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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

Dicers and dicer-like enzymes play an essential role in small RNA processing in eukaryotes. Nematodes are thought to encode one dicer, DCR-1; only that for Caenorhabditis spp. is well-characterised. Using genomic sequences of eight root-knot nematodes (Meloidogyne spp.), we identified putative coding sequences typical of eukaryotic DICERS. We noted that the primary and secondary structures of DICERS they encode were different for different Meloidogyne species and even for isolates of the same species, suggesting paralogy for the gene. One of the genes for M. incognita (Midcr-1.1) expressed in eggs, juvenile stage 2 and adults, with the highest expression in the adult females. All the Meloidogyne DICERS had seven major domains typical of those for Caenorhabditis spp. and humans with very similar protein folding. RNAi of Midcr-1.1 in J2s using seven dsRNAs, each based on sequences encoding the domains, induced mild paralysis but measurable knockdown was detected in J2s treated with five of the dsRNAs. For four of the dsRNAs, the RNAi effect lasted and reduced the nematode’s infectivity. Also, host plant delivery of dsRNAs complementary to coding sequences of the Dicer Dimerisation domain impaired development, reducing nematode infection by 71%. These results confirm the importance of the gene to nematode health.

Conventional nested PCR and Sanger sequencing methods are currently the gold standards for detecting trypanosomes in wildlife. However, these techniques are time-consuming and can often overlook mixed infections. True trypanosome prevalence can thus be underrepresented. Here, we designed an 18S rDNA-based real-time quantitative PCR (qPCR) assay coupled with High-Resolution Melting Analysis (HRMA) to detect and discriminate three Trypanosoma species (T. copemani, T. noyesi, and T. vegrandis) commonly infecting Australian marsupials. A total of 68 genetically characterised samples from blood and tissue were used to validate the High-Resolution Melting - Real Time Quantitative Polymerase Chain Reaction (HRM-qPCR) assay. A further 87 marsupial samples consisting of blood, tissue and in vitro cultures derived from wildlife blood samples, were screened for the first time using this assay, and species identity confirmed using conventional PCR and Sanger sequencing. All three Trypanosoma species were successfully detected in pure cultures using the HRM-qPCR assay, and in samples containing mixed trypanosome infections. Of the 87 marsupial samples screened using the HRM-qPCR assay, 93.1% were positive for trypanosomes, and 8.0% contained more than one trypanosome species. In addition to the three targeted Trypanosoma species, this assay was also able to detect and identify other native and exotic trypanosomes. The turnaround time for this assay, from sample preparation to obtaining results, was less than 2 h, with a detection limit of 10 copies of the amplicon in a reaction for each of the targeted trypanosome species. This more rapid and sensitive diagnostic tool provides a high throughput platform for the detection, identification and quantification of trypanosome infections. It will also improve understanding of host diversity and parasite relationships and facilitate conservation management decisions., Graphical abstract Image 1

Pratylenchus curvicauda , which was first described in metropolitan Perth in 1991, was recently identified in grain‐growing areas in Western Australia. The biology of this root‐lesion nematode, and especially its pest status, is unknown. We investigated its life cycle and interaction with host plants, because such information is essential for its management. The life cycle took 45 days to complete in a wheat cultivar maintained at 23°C. Over 10 weeks, the nematode multiplied in 26 of 61 genotypes; these host plants were all cereals and included widely grown cultivars of wheat and barley. Eighteen other cereal genotypes and 13 cultivars including canola, chickpea, ryegrass, lupin, soybean, and tomato, sustained the nematodes to different degrees without multiplication. Four cover crops were not suitable hosts. The patterns of attraction of the nematodes and penetration into roots of the host and tolerant plants were similar. The nonhosts attracted fewer nematodes, none of which penetrated the roots. Browning of infected roots was atypical—it occurred late in some roots, 55 days after inoculation, and in the presence of a fungus. The nematodes were confined to, and fed from, cortical cells. The ultrastructure of infected wheat and barley cells showed typical signs of damage caused by Pratylenchus spp. and included cell disorganization and lack of membrane integrity, disintegration of cytoplasm, hypertrophy of some nuclei, and deposition of tannin‐like granules. This detailed characterization of P . curvicauda– host interaction indicates the nematode is likely to be a pest of major crops, and attention should be given to its management.

Large amounts of food products are disposed of around the world because they are below market standards. In Australia, low value, non-marketable carrots (Daucus carota) are ploughed into farmlands as green manure or are treated as waste. In recent years significant research interest has focused on developing waste valorisation strategies using new green chemistry-based sustainable processes. More importantly, strategies that also provide solutions for emerging challenges like the rising reports of resistance of bacteria to existing microbes are favourable. This study explored a facile synthesis process to reduce aqueous silver ions in aqueous carrot extracts to form silver nanoparticles that may have antibacterial properties. The synthesis process produced particles with surface plasmon resonance peaks typical of crystalline silver. The silver nanoparticles produced from pure carrot extracts were spherical and pseudo-spherical, 2 to 25 nm wide. However, with polyvinylpyrrolidone, much wider (10-50 nm), well-dispersed silver nanoparticles of various shapes including spherical, polygonal, rod-like and triangular types were produced. Several biomolecules which may act as reducing and capping agents for the process were identified; they included ascorbic, gallic and chlorogenic acids. The Ag nanoparticles produced significant zones of inhibition against the gram-negative E. coli and gram-positive S. epidermidis, indicating they had bacteriostatic properties. The study demonstrates that producing Ag nanoparticles with antibiotic properties from carrots is a good valorisation strategy because other uses for rejected carrot produce such as application as green manure may not be interrupted.

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.

Identifying genes responsive to insecticide treatment is the first step towards understanding the mechanism(s) of insecticide resistance and the development of effective insecticides against economic insect pests such as the Green peach aphid (GPA). Functional and Reverse Genetics approaches such as the RNA interference (RNAi) technology can be used to assess the possible involvement of genes whose expression is associated with an insecticide treatment. For GPA, this can be done by comparing the behavior and development of the insect following RNAi of a putative gene associated with insecticide treatment and exposure of the RNAi-treated insects to lethal doses of insecticides. In a case where knockdown of a gene or genes increases the susceptibility of RNAi-treated populations compared to controls, the target gene may have a direct role in the development of resistance to the insecticide or the gene may be involved in other metabolic processes that may be required for resilience against the insecticide.

Control of plant-parasitic nematodes (PPNs) via host-induced gene silencing (HIGS) involves rational selection of genes and detailed assessment of effects of a possible knockdown on the nematode. Some genes by nature may be very important for the survival of the nematode that knockdown may be resisted. Possible silencing and effects of 20 such genes involved in the RNA interference (RNAi) pathways of Meloidogyne incognita were investigated in this study using long double-stranded RNAs (dsRNAs) as triggers. Two of the genes, ego-1 and mes-2, could not be knocked down. Expression of six genes (xpo-1, pash-1, xpo-2, rha-1, ekl-4, and csr-1) were significantly upregulated after RNAi treatment whereas for 12 of the genes, significant knockdown was achieved and with the exception of mes-2 and mes-6, RNAi was accompanied by defective phenotypes in treated nematodes including various degrees of paralysis and abnormal behaviors and movement such as curling, extreme wavy movements, and twitching. These abnormalities resulted in up to 75% reduction in infectivity of a tomato host, the most affected being the J2s previously treated with dsRNA of the gfl-1 gene. For 10 of the genes, effects of silencing in the J2s persisted as the adult females isolated from galls were under-developed, elongated, and transparent compared to the normal saccate, white adult females. Following RNAi of ego-1, smg-2, smg-6, and eri-1, reduced expression and/or the immediate visible effects on the J2s were not permanent as the nematodes infected and developed normally in tomato hosts. Equally intriguing was the results of RNAi of the mes-2 gene where the insignificant change in gene expression and behavior of treated J2s did not mean the nematodes were not affected as they were less effective in infecting host plants. Attempt to silence drsh-1, mut-7, drh-3, rha-1, pash-1, and vig-1 through HIGS led to reduction in nematode infestation by up to 89%. Our results show that genes may respond to RNAi knockdown differently so an exhaustive assessment of target genes as targets for nematode control via RNAi is imperative.

A Pratylenchus species identified during a survey of Pratylenchus quasitereoides incidence at four locations of the grainbelt of Western Australia is described. Morphological and morphometric features indicated the previously undescribed morphotypes in nematode mixtures encountered were conspecific to P. curvicauda, and were clearly distinguishable from nine common Pratylenchus spp. Typical features of P. curvicauda were its body length (415–540 µm), which was curved to a c-shaped with a maximum body diameter of 20 µm, and the nature of its tail; 34 µm long, 2.8 µm wide at the anus and a typical ventrally arcuate with a round terminus. Sequenced for the first time, the sequences of the partial 18S-ITS1-5.8S-ITS2-partial 28S (80 clones, 14 individual nematodes) and the 28S-D3 (17 clones) regions of the rDNA of P. curvicauda had overall mean distances of 0.013 and 0.085, respectively. Phylogenetic analyses with sequences of both segments of the rDNA clearly showed the P. curvicauda isolates as monophyletic, distinct from ca 40 Pratylenchus species. Notably, it was distinct from Pratylenchus species present in Australia including P. quasitereoides and a Western Australia isolate of P. thornei. Further research into the biology of P. curvicauda is needed to facilitate development of strategies for its management, if it is an important pest.

The discovery of RNA interference (RNAi) as an endogenous mechanism of gene regulation in a range of eukaryotes has resulted in its extensive use as a tool for functional genomic studies. It is important to study the mechanisms which underlie this phenomenon in different organisms, and in particular to understand details of the effectors that modulate its effectiveness. The aim of this study was to identify and compare genomic sequences encoding genes involved in the RNAi pathway of four parasitic nematodes: the plant parasites Meloidogyne hapla and Meloidogyne incognita and the animal parasites Ascaris suum and Brugia malayi because full genomic sequences were available-in relation to those of the model nematode Caenorhabditis elegans. The data generated was then used to identify some potential targets for control of the root knot nematode, M. incognita. Of the 84 RNAi pathway genes of C. elegans used as model in this study, there was a 42-53 % reduction in the number of effectors in the parasitic nematodes indicating substantial differences in the pathway between species. A gene each from six functional groups of the RNAi pathway of M. incognita was downregulated using in vitro RNAi, and depending on the gene (drh-3, tsn-1, rrf-1, xrn-2, mut-2 and alg-1), subsequent plant infection was reduced by up to 44 % and knockdown of some genes (i.e. drh-3, mut-2) also resulted in abnormal nematode development. The information generated here will contribute to defining targets for more robust nematode control using the RNAi technology.

The Root-lesion nematode, Pratylenchus loosi Loof inhabits all tea growing regions of Sri Lanka depicting a variety of symptoms and damage severities to affected tea. Though much has been researched on biology and control measures, isolate characterization has not been attempted. Hence, male and female morphometric variations and molecular characterization of P. loosi populations isolated from different agro-ecological regions in Sri Lanka viz. PL1 (Cecilton, Balangoda), PL2 (Delmar, Halgranoya), PL3 (Hapugastenna, Ratnapura) PL4 (Mahadowa, Passara), PL5 (Nawalapitiya) and PL6 (Richiland, Deniyaya) were studied. Female morphometrics of P. loosi showed intraspecific variation and clustered in four groups in Principal Component Analysis where PL1 and PL5 were closely-related while PL3 and PL6 clustered separately with the exception of PL2 and PL4. Sequence analysis of the D2/D3 expansion segments of the 28S rDNA gene of the P. loosi populations revealed that PL3 and PL6 were closelyrelated while PL1, PL4 and PL5 were relatively distant. Sequences of the ITS region of rDNA placed PL3 and PL6 in a single clade. The isolates PL1, PL2, PL4 and PL5 were relatively distantly-related and PL2 and PL4 were relatively distant from the other populations. Molecular characterization further validated the relatedness of PL1 and PL5, PL3 and PL6 and, PL2 and PL4 obtained from the morphometric data. The divergence in P. loosi populations shown in this study supports evidence of intra specific isolates resulting different symptomological expressions and thus implies need of specific management strategies in managing nematode infestations in tea plantations in Sri Lanka.

The root lesion nematode Pratylenchus zeae, a migratory endoparasite, is an economically important pest of major crop plants (e.g. cereals, sugarcane). It enters host roots, migrates through root tissues and feeds from cortical cells, and defends itself against biotic and abiotic stresses in the soil and in host tissues. We report de novo sequencing of the P. zeae transcriptome using 454 FLX, and the identification of putative transcripts encoding proteins required for movement, response to stimuli, feeding and parasitism. Sequencing generated 347,443 good quality reads which were assembled into 10,163 contigs and 139,104 singletons: 65% of contigs and 28% of singletons matched sequences of free-living and parasitic nematodes. Three-quarters of the annotated transcripts were common to reference nematodes, mainly representing genes encoding proteins for structural integrity and fundamental biochemical processes. Over 15,000 transcripts were similar to Caenorhabditis elegans genes encoding proteins with roles in mechanical and neural control of movement, responses to chemicals, mechanical and thermal stresses. Notably, 766 transcripts matched parasitism genes employed by both migratory and sedentary endoparasites in host interactions, three of which hybridized to the gland cell region, suggesting that they might be secreted. Conversely, transcripts for effectors reported to be involved in feeding site formation by sedentary endoparasites were conspicuously absent. Transcripts similar to those encoding some secretory-excretory products at the host interface of Brugia malayi, the secretome of Meloidogyne incognita and products of gland cells of Heterodera glycines were also identified. This P. zeae transcriptome provides new information for genome annotation and functional analysis of possible targets for control of pratylenchid nematodes.

Plant-parasitic nematodes of the genus Pratylenchus are among the top three most significant nematode pests of crop and horticultural plants worldwide. There are more than 70 described species, most are polyphagous with a wide range of host plants. Because they do not form obvious feeding patterns characteristic of sedentary endoparasites (e.g. galls or cysts), and all worm-like stages are mobile and can enter and leave host roots, it is more difficult to recognise their presence and the damage they cause. As a result their study has been relatively neglected, despite being the major nematode pests of staple crops such as wheat in dryland agriculture in Australia and the Pacific Northwest of the USA. The current status of molecular research on Pratylenchus spp. is described, including molecular diagnostics for their detection in soil samples, the status of breeding for resistance or tolerance, transcriptome data from deep sequencing, and their amenability to powerful new transgenic strategies for control using gene silencing (RNAi) technology. A comparison of the repertoire of enzymes and putative effectors used by Pratylenchus species to enter roots, migrate from cell-to-cell, suppress host defence responses and feed successfully with those present in sedentary endoparasites provides new insights into which effectors are required for migratory phases versus feeding site induction for sedentary endoparasites. The genomics era has finally arrived for Pratylenchus species, and it can be predicted that new genomic information will lead to new strategies to control this neglected but economically important group of plant pests.

Root lesion nematodes (RLNs, Pratylenchus species) are a group of economically important migratory endoparasitic plant pathogens that attack host roots of major crops such as wheat and sugarcane, and can reduce crop yields by 7-15%. Pratylenchus thornei and Pratylenchus zeae were treated with double stranded RNA (dsRNA) to study gene silencing, (RNA interference, RNAi), as a potential strategy for their control. Mixed stages of nematodes of both species ingested dsRNA when incubated in a basic soaking solution in the presence of the neurostimulant octopamine. Incubation for up to 16 h in soaking solutions containing 10-50 mM octopamine, 0.1-1.0 mg/mL FITC, and 0.5-6 mM spermidine did not affect vitality. Spermidine phosphate salt hexahydrate rather than spermidine or spermidine trihydrochloride increased uptake of FITC by nematodes, and this resulted in more effective gene silencing. Silencing pat-10 and unc-87 genes of P. thornei and P. zeae resulted in paralysis and uncoordinated movements in both species, although to a higher degree in P. thornei. There was also a greater reduction in transcript of both genes in P. thornei indicating that it may be more susceptible to RNAi. For P. thornei treated with dsRNA of pat-10 and unc-87 there was a significant reduction (77-81%) in nematode reproduction on carrot mini discs over a 5 week period. The results show that RLNs are clearly amenable to gene silencing, and that in planta delivery of dsRNA to target genes in these nematodes should confer host resistance. Moreover, for the two genes, dsRNA derived from either nematode species silenced the corresponding gene in both species. This implies cross-species control of nematodes via RNAi is possible.

Root lesion nematodes (RLNs) are one of the most economically important groups of plant nematodes. As migratory endoparasites, their presence in roots is less obvious than infestations of sedentary endoparasites; nevertheless, in many instances, they are the major crop pests. With increasing molecular information on nematode parasitism, available data now reflect the differences and, in particular, similarities in lifestyle between migratory and sedentary endoparasites. Far from being unsophisticated compared with sedentary endoparasites, migratory endoparasites are exquisitely suited to their parasitic lifestyle. What they lack in effectors required for induction of permanent feeding sites, they make up for with their versatile host range and their ability to move and feed from new host roots and survive adverse conditions. In this review, we summarize the current molecular data available for RLNs and highlight differences and similarities in effectors and molecular mechanisms between migratory and sedentary endoparasitic nematodes.

The beet cyst nematode, Heterodera schachtii, is a major root pest that significantly impacts the yield of sugar beet, brassicas and related species. There has been limited molecular characterisation of this important plant pathogen: to identify target genes for its control the transcriptome of the pre-parasitic J2 stage of H. schachtii was sequenced using Roche GS FLX. Ninety seven percent of reads (i.e., 387,668) with an average PHRED score > 22 were assembled with CAP3 and CLC Genomics Workbench into 37,345 and 47,263 contigs, respectively. The transcripts were annotated by comparing with gene and genomic sequences of other nematodes and annotated proteins on public databases. The annotated transcripts were much more similar to sequences of Heterodera glycines than to those of Globodera pallida and root knot nematodes (Meloidogyne spp.). Analysis of these transcripts showed that a subset of 2,918 transcripts was common to free-living and plant parasitic nematodes suggesting that this subset is involved in general nematode metabolism and development. A set of 148 contigs and 183 singletons encoding putative homologues of effectors previously characterised for plant parasitic nematodes were also identified: these are known to be important for parasitism of host plants during migration through tissues or feeding from cells or are thought to be involved in evasion or modulation of host defences. In addition, the presence of sequences from a nematode virus is suggested. The sequencing and annotation of this transcriptome significantly adds to the genetic data available for H. schachtii, and identifies genes primed to undertake required roles in the critical pre-parasitic and early post-parasitic J2 stages. These data provide new information for identifying potential gene targets for future protection of susceptible crops against H. schachtii.

The biology of migratory plant parasitic nematodes has been less studied than that of the sedentary endoparasites. The damage they cause is less obvious, their presence and number are more difficult to quantify and they are difficult organisms to study. Nevertheless, they are economically serious pests of many crops, from wheat and barley grown in low rainfall areas to horticultural crops (e.g. Lilium longiflorum) and tropical crops such as coffee, banana and sugarcane. The most studied migratory nematodes are the root lesion nematodes, Pratylenchus spp., the burrowing nematode Radopholus similis and the rice root nematode Hirschmanniella oryzae. In the life cycle of migratory nematodes apart from the egg, all stages of juveniles and adults are motile and can enter and leave host roots. They do not induce the formation of a permanent feeding site, but feed from individual host cells. They create pathways for entry of other root pathogens, often resulting in lesions, stunted roots, yellowing of leaves and plants showing symptoms of water stress, leading to yield loss and decreased quality of produce. In terms of genetic plant defences, no major genes for resistance to migratory nematodes have been found, and resistance breeding is usually based on QTL analysis and marker-assisted selection to combine the best minor resistance genes. Feeding damage reduces root function, and root damage and necrotic lesions the nematodes cause can then make them leave the root and seek others to parasitise. Infestation induces classical plant defence responses and changes in host metabolism which reflects the damage they cause, although detailed studies are lacking. New genomic resources are becoming available to study migratory endoparasites, and the knowledge gained can contribute to improved understanding of their interactions with hosts. Notably transcriptomes of Pratylenchus coffeae, Pratylenchus thornei, Pratylenchus zeae, R. similis and H. oryzae and the first genomic sequence, for P. coffeae, are now available. From these data, some candidate effector genes required for parasitism have been identified: many effectors similar to those found in sedentary endoparasites are present, with the exception of those thought to be involved in formation of feeding sites induced by the sedentary parasites. Belowground defence, in the form of enhanced resistance to migratory parasites, may also be achieved by transgenic expression of modified cysteine protease inhibitors (cystatins), anti-root invasion peptides and host-induced gene silencing (RNAi) strategies, demonstrating that migratory nematodes are amenable to control by these technologies. New more environmentally friendly nematicides, combined with better biological control agents, can be applied or used in seed coatings in integrated pest management approaches to defend roots from attack by migratory nematodes.

The migratory endoparasitic root lesion nematode Pratylenchus thornei is a major pest of the cereals wheat and barley. In what we believe to be the first global transcriptome analysis for P. thornei, using Roche GS FLX sequencing, 787,275 reads were assembled into 34,312 contigs using two assembly programs, to yield 6,989 contigs common to both. These contigs were annotated, resulting in functional assignments for 3,048. Specific transcripts studied in more detail included carbohydrate active enzymes potentially involved in cell wall degradation, neuropeptides, putative plant nematode parasitism genes, and transcripts that could be secreted by the nematode. Transcripts for cell wall degrading enzymes were similar to bacterial genes, suggesting that they were acquired by horizontal gene transfer. Contigs matching 14 parasitism genes found in sedentary endoparasitic nematodes were identified. These genes are thought to function in suppression of host defenses and in feeding site development, but their function in P. thornei may differ. Comparison of the common contigs from P. thornei with other nematodes showed that 2,039 were common to sequences of the Heteroderidae, 1,947 to the Meloidogynidae, 1,218 to Radopholus similis, 1,209 matched expressed sequence tags (ESTs) of Pratylenchus penetrans and Pratylenchus vulnus, and 2,940 to contigs of Pratylenchus coffeae. There were 2,014 contigs common to Caenarhabditis elegans, with 15.9% being common to all three groups. Twelve percent of contigs with matches to the Heteroderidae and the Meloidogynidae had no homology to any C. elegans protein. Fifty-seven percent of the contigs did not match known sequences and some could be unique to P. thornei. These data provide substantial new information on the transcriptome of P. thornei, those genes common to migratory and sedentary endoparasitic nematodes, and provide additional understanding of genes required for different forms of parasitism. The data can also be used to identify potential genes to study host interactions and for crop protection.

Terminal drought tolerance of wheat is a major target in many areas in the world and is a particular focus in Western Australia. It is widely considered to relate to water soluble carbohydrate (WSC) levels such as fructan in the stem, as the head is maturing. Fructan exohydrolases are key enzymes during both fructan biosynthesis and mobilization. The wheat genome sequences of three fructan 1-exohydrolase (1-FEH) genes with seven exons and six introns were isolated by using the available 1-FEH w2 cDNA sequence. The major size differences among the three genes were located in intron 1 and intron 4. The three 1-FEH genes were mapped to Chinese Spring chromosome 6A, 6B and 6D based on polymerase chain reaction (PCR) polymorphisms and Southern hybridization. 1-FEH-6A, -6B and -6D corresponded to published cDNA sequences 1-FEH w1, w3 and w2, respectively. The overall correlation of the mRNA accumulation profile for the 1-FEH genes in stem and sheath leaf tissue in relation to the profile of soluble carbohydrate accumulation was consistent with their postulated role in stem soluble carbohydrate accumulation. The accumulation of the 1-FEH-6B (1-FEH w3) mRNA was 300 fold greater than that of 1-FEH-6A and -6D. The mRNA accumulation continued after the stem water soluble carbohydrate concentrations reached a peak, consistent with a role of 1-FEH-6B in the breakdown of soluble carbohydrate. The relationship between the 1-FEH genes and soluble carbohydrate accumulation is discussed and the 1-FEH-6B gene in particular is suggested to provide a new class of molecular marker for this trait.

Effective control of plant parasitic nematodes in crop plants will contribute hundreds of millions of dollars to global agriculture and help underpin future food security. Natural nematode resistance genes present in gene pools of crop species and their relatives have long been exploited with the aim of transferring such traits into economically important crops where effective resistance is lacking. Biotechnology also contributes to this process via marker-assisted selection to identify and combine the best nematode resistance genes, and increasingly in providing new knowledge of target genes, and the potential to exploit this knowledge using transgenic technology. Thus recent advances now make it possible to exploit specific aspects of nematode-host plant interactions to design control strategies that include enabling plants to prevent nematode invasion, reducing effectiveness of nematode migration through tissues, preventing successful establishment or reducing feeding ability or nematode fecundity. The knowledge of what genes are vital for successful nematode parasitism can also be used to develop new chemical control agents. These new strategies may either be available for public use or be delivered commercially. For transgenic technologies, both modes of delivery face the same issues in terms of deployment, such as substantial field testing, meeting environmental and human safety regulations, adequate funding to complete statutory requirements, and public acceptance of GMOs when the product is to be marketed. However, as technology develops, new strategies for nematode control are emerging, both for transgenic approaches and in genome editing, which should be regarded by regulators as a form of mutation rather than ‘genetic modification’. With such advances in biotechnology, the release of commercial varieties of major crops with new forms of nematode resistance, or new modes of delivery of control agents, is likely to become a commercial reality. To improve durability, transgenic traits could be based on resistance with different modes of action: for example, RNAi-based technology combined with expression of peptides which disrupt sensory activities. Ideally such traits would be added to existing crop genotypes with the best conventional or natural nematode resistance, to increase the effectiveness and durability of the nematode resistance trait. Biotech trait expression could also be limited to roots to minimise expression in harvested parts, and this could improve public acceptability.

We report the first gene-based linkage map of Lupinus angustifolius (narrow-leafed lupin) and its comparison to the partially sequenced genome of Medicago truncatula. The map comprises 382 loci in 20 major linkage groups, two triplets, three pairs and 11 unlinked loci and is 1,846 cM in length. The map was generated from the segregation of 163 RFLP markers, 135 gene-based PCR markers, 75 AFLP and 4 AFLP-derived SCAR markers in a mapping population of 93 recombinant inbred lines, derived from a cross between domesticated and wild-type parents. This enabled the mapping of five major genes controlling key domestication traits in L. angustifolius. Using marker sequence data, the L. angustifolius genetic map was compared to the partially completed M. truncatula genome sequence. We found evidence of conserved synteny in some regions of the genome despite the wide evolutionary distance between these legume species. We also found new evidence of widespread duplication within the L. angustifolius genome.

The complete nucleotide sequence of Subterranean clover mottle virus (SCMoV) genomic RNA has been determined. The SCMoV genome is 4,258 nucleotides in length. It shares most nucleotide and amino acid sequence identity with the genome of Lucerne transient streak virus (LTSV). SCMoV RNA encodes four overlapping open reading frames and has a genome organisation similar to that of Cocksfoot mottle virus (CfMV). ORF1 and ORF4 are predicted to encode single proteins. ORF2 is predicted to encode two proteins that are derived from a -1 translational frameshift between two overlapping reading frames (ORF2a and ORF2b). A search of amino acid databases did not find a significant match for ORF1 and the function of this protein remains unclear. ORF2a contains a motif typical of chymotrypsin-like serine proteases and ORF2b has motifs characteristically present in positive-stranded RNA-dependent RNA polymerases. ORF4 is likely to be expressed from a subgenomic RNA and encodes the viral coat protein. The ORF2a/ORF2b overlapping gene expression strategy used by SCMoV and CfMV is similar to that of the poleroviruses and differ from that of other published sobemoviruses. These results suggest that the sobemoviruses could now be divided into two distinct subgroups based on those that express the RNA-dependent RNA polymerase from a single, in-frame polyprotein, and those that express it via a -1 translational frameshifting mechanism.

In wheat (Triticum aestivum L.) drought-induced pollen sterility is a major contributor to grain yield loss and is caused by the downregulation of the cell wall invertase gene IVR1. The IVR1 gene catalyses the irreversible hydrolysis of sucrose to glucose and fructose, the essential energy substrates which support pollen development. Downregulation of IVR1 in response to drought is isoform specific and shows variation in temporal and tissue-specific expression. IVR1 is now prompting interest as a candidate gene for molecular marker development to screen wheat germplasm for improved drought tolerance. The aim of this study was to define the family of IVR1 genes to enable: (1) individual isoforms to be assayed in gene expression studies; and (2) greater accuracy in IVR1 mapping to the wheat genetic map and drought tolerance QTL analysis. Using a cell wall invertase-specific motif as a probe, wheat genomics platforms were screened for the presence of unidentified IVR1 isoforms. Wheat genomics platforms screened included the IWGSC wheat survey sequence, the wheat D genome donor sequence from Aegilops tauschii Coss, and the CCG wheat chromosome 3B assembly: contig506. Chromosome-specific sequences homologous to the query motif were isolated and characterised. Sequence annotation results showed five previously unidentified IVR1 isoforms exist on multiple chromosome arms and on all three genomes (A, B and D): IVR1–3A, IVR1–4A, IVR1–5B, IVR1.2–3B and IVR1-5D. Including three previously characterised IVR1 isoforms (IVR1.1–1A, IVR1.2–1A and IVR1.1–3B), the total number of isoform gene family members is eight. The IVR1 isoforms contain two motifs common to cell wall invertase (NDPN and WECPDF) and a high degree of conservation in exon 4, suggesting conservation of functionality. Sequence divergence at a primary structure level in other regions of the gene was evident amongst the isoforms, which likely contributes to variation in gene regulation and expression in response to water deficit within this subfamily of IVR1 isoforms in wheat.

Laser microdissection (LM) has become an important tool for isolating individual cells or cell types from suitably prepared tissue samples. The technique can be used to isolate both fungal and host plant cells after pathogen infection for molecular studies. Sample preparation is a crucial step in LM and involves fixing samples with appropriate fixatives to preserve the integrity of cell morphology and target metabolites (e.g., RNA), and embedding the fixed tissue in paraffin wax for sectioning onto microscope slides. The sample sections are then deparaffinised, rehydrated, and cells are dissected by a laser focused through a microscope. LM samples are collected into protective (e.g., RNAse-free) medium for isolation of RNA. The RNA can then be subjected to gene expression studies such as quantitative RT-PCR and microarray analysis after a linear RNA amplification process.

Laser microdissection (LM) has become an important tool for isolating individual cells or cell types from suitably prepared tissue samples. The technique can be used to isolate both fungal and host plant cells after pathogen infection for molecular studies. Sample preparation is a crucial step in LM and involves fixing samples with appropriate fixatives to preserve the integrity of cell morphology and target metabolites (e.g., RNA), and embedding the fixed tissue in paraffin wax for sectioning onto microscope slides. The sample sections are then deparaffinised, rehydrated, and cells are dissected by a laser focused through a microscope. LM samples are collected into protective (e.g., RNAse-free) medium for isolation of RNA. The RNA can then be subjected to gene expression studies such as quantitative RT-PCR and microarray analysis after a linear RNA amplification process.

The root-knot nematode Meloidogyne javanica induces giant cells and feeds from them during its development and reproduction. To study the cellular processes underlying the formation of giant cells, laser microdissection was used to isolate the contents of early-stage giant cells 4 and 7 days post-infection (dpi) from tomato, and cDNA libraries from both stages were generated with 87 [250 expressed sequence tag (EST) clones] and 54 (309 EST clones) individual transcripts identified, respectively. These transcripts have roles in metabolism, stress response, protein synthesis, cell division and morphogenesis, transport, signal transduction, protein modification and fate, and regulation of cellular processes. The expression of 25 selected transcripts was studied further by real-time quantitative reverse transcriptase-polymerase chain reaction. Among them, 13 showed continuous up-regulation in giant cells from 4 to 7 dpi. The expression of two transcripts was higher than in controls at 4 dpi and remained at the same level at 7 dpi; a further five transcripts were highly expressed only at 7 dpi. The Phi-1 protein gene, a cell cycle-related homologue in tobacco, was expressed 8.5 times more strongly in giant cells than in control cells at 4 dpi, but was reduced to 6.7 times at 7 dpi. Using in situ hybridization, the expression of the Phi-1 gene was preferentially localized in the cytoplasm of giant cells at 4 dpi, together with a pectinesterase U1 precursor gene. The identification of highly expressed transcripts in developing giant cells adds to the knowledge of the plant genes responsive to nematode infection, and may provide candidate genes for nematode control strategies.

Seventeen accessions of ground nuts (Arachis hypogaea L.) were characterized at the University of Ghana, Legon, 10 identity useful variation tor the genetic improvement of the crop. Using morphological and agronomic markers based on six qualitative and: 11 quantitative descriptors, wide variations were shown among the accessions. Each of the accessions was distinct except four, which fell into two groups of two each. Starch gel electrophoresis of crude protein extracted from the root-tip tissue of the accessions to study variation in esterase, peroxidase and acid phosphatase, also exposed variations among the accessions. Acid phosphatase showed the most variation while peroxidase showed the least variation. When all of the three isozymes were considered, it was possible to unambiguously distinguish 13 of the 17 accessions. The remaining four tell into two groups of two each. The results of the study indicated that isozymes are useful in the characterization of groundnuts.

Fifty one plant species from 25 different genera in seven families were challenged with Subterranean clover mottle virus (SCMoV) by inoculation with infective sap. These included 39 legume species and 12 species belonging to the families Amaranthaceae, Apiaceae, Chenopodiaceae, Cruciferae, Cucurbitaceae and Solanaceae. Twenty one new host species belonging to eight different legume genera were found for SCMoV, nine of which were alternative annual pasture or forage species and 12 were crop legumes. Of these, seven pasture or forage species and six crop species were infected systemically. Two Chenopodium spp. were also hosts. The host status of eight previously tested legume species was also re-examined. The implications of these results for paddocks sown with alternative pasture or forage crop legumes, and for plant improvement programs evaluating such species for commercial use, are discussed.

Despite prominent differences in lifestyles of obligate parasitic and free-living nematodes, the intensive research in characterizing genes and proteins of Caenorhabditis elegans using RNA silencing has inspired research and technological applications for parasitic nematodes. This is exemplified in dsRNA-mediated silencing of plant-parasitic nematode (PPN) genes to study their function, and demonstration of the existence of similar functional mechanisms of RNA silencing in these species. Armed with this knowledge, it then became possible to explore and identify appropriate genes of PPNs as targets for new strategies of nematode control. This chapter focuses on the methods and applications of RNA silencing to PPNs. There is no doubt that commercial implementation of biotechnology-based control strategies for nematode pests via host-induced gene silencing, for example, is now closer to reality. This advance is based on RNA silencing technology, in which research into the free-living nematode C. elegans has played a major role. Intensive efforts to address practical issues of implementation will make gene silencing technology capable of delivering effective nematode resistance for crops, either as a stand-alone approach, or in combination with alternative nematode control options.