STUDIES ON THE APHID TRANSMISSION OF A STRAIN OF HENBANE MOSAIC VIRUS

A virus causing a wilt of Datura stramonium was identified as a strain of henbane mosaic virus. It causes necrotic local lesions in Nicotiana rustica , and local lesions are demonstrable in tobacco by staining with iodine. Some of the factors affecting its transmission by Myzus persicae (Sulz.) were studied quantitatively using these lesions.
Infective aphids differed little in their ability to cause infection, and usually produced two or three lesions. The duration of the feeding puncture did not affect the number of infections and had little effect on the percentage of aphids becoming infective. Transmissible virus did not seem to be continually imbibed while aphids fed on infected plants, and there were indications that it was acquired immediately before aphids withdrew their stylets from the leaf. Aphids became infective when allowed to make feeding punctures into epidermis stripped from infected leaves.
M. persicae transmitted during feeding punctures as brief as 5–10 sec; the probability of single feeding punctures resulting in infection reached a maximum with those lasting from 20 to 30 sec, during which the stylets did not penetrate as far as the centre of the epidermal cell and little or no saliva appeared to be ejected. M. persicae did not transmit the virus when its stylets were artificially wetted with infective sap.
Periods of darkness before inoculation with datura wilt virus increased the susceptibility of Nicotiana rustica to infection by rubbing, but not to infection by aphids.     

THE DISTRIBUTION OF VIRUSES IN LEAVES AND THEIR INACTIVATION BY ULTRA-VIOLET IRRADIATION

The infectivity of sap expressed from the lower epidermis stripped from leaves systemically infected with potato virus Y , henbane mosaic virus or tobacco mosaic virus was compared with that of sap from the underlying mesophyll. Results suggested that the concentration of virus in each of the two tissues was about the same.
Ultra-violet irradiation of leaves infected with potato virus Y or henbane mosaic virus greatly reduced the infectivity of sap expressed from subepidermal tissues.     

THE DIFFERENT DISTRIBUTION OF TWO BRASSICA VIRUSES IN THE PLANT AND ITS INFLUENCE ON SPREAD IN THE FIELD

Previous knowledge provided no explanation for the greater prevalence of cauliflower mosaic than of cabbage black ring spot in field crops of cauliflower. Both viruses are spread principally by Myzus persicae and Brevicoryne brassicae , and both are transmitted equally readily from infected seedlings. Cabbage black ring spot virus has a much wider host range, and sap from infected leaves has a higher dilution end-point than sap from leaves infected with cauliflower mosaic virus.
At least part of the difference between the rate at which the two viruses spread in the field may be accounted for by the different manner in which they are distributed in old infected plants, and the effect this has on transmission by aphids. Cauliflower mosaic virus occurs in high concentration in all the new leaves produced by infected plants. Cabbage black ring spot virus, on the other hand, occurs mainly in the older leaves, and even there is localized in parts that show symptoms. Only in recently infected plants does cabbage black ring spot virus occur in young leaves.
After flying, most aphids alight on the upper parts of plants; they are therefore less likely to acquire cabbage black ring spot virus than cauliflower mosaic virus. It may be significant that cabbage, a host in which old leaves are in a more favourable position for alighting aphids than are those of cauliflower, is also often extensively infected with cabbage black ring spot virus.     

Aphid-injection experiments with carrot mottle virus and its helper virus, carrot red leaf

Carrot mottle virus (CMotV) and its helper virus, carrot red leaf (CRLV), were not transmitted by aphids (Cavariella aegopodii) that had fed through membranes on, or had been injected with, sap from mixedly infected chervil plants or partially purified preparations of CMotV. However, the viruses were transmitted by recipient aphids injected with haemolymph from donor aphids that had fed on mixedly infected plants but not by a second series of recipients injected with haemolymph from the first series. Some of the first series of recipients transmitted both viruses for up to 11 days but others transmitted erratically and many lost ability to transmit after a few days. The results confirm that both viruses are circulative but provide no evidence for multiplication in the vector. Non-viruliferous aphids, or aphids that had acquired CRLV by feeding, did not transmit CMotV when they were injected with haemolymph from aphids that had fed on a source of CMotV alone, confirming that they can only transmit CMotV when they acquire it from a mixedly infected plant. When extracts from donor aphids were treated with ether before injection, recipient aphids transmitted both CRLV and CMotV, although the infectivity of CMotV grown in Nicotiana clevelandii in the absence of CRLV is destroyed by ether treatment. CMotV particles acquired by aphids from mixedly infected plants therefore differed in some way from those in singly infected plants. A plausible explanation of these results, and of the dependence of CMotV on CRLV for aphid transmission, is that doubly infected plants contain some particles that consist of CMotV nucleic acid coated with CRLV protein.     

THE TRANSMISSION OF PLANT VIRUSES BY BITING INSECTS, WITH PARTICULAR REFERENCE TO COWPEA MOSAIC

Experiments on the virus-vector relationship of the Trinidad cowpea mosaic virus, transmitted by Ceratoma ruficornis , gave the following results: ability to infect decreased with increasing time after ceasing to feed on infected plants, but vectors remained infective for 14 days (much longer than the longevity in vitro of the virus at glasshouse shade temperatures of 23–31°C.); the beetles transmitted more consistently after longer feeding on infected plants, though feeds of under 5 min. made them efficient vectors; the proportion of plants infected increased with the amount of feeding damage on them; fasting the vectors before feeding on infected plants increased voracity but had no effect on their ability to transmit; beetles became infective immediately after feeding on infected plants. Cowpeas were infected by inoculation with macerated infective vectors or with juice regurgitated by vectors. There is no evidence that aphids or other sucking insects can transmit the virus. It seems similar to squash mosaic and turnip yellow mosaic, for vectors of all three viruses probably transmit by regurgitating infective juice during feeding.     

INFECTIVITY OF APHIDS BRED ON VIRUS-INFECTED CAULIFLOWER PLANTS

Caged cauliflower plants infected with either cabbage black ring spot virus (CBRSV) or cauliflower mosaic virus (CIMV) were colonized with Myzus persicae or Brevicoryne brassicae. Winged and wingless aphids that voluntarily flew or walked from these plants were transferred singly to healthy cauliflower or other brassica seedlings to compare their feeding behaviour and ability to transmit the viruses. Wingless aphids settled to probe more readily than winged, and B. brassicae was initially more restless than M. persicae. CIMV was more readily transmitted than CBRSV by both species, and B. brassicae rarely transmitted CBRSV. Wingless aphids transmitted less often than winged ones, and no wingless B. brassicae transmitted CBRSV, although they did CIMV. Fewer aphids transmitted CBRSV from old plants than from young ones, but plant age had little effect on CIMV transmission.     

Aphid transmission of Beet Yellows Virus affected by homologous antiserum

A thin layer of homologous antiserum (against the beet yellows virus - BYV) between the leaf surface and a Parafilm membrane totally inhibited the acquisition of BYV by aphidsMyzus persicae (Sulz.), but it did not affect the inoculation of BYV by infective aphids. BYV transmission decreased with aphids picking up the virus from leaves coated with a normal rabbit serum. Aphids sucking on purified BYV suspension through the Parafilm membrane as well as aphids allowed to probe into leaves of healthy plants spread with an infectious purified BYV suspension failed to transmit BYV. No BYV particles could be detected in eluates from stylets and labia cut off from aphids which had probed on BYV infected plants by electron microscopic examination. The acquisition seems to be the most important phase for the aphid transmission of BYV which is apparently carried on the stylet surface.     

STUDIES ON TWO APHID-TRANSMITTED VIRUSES OF LEGUMINOUS CROPS

Pea mosaic virus was transmitted by Myzus persicae Sulz., Macrosiphum pisi Kalt., M. solanifolii Ash. and Aphis fabae Scop., but not by Hyperomyzus staphyleae Koch. It is a 'non-persistent' virus (Watson & Roberts, 1939), and is most readily transmitted when vectors are fasted and then given a short infection feeding. Vector efficiency was not increased by increases in preliminary fasting beyond 15 min. or with increasing infection feeding beyond 1 hr. Most aphids became non-infective within 15 min. when feeding, but fasting aphids remained infective for 3 hr. Species that fed readily on the infected plants were less efficient vectors than those which did not. Seed set by infected plants produced healthy seedlings.
Pea enation mosaic virus persisted in Myzus persicae and Macrosiphum pisi for more than 140 hr.; its transmission was unaffected by preliminary treatments of aphids. No transmission was obtained until at least 4 hr. after aphids had left infected plants; usually the 'latent' period exceeded 1 day and its duration was apparently unaffected by the length of the infection feeding.     

Studies on the transmission of velvet tobacco mottle virus by the mirid, Cyrtopeltis nicotianae

The minimum acquisition period of velvet tobacco mottle virus (VTMoV) by its mirid vector Cyrtopeltis nicotianae was about 1 min, with an increase in the rate of transmission (i.e. proportion of test plants infected) for acquisition periods up to 1000 min. Pre-acquisition starvation periods up to 18 h did not affect the rate of transmission. After an acquisition access period of 2 days, the minimum inoculation period was between 1 and 2 h and the rate of transmission increased with increasing inoculation time; when the acquisition access period was 1 h, or if vectors were fasted for 16 h after the 2 day acquisition, the rate of transmission was significantly lower. When mirids were transferred sequentially each day to a healthy plant after a 24 h acquisition feed, they transmitted intermittently for up to 10 days. Up to 50% of mirids transmitted after a moult and this was not due to the mirids probing the shed cuticles or exudates of infective insects. Mirids transmitted after a moult, following acquisition periods of 10, 100 or 1000 min. C. nicotianae transmitted solanum nodiflorum mottle virus (SNMV), sowbane mosaic virus (SoMV) and southern bean mosaic virus (SBMV), but not subterranean clover mottle virus (SCMoV), lucerne transient streak virus (LTSV), tobacco ringspot virus (TRSV), galinsoga mosaic virus (GMV), nor nicotiana velutina mosaic virus (NVMV). Tomato bushy stunt virus (TBSV) was transmitted to 1/58 test plants.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

The role of the helper virus, anthriscus yellows, in the transmission of parsnip yellow fleck virus by the aphid Cavariella aegopodii

Transmission of parsnip yellow fleck virus (PYFV) by the aphid Cavariella aegopodii occurs only when the aphids are also carrying the helper virus, anthriscus yellows (AYV). None of five other viruses tested was able to act as helper. In experiments in which aphids were allowed to feed through membranes on crude or treated extracts from infected plants, aphids already carrying AYV acquired PYFV, but virus-free aphids failed to acquire either AYV or PYFV. PYFV was not transmitted by insects injected with haemolymph from aphids carrying both viruses, or with purified preparations of PYFV. PYFV was transmitted when AYV-carrying aphids, except those whose stylets had been removed, were contaminated externally with PYFV preparations. Ultraviolet irradiation of infected leaves did not prevent aphids from acquiring AYV, presumably because it is confined to deeply-lying tissues. AYV-carrying aphids could acquire PYFV from u.v.-irradiated leaves after acquisition access times of 2 h but not after feeds of only 2 or 15 min (which are adequate on unirradiated leaves), suggesting that PYFV is present in all parts of the leaf. No ‘helper agent’ distinct from AYV itself was detected in these experiments or in experiments on minimum acquisition feeding time or maximum period of persistence in the aphid. U.v.-inactivated PYFV competed with infective PYFV for retention sites in AYV-carrying aphids, whereas AYV apparently did not. It is suggested that there is no helper agent for PYFV, other than AYV particles. The possibility that there is one for AYV is not excluded.     

Properties of some defective strains of tobacco mosaic virus and their behaviour as affected by inhibitors during storage in sap

From the type strain of tobacco mosaic virus, defective strains were isolated that produced chlorotic or ringspot type symptoms in tobacco and were difficult to transmit without carborundum in the inoculum. Their concentration was less than 0–1 μg/ml of sap instead of the usual 2 mg/ml with the type strain. Phenol extracts of infected leaves were a little more infective than extracts in buffer, whereas phenol extracts of leaves infected with type strain were very much less infective than extracts in buffer. Electron microscopy of infective sap rarely showed any virus particles, but preparations concentrated by ultracentrifugation contained virus particles, many of which were broken or seemed inadequately assembled. Changing the ambient temperature at which infected plants were kept from 20 to 35°C did not increase the amount or improve the appearance of the virus. Some of the strains were inactivated during heating for 10 min between 70 and 80 °C. Undiluted sap lost its infectivity in 3 days at 20 °C, as did the type strain when diluted to 0–1 μg/ml in sap from healthy leaves. This is because substances that inhibit infection were produced by microbes in the sap. The ability of sap from healthy leaves to inhibit infection increased by more than twenty-five times when left 3 days at 20 °C. Infectivity of appropriate mixtures of type strain and aged sap was restored by diluting them in buffer. Sodium azide at 0·02% in sap prevented formation of the inhibitor. The infectivity of the defective strains increased when inoculated together with the type strain.     

THE MANNER OF TRANSMISSION OF SOME BARLEY YELLOW-DWARF VIRUSES BY DIFFERENT APHID SPECIES

Some barley yellow-dwarf (BYD) viruses isolated from cereal crops in Great Britain were transmitted by Rhopalosiphum padi , L. and others were not. Sitobion fragariae (Walker), S. avenae (Fabricius), and Metopolophium dirhodum (Walker) all transmitted viruses of both types, but they usually transmitted those of which Rhopalosiphum was a vector less readily than did R. padi. The transmissibility of a virus by a given aphid species was not affected by transmission with another, less efficient, vector species. Neomyzus circumflexus (Buckt.) and Rhopalosiphum maidis (Fitch) transmitted the few viruses with which they were tested.
A few R. padi acquired virus from infected leaves during 30 min. feeding and inoculated healthy seedlings during 15 min. feeding, but the minimum total time taken to acquire and transmit was 10 hr. and 32 hr. were needed for about half the aphids that were able to acquire and transmit virus to do so. This may indicate the existence of a short latent period of the virus in the vector, although the evidence is not conclusive. The times spent on infected plants influenced the results more than those spent on healthy ones; many transmissions occurred with short feeding times on healthy plants so long as the time spent on infected leaves was long, but the reverse was not true. Nymphs of R. padi that moulted after they left infected plants on which they fed long enough to become infective, infected slightly fewer plants than adults fed for the same times.     

SOME EFFECTS OF HOST-PLANT NUTRITION ON THE MULTIPLICATION OF VIRUSES

The amounts of tobacco mosaic virus present in systemically infected tobacco plants varied greatly with the mineral nutrition of the plants and were related to the effects on plant growth. With plants in soil, supplements of phosphorus produced the greatest increases in plant size, in virus concentration of expressed sap, and in total virus per plant; nitrogen increased plant size only when phosphorus was also added, and only then increased virus concentration and total virus per plant. Combined supplements of phosphorus and nitrogen doubled the virus concentration of sap and increased the total virus per plant by factors up to forty. Potassium slightly reduced the virus concentration of sap, though it usually increased plant size and total virus per plant. From all plants, only about one-third of the virus contained in leaves was present in sap. Virus production seemed to occur at the expense of normal plant proteins, and the ratio of virus to other nitrogenous materials was highest in plants receiving a supplement of phosphorus but not of nitrogen.
The effects of host nutrition on the production of virus in inoculated leaves resembled those in systemically infected leaves, but were more variable.
No evidence was obtained, with plants grown in soil or sand, that host nutrition had any consistent effect on the intrinsic infectivity of tobacco mosaic virus.
The concentration of virus in sap from potato plants systemically infected with two strains of potato virus X was not consistently affected by fertilizers; the chief effect of host nutrition on virus production was indirect by altering plant size.     

Association of a potyvirus with mosaic disease of gherkin (<Emphasis Type="Italic">Cucumis anguria</Emphasis> L.) in India

A virus associated with severe mosaic disease of gherkin (Cucumis anguria L.) in south India was identified. The infected plants showed mosaic, vein banding, blistering on malformed leaves and fruits. Host range, transmission, serological and electron microscopic studies were carried out to identify the virus. The virus was readily transmitted by Sap inoculation and by aphids in a non-persistent manner. The host range of the virus was mainly limited to cucurbitaceous and chenopodium species. The virus showed positive serological relationships with members of potyvirus genus but not with cucumo, ilar and taspoviruses. Electron microscopy of leaf dip preparation of infected leaves revealed long flexuous filamentous virus particles measuring 750 × 12 nm. On the basis of symptomotology, host range, transmission, serology and particle morphology the virus associated with mosaic disease of gherkin might be the member of potyvirus genus.     

SOME PROPERTIES OF FOUR STRAINS OF CUCUMBER MOSAIC VIRUS

Different strains of cucumber mosaic virus differ in their host range, symptoms caused, virulence towards different plants, transmissibility by aphids, dilution end-point and thermal inactivation point.
There are seasonal variations in the susceptibility of some host species; French bean is apparently immune during the summer but during the winter produces countable local lesions suitable for quantitative assays.
Different host species differ in the ease with which cucumber mosaic virus is transmitted to and from them; systemic infection in beet rarely occurs unless the virus is introduced into young tissues. Inhibitors of infectivity in sap of sugar beet and Phytolacca sp. make mechanical transmission from these to other hosts difficult; the inhibitors interfere less with the infection of hosts in which they occur than with the infection of tobacco.
Cucumber mosaic virus has a low temperature coefficient of thermal inactivation and much infectivity is destroyed by heating at temperatures below the thermal inactivation point.
Myzus persicae (Sulz.) is a more efficient vector than M. ornatus Laing which is more efficient than Macrosiphum euphorbiae (Thomas); although individual aphids can cause more than one infection, most cease to be infective in feeding periods of from one to five minutes.     

ANEMONE MOSAIC–A VIRUS DISEASE

The name anemone mosaic is proposed for a previously unrecorded virus disease of Anemone coronaria L.; infected plants have mottled leaves, and broken and distorted flowers. This virus can cause winter browning, and can contribute to crinkle in anemones.
The virus infected forty-seven out of ninety plant species tested; it was transmitted by mechanical inoculation, and by four of the six aphid species tested. Most aphids ceased to be infective within 30 min. when continuing to feed after leaving an infected plant.
Properties in vitro varied according to conditions of the tests; the thermal inactivation point was always below 62°C., the dilution end-point did not exceed 1/2500, and the virus inactivated at 18°C., the fewer than 72 hr.
Intracellular inclusion bodies were produced in all hosts examined.
Anemone mosaic virus is very similar to viruses placed in the turnip virus 1 group of Hoggan & Johnson, and is serologically related to cabbage black ringspot virus, although AMV infection did not protect plants against infection with cabbage black ring-spot virus.
Weeds naturally infected with AMV were found in anemone plantations, and this virus was detected, together with cucumber mosaic and tobacco necrosis viruses, in corms imported into this country.     

QUANTITATIVE STUDIES ON THE TRANSMISSION OF CABBAGE BLACK RING SPOT VIRUS BY MYZUS PERSICAE (SULZ.)

Factors affecting the transmission of cabbage black ring spot virus by Mysus persicae (Sulz.) were studied quantitatively using the local lesions produced on tobacco leaves. Aphids prevented from feeding for 15 min. or more, before feeding for a few minutes on an infected plant, caused more infections than unfasted aphids. Fasted aphids acquired virus from infected plants in feeding times as short as 10 sec., and infected healthy plants in test-feeding times of 5 sec. Increasing test-feeding times to 30 min. increased the numbers of infections. Increasing infection- feeding times from 10 sec. to 5 min. had little effect, but increasing to more than 5 min. greatly reduced the number of transmissions. This reduction was partly offset if the aphids were prevented from feeding continuously while on the infected plants. With undisturbed infection-feeding periods of 15 min. or longer, previously fasted aphids caused no more infections than unfasted aphids.
Infective aphids lost their ability to produce lesions more rapidly when feeding than when fasting.
Winged and wingless aphids were equally efficient vectors.     

The transmission of potato virus Y by aphids of different vectoring abilities

Adult apterae of Myzus persicae and Macrosiphum euphorbiae that did not transmit potato virus Y^N (PVY^N) in a first test were as likely to transmit the virus in a subsequent test as those that did transmit on the first occasion. Only 16% of M. persicae that were allowed a single acquisition probe into a leaf infected with both PVY^O and PVY^N transmitted both strains, 37% transmitted either PVY^O or PVY^N and 47% did not transmit. There was no difference in the duration of probes that did or did not result in virus transmission. Statistical models were fitted to data on the frequency of transmission of PVY^O, PVY^N or both PVY^O and PVY^N by M. persicae and by aphids of poorer vector species, M. euphorbiae and Rhopalosiphum padi. Transmission of the two viruses ocurred independently of each other and consequently transmission of both was rare with M. euphorbiae and R. padi. Mineral oil applied to leaves infected with both strains diminished the frequency of transmission by M. persicae. Fitted models suggested that the aphids that probed through the oil droplets on leaves treated 30 min previously did not transmit virus, and that 24 h later, when the droplets had spread, aphids probing through them could transmit but with a decreased ability.