Host-delivered RNAi: An effective strategy to silence genes in plant parasitic nematodes

Root-knot nematodes (Meloidogyne spp.) are obligate, sedentary endoparasites that infect many plant species causing large economic losses worldwide. Available nematicides are being banned due to their toxicity or ozone-depleting properties and alternative control strategies are urgently required. We have produced transgenic tobacco (Nicotiana tabacum) plants expressing different dsRNA hairpin structures targeting a root-knot nematode (Meloidogyne javanica) putative transcription factor, MjTis11. We provide evidence that MjTis11 was consistently silenced in nematodes feeding on the roots of transgenic plants. The observed silencing was specific for MjTis11, with other sequence-unrelated genes being unaffected in the nematodes. Those transgenic plants able to induce silencing of MjTis11, also showed the presence of small interfering RNAs. Even though down-regulation of MjTis11 did not result in a lethal phenotype, this study demonstrates the feasibility of silencing root-knot nematode genes by expressing dsRNA in the host plant. Host-delivered RNA interference-triggered (HD-RNAi) silencing of parasite genes provides a novel disease resistance strategy with wide biotechnological applications. The potential of HD-RNAi is not restricted to parasitic nematodes but could be adapted to control other plant-feeding pests.

Effect of microwave radiation on seed mortality of rubber vine (Cryptostegia grandiflora R.Br.), parthenium (Parthenium hysterophorous L.) and bellyache bush (Jatropha gossypiifolia L.)

A trial was undertaken to evaluate the effect of microwaves on seed mortality of three weed species. Seeds of rubber vine (Cryptostegia grandiflora R.Br.), parthenium (Parthenium hysterophorous L.) and bellyache bush (Jatropha gossypiifolia L.) were buried at six depths (0, 2.5, 5, 10, 20 and 40 cm) in coarse sand maintained at one of two moisture levels, oven dry or wet (field capacity), and then subjected to one of five microwave radiation durations of (0, 2, 4, 8 and 16 min). Significant interactions between soil moisture level, microwave radiation duration, seed burial depth and species were detected for mortality of seeds of all three species. Maximum seed mortality of rubber vine (88%), parthenium (67%) and bellyache bush (94%) occurred in wet soil irradiated for 16 min. Maximum seed mortality of rubber vine and bellyache bush seeds occurred in seeds buried at 2.5 cm depth whereas that of parthenium occurred in seeds buried at 10 cm depth. Maximum soil temperatures of 114.1 and 87.5°C in dry and wet soil respectively occurred at 2.5 cm depth following 16 min irradiation. Irrespective of the greater soil temperatures recorded in dry soil, irradiating seeds in wet soil generally increased seed mortality 2.9-fold compared with dry soil. Moisture content of wet soil averaged 5.7% compared with 0.1% for dry soil. Results suggest that microwave radiation has the potential to kill seeds located in the soil seed bank. However, many factors, including weed species susceptibility, determine the effectiveness of microwave radiation on buried seeds. Microwave radiation may be an alternative to conventional methods at rapidly depleting soil seed banks in the field, particularly in relatively wet soils that contain long lived weed seeds.

Molecular discrimination of Perna (Mollusca: Bivalvia) species using the polymerase chain reaction and species-specific mitochondrial primers

This work was prompted by the need to be able to identify the invasive mussel species, Perna viridis, in tropical Australian seas using techniques that do not rely solely on morphology. DNA-based molecular methods utilizing a polymerase chain reaction (PCR) approach were developed to distinguish unambiguously between the three species in the genus Perna. Target regions were portions of two mitochondrial genes, cox1 and nad4, and the intergenic spacer between these that occurs in at least two Perna species. Based on interspecific sequence comparisons of the nad4 gene, a conserved primer has been designed that can act as a forward primer in PCRs for any Perna species. Four reverse primers have also been designed, based on nad4 and intergenic spacer sequences, which yield species-specific products of different lengths when paired with the conserved forward primer. A further pair of primers has been designed that will amplify part of the cox1 gene of any Perna species, and possibly other molluscs, as a positive control to demonstrate that the PCR is working.

Growth and species interactions of Eucalyptus pellita in a mixed and monoculture plantation in the humid tropics of north Queensland

This study investigated whether mixed-species designs can increase the growth of a tropical eucalypt when compared to monocultures. Monocultures of Eucalyptus pellita (E) and Acacia peregrina (A) and mixtures in various proportions (75E:25A, 50E:50A, 25E:75A) were planted in a replacement series design on the Atherton Tablelands of north Queensland, Australia. High mortality in the establishment phase due to repeated damage by tropical cyclones altered the trial design. Effects of experimental designs on tree growth were estimated using a linear mixed-effects model with restricted maximum likelihood analysis (REML). Volume growth of individual eucalypt trees were positively affected by the presence of acacia trees at age 5 years and this effect generally increased with time up to age 10 years. However, the stand volume and basal area increased with increasing proportions of E. pellita, due to its larger individual tree size. Conventional analysis did not offer convincing support for mixed-species designs. Preliminary individual-based modelling using a modified Hegyi competition index offered a solution and an equation that indicates acacias have positive ecological interactions (facilitation or competitive reduction) and definitely do not cause competition like a eucalypt. These results suggest that significantly increased in growth rates could be achieved with mixed-species designs. This statistical methodology could enable a better understanding of species interactions in similarly altered experiments, or undesigned mixed-species plantations.

Review of Nezara viridula (L.) management strategies and potential for IPM in field crops with emphasis on Australia

Nezara viridula (L.) is a cosmopolitan, polyphagous heteropteran that causes economic damage to many crop species. At present, control of N. viridula in Australia and other countries relies heavily upon insecticides, most of which are disruptive to beneficial insects, constituting a constraint on integrated pest management (IPM). Much research has been conducted into non-chemical control methods for N. viridula. This paper reviews the potential for and limitations of sterile insect technique, classical, inundative and conservation biological control, and trap cropping. None of these techniques appear to be adequate for control of N. viridula when used alone but there is scope for these non-chemical approaches to be adopted for use in integrated management of this pest. A proposal is given for one such integrated approach for future development. It includes biopesticides, trap crops and carefully targeted habitat manipulation to enhance arthropod natural enemies as well as area-wide management and grower education.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Bitou bush and boneseed eradication and containment in Australia

Bitou bush and boneseed (Chrysanthemoides monilifera subsp. rotundata (DC.) T.Norl. and C. monilifera subsp. monilifera (L.) T.Norl., respectively) are highly invasive environmental weeds that pose a serious threat to Australia’s natural ecosystems and biota. Bitou bush threatens coastal plant communities in New South Wales (NSW), eastern Victoria and southeast Queensland (Qld), while boneseed threatens inland and coastal native plant communities across NSW, South Australia (SA), Tasmania, Victoria, and Western Australia (WA). Over 200 plant species and ecological communities in Australia are negatively impacted by these weeds (ARMCANZ et al. 2000, DEC 2006) and over 15% (approx. 120 million ha) of Australia is susceptible to invasion (see maps in Weiss et al 2008). In 2000, the National Bitou Bush and Boneseed Strategic Plan (ARMCANZ et al. 2000) was approved as part of the Commonwealth’s Weeds of National Significance initiative. A key goal of this plan is to prevent the spread of bitou bush and boneseed in Australia. A national program sponsored by the Australian Government and the affected states has resulted in the development of national containment and eradication zones that prevent the spread of bitou bush and boneseed. This paper presents an overview of these bitou bush and boneseed containment and eradication programs.

Validation of in silico-predicted genic SNPs in white clover (Trifolium repens L.), an outbreeding allopolyploid species

White clover (Trifolium repens L.) is an obligate outbreeding allotetraploid forage legume. Gene-associated SNPs provide the optimum genetic system for improvement of such crop species. An EST resource obtained from multiple cDNA libraries constructed from numerous genotypes of a single cultivar has been used for in silico SNP discovery and validation. A total of 58 from 236 selected sequence clusters (24.5%) were fully validated as containing polymorphic SNPs by genotypic analysis across the parents and progeny of several two-way pseudo-testcross mapping families. The clusters include genes belonging to a broad range of predicted functional categories. Polymorphic SNP-containing ESTs have also been used for comparative genomic analysis by comparison with whole genome data from model legume species, as well as Arabidopsis thaliana. A total of 29 (50%) of the 58 clusters detected putative ortholoci with known chromosomal locations in Medicago truncatula, which is closely related to white clover within the Trifolieae tribe of the Fabaceae. This analysis provides access to translational data from model species. The efficiency of in silico SNP discovery in white clover is limited by paralogous and homoeologous gene duplication effects, which are resolved unambiguously by the transmission test. This approach will also be applicable to other agronomically important cross-pollinating allopolyploid plant species.

Identification of viral and non-viral reverse transcribing elements in pineapple ( Ananas comosus ), including members of two new badnavirus species

A previously published partial sequence of pineapple bacilliform virus was shown to be from a retrotransposon (family Metaviridae) and not from a badnavirus as previously thought. Two newly discovered sequence groups isolated from pineapple were associated with bacilliform virions and were transmitted by mealybugs. Phylogenetic analyses indicated that they were members of new badnavirus species. A third caulimovirid sequence was also amplified from pineapple, but available evidence suggests that this DNA is not encapsidated, but more likely derived from an endogenous virus.

New microsatellite loci for Carcharhinid sharks (Carcharhinus tilstoni and C. sorrah) and their cross-amplification in other shark species

Twelve microsatellite DNA markers were isolated in the spot-tail shark (Carcharhinus sorrah) and nine were isolated in Australian black-tip shark (Carcharhinus tilstoni). These loci plus 18 others developed for sharks from the genera Negaprion, Ginglymostoma, Carcharodon and Isurus were tested for amplification success on four species of Carcharhinus (including C. sorrah and C. tilstoni) and four other species representing three diverse families. Cross-amplification was most common within families. Five loci were subsequently tested for polymorphism on 50 C. sorrah and 60 C. tilstoni. The number of alleles per locus was two to 24 and the average heterozygosity was 0.54 (range 0.16-0.87) for C. sorrah and 0.64 (range 0.44-0.78) for C. tilstoni. These loci may be useful tools for genetic analyses of the Carcharhinidae.

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.

Biological control of parthenium (Parthenium hysterophorus) in Australian rangeland translates to improved grass production.

Biological control of parthenium, a major weed in grazing areas in Australia, was initiated in the mid 1970s. Since then, nine species of insects and two rust fungi have been introduced. Evaluation using pesticide exclusion at two sites (Mt. Panorama and Plain Creek) in Queensland, Australia, revealed that classical biological control had a significant negative effect on the target weed, but the impact varied between years. In this study, I quantified the effects of biological control of parthenium on grass production. Grass production declined with the increase in parthenium biomass. Significant increase in grass production due to biological control was observed, but only in 1 of 4 yr at Mt. Panorama and 2 of 4 yr at Plain Creek. At Mt. Panorama, there was a 40% increase in grass biomass in 1997 because of defoliation by Zygogramma bicolorata and galling by Epiblema strenuana. At Plain Creek, grass biomass increased by 52% in 1998 because of E. strenuana and by 45% in 2000 because of combined effects of E. strenuana and the summer rust Puccinia melampodii. This study provides evidence on the beneficial effects of biological control of parthenium in areas under limited grazing.

Achievements in forest tree improvement in Australia and New Zealand 6: Genetic improvement and conservation of Araucaria cunninghamii in Queensland

Araucaria cunninghamii (hoop pine) typically occurs as an emergent tree over subtropical and tropical rainforests, in a discontinuous distribution that extends from West Irian Jaya at about 0°30'S, through the highlands of Indonesian New Guinea and Papua New Guinea, along the east coast of Australia from 11°39'S in Queensland to 30°35'S in northern New South Wales. Plantations established in Queensland since the 1920s now total about 44000 ha, and constitute the primary source for the continuing supply of hoop pine quality timber and pulpwood, with a sustainable harvest exceeding 440 000 m3 y-1. Establishment of these managed plantations allowed logging of all native forests of Araucaria species (hoop pine and bunya pine, A. bidwillii) on state-owned lands to cease in the late 1980s, and the preservation of large areas of araucarian forest types within a system of state-owned and managed reserves. The successful plantation program with this species has been strongly supported by genetic improvement activities since the late 1940s - through knowledge of provenance variation and reproductive biology, the provision of reliable sources of improved seed, and the capture of substantial genetic gains in traits of economic importance (for example growth, stem straightness, internode length and spiral grain). As such, hoop pine is one of the few tropical tree species that, for more than half a century, has been the subject of continuous genetic improvement. The history of commercialisation and genetic improvement of hoop pine provides an excellent example of the dual economic and conservation benefits that may be obtained in tropical tree species through the integration of gene conservation and genetic improvement with commercial plantation development. This paper outlines the natural distribution and reproductive biology of hoop pine, describes the major achievements of the genetic improvement program in Queensland over the past 50+ y, summarises current understanding of the genetic variation and control of key selection traits, and outlines the means by which genetic diversity in the species is being conserved.

Completion of the genome sequence of Lettuce necrotic yellows virus, type species of the genus Cytorhabdovirus

We completed the genome sequence of Lettuce necrotic yellows virus (LNYV) by determining the nucleotide sequences of the 4a (putative phosphoprotein), 4b, M (matrix protein), G (glycoprotein) and L (polymerase) genes. The genome consists of 12,807 nucleotides and encodes six genes in the order 3′ leader-N-4a(P)-4b-M-G-L-5′ trailer. Sequences were derived from clones of a cDNA library from LNYV genomic RNA and from fragments amplified using reverse transcription-polymerase chain reaction. The 4a protein has a low isoelectric point characteristic for rhabdovirus phosphoproteins. The 4b protein has significant sequence similarities with the movement proteins of capillo- and trichoviruses and may be involved in cell-to-cell movement. The putative G protein sequence contains a predicted 25 amino acids signal peptide and endopeptidase cleavage site, three predicted glycosylation sites and a putative transmembrane domain. The deduced L protein sequence shows similarities with the L proteins of other plant rhabdoviruses and contains polymerase module motifs characteristic for RNA-dependent RNA polymerases of negative-strand RNA viruses. Phylogenetic analysis of this motif among rhabdoviruses placed LNYV in a group with other sequenced cytorhabdoviruses, most closely related to Strawberry crinkle virus.

Resistance of Brassicaceae plants to root-knot nematode (Meloidogyne spp.) in northern Australia

Brassicaceae plants have the potential as part of an integrated approach to replace fumigant nematicides, providing the biofumigation response following their incorporation is not offset by reproduction of plant-parasitic nematodes on their roots. Forty-three Brassicaceae cultivars were screened in a pot trial for their ability to reduce reproduction of three root-knot nematode isolates from north Queensland, Australia: M. arenaria (NQ1), M. javanica (NQ2) and M. arenaria race 2 (NQ5/7). No cultivar was found to consistently reduce nematode reproduction relative to forage sorghum, the current industry standard, although a commercial fodder radish (Raphanus sativus) and a white mustard (Sinapis alba) line were consistently as resistant to the formation of galls as forage sorghum. A second pot trial screened five commercially available Brassicaceae cultivars, selected for their biofumigation potential, for resistance to two nematode species, M. javanica (NQ2) and M. arenaria (NQ5/7). The fodder radish cv. Weedcheck, was found to be as resistant as forage sorghum to nematode reproduction. A multivariate cluster analysis using the resistance measurements, gall index, nematode number per g of root and multiplication for two nematode species (NQ2 and NQ5/7) confirmed the similarity in resistance between the radish cultivar and forage sorghum. A field trial confirmed the resistance of the fodder radish cv. Weedcheck, with a similar reduction in the number of Meloidogyne spp. juveniles recovered from the roots 8 weeks after planting. The use of fodder radish cultivars as biofumigation crops to manage root-knot nematodes in tropical vegetable production systems deserves further investigation.