Phage cocktail containing Podoviridae and Myoviridae bacteriophages inhibits the growth of Pectobacterium spp. under in vitro and in vivo conditions.

Globally, there is a high economic burden caused by pre- and post-harvest losses in vegetables, fruits and ornamentals due to soft rot diseases. At present, the control methods for these diseases are limited, but there is some promise in developing biological control products for use in Integrated Pest Management. This study sought to formulate a phage cocktail which would be effective against soft rot Pectobacteriaceae species affecting potato (Solanum tuberosum L.), with potential methods of application in agricultural systems, including vacuum-infiltration and soil drench, also tested. Six bacteriophages were isolated and characterized using transmission electron microscopy, and tested against a range of Pectobacterium species that cause soft rot/blackleg of potato. Isolated bacteriophages of the family Podoviridae and Myoviridae were able to control isolates of the Pectobacterium species: Pectobacterium atrosepticum and Pectobacterium carotovorum subsp. carotovorum. Genomic analysis of three Podoviridae phages did not indicate host genes transcripts or proteins encoding toxin or antibiotic resistance genes. These bacteriophages were formulated as a phage cocktail and further experiments showed high activity in vitro and in vivo to suppress Pectobacterium growth, potentially indicating their efficacy in formulation as a microbial pest control agent to use in planta.

Genomic Characterization, Formulation and Efficacy in Planta of a Siphoviridae and Podoviridae Protection Cocktail against the Bacterial Plant Pathogens Pectobacterium spp.

In the face of global human population increases, there is a need for efficacious integrated pest management strategies to improve agricultural production and increase sustainable food production. To counteract significant food loses in crop production, novel, safe and efficacious measures should be tested against bacterial pathogens. Pectobacteriaceae species are one of the causative agents of the bacterial rot of onions ultimately leading to crop losses due to ineffective control measures against these pathogens. Therefore, the aim of this study was to isolate and characterize bacteriophages which could be formulated in a cocktail and implemented in planta under natural environmental conditions. Transmission electron microscopy (TEM) and genome analysis revealed Siphoviridae and Podoviridae family bacteriophages. To test the protective effect of a formulated phage cocktail against soft rot disease, three years of field trials were performed, using three different methods of treatment application. This is the first study to show the application of a phage cocktail containing Podoviridae and Siphoviridae bacteriophages capable of protecting onions against soft rot in field conditions.

Nematode neuropeptides as transgenic nematicides.

Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars.

Chemosensory Responses of Plant Parasitic Nematodes to Selected Phytochemicals Reveal Long-Term Habituation Traits.

Plant parasitic nematodes (PPN) are important crop pests within the global agri-sector. Critical to their success is a complex and highly sensitive chemosensory system used to locate plants by detecting host cues. In addition to this, the nematode neuronal system has evolved mechanisms to allow adaptation to a changing environment. Clearly, there is a need to better understand the host-parasite relationship and the mechanisms by which PPN successfully locate and infect host plants. Here, we demonstrate the chemotactic response of two economically important PPN species, Meloidogyne incognita and Globodera pallida to selected phytochemicals. We further reveal an adapted chemotactic response in M. incognita second-stage juveniles preexposed to ethephon (Eth), potato root diffusate (PRD), and salicylic acid (SA), and present pharmacological evidence supporting the existence of long-term habituation traits acting via serotonergic-dependent neurotransmission.

Exogenous RNA interference exposes contrasting roles for sugar exudation in host-finding by plant pathogens.

Plant parasitic nematodes (PPN) locate host plants by following concentration gradients of root exudate chemicals in the soil. We present a simple method for RNA interference (RNAi)-induced knockdown of genes in tomato seedling roots, facilitating the study of root exudate composition, and PPN responses. Knockdown of sugar transporter genes, STP1 and STP2, in tomato seedlings triggered corresponding reductions of glucose and fructose, but not xylose, in collected root exudate. This corresponded directly with reduced infectivity and stylet thrusting of the promiscuous PPN Meloidogyne incognita, however we observed no impact on the infectivity or stylet thrusting of the selective Solanaceae PPN Globodera pallida. This approach can underpin future efforts to understand the early stages of plant-pathogen interactions in tomato and potentially other crop plants.

A First Report of Anguina pacificae in Ireland.

Anguina pacificae is a significant pest of Poa annua golf course greens in northern California. This study presents the first confirmed case of an A. pacificae infestation outside of North America, where the nematode's distribution is further restricted to a relatively limited coastal region. Species confirmation was made by morphometric and molecular methods and comparisons to closely related species including the European species, Anguina agropyri. The A. pacificae population detected on an Irish golf course was monitored over a 2-yr period and the life cycle compared with Californian population dynamics. A. pacificae was assessed for the potential risk of spreading to the local agricultural sector, in addition, the biosecurity risks from A. pacificae and plant parasitic nematodes in general were reviewed for northwest Europe.

RNAi effector diversity in nematodes.

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes.

Short interfering RNA-mediated knockdown of drosha and pasha in undifferentiated Meloidogyne incognita eggs leads to irregular growth and embryonic lethality.

Micro-(mi)RNAs play a pivotal role in the developmental regulation of plants and animals. We reasoned that disruption of normal heterochronic activity in differentiating Meloidogyne incognita eggs may lead to irregular development, lethality and by extension, represent a novel target for parasite control. On silencing the nuclear RNase III enzyme drosha, a critical effector of miRNA maturation in animals, we found a significant inhibition of normal development and hatching in short interfering (si)RNA-soaked M. incognita eggs. Developing juveniles presented with highly irregular tissue patterning within the egg, and we found that unlike our previous gene silencing efforts focused on FMRFamide (Phe-Met-Arg-Phe-NH(2))-like peptides (FLPs), there was no observable phenotypic recovery following removal of the environmental siRNA. Aberrant phenotypes were exacerbated over time, and drosha knockdown proved embryonically lethal. Subsequently, we identified and silenced the drosha cofactor pasha, revealing a comparable inhibition of normal embryonic development within the eggs to that of drosha-silenced eggs, eventually leading to embryonic lethality. To further probe the link between normal embryonic development and the M. incognita RNA interference (RNAi) pathway, we attempted to examine the impact of silencing the cytosolic RNase III enzyme dicer. Unexpectedly, we found a substantial up-regulation of dicer transcript abundance, which did not impact on egg differentiation or hatching rates. Silencing of the individual transcripts in hatched J2s was significantly less successful and resulted in temporary phenotypic aberration of the J2s, which recovered within 24h to normal movement and posture on washing out the siRNA. Soaking the J2s in dicer siRNA resulted in a modest decrease in dicer transcript abundance which had no observable impact on phenotype or behaviour within 48h of initial exposure to siRNA. We propose that drosha, pasha and their ancillary factors may represent excellent targets for novel nematicides and/or in planta controls aimed at M. incognita, and potentially other parasitic nematodes, through disruption of miRNA-directed developmental pathways. In addition, we have identified a putative Mi-eri-1 transcript which encodes an RNAi-inhibiting siRNA exonuclease. We observe a marked up-regulation of Mi-eri-1 transcript abundance in response to exogenously introduced siRNA, and reason that this may impact on the interpretation of RNAi-based reverse genetic screens in plant parasitic nematodes.

Short interfering RNA-mediated gene silencing in Globodera pallida and Meloidogyne incognita infective stage juveniles.

The analysis of gene function through RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) remains inexplicably reliant on the use of long double-stranded RNA (dsRNA) silencing triggers; a practice inherently disadvantageous due to the introduction of superfluous dsRNA sequence, increasing chances of aberrant or off-target gene silencing through interactions between nascent short interfering RNAs (siRNAs) and non-cognate mRNA targets. Recently, we have shown that non-nematode, long dsRNAs have a propensity to elicit profound impacts on the phenotype and migrational abilities of both root knot and cyst nematodes. This study presents, to our knowledge for the first time, gene-specific knockdown of FMRFamide-like peptide (flp) transcripts, using discrete 21bp siRNAs in potato cyst nematode Globodera pallida, and root knot nematode Meloidogyne incognita infective (J2) stage juveniles. Both knockdown at the transcript level through quantitative (q)PCR analysis and functional data derived from migration assay, indicate that siRNAs targeting certain areas of the FMRFamide-like peptide (FLP) transcripts are potent and specific in the silencing of gene function. In addition, we present a method of manipulating siRNA activity through the management of strand thermodynamics. Initial evaluation of strand thermodynamics as a determinant of RNA-Induced Silencing Complex (RISC) strand selection (inferred from knockdown efficacy) in the siRNAs presented here suggested that the purported influence of 5' stand stability on guide incorporation may be somewhat promiscuous. However, we have found that on strategically incorporating base mismatches in the sense strand of a G. pallida-specific siRNA, we could specifically increase or decrease the knockdown of its target (specific to the antisense strand), presumably through creating more favourable thermodynamic profiles for incorporation of either the sense (non-target-specific) or antisense (target-specific) strand into a cleavage-competent RISC. Whilst the efficacy of similar approaches to siRNA modification has been demonstrated in the context of Drosophila whole-cell lysate preparations and in mammalian cell cultures, it remained to be seen how these sense strand mismatches may impact on gene silencing in vivo, in relation to different targets and in different sequence contexts. This work presents the first application of such an approach in a whole organism; initial results show promise.

Non-nematode-derived double-stranded RNAs induce profound phenotypic changes in Meloidogyne incognita and Globodera pallida infective juveniles.

Nine non-nematode-derived double-stranded RNAs (dsRNAs), designed for use as controls in RNA interference (RNAi) screens of neuropeptide targets, were found to induce aberrant phenotypes and an unexpected inhibitory effect on motility of root knot nematode Meloidogyne incognita J2s following 24h soaks in 0.1 mg/ml dsRNA; a simple soaking procedure which we have found to elicit profound knockdown of neuronal targets in Globodera pallida J2s. We have established that this inhibitory phenomenon is both time- and concentration-dependent, as shorter 4h soaks in 0.1 mg/ml dsRNA had no negative impact on M. incognita J2 stage worms, yet a 10-fold increase in concentration to 1 mg/ml for the same 4h time period had an even greater qualitative and quantitative impact on worm phenotype and motility. Further, a 10-fold increase of J2s soaked in 0.1 mg/ml dsRNA did not significantly alter the observed phenotypic aberration, which suggests that dsRNA uptake of the soaked J2s is not saturated under these conditions. This phenomenon was not initially observed in potato cyst nematode G. pallida J2s, which displayed no aberrant phenotype, or diminution of migratory activity in response to the same 0.1 mg/ml dsRNA 24h soaks. However, a 10-fold increase in dsRNA to 1mg/ml was found to elicit comparable irregularity of phenotype and inhibition of motility in G. pallida, to that initially observed in M. incognita following a 24h soak in 0.1 mg/ml dsRNA. Again, a 10-fold increase in the number of G. pallida J2s soaked in the same volume of 1 mg/ml dsRNA preparation did not significantly affect the observed phenotypic deviation. We do not observe any global impact on transcript abundance in either M. incognita or G. pallida J2s following 0.1 mg/ml dsRNA soaks, as revealed by reverse transcriptase-PCR and quantitative PCR data. This study aims to raise awareness of a phenomenon which we observe consistently and which we believe signifies a more expansive deficiency in our knowledge and understanding of the variables inherent to RNAi-based investigation.

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference.

The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well-established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp-flp-1, -6, -12, -14, or -18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double-stranded RNA exposure time (> or = 18 h) and concentration (> or = 0.1 microg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomotory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi-mediated flp gene silencing as a novel plant parasite control strategy.

High nuclear genetic diversity, high levels of outcrossing and low differentiation among remnant populations of Quercus petraea at the margin of its range in Ireland.

BACKGROUND AND AIMS: Quercus petraea colonized Ireland after the last glaciation from refugia on mainland Europe. Deforestation, however, beginning in Neolithic times, has resulted in small, scattered forest fragments, now covering less than 12,000 ha. METHODS: Plastid (three fragments) and microsatellite variation (13 loci) were characterized in seven Irish populations sampled along a north-south gradient. Using Bayesian approaches and Wright's F-statistics, the effects of colonization and fragmentation on the genetic structure and mating patterns of extant oak populations were investigated. KEY RESULTS: All populations possessed cytotypes common to the Iberian Peninsula. Despite the distance from the refugial core and the extensive deforestation in Ireland, nuclear genetic variation was high and comparable to mainland Europe. Low population differentiation was observed within Ireland and populations showed no evidence for isolation by distance. As expected of a marker with an effective population size of one-quarter relative to the nuclear genome, plastid variation indicated higher differentiation. Individual inbreeding coefficients indicated high levels of outcrossing. CONCLUSIONS: Consistent with a large effective population size in the historical migrant gene pool and/or with high gene flow among populations, high within-population diversity and low population differentiation was observed within Ireland. It is proposed that native Q. petraea populations in Ireland share a common phylogeographic history and that the present genetic structure does not reflect founder effects.

Localisation of Globodera pallida FMRFamide-related peptide encoding genes using in situ hybridisation.

The present study employed an in situ hybridisation technique to detect the expression of a number of FMRFamide-like peptide encoding (flp) genes, previously identified from Globodera pallida, in whole-mount preparations of the J(2) stage of this worm. gpflp-1, encoding the FMRFamide-related peptide (FaRP) KSAYMRFamide, was expressed in neurones associated with the circumpharyngeal nerve ring and specifically in a number of cell bodies in the lumbar ganglia of the perianal nerve ring. The lumbar ganglia and pre-anal ganglia along with the BDU neurones and a number of cells in the retrovesicular ganglion were observed to express gpflp-2, encoding KNKFEFIRFamide. gpflp-3 (encoding KHEYLRFamide) expression was localised to the anterior ganglion and a number of paired cells posterior to the circumpharyngeal nerve ring whilst expression of gpflp-4, encoding a number of -P(G/Q)VLRFamides, was localised to the retrovesicular ganglion. No expression of gpflp-5 was observed. Identification of the reactive cells has implicated distinct roles for the FaRPs encoded on these genes in regulation of both dorsal and ventral body wall muscles, the musculature of the vulva and in the function of a number of sensory structures in both the head and tail of G. pallida. Comparison with the expression patterns of analogous genes in Caenorhabditis elegans suggests that, whilst some of the encoded peptides are conserved between nematode species, their functions therein are distinct. Furthermore, the expression of some of these genes in a number of interneurones supports the idea that FaRPs fulfil neuromodulatory as well as neurotransmitter roles.