An efficient low‐cost xylem sap isolation method for bacterial wilt assays in tomato

Longchar, Bendangchuchang; Phukan, Tarinee; Yadav, Sarita; Senthil‐Kumar, Muthappa

doi:10.1002/aps3.11335

Cited by 8 publications

(7 citation statements)

References 26 publications

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“…One of the simplest approaches for collecting xylem exudates is to make a cross-section excision in the stem and harvest the sap oozing out through capillary movement by gravity and root pressure [ 40 , 61 ]. Additionally, a portable simple method has been developed for the rapid extraction of xylem sap from stems and petioles using negative pressure generated from handheld needleless syringes [ 62 ]. However, these methods do not work for most woody plants due to their high negative stem water potential [ 63 ].…”

Section: Methodological Approaches To Study the Xylem Microbiotamentioning

confidence: 99%

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Anguita-Maeso

Navas‐Cortés

Landa

2023

Plants

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Vascular pathogens are the causal agents of some of the most devastating plant diseases in the world, which can cause, under specific conditions, the destruction of entire crops. These plant pathogens activate a range of physiological and immune reactions in the host plant following infection, which may trigger the proliferation of a specific microbiome to combat them by, among others, inhibiting their growth and/or competing for space. Nowadays, it has been demonstrated that the plant microbiome can be modified by transplanting specific members of the microbiome, with exciting results for the control of plant diseases. However, its practical application in agriculture for the control of vascular plant pathogens is hampered by the limited knowledge of the plant endosphere, and, in particular, of the xylem niche. In this review, we present a comprehensive overview of how research on the plant microbiome has evolved during the last decades to unravel the factors and complex interactions that affect the associated microbial communities and their surrounding environment, focusing on the microbial communities inhabiting the xylem vessels of olive trees (Olea europaea subsp. europaea), the most ancient and important woody crop in the Mediterranean Basin. For that purpose, we have highlighted the role of xylem composition and its associated microorganisms in plants by describing the methodological approaches explored to study xylem microbiota, starting from the methods used to extract xylem microbial communities to their assessment by culture-dependent and next-generation sequencing approaches. Additionally, we have categorized some of the key biotic and abiotic factors, such as the host plant niche and genotype, the environment and the infection with vascular pathogens, that can be potential determinants to critically affect olive physiology and health status in a holobiont context (host and its associated organisms). Finally, we have outlined future directions and challenges for xylem microbiome studies based on the recent advances in molecular biology, focusing on metagenomics and culturomics, and bioinformatics network analysis. A better understanding of the xylem olive microbiome will contribute to facilitate the exploration and selection of specific keystone microorganisms that can live in close association with olives under a range of environmental/agronomic conditions. These microorganisms could be ideal targets for the design of microbial consortia that can be applied by endotherapy treatments to prevent or control diseases caused by vascular pathogens or modify the physiology and growth of olive trees.

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Section: Methodological Approaches To Study the Xylem Microbiotamentioning

confidence: 99%

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Anguita-Maeso

Navas‐Cortés

Landa

2023

Plants

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“…The sampling of stem xylem fluids from infected plants is a well-known, robust and fast method to identify pathogens and to estimate the bacterial load. The rapid extraction of xylem sap is crucial for diagnosing the xylem-colonizing bacteria as Rs and Cmm [24]. Starting from the availability of naturally infected tomato plants, the potentialities of the assays in practical applications were tested.…”

Section: Chip Digital Pcr For Cmm and Rs Diagnosticsmentioning

confidence: 99%

Molecular Diagnostics in Tomato: Chip Digital PCR Assays Targeted to Identify and Quantify Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum in planta

Morcia¹,

Piazza²,

Ghizzoni³

et al. 2023

Horticulturae

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Clavibacter michiganensis subsp. michiganensis (Cmm) and Ralstonia solanacearum (Rs) are important bacterial pathogens of tomatoes (Solanum lycopersicum), are included in A2 list in the EPPO (European and Mediterranean Plant Protection Organization) region and are recommended for regulation as quarantine pests. The control of quarantine pathogens requires accurate and rapid detection tools. In this study, a method based on chip digital PCR (cdPCR) was developed to identify and quantify Cmm and Rs. The assays were tested on pure bacteria samples and on tomato samples naturally contaminated or spiked with bacteria DNA. For a better estimation of infection level in host plants, duplex assays that are able to simultaneously amplify plant and bacteria DNA were developed. The two cdPCR assays proposed can be used for the rapid and timely detection of this group of high-risk quarantine bacteria to prevent the spread of pathogens and the occurrence of disease in other areas.

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“…The method of homogenization depends on the stiffness of the plant tissues. Soft leaves and crops or sap can be homogenized without much mechanical treatment, by shaking in appropriate buffer and can be performed at any site [25,26]. Soft parts of a plant can be macerated by plastic mesh in a zip-lock bag (e.g., Sample Extraction Bag 1, Agdia) in appropriate buffer [27], even in field conditions.…”

Section: Pre-treatment Of Plant Tissue For Nucleic Acid Extractionmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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An efficient low‐cost xylem sap isolation method for bacterial wilt assays in tomato

Cited by 8 publications

References 26 publications

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Insights into the Methodological, Biotic and Abiotic Factors Influencing the Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees

Molecular Diagnostics in Tomato: Chip Digital PCR Assays Targeted to Identify and Quantify Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum in planta

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

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