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This is a response to a recent Letter to the Editor of Phytopathology, in which Gupta et al. (2019) caution against the indiscriminate use of the MoT3 diagnostic assay that distinguishes isolates of Magnaporthe oryzae in the Triticum lineage from those that do not cause aggressive wheat blast. We confirm that the assay does reliably distinguish between wheat and rice isolates from Bangladesh and worldwide, as described in the original paper by Pieck et al. (2017) . We have been unable to reproduce the equally intense amplification of WB12 and WB12-like sequences reported in Figure 1 of the Letter. Other data presented by Gupta et al. (2019) support the specificity of the MoT3 assay. Therefore, cautions beyond those always associated with accurate reproduction of diagnostic assays are unwarranted.
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Wheat blast, caused by the Magnaporthe oryzae Triticum pathotype, is an economically important fungal disease of wheat. Wheat blast symptoms are similar to Fusarium head scab and can cause confusion in the field. Currently, no in-field diagnostic exists for M. oryzae Triticum. Loop-mediated isothermal amplification (LAMP) primers were designed to target the PoT2 and MoT3 loci, previously shown to be specific for M. oryzae and M. oryzae Triticum, respectively. Specificity was determined using 158 M. oryzae strains collected from infected wheat and other grasses and representing geographic and temporal variation. Negative controls included 50 Fusarium spp. isolates. Sensitivity was assessed using 10-fold serial dilutions of M. oryzae Triticum gDNA. PoT2- and MoT3-based assays showed high specificity for M. oryzae and M. oryzae Triticum, respectively, and sensitivity to approximately 5 pg of DNA per reaction. PoT2 and MoT3 assays were tested on M. oryzae Triticum-infected wheat seed and spikes and identified M. oryzae and M. oryzae Triticum, respectively, using a field DNA extraction kit and the portable Genie II system. The mitochondrial NADH-dehydrogenase (nad5) gene, an internal control for plant DNA, was multiplexed with PoT2 and MoT3 and showed results comparable with individual assays. These results show applicability for M. oryzae Triticum field surveillance, as well as identifying nonwheat species that may serve as a reservoir or source of inoculum for nearby wheat fields.
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Magnaporthe/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedades de las Plantas/microbiología , Triticum/microbiología , Cartilla de ADN/genética , ADN de Hongos/genética , ADN Mitocondrial/genética , ADN de Plantas/genética , Flores/microbiología , Fusarium/genética , Fusarium/aislamiento & purificación , Sitios Genéticos , Magnaporthe/genética , Semillas/microbiología , Sensibilidad y Especificidad , Especificidad de la EspecieRESUMEN
Wheat blast has emerged as a major threat to wheat production in South America. Although originally restricted to Brazil, the disease has since been observed in the neighboring countries of Argentina, Bolivia, and Paraguay and recently the pathogen, Magnaporthe oryzae Triticum pathotype, was isolated from infected wheat in Bangladesh. There is growing concern that the pathogen may continue to spread to other parts of the world, including the United States, where several M. oryzae pathotypes are endemic. M. oryzae pathotypes are morphologically indistinguishable and, therefore, must be characterized genotypically. Symptoms of wheat blast include bleaching of the head, which closely resembles the symptoms of Fusarium head blight, further complicating efforts to monitor for the presence of the pathogen in the field. We used a genomics-based approach to identify molecular markers unique to the Triticum pathotype of M. oryzae. One of these markers, MoT3, was selected for the development of a polymerase chain reaction (PCR)-based diagnostic assay that was evaluated for specificity using DNA from 284 M. oryzae isolates collected from a diverse array of host species. Conventional PCR primers were designed to amplify a 361-bp product, and the protocol consistently amplified from as little as 0.1 ng of purified DNA. The specificity of the MoT3-based assay was also evaluated using Fusarium spp. DNA, from which no amplicons were detected.
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Following the discovery of two new wheat virus diseases in the United States, the Great Plains region (Colorado, Kansas, Montana, Nebraska, North Dakota, Oklahoma, South Dakota, Texas, and Wyoming) of the National Plant Diagnostic Network (NPDN) initiated a project to measure the prevalence of five wheat diseases using indirect ELISA. Wheat streak mosaic virus (WSMV), Wheat mosaic virus (WMoV), and Triticum mosaic virus (TriMV) were found in all nine states. WSMV was the most prevalent, averaging 23 to 47% of samples each year. TriMV and WMoV were detected with WSMV (in up to 76% of the samples). All three mite-transmitted viruses were present in 26% or fewer of the samples. Aphid-transmitted viruses in the barley yellow dwarf complex Barley yellow dwarf virus, and Cereal yellow dwarf virus-RPV were less frequent (fewer than 65% of the samples). This paper presents the first case-control methodology paper using plant diagnostic laboratory data and the first signed diagnostic data-sharing agreement between the NPDN and its regulatory stakeholders. Samples collected when <700 cumulative degree-days base 0°C, were twice as likely to be virus negative. This proof-of-concept effort highlights the potential of the NPDN and its National Data Repository to develop knowledge about emerging diseases.
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Wheat blast, caused by the Triticum pathotype of Magnaporthe oryzae, is an emerging disease considered to be a limiting factor to wheat production in various countries. Given the importance of wheat blast as a high-consequence plant disease, weather-based infection models were used to estimate the probabilities of M. oryzae Triticum establishment and wheat blast outbreaks in the United States. The models identified significant disease risk in some areas. With the threshold levels used, the models predicted that the climate was adequate for maintaining M. oryzae Triticum populations in 40% of winter wheat production areas of the United States. Disease outbreak threshold levels were only reached in 25% of the country. In Louisiana, Mississippi, and Florida, the probability of years suitable for outbreaks was greater than 70%. The models generated in this study should provide the foundation for more advanced models in the future, and the results reported could be used to prioritize research efforts regarding the biology of M. oryzae Triticum and the epidemiology of the wheat blast disease.
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The National Plant Diagnostic Network (NPDN) has developed into a critical component of the plant biosecurity infrastructure of the United States. The vision set forth in 2002 for a distributed but coordinated system of plant diagnostic laboratories at land grant universities and state departments of agriculture has been realized. NPDN, in concept and in practice, has become a model for cooperation among the public and private entities necessary to protect our natural and agricultural plant resources. Aggregated into five regional networks, NPDN laboratories upload diagnostic data records into a National Data Repository at Purdue University. By facilitating early detection and providing triage and surge support during plant disease outbreaks and arthropod pest infestations, NPDN has become an important partner among federal, state, and local plant protection agencies and with the industries that support plant protection.
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Schilbach et al.'s model assumes that the ability to "experience" minds is already present in human infants and therefore falls foul of the very intellectualist problems it attempts to avoid. We propose an alternative relational, action-based account, which attempts to grasp how the individual's construction of knowledge develops within interactions.
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Cognición/fisiología , Relaciones Interpersonales , Neuronas Espejo/fisiología , Percepción Social , Teoría de la Mente/fisiología , HumanosRESUMEN
Magnaporthe oryzae is the causal agent of blast disease on several graminaceous plants. The M. oryzae population causing wheat blast has not been officially reported outside South America. Wheat production in the United States is at risk to this pathogen if it is introduced and established. Proactive testing of U.S. wheat cultivars for their reaction to blast and identification of resistance resources is crucial due to the national and global importance of the U.S. wheat industry. In this preliminary study, the phenotypic reaction of 85 U.S. wheat cultivars to M. oryzae (Triticum pathotype) was determined. Although there was a significant correlation in the reaction to blast at the seedling and adult plant stages, only 57% of the head reaction was explained by the seedling reaction. Because of the importance of disease development at the head stage in the field, assessment of all 85 cultivars occurred at the head stage. Among cultivars tested, a continuum in severity to head blast was observed; cultivars Everest and Karl 92 were highly susceptible with more than 90% disease severity, while cultivars Postrock, JackPot, Overley, Jagalene, Jagger, and Santa Fe showed less than 3% infection. No evidence of the presence of physiological races among isolates T-7, T-12, T-22, and T-25 was found.
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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.
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Orchidaceae , Pectobacterium , Dickeya , Técnicas de Amplificación de Ácido Nucleico/métodos , Genómica , Enterobacteriaceae/genética , Pectobacterium/genética , Orchidaceae/genética , Sensibilidad y EspecificidadRESUMEN
Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Goss's wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg (~ 3000 copies) and 100 fg (~ 30 copies) was determined for Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1 pg (~ 300 copies) when 1 µL of host sap was added into the RPA reaction containing tenfold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA assay for any plant pathogen.
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Clavibacter/genética , Genómica , Técnicas de Amplificación de Ácido Nucleico/métodos , Nucleotidiltransferasas/genética , Zea mays/microbiología , Temperatura Corporal , Simulación por Computador , ADN Bacteriano/genética , Límite de Detección , Microbiología , Filogenia , Enfermedades de las Plantas/microbiología , Reacción en Cadena de la Polimerasa , Recombinasas/genética , Sensibilidad y EspecificidadRESUMEN
Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.
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Actinobacteria/genética , Actinobacteria/aislamiento & purificación , Productos Agrícolas/microbiología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN Bacteriano , Genoma Bacteriano , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Rathayibacter toxicus is a toxigenic bacterial pathogen of several grass species and is responsible for massive livestock deaths in Australia and South Africa. Due to concern for animal health and livestock industries, it was designated a U.S. Select Agent. A rapid, accurate, and sensitive in-field detection method was designed to assist biosecurity surveillance surveys and to support export certification of annual ryegrass hay and seed. Complete genomes from all known R. toxicus populations were explored, unique diagnostic sequences identified, and target-specific primers and a probe for recombinase polymerase amplification (RPA) and endpoint PCR were designed. The RPA reaction ran at 37 °C and a lateral flow device (LFD) was used to visualize the amplified products. To enhance reliability and accuracy, primers and probes were also designed to detect portions of host ITS regions. RPA assay specificity and sensitivity were compared to endpoint PCR using appropriate inclusivity and exclusivity panels. The RPA assay sensitivity (10 fg) was 10 times more sensitive than endpoint PCR with and without a host DNA background. In comparative tests, the RPA assay was unaffected by plant-derived amplification inhibitors, unlike the LAMP and end-point PCR assays. In-field validation of the RPA assay at multiple sites in South Australia confirmed the efficiency, specificity, and applicability of the RPA assay. The RPA assay will support disease management and evidence-based in-field biosecurity decisions.
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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.
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Genoma Bacteriano , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Pectobacterium/aislamiento & purificación , Microbiología del Suelo , Solanum tuberosum/microbiología , Simulación por Computador , ADN Bacteriano/genética , Límite de Detección , Pectobacterium/genética , Reproducibilidad de los ResultadosRESUMEN
Non-windfall approaches to sharing demonstrate pre-schoolers' sensitivity to merit-based distributions of resources. However, such studies have not considered (1) whether epistemic aspects of task performance, such as the relative accuracy of a co-worker, influences pre-schoolers' rates of sharing; and (2) how children's emerging social understanding may impact resource allocations in high- and low-merit situations. These issues are of theoretical importance as they may provide new information about the scope of pre-schooler's merit-based sharing behaviours. Moreover, as social understanding has been related to both increases and decreases in pre-schoolers' levels of sharing, providing a merit-based assessment of this relationship would allow for a concurrent assessment of recent conflicting findings. In this study, three- and four-year-olds (N = 131) participated in an unexpected transfer task which was followed by a resource generation picture card naming task with a reliable or unreliable (high- or low-merit) co-worker (a hand puppet). The results showed that children engage in more generous rates of sharing with a high-merit co-worker. This suggests that merit-based sharing is apparent in young children and extends to epistemic aspects of task performance. However, such sharing was constrained by a self-serving bias. Finally, we were not able to detect an effect of children's performance on the false belief task on sharing behaviours in the high- or low-merit trials, suggesting that these behaviours may not be modulated by social understanding during early childhood.
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Desarrollo Infantil , Psicología Infantil , Asignación de Recursos , Conducta Social , Preescolar , Femenino , Humanos , MasculinoRESUMEN
Rathayibacter toxicus is a Gram-positive, nematode-vectored bacterium that infects several grass species in the family Poaceae. Unique in its genus, R. toxicus has the smallest genome, possesses a complete CRISPR-Cas system, a vancomycin-resistance cassette, produces tunicamycin, a corynetoxin responsible for livestock deaths in Australia, and is designated a Select Agent in the United States. In-depth, genome-wide analyses performed in this study support the previously designated five genetic populations, with a core genome comprising approximately 80% of the genome for all populations. Results varied as a function of the type of analysis and when using different bioinformatics tools for the same analysis; e.g., some programs failed to identify specific genomic regions that were actually present. The software variance highlights the need to verify bioinformatics results by additional methods; e.g., PCR, mapping genes to genomes, use of multiple algorithms). These analyses suggest the following relationships among populations: RT-IV â RT-I â RT-II â RT-III â RT-V, with RT-IV and RT-V being the most unrelated. This is the most comprehensive analysis of R. toxicus that included populations RT-I and RT-V. Future studies require underrepresented populations and more recent isolates from varied hosts and geographic locations.
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Clavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Goss's wilt/blight of corn, accounts for high yield losses and is listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5' end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5' position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels-no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5' AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5' AT-rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; an increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize reaction efficiency and to harmonize diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.
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Actinobacteria/genética , ADN Bacteriano/genética , Reacción en Cadena de la Polimerasa Multiplex , Filogenia , Reacción en Cadena en Tiempo Real de la Polimerasa , Clavibacter , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Zea mays/microbiologíaRESUMEN
In 2006, a mechanically-transmissible and previously uncharacterized virus was isolated in Kansas from wheat plants with mosaic symptoms. The physiochemical properties of the virus were examined by purification on cesium chloride density gradients, electron microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), sequencing of the nucleotides and amino acids of the coat protein, and immunological reactivity. Purified preparations contained flexuous, rod-shaped particles that resembled potyviruses. The coat protein was estimated from SDS-PAGE to have a mass of approximately 35 kDa. Its amino acid sequence, as deduced from DNA sequencing of cloned, reverse-transcribed viral RNA and separately determined by time-of-flight mass spectrometry, was most closely related (49% similarity) to Sugarcane streak mosaic virus, a member of the Tritimovirus genus of the family Potyviridae. The virus gave strong positive reactions during enzyme-linked immunosorbent assays using polyclonal antibodies raised against purified preparations of the cognate virus but gave consistent negative reactions against antibodies to Wheat streak mosaic virus (WSMV), other wheat potyviruses, and the High Plains virus. When the virus was inoculated on the WSMV-resistant wheat cv. RonL, systemic symptoms appeared and plant growth was diminished significantly in contrast with WSMV-inoculated RonL. Taken together, the data support consideration of this virus as a new potyvirus, and the name Triticum mosaic virus (TriMV) is proposed.
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ABSTRACT Pseudomonas spp. have been studied for decades as model organisms for biological control of plant disease. Currently, there are three commercial formulations of pseudomonads registered with the U.S. Environmental Protection Agency for plant disease suppression, Bio-Save 10 LP, Bio-Save 11 LP, and BlightBan A506. Bio-Save 10 LP and Bio-Save 11 LP, products of Jet Harvest Solutions, Longwood, FL, contain Pseudomonas syringae strains ESC-10 and ESC-11, respectively. These products are applied in packinghouses to prevent postharvest fungal diseases during storage of citrus, pome, stone fruits, and potatoes. BlightBan A506, produced by NuFarm Americas, Burr Ridge, IL, contains P. fluorescens strain A506. BlightBan A506 is applied primarily to pear and apple trees during bloom to suppress the bacterial disease fire blight. Combining BlightBan A506 with the antibiotic streptomycin improves control of fire blight, even in areas with streptomycin-resistant populations of the pathogen. BlightBan A506 also may reduce fruit russet and mild frost injury. These biocontrol products consisting of Pseudomonas spp. provide moderate to excellent efficacy against multiple production constraints, are relatively easy to apply, and they can be integrated with conventional products for disease control. These characteristics will contribute to the adoption of these products by growers and packinghouses.
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Disease phenotypes for winter wheat cultivars were determined in numerous inoculated greenhouse and field experiments over many years. For four diseases, Fusarium head blight, tan spot, Septoria leaf blotch, and Stagonospora leaf blotch, at least 20 cultivars each had been evaluated in a minimum of five experiments. Reference cultivars of known disease reaction were included in each experiment, which allowed transformation of the percent disease severity data to a 1-to-9 scale for comparisons between experiments. Variations in scale values obtained for individual cultivars among the different experiments were used to calculate standard deviations for disease phenotype data. Standard deviations were used to calculate the number of experiment repetitions needed within each disease to achieve different levels of accuracy (margins of error). A margin of error of ±1.5 for the 1-to-9 scale was chosen as the best level of accuracy. Rounding values within this range would put the estimated disease phenotype within ±1 unit of the actual phenotype. To achieve a margin of error of ±1.5 for Fusarium head blight, tan spot, Septoria leaf blotch, and Stagonospora leaf blotch would require a mean that was calculated from a minimum of five, five, seven, and eight experiments, respectively. Personnel who report disease phenotype data to wheat producers or breeders should be aware of the number of experiments upon which they are basing their reports and adjust any disclaimers accordingly. Similarly, wheat breeders should be aware of the inherent variability in phenotyping these four wheat diseases and make appropriate adjustments to their selection protocols. With a minimum of five experimental repetitions, disease phenotype values obtained from inoculated greenhouse and field experiments had very high correlations (r = 0.81 to 0.92, P < 0.0001) with published Kansas State University Research and Extension ratings obtained from commercial fields.
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Rathayibacter toxicus is a gram-positive bacterium that infects the floral parts of several Poaceae species in Australia. Bacterial ooze is often produced on the surface of infected plants and bacterial galls are produced in place of seed. R. toxicus is a regulated plant pathogen in the U.S. yet reliable detection and diagnostic tools are lacking. To better understand this geographically-isolated plant pathogen, genetic variation as a function of geographic location, host species, and date of isolation was determined for isolates collected over a forty-year period. Discriminant analyses of recently collected and archived isolates using Multi-Locus Sequence Typing (MLST) and Inter-Simple Sequence Repeats (ISSR) identified three populations of R. toxicus; RT-I and RT-II from South Australia and RT-III from Western Australia. Population RT-I, detected in 2013 and 2014 from the Yorke Peninsula in South Australia, is a newly emerged population of R. toxicus not previously reported. Commonly used housekeeping genes failed to discriminate among the R. toxicus isolates. However, strategically selected and genome-dispersed MLST genes representing an array of cellular functions from chromosome replication, antibiotic resistance and biosynthetic pathways to bacterial acquired immunity were discriminative. Genetic variation among isolates within the RT-I population was less than the within-population variation for the previously reported RT-II and RT-III populations. The lower relative genetic variation within the RT-I population and its absence from sampling over the past 40 years suggest its recent emergence. RT-I was the dominant population on the Yorke Peninsula during the 2013-2014 sampling period perhaps indicating a competitive advantage over the previously detected RT-II population. The potential for introduction of this bacterial plant pathogen into new geographic areas provide a rationale for understanding the ecological and evolutionary trajectories of R. toxicus.