An Effective Method for Bacterial Leaf Streak Disease Severity Estimation in Controlled and Field Environments in Small Grains

Background Wheat ( Triticum aestivum ) is one of the most economically important crops in the United States. However, over the past two years, wheat production has suffered up to a 40% reduction in final yield due to pathogen infections worldwide. A major emerging threat in the Great Plains and Canadian Prairies, including South Dakota, North Dakota, and Minnesota, is bacterial leaf streak (BLS)/black chaff disease caused by Xanthomonas translucens spp., which has led to substantial yield losses in the last decade. Absence of both effective chemical controls and competitive highly resistant varieties makes BLS disease management very difficult. A critical step missing in this process is the establishment of a reliable and reproducible infection protocol for resistance evaluation under both controlled and field conditions. Currently, no protocols are published, and the methods published as part of research manuscripts lack detailed procedures, equipment specifications, and have major drawbacks for applications limited to controlled environment and discrepancies in field disease ratings scales. Therefore, we are presenting here a robust and reproducible BLS disease infection protocol and, disease severity rating scale for estimation of BLS disease in both controlled and field conditions. Results After Three days of inoculation with X. translucens pv. undulosa ( Xtu ), wheat plants developed initial water-soaked symptoms at inoculation sites. Over seven days, symptoms progressed to chlorosis and necrosis, frequently covering entire leaves of highly susceptible genotypes, whereas limited to no symptoms on resistant genotypes. Disease severity was consistently scored on a 1–9 scale, enabling clear differentiation of resistant, moderately resistant, and susceptible genotypes. Pathogen re-isolation confirmed infection fidelity. Field validation at the booting stage produced comparable symptom progression on flag leaves, with severity scored at 7, 14, and 21 days post-inoculation. The same protocol was successfully adapted for Pantoea ananatis and Xanthomonas prunicola , demonstrating the adaptability of the method. The protocol was repeated across five independent trials and produced reproducible results in both controlled and field environments. Conclusion We describe a simple, reproducible, and cost-effective inoculation protocol for evaluating BLS severity in wheat. The method reliably distinguishes resistance responses across environments and can be extended to other bacterial pathogens affecting small grains. Its affordability, accessibility, and reproducibility make it a valuable tool for large-scale germplasm screening and resistance breeding. Key Features • A detailed and systemic infection protocol is devised for different cultivars of wheat. • Plants can screen at seedling and adult-plant stage. • No specific equipment required. • Using an inexpensive pipeline to ensure uniform symptoms. • This protocol is validated for other bacterial species that are reported to cause bacterial leaf streak symptoms on small grains ( Pantoea spp and Xanthomonas prunicola on small grains (wheat and Barley).