Sugar plays a vital role in our daily lives, serving as a critical energy source and fundamental ingredient in various foods and beverages. Beyond its culinary uses, sugar is essential in preserving food, as a high concentration of it inhibits microbial growth. Additionally, sugar is used in the pharmaceutical industry, which aids in the formulation of medicines. Its versatility extends to uses in bioplastics and biofuel production. Sugar is a critical component of our diet and a significant contributor to global economic growth.

80% of the sugar we use today comes from Sugarcane, and the rest comes from sugar beets[1]. Sugarcane is the primary source of sugar in many countries due to its high yield and efficiency in sugar production compared to sugar beets.

The life cycle of sugarcane plants is affected by approximately 240 sugarcane diseases. Approximately 100 fungi, 10 bacteria, 50 nematodes, and 10 viruses have been identified as sugarcane pathogens worldwide[1]. Out of the many biotic stresses of the Sugarcane, the fungi Colletotrichum falcatum Went responsible for Red Rot stands out as one of the most destructive sugarcane pathogens, causing significant reduction in the quality and yield of susceptible sugarcane cultivars. The red rot occurs in 77 sugarcane-producing countries. This disease decreases sugarcane yield by up to 50%. The loss results in only around 30% sugar recovery[1]. Besides reducing yield attributes, the red rot reduces the sugarcane juice quality (such as sucrose content, purity, Brix, Pol) and commercial cane sugar. Moreover, managing red rot disease in the field is difficult as the genetic makeup of this fungus keeps changing. Therefore, accurate and rapid identification of Colletotrichum is essential. We identified specific DNA regions, ITS rDNA, that are highly conserved and indicative of the presence of C. falcatum in sugarcane tissue. Despite its conservation, it contains enough variability to distinguish between different fungal species within the Colletotrichum family, making it an ideal target for molecular identification[4].

The project we’re developing this year is a Microfluidic Phage Display kit that enables rapid and convenient detection of ITS rDNA in Sugarcane, facilitating early identification and management of red rot disease. We engineered a protein called ZAP1, which contains a DNA binding domain (DBD) specific to a motif in the ITS rDNA. We have fused the gene for this DBD with a phage coat protein known as P3, allowing our engineered protein to be displayed on the surface. This setup enables effective interaction with the target DNA, leading to the identification of the presence of this pathogen in samples.

Our kit fundamentally works on the principle of detecting the binding event when the DBD of ZAP1 binds to its target motif sequence in ITS rDNA, converting this binding event into a quantifiable optical signal. This approach promises a field-deployable, easy-to-use diagnostic tool that can empower farmers to promptly identify and manage red rot disease, reducing crop losses and economic burden.

Early detection and elimination of C. falcatum in the plant would help prevent the spread of red rot to other fields and regions. Farmers can identify the diseased stalks, remove them promptly, and apply fungicides to purify nearby areas, thereby lessening the spread of C. falcatum. The sugarcane industry as a whole would benefit economically from reduced crop losses. Farmers would save on the costs associated with treating infected crops and managing disease outbreaks during the growing season. This method provides a rapid, accurate, and field-deployable solution to manage red rot disease effectively.

“Ganne ka ras” is pure sugarcane juice skillfully extracted by street vendors using rustic wooden presses. Bursting with natural flavors and enriched with lime or ginger, it's a refreshing drink rich in vitamins, minerals, and antioxidants, making it a beloved, health-boosting elixir nationwide. As we quenched our thirst with the sweet, refreshing juice, our eyes wandered to a nearby sugarcane cart where a farmer was unloading his harvest. We noticed that among the vibrant green stalks, there were a few withered and reddened ones being discarded to the side.

Curiosity piqued, we approached the farmer and inquired about the red stalks. With a heavy sigh, the farmer explained that they were infected with “Lal Rog” which we later found to be the local name for Red Rot disease, a devastating fungal disease that plagued sugarcane crops. He lamented the loss of his harvest and the struggles he faced in combating the disease year after year.

As we bid farewell to the farmer and continued on our way, the image of the discarded red stalks lingered in their minds. Inspiration struck like lightning. What if we could develop a way to diagnose red rot early before it wreaked havoc on the crops? What if we could help farmers with the knowledge and tools to combat the disease? Excited by the possibility of making a real difference, we rushed back to our lab to learn more about Red Rot.

Red rot in Sugarcane caused by Colletotrichum falcatum is a widespread fungal disease that has plagued sugarcane crops in India for over a century. Characterized by the reddening and rotting of the cane's internal tissues, this disease leads to significant yield losses and deterioration in cane quality. It attacks the valuable stalk tissues and can even significantly reduce the sugar content in the cane. The fungus produces enzymes that break down sucrose into simpler sugars, reducing overall sugar yield and quality. This disease has been responsible for eliminating many commercial sugarcane varieties worldwide. Managing red rot disease in the field is difficult as the genetic makeup of this fungus keeps changing. The disease spreads through infected planting material(Sugarcane is also grown vegetatively), wounds(lesions), and conducive environmental conditions, such as high humidity and temperature. The secondary spread of disease happens through conidial dissemination through irrigation water, rain splashing, dew washings from mid-rib lesions, and wind dispersal. The resting structures of the fungus, such as appressoria, chlamydospores, thick-walled-hyphae, and setae, play a vital role in soil-borne transmission. The disease progress in terms of visual symptoms is divided into four divisions, namely tiller, lamina, midrib, and stem red rot[1].

The worldwide occurrence of Sugarcane is approximately 26.3 M hectares, and the gross production is approximately 1.9 billion tons. The major sugarcane-producing countries are Brazil, India, Thailand, Pakistan, China, Mexico, the United States of America and Australia[8].

More than 100 fungi have been reported to cause diseases in Sugarcane. The damage share of Red Rot is approximately 20-30% of the total damage caused by major fungal diseases, which are whip smut, red rot, leaf blast, sugarcane mosaic virus, ratoon stunting disease, leaf scald, mottled stripe, pokkah boeng, and wilt[8].

These scores change with time as C. Falcatum undergoes genetic changes. India recently faced one of the worst crop losses due to the sudden failure of the most popular Red Rot-Resistant variety Co 0238 in the states of Uttar Pradesh and Bihar. In UP state alone, nearly 0.5 M ha out of 2.6 M ha area has severe red rot during the 2020–2021 season[6]. The crop losses due to red rot in the state have increased from a few thousand ha during the 2016–2017 season to a mammoth figure. The total loss caused by this disease outbreak works out to be 1.0 to 1.414 billion US$ during the current season alone. The previous year’s loss could be at least 40%–50% of the current loss. Overall, the disease has affected nearly 10% of the cane area in the country, and it may increase further in the coming seasons. Hence, it is called as ‘cancer of sugarcane’[6].

1.)The most effective and sustainable method is developing and planting red rot-resistant sugarcane varieties. Breeding programs focus on selecting and cultivating strains that show natural resistance to Colletotrichum Falcatum.

2.)Preventive strategies include crop rotation to break the disease cycle, field sanitation to remove the debris of infected plants, proper drainage to avoid water logging as they favor fungal growth and balanced fertilisation.

3.)Applying fungicides such as carbendazim, mancozeb, and propiconazole can help control the disease. These are used as a protective measure, especially during favorable conditions for the disease. (Sugarcane is also grown vegetatively through their sett, and sett-borne inoculum is the primary mode of red rot transmission hence the sett is also pretreated with a fungicide to minimize the risk)

4.)It utilises beneficial microorganisms like Trichoderma spp. and Pseudomonas fluorescens that antagonize the red rot pathogen and reduce its impact.

Though researchers focused on finding and generating disease-resistant sugarcane varieties and introduced them into the field, they have grown in large numbers and become a dominant part of the cultivation. But when these resistant strains succumb to red rot, a lot of sugarcane harvest is wasted, resulting in immense loss.

Of the 5.2 M ha land in cultivation in India 2.6 M ha (50%) area of sugarcane production is done in the state Uttar Pradesh(UP).

Cases: -

1.)Uttar Pradesh: Co0238 was a popular moderately disease-resistant variant of Sugarcane, and its popularity increased from 19.8% of the cultivation area in 2015-16 to 86.67% of the cultivation area in 2020-21. The journey of red rot was initiated from 5 to 100 percent in variety Co 0238 during 2015-16 and 2020-21, respectively. The primary source of this swift disease spread was infected soil as well as setts at farmer fields[9].

2.)Bihar:- CoS8436 was a popular moderately disease-resistant variant of Sugarcane.

The cane production dipped almost 1/3rd over 3 seasons of Sugarcane Production as Variety CoS8436 succumbed to Red Rot[10].

1.)Visual Inspection:- Yellowing and drying of leaves, red streaks on the rind, and the presence of reddish lesions in the internal tissues of the cane are some Field Symptoms of Red Rot. Splitting the cane longitudinally to observe the internal red discoloration and rotting, often accompanied by a sour smell, is also a method.

2.)Microscopic Inspection:- Observing fungal structures under a microscope to identify Colletotrichum falcatum.

3.)Enzyme Activity:- Conducting tests to detect specific enzyme activities associated with C. falcatum, such as pectinases, which degrade plant cell walls.

4.)ELISA (Enzyme-Linked Immunosorbent Assay):- Using antibodies specific to C. falcatum to detect the presence of the pathogen in plant tissues.

5.)Polymerase Chain Reaction(PCR):- Employing PCR to amplify DNA specific to C. falcatum. This technique is highly sensitive and can detect the pathogen even in the early stages of infection.

6.)DNA Barcoding:- Sequencing specific regions of the pathogen's DNA to accurately identify and confirm its presence.

The visual symptoms of the disease appear too late when the fungus has already multiplied and started producing spores. While other diagnostic methods are specific, they are complex and require laboratory conditions. We need a point-of-care diagnostic tool that can detect the presence of C. falcatum in the environment at the early stages of disease progression or even before planting Sugarcane. This diagnostic method should be simple enough for a farmer to use in testing whether the fungus is present in their field.

A biomarker is a molecule found in an organism that can indicate the presence or progression of a particular disease. In scientific communities, proteins and nucleic acids are commonly used as biomarkers for species identification. After conducting an extensive literature review, we identified three promising biomarkers that effectively signal the presence of Colletotrichum falcatum.

1.)PKS1 Gene: Polyketide synthase1 has a functional role in melanin biosynthesis. It is involved in the production of dihydroxy naphthalene (DHN) melanin to determine its role in virulence in C. falcatum [11]

2.)EPL1 Protein: Eliciting Plant like Protein is a protein present in C. falcatum, which elicits the defence mechanism of Sugarcane as C. falcatum starts to infect the Plant.[12]

3.)ITS rDNA Gene: Internal Transcribed Spacer Ribosomal DNA acts as a spacer region for the gene sequence coding for the ribosome protein of C. falcatum species.[3]

To be a reliable biomarker, it should be uniquely present in the specific species and capable of distinguishing it from other closely related species, even within the same family. Additionally, it should be conserved across different strains of the species. PKS1 Gene was conserved across different strains but could not distinguish C. falcatum species from other Colletotrichum family species [11]. EPL1 Protein is unique to C. falcatum, but it has many variations among different strains of the same species [12]. ITS region, which is present in all the species, has a speciality in that they are highly conserved across different species strains but vary significantly even among closely related species from the same family [3].

After thoroughly considering the above three as potential candidates for biomarkers in our project, we discovered that ITS (Internal Transcribed Spacer) rDNA could be a potential biomarker in detecting disease/fungi. It is promising to detect Red Rot in Sugarcane caused by Colletotrichum Falcatum as the ITS rDNA sequence is highly conserved across different strains of C. falcatum but varies significantly even among closely related Colletotrichum species. This uniqueness provides a significant advantage for precise detection. The phylogenetic analysis based on ITS sequences revealed distinct clustering of C. falcatum isolates into three central regions: region I representing Bangladesh, region II representing India, and region III encompassing isolates from China, Japan, Thailand, USA, Mexico, and the Netherlands. Notably, the isolates from Bangladesh and India formed separate but closely related regions, indicating regional genetic differentiation within C. falcatum populations[3].

Further examination of ITS rDNA sequences identified specific nucleotide substitutions unique to Indian C. falcatum isolates at positions 132, 136, 138, 388, and 389 (T/G/C/TC), distinguishing them from isolates from other countries. This genetic variation underscores the regional diversity and evolutionary dynamics within C. falcatum populations in the Indian subcontinent [3].

We did MSA(Multiple Sequence Aligning) on the top 20 hits of NCBI Blast, which were different strains of C. falcatum (viz. KP205442.1, MN636354.1, PP663265.1, etc. ), and the obtained result is as follows:-

After doing MSA for different Strains of C falcatum, the most Conserved sequence(430 bp) we obtained is

The proposed diagnosis employs a three-step approach for the rapid and specific detection of Red Rot disease in Sugarcane. The first step involves sample preparation, where we collect and create a solution from the sample before loading it into the kit. This step is crucial, as it's essential to use an adequate amount of the sample to ensure accurate disease detection and minimize the likelihood of false results.

The diagnostic kit, featuring two chambers, is activated in the next phase. The sample is introduced into the first chamber, where cell lysis occurs, releasing genetic material. This material then moves to the second chamber, where detection occurs as the DNA motif binds to the DNA-binding domain (DBD) of the ZAP1 protein. This interaction alters the refractive index at the metal surface(gold sheet of iSPR system) , generating a quantifiable optical signal through iSPR (portable smartphone imaging Surface Plasmon Resonance) technology, enhancing measurement precision.

Once the optical signal is generated from the binding of the DNA motif to the ZAP1 protein's DNA-binding domain (DBD), the next step is data acquisition and analysis. The iSPR system captures the change in refractive index at the metal surface and converts these optical signals into electronic data. This data is then processed to quantify the binding events, enabling the determination of the target DNA sequence's concentration. Final results are displayed in real-time on a smartphone application, providing users with immediate access to quantitative values that indicate both the presence and concentration of the target genetic material.

Early Detection: - The presence of ITS rDNA sequences specific to Colletotrichum Falcatum, the pathogen causing red rot in Sugarcane, can be detected in the soil (before plantation) and infected plants far before visible symptoms appear. This allows for timely intervention, enabling farmers to take action to manage and contain the disease before it spreads extensively.

Point-Of-Care:- Our innovative and cost-effective diagnostic approach uses a simple soil sample from the field or a leaf/stem sample from the sugarcane plant and an aptamer-based biosensor. Enclosed in a compact device, the sensor can be easily operated by anyone with minimal training. The accessibility and rapid results make our diagnostic kit a valuable tool for the early detection and management of red rot in Sugarcane, providing an efficient alternative to traditional diagnostic methods.

5. Viswanathan, R. (2021). Red Rot of sugarcane ( Colletotrichum Falcatum Went). In CABI Reviews. CABI Publishing. https://doi.org/10.1079/pavsnnr202116023

6. Minnatullah, Md., & Kamat, D. N. (2018). Losses Due to Red Rot Pathogen in Cane Juice Quality. In International Journal of Current Microbiology and Applied Sciences (Vol. 7, Issue 2, pp. 13–16). Excellent Publishers. https://doi.org/10.20546/ijcmas.2018.702.003

7. Rajput, M. A., Rajput, N. A., Syed, R. N., Lodhi, A. M., & Que, Y. (2021). Sugarcane Smut: Current Knowledge and the Way Forward for Management. In Journal of Fungi (Vol. 7, Issue 12, p. 1095). MDPI AG. https://doi.org/10.3390/jof7121095

10. Scindiya, M., Malathi, P., Kaverinathan, K. et al. RNA-mediated silencing of PKS1 gene in Colletotrichum Falcatum causing red rot in Sugarcane. Eur J Plant Pathol 153, 371–384 (2019). doi: https://doi.org/10.1007/s10658-018-1563-z

11. Ashwin NMR, Barnabas L, Ramesh Sundar A, et al. Comparative secretome analysis of Colletotrichum Falcatum identifies a cerato-platanin protein (EPL1) as a potential pathogen-associated molecular pattern (PAMP) inducing systemic resistance in Sugarcane. J Proteomics. 2017;169:2-20. doi: https://doi.org/10.1016/j.jprot.2017.05.020

12. Hossain MI, Ahmad K, Vadamalai G, Siddiqui Y, Saad N, Ahmed OH, Hata EM, Adzmi F, Rashed O, Rahman MZ, Kutawa AB. Phylogenetic Analysis and Genetic Diversity of Colletotrichum Falcatum Isolates Causing Sugarcane Red Rot Disease in Bangladesh. Biology (Basel). 2021 Sep 3;10(9):862. doi: https://doi.org/10.3390/biology10090862 .