A Response to Gupta et al. (2019) Regarding the MoT3 Wheat Blast Diagnostic Assay.

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.

Genomics-Based Marker Discovery and Diagnostic Assay Development for Wheat Blast.

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.

Preliminary Assessment of Resistance Among U.S. Wheat Cultivars to the Triticum Pathotype of Magnaporthe oryzae.

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.

Analysis of Induction and Establishment of Dwarf Bunt of Wheat Under Marginal Climatic Conditions.

Dwarf bunt caused by Tilletia contraversa is a disease of winter wheat that has a limited geographic distribution due to specific winter climate requirements. The pathogen is listed as a quarantine organism by several countries that may have wheat production areas with inadequate or marginal climate for the disease-in particular the People's Republic of China. Field experiments were conducted in the United States in an area of Kansas that is a climatic analog to the northern winter wheat areas of China to evaluate the risk of disease introduction into such areas. The soil surface of four replicate 2.8 × 9.75 m plots, planted with a highly susceptible cultivar, was inoculated with six teliospore concentrations ranging from 0.88 to 88,400 teliospores/cm2. A single initial inoculation was done in each of three nurseries planted during separate seasons followed by examination for disease for 4 to 6 years afterward. Any diseased spikes produced were crushed and returned to the plots where they were produced. One nursery had no disease during all six seasons. In two nurseries, the disease was induced at trace levels at the three highest inoculation rates. Disease carryover to the second year occurred during one year in one nursery in plots at the highest inoculation rate, but no disease occurred the following three seasons. A duplicate nursery planted in a disease conducive area in Utah demonstrated that the highest rate of inoculum used in the experiments was sufficient to cause almost 100% infection. This study demonstrated that in an area with marginal climatic conditions it was possible to induce transient trace levels of dwarf bunt, but the disease was not established even with a highly susceptible cultivar and high levels of inoculum. Our results support the conclusions of the 1999 Agreement on U.S.-China Agricultural Cooperation which set a tolerance for teliospores in grain, and supports the Risk Assessment Model for Importation of United States Milling Wheat Containing T. contraversa.

Detection and characterization of fungus (Magnaporthe oryzae pathotype Triticum) causing wheat blast disease on rain-fed grown wheat (Triticum aestivum L.) in Zambia.

Wheat blast caused by Magnaporthe oryzae pathotype Triticum (MoT) is a threat to wheat production especially in the warmer-humid environments. In Zambia, wheat blast symptoms were observed for the first time on wheat (Triticum aestivum L.) grown in experimental plots and five farmers' fields in Mpika district of Muchinga Province during the 2017-18 rainy season. Infected plants showed the typical wheat blast symptoms with the spike becoming partially or completely bleached with the blackening of the rachis in a short span of time. Incidence of blast symptoms on nearly all wheat heads was high and ranged from 50 to 100%. Examination of diseased plant leaves showed the presence of elliptical, grayish to tan necrotic lesions with dark borders on the leaf often mixed with other foliar diseases. A study was conducted to isolate and identify the causal pathogen(s) using classical and molecular methods and determine the pathogenicity of the detected disease causal agent. Morphobiometrical determination of causal pathogen revealed conidia with characteristic pear shaped 2-septate hyaline spores associated with M. oryzae species. Preliminary polymerase chain reaction screening of six isolates obtained from wheat blast infected samples with diagnostic primers (MoT3F/R) was conducted at ZARI, Zambia, and subsequent analysis of two isolates with MoT3F/R and C17F/R was performed at USDA-ARS, USA. Both experiments confirmed that MoT is the causal agent of wheat blast in Zambia. Further, pathogenicity tests performed with pure culture isolates from samples WS4 and WS5 produced typical blast symptoms on all the six inoculated wheat genotypes. Results of this study indicate that MoT is causing wheat blast in rain-fed wheat grown in Zambia, thus making it the first report of MoT in Zambia and Africa. This inter-continental movement of the pathogen (disease) has serious implication for wheat production and trade that needs to be urgently addressed.

Polymerase Chain Reaction Assays for the Detection and Discrimination of the Soybean Rust Pathogens Phakopsora pachyrhizi and P. meibomiae.

ABSTRACT Soybean rust occurs in Australia and many countries throughout Africa, Asia, and South America. The causal agents of soybean rust are two closely related fungi, Phakopsora pachyrhizi and P. meibomiae, which are differentiated based upon morphological characteristics of the telia. Determination of the nucleotide sequence of the internal transcribed spacer (ITS) region revealed greater than 99% nucleotide sequence similarity among isolates of either P. pachyrhizi or P. meibomiae, but only 80% sequence similarity between the two species. Utilizing differences within the ITS region, four sets of polymerase chain reaction (PCR) primers were designed specifically for P. pachyrhizi and two sets for P. meibomiae. Classical and real-time fluorescent PCR assays were developed to identify and differentiate between P. pachyrhizi and P. meibomiae. Identification of P. pachyrhizi from infected soybean leaves using the real-time PCR assay will allow for more rapid diagnoses.

Relationship Between Soilborne and Seedborne Inoculum Density and the Incidence of Dwarf Bunt of Wheat.

The incidence of dwarf bunt of wheat as a function of inoculum density was studied in a susceptible and a partially resistant cultivar at three disease-conducive locations for three seasons. Prior to seeding, plots were fumigated with methyl bromide to eliminate residual inoculum. Each cultivar was seeded into two 1.2-m rows in four replicates. The soil surface was inoculated with 0, 16 × 102, 16 × 103, 16 × 104, 16 × 105, and 16 × 106 teliospores of Tilletia controversa per row, or seed was inoculated with 0, 2 × 102, 2 × 103, 2 × 104, 2 × 105, and 2 × 106 teliospores per gram. To determine maximum possible infection, two 3.1-m rows of each cultivar were soil-surface inoculated at 10× the highest treatment rate. In the soil-inoculated plots, a minimum of 16 × 103 teliospores/row was needed to cause trace amounts of disease (0.6% maximum), even when the positive indicator treatment had up to 88% incidence. Only trace amounts or no disease occurred below the 16 × 105 rate. In the seed-inoculated plots, infection was rare and occurred only at inoculation rates of 2 × 105 teliospores/g or higher; the highest incidence was 0.4%.