Detection of bacteriosis pathogens significant for grain export and a complex of associated microorganisms in grain crops (on the example of Timiryazevskaya Field Experimental Station)

According to official statistics, about 130 million tons of cereals are produced annually in Russia. In the Unified List of quarantine objects of the Eurasian Economic Union is the causative agent of wheat yellow mucous bacteriosis Rathayibacter tritici . This species is subject to detection during import and, if the importer requires, during export of wheat. Due to the need for regulation, there is a diagnostic method for Rathayibacter tritici in quarantine phytosanitary laboratories. For other pathogens of bacteriosis in grain crops, such as Rathayibacter rathayi , Pseudomonas fuscovaginae , Pseudomonas cichorii , Pseudomonas fluorescens , Pseudomonas syringae, Acidovorax avenae, E rwinia rhapontici , Xanthomonas translucens , Clavibacter tessellarius , etc., there are no diagnostic methods, due to which no detections have been recorded in the practice of diagnostic phytosanitary laboratories. The listed types are regulated by importing countries that purchase more than half of all grain products intended for export in Russia. Bacterioses pose a serious threat to grain production, and the possible damage they cause to the crop is estimated at 10-40 %. The bacteria can cause disease outbreaks or be latent in plants depending on environmental conditions and almost never cause symptoms on grain. In this regard, it is possible to detect causative agents of bacteriosis only in the laboratory using the method of inoculation on nutrient media, which often takes a week or more. Reliable identification of each type of bacteria is possible only with the use of molecular methods. It is required to develop PCR tests that allow the identification of target bacteria directly in samples without using the cultural method, which will significantly simplify and speed up the procedure for confirming the compliance of the state of Russian grain batches with the requirements of importers. The development of molecular methods for diagnosing causative agents of bacterioses in grain crops is possible only after studying their species composition in plants and grain, while the diversity of living bacteria in vegetative plants is significantly higher than in grain. Information on the species composition of bacteria on grain crops will make it possible, using genomic analysis, to detect species-specific PCR targets and develop diagnostic PCR tests for the rapid identification of bacterial species that are especially dangerous and important for grain export. Previously, a large-scale study of the bacterial composition in grain crops was not carried out, and therefore, there is no list of bacteria that can be found together in one sample. There is also no complete list of all bacteria that can be found in cereals. At the same time, for bioinformatic prediction of a species-specific PCR target, it is necessary to know all the species that can be found in the analyzed sample, from which the target species should be distinguished. The composition of the bacterial microbiota may differ depending on the crop and variety, so the maximum diversity of different crops and varieties will provide more complete information. Humid and moderately warm summer conditions in the Central region are ideal for the development of bacteriosis. In connection with the foregoing, sampling was carried out on the territory of the Timiryazevskaya field experimental station (Moscow), where hybridization, selection and variety testing of several hundred varieties of grain crops are carried out annually. The work is devoted to the detection and identification of bacteria in samples of grain crops of the Timiryazevskaya field experimental station (Moscow). The objects of the study were bacterial isolates from grain samples in 2020. Bacteria were identified by sequencing the amplicons obtained by PCR with primer pairs PSF/PSR, SyD1/SyD2, and 8UA/519B and comparing the resulting sequences using the BLAST service with sequences posted in GenBank (https://blast.ncbi.nlm. nih.gov). As a result, 55 samples of grain crops were collected, 171 bacterial isolates were isolated and identified, including 34 isolates identified to species. Bacterial diversity is represented by 14 species. Among them, there are phytopathogens Pantoea ananatis , Clavibacter michiganensis , Rhodococcus fascians , Pseudomonas trivialis , Pseudomonas viridiflava and Pseudomonas syringae. The highest frequency of occurrence, 70.9 %, was noted in species belonging to the genus Pseudomonas . Representatives of the genera Frigoribacterium (36.4 %), Clavibacter (16.4 %), Arthrobacter (12.7 %) and Rhodococcus (10.9%) also have a high frequency of occurrence.