Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling

In the plant pathogen Phytophthora infestans, nuclear integration of inf1 transgenic DNA sequences results in internuclear gene silencing of inf1. Although silencing is regulated at the transcriptional level, it also affects transcription from other nuclei within heterokaryotic cells of the mycelium. Here we report experiments exploring the mechanism of internuclear gene silencing in P. infestans. The DNA methylation inhibitor 5-azacytidine induced reversion of the inf1-silenced state. Also, the histone deacetylase inhibitor trichostatin-A was able to reverse inf1 silencing. inf1-expression levels returned to the silenced state when the inhibitors were removed except in non-transgenic inf1-silenced strains that were generated via internuclear gene silencing, where inf1 expression was restored permanently. Therefore, inf1-transgenic sequences are required to maintain the silenced state. Prolonged culture of non-transgenic inf1-silenced strains resulted in gradual reactivation of inf1 gene expression. Nuclease digestion of inf1-silenced and non-silenced nuclei showed that inf1 sequences in silenced nuclei were less rapidly degraded than non-silenced inf1 sequences. Bisulfite sequencing of the endogenous inf1 locus did not result in detection of any cytosine methylation. Our findings suggest that the inf1-silenced state is based on chromatin remodelling

Infection of the brown alga Ectocarpus siliculosus by the oomycete Eurychasma dicksonii induces oxidative stress and halogen metabolism

Acknowledgments We would like to thank the Aberdeen Proteome Facility, especially Phil Cash, David Stead and Evelyn Argo for assistance with 2D electrophoresis and mass spectrometry. M.S. gratefully acknowledges a Marie Curie PhD fellowship from the European Commission (ECOSUMMER, MEST-CT-2005-20501), a joint FEMS/ESCMID Research Fellowship and the Genomia Fund. C.M.M.G. is supported by a Marie Curie postdoctoral fellowship (MEIF-CT-2006-022837), a Marie Curie Re-Integration Grant (PERG03-GA-2008-230865) and a New Investigator grant from the UK Natural Environment Research Council (NERC, grant NE/J00460X/1). F.C.K. would like to thank NERC for funding (grants NE/D521522/1, NE/F012705/1 and Oceans 2025 / WP 4.5). L.J.G.-B., C.M.M.G., F.C.K. and P.W. would like to acknowledge funding from NERC for a Strategic Ocean Funding Initiative award (NE/F012578/1). Funding from the MASTS pooling initiative (Marine Alliance for Science and Technology for Scotland, funded by the Scottish Funding Council and contributing institutions; grant reference HR09011) and from the TOTAL Foundation (Paris) to F.C.K. is gratefully acknowledged. Finally, we would like to thank the two anonymous referees for constructive suggestions to improve our manuscript.Peer reviewedPublisher PD

Rapid emergence of boscalid resistance in Swedish populations of Alternaria solani revealed by a combination of field and laboratory experiments

Early blight, caused by Alternaria solani, is a common potato disease worldwide. Reduced field efficacy of the fungicide boscalid against this disease has been reported in several countries. Boscalid resistance has been mostly studied with in-vitro and/or greenhouse experiments. Field studies validating this phenomenon are largely missing. Here, for the first time in Scandinavia, we validated boscalid resistance in a Swedish population of A. solani both in the field and in the laboratory. Field trials between 2014 and 2017 in Nymo showed significant efficacy reduction by year. The target regions of the A. solani genes encoding the succinate dehydrogenase subunits (Sdh) B, C and D of samples collected from Nymo, and additional fields in south-eastern and central Sweden, were analysed for substitutions associated with loss of boscalid sensitivity. In 2014, the SdhC-H134R mutation was found at several sites at a low frequency, while, in 2017, the majority of the samples had either the SdhB-H278Y or the SdhC-H134R substitution. No mutations were detected in the gene encoding the SdhD subunit. Spore germination tests showed a high sensitivity (EC50 100 mu g mL(-1) and their growth rates hardly decreased at concentrations above 1-10 mu g mL(-1). These results add to the current knowledge of fungicide resistance development in field and indicate that early blight management in southeast Sweden should no longer rely on boscalid

Horizontal Gene Transfer and Tandem Duplication Shape the Unique CAZyme Complement of the Mycoparasitic Oomycetes Pythium oligandrum and Pythium periplocum

Crop protection strategies that are effective but that reduce our reliance on chemical pesticides are urgently needed to meet the UN sustainable development goals for global food security. Mycoparasitic oomycetes such as Pythium oligandrum and Pythium periplocum, have potential for the biological control of plant diseases that threaten crops and have attracted much attention due to their abilities to antagonize plant pathogens and modulate plant immunity. Studies of the molecular and genetic determinants of mycoparasitism in these species have been less well developed than those of their fungal counterparts. Carbohydrate-active enzymes (CAZymes) from P. oligandrum and P. periplocum are predicted to be important components of mycoparasitism, being involved in the degradation of the cell wall of their oomycete and fungal prey species. To explore the evolution of CAZymes of these species we performed an in silico identification and comparison of the full CAZyme complement (CAZyome) of the two mycoparasitic Pythium species (P. oligandrum and P. periplocum), with seven other Pythium species, and four Phytophthora species. Twenty CAZy gene families involved in the degradation of cellulose, hemicellulose, glucan, and chitin were expanded in, or unique to, mycoparasitic Pythium species and several of these genes were expressed during mycoparasitic interactions with either oomycete or fungal prey, as revealed by RNA sequencing and quantitative qRT-PCR. Genes from three of the cellulose and chitin degrading CAZy families (namely AA9, GH5_14, and GH19) were expanded via tandem duplication and predominantly located in gene sparse regions of the genome, suggesting these enzymes are putative pathogenicity factors able to undergo rapid evolution. In addition, five of the CAZy gene families were likely to have been obtained from other microbes by horizontal gene transfer events. The mycoparasitic species are able to utilize complex carbohydrates present in fungal cell walls, namely chitin and N-acetylglucosamine for growth, in contrast to their phytopathogenic counterparts. Nonetheless, a preference for the utilization of simple sugars for growth appears to be a common trait within the oomycete lineage

A molecular insight into algal-oomycete warfare: cDNA analysis of <i>Ectocarpus siliculosus</i> infected with the basal oomycete <i>Eurychasma dicksonii</i>

Brown algae are the predominant primary producers in coastal habitats, and like land plants are subject to disease and parasitism. Eurychasma dicksonii is an abundant, and probably cosmopolitan, obligate biotrophic oomycete pathogen of marine brown algae. Oomycetes (or water moulds) are pathogenic or saprophytic non-photosynthetic Stramenopiles, mostly known for causing devastating agricultural and aquacultural diseases. Whilst molecular knowledge is restricted to crop pathogens, pathogenic oomycetes actually infect hosts from most eukaryotic lineages. Molecular evidence indicates that Eu. dicksonii belongs to the most early-branching oomycete clade known so far. Therefore Eu. dicksonii is of considerable interest due to its presumed environmental impact and phylogenetic position. Here we report the first large scale functional molecular data acquired on the most basal oomycete to date. 9873 unigenes, totalling over 3.5Mb of sequence data, were produced from Sanger-sequenced and pyrosequenced EST libraries of infected Ectocarpus siliculosus. 6787 unigenes (70%) were of algal origin, and 3086 (30%) oomycete origin. 57% of Eu. dicksonii sequences had no similarity to published sequence data, indicating that this dataset is largely unique. We were unable to positively identify sequences belonging to the RXLR and CRN groups of oomycete effectors identified in higher oomycetes, however we uncovered other unique pathogenicity factors. These included putative algal cell wall degrading enzymes, cell surface proteins, and cyclophilin-like proteins. A first look at the host response to infection has also revealed movement of the host nucleus to the site of infection as well as expression of genes responsible for strengthening the cell wall, and secretion of proteins such as protease inhibitors. We also found evidence of transcriptional reprogramming of E. siliculosus transposable elements and of a viral gene inserted in the host genome

Pythium oligandrum induces growth promotion in starch potato without significantly altering the rhizosphere microbiome

Plant health promoting organisms, including microbial biological control agents, are of increasing importance for the development of more sustainable agriculture. To understand the function of these microbes as biological control agents under field conditions and their overall impact on soil and plant health, we need to learn more about the impact of plant beneficial microbes on the rhizosphere microbiome of crops such as potato. The plant beneficial oomycete Pythium oligandrum has previously been reported both as a biocontrol agent and as a plant growth promoter, or biostimulant, in several crop species. To investigate the potential of P. oligandrum as a biostimulant in potato, we performed a series of controlled -environment bioassays in three cultivars. We showed that biostimulation of potato by P. oligandrum is plant genotype -specific. We confirmed the biostimulation by P. oligandrum in the starch potato cultivar Kuras under field conditions. We further investigated the effects of P. oligandrum on the potato rhizosphere microbiome, sampling individual potato plants at three time points over the growing season (representing the vegetative growth phase, flowering, and the onset of senescence). Metabarcoding using ITS and 16S amplicon sequencing revealed no significant overall effect of P. oligandrum application on the bacterial and fungal rhizosphere communities. However, some genera were significantly differentially abundant after P. oligandrum application, including some classified as plant -beneficial microbes. We conclude that P. oligandrum has a cultivar-dependent growth -promoting effect in potato and only minor effects on the rhizosphere microbiome

Reduced efficacy of biocontrol agents and plant resistance inducers against potato early blight from greenhouse to field

Early blight in potato, caused by Alternaria solani, is mainly controlled by frequent applications of synthetic fungicides. Reducing the use of synthetic fungicides in agriculture is desired to reach an overall sustainable development since the active components can be harmful for humans and for the ecosystem. In integrated pest management, IPM, the idea is to combine various measures, including optimized crop management, crop rotation, use of resistant cultivars, biological control agents (BCAs), plant resistance inducers, and fertilizers, to decrease the dependence on traditional chemical fungicides. In this paper, we present the results from greenhouse and field trials where we evaluated the effect of strategies aimed at reducing our reliance on synthetic fungicides including treatments with biological control agents (BCAs) (Pythium oligandrum, Polygandron (R), and Bacillus subtilis, Serenade (R)) and plant resistance inducers (silicon products HortiStar (R) and Actisil (R)) for early blight in potato. The agents were applied separately or in combination with each other or with synthetic fungicides. In the greenhouse, trials application of these agents resulted in 50-95% reduction of infection by A. solani, but their combination did not generally improve the outcome. However, the effects were much smaller in the hand-sprayed field trials, 20-25% disease reduction and almost disappeared in full-scale field trials where application was done with tractor sprayers. In this article, we discuss possible reasons behind the drop in efficacy from greenhouse trials to full-size field evaluation

The hunt for sustainable biocontrol of oomycete plant pathogens, a case study of Phytophthora infestans

Late blight caused by the oomycete Phytophthora infestans is considered to be one of the most severe diseases of potato and tomato worldwide. Whilst current synthetic fungicides are efficient at controlling this disease, they are an environmental and economic burden. In line with EU directives to reduce the use of synthetic pesticides and increase the use of sustainable alternative disease control strategies that can form part of integrated pest management systems, practical biological control solutions are urgently needed. Despite the fact that there has been a large body of scientific research into microorganisms with potential for the biological control of late blight disease, relatively few commercial biocontrol agents, licensed to control late blight, exist. Furthermore, the practical uptake of those in Europe is lower than might be expected, suggesting that such solutions are not yet feasible, or effective. Here we review the scientific literature, focusing on the most recent developments in the hunt for efficient and sustainable biological control of late blight disease. We discuss the progress in our mechanistic understanding of mycoparasite–prey interactions, in the context of late blight and the challenges and limitations to the use of such knowledge in practical disease control within a European context

Earlier occurrence and increased explanatory power of climate for the first incidence of potato late blight caused by Phytophthora infestans in Fennoscandia

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Double trouble: Co-infection of potato with the causal agents of late and early blight

Global potato production is plagued by multiple pathogens, amongst which are Phytophthora infestans and Alternaria solani, the causal agents of potato late blight and early blight, respectively. Both these pathogens have different lifestyles and are successful pathogens of potato, but despite observations of both pathogens infecting potato simultaneously in field conditions, the tripartite interactions between potato and these two pathogens are so far poorly understood. Here we studied the interaction of A. solani and P. infestans first in vitro and subsequently in planta both in laboratory and field settings. We found that A. solani can inhibit P. infestans in terms of growth in vitro and also infection of potato in both laboratory experiments and in an agriculturally relevant field setting. A. solani had a direct inhibitory effect on P. infestans in vitro and compounds secreted by A. solani had both an inhibitory and disruptive effect on sporangia and mycelium of P. infestans in vitro. In planta infection bioassays revealed that simultaneous co-inoculation of both pathogens resulted in larger necrotic lesions than single inoculations; however, consecutive inoculations only resulted in larger lesions when A. solani was inoculated after P. infestans. These results indicate that the order in which these pathogens attempt to colonize potato is important for the disease outcome and that the influence of plant pathogens on each other should be accounted for in the design of future disease control strategies in crops such as potato