The Food and Agriculture Organization of the United Nations (FAO) estimates that 98% of the world’s reported 870 million undernourished people reside in developing countries, where nearly 15% of the population are undernourished. In these regions, smallholder farmers are dominant factors in meeting food demands. Therefore, in order to alleviate recurrent food shortages worldwide, low-cost and robust solutions must be provided to protect and prevent the loss of smallholder crop yield. This research reports on the development of in vitro and in vivo diagnostic devices for in-field crop diagnostics. In particular, we employ chromatic polydiacetylenes in the fabrication of analyte-sensitive strips for plant testing in vitro. Specifically, two sets of strips were developed that exhibited blue to pink/red transitions when incubated in solutions containing plant nutrient, Zn2+, or xylem-limited bacterium, X. fastidiosa respectively. These strips demonstrated one-step, equipment-free detection appropriate for resource-limited settings. Additionally, a poof-of-concept in vivo detection platform was evaluated and subsequently, injectable poydiacetylene liposome sensors were designed for specific colorimetric detection of apoplast-colonizing bacterium P. stewartii. An in vivo detection system would provide continuous monitoring of plant pathogen transmission for early detection of crop diseases. Such a platform would be ideal for disease prevention and management on smallholder farms by eliminating the need for user-initiated testing for disease diagnostics. Both of our in vitro and in vivo detection systems provide great potential to improve in-field plant diagnostics in low-resource settings.