First report of leaf blight caused by Pantoea agglomeranson wheat in China.

Abstract

Wheat (Triticum aestivum L.) is the main grain crop in Ningxia Hui Autonomous Region, China. A new leaf blight disease of wheat was observed in many wheat fields in Yinchuan City and Wuzhong City of Ningxia during 2020-2021. The average disease incidence of the cultivar Ningchun 50 was 5 to 15%, and there appeared the evident disease symptoms from the heading stage, then the symptoms got more serious until the mature stage. The tips of the leaves were chlorotic and turned bright yellow at the early stage of the disease. Later on, the yellow leaf spots were further spread from the tip to the petiole, and the yellow-colored necrotic lesions emerged, resulted in withering and death of leaves (e-Xtra 1, a-d). To isolate and identify the pathogenic agent, diseased leaves were cut into 0.5 cm × 0.5 cm small pieces, and sterilized in 5% NaOCl solution for 5 min, and were rinsed three times in sterile water, then crushed with tweezers in 2 mL sterilized water and streaked three times onto Nutrient Agar (NA) medium. and 15 single colonies which had the same colony morphology were obtained. Of the 15 colonies, 3 (named WH1, Cx1 and HJ1) were randomly selected for further morphological, biochemical and molecular characterization. The resulting bacterial colonies were incubated at 29±1°C in the dark for 3 days, colony morphology was raised, mucoid texture, round, and smooth with entire margin; the color of these colonies was white at the beginning and turned yellow later. These bacteria were rod-shaped gram-negative cells with peritrichous flagella. Based on the physiological and biochemical assay results (e-Xtra 2), the three strains were initially identified as Pantoea agglomerans (Wang, D H., et al. 2021; Wang, J J., et al. 2021). 16S rDNA and gyrB of the three strains were amplified and sequenced by ABI3730XL sequencer in GENEWIZ (Suzhou, China). The sequences of 16S rDNA and gyrB of these strains were submitted to GenBank with the accession numbers ON428446, ON428461 and ON428462 for 16S rDNA; ON461799, ON461801, ON461803 for gyrB. 16S rDNA and gyrB sequences homology analysis showed that the three strains had the highest homology which were over 99.5% with the sequences of the reported P. agglomerans (e-Xtra 1, g) . A phylogenetic analysis based on 16S rDNA and gyrB gene sequences was performed using the MEGA6.0 proximity method, and the results of the phylogenetic tree showed that strains Cx1, WH1 and HJ1 clustered on the same clade with the reported P. agglomerans strains (e-Xtra 1, h-j). Thus, Cx1, WH1 and HJ1 were identified as P. agglomerans. Pathogenicity test was performed to complete Koch's postulates, Ningchun 50 was planted in pots, four-week-old healthy wheat seedlings were inoculated with 107 CFU/mL bacterial suspension using two inoculation methods: 1) Leaf surface was poked with disposable syringe needle, and 50 µL of suspension was injected into each of the pinholes (Suraj, S., et al. 2020); 2) Leaf was cut at 45° at the lower 2-3 cm of the leaf tip with scissors dipped in the bacterial suspension. Wheat leaves inoculated with sterile distilled water were regarded as controls. The inoculated wheat was cultivated in a greenhouse (temperature 28 ± 2°C, humidity 40 ± 2%) and covered in transparent polyethylene bags at first 96 h. Symptoms appeared at 3 days after inoculation, and after 7 days, the acupunctured wheat leaves turned chlorotic and yellow around the pinholes and some were necrotic; the leaf-cutting wheat turned yellow and necrotic from the clipping point to the leaf base; the acupunctured and cut leaves totally died after 15 days, and all of the control leaves were healthy (e-Xtra 1, e-f). Subsequently, pathogens were reisolated from inoculated leaves, and identified as P. agglomerans according to molecular identification described above. To our knowledge, this is the first report of leaf blight disease of wheat caused by Pantoea agglomeransglobally as well as in China. Identifying the cause of the disease will support efforts for its future control and management.