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181 THE EFFECT OF IN PLANTA PRODUCED HUMAN INTERFERON ON TYMY PRODUCTION OF TRANSGENIC TOBACCO CONTAINING COWPEA MOSAIC INFECTIONT VIRUS (CPMV) COAT PROTEIN GENES. D. L. NIDA and S. A. G.IA. de Zoeten, J. R. Penwick and T. Hohn. Friederich Ghabrial, Dept. of Plant Pathology, University of Kentucky, Giescher-Institut, P. 0. Box 2543, CH-4002, Basel, Lexington, KY 40546-0091 Switzerland Preliminary data in our laboratory indicate that tobacco may serve as a model to study the potential of cross-protection against CPMV. ELISA results show that tobacco supports virus multiplication and cell-to-cell movement. Because CPMV coat proteins VP37 and VP23 are derived from a 60k polyprotein Cauliflower mosaic virus (CaMV) DNA was engineered to carry the human IFN ciD gene to the infected plant. Inoculation of turnip (Brassica rapa cv "Just Right") with CaMV strains carrying the IFN cLD gene resulted in the production of biologically active IFN oD in infected plants precursor by proteolytic processing, it was of interest to determine whether constitutive expression of the 60k polypeptide in transgenic plants would interfere with CPMV infection. Therefore, plant expression vectors were constructed by inserting cDNA representing the CPMV 60k biol (ca. 2pg/g g Fwt.) activ In nplanta In plan produced infeced IFN plants ciD did not hamper superinfection with a single stranded (+) RNA plant virus, turnip yellow mosaic virus (TYMV). precursor into the binary Ti vector pMON530. Constructs containing CPMV coat protein genes in sense and antisense 186 orientations were generated. Transgenic plants are being produced via Agrobacterium cocultivation and selection on kanamycin-containing medium. GENOMIC CHARACTERIZATION OF BEET CURLY TOP VIRUS ISOLATES. Stenger and J. E. Duffus, USDA-ARS, Salinas, CA, 93905. 0. C. Full-length, infectious DNA clones have been constructed for three distinct isolates of beet curly top virus (BCTV). 182 Progeny virus derived from cloned genomes of the Logan (sev CELL-FREE STUDIES OF TEV 49KDA PROTEINASE PROCESSING: on Beta vulgaris, wide host range), Worland (mild on B. DIFFERENTIAL CLEAVAGE RATES AT DIFFERENT POLYPROTEIN vulgaris, wide host range), and Horseradish (narrow host range) JUNCTIONS. W, G, Dougherty and T. D. Parks, Dept. of isolates were transmitted by Circulifer tenellus, and displayed Microbiology, Oregon State Univ., Corvallis, OR 97331-3804. the same phenotypes as the original isolates. A fourth BCTV genome (severe, wide host range) was inadvertently cloned as a Tobacco etch virus (TEV) 49kDa proteinase recognizes a contaminant of the Horseradish isolate. Southern hybridization specific heptapeptide sequence at five locations on the TEV assays indicated that each cloned genome shared sequence polyprotein. This consensus cleavage sequence contains both relatedness with a full-length, infectious BCTV DNA clone conserved and nonconserved positions. In cell free studies, previously characterized by Stanley et al (EMBO J 5:1761). cleavage at the 50kDa/7lkDa protein junction proceeded at a Endonuclease restriction maps developed for the cloned BCTV slow rate relative to processing at the 58kDa/3OkDa cleavage genomes were distinct from one another. Infectivity assays site. Site directed mutagenesis of TEV cDNA sequences was determined that plasmids containing tandem repeats of BCTV performed, such that the nonconserved positions of the genomes were generally more infectious than excised linear 50kDa/71kDa site were converted into those amino acids found BCTV DNA inserts. at the 58kDa/3OkDa cleavage site. Reciprocal mutations of the 58kDa/3OkDa site were also tested. In each case, processing of the 58kDa/3OkDa sequence proceeded at a faster 187 rate. These data suggest that post translational regulation of gene expression in TEV may be possible via differential MUTAGENESIS OF GENE VI of CAULIMOVIRUSES RESULTS IN CHANGES IN proteolytic processing at various gene product junctions. THE DISEASE PHENOTYPE. E. P. Broglio and R. J. Shepherd. University of Kentucky, Lexington, KY 40506. A CaMV hybrid genome was constructed which contained unique 183 restriction sites bordering gene VI that allowed its sequences DETECTION OF PLANT RNA VIRUS-SPECIFIC RIBONUCLEOPROTEIN to be subcloned, mutagenized and then ligated into the remain- COMPLEXES IN INFECTED CELL EXTRACTS. R French. USDA, ARS, der of the genome. Changes in the primary structure of gene VI Department of Plant Pathology, University of Nebraska, Lincoln, NE were made by linker-insertion mutagenesis. Of the several 68583. mutants constructed, three were found to be infectious and were examined for disease phenotype on Brassica campestris and several solanaceous hosts. The infectious mutants and the parent Within cells, both cellular and viral RNAs probably exist as complex ribo- virus displayed a similar phenotype on B. campestris and two nucleoproteins. In order to detect RNA virus-specific ribonucleoproteins in Nicotiana species, N_. edwardsonii and N. bigelovii. In Datura a general way, antiserum to double-stranded RNA (dsRNA) was used to stramonium one of the mutants induced a-distinctly differen immunoprecipitate complexes fror barley stripe mosaic virus (BSMV, phenotype. This result supports previous reports that gene VI brome mosaic virus, or tobacco mosaic virus-infected barley protoplasts. is largely responsible for disease induction. Radiolabeled protein components of the complexes were then detected by SDS-PAGE and fluorography. Immunoprecipitates from mock-inoculated protoplasts contained little protein while those from infected cells revealed several proteins unique for each virus, including coat protein. Extracts from BSMV-infected cells contained polypeptides of ca. 140, 60, 25 (coat), 20 and 18 kD. UV irradiation of cells prior to extraction increased the yield of protein suggesting that the immunoprecipitated proteins are intimately associated with RNA in yiVQ. Several types of complexes may be precipitated because synthetic dsRNA and BSIMV virion RNA were both partially effective in competition assays. 184 COMPARISON OF SYMPTOMS ASSOCIATED WITH EXPRESSION OF CaNV GENE VI WITH TWO PROMOTERS IN TRANSGENIC PLANTS. K-B. Goldberg, J.M. Kiernan, and R.J. Shepherd. Dept. of Plant Pathology, Univ. of Kentucky, Lexington, KY 40546. Datura innoxia and Nicotiana edwardsonii were transformed with gene VI of cauliflower mosaic virus (CaMV) strains CM1841 and 04 with their homologous 19S promoters or a chimeric 35S promoter-gene VI of CM1841 using the Ti-plasmid vectors pGA472 or pKYLX-7, respectively. These plants are not hosts for CH1841. Expression of P62, the gene VI protein, in 0. innoxia was associated with stunting accompanied by chlorosis or necrosis. The maximum levels of P62 in these plants with either a 19S or 35S promoter were similar, as determined by western blot analysis using antiserum to P62. The transgenic N. 1B8a edwardsonii were mildly chlorotic when expressing gene VI of MOLECULAR CHARACTERIZATION OF NON-ENVELOPED BACILLIFORM VIRUSES. CM1841 hut overall the levels of P62 were much lower than N. E. Olszewski, S. L. Medberry and B. E. L. Lockhart, observed with 0. innoxia. The accumulation of P62 in N." ... edwardsonii was at least ten times greater using gene VI in Uiest fMneoa t al N518 combination with a 35S promoter compared to the 19S promoters. Gienoses of several non-enveloped bacilliform viruses have been 1158 PHYTOPATHOLOGY 185
shown to be double-stranded circular DNA. We have constructed Penicilliwm funiculosum, which showed antagonism to several genomic clones of two non-enveloped bacilliform viruses, Phytophthora spp. in vitro, suppressed azalea root rot caused kalanchoe top-spot virus (KTSV) and Commelina yellow mottle by Ph. cinnamnomi or Ph. parasitica in some, but not all, virus (CoYMV), and are using these clones to characterize these greenhouse experiments. The antagonist was grown in a genomes and the transcripts they encode. The CoYMV genome is 7.5 bran/peat medium and mixed into a planting medium [peat/perlite kb in size and each strand contains a single-stranded region (3:1)]; this was followed by inoculation with Phytophthora at (discontinuity). The CoYMV genome encodes a 7.6-7.7 kb 5-7 days. Results obtained over a 2-month period showed that polyadenylated transcript. DNA sequencing has identified a suppression of Ph. parasitica was in general greater than Ph. region of the genome with homology to the '-end of the cinnamomi. Introduction of the biocontrol agent by dipping the initiator tRNAmeT of wheat and bean. The tRNAme, homology and roots in a spore suspension was not effective. P. funiculosum, one discontinuity map to the same region. These features of however, improved the growth of azalea plants even in experi- CoYMV suggest that this virus replicates by a mechanism similar ments where Phytophthora was not suppressed. Plants in the to that of caulimoviruses. The host for CoYMV, Commelina antagonist-containing treatment grew better (70-220%) and had diffusa, contains a 1 kb transcript which shares homology with greener foliage than those in the control without the antagon- CoYMV DNA. This transcript occurs in both healthy and infected ist, possibly due to suppression of minor root pathogens leaves but its significance is unknown. present in the planting medium. 189 193 PROXIMITY OF PSEUDOMONAS FLUORESCENS STRAIN PRA25rif TO PEA INCREASE IN RHIZOSPHERE COMPETENCE OF TRICHODERMA HARZIANUM TAPROOTS. J. L. Parke and C. M. Liddell, Dept. of Plant FOLLOWING PROTOPLAST FUSION. A. Sivan and G.E. Harman, Dept. of Pathology, University of Wisconsin, Madison 53706. Horticultural Sciences, Cornell Univ., Geneva, NY, 14456. Pea seeds coated with a spontaneous mutant strain of P. Rhizosphere competence of strains of Trichoderma harzianum was fluorescens, PRA25rif, resistant to rifampicin, were sown in tested by treating seeds with conidial suspensions and assesssoil held at -6 kPa matric potential. Seven days later, ing colonization of roots of plants grown from inoculated seeds. PRA25rif populations associated with the pea taproot segment A strain (1295-22) derived by protoplast fusion exhibited a 0.5-1.5 cm below the seed were assessed from dilution plate greater degree of rhizosphere competence than the parental counts. Root segments with various amounts of adhering soil strains (T12 and T95). T12 colonized mainly the upper third of (0-1200 mg cm" 1 ) were sampled to determine if soil mass the roots, while T95 was found primarily on the upper and the affects quantification of root-colonizing bacteria. The lower parts of the roots. However, 1295-22 showed a greater and amount of soil adhering to roots did not significantly affect more uniform colonization of the whole rhizosphere than did T12 the number of bacteria recovered, and the density of PRA25rif or T95. Colony forming units of T95 and T12 along the lower half was greatest in the inner rhizosphere. After sonication of of corn roots were primarily confined to the rhizosphere. Conthe samples, 99.8% of the bacteria recovered were isolated versely, 1295-22 colonized both the rhizosphere and rhizoplane from the soil suspension, and only 0.2% were recovered from equally. Although population densities of the tested strains macerated roots. were, in general, lower in cotton rhizosphere than in corn, 1295-22 colonized more cotton root segments than the parental types. 190 EFFECTS OF SOIL PH ON SUPPRESSION OF TAKE-ALL BY PSEUDOMONAS FLUORESCENS 2-79. B.H. Ownley, D.M. Weller, and L.S. 194 Thomashow. USDA-ARS, Pullman, WA 99164-6430 INHIBITION BY PSEUDOMONAS CEPACIA, A POTENTIAL BIOCONTROL Pseudomonas AGENT, OF fluorescens SELECTED SOILBORNE 2-79 suppresses PATHOGENS. Gaeumannomyces K. E. Conway, graminis C. J. Foor, D. Malvick, and C. Bender, Department of Plant Pathology, var. tritici (Ggt) which causes take-all of wheat. The major Oklahoma State University, Stillwater, OK 74078-9947. mechanism of suppression is phenazine-1-carboxylate; fluorescent siderophore and a second iron regulated factor have only A bacterium isolated from soil at the Oklahoma Forest minor roles. Mycelial growth of Ggt was inhibited by 2-79 on Regeneration Center in Washington, OK, exhibited strong anti- Kanner's agar (used for phenazine production) adjusted to pH biotic activity in culture against macrophomina phaseolina. 4.5, 5.5, 6.5, 7.2, 7.6, or 8.5. To determine the effect of The bacterium was identified as Pseudomonas cepacia soil (Pc) pH by on take-all suppression, seeds were treated with 2-79 fatty acid profile analysis. In-dual culture on PDA, or mutants-deficient in production of phenazine (Phz-), sidero- strongly inhibited M. phaseolina, Rhizoctonia solani AG 1, AG phore (Sid ), antifungal factor (Aff-), or a combination and type 1, AG 3 and AG 4, FusariumR oxyporum, PythiumA irregula sown in a steamed Ritzville silt loam (pH 7.6, 29.6% sand, and Laetisaria arvalis, but not Sclerotium rolfsiir 64.0% silt, 6.4% clay) with bulk soil pH adjusted to 4.9, 5.7, Trichoderma harzianum. However, Pc strongly inhibited S. 6.2, 7.3, 7.6 or 8.0. Strain 2-79 and a Phz Sid Aff mutant - rolfsii in pairings on KMB. This indicated possible significantly suppressed take-all at all soil pH values. Phz siderophore production in addition to antibiotic production mutants were less suppressive and generally failed to suppress Eg. Potential of the bacterium as a biocontrol agent is being take-all at low (7.6) soil pH. assessed in soil systems. 191 195 BIOLOGICAL CONTROLOFRHIZOCTONIASEEDLINGDISEASES IN INFLUENCE OF WHEAT ROOT PATHOGENS ON MAINTENANCE OF POPUl.ATiONS BEDDING PLANT NURSERIES WITH THOUGHTS TOWARDS OF PSEUDOMONAS FLUORESCENS Q72a-80 AND 2-79 IN THE WHEAT COMIMERCIAL DEVELOPMENT. D.A. Schisler 1 , M.H. Ryder', R.G. RHIZOSPHERE. N. Mazzola and R.J. Cook, Dept. of Plant Path- Rowden 2 , 2 1CSIRO-Division of Soils, Glen Osmond, South Austraia and 1n citec L td ., G ib so n Islan d , B ri sb an e, Q u een slan d , A u stralia.ol olg.n. g a n U S A R , W sh SaeUiesty .AA.,Wsigo ula g t n t t e n v r s y , P l m n 96 )9 6 A cooperative research project between CSLRO and IncitecLtd. was iniiated to investigate tbe feasibility of using antagonistic microorganisms to control Rhizoctoniadiseases of seedlings in bedding plant nurseries. Fifty soil samples from 30 nursenies in South Australia were assayed for suppressiveness to Rhizoctonia SOlni. From these assays, 13 highly suppressive soils were selected and fungi, bacteria and actinomycetes were isolated from bulk and rhizosphere soils. Ninety-fsve prokaryotic antagonists were assayed against R. solani (anastomosis group 8) on Capsicum annuum cv "Green Giant" and Celosia argentea using commercial potting, seeding and watering equipment. More than one-third of the organisms significantly decreased disease (p
Page 1 and 2: Abstracts of Presentations at the 1
Page 3 and 4: Twenty Fusarium solani (Mart.) Sacc
Page 5 and 6: means used to derive them were not
Page 7 and 8: 46 A COMPARISON OF THE EPIPRE AND K
Page 9 and 10: 62 THE INCIDENCE AND IMPACT OF SPHA
Page 11 and 12: 79 83 82 SELECTION AND USE OF OXALA
Page 13 and 14: nificantly among plots but there wa
Page 15 and 16: Chrysanthemum morifolium (florists'
Page 17 and 18: anine ammonia lyase (PAL) and hydro
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Page 27 and 28: in the greenhouse for resistance to
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Page 31 and 32: 236 240 INDUCTION OF RESISTANCE TO
Page 33 and 34: y suppression of Mn oxidizers in th
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Page 37 and 38: 284 BASELINE-SENSITIVITY OF THREE P
Page 39 and 40: Department of Agronomy, University
Page 41 and 42: 316 SEROLOGICAL NONIDENTITY OF IRIS
Page 43 and 44: amended with 25 g/L of KC1O Prelimi
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Page 47 and 48: Department of Plant Pathology, Univ
Page 49 and 50: causative agent of greening, howeve
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Page 55 and 56: M. Laakso and J. W. Moyer, Dept. of
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Page 59 and 60: seed decay by 10%. Phomopsis seed d
Page 61 and 62: LARYAFD, QMKAA, FGLDG and TERH, in
Page 63 and 64: In an initial survey of ten commerc
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Page 67 and 68: 523 AVIRULENCE OF WHEAT LEAF RUST T
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Page 71 and 72: verified. A translocation linked to
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587 591 MEASURING VIRULENCE ASSCIAT
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603 PHYSICAL CHARACTERIZATION OF MU
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agar diffusion tests and spore germ
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Spring beauty latent virus (SBLV) h
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SOYBEANS. Barry M. Cunfer and Juju
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666 670 COMPUTER-ASSISTED WHITE MOL
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682 PHYTOPHTHORA CINNAMOMI. Univers
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698 SCREENING OF VESICULAR-ARBUSCUL
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