De novo genome assembly and annotation of rice sheath rot fungus Sarocladium oryzae reveals genes involved in Helvolic acid and Cerulenin biosynthesis pathways
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- Hittalmani, S., Mahesh, H.B., Mahadevaiah, C. et al. BMC Genomics (2016) 17: 271. doi:10.1186/s12864-016-2599-0
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Sheath rot disease caused by Sarocladium oryzae is an emerging threat for rice cultivation at global level. However, limited information with respect to genomic resources and pathogenesis is a major setback to develop disease management strategies. Considering this fact, we sequenced the whole genome of highly virulent Sarocladium oryzae field isolate, Saro-13 with 82x sequence depth.
The genome size of S. oryzae was 32.78 Mb with contig N50 18.07 Kb and 10526 protein coding genes. The functional annotation of protein coding genes revealed that S. oryzae genome has evolved with many expanded gene families of major super family, proteinases, zinc finger proteins, sugar transporters, dehydrogenases/reductases, cytochrome P450, WD domain G-beta repeat and FAD-binding proteins. Gene orthology analysis showed that around 79.80 % of S. oryzae genes were orthologous to other Ascomycetes fungi. The polyketide synthase dehydratase, ATP-binding cassette (ABC) transporters, amine oxidases, and aldehyde dehydrogenase family proteins were duplicated in larger proportion specifying the adaptive gene duplications to varying environmental conditions. Thirty-nine secondary metabolite gene clusters encoded for polyketide synthases, nonribosomal peptide synthase, and terpene cyclases. Protein homology based analysis indicated that nine putative candidate genes were found to be involved in helvolic acid biosynthesis pathway. The genes were arranged in cluster and structural organization of gene cluster was similar to helvolic acid biosynthesis cluster in Metarhizium anisophilae. Around 9.37 % of S. oryzae genes were identified as pathogenicity genes, which are experimentally proven in other phytopathogenic fungi and enlisted in pathogen-host interaction database. In addition, we also report 13212 simple sequences repeats (SSRs) which can be deployed in pathogen identification and population dynamic studies in near future.
Large set of pathogenicity determinants and putative genes involved in helvolic acid and cerulenin biosynthesis will have broader implications with respect to Sarocladium disease biology. This is the first genome sequencing report globally and the genomic resources developed from this study will have wider impact worldwide to understand Rice-Sarocladium interaction.
KeywordsRice Sheath rot Sarocladium Genome Helvolic acid Cerulenin
Core Orthologous Groups
Glycosyl Phosphatidyl Inositol
Indole-3 acetic acid
Internal Transcribed Spacer
Major facilitator superfamily
Multiple Sequence Alignments
Nuclear Magnetic Resonance
nonribosomal peptides synthases
Potato Dextrose Agar
Potato Dextrose Broth
Quantitative Trait Loci
Short chain dehydrogenase
Simple Sequence Repeats
Sarocladium oryzae [(Sawada) W. Gams & D. Hawksw] is an Ascomycetes fungus causing sheath rot disease in rice. It has recently emerged as a major threat for rice production in rice growing ecosystems in the world. In addition to rice, this fungus infects other important cereal food crops such as maize, sorghum, pearl millet, finger millet, and foxtail millet . The commonly occurring weedy species in rice fields also acts as collateral hosts and source of natural inoculum in endemic areas .
S. oryzae produces white, sparsely branched and septate mycelium. Conidiophores are branched once or twice with 3–4 phialades in a whorl. The conidium is a aseptate, hyaline, cylindrical in shape and located on tip of phialades . The conidium germinates and invades rice through the stomata and wounds caused by insects. Later mycelium grows intercellularly within vascular and mesophyll tissues . The pathogen infects the uppermost leaf sheath enclosing young panicle and lesion length may range from 1 to 5 cm and lesion may enlarge to whole flag leaf sheath in severe cases. The necrotic lesions on flag leaf retards translocation of nutrients from foliage to panicle leading to complete suppression of panicle exertion. This results in production of partially filled chaffy grains, and yield loss ranging from of 3 to 85 % [5, 6]. Despite the considerable loss caused by this fungus, the life cycle and infection biology has been meagerly studied. Sheath rot symptom is also induced by application of Cerulenin which was demonstrated by developing Cerulenin negative mutants, which did not produce rot symptoms . Also virulent strains of the fungi known to secrete proteinases at significantly higher levels compared to less virulent strains indicating the possible roles of fungal proteinases in plant pathogenicity.
The genomic resources for S. oryzae in public databases (NCBI) are limited to internal transcribed spacer (ITS) region sequences of ribosomal DNA and our previous QTL mapping study . Due to lack of information on genes involved in pathogenicity/virulence, host–pathogen interactions and microsatellites markers, rice-Sarocladium pathosystem has not been studied well at global level. Considering these facts, we sequenced whole genome of highly virulent isolate of S. oryzae (Saro-13) from major rice growing region of South India. This is the first report of de novo genome assembly and annotation of S. oryzae. We carried out detailed analyses of gene families, secondary metabolite gene clusters, pathogenicity related genes, transposable repeat elements, phylogenetic relationship with other fungi and microsatellites. In addition, we analysed putative genes involved in helvolic acid and cerulenin biosynthesis pathways, which are very important in Sarocladium disease biology. The genomic resources generated from this study can be translated into designing better disease management strategies to mitigate sheath rot disease epidemics globally and widen the understanding of rice-Sarocladium pathosystem.
Isolation of fungus and confirmation
Diseased flag leaf sheath sampled over 25 locations in major rice growing regions of Karnataka state, India was used for isolation of fungus. Diseased sheath was surface sterilized using 0.05 % mercuric chloride solution followed by three times washing with sterile water. Sterilized diseased sheath pieces were incubated at room temperature for 4–5 days and germinating spores were transferred to potato dextrose agar (PDA) medium. Based on morphological features of conidiophores, phialades and conidiospores , the fungus was identified as S. oryzae. The virulence test of S. oryzae was carried out by standard mycelial inoculation  and detached tiller assay. The virulent field isolate Saro-13 isolated from Shrirangapatna (12.401035° N, 76.695754° E), Mandya District, a major rice growing region under cauvery command area was selected for whole genome sequencing. The fungus was characterized for internal transcriber region using ITS-4 and ITS-5 markers  to confirm the fungus as S. oryzae.
DNA isolation, Illumina library preparation and sequencing
The virulent strain of S. oryzae (Saro-13) was grown on potato dextrose broth (PDB) medium for three days. The mycelium was grinded using liquid nitrogen and genomic DNA was isolated using nucleo-pore gDNA fungal and bacterial mini kit (Genaxy, Catalogue# NP-7006D). The DNA quality and quantity was assessed by Nanodrop and Qubit (Applied Biosystems), respectively. The genomic DNA was sheared to generate fragments of approximately 400–600 bp in Covaris microtube with the E220 system (Covaris, Inc., Woburn, MA, USA). The fragment size distribution was checked using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) with high sensitivity DNA Kit (Agilent Technologies). The fragmented DNA was cleaned up using HighPrep beads (MagBio Genomics Inc, Gaithersburg, Maryland). The Illumina paired-end library was prepared as per manufacturers instruction using NEXTflex DNA sequencing Kit (Catalogue # 5140–02, Bioo Scientific). The paired end library was sequenced using Illumina NextSeq 500 in Genotypic Technologies, Bengaluru and the length of read sequence was 151 nts from both the ends of the fragment.
Preprocessing of raw sequence reads
The low quality bases with Phred score less than Q30 (accuracy less than 99.99 % of the base called) and adapter sequence contamination in raw sequence reads of Illumina was discarded using FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html).
Genome assembly and functional annotation
De novo assembly of S. oryzae was performed using SPAdes assembler . SPAdes assembler corrected sequencing errors in reads and performed scaffolding to output de novo assembled scaffolds. The assembled scaffolds were screened for sequences of mitochondrial genome contaminants. The gene prediction was performed using Augustus 3.0.3 (−−species = fusarium_graminearum --strand = both --genemodel = complete) [12, 13]. Functional annotation of genes was done by searching homology with Ascomycetes protein sequences of SwissProt (http://www.uniprot.org) using BLASTP with an e-value threshold of 1e-10. The annotation of protein domain structures was performed using InterProScan5 software . The gene ontology (GO) terms were assigned by KAAS server .
Analysis of orthologous gene families in Ascomycetes fungi
Gene families were inferred using orthoMCL (with default parameters like percentMatchCutoff = 50 and evalueExponentCutoff = −5)  by comparing proteins of S. oryzae with other Ascomycetes fungi like Magnaporthe oryzae (strain 70–15, http://www.broadinstitute.org/), Fusarium graminearum (strain PH-1, http://www.broadinstitute.org/), Acremonium chrysogenum (strain ATCC 11550, NCBI Accession number JPKY01000001), and Fusarium oxysporum (strain 4287,http://www.broadinstitute.org). The groups having at least one copy from each genome were considered as core orthologous groups (COGs).
Based on orthoMCL clustering, 100 single copy ortholog gene groups from five fungal species were selected randomly and aligned separately using MUSCLE  version 3.8.31 with default parameters. Poorly aligned regions were removed by Gblocks  and all hundred Multiple Sequence Alignments (MSA) were concatenated. Then, 1000 bootstrap replicates were performed using SEQBOOT program in Phylip package version 3.696 . The maximum-likelihood tree was constructed by PhyML  V3.1 (−−datatype aa --model WAG --bootstrap 1000) with 1000 bootstrap replicates to infer phylogenetic relationship of S. oryzae to other Ascomycetes fungi (M. oryzae, F. graminearum, A. chrysogenum, and F. oxysporum). The consensus tree was drawn using FigTree V1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/). The phylogenetic analysis results are deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S19046) and Dryad Digital Repository (doi:10.5061/dryad.674p4).
Pathogenicity genes in S. oryzae
The fungal pathogenicity genes were retrieved from the Pathogen-Host Interaction (PHI) database  (http://www.phi-base.org) and BLASTP was performed against S. oryzae proteome. Protein alignments with more than 40 % identity and 70 % query coverage were considered as putative pathogenicity genes in S. oryzae.
Signal peptides and cleavage sites of S. oryzae proteins were predicted using SignalP version 4.1 (http://www.cbs.dtu.dk/services/SignalP/) and all proteins with signal peptides were analysed for presence of transmembrane (TM) domains using web servers like Phobius (http://phobius.sbc.su.se) and TMHMM version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). Subsequently, mitochondrial and chloroplast targeting signal containing S. oryzae proteins were removed based on prediction by TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/). Finally, proteins containing a potential GPI (glycosyl phosphatidyl inositol)-anchor signal identified by PredGPI (http://gpcr.biocomp.unibo.it/predgpi/) web server were discarded.
Analysis of carbohydrate-active (CAZy) enzymes in S. oryzae proteome
The S. oryzae proteins from secretome analysis were subjected for CAZy annotation using CAT  and dbCAN  servers, which are based on the CAZy (Carbohydrate-Active Enzyme) database classification . The results from both were combined when the e-value less than 10−05 and classified as per type of reaction catalyzed like Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs), Carbohydrate Esterases (CEs), Carbohydrate-Binding Modules (CBMs), and Auxillary Activities (AAs) as described in CAZY database (http://www.cazy.org/Welcome-to-the-Carbohydrate-Active.html).
Secondary metabolite gene cluster analysis
The scaffold sequences of S. oryzae were analysed for secondary metabolites gene clusters using antiSMASH .
Cytochrome P450 family and transcription factors (TFs) analyses
Pathway analysis of helvolic acid and Cerulenin biosynthesis
We retrieved amino acid sequences of putative genes involved in helvolic acid biosynthesis from Aspergillus genome database (AspDB; http://www.aspgd.org) and protein homology search was carried out with S. oryzae genes. The genes with minimum 50 % identity and 70 % query coverage were considered as putative candidates in helvolic acid biosynthesis pathway. In addition, a homology search was also performed against NCBI non-redundant protein database to obtain homologous sequences in closely related fungal species. The protein domain based search was performed to identify putative genes involved in Cerulenin biosynthesis.
Prediction of repeats and simple sequence repeats (SSRs)
The S. oryzae scaffold sequences were subjected for de novo repeat prediction using RepeatMasker . Reference based repeats analysis was done by comparing to reference repeat library database of RepBase (http://www.girinst.org/repbase/). The whole genome of S. oryzae was analyzed to determine the distribution and frequency of various types of SSRs using Microsatellite Identification tool (MISA)  (http://pgrc.ipk-gatersleben.de/misa/). The minimum length of SSR motif was set as 10 for mono, 6 for di, 5 for tri, tetra, penta and hexa motifs.
Results and discussion
Genome assembly and annotation
The S. oryzae isolate, Saro-13 was selected for whole genome sequencing based on virulence study, and was confirmed by mycelial morphology, colony characteristics and ITS sequencing. S. oryzae (Saro-13) produced sparsely branched mycelium with orange pigmentation on potato dextrose agar (PDA) medium. The conidium was single-celled, cylindrical and hyaline in structure (Additional file 1). The ribosomal DNA internal transcribed spacer (ITS) region of S. oryzae isolate Saro- 13 was sequenced using Sanger sequencing platform. Then, the ITS sequence was analysed by BLASTN to confirm the identity of Saro-13. The top 20 hits with e-value of 0 confirmed the identity of Saro-13 isolate as S. oryzae (Additional file 2).
Whole genome assembly features of S. oryzae
A. Assembly parameters
Total genome size (bp)
Number of contigs
Maximum contig length (bp)
Minimum contig length (bp)
Average contig length (bp)
Total number of non-ATGC characters
Depth of genome coverage (x)
N50 value (bp)
B. Protein annotation
Total number of proteins/genes predicted
Total number of annotated proteins/genes
Total number of un-annotated proteins/genes
Details of coding genes, exon and intron in genome of S. oryzae
Number of coding genes
Average gene length (bp)
Gene density (number of genes per Mb)
Average distance between genes (Kb)
GC content (%) in coding region
Mean protein length (amino acids)
Number of exon
Total exon length (Mb)
Average exon length (bp)
Number of exons per gene
Number of intron
Total intron length (Mb)
Average intron length (bp)
Protein family (pfam) domains and gene ontology (GO) annotation
An InterProScan pfam analysis identified 2,820 protein families containing 7718 proteins in S. oryzae. Large number of major facilitator superfamily (212 proteins), fungal specific transcription factor (175 proteins), protein kinase (137 proteins), fungal Zn(2)-Cys(6) binuclear cluster (122 proteins), sugar transporters (120 proteins), short chain dehydrogenase (97 proteins), cytochrome P450 (93 proteins), WD domain G-beta repeat (72 proteins), FAD binding (67 proteins), methyltransferase (57 proteins), and pyridine nucleotide-disulphide oxidoreductases (50 proteins) domain containing proteins were enriched in the S. oryzae genome. Majority of these gene families are known to be involved in host-pathogen interactions, indicating S. oryzae emerging as a very important plant pathogen to study arsenal of pathogenicity genes.
Orthology, multigene families and phylogenetic relationship of Ascomycetes fungi
Pathogenicity associated genes/factors in S. oryzae
To our knowledge, pathogenicity genes/factors are not determined so far in S. oryzae genome due to lack of genomic resources. The S. oryzae infect aerial parts of the rice plant, especially uppermost leaf sheath enclosing the young panicles. To identify putative genes involved in pathogenicity, we analysed S. oryzae proteomes for pathogen-host interaction (PHI) gene database, secretary proteins, carbohydrate-active enzymes (CAZymes), secondary metabolites, transporters, and transcription factors that are required to colonize in the host tissue.
a. Putative Pathogen-Host Interaction (PHI) genes
The PHI database has collection of experimentally verified virulence associated genes from fungi, oomycetes and bacteria . All 10,526 protein sequences of S. oryzae were aligned to PHI fungal genes using BLASTP (e-value 10−10). We identified 953 (9.06 % of total genes of S. oryzae) putative PHI genes in S. oryzae spanning across 59 different fungal species. Highest number of homologs was found in Fusarium graminearum (483 genes), followed by Magnaporthe oryzae (145 genes), Aspergillus fumigatus (66 genes), Candida albicans (36 genes), Botrytis cinerea (20 genes), Cryptococcus neoformans (18 genes), Fusarium oxysporum (18 genes), and other fungal species (167 genes) (Additional files 3 and 4). We assume that these genes are putative candidate pathogenicity determinants to induce pathogenicity in S. oryzae as their role in pathogenesis is already proven in their respective host species (cross-species pathogenicity) . These preliminary results pave the way for future researchers to dissect pathogenicity genes in S. oryzae.
b. Secretory proteins
The secretome analysis of S. oryzae proteome revealed 391 proteins harboring signal peptides (SPs) (Additional file 5). The aspartyl protease domain (Asp) containing secretory proteins were enriched in the S. oryzae genome and are mainly involved in proteolytic activity (hydrolysis of peptide bonds). Another class of secretory proteins like Tyrosinase is known to be involved in melanin production. Other important domain containing secretory proteins like hydrophobic surface binding protein A (HsbA), cupin, fungal hydrophobin, and lipase were enriched in the S. oryzae genome (Additional file 6).
Overview of CAZyme and number of gene families in each CAZyme categories
Number of CAZymes proteins
Number of families
With signal peptide
Without signal peptide
d. Transcription factors (TFs)
Distribution of transcription factors in S. oryzae genome
Type of TFs
Number of TFs
C2H2 zinc finger
Heteromeric CCAAT factors
e. Cytochrome P450 enzymes and membrane transporters
The cytochrome P450 enzymes in fungi carry out a wide range of bioconversions of complex polyaromatic hydrocarbons (PAHs) and steroid compounds mediated by monooxygenase enzymes . There were 93 genes distributed across 82 various cyp gene families  in S. oryzae based on fungal cytochrome P450 database (FCPD) (Additional files 8 and 9). Genes encoding for plasma membrane transporters will help in assimilating the products degraded by CAZymes. The protein family classification of S. oryzae proteome revealed 212 genes encoding for major facilitator superfamily (MFS) and 120 genes encoding for sugar and other transporters. As compared to other gene families, MFS membrane transporters were high indicating their role in transporting small solutes in response to chemiosmotic ion gradients during pathogenesis.
f. Pathway analysis of helvolic acid and cerulenin secondary metabolites production
Another important SM produced by S. oryzae is Cerulenin and its biosynthesis is closely related to fattyacid synthesis . The structure of Cerulenin is (2S), (3R) 2,3-epoxy-4-oxo-7, 10-do-decadienoyl amide concluded based on mass and NMR spectroscopic methods . We looked at the enzymes involved in Dodecanoic acid pathway under fatty acid biosynthesis. There are six enzymes (FabD, FabB, FabF, FabG, FabA and FabZ) involved in biosynthesis of trans-dodeca-2-enoyl-[acp], an intermediary product of dodecanoic acid pathway (Additional file 11). The major protein domains of these enzymes are acyltransferase, oxidoreductase, and lyases. We identified putative candidate genes involved in Cerulenin biosynthesis based on protein domain annotation. There were 97 short chain dehydrogenase (SDR), 24 enoyl-(acyl carrier protein) reductases, 12 acyltransferase, seven beta-ketoacyl synthase, and 25 oxidoreductases genes found in S. oryzae genome. These genes are of future interest to understand its biosynthesis since Cerulenin is mainly used as antifungal antibiotic and anticancer agent that inhibits fatty acid and steroid biosynthesis [60, 61]. The knowledge of these pathway genes can be utilized for therapeutic and industrial uses, by exploring genetic engineering approaches to convert pathogenic strain to non-pathogenic strain for commercial purpose.
Repetitive DNA content
Repetitive DNA is an integral part of fungal genomes. Repeat sequences play a vital role in generating genetic diversity, genome expansion and might also be detrimental to genome with respect to genome stability . Repetitive DNA analysis was carried out on contig sequences of S. oryzae. The de novo and reference (by taking Sarocladium repeat library from repbase) based repeat analysis showed that 1.09 % of genome was repetitive. Interestingly, retro and DNA transposons were absent. However, repetitive DNA was limited to small RNA (0.03 %), simple repeats (0.70 %) and low complexity repeats (0.37 %). Then, we performed reference based repeat analysis by choosing repeat databases of closely related Ascomycetes fungi like Aspergillus, Colletotrichum, Fusarium, Gibberella, Magnaporthe and Sarocladium zeae. However, we did not observe significant increase in content of repeat elements. Many genome drafts of Ascomycetes have been assembled using short read technologies, and have reported repeat percentage in the range of 3-10 % [30, 32, 62, 63]. Thus, we believe that sequence read length (151 bp) might have not imposed a significant bias in repeat resolution. Low content of repeat elements is surprising since most of the pathogenic Ascomycetes fungi are known to harbor little higher percentage of repeat elements as compared to non-pathogenic counterparts. The low percentage of repeats can also be attributed to repeat induce point (RIP) mutations operating in the genome [64, 65]. Genome sequencing of other Ascomycetes fungi like N. crassa, F. oxysporum, A. nidulans and A. fumigatus showed lower repeat content coupled with RIP mechanism. Thus, these fungal species and S. oryzae which are closely related, might be sharing similar phenomenon like RIP in their genomes .
Identification of microsatellites in S. oryzae genome
Microsatellites or SSRs markers are highly useful for molecular identification, genetic differentiation among individuals and populations in fungi. The genome-wide identification of SSRs in S. oryzae was performed in order to enrich genomic resources for population characterization. The scanning of 32.78 Mb S. oryzae genome revealed presence of 13,212 SSRs. Of which, 10650 were simple and remaining 2562 were complex types. The major proportion of simple SSRs was mononucleotide repeats occupying 50 % of total SSRs, followed by 22.06 % dinucleotides (2349), 18.49 % trinucleotides (1969), and 3.16 % tetranucleotides (337) repeats. The remaining SSRs were complex type, with 0.82 % of penta and 0.57 % of hexa nucleotides.
Rice sheath rot disease caused by S. oryzae is an emerging disease in rice growing regions. Lack of genomic resource for S. oryzae motivated us to takeup this sequencing effort and report the first ever genome draft of S. oryzae. The whole genome sequencing and de novo assembly revealed 32.78 Mb is the genome size of S. oryzae. This genome of this fungus codes for 10,526 proteins based on ab initio gene prediction algorithm. Furthermore, functional annotation of proteins showed that 73.23 % of total genes distributed across 2,820 protein families. The gene ontology annotation showed 12.21, 39.1 and 47.33 % of genes were involved in biological, cellular and molecular functions, respectively. Comparative orthology studies revealed 8,400 genes were orthologous to other Ascomycetes fungi and remaining (2126) genes were unique to S. oryzae. Multigene families such as polyketide synthase, ABC transporters and other pathogenicity related genes were distributed across 480 orthologous groups. The expansion of these gene families through natural selection denotes survival advantage of this pathogen for acclimatization to diverse environmental conditions. The overall analysis showed that S. oryzae has large sets of pathogenicity-related genes encoding secreted effectors, proteinases, secondary metabolism enzymes, transporters, carbohydrate-active enzymes, cytochrome P450 enzymes and transcription factors. This diversification and maintenance of more number of arsenal of diverse virulence factors may be required to colonize a wider range of host species by S. oyzae. More interestingly, helvolic acid biosynthesis pathway genes were found in a single cluster encoding for cytochrome P450 monooxygenase, transferase, short chain dehydrogenase (SDR), qualene-hopene-cyclase, and 3-ketosteroid-delta-1-dehydrogenase. Genome-wide identification of microsatellites revealed that around 43.71 % of SSRs were di, tri and tetra types, which could be explored in pathogen identification and population dynamic studies. Prior to elucidation of this draft genome sequence, very little was known about molecular mechanisms involved in pathogenicity and research in this area was limited to metabolite studies. Indeed, the availability of this genome in the public domain from our sequencing effort will now allow the researchers to carry out accelerated and rational experiments to dissect Rice-Sarocladium interaction that may help to articulate better disease control measures.
The genome assembly/contigs are deposited in NCBI/DDBJ/Genbank genome database under the accession number LOPT01000000. The raw sequence reads are deposited in NCBI SRA database under accession number SRX1639538.
This project was supported by revolving fund generated by conducting training programmes in our laboratory. We acknowledge Texas A&M for awarding Monsanto’s Beachell Borlaug International Fellowship to H. B. Mahesh for his Ph.D degree programme. We also acknowledge ICAR-Sugarcane Breeding Institute, Coimbatore, India for granting three years Ph. D study leave to Mahadevaiah.
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