A biomarker is a molecular indicator used to identify the presence or progression of a disease, and its effectiveness depends on its ability to distinguish the target species from other closely related species. Proteins and nucleic acids are commonly employed as biomarkers in disease detection and species identification. After conducting an extensive literature review, we identified three promising biomarkers associated with Colletotrichum falcatum, the fungal pathogen responsible for Red Rot in Sugarcane:

PKS1 Gene: The Polyketide synthase 1 (PKS1) gene plays a crucial role in melanin biosynthesis, which is linked to virulence in C. falcatum. PKS1 is involved in producing dihydroxy naphthalene (DHN) melanin, an essential factor in the pathogen's ability to infect sugarcane plants. Although conserved across different strains, PKS1 was found to be non-specific to C. falcatum, as it is present in other closely related Colletotrichum species, making it less reliable as a unique biomarker for Red Rot detection [1].

EPL1 Protein: The Eliciting Plant-like Protein (EPL1) is a protein that triggers Sugarcane’s defence mechanisms upon C. falcatum infection. This protein elicits a response from the plant’s immune system as the pathogen starts its invasion process. Although EPL1 is unique to C. falcatum, it exhibits variations across different strains, which complicates its use as a consistent and reliable biomarker for all strains of the pathogen [2].

ITS rDNA Gene: The Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) is a highly conserved genetic region in C. falcatum that serves as a spacer between ribosomal RNA genes. While the ITS region is present in all species of Colletotrichum, its sequence in C. falcatum shows significant variation compared to closely related species, making it an ideal candidate for accurate species identification [3].

Among these biomarkers, the ITS rDNA gene stands out as the most promising for our diagnostic project, primarily due to its high conservation across different strains of C. falcatum and its ability to differentiate the pathogen from other species within the Colletotrichum genus. This specificity is critical for the precise detection of Red Rot in Sugarcane. Phylogenetic analysis based on ITS sequences has further revealed distinct clustering of C. falcatum isolates into three primary regions: region I (Bangladesh), region II (India), and region III (China, Japan, USA, Mexico, and others). Notably, isolates from Bangladesh and India, while closely related, form separate clusters, indicating regional genetic differentiation within C. falcatum populations [3].

Additionally, a deeper examination of ITS rDNA sequences identified unique nucleotide substitutions at positions 132, 136, 138, 388, and 389 (T/G/C/TC) that are specific to Indian isolates of C. falcatum. These genetic markers further distinguish the Indian strains from isolates from other countries, highlighting the evolutionary dynamics and regional diversity of C. falcatum [3].

To validate the robustness of ITS rDNA as a biomarker, we performed a Multiple Sequence Alignment (MSA) on the top 20 hits from an NCBI BLAST search, which included different strains of C. falcatum (e.g., KP205442.1, MN636354.1, PP663265.1). The analysis revealed a highly conserved 430 bp sequence across these strains, confirming its suitability as a universal marker for the detection of C. falcatum.

We are collecting samples from both soil and sugarcane stalks to ensure comprehensive detection of C. Falcatum. Soil samples are essential because the fungus often persists in the soil, serving as a primary source of infection, particularly through infected setts and plant debris. By analyzing soil samples, we can detect the presence of the pathogen before it infects the plant, enabling proactive disease management. Stalk samples are equally important as they allow us to identify the presence of the pathogen within the plant tissue itself, especially during the early stages of infection before visible symptoms appear. Collecting from both sources provides a broader, more accurate diagnostic approach, helping to reduce the spread of the disease.

We are choosing this chemical method for DNA extraction in our Point-of-Care (POC) red rot detection system due to its proven efficiency, simplicity, and ability to yield high-quality DNA from C. falcatum, the pathogen responsible for red rot in Sugarcane. The method employs a detergent-based lysis buffer, which efficiently disrupts the tough fungal cell walls, followed by organic solvent extraction (phenol-chloroform-isoamyl alcohol) to purify the DNA. This approach provides a balance between cost-effectiveness and reliability, making it ideal for a rapid, field-ready diagnostic tool. By ensuring that the extracted DNA is free from contaminants, the method supports sensitive and accurate detection, crucial for early-stage pathogen identification. The overall protocol involves cell lysis with SDS, protein denaturation using phenol-chloroform, and subsequent purification, ensuring high-purity DNA suitable for amplification in our system. For a detailed breakdown of the protocols, please refer WetLabs Protocols [4].