Viruliferous Insects Research Articles

Comparison of Four Serological Methods for Practical Detection of Ten Viruses of Rice in Plants and Insects

Four serological methods (ELISA, simplified ELISA, latex flocculation test [LF], and passive hemagglutination test [PHA]) were compared for practical detection of 10 viruses of rice in plants and viruliferous insects. All of the viruses were detected by ELISA and simplified ELISA. The detection of the viruses in plants with systemic infections was easier than the detection of phloem-restricted viruses. Some viruses in plants could not be detected in LF and PHA because of inhibition or nonspecific reactions. All of the viruses that are transmitted in a persistent manner were easily detected in individual insect vectors by ELISA and simplified ELISA, but some viruses were unable to detect by LF and PHA (.)

イネ縞葉枯抵抗性品種に対するイネウイルスの感染性

Rice varieties “Musashi-kogane”, “Tamaminori” and “Tamahonami” which have St2igene for resistance to rice stripe virus (RSV), showed resistance to RSV infection, but no resistance to virus multiplication. Furthermore, the vector Laodelphax striatellus acquired the virus readily on infected “Musashi-kogane” plants as did on susceptible “Nipponbare” plants. Sharp decline in the percentage of viruliferous insects in the overwintering population with the increase in the percentage areas planted with RSV-resistant varieties can be explained by the decrease of virus source plants for the secondary infection. The three RSV-resistant varieties did not show resistance to infection and multiplication of rice black-streaked dwarf virus (RBSDV) and rice dwarf virus (RDV). “Musashi-kogane” as well as “Nipponbare” served as the source of the three viruses for the transmission by their vectors.

Influence of introduction of resistant cultivars to rice stripe disease on fluctuation of the viruliferous insect vector, small brown plant hopper, and occurrence of the disease

Influence of introduction of resistant cultivars to rice stripe disease on fluctuation of the viruliferous insect vector, small brown plant hopper, and occurrence of the disease

Infection by Rice Gall Dwarf Virus of Cultured Monolayers of Leafhopper Cells

Summary Synchronous multiplication of rice gall dwarf virus (RGDV) and an assay of its infectivity in cell monolayers of its vector, the green rice leafhopper Nephotettix cincticeps, are described. The number of foci of infected cells was linearly related to the concentration of virus. The method was about 103 times more sensitive than enzymelinked immunosorbent assay. All the vector cells in monolayers were infected when inocula were dilutions of 10-3.5 of sap from infected plants, 10-4.5 of extracts of viruliferous insects, 10-5.5 of infected monolayer cells or purified virus with A 1cm 260nm = 10-5. RGDV was first detected in monolayer cells 10 h after inoculation, and multiplied 105-fold in the subsequent 40 h. Vector cell monolayers are thus an excellent experimental system for the study of RGDV.

Maize rough dwarf virus (Reoviridae) in its planthopper vector Laodelphax striatellus in relation to vector infectivity

SUMMARYThe infectivity of females of the planthopper vector Laodelphax striatellus given access to maize rough dwarf virus (MRDV) infected plants was assessed for up to 55 days from the end of the access period. A 3‐day inoculation access period was used, and this avoided intermittent transmission. Maximum infectivity was reached c. 30 days after acquisition access and the proportion of transmitter insects then remained constant.There was no difference in the efficiency of female L. striatellus in acquiring MRDV as third instar nymphs or as adults when compared in transmission tests 24, 30, 35 and 40 days after access to the virus.ELISA tests for MRDV subviral particles (SVPs) discriminated between individual viruliferous and non‐viruliferous insects from the 30th day after access.Of the viruliferous (ELISA positive) insects about 30% did not transmit MRDV and the proportion remained similar from 30 to 55 days after access. None of the non‐transmitter insects tested in serial transfer transmission tests was positive in ELISA.The concentration of SVPs detected by ELISA in the transmitter hoppers continued to increase exponentially, even after maximum infectivity was reached.

Production of monoclonal antibodies against rice stripe virus for the detection of virus antigen in infected plants and viruliferous insects.

Hybridoma cells that secrete monoclonal antibodies against rice stripe virus (RSV) were produced by fusing spleen cells of a BALB/c mouse immunized with RSV to a mouse myeloma cell line P3-X63-Ag8-U1 (P3U1). A single stable cell line with the highest titer in the avidin-biotin-peroxidase system to the homologous antigen was screened. The titer of the hybridoma ascitic fluid increased with the number of days after the injection of hybridoma cells to mice. The titers of the ascitic fluid were 800-1, 600 in precipitin ring interface test. The yield of the ascitic fluid was 11.4-22.5ml for a single mouse and was ca. 100ml for 6 mice. The titer and the yield of the ascitic fluid decreased with the serial passage of the hybridoma to new mice and the titers became about half that of the initial value after one to two passages through mice. The monoclonal antibodies were successfully used to detect virus antigen in virus infected plants and viruliferous insects by the latex flocculation test and enzyme-linked immunosorbent assay.

Detection of rice stripe virus from infected rice and viruliferous insects by latex flocculation test

Detection of rice stripe virus from infected rice and viruliferous insects by latex flocculation test

Rice black-streaked dwarf virus: Its properties, morphology and intracellular localization

The properties of rice black-streaked dwarf virus (RBSDV) were investigated by injection into insect vectors. The virus was inactivated by heating to 60°or 10 min, and no infectivity resulted when inocula prepared from either infected rice leaf or viruliferous insect extracts were diluted to 10 −5 and 10−6, respectively. Infectivity was lost by holding sap from infected leaves for 7 days at 4° or 6 days at 20°. No reduction of infectivity occurred after 5 days at 4° in insect sap or 232 days in frozen leaf tissues or after freezing and thawing three times. Carbon tetrachloride and fluorocarbon (Daifron S-3, trichlorotrifluoroethane) did not destroy infectivity. Chloroform-treated crude extracts lost all infectivity, but the purified virus was not affected by chloroform. The virus, purified by carbon tetrachloride and fluorocarbon treatment followed by differential centrifugation and sucrose density gradient centrifugations, was highly infectious and was seen as spherical particles 60 nm in diameter without any outer projections when viewed in 1% phosphotungstic acid (PTA)-stained preparations, which corresponds to the size of small particles (incomplete particles, 50–55 nm in diameter) which appeared within the viroplasm in the infected cells. High-titer antisera were obtained from these purified virus. Dip preparations made from paraformaldehyde- or osmic acid-fixed leaves and negatively stained in 1% PTA revealed complete virions 75–85 nm in diameter, which correspond to the large particles that appeared in the infected cell cytoplasm outside of the viroplasm. The large particles probably represent the complete virions of RBSDV, but the outer coat may be easily removed by the purification procedures or by PTA staining. Multiplication of the virus in its insect vectors was ascertained by the serial injection technique and by electron microscopic examination of insect organs. The virus multiplied in the fat body, salivary gland, intestine, and trachea. In the diseased plants, the virus was restricted to the veins. Italian cultivars of rice, wheat, barley, and maize plants were highly susceptible to RBSDV.

Electrophoretic separation of dsRNA genome segments from maize wallaby ear virus and its relationship to other phytoreoviruses

Partially purified maize wallaby ear virus (MWEV) preparations were shown to contain 10 double-stranded (ds) RNA segments. Electrophoretic mobilities of the dsRNAs of MWEV and of other phytoreoviruses have been compared in two buffers. The total genome molecular weight of maize wallaby ear virus was ∼18.92 × 10 6 and ∼19.33 × 10 6 in PTE and Loening's buffer, respectively. Extracts from the virus-free leafhopper vector, Cicadulina bimaculata, contained two dsRNA segments of molecular weight 1.15 × 10 6 and 1.08 × 10 6 in PTE buffer. Extracts from viruliferous insects also contained these same 2 dsRNA segments in addition to the 10 segments of maize wallaby ear virus. The electrophoretic pattern of dsRNA components of maize wallaby ear virus was similar to those of maize rough dwarf virus and Fiji disease virus suggesting a relationship between them. The degrees of relationship among these viruses and rice black-streaked dwarf virus, wound tumor virus, and rice dwarf virus were indicated by the electrophoretic patterns of their genome segments in the two buffers.

Multiplication of Rice Black-Streaked Dwarf Virus in its Plant and Insect Hosts

In a serial transfer from insect to insect by means of injecting inoculation of plant-hopper, Laodelphax striatellus Fallen, rice black-streaked dwarf virus (RBSV) was successfully carried to the 6th group of insects. The calculated dilution of the final inoculum would be 10-17, but the dilution end-point of RBSV in viruliferous insect juices was found to be below 10-5. The serial passage provided direct evidence for multiplication of RBSV in its insect vectors. The infectivity of RBSV was detected in salivary glands, intestine, fat body, hemolymph and ovarian tubules in viruliferous insects, but not in brain, Malpighian tubules, and testis. The virus was recovered first from the intestine 10 days after acquisition feeding on diseased rice plants, from aslivary glands and hemolymph after 13 days, and from fat body after 16 days.Two kinds of spherical particles were found mixed in the virus fractions isolated from viruliferous insects. One of which, the isometric particles of 70nm in diam., was also isolated from healthy insects and seemed to be latent virus of this insect vector. The other was 60nm in diam. being morphologically identical with RBSV isolated from the diseased plants.The virus was recovered from roots as well as from leaves of the diseased rice plants. Infectivity of the expressed juice from vein portions of the diseased rice leaves was much higher than from interveinal portions. When rice plants were inoculated with this virus, the infectivity of the expressed juice of the infected plants rapidly increased within 2 weeks and reached a maximum in 22 to 30 days, and was stabilized at high level for a period from 40 to 110 days after inoculation. In contrast, the amount of virus particles isolated from infected rice plants increased gradually to reach a maximum in 63 to 90 days after inoculation. Rice and corn plants were highly susceptible to RBSV, but the virus contents, when purified from diseased rice or corn plants, were extremely low. With same purification procedures, the contents in diseased barley, wheat, and oat plants were much higher than those in diseased rice and corn plants.

Latent Period and Retention of Potato Leaf-Roll Virus in its Vector, Myzus persicae

Green peach aphids, Myzus persicae Sulz. fed on the Physalis floridana Rydb. plants infected with potato leaf-roll virus (PLRV) for 3 hours, began to transmit the virus 9 hours after leaving the source plants. Aphids injected with extracts or blood from the viruliferous insects were also enable to transmit PLRV 6 hours after injection. The length of the latent period and the frequency of virus transmission in its aphid vector appeared to depend on the virus concentration within the vector. Inoculativity in the transmission of aphids tended gradually to increase till 24 hours after acquisition feeding or injection of extracts and blood from viruliferous insects.PLRV was detected in extracts and blood from aphids fed on the infected P. floridana plants for more than 3 hours, but no virus was recognized in them from insects given an hour's acquisition feeding. Even though the concentration of PLRV in the aphid vectors increased with the prolonged acquisition feeding, it decreased gradually with the time after leaving the source plants. The frequency of virus recovery from blood of the injected aphids also tended similarly to decrease gradually with the time after injection of the virus into aphids.The latent period of PLRV in the aphids vectors appeared to represent the time required for the virus to circulate within the insect body.

Transmission of rayado fino virus of maize (Zea mays) by Dalbulus maidis.

SUMMARYRayado fino virus (RFV) of maize (Zea mays) was transmitted by the leaf‐hopper Dalbulus maidis in a manner characteristic of viruses that multiply in their insect vectors. Individual insects fed on infected plants transmitted the virus after incubation periods of 8–22 days; males had shorter incubation periods than females but died sooner. Insects retained infectivity for 1–20 days. Transmission by most insects was intermittent. Inoculativity by D. maidis decreased with time, but the virus was recovered from insects that had lost their ability to transmit. Extracts of plants infected with RFV and viruliferous insects were injected into healthy insects, which became viruli‐ferous. Infectivity of the extracts was not affected by tetracycline hydrochloride (Achromycin). D. maidis was able to transmit simultaneously RFV and the corn stunt agent. Other than maize, Teosinte (Euchlaena mexicana) was the only plant susceptible to the virus, among a number of species of Gramineae tested.

The effect of insecticides on the spread of potato leaf roll virus in seed potato fields at Pukekohe

Abstract In recent years potato seed crops at Pukekohe have shown high levels of leaf roll virus infection, in spite of the use of insecticides. Laboratory and field tests have shown that although insecticides control aphid populations, they do not kill viruliferous insects quickly enough to prevent transmission of leaf roll to potato. High levels of leaf roll were seen in plots of potatoes that had emerged in a peak of aphid flight activity in the second half of November 1969, whereas plots emerging later were relatively free of virus. The evidence suggests that most of the virus seen in seed potato fields is brought there by viruliferous aphids migrating from infected crops in the neighbourhood. Isolation of seed fields from the Pukekohe district is advocated.

Screening Rice for Resistance to the Planthopper, Sogatodes oryzicola (Muir)1

Studies were conducted on resistance in rice (Oryza sativa L.) to direct damage caused by the planthopper, Sogatodes oryzicola (Muir). Reistance was expressed throughout the growing period of the plant. Nymphs, males, and females all caused damage and killing of susceptible rice varieties. Resistant to the insect was independent of resistance to the hoja blanca virus (HBV) vectored by the same species. A mass screening technique for evaluation of large numbers of varieties or plant selections was developed along with a scale for evaluation of damage.A survey of 534 varieties indicated that about 20% were highly resistant or resistant, 40% were intermediate, and 40% were susceptible. All resistant varieties were indicas from southeast Asia which showed little or no damage while susceptible ones were killed. Resistance appears to be highly heritable, is easily recombined with other agronomic traits, and is unassociated with plant pubescence. It is believed that resistance to the planthopper will protect varieties in the field from appreciable virus damage, even though the variety is susceptible to hoja blanca when the viruliferous insects are obliged to feed on it.

Effect of Resistant Rice Plants on Multiplication of the Planthopper, Sogatodes ozyzicola (Muir)1

Studies were conducted to determine the effect of resistant rice plants (Oryza sativa L.) on the multiplication of the planthopper Sogatodes oryzicola (Muir). Plant resistance affected all phases of the life cycle of both nonvirulifcrous and viruliferous insects. Resistance reduced the number of eggs deposited and the number of nymphs hatched, lowered nymphal survival, prolonged the nymphal period, and reduced adult longevity. Because of more eggs deposited per day, greater egg fertility, and increased nymphal survival, susceptible plants supported about 10 to 12 times as many nymphs as did resistant plants. Of these, about 3 to 5 times as many nymphs became adults on susceptible rice as on resistant rice.

The infection of leafhoppers by Western X-disease virus: V. Properties of the infectious agent

The concentration of extractable Western X-disease virus (WXV) in a vector, Colladonus montanus (the mountain leafhopper), was much higher than in any of several herbaceous plants tested. Leafhoppers were therefore used as virus source material for a survey of viral properties in crude extracts. A new method of bioassay was employed, based on the concentration dependence of the incubation period, in the insect, between injection of virus and transmission of WXV to plants. This bioassay permitted more accurate study of viral properties than has heretofore been possible with leafhopper-borne viruses. WXV activity was not recovered after saturation of saline extracts from leafhoppers with butanol. However, WXV survived brief ultrasonic treatments, brief emulsification with Genetron 113, and in limited trials, emulsification with chloroform. Freezing of whole viruliferous insects or saline extracts from such insects did not destroy the virus. Infectivity was present in centrifugal supernatants after 10 min at 8,000 g, but was almost completely sedimented after 10 min at 25,000 g. After rate zonal density gradient centrifugation for 20 or 25 min at 20,000 or 25,000 rpm, infectivity was found throughout the gradient column, but was most concentrated in the bottom one-third of the column. Viral infectivity was greatly reduced by overnight dialysis against saline, but not by dialysis against a buffer containing glycine, divalent cations, and cysteine, or by buffer transfer through Sephadex G-75 into a protective buffer containing Mg 2+ and glycine. Infectious WXV was recovered after brief exposure to various buffers at pH values from 5.1 to 9.7. Brief exposure to 0.01 m glycine or 0.01 m sodium borate buffers at pH 8.2 and 8.9, respectively, increased viral yield, but longer exposures to these buffers decreased the virus yield. Stability seemed best between pH 6.0 and 7.1. Recovery of virus from resuspended pellets after centrifugation at 80,000 g for 30 min was much better when 0.3 m glycine was used than any lower concentration of glycine whether the pH was 6.1, 7.5, or 8.5.

Multiplication of the Rice Stripe Virus in its Insect Vector, Laodelphax striatellus Fallen

The present paper describes the results of experiments carried out with the aim of obtaining evidences other than the transovarial virus transmission, of multiplication of the rice stripe virus in its insect vector, the smaller brown planthopper Laodelphax striatellus Fallen, and information about the possible site of virus multiplication and pathological effects of the virus on the insect vector.Serial transfers of the rice stripe virus from insect to insect were made using micro-injection method described by Maramorosch (1952). The virus was successfully carried in 4 serial passages in the smaller brown planthopper. The final dilution of the inoclum used to inject the last group of the series is calculated to be at least 1.25×10-6 of the original inoculum, while the virus derived from viruliferous insects was shown to have a dilution end-point between 10-2 and 10-3. Groups of planthoppers which had been injected with the juice from viruliferous insects, were macerated 2, 5, 10 and 15 days after the injection, and the preparations were tested for infectivity by inoculating to virus free insects. The inocula prepared from the planthoppers 2 and 5 days after injection were found noninfective, in coincidence with the incubation period in the vector. A 1:10 dilution of the macerate from planthoppers 10 days after injection and a 1:50 dilution of those from planthoppers 15 days after injection proved infective. This suggests that the virus may have multiplied in its vector at least fivehold during the period from 5th to 15th day after injection.Blood and various organs from viruliferous planthoppers were tested for the presence of virus by the injection method. Virus was recovered from eggs, male reproductive organs, and blood and fat body, while no virus was recovered from alimentary canal.Male planthoppers following 2 days of acquisition feeding on diseased rice plants were dissected to remove legs, wings, alimentary canal, Malpighian tubules and reproductive organ, and the remaining part was separated into head, thorax, and abdomen. These body parts were incubated for 13 days at 25-27°C in hanging drops of a medium consisting of Takami's salt solution, D-glucose and extract from their respective part. The virus was not recovered from fresh material of these parts, but was recovered from the abdominal part incubated in vitro for 13 days. Female and male reproductive organs excised from planthoppers which had fed for 3 days on diseased plants, were incubated for 12 days in vitro. From both organs virus was recovered after having been incubated for 12 days, but not before incubation.Cytological examinations of blood corpsucles, reproductive cells, salivary glands, alimentary canal, dermis, and fat body of viruliferous planthoppers showed no consistent, conspicuous changes in these tissues. Histochemical tests for hyaluronic acid, RNA, DNA, lipid, and phospholipid, and phosphatase activity showed no distinct difference between viruliferous and virus-free planthoppers. Relative amount of glycogen and of polysaccharides in the fat body and of mycetome, as determined by Bauer-Feulgen stain and by Lillie method, appeared to be somewhat less in viruliferous planthoppers than in virusfree ones.

Electron microscopy of wound tumor virus assembly sites in insect vectors and plants

Thin sections were prepared for electron microscopy from Agallia constricta insect vectors and from Trifolium incarnatum and Melilotus alba plants infected with wound tumor virus. Insects acquired the virus by feeding on wound tumor diseased plants. Electron microscopy of thin sections of various organs of viruliferous insects indicated that the virus is assembled in accumulations of electron-dense aggregates, designated viroplasms, formed in the cytoplasm of fat body, muscle, midgut, Malpighian tubules, trachea, salivary gland, and epidermal cells. The presence of viroplasms in the cells of the mycetome was not ascertained. The viroplasms are considered the sites of virus assembly. Careful examination of infected plants during early stages of disease revealed the formation of viroplasms in the root tumor cell cytoplasm and in the cytoplasm of cells of enlarged leaf veinlets. In plant cell sections prepared shortly after the appearance of first signs of the wound tumor disease, mature particles were found first at the periphery of the viroplasmic zone. A few days later virions were detected in increasing number inside the viroplasmic area. These morphological findings indicate that wound tumor virus is assembled in plant and insect host cells in viroplasms, formed within the cytoplasmic matrix proper. The similarity of wound tumor viroplasms with those of several viruses infecting higher animals, and the significance of the observations in the elucidation of the precise foci of virus assembly in cells and organs of plant and invertebrate animal hosts of the virus are discussed.

The Effect of Barley Yellow Dwarf Virus on the Biology of Its Vector the English Grain Aphid, Macrosiphum granarium12

The effect of the barley yellow dwarf virus on the biology of its vector, the English grain aphid, Macrosiphum granariiun (Kirby), was investigated. oxygen consumption of viruliferous and nonviruliferous aphids was measured with a Warburg respirometer. A comparison of the results indicated a reduction of 13.8% in the oxygen consumption of the viruliferous insects. The alteration in the metabolism of the infected individuals was apparent in their life histories. Longevity reproduction observations were conducted with 3 experimental populations. One group (NV-NV) consisted of nonviruliferous aphids that fed on virus-free leaves. A second group of aphids (V-V) was maintained exclusively on leaves from virus-infested plants. The nutritional factor was minimized by the third group of insects (V-NV) that was fed for 48 hr on virus-infected leaves and then reared on virus-free material. The 2 groups of viruliferous aphids tended to (1) have an increased rate of development from birth to adult, (2) live longer, (3) have a longer reproductive period, and (4) therefore produce greater total numbers of progeny. The observed differences are considered as evidence of an active biological interaction between the virus and its vector, which is difficult to classify as either beneficial or pathological.

SOME PROPERTIES OF WHEAT STRIATE MOSAIC VIRUS

Suspensions of wheat striate mosaic virus prepared with the sediments from high-speed centrifugation (105,000 g) of liquid extracts from viruliferous leafhoppers (Endria inimica Say), infected 63%, 46%, 33%, 5.5%, 3.5%, and 1.6% of the leafhoppers into which they were injected after dilution to 10−0.7, 10−1, 10−2, 10−3, 10−4, and 10−5 respectively. Suspensions of the virus were still infective after they were heated for 10 minutes at 45 °C, but not at 55 °C. Suspensions stored at 4 °C were highly infective after 3 days, but were toxic to the injected insects by the fourth day. Most infectivity was lost after 3 days at −10 °C. Wheat plants did not become infected when mechanically inoculated with suspensions which were infectious to leafhoppers. A specific particle was not correlated with infectivity when suspensions from viruliferous and healthy insects were compared in the electron microscope.