John Fosu-Nyarko

Root-lesion nematodes (Pratylenchus spp.) reduce the yield and quality of cereal crops in Australia. Eleven of the ~90 species characterised are present in Aus-tralia, with those determined as economic pests of broadacre agriculture costing an estimated AUD 250 million annually. Two species, P. curvicauda and P. quasitereoides, recently re-described, were isolated from fields located in the grainbelt of Western Australia, but little is known about their distribution in the region surveyed in this study. To investigate this and possible co-infestations with other Pratylenchus spp., we surveyed seven commercial wheat, barley, and oat farms near Katanning, Cancanning, Kenmare, Duranillin, Darkan, and a barley seed-bulk nursery near Manjimup, all in the southwest grainbelt of Western Australia. Morphological and molecular charac-terisation of Pratylenchus spp. extracted from soil and plant roots indicated all fields surveyed were infested. Both P. quasitereoides and P. curvicauda were present as single or mixed populations with P. penetrans and/or P. neglectus, although they were not found in the same field. Analyses of the D2–D3 sequences of the identified nematodes indicated that the species found in Australia were distinct, particularly P. quasitereoides and P. curvicauda. This work suggests P. curvicauda is likely to be present more widely in the WA grainbelt. Expanding molecular diagnostic testing for Pratylenchus species in the region to account for both nematodes is urgently needed so effective management can be implemented.

Plastic pollution in terrestrial environments is a growing concern, with an increasing focus on the impact of plastic additives on soil ecosystems. We evaluated the impact of additives from conventional plastics (ACP) and biodegradable plastics (ABP) on the soil nematode, Pratylenchus neglectus. The additives represented five functional classes (antioxidants, colourants, flame retardants, nucleating agents, and plasticisers). P. neglectus exhibited concentration-dependent mortality when exposed to the additives, with Tartrazine, an ABP colourant, inducing higher mortality compared to the conventional counterpart. No significant changes in the locomotory patterns of P. neglectus were observed, whereas oxidative stress significantly increased in response to all assistive treatments. Exposure to most of the additives resulted in a significant decline in nematode reproduction; ACPs generally caused more severe effects than ABPs. Our findings highlight a complexity in how plastic additives impact soil organisms and challenge the assumption that ABPs may be universally safer for ecosystems. The study emphasises the importance of conducting ecotoxicological assessments of specific ABPs on important species to inform the design of environmentally sustainable plastics. The results also suggest that P. neglectus could serve as a valuable sentinel organism for evaluating the ecological impacts of plastic pollution in soil.

Storing potato tubers at cold temperatures, either for transport or continuity of supply, is associated with the conversion of sucrose to reducing sugars. When cold-stored cut tubers are processed at high temperatures, with endogenous asparagine, acrylamide is formed. Acrylamide is classified as a carcinogen. Potato processors prefer cultivars which accumulate fewer reducing sugars and thus less acrylamide on processing, and suitable processing cultivars may not be available. We used CRISPR-Cas9 to disrupt the genes encoding vacuolar invertase (VInv) and asparagine synthetase 1 (AS1) of cultivars Atlantic and Desiree to reduce the accumulation of reducing sugars and the production of asparagine after cold storage. Three of the four guide RNAs employed induced mutation frequencies of 17-98%, which resulted in deletions, insertions and substitutions at the targeted gene sites. Eight of ten edited events had mutations in at least one allele of both genes; for two, only the VInv was edited. No wild-type allele was detected in both genes of events DSpco7, DSpFN4 and DSpco12, suggesting full allelic mutations. Tubers of two Atlantic and two Desiree events had reduced fructose and glucose concentrations after cold storage. Crisps from these and four other Desiree events were lighter in colour and included those with 85% less acrylamide. These results demonstrate that multiplex CRISPR-Cas9 technology can generate improved potato cultivars for healthier processed potato products.

Australia has a well-developed system for the regulation of genetically manipulated (GM) and gene-edited (GEd) organisms, including grains and horticultural crop plants. The aim is to protect the health and safety of people and the environment by identifying any risks posed by gene technology. It is based on two Commonwealth legislative acts, the Gene Technology (GT) Act 2000 and the Gene Technology Regulations 2001, with corresponding State and Territory laws. The GT Act included establishing the Office of the Gene Technology Regulator (OGTR), which is overseen by the Gene Technology Regulator, who takes advice and consults with a range of bodies, and is responsible for ensuring the monitoring and compliance of the GT legislation. There have been a series of reviews of the National Gene Technology Scheme to modernize and future-proof it in response to new scientific developments. Following a consultative review process (2016–2019), to address national policy on the products of various new GEd technologies (including site-directed nucleases: SDN-1, SDN-2, SDN-3 and olignucleotide-directed mutagenesis—ODM), in October 2019 the decision was released to deregulate SDN-1 products not generated via transgenic approaches, but not those which used SDN-2, ODM or SDN-3 technologies, which remain captured as GMOs. Thus only products of SDN-1 technology which do not contain any externally introduced DNA bases can be grown in the same way as products of conventional breeding activities.

In New Zealand, GM and GEd organisms are regulated by the Hazardous Substances and New Organisms (HSNO) Act 1996 administered by the Environmental Protection Agency (EPA). In an initial determination in 2012, the EPA determined that organisms produced using GEd methods, where no foreign DNA remained in the edited plant, were not GMOs. However, in 2014, following an appeal to this decision by the Sustainability Council of New Zealand Trust (Sustainability Council) in the High Court, the determination of the presiding judge was to overturn the EPA’s decision. Thus the current status of all GEd products in New Zealand is that they are regulated as GMOs.

Nonliving food products of GM or GEd technology are overseen by Food Standards Australia New Zealand (FSANZ); the Australia New Zealand Food Standards Code applies to GM and GEd foods. FSANZ is currently reviewing its position on GEd foods, and at present considers applications for GEd food on a case-by-case basis.

Next-generation sequencing and analyses of whole-genome transcripts can be used to identify genes and potential mechanisms that may be responsible for the development of resistance to insecticides. Such genes can be identified by isolating and sequencing high-quality messenger RNA and identifying differentially expressed genes (DEGs), and gene variants from insecticide-treated and untreated colonies of the Green peach aphid (GPA) or resistant and susceptible GPA populations. Datasets generated would reveal a set of genes whose expression may be associated with the insecticide treatment. The DEGs can then be validated using quantitative PCR assays.

Genome- or gene-editing (abbreviated here as ‘GEd’) presents great opportunities for crop improvement. This is especially so for the countries in the Asia-Pacific region, which is home to more than half of the world’s growing population. A brief description of the science of gene-editing is provided with examples of GEd products. For the benefits of GEd technologies to be realized, international policy and regulatory environments must be clarified, otherwise non-tariff trade barriers will result. The status of regulations that relate to GEd crop products in Asian countries and Australasia are described, together with relevant definitions and responsible regulatory bodies. The regulatory landscape is changing rapidly: in some countries, the regulations are clear, in others they are developing, and some countries have yet to develop appropriate policies. There is clearly a need for the harmonization or alignment of GEd regulations in the region: this will promote the path-to-market and enable the benefits of GEd technologies to reach the end-users.

Plant-parasitic nematodes are found in most places in the world. The most prevalent ones have a broad host range, so wherever they are located, whether in a backyard garden or on a commercial farm, they are likely to be pests. For this reason, readily accessible, relatively inexpensive, and environmentally friendly methods of control have been sought and applied to control many species of these pests. One such control strategy is organic amendments, of which many types have been demonstrated as a potentially successful method for controlling different types of plant nematodes. This chapter discusses the biochemical and molecular mechanisms underlying the efficacy of organic amendments, the direct effects of active compounds, and the indirect adverse impact on various aspects of the life cycle of different plant-parasitic nematodes. Caveats in interpreting data on the applications of organic amendments to control nematodes and other factors that may dictate their efficacy are also discussed.

Herbicide and antibiotic tolerance genes serve as useful selectable markers for the development of transgenic plants expressing other transgenes. It may be desirable for regulatory or safety reasons to silence the herbicide tolerance trait after transformants have been selected. However, because the genes of interest and the marker gene are usually tightly linked, traditional segregation-based strategies for elimination of undesirable transgenes are usually unsuccessful. Here, we created Nicotiana tabacum plants that carry a single copy of a Cas9 gene, a nuclease in the clustered regularly interspaced short palindromic repeats (CRISPR) system, physically linked to the selectable marker gene bar for tolerance to the herbicide glufosinate (Basta, Liberty). Here, bar was targeted within the genome by introducing bar-specific single guide RNAs (sgRNAs) to the N. tabacum line in vitro, resulting in abolishment of the glufosinate-tolerance trait in mature plants. Sequence analysis of the bar gene revealed a frame-shift mutation at a sgRNA target site, confirming efficacy of the strategy.

We report comparisons between the complete genomic sequences of five historical Western Australian isolates of subterranean clover mottle virus (SCMoV) from 1989–2000, and an infectious clone of its 1989 isolate. Sanger Sequencing (SS) and High Throughput Sequencing (HTS), or both, were used to obtain these genomes. Four of the SCMoV isolates were sequenced by SS in 1999–2002, but re-sequenced again by HTS in 2020. The pairs of sequences obtained from these four isolates differed by only 18–59 nucleotides. This small difference resulted from the different sequencing methods, the < 1–5 years each isolate was host passaged before freeze-drying prior to HTS sequencing, or a combination of both. Since SCMoV has not been reported outside Australia, this similarity suggests the population sequenced represents the progeny of either an indigenous virus that spread from a native legume to subterranean clover after its introduction or a recent seed-borne incursion from elsewhere. The ORF1 was the most variable, and the phylogenetic tree constructed with ORF1s showed the isolates grouped according to their symptom severity in subterranean clover, indicating the probability that ORF1-encoded P1 protein is a symptom determinant. A satellite RNA was associated with all SCMoV genomes obtained by HTS but none derived by SS.

Plant hosts can be engineered to disrupt the development of sedentary plant-parasitic nematodes or proper functioning of the feeding sites the nematodes induce. The use of constitutive promoters to express dsRNAs or nematode inhibitor proteins may be unreliable because of possible silencing or yield penalty from continuous expression in a plant host. This ill-effect can be avoided if a root-specific, nematode-responsive promoter (NRP) is used to drive the target nematode-inhibitory message. This study used the In Plant Activation (INPACT) system to express a barstar-controlled barnase in galls of Meloidogyne javanica and assessed how the engineered tobacco lines affected the growth and development of the nematodes. Of the 11 combinations of four NRPs and the CaMV 35S promoter assessed, the AtCel1 and TobRB7 combinations activated specific expression of split β-glucuronidase (GUS) and barnase genes in and around giant cells. The same NRP combination directed expression of the barnase gene in tobacco roots also constitutively expressing the barstar gene (SPBB transgenic lines). On roots of six T1 SPBB lines, there was up to 94% reduction in the number of galls with significantly smaller adult females compared to those on wild-type plants. Some of the females on lines SPBB4-1 and SPBB-4-2, for example, were not associated with galls. The results indicated the engineered plants disrupted M. javanica development and demonstrate the potential for controlled and localized expression of peptides, such as those that could block specific effectors, to disrupt initiation, formation, establishment, or proper functioning of feeding cells induced by damaging sedentary nematodes.