Next-generation methods for early disease detection in crops.

Plant pathogens are commonly identified in the field by the typical disease symptoms that they can cause. The efficient early detection and identification of pathogens are essential procedures to adopt effective management practices that reduce or prevent their spread in order to mitigate the negative impacts of the disease. In this review, the traditional and innovative methods for early detection of the plant pathogens highlighting their major advantages and limitations are presented and discussed.

Real-Time Portable LAMP Assay for a Rapid Detection of Xylella fastidiosa In-Field.

Early diagnosis is part of a decision-making process which in the case of plant diseases may prevent the spread of invasive plant pathogens and assist in their eradication. Significant advantages could be obtained from moving testing technology closer to the sampling site, thereby reducing the detection time. This chapter describes a portable real-time LAMP assay for a specific detection of Xylella fastidiosa in-field.

Plant Sampling for Production of Essential Oil and Evaluation of Its Antimicrobial Activity In Vitro.

Essential oils (EOs) and oleoresins are complex mixtures mainly made up of terpenes, synthesized by a wide variety of plants. Individual terpenes may show broad-spectrum activity against different plant pathogens, and their combination into EO and oleoresin mixtures enhances plant chemical defense. The interest in EOs has significantly increased due to the trend of using natural products as herbicides, insecticidal and antimicrobial agents.

A Rapid Method for Extracting High-Quality DNA from Soils.

Metagenomics offers the possibility to study the microbial community of soils at large scale, allowing the characterization also of unculturable microorganisms. Availability of high-quality DNA is a prerequisite of this approach. However, since soils have highly heterogeneous physicochemical properties, several parameters need to be considered for the development of the best molecular practices.

DNA Extraction Methods to Obtain High DNA Quality from Different Plant Tissues.

DNA extraction from plant samples is very important for a good performance of diagnostic molecular assays in phytopathology. The variety of matrices (such as leaves, roots, and twigs) requires a differentiated approach to DNA extraction. Here we describe three categories of matrices: (a) symptomatic bark/wood tissue; (b) residues of frass resulting from insect woody trophic activities, portions of the galleries produced in the wood, and tissues surrounding exit holes; and (c) leaves of different plant species.

Loop-Mediated Isothermal Amplification (LAMP) and SYBR Green qPCR for Fast and Reliable Detection of (Kolařik) in Infected Walnut.

Walnut species ( spp.) are multipurpose trees, widely employed in plantation forestry for high-quality timber and nut production, as well as in urban greening as ornamental plants. These species are currently threatened by the thousand cankers disease (TCD) complex, an insect-fungus association which involves the ascomycete (GM) and its vector, the bark beetle .

Rapid diagnostics for Gnomoniopsis smithogilvyi (syn. Gnomoniopsis castaneae) in chestnut nuts: new challenges by using LAMP and real-time PCR methods.

Nuts of the sweet chestnut (Castanea sativa) are a widely appreciated traditional food in Europe. In recent years producers and consumers reported a drop of nut quality due to the presence of rot diseases caused by Gnomoniopsis smithogilvyi. Early detection of this pathogen is fundamental to the economic viability of the chestnut industry.

Rapid Detection of (Blackman) (Coleoptera, Curculionidae) with the Loop-Mediated Isothermal Amplification (LAMP) Method.

The walnut twig beetle is a phloem-boring bark beetle responsible, in association with the ascomycete , for the Thousand Cankers Disease (TCD) of walnut trees. The recent finding of TCD in Europe prompted the development of effective diagnostic protocols for the early detection of members of this insect/fungus complex. Here we report the development of a highly efficient, low-cost, and rapid method for detecting the beetle, or even just its biological traces, from environmental samples: the loop-mediated isothermal amplification (LAMP) assay.

Development of a loop-mediated isothermal amplification (LAMP) assay for the identification of the invasive wood borer (Coleoptera: Cerambycidae) from frass.

The red-necked longhorn beetle (Faldermann, 1835) (Coleoptera: Cerambycidae) is native to east Asia, where it is a major pest of cultivated and ornamental species of the genus . Morphological or molecular discrimination of adults or larval specimens is required to identify this invasive wood borer. However, recovering larval stages of the pest from trunks and branches causes extensive damage to plants and is timewasting.

, a new species for a fungus long confused with the pine pathogen .

The genus (Eurotiomycetes, Coryneliales) includes saprobic and plant pathogenic species. Caliciopsis canker is caused by Peck, a species first reported in the 19 century in North America. In recent years, increasing numbers of outbreaks of Caliciopsis canker have been reported on different spp.

Molecular Identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) From Frass by Loop-Mediated Isothermal Amplification.

Anoplophora glabripennis (Motschulsky, 1853), native to eastern Asia, is a destructive woodborer of many ornamental species, leading to the decline and the death of the attacked trees. In outbreak areas as Europe or North America, this pest is usually identified using morphological or molecular analyses of adult or larval specimens. However, the procedures for collecting A.

Real-time loop-mediated isothermal amplification assay for rapid detection of .

is the causal agent of pitch canker, a lethal disease of pine and other conifers. Since is a quarantine organism, its timely detection could efficiently prevent its introduction into new areas or facilitate spread management in already infected sites. In this study, we developed a sequence-specific probe loop-mediated isothermal amplification (LAMP) assay for using a field-deployable portable instrument.

Fast and reliable molecular methods to detect fungal pathogens in woody plants.

Plant diseases caused by pathogenic microorganisms represent a serious threat to plant productivity, food security, and natural ecosystems. An effective framework for early warning and rapid response is a crucial element to mitigate or prevent the impacts of biological invasions of plant pathogens. For these reasons, detection tools play an important role in monitoring plant health, surveillance, and quantitative pathogen risk assessment, thus improving best practices to mitigate and prevent microbial threats.

Early Detection of Fungal Plant Pathogens by Real-Time Quantitative PCR: The Case of Diplodia sapinea on Pine.

This chapter reports the use of real-time quantitative PCR to detect Diplodia sapinea, a fungal plant pathogen that causes shoot tip dieback and tree mortality on pine trees. This molecular approach represents a reliable and sensitive tool to detect fungal pathogens in DNA extracted from plant tissues and its use can be also recommended to study fungal behavior in host tissues by quantifying fungal growth in the latent phase, when symptoms in the host are not present yet.

Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum.

Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F.

Real-time loop-mediated isothermal amplification: an early-warning tool for quarantine plant pathogen detection.

An effective framework for early warning and rapid response is a crucial element to prevent or mitigate the impact of biological invasions of plant pathogens, especially at ports of entry. Molecular detection of pathogens by using PCR-based methods usually requires a well-equipped laboratory. Rapid detection tools that can be applied as point-of-care diagnostics are highly desirable, especially to intercept quarantine plant pathogens such as Xylella fastidiosa, Ceratocystis platani and Phytophthora ramorum, three of the most devastating pathogens of trees and ornamental plants in Europe and North America.

Diplodia Tip Blight on Its Way to the North: Drivers of Disease Emergence in Northern Europe.

Disease emergence in northern and boreal forests has been mostly due to tree-pathogen encounters lacking a co-evolutionary past. However, outbreaks involving novel interactions of the host or the pathogen with the environment have been less well documented. Following an increase of records in Northern Europe, the first large outbreak of on was discovered in Sweden in 2016.

Comparative transcriptional and metabolic responses of Pinus pinea to a native and a non-native Heterobasidion species.

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naïve host tree and the ecosystem in which it resides, requiring a better understanding of the system.

Duplex real-time PCR assay for the simultaneous detection of Caliciopsis pinea and Fusarium circinatum in pine samples.

Fusarium circinatum and Caliciopsis pinea are the causal agents of Pitch canker and Caliciopsis canker, respectively. These diseases affect pines and other conifers both in Europe and North America. The two pathogens cause similar bleeding cankers, especially at the early stage of colonization.

Powerful qPCR assays for the early detection of latent invaders: interdisciplinary approaches in clinical cancer research and plant pathology.

Latent invaders represent the first step of disease before symptoms occur in the host. Based on recent findings, tumors are considered to be ecosystems in which cancer cells act as invasive species that interact with the native host cell species. Analogously, in plants latent fungal pathogens coevolve within symptomless host tissues.

Early physiological responses of Pinus pinea L. seedlings infected by Heterobasidion sp.pl. in an ozone-enriched atmospheric environment.

The presence of the American root-rot disease fungus Heterobasidion irregulare Garbel. & Otrosina was detected in Italian coastal pine forests (Pinus pinea L.) in addition to the common native species Heterobasidion annosum (Fries) Brefeld.

Rapid detection of Ceratocystis platani inoculum by quantitative real-time PCR assay.

Ceratocystis platani is the causal agent of canker stain of plane trees, a lethal disease able to kill mature trees in one or two successive growing seasons. The pathogen is a quarantine organism and has a negative impact on anthropogenic and natural populations of plane trees. Contaminated sawdust produced during pruning and sanitation fellings can contribute to disease spread.

Upward movement of Verticillium dahliae from soil to olive plants detected by qPCR.

Olive trees play an important role in cultural, ecological, environmental and social fields, constituting in large part the Mediterranean landscape. In Tuscany, an important economic activity is based on olive. Unfortunately, the Verticillium wilt affects this species and causes vascular disease.

High-resolution melting analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine.

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D.

New proteins orthologous to cerato-platanin in various Ceratocystis species and the purification and characterization of cerato-populin from Ceratocystis populicola.

Natural variants of cerato-platanin (CP), a pathogen associated molecular pattern (PAMP) protein produced by Ceratocystis platani (the causal agent of the plane canker stain), have been found to be produced by other four species of the genus Ceratocystis, including five clones of Ceratocystis fimbriata isolated from different hosts. All these fungal strains were known to be pathogenic to plants with considerable importance in agriculture, forestry, and as ornamental plants. The putative premature proteins were deduced on the basis of the nucleotide sequence of genes orthologous to the cp gene of C.