Toward Accurate Detection: Protocol Adjustment for Ascochyta rabiei in Chickpea Seeds

This set of protocols was developed to detect Ascochyta rabiei, the fungus responsible for chickpea blight, in seeds. Given the significant impact of this disease and its potential to cause complete crop loss, even at low inoculum levels, optimizing detection protocols is crucial. This article proposes adjustments to key variables of a basic agar plate test-disinfection method, incubation, illumination, and culture medium-to improve microscopic identification (mainly using a stereomicroscope) and enhance differential growth of A. rabiei relative to other seed-borne fungi. Each step was carefully validated. Detection was verified through morphological and molecular confirmation of A. rabiei, as well as interlaboratory repeatability tests. In addition, the first distribution map of A. rabiei in Argentina was generated based on chickpea seed analyses conducted in the past decade and using the protocols outlined in this article. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Seed sampling procedure Basic Protocol 2: Preparation of culture medium Support Protocol: Sterilization of antibiotic Basic Protocol 3: Seed disinfection by washing Basic Protocol 4: Seed sowing and incubation Basic Protocol 5: Detection of infected seeds.