Detection Of Plant Pathogens Research Articles

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

Comprehensive account of present techniques for in-field plant disease diagnosis.

The early detection of plant pathogens is an appropriate preventive strategy for the management of crop yield and quality. For this reason, effective diagnostic techniques and tools, which are simple, specific, rapid and economic, are needed to be developed. Although several such technologies have been developed still most of them suffer one or the other limitation. Major limitations of the widely used diagnostic methods are requirement of trained staff and laboratory setup. Development of point-of-care diagnostic devices (handy portable devices) that require no specialized staff and can directly be used in fields is need of the hour. The aim of this review is to compile the information on current promising techniques that are in use for plant-pathogen diagnosis. Additionally, it focuses on the latest in-field pathogen diagnostic techniques with associated advantages and limitations.

Latest Developments and Applications of Nanotechnology in Agriculture Sector: A Review

Nanotechnology, a term coined by Norio Taniguchi first time in 1974, pertains to controlling, building, designing, restructuring materials and devices on the scale of atoms and molecules. Till today nanotechnology is used in the entire spectrum of the agriculture sector, such as agriculture production, single-molecule detection for the determination of enzyme-substrate interactions, nanocapsules for efficient and timely delivery of agrochemicals and growth hormones in a controlled fashion, nanosensors for monitoring soil conditions, crop growth and quality and for detection of animals and plant pathogens, nanochips for the preservation of identity and tracking, nanocapsules for vaccine delivery and nanoparticles to deliver DNA (Deoxyribonucleic Acid) to plants, a process known as “targeted genetic engineering.” Especially in the agriculture sector, research and technical innovation are important, addressing the crucial global challenges like population growth, drastic climate change and the limited availability of prime plant elemental nutrients such as phosphorus and potassium. Despite these tremendous advantages, uses of nanotechnology are still comparably marginal in the agricultural sector and research has not been commercialized and reached to market to any larger extent in comparision to the other sectors of nanotechnology application. Major safety concerns and stringent laws related to human health and the environment were raised towords industrial-scale manufacturing and use of nanomaterials hinders its use in the agriculture sector as well.

Biosensor Technologies for Early Detection and Quantification of Plant Pathogens.

Plant pathogens are a major reason of reduced crop productivity and may lead to a shortage of food for both human and animal consumption. Although chemical control remains the main method to reduce foliar fungal disease incidence, frequent use can lead to loss of susceptibility in the fungal population. Furthermore, over-spraying can cause environmental contamination and poses a heavy financial burden on growers. To prevent or control disease epidemics, it is important for growers to be able to detect causal pathogen accurately, sensitively, and rapidly, so that the best practice disease management strategies can be chosen and enacted. To reach this goal, many culture-dependent, biochemical, and molecular methods have been developed for plant pathogen detection. However, these methods lack accuracy, specificity, reliability, and rapidity, and they are generally not suitable for in-situ analysis. Accordingly, there is strong interest in developing biosensing systems for early and accurate pathogen detection. There is also great scope to translate innovative nanoparticle-based biosensor approaches developed initially for human disease diagnostics for early detection of plant disease-causing pathogens. In this review, we compare conventional methods used in plant disease diagnostics with new sensing technologies in particular with deeper focus on electrochemical and optical biosensors that may be applied for plant pathogen detection and management. In addition, we discuss challenges facing biosensors and new capability the technology provides to informing disease management strategies.

Graphene Based Electrochemical DNA Biosensor for Detection of False Smut of Rice (Ustilaginoidea virens).

False smut caused by Ustilaginoidea virens is an important rice fungal disease that significantly decreases its production. In the recent past, conventional methods have been developed for its detection that is time-consuming and need high-cost equipments. The research and development in nanotechnology have made it possible to assemble efficient recognition interfaces in biosensors. In this study, we present a simple, sensitive, and selective oxidized graphene-based geno-biosensor for the detection of rice false smut. The biosensor has been developed using a probe DNA as a biological recognition element on paper electrodes, and oxidized graphene to enhance the limit of detection and sensitivity of the sensor. Probe single-stranded DNA (ssDNA) and target ssDNA hybridization on the interface surface has been quantitatively measured with the electrochemical analysis tools namely, cyclic voltammetry, and linear sweep voltammetry. To confirm the selectivity of the device, probe hybridization with non-complementary ssDNA target has been studied. In our study, the developed sensor was able to detect up to 10 fM of target ssDNA. The paper electrodes were employed to produce an effective and cost-effective platform for the immobilization of the DNA and can be extended to design low-cost biosensors for the detection of the other plant pathogens.

Ultrasensitive isothermal detection of a plant pathogen by using a gold nanoparticle-enhanced microcantilever sensor

• GNP-enhanced dynamic microcantilever biosensor is constructed for pathogen analysis. • Combined with RPA, a LOD as low as one copy genomic DNA could be achieved. • This RPA-MCL could detect 57 ppm pathogen genomic DNA out of oilseed plant. Sensitive and rapid detection of plant pathogens, particularly for on-site diagnosis during custom inspections, is essential to prevent and control the spread of these pathogens. To address the need, a combined assay for detecting plant pathogens was developed by using gold nanoparticle-enhanced dynamic microcantilever (MCL) biosensing and isothermal recombinase polymerase amplification (RPA). In this study, we used Leptosphaeria maculans (L . maculans) , an aggressive fungus responsible for major losses in oilseed rape yield worldwide, as the target pathogen to demonstrate the merits of the combined RPA and MCL strategy (named as the RPA-MCL assay). The results indicated that the sensitivity of the RPA-MCL assay is higher than that of the fluorescence RPA assay, with the detection limit of only one copy of L . maculans DNA. In the practical assay, the newly developed RPA-MCL method was found to detect 57 ppm L . maculans genomic DNA in the oilseed rape seeds genomic DNA sample. Considering the high sensitivity and specificity of this strategy, we envision that the proposed RPA-MCL assay could have wide applications in nucleic acid diagnostics for on-site plant pathogen detection and species identification.

Fantastic Downy Mildew Pathogens and How to Find Them: Advances in Detection and Diagnostics.

Downy mildews affect important crops and cause severe losses in production worldwide. Accurate identification and monitoring of these plant pathogens, especially at early stages of the disease, is fundamental in achieving effective disease control. The rapid development of molecular methods for diagnosis has provided more specific, fast, reliable, sensitive, and portable alternatives for plant pathogen detection and quantification than traditional approaches. In this review, we provide information on the use of molecular markers, serological techniques, and nucleic acid amplification technologies for downy mildew diagnosis, highlighting the benefits and disadvantages of the technologies and target selection. We emphasize the importance of incorporating information on pathogen variability in virulence and fungicide resistance for disease management and how the development and application of diagnostic assays based on standard and promising technologies, including high-throughput sequencing and genomics, are revolutionizing the development of species-specific assays suitable for in-field diagnosis. Our review provides an overview of molecular detection technologies and a practical guide for selecting the best approaches for diagnosis.

Research advances and applications of biosensing technology for the diagnosis of pathogens in sustainable agriculture.

Plant diseases significantly impact the global economy, and plant pathogenic microorganisms such as nematodes, viruses, bacteria, fungi, and viroids may be the etiology for most infectious diseases. In agriculture, the development of disease-free plants is an important strategy for the determination of the survival and productivity of plants in the field. This article reviews biosensor methods of disease detection that have been used effectively in other fields, and these methods could possibly transform the production methods of the agricultural industry. The precise identification of plant pathogens assists in the assessment of effective management steps for minimization of production loss. The new plant pathogen detection methods include evaluation of signs of disease, detection of cultured organisms, or direct examination of contaminated tissues through molecular and serological techniques. Laboratory-based approaches are costly and time-consuming and require specialized skills. The conclusions of this review also indicate that there is an urgent need for the establishment of a reliable, fast, accurate, responsive, and cost-effective testing method for the detection of field plants at early stages of growth. We also summarized new emerging biosensor technologies, including isothermal amplification, detection of nanomaterials, paper-based techniques, robotics, and lab-on-a-chip analytical devices. However, these constitute novelty in the research and development of approaches for the early diagnosis of pathogens in sustainable agriculture.

Metagenomics Approaches for the Detection and Surveillance of Emerging and Recurrent Plant Pathogens.

Globalization has a dramatic effect on the trade and movement of seeds, fruits and vegetables, with a corresponding increase in economic losses caused by the introduction of transboundary plant pathogens. Current diagnostic techniques provide a useful and precise tool to enact surveillance protocols regarding specific organisms, but this approach is strictly targeted, while metabarcoding and shotgun metagenomics could be used to simultaneously detect all known pathogens and potentially new ones. This review aims to present the current status of high-throughput sequencing (HTS) diagnostics of fungal and bacterial plant pathogens, discuss the challenges that need to be addressed, and provide direction for the development of methods for the detection of a restricted number of related taxa (specific surveillance) or all of the microorganisms present in a sample (general surveillance). HTS techniques, particularly metabarcoding, could be useful for the surveillance of soilborne, seedborne and airborne pathogens, as well as for identifying new pathogens and determining the origin of outbreaks. Metabarcoding and shotgun metagenomics still suffer from low precision, but this issue can be limited by carefully choosing primers and bioinformatic algorithms. Advances in bioinformatics will greatly accelerate the use of metagenomics to address critical aspects related to the detection and surveillance of plant pathogens in plant material and foodstuffs.

REAL TIME PCR METHOD FOR PPV DIAGNOSTIC ON PLUMS AND APRICOT IN THE REPUBLIC OF NORTH MACEDONIA

Real-time PCR (Polymerase Chain Reaction) or qPCR is a method by which the amount of the PCR product can be determined in real-time, and is very useful for investigating gene expression. The main advantages of qPCR are that it provides fast and high-throughput detection and quantification of target DNA sequences in different matrices. The lower time of amplification is facilitated by the simultaneous amplification and visualization of newly formed DNA amplicons. The development and application of molecular methods for the detection of pathogens has significantly changed the diagnosis and control of plant diseases, various environmental samples, including hosts tissues, soil, water and air. With real time PCR method, it is possible not only to identify and detect the presence or absence of the target pathogen, but it is also possible to quantify the amount present in the sample allowing the quantitative assessment of the number of the pathogen in the sample. Detection and accurate identification of plant pathogens is one of the most important strategies for controlling plant diseases to initiate preventive or curative measures. Plum pox virus (PPV), the agent of Sharka, is the most devastating virus infecting stone fruits. The PPV control is mainly based on prevention, and its quick and reliable detection is considered crucial in this strategy. In this study DAS-ELISA and real-time PCR were compared for evaluating their potentialities and limits for large scale surveys. Plum hosts (Prunus domestica) and apricot samples (Prunus armeniaca) from several different place are included for laboratory test analyzes, plant organs (phloem, buds, flowers, leaves and fruits) and parts of them, different periods of the year (spring, summer and winter period 2017/20), the presence or absence of symptoms were considered for comparison. Using DAS-ELISA tests, and use a universal set of antibodies (BIOREBA), has proved the presence of virus of Plum pox in all examined samples, especially from samples collected in summer, but also in checking virus status in winter and early spring season. Testing found high concentrations of viral antigens in plant samples (OD 2.912-2.752, for 30 min / 405 nm). Real time PCR show amplification plot for positive PPV samples on plums and apricot.

Rapid Loop-Mediated Isothermal Amplification for Detection of the Ralstonia solanacearum Species Complex Bacteria in Symptomatic Potato Tubers and Plants.

The Ralstonia solanacearum species complex (RSSC) is composed of several Ralstonia species and strains that are little related and show varied host range and distinct geographic distributions. The RSSC causes wilt disease, and can thus have severe economic consequences for many important crops and ornamental plants. One such is potato (Solanum tuberosum), where infection causes brown rot of the tubers. It is important that symptomatic tubers and plants can be rapidly and easily tested, as exclusion of infected material is a cornerstone of management of bacterial diseases. A suitable method is loop-mediated isothermal amplification, a rapid, DNA-based method that can be used for specific detection of plant pathogens in infected materials. The combination of this loop-mediated isothermal amplification assay for the RSSC with a simple sample preparation method is fit for purpose for identification of this devastating disease in symptomatic tubers and plants. This methodology is rapid and cost efficient, and can be carried out outside of conventional laboratory facilities.

Efficient, Rapid, and Sensitive Detection of Plant RNA Viruses With One-Pot RT-RPA-CRISPR/Cas12a Assay.

Most viruses that infect plants use RNA to carry their genomic information; timely and robust detection methods are crucial for efficient control of these diverse pathogens. The RNA viruses, potexvirus (Potexvirus, family Alphaflexiviridae), potyvirus (Potyvirus, family Potyviridae), and tobamovirus (Tobamovirus, family Virgaviridae) are among the most economically damaging pathogenic plant viruses, as they are highly infectious and distributed worldwide. Their infection of crop plants, alone or together with other viruses, causes severe yield losses. Isothermal nucleic acid amplification methods, such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and others have been harnessed for the detection of DNA- and RNA-based viruses. However, they have a high rate of non-specific amplification and other drawbacks. The collateral activities of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems such as Cas12 and Cas14 (which act on ssDNA) and Cas13 (which acts on ssRNA) have recently been exploited to develop highly sensitive, specific, and rapid detection platforms. Here, we report the development of a simple, rapid, and efficient RT- RPA method, coupled with a CRISPR/Cas12a-based one-step detection assay, to detect plant RNA viruses. This diagnostic method can be performed at a single temperature in less than 30 min and integrated with an inexpensive commercially available fluorescence visualizer to facilitate rapid, in-field diagnosis of plant RNA viruses. Our developed assay provides an efficient and robust detection platform to accelerate plant pathogen detection and fast-track containment strategies.

Comparison of Nanopore Sequencing Protocols and Real-Time Analysis for Phytopathogen Diagnostics

Next-generation sequencing technologies have aided molecular detection and diagnosis of plant pathogens. Short read sequencing technologies can be expensive, time consuming, and lack portability. The Oxford Nanopore Technologies (ONT) MinION sequencer offers plant diagnosticians an alternative for whole-genome and transcriptome sequencing with the advantages of portability, rapid results and access to sequences generated, and relatively low cost. Although this company offers multiple sequencing products, most previous articles focused on comparing this technology with other sequencing technology (Sanger, Illumina) and not comparing ONT product performance. In this project, we explored the potential of using ONT’s kits without modifications for the sequencing of plant pathogens. We compared the performance of DNA library preparation protocols (1D versus 1D2 versus Rapid) and RNA library preparation protocols (cDNA PCR Sequencing versus Direct RNA) on the MinION sequencer and examined different data analysis approaches for two well-characterized plant pathogens of agricultural concern, Ralstonia solanacearum and plum pox virus. For bacterial DNA sequencing, the Rapid Sequencing kit had the lowest throughput but offered the shortest sample-to-sequence turnaround time. The cDNA PCR Sequencing kit was better suited for plant RNA viruses than the Direct RNA. Whole-genome assemblies using all tested kits provided accurate identification of the target pathogen when pure cultures or infected plant material was used. This technology offers a shorter sample-to-sequence timeline than previous technologies for both DNA and RNA samples. Data analysis can still be time- and resource-consuming, but we found that 1 h of data generation was sufficient for accurate identification of the target pathogens, which can alleviate computing resources because a full 48-h run of sequencing data is not required. With the decreasing costs of flow cells, MinION is now an additional tool to be considered by plant diagnosticians for whole-genome and transcriptome-based diagnostics.

Electrochemical investigations for COVID-19 detection-A comparison with other viral detection methods.

Virus-induced infection such as SARS-CoV-2 is a serious threat to human health and the economic setback of the world. Continued advances in the development of technologies are required before the viruses undergo mutation. The low concentration of viruses in environmental samples makes the detection extremely challenging; simple, accurate and rapid detection methods are in urgent need. Of all the analytical techniques, electrochemical methods have the established capabilities to address the issues. Particularly, the integration of nanotechnology would allow miniature devices to be made available at the point-of-care. This review outlines the capabilities of electrochemical methods in conjunction with nanotechnology for the detection of SARS-CoV-2. Future directions and challenges of the electrochemical biosensors for pathogen detection are covered including wearable and conformal biosensors, detection of plant pathogens, multiplexed detection, and reusable biosensors for on-site monitoring, thereby providing low-cost and disposable biosensors.

Molecular Approaches for Low-Cost Point-of-Care Pathogen Detection in Agriculture and Forestry.

Early detection of plant diseases is a crucial factor to prevent or limit the spread of a rising infection that could cause significant economic loss. Detection test on plant diseases in the laboratory can be laborious, time consuming, expensive, and normally requires specific technical expertise. Moreover, in the developing countries, it is often difficult to find laboratories equipped for this kind of analysis. Therefore, in the past years, a high effort has been made for the development of fast, specific, sensitive, and cost-effective tests that can be successfully used in plant pathology directly in the field by low-specialized personnel using minimal equipment. Nucleic acid-based methods have proven to be a good choice for the development of detection tools in several fields, such as human/animal health, food safety, and water analysis, and their application in plant pathogen detection is becoming more and more common. In the present review, the more recent nucleic acid-based protocols for point-of-care (POC) plant pathogen detection and identification are described and analyzed. All these methods have a high potential for early detection of destructive diseases in agriculture and forestry, they should help make molecular detection for plant pathogens accessible to anyone, anywhere, and at any time. We do not suggest that on-site methods should replace lab testing completely, which remains crucial for more complex researches, such as identification and classification of new pathogens or the study of plant defense mechanisms. Instead, POC analysis can provide a useful, fast, and efficient preliminary on-site screening that is crucial in the struggle against plant pathogens.

Optimising risk-based surveillance for early detection of invasive plant pathogens.

Emerging infectious diseases (EIDs) of plants continue to devastate ecosystems and livelihoods worldwide. Effective management requires surveillance to detect epidemics at an early stage. However, despite the increasing use of risk-based surveillance programs in plant health, it remains unclear how best to target surveillance resources to achieve this. We combine a spatially explicit model of pathogen entry and spread with a statistical model of detection and use a stochastic optimisation routine to identify which arrangement of surveillance sites maximises the probability of detecting an invading epidemic. Our approach reveals that it is not always optimal to target the highest-risk sites and that the optimal strategy differs depending on not only patterns of pathogen entry and spread but also the choice of detection method. That is, we find that spatial correlation in risk can make it suboptimal to focus solely on the highest-risk sites, meaning that it is best to avoid 'putting all your eggs in one basket'. However, this depends on an interplay with other factors, such as the sensitivity of available detection methods. Using the economically important arboreal disease huanglongbing (HLB), we demonstrate how our approach leads to a significant performance gain and cost saving in comparison with conventional methods to targeted surveillance.

Development of Real-Time and Colorimetric Loop Mediated Isothermal Amplification Assay for Detection of Xanthomonas gardneri

Xanthomonas gardneri is one of the causal agents of bacterial spot (BS), an economically important bacterial disease of tomato and pepper. Field-deployable and portable loop-mediated isothermal amplification (LAMP)-based instruments provide rapid and sensitive detection of plant pathogens. In order to rapidly and accurately identify and differentiate X. gardneri from other BS-causing Xanthomonas spp., we optimized a new real-time monitoring LAMP-based method targeting the X. gardneri-specific hrpB gene. Specificity and sensitivity of real-time and colorimetric LAMP assays were tested on the complex of bacterial strains pathogenic to tomato and pepper and on plants infected by the pathogen. The assay detection limit was 1 pg/μL of genomic DNA with an assay duration of only 30 min. The use of portable and handheld instruments allows for fast analysis, reducing the diagnosis time, and can contribute to proper disease management and control of X. gardneri. Due to the high efficiency of this method, we suggest its use as a standard diagnostic tool during phytosanitary controls.

Examining the Utility of Visible Near-Infrared and Optical Remote Sensing for the Early Detection of Rapid ‘Ōhi‘a Death

The early detection of plant pathogens at the landscape scale holds great promise for better managing forest ecosystem threats. In Hawai‘i, two recently described fungal species are responsible for increasingly widespread mortality in ‘ōhi‘a Metrosideros polymorpha, a foundational tree species in Hawaiian native forests. In this study, we share work from repeat laboratory and field measurements to determine if visible near-infrared and optical remote sensing can detect pre-symptomatic trees infected with these pathogens. After generating a dense time series of laboratory spectral reflectance data and red green blue (RGB) images for inoculated ‘ōhi‘a seedlings, seedlings subjected to extreme drought, and control plants, we found few obvious spectral indicators that could be used for reliable pre-symptomatic detection in the inoculated seedlings, which quickly experienced complete and total wilting following stress onset. In the field, we found similar results when we collected repeat multispectral and RGB imagery over inoculated mature trees (sudden onset of symptoms with little advance warning). We found selected vegetation indices to be reliable indicators for detecting non-specific stress in ‘ōhi‘a trees, but never providing more than five days prior warning relative to visual detection in the laboratory trials. Finally, we generated a sequence of linear support vector machine classification models from the laboratory data at time steps ranging from pre-treatment to late-stage stress. Overall classification accuracies increased with stress stage maturity, but poor model performance prior to stress onset and the sudden onset of symptoms in infected trees suggest that early detection of rapid ‘ōhi‘a death over timescales helpful for land managers remains a challenge.

Nanotechnological interventions for plant health improvement and sustainable agriculture.

Agriculture is the source of food for both humans and animals. With the growing population demands, agricultural production needs to be scaled up where nanotechnology can play a significant role. The use of nanotechnology in agriculture can manage plant disease and growth for better and quality output. Therefore, this review focuses on the use of various nanoparticles for detection of nutrients and contaminants, nanosensors for monitoring the environmental stresses and crop conditions as well as the use of nanotechnology for plant pathogen detection and crop protection. In addition, the delivery of plant growth regulators and agrichemicals like nanopesticides and nanofertilizers to the plants along with the delivery of DNA for targeted genetic engineering and production of genetically modified (GM) crops are discussed briefly. Further, the future concerns regarding the use of nanoparticles and their possible toxicity, impact on the agriculture and ecosystem needs to be assessed along with the assessment of the nanoparticles and GM crops on the environment and human health.

Specific and sensitive PCR detection of Cadophora luteo-olivacea associated with grapevine trunk diseases

Grapevine trunk diseases, caused by different fungal pathogens, are the most relevant group of diseases that affect vineyards, causing important economic losses. Quality control for sanitary purposes should be applied for different diseases in every stages of the plant development, from the nursery during propagation to the vineyard during grape production. Petri disease has been identified as one of the most dangerous for vine plants causing economic losses in cellars worldwide. Petri disease is caused by the infection of different fungal pathogens, mainly, Phaeomoniella chlamydospora, Phaeocremonium minimum and Cadophora luteo-olivacea. The molecular detection of plant pathogens is topical due to its facility, reproducibility and accuracy. Here we have described a PCR protocol based in a pair of new designed specific primers for the rapid detection of C. luteo-olivacea. The described procedure is intended for the direct inspection of plants suspected of being infected. Bovine serum albumin and MgCl2 were used as PCR adjuvants in order to cope with the presence of polymerase inhibitors from wood samples. Several assays have been settled out in order to assign the sensitivity and specificity of the developed technique. Under the conditions tested, the procedure has a low detection limit (10 pg) and it is specific for C. luteo-olivacea detection among other Cadophora species and fungal endophytes of Vitis vinifera.