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Ensuring the reliability of diagnostic activities is an essential cornerstone of plant health strategies to reduce the risk of entry and spread of plant pests in a region and ultimately their impacts. Diagnostic tests should be validated to ensure that they are fit for purpose. Validation is usually done by diagnostic laboratories, although companies commercializing diagnostic kits also produce validation data for their products. Due to the high number of pest, matrix, and method combinations and given the significant resources required to validate tests, it is essential that validation data are shared with the entire diagnostic community and produced in a harmonized way to facilitate their use by different stakeholders. Indeed, the selection of tests to be used in specific contexts is not the sole responsibility of diagnostic laboratories but also involves national plant protection organizations. The VALITEST EU project (2018 to 2021) was established to tackle all these issues. New validation data for tests targeting important pests for the European and Mediterranean Plant Protection Organization region were produced. Guidelines to improve and harmonize the validation framework were developed. Sharing of validation data and experience was ensured through the development of new or existing databases, the organization of training courses, and the dissemination of the project outputs in scientific publications and standards. Finally, the involvement of researchers, diagnosticians, policy makers, inspectors, and industries and the establishment of the European Plant Diagnostic Industry Association were important actions to strengthen the interactions between plant health stakeholders. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, mycetes, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health., Scientific publication adapted from the deliverable 2.2 of the Valitest project - www.valitest.eu

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material., This article is based upon work from the work package 2 of the project VALITEST (https://www.valitest.eu/), supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 773139.

The organisation of a test performance study (TPS) requires significant inputs in finances, time, and a high level of expertise from the TPS organiser to ensure a smooth process and reliable results. In this book authors have used collective experience gained in the organization of 12 TPS studies, as well as previous extensive experience in the organization of similar studies to prepare a ‘textbook’ for organisers of future TPS not only in the field of plant pest detection, but also in other areas of microbiology. This chapter summarises the main challenges which can occur during the TPS organisation and provides the recommendations how to foresee them and successfully organize TPS.

The organisation of a test performance study (TPS) involves different steps that are mostly sequential, but some may be conducted simultaneously. This chapter details the following: the steps regarding the selection of the tests to be validated; the selection of the laboratories to participate in the TPS; the preparation of the materials and the dispatch of the samples; and the completion of the TPS (including the collection and analysis of the TPS results). The reader will be able to get the detailed information on how to define and plan timeline of the TPS, the appropriate number of samples (including replicates) and of laboratories that should be included in the TPS to ensure an appropriate statistical analysis, and how to perform basic analyses of the obtained data. In addition, this chapter covers the most important critical points which can endanger successful TPS organization providing the future TPS organisers in the field of plant health (but also in other similar fields) with the possibility to identify them in advance and carry-out successful TPS.

There are two types of interlaboratory comparison studies: proficiency tests, which aim at monitoring the proficiency of laboratories, and test performance studies (TPS), which aim at evaluating the performance of (a) specific test(s) and whether it (they) is (are) fit for purpose. This booklet covers only TPS organisation. A TPS is usually organised to monitor the performance of a newly developed test to detect and/or identify pests or to compare the performance of different tests. The results of TPS also provide information on how (a) test(s) perform(s) in different laboratories. This allows a better estimation of the accuracy and reproducibility of tests. Organising a TPS is a complex process that requires considerable effort from the organisers in terms of time, expertise, and finances. Therefore, the selection of e.g. pests and of participants are important steps which are described in this chapter It also discusses the minimum criteria that the TPS organiser should meet.

The recent COVID-19 pandemic highlighted the importance of rapid and reliable pathogen detection. In the field of plant health accurate and timely detection is a corner stone for successful pest control. Plant pests can be diagnosed with a variety of tests, which may be developed by commercial companies or research institutions. The reliability of diagnostic tests depends on the intended use of the tests, their performance characteristics and associated uncertainty obtained from validation studies and the experience of the laboratories. The performance characteristics of tests are obtained during a process called validation. Performance characteristics that are frequently used to characterise tests include: analytical sensitivity, analytical specificity (inclusivity and exclusivity), selectivity, repeatability and reproducibility. The validation process requires significant investment in terms of human and financial resources and can be conducted by a single diagnostic laboratory or through interlaboratory comparisons. The purpose of this book is to provide practical and technical guidance for the organisation of test performance studies (TPS), which are one type of interlaboratory comparison where the performance of (a) test(s) (is)are assessed by two or more laboratories. The major steps and challenges faced during the preparation, organisation and reporting of TPS are identified and can be used by organisers of future TPS not only in the field of plant pest detection, but also in other areas.

ISBN : 978-0-8243-0668-7; Quantitative genetics has a long history in plants: It has been used to study specific biological processes, identify the factors important for trait evolution, and breed new crop varieties. These classical approaches to quantitative trait locus mapping have naturally improved with technology. In this review, we show how quantitative genetics has evolved recently in plants and how new developments in phenotyping, population generation, sequencing, gene manipulation, and statistics are rejuvenating both the classical linkage mapping approaches (for example, through nested association mapping) as well as the more recently developed genome-wide association studies. These strategies are complementary in most instances, and indeed, one is often used to confirm the results of the other. Despite significant advances, an emerging trend is that the outcome and efficiency of the different approaches depend greatly on the genetic architecture of the trait in the genetic material under study.

Many important crops are members of the Poaceae family, which develop root systems characterized by a high degree of root initiation from the belowground basal nodes of the shoot, termed the crown. Although this postembryonic shoot-borne root system represents the major conduit for water uptake, little is known about the effect of water availability on its development. Here we demonstrate that in the model C4 grass Setaria viridis, the crown locally senses water availability and suppresses postemergence crown root growth under a water deficit. This response was observed in field and growth room environments and in all grass species tested. Luminescence-based imaging of root systems grown in soil-like media revealed a shift in root growth from crown-derived to primary root-derived branches, suggesting that primary root-dominated architecture can be induced in S. viridis under certain stress conditions. Crown roots of Zea mays and Setaria italica, domesticated relatives of teosinte and S. viridis, respectively, show reduced sensitivity to water deficit, suggesting that this response might have been influenced by human selection. Enhanced water status of maize mutants lacking crown roots suggests that under a water deficit, stronger suppression of crown roots actually may benefit crop productivity.

The root endodermis is characterized by the Casparian strip and by the suberin lamellae, two hydrophobic barriers that restrict the free diffusion of molecules between the inner cell layers of the root and the outer environment. The presence of these barriers and the position of the endodermis between the inner and outer parts of the root require that communication between these two domains acts through the endodermis. Recent work on hormone signaling, propagation of calcium waves, and plant-fungal symbiosis has provided evidence in support of the hypothesis that the endodermis acts as a signaling center. The endodermis is also a unique mechanical barrier to organogenesis, which must be overcome through chemical and mechanical cross talk between cell layers to allow for development of new lateral organs while maintaining its barrier functions. In this review, we discuss recent findings regarding these two important aspects of the endodermis.

The TCP family is an ancient group of plant developmental transcription factors that regulate cell division in vegetative and reproductive structures and are essential in the establishment of flower zygomorphy. In-depth research on eudicot TCPs has documented their evolutionary and developmental role. This has not happened to the same extent in monocots, although zygomorphy has been critical for the diversification of Orchidaceae and Poaceae, the largest families of this group. Investigating the evolution and function of TCP-like genes in a wider group of monocots requires a detailed phylogenetic analysis of all available sequence information and a system that facilitates comparing genetic and functional information.The phylogenetic relationships of TCP-like genes in monocots were investigated by analysing sequences from the genomes of Zea mays, Brachypodium distachyon, Oryza sativa and Sorghum bicolor, as well as EST data from several other monocot species.All available monocot TCP-like sequences are associated in 20 major groups with an average identity ≥64 % and most correspond to well-supported clades of the phylogeny. Their sequence motifs and relationships of orthology were documented and it was found that 67 % of the TCP-like genes of Sorghum, Oryza, Zea and Brachypodium are in microsyntenic regions. This analysis suggests that two rounds of whole genome duplication drove the expansion of TCP-like genes in these species.A system of classification is proposed where putative or recognized monocot TCP-like genes are assigned to a specific clade of PCF-, CIN- or CYC/tb1-like genes. Specific biases in sequence data of this family that must be tackled when studying its molecular evolution and phylogeny are documented. Finally, the significant retention of duplicated TCP genes from Zea mays is considered in the context of balanced gene drive.

Root systems develop different root types that individually sense cues from their local environment and integrate this information with systemic signals. This complex multi-dimensional amalgam of inputs enables continuous adjustment of root growth rates, direction, and metabolic activity that define a dynamic physical network. Current methods for analyzing root biology balance physiological relevance with imaging capability. To bridge this divide, we developed an integrated-imaging system called Growth and Luminescence Observatory for Roots (GLO-Roots) that uses luminescence-based reporters to enable studies of root architecture and gene expression patterns in soil-grown, light-shielded roots. We have developed image analysis algorithms that allow the spatial integration of soil properties, gene expression, and root system architecture traits. We propose GLO-Roots as a system that has great utility in presenting environmental stimuli to roots in ways that evoke natural adaptive responses and in providing tools for studying the multi-dimensional nature of such processes. DOI: http://dx.doi.org/10.7554/eLife.07597.001, eLife digest Most plants absorb water and nutrients from the soil via structures called roots. The shape, size, and structure of a plant's root system can change over its lifetime as the plant responds to changes in their local environment. For example, if water is scarce, a plant may develop a very deep root system that is more efficient at capturing water. Understanding how root systems respond to environmental cues may help us to identify the genes and processes involved. In this study, Rellán-Álvarez et al. report a new live-imaging platform for analyzing root architecture and its regulation. This platform is called Growth and Luminescence Observatory for Roots (or GLO-Roots for short) and uses ‘luminescent’ markers that allow growing roots to be visualized when plants are grown in thin, soil-filled, transparent pots. GLO-Roots can track the growth of the plant roots as well as the activity of genes that respond to environmental stress. Rellán-Álvarez et al. developed a software tool called GLO-RIA (GLO-Roots Image Analysis) to analyze the resulting images. GLO-RIA performs several different types of image analysis, including one that detects the position, length, and direction of roots, as well as their shape and depth. Rellán-Álvarez et al. tested the GLO-Roots techniques in various ways, for example, by analyzing the effects that different conditions have on the growth of the roots of the model plant known as Arabidopsis thaliana. Depriving the plants of a nutrient called phosphorous caused the roots to grow more horizontally than when phosphorus is plentiful, presumably to allow the plants to expand their search for phosphate in the upper layers of the soil, where this nutrient is usually more abundant. On the other hand, a shortage of water caused the roots to grow more vertically to access water stored deeper in the soil. GLO-Roots can also be used to measure the water content of soil at different depths and how this influences the architecture of the root. Further experiments on tomato plants and a grass species called Brachypodium distachyon revealed the different architectures of their root systems. Rellán-Álvarez et al. propose that this system could be used to study the roots of other plant species in a variety of environmental conditions. This will provide a more detailed understanding of the ways that different plants adapt in response to changes in their environment. DOI: http://dx.doi.org/10.7554/eLife.07597.002

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5′end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation., Author Summary Epigenetics is defined as the study of heritable changes in gene expression that are not linked to changes in the DNA sequence. In plants, these heritable variations are often associated with differences in DNA methylation. So far, very little is known about the extent and stability of epigenetic variation in nature. In this study, we report a case of extensive epigenetic variation in natural populations of the flowering plant Arabidopsis thaliana, which concerns a gene involved in starch metabolism, named Qua-Quine Starch (QQS). We show that in the wild QQS expression varies extensively and concomitantly with DNA methylation of the gene promoter. We also demonstrate that these variations are independent of DNA sequence changes and are stably inherited for several generations. In view of the recent evolutionary origin of QQS, we speculate that genes that emerge from scratch could be particularly prone to epigenetic variation. This would in turn endow epigenetic variation with a unique adaptive role in enabling de novo originated genes to adjust their expression pattern.

As sessile organisms, plants have to cope with diverse environmental constraints that may vary through time and space, eventually leading to changes in the phenotype of populations through fixation of adaptive genetic variation. To fully comprehend the mechanisms of evolution and make sense of the extensive genotypic diversity currently revealed by new sequencing technologies, we are challenged with identifying the molecular basis of such adaptive variation. Here, we have identified a new variant of a molybdenum (Mo) transporter, MOT1, which is causal for fitness changes under artificial conditions of both Mo-deficiency and Mo-toxicity and in which allelic variation among West-Asian populations is strictly correlated with the concentration of available Mo in native soils. In addition, this association is accompanied at different scales with patterns of polymorphisms that are not consistent with neutral evolution and show signs of diversifying selection. Resolving such a case of allelic heterogeneity helps explain species-wide phenotypic variation for Mo homeostasis and potentially reveals trade-off effects, a finding still rarely linked to fitness., Author Summary Plants are studied for their ability to adapt to their environment and especially to the physical constraints to which they are subjected. It is expected that they evolve in promoting genetic variants favorable under their native conditions, which could lead to negative consequences in other conditions. One approach to study the mechanisms and dynamics of these adaptations is to discover genetic variants that control potentially adaptive traits, and to study directly these variants in wild populations to try to reveal their evolutionary trajectory. We have identified a new polymorphism in a gene coding for a transporter of molybdenum (an essential micronutrient for the plant) in Arabidopsis; we show that this variant has strong phenotypic consequences at the level of plant growth and reproductive value in specific conditions, and that it explains a lot of the species diversity for these traits. Especially, the variant is associated with a clear negative effect under molybdenum-deficient conditions (caused by soil acidity) and with a subtle positive effect under molybdenum-plethoric conditions. Interestingly, the landscape distribution of the variant is not random among Asian populations and correlates well with the availability of molybdenum in the soil at the precise location where the plants are growing in the wild.

Sessile organisms such as plants have to develop adaptive responses to face environmental change. In Arabidopsis thaliana populations, natural variation for stress responses have been observed at different levels of integration and the genetic bases of those variations have been analysed using two strategies: classical linkage and association (LD) mapping. The strength of Arabidopsis resides in the huge amount of genomic data and molecular tools available leading to the identification of many polymorphisms responsible for phenotypic variation. Remaining limitations to clearly understand how Arabidopsis adapts to its environment, that is the complexity of the genetic architecture and the lack of ecological data, should be partially solved thanks to the development of new methods and the acquisition of new data.