Diversity of Phytophthora clandestina isolated from subterranean clover in southern australia: Analysis of virulence and RAPD profiles

The virulence spectrum of 112 isolates of Phytophthora clandestina collected from 56 sites in four subterranean clover-growing states in southern Australia was determined using differential cultivars of subterranean clover. Five races were detected, with race 0 in all states except New South Wales, race 1 in all states, race 2 only in Victoria, race 3 only in New South Wales, and race 4 in Victoria and Western Australia. The level of genotypic diversity among the different P. clandestina populations was investigated using five RAPD primers. Among 30 bands amplified, only two were polymorphic. This enabled identification of four multilocus RAPD genotypes. Three of the four genotypes occurred in all four states. Races 2 and 3 occurred with RAPD genotypes 1 and 2 only whereas races 0 and 1 occurred in all four multilocus RAPD genotypes. These results indicate that the pathogenicity spectrum of P. clandestina can change rapidly.

Evaluation of a viral vector system, based on a defective interfering RNA of tomato bushy stunt virus, for protein expression and the induction of gene silencing

A viral vector system was developed based on a DI-RNA, a sub-viral particle derived from TBSV-BS3-statice. This newly designed vector system was tested for its applicability in protein expression and induction of gene silencing. Two strategies were pursued. The first strategy was replication of the DI-RNA by a transgenically expressed TBSV replicase and the second was the replication by a so called helper virus. It could be demonstrated by northern blot analysis that the replicase, expressed by the transgenic N. benthamiana plant line TR4 or supplied by the helper virus, is able to replicate DI-RNA introduced into the plant cells. Various genes were inserted into different DI constructs in order to study the vector system with regard to protein expression. However, independent of how the replicase was provided no detectable amounts of protein were produced in the plants. Possible reasons for this failure are identified: the lack of systemic movement of the DI-RNA in the transgenic TR4 plants and the occurrence of deletions in the inserted genes in both systems. As a consequence the two strategies were considered unsuitable for protein expression. The DI-RNA vector system was able to induce silencing of transgenes as well as endogenous genes. Several different p19 deficient helper virus constructs were made to evaluate their silencing efficiency in combination with our DI-RNA constructs. However, it was found that our vector system can not compete with other existing VIGS (virus induced gene silencing) systems in this field. Finally, the influence of DI sequences on mRNA stability on transient GUS expression experiments in GUS silenced plants was evaluated. The GUS reporter gene system was found to be unsuitable for distinguishing between expression levels of wild type plants and GUS silenced transgenic plants. The results indicate a positive effect of the DI sequences on the level of protein expression and therefore further research into this area is recommended.

Effects of dark or of red, blue or white light on germination of subterranean clover seeds

Dry or imbibed seeds of the negatively photoblastic burr burying subterranean clover cv. ‘Seaton Park’ were treated with dark or with red, blue or white light to evaluate the effects of light on seed germination. Dry seeds treated with constant white light, red light or blue light during 8 days and subsequently incubated in dark had final germination and duration of germination reduced, and the distribution of germination changed from highly asymmetric to symmetric respectively. Imbibing seeds incubated under constant blue or white light had final germination strongly reduced seven days after sowing (7.3% and 50.1% of the germination under dark) with significant differences between them. After transferral to dark, true complete recovery of germination of seeds treated with white light was observed 19 days after sowing, but only partial recovery in seeds treated with blue light. Results of dry and imbibed seeds are consistent with no activity of phytochromes, as expected in negatively photoblastic seeds. Results of dry seeds are seemingly contradictory because total germination data imply the inactivity of red and blue light photoreceptors, the opposite being implied by duration and shape of germination. A tentative hypothetical solution for the contradiction is presented. Results of imbibed seeds are fully consistent with cryptochromes but not with phototropins mediation of responses to light of seed germination in ‘Seaton Park’. The ecological and adaptive significance of such responses are discussed in the framework of light attenuation in soil and the requirement and ability of subterranean clover ‘Seaton Park’ to bury seeds.

Host plant recognition by the root feeding clover weevil, Sitona lepidus (Coleoptera : Curculionidae)

This study investigated the ability of neonatal larvae of the root-feeding weevil, Sitona lepidus Gyllenhal, to locate white clover Trifolium repens L. (Fabaceae) roots growing in soil and to distinguish them from the roots of other species of clover and a co-occurring grass species. Choice experiments used a combination of invasive techniques and the novel technique of high resolution X-ray microtomography to non-invasively track larval movement in the soil towards plant roots. Burrowing distances towards roots of different plant species were also examined. Newly hatched S. lepidus recognized T. repens roots and moved preferentially towards them when given a choice of roots of subterranean clover, Trifolium subterraneum L. (Fabaceae), strawberry clover Trifolium fragiferum L. (Fabaceae), or perennial ryegrass Lolium perenne L. (Poaceae). Larvae recognized T. repens roots, whether released in groups of five or singly, when released 25 mm (meso-scale recognition) or 60 mm (macro-scale recognition) away from plant roots. There was no statistically significant difference in movement rates of larvae.

A single chain antibody against a viral RNA polymerase (TBSV-BS3-Statice)

Expression of antibodies in plant against essential viral proteins could provide an alternative approach to engineered viral resistance. Engineered single chain Fv antibodies scFV are particularly suitable for expression in plant because of their small size and the lack of assembly requirements. RNA-dependent RNA polymerases (RdRps) function as the catalytic subunit of viral replicases required for the replication of all positive strand RNA viruses. By using Phage technology we selected scFvs from a phage library using purified E.coli expressed TBSV(Tomato bushy stunt virus) replicase as antigen. The scFvs mediated-inhibition of RdRp activity was studied in vitro and in planta. In vitro experiments showed the inhibition of CNV(Cucumber necrosis virus) and TCV(Turnip crinkle virus) RdRp. Transient in planta assays based on agroinfiltration and an infectious clone of TBSV demonstrated the inhibition of the replication of TBSV(Tomato bushy stunt virus). Epitope mapping showed that the selected scFvs target the motif E of RdRp which is involved in template binding.Moreover T1 plants of transgenic lines of N. benthamiana expressing different scFvs either in the cytoplasm or the ER (endoplasmic reticulum) showed a high level of resistance against infection with TBSV and RCNMV(Red clover necrotic mosaic virus) upon inoculation with virus particles. This is the first report that scFvs against a RdRp of a plant viruses can inhibit viral replication in vivo. The resistance is even efficient against viruses belonging to different virus families.

Characterization and greenhouse evaluation of Brazilian calcined nonapatite phosphate rocks for rice

Little information is available on the agronomic effectiveness of calcined nonapatite phosphate rock (PR) sources containing crandallite minerals in the form of Ca-Fe-Al-P for flooded and upland rice (Oryza sativa L.). We conducted laboratory and greenhouse studies to (i) characterize the mineralogical composition, (ii) investigate the solubility and dissolution behavior, and (iii) evaluate the agronomic effectiveness of two nonapatite PR sources (Juquia and Sapucaia) from Brazil and compared them with (i) a highly reactive Gafsa PR (Tunisia) containing apatite in the form of Ca-P and (ii) a reference water-soluble triple superphosphate (TSP) for flooded and upland rice. After calcination at 500 degrees C for 4 h, the solubility of Juquia PR and Sapucaia PR in neutral ammonium citrate (NAC) significantly increased from almost nil to a maximum of 39.3 and 114 g P kg(-1), respectively. X-ray diffraction showed that crystalline crandallite mineral was transformed to an amophorus form after calcination. The solubility behavior of the two calcined PR sources followed the same trend as Gafsa PR, that is, P release decreased with increasing equilibrium pH in the 0.01 M KCl solution (PH 3.0-8.0). At PH 3, the solubility followed: Gafsa PR > calcined Sapucaia PR > calcined Juquia PR. No P release was detected from any of the PR sources at pH >= 5.0 in the solution, indicating the Ca-P characteristic of the Ca-Fe-Al-P mineral controlled P dissolution of the calcined PR. Without calcination, both Juquia PR and Sapucaia PR were totally ineffective for upland rice grown on a Hiwassee clay loam (fine, kaolinitic, thermic Rhodic Kanhapludult) with pH 5.4 whereas a significant P response was observed with the calcined PR samples. For flooded rice grown on Hiwassee soil, the calcined Juquia PR and Sapucaia PR were 66 and 72%, respectively, as effective as TSP in increasing rice grain yield whereas Gafsa PR was ineffective. For upland rice grown on the unlimed soil, Gafsa PR was as effective as TSP in increasing rice grain yield whereas calcined Juquia PR and Sapucaia PR were 89 and 83% of TSP. The effectiveness of Gafsa PR was reduced to 0% after the soil was limed to pH 7.0 whereas the two calcined PR sources were reduced to 49% of TSP. Soil available P extracted by iron oxide impregnated filter paper (Pi test) or anion-exchange resin after rice harvest correlated well with P uptake by rice grain for flooded and upland rice.

Reverse transcription of a naturally occurring nonretroviral RNA produces a precise deletion in the majority of its cDNA products

A precise, reproducible deletion made during in vitro reverse transcription of RNA2 from the icosahedral positive-stranded Helicoverpa armigera stunt virus (Tetraviridae) is described. The deletion, located between two hexamer repeats, is a 50-base sequence that includes one copy of the hexamer repeat. Only the Moloney murine leukemia virus reverse transcriptase and its derivative Superscript I, carrying a deletion of the carboxy-terminal RNase H region, showed this response, indicating a template-switching mechanism different from one proposed that involves a RNase H-dependent strand transfer, Superscript II, however, which carries point mutations to reduce RNase H activity, does not cause a deletion. A possible mechanism involves the enzyme pausing at the 3' side of a stem-loop structure and the 3' end of the nascent DNA strand separating from the template and reannealing to the upstream hexamer repeat.

Caracterización biológica y molecular de un rhabdovirus causal de mosaico estriado amarillo, en maíz (Zea mays L.) en la provincia de Córdoba.

La familia Rhabdoviridae incluye varios patógenos económicamente importantes de cultivos, entre los más de 70 virus que afectan plantas. Estos últimos se clasifican en los géneros Cytorhabdovirus y Nucleorhabdovirus, dependiendo de si producen inclusiones en el espacio perinuclear, o si desarrollan viriones citoplasmáticos. Los integrantes de esta familia infectan gran cantidad de monocotiledóneas y dicotiledóneas y la mayoría son dependientes de transmisión por insectos. Las interacciones virus-vector son altamente específicas, y se ha registrado la replicación en insectos, del rhabdovirus que transmiten a las plantas. Cada especie de rhabdovirus induce un amplio espectro de síntomas en sus plantas huéspedes, y estos van desde la falta de efectos discernibles hasta la muerte total de la planta. El maíz (Zea mays L.) es el cultivo más ampliamente distribuido a nivel mundial y uno de los principales cultivos de cereales, ubicándose tercero en el ranking de producción en el mundo. En maíz se ha citado la presencia de varios rhabdovirus, entre estos American wheat striate mosaic virus (AWSMV), Cereal chlorotic mottle virus (CCMV), Maize mosaic virus (MMV), Maize sterile stunt virus (strains of Barley yellow striate virus), Northern cereal mosaic virus (NCMV) y Maize fine streak virus (MFSV). Ninguno de ellos reportado en Argentina. Desde 2001 un rhabdovirus es observado, por sintomatología y microscopía electrónica, en plantas de maíz de diferentes localidades de la provincia de Córdoba. Esta virosis pudo ser transmitida en dos oportunidades a plantas de maíz sanas mediante Peregrinus maidis y logró amplificarse mediante RT-PCR con iniciadores degenerados, el gen de la polimerasa L. Nuestra hipótesis es que el agente causal de la sintomatología de mosaico estriado amarillo en maíz sería un rhabdovirus emergente en Argentina, diferente de Maize mosaic virus (MMV), transmitido por delfácidos, que puede aislarse y mantenerse en condiciones controladas. El objetivo del presente trabajo es generar conocimientos biológicos, moleculares y epidemiológicos sobre el agente causal de la sintomatología en maíz de mosaico estriado amarillo. Para ello se colectarán plantas de maíz con sintomatología de mosaico estriado amarillo, en distintas localidades donde se presente la sintomatología. Las muestras se observarán al microscopio electrónico en cortes ultrafinos y en “leaf dip". Los viriones se purificarán, extraerá el RNA de los mismos, y obtendrá la secuencia de nucleótidos, para compararla con otras publicadas de virosis vegetales y se obtendrán homologías. Se realizarán transmisiones experimentales de esta virosis, por incisiones vasculares y mediante el empleo de diferentes especies de insectos vectores. Importancia del proyecto El avance de patógenos tropicales hacia zonas templadas es una de las causas de la aparición de las virosis emergentes, que se caracterizan por producir epifítias al ingresar a nuevos ecosistemas. El Maize mosaic virus (MMV) es un rhabdovirus que produce una de las virosis más importantes del maíz en el continente americano. Determinar la identidad del agente etiológico del mosaico estriado amarillo y establecer su relación con MMV es fundamental para desarrollar medidas proactivas y diseñar estrategias de manejo de esta nueva enfermedad.

Rhizoctonia crown and root rot of the pasture legume, sulla (Hedysarum coronarium)

Rhizoctonia solani AG-2-2 was isolated from wilting and dying plants of sulla ( Hedysarum coronarium), which is currently being assessed in eastern and southern Australia for its potential as a pasture and forage legume. Infected plants in the field had extensive rotting of the taproot, lateral roots and crown. Koch's postulates were fulfilled using three inoculation methods. The disease may pose a considerable threat to the potential use of H. coronarium in the dryland, grazing farming systems of Australia, with resistance offering the most viable option for minimising its impact.

Defensive role of allelopathic secondary compounds in plants I: testing two independent general hypotheses

This study tests two general and independent hypotheses with the basic assumption that phytoactive secondary compounds produced by plants evolved primarily as plant defences against competitor plant species. The first hypothesis is that the production and main way of release of phytoactive compounds reflect an adaptive response to climatic conditions. Thus, higher phytoactivity by volatile compounds prevails in plants of hot, dry environments, whereas higher phytoactivity by water-soluble compounds is preponderant in plants from wetter environments. The second hypothesis is that synergy between plant phytoactive compounds is widespread, due to the resulting higher energy efficiency and economy of resources. The first hypothesis was tested on germination and early growth of cucumber treated with either water extracts or volatiles from leaves or vegetative shoot tops of four Mediterranean-type shrubs. The second hypothesis was tested on germination of subterranean clover treated with either water extracts of leaves or vegetative shoot tops of one tree and of three Mediterranean-type shrubs or with each of the three fractions obtained from water extracts. Our data do not support either hypotheses. We found no evidence for higher phytoactivity in volatile compounds released by plants that thrive in hot, dry Mediterranean-type environments. We also found no evidence for the predominance of synergy among the constituents of fractions. To the contrary, we found either antagonism or no interaction of effects among allelopathic compounds.

Epidemiological significance of subterranean Aedes aegypti (Diptera : Culicidae) breeding sites to dengue virus infection in charters towers, 1993

The objective of this study was to determine the epidemiological significance of subterranean mosquito breeding sites to the 1993 outbreak of dengue fever (type 2) in the northern Queensland town of Charters Towers, Australia. In recent studies on subterranean mosquito breeding, containers such as wells and service manholes have been shown to be important breeding sites to Australia's only dengue vector, Aedes aegypti (L.). This study demonstrates a direct epidemiological association between subterranean breeding sites and dengue virus infection. The mean distance between residents seropositive for dengue 2 and the nearest subterranean container (113 m) was significantly less than for a randomly selected control (191 m), (F = 81.9; df = 1, 478; P < 0.001). Residents positive for dengue 2 antibodies was 2.47 (95% confidence interval 1.88-3.24) times higher for those living within 160 m of a well or service manhole, compared with those residing further away. These findings emphasize the importance of including subterranean water containers in Ae. aegypti surveillance and control programs.

A List of references: maize virus diseases and corn stunt.

Each list is an addendum to the previous list.

Bioassay of clover wound tumor virus and the mycoplasmalike organisms of peach Western X and aster yellows /

Literature cited: p. 28-32.

Biological and molecular characterization of an isolate of Tobacco streak virus obtained from soybeans in Brazil

A virus was isolated from soybean (Glycine max) plants with symptoms of dwarfing and bud blight in Wenceslau Braz County, Paraná, Brazil. The host range and properties resembled those of Tobacco streak virus (TSV). The purified virus showed three peaks in a frozen sucrose gradient. Antiserum was produced and the virus was serologically related to TSV. Electron microscopy detected 28 nm spherical particles. Coat protein (CP) had a Mr of 29.880 Da. A fragment of 1028 nt was amplified, cloned and sequenced. One open reading frame with 717 nt was identified and associated to the CP. The CP gene shared 83% identity with the sequence of TSV CP from white clover (Trifolium repens) (GenBank CAA25133). This is the first report of the biological and molecular characterization of TSV isolated from soybeans. It is proposed that this isolate be considered a strain of TSV named TSV-BR.

Dispersal of soil-dwelling clover root weevil (Sitona lepidus Gyllenhal, Coleoptera: Curculionidae) larvae in mixed plant communities

Insect pests that have a root-feeding larval stage often cause the most sustained damage to plants because their attrition remains largely unseen, preventing early diagnosis and treatment. Characterising movement and dispersal patterns of subterranean insects is inherently difficult due to the difficulty in observing their behaviour. Our understanding of dispersal and movement patterns of soil-dwelling insects is therefore limited compared to above ground insect pests and tends to focus on vertical movements within the soil profile or assessments of coarse movement patterns taken from soil core measurements in the field. The objective of this study was to assess how the dispersal behaviour of the clover root weevil (CRW), Sitona lepidus larvae was affected by differing proportions of host (clover) and non-host (grass) plants under different soil water contents (SWC). This was undertaken in experimental mini-swards that allowed us to control plant community structure and soil water content. CRW larval survival was not affected either by white clover content or planting pattern or SWC in either experiment; however, lower clover composition in the sward resulted in CRW larvae dispersing further from where they hatched. Because survival was the same regardless of clover density, the proportion of infested plants was highest in sward boxes with the fewest clover plants (i.e. the low host plant density). Thus, there is potential for clover plants over a larger area to be colonised when the clover content of the sward is low.