Effect of Diaporthe RNA virus 1 (DRV1) on growth and pathogenicity of different <Emphasis Type="Italic">Diaporthe</Emphasis> species

A 4.1 kbp positive-strand RNA virus known as Diaporthe RNA virus 1 (DRV1) occurs in hypovirulent, non-sporulating isolates of the fungal pathogen Diaporthe perjuncta. A full-length cDNA clone of DRV1 was developed and RNA transcribed from the cDNA clone used to transfect different Diaporthe spp. The transfected species included three D. ambigua isolates and an unidentified Phomopsis asexual state of a Diaporthe sp. Successful transfections were confirmed using RT-PCR. Although the in vitro-transcribed positive sense single-stranded RNA used for transfection included vector sequences at both ends, the genomes of progeny virus from DRV1-transfected isolates were free of the vector sequences. Transfection resulted in morphological changes in these fungal pathogens. However, the presence of DRV1 did not reduce growth rate in two of the three D. ambigua or the Phomopsis sp. significantly. Pathogenicity studies showed that the transfected isolates have reduced aggresiveness.     

Using metabolic profiling to assess plant-pathogen interactions: an example using rice (<Emphasis Type="Italic">Oryza sativa)</Emphasis> and the blast pathogen <Emphasis Type="Italic">Magnaporthe grisea</Emphasis>

A metabolomics based approach has been used to study the infection of the Hwacheong rice cultivar (Oryza sativa L. cv. Hwacheong) with compatible (KJ201) and incompatible (KJ401) strains of the rice blast fungal pathogen Magnaporthe grisea. The metabolic response of the rice plants to each strain was assessed 0, 6, 12, 24, 36, and 48 h post inoculation. Nuclear Magnetic Resonance (NMR) spectroscopy and Gas and Liquid Chromatography Tandem Mass spectrometry (GC/LC-MS/MS) were used to study both aqueous and organic phase metabolites, collectively resulting in the identification of 93 compounds. Clear metabolic profiles were observed at each time point but there were no significant differences in the metabolic response elicited by each pathogen strain until 24 h post inoculation. The largest change was found to be in alanine, which was ~30% (±9%) higher in the leaves from the compatible, compared to the resistant, plants. Together with several other metabolites (malate, glutamine, proline, cinnamate and an unknown sugar) alanine exhibited a good correlation between time of fungal penetration into the leaf and the divergence of metabolite profiles in each interaction. The results indicate both that a wide range of metabolites can be identified in rice leaves and that metabolomics has potential for the study of biochemical changes in plant-pathogen interactions.     

Quantitative disease resistance assessment by real-time PCR using the Stagonospora nodorum-wheat pathosystem as a model

Measuring disease resistance accurately and reproducibly is a key requirement for the introgression of partial resistance genes into breeding lines. Here, a qPCR protocol is used to measure fungal biomass, using the wheat- Stagonospora nodorum pathosystem as a model. Seven cultivars of differing reported resistance levels were used. Fungal biomass taken at 220°C thermal days after inoculation accurately predicts the final grain weight loss. It is concluded that a test based on qPCR methods is specific, quantitative, rapid and objective. Such tests could provide useful and economic tools in the development of robustly resistant crop cultivars.     

Structural Characterisation of the Interaction between Triticum aestivum and the Dothideomycete Pathogen Stagonospora nodorum

The interaction between Stagonospora nodorum and a susceptible wheat cultivar was investigated using a range of microscopic techniques. Germination of pycnidiospores occurred approximately 3 h after making contact with the leaf surface and was followed by attempted penetration 8–12 h later. Penetration was observed through stomata and also directly through periclinal and anticlinal epidermal cell walls. Penetration down the anticlinal cell walls appeared to occur without a differentiated penetrating structure whilst structures identified as either lateral appressoria or hyphopodia were typically present when penetrating over a periclinal cell wall. Once inside the leaf, the fungus continued to grow for the next 4–5 days colonising all parts of the leaf except the vascular bundles. Only in the later phase of the infection was total host cell collapse apparent. Evidence of polyphenolic compounds was observed. The infection cycle was completed within 7 days as indicated by sporulation on the leaf surface. These results have allowed us to understand how the fungus physically interacts with the leaf and will help the overall understanding of the infection process.     

On-farm management practices to minimise off-site movement of pesticides from furrow irrigation

Off-site movement of pesticides from furrow-irrigated agriculture has been a concern in the Ord River Irrigation Area, Western Australia. This paper reports on the effectiveness of incorporation of pesticides by cultivator or power harrows before irrigating, and spraying pesticides only onto beds to minimise off-site transport. Incorporation of pesticides by power harrows prior to irrigation was found to be more effective in decreasing the off-site transport of a more strongly sorbed pesticide, endosulfan. The average load of total endosulfan (alpha + beta + sulfate) decreased by 74% (P < 0.01) from 11.41 g ha(-1) from the conventionally treated bays to 2.96 g ha(-1) from the incorporated irrigation bays. The total average load of atrazine leaving the irrigation bays was decreased by 81% (P < 0.05) from 87.82 g ha(-1) under the conventional practice of spraying the whole field to 16.95 g ha(-1) by spraying the beds only. A reduction of 52% in total average load of metolachlor was observed following incorporation with power harrows, but this was not significant. Incorporation by cultivator or by power harrows decreased the total load of atrazine or metolachlor leaving the irrigation bays over the whole irrigation period, but these treatments were not shown to be statistically significant, which may have been due to the limited number of field replicates. Incorporation of strongly sorbed pesticides (e.g. endosulfan) prior to irrigation significantly decreased the off-site transport of these pesticides in a furrow irrigation system and may be a useful practice to minimise off-site transport of other similar pesticides. Minimising off-site transport of weakly sorbed pesticides (e.g. atrazine and metolachlor) from a furrow irrigation system is more difficult. The nature of furrow irrigation makes it highly conducive to pesticide transport, particularly of weakly sorbed pesticides, and further work is needed to develop strategies to minimise the movement of this group of pesticides to water bodies.     

Identification of Sources of Resistance to Phoma medicaginis Isolates in Medicago truncatula SARDI Core Collection Accessions, and Multigene Differentiation of Isolates

ABSTRACT Phoma medicaginis is a necrotrophic fungal pathogen, commonly found infecting the annual medic Medicago truncatula. To differentiate eight P. medicaginis isolates, five gene regions were examined: actin, beta-tubulin, calmodulin, translation elongation factor 1-alpha (EF-1alpha), and the internal transcribed spacer ribosomal DNA. Sequence comparisons showed that specimens isolated from M. truncatula in Western Australia formed a group that was consistently different from, but allied to, a P. medicaginis var. medicaginis type specimen. EF-1alpha contained a hyper-variable 55-bp repeat unit, which forms the basis of a rapid polymerase chain reaction-based method of reliably distinguishing isolates. Characterization of three isolates showed that all exhibited a narrow host range, causing disease only in M. sativa and M. truncatula among eight commonly cultivated legume species sampled. Infection of 86 M. truncatula single-seeded accessions showed a continuous distribution in disease phenotypes, with the majority of accessions susceptible. On a 1-to-5 disease reaction scale increasing in severity, individual fungal isolates showed means of 2.6 to 3.2, and scores ranged from 1 to 4.8 among accessions. The results presented here suggest that M. truncatula harbors specific and diverse sources of resistance to individual P. medicaginis genotypes.     

Aerial applications of pesticides in the United Kingdom: 1978 to 1998

The aerial application of pesticides increased rapidly following the end of the Second World War, as, along with the development of a wider range of synthetic pesticides, more planes and pilots became available. It also became clear that new and previously inaccessible areas could be sprayed from the air. By the 1980s there were around 100 planes in the United Kingdom (UK) capable of spraying pesticides. However, increasing environmental pressure brought about the introduction of tougher legislation to control pesticide usage. The Food and Environment Protection Act 1985 and the Control of Pesticides Regulations 1986 (as amended) introduced tight controls over the application of pesticides from the air. Aerial application of pesticides has been monitored each year by the UK Ministry of Agriculture, Fisheries and Food (MAFF) since 1978, during which time the area treated has decreased significantly. This has been partly as a result of changes in legislation but also due to technological improvements in conventional crop‐protection methods. The area treated from the air in the UK has never risen above 2.5% of the area treated from the ground and in 1998 had fallen to less than 0.04% of the total area treated with pesticides. It is likely that this figure will continue to fall in the future. By 1998, the only pesticides to be sprayed from the air comprised fungicides, herbicides and insecticides. As a group, herbicides comprised the greatest area of treatment and the only target to show an increase in area treated in recent years has been bracken (Pteridium aquilinum (L) Kuhn). © Crown copyright 2000. Reproduced with the permission of Her Majesty's Stationery Office.     

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

Identification and properties of a deviant isolate of the broad bean yellow band serotype of pea early-browning virus from faba bean (Vicia faba) in Algeria

A virus, isolated from faba bean (Vicia faba) obtained from Algeria, was readily recognized as a tobravirus by its particle sizes and morphology. Pea (Pisum sativum) and French bean (Phaseolus vulgaris) characteristically reacted to the isolate like pea early-browning virus (PEBV), but faba bean,Antirrhinum majus, Nicotiana rustica, andN. tabacum reacted with line-pattern symptoms which were unusually brilliant on theNicotiana species. In electronmicroscope decoration tests, the isolate did not react with an antiserum to the Dutch type strain of PEBV, but with one to the broad bean yellow band (BBYB) serotype from Italy. It resembles this serotype in reaction on faba bean, but seems to differ appreciably onN. rustica, N. tabacum, andPetunia hybrida. It is described as a deviant isolate of the BBYB serotype of PEBV.All thirteen faba-bean genotypes tested were found to be susceptible to the Algerian isolate and two Dutch type strain isolates of the virus, and to react with erratic line-pattern symptoms to the Algerian isolate only. All ten genotypes of chickpea (Cicer arietinum) tested reacted hypersensitively, and four out of ten genotypes of lentil (Lens culinaris) were susceptible to the virus but reacted differentially to the three isolates. Seed transmission of PEBV, including the new isolate, in faba bean is confirmed (9% for the Algerian isolate, and over 45% for one of the Dutch type strain isolates), and seed transmission of the virus in a non-legume (N. rustica, 4%) is herewith first reported. This is the first report on the occurrence of the BBYB serotype of PEBV outside Italy, and of PEBV outside Morocco in North Africa.     

Calibrating the liana crown occupancy index in Amazonian forests

Lianas hold an important, but understudied, role in forest dynamics, however they are difficult to measure and detailed liana measurements are time consuming. Many researchers have therefore used an ordinal scale index, the crown occupancy index (COI), to describe the liana load carried by trees. Here we assess the overall effectiveness, in terms of accuracy, precision, repeatability and efficiency, of the COI in tropical forests. We relate the COI to more detailed liana measurements at the individual tree-level and site-level, comparing sites with different levels of liana infestation. Our results show (1) that the COI accurately measures individual tree and plot level liana loads, indicated by the strong correlations between the COI and the number and basal area of lianas. However, (2) as expected, the COI is only weakly related to the basal area of lianas rooted close to the tree, which is a proxy for competition for below-ground resources. The COI is also (3) an efficient measure of liana loads, as the input time needed for a COI survey is considerably less than that of a detailed liana survey. We also (4) found a high degree of repeatability in COI classification between observers. Additionally (5), the COI can be used to differentiate between sites in terms of their overall liana canopy competition (precision), but (6) may not be a precise indicator of the site-level mean basal area of lianas in tree crowns.