Tools to kill: Genome of one of the most destructive plant pathogenic fungi Macrophomina phaseolina

Genome sequencing and assembly

We examined the homology between M. phaseolina and 12 other fungal genomes under the classes of Saccharomycetes, Sordariomycetes, Agaricomycetes, and Eurotiomycetes. The results revealed that 71% of the genes in the M. phaseolina genome have homologs in other fungal genomes and the remaining 29% are orphan genes (Figure 2a). Among the orphan genes, 51.11% are found in the transcriptome.

Conserved syntenic and phylogenetic relationship

Pairwise comparison revealed that the genome structures of M. phaseolina and Fusarium oxysporum, one of the most important phytopathogenic and toxigenic fungi, have large areas of synteny (Figure 2b). Among the 14,249 M. phaseolina genes, 7,767 (54.10%) are shared with F. oxysporum (Additional file 2: Figure S1). The large number of shared genes may reflect common strategies for infecting a remarkably broad host range. Ninety seven percent of the M. phaseolina genome comprises non-repetitive sequences, and the orthologs shared with the F. oxysporum genome display an average 52% identity. A phylogenetic analysis revealed the evolutionary relationship among fungal taxa and the positioning of M. phaseolina within the pezizomycotina (Figure 2c).

Global paralog network

Of the 14,249 predicted proteins, we found homologs for 7,999 proteins in nine fungal genomes. To characterize functional protein families in the M. phaseolina genome, we constructed a database in Pathway Studio (Ariadne Genomics Inc.) containing M. phaseolina proteins with functional annotation from the orthologs in nine other fungal genomes, predicted interologs, predicted pathways, and paralog network. The largest paralog families were identified by clustering the paralog network consisting of 6,210 paralog links representing paralog pairs with sequence homology above 30%. We identified 77 paralog families having more than six proteins (Additional file 2: Figure S2). The larger paralog families were the cytochrome P450 (151), MFS type membrane transporters (222), and transposases (101). The large number of transposons in the M. phaseolina genome suggests that they could be the primary mechanism for mutagenesis and gene duplications, which in turn may promote the ability of M. phaseolina to infect new plant species.

There was also a high number of paralogs for the oxido-reductase class of enzymes which includes dehydrogenases, aldehyde dehydrogenases, choline dehydrogenases, cytochrome P450 and aldose reductases. These oxido-reductase enzymes produce and utilize a large variety of secondary metabolites [22] which may trigger M. phaseolina to survive in a wide range of physical environments and facilitate to infect new plant species. The PTH11 paralog family consists of a large number of G-protein coupled receptors (GPCR; 44), which contain a cysteine-rich fungal extracellular membrane domain. This may facilitate M. phaseolina to be more virulent.

Repetitive DNA and transposable elements

Carbohydrate degrading enzymes

Phytopathogenic fungi secrete a cocktail of hydrolytic enzymes (including carbohydrate-active enzymes; CAZymes) for degrading the plant cell wall and penetrating into the host tissue [25]. The M. phaseolina genome encodes 362 putative CAZymes including 219 glycoside hydrolases (GH), 56 glycosyltransferases (GT), 65 carbohydrate esterases (CE), 6 carbohydrate binding modules (CBM), and 16 polysaccharide lyases (PL) comprising more than 80 distinct families. These enzymes do not appear to be tightly clustered, but are distributed throughout the genome ( Additional file 2: Figure S4).

The number of GHs possessed by M. phaseolina is higher than the average for plant pathogenic fungi and is nearly four times more abundant than the GTs, consistent with the importance of carbohydrate degradation rather than formation. There are 25 putative endoglucanases (GH5, GH12, GH45, GH61), 7 exocellobiohydrolases (GH6, GH7, GH81), and 28 β-glucosidases (GH1, GH3, GH17) for the hydrolysis of cellulose. The cellulolytic activity of M. phaseolina was shown to be significantly higher than that of other fungal species (i.e., Aspergillus niger and Trichoderma reesei) [26], reflecting the pathogenicity potency of this fungus.

Genes involved in lignin degradation

Virulence associated genes

As a wide host ranged pathogenic fungus, M. phaseolina is expected to possess a significant number of pathogen-host interaction genes. We searched the genome using the pathogen-host interaction database (PHI-base) [28] and identified 537 putative PHI genes (Additional file 1: Table S5). These genes play diverse roles in pathogenesis including adhesion, signal transduction, cell wall breakdown, purine biosynthesis, and biosynthesis of the potent mycotoxin patulin. ATP-binding cassette (ABC) transporters aid in defending the pathogen from host-produced secondary metabolites as well as provide essential nutrients [29]. A number of detoxification genes are present, such as those encoding cytochrome P450 (IPR001128), Cof protein (IPR000150), and superoxide dismutase, Cu/Zn binding (IPR001424). In addition, several beta-ketoacyl synthases (IPR000794) involved in the biosynthesis of a polyketide antibiotic [30] as well as some tetracycline resistance genes have been identified in M. phaseolina. These data suggest that the M. phaseolina genome encodes a large repertoire of pathogenicity-associated genes which may be involved in the pathogenesis of this organism.

Signal transduction

The perception of environmental cues through cell-surface receptors and relaying the information to intracellular signaling pathways is essential for pathogenicity. The PTH11-like GPCR is a PHI protein shown to regulate Magnaporthe grisea appressorium differentiation in response to the plant surface [31]. The M. phaseolina genome has 44 putative PTH11-like GPCRs compared to an average of 34 in other pathogenic fungi (Table 2). The putative PTH11-like GPCRs are grouped into seven subfamilies. Three G-protein alpha subunits are present to transduce the extracellular signals leading to infection specific development, which is required for pathogenicity [32, 33]. The 140 protein kinases in M. phaseolina is above the average (131) found in other ascomycete fungi (Table 2). Since signal transduction is a crucial part of fungal development and the infection process, indeed most of the kinases had orthologs in PHI-base (134/140). This result indicates that protein kinases in M. phaseolina might play a functional role in pathogen-host interaction.

Transport and detoxification of compounds

Plant pathogenic fungi use a wide range of strategies to gain access to the carbon sources of their host plants and counter the plant defense response. The M. phaseolina genome encodes 839 transporter genes comprising 106 families ( Additional file 1: Table S6). Majority of the transporter genes (782/839) were similar to those cataloged in PHI-base. A large proportion of transporters belong to the MFS family (270), but the ABC superfamily (59) and Amino Acid-Polyamine-Organocation (APC) family (40) are also well represented in the genome ( Additional file 1: Table S6). M. phaseolina has more amino acid transporters (54) than other pathogenic fungi (29 to 38), revealing that this fungus might be able to access a wide range of protein degradation products from host sources. The sucrose and galactoside transporter (MFS superfamily) is required by Metarhizium anisopliae for rhizosphere competence but not for virulence [34]. The M. phaseolina genome has 15 sucrose and galactoside transporters, whereas Fusarium graminearum contains 12, suggesting these genes could be generally important for establishing plant-fungus relationships.

We found a relatively large number of genes involved in detoxification (Table 2). The dehydrogenases (411), acyl-CoA N-acetyltransferases (7), monooxygenases (104), and cytochrome P450s (256) were preferentially expanded in M. phaseolina. P450s play an important role in various hydroxylation and oxidation processes including secondary metabolism as well as the breakdown of toxins and other xenobiotic compounds [35]. For example, pisatin demethylase, a P450 from the plant pathogenic fungus Nectria haematococca, detoxifies a specific class of plant defense compounds [36]. The genome was particularly enriched in zinc-containing alcohol dehydrogenases (92) required for the biosynthesis of mannitol, a crucial factor for stress tolerance and virulence in the animal pathogen Cryptococcus neoformans[37]. The monooxygenases are generally involved in rapid elimination of plant polyphenols (which can act as antifungal agents), thus reducing the plant defense [38–40].

Secondary metabolic pathways

Phenotypic response in relation to different environmental stimuli

We used Phenotype Microarray (PM) analysis (Biolog Inc.) to evaluate M. phaseolina against ~960 different carbon, nitrogen, phosphorus, sulfur, nutrient supplement, peptide nitrogen, osmolytes, and pH sources (Figure 3; Additional file 2: Figures S6-S11). Of particular interest, we found that the adaptability to wide osmotic and pH ranges could be a contributing factor to this organism’s pervasive nature.

PM analysis revealed that M. phaseolina is capable of growing in sodium salt concentrations of 1-8%, with maximal growth at 2-4% (Figure 3a). In comparison, 3 % NaCl is toxic for Saccharomyces cerevisiae but is close to the optimum for growth of the halophilic black yeast Hortaea werneckii, one of the most salt-tolerant eukaryotic organisms so far described [45]. The efficient utilization of various osmolytes provides clues to M. phaseolina’s osmoadaptation strategy. It was recently shown that salinity increases the disease severity caused by M. phaseolina on Phaseolus vulgaris (common bean), by enhancing the growth rate of the pathogen as well as weakening the plant due to ion (K⁺ and Na⁺) imbalance [46]. This indicates that M. phaseolina could be a greater threat in areas with saline soils.

pH is one of the major environmental factors affecting pathogenicity. The M. phaseolina genome contains 2 putative PalH and 5 PalI proteins which are responsible for sensing ambient pH [47]. Moreover, there are several pH-regulated proteins including 19 acid phosphatases, 2 α-L-arabinofuranosidases, and 7 alkaline phosphatases. The presence of acid and alkaline phosphatases indicates that M. phaseolina has an extraordinary capability to neutralize both acidic and alkaline environments for its growth. This is clearly evident from our PM analysis. The results revealed that M. phaseolina can grow in pH ranging from strongly acidic to alkaline (pH 3.5 to 10), with maximum growth between pH 5 to 7 (Figure 3b). Therefore, M. phaseolina has a robust pH sensing system which enables it to adapt to adverse conditions.

Pathogenesis of M. phaseolina

The array of metabolic genes within the M. phaseolina genome reflects its pathogenic lifestyle (Figure 4; Additional file 1: Table S7).

The production and dispersal of conidia is important for fungal survival and infection of new hosts. Hyperosmotic stress is one of the environmental stimuli that often trigger conidiation [48]. The M. phaseolina genome encodes MPH_01444, a homolog of the MAP kinase OSM1, which regulates the osmotic stress response (to maintain cellular turgor) along with MPH_10325 and MPH_03305 (Figure 5; Additional file 1: Table S8). In M. grisea, deletion of OSM1 has pleiotropic effects including osmotic sensitivity, reduced conidiation, and overproduction of appressoria [49].

Adhesion of fungal propagules to the plant surface is the prerequisite to establish disease. We identified 8 homologs of Cellulose-Binding Elicitor Lectin (CBEL), a cell surface glycoprotein that plays a role in adhesion to host wall components [50], as well as a Class II hydrophobic protein which mediates contact and communication between the fungus and its environment [51]. M. phaseolina also has three transglutaminase-like proteins containing a 13-amino acid motif (Pep-13) that is able to stimulate the plant defense response. Presence of these cell surface proteins suggests that M. phaseolina produces PAMPs, which can be efficiently perceived by a wide range of plant species. Activation of the plant immune system results in the synthesis of antifungal peptides, inhibitors of cell wall degrading enzymes, and phytoalexins. At the same time, the fungus responds to surface inductive cues via a cAMP-dependent pathway in order to initiate the infection process and combat the plant defense response (e.g., production of salicylate-1-monooxygenase).

Invasion begins with the emergence of a germ tube from the conidium, followed by appressorium formation. The developing appressorium swells as the cytoskeleton and Golgi vesicles accumulate in the tip. In bean rust pathogen Uromyces appendiculatus, the cytoskeleton and vesicles in the apex of the hypha are reorganized along the cell wall within 4 minutes of signal perception [52]. Enzymes for fungal cell wall synthesis (e.g., chitin synthase and β-1,3-glucan synthase) as well as cell wall degradation (e.g., cutinase and β-1,4-endoglucanase) are contained within different vesicles. M. phaseolina also synthesizes phytosphingosine and phytoceramide to protect the cell membrane from mechanical damage during penetration of the host cell. High turgor pressure is generated within the appressorium, allowing the penetration peg to break through the plant epidermis. Secretion of a variety of cell wall degrading enzymes and other toxins aid in the invasion of the host. These hyphae penetrate epidermal walls directly and subsequently colonize the tissue by intra- and intercellular growth (Figure 1c).

Regulation of these invasion processes involves both cAMP-dependent and MAP kinase pathways (Figure 5). Surface recognition and appressoria initiation is dependent on the adenylate cyclase MAC1 (MPH_00975). Early research revealed that deletion of the MAC1 gene blocked appressorium formation in M. grisea[53]. The catalytic subunit of cAMP-dependent protein kinase A (MPH_07566 and MPH_00397) is also required for appressoria formation and penetration. Furthermore, the M. phaseolina genome contains heterotrimeric G-proteins (MPH_02326, MPH_07066, MPH_00770, MPH_05860 and MPH_09815), which could also contribute to the signaling pathways for pathogenesis. The later stage of plant penetration is regulated by the MAP kinase PMK1 (MPH_06596). A PMK1 mutant of M. grisea is unable to make appressoria in rice plants and is non-pathogenic. Therefore, PMK1 is specifically involved in the regulation of appressorium formation in response to surface signals and is also necessary for invasive growth [54].

Strain, growth condition, and nucleic acid isolation

M. phaseolina strain MS6 was isolated from an infected jute plant at Bangladesh Jute Research Institute (BJRI), Dhaka. Strain MS6 is the most virulent among the 19 isolates so far isolated in BJRI. This strain has mycelium that is grey-white at the initial stage and turns dark green at the mature stage. It is coarse with feathery strand. The sclerotia are embedded in the strand. The fungus was cultured at 30°C in liquid potato dextrose medium. DNA and RNA were extracted as previously described [57, 58], respectively.

Genome sequencing and assembly

Whole-genome shotgun sequencing of the M. phaseolina MS6 strain was performed using the 454 and Illumina sequencing platforms. We generated a total of 6.92 Gb raw data, having 40.56 millions of raw reads. In the 454 sequencing strategy, both single-end (SE) and paired-end (PE) genomic libraries were constructed, of which 2.38 Gb of shotgun sequences provided 48.29x coverage and 1.57 Gb of 8 kb, 15 kb, and 20 kb PE sequences provided 31.95x coverage of the M. phaseolina genome. In the Illumina sequencing strategy, 2.97 Gb of PE libraries were generated, of which 1.73 Gb of 500 bp PE sequences provided 35.11x coverage and 1.24 Gb of 3 kb mate-paired sequences provided 25.14x coverage of the M. phaseolina genome.

We produced a high quality assembly of the M. phaseolina genome using Newbler assembly program version 2.5.3 (http://my454.com/products/analysis-software/index.asp). For both de novo assembly and reference mapping of the M. phaseolina genome, we only used raw data generated from 454 pyrosequencing. For de novo assembly, we fed the Newbler GS de novo assembler first with the shotgun sequences in one-step form and later with paired-end sequences incrementally in order to get better contigging and scaffolding. About 96.50% raw reads were assembled into 3,036 contigs and 94 scaffolds having 98.92% bases with Q40 plus bases. For reference mapping, we used Newbler GS reference mapper to map the raw sequence files onto the all contigs file generated that gave 98.89% reads and 99.11% bases mapped to the reference.

We also used Illumina PE sequences with GapCloser version 1.10, a tool from SOAP de novo (http://soap.genomics.org.cn/soapdenovo.html), in order to close the gaps inside the scaffolds that were generated from the Newbler scaffolding process. A total of 785 gaps were detected by GapCloser that cover 1.5 Mb residues of which 197 gaps were completely filled up, leaving only 1.47% (0.73 Mb) gaps inside the 94 scaffolds.

We checked the relative completeness of the M. phaseolina MS6 draft assembly version 1.0 by performing core gene annotation using the CEGMA pipeline [59]. The resulting contigs as well as scaffolds from the M. phaseolina MS6 assembly were independently analyzed through this pipeline. In both cases we have found 245 (98.79%) complete gene models out of 248 ultra-conserved core eukaryotic genes (CEGs) present in the M. phaseolina genome.

This Whole Genome Shotgun project has been deposited at GenBank under the accession AHHD00000000. The version described in this paper is the first version, AHHD01000000.

Genome annotation

We used Program to Assemble Spliced Alignments (PASA), a eukaryotic genome annotation pipeline [60], to generate potential training gene sets that were used to train other ab initio gene prediction software like Augustus v. 2.5.5 [61] and Glimmer HMM v. 3.0.1 [62] for predicting M. phaseolina genes.

A total of 13,481 gene assemblies under 11,414 gene clusters predicted by PASA, along with cDNA of M. phaseolina and other 4 closely related species (Aspergillus nidulans, M. grisea, P. marneffei, and S. cerevisiae) were used for this training purpose. Augustus and Glimmer independently predicted 12,231 and 11,432 ORFs, respectively, which were then subjected to correct gene structure annotation by EVidenceModeler (EVM) [63]. EVM, when combined with PASA, yields a comprehensive, configurable annotation system that predicts protein-coding genes and alternatively spliced isoforms.

We also used Analysis and Annotation Tool (AAT) [64] as a pipeline for transcript and protein alignments. To align the M. phaseolina genome independently, we used cDNA of its own with cDNA of other 4 related fungal species (described earlier) and fungal protein databases downloaded from Fungal Genome Research (http://fungalgenome.org/data/PEP/). These two along with PASA transcript alignments were used as transcript and protein evidences for EVM. Finally, a total of 14,249 ab initios were predicted of which 11,975 were corrected gene structures from EVM along with 2,274 genes from Augustus and Glimmer underlying the intergenic region of EVM predictions.

The ab initios were then subjected to InterProScan [65] and nr BLAST (threshold value of E < 10^-5) [66] for searching functional domains and homologies. Results revealed a total of 10,250 genes with either potential domains or homologies with other fungal proteins and 3,999 novel genes out of 14,249 predicted protein coding genes of M. phaseolina.

Transfer RNA-coding regions were searched using tRNAscan-SE [67] and rRNA was searched using RNAmmer [68]. Repetitive elements were predicted by using RepeatMasker (http://www.repeatmasker.org/) and Putative Transposon elements were identified by Transposon-PSI (http://transposonpsi.sourceforge.net), a program that performs tBLASTn searches using a set of position specific scoring matrices (PSSMs) specific for different TE families.

The genomes of M. phaseolina and F. oxysporum were compared using MUMmer [69] tools to identify regions of synteny, with Aspergillus fumigatus used as a reference genome. For visualizing multiple genome comparisons, SyntenyMiner (http://syntenyminer.sourceforge.net/) was also used to visualize orthologous gene clusters among the organisms.

To identify proteins involved in carbohydrate metabolism, we used the Carbohydrate Active Enzymes (CAZy) database (http://www.cazy.org/). All CAZy related GenBank accession numbers were first downloaded from the CAZy website and then sequences were downloaded from NCBI using a custom python script. These sequences were searched by RPS-BLAST against the Pfam database to reveal protein domain architectures and compared against the Pfam domains identified in M. phaseolina proteins. The sequences were also compared by BLASTp against all M. phaseolina proteins to confirm the Pfam database matches.

Putative secondary metabolites (PKS and NRPS) were identified by using antiSMASH [70]. Pathogenicity and virulence associated genes were identified using the PHI-base database (http://www.phibase.org/), a database that catalogs experimentally verified pathogenicity, virulence, and effector genes from fungal, Oomycete, and bacterial pathogens which infect animal, plant, fungal and insect hosts. Briefly, all sequences were first downloaded from the PHI-base database and then compared by BLASTp against all M. phaseolina proteins to confirm the presence of homologous genes in the M. phaseolina genome.

The in silico predictions were manually curated and tested experimentally with several larger gene families such as CAZymes and lignin degrading protein coding genes by PCR.

Construction of phylogenetic tree

Orthologous relationships were determined for a 14-way clustering from the complete genomes of the 14 fungal taxa: Aspergillus nidulans, Fusarium oxysporum, Penicillium chrysogenum, Grosmannia clavigera, Magnaporthe grisea, Podospora anserina, M. phaseolina, Botryosphaeria dothidea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Yarrowia lipolytica, Saccharomyces cerevisiae, and Trichoderma reesei. All predicted protein sequences for the genomes of these fungi were searched against each other using BLASTp and clustered into orthologous groups using MCL-10-201. Single-copy orthologs were identified as the clusters with exactly one member per species. Phylogenetic relationships were determined from these single-copy orthologs and were aligned with MAFFT [71]. Alignments were pruned with Gblocks [72].

The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model [73] and bootstrap value was 1,000. Evolutionary analyses were conducted in MEGA5 [74].

Metabolic pathway reconstruction

The Yeast-5 Pathway Studio database (Ariadne Genomics) contains molecular interactions extracted by MedScan natural processing technology for all fungal species from over 1,000,000 Pubmed abstracts annotated with Medical Subject Headings (MeSH) term “Yeast OR Fungi” and from more than 100,000 full-length open access articles. Proteins in the Yeast-5 database are annotated with Entrez Gene and GenBank identifiers from six fungal genomes: S. cerevisiae, Schizosaccharomyces pombe, Cryptococcus neoformans var. neoformans JEC21, A. fumigatus Af293, A. nidulans FGSC A4, and Aspergillus niger CBS 513.88. To facilitate analysis of the M. phaseolina genome, we have added to the Yeast-5 database annotated proteins from recently sequenced genomes for Ustilago maydis 52 [75], Metarhizium anisopliae ARSEF 23, and Metarhizium acridum CQMa 102 [76].

The Yeast-5 database contains a collection of 303 metabolic pathways copied from MetaCyc. Pathways are represented as a collection of complex database entities called functional classes (enzymes) and a set of corresponding Chemical reactions. Every functional class in the database can contain an unlimited number of protein members performing corresponding enzymatic activity. Usually a set of members includes paralogs of catalytic and regulatory subunits necessary to perform enzymatic activity. The original Yeast-5 database has 444 functional classes with members out of a total of 769 pathways. We augmented the Yeast-5 database with an additional 168 metabolic pathways from RiceCyc and PoplarCyc to increase the pool of candidate enzymatic reactions for metabolic reconstruction of M. phaseolina.

The database of M. phaseolina interologs (predicted interactions) and reconstructed pathways was created by annotating proteins in the Yeast-5 database with M. phaseolina ortholog identifiers. Orthologs for M. phaseolina proteins in other fungal organisms were calculated using the best reciprocal hit method from full length protein sequence similarities calculated from BLAST alignments as described previously [77]. First, orthologs were calculated between M. phaseolina and each of the nine fungal genomes supported in the Yeast-5 database. The best ortholog was then chosen for each M. phaseolina protein among the nine possible ortholog pairs. All interactions extracted for M. phaseolina orthologs were exported from the Yeast-5 database along with pathways containing M. phaseolina orthologs. M. phaseolina interologs and predicted pathways were imported into a new Pathway Studio database for manual pathway reconstruction and genome analysis. Pathways that contained at least one functional class with no M. phaseolina orthologs were manually curated to achieve one of the following three outcomes: a) close the gap by finding members in the M. phaseolina genome and adding them to empty functional classes, b) dismiss entire pathway if gap cannot be closed, or c) remove enzymatic step if empty functional class represents redundant path in the pathway.

To identify paralog families in the M. phaseolina genome, we used BLASTP to calculate all possible protein homologs in the M. phaseolina genome and then selected only homologs that have 30% shared amino acid similarity calculated as the average sequence similarity between two homologs. Paralog pairs were imported into the M. phaseolina database as a new type of interaction called “Paralog”. Protein functional families were identified as clusters in the global Paralog network using the direct force layout algorithm. To assign biological function to each Paralog cluster we found Gene Ontology groups enriched by the proteins in the cluster or simply inspected available functional annotation for proteins in the cluster.

Phenotype microarray analysis

Phenotype microarray (PM) is a high-throughput technique for screening the response of an organism against various substrates. M. phaseolina was evaluated using panels PM1 to PM10 (Biolog Inc.). The PM plates are denoted as PM1 and PM2A MicroPlates for Carbon sources; PM3B MicroPlate for Nitrogen sources; PM4A MicroPlate for Phosphorus and Sulfur sources; PM5 MicroPlate for Nutrient supplements; PM6, PM7, and PM8 MicroPlates for Peptide nitrogen sources; PM9 MicroPlate for Osmolytes; and PM10 MicroPlate for pH. There are 96 wells in each plate, so the substrate utilization patterns by the fungus were evaluated against a total of 960 substrates including 9 negative and 4 positive controls (see Additional file 3 for list of substrates).

M. phaseolina was grown on potato dextrose agar (PDA) at 30°C for 72 hr. Active hyphae was inoculated into 30 ml of liquid potato dextrose medium and incubated at 30°C for 60 hr. The mycelia from the liquid culture (~2 g) were washed with physiological buffer solution (10 mM sodium phosphate, pH 7.0, filter sterilized) at least 7 times to remove nutrient contamination. After washing, the mycelia were aliquoted into two 1.5 ml microcentrifuge tubes and macerated by a pellet pestle motor for 15 minutes. One ml of filamentous fungi inoculating fluid (FF-IF, Biolog) was added into each tube. The solution was transferred into a 15 ml falcon tube and 6 ml of FF-IF was added. The macerated mycelia were centrifuged at 3500 rpm for 5 minutes and the supernatant was discarded. Six to 8 ml of FF-IF was added to the tube and let stand for at least 40 minutes to settle down the bigger mycelial clumps. Approximately 2 ml from the clear upper portion was harvested for measuring the transmittance at OD_590nm. The transmittance was adjusted to 62% for usable concentration of inoculums.

The inoculum suspensions along with different stock solutions were prepared as per the protocol standardized by Biolog (“PM Procedures for Filamentous Fungi”, 25-Aug-07). All the wells of PM 1–10 were inoculated with 100 μl of the inoculum suspensions and incubated in the OmniLog machine at 30°C for 96 hr. The instrument was programmed for recording data from each well in 15 minute intervals. After completion of incubation, the recorded data were extracted and analyzed using TIBCO Spotfire v23.0.0.320. The experiment was replicated three times.

It was observed that there was almost no growth in all the plates up to 40 hr of incubation. We therefore considered the 40 hr incubation readings as the baseline in our analysis. The data for each 8 hrs from three replications were averaged for analysis. Based on the 96 hr reading, all the figures (Figure 3; Additional file 2: Figures S6-S11) were constructed using OmniLog value ≥ 200.

Verification of lignin degradation

We measured the ability of M. phaseolina to degrade lignin on modified Boyd and Kohlmeyer (B&K) agar medium containing 4 mM guaiacol along with 0.001% azure B dye [78]. The medium was inoculated with this fungus and incubated at 30°C in the dark. After 4 days of incubation, a halo of intense brownish white color was formed under and around the fungal colony, and the azure B dye turned from blue to white (Additional file 2: Figure S5). The growth of intense brownish white color fungal colony indicates a positive reaction resulting from guaiacol oxidation [79]. The disappearance of the blue colored medium is also evidence of peroxidase production ( Additional file 2: Figure S5). The discoloration of azure B dye has been positively correlated with the production of lignin peroxidase and Mn dependent peroxidase, but it does not indicate the presence of laccase [80].