Seasonal phenology of Aphis glycines (Hemiptera: Aphididae) and other aphid species in cultivated bean and noncrop habitats in Wisconsin

The occurrence of aphid-transmitted viruses in agricultural crops of the Midwest and northeastern United States has become more frequent since the arrival and establishment of the soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae). A. glycines is a competent vector of plant viruses and may be responsible for recent virus epidemics in Wisconsin snap bean, Phaseolus vulgaris L., fields. To determine whether vegetation surrounding crop fields could serve as sources of virus inocula, we examined the settling activity ofA. glycines and other aphid species in agricultural crops and noncrop field margins adjacent to snap bean fields. Noncrop field margins were made up of numerous virus-susceptible plant species within 10 m from snap bean field edges. During summers 2006 and 2007, horizontal pan traps were placed in commercial soybean [Glycine max (L.) Merr.], snap bean, and surrounding field margins to characterize aphid flight activity patterns in the different habitat types. Alate abundance and peak occurrence across years varied between crop and noncrop field margins and differed among patches of plants in field margins. Overall aphid activity peaked late in the season (21 August in 2006 and 28 July in 2007); with the majority (52%) of total aphids trapped in all habitats being A. glycines. Susceptibility to viral infection and confirmed visitation of A. glycines to these forage plants suggests the importance ofnoncrop habitats as potential sources of primary virus inoculum. Viral disease onset followed peak aphid flights and further implicates A. glycines as a likely vector of viruses in commercial bean and other crops in Wisconsin.     

Taxonomic patterns in the host ranges of viruses among grasses, and suggestions on generic sampling for host-range studies

Analyses of published host-range data for certain viruses reveal correlations with taxonomic groupings of grasses. Barley yellow dwarf virus (BYDV), cocksfoot mottle and phleum mottle viruses are found to have infected greater proportions of the festucoid grasses than of the non-festucoids to which they were inoculated. By contrast, all strains of sugarcane mosaic virus (SCMV) and of the closely related maize dwarf mosaic virus (MDMV) infected more non-festucoids than festucoids. In addition, infected plants from grass groups containing higher concentrations of genera susceptible to BYDV, SCMV and MDMV usually show clear symptoms, whereas infected plants from less susceptible groups are frequently symptomless. Some viruses, such as barley stripe mosaic, brome mosaic, cocksfoot streak and ryegrass mosaic, show no apparent preferences for particular grass groups. Samples of grasses employed in host-range studies are usually strongly biased towards festucoids. It is suggested that viruses ought to be adequately tested against genera from all the major groups, and a classified list of grass genera suitable for host-range studies is provided.     

Effect of sowing date and straw mulch on virus incidence and aphid infestation in organically grown faba beans (Vicia faba)

The effect of sowing date on aphid infestation and the incidence of aphid-transmitted viruses were investigated in organically managed, small-scale field experiments with two faba bean cultivars over 3 years (2002–04). As an additional factor, straw mulch was applied in 2 of the 3 years shortly before the start of vector activity in May. Virus incidence was determined using enzyme-linked immunosorbent assay and immunoelectron microscopy. Aphid flight activity was monitored using standard yellow water traps. Bean colonising aphids were assessed throughout the vegetation period by counting the number of plants infested with Acyrthosiphon pisum , Megoura viciae and Aphis fabae . Pea enation mosaic virus and bean yellow mosaic virus were the most abundant aphid-transmitted viruses, being detected in 22–54% and 9–69%, respectively, of the total number of virus-infected plants analysed per year. Further aphid-transmitted viruses found in faba bean were bean leaf roll virus, beet western yellows virus, clover yellow vein virus (in 2002) and soybean dwarf virus (in 2004). A. pisum was the predominant aphid species colonising faba bean plants. Early sowing compared with late sowing led to a significant reduction of the total virus incidence in faba bean in all 3 years. However, significantly decreased levels of A. pisum colonisation as a result of early sowing were observed only in 1 year and one cultivar. Irrespective of sowing date, straw mulching had no significant effects on virus incidence and aphid colonisation. Compared with late sowing, early sowing significantly increased bean yield in all 3 years and kernel weight in 2 years, whereas straw mulching had no effect on yield.     

Impact of 9 years of Bt-maize cultivation on the distribution of maize viruses

This study assesses the effect of Bt-maize on the distribution of maize viruses. Random surveys were conducted in Spain between 2001 and 2006 to evaluate the occurrence of maize viruses in Bt-maize cultivation areas and in areas where this crop had not been introduced. Maize dwarf mosaic virus (MDMV) was the predominant virus in Bt-areas, and Maize rough dwarf virus (MRDV) was the most predominant one in non-Bt-areas, with MRDV an emergent virus in both types of areas. A decline in the occurrence of MDMV and an increase in that of Sugarcane mosaic virus was observed in Bt-areas. Additionally, data obtained over 6 years in experimental fields showed non-significant differences between the infection rates exhibited by two generations of Bt varieties and the non-transformed isogenics varieties for any of the viruses. Our data suggest that differences in virus distribution are linked to the genetic background of the maize varieties and the distribution of virus reservoirs rather than to Bt-maize cultivation.     

Neem seed oil inhibits aphid transmission of potato virus Y to pepper*

Transmission of potato vims Y to sweet pepper by the green peach aphid, Myzus persicae (Sulzer), was inhibited by foliar applications of 1.0% or 2.0% neem seed oil to infected source plants or to uninfected recipient plants. Neem seed oil interfered with virus acquisition and inoculation in a manner comparable to that of a commercial horticultural oil, while an oil-free neem seed extract did not reduce rates of transmission compared with controls. The finding that neem seed oil inhibits virus transmission, while oil-free neem seed extract does not, suggests that the presence of the oil rather than biologically active limonoids such as azadirachtin interfere with virus transmission. None of the treatments affected rates of infection when potato virus Y was transmitted mechanically, or the resulting virus titre and symptom expression. In addition to direct control of insect pests, formulated neem oils may help reduce or delay the spread of non-persistent plant viruses.     

Aphid retention of maize dwarf mosaic virus (potyvirus): epidemiological implications

In total, 17 589 aphids were assayed for rate of loss of inoculativity and maximum retention times of maize dwarf mosaic (MDMV). The Standard-treatment, involved acquisition access to MDMV-infected tissue followed by confinement of active aphids in Petri dishes. In addition various aphid immobilisation treatments were used to prevent probing on solid surfaces after acquisition access to simulate conditions experienced by wind-borne aphids when aloft. Immobilisation treatments, using nitrogen or argon gases at 25°C, or cold treatments at 6°C after acquisition access greatly increased the efficiency of MDMV transmission by greenbugs, Schizaphis graminum, in an experimental design where insects were individually assayed for transmission over a 7 h period. Further tests in which groups of greenbugs were assayed for MDMV transmission revealed that MDMV strains may be retained for over 21 h, regardless of post-acquisition access treatment. Experiments with other aphid vectors of MDMV (Dactynotus ambrosiae, Macrosiphum euphorbiae, Rhopalosiphum maidis and Myzus persicae) also demonstrated MDMV retention times exceeding 18 h. These results show that the rate at which aphids lose MDMV inoculativity is lower when solid surface probing behaviour is denied, and that MDMV retention times are longer than those previously published. The findings are discussed in relation to the epidemiology of nonpersistent viruses and their dispersal over great distances.     

Evaluation of hairy nightshade as an inoculum source for aphid-mediated transmission of potato leafroll virus

Potato leafroll virus (PLRV) causes one of the most serious aphid-transmitted diseases affecting yield and quality of potatoes, Solanum tuberosum (L.), grown in the United States. The green peach aphid, Myzus persicae (Sulzer), is considered to be by far the most efficient vector of this virus. Even the most strict aphid control strategy may not prevent the spread of PLRV unless measures also are taken to keep virus source plants within and outside the crop at a minimum. Hairy nightshade, Solanum sarrachoides (Sendtner), is one of the preferred weed hosts for green peach aphid. The potential of this weed as an aphid reservoir and virus source and its spread or perpetuation were investigated. With the use of double antibody sandwich enzyme-linked immunosorbent assay, it was confirmed that green peach aphid can transmit PLRV to hairy nightshade and that aphids can become viruliferous after feeding on infected hairy nightshade plants. Transmission from hairy nightshade to potato is 4 times the rate of potato to potato or potato to hairy nightshade. The green peach aphid preferred hairy nightshade over potato plants and reproduced at a higher rate on hairy nightshade than on potato. Therefore, a low level of PLRV-hairy nightshade infection could enhance the disease spread in the field.     

The Natural Occurrence of Turnip Mosaic Potyvirus in Allium ampeloprasum

An isolate of turnip mosaic potyvirus (TuMV) was obtained from Allium ampeloprasum grown in commercial greenhouses in Israel. Symptoms on infected plants include systemic chlorosis and yellow stripes, accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically, and in a non-persistent manner by aphids, among Allium, Chenopodium. Gomphrena and some Nicotiana spp. Purified preparations contained numerous filamentous particles similar to those observed in crude extracts of infected leaves. Particles from crude plant extracts had a normal length of 806 nm. Cells of infected plants contained cylindrical cytoplasmic inclusions with pinwheel, scrolls and laminated aggregates which indicated the presence of a potyvirus of Edwardson's subgroup III. and which resemble those of turnip mosaic virus (TuMV), The virus reacted strongly with antiserum to typical isolates of TuMV in immunoelectron microscopy and western blotting but not with antisera to several other potyviruses. Based on serological reactivity, electron microscopy, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV.     

Temporal and spatial spread of Lettuce mosaic virus in lettuce crops in central Spain: factors involved in Lettuce mosaic virus epidemics

Lettuce mosaic virus (LMV) is transmitted by aphid vectors in a nonpersistent manner as well as by seeds. The virus causes severe disease outbreaks in commercial lettuce crops in several regions of Spain. The temporal and spatial patterns of spread of LMV were studied in autumn 2002 in the central region of Spain. Symptomatic lettuce (var. Cazorla) plant samples were collected weekly, first at the seedling stage from the greenhouse nursery and later outdoors after transplantation. The exact position of symptomatic plants sampled in the field was recorded and then material was tested by enzyme‐linked immunosorbent assay to assess virus infection. Cumulative spatial data for infected plants at different growth stages were analysed using spatial analysis by distance indices. For temporal analysis, the monomolecular, Gompertz, logistic and exponential models were evaluated for goodness of fit to the entire set of disease progress data obtained. The results indicated that the disease progress curve of LMV epidemics in the selected area is best described by a Gompertz model and that the epidemic follows a polycyclic disease progression. Our data suggest that secondary cycle of spread occurs when noncolonising aphid species land on the primary infected plants (probably coming from infected seed) and move to adjacent plants before leaving the crop. The role of weeds growing close to lettuce fields as potential inoculum sources of virus and the aphid species most likely involved in the transmission of LMV were also identified.     

The role of visual and olfactory plant cues in aphid behaviour and the development of non-persistent virus management strategies

Non-persistent viruses are transmitted by aphids in short feeding probes during the initial stages of aphid host plant selection behaviour. To control the transmission of these viruses, farmers rely on pesticides and cultural control practices, with varying success rates. As a result, there is a need for novel management practices that are more robust and specific to reducing aphid landing rates in crops. Aphid–plant–virus interactions involve a number of behaviours and processes to ensure survival of the insect vector and virus. So far, virus management tactics focused on reducing immigrating aphids in crops have emphasized the manipulation of visual rather than olfactory stimuli. An improved understanding of the synergistic or additive effects in which aphids use visual and olfactory stimuli to locate host plants could be used to improve on current non-persistent virus management tactics and develop novel strategies. The aim of this review is to evaluate current understanding of aphid vector behaviour and the ways that these behaviours have been exploited to develop management strategies, and to identify areas of research needed to further improve virus management.     

THE USE OF SOIL INSECTICIDES TO CONTROL POTATO APHIDS AND VIRUS DISEASES

A replicated trial was done to find whether the insecticides Thimet and Rogor applied in the soil affected the spread of aphid-transmitted viruses from infected to healthy plants with potato crops. The insecticides were applied at planting as activated carbon formulations at rates equal in cost to three sprays with DDT emulsion at 2 lb. DDT per acre. The infected plants were removed (rogued) in late June.
Thimet applied along the furrows with the fertilizer, and Rogor applied in individual doses beneath each tuber, kept the plants free from aphids from a week after the plants emerged until early August. Thimet in individual doses was less effective but greatly decreased the aphid infestation. All treatments prevented or greatly decreased the spread of leaf-roll virus, but they only slightly decreased the spread of virus Y. No treatment damaged the plants or depressed yields significantly.
Tubers harvested from the plots treated with insecticides contained only very small quantities of the insecticides, but shoots from them, when infested with adult Myzus persicae (Sulz.), carried fewer aphids a week after infestation than did shoots from control tubers. Shoots of tubers from treated plots also grew more slowly than those from the controls.
The aphicidal efficiency of Thimet applied as individual doses separated from the tubers by distances of up to 6 in., decreased as the distance increased, but the effect of distance became less as time passed.
Reasons for the differences in the behaviour of the insecticides are discussed, and the possibilities that the method offers to control virus diseases. The application of insecticides to soil promises to be a useful way of controlling the spread of viruses, provided the harvested crop is free from toxic residues.     

Epidemiology of an aphid nontransmissible potyvirus in fields of nontransgenic and coat protein transgenic squash

Spread of the aphid nontransmissible Zucchini yellow mosaicvirus virus (ZYMV) strain MV was monitored over two consecutive years in field plots of nontransgenic and transgenic squash expressing the coat protein (CP) gene of the aphid transmissible strain FL of Watermelon mosaic virus (WMV). The experimental approach was to mechanically inoculate plants with ZYMV strain MV and to assess subsequent transmissions, assumed to be vectored by aphids, of this strain to nonmechanically inoculated plants. Strain MV was distinguished from other ZYMV isolates by a threonine at position 10 of the CP or by a distinct electrophoretic pattern of a Nla IV-digested genomic cDNA fragment generated by RT-PCR. ZYMV strain MV was not detected in fields of nontransgenic plants, but was apparently aphid transmitted to 77 of 3,700 plants (2%) in transgenic fields. Despite the availability of numerous test plants and conditions of high disease pressure but low selection pressure, an epidemic of ZYMV strain MV did not develop in fields of transgenic plants. In contrast, the aphid transmissible ZYMV strain NY was aphid-transmitted to 99% (446/450) of transgenic plants under similar conditions. The relevance of these results in assessing environmental risks of transgenic plants expressing CP transgenes is discussed.     

Rate of Loss of Infectivity of Maize Dwarf Mosaic Virus by Schizaphis graminum Rondani After Different Acquisition Access Periods

The rate of loss of infectivity for greenbugs, Schizaphis graminum Rondani, carrying maize dwarf mosaic virus (MDMV) was similar for adult apterae and alates regardless of the acquisition access period (AAP) given. The rate was rapid during the first 2 h post-acquisition and then decreased. Weighted least squares linear regression of log % transmission were plotted against retention-time intervals to obtain regression slopes for each of the AAP groups tested (1, 3, 17, and 30 min). There were no significant differences among the regression slopes using F-test comparisons. In serial transmission trials, transmission frequencies among aphids allowed 1-min or 30-min timed acquisition probes followed by 30-see or 1-min timed inoculation probes were not significantly different when statistical tests that quantify categorical data were applied. This suggests little difference in the amount of virus obtained by these groups of aphids. Transmission and retention data are discussed in relation to the two principal theories of the mechanism of non-persistent virus transmission by aphids. Transmission of MDMV by the greenbug by a two mechanism process (stylet-borne and ingestion-egestion) is not confirmed.     

Aphid-transmitted viruses from lilies in Britain

Three aphid-transmitted viruses, tulip breaking, lily symptomless and cucumber mosaic, were obtained from lilies in Britain. Tulip breaking virus was detected by the leaf mottle produced in Lilium formosanum, cucumber mosaic virus by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and lily symptomless virus by electron microscopy of crude leaf extracts from symptomless L. formosanum. Liiy symptomless virus was transmitted by Myzus persicae, Macrosiphum euphorbiae, Aulacorthum solani and Aphis fabae. M. persicae, which in a small experiment appeared a more efficient vector than A. fabae, transmitted the virus in a non-persistent manner. This conflicts with the original report of transmission in a persistent manner by A. gossypii but no transmission by M. persicae. The possibility that there are two distinct viruses with similar pathological effects is discussed. Tulip breaking and lily symptomless viruses spread to bait plants of L. formosanum within a field planting of lilies in Scotland especially during July to September; lily symptomless virus was the more prevalent. No spread of cucumber mosaic virus was detected.     

Improvement of resistance to maize dwarf mosaic virus mediated by transgenic RNA interference

To overcome the low efficiency of agronomic protection from maize dwarf mosaic disease, susceptible maize inbred line was transformed by Agrobacterium tumefaciens harboring hpRNA expression vectors containing inverted-repeat sequences of different lengths targeting coat protein gene (CP) of maize dwarf mosaic virus (MDMV). After PCR screening and Southern blotting, the flanking sequences of the integration sites were amplified by thermal asymmetric interlaced PCR (TAIL-PCR) and used for analysis of T-DNA integration patterns. The T? plant lines were evaluated for their MDMV resistance in field inoculation trials under two environments. Of the nineteen T? plant lines positive in Southern blotting, six were evaluated as resistant to MDMV, and four of them had resistance non-significantly different from the highly resistant control "H9-21", while the resistance of the other eleven was proved to be significantly improved when compared to their non-transformed parent line. These improvements in MDMV resistance were verified by the relative amount of virus CP gene expression measured by quantitative real time PCR. Comparing the results of Southern blotting and TAIL-PCR analysis, different integration patterns of one or two copies of the inverted-repeat sequences were identified from non-repetitive and repetitive sequences of the maize genome. The MDMV resistance mediated by RNA interference is relative to the length of the inverted-repeat sequence, the copy number of T-DNA integration and the repeatability of integration sites. A longer hpRNA expression construct shows more efficiency than a shorter one.     

Location of the mechanism of resistance to Amphorophora agathonica (Hemiptera: Aphididae) in red raspberry

The aphid Amphorophora agathonica Hottes (Hemiptera: Aphididae) is an important virus vector in red (Rubus idaeus L.) and black (Rubus occidentalis L.) raspberries in North America. Raspberry resistance to A. agathonica in the form of a single dominant gene named Ag1 has been relied upon to help control aphid-transmitted plant viruses; however, the mechanism of resistance to the insect is poorly understood. Aphid feeding was monitored using an electrical penetration graph on the resistant red raspberry 'Tulameen' and compared with a susceptible control, 'Vintage'. There were no differences in pathway feeding behaviors of aphids as they moved toward the phloem. Once in the phloem, however, aphids feeding on resistant plants spent significantly more time salivating than on susceptible plants, and ingested significantly less phloem sap. This suggests that a mechanism for resistance to A. agathonica is located in the phloem. Reduced ingestion of phloem may result in inefficient acquisition of viruses and is a likely explanation for the lack of aphid-transmitted viruses in plantings of resistant cultivars.     

Alfalfa mosaic and cucumber mosaic virus infection in chickpea and lentil: incidence and seed transmission

Samples collected in 1994 and 1995 from commercial crops of chickpeas and lentils growing in the agricultural region of south-west Western Australia were tested for infection with alfalfa mosaic (AMV) and cucumber mosaic (CMV) viruses, and for members of the family Potyviridae using enzyme-linked immunosorbent assay (ELISA). In 1994 no virus was detected in the 21 chickpea crops tested but in 1995, out of 42 crops, AMV was found in two and CMV in seven. With lentils, AMV and/or CMV was found in three out of 14 crops in 1994 and 4 out of 13 in 1995, both viruses being detected in two crops in each year. Similar tests on samples from chickpea and lentil crops and plots growing at experimental sites, revealed more frequent infection with both viruses. No potyvirus infection was found in chickpeas or lentils in agricultural areas either in commercial crops or at experimental sites. However, bean yellow mosaic virus (BYMV) was detected along with AMV and CMV in irrigated plots of chickpeas and lentils at a site in Perth. When samples of seed from infected crops or plots of chickpeas and lentils were germinated and leaves or roots of seedlings tested for virus infection by ELISA, AMV and CMV were found to be seed-borne in both while BYMV was seed-borne in lentils. The rates of transmission found through seed of chickpea to seedlings were 0.1–1% with AMV and 0.1–2% with CMV. Seed transmission rates with lentil were 0.1–5% for AMV, 0.1–1% for CMV and 0.8% for BYMV. Individual seed samples of lentil and chickpea sometimes contained both AMV and CMV. With both species, infection with AMV and CMV was sometimes found in commercial seed stocks or seed stocks from multiplication crops of advanced selections nearing release as new cultivars. Seed-borne virus infection has important practical implications, as virus sources can be re-introduced every year to chickpea and lentil crops or plots through sowing infected seed stocks leading to spread of infection by aphid vectors, losses in grain yield and further contamination of seed stocks.     

Identification of a Strain of Peanut Chlorotic Streak Virus Causing Chlorotic Vein Banding Disease of Groundnut in India

A virus disease characterized by chlorotic vein banding, chlorotic line pattern along the margins or midrib of mature leaflets and chlorotic spots/rings was observed on commercial groundnut crops in Rayalaseema area of Andhra Pradesh with an incidence from 1% to nearly 60%. The virus was transmitted by mechanical inoculation in extracts prepared with 0.01 M potassium phosphate butter, pH 8.0 to 21 species from the Chenopodiaceae, Cruciferae, Leguminosae and Solanaceae, Chenopodium quinoa was found to be a good local lesion host. The virus was neither seed-transmitted through 1591 groundnut seeds nor aphid-transmitted by Aphis craccivora, Myzus persicae and Rhopalosiphum maidis either in non-persistent or semi-persistent manner. The virus remained infective in buffered tobacco leaf sap at a dilution of 10^?5; in a 10^?1 dilution of buffered sap the virus was infective for 2–3 days at 22–29°C or when heated to 65°C for 10 min but not to 70°C. Clarification treatments with organic solvents with 10% chloroform was least damaging. The virus was purified from Nicotiana rustica leaves. Purified virus contained isometric particles of 51 nm in diameter with an electron dense core of 22 nm and two major polypeptides of 76 kDa and 36 kDa. A polyclonal antiserum to this virus was produced. In agar gel double diffusion, enzyme-linked immunosorbent assay and in electro-blot immunoassay rests the virus was related to peanut chlorotic streak virus and not to cauliflower mosaic, figwort mosaic and soybean chlorotic mottle viruses.     

Seed Transmission of Maize Dwarf Mosaic Virus in Sweet Corn

Sweet corn seed from several maize dwarf mosaic virus (MDMV)-infected hybrids grown in the field were tested for transmission of MDMV through the seed. Seeds collected in 1979, 1980, 1981, and 1982, were germinated in the greenhouse the following winters. Only one seedling of 22,189 was MDMV-infected During the last three years, seed were dissected at different maturities and the seed parts tested for the presence of MDMV by both infectivity and enzyme-linked immunosorbent assay (ELISA). At 21 days after pollination, MDMV always was detected in the pericarp, but rarely in the endosperm or embryo. No MDMV was detected in the embryo of mature kernels, but virus occasionally was detected in the endosperm and pericarp. MDMV was regularly detected in unfertilized kernels and whole silks, but not in pollen by infectivity, ELISA or serological specific electron microscopy. MDMV was detected in glumes and whole anthers.     

RNA interference-mediated resistance to maize dwarf mosaic virus

Maize dwarf mosaic virus (MDMV) is a widespread pathogen that causes serious yield loss to maize crops. A hairpin RNA expression vector was constructed herein to overcome the low efficiency of cultural protection against MDMV and to improve the MDMV resistance mediated by a shorter transgenic inverted-repeat sequence. This expression vector contained a 451 bp inverted-repeat sequence, homologous to the protease gene (P1) of MDMV. It was used for the Agrobacterium tumefaciens-mediated transformation of maize calli induced from a susceptible inbred line. A total of 17 T₂ transgenic lines were identified by both specific PCR amplification and Southern blot hybridization. Of these lines, 15 were evaluated for MDMV resistance in inoculation field trials under two environments. The relative replication levels of the P1 gene were analyzed by quantitative real-time (qRT)-PCR. Results demonstrated that all of the 15 T₂ lines showed an enhanced resistance to MDMV in comparison with that of the non-transformed parent line. Six lines were deemed to be ‘resistant’ with an average disease index below 25 %, which was not significantly different from that of the resistant control. The relative replication levels of the virus gene were significantly reduced in these resistant T₂ transgenic lines. The efficiency of virus gene silencing was directly related to the transgene copy numbers presented in these transgenic lines.