Recent advances in biodegradable matrices for active ingredient release in crop protection: Towards attaining sustainability in agriculture.

Climate changes, emerging species of plant pests, and deficits of clean water and arable land have made availability of food to the ever-increasing global population a challenge. Excessive use of synthetic pesticides to meet ever-increasing production needs has resulted in development of resistance in pest populations, as well as significant ecotoxicity, which has directly and indirectly impacted all life-forms on earth. To meet the goal of providing safe, sufficient, and high-quality food globally with minimal environmental impact, one strategy is to focus on targeted delivery of pesticides using eco-friendly and biodegradable carriers that are derived from naturally available materials. Herein, we discuss some of the recent approaches to use biodegradable matrices in crop protection, while exploring their design and efficiency. We summarize by discussing associated challenges with the existing approaches and future trends that can lead the world to more sustainable agricultural practices.

A Draft Genome Sequence of the Burrowing Nematode Radopholus similis.

Radopholus similis also known as the burrowing nematode is a devastating pest of banana (Musa spp.) and many economically important crops and ornamentals. In this publication, we present the genome assembly of R. similis.

Signatures of adaptation to plant parasitism in nematode genomes.

Plant-parasitic nematodes cause considerable damage to global agriculture. The ability to parasitize plants is a derived character that appears to have independently emerged several times in the phylum Nematoda. Morphological convergence to feeding style has been observed, but whether this is emergent from molecular convergence is less obvious. To address this, we assess whether genomic signatures can be associated with plant parasitism by nematodes. In this review, we report genomic features and characteristics that appear to be common in plant-parasitic nematodes while absent or rare in animal parasites, predators or free-living species. Candidate horizontal acquisitions of parasitism genes have systematically been found in all plant-parasitic species investigated at the sequence level. Presence of peptides that mimic plant hormones also appears to be a trait of plant-parasitic species. Annotations of the few genomes of plant-parasitic nematodes available to date have revealed a set of apparently species-specific genes on every occasion. Effector genes, important for parasitism are frequently found among those species-specific genes, indicating poor overlap. Overall, nematodes appear to have developed convergent genomic solutions to adapt to plant parasitism.

Loading and release mechanism of red clover necrotic mosaic virus derived plant viral nanoparticles for drug delivery of doxorubicin.

Loading and release mechanisms of Red clover necrotic mosaicvirus (RCNMV) derived plant viral nanoparticle (PVN) are shown for controlled delivery of the anticancer drug, doxorubicin (Dox). Previous studies demonstrate that RCNMV's structure and unique response to divalent cation depletion and re-addition enables Dox infusion to the viral capsid through a pore formation mechanism. However, by controlling the net charge of RCNMV outer surface and accessibility of RCNMV interior cavity, tunable release of PVN is possible via manipulation of the Dox loading capacity and binding locations (external surface-binding or internal capsid-encapsulation) with the RCNMV capsid. Bimodal release kinetics is achieved via a rapid release of surface-Dox followed by a slow release of encapsulated Dox. Moreover, the rate of Dox release and the amount of released Dox increases with an increase in environmental pH or a decrease in concentration of divalent cations. This pH-responsive Dox release from PVN is controlled by Fickian diffusion kinetics where the release rate is dependent on the location of the bound or loaded active molecule. In summary, controllable release of Dox-loaded PVNs is imparted by 1) formulation conditions and 2) driven by the capsid's pH- and ion- responsive functions in a given environment.

Plant-biomass-based hybrid seed wraps mitigate yield and post-harvest losses among smallholder farmers in sub-Saharan Africa.

Sustainable practices that reduce food loss are essential for enhancing global food security. We report a 'wrap and plant' seed treatment platform to protect crops from soil-borne pathogens. Developed from the abundantly available wastes of banana harvest and recycled old, corrugated cardboard boxes via chemical-free pulping, these paper-like biodegradable seed wraps exhibit tunable integrity and bioavailability of loaded moieties. These wraps were used for nematode control on yam (Dioscorea cayenensis-rotundata) seed pieces in Benin, a major producer of this staple crop in the sub-Saharan African 'yam belt'. Our seed wraps loaded with ultra-low-volume abamectin (1/100 ≤ commercial formulation) consistently controlled yam nematode (Scutellonema bradys) populations while considerably increasing the yield at various locations over 2015-2018. Substantial reduction in post-harvest tuber weight loss and cracking was observed after 3 and 5 months of storage, contributing to increased value, nutrition and stakeholders' preference for the wrap and plant treatment.

Sequence and genetic map of Meloidogyne hapla: A compact nematode genome for plant parasitism.

We have established Meloidogyne hapla as a tractable model plant-parasitic nematode amenable to forward and reverse genetics, and we present a complete genome sequence. At 54 Mbp, M. hapla represents not only the smallest nematode genome yet completed, but also the smallest metazoan, and defines a platform to elucidate mechanisms of parasitism by what is the largest uncontrolled group of plant pathogens worldwide. The M. hapla genome encodes significantly fewer genes than does the free-living nematode Caenorhabditis elegans (most notably through a reduction of odorant receptors and other gene families), yet it has acquired horizontally from other kingdoms numerous genes suspected to be involved in adaptations to parasitism. In some cases, amplification and tandem duplication have occurred with genes suspected of being acquired horizontally and involved in parasitism of plants. Although M. hapla and C. elegans diverged >500 million years ago, many developmental and biochemical pathways, including those for dauer formation and RNAi, are conserved. Although overall genome organization is not conserved, there are areas of microsynteny that may suggest a primary biological function in nematodes for those genes in these areas. This sequence and map represent a wealth of biological information on both the nature of nematode parasitism of plants and its evolution.

The genomes of root-knot nematodes.

Plant-parasitic nematodes are the most destructive group of plant pathogens worldwide and are extremely challenging to control. The recent completion of two root-knot nematode genomes opens the way for a comparative genomics approach to elucidate the success of these parasites. Sequencing revealed that Meloidogyne hapla, a diploid that reproduces by facultative, meiotic parthenogenesis, encodes approximately 14,200 genes in a compact, 54 Mpb genome. Indeed, this is the smallest metazoan genome completed to date. By contrast, the 86 Mbp Meloidogyne incognita genome encodes approximately 19,200 genes. This species reproduces by obligate mitotic parthenogenesis and exhibits a complex pattern of aneuploidy. The genome includes triplicated regions and contains allelic pairs with exceptionally high degrees of sequence divergence, presumably reflecting adaptations to the strictly asexual reproductive mode. Both root-knot nematode genomes have compacted gene families compared with the free-living nematode Caenorhabditis elegans, and both encode large suites of enzymes that uniquely target the host plant. Acquisition of these genes, apparently via horizontal gene transfer, and their subsequent expansion and diversification point to the evolutionary history of these parasites. It also suggests new routes to their control.

Proteomic and bioinformatic analysis of the root-knot nematode Meloidogyne hapla: the basis for plant parasitism.

On the basis of the complete genome sequence of the root-knot nematode Melodogyne hapla, we have deduced and annotated the entire proteome of this plant-parasite to create a database of 14,420 proteins. We have made this database, termed HapPep3, available from the Superfamily repository of model organism proteomes (http://supfam.mrc-lmb.cam.ac.uk/SUPERFAMILY). To experimentally confirm the HapPep3 assignments using proteomics, we applied a data-independent LC/MS(E) analysis to M. hapla protein extracts fractionated by SDS-PAGE. A total of 516 nonredundant proteins were identified with an average of 9 unique peptides detected per protein. Some proteins, including examples with complex gene organization, were defined by more than 20 unique peptide matches, thus, providing experimental confirmation of computational predictions of intron/exon structures. On the basis of comparisons of the broad physicochemical properties of the experimental and computational proteomes, we conclude that the identified proteins reflect a true and unbiased sampling of HapPep3. Conversely, HapPep3 appears to broadly cover the protein space able to be experimentally sampled. To estimate the false discovery rate, we queried human, plant, and bacterial databases for matches to the LC/MS(E)-derived peptides, revealing fewer than 1% of matches, most of which were to highly conserved proteins. To provide a functional comparison of the acquired and deduced proteomes, each was subjected to higher order annotation, including comparisons of Gene Ontology, protein domains, signaling, and localization predictions, further indicating concordance, although those proteins that did deviate seem to be highly significant. Approximately 20% of the experimentally sampled proteome was predicted to be secreted, and thus potentially play a role at the host-parasite interface. We examined reference pathways to determine the extent of proteome similarity of M. hapla to that of the free-living nematode, Caenorhabditis elegans, revealing significant similarities and differences. Collectively, the analyzed protein set provides an initial foundation to experimentally dissect the basis of plant parasitism by M. hapla.

Current Insights into Migratory Endoparasitism: Deciphering the Biology, Parasitism Mechanisms, and Management Strategies of Key Migratory Endoparasitic Phytonematodes.

Despite their physiological differences, sedentary and migratory plant-parasitic nematodes (PPNs) share several commonalities. Functional characterization studies of key effectors and their targets identified in sedentary phytonematodes are broadly applied to migratory PPNs, generalizing parasitism mechanisms existing in distinct lifestyles. Despite their economic significance, host-pathogen interaction studies of migratory endoparasitic nematodes are limited; they have received little attention when compared to their sedentary counterparts. Because several migratory PPNs form disease complexes with other plant-pathogens, it is important to understand multiple factors regulating their feeding behavior and lifecycle. Here, we provide current insights into the biology, parasitism mechanism, and management strategies of the four-key migratory endoparasitic PPN genera, namely Pratylenchus, Radopholus, Ditylenchus, and Bursaphelenchus. Although this review focuses on these four genera, many facets of feeding mechanisms and management are common across all migratory PPNs and hence can be applied across a broad genera of migratory phytonematodes.

Tailored Lignocellulose-Based Biodegradable Matrices with Effective Cargo Delivery for Crop Protection.

Controlled release and targeted delivery of agrochemicals are crucial for achieving effective crop protection with minimal damage to the environment. This work presents an innovative and cost-effective approach to fabricate lignocellulose-based biodegradable porous matrices capable of slow and sustained release of the loaded molecules for effective crop protection. The matrix exhibits tunable physicochemical properties which, when coupled with our unique "wrap-and-plant" concept, help to utilize it as a defense against soil-borne pests while providing controlled release of crop protection moieties. The tailored matrix is produced by mechanical treatment of the lignocellulosic fibers obtained from banana plants. The effect of different extents of mechanical treatments of the lignocellulosic fibers on the protective properties of the developed matrices is systematically investigated. While variation in mechanical treatment affects the morphology, strength, and porosity of the matrices, the specific composition and structure of the fibers are also capable of influencing their release profile. To corroborate this hypothesis, the effect of morphology and lignin content changes on the release of rhodamine B and abamectin as model cargos is investigated. These results, compared with those of the matrices developed from non-banana fibrous sources, reveal a unique release profile of the matrices developed from banana fibers, thereby making them strong candidates for crop protection applications.

The genome of the migratory nematode, Radopholus similis, reveals signatures of close association to the sedentary cyst nematodes.

Radopholus similis, commonly known as the burrowing nematode, is an important pest of myriad crops and ornamentals including banana (Musa spp.) and Citrus spp. In order to characterize the potential role of putative effectors encoded by R. similis genes we compared predicted proteins from a draft R. similis genome with other plant-parasitic nematodes in order to define the suite of excreted/secreted proteins that enable it to function as a parasite and to ascertain the phylogenetic position of R. similis in the Tylenchida order. Identification and analysis of candidate genes encoding for key plant cell-wall degrading enzymes including GH5 cellulases, PL3 pectate lyases and GH28 polygalactouranase revealed a pattern of occurrence similar to other PPNs, although with closest phylogenetic associations to the sedentary cyst nematodes. We also observed the absence of a suite of effectors essential for feeding site formation in the cyst nematodes. Clustering of various orthologous genes shared by R. similis with other nematodes showed higher overlap with the cyst nematodes than with the root-knot or other migratory endoparasitic nematodes. The data presented here support the hypothesis that R. similis is evolutionarily closer to the cyst nematodes, however, differences in the effector repertoire delineate ancient divergence of parasitism, probably as a consequence of niche specialization. These similarities and differences further underscore distinct evolutionary relationships during the evolution of parasitism in this group of nematodes.

Plant Virus-Based Nanoparticles for the Delivery of Agronomic Compounds as a Suspension Concentrate.

Nanoparticle formulations of agrichemicals may enhance their performance while simultaneously mitigating any adverse environmental effects. Red clover necrotic mosaic virus (RCNMV) is a soil-transmitted plant virus with many inherent attributes that allow it to function as a plant virus-based nanoparticle (PVN) when loaded with biologically active ingredients. Here we describe how to formulate a PVN loaded with the nematicide abamectin (Abm) beginning with the propagation of the virus through the formulation, deactivation, and characterization of the finished product.

Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses.

Five viruses were previously discovered infecting soybean cyst nematodes (SCN; Heterodera glycines) from greenhouse cultures maintained in Illinois. In this study, the five viruses [ScNV, ScPV, ScRV, ScTV, and SbCNV-5] were detected within SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN from 43 greenhouse cultures and 25 field populations were analyzed using qRT-PCR. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Viruses were found at similar or lower levels within field populations of SCN compared with greenhouse cultures of North Carolina populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mixture of fewer viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Several of these SCN viruses were also detected in Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst), but not the other cyst, root-knot, or reniform nematode species tested. Viruses were not detected within soybean host plant tissue. If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation.

Disparate gain and loss of parasitic abilities among nematode lineages.

Plant parasitism has arisen time and again in multiple phyla, including bacteria, fungi, insects and nematodes. In most of these organismal groups, the overwhelming diversity hampers a robust reconstruction of the origins and diversification patterns of this trophic lifestyle. Being a moderately diversified phylum with ≈ 4,100 plant parasites (15% of total biodiversity) subdivided over four independent lineages, nematodes constitute a major organismal group for which the genesis of plant parasitism could be mapped. Since substantial crop losses worldwide have been attributed to less than 1% of these plant parasites, research efforts are severely biased towards this minority. With the first molecular characterisation of numerous basal and supposedly harmless plant parasites as well as their non-parasitic relatives, we were able to generate a comprehensive molecular framework that allows for the reconstruction of trophic diversification for a complete phylum. In each lineage plant parasites reside in a single taxonomic grouping (family or order), and by taking the coverage of the next lower taxonomic level as a measure for representation, 50, 67, 100 and 85% of the known diversity was included. We revealed distinct gain and loss patterns with regard to plant parasitism per se as well as host exploitation strategies between these lineages. Our map of parasitic nematode biodiversity also revealed an unanticipated time reversal in which the two most ancient lineages showed the lowest level of ecological diversification and vice versa.

A Method for Isolation of Pasteuria penetrans Endospores for Bioassay and Genomic Studies.

A rapid method for collection of Pasteuria penetrans endospores was developed. Roots containing P. penetrans-infected root-knot nematode females were softened by pectinase digestion, mechanically processed, and filtered to collect large numbers of viable endospores. This method obviates laborious handpicking of Pasteuria-infected females and yields endospores competent to attach to and infect nematodes. Endospores are suitable for morphology studies and DNA preparations.

A Method for Generating Meloidogyne incognita Males.

A method for producing mass quantities of Meloidogyne incognita males free from other developmental stages was developed. Host plants were grown hydroponically to facilitate nematode harvest. Pruning stress was shown to cause a higher percentage of juveniles to develop as males vs. a no-stress control. Application of pruning stress in the first 48 hr post-inoculation was also shown to be more effective at driving male development than at later times.

Genetic Drift, Not Life History or RNAi, Determine Long-Term Evolution of Transposable Elements.

Transposable elements (TEs) are a major source of genome variation across the branches of life. Although TEs may play an adaptive role in their host's genome, they are more often deleterious, and purifying selection is an important factor controlling their genomic loads. In contrast, life history, mating system, GC content, and RNAi pathways have been suggested to account for the disparity of TE loads in different species. Previous studies of fungal, plant, and animal genomes have reported conflicting results regarding the direction in which these genomic features drive TE evolution. Many of these studies have had limited power, however, because they studied taxonomically narrow systems, comparing only a limited number of phylogenetically independent contrasts, and did not address long-term effects on TE evolution. Here, we test the long-term determinants of TE evolution by comparing 42 nematode genomes spanning over 500 million years of diversification. This analysis includes numerous transitions between life history states, and RNAi pathways, and evaluates if these forces are sufficiently persistent to affect the long-term evolution of TE loads in eukaryotic genomes. Although we demonstrate statistical power to detect selection, we find no evidence that variation in these factors influence genomic TE loads across extended periods of time. In contrast, the effects of genetic drift appear to persist and control TE variation among species. We suggest that variation in the tested factors are largely inconsequential to the large differences in TE content observed between genomes, and only by these large-scale comparisons can we distinguish long-term and persistent effects from transient or random changes.

Development of abamectin loaded plant virus nanoparticles for efficacious plant parasitic nematode control.

Plant parasitic nematodes are one of the world's major agricultural pests, causing in excess of $157 billion in worldwide crop damage annually. Abamectin (Abm) is a biological pesticide with a strong activity against a wide variety of plant parasitic nematodes. However, Abm's poor mobility in the soil compromises its nematicide performance because of the limited zone of protection surrounding the growing root system of the plant. In this study, we manipulated Abm's soil physical chemistry by encapsulating Abm within the Red clover necrotic mosaic virus (RCNMV) to produce a plant virus nanoparticle (PVN) delivery system for Abm. The transmission electron microscopic and dynamic light scattering characterization of Abm-loaded PVN (PVN(Abm)) indicated the resultant viral capsid integrity and morphology comparable to native RCNMV. In addition, the PVN(Abm) significantly increased Abm's soil mobility while enabling a controlled release strategy for Abm's bioavailability to nematodes. As a result, PVN(Abm) enlarged the zone of protection from Meloidogyne hapla root knot nematodes in the soil as compared to treating with free Abm molecules. Tomato seedlings treated with PVN(Abm) had healthier root growth and a reduction in root galling demonstrating the success of this delivery system for the increased efficacy of Abm to control nematode damage in crops.

Interactions between bacteria and plant-parasitic nematodes: now and then.

Based on genome-to-genome analyses of gene sequences obtained from plant-parasitic, root-knot nematodes (Meloidogyne spp.), it seems likely that certain genes have been derived from bacteria by horizontal gene transfer. Strikingly, a common theme underpinning the function of these genes is their apparent direct relationship to the nematodes' parasitic lifestyle. Phylogenetic analyses implicate rhizobacteria as the predominant group of 'gene donor' bacteria. Root-knot nematodes and rhizobia occupy similar niches in the soil and in roots, and thus the opportunity for genetic exchange may be omnipresent. Further, both organisms establish intimate developmental interactions with host plants, and mounting evidence suggests that the mechanisms for these interactions are shared too. We propose that the origin of parasitism in Meloidogyne may have been facilitated by acquisition of genetic material from soil bacteria through horizontal transfer, and that such events represented key steps in speciation of plant-parasitic nematodes. To further understand the mechanisms of horizontal gene transfer, and also to provide experimental tools to manipulate this promising bio-control agent, we have initiated a genomic sequence of the bacterial hyper-parasite of plant parasitic nematodes, Pasteuria penetrans. Initial data have established that P. penetrans is closely related to Bacillus spp., to the extent that considerable genome synteny is apparent. Hence, Bacillus serves as a model for Pasteuria, and vice versa.

Development and Evaluation of a Standard Method for Screening for Resistance to Radopholus similis in Bananas.

The description and evaluation of a standard assay method for screening for resistance of bananas to the burrowing nematode (Radopholus similis) under greenhouse conditions is presented. Seven banana genotypes, ranging from susceptible to resistant, were used to evaluate the method. Banana plants from tissue culture, grown in 0.4-liter Styrofoam cups containing sterilized sand as substrate, were maintained in the greenhouse for 4 weeks before inoculation. Two hundred burrowing nematodes, reared in monoxenic carrot-disk culture, were used as inoculum for each container. Plants were kept in the greenhouse for an additional 8 weeks at about 27°C and 80% relative humidity after inoculation. Burrowing nematodes reproduced well in the susceptible cultivars False Horn, Grande Naine, Valery, and Lacatan, whereas the reproductive fitness was very low in the resistant cultivars Pisang Jari Buaya and Yangambi. An intermediate reaction between these two groups was observed with Pisang mas. A similar trend was obtained in a follow-up field test, which indicated that the method is accurate and reliable. Assessments of total-root necrosis associated with this pathogen were also comparable between greenhouse and field conditions. However, nematode effects on the roots were more severe in the greenhouse test than in the field. In spite of low nematode reproductive fitness, root necrosis was relatively high in the two resistant cultivars tested in the greenhouse trial.