Attempt to silence genes of the RNAi pathways of the root-knot nematode, Meloidogyne incognita results in diverse responses including increase and no change in expression of some genes

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

Comparisons between sequenced and re-sequenced genomes of historical subterranean clover mottle virus isolates

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

Sustainable green chemical synthesis of discrete, well-dispersed silver nanoparticles with bacteriostatic properties from carrot extracts aided by polyvinylpyrrolidone

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

De novo analysis of the transcriptome of Pratylenchus zeae to identify transcripts for proteins required for structural integrity, sensation, locomotion and parasitism

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

Analysis of the transcriptome of the infective stage of the beet cyst nematode, H. schachtii

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

Enabling trade in Gene-Edited produce in Asia and Australasia: The developing regulatory landscape and future perspectives

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

Analysis of Gene expression in soybean (Glycine max) roots in response to the root knot nematode Meloidogyne incognita using microarrays and KEGG pathways

<p>Abstract</p> <p>Background</p> <p>Root-knot nematodes are sedentary endoparasites that can infect more than 3000 plant species. Root-knot nematodes cause an estimated $100 billion annual loss worldwide. For successful establishment of the root-knot nematode in its host plant, it causes dramatic morphological and physiological changes in plant cells. The expression of some plant genes is altered by the nematode as it establishes its feeding site.</p> <p>Results</p> <p>We examined the expression of soybean (<it>Glycine max</it>) genes in galls formed in roots by the root-knot nematode, <it>Meloidogyne incognita</it>, 12 days and 10 weeks after infection to understand the effects of infection of roots by <it>M. incognita</it>. Gene expression was monitored using the Affymetrix Soybean GeneChip containing 37,500 <it>G. max </it>probe sets. Gene expression patterns were integrated with biochemical pathways from the Kyoto Encyclopedia of Genes and Genomes using PAICE software. Genes encoding enzymes involved in carbohydrate and cell wall metabolism, cell cycle control and plant defense were altered.</p> <p>Conclusions</p> <p>A number of different soybean genes were identified that were differentially expressed which provided insights into the interaction between <it>M. incognita </it>and soybean and into the formation and maintenance of giant cells. Some of these genes may be candidates for broadening plants resistance to root-knot nematode through over-expression or silencing and require further examination.</p

Knowledge Economy Gaps, Policy Syndromes and Catch-up Strategies: Fresh South Korean Lessons to Africa

Africa’s overall knowledge index fell between 2000 and 2009. South Korea’s economic miracle is largely due to a knowledge-based development strategy that holds valuable lessons for African countries in their current pursuit towards knowledge economies. Using updated data (1996-2010), this paper presents fresh South Korean lessons to Africa by assessing the knowledge economy (KE) gaps, deriving policy syndromes and providing catch-up strategies. The 53 African frontier countries are decomposed into fundamental characteristics of wealth, legal origins, regional proximity, oil-exporting, political stability and landlockedness. The World Bank’s four KE components are used: education, innovation, information & communication technology (ICT) and economic incentives & institutional regime. Absolute beta and sigma convergence techniques are employed as empirical strategies. With the exception of ICT for which catch-up is not very apparent, in increasing order it is visible in: innovation, economic incentives, education and institutional regime. The speed of catch-up varies between 8.66% and 30.00% per annum with respective time to full or 100% catch-up of 34.64 years and 10 years. Based on the trends and dynamics in the KE gaps, policy syndromes and compelling catch-up strategies are discussed. Issues standing on the way to KE in Africa are dissected with great acuteness before South Korean relevant solutions are provided. The paper is original in its provision of practical policy initiatives drawn from the Korean experience to African countries embarking on a transition to KE

The <i>Pratylenchus penetrans</i> transcriptome as a source for the development of alternative control strategies:mining for putative genes involved in parasitism and evaluation of <i>in planta</i> RNAi

The root lesion nematode Pratylenchus penetrans is considered one of the most economically important species within the genus. Host range studies have shown that nearly 400 plant species can be parasitized by this species. To obtain insight into the transcriptome of this migratory plant-parasitic nematode, we used Illumina mRNA sequencing analysis of a mixed population, as well as nematode reads detected in infected soybean roots 3 and 7 days after nematode infection. Over 140 million paired end reads were obtained for this species, and de novo assembly resulted in a total of 23,715 transcripts. Homology searches showed significant hit matches to 58% of the total number of transcripts using different protein and EST databases. In general, the transcriptome of P. penetrans follows common features reported for other root lesion nematode species. We also explored the efficacy of RNAi, delivered from the host, as a strategy to control P. penetrans, by targeted knock-down of selected nematode genes. Different comparisons were performed to identify putative nematode genes with a role in parasitism, resulting in the identification of transcripts with similarities to other nematode parasitism genes. Focusing on the predicted nematode secreted proteins found in this transcriptome, we observed specific members to be up-regulated at the early time points of infection. In the present study, we observed an enrichment of predicted secreted proteins along the early time points of parasitism by this species, with a significant number being pioneer candidate genes. A representative set of genes examined using RT-PCR confirms their expression during the host infection. The expression patterns of the different candidate genes raise the possibility that they might be involved in critical steps of P. penetrans parasitism. This analysis sheds light on the transcriptional changes that accompany plant infection by P. penetrans, and will aid in identifying potential gene targets for selection and use to design effective control strategies against root lesion nematodes