Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

Tospoviruses - An Australian perspective

The detection, distribution, molecular and biological properties, vector relations and control of tospoviruses present in Australia, including Tomato spotted wilt virus (TSWV), Capsicum chlorosis virus (CaCV) and Iris yellow spot virus (IYSV), are reviewed. TSWV occurs throughout Australia where it has caused serious sporadic epidemics since it was first described in the 1920s. The frequency and distribution of outbreaks has increased in the 1990s, with the arrival and dispersal of the western flower thrips (Frankliniella occidentalis) being one factor favouring this situation. The crops most frequently and severely affected are capsicum, lettuce, tomato, potato and several species of ornamentals. Minimal differences were found between the nucleocapsid (N) gene amino acid sequences of Australian isolates and these were most closely related to a clade of northern European isolates. CaCV was first detected in Australia in 1999 and is most closely related to Watermelon silver mottle virus, a serogroup IV tospovirus. The natural hosts include capsicum, tomato, peanut and Hoya spp. The virus also occurs in Thailand and Taiwan. IYSV was first found in Australia in 2003, infecting onion and leek, with the distribution in three States suggesting that the virus has been present for some time.

Oidium perseae-americanae sp. nov. on avocado

Oidium perseae-americanae is described causing powdery mildew on avocado (Persea americana) in New Caledonia and Brazil.

Taxonomic notes on two powdery mildews: Phyllactinia chorisiae and Ovulariopsis wissadulae (Erysiphaceae: Phyllactinieae)

The powdery mildew Phyllactinia chorisiae has been considered conspecific with P. guttata. A re-examination of the type material of P. chorisiae and another specimen showed that this fungus, unlike P. guttata, has dimorphic conidia and its anamorph does not belong to the genus Ovulariopsis, which is the typical anamorph for Phyllactinia species. This suggests that P. chorisiae is morphologically distinct from P. guttata and should no longer be accepted as a synonym. Re-evaluation of type material of Oidiopsis wissadulae revealed that it has monomorphic conidia (mostly lemon-shaped) and hemiendophytic mycelium, a combination of characters that clearly places this fungus in the genus Ovulariopsis. Emended descriptions of P. chorisiae and Ovulariopsis wissadulae are presented.

Leaf-clearing and staining techniques for the observation of conidiophores in the Phyllactinioideae (Erysiphaceae)

Some whole leaf-clearing and staining techniques are described for the microscopic observation of the origin of powdery mildew conidiophores, whether from external mycelium or internal mycelium, emerging through stomata. These techniques enable separation of the two genera, Oidiopsis and Streptopodium, in the Erysiphaceae.

Oidiopsis (Erysiphaceae) on Euphorbia spp. in Australia and Vanuatu

One specimen of powdery mildew on Euphorbia cyathophora from Vanuatu and 11 specimens on E. cyathophora, E. dentata, E. heterophylla and E. leucocephala from Australia were studied. All were shown to represent the Oidiopsis anamorph of Leveillula taurica, which is described. This is the first record of Oidiopsis in Vanuatu. E. leucocephala is a new host record for this powdery mildew.

Controlling plant and fruit diseases in strawberry fields

Grey mould, powdery mildew and stem-end rot are major diseases affecting the strawberry industry. Some of the chemicals used are ineffective under wet weather, have limits to the number of applications allowed in a season or may become ineffective in the long-term because of the development of resistance in the fungi. We will assess the effectiveness of the chemicals currently used by the strawberry industry and whether the fruit rot fungi are resistant to these fungicides. We will screen other chemicals that are used to control these diseases in related crops. We will also evaluate new chemicals in collaboration with the crop protectant industry. We will also undertake similar work to control nematodes in strawberry fields.

Driving better vegetable irrigation through profitable practice change

The availability and quality of irrigation water has become an issue limiting productivity in many Australian vegetable regions. Production is also under competitive pressure from supply chain forces. Producers look to new technologies, including changing irrigation infrastructure, exploring new water sources, and more complex irrigation management, to survive these stresses. Often there is little objective information investigating which improvements could improve outcomes for vegetable producers, and external communities (e.g. meeting NRM targets). This has led to investment in inappropriate technologies, and costly repetition of errors, as business independently discover the worth of technologies by personal experience. In our project, we investigated technology improvements for vegetable irrigation. Through engagement with industry and other researchers, we identified technologies most applicable to growers, particularly those that addressed priority issues. We developed analytical tools for ‘what if’ scenario testing of technologies. We conducted nine detailed experiments in the Lockyer Valley and Riverina vegetable growing districts, as well as case studies on grower properties in southern Queensland. We investigated root zone monitoring tools (FullStop™ wetting front detectors and Soil Solution Extraction Tubes - SSET), drip system layout, fertigation equipment, and altering planting arrangements. Our project team developed and validated models for broccoli, sweet corn, green beans and lettuce, and spreadsheets for evaluating economic risks associated with new technologies. We presented project outcomes at over 100 extension events, including irrigation showcases, conferences, field days, farm walks and workshops. The FullStops™ were excellent for monitoring root zone conditions (EC, nitrate levels), and managing irrigation with poor quality water. They were easier to interpret than the SSET. The SSET were simpler to install, but required wet soil to be reliable. SSET were an option for monitoring deeper soil zones, unsuitable for FullStop™ installations. Because these root zone tools require expertise, and are labour intensive, we recommend they be used to address specific problems, or as a periodic auditing strategy, not for routine monitoring. In our research, we routinely found high residual N in horticultural soils, with subsequently little crop yield response to additional nitrogen fertiliser. With improved irrigation efficiency (and less leaching), it may be timely to re-examine nitrogen budgets and recommendations for vegetable crops. Where the drip irrigation tube was located close to the crop row (i.e. within 5-8 cm), management of irrigation was easier. It improved nitrogen uptake, water use efficiency, and reduced the risk of poor crop performance through moisture stress, particularly in the early crop establishment phases. Close proximity of the drip tube to the crop row gives the producer more options for managing salty water, and more flexibility in taking risks with forecast rain. In many vegetable crops, proximate drip systems may not be cost-effective. The next best alternative is to push crop rows closer to the drip tube (leading to an asymmetric row structure). The vegetable crop models are good at predicting crop phenology (development stages, time to harvest), input use (water, fertiliser), environmental impacts (nutrient, salt movement) and total yields. The two immediate applications for the models are understanding/predicting/manipulating harvest dates and nitrogen movements in vegetable cropping systems. From the economic tools, the major influences on accumulated profit are price and yield. In doing ‘what if’ analyses, it is very important to be as accurate as possible in ascertaining what the assumed yield and price ranges are. In most vegetable production systems, lowering the required inputs (e.g. irrigation requirement, fertiliser requirement) is unlikely to have a major influence on accumulated profit. However, if a resource is constraining (e.g. available irrigation water), it is usually most profitable to maximise return per unit of that resource.

Bacterial Community Structure in the Hindgut of Wild and Captive Dugongs (Dugong dugon)

Dugongs (Dugong dugon) are marine mammals that obtain nutrients through hindgut fermentation of seagrass, however, the microbes responsible have not been identified. This study used denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing to profile hindgut bacterial communities in wild dugongs. Faecal samples obtained from 32 wild dugongs representing four size/maturity classes, and two captive dugongs fed on cos lettuce were screened using DGGE. Partial 16S rRNA gene profiles of hindgut bacteria from wild dugong calves and juveniles were grouped together and were different to those in subadults and adults. Marked differences between hindgut bacterial communities of wild and captive dugongs were also observed, except for a single captive whose profile resembled wild adults following an unsuccessful reintroduction to the wild. Pyrosequencing of hindgut communities in two wild dugongs confirmed the stability of bacterial populations, and Firmicutes (average 75.6% of Operational Taxonomic Units [OTUs]) and Bacteroidetes (19.9% of OTUs) dominated. Dominant genera were Roseburia, Clostridium, and Bacteroides. Hindgut microbial composition and diversity in wild dugongs is affected by ontogeny and probably diet. In captive dugongs, the absence of the dominant bacterial DNA bands identified in wild dugongs is probably dependent upon prevailing diet and other captive conditions such as the use of antibiotics. This study represents a first step in the characterisation of a novel microbial ecosystem-the marine hindgut of Sirenia.

Powdery mildews on Clitoria in Australia

An examination of Australian herbarium specimens of powdery mildew on Clitoria revealed two species. Oidium clitoriae is reported for the first time in Australia causing powdery mildew on Clitoria sp. in northern Queensland. Golovinomyces cf. orontii is reported for the first time on the genus Clitoria, from Clitoria ternatea in Queensland and the Northern Territory. © 2012 Australasian Plant Pathology Society Inc.

Pathogenicity of Blumeria graminis f. sp. hordei in Australia in 2010 and 2011

A survey of the Australian barley powdery mildew (Blumeria graminis f. sp. hordei) population was conducted in 2010 and 2011. Three hundred and sixty-two isolates of the pathogen were collected from 18 locations across all six states of Australia. Thirty-two barley differentials were used and 11 genotypes were able to differentiate the population with virulence frequencies varying from 14.5 % to 96.6 %. Twenty-seven pathotypes were detected. Fifteen of them were found in both years and they represented 92.0 % of all isolates examined. No virulence was found on a further 16 major genes for resistance (Mla1, Mla3, Mla6, Mla7, Mla9, Mla10, Mla12, Mla13, Mla23, MlaN81, Mlh, MlLa, Mlp1, Ml(IM9), Ml(St) and mlo) indicating a relatively simple population and the ready availability of diverse sources of resistance. This paper reports the powdery mildew virulences present in Australia, provides intelligence for future resistance breeding and sets a basis for further virulence studies.

Cytorhabdovirus phosphoprotein shows RNA silencing suppressor activity in plants, but not in insect cells

RNA silencing in plants and insects provides an antiviral defense and as a countermeasure most viruses encode RNA silencing suppressors (RSS). For the family Rhabdoviridae, no detailed functional RSS studies have been reported in plant hosts and insect vectors. In agroinfiltrated Nicotiana benthamiana leaves we show for the first time for a cytorhabdovirus, lettuce necrotic yellows virus (LNYV), that one of the nucleocapsid core proteins, phosphoprotein (P) has relatively weak local RSS activity and delays systemic silencing of a GFP reporter. Analysis of GFP small RNAs indicated that the P protein did not prevent siRNA accumulation. To explore RSS activity in insects, we used a Flock House virus replicon system in Drosophila S2 cells. In contrast to the plant host, LNYV P protein did not exhibit RSS activity in the insect cells. Taken together our results suggest that P protein may target plant-specific components of RNA silencing post siRNA biogenesis.