This article is part of the Top 10 Unanswered Questions in MPMI invited review series.The past few decades have seen major discoveries in the field of molecular plant-microbe interactions. As the result of technological and intellectual advances, we are now able to answer questions at a level of mechanistic detail that we could not have imagined possible 20 years ago. The MPMI Editorial Board felt it was time to take stock and reassess. What big questions remain unanswered? We knew that to identify the fundamental, overarching questions that drive our research, we needed to do this as a community. To reach a diverse audience of people with different backgrounds and perspectives, working in different areas of plant-microbe interactions, we queried the more than 1,400 participants at the 2019 International Congress on Molecular Plant-Microbe Interactions meeting in Glasgow. This group effort resulted in a list of ten, broad-reaching, fundamental questions that influence and inform our research. Here, we introduce these Top 10 unanswered questions, giving context and a brief description of the issues. Each of these questions will be the subject of a detailed review in the coming months. We hope that this process of reflecting on what is known and unknown and identifying the themes that underlie our research will provide a framework to use going forward, giving newcomers a sense of the mystery of the big questions and inspiring new avenues and novel insights.[ ] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Section Key Message Using disease bioassays and transcriptomic analysis we show that intact SA-signalling is required for potato defences against the necrotrophic fungal pathogen Alternaria solani. AbstractSection Abstract Early blight, caused by the necrotrophic fungus Alternaria solani, is an increasing problem in potato cultivation. Studies of the molecular components defining defence responses to A. solani in potato are limited. Here, we investigate plant defence signalling with a focus on salicylic acid (SA) and jasmonic acid (JA) pathways in response to A. solani. Our bioassays revealed that SA is necessary to restrict pathogen growth and early blight symptom development in both potato foliage and tubers. This result is in contrast to the documented minimal role of SA in resistance of Arabidopsis thaliana against necrotrophic pathogens. We also present transcriptomic analysis with 36 arrays of A. solani inoculated SA-deficient, JA-insensitive, and wild type plant lines. A greater number of genes are differentially expressed in the SA-deficient mutant plant line compared to the wild type and JA- insensitive line. In wild type plants, genes encoding metal ion transporters, such as copper, iron and zinc transporters were upregulated and transferase-encoding genes, for example UDP-glucoronosyltransferase and Serine-glyoxylate transferase, were downregulated. The SA-deficient plants show upregulation of genes enriched in GO terms related to oxidoreductase activity, respiratory chain and other mitochondrial-related processes. Pathogenesis-related genes, such as genes encoding chitinases and PR1, are upregulated in both the SA-deficient and wild type plants, but not in the JA-insensitive mutants. The combination of our bioassays and the transcriptomic analysis indicate that intact SA signalling, and not JA signalling, is required for potato defences against the necrotrophic pathogen A. solani.

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^®, and Bacillus subtilis, Serenade^®) and plant resistance inducers (silicon products HortiStar^® and Actisil^®) 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.

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.

Potato early blight is caused by the necrotrophic fungus Alternaria solani and can result in yield losses of up to 50% if left uncontrolled. At present, the disease is controlled by chemical fungicides, yet rapid development of fungicide resistance renders current control strategies unsustainable. On top of that, a lack of understanding of potato defences and the quantitative nature of resistance mechanisms against early blight hinders the development of more sustainable control methods. Necrotrophic pathogens, compared to biotrophs, pose an extra challenge to the plant, since common defence strategies to biotic stresses such as the hypersensitive response and programmed cell death are often beneficial for necrotrophs. With the aim of unravelling plant responses to both the early infection stages (i.e., before necrosis), such as appressorium formation and penetration, as well as to later responses to the onset of necrosis, we present here a transcriptome analysis of potato interactions with A. solani from 1 h after inoculation when the conidia have just commenced germination, to 48 h post inoculation when multiple cell necrosis has begun. Potato transcripts with putative functions related to biotic stress tolerance and defence against pathogens were upregulated, including a putative Nudix hydrolase that may play a role in defence against oxidative stress. A. solani transcripts encoding putative pathogenicity factors, such as cell wall degrading enzymes and metabolic processes that may be important for infection. We therefore identified the differential expression of several potato and A. solani transcripts that present a group of valuable candidates for further studies into their roles in immunity or disease development.

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.