Selection, biological properties and fitness of resistance-breaking strains of Tomato spotted wilt virus in pepper

In glasshouse tests, infective sap from plants infected with 17 different isolates of Tomato spotted wilt virus (TSWV) from four Australian states was inoculated to three Capsicum chinense accessions (PI 152225, PI 159236 and C00943) carrying single genes that confer hypersensitive resistance to TSWV. The normal response to inoculation was development of necrotic (hypersensitive) local lesions in inoculated leaves without systemic invasion, but 3/1386 infected plants also developed systemic susceptible reactions in addition to hypersensitive ones. Similarly when two isolates were inoculated to C. chinense backcross progeny plants, 1/72 developed systemic susceptible reactions in addition to localised hypersensitive ones. Using cultures from the four plants with susceptible reactions and following three to five further cycles of serial subculture in TSWV‐resistant C. chinense plants, four isolates were obtained that gave systemic susceptible type reactions in the three TSWV‐resistant accessions, and in TSWV‐resistant cultivated pepper (C. annuum). When three of these isolates were inoculated to tomato (Lycopersicon esculentum) breeding lines with single gene resistance to TSWV, resistance was not overcome. Similarly, none of the four isolates overcame partial resistance to TSWV in Lactuca virosa. When TSWV isolates were inoculated to tomato breeding lines carrying partial resistance from L. chilense, systemic infection developed which was sometimes followed by ‘recovery’. After four successive cycles of serial passage in susceptible cultivated pepper of a mixed culture of a resistance‐breaking isolate with the non resistance‐breaking isolate from which it came, the resistance‐breaking isolate remained competitive as both were still found. However, when the same resistance‐ breaking isolate was cultured alone, evidence of partial reversion to wild‐type behaviour was eventually obtained after five but not four cycles of long term serial subculture in susceptible pepper, as by then the culture had become a mixture of both types of strain. This work suggests that resistance‐breaking strains of TSWV that overcome single gene hypersensitive resistance in pepper are relatively stable. The findings have important implications for situations where resistant pepper cultivars are deployed widely in the field without taking other control measures as part of an integrated TSWV management strategy.     

Comparison of resistance to potyviruses within Solanaceae: infection of potatoes with tobacco etch potyvirus and peppers with potato A and Y potyviruses

The reaction of several cultivated potato varieties (Solarium tuberosum L.) to three strains of tobacco etch potyvirus (TEV-F, TEV-Mex21 and TEV-ATCC) and the reaction of several pepper lines (Capsicum annuum L. and C. chinense L.) to two strains of potato Y potyvirus (PVY^O and PVY^N) and one strain of potato A potyvirus (PVA-M) was tested. The potato varieties included in this study carried resistance genes against PVY, PVA and potato V potyvirus, but all were susceptible to TEV and developed mottle and mosaic symptoms. TEV was readily transmitted by mechanical inoculation from tobacco and potato to potato, whereas transmission from pepper to potato occurred infrequently. TEV was transmitted through potato tubers, and from pepper to potato plants by aphids. Lack of detectable systemic infection following graft-inoculation indicated extreme resistance to PVY^O and PVA in several pepper lines. No pepper line was systemically infected with PVY^N following mechanical inoculation (graft-inoculation was not carried out with PVY^N). The development of necrotic lesions following mechanical and graft-inoculation indicated hypersensitive response to PVY^O in several pepper lines which resembled the resistance responses to these potyvirus strains in potato. Results of this study together with previous work indicate that C. annuum cv. Avelar is resistant to four potyviruses [PVY, PVA, pepper mottle potyvirus (PepMoV) and some isolates of TEV]; C. annuum cv. Criollo de Morelos and C. chinense PI 152225 and PI 159236 are resistant to three potyviruses (PVY, PepMoV and PVA; and PVY, PepMoV and TEV, respectively); C. annuum 9093–1 and 92016–1 are resistant to PVY and PepMoV; and C. annuum cv. Jupiter and C. annuum cv. RNaky are resistant to PVY^N and PVA.     

Systemic Infection of Tobacco by Pepper Veinal Mottle Potyvirus (PVMV) Depends on the Presence of Potato Virus Y (PVY)

In single inoculations, both PVY and PVMV replicated in inoculated leaves of Nicotiana tabacum cv. ‘Xanthi nc’ plants, but only PVY infected the tobacco plants systemically, whereas PVMV caused localized infection. A mixed infection by the PVY-To72 and PVMV-type strains was experimentally realized in ‘Xanthi nc’ plants. In the presence of PVY, PVMV migrated systemically into the upper leaves of the tobacco plant, as was proved by back inoculation. It would appear that in tobacco, PVY acts as a “helper” virus, providing PVMV with the necessary component factor for migration. In extracts from the co–infected leaves. Immune Electron Microscopy (IEM) revealed phenotypic mixed particles which contained a mixture of coat proteins of PVY and PVMV. The role of the structural and functional interactions between the two viruses, which enable PVMV to migrate systemically in tobacco plants, is discussed.     

Expression of genes for resistance to potato virus Y in potato plants and protoplasts

Plants of several potato clones with major gene resistance to potato virus Y (PVY) developed necrotic local lesions and systemic necrosis after manual inoculation with common (PVY_o) or veinal necrosis (PVY_N) strains of the virus. The clones reacted similarly, although their resistance genes are thought to be derived from four different wild species of Solarium. Mesophyll protoplasts from each clone became infected when inoculated with RNA of PVY_o by the polyethylene glycol method. The proportion of protoplasts infected, assessed by staining with fluorescent antibody to virus particles, was similar to that of protoplasts of susceptible potato cultivars. In contrast, plants of potato cultivars Corine and Pirola, which possess gene Ry from S. stoloniferum, developed few or no symptoms when manually inoculated or grafted with PVY_o. Moreover, only very few protoplasts of these cultivars produced virus particle antigen after inoculation with PVY_o RNA. The extreme resistance to PVY of cvs Corine and Pirola was therefore expressed by inoculated protoplasts whereas the resistance of the necrotic-reacting potato clones was not.     

Potato virus-Y multiplication in susceptible tobacco cultivar and transgenic breeding line producing coat protein mRNA

Changes in ribonucleases (RNases) and glucose-6-phosphate dehydrogenase (G6P DH) activities, their content and subcellular localisation were studied in relation to virus multiplication in susceptible (cv. Samsun) or resistant (transgenic breeding line NCTG 83) tobacco plants infected with the potato virus Y^N (necrotic strain of PVY). Activities of RNases and G6P DH from diseased susceptible tobacco plants were markedly increased during the experimental period and significantly correlated with the multiplication curve of the PVY^N. In contrast, the activities of RNases and G6P DH were not changed after PVY inoculation of resistant breeding line NCTG 83 producing the CP mRNA of PVY. Changes in the content and in the subcellular localisation of RNases and G6P DH isozymes were also determined in mesophyll protoplasts isolated from healthy as well as PVY^N infected plants of both cultivars by differential centrifugation of broken protoplasts on day eight post inoculation (the culmination of multiplication curve of PVY and enhanced activity of both enzymes). The chloroplasts fraction from infected protoplasts showed an enhanced content of RNases (192.4% when compared with that from healthy control ones), and of G6P DH (174.4 %). The cytosol fraction from infected protoplasts contained slightly enhanced levels of G6P DH (117.4 %) and considerably enhanced levels of RNases (141.7 %). No significant differences in the activities, contents and subcellular localisation of RNases and/or G6P DH isozymes were observed in the resistant line NCTG 83. This is in accordance with no detectable contents of PVY. This revised version was published online in July 2006 with corrections to the Cover Date.     

Subterranean clover mottle sobemovirus: its host range, resistance in subterranean clover and transmission through seed and by grazing animals

Subterranean clover mottle sobemovirus (SCMV) was transmitted by manual inoculation of sap to 27 cultivars belonging to three sub-species of subterranean clover. The virus readily infected systemically all inoculated plants of five susceptible cultivars of ssp. subterraneum. Ten others showed partial resistance as not all infected plants developed systemic infection; cold winter conditions further delayed or prevented systemic movement in four of them. Two cultivars of spp. brachycalycinum and four of spp. yanninicum failed to develop systemic infection following inoculation and were considered highly resistant. Resistance to SCMV in three of the spp. yanninicum was further confirmed by the failure to establish detectable primary infections in most of the inoculated leaves. Moreover, when the four ssp. yanninicum cultivars were graft-inoculated with SCMV, systemic infection eventually developed in them but the virus concentration was low. SCMV was also transmitted by manual inoculation of sap to a further 23 species of Trifolium, Medicago or Pisum. Three species were non-hosts, five were infected only in inoculated leaves and 18 others developed systemic infection in some or all plants. SCMV reached very high concentrations and was stable in subterranean clover sap. It was transmitted experimentally between subterranean clover plants by brushing infected leaves against healthy ones and in swards was readily transmitted by the trampling and grazing of sheep, but only poorly by mowing. Seed transmission of SCMV to seedlings of five cultivars of subterranean clover was low (0–0.12%). SCMV was not transmitted by Myzus persicae.     

辣椒种质资源抗青枯病的鉴定与评价

采用青枯菌FJC100301菌株对田间辣椒（Capsicum annuum）抗病品种76a和感病品种TW-1分别作了不同温度、不同接种量和不同接种方法的接种试验。结果表明,辣椒青枯病抗性的室内鉴定以接种温度28℃、浸根20 min和3×10^8cfu/mL接种浓度为宜;辣椒种质田间抗青枯病接种鉴定宜选择5月上旬进行,浸根20 min,接种浓度为3×10^8cfu/mL。采用田间抗性接种鉴定的方法,用青枯菌FJC100301菌株对106份辣椒材料进行了抗性鉴定。田间接种后每隔10 d统计病情指数,划分辣椒抗青枯病鉴定分级标准,获得了高抗材料14份、抗病材料8份、中抗材料23份、中感材料23份、感病材料20份、高感材料18份;采用离体叶片接种法对田间筛选得到的高抗和高感纯度较高品种进行抗性分析,结果与田间鉴定一致。     

Application of Vascular Puncture for Evaluation of Curtovirus Resistance in Chile Pepper and Tomato

Curtoviruses cause severe damage to tomatoes and peppers. Functional field resistance to curtoviruses in these plants is desirable but difficult to produce and difficult to screen for because it is time‐consuming and resistance could be achieved by developing resistance either to the virus or to insect feeding. To improve and speed curtovirus resistance testing in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants, two puncture methods were developed and compared to leafhopper inoculation and feeding preference assays. The two puncture methods were adapted to introduce a modified Agrobacterium tumefaciens plasmid carrying a recombinant curtovirus into the meristem tissue of tomato plants and into newly germinated chile pepper seedlings. The puncture techniques were used to screen for resistance to curtoviruses in chile pepper and tomato breeding lines and varieties. Similarly, the peppers and tomatoes were assayed for curtovirus resistance using leafhopper inoculation and feeding preference, which was assessed by stylet sheath staining. Virus infection by puncture and leafhopper feeding was monitored using PCR and ELISA. ELISA was performed using an antibody to bacterially expressed coat protein. While pepper cvs Tabasco, NuMex Las Cruces cayenne and New Mexico 6‐4 were infected using both puncture and leafhopper inoculation methods, New Mexico 6‐4 had higher infection rates than the other two cultivars. Stylet sheath staining results suggest that leafhoppers prefer to feed on New Mexico 6‐4 rather than Tabasco and NuMex Las Cruces cayenne. Eight tomato cultivars were infected using meristem removal injection inoculation. Three tomatoes cultivars (CVF‐11, Saladmaster and Supersteak) were infected using leafhopper inoculation, although stylet sheath staining results suggested that the first two cultivars were not preferred by the insect vector. Our results suggest that puncture methods and leafhopper inoculation are successful in resistance screening, and both methods should be used as part of screening, because they assess different types of resistance.     

Factors affecting aphid acquisition and transmission of soybean mosaic virus

Myzus persicae transmitted soybean mosaic virus (SMV) most efficiently following 30 or 60 s acquisition probes on infected plants. There were no differences in susceptibility to SMV infection of soybean plants 1 to 12 wk old, but symptoms were more severe in plants inoculated when young than when old. Soybeans inoculated between developmental stages R3 and R6 only showed yellowish-brown blotching on one or more leaves. There were no observable differences in the time of appearance or type of symptoms shown by soybean seedlings inoculated either by sap or by aphids; infected plants became acquisition hosts for aphids 5–6 days after inoculation. There was no change in the efficiency with which M. persicae transmitted SMV from source plants up to 18 wk after inoculation. M. persicae transmitted SMV from leaves of field-grown soybeans when plants were inoculated at developmental stages V6, R2, and R3 and tested as sources 57–74 days after inoculation but not from plants inoculated at R5 and tested as sources 14 to 32 days after inoculation. M. persicae acquired SMV from soybean buds, flowers, green bean pods, and unifoliolate, trifoliolate, and senescent leaves. Middle-aged and deformed leaves were better sources of the virus than buds, unfolding and old symptomless leaves. The results are being incorporated into a computer model of SMV epidemiology.     

Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Effects of Inoculation with a Mild Strain of Tomato Aspermy Virus on the Growth and Yield of Tomatoes and the Potential for Cross Protection

Mechanical inoculation of seedlings of 26 tomato cultivars with an attenuated strain of tomato aspermy virus (TAV-M, ATCC # PV361) resulted in development of only mild symptoms, which had largely disappeared by the time the infected plants had begun to set fruit. Yield trials revealed that plants inoculated with TAV-M when transplanted at the 6–8 leaf stage displayed no delay in fruit set and had yields only slightly less than uninoculated plants. Cross protection studies carried out in the greenhouse revealed that TAV-M has the potential to interfere with infection of tomato seedlings by a more severe TAV strain (ATCC # PV127). ‘Pik-Red’ seedlings inoculated with TAV-M showed a significant reduction in symptom expression when challenged with the Blencowe strain of TAV 21 days after the initial inoculation with TAV-M; interference was incomplete at 7 and 14 days.     

Occurrence of symptomless source of Piper yellow mottle virus in black pepper (Piper nigrum L.) varieties and a wild Piper species

Symptomless nature of Piper yellow mottle virus (PYMoV) infection in three varieties of black pepper (Piper nigrum) (Panniyur 1, Panniyur 5 and Panchami) and a wild species of Piper (Piper colubrinum) was confirmed by polymerase chain reaction (PCR) using PYMoV specific primers. The virus could be transmitted from these PYMoV-infected symptomless plants onto symptom producing black pepper cv. Karimunda through mealybug vector, Ferrisia virgata and by graft transmission. About 20–50% seedlings showed typical symptoms of the PYMoV at 30 days after mealybug inoculations while it was 75–94% at 90 days after inoculation. PCR test of the inoculated seedlings confirmed the presence of PYMoV in 50–64%, 76–100% and 80–100% of plants in 30, 60 and 90 days after inoculation, respectively. Similarly, 50–66%, 91–100% and 100% of graft-transmitted plants showed typical symptoms of the disease at 30, 60 and 90 days after grafting. PCR test of the graft-transmitted plants showed 100% PYMoV infection at 60 days after grafting. The results clearly demonstrated the existence of PYMoV-infected symptomless plants that can act as source for secondary spread of the virus in the field.     

Ultrastructural events during hypersensitive response of potato cv. Rywal infected with necrotic strains of potato virus Y

Potato plants cv. Rywal with hypersensitivity gene Ny-1 infected with PVY^N or PVY^NTN reacted in local necroses 3 days after infection. Potato virus Y (PVY) particles were found in epidermis, mesophyll, phloem and xylem cells in inoculated leaves. Noncapsidated virus particles (without capsid protein) were observed already 10 h after infection by using electron microscopy in situ. Capsid protein on one terminus of noncapsidated virus particles was located 5 days after inoculation with the use of immunogold labeling method. Whereas cytoplasmic inclusions were observed for the first time 24 days after infection during hypersensitive response. Ultrastructural studies showed that ER may take part in PVY RNA replication and capsidation of Potyvirus particles. Observed cytopathological changes and virus particles indicate that cell nucleus and mitochondrion might participate in PVY life cycle. During hypersensitive response PVY particles were found in plasmodesmata as well as in phloem and xylem.     

Some pea cultivars as local lesion hosts for potato virus X

SixteenPisum sativum cultivars were mechanically inoculated with two isolates of PVX. Distinct necrotic local lesions on the inoculated leaves were found in six cultivars 5 to 6 days after inoculation. The best results were obtained with the Meteor and Raman cultivars, 17 to 23 days old.     

Duration of effects of benomyl on growth and wilt (Verticillium dahliae) development in the strawberry, cv. Cambridge Vigour, in relation to time of inoculation

Single doses of benomyl, 0–4 g/plant in 1972 or 0–04 g in 1973, were applied in 100 ml water to the surface of the soil in 12-5 cm pots containing Cambridge Vigour strawberry plants, either before inoculation with Verticillium dahliae or at various times up to 56 days afterwards. Pre-inoculation treatments were terminated by washing the potting medium from the roots at the time of inoculation and their effects on pathogenesis and growth were relatively small. Post-inoculation treatments with benomyl prevented or suspended pathogenesis for at least as long as fungistatic activity could be detected in petiole segments by bioassay; this was for more than 150 days after the larger dose, 50–60 days after the smaller dose applied 7–28 days after inoculation and 30 days when applied 56 days after inoculation. There were no consistent differences in the uptake and persistence of benomyl in inoculated and non-inoculated plants. Early post-inoculation treatment caused some retardation of plant growth, but benomyl-treated inoculated plants were generally comparable in size with similarly treated non-inoculated ones, and much larger than untreated inoculated plants. Increasing the dose of benomyl applied to the soil apparently had little influence on the intensity of its effects but greatly increased their duration, probably because of the low solubility of the systemic chemical.     

Use of morphometric analysis for characterization of tobacco root growth in relation to infection byPhytophthora parasitica var.nicotianae

Development of tobacco root systems was characterized under controlled environmental conditions by use of morphometric root analysis. According to the classification scheme of this system, roots terminating in apical meristems are defined as first-order roots. Elements of second-order roots begin where two first-order roots merge, and so forth. Growth of root systems was similar for susceptible and resistant tobacco cultivars in nonautoclaved and autoclaved soils. During 15 days of growth subsequent to transplanting of 2-week-old plants, relative multiplication and extension rates of first-order and second-order roots were constant. Apparent unit extension rates of first-order and second-order root elements increased through 15 days of root system growth. Classification of tobacco root systems by the morphometric scheme provided a useful means of partitioning susceptibility of tissues to infection byPhytophthora parasitica var.nicotianae. Zoospores applied at the tips of first-order roots were most successful in causing infections; 73.3% of the roots inoculated with 16 zoospores per root tip became infected. Percentages of infections after inoculation of first-order root tissues 2 cm behind root tips or after inoculation of second-order roots were 10 and 4.3%, respectively.Florida Agricultural Experiment Station, Journal Series Paper 8106.     

The identification of mild and severe strains of tobacco mosaic virus in doubly inoculated tomato plants

Using specific antisera, it was possible to identify mild (MII-16) and the 0 and 1 strains of tobacco mosaic virus (TMV) in tomato plants infected with the mild strain following challenge inoculation with the wild strains. In addition, with single lesion isolates of the MII-16 and o strains it has been shown that both strains can be assayed simultaneously in mixed isolates on Nicotiana glutinosa based on differences in local lesion size. The accuracy of this differential local lesion assay was c. 90% as determined by back inoculation of single lesion isolates into susceptible tomato cultivars.     

Chemical treatment of soil to prevent transmission of tobacco rattle virus to potatoes by Trichodorus spp

Alternaria longipes (Ell. &Ev.) Mason survived on autoclaved maize stems for 6 months without losing its pathogenicity, but rapidly lost viability on non-autoclaved stems and could not be re-isolated 4 months after inoculation. In laboratory tests it infected both living and dead maize leaves. Some Alternaria isolates from non-solanaceous hosts infected tobacco leaves kept at high humidities for 10 days after inoculation, but not when this incubation period was reduced to 48 h. In the field, perennation on plants other than tobacco is unlikely to be important as a source of inoculum. Pathogenicity of Alternaria isolates was maintained from one season to the next when stored as conidia in sterile soil, or as dried, infected tobacco leaves; some isolates maintained on agar slopes under oil were still pathogenic after 5 years. Alternaria conidia collected from the surface of tobacco seedlings, and isolates from apparently healthy seedling leaves were pathogenic to mature tobacco. In the field conidia were detected on tobacco leaves soon after these emerged, and epiphytic colonies were occasionally found well in advance of symptoms. Many latent infections were also detected up to 5 weeks in advance of symptoms. Visual development of latent infections closely coincided with the end of leaf expansion.     

Molecular Analysis of the Coat Protein of Potato virus Y Isolates in Greece Suggests Multiple Introduction from Different Genetic Pools

The phylogenetic relationships among Potato virus Y (PVY) isolates from northern and southern Greece were investigated. A large part of coat protein gene of 49 tobacco isolates and three from pepper was examined. The analysis showed that all 52 isolates consisted of 34 distinct haplotypes, with only one haplotype found in both northern and southern regions. The southern population was more diverse than that from the north. The phylogenetic analyses of the Greek haplotypes alone or in combination with isolates from other countries using the maximum likelihood method classified unambiguously almost all the haplotypes examined. Nine tobacco haplotypes from the south were classified as C‐like (particularly C1), whereas 22 haplotypes from tobacco and two from pepper from both north and south were classified as N‐like. One tobacco haplotype from the south was found recombinant between N‐like and C1 lineages. The pattern of molecular evolution was examined using the fixed‐effects likelihood and the single‐likelihood ancestor counting methods. The analysis indicated that the evolution of PVY isolates appeared to be conservative (purifying selection and neutral evolution). These findings are discussed in relation to the introduction of PVY in the tobacco crop in Greece and the between region dispersal. A scenario of multiple introductions of PVY isolates in north and south Greece from different genetic pools and low or nil between region spread of the virus isolates was proposed.