Red rot resistant varieties are deployed in the field to avoid serious losses caused by the red rot pathogen, Colletotrichum falcatum. However, frequent breakdown of resistance occurs due to the development of newer pathotypes and in certain situations, elite varieties that are moderately susceptible to red rot are being cultivated. Under such situations, adopting novel strategies to manage the disease is of paramount importance. Since protection of crop at different stages of crop growth from red rot incidence is essential to maintain good crop stand and to achieve higher productivity, attempts have been made by following conventional and molecular approaches. Conventionally, effective fungicides, bioagents and their integration were identified and also standardized effective delivery methods for successful management. In addition, attempts have been made at molecular level to identify virulence related genes of C. falcatum to monitor pathogen population under field conditions and to identify newer pathotypes with high virulence for effective screening programme. Besides, established the possibility of identifying potential target sites as genes/ proteins for fungicidal management of red rot in the field. Findings of both conventional and molecular approaches attempted for red rot management under field conditions are summarized in this review.

Red rot caused by Colletotrichum falcatum, is the major constraint in sugarcane production in India and varietal resistance plays an important role in managing the disease. However, breakdown of resistance in sugarcane varieties to new pathogen variants which co-evolve concurrently is a serious concern and warrants a clear understanding on pathogenic variation in response to virulence. Hence, a detailed study has been carried out through genomic and proteomic approaches to characterize representative group of C. falcatum pathotypes and isolates varying phylogenetically based on 5.8S-ITS sequence analysis. At genomic level the isolates were characterized using conserved gene sequences viz., actin, calmodulin and GPDH and molecular markers viz., RAPD and ISSR. Molecular analyses with conserved gene sequences highly implicated the existence of one major group of virulent isolates and a minor group of least virulent isolates which was confirmed by definite nucleotide variation. Further studies with molecular markers viz., RAPD and ISSR also confirmed separate grouping of least virulent isolates and different groups among the virulent isolates. Among the markers, ISSR was more efficient in grouping the isolates based on virulence, since it clearly differentiated the least virulent isolates like conserved sequences and some of its primers were able to differentiate the virulent isolates with specific markers. For proteomic analysis, a protocol has been standardized to develop C. falcatum proteome profiles of phylogenetically differentiated virulent and least virulent isolates and demonstrated unique and differential expression of spots related to pathogen virulence. Some of the unique proteins viz., serine protease, DJ-1/PfpI family protein and glutathione S-transferase identified from C. falcatum were closer to well characterized pathogenicity related genes in Colletotrichum spp and other fungi. Outcome of the study formed a basis for characterizing virulence related genes in C. falcatum.

In continuation of our earlier findings on suppression of C. falcatum by T. harzianum , the present study was attempted to characterize differentially expressed proteins from T. harzianum during its growth over C. falcatum metabolites. Proteomic analysis clearly indicated the degradation of C. falcatum proteins and expression of newer proteins by T. harzianum through SDS–PAGE. Two-Dimensional Electrophoresis (2-DE) of individual protein bands eluted from SDS–PAGE resulted in clear separation of 15 protein spots from 3 differentially expressed protein bands viz., DEPb1, DEPb2 and DEPb3. MALDI-TOF analysis of these proteins revealed that the identified 8 proteins were belonging to two functional categories viz., stress and defence (choline dehydrogenase, FKBP-type peptidyl-prolyl cis–trans isomerase, superoxide dismutase (Cu/Zn) and Hex1) and unknown function (Hypothetical proteins). The stress-related proteins were reported to be involved in biological suppression of various pathogens in other crops. In addition to this, 7 unknown function proteins expressed from T. harzianum during interaction with C. falcatum may play a major role in degradation of metabolites. Role of identified proteins was further confirmed during in-planta expression analysis by qRT-PCR, which showed the upregulation of selected proteins with distinct patterns at all the time interval during the tritrophic interaction of host, pathogen and antagonist and corresponding reduction in symptom production.

Twenty bacterial and 27 Trichoderma isolates obtained from sugarcane ecosystem were identified at species level based on ITS-rDNA sequencing. Phylogenetic analysis based on 16S rDNA gene sequencing of bacterial isolates resulted in four groups, viz. Paenibacillus alvei, Alcaligenes faecalis, Pseudomonas fluorescenes and uncultured bacterium. Also, sequencing ITS region of rDNA (ITS1, 2 and 5.8S rDNA) in Trichoderma isolates led to clustering into four taxa, viz. T. asperellum, T. harzianum, T. aureoviride and T. atroviride. All the fungal and bacterial antagonistic isolates were screened against C. falcatum in vitro and effective ones were selected. The sequence results revealed that four effective bacterial antagonistic isolates (BA2, BA8, BA10 and BA19) have high similarity with Paenibacillus alvei. Among the effective Trichoderma isolates, T1, T3 and T18 showed similarity with T. asperellum while T17, T22 and T20 were confirmed as T. harzianum. The performance of all these antagonistic isolates was evaluated individually, in comparison with fungicide alone and in combination with best bacterial antagonist against red rot under pot culture and field conditions. Among the bacterial and fungal antagonists, P. alvei (BA2) and T. harzianum (T20) stand superior to other isolates as they augmented percent germination and plant survival against soil borne inoculum of red rot significantly as compared to pathogen inoculated plots without treatment. However, the combination of bacterial antagonist and fungicide was found to be the best under field conditions. Hence, a combination of P. alvei and fungicide could be recommended for red rot management by conventional means whereas the best antagonistic isolates could be selected for molecular analysis of suppression of C. falcatum and characterization of antifungal proteins.

Detailed tritrophic interaction studies were conducted under in vitro and in vivo conditions among sugarcane, Colletotrichum falcatum, the red rot pathogen of sugarcane and its antagonist Trichoderma harzianum. Two way interaction under in vitro conditions clearly indicated that T. harzianum was able to inhibit the growth of C. falcatum which was evidenced by degradation of C. falcatum proteins and expression of new proteins by Trichoderma both at intra and extra cellular level. Similar suppression of the pathogen by the antagonist was also observed during three way interaction with sugarcane tissue in vitro and reduction of symptom production in stalk tissue in vivo. At the molecular level, SDS-PAGE analysis of proteins extracted from different samples of three way interaction in vitro revealed that there were differentially expressed proteins (ca. 100-150 kDa) when C. falcatum was inoculated in sugarcane tissue followed by Trichoderma at different intervals, particularly 72 h after pathogen inoculation. Under field conditions, C. falcatum symptoms in the stalk tissue were inhibited by simultaneous inoculation of Trichoderma and C. falcatum. Protein profiles of these samples in SDS-PAGE indicated that the sugarcane proteins (ca. 80-100 kDa) were degraded during the host-pathogen interaction and the same proteins were retained during three way interaction of pathogen along with the antagonist on sugarcane. Furthermore, the growth and proliferation of T. harzianum over C. falcatum inside the stalk tissue was shown by duplex PCR, tissue bioassay, and microscopic observations. All these results confirm that T. harzianum was able to suppress C. falcatum in sugarcane by antagonistic mechanism and expression of newer proteins that could be related to defence / antifungal nature.