KEY MESSAGE The differential compatibility responses of sugarcane to Colletotrichum falcatum path... more KEY MESSAGE The differential compatibility responses of sugarcane to Colletotrichum falcatum pathotypes depend on the nature of both host primary defence signalling cascades and pathogen virulence. The complex polyploidy of sugarcane genome and genetic variations in different cultivars of sugarcane remain a challenge to identify and characterise specific genes controlling the compatible and incompatible interactions between sugarcane and the red rot pathogen, Colletotrichum falcatum. To avoid host background variation in the interaction study, suppression subtractive hybridization (SSH)-based next-generation sequencing (NGS) technology was used in a sugarcane cultivar Co 7805 which is compatible with one C. falcatum pathotype but incompatible with another one. In the incompatible interaction (ICI-less virulent) 10,038 contigs were assembled from ~ 54,699,263 raw reads, while 4022 contigs were assembled from ~ 52,509,239 in the compatible interaction (CI-virulent). The transcripts homologous to CEBiP receptor and those involved in the signalling pathways of ROS, Ca2+, BR, and ABA were expressed in both interaction responses. In contrast, MAPK, ET, PI signalling pathways and JA amino conjugation related transcripts were found only in ICI. In temporal gene expression assays, 16 transcripts showed their highest induction in ICI than CI. Further, more than 17 transcripts specific to the pathogen were found only in CI, indicating that the pathogen colonizes the host tissue whereas it failed to do so in ICI. Overall, this study has identified for the first time that a probable PAMP triggered immunity (PTI) in both responses, while a more efficient effector triggered immunity (ETI) was found only in ICI. Moreover, pathogen proliferation could be predicted in CI based on transcript expression, which were homologous to Glomerella graminicola, the nearest clade to the perfect stage of C. falcatum (G. tucumanensis).
ABSTRACT A suppression subtractive hybridization technique was used to identify differentially ex... more ABSTRACT A suppression subtractive hybridization technique was used to identify differentially expressed genes in sugarcane in response to Colletotrichum falcatum, the fungal pathogen causing red rot in sugarcane. Messenger RNA (mRNA) samples collected from red rot resistant cv. Co 93009 at 6, 12, 24 and 48 h after pathogen challenge were pooled together and used as tester population. mRNA samples collected from red rot susceptible cv. CoC 671 at 6, 12, 24 and 48 h after pathogen challenge and control of samples of cv. Co 93009 and cv. CoC 671 were pooled together and used as the driver population. A forward subtraction enriched for differentially expressed ESTs in the resistant cultivar was carried out. At the end of subtraction, cloning and sequencing, 136 EST sequences were assembled into ten clusters/contigs. Based on TIGR homology search, the clusters were found to be involved in reactive oxygen species signaling, defense and the secretory pathway of plant innate immunity associated with hypersensitive response—mediated programmed cell death. Temporal gene expression pattern of the EST clusters by quantitative real time PCR revealed that, resistant cultivar had a higher level of gene expression than the susceptible cultivar in all the clusters till 48 h after pathogen inoculation. In conclusion, this study identified for the first time a set of differentially expressed EST clusters in red rot resistant sugarcane cultivar in response to C. falcatum infection.
The microRNAs role in various cellular and metabolic functions is gaining more limelight in line ... more The microRNAs role in various cellular and metabolic functions is gaining more limelight in line with second-generation NGS technology. For the validation of candidate miRNA genes, the quantitative real-time PCR is the widely trusted and efficient method to follow. Sugarcane miRNAs are less explored in sugarcane defense response during their interaction with Colletotrichum falcatum inciting red rot. Further, for RT-qPCR experiments involving sugarcane miRNA expression studies, a stable internal reference gene is required. Hence, we have taken a study involving 20 candidate genes to identify stable expressing reference genes using NormFinder, geNorm, BestKeeper, and deltaCt statistical algorithms. The candidate reference genes included miRNAs and protein-coding genes. The results indicated that there is a variation in ranking among the algorithms. We found miR1862c as the stably expressed miRNA reference gene among the candidates and miR444b.2 along miR1862c formed the best reference gene pair combination, which can be used in the experiments aiming to explore sugarcane miRNAs in the defense mechanism against C. falcatum. The stable miRNA reference gene was further validated with other lesser stable reference gene candidates to assess the effect of stable reference genes during normalization. The present study evaluating the sugarcane miRNAs as reference genes for normalizing RT-qPCR expression data involving miRNAs during sugarcane × C. falcatum interaction is the first of its kind. Further, this systematic approach can be followed to assess the reference gene in various experimental conditions involving sugarcane miRNAs.
Colletotrichum falcatum, an intriguing pathogen causing red rot in sugarcane, exhibits enormous v... more Colletotrichum falcatum, an intriguing pathogen causing red rot in sugarcane, exhibits enormous variation for pathogenicity under field conditions. A species-specific marker is very much needed to classify the virulence among the varying population and to identify the potential of a pathotype by mining the microsatellites, which are considered to be the largest genetic source to develop molecular markers for an organism. In this study, we have mined the C. falcatum genome using MISA database which yielded 12,121 SSRs from 48.1 Mb and 2745 SSRs containing sequences. The most frequent SSR types from the genome of C. falcatum was di-nucleotide which constitutes 50.89% followed by tri-nucleotide 39.60%, hepta-nucleotide 6.7%, hexa-nucleotide 1.38% and penta-nucleotide 1.3%. Over 90 SSR containing sequences from the genome were predicted using BlastX which are found to be non-homologs. Most of the annotated SSR containing sequences fell in CAZy superfamilies, proteases, peptidases, plant cell wall degrading enzymes (PCDWE) and membrane transporters which are considered to be pathogenicity gene clusters. Among them, glycosyl hydrolases (GH) were found to be abundant in SSR containing sequences which again proved our previous transcriptome results. Our in-silico results suggested that the mined microsatellites from C. falcatum genome show absence of homolog sequences which suggests that these markers could be used as an ideal species-specific molecular marker. Two virulence specific markers were characterized using conventional PCR assays from C. falcatum along with virulent species-specific (VSS) marker developed for C. gloeosporioides. The study lays the foundation for the development of C. falcatum specific molecular marker to phenotype the pathotypes based on virulence.
Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most de... more Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most devastating diseases that affect sugarcane production, globally. At present, the most practical and effective management strategy for the disease is the cultivation of resistant cultivars. In this connection, a detailed understanding of the host’s defense mechanism in response to smut isolates with varying degrees of virulence at the molecular level would facilitate the development of reliable and durable smut-resistant sugarcane varieties. Hence, in this study, a comparative whole transcriptome analysis was performed employing Illumina RNA-seq in the smut susceptible cultivar Co 97009 inoculated with two distinct S. scitamineum isolates, Ss97009 (high-virulent) and SsV89101 (low-virulent) during the early phases of infection (2 dpi and 5 dpi) and at the phase of sporogenesis (whip emergence) (60 dpi). Though the differential gene expression profiling identified significant transcriptional...
Wilt of sugarcane caused by Fusarium sacchari is a serious disease affecting cane stalks in the c... more Wilt of sugarcane caused by Fusarium sacchari is a serious disease affecting cane stalks in the crop. The disease affects the plant during any period of the crop cycle, decreasing agricultural and industrial production. Managing the disease through resistant varieties is considered as the most sustainable and economically viable approach. However, there is a lack of a versatile disease rating system for F. sacchari resistance in sugarcane. To address the issue, we developed a mean wilt severity index on a 0–9 rating scale. In the revised wilt severity system, essential criteria governing wilt were taken into account to develop 0–9 rating scale as resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible corresponding to 0–2.0, 2.1–4.0, 4.1–6.0, 6.1–8.0 and 8.1–9.0 scores, respectively, based on overall wilt expression in sugarcane. This new scale clearly discerns the varietal reactions based on internal symptoms, especially pith region cavities, cane and spindle leaf phenotype and disease progress through nodes and roots. This system of grading based on 0–9 scale was found better than the previous numeric index system which grouped clones into 4 grades, ignoring the relative importance of nodal transgression and pith cavity development. This new scale is applicable to grade sugarcane genotypes after plug method of inoculation or disease development screening through soil application of F. sacchari inoculum.
India is a major supplier of turmeric to the world with more than 60 per cent share in turmeric t... more India is a major supplier of turmeric to the world with more than 60 per cent share in turmeric trade. The production and export performance of turmeric in India have been examined using secondary data for the period from 1974-75 to 2007-08 and exponential form of growth function has been used for the analysis. The growth in production and export of turmeric has been reported significant, because of the high demand coupled with inflation. Instability index has been worked for the production and export for preliberalization and post-liberalization periods. Instability has been observed high for production, export and prices of domestic and international markets and domestic and international prices have shown high integration. For the assessment of direction of trade, the Markov chain model has been used. The data regarding country-wise export of turmeric has shown that the previous export share retention for Indian turmeric has been high in minor importing countries (pooled under ot...
Colletotrichum falcatum, an ascomycete pathogen causes red rot of sugarcane which is specialized ... more Colletotrichum falcatum, an ascomycete pathogen causes red rot of sugarcane which is specialized to infect cane stalks. Cellulolytic and pectinolytic enzymes are necessary for degradation of plant cell wall which stands as barrier for successful fungal pathogenesis. In the study, we have confined to the CAZy genes that regulate cellulolytic and pectinolytic enzymes in two distinctive pathotypes of C. falcatum. Comparative transcriptome analysis revealed that a number of CAZy genes producing cellulolytic and pectinolytic enzyme were present in the virulent (Cf671) and least virulent (RoC) pathotypes. Two consecutive transcriptome analyses (in vitro) were performed using Illumina Hi Seq 2500, further analysis was done with various bioinformatic tools. In vitro expression analysis of cutinase, glycoside hydrolyase and pectin-related genes revealed number of genes that attributes virulence. Numerous pectin-related genes involved in degradation of plant cell wall, pectinase and pectin lyase are considered to be key precursor in degradation of pectin in sugarcane. These results suggest that cellulolytic enzymes, cutinase and pectin-related genes are essential for degradation of sugarcane cell wall and considered to be an important pathogenic factor in C. falcatum. This is the first detailed report on sugarcane cell wall-degrading enzymes during its interaction with C. falcatum and also this comparative transcriptome analysis provided more insights into pathogen mechanism on C. falcatum.
Detailed tritrophic interaction studies were conducted under in vitro and in vivo conditions amon... more 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 afte...
KEY MESSAGE The differential compatibility responses of sugarcane to Colletotrichum falcatum path... more KEY MESSAGE The differential compatibility responses of sugarcane to Colletotrichum falcatum pathotypes depend on the nature of both host primary defence signalling cascades and pathogen virulence. The complex polyploidy of sugarcane genome and genetic variations in different cultivars of sugarcane remain a challenge to identify and characterise specific genes controlling the compatible and incompatible interactions between sugarcane and the red rot pathogen, Colletotrichum falcatum. To avoid host background variation in the interaction study, suppression subtractive hybridization (SSH)-based next-generation sequencing (NGS) technology was used in a sugarcane cultivar Co 7805 which is compatible with one C. falcatum pathotype but incompatible with another one. In the incompatible interaction (ICI-less virulent) 10,038 contigs were assembled from ~ 54,699,263 raw reads, while 4022 contigs were assembled from ~ 52,509,239 in the compatible interaction (CI-virulent). The transcripts homologous to CEBiP receptor and those involved in the signalling pathways of ROS, Ca2+, BR, and ABA were expressed in both interaction responses. In contrast, MAPK, ET, PI signalling pathways and JA amino conjugation related transcripts were found only in ICI. In temporal gene expression assays, 16 transcripts showed their highest induction in ICI than CI. Further, more than 17 transcripts specific to the pathogen were found only in CI, indicating that the pathogen colonizes the host tissue whereas it failed to do so in ICI. Overall, this study has identified for the first time that a probable PAMP triggered immunity (PTI) in both responses, while a more efficient effector triggered immunity (ETI) was found only in ICI. Moreover, pathogen proliferation could be predicted in CI based on transcript expression, which were homologous to Glomerella graminicola, the nearest clade to the perfect stage of C. falcatum (G. tucumanensis).
ABSTRACT A suppression subtractive hybridization technique was used to identify differentially ex... more ABSTRACT A suppression subtractive hybridization technique was used to identify differentially expressed genes in sugarcane in response to Colletotrichum falcatum, the fungal pathogen causing red rot in sugarcane. Messenger RNA (mRNA) samples collected from red rot resistant cv. Co 93009 at 6, 12, 24 and 48 h after pathogen challenge were pooled together and used as tester population. mRNA samples collected from red rot susceptible cv. CoC 671 at 6, 12, 24 and 48 h after pathogen challenge and control of samples of cv. Co 93009 and cv. CoC 671 were pooled together and used as the driver population. A forward subtraction enriched for differentially expressed ESTs in the resistant cultivar was carried out. At the end of subtraction, cloning and sequencing, 136 EST sequences were assembled into ten clusters/contigs. Based on TIGR homology search, the clusters were found to be involved in reactive oxygen species signaling, defense and the secretory pathway of plant innate immunity associated with hypersensitive response—mediated programmed cell death. Temporal gene expression pattern of the EST clusters by quantitative real time PCR revealed that, resistant cultivar had a higher level of gene expression than the susceptible cultivar in all the clusters till 48 h after pathogen inoculation. In conclusion, this study identified for the first time a set of differentially expressed EST clusters in red rot resistant sugarcane cultivar in response to C. falcatum infection.
The microRNAs role in various cellular and metabolic functions is gaining more limelight in line ... more The microRNAs role in various cellular and metabolic functions is gaining more limelight in line with second-generation NGS technology. For the validation of candidate miRNA genes, the quantitative real-time PCR is the widely trusted and efficient method to follow. Sugarcane miRNAs are less explored in sugarcane defense response during their interaction with Colletotrichum falcatum inciting red rot. Further, for RT-qPCR experiments involving sugarcane miRNA expression studies, a stable internal reference gene is required. Hence, we have taken a study involving 20 candidate genes to identify stable expressing reference genes using NormFinder, geNorm, BestKeeper, and deltaCt statistical algorithms. The candidate reference genes included miRNAs and protein-coding genes. The results indicated that there is a variation in ranking among the algorithms. We found miR1862c as the stably expressed miRNA reference gene among the candidates and miR444b.2 along miR1862c formed the best reference gene pair combination, which can be used in the experiments aiming to explore sugarcane miRNAs in the defense mechanism against C. falcatum. The stable miRNA reference gene was further validated with other lesser stable reference gene candidates to assess the effect of stable reference genes during normalization. The present study evaluating the sugarcane miRNAs as reference genes for normalizing RT-qPCR expression data involving miRNAs during sugarcane × C. falcatum interaction is the first of its kind. Further, this systematic approach can be followed to assess the reference gene in various experimental conditions involving sugarcane miRNAs.
Colletotrichum falcatum, an intriguing pathogen causing red rot in sugarcane, exhibits enormous v... more Colletotrichum falcatum, an intriguing pathogen causing red rot in sugarcane, exhibits enormous variation for pathogenicity under field conditions. A species-specific marker is very much needed to classify the virulence among the varying population and to identify the potential of a pathotype by mining the microsatellites, which are considered to be the largest genetic source to develop molecular markers for an organism. In this study, we have mined the C. falcatum genome using MISA database which yielded 12,121 SSRs from 48.1 Mb and 2745 SSRs containing sequences. The most frequent SSR types from the genome of C. falcatum was di-nucleotide which constitutes 50.89% followed by tri-nucleotide 39.60%, hepta-nucleotide 6.7%, hexa-nucleotide 1.38% and penta-nucleotide 1.3%. Over 90 SSR containing sequences from the genome were predicted using BlastX which are found to be non-homologs. Most of the annotated SSR containing sequences fell in CAZy superfamilies, proteases, peptidases, plant cell wall degrading enzymes (PCDWE) and membrane transporters which are considered to be pathogenicity gene clusters. Among them, glycosyl hydrolases (GH) were found to be abundant in SSR containing sequences which again proved our previous transcriptome results. Our in-silico results suggested that the mined microsatellites from C. falcatum genome show absence of homolog sequences which suggests that these markers could be used as an ideal species-specific molecular marker. Two virulence specific markers were characterized using conventional PCR assays from C. falcatum along with virulent species-specific (VSS) marker developed for C. gloeosporioides. The study lays the foundation for the development of C. falcatum specific molecular marker to phenotype the pathotypes based on virulence.
Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most de... more Sugarcane smut caused by the basidiomycetous fungus Sporisorium scitamineum is one of the most devastating diseases that affect sugarcane production, globally. At present, the most practical and effective management strategy for the disease is the cultivation of resistant cultivars. In this connection, a detailed understanding of the host’s defense mechanism in response to smut isolates with varying degrees of virulence at the molecular level would facilitate the development of reliable and durable smut-resistant sugarcane varieties. Hence, in this study, a comparative whole transcriptome analysis was performed employing Illumina RNA-seq in the smut susceptible cultivar Co 97009 inoculated with two distinct S. scitamineum isolates, Ss97009 (high-virulent) and SsV89101 (low-virulent) during the early phases of infection (2 dpi and 5 dpi) and at the phase of sporogenesis (whip emergence) (60 dpi). Though the differential gene expression profiling identified significant transcriptional...
Wilt of sugarcane caused by Fusarium sacchari is a serious disease affecting cane stalks in the c... more Wilt of sugarcane caused by Fusarium sacchari is a serious disease affecting cane stalks in the crop. The disease affects the plant during any period of the crop cycle, decreasing agricultural and industrial production. Managing the disease through resistant varieties is considered as the most sustainable and economically viable approach. However, there is a lack of a versatile disease rating system for F. sacchari resistance in sugarcane. To address the issue, we developed a mean wilt severity index on a 0–9 rating scale. In the revised wilt severity system, essential criteria governing wilt were taken into account to develop 0–9 rating scale as resistant, moderately resistant, moderately susceptible, susceptible and highly susceptible corresponding to 0–2.0, 2.1–4.0, 4.1–6.0, 6.1–8.0 and 8.1–9.0 scores, respectively, based on overall wilt expression in sugarcane. This new scale clearly discerns the varietal reactions based on internal symptoms, especially pith region cavities, cane and spindle leaf phenotype and disease progress through nodes and roots. This system of grading based on 0–9 scale was found better than the previous numeric index system which grouped clones into 4 grades, ignoring the relative importance of nodal transgression and pith cavity development. This new scale is applicable to grade sugarcane genotypes after plug method of inoculation or disease development screening through soil application of F. sacchari inoculum.
India is a major supplier of turmeric to the world with more than 60 per cent share in turmeric t... more India is a major supplier of turmeric to the world with more than 60 per cent share in turmeric trade. The production and export performance of turmeric in India have been examined using secondary data for the period from 1974-75 to 2007-08 and exponential form of growth function has been used for the analysis. The growth in production and export of turmeric has been reported significant, because of the high demand coupled with inflation. Instability index has been worked for the production and export for preliberalization and post-liberalization periods. Instability has been observed high for production, export and prices of domestic and international markets and domestic and international prices have shown high integration. For the assessment of direction of trade, the Markov chain model has been used. The data regarding country-wise export of turmeric has shown that the previous export share retention for Indian turmeric has been high in minor importing countries (pooled under ot...
Colletotrichum falcatum, an ascomycete pathogen causes red rot of sugarcane which is specialized ... more Colletotrichum falcatum, an ascomycete pathogen causes red rot of sugarcane which is specialized to infect cane stalks. Cellulolytic and pectinolytic enzymes are necessary for degradation of plant cell wall which stands as barrier for successful fungal pathogenesis. In the study, we have confined to the CAZy genes that regulate cellulolytic and pectinolytic enzymes in two distinctive pathotypes of C. falcatum. Comparative transcriptome analysis revealed that a number of CAZy genes producing cellulolytic and pectinolytic enzyme were present in the virulent (Cf671) and least virulent (RoC) pathotypes. Two consecutive transcriptome analyses (in vitro) were performed using Illumina Hi Seq 2500, further analysis was done with various bioinformatic tools. In vitro expression analysis of cutinase, glycoside hydrolyase and pectin-related genes revealed number of genes that attributes virulence. Numerous pectin-related genes involved in degradation of plant cell wall, pectinase and pectin lyase are considered to be key precursor in degradation of pectin in sugarcane. These results suggest that cellulolytic enzymes, cutinase and pectin-related genes are essential for degradation of sugarcane cell wall and considered to be an important pathogenic factor in C. falcatum. This is the first detailed report on sugarcane cell wall-degrading enzymes during its interaction with C. falcatum and also this comparative transcriptome analysis provided more insights into pathogen mechanism on C. falcatum.
Detailed tritrophic interaction studies were conducted under in vitro and in vivo conditions amon... more 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 afte...
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Papers by Ramesh Sundar