First report of rice bacterial leaf blight disease caused by Pantoea ananatis in the United States.

In August 2021, bacterial leaf blight-like symptoms were observed on 14 out of 570 rice genotypes (Oryza sativa) in research field plots of global rice germplasm grown in Arkansas (eXtra Figure S1. A & B). The disease was characterized by spreading lesions on leaves, panicle sterility and reduced yield in highly susceptible, mature rice germplasm. No spread of disease to nearby plants was observed. Isolations were performed at Colorado State University, where soakates from symptomatic leaves were spread onto nutrient agar. After 72 h at 28°C, uniform, distinct, yellow-colored bacterial colonies were observed. To screen for the presence of common rice bacterial pathogens, PCR amplification directly from colonies or from DNA isolated from symptomatic field-collected leaves was performed. Primers specific for Xanthomonas oryzae pvs. oryzae and oryzicola (Lang et al., 2010), Burkholderia glumae (Echeverri-Rico et al., 2021), and Pseudomonas fuscovaginae (Ash et al., 2014) did not amplify indicating these organisms were not present. Sequencing of 16S rRNA gene (Weisburg et al., 1991) amplicons suggested the bacteria belonged to the genera Pantoea and Sphingomonas (NCBI accession no. OP683332 and OP683333, respectively). Amplicons resulting from primers specific to the gyrB gene region of P. ananatis (Kini et al., 2021) were sequenced and the fragment was compared to the P. ananatis PA13 reference genome using a BLAST analysis. One candidate (AR358) showed 100% identity with the P. ananatis gyrB region. Primers specific for Sphingomonas sp. (Bangratz et al., 2020) confirmed the second candidate (AR359) as a Sphingomonas sp. The identity of P. ananatis was confirmed by the Plant Pathogen Confirmatory Diagnostics Laboratory (Beltsville, MD, USA). To determine pathogenicity, leaves from 7-day-old seedlings of rice (Oryza sativa) cultivar Kitaake were scissor-clip inoculated (Kauffman et al., 1973) with four different treatments and compared to control leaves inoculated with sterile water. Treatments for the experiment consisted of bacterial suspensions (108 CFU/ml) of the two candidate organisms, P. ananatis (strain AR358) or Sphingomonas sp. (strain AR359), individually or in a 1:1 ratio of P. ananatis:Sphingomonas sp., or soakate from infected field tissue. Lesions similar to those observed in the field were only detected on leaves inoculated with P. ananatis or infected field tissue soakate at 7-days post-inoculation (eXtra Figure S1. C). Bacteria were recovered from the leaves of the artificially inoculated seedlings from three treatments (P. ananatis, P. ananatis:Sphingomonas sp. and soakate from the infected field tissue) and were determined to be P. ananatis based on colony morphology, amplification of 16s rRNA, and gyrB sequence data. Our results confirm the pathogenicity of P. ananatis to rice and fulfill Koch's postulates. P. ananatis was also recovered from several similarly diseased rice breeding lines at the University of Arkansas System Division of Agriculture Rice Research and Extension Center. We conclude that P. ananatis is the causal pathogen for leaf blight-like symptoms observed in the global rice cultivars grown in Arkansas. P. ananatis was previously reported as a pathogen on rice in several rice growing regions, including China (Yu et al., 2021), India (Reshma et al., 2022), and Africa (Kini et al., 2017), however, this is the first report of P. ananatis as a pathogen of rice in the United States.

Harnessing Pseudomonas protegens to Control Bacterial Panicle Blight of Rice.

Bacterial panicle blight of rice is a seedborne disease caused by the bacterium Burkholderia glumae. This disease has affected rice production worldwide and its effects are likely to become more devastating with the continuous increase in global temperatures, especially during the growing season. The bacterium can cause disease symptoms in different tissues and at different developmental stages. In reproductive stages, the bacterium interferes with grain development in the panicles and, as a result, directly affects rice yield. Currently, there are no methods to control the disease because chemical control is not effective and completely resistant cultivars are not available. Thus, a promising approach is the use of antagonistic microorganisms. In this work, we identified one strain of Pseudomonas protegens and one strain of B. cepacia with antimicrobial activity against B. glumae in vitro and in planta. We further characterized the antimicrobial activity of P. protegens and found that this activity is associated with bacterial secretions. Cell-free secretions from P. protegens inhibited the growth of B. glumae in vitro and also prevented B. glumae from causing disease in rice. Although the specific molecules associated with these activities have not been identified, these findings suggest that the secreted fractions from P. protegens could be harnessed as biopesticides to control bacterial panicle blight of rice.

Dynamic Changes in the Rice Blast Population in the United States Over Six Decades.

Rice blast disease caused by Magnaporthe oryzae is one of the most destructive diseases of rice. Field isolates of M. oryzae rapidly adapt to their hosts and climate. Tracking the genetic and pathogenic variability of field isolates is essential to understand how M. oryzae interacts with hosts and environments. In this study, a total of 1,022 United States field isolates collected from 1959 to 2015 were analyzed for pathogenicity toward eight international rice differentials. A subset of 457 isolates was genotyped with 10 polymorphic simple sequence repeat (SSR) markers. The average polymorphism information content value of markers was 0.55, suggesting that the SSR markers were highly informative to capture the population variances. Six genetic clusters were identified by both STRUCTURE and discriminant analysis of principal components methods. Overall, Nei's diversity of M. oryzae in the United States was 0.53, which is higher than previously reported in a world rice blast collection (0.19). The observed subdivision was associated with collection time periods but not with geographic origin of the isolates. Races such as IC-17, IE-1, and IB-49 have been identified across almost all collection periods and all clusters; races such as IA-1, IB-17, and IH-1 have a much higher frequency in certain periods and clusters. Both genomic and pathogenicity changes of United States blast isolates were associated with collection year, suggesting that hosts are a driving force for the genomic variability of rice blast fungus.

The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance.

Plant resistance genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. Here we show that Ptr is an atypical resistance gene encoding a protein with four Armadillo repeats. Ptr is required for broad-spectrum blast resistance mediated by the NLR R gene Pi-ta and by the associated R gene Pi-ta2. Ptr is expressed constitutively and encodes two isoforms that are mainly localized in the cytoplasm. A two base pair deletion within the Ptr coding region in the fast neutron-generated mutant line M2354 creates a truncated protein, resulting in susceptibility to M. oryzae. Targeted mutation of Ptr in a resistant cultivar using CRISPR/Cas9 leads to blast susceptibility, further confirming its resistance function. The cloning of Ptr may aid in the development of broad spectrum blast resistant rice.

Genes for Adult-Plant Resistance to Leaf Rust in Soft Red Winter Wheat.

Host plant resistance in wheat (Triticum aestivum) has been the principal means of managing leaf rust caused by Puccinia triticina. The need for durable resistance has changed the focus from the use of seedling resistance to adult-plant resistance. The objectives of this study were to determine the genetic basis for adult-plant resistance and to determine the most effective method to identify adult-plant resistance genes Lr12, 13, and 34 among 116 contemporary soft red winter wheat cultivars and breeding lines. Adult-plant resistance was detected by inoculating flag leaves with a race that was virulent on seedlings. Approximately 90% of the lines expressed resistance under controlled conditions. It was postulated that the adult-plant resistance in 67 lines was due in part to either Lr12, 13, or 34; the adult-plant resistance detected in 17 lines was attributed to Lr12 based on a distinctive low infection type very similar to that on the isoline TcLr12; the adult-plant resistance in 27 lines was attributed to Lr34, as all of these lines expressed a "leaf tip necrosis" in the field (a phenotype controlled by a gene known to be tightly linked with Lr34); and the adult-plant resistance in 23 lines was attributed to Lr13 based on a high infection type at 18.1°C and low infection type at 25.5°C with one or more pathogen isolates that were virulent on Lr13 at 18.1°C and avirulent on Lr13 at 25.5°C. The adult-plant resistance detected in the remaining 40% of the lines was due to one or more unidentified genes for adult-plant resistance. In a 4-year field study at several locations, nearly 29% of the lines were resistant at all locations, no line was susceptible at all locations, and only 30% of the lines were susceptible at one or more locations. Given that many of the lines in this study were resistant to all known races of P. triticina before being released as cultivars, the high frequency of adult-plant resistance in this study demonstrates that adult-plant resistance can be incorporated even in the presence of highly effective seedling resistance.

Seedling Resistance Genes to Leaf Rust in Soft Red Winter Wheat.

Seedling and adult-plant resistance have been used to manage leaf rust, caused by Puccinia triticina, but there is little information on resistance genes in contemporary cultivars and advanced breeding lines of soft red winter wheat (Triticum aestivum). Lack of information on the genetic basis for resistance leads to uncertainty about durability of resistance and makes pyramiding resistance genes more difficult. The objective of this study was to determine the genetic basis for race-specific seedling resistance to leaf rust among the 116 contemporary lines from the 1998-99 Arkansas Wheat Cultivar Performance Test and the Uniform Eastern and Southern Soft Red Winter Wheat Nurseries. To postulate the presence of genes for leaf rust resistance (Lr genes), seedlings of each line and 24 isolines in a Thatcher background were evaluated for infection type in growth chambers at 22/18°C (day/night) or constant 17 or 18°C using 22 races of P. triticina. A computer program was used to analyze infection type data and facilitate identification of Lr genes. Genes Lr1, 2a, 2c, 3, 3ka, 9, 10, 11, 14a, 18, 20, 23, 24, and 26 were identified among the lines tested. Genes Lr3, 10, and 11 were the most common. Genes Lr15, 28, and 30 were postulated as possibly present in some lines but were not likely to be important among the lines. Genes Lr16, 17, 21, 32, 36, 38, and 39 were not detected. Fifty-four lines had one or more unidentified Lr genes that were not included in the set of 24 isolines. Only four lines (Agripro Marion, APD94-5282, NC94-7197, and VA97W-375) were resistant to all races used in this study, and these were postulated to have the combination of Lr9, 24, and 26.

A Computer Program to Improve the Efficiency and Accuracy of Postulating Race-Specific Resistance Genes.

Gene postulation has been the most widely used technique to determine the presence of particular rust resistance genes in lines of small grains. It applies the principles of gene-for-gene specificity to determine the most probable race-specific resistance genes present in host lines. As the numbers of lines, resistance genes, and races increase, postulation based on visual comparisons of infection types becomes more complex and laborious, and errors may occur. A computer program was developed to facilitate identification of race-specific leaf rust (Lr) genes in wheat (Triticum aestivum). Seedlings of 116 contemporary lines of soft red winter wheat and 24 Thatcher isolines (each Thatcher isoline with a single Lr gene) were inoculated with 22 races of Puccinia triticina. Infection types were recorded on the standard 0 to 4 scale where infection types 3 and 4 were considered high (line was susceptible; race was virulent) and others were low (line was resistant; race was avirulent). Based on the gene-for-gene concept, lines susceptible to a particular race cannot have an Lr gene for which the race is avirulent. For each line, step 1 of the program summarized results from races that were virulent on the line to definitively exclude Lr genes from the line, and this exclusion resulted in a relatively short list of Lr genes that could be present. Step 2 of the program utilized data from races that were avirulent on the line, and the output listed the low infection types produced on the line and the isolines with Lr genes that were not excluded in step 1. Of these Lr genes, a gene was considered present if the low infection type produced on the line by one or more races matched the low infection type on the corresponding isoline. Otherwise, the gene was considered possibly present. Epistatic effects of one or more Lr genes prevented definitive inclusion or exclusion of genes considered possibly present. If the low infection type produced on the line was lower than that on any of the isolines listed in step 2, then the line was considered to have an unidentified Lr gene; i.e., a gene that was not in the set of 24 isolines. This program facilitated the objective and accurate postulation of Lr genes and could be adapted to other host-pathogen systems.