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.

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.

Fusarium proliferatum and F. verticillioides are mycotoxin-producing, seedborne pathogens of maize. They are often asymptomatic in seed, eluding symptom-based detection. Experiments were conducted in nonsterile soil to determine whether interspecific competition influenced establishment in maize plants of an introduced isolate of F. proliferatum or F. verticillioides. Hygromycin-resistant, green fluorescent protein (GFP) transformed (GFP-tagged) F. proliferatum (F. proliferatum-green) and hygromycin-resistant, monomeric red fluorescent protein (mRFP) transformed (mRFP-tagged) F. verticillioides (F. verticillioides-red) strains were developed to provide molecular markers to track fungal establishment. Heat-killed Fusarium-free maize seed, colonized with F. proliferatum-green or F. verticillioides-red by immersion in a spore suspension for 16 h, served as the source of inoculum. The ability of F. proliferatum-green and F. verticillioides-red to colonize viable maize plants already colonized by the other species was determined. Maize plants were retrieved from soil after 14 days and DNA was extracted from three consecutive root segments and three consecutive stem segments. A TaqMan multiplex real-time quantitative PCR protocol was developed to identify and quantify F. proliferatum-green and F. verticillioides-red from each plant segment from each treatment; the experiment was repeated three times. This experiment confirmed that F. proliferatum-green and F. verticillioides-red effectively colonized roots and stems of the maize plant already colonized with the other species. Prior colonization of maize tissues by F. verticillioides-red (P = 0.6749) and other seedborne microorganisms (P = 0.1910) reduced but did not prevent subsequent colonization by F. proliferatum-green. Similarly, prior colonization of maize tissues by F. proliferatum-green (P = 0.7032) and other seedborne microorganisms (P = 0.1447) reduced but did not prevent subsequent colonization by F. verticillioides-red.

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.

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.

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.