No description available.

A two year study was conducted to investigate the effect of intercropping corn with alfalfa or red clover with or without an initial application of the herbicide EPTC$ sp+$ (S-ethyl dipropylthiocarbamate + R-25788 antidote) on the weed yellow nutsedge (Cyperus esculentus L.). EPTC$ sp+$ applied prior to crop seeding in 1984 significantly reduced yellow nutsedge shoot growth in the first year but did not significantly reduce tuber populations. EPTC$ sp+$ significantly improved corn silage and grain yield and alfalfa establishment in the first year but did not significantly improve red clover establishment. In the second year, a trend of superior forage legume establishment and lower yellow nutsedge shoot production was observed in those treatments established the previous year with EPTC$ sp+$. Over the two year period, yellow nutsedge tuber populations in treatments established with EPTC$ sp+$ were significantly greater in the monocropped corn treatment than in any other treatment. In treatments established without a herbicide, yellow nutsedge tuber populations were also greatest in the monocropped corn treatment.

Potato leafhopper causes considerable damage in red clover. The main objectives of this study were to clarify the inheritance of pubescence and to evaluate the relationship between pubescence and potato leafhopper (Empoasca fabae (Harris) resistance. Thirteen red clover clones of diverse origin, including both pubescent and non-pubescent types were used as parents. A series of crosses were made in all possible combinations among the 13 parental clones. Seedlings of F$ sb1$ progeny and stem cuttings from parents were planted in the field in the summer of 1993 in a randomized complete block design. Based on the results, the inheritance of pubescence type on red clover stems, petioles and abaxial leaf surfaces was best explained individually by two-locus models showing dominant and recessive interaction. A two locus model with recessive epistasis was proposed for pubescence on stipules and basal internodes, but there were a number of crosses that deviated from expected ratios. There was quantitative variation for trichome density on red clover and it appeared to be inherited as a quantitative trait. Based on mid-parent offspring regression, the heritability estimates of trichome density on petioles, stems, abaxial leaf surfaces, and adaxial leaf surfaces were 0.16, 0.77, 0.50 and 0.48, respectively. Pubescence was apparently associated with potato leafhopper resistance. Visual ratings of feeding injury, the numbers of leafhopper nymphs per plant and the numbers of nymphs per gram of dry plant material were higher on glabrous plants than on pubescent plants. (Abstract shortened by UMI.)

The objectives of the study were to determine whether intercropped alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.) or rye grass (Lolium multiflorum Lmk.) could simultaneously contribute to the nitrogen regime of grain-corn (ie: increased yield), improve edaphic quality, and decrease the level of inorganic nitrogen present in the soil after corn harvest. To achieve these objectives the seeding of the intercrops was delayed, the corn population was increased, and the red clover was spring-ploughed. / Fall-ploughed red clover maintained the mean weight diameter (MWD) of aggregates on the Ste-Rosalie clay. Spring-ploughed red clover on the other hand caused a decrease in bulk density in the top 10 cm of the Chicot loam. / The nitrogen-response of corn indicated that the intercrops did not contribute to the nitrogen regime of grain-corn. There were also indications that intercrops competed with corn for available inorganic nitrogen and water. The intercrops, however, did reduce the levels of soil inorganic nitrogen in the fall allowing for a possible decrease in the nitrogen load of soil percolates. / Despite the beneficial effect of nitrogen fertilisation on the organic nitrogen content of the soil, the presence of leguminous intercrops did not prevent the increase of the soil CN ratio.

Trifolium ambiguum (Caucasian clover) is being released in New Zealand for use in areas where growth of T. repens (white clover) is marginal. Although closely related to T. repens, T. ambiguum has unique and highly specific nodulation requirements and as rhizobial strains capable of effectively nodulating T. ambiguum are not naturally found in New Zealand soils, they must be introduced with the seed. Rhizobium leguminosarum bv. trifolii strains such as ICC105 form effective nodules on T. ambiguum but ineffective (Fix⁻) nodules on T. repens. The T. repens nodules nevertheless develop normally and contain bacteroids. R. l. bv. trifolii strains that are effective on T. repens such as NZP561, fail to nodulate T. ambiguum. As the host-specific nitrogen fixation defect of Caucasian clover rhizobia on T. repens has potentially adverse agronomic implications, the genetic basis for this Fix⁻ phenotype was investigated. Rhizobium leguminosarum bv. trifolii strain ICC105 was converted to Fix⁺ on T. repens by the introduction of an 18-kb fragment of DNA from a white clover rhizobial strain (NZP514) symbiotic plasmid. This fragment contained several nif and fix genes, including nifHDKEN, fixABCX, nifA, nifB, fdxN and fixU. Tn5 mutation of these white clover rhizobial genes demonstrated that most were required to impart the Fix⁺ phenotype on T. repens to ICC105, with the exception of nifA. Mutagenesis of the ICC105 nifA gene and subsequent complementation with various combinations of the white clover rhizobia nif/fix genes as well as transcriptional lacZ fusion studies of the ICC105 nifA and nifH genes demonstrated that ICC105 nifA is expressed and functional during the ineffective nodulation of T. repens and able to activate expression of nifHDKEN and fixABCX operons derived from white clover rhizobium but not from ICC105. Sequence analysis and comparison of the intergenic region between the divergently transcribed nif/fix operons revealed a conserved 111-bp region found between the nifH/fixA promoters of Caucasian clover rhizobia, but not in white clover rhizobia. Attempts to modify this region in ICC105 failed in creating a strain which was Fix⁺ on T. repens; however recombination of the nifHD/fixAB region from a white clover rhizobium into the ICC105 genome produced several strains with a �swapped� nitrogen fixation phenotype (i.e. Fix⁺ on T. repens and Fix⁻ on T. ambiguum). A hypothesis was therefore proposed by which differences in the nifH/fixA promoter regions of Caucasian clover rhizobia and white clover rhizobia modulate the expression of the upstream genes in response to the particular plant host they are nodulating. The incompatibility between the symbiotic plasmid of R. l. bv. trifolii ICC105 and the white clover rhizobium symbiotic plasmid cointegrate, pPN1, was also investigated and potential regions of each plasmid involved in this incompatibility were identified. The research presented in this thesis has contributed to the genetic knowledge of the nitrogen fixation genes, and regulation of these genes in R. l. bv. trifolii. It has also provided progress towards the goal of creating a suitable inoculant strain for T. ambiguum that is able to fix nitrogen in symbiosis with both T. repens and T. ambiguum.

Bibliography: leaves 108-152 / viii, 152 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study investigates the induction of resistance to the clover cyst nematode, Heterodera trifolii Goffart, an economic pest in white clover pastures that are a key to high milk yields in dairy cattle...it explores the potential of soil and rhizosphere bacteria to induce systemic resistance in white clover, Trifolium repens L. (summary) / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2000

Bibliography: last 24 unnumbered leaves / 161, [32] leaves : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1985

Subterranean clover mottle virus (SCMoV) is a positive sense, single-stranded RNA virus that infects subterranean clover (Trifolium subterraneum) and a number of related legume species. The ultimate aim of this research was to investigate aspects of SCMoV that would support its use as a gene silencing vector for legume species, since RNA (gene) silencing is now a potential tool for studylng gene function. The ability of viruses to induce an antiviral defense system is being explored by virus-induced gene silencing (VIGS), in which engmeered viral genomes are used as vectors to introduce genes or gene ii-agments to understand the function of endogenous genes by silencing them. To develop a gene silencing vector, a number of aspects of SCMoV host range and molecular biology needed to be studied. A requirement for a useful viral vector is a suitably wide host range. Hence the first part of this work involved study of the host range and symptom development of SCMoV in a range of leguminous and non-leguminous plants. The aim of this work was to identify new and most suitable hosts among economically important crop and model legumes for functional genomic studies, and also to study symptom development in these hosts for comparison with host responses to any SCMoV-based viral vectors that might be used in later infection studies. A total of 61 plant genotypes representing 52 species from 25 different genera belonging to 7 families were examined for their response to SCMoV infection, including established and new crop legumes, established pasture, and novel pasture and forage legumes, and 12 host indicator plants belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Following mechanical inoculation, plants were examined for symptoms and tested for primary and secondary infection by RT-PCR andlor ELISA after 2-3 weeks and 3-9 weeks, respectively. Thirty-six legume hosts belonging to eight different genera of legumes were identified as suitable hosts of SCMoV, 22 of them systemic hosts and 15 were infected locally. Only two non-legumes were infected with SCMoV-P23, one systemically and one as a local host, so confirming that SCMoV is essentially a legume-infecting virus. This work considerably expanded knowledge of the host range of SCMoV. To provide the information needed to modify the SCMoV genome to develop gene vectors, the virus was characterized in detail. The complete genomes of four isolates, SCMoV-AL, SCMoV-MB, SCMoV-MJ and SCMoV-pFL, were sequenced using high fidelity RT-PCR and molecular cloning, and compared to the first sequenced isolate (SCMoV-P23) to give a complete picture of the genome organisation of the virus. The 4,258 nucleotide (nt) sequence of SCMoV RNA is not polyadenylated. The 5' non-coding region (NCR) is 68 nt in length and the 3' NCR is 174 nt. The coding regon contains four overlapping open reading fi-ames (ORFs). The first, OW1 (nt 68-608), encodes a putative protein containing 179 amino acids with a calculated molecular mass (Ma,) of 20.3 kDa. It overlaps with the next ORF, ORF2a, by four bases. ORF2a (nt 605-2347) encodes a putative protein of 580 amino acids with a Ma, of 63.7 kDa and contains a motif characteristic of chymotrypsin-like serine proteases. The ORF2b is probably translated as part of a polyprotein by -1 ribosomal fiameshifting in ORF2a. The transfiame product (Ma, = 107.5 kDa) is made up of 966 amino acids. A GDD motif typical of RNA virus polymerases is present in ORF2b. The 3' terminal ORF3 (nt 3323-4084) encodes the 27.3 kDa coat protein (CP). Nucleotide variation between the complete sequences of the isolates was two to three orders of magnitude larger than base misincoporation rates of the polymerases used in RT-PCR. Molecular relationship analysis between all five isolates, undertaken with the complete nucleotide sequences, clearly separated them into three groups. These groups reflect similar significantly diverse groupings based on the symptoms and their severity in subterranean clover. Intra-isolate sequence variability is therefore a possible cause of the differences in symptom severity. The analysis also showed that there were more nucleotide substitutions at the 5' terminal half of SCMoV than at the 3' end. This observation was confirmed by the higher value of nucleotide diversities at nonsynonymous versus synonymous sites (dN/ds ratio) estimated for the ORF1, compared to the near conservation of sequences of the other ORFs. These results, together with functional and comparative sequence analysis with other sobemoviruses, implicate the ORFl gene product in pathogenicity of SCMoV, possibly as a severity determinant or as a viral suppressor of RNA silencing in plants. Because more information on SCMoV genome function was required, the possible involvement of the ORFl gene product (PI) and the CP in movement of SCMoV was studied in cells of grasspea (Lathyrus clymenum L) and chickpea as C-terminal fusion constructs with jellyfish (Aequorea victoriae) green fluorescent protein (GFP). A transient expression vector, pTEV, for in planta synthesis of reporter gene constructs was developed. The vector was based on pGEM-T with 35s RNA transcriptional promoter of Caulzjlower Mosaic virus (CaMV) and nopaline synthase gene transcription terminator signal (T-Nos) separated by a multiple subcloning site. A custom-made particle inflow gun was used to introduce the constructs into plant cells. The bombardment conditions were fxst optimised for efficient delivery of DNAcoated particles. Transient gene expression of GFP was monitored 24-96 hours after particle bombardment. Fluorescence from GFP alone, GFP:CP and GFP:Pl constructs was observed in the nucleus of single cells, cytoplasm and cell periphery of neighbouring cells. There was limited spread of these fusion proteins from one cell to another 36-48 hours after transformation. These results indicate that the P 1 and CP cannot move independently from cell to cell. Other viral/cellular components might be needed to form a complex with these proteins to transport the viral genome. Putative nuclear export signals in the P1 and CP sequences of SCMoV were identified by sequence comparison. These could be tested by mutagenesis using full-length infectious clones. To determine the possibility of gene expression of vectors based on SCMoV, three forms of a full-length cDNA clone of SCMoV-pFL were developed: one with no heterologous transcriptional factors (pFL), a second under the control of only 35s (p35SFL) and a third with 35s and T-Nos (pTEVFL). Fifteen day-old in vitro-cultured chickpea, grasspea and subterranean clover seedlings were inoculated by particle bombardment using gold particles coated with plasmid pTEVFL. In vivo-transcribed RNA transcripts were detected by RT-PCR after two weeks in grasspea but not in subterranean clover and chickpea. Experiments were undertaken towards developing the SCMoV genome into a VIGS vector. Three forms each of five major GFP chimeric constructs of pFL (the full length SCMoV cDNA clone) were generated from which in vitro- and in vivo-transcribed RNA transcripts could be derived. The rationale used in developing these constructs was gene insertion andlor replacement with d p , and duplication of the putative subgenomic RNA promoter (sgPro) of SCMoV. The major constructs were as follows: pFLCPgfp, pFL with the d p gene fused to the 3' end of the CP gene, pFLP 1 gfp, pFL with gj27 gene fused to the 3 ' end of the ORF 1, pFLCPsgprogfp, pFL with a putative sgPro sequence and a translatable & gene cloned in tandem between the CP gene and the 3' NCR of SCMoV, pFLCPVsgprogf$, pFL with a putative sgPro sequence and a translatable gfp gene cloned in tandem between a truncated CP gene and the 3' NCR and pFLREPsgprog@, pFL with the ORF2b, a putative sgPro sequence and a translatable &fP gene cloned in tandem between a truncated CP gene and the 3' NCR These constructs were all made, but a detailed assessment of their vector potential could not be done because there was a delay of about one year whilst the Office of the Gene Technology Regulator processed the application for permission for glasshouse testing. Although additional work needs to be undertaken to complete development of a final RNA silencing vector, this study has contributed to new knowledge in terms of extending understanding of SCMoV host range, symptoms, sequence variation and control of gene expression. The constructs made have also laid the groundwork for development of a legume gene silencing vector based on SCMoV.

Thesis (Ph. D.)--University of Adelaide, 1984. / Some mounted ill. Includes bibliographical references (leaves 156-186).

Thesis (Ph.D.)--Murdoch University, 2005. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 184-207.