title: Developing an Integrated Device for On-Farm Detection of Sugarcane Diseases
Creators: name: Strachan, Simon M
Publisher: name: Griffith University
Year: 2024
Subjects: subject: sugarcane
subject: diagnostics
subject: plant pathogens

description: Plant diseases are a major cause of reduced crop yields and economic losses worldwide. Detecting plant pathogens is crucial for managing diseases, and portable on-site diagnostic devices can play a vital role in this effort. The market for diagnostic devices is expected to grow rapidly, especially with the increase in rapid manufacturing processes and improved public access. As a result, technological advancements could help resource-limited economies in disease diagnostics and create job opportunities locally. Point-of-care (POC) diagnostics, particularly those using embedded microcontrollers, have gained attention in the research community for their ability to evaluate biological markers in various sample types. By developing precise, rapid, and sensitive technologies for POC diagnostics, user-friendly platforms could effectively simplify disease diagnosis in the field and reduce the need for skilled personnel. This thesis begins with a comprehensive review of sample preparation and diagnostics literature, focusing on their potential impact on sugarcane disease detection. The review not only covers the methodology, results, and potential implementation of these techniques in a portable device but also underscores the significant strides this research can make in the field of sugarcane disease management. In the study of portable diagnostics, a focus was placed on how an Arduino nano can be used to assist sugarcane agronomists. It was found that molecular and electrochemical diagnostics show the most promise. This thesis introduces a new design for both a molecular and electrochemical device. Research showed that the loop-mediated isothermal amplification (LAMP) technique is the appropriate molecular diagnostic method. Traditional methods like polymerase chain reaction (PCR) require a highly sensitivity and powerful thermocycler, which LAMP does not. This, therefore, allows for a more robust, low-power and smaller portable device. Additionally, a real-time colorimetric method was developed to provide a more detailed result compared to the conventional positive or negative outcomes of colorimetric LAMP. This new method offers cost-effective components as an alternative to the more technologically sensitive fluorescent LAMP. The handheld LAMP diagnostic platform was used to diagnose the two sugarcane diseases leaf scald and ratoon stunting disease (RSD), caused by the bacteria Xanthomonas albilineans (Xalb) and Leifonsia xyli subsp. xyli (Lxx) respectively. This approach enabled a sample-in-result-out diagnostic procedure, involving a single chamber and a single-step sample preparation process. Within the single chamber, sample preparation, target nucleic acid amplification, and diagnosis can all be conducted. The method involves a 95°C heat lysis step of the xylem sap sample to extract the nucleic acid target sequences of the target pathogen. A heating block incorporated into a coil of nichrome wire embedded within a polydimethylsiloxane (PDMS) mould performs both the heat lysis and amplification. The presence and quantification of the target organism are then analysed through colorimetric readout. This innovative method offers several advantages over existing plant diagnostic approaches, including ease of on-farm application, reduced sampling to result out time, equipment-less operation, minimal assay time due to heat lysis for sample preparation, and a simplistic design that allows for mass production of affordable devices, thus widening the user base. In addition, field trials have demonstrated the device's effectiveness in real-world conditions, especially in the detection of Xalb, the pathogen that causes leaf scald in sugarcane. The LAMP device was tuned to detect Xalb within a dynamic range from 108 to 102 cells, with a detection limit of 100 cells/μL. Testing of field samples from eight different sugarcane varieties showed a strong correlation with laboratory-based methods (Spearman correlation r = 0.857), confirming the accuracy and reliability of the device. Additionally, a portable electrochemical biosensor was developed with a customizable analog front-end potentiostat integrated chip (IC), LMP91000, an Arduino Nano, and custom-printed circuit boards (PCBs), all enclosed within a compact 3Dprinted casing. This device utilized screen-printed electrodes (SPEs) for electrochemical detection using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Emphasizing miniaturization and portability, the construction of the device aimed to make it suitable for field applications. It was designed to offer rapid and sensitive detection of the sugarcane pathogen Xalb, with performance metrics comparable to traditional laboratory potentiostats such as the CH660E. Through proof-of-concept tests, the device's capability to detect specific DNA sequences, particularly Xalb immobilized on the electrode surface, was confirmed, establishing it as a reliable and effective tool for on-site diagnostics. The primary goal of this study is to address the engineering challenges and negative perceptions regarding portable diagnostics within the sugarcane community. As a solution, two affordable portable diagnostic devices were developed to rapidly detect sugarcane pathogens, specifically focusing on leaf scald and ratoon stunting disease (RSD). These innovative devices signify significant progress in agricultural diagnostics, providing farmers with accessible, cost-effective tools for early disease detection. By facilitating prompt intervention, these technologies have the potential to significantly improve disease management, minimise crop losses, and enhance the sustainability and profitability of sugarcane cultivation.