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

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

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

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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Pathogen-Mediated Stomatal Opening: A Previously Overlooked Pathogenicity Strategy in the Oomycete Pathogen Phytophthora infestans

Phytophthora infestans, the most damaging oomycete pathogen of potato, is specialized to grow sporangiophore through opened stomata for secondary inoculum production. However, it is still unclear which metabolic pathways in potato are manipulated by P. infestans in the guard cell-pathogen interactions to open the stomata. Here microscopic observations and cell biology were used to investigate antagonistic interactions between guard cells and the oomycete pathogen. We observed that the antagonistic interactions started at the very beginning of infection. Stomatal movement is an important part of the immune response of potato to P. infestans infection and this occurs through guard cell death and stomatal closure. We observed that P. infestans appeared to manipulate metabolic processes in guard cells, such as triacylglycerol (TAG) breakdown, starch degradation, H2O2 scavenging, and NO catabolism, which are involved in stomatal movement, to evade these stomatal defense responses. The signal transduction pathway of P. infestans-induced stomatal opening likely starts from H2O2 and NO scavenging, along with TAG breakdown while the subsequent starch degradation reinforces the opening process by strengthening guard cell turgor and opening the stomata to their maximum aperture. These results suggest that stomata are a barrier stopping P. infestans from completing its life cycle, but this host defense system can be bypassed through the manipulation of diverse metabolic pathways that may be induced by P. infestans effector proteins

Draft genome of the oomycete pathogen <i>Phytophthora cactorum</i> strain LV007 isolated from European beech (<i>Fagus sylvatica</i>)

Phytophthora cactorum is a broad host range phytopathogenic oomycete. P. cactorum strain LV007 was isolated from a diseased European Beech (Fagus sylvatica) in Malmö, Sweden in 2016. The draft genome of P. cactorum strain LV007 is 67.81 Mb. It contains 15,567 contigs and 21,876 predicted protein-coding genes. As reported for other phytopathogenic Phytophthora species, cytoplasmic effector proteins including RxLR and CRN families were identified. The genome sequence has been deposited at DDBJ/ENA/GenBank under the accession NBIJ00000000. The version described in this paper is version NBIJ01000000.</p

Altitudinal Heterogeneity of UV Adaptation in Phytophthora infestans Is Associated with the Spatial Distribution of a DNA Repair Gene

Climate change is considered a major threat to society and nature. UV irradiation is the most important environmental genotoxic agent. Thus, how elevated UV irradiation may influence human health and ecosystems has generated wide concern in the scientific community, as well as with policy makers and the public in general. In this study, we investigated patterns and mechanisms of UV adaptation in natural ecosystems by studying a gene-specific variation in the potato late blight pathogen, Phytophthora infestans. We compared the sequence characteristics of radiation sensitive 23 (RAD23), a gene involved in the nucleotide excision repair (NER) pathway and UV tolerance, in P. infestans isolates sampled from various altitudes. We found that lower genetic variation in the RAD23 gene was caused by natural selection. The hypothesis that UV irradiation drives this selection was supported by strong correlations between the genomic characteristics and altitudinal origin (historic UV irradiation) of the RAD23 sequences with UV tolerance of the P. infestans isolates. These results indicate that the RAD23 gene plays an important role in the adaptation of P. infestans to UV stress. We also found that different climatic factors could work synergistically to determine the evolutionary adaptation of species, making the influence of climate change on ecological functions and resilience more difficult to predict. Future attention should aim at understanding the collective impact generated by simultaneous change in several climate factors on species adaptation and ecological sustainability, using state of the art technologies such as experimental evolution, genome-wide scanning, and proteomics