Seed Certification Programs Research Articles

A New Multiplex TaqMan qPCR for Precise Detection and Quantification of Clavibacter michiganensis in Seeds and Plant Tissue.

Bacterial canker of tomato caused by Clavibacter michiganensis (Cm) is one of the most devastating bacterial diseases affecting the tomato industry worldwide. As the result of Cm colonization of the xylem, the susceptible host shows typical symptoms of wilt, marginal leaf necrosis, stem cankers, and ultimately plant death. However, what makes Cm an even more dangerous pathogen is its ability to infect seeds and plants without causing symptoms. Unfortunately, there are no resistant cultivars or effective chemical or biological control methods available to growers against Cm. Its control relies heavily on prevention. The implementation of a rapid and accurate detection tool is imperative to monitor the presence of Cm and prevent its spread. In this study, we developed a specific and sensitive multiplex TaqMan qPCR assay to detect Cm and distinguish it from related bacterial species that affect tomato plants. Two Cm chromosomal virulence-related genes, rhuM and tomA, were used as specific targets. The plant internal control tubulin alpha-3 was included in each of the multiplexes to improve the reliability of the assay. Specificity was evaluated with 37 bacterial strains including other Clavibacter spp. and related and unrelated bacterial pathogens from different geographic locations affecting a wide variety of hosts. Results showed that the assay is able to discriminate Cm strains from other related bacteria. The assay was validated on tissue and seed samples following artificial infection, and all tested samples accurately detected the presence of Cm. The tool described here is highly specific, sensitive, and reliable for the detection of Cm and allows the quantification of Cm in seeds, roots, stems, and leaves. The diagnostic assay can also be adapted for multiple purposes such as seed certification programs, surveillance, biosafety, the effectiveness of control methods, border protection, and epidemiological studies.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Concept Note: Toward Metagenomic Sequencing for Rapid, Sensitive, and Accurate Detection of Bacterial Pathogens in Potato Seed Production

Disease-free planting stocks and formal certification systems are critical for the profitable production of vegetatively propagated crops. Propagation through seed tubers is the standard and preferred process for potato production. Seed tuber certification systems are used in the United States and throughout the world to mitigate degeneration and promote the use of high-quality planting stocks. Seed certification uses molecular and serological diagnostic methods for stock testing. While the molecular assays available are well-validated and offer high sensitivity, the rapidly evolving pathogens they target routinely develop mutations that allow them to evade detection. Examples of these limitations are the potato diseases soft rot and blackleg, caused by Pectobacterium and Dickeya species. These diseases are challenging for current diagnostics tools because of high pathogen genomic variability, identical symptom development, and co-infections. Given the rapidly changing genetics of soft rot and blackleg bacteria, metagenomic sequencing should be adopted as a tool for routine validation of currently accepted diagnostic methods, and for the development of new diagnostic assays. We propose metagenome sequencing usage to enhance the diagnostic capabilities and complement molecular epidemiology approaches. We envision that metagenomics will accelerate diagnosis, shorten response time to outbreaks caused by unknown microorganisms, and provide more accurate and sustainable management strategies for seed certification programs. [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 .

Loop-mediated isothermal amplification (LAMP) assay for specific and rapid detection of Dickeya fangzhongdai targeting a unique genomic region

Dickeya fangzhongdai, a bacterial pathogen of taro (Colocasia esculenta), onion (Allium sp.), and several species in the orchid family (Orchidaceae) causes soft rot and bleeding canker diseases. No field-deployable diagnostic tool is available for specific detection of this pathogen in different plant tissues. Therefore, we developed a field-deployable loop-mediated isothermal amplification (LAMP) assay using a unique genomic region, present exclusively in D. fangzhongdai. Multiple genomes of D. fangzhongdai, and other species of Dickeya, Pectobacterium and unrelated genera were used for comparative genomic analyses to identify an exclusive and conserved target sequence from the major facilitator superfamily (MFS) transporter gene region. This gene region had broad detection capability for D. fangzhongdai and thus was used to design primers for endpoint PCR and LAMP assays. In-silico validation showed high specificity with D. fangzhongdai genome sequences available in the NCBI GenBank genome database as well as the in-house sequenced genome. The specificity of the LAMP assay was determined with 96 strains that included all Dickeya species and Pectobacterium species as well as other closely related genera and 5 hosts; no false positives or false negatives were detected. The detection limit of the assay was determined by performing four sensitivity assays with tenfold serially diluted purified genomic DNA of D. fangzhongdai with and without the presence of crude host extract (taro, orchid, and onion). The detection limit for all sensitivity assays was 100 fg (18–20 genome copies) with no negative interference by host crude extracts. The assays were performed by five independent operators (blind test) and on three instruments (Rotor-Gene, thermocycler and dry bath); the assay results were concordant. The assay consistently detected the target pathogen from artificially inoculated and naturally infected host samples. The developed assay is highly specific for D. fangzhongdai and has applications in routine diagnostics, phytosanitary and seed certification programs, and epidemiological studies.

Evaluation of artificial neural network performance for classification of potato plants infected with potato virus Y using spectral data on multiple varieties and genotypes

Potato virus Y (Potyviridae, PVY) is a plant virus that poses a significant threat to potato producers on a global basis. The pathogen has disrupted seed potato supplies and negatively impacted yield and quality of commercial potato crops. The potato industry currently manages PVY infection levels via insecticide applications, regional seed certification programs that rely on field scouting to visually assess individual plants for infection status, and destructive and costly tissue sampling coupled with laboratory assays. Despite these efforts, PVY continues to confound potato industry stakeholders resulting in economic harm. Remote sensing and machine learning provide for the development of new tools to more accurately detect and spatially quantify PVY-infected plants versus the current state of the art. However, there is a need to understand how the occurrence of many different potato varieties impact the dynamics of developing models to detect potato plants impacted with PVY and their potential effectiveness. This study evaluates classification modelling outcomes using spectral datasets collected in different temporal and spatial environments (greenhouse and a production field) on multiple potato varieties consisting of labelled instances of plants infected with PVY and those not infected with the virus. A modelling framework was developed to support iterative modelling runs using artificial neural network (ANN) architectures configured as binary classifiers to develop sample populations to support statistical analysis on model performance using specific spectral subsets. When using spectral data to detect PVY-infected plants, ANN models achieved the highest mean accuracy of 0.894 on a single variety. Conversely, the same ANN model architecture only achieved a mean accuracy of 0.575 on a spectral data set representing 29 potato breeding lines. Additionally, statistical analysis indicates spectral regions including the red edge, near infrared and shortwave infrared contain more important spectral features for the ANN classifier introduced in this research.

Development of a multiplex TaqMan qPCR targeting unique genomic regions for the specific and sensitive detection of Pectobacterium species and P. parmentieri.

The newly defined species Pectobacterium parmentieri has emerged as an aggressive pathogen that causes soft rot and blackleg diseases on potato and has been widely disseminated across the globe, jeopardizing the productivity and potato food safety. The implementation of a fast and accurate detection tool is imperative to control, monitor and prevent further spread of these pathogens. The objective of this work was to develop a specific and sensitive multiplex TaqMan qPCR to detect P. parmentieri and distinguish it from all known Pectobacterium species. A universal internal control was included to enhance the reliability of the assay. A comparative genomics approach was used to identify O-acetyltransferase and the XRE family transcriptional regulator as specific targets for primers/probe design for the detection of the Pectobacterium genus and P. parmentieri, respectively. Specificity was assessed with 35 and 25 strains included in the inclusivity and exclusivity panels, respectively, isolated from different geographical locations and sources. The assay specifically detected all 35 strains of Pectobacterium sp. and all 15 P. parmentieri strains. No cross-reactivity was detected during assay validation. Our assay detected up to 10fg genomic DNA and 1CFUml-1 bacterial culture. No change in the detection threshold (1CFUml-1 ) was observed in spiked assays after adding host tissue to the reactions. The assay was validated with naturally and artificially infected host tissues and soil rhizosphere samples. All infected plant samples containing the target pathogens were accurately amplified. The presented multiplex TaqMan qPCR diagnostic assay is highly specific, sensitive, reliable for the detection of Pectobacterium species and P. parmentieri with no false positives or false negatives. The developed assay can be adopted for multiple purposes such as seed certification programmes, surveillance, biosecurity, microbial forensics, quarantine, border protection, inspections and epidemiology.

Genome-informed loop-mediated isothermal amplification assay for specific detection of Pectobacterium parmentieri in infected potato tissues and soil

Pectobacterium parmentieri (formerly Pectobacterium wasabiae), which causes soft rot disease in potatoes, is a newly established species of pectinolytic bacteria within the family Pectobacteriaceae. Despite serious damage caused to the potato industry worldwide, no field-deployable diagnostic tests are available to detect the pathogen in plant samples. In this study, we aimed to develop a reliable, rapid, field-deployable loop-mediated isothermal amplification (LAMP) assay for the specific detection of P. parmentieri. Specific LAMP primers targeting the petF1 gene region, found in P. parmentieri but no other Pectobacterium spp., were designed and validated in silico and in vitro using extensive inclusivity (15 strains of P. parmentieri) and exclusivity (94 strains including all other species in the genus Pectobacterium and host DNA) panels. No false positives or negatives were detected when the assay was tested directly with bacterial colonies, and with infected plant and soil samples. Sensitivity (analytical) assays using serially diluted bacterial cell lysate and purified genomic DNA established the detection limit at 10 CFU/mL and 100 fg (18–20 genome copies), respectively, even in the presence of host crude DNA. Consistent results obtained by multiple users/operators and field tests suggest the assay’s applicability to routine diagnostics, seed certification programs, biosecurity, and epidemiological studies.

Analysis of methods of diagnosis of bacterial diseases of tomatoes in Ukraine

Effective and economical methods of detection and identification necessary for the study of ecology, pathogenesis and limiting the spread of phytopathogenic bacteria, as well as for seed certification programs. The available methods for the detection and identification of four main causative agents of bacterial diseases of tomatoes Xanthomonas vesicatonia, Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato, Pectobacterium carotovorum subsp. carotovorum, which are common in Ukraine. These methods include microbiological using selective media, serological, molecular methods based on the amplification of unique DNA sequences. The use of modern biochemical test systems has eliminated the need to identify phytopathogenic bacteria using long-term routine methods. Commercially available commercial test systems provide rapid identification at the species level and also detect new and quarantine pathogens. PCR-based methods have advantages over traditional diagnostic tests because isolates do not need to be cultured before detection and protocols are highly sensitive and fast.

Potato virus Y and Potato leafroll virus management under climate change in sub-Saharan Africa

Potato has increased in importance as a staple food in sub-Saharan Africa, where its production is faced with a multitude of challenges, including plant disease development and spread under changing climatic conditions. The economically most important plant viruses affecting potatoes globally are Potato virus Y (PVY) and Potato leafroll virus (PLRV). Disease management relies mostly on the use of insecticides, cultural control and seed certification schemes. A major obstacle in many sub-Saharan Africa countries is the availability of disease-free quality seed potatoes. Establishment and implementation of quality control through specialised seed production systems and certification schemes is critical to improve seed potato quality and reduce PVY and PLRV sources. Seed could be further improved by breeding virus-resistant varieties adapted to different environmental conditions combined with management measures tailored for smallholder or commercial farmers to specific agricultural requirements. Innovative technologies – including more sensitive testing, remote sensing, machine learning and predictive models – provide new tools for the management of PVY and PLRV, but require support for adoption and implementation in sub-Saharan Africa.
 Significance:
 
 Potato virus Y (PVY) and Potato leafroll virus (PLRV) are the two major potato viruses threatening profitable seed potato production.
 High-quality seed shortage in many sub-Saharan Africa countries has been identified as a constraint to increasing yield.
 Specialised seed grower or seed certification programmes should be implemented to prevent virus transmission from seed to daughter tubers.
 Sustainable PVY and PLRV management in seed potatoes requires specific regional approaches to growth, farming and climatic conditions.
 Future research should include predictive models and new innovative technologies such as more sensitive testing, machine learning and remote sensing.

Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10−3–10−4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.

Specificity and sensibility of primer pair in the detection of Colletotrichum gossypii var. cephalosporioides in cotton seeds by PCR technique

Abstract: Cotton Ramulosis (Gossypium hirsutum) is an important disease affecting cotton plantations in Brazil, and its causal agent, Colletotrichum gossypiivar.cephalosporioides(Cgc), according to the Brazilian phytosanitary authority, was considered a regulated non quarantine pest. It makes this microorganism subject to standardization in seed certification programs. The current seed health testing for detecting that pathogen in seed samples does not provide reliable results for routine analysis. On this paper, attempts were made to design specific primers for detection of Cgc associated with cotton seed. Two primer sets were selected based on the analysis of a multiple alignment of gene’s sequence encoding the glyceraldehyde 3-phosphate dehydrogenase from Cgc, C. gossypii and reference strains of the C. gloeosporioides species complex. The conserved sites unique to Cgc strains were used to design specific fragment of 140 bp. The primer specificity was confirmed by using other fungi. The primers produced a detectable band of target DNA of Cgc in all inoculum potentials of the pathogen artificially inoculated by the water restriction technique. The developed primer pair represents, therefore, a reliable and rapid mean to diagnose the Ramulosis agent in cotton seed.

Diseño de cebadores específicos para la detección por RT-PCR del <i>Potato Virus</i> Y (PVY)

El Potato virus Y (PVY) es uno de los virus más limitantes para la producción de papa (Solanum tuberosum y S. phureja) en el mundo. Este virus es transmitido por tubérculo-semilla de papa y por diferentes especies de áfidos. Para su manejo es fundamental la siembra de tubérculos certificados por su sanidad viral, para lo que se requieren metodologías de detección altamente sensibles como ELISA y RT-PCR. Para éstas últimas pruebas, es necesario disponer de cebadores específicos que permitan el diagnóstico del virus en tejidos asintomáticos. En este estudio se reportan los cebadores PVY_Col para la detección del PVY en RT-PCR convencional y en tiempo real (RT-qPCR). Estos cebadores fueron diseñados con base en las secuencias de este virus que se han reportado en Colombia sobre diferentes hospedantes, así como de las diferentes variantes encontradas en el mundo. Una particularidad adicional de estos cebadores es que no presentan reacción cruzada con el genoma del Potato virus V (PVV), otro potyvirus que recientemente se ha encontrado afectando cultivos de papa en Colombia. Se espera que los cebadores PVY_Col sean utilizados para apoyar los programas de certificación de material de siembra de papa, así como para adelantar estudios epidemiológicos y de manejo fitosanitario de este virus.

Seed certification as a means of curbing emerging diseases: A case study of Maize Lethal Necrosis (MLN) in Kenya

Maize lethal necrosis (MLN) is an important constraint in maize production in sub-Sahara Africa that threatens food security and poses challenge in trade. It was first reported in Kenya in 2011 and has since spread to other countries in the region. In Kenya, the disease is caused by a combination of Sugarcane mosaic virus (SCMV) and Maize chlorotic mottle virus (MCMV), which are vectored by aphids and thrips, respectively. Maize is the main staple food in Kenya and therefore, with the advent of the MLN, there was need to establish mechanisms aimed at combating the spread of the disease. This resulted in the amendment of seed certification protocol which included testing of seed. In consultation with seed stakeholders, KEPHIS incorporated guidelines for MLN inspection in maize seed certification program where all maize seed crops should be inspected four times including preliminary, first, second and third inspections. A seed sample was taken before seed dressing for laboratory test to ensure the lot is free from MLN. Imported seeds were also tested for MLN before being accepted into the country. Furthermore, there have been concerted efforts by breeders and researchers to develop and screen maize lines for resistance to MLN. Other strategies that have been put in place include control of vectors and use of certified seed which are free from MLN. Amendments in seed certification program that were implemented in Kenya have led to drastic reduction of MLN. It is further hoped that resistant maize lines from the breeding program will further support the effort to manage the disease. Keywords: Maize, MLN, Resistance, Screening, Seed certification, seed testing

Influence of Bacterial Blight on different phenological stages of cowpea

The cowpea bacterial blight is a common disease, but some of its epidemiological parameters have not been totally elucidated. The objective of this study was to determine the influence of the inoculation of the bacterial blight pathogen (Xanthomonas citri pv. vignicola - Xcv) at different phenological stages of the cowpea, as well the effect on its yield components, and the transmission of the bacterium to the seed. Under greenhouse conditions, plant defoliation was observed on the cowpea cultivar BRS Bragança inoculated with 107 CFU of Xcv/mL at the four phenological stages, - VC (plants with primary leaves at nodes 1 & 2), V3 (3rd trifoliolate leaf unfolded at node 5), R2 (50–100% of open flowers), and R3 (one pod at maximum lenght). There was a difference in the relative area under the disease progress curve (RAUDPC) between the phenological stages and the reduction of some production components, such as dry seed mass. The pathogen was isolated from seeds produced in infected plants in all phenological stage, with the highest values ​​of CFU per gram of seeds in the V3 and R3. Under field conditions, two experiments were conducted using the cv. BRS Vita-7 (resistant) and cv. BRS Bragança (susceptible) inoculated with Xcv at the V3 and R2. The disease was more severe in cv. BRS Bragança, with the RAUDPC being higher in plants inoculated at V3 with a reduction of dry seed mass in inoculated R2 and inoculated R3 when compared to their uninoculated counterparts across two growing seasons. Regardless of the phenological stage in which the plant was inoculated, it was possible to detect Xcv in cv. BRS Bragança and cv. BRS Vita-7 seeds. The highest values of CFU per gram were recorded in samples from cv. BRS Bragança inoculated at V3 stage. Results on the presence cowpea's seeds infected by Xcv show that bacterial blight disease is highly seed transmitted and demonstrate the current need to avoid additional spread of the pathogen to new areas where there are favorable climatic conditions in Brazil or to disease-free countries that can result in considerable losses. Thus, the disease management should be used based on seed certification programmes, realizing sanitary tests of cowpea seed to avoid the spread of Xcv and the maintenance of disease-free seed stocks.

Estado actual de ciencia de las arvenses en Nicaragua

En Nicaragua el estudio de las arvenses, componentes importantes de los agro ecosistema, ha sido relegado a un segundo plano. En el periodo comprendido entre agosto y septiembre del año 2010 se llevó a cabo un estudio con el objetivo de actualizar la información sobre las arvenses en Nicaragua. La investigación fue de corte transversal, la información se obtuvo a través de análisis documental y entrevistas personalizadas con actores clave de las instituciones estatales y líderes de instituciones regionales, asociaciones de productores y académicos. La mayoría de los sistemas de cultivos se ven afectados por la presencia de arvenses y existe predominio de prácticas de manejo convencional que propician mayor infestación de arvenses. Trece especies de malezas son consideradas altamente problemáticas en la agricultura nicaragüense, y trece muy problemáticas. La práctica de manejo de arvenses utilizada por pequeños agricultores es manual, a través de la utilización de implementos livianos y arranque a mano. Agricultores medianos y grandes utilizan herbicidas como paraquat, glifosato y 2,4 D, algunos de ellos de uso restringido en muchos países. En los últimos diez años ha habido un incremento en el uso de herbicidas, y hay sistemas de cultivo como arroz, sorgo, caña de azúcar, café y cultivos oleaginosos (soja, maní y ajonjolí) que dependen de los herbicidas para reducir las arvenses. Son pocos los profesionales que trabajan en ciencia de las malezas, así como los programas y proyectos de investigación en curso que consideran la temática. No existe información sobre arvenses acuáticas, la práctica de control biológico es inexistente, y pocos programas de certificación de semillas cumplen con las normas establecidas que incluyen un especial manejo de arvenses. A nivel nacional existen documentos de reglamentación, así como convenios o tratados internacionales que influyen en las opciones de manejo de las arvenses.

A MULTIPLEX RT-PCR ASSAY FOR SIMULTANEOUS DETECTION OF FIVE VIRUSES IN POTATO

Potato viruses including Potato aucuba mosaic virus (PAMV), Potato leafroll virus (PLRV), Potato virus M (PVM), Potato virus S (PVS) and Potato virus X (PVX) are well known viruses infecting potato and can cause serious economic losses. In this study, a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay was developed and standardized for simultaneous detection of PAMV, PLRV, PVM, PVS and PVX with elongation factor 1-α (ef1α) as plant internal control. At least three primer pairs were designed within the conserved regions of genomic sequences of five potato viruses and ef1α of potato for single and/or multiplex RT-PCR. The reaction conditions were initially optimized by selecting primer pairs and standardized the individual RT-PCR. Then, one pair of primer for each virus including ef1α was selected for multiplex RT-PCR producing distinct fragments of 305, 452, 408, 363, 565, 803 bp, representing PAMV, PLRV, PVM, PVS, PVX and ef1α, respectively. All the primers were designed based on specificity and compatibility and the reliability was confirmed by cloning and sequencing followed by BLAST analysis. The sensitivity of single as well as multiplex RT-PCR was evaluated. The assay could detect all five viruses including ef1α even at total RNA dilutions of 10−3 and total cDNA dilution of 10- 7 . The multiplex RT-PCR assay was then used to test virus infections from field samples of potato (leaves and tubers) collected from different states of India along with plant internal control. This multiplex RT-PCR was equally enough in detecting mixed infection of five viruses in foliage as well as tubers. The results indicate the robustness and reliability of the assay for virus indexing of mother stocks for five viruses simultaneously in potato seed production and will be useful for seed certification programme, breeding and epidemiological studies.

Detección serológica y molecular del Potato virus X (PVX) en tubérculos-semilla de papa (Solanum tuberosum L. y Solanum phureja Juz. Bukasov) en Antioquia, Colombia

<p>El Potato virus (PVX) es uno de los virus más limitantes del cultivo de la papa en el mundo. Es transmitido solamente por contacto y por tubérculo-semilla. Su control se fundamenta en la siembra de tubérculos certificados por su sanidad viral y en la disponibilidad de metodologías de diagnóstico altamente sensibles. En este trabajo se evaluó la prevalencia del PVX en cuatro diferentes tejidos de tubérculos de Solanum tuberosum subsp. andigena var. Diacol-Capiro y S. phureja var. Criolla Colombia utilizando pruebas de DAS-ELISA para 128 submuestras y de RT-qPCR para 32 grupos de submuestras (4 submuestras/grupo). Los resultados de las pruebas serológicas indicaron la presencia de PVX en el 6,25 y 50% de las submuestras analizadas para la variedad Diacol-Capiro y Criolla Colombia, respectivamente; mientras que los niveles de prevalencia del PVX utilizando la detección por RT-qPCR fueron del 93,75%, independientemente de la variedad de papa y del tejido evaluado. Los valores promedio del ciclo umbral (Ct) en las RT-qPCR fueron de 25,6 (Ct=18,02 a 34,49) y el análisis de las curvas de desnaturalización permitió identificar dos variantes del virus con valores de Tm de 79,5±1°C y 83,7±1°C. La secuenciación de los amplicones obtenidos por RT-qPCR para los controles positivos y para dos de las muestras, confirmó su naturaleza viral. Estos resultados señalan unos muy altos niveles de prevalencia de PVX en el material de siembra de papa en Antioquia y la necesidad de fortalecer los programas de certificación de semilla con pruebas de detección como RT-qPCR.</p>

Diagnóstico serológico y molecular del Potato leafroll virus (PLRV) en tubérculos-semilla de papa en Antioquia, Colombia

<p>Las virosis son uno de los problemas fitosanitarios más limitantes para la producción de papa, siendo los virus Potato leafroll virus (PLRV), Potato virus Y (PVY), Potato virus S (PVS) y Potato ye-llow vein virus (PYVV) los que generan mayores pérdidas económicas en la región andina. Gene-ralmente estos virus son transmitidos por insectos hemípteros y por tubérculos-semilla. En esta investigación se evaluó la presencia y distribución del PLRV en tubérculos de <em>Solanum tuberosum</em> variedad Diacol-Capiro y S. phureja variedad Criolla-Colombia obtenidos en Antioquia, utilizando pruebas serológicas y moleculares. Mediante DAS-ELISA se evaluaron 128 muestras consisten-tes en tres tipos de tejidos (piel, yemas latentes y brotes), mientras que con RT-PCR en tiempo real (RT-qPCR), se utilizó un subgrupo de 12 muestras de cada variedad. Utilizando DAS-ELISA, el PLRV se encontró en el 3,1% y 10,1% de las muestras de Diacol-Capiro y Criolla-Colombia, respectivamente; siendo evidente una mayor detección del virus en la piel de los tubérculos de ésta última variedad. Las pruebas de RT-qPCR detectaron el PLRV en 83,3% y 91,6% de las 12 muestras de Diacol-Capiro y de Criolla-Colombia, respectivamente; con valores de Ciclo umbral (CT) entre 17,72 y 30,59. La naturaleza de los amplicones obtenidos por RT-qPCR fue confirmada por secuenciación como parte de una región codificante de la cápside del PLRV (Identidad=99%-100%). Estos resultados enfatizan en la necesidad de utilizar tubérculo-semilla certificados por su sanidad viral en los cultivos de papa del país y de fortalecer dichos programas a partir del empleo de técnicas de detección altamente sensibles como RT-qPCR.</p>

Forecasting virus disease in seed potatoes using flight activity data of aphid vectors

AbstractWe compiled data from the Swiss seed certification programme for the country‐wide incidence of viruses in seed potato crops for the years 1989–2012. Model selection techniques were used to regress year‐to‐year variation in the incidence of potato viruses – largely dominated by Potato virus Y (PVY) – in three susceptible varieties against the abundance of virus vectors (winged aphids), obtained in a suction trap, to identify the most important vector species. The ultimate aim of this study was to develop a decision‐support system capable of forecasting virus spread during the current season using trap data of aphid flights. The average virus incidence in the varieties Bintje, Sirtema and Charlotte varied considerably among years, ranging from 1.0% in 2009 to 13.6% in 1989 (N = 150–611 seed lots per year). A linear regression model including the cumulative sums (until mid‐June) of two aphid species (Brachycaudus helichrysi and Phorodon humuli) as predictor variables for virus disease was remarkably well supported by the data (R2 = 0.86). Similarly, using counts of B. helichrysi alone resulted in a good model fit (R2 = 0.81). Cross‐validation revealed high predictive accuracy of the model. Although prediction root mean squared errors (RMSE) calculated for different timings of forecasts were high for extremely early forecasts, they rapidly declined for forecasts conducted by the end of May (i.e. 2–4 weeks after potato emergence). Winter temperature (January–February) was positively correlated with the abundance of B. helichrysi in early summer as well as with post‐harvest virus incidence. Remarkably, the abundance of Myzus persicae, often considered the main vector of PVY, was not correlated with virus incidence. Taken together, our analysis suggests that the early migrating aphid B. helichrysi, rather than M. persicae, is the main vector of PVY in Switzerland, and that suction trap data are useful for the design of decision‐support systems aimed to optimise virus control in seed potato production.

Detection, identification and differentiation of Pectobacterium and Dickeya species causing potato blackleg and tuber soft rot: a review.

The soft rot Enterobacteriaceae (SRE) Pectobacterium and Dickeya species (formerly classified as pectinolytic Erwinia spp.) cause important diseases on potato and other arable and horticultural crops. They may affect the growing potato plant causing blackleg and are responsible for tuber soft rot in storage thereby reducing yield and quality. Efficient and cost-effective detection and identification methods are essential to investigate the ecology and pathogenesis of the SRE as well as in seed certification programmes. The aim of this review was to collect all existing information on methods available for SRE detection. The review reports on the sampling and preparation of plant material for testing and on over thirty methods to detect, identify and differentiate the soft rot and blackleg causing bacteria to species and subspecies level. These include methods based on biochemical characters, serology, molecular techniques which rely on DNA sequence amplification as well as several less-investigated ones.

Neck blast disease influences grain yield and quality traits of aromatic rice

A critical investigation was conducted to find out the effect of neck blast disease on yield-contributing characters, and seed quality traits of aromatic rice in Bangladesh. Both healthy and neck-blast-infected panicles of three aromatic rice cultivars (high-yielding and local) were collected and investigated at Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. All of the tested varieties were highly susceptible to neck blast disease under natural conditions, though no leaf blast symptoms appear on leaves. Neck blast disease increased grain sterility percentages, reduced grain size, yield and quality traits of seeds. The degrees of yield and seed quality reduction depended on disease severity and variety's genetic make-up. Unfilled grains were the main source of seed-borne pathogen, especially for blast in the seed lot. Transmission of blast pathogen from neck (panicle base) to seed was very poor. These findings are important, especially concerning the seed certification programme in which seed lots are certified on the basis of field inspection. Finally, controlled experiments are needed to draw more critical conclusions.