracted inhibitor and the aged inhibitor.

The juice of A. repens stored at either room temperature (22°C), or 4^G for 64 days did not show any decline in its inhibitory activity, when compared with that of the freshly extracted juice (Table 13 and Pig. 5)»

The coconut meat extract stored at either room temperature or at 4°G for 64 days had almost the same inhibitory


62

activity as that of the freshly extracted inhibitor (Table 14 and Pig. 6).

Aging ^ vitro for 2 months had no effect on the TMV inhibitory activity of either juice of A, repena or the coconut meat extract.


63

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67

G. Effect of Dilution and Concentrât ion of the Inhibitor Experiments were designed to study the effect of

dilution and concentration of both the juice of A. repena and the coconut meat extract. The inhibitor was diluted lî5» lî50, 1:500, and 1:^000 with distilled water. One volume of the sap containing the TMV was mixed with an equal volume of the diluted inhibitor. For control, the sap containing the TMV (same stock inoculum as used with the inhibitor) was mixed with an equal volume of distilled water. The inhibitor was also tested in the concentrated form.Four volumes of the inliibitor were mixed with 1 volume of the sap containing the TMV. In a higher concentration 9 volumes of the inhibitor was mixed with 1 volume of the sap containing the TMV, The controls were prepared by mixing virus and distilled water in the same proportions as above* The virus-inhibitor mixture was inoculated to one-half leaf of N. glutinoaa, while the control mixture was inoculated to the opposite half leaf. Fifteen leaves on 3 plants were inoculated for each treatment. The relative percentage inhibition was calculated by taking the percentage inhibition obtained by the 1:1 virus-undiluted inhibitor mixture as 100,

The juice of A, repens.^The undiluted Inhibitor and the inhibitor concentrated 4 times produced nearly the same inhibitory effect. The inhibitor concentrated 9 times, however produced 10^ more inhibition than the undiluted inhibitor, The dilution of the inhibitor resulted in a rapid decline in inhibitory activity. The inhibitor dilution of


68

1:5 declined 3d% in inhibitory activity when compared with the undiluted inhibitor. The inhibitor dilution of 1:50 declined in inhibitory effect by while that of 1:500declined 88j2, However, the effect of further dilution increased inhibitory activity when compared with that of 1:500 dilution (Table 15 and Pig. ?)• In contrast to results obtained by Blaszcak et al. (1959) with Datura metel, Capgictun frutacens and Chenopodium amarantIcolor. moderate dilution of A. repens did not increase virus infactivity.

The coconut meat extract.-The relative percentages of infection obtained by 1:5 inhibitor dilution, the undiluted inhibitor, and the inhibitor concentrated 1|. tiroes and 9 times were nearly the same. However, the dilution of the inhibitor, beyond 1:5 produced a marked decline in the inhibitory activity. The inhibitory activity obtained from the 1:500 dilution was 71^ less than that of the undiluted inhibitor. In contrast to Juice of A. repens, the 1:500 dilution of the coconut meat extract produced less inhibitory activity than that of 1:500 dilution (Table 16 Fig. 8).


69

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73

D. Effect of Heating of the InhibitorsExperiments were designed to study the effect of

heating of both the Juice of A. repena and the coconut meat extract on their virus inhibitory properties. Ten ml of the inhibitor, obtained after centrifugation, was maintained in a thin-walled 25 ml Erlenraeyer flask. The flask was submerged 3/h of its height in a continually- agitated water bath, held within 3°C of the desired temperature. The mouth of the flask was always maintained above the water level. Prom time to time the temperature of the solution in the flask was recorded. After holding the flask for 10 minutes at the desired tmiperature, the flask was allowed to cool. By this method, a range of treatments at temperatures of 30°C, i+O C, 50°C, TO^G, and 90^0 was carried out. One volume of the heated inhibitor was mixed with a equal volume of the sap containing TMV. For the control, one volume of the sap containing TMV was mixed with an equal volume of distilled water. The vlrus- inhibitor mixture was inoculated on one-half leaf of M. glutinosa. while the control mixture was inoculated on the opposite half leaf. Fifteen leaves on 3 plants were inoculated for each treatment.

Heating the Juice of A. repens at ij.O®C produced a slight decline in inhibitory activity. However, heating of the inhibitors at temperatures of 50®C, 70®C, and 90®G produced nearly the same percentage inhibition of TMV infection as that of the unheated inhibitor (Table 17 and


7k

Pig. 9). In contrast to the results obtained by several workers with various plant Juices (Kuntz and Walker, 1947; Blaszczak et al.. 1959» Weintraub and Kilpatrick, 1952), the inhibitor in A.repens does not appear to be thermolabile as heating of the inhibitor for 10 minutes at 90^0 bad little or no effect on Its Inhibitory property. Heating of the coconut meat extract at 40^0 and 50^0 for 10 minutes reduced the inhibitory activity markedly. At 50®C the inhibitory activity was reduced by almost half, when compared to unheated inhibitor. However further increase in temperature enhanced the inhibitory activity. Wben the inhibitor was heated at 90®0 for 10 minutes the inhibitory activity was the same as that of unheated inhibitor (Table iB, Fig. 10).


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79

SECTION V

DISCUSSION AND CONCLUSIONS

Eight species of dicotyledons were tested for TMV inhibition of infection. Three of the species tested, E. Cbaraolas» D. Caryophyllus and Mesembryantherouin sp. were effective inhibitors, D. Caryophyllus inhibited TMV infection by 95^» Weintraub and Gilpatrick (1952) reported that extracts of JD, barbatua reduced infactivity of TMV, cucumber mosaic and tobacco etch viruses. They found that the inhibitor had no effect in preventing virus infection of D. barbatua and that its effectiveness was not lost on dialysis. Although no detailed study of the properties of D. Caryophyllus inhibitor was carried out its properties may be similar to those of D, barbatus. These results warrant further research in the genus ]%^thU8 for possible virus inhibitors.

Thirty seven species of Monocotyledons were tested for inhibition of TMV infection. Only 30^ of the species tested Inhibited TMV infection by 90^ or more.

Grant (1934) obtained no TMV infection in any of the 16 species of monocotyledons inoculated. Grant * s publication lacks sufficient details to ascertain the names of the species which he used. Also, there is very little information available in the literature to ascer


80

tain the degree of susceptibility to TMV infection of the species of monocotyledons used in this investigation. Assuming that roost of the species of monocotyledons used in this investigation are resistant to TMV infection, the evidence available is Insufficient to conclude the existence of any inverse correlation between susceptibility of plants to virus infection and the presence of inhibitors, because only a small percentage of the species tested inhibited TMV infection appreciably.

All J4 species of marine algae tested were effective in inhibition of TMV, These results warrant further research for possible virus inhibitors in this group of plants.

Three commercially available growth regulators, lAA, ISA and P-CP x AA tested were ineffective as TMV inhibitors. Chlorophenoxyacetic acid stimulated TMV infection by 3^. Nour-Eldin (1953) reported that by floating TMV-infected tobacco discs in solutions of growth regulators that lAA and IBA had no effect on TMV multiplication, while all growth regulators in the phenoxyacetic group stimulated virus infection. Although Hour-Eldin (1953) tested these compounds for their effect as inhibitors of virus Increase, the results are in agreement with the results of this investigation.

All 3 systemic insecticides tested, Dimethoate,Systox and OMPA were of no value as inhibitors of TMV infection. On the other hand, Dimethoate and Systox stimulated TMV infection by 70% and 77^ respectively. This observed stimulation of TMV infection by Dimethoate and


81

Systox may be a limiting factor in the use of these insecticides in pest control work.

The juice of A. repens and coconut meat extract effectively inhibited TMV infection in all the trials.When A. repens juice was heated at 90®C for 10 minutes its inhibitory activity was not reduced. This property suggests that the inhibitor is not protein in nature. However, to reach the conclusion that the inhibitor was not proteinaoeous further tests such as dialysis and precipitation with 95^ ethanol are necessary to be carried out (McKeen, 1956| Kaasanis and Kleozkowski, 1948)« The effect of heating on coconut meat extract is difficult to interpret, l«0)en the inhibitor was heated at 50®C for 10 minutes, the inhibitor activity was reduced almost by half, compared to that of the unheated inhibitor. However, a further increase in temperature enhanced the inhibitory activity. Thus, when the inhibitor was heated at 90®C for 10 minutes the percentage inhibition was the same as that of the unheated inhibitor. Assuming that the reduction In percentage Inhibition obtained at 40°C and 50* C is not due to experimental error, it can be postulated that the coconut meat extract has more than one inhibitor, one of which is thermolabile, and in the other (or others) the inhibition is activated at higher temperatures.

Aging of A. repens juice and coconut meat extract in vitro for 61+ days had no effect on their inhibitory properties.


82

When the juice of A. repens was diluted 1:5» the inhibitory activity was reduced by nearly 35^. Wien the juice was concentrated 1+ times the inhibitory activity obtained was the same as that of the undiluted inhibitor.The dilution of inhibitor beyond 1:5 reduced the inhibitory activity markedly. The coconut meat extract was diluted 1:5» and concentrated 4 and 9 times without any appreciable effect on its inhibitory activity.

This behaviour of the juice of A. repens and coconut meat extract on dilution follow the same pattern as that of spinach extract, normal blood serum, milk, and pepper extract (Kuntz and Walker, 1947» Chester, 1934» Weintraub and Gilpatrick, 1952). These results suggest that for virus Inhibition to occur, there is a certain minimum concentration of inhibitor necessary to be present. This phenomenon could also answer the question why most plant viruses are transmitted by insects but only a few viruses are transmissible by sap inoculation. Bawden (1954) stated :

The inhibitors seem to operate only when tranamls- sions are by the mechanical inoculation of expressed sap, which is not for most viruses a usual method of spread. The usual method, except for tobacco mosaic and perhaps a few others, is by insect vectors, whose ability to transmit seems not at all affected by the presence in infected plants of these substances.This presumably means either that the viruses in infected cells occur separate from these substances, the two being brought together only when the tissues are macerated, or that the insect vectors can separate the viruses from the inhibitors.

Since several workers have reported that the dilution of inhibitor reduced inhibitory action it appears that most viruses are not mechanically transmissible because


83

in the extracted sap the inhibitors are present in sufficient concentration to prevent infection. The insect vectors are able to transmit the virus because the inhibitor is not present in sufficient concentration in the inoculum of the insect. However, this explanation has several drawbacks. It does not explain the reason for complete absence of mechanical transmission of certain viruses under all conditions thus far attempted. It also fails to explain the absence of mechanical transmission of insect-transmitted viruses by the sap of the diseased plant to healthy plants of the same species because it has been reported by several workers that inhibitors are ineffective in preventing the infection of the species from which the Inhibitor comes.

Many inhibitors combine with viruses reversibly, and the dilution of the inhibitor-virus mixture results in reinfactivity of the viruses. This phenomenon suggests that the combination of the virus and the inhibitor is temporary. It does not Involve any alteration of the virus, end probably no change occurs in the inhibitor either, (It seems to follow the same principle of antibody antigen reaction) Boyd (1956). -- -

The reason that the inhibitor combines with the virus in a fixed ratio led various workers to the conclusion that the inhibitor affects the virus directly but has no effect on the susceptibility of the host to virus infection. However, to conclude that an inliibitor affects the


84

virus only, it is necessary to carry out tests with inhibitor application before virus inoculation. Further when it can be demonstrated that the virus regains its infectivity on dilution of the virus-inhibitor mixture it is erroneous to conclude that the inhibitor affects the virus only.

The results obtained from the tests, where juice of A. repena and coconut meat extract were applied before and after virus inoculation suggest that the effect of the inhibitor is on the host rather than on the virus,

Bawden (1954) divided Inhibitors into two categories : (a) inhibitors of virus infection, and (b) inhibitors of virus increase. Bae inhibitors of infection are, according to Bawden, substances that when inoculated to leaves simultaneously with the viruses prevent infection from occurring. The inhibitors of virus increase are substances, that when applied to leaves already infected, retard the rate at which the infecting viruses multiply.It seems more reasonable to define Inhibitors of infection as substances, that when Inoculated to leaves simultaneously with the viruses or substances when applied to the host before virus inoculation, prevent infection from occurring.In view of the results obtained in this investigation, it is evident that both the juice of A. repena and coconut meat extract belong to the group of inhibitors of virus infection.

According to Bawden (1954)» there are 2 rival schools


85

of thought concernlxig the mechanism of the Inhibitory activity of the inhibitors of virus infection. One school maintains that the inhibitor affects the resistance of the host and the other maintains that the inhibitor acts directly on the virus. The reason that a constant concentration of the inhibitors produced the same relative decrease in infectlvity, regardless of the virus concentration, led Stanley (1934^), Chester (1934)» Kassanls and Kleozkowski (194®)» Bawden and Freeman (1952), and McKeen (1956) to conclude that trypsin, normal serum, extract of P. esculenta» the culture filtrates of T. roseum. and the pepper extract affected the resistance of the host.

Bawden (1954) stated that :On present evidence the conclusion seems inescapable that most, if not all, inhibitors act on the host plant rather than directly on the virus particles,

Stanley (1934c) &od Cadman (1959) showed that pepsin, the raspberry inhibitor and tannic acid combined with the virus in a fixed ratio and that the dilution of the virus- inhibitor mixture resulted in no regaining of the infectivlty of the viruses. Hence to conclude that all inhibitors act on the host plant seems to be erroneous. In view of these results the writer believes that it would be reasonable to divide inhibitors of infection into 2 categories :(a) the inhibîtors of infection that affect the resistance of the host, and (b) the inhibitors of infection that act directly on the virus. These 2 ways in which inhibitors


86

of infection act are not 2 rival schoola of thought, but they are 2 separate mechanisms of inhibition of infection specific to the inhibitor.

Two schools of thought also exist concerning the mechanism of action by which inhibitors of infection affect the resistance of the host. One school maintains that the inhibitor combines with some specicic receptors in the host cell (It has been postulated that the virus combines with some specific receptors in the host cell as a first step toward infection.) , and the other maintains that the inhibitor alters the physiology of the host cell so that it no longer supports virus multiplication. There seems to be more evidence in favor of the latter school of thought than the former. Certain observed phenomena in animal viruses can be sighted in support of the hypothesis that the Inhibitor combines with the virus receptors of the host cell. luria (1953) » discussing the reports of several workers, stated that when the allantoic fluid containing influenza virus came into contact with blood, the erythrocytes were agglutinated. This phenomenon was referred to as hemagglutination. The virus that was agglutinated was released from the reed blood cells on incubation at 37^0. The red blood cells that had released or eluted the virus were not agglutinable either by the same virus of newly added viruses of the same strain. The red blood cells that had eluted the mumps virus were agglutinable by influenza virusj influenza A strain of


87

the virus left the red blood cells agglutinable by influenza B strain of the virus. On this basis the virus is supposed to destroy a certain fraction of the receptors on the red blood cells. A receptor gradient can be constructed on the basis mentioned above. Several workers have reported that inhibitors are ineffective in preventing the infection of the species in which they occur. Inhibitors are specific in their action in that they inhibit infection of only certain viruses. The hypothesis that the inhibitor altera the physiology of the host cells in such a way that they no longer support virus multiplication can explain the inability of inhibitors to prevent infection of the species in which they occur. However, this does not explain the observed specificity of the inhibitors.If the inhibitor alters the physiology of the host cells, it is difficult to explain why this physiologic change inhibits multiplication of only certain viruses and not others. This hypothesis also fails to explain the reason for the localization of the physiologic effect only to those portions of the host to which the inhibitor had been applied. If one assumes that the Inhibitor competes with the virus for combination with specific receptors of the host cells and prevents the virus from accomplishing the first step of infection, an explanation can be found for both phenomena. In an analogy with the receptor gradient reported in hemagglutination, there is reason to assume that the inhibitor competes with viruses only for the


88

receptors for viruses which it acts as the inhibitor and not the others. Probably the inhibitor does not affect the virus directly and the virus remains potentially infective.

The writer is of the opinion that the inhibitor combines with some specific receptors in the host cell for the virus and prevents the first step in virus infection.

The reaction of the inhibitor with the virus receptors in the host cell is probably an independent reaction from the inhibition reaction. If this assumption is made an explanation also could be found for the ineffectiveness of inhibitors in preventing the infection of the species from which they originate.

Bawden (1954)» in explaining that inhibitors do not affect the resistance of the host to virus infection stated :

' It seems highly unlikely either that those substances do normally affect the spread of the viruses or that they are in any way correlatedwith the host resistance.

In support of this argument he states that :It would be an odd piece of evolutionary benevolence, with no obvious selection mechanism, that would encourage the maintenance of substances that serve only to prevent other species from contracting infection.

The writer agrees with the first statement of Bawden thatinhibitors are not related to the resistance of the host.However, as to the second statement it is suggested thatinhibitors do not exist for the purpose of defenseagainst virus infection, but that inhibition is only a


89

secondary reaction which takes place as a result of the first reaction; the destruction of the virus receptors of the host cell by the inhibitors. The 2 processes.of the action of the inhibitors on the receptors and the prevention of virus infection are 2 independent processes.In the process of evolution it is unlikely that an organism would produce a compound that can be antagonistic to its own tissues. Qû the same basis one cannot expect inhibitors to destroy a part of their own cell. However, it is very likely that they could do so in another organism.

In conclusion the writer wishes to make the following suggestions as to the mechanism of the virus inhibition :

Inhibitors should be divided into two categories.(a) Inhibitors of virus infection(b) Inhibitors of virus increase (Bawden)

Inhibitors of virus infection are substances that when inoculated to leaves simultaneously with the virus or substances when applied to host before virus inoculation prevent infection from occurring, Inhibitors of virus Increase are substances, that when applied to leaves already Infected, retard the rate at which the infecting viruses multiply.

There are two ways in which inhibitors of infectionact.

(a) Some inhibitors siffect the resistance of the host. The inhibitors that affectthe resistance of the host do so by


90

competing with the virus for some specific receptors for the virus in the host cells and prevent the first step in virus infection.

(b) Some inhibitors act directly on the virus either by combination with the virus irreversibly or by bringing about a chemical or physical change in the virus.


91

SECTION IV

SDMMAHÏI

Fifty two plant juices, i+ growth regulators, and 3 systemic insecticides were tested for the inhibition of tobacco mosaic virus Infection. The juices of Agrouvron repenss Dlanthus Caryophyllus, Euphorbia Characia, Avena satlva, Mesembryanthemum sp., Plnus sylvestrls» Ulva lactuca and a species of Rhodophyta inhibited TMV infection by 90^ or more. The juices of Monstera dellclosa, Sansevlera zeylanica, Phlomis fruticosa. Aloe succotrlna. So111a sp., Barobusa arundinaceae, Spartina patens. Festuca rubra, Enteroroorpha sp.. and Cutleria sp. inhibited TMV Infection between 8o^ and 90^. All 1+ species of marine algae tested Inhibited tobacco mosaic virus infection by 8o^ or more.

The commercially available growth regulators tested, Indoleacetic acid, Indolebutyric acid and p-Chlorophonoxy- acetic acid were not effective as inhibitors of TMV Infee- tion. The naturally occurring growth regulator tested, coconut meat extract, inhibited TMV infection by more than 90#.

Three systemic insecticides, Dimethoate, Systox, and OMPA tested for inhibition of TMV infection were not effective as inhibitors. In fact Dimethoate and Systox stimulated TMV infection.

Aging in vitro for 64 days did not reduce the inhibitory effect of either juice of A. repens or coconut meat extract.


92.

The Juice of A. repena and the coconut meat extract reduced TMV infectlvity when applied to host leaves before virus inoculation. However, they did not reduce TMV Infection when they were applied to host leaves after virus inoculation The dilution of both inhibitors reduced their inhibitory activity. However, the increase in the inhibitory activity obtained by concentration of the inhibitors was not significant. The inhibitor in the juice of A. repens is heat- stable and probably non protein in nature. The coconut meat extract lost nearly half of its inhibitory activity when it was heated at 50®C. However, further increase in temperature enhanced the inhibitory activity. The probable mechanism of inhibition is discussed.


93

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STUDIES ON THE INHIBITION OF TOBACCO MOSAIC VIRUS