MALDI-TOF MS profiling of maize isolation plates: from background flora to overlooked pathogens (EPPO Conference on Diagnostics of Plant Pests, 2026)

The isolation of Pantoea stewartii subsp. stewartii from maize is complicated by mixed infections and an abundance of background flora, which makes selecting colonies on non-selective media challenging. The isolation of Pantoea stewartii subsp. stewartii from maize and related hosts is typically carried out on King's B medium, but this process is often complicated by heterogeneous symptoms, mixed infections and an abundance of background flora. Colonies of non-target bacteria can closely resemble the target pathogen, which makes selecting the correct colonies difficult, especially in composite samples. To better characterise and address this diagnostic challenge, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) was systematically applied to colonies recovered during routine isolation. The resulting dataset included the expected pathogens, as well as recurrent non-target taxa such as Sphingomonas, Curtobacterium, Microbacterium, Agrobacterium and various Pantoea species. Several taxa, including Pantoea ananatis and Microbacterium spp., exhibited colony morphologies similar to those of P. stewartii, which complicated interpretation. MALDI-TOF MS enabled the rapid identification of colonies and supported the development of diagnostic expertise by linking colony morphology to species identity. When combined with systematic subsampling, this approach improved colony selection and increased isolation success in complex plant samples. The poster illustrates the temporal development of colony morphology and the increasing complexity of the microbial community on isolation plates, as demonstrated by image panels and a time-lapse video. This work contributes to the improvement of routine diagnostic workflows and the training of diagnosticians, as well as the development of realistic reference materials for colony-based identification. This poster was presented at the EPPO Conference on Diagnostics of Plant Pests: Recent Developments and Future Trends, held at the Austrian Agency for Health and Food Safety (AGES) in Vienna, Austria from 22 to 24 April 2026. This work builds on a previous version presented at a national conference and is available at: https://zenodo.org/records/18780441. Supplementary time-lapse video available at: 10.5281/zenodo.19593528 and YouTube: https://youtu.be/1oECSotA8Ns We acknowledge the contribution of the diagnostic team in generating and interpreting isolation data.