High-Resolution Disease Phenotyping Reveals Distinct Resistance Mechanisms of Tomato Crop Wild Relatives against Sclerotinia sclerotiorum.

Besides the well-understood qualitative disease resistance, plants possess a more complex quantitative form of resistance: quantitative disease resistance (QDR). QDR is commonly defined as a partial but more durable form of resistance and, therefore, might display a valuable target for resistance breeding. The characterization of QDR phenotypes, especially of wild crop relatives, displays a bottleneck in deciphering QDR's genomic and regulatory background. Moreover, the relationship between QDR parameters, such as infection frequency, lag-phase duration, and lesion growth rate, remains elusive. High hurdles for applying modern phenotyping technology, such as the low availability of phenotyping facilities or complex data analysis, further dampen progress in understanding QDR. Here, we applied a low-cost (<1.000 €) phenotyping system to measure lesion growth dynamics of wild tomato species (e.g., Solanum pennellii or Solanum pimpinellifolium). We provide insight into QDR diversity of wild populations and derive specific QDR mechanisms and their cross-talk. We show how temporally continuous observations are required to dissect end-point severity into functional resistance mechanisms. The results of our study show how QDR can be maintained by facilitating different defense mechanisms during host-parasite interaction and that the capacity of the QDR toolbox highly depends on the host's genetic context. We anticipate that the present findings display a valuable resource for more targeted functional characterization of the processes involved in QDR. Moreover, we show how modest phenotyping technology can be leveraged to help answer highly relevant biological questions.

Network analyses predict major regulators of resistance to early blight disease complex in tomato.

BACKGROUND: Early blight and brown leaf spot are often cited as the most problematic pathogens of tomato in many agricultural regions. Their causal agents are Alternaria spp., a genus of Ascomycota containing numerous necrotrophic pathogens. Breeding programs have yielded quantitatively resistant commercial cultivars, but fungicide application remains necessary to mitigate the yield losses. A major hindrance to resistance breeding is the complexity of the genetic determinants of resistance and susceptibility. In the absence of sufficiently resistant germplasm, we sequenced the transcriptomes of Heinz 1706 tomatoes treated with strongly virulent and weakly virulent isolates of Alternaria spp. 3 h post infection. We expanded existing functional gene annotations in tomato and using network statistics, we analyzed the transcriptional modules associated with defense and susceptibility. RESULTS: The induced responses are very distinct. The weakly virulent isolate induced a defense response of calcium-signaling, hormone responses, and transcription factors. These defense-associated processes were found in a single transcriptional module alongside secondary metabolite biosynthesis genes, and other defense responses. Co-expression and gene regulatory networks independently predicted several D clade ethylene response factors to be early regulators of the defense transcriptional module, as well as other transcription factors both known and novel in pathogen defense, including several JA-associated genes. In contrast, the strongly virulent isolate elicited a much weaker response, and a separate transcriptional module bereft of hormone signaling. CONCLUSIONS: Our findings have predicted major defense regulators and several targets for downstream functional analyses. Combined with our improved gene functional annotation, they suggest that defense is achieved through induction of Alternaria-specific immune pathways, and susceptibility is mediated by modulating hormone responses. The implication of multiple specific clade D ethylene response factors and upregulation of JA-associated genes suggests that host defense in this pathosystem involves ethylene response factors to modulate jasmonic acid signaling.

Alternaria diseases on potato and tomato.

Alternaria spp. cause different diseases in potato and tomato crops. Early blight caused by Alternaria solani and brown spot caused by Alternaria alternata are most common, but the disease complex is far more diverse. We first provide an overview of the Alternaria species infecting the two host plants to alleviate some of the confusion that arises from the taxonomic rearrangements in this fungal genus. Highlighting the diversity of Alternaria fungi on both solanaceous hosts, we review studies investigating the genetic diversity and genomes, before we present recent advances from studies elucidating host-pathogen interactions and fungicide resistances. TAXONOMY: Kingdom Fungi, Phylum Ascomycota, Class Dothideomycetes, Order Pleosporales, Family Pleosporaceae, Genus Alternaria. BIOLOGY AND HOST RANGE: Alternaria spp. adopt diverse lifestyles. We specifically review Alternaria spp. that cause disease in the two solanaceous crops potato (Solanum tuberosum) and tomato (Solanum lycopersicum). They are necrotrophic pathogens with no known sexual stage, despite some signatures of recombination. DISEASE SYMPTOMS: Symptoms of the early blight/brown spot disease complex include foliar lesions that first present as brown spots, depending on the species with characteristic concentric rings, which eventually lead to severe defoliation and considerable yield loss. CONTROL: Good field hygiene can keep the disease pressure low. Some potato and tomato cultivars show differences in susceptibility, but there are no fully resistant varieties known. Therefore, the main control mechanism is treatment with fungicides.

Untargeted metabolomics reveals PTI-associated metabolites.

Plants employ a multilayered immune system to combat pathogens. In one layer, recognition of Pathogen- or Microbe-Associated Molecular Patterns or elicitors, triggers a cascade that leads to defence against the pathogen and Pattern Triggered Immunity. Secondary or specialised metabolites (SMs) are expected to play a role, because they are potentially anti-fungal compounds. Tomato (Solanum lycopersicum) plants inoculated with Alternaria solani s.l. show symptoms of infection after inoculation. Plants inoculated with Alternaria alternata remain symptomless. We hypothesised that pattern-triggered induction of resistance related metabolites in tomato contributes to the resistance against A. alternata. We compared the metabolomic profile (metabolome) of tomato after treatments with A. alternata, A. solani and the fungal elicitor chitin, and identified SMs involved in early defence of tomato plants. We revealed differential metabolome fingerprints. The composition of A. alternata and chitin induced metabolomes show larger overlap with each other than with the A. solani induced metabolome. We identify 65 metabolites possibly associated with PTI in tomato plants, including NAD and trigonelline. We confirm that trigonelline inhibits fungal growth in vitro at physiological concentrations. Thus, a true pattern-triggered, chemical defence is mounted against A. alternata, which contains anti-fungal compounds that could be interesting for crop protection strategies.

Barley shows reduced Fusarium head blight under drought and modular expression of differentially expressed genes under combined stress.

Plants often face simultaneous abiotic and biotic stress conditions; however, physiological and transcriptional responses under such combined stress conditions are still not fully understood. Spring barley (Hordeum vulgare) is susceptible to Fusarium head blight (FHB), which is strongly affected by weather conditions. We therefore studied the potential influence of drought on FHB severity and plant responses in three varieties of different susceptibility. We found strongly reduced FHB severity in susceptible varieties under drought. The number of differentially expressed genes (DEGs) and strength of transcriptomic regulation reflected the concentrations of physiological stress markers such as abscisic acid or fungal DNA contents. Infection-related gene expression was associated with susceptibility rather than resistance. Weighted gene co-expression network analysis revealed 18 modules of co-expressed genes that reflected the pathogen- or drought-response in the three varieties. A generally infection-related module contained co-expressed genes for defence, programmed cell death, and mycotoxin detoxification, indicating that the diverse genotypes used a similar defence strategy towards FHB, albeit with different degrees of success. Further, DEGs showed co-expression in drought- or genotype-associated modules that correlated with measured phytohormones or the osmolyte proline. The combination of drought stress with infection led to the highest numbers of DEGs and resulted in a modular composition of the single-stress responses rather than a specific transcriptional output.

Small-spored Alternaria spp. (section Alternaria) are common pathogens on wild tomato species.

The wild relatives of modern tomato crops are native to South America. These plants occur in habitats as different as the Andes and the Atacama Desert and are, to some degree, all susceptible to fungal pathogens of the genus Alternaria. Alternaria is a large genus. On tomatoes, several species cause early blight, leaf spots and other diseases. We collected Alternaria-like infection lesions from the leaves of eight wild tomato species from Chile and Peru. Using molecular barcoding markers, we characterized the pathogens. The infection lesions were caused predominantly by small-spored species of Alternaria of the section Alternaria, like A. alternata, but also by Stemphylium spp., Alternaria spp. from the section Ulocladioides and other related species. Morphological observations and an infection assay confirmed this. Comparative genetic diversity analyses show a larger diversity in this wild system than in studies of cultivated Solanum species. As A. alternata has been reported to be an increasing problem in cultivated tomatoes, investigating the evolutionary potential of this pathogen is not only interesting to scientists studying wild plant pathosystems. It could also inform crop protection and breeding programs to be aware of potential epidemics caused by species still confined to South America.

Laminarin-triggered defence responses are geographically dependent in natural populations of Solanum chilense.

Natural plant populations are polymorphic and show intraspecific variation in resistance properties against pathogens. The activation of the underlying defence responses can depend on variation in perception of pathogen-associated molecular patterns or elicitors. To dissect such variation, we evaluated the responses induced by laminarin (a glucan, representing an elicitor from oomycetes) in the wild tomato species Solanum chilense and correlated this to observed infection frequencies of Phytophthora infestans. We measured reactive oxygen species burst and levels of diverse phytohormones upon elicitation in 83 plants originating from nine populations. We found high diversity in basal and elicitor-induced levels of each component. Further we generated linear models to explain the observed infection frequency of P. infestans. The effect of individual components differed dependent on the geographical origin of the plants. We found that the resistance in the southern coastal region, but not in the other regions, was directly correlated to ethylene responses and confirmed this positive correlation using ethylene inhibition assays. Our findings reveal high diversity in the strength of defence responses within a species and the involvement of different components with a quantitatively different contribution of individual components to resistance in geographically separated populations of a wild plant species.

Metabolomics in Plant Pathogen Defense: From Single Molecules to Large-Scale Analysis.

Plants produce a high diversity of secondary metabolites that are involved in a wide range of different functions, including stress tolerance, signaling molecules for interactions with other species (allelopathy), and protecting plants against herbivores and pathogens. With the rise of more accessible, high-throughput mass spectrometry and new analytical tools, it becomes feasible to identify and validate new secondary metabolites involved in pathogen resistance or assign new roles to previously detected compounds. In this review, we provide a brief overview of the major pathogen defense-associated classes of secondary metabolites, with a focus on those with direct anti-pathogen function. For each class, we highlight one or more typical examples representing the class to give a comprehensive summary of some of the work done to date. In the second part of this review, we highlight how new technological advances and high-throughput experiments in combination with other sources of -omics data, such as genomics and transcriptomics, can accelerate the studies on secondary metabolites and help to link these compounds to genotypes. Employing such approaches will improve our understanding of chemical defenses against plant pathogens and allow for rapid development of markers for resistance breeding.

Distinct Phyllosphere Microbiome of Wild Tomato Species in Central Peru upon Dysbiosis.

Plants are colonized by myriads of microbes across kingdoms, which affect host development, fitness, and reproduction. Hence, plant microbiomes have been explored across a broad range of host species, including model organisms, crops, and trees under controlled and natural conditions. Tomato is one of the world's most important vegetable crops; however, little is known about the microbiota of wild tomato species. To obtain insights into the tomato microbiota occurring in natural environments, we sampled epiphytic microbes from leaves of four tomato species, Solanum habrochaites, S. corneliomulleri, S. peruvianum, and S. pimpinellifolium, from two geographical locations within the Lima region of Peru over 2 consecutive years. Here, a high-throughput sequencing approach was applied to investigate microbial compositions including bacteria, fungi, and eukaryotes across tomato species and geographical locations. The phyllosphere microbiome composition varies between hosts and location. Yet, we identified persistent microbes across tomato species that form the tomato microbial core community. In addition, we phenotypically defined healthy and dysbiotic samples and performed a downstream analysis to reveal the impact on microbial community structures. To do so, we compared microbial diversities, unique OTUs, relative abundances of core taxa, and microbial hub taxa, as well as co-occurrence network characteristics in healthy and dysbiotic tomato leaves and found that dysbiosis affects the phyllosphere microbial composition in a host species-dependent manner. Yet, overall, the present data suggests an enrichment of plant-promoting microbial taxa in healthy leaves, whereas numerous microbial taxa containing plant pathogens occurred in dysbiotic leaves.Concluding, we identify the core phyllosphere microbiome of wild tomato species, and show that the overall phyllosphere microbiome can be impacted by sampling time point, geographical location, host genotype, and plant health. Future studies in these components will help understand the microbial contribution to plant health in natural systems and can be of use in cultivated tomatoes.

Whole-genome sequencing elucidates the species-wide diversity and evolution of fungicide resistance in the early blight pathogen Alternaria solani.

Early blight of potato is caused by the fungal pathogen Alternaria solani and is an increasing problem worldwide. The primary strategy to control the disease is applying fungicides such as succinate dehydrogenase inhibitors (SDHI). SDHI-resistant strains, showing reduced sensitivity to treatments, appeared in Germany in 2013, shortly after the introduction of SDHIs. Two primary mutations in the SDH complex (SdhB-H278Y and SdhC-H134R) have been frequently found throughout Europe. How these resistances arose and spread, and whether they are linked to other genomic features, remains unknown. For this project, we performed whole-genome sequencing for 48 A. solani isolates from potato fields across Europe to better characterize the pathogen's genetic diversity in general and understand the development and spread of the genetic mutations that lead to SDHI resistance. The isolates can be grouped into seven genotypes. These genotypes do not show a geographical pattern but appear spread throughout Europe. We found clear evidence for recombination on the genome, and the observed admixtures might indicate a higher adaptive potential of the fungus than previously thought. Yet, we cannot link the observed recombination events to different Sdh mutations. The same Sdh mutations appear in different, non-admixed genetic backgrounds; therefore, we conclude they arose independently. Our research gives insights into the genetic diversity of A. solani on a genome level. The mixed occurrence of different genotypes, apparent admixture in the populations, and evidence for recombination indicate higher genomic complexity than anticipated. The conclusion that SDHI tolerance arose multiple times independently has important implications for future fungicide resistance management strategies. These should not solely focus on preventing the spread of isolates between locations but also on limiting population size and the selective pressure posed by fungicides in a given field to avoid the rise of new mutations in other genetic backgrounds.

Steroidal Saponins─New Sources to Develop Potato (Solanum tuberosum L.) Genotypes Resistant against Certain Phytophthora infestans Strains.

Plant pathogens such as Phytophthora infestans that caused the Irish Potato Famine continue to threaten local and global food security. Genetic and chemical plant protection measures are often overcome by adaptation of pathogen population structures. Therefore, there is a constant demand for new, consumer- and environment-friendly plant protection strategies. Metabolic alterations induced by P. infestans in the foliage and tubers of six different potato cultivars were investigated. Using a combination of untargeted metabolomics, isolation techniques, and structure elucidation by MS and 1D/2D-NMR experiments, five steroidal glycoalkaloids, five oxylipins, and four steroidal saponins were identified. As the steroidal saponins showed antioomycete but no hemolytic activity, they may thus be considered as probably safe target substances for enrichment in breeding programs for disease resistance and as chemical lead structures for the production of nature-derived synthetic antioomycetes.

Evolution of resistance (R) gene specificity.

Plant resistance (R) genes are members of large gene families with significant within and between species variation. It has been hypothesised that a variety of processes have shaped R gene evolution and the evolution of R gene specificity. In this review, we illustrate the main mechanisms that generate R gene diversity and provide examples of how they can change R gene specificity. Next, we explain which evolutionary mechanisms are at play and how they determine the fate of new R gene alleles and R genes. Finally, we place this in a larger context by comparing the diversity and evolution of R gene specificity within and between species scales.

Quantitative resistance differences between and within natural populations of Solanum chilense against the oomycete pathogen Phytophthora infestans.

The wild tomato species Solanum chilense is divided into geographically and genetically distinct populations that show signs of defense gene selection and differential phenotypes when challenged with several phytopathogens, including the oomycete causal agent of late blight Phytophthora infestans. To better understand the phenotypic diversity of this disease resistance in S. chilense and to assess the effect of plant genotype versus pathogen isolate, respectively, we evaluated infection frequency in a systematic approach and with large sample sizes. We studied 85 genetically distinct individuals representing nine geographically separated populations of S. chilense. This showed that differences in quantitative resistance can be observed between but also within populations at the level of individual plants. Our data also did not reveal complete immunity in any of the genotypes. We further evaluated the resistance of a subset of the plants against P. infestans isolates with diverse virulence properties. This confirmed that the relative differences in resistance phenotypes between individuals were mainly determined by the plant genotype under consideration with modest effects of pathogen isolate used in the study. Thus, our report suggests that the observed quantitative resistance against P. infestans in natural populations of a wild tomato species S. chilense is the result of basal defense responses that depend on the host genotype and are pathogen isolate-unspecific.

Polymorphisms in plants to restrict losses to pathogens: From gene family expansions to complex network evolution.

Genetic polymorphisms are the basis of the natural diversity seen in all life on earth, also in plant-pathogen interactions. Initially, studies on plant-pathogen interaction focused on reporting phenotypic variation in resistance properties and on the identification of underlying major genes. Nowadays, the field of plant-pathogen interactions is moving from focusing on families of single dominant genes involved in gene-for-gene interactions to an understanding of the plant immune system in the context of a much more complex signaling network and quantitative resistance. Simultaneously, studies on pathosystems from the wild and genome analyses advanced, revealing tremendous variation in natural plant populations. It is now imperative to place studies on genetic diversity and evolution of plant-pathogen interactions in the appropriate molecular biological, as well as evolutionary, context.

Allelic variants of the NLR protein Rpi-chc1 differentially recognize members of the Phytophthora infestans PexRD12/31 effector superfamily through the leucine-rich repeat domain.

Phytophthora infestans is a pathogenic oomycete that causes the infamous potato late blight disease. Resistance (R) genes from diverse Solanum species encode intracellular receptors that trigger effective defense responses upon the recognition of cognate RXLR avirulence (Avr) effector proteins. To deploy these R genes in a durable fashion in agriculture, we need to understand the mechanism of effector recognition and the way the pathogen evades recognition. In this study, we cloned 16 allelic variants of the Rpi-chc1 gene from Solanum chacoense and other Solanum species, and identified the cognate P. infestans RXLR effectors. These tools were used to study effector recognition and co-evolution. Functional and non-functional alleles of Rpi-chc1 encode coiled-coil nucleotide-binding leucine-rich repeat (CNL) proteins, being the first described representatives of the CNL16 family. These alleles have distinct patterns of RXLR effector recognition. While Rpi-chc1.1 recognized multiple PexRD12 (Avrchc1.1) proteins, Rpi-chc1.2 recognized multiple PexRD31 (Avrchc1.2) proteins, both belonging to the PexRD12/31 effector superfamily. Domain swaps between Rpi-chc1.1 and Rpi-chc1.2 revealed that overlapping subdomains in the leucine-rich repeat (LRR) domain are responsible for the difference in effector recognition. This study showed that Rpi-chc1.1 and Rpi-chc1.2 evolved to recognize distinct members of the same PexRD12/31 effector family via the LRR domain. The biased distribution of polymorphisms suggests that exchange of LRRs during host-pathogen co-evolution can lead to novel recognition specificities. These insights will guide future strategies to breed durable resistant varieties.

ONT-Based Draft Genome Assembly and Annotation of Alternaria atra.

Species of Alternaria (phylum Ascomycota, family Pleosporaceae) are known as serious plant pathogens, causing major losses on a wide range of crops. Alternaria atra (previously known as Ulocladium atrum) can grow as a saprophyte on many hosts and causes Ulocladium blight on potato. It has been reported that it can also be used as a biocontrol agent against Botrytis cinerea. Here, we present a scaffold-level reference genome assembly for A. atra. The assembly contains 43 scaffolds with a total length of 39.62 Mbp, with scaffold N50 of 3,893,166 bp, L50 of 4, and the longest 10 scaffolds containing 89.9% of the assembled data. RNA-sequencing-guided gene prediction using BRAKER resulted in 12,173 protein-coding genes with their functional annotation. This first high-quality reference genome assembly and annotation for A. atra can be used as a resource for studying evolution in the highly complicated Alternaria genus and might help in understanding the mechanisms defining its role as pathogen or biocontrol agent.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector.

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologues and paralogues. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogues and alleles of the recognized effector, facilitating durable resistance against P. infestans.

Population Genomic- and Phylogenomic-Enabled Advances to Increase Insight Into Pathogen Biology and Epidemiology.

Population genetics has been a key discipline in phytopathology for many years. The recent rise in cost-effective, high-throughput DNA sequencing technologies, allows sequencing of dozens, if not hundreds of specimens, turning population genetics into population genomics and opening up new, exciting opportunities as described in this Focus Issue. Without the limitations of genetic markers and the availability of whole or near whole-genome data, population genomics can give new insights into the biology, evolution and adaptation, and dissemination patterns of plant-associated microbes.

Population Genomics of Filamentous Plant Pathogens-A Brief Overview of Research Questions, Approaches, and Pitfalls.

With ever-decreasing sequencing costs, research on the population biology of plant pathogens is transitioning from population genetics-using dozens of genetic markers or polymorphism data of several genes-to population genomics-using several hundred to tens of thousands of markers or whole-genome sequence data. The field of population genomics is characterized by rapid theoretical and methodological advances and by numerous steps and pitfalls in its technical and analytical workflow. In this article, we aim to provide a brief overview of topics relevant to the study of population genomics of filamentous plant pathogens and direct readers to more extensive reviews for in-depth understanding. We briefly discuss different types of population genomics-inspired research questions and give insights into the sampling strategies that can be used to answer such questions. We then consider different sequencing strategies, the various options available for data processing, and some of the currently available tools for population genomic data analysis. We conclude by highlighting some of the hurdles along the population genomic workflow, providing cautionary warnings relative to assumptions and technical challenges, and presenting our own future perspectives of the field of population genomics for filamentous plant pathogens.