New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

Fungal diversity in canopy soil of silver beech, Nothofagus menziesii (Nothofagaceae).

Adventitious roots in canopy soils associated with silver beech (Nothofagus menziesii Hook.f. (Nothofagaceae)) form ectomycorrhizal associations. We investigated the extent to which canopy ectomycorrhizal communities contribute to overall diversity of ectomycorrhizal fungi associated with silver beech. Hyphal ingrowth bags were buried for 12 months in canopy and terrestrial soils of five trees at one site. We used amplicon sequencing of the nuclear ribosomal internal transcribed spacer 2 region (ITS2) to assess diversity of both ectomycorrhizal and non-ectomycorrhizal OTUs in hyphal ingrowth bags. There was a significant difference in ectomycorrhizal fungal community diversity between the terrestrial and canopy hyphal ingrowth bag communities. Ectomycorrhizal community composition of the terrestrial and canopy environments was also significantly different. Some ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The hyphal ingrowth bags also accumulated non-ectomycorrhizal species. The non-ectomycorrhizal fungi also had significantly different diversity and community composition between the canopy and terrestrial environments. Like the ectomycorrhizal community, some non-ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The canopy soil microhabitat provides a novel environment for growth of ectomycorrhizal adventitious roots and enables the spatial partitioning of ectomycorrhizal and non-ectomycorrhizal fungal diversity in the forest.

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand.

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non-heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%-42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.

Prospects for analyzing ancient RNA in preserved materials.

An ever increasing wealth of ancient biological material is providing opportunities to study biomolecules. Animal, plant, and microbial samples dating back hundreds, thousands, and even millions of years have been preserved in a dry state under climatic conditions ranging from the arctic to hot deserts. Various small molecules, often crystalized or polymerized, have improved preservation. Modern methods such as polymerase chain reaction (PCR), mass spectrometry, and shotgun sequencing have detected and characterized ancient biomolecules. Modern sequencing has the capacity not only to assemble the whole genome of the target host but also those of the host's parasites, mutualists, and commensals. The study of ancient RNA has barely begun. Several studies show that RNA has been preserved for decades to hundreds of years and the germination of ancient seeds implies that messenger RNA can be preserved for thousands of years. This review briefly examines the types of ancient materials available and assesses their suitability for the study of ancient RNA. Sequencing RNA from this material has the potential not only to illuminate the target host's transcriptome and small RNAs but also to characterize the host's RNA parasites: viruses and viroids.

Ancient RNA? RT-PCR of 50-year-old RNA identifies peach latent mosaic viroid.

The preservation of macromolecules is at best haphazard. Modern techniques have improved the detection of ancient DNA and proteins, but there is little information on the preservation of RNA. Fifty-year-old dried leaf material showing symptoms of peach calico disease was used successfully in RT-PCRs to amplify peach latent mosaic viroid (PLMVd) RNA and the mRNA for the large subunit of ribulose 1,5-bisphosphate carboxylase (rubisco). These results indicate that naked RNA may be preserved, under suitable conditions, for at least 50 years. The results are discussed in the context of ancient DNA and proteins and the process of fossilization.

Detection and analysis of endogenous badnaviruses in the New Zealand flora.

BACKGROUND AND AIMS: Badnaviruses and their host-integrated DNA occur in tropical crops and a few northern temperate species. Following the discovery of a badnavirus on a subantarctic island with floristic links to New Zealand, we postulated that badnaviruses exist in the New Zealand flora. Badnavirus reverse transcriptase (RT) sequences consist of variable regions flanked by highly conserved regions. This study used RT sequences to detect and characterize badnavirus sequences in the New Zealand flora and to investigate their utility for the study of broader aspects of plant biology. METHODOLOGY: Molecular diversity of RT sequences was analysed using polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). In a study of the genus Melicytus, internal transcribed spacer (ITS) sequences were compared with the RT data. PRINCIPAL RESULTS: No freely replicating badnaviruses were detected but more than half of the species (37/60) contained RT sequences. Phylogenetic analysis of 21 RT sequences formed monophyletic groups distinct from other species and from badnaviruses. No frameshift mutations occurred in any of the sequences translated in silico. More detailed study of the genus Melicytus indicated broader applications for our approach. Analysis of RT sequences revealed the presence of a previously unrecognized species (confirmed using ITS). Inheritance of DGGE profiles by Melicytus ramiflorus seedlings suggested that this species may undergo apomixis. CONCLUSIONS: The presence of integrated badnavirus sequences in a wide range of taxa from this Southern Hemisphere flora indicates that these sequences may be common in many temperate regions. Potential to activate viruses from these sequences should be considered when placing these species in tissue culture or under other forms of abiotic or genomic stress. Analysis of endogenous RT sequences shows potential for the study of systematics, phylogenetics and plant reproductive biology.

Changes in the activities of antioxidant enzymes in response to virus infection and hormone treatment.

Activities of enzymes involved in the detoxification of reactive oxygen species (catalase, glutathione reductase, peroxidase and superoxide dismutase (SOD)) were examined in the leaves of Phaseolus vulgaris L. var. Top Crop treated with plant hormones and infected with a non-lesion-forming isolate of white clover mosaic potexvirus (WClMV). The activities of catalase, glutathione reductase and SOD rapidly declined after infection while peroxidase activity was enhanced. These changes occurred before the rapid increase (5 days) in WClMV replication. A mild chlorosis appeared 7-10 days after inoculation but necrosis was never observed on inoculated leaves. Plants treated with dihydrozeatin, salicylic acid and jasmonic acid prior to WClMV inoculation showed elevated catalase, glutathione reductase, and peroxidase activity, while SOD activities remained the same as in water-treated controls. These treatments all inhibited virus replication with enzyme activities remaining near control levels. We propose that a decline in free radical scavenging capacity may be required before a rapid increase in virus replication can take place. Treatments increasing the ability of the plant to scavenge reactive oxygen species may hinder virus replication. A possible role for reactive oxygen species as a requirement for virus replication is discussed.