Transcriptomic Analyses of Differential Host Responses to Red Rot Pathogen Colletotrichum Falcatum in Sugarcane Through Subtractive Library and NGS Approach

The fungal pathogen Colletotrichum falcatum causes the stalks, the economically important for sugar extraction. Although, disease management is achieved by cultivating resistant cultivars, the complex polyploidy of sugarcane genome complicates understanding the inheritance of disease resistance. Earlier attempts of using resistant and susceptible varieties to understand host-pathogen interaction resulted in cultivar specific expression of genes due to different genomic background of the varieties. To avoid host background variation in the interaction, suppression subtractive hybridization (SSH) based next generation sequencing technology was utilized in the same cv Co 7805 which behaves differently as incompatible and compatible to two different C. falcatum pathotypes. In the incompatible interaction (ICI) with C. falcatum pathotype Cf87012 (Less virulent, LVir) 10,038 contigs were assembled from ~54,699,263 raw reads. In the compatible interaction (CI) to the C. falcatum pathotype Cf94012 (Virulent, Vir) 4022 contigs were assembled from ~52,509,239 raw reads. The transcripts homologous to CEBiP receptor and transcripts involved in the signals ROS, Ca2+, BR, JA and ABA were exhibited in both the responses. Additionally, MAPK, ET, PI signals and JA amino conjugation related transcripts were found only in ICI. Finally, the temporal gene expression of a total number of 16 transcripts was monitored in qRT-PCR. Most of the transcripts exhibited highest induction in ICI in comparison with 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 to do so in ICI. Overall, this study has identified for the first time, the differential responses of a single sugarcane host to two different C. falcatum pathotypes and PAMP triggered immunity (PTI) is exhibited in both the responses, but the more efficient effector triggered immunity (ETI) was found only in ICI at the molecular level.

Antimicrobial and Antioxidant properties of medicinal mushroom Ganoderma P. Karst

Ganoderma species have been known all over the world as highly medicinal mushrooms. Antimicrobial activity of it is an attractive approach which raises the global interests from the scientific community. In this study, the antimicrobial assay of ethanol and methanol extracts of Ganoderma were prepared by using the dried mycelial powder obtained from five different liquid media; was performed against seven plant pathogenic fungi viz., Alternaria macrospora, Aspergilus niger, A. flavus, Colletotrichum falcatum, Fusarium oxysporum, Pestalotiopsis mangiferae and Penicillium sp. and two plant pathogenic bacteria Xanthomonas oryzae and Ralstonia solanacearum. The extracts of mycelia obtained from Yeast Wine Media exhibited the highest inhibition percentage as compared to rest. At concentration 1000ppm, there was complete inhibition of mycelial growth for Alternaria macrospora, Aspergilus niger and A. flavus while for Fusarium oxysporum and Penicillum sp. complete inhibition was observed at 500ppm but for Colletotrichum falcatum and Pestalotiopsis mangiferae, more than 80% mycelial inhibition at concentration 1000ppm in both ethanol and methanol extracts. In the case of Xanthomonas oryzae and Ralstonia solanacearum, at concentration 1000ppm, methanol extract showed the highest inhibition zone (3.50mm, 3.75mm). Ganoderma exhibited antagonistic effect against plant pathogenic fungi could add to the interest of developing Ganoderma as a successful bioagent in the near future. Antioxidant activities were evaluated by using DPPH radical scavenging assay and Metal chelating activity on ferrous ions. The DPPH radical scavenging effect was detected in methanol extract (Inhibition% = 27.312%) was higher than that of the ethanol extract (Inhibition% = 24.79%) and also Ferrous ion chelating ability of methanol extract (Inhibition% =22.27%) was higher than the ethanol extract (Inhibition% =12.55%). It is clearly indicated that both methanol and ethanol extract of the Ganoderma show antioxidant properties and Ganoderma extracts act as an effective antioxidant agents.

Transcriptome Analysis of Sugarcane in Response to Colletotrichum Falcatum Infection Reveals Repertoire of Transcription Factors and Host Targets

Background - Sugarcane (Saccharum spp hybrid), an important C4 perennial plantation crop, globally grown for white sugar and ethanol production. Red rot caused by Colletotrichum falcatum is one of the most important threats affecting sugarcane productivity in many countries including India.Materials and Methods - Comprehensive understanding is very much needed to define their transcription level differences and their key regulatory genes during interaction of sugarcane with C. falcatum. To compute and evaluate the molecular mechanism in sugarcane, transcriptome analysis of sugarcane challenged with C. falcatum was sequenced using Hi-Seq 2500 and gene expression profiles were generated by qRT-PCR assays in both compatible and incompatible interactions after challenge inoculation of C. falcatum in sugarcane.Results - A total of 15,728,914 reads were aligned to 48,935 unigenes using BOWTIE 2; the unigenes were annotated using BLASTX and found that 39,895 unigenes were annotated and 22,025 were unigenes with respect to host species, 8,830 with respect to Colletotrichum spp and 9,040 were found to be novel genes. A total of 243 transcription factors (TFs) were found to be predicted in sugarcane challenged with C. falcatum and those TFs were divided into 45 specific families. WRKY, MYB, NAC, bHLH and AUX/IAA transcription factors were found to be abundant which are considered to be key regulators in controlling wide range of molecular events such as defense response, oxidative stimuli, host signalling and triggering disease resistance. In addition, a lot of stress related genes and genes involved in gene ontological and KEGG pathway were significantly affected due to C. falcatum infection. Quantative real time PCR assays carried out to validate reliability of observed expression patterns in sugarcane in response to C. falcatum infection illustrates first transcriptome wide in planta identification and analysis of TF repertoire in the host pathogen interaction.Conclusion - The results of this study provide a benchmark discovery in finding host targets and provide tissue specific data set of genes that express in response to C. falcatum in sugarcane and also a complete analysis of main group of genes that significantly enriched under this condition. This is the first comprehensive work provides basis for the further studies to dissect role of TFs at molecular level in sugarcane defense to fungal pathogens.

Phylogenetic Analysis and Genetic Diversity of Colletotrichum falcatum Isolates Causing Sugarcane Red Rot Disease in Bangladesh

Colletotrichum falcatum Went causes red rot disease in sugarcane farming in the tropical and sub-tropical regions. This disease causes significant economic loss to the sugarcane production industry. Successful disease management strategies depend on understanding the evolutionary relationship between pathogens, genetic diversity, and population structure, particularly at the intra-specific level. Forty-one isolates of C. falcatum were collected from different sugarcane farms across Bangladesh for molecular identification, phylogeny and genetic diversity study. The four genes namely, ITS-rDNA, β-tubulin, Actin and GAPDH sequences were conducted. All the 41 C. falcatum isolates showed a 99–100% similarity index to the conserved gene sequences in the GenBank database. The phylogram of the four genes revealed that C. falcatum isolates of Bangladesh clustered in the same clade and no distinct geographical structuring were evident within the clade. The four gene sequences revealed that C. falcatum isolates from Bangladesh differed from other countries´ isolates because of nucleotides substitution at different loci. The genetic structure of C. falcatum isolates were determined using ISSR marker generated 404 polymorphic loci from 10 selected markers. The percentage of polymorphic loci was 99.01. The genetic variability at species level was slightly higher than at population level. Total mean gene diversity at the species level was 0.1732 whereas at population level it was 0.1521. The cluster analysis divided 41 isolates into four main genetic groups and the principal component analysis was consistent with cluster analysis. To the best of our knowledge, this is the first finding on characterizing C. falcatum isolates infesting sugarcane in Bangladesh. The results of this present study provide important baseline information vis a vis C. falcatum phylogeny analysis and genetic diversity study.

Assessment of Sugarcane Genotypes for Red Rot Resistance and Antifungal Activity of Rhizosphere Microbiota against Colletotricum falcatum

The aim of this study was to screen the potential of locally grown sugarcane genotypes for red rot resistance and activity of native microbial strains against the pathogen. Field trials were conducted with 70 genotypes for consecutive years and results showed that only two genotypes viz., SSRI-1 and CO-0238 showed resistant behavior towards red rot of sugarcane. The red rot pathogen Colletotrichum falcatum Went was characterized and pathogenicity tests on two susceptible genotypes (NSG-59 and CPSG-2923) showed high virulence of SUCF04 isolate to develop severe disease lesions. The native rhizospheric microbiota was screened for microbial consortia exhibiting fine antifungal activity against the highly virulent pathogenic strain. The antagonism assay exposed that 10 bacterial isolates out of 46 showed great potential for antifungal activity. The selected bacterial isolates revealed 68–99% pathogen inhibition during the assay. The fungal strains with biological control potential inhibited the pathogen growth by 20–80% and a group of three strains with more than 50% antifungal activity were characterized. The molecular characterization of these microbes revealed that the isolates were belonging to Bacillus subtilus, Pseudomonas putida, Pseudomonas fluorescence, Trichoderma harzianum and several other important taxa. This study revealed that only two sugarcane genotypes were found as resistant against red rot pathogen, while most of the genotypes showed susceptible to moderately susceptible response. Moreover, the native residential microbiota associated with sugarcane exhibited great antifungal potential and can be utilized for disease protection and improved crop productivity. © 2021 Friends Science Publishers

Use of Liming and Incidence of Diseases in Sugarcane

This study aimed to evaluate the incidence of red rot, brown leaf spot, and smut in ten sugarcane genotypes during two consecutive cycles, in the absence and presence of limestone. The experimental design consisted of randomized blocks with four replications, in the presence and absence of liming in the following sugarcane genotypes: G1 (RB002754), G2 (RB021754), G3 (RB041443), G4 (RB863129), G5 (RB93509), G6 (RB951541), G7 (RB962962), G8 (RB992506), G9 (SP79-1011), and G10 (VAT90-212) for genotype x environment interaction. The lowest incidences of red rot were observed in G3 (RB041443), G4 (RB863129), G8 (RB992506), and G9 (SP79-1011) for plant cane, and in G3 (RB041443), G4 (RB863129), G5 (RB93509), G8 (RB992506), and G9 (SP79-1011) for ratoon. All genotypes were susceptible to Colletotrichum falcatum, but limestone reduced its incidence in G3 (RB041443), G6 (RB951541), and G10 (VAT90-212) during the first growth cycle, and in G1 (RB002754), G2 (RB021754), G5 (RB93509), G6 (RB951541), G7 (RB962962), and G10 (VAT90-212) in the ratoon crop. Liming also reduced the incidence of brown leaf spot in G4 (RB863129), G6 (RB951541), and G9 (SP79-1011) in plant cane and G6 (RB951541) and G7 (RB962962) in the ratoon crop. Only the G9 genotype (SP79-1011) showed an incidence of smut. The genotypes had different incidence levels of red rot, brown leaf spot, and smut diseases, which varied in the presence of limestone. Limestone use reduced disease incidence as a function of genotype and cutting cycle.

Over-Expression of Endogenous SUGARWIN Genes Exalted Tolerance against Colletotrichum Infection in Sugarcane

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens.