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In the 1995 to 1996 season, severe viral disease symptoms were observed on carnations (Dianthus caryophyllus [hybrid Kooij Echo kgr]) propagated from imported seedlings on farms in central Taiwan. Disease symptoms began on upper leaves as numerous yellow spots that enlarged and fused into large chlorotic patches and expanded to cover entire leaves, which eventually became necrotic. Electron microscopy of crude extracts, purified preparations, and ultrathin sections of diseased tissues revealed the presence of isometric particles ≈32 to 33 nm in diameter. Earlier, in the 1994 to 1995 season, a strain of Lisianthus necrosis virus (LNV-L) was identified in lisianthus (Eustoma russellianum (Don.) Griseb) in a nearby nursery propagating seedlings (1). Both the lisianthus and carnations were imported from Europe. Chlorotic leaves from carnations reacted strongly with antiserum prepared against LNV-L in tissue blot immunoassay. Extracts of diseased leaves also reacted positively to LNV-L antiserum in both immunodiffusion and doubleantibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) tests. Mouse monoclonal antibodies prepared against LNV-L reacted positively based on indirect ELISA with extracts of chlorotic carnation leaves. The capsid protein of the carnation virus (LNV-D) was ≈38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, similar to the LNV-L coat protein (1), and reacted with LNV-L antiserum in western blot analysis. LNV-D differs biologically from LNV-Japan and LNV-L isolates previously reported in Japan and Taiwan, respectively (1,2). In experiments, LNV-D has induced systemic infection in many hosts that are either nonhosts or local-lesion hosts for LNV-Japan or LNV-L. D. caryophyllus L. and D. chinensis L. are susceptible to systemic invasion by LNV-D but are nonhosts for LNV-Japan and LNV-L. D. barbatus L. is a systemic host for LNV-D but a nonhost for LNV-L and has not been tested as a host for LNV-Japan. Chenopodium amaranticolor Coste & Reyn. and C. quinoa Willd. are systemic hosts for LNV-D but are local-lesion hosts for both LNV-Japan and LNV-L. Capsicum annuum L. is a systemic host for LNV-D and LNV-L but is not susceptible to LNV-Japan. Lycopersicon esculentum Mill. is a systemic host for LNV-D, a local-lesion host for LNV-L, and a nonhost for LNV-Japan. All three isolates systemically infect E. russellianum, the only systemic host for all three isolates tested. The first reports of LNV in Japan and later in Taiwan were in lisianthus. To our knowledge, this is the first report of the natural occurrence of LNV in imported carnation seedlings in Taiwan. LNV infection in Taiwan was only noticed once in lisianthus (1994 to 1995 season) and once in carnation (1995 to 1996 season) in farms propagating imported seedlings. LNV is transmitted by Olpidium sp. (2). Olpidium-like structures were not observed in Taiwan in rootlets of diseased carnation and lisianthus nor were they isolated from soil around diseased plants. Surveys of LNV in the nurseries and nearby areas in subsequent years have not found a new case of infection. We believe that LNV disease is not endemic in Taiwan and that its occurrence in lisianthus and carnation are one-time incidents caused by the importation of infected seedlings or contaminated culture matrices associated with the seedlings. References: (1) C. C. Chen et al. Plant Dis. 84:506, 2000. (2) M. Iwaki et al. Phytopathology 77:867, 1987.

Calla lilies are ornamental plants of major economic importance in Taiwan. They are grown in the central and northern areas of the island, and ≈3 million stems are shipped annually. Calla lilies are susceptible to several viruses (1). Infections by Cucumber mosaic virus, Dasheen mosaic virus, Turnip mosaic virus, and Watermelon silver mottle virus were reported in Taiwan. Recently, virus-like symptoms including yellow mottling, light yellow spot, yellow ringspot, and mosaic were observed on leaves of field-grown calla lilies from Changhua County, located in central Taiwan. In March 2001, a virus culture was isolated from diseased calla lilies and established in Chenopodium quinoa Willd. and Nicotiana benthamiana Domin. When inoculated with the virus, healthy calla lilies developed chlorotic spots that enlarged and fused to form large, yellow patches on inoculated leaves. Symptoms were similar to those on the naturally infected plants observed in the fields. The virus induced chlorotic local lesions on C. quinoa, C. ficifolium Sm., C. amaranticolor Coste & Reyn, Cucurbita moschata Duchesne ex Poir, Lisianthus russellianum (Don.) Griseb, Phaseolus angularis Wight, Vigna angularis Willd., and V. radiata (L.) Wilczek. In addition to the localized chlorotic spots on inoculated leaves, systemic invasion of the virus was also observed 8 to 10 days postinoculation in Dianthus caryophyllus L., D. chinensis L., and Glycine max Merr. In N. benthamiana, the only symptom observed was systemic wilting. Examination of 2% of uranyl-acetate-stained samples using electron microscopy revealed the presence of spherical particles ≈34 to 35 nm in diameter in crude extracts of leaves of diseased calla lilies, or infected C. quinoa. Similar particles were also observed in the cytoplasm but not in the nuclei in ultrathin sections of virus-infected leaf tissues of C. quinoa and N. benthamiana. Differential centrifugation followed by sucrose density gradient centrifugation of tissue extracts of infected C. quinoa yielded virions with similar size. Sodium dodesyl sulfate polyacrylamide gel electrophoresis of the purified virus showed a single structural polypeptide ith a Mr of 41.6 kDa. The viral antigen reacted positively with its homologous antiserum and an antiserum against Carnation mottle virus (CarMV; Agdia, Inc., Elkhart, IN) in double antibody sandwich enzyme-linked immunosorbent assay. Using primers 5′-CTCCATGGTCATGGAA(A/G)ATAAA GGAGAA and 3′-CAACAAATATCCTACACTGTCCTAGGTG specific to the coat protein (CP) gene of CarMV, an expected viral CP gene product of 1.05 kb was amplified by reverse transcription-polymerase chain reaction from total RNA isolated from infected N. benthamiana. Comparisons of the 1,047-nucleotide CP gene with those of 15 CarMV isolates available in GenBank showed 94.6 to 98.2% nucleotide identity and 94.8 to 96.8% amino acid identity. Results from current studies indicate that the virus infecting calla lilies is an isolate of CarMV. To our knowledge, this is the first report of CarMV infection in calla lilies. The occurrence of CarMV in calla lilies has direct implication for the economically important nursery and floral industry in Taiwan. Reference: (1) F. W. Zettler and R. D. Hartman. Dieffenbachia, Caladium, and Zantedeschia. Pages 464–470 in: Virus and Virus-Like Diseases of Bulb and Flower Crops. G. Loebenstein, R. H. Lawson, and A. A. Brunt, eds. John Wiley and Sons, West Sussex, U.K., 1995.

A new tospovirus, Calla lily chlorotic spot virus (CCSV), was isolated from calla lilies (Zantedeschia spp.) in Taiwan. Chlorotic spots, ranging from light green to yellow, appear on the middle leaves of the affected plants. Virions measuring 75 to 105 nm, similar in size to tospovirus particles, were present in crude extracts and ultrathin sections of diseased leaves. Of 35 plant species inoculated mechanically, 24, including wax gourd (Benincasa hispida) and zucchini squash (Cucurbita pepo), were susceptible to the virus. CCSV was transmitted from infected wax gourd by Thrips palmi to healthy wax gourd and zucchini squash. The virus was weakly related to Watermelon silver mottle virus (WSMoV) in enzyme-linked immunosorbent assay (ELISA) and western blot tests. WSMoV-specific N gene primers, however, failed to produce DNA fragments from total RNA extracts of CCSV-infected plants in reverse transcription-polymerase chain reaction (RT-PCR). Results of RT-PCR show that the conserved regions of the L genes of tospoviruses are present in CCSV.

Allamanda (Allamanda cathartica L., family Apocynaceae) is native to Brazil and is a popular perennial shrub or vine ornamental in Taiwan. Plants showing severe mosaic, rugosity, and leaf distortion symptoms on leaves are common in commercial nurseries and private gardens. Examination of crude sap prepared from symptomatic leaves using an electron microscope revealed the presence of spherical virus particles with a diameter of approximately 28 nm. The virus was mechanically transmitted to indicator plants and induced symptoms similar to those incited by Cucumber mosaic virus (CMV). The virus caused local lesions on inoculated leaves of Chenopodium quinoa and C. amaranticolor and systemic mosaic in Cucumis sativus, Lycopersicon esculentum, Nicotiana benthamiana, N. glutinosa, N. rustica, and N. tabacum. On N. tabacum, necrotic ringspots developed on inoculated leaves followed by systemic mosaic. Tests of leaf sap extracted from naturally infected allamanda and inoculated indicator plants using enzyme-linked immunosorbent assay were positive to rabbit antiserum prepared to CMV. Viral coat protein on transblots of sodium dodecyl sulfate-polyacrylamide gel electrophoresis reacted with CMV subgroup I specific monoclonal antibodies (2). With primers specific to the 3′-half of RNA 3 (1), amplicons of an expected size (1,115 bp) were obtained in reverse transcription-polymerase chain reaction (RT-PCR) using total RNA extracted from infected allamanda and N. benthamiana. The amplified fragment (EMBL Accession No. AJ871492) was cloned and sequenced. It encompasses the 3′ part of the intergenic region of RNA 3 (158 nt), CP ORF (657 nt), and 3′ NTR (300 nt) showing 91.8–98.9% and 71.4–72.8% identities to those of CMV in subgroups I and II, respectively. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and the nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB, which is predominant on the island. To our knowledge, CMV is the only reported virus that infects allamanda and was first detected in Brazil (3), and this is the first report of CMV infection in allamanda plants occurring in Taiwan. References: (1) Y. K. Chen et al. Arch. Virol. 146:1631, 2001. (2) H. T. Hsu et al. Phytopathology 90:615, 2000. (3) E. W. Kitajima. Acta. Hortic. 234:451, 1988.

Lisianthus necrosis virus (LNV) was first identified as a fungus-borne virus that induced systemic necrosis in lisianthus (Eustoma russellianum) in Japan (2). In Taiwan, LNV causes systemic bright yellow chlorosis followed by necrosis in lisianthus (1). The disease was able to spread through the infested soil. Isolation of a fungus vector was attempted but was not successful (1). Calla lilies (Zantedeschia spp.) showing symptoms of systemic necrosis were observed in the fields of central Taiwan. A virus culture was established through single-lesion isolation from a local lesion host, Chenopodium quinoa, and maintained in Nicotiana benthamiana. Mechanical inoculation of the virus resulted in systemic infection in E. russellianum and Datura stramonium and local infection in Celosia argentea, Gomphrena globosa, Chenopodium amaranticolor, Zinnia elegans, Cucumis melo, Cucumis sativus, Cucurbita pepo, Vigna angularis, and Petunia hybrida. Electron microscopic examination of ultrathin sections of infected plant tissues revealed the presence of spherical viral particles approximately 33 nm in diameter. Scattered and aggregated virion particles were frequently observed in the cytoplasm of infected cells. Results of enzyme-linked immunosorbent assay, western blotting, and immunoelectron microscopy indicate that the virus is serologically related to LNV (1). Reverse transcription-polymerase chain reaction (RT-PCR) with degenerate primers (forward primer 5′-ATGGAAATCGTTAGG and reverse primer 5′-CTATAGCAATGTTGC) for LNV coat protein gene produced a cDNA of approximately 1.1 kb. The RT-PCR product was cloned into pGEM-T vector (Promega, Madison, WI) and sequenced. Sequence analysis showed that the cloned fragment (GenBank Accession No. DQ523229) was 1,167 bp long and shared 99% identity at the nucleotide and deduced amino acid levels with that of the LNV isolated from lisianthus (GenBank Accession No. DQ011234). To our knowledge, this is the first report of the natural occurrence of LNV infection in calla lily. References: (1) C. C. Chen et al. Plant Dis. 84:506, 2000. (2) M. Iwaki et al. Phytopathology 77:867, 1987.

Plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were transformed with single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or sugarcane Ubi9 promoters. Integration of the transgenes was verified by Southern hybridization. Plants were challenged in vitro by inoculating with purified CMV using the Helios hand-held gene gun. An initial pathogen challenge was performed using six plants/plant line for the 51 lines containing the CMV subgroup I and 37 lines with the CMV subgroup II scFv fragments. Four plant lines with the CMV subgroup I scFv and four lines with the CMV subgroup II scFv were selected for further challenging. Less than 30% of the plants challenged from two plant lines with the CMV subgroup I scFv and three plant lines with the CMV subgroup II scFv contained CMV as compared to 70% for the non-transformed control plants. Less than 40% of the plants from two other plant lines with the CMV subgroup I scFv contained CMV 1–2 months after challenge. These eight plant lines that showed resistance and two susceptible lines expressed the transgene as determined by Northern hybridization and reverse transcriptase PCR.

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.

Viral, bacterial, and fungal diseases of ornamental plants cause major losses in productivity and quality. Chemical methods are available for control of fungal diseases, and to a lesser extent for bacterial diseases, but there are no economically effective chemical controls for viral diseases except to control vector species. Host plant resistance is an effective means of controlling plant diseases, and minimizing the necessity for the application of pesticides; however, there are many ornamentals in which no natural disease resistance is available. It is possible to introduce resistance derived from other species, or even from the pathogen itself, by genetic engineering. This allows the introduction of specific, or in some instances broad spectrum, disease resistance into plant genotypes that have been selected for desirable horticultural characters; in contrast, introduction of natural resistance by traditional breeding may take many cycles of breeding to combine disease resistance with desirab...

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.

A serologically and cytologically distinct gloxinia tospovirus (HT-1) previously isolated from a gloxinia plant infected with Impatiens necrotic spot virus (INSV) when propagated in a high-temperature environment was characterized. Rabbit antisera produced for INSV and Tomato spotted wilt virus (TSWV) nucleocapsids (N) failed to react with HT-1 proteins in western blot analysis. The HT-1 antibodies reacted strongly with homologous antigen but failed to react with INSV and TSWV. However, the HT-1 antiserum reacted in ELISA with Watermelon silver mottle virus (WSMV) from Taiwan and in western blot analysis with the WSMV N protein. A reciprocal test showed that the antiserum prepared against the N protein of WSMV also reacted with the HT-1 N protein in both ELISA and western blot analysis. DNA probes derived from the N gene of HT-1 or WSMV hybridized to RNAs prepared from plants infected with either virus. Stronger signals were obtained with homologous than with heterologous reactions. Neither probe detected INSV or TSWV. The M and S RNAs of HT-1 were sequenced. The M RNA contains two open reading frames (ORF) ; one in the sense orientation encoding a nonstructural (NSm) protein of 308-amino-acids (aa) and the other in the ambisense orientation, a 1122-aa precursor of Gl and G2 glycoproteins. The S RNA also contains two ORFs ; one in the sense orientation encoding a nonstructural (NSs) protein of 439 aa and the other in the ambisense orientation, an N protein of 277 aa. HT-1 is distantly related to INSV and TSWV as shown by low nucleotide (40–52%) and amino acid (28–48%) similarities in the four ORF sequences. The HT-1 virus shares high nucleotide (76–81%) and amino acid (85–92%) similarities with WSMV and peanut bud necrosis virus (PBNV). Based on the serological properties and sequence data, we propose that HT-1 is a distinct species of serogroup IV in the genus Tospovirus. This is the first time that a tospovirus similar to those found in the Far East and in Southeast Asia has been identified in the US.

Acidification of endomembrane compartments by the vacuolar-type H 1 -ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V 1 ) and a membrane integral sector (V o ); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein‐protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V 1 V o -ATPases at the endoplasmic reticulum.

Development time and life table parameters of Thrips palmi Karny, a pest new to the continental United States, were measured at 3 temperatures on 4 host plants. Net reproductive rate was the highest because of greatest survival and highest egg production when thrips were reared at 26°C on winter melon, Benincasa hispida (Thunb.) Cogn., eggplant, Solanum melongena L., and cucumber, Cucumis sativus L. , compared with bell pepper. However, because of shorter development time, the intrinsic rates of natural increase for T. palmi were highest when individuals were reared at 32°C on these 3 host plants. Although development times were similar on all 4 hosts at 26°C, survival and reproduction were much lower for thrips reared on bell pepper, Capsicum annuum L. , leaves. At 15 and 32°C, these differences were even greater, with only 40 and 48% of thrips reared on bell pepper surviving at the respective temperatures. T. palmi was able to tolerate the low temperature (56% mortality when held at O°C for 15 h) much better than the high temperature (100% mortality at 40°C for 15 h), which may partially explain why T. palmi populations are high in winter and spring and low in the summer in Florida. This study provides information needed to understand population dynamics of thrips in important vegetable crops, information which will be valuable in developing and evaluating cultural and biological controls for this extremely damaging pest.

To understand the origin of vacuolar H+ -ATPases (V-ATPases) and their cellular functions, the subcellular location of V-H+ -ATPases was examined immunologically in root cells of oat seedlings. A V-ATPase complex from oat roots consists of a large peripheral sector (V1) that includes the 70-kD (A) catalytic and the 60-kD (B) regulatory subunits. The soluble V1 complex, thought to be synthesized in the cytoplasm, is assembled with the membrane integral sector (V0) at a yet undefined location. In mature cells, V-ATPase subunits A and B, detected in immunoblots with monoclonal antibodies (Mab) (7A5 and 2E7), were associated mainly with vacuolar membranes (20–22% sucrose) fractionated with an isopycnic sucrose gradient. However, in immature root tip cells, which lack large vacuoles, most of the V-ATPase was localized with the endoplasmic reticulum (ER) at 28 to 31% sucrose where a major ER-resident binding protein equilibrated. The peripheral subunits were also associated with membranes at 22% sucrose, at 31 to 34% sucrose (Golgi), and in plasma membranes at 38% sucrose. Immunogold labeling of root tip cells with Mab 2E7 against subunit B showed gold particles decorating the ER as well as numerous small vesicles (0.1–0.3 [mu]m diameter), presumably pro-vacuoles. The immunological detection of the peripheral subunit B on the ER supports a model in which the V1 sector is assembled with the V0 on the ER. These results support the model in which the central vacuolar membrane originates ultimately from the ER. The presence of V-ATPases on several endomembranes indicates that this pump could participate in diverse functional roles.

Conditions for the dissociation and reassembly of the multi-subunit vacuolar proton-translocating ATPase (H(+)-ATPase) from oat roots (Avena sativa var Lang) were investigated. The peripheral sector of the vacuolar H(+)-ATPase is dissociated from the membrane integral sector by chaotropic anions. Membranes treated with 0.5 molar KI lost 90% of membrane-bound ATP hydrolytic activity; however, in the presence of Mg(2+) and ATP, only 0.1 molar KI was required for complete inactivation of ATPase and H(+)-pumping activities. A high-affinity binding site for MgATP (dissociation constant = 34 micromolar) was involved in this destabilization. The relative loss of ATPase activity induced by KI, KNO(3), or KCl was accompanied by a corresponding increase in the peripheral subunits in the supernatant, including the nucleotide-binding polypeptides of 70 and 60 kilodaltons. The order of effectiveness of the various ions in reducing ATPase activity was: KSCN > KI > KNO(3) > KBr > K-acetate > K(2)SO(4) > KCl. The specificity of nucleotides (ATP > GTP > ITP) in dissociating the ATPase is consistent with the participation of a catalytic site in destabilizing the enzyme complex. Following KI-induced dissociation of the H(+)-ATPase, the removal of KI and MgATP by dialysis resulted in restoration of activity. During dialysis for 24 hours, ATP hydrolysis activity increased to about 50% of the control. Hydrolysis of ATP was coupled to H(+) pumping as seen from the recovery of H(+) transport following 6 hours of dialysis. Loss of the 70 and 60 kilodalton subunits from the supernatant as probed by monoclonal antibodies further confirmed that the H(+)-ATPase complex had reassembled during dialysis. These data demonstrate that removal of KI and MgATP resulted in reassociation of the peripheral sector with the membrane integral sector of the vacuolar H(+)-ATPase to form a functional H(+) pump. The ability to dissociate and reassociate in vitro may have implications for the regulation, biosynthesis, and assembly of the vacuolar H(+)-ATPase in vivo.

Immunoassays are among the most powerful and useful techniques for analysis of biological materials. There are numerous variations in which immunoassays can be performed. Coupled with enzyme, using chromogenic substrates, the enzyme immunoassay technique is used to trace the target antigen in tissues. The technique also is used to measure the concentration of antigen in tissue extracts. This chapter provides fundamental information that is needed to carry out the routinely used procedures in plant virus research.

The quality of reagents greatly affects the interpretation of serological tests. Methods used in conventional viral purification and molecular cloning and expression of target viral proteins to obtain antigens for immunization are presented. Immunization of rabbits, mice and chickens and isolation of immunoglobulin from immunized animals also are described.

Immunoassays are among the most powerful and useful techniques for analysis of biological materials. There are numerous variations in which immunoassays can be performed. Coupled with enzyme, using chromogenic substrates, the enzyme immunoassay technique is used to trace the target antigen in tissues. The technique also is used to measure the concentration of antigen in tissue extracts. This chapter provides fundamental information that is needed to carry out the routinely used procedures in plant virus research.

The quality of reagents greatly affects the interpretation of serological tests. Methods used in conventional viral purification and molecular cloning and expression of target viral proteins to obtain antigens for immunization are presented. Immunization of rabbits, mice and chickens and isolation of immunoglobulin from immunized animals also are described.

Calla lily chlorotic spot virus (CCSV) isolated from central Taiwan was recently identified as a tospovirus serologically but distantly related to Watermelon silver mottle virus (WSMoV). To clarify the serological relationship between the two viruses, rabbit polyclonal antibody (PAb) to CCSV and mouse monoclonal antibodies (MAbs) to WSMoV NP or CCSV NP were produced in this investigation, using purified nucleocapsid protein (NP) as immunogens. The PAb to CCSV NP reacted stronger with the homologous antigen than with the heterologous antigen, with much lower A405 readings in indirect enzyme-linked immunosorbent assay (ELISA) and low-intensity banding in immunoblotting. MAbs produced to CCSV NP or WSMoV NP reacted specifically with the homologous antigens but not with the heterologous antigens in both ELISA and immunoblot analyses. The CCSV S RNA was determined to be 3,172 nucleotides in length, with an inverted repeat at the 5′ and 3′ ends and two open reading frames encoding the NP and a nonstructural (NSs) protein in an ambisense arrangement. A typical 3′-terminal sequence (5′-AUUGCUCU-3′) that is shared by all members of the genus Tospovirus also is present in the CCSV S RNA. The CCSV NP and NSs protein share low amino acid identities of 20.1 to 65.1% and 19.9 to 66.1%, respectively, with those of reported tospoviruses. Phylogenetic dendrogram analysis indicates that CCSV is a distinct member in the genus Tospovirus. The results provide evidence that CCSV is a new species in the genus Tospovirus and belongs to WSMoV serogroup.

A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.

A new method of inoculation of gladiolus with cucumber mosaic virus (CMV) was developed using the Bio-Rad Helios Gene Gun System. This method circumvents the traditional use of aphids to transmit CMV, a virus that is mechanically transmissible to many plant species but only with difficulty to gladiolus. Cartridges containing virus-coated gold microcarriers were prepared and the virus shot into Nicotianabenthamiana leaves and gladiolus corms and cormels. The biolistic procedure successfully transmitted three CMV isolates, two from serogroup I and one from serogroup II. Survival rates of two cultivars of gladiolus cormels and corms in sterile and non-sterile environments were compared. Infection rates of 100% were obtained when as little as 2 microg of virus was used in cartridge preparation. CMV remained viable after the cartridges were stored for many months at 4 degrees C.

Objectives. The major aim of the proposed research was to establish an efficient and reproducible genetic transformation system for Easter lily and gladiolus using either biolistics or Agrobacterium. Transgenic plants containing pathogen-derived genes for virus resistance were to be developed and then tested for virus resistance. The proposal was originally aimed at studying cucumber mosaic virus (CMV) resistance in plants, but studies later included bean yellow mosaic virus (BYMV). Monoclonal antibodies were to be tested to determine their effectiveness in interning with virus infection and vector (aphid) transmission. Those antibodies that effectively interfered with virus infection and transmission were to be cloned as single chain fragments and used for developing transgenic plants with the potential to resist virus infection. Background to the topic. Many flower crops, as lily and gladiolus are propagated vegetatively through bulbs and corms, resulting in virus transmission to the next planting generation. Molecular genetics offers the opportunity of conferring transgene-mediated disease resistance to flower crops that cannot be achieved through classical breeding. CMV infects numerous plant species worldwide including both lilies and gladioli. Major conclusions, solutions and achievements. Results from these for future development of collaborative studies have demonstrated the potential transgenic floral bulb crops for virus resistance. In Israel, an efficient and reproducible genetic transformation system for Easter lily using biolistics was developed. Transient as well as solid expression of GUS reporter gene was demonstrated. Putative transgenic lily plantlets containing the disabled CMV replicase transgene have been developed. The in vitro ability of monoclonal antibodies (mAbs) against CMV to neutralize virus infectivity and block virus transmission by M. persicae were demonstrated. In the US, transgenic Gladiolus plants containing either the BYMV coat protein or antisense coat protein genes have been developed and some lines were found to be virus resistant. Long-term expression of the GUS reporter gene demonstrated that transgene silencing did not occur after three seasons of dormancy in the 28 transgenic Gladiolus plants tested. Selected monoclonal antibody lines have been isolated, cloned as single chain fragments and are being used in developing transgenic plants with CMV resistance. Ornamental crops are multi-million dollar industries in both Israel and the US. The increasing economic value of these floral crops and the increasing ban numerous pesticides makes it more important than ever that alternatives to chemical control of pathogens be studied to determine their possible role in the future. The cooperation resulted in the objectives being promoted at national and international meetings. The cooperation also enabled the technology transfer between the two labs, as well as access to instrumentation and specialization particular to the two labs.

Cultured cells offer a wide range of usages for studies of insect physiology, toxicology and pathology. Embryonic eggs from western flower thrips (WFT) were used to develop primary cell cultures for studies of the replication of tomato spotted wilt virus (tospoviruses) in their thrips vectors. Development of a thrips cell culture allows examination of virus-vector interactions at the cellular level. Several insect cell culture media were evaluated for suitability to establish primary thrips cell cultures. The best combination of media allowed cells to survive for 100 d. Two types of cells were readily observed. Within 1-2 d after initiating a culture, fibroblast-like cells become the dominant cell type, and at ≈20 d, epithelial-like cells were observed. These cells were subsequently subcultured to develop a thrips-cell line.

Pelargonium (Geranium) is the number one pot plant in many areas of the United States and Europe. Israel and the U.S. send to Europe rooted cuttings, foundation stocks and finished plants to supply a certain share of the market. Geraniums are propagated mainly vegetatively from cuttings. Consequently, viral diseases have been and remain a major threat to the production and quality of the crop. Among the viruses isolated from naturally infected geraniums, 11 are not specific to Pelargonium and occur in other crops while 6 other viruses seem to be limited to geranium. However, several of these viruses are not sufficiently characterized to conclude that they are distinct agents and their nomenclature and taxonomy are confusing. The ability to separate, distinguish and detect the different viruses in geranium will overcome obstacles te developing effective detection and certification schemes. Our focus was to further characterize some of these viruses and develop better methods for their detection and control. These viruses include: isolates of pelargonium line pattern virus (PLPV), pelargonium ringspot virus (PelRSV), pelargonium flower break virus (PFBV), pelargonium leaf curl (PLCV), and tomato ringspot virus (TomRSV). Twelve hybridoma cell lines secreting monoclonal antibodies specific to a geranium isolate of TomRSV were produced. These antibodies are currently being characterized and will be tested for the ability to detect TomRSV in infected geraniums. The biological, biochemical and serological properties of four isometric viruses - PLPV, PelRSV, and PFBV (and a PelRSV-like isolate from Italy called GR57) isolated from geraniums exhibiting line and ring pattern or flower break symptoms - and an isolate ol elderbeny latent virus (ELV; which the literature indicates is the same as PelRSV) have been determined Cloned cDNA copies of the genomic RNAs of these viruses were sequenced and the sizes and locations of predicted viral proteins deduced. A portion of the putative replicase genes was also sequenced from cloned RT-PCR fragments. We have shown that, when compared to the published biochemical and serological properties, and sequences and genome organizations of other small isometric plant viruses, all of these viruses should each be considered new, distinct members of the Carmovirus group of the family Tombusviridae. Hybridization assays using recombinant DNA probes also demonstrated that PLPV, PelRSV, and ELV produce only one subgenomic RNA in infected plants. This unusual property of the gene expression of these three viruses suggests that they are unique among the Carmoviruses. The development of new technologies for the detection of these viruses in geranium was also demonstrated. Hybridization probes developed to PFBV (radioactively-labeled cRNA riboprobes) and to PLPV (non-radioactive digoxigenin-labeled cDNAs) were generally shown to be no more sensitive for the detection of virus in infected plants than the standard ELISA serology-based assays. However, a reverse transcriptase-polymerase chain reaction assay was shown to be over 1000 times more sensitive in detecting PFBV in leaf extracts of infected geranium than was ELISA. This research has lead to a better understanding of the identity of the viruses infecting pelargonium and to the development of new tools that can be used in an improved scheme of providing virus-indexed pelargonium plants. The sequence information, and the serological and cloned DNA probes generated from this work, will allow the application of these new tools for virus detection, which will be useful in domestic and international indexing programs which are essential for the production of virus-free germplasm both for domestic markets and the international exchange of plant material.

Hybridoma cell lines secreting monoclonal antibodies (MAbs) specific to a 31 000 molecular weight viral protein or a 31 000 molecular weight polyhedrin protein of Lymantria dispar nuclear polyhedrosis virus (LdNPV) were developed. The two polypeptides were shown to be different by comparing their amino acid compositions. Immuno-electron microscopy was used to verify specific binding of the MAbs to their respective targets. Specific MAbs were used to develop an ELISA procedure to monitor the development of LdNPV virus and polyhedrin in vivo. Results indicated that in hemolymph of larvae fed 106 polyhedral inclusion bodies, the concentration of virus began to increase 16 h after inoculation and continued to increase for the next 5 days. By 36 h, the concentration of polyhedrin increased and was maintained at a high level in the later stages of infection. One-third of this group of infected larvae survived the infection. In these individuals, the concentrations of virus and polyhedrin declined to a low level 5 days after infection. This suggests the presence of a host mechanism for clearing the virus from the hemolymph. Key words: infection mechanism, monoclonal antibody, in vitro immunization, Lymantria dispar nuclear polyhedrosis virus, ELISA.

Tomato spotted wilt virus (TSWV) and Calla lily chlorotic spot virus (CCSV) are two recognized species of the Tospovirus genus in the family Bunyaviridae infecting calla lily (Zantedeschia spp.). During 2005, 15 virus isolates were collected from different calla lily plants exhibiting yellow stripes on their leaves in Ho-Li, a major calla lily-production township in Taiwan. After three successive local lesion passages on Chenopodium quinoa Willd., diseased leaf tissues individually infected by these isolates were preserved in liquid nitrogen and used for subsequent identification studies. Using the tospovirus genus-specific primers gL3637 and gL4435c designed from the L RNA, an 800-bp DNA fragment was amplified in reverse transcription-PCR from all 15 isolates. Moreover, leaf extracts of the diseased calla lilies and the C. quinoa plants inoculated with the 15 virus isolates reacted with antisera against the nucleocapsid proteins (NP) of Capsicum chlorosis virus (CaCV)-gloxinia and Watermelon silver mottle virus (WSMoV), but not to monoclonal antibodies against the NP of TSWV, CCSV, Peanut chlorotic fan-spot virus (PCFV), or Impatiens necrotic spot virus (INSV) in indirect ELISA. These results indicate that the 15 virus isolates are tospoviruses belonging to the WSMoV serogroup. Additionally, we amplified and sequenced the full-length N gene from these tospovirus isolates using primers WN2328 (5′-CCATTGGTTTGCCTCCG-3′) and WN3534 (5′-CGTCGACAGAGCAATCGAGGC-3′) designed from the S RNA of WSMoV. The deduced amino acid sequences of the N protein from these 15 tospovirus isolates showed a greater than 92% identity to that of CaCV (GenBank Accession No. NC-008301). Furthermore, results of phylogenetic analysis of the 15 isolates on the basis of amino acids sequences, both genetic distance and parsimony trees indicated that they were all genetically clustered within CaCV using INSV, TSWV, and WSMoV as outgroups. The results indicate that the virus causing yellow stripes in calla lilies is a strain of CaCV. To our knowledge, this is the first evidence that CaCV can naturally infect calla lilies and cause yellow stripe symptoms. Reference: (1) F.-H. Chu et al. Phytopathology 91:361, 2001.

A new potyvirus designated as Calla lily latent virus (CLLV) was isolated from apparently healthy calla lilies (Zantedeschia spp.) collected from nurseries in Taichung County, Taiwan. Different from most calla lily-infecting potyviruses, CLLV infects Chenopodium quinoa and develops local lesions on inoculated leaves (3). Typical potyvirus particles approximately 780 nm long were detected from CLLV-induced C. quinoa local lesions. CLLV was transmitted readily to and established in C. quinoa. Attempts to establish CLLV infection in calla lilies from extracts of C. quinoa lesions were not successful. The virus was transmitted from infected to healthy calla lilies with difficulty. A 1.3-kb cDNA product was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from CLLV-infected calla lilies and C. quinoa using potyvirus degenerate primers (2). The PCR product was cloned and sequenced. It was found to consist of 1,339 nucleotides (nt) (GenBank Accession No. AF469171) corresponding to the genome organization of the 3′terminal region of potyviruses. The deduced amino acid sequence contains 362 residues encoding the 3′terminal region of the nuclear inclusion b gene (80 residues) and the complete coat protein (CP) gene (282 residues). A 253-nt noncoding region (NCR) was found at the 3′terminal region of the cDNA. By comparing with known sequences of potyviruses, CLLV was identified as a new species of Potyvirus based on the uniqueness in the CP gene and 3′ NCR. Soybean mosaic virus and Watermelon mosaic virus 2 are the potyviruses most similar to CLLV, but they share only approximately 80% nucleotide identity with CLLV in the CP and NCR regions. Attempts to purify sufficient CLLV from C. quinoa for antiserum preparation were not successful. Alternatively, polyclonal antibodies were produced using E. coli-expressed CLLV CP (1). The antibodies were useful for detection of CLLV and its CP in calla lilies using enzyme-linked immunosorbent assay, sodium dodecyl sulfate-immunodiffusion, immuno-specific electron microscopy, and western blot. Field surveys showed that calla lily plants found positive for CLLV by serological methods always remained symptomless throughout the six-month growing season. Occasionally, CLLV was detected in symptomatic calla lilies, but these plants were consistently confirmed dually infected by other viruses (Dasheen mosaic virus and Konjak mosaic virus found most commonly). Infection of CLLV alone in calla lilies may not have a direct impact on the production and marketing of the crop. Synergism is not currently known when calla lilies are coinfected with other viruses. CLLV is spread by vegetative propagation through infected rhizomes or tubers. References: (1) C. C. Chen et al. Plant Dis. 87:901–905, 2003. (2) S. S. Pappu et al. Plant Dis. 82:1121–1125, 1998. (3) F. W. Zettler and R. D. Hartman. Pages 464–470 in: Virus and Virus-like Diseases of Bulb and Flower Crops. G. Loebenstein et al., eds. John Wiley and Sons Inc., UK, 1995.

Mice and rabbits were injected with various forms of a 16 amino acid synthetic peptide representing PSTAIR, the evolutionarily conserved region of the protein kinase p34cdc2, for polyclonal antisera and hybridoma-monoclonal antibody production. Antisera from mice injected with an unconjugated monomeric form of the peptide showed no reaction to the peptide. Of four animals injected with the monomeric form of the peptide conjugated to keyhole limpet hemocyanin via m-maleimidobenzoyl-N-hydroxysulfosuccinimide (MBS), antisera from only one mouse had a very low titer to the peptide, and all four animals produced antibody to the MBS bridge. Both mice injected with an octameric multiple antigen peptide (MAP) of PSTAIR produced antisera reactive to the octameric MAP form of the peptide in ELISA and also to the cdc2 protein expressed in bacteria in an immunoblotting assay. Splenocytes from one mouse injected with the octameric MAP form of the peptide were successfully used for hybridoma-monoclonal antibody production. A monoclonal antibody was produced that reacted with octamer, monomer and cdc2-expressed protein and specifically with the carboxyl terminus of the 16 amino acid peptide.