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Talaromyces trachyspermus, an endophyte from Withania somnifera with plant growth promoting attributes

  • Sharda Sahu
  • Anil PrakashEmail author
  • Kishor Shende
Original Article
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Abstract

The medicinal plant, Withania somnifera is attributed by valuable medicinal properties and is widely cultivated. It is a need to take care of this plant from synthetic agrochemicals that may be hazardous for health and environment. The aim of the present study was to isolate and screen the endophytic fungi of W. somnifera that  have potential of plant growth promotion and antagonism against plant pathogens. In this study, 22 potential fungal endophytes comprising of species of Alternaria, Aspergillus, Fusarium, Nigrospora, Colletotrichum and Talaromyces identified at National Fungal Culture Collection of India (NFCCI), Pune were isolated. The potential isolate, Talaromyces trachyspermus was confirmed by BLAST and phylogenetic analysis of sequences of rDNA ITS, LSU (D1 D2) and β-tubulin genes. Among all the isolates, T. trachyspermus exhibited comparatively higher activity for hydrolytic enzymes, protease, chitinase, amylase, cellulase and pectinase that are required for antagonistic property. It was observed to be a promising biocontrol agent against plant pathogen, Sclerotinia sclerotiorum. This strain is also characterized with high level of indole acetic acid (IAA), siderophore synthesis, and phosphate solubilization activities that are important for plant growth promotion. This is the first report on endophyte, T. trachyspermus from W. somnifera having potential plant growth promoting traits and biocontrol, which can be further exploited to enhance the medicinal value of the plant.

Keywords

Talaromyces trachyspermus Sclerotinia sclerotiorum Siderophore Phosphate solubilization 

Introduction

The genus Talaromyces belongs to fungal species producing asci in chains. Benjamin (1955) reported Talaromyces as the species transferred from the imperfect genus Penicillium. Talaromyces differs from the other perfect penicillate genus Eupenicillium by its soft ascomata and form a monophyletic group that is distinct from the genus Penicillium based on the nuclear ribosomal internal transcribed spacer (ITS) regions, small subunit nuclear ribosomal DNA, and/or large subunit ribosomal DNA (Berbee et al. 1995; Ogawa et al. 1997; Peterson 2000). Samson et al. (2011) described Penicillium subgenus Biverticillium and the genus Talaromyces as a taxonomically unified group, and transferred all accepted species of Penicillium subgenus Biverticillium to the genus Talaromyces in accordance with the concept of unified nomenclatural system of fungi. Talaromyces species have been isolated from diversified resources like soil samples (Fang and Shi 2016; Adhikari et al. 2015), food samples (Tranquillini et al. 2017), marine sponges of coral reefs (Dethoup et al. 2015), coastal plant roots (Kim et al. 2014) and as plant endophytes (Qadri et al. 2013; Romão-Dumaresq et al. 2016). Endophytism, the unique associations of plant and its internally residing symptomless microbes can result in positive effects with respect to plant growth and tissue differentiation, as well as the biotic and abiotic stresses to which the host plants are subjected to (Saikkonen et al. 1998; Schulz and Boyle 2005). Moreover, endophytic fungi can benefit the plant host by providing nutrients and competing with pathogenic organisms (Singh et al. 2011) resulting in the reduction of chemical use in the agriculture, protection of agro-ecosystems and biological resources which are very important factors for a sustainable agricultural system.

Withania somnifera, a medicinal perennial shrub known for its ability to synthesize numerous bioactive secondary metabolites, is a widely growing species with remarkable ecological adaptability; it thrives in many parts of Asia, Europe, and the Americas. In addition to its economic importance as a medicinal plant, the endophytic fungi that associates with W. somnifera has also been studied for its numerous applications (Pandey et al. 2008). The endophytic fungi symbiotically produce phytohormones and valuable secondary metabolites responsible for plant growth promotion (Mane and Vedamurthy 2018). Present study was carried out to isolate the fungal endophytes that harbour the parts of medicinal plant, W. somnifera. The aim of the study was to examine the plant growth-promoting (PGP) properties such as indole acetic acid (IAA) and siderophore production, phosphate solubilization and antagonism against fungal phytopathogens of W. somnifera. This study was also focused on the isolation of agriculturally important fungal species, Talaromyces trachyspermus from the leaves of the W. somnifera. Many studies have shown that T. trachyspermus is capable to control some important soil borne pathogens such as Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum and Sclerotinia rolfsii in several crops including cotton, potato, tomato, eggplant and beans (Dutta 1981; Madi et al. 1997; Tjamos and Fravel 1997; Menendez and Godeas 1998; Naraghi et al. 2006, 2012).

Materials and methods

Isolation and culture of endophytic fungi

Samples of plant parts were collected from wild growing W. somnifera commonly known as Ashwagandha, a medicinal plant from Bhopal region, Madhya Pradesh (India). The parts, leaves, root, stem and branches of plant were brought to the laboratory in sterilized bags and processed within a few hours after sampling. Prior to isolation of endophytic fungi, the fresh and healthy parts of the plants were washed with tap water to remove soil, dust and debris and then cut into small pieces by a sterilized blade under aseptic conditions. Each sample was surface sterilized by 70% ethanol for 1 min and after that immersed in 4% sodium hypochlorite (NaOCl) solution for 30 s to 1 min. The samples were rinsed with sterile distilled water for 1 min and then allowed to surface dry on filter paper. After proper drying, pieces of plant parts were inoculated in Potato Dextrose Agar (PDA) plate (Hawksworth et al. 1995) supplemented with the antibiotic, streptomycin sulphate and incubated at 28 ± 2 °C for 7 days. The pure colonies obtained after incubation were transferred to PDA slant.

In vitro antagonistic activity of fungal endophytes

The isolated endophytic fungal strains were screened for antagonistic properties against the phytopathogen S. sclerotiorum AP301 (Accession no: TF-2363) by dual culture technique (Szekeres et al. 2005). The culture of phytopathogen S. sclerotiorum AP301 (Accession no: TF-2363) was obtained from National Bureau of Agriculturally Important Microbial Culture Collection (NBAIMCC), Mau India. The plates were observed regularly and antagonism was expressed in terms of inhibition zone at the point of interaction.

Plant growth promoting characteristics of fungal endophytes

The fungal endophytes were analyzed for their plant growth promoting characteristics viz., qualitative and quantitative production of IAA (Bric et al. 1991) and their ability to solubilize phosphates (Pikovskaya 1948). Also siderophore production was measured (Schwyn and Neilands 1987; Sayyed et al. 2007) in terms of  percent of siderophore unit (%SU) after measurement of optical density of sample (S) and reference (R) using 630 nm UV spectrophotometer (Patel et al. 2018):
$$\% {\text{SU}}\, = \,\left( {{\text{S}} - {\text{R}}} \right)\, \times \, 100/{\text{R}}$$

Characterization of potential endophytes

The potential endophytic fungi were identified at National Fungal Culture Collection of India (NFCCI), Agharkar Research Institute, Pune by examining their macroscopic characteristics like morphology of fruiting bodies and microscopic characteristics like spore morphology. Morphological characterization was done on the basis of color, margin, reverse pigmentation and texture (Rifai 1969). Antagonistic endophytic fungi were characterized by functionally employing plate assays for protease (Shakeri and Howard 2007), lipase (Sierra 1957), chitinase (Thirumurugan et al. 2015), amylase (Paterson and Bridge 1994) cellulase (Teather and Wood 1982) and pectinase (Paterson and Bridge 1994).

Morphological analysis of selected fungus

The obtained most potential endophytes were also characterized by polyphasic approach according to Yilmaz et al. (2014). The macroscopic characters were observed on various growth medium with different conditions. Inoculation of culture was done on Czapek yeast extract agar (CYA), CYA with 5% NaCl (CYAS), creatine sucrose agar (CREA), dichloran 18% glycerol agar (DG18), oatmeal agar (OA), malt extract agar (Oxoid; MEA) and yeast extract sucrose agar (YES) medium (Samson et al. 2010). All the plates were incubated at 25 °C for 7 days for development of ascomata. Further, according to Samson et al. (2011) the cultures on CYA and MEA media were incubated at 30 °C for the ascomata development. The diameters of the colony, degree of sporulation, production of soluble pigments and reverse colony colors were observed and noted with reference to Kornerup and Wanscher (1967). 1–2 week old colonies on MEA were used for microscopic preparations. The observation of asci, ascospores and ascomata were done with the OA plate by using lactic acid (60%) as the mounting fluid.

DNA Extraction, PCR amplification and sequencing

The ten potential fungal isolates were identified on molecular basis at National Fungal Culture Collection India (NFCCI), Pune. Total genomic DNA of the chosen endophytic fungus was extracted directly from actively growing mycelium in potato dextrose broth (PDB), using cetyl trimethyl ammonium bromide (CTAB) method (Sambrook and Russell 2001). DNA amplification was performed by PCR using primer pair ITS4 and ITS5. The LSU (D1 D2) region of rDNA was successfully amplified using fungal universal primers LROR and LR7 and β- tubulin gene was successfully amplified using primers Btu-F and Btu-R. PCR was set up with ABI-BigDye Terminator v3, 1 Cycle Sequencing Kit. The raw sequence from ABI 3100 automated DNA sequencer was manually edited for inconsistency.

Sequence homology and phylogenetic analysis

The study was focused on fungal isolate Talaromyces trachyspermus 4014 for further analysis. The ITS, LSU and β-tubulin gene sequences were compared against RefSeq nucleotide sequence database of NCBI (National Center for Biotechnology Information) by BLASTN (Altschul et al. 1990) homology search tool. The taxonomical status and phylogenetic relationship is confirmed by phylogenetic analysis of ITS, LSU and β-tubulin sequences of T. trachyspermus 4014 with the respective set of homologous sequences of maximum similarity (> 97%) in BLAST analysis. The respective set homologous sequences were retrieved and aligned using ClustalW (Higgins et al. 1994) program of MEGA6 software (Tamura et al. 2013). The clustering of the selected sequences in each gene set were done by Maximum-Likelihood method (Felsenstein 1985) using Kimura’s 2—parameter distance model (Kimura 1980) in MEGA6 software. Test of phylogeny was applied by BootStrap method with 1000 replicates (Sneath and Sokal 1973).

Results and discussion

Isolation and PGP characterization

The population of endophytes of W. somnifera plant has been studied widely by many researchers. The endophytes are considered as the unexplored potential source of bioactive compounds (Strobel et al.1996; Strobel and Daisy 2003). Khan and Tenguria (2015) reported endophytes, Alternaria sp., and Aspergillus sp. Pandey et al. (2018) reported the endophytes such as, Penicillium sp, Aspergillus sp., Trametes sp, Sarocladium sp., Penicillium sp, Colletotrichum sp., Ceratobasidium sp. and Hypocrea sp. Sathiyabama and Parthasarathy (2018) reported endophytic Talaromyces pinophilus from leaves of W. somnifera which is capable of producing withanolides, secondary metabolites important in treatment of cardiovascular disease. In the present study 22 plant growth promoting fungal (PGPF) endophytes of W. somnifera (L), comprising of species of Alternaria, Aspergillus, Fusarium, Nigrospora, Colletotrichum and Talaromyces which have been identified at NFCCI Pune, are reported. All the fungal isolates were screened for multiple plant growth promoting traits i.e., IAA synthesis, siderophore production, phosphate solubilization, antagonistic property, synthesis of enzymes, protease, lipase, chitinase, amylase, and cellulase (Table 1). Most of them exhibited one or more plant growth- promoting activity (Table 1) out of which T. trachyspermus was the most potent for PGP attributes. It produced significant amount of plant growth regulating metabolites, IAA (69 ± 0.05), siderophore (80%SU) and phosphate solubilization (550 ± 0.4 µg/ml) and showed inhibition of phytopathogen (19 mm zone of inhibition in 24 h). Sayyed et al. (2007) observed the growth promotion in W. somnifera plant by siderophore producing PGPR, Alcaligenes faecalis. The lack of resistant germplasms of economically important crops leads to serious diseases in them caused by phytopathogens such as S. sclerotiorum (Verma et al. 2014). Such plant can be protected by symbiotic endophytes, such as T. trachyspermus by releasing antagonistic compounds that are active against various plant pathogens (Dethoup et al. 2015). In this study we observed that T. trachyspermus showed a good result in inhibiting the growth of S. sclerotiorum AP301 (Fig. 1) and further this genus has never been reported for plant pathogenicity. Out of 22 isolates T. trachyspermus exhibited positive results for synthesis of enzyme protease, lipase, chitinase, amylase, cellulase and pectinase. The enzymes, chitinase, protease and cellulase are hydrolytic enzymes that carry out the cell wall lysis. This attributes of fungal isolate accounts for potential defence mechanism against the phytopathogens by degradation of their cell wall and inhibition of growth (Jadhav and Sayyed 2016).
Table 1

Plant growth promoting attributes of endophytic fungal isolates

Isolates

IAA (mg/ml)

Siderophore (S.U%)

Phosphate

solubilizing efficiency

% Growth inhibition (24 h)

Protease

Lipase

Chitinase

Amylase

Cellulase

Pectinase

Alternaria sp.

6.2 ± 0.8

56

52 ± 0.07

13

++

+

+

+

+

+

Alternaria sp.

4.6 ± 0.02

20

20 ± 0.03

10

+

+

+

++

++

Alternaria sp.

11

5.4 ± 0.1

10.4

+

Alternaria alternate a

49.8 ± 0.06

71

17.3 ± 0.00

11.3

++

++

+++

+++

+

Alternaria alternate a

55.5 ± 0.03

61

22 ± 0.1

15

++

++

+++

++

+

Alternaria alternate a

63.2 ± 0.06

69

71.4 ± 0.09

12

++

++

+++

++

+

Alternaria pluriseptata a

55 ± 0.08

56

310 ± 0.07

16.4

+

+

+

+++

++

+

Aspergillus sp.

6.3 ± 0.05

32

32 ± 0.05

12.2

+

+

+

+

+

Aspergillus sp.

6.7 ± 0.05

45

14 ± 0.01

9.4

+

+

+

Aspergillus sp.

4.6 ± 0.04

69

22.2 ± 0.04

10

++

+

Aspergillus sp.

14

+

++

+++

++

+

Collectotricum gloesporioides a

43 ± 0.04

68

60 ± 0.03

11.3

++

+

+

+

Fusarium sp.

8 ± 0.03

12

7 ± 0.03

14

++

+

Fusarium sp.

63

50 ± 0.02

10

+

+

++

+

Fusarium moniliform a

1.9 ± 0.04

70

110 ± 0.01

23.4

+

++

+

+++

+

Hyaline vegetative a

43.8 ± 0.01

26

8 ± 0.2

15

+

Nigrospora sp.

6.3 ± 0.06

72

70 ± 0.03

10.2

+

+++

++

+

Nigrospora oryzae a

59 ± 0.2

70

75 ± 0.1

17

+

+

++

+++

+

+

Talaromyces trachyspermus a

69 ± 0.05

80

550 ± 0.04

19

+

+

+

+++

+++

+

Vegetative forma

44.8 ± 0.01

79

205 ± 0.03

10

+

Vegetative form

6.5 ± 0.02

11 ± 0.1

10

+

++

Vegetative form

5.3 ± 0.03

56

36 ± 0.01

9

aThe fungal species were found most potential and were identified from NFCCI Pune on the basis of morphological identification and deposited at national fungal culture collection centre Pune

Where − = negative; + = good positive; ++ = very good; +++ = excellent

Fig. 1

Plant growth promoting characteristics of T. trachyspermus, phosphate solubilization on pikovskaya medium (a), Siderophore production on CAS agar plate (b), antagonistic effect against S. sclerotiorum AP301 (c) and inhibition effect of extract of T. trachyspermus against S. sclerotiorum AP301 (d)

Morphological Identification

The identification of the potential isolates were done on the basis of culture morphology and culture was submitted in NFCCI Pune and the accession number was assigned to T. trachyspermus is 4014. The culture was confirmed by physiological characterization in the laboratory, as the colonies were growing rapidly on malt agar, attaining a diameter of about 3.5 cm within 2 weeks at 25° C. It consisted of a basal felt in which after 1 week numerous white to creamish ascomata developed (Fig. 2), overgrown by a floccose to funiculose mycelium. Ascomata initially white, became creamish to yellowish with age, globose, 50–350 µm in diameter, occasionally giving a greenish appearance to the colony. T. trachyspermus has restricted growth on media CYA, YES and DG18 and has slightly faster growth on MEA, and unable to grow on CREA media. Specific species produced abundant red pigments. Conidiophores are generally biverticillate and ascomata, when produced, have a creamish white or yellow colour. Talaromyces atroroseus, T. minioluteus and T. albobiverticillus are biotechnologically important, with their pigments being used as colorants in the food industry (Frisvad et al. 2013).
Fig. 2

Polyphasic approach for identification. Isolated T. trachyspermus in PDA medium (a), microscopic observation under 100X (b), colony on CYA medium (c), colony on MEA medium (d), colony on DG18 (e), colony on OA medium (f), colony texture and ascomata on CYA medium and OA medium, respectively after 2 week incubation (g and h)

Molecular identification and phylogeny of isolated Talaromyces species

Total 10, out of 22 potential endophytic fungal species isolated from W. somnifera were identified in NFCCI, Pune laboratory. The potential endophytic fungus, T. trachyspermus 4014 was further characterized to confirm its identity by sequence homology method using ITS (KX641007), LSU (KX668277) and β-tubulin gene (accession number MF509777) sequence.

ITS sequences homology based Identification and Phylogeny

Total 50 representative homologous sequences of ITS rDNA above 97% similarity were selected for cluster analysis. These comprises of species T. trachyspermus, T. assiutensis, T. gossypii, T. aurantiacus, T. stipitatus, T. purpureus, T. udagawae, T. minioluteus, Paecilomyces tenuis, Penicillium rubicundum, P. cecidocola, P. marneffei, P. diversum, P. purpureus and P. minioluteum and T. trachyspermus 4014 (accession number KX641007). The phylogenetic tree (Supplementary Fig. 3) is divided into two main clusters as, Talaromyces species and Paecilomyces and second one is of Penicillium and few species of Talaromyces. Talaromyces genus is divided from Penicillium (Samson et al. 2011) but still many sequences of Penicillium species are similar to Talaromyces species. Clade one is clearly divided into two sub-clades. First sub-clade comprises of T. trachyspermus and T. assiutensis inclusive of T. trachyspermus 1014 isolated from W. somnifera with 99% similarity. Second sub-clade comprises of T. trachyspermus, T. assuitensis, T. gossypii, T. aurantiacus, P. tenuis isolated from different sources and different countries and different parts of India. Taxonomy of Talaromyces species is always a controversial point and many researchers supported theme of classification (Frisvad et al. 2013; Yilmaz et al. 2014; Samson et al. 2014, 2016). In this study, based on ITS regions homology, the endophytic fungal isolate is confirmed as T. trachyspermus with PGP properties, as also proposed by various researchers (Schocha et al. 2012; Samson et al. 2014, 2016).

T. trachyspermus, T. siamensis and T. cnidii have identical ITS sequences, while there is lack of support in most branches of the phylogeny. Another example is T. assiutensis and T. trachyspermus, classified in section Trachyspermi which also have identical ITS sequence. Overall ITS does, however, perform very well for species recognition in the genus, even though it should be used cautiously since variability is low in a few clades (Yilmaz et al. 2014). A set of total 66 sequences of ITS rDNA of 21 different species comprised of 10 Talaromyces sp., eight Penicillium sp; two Paecilomyces and one Sagenomella sp. were retrieved from NCBI nucleotide RefSeq database. This was included with ITS rDNA sequence of T. trachyspermus isolated from W. somnifera plant. In the Figs. 3 and 4, tree is divided into two main clusters. One cluster of 99% similar and second one is 100% similar. First clade comprises of 21 sequences of different strains of T. trachyspermus from different geographical areas (wherever geographical data is available), two Talaromyces gossypii strains, seven Talaromyces assientensis strains, four Paecilomyces tenuis strains, one Paecilomyces cateniannulatus strains and four Termitomyces aurantiacus. T. trachyspermus isolated from W. somnifera plant from Bhopal region of India is also clustered closely with this clade of T. trachyspermus, T. assientensis and T. gossypii.

Second cluster comprises of species of genus Penicillium and Talaromyces. Many of these Talaromyces species were previously classified under the genus Penicillium and are divided into three sub-clusters, in first; strains of Penicillium marneffei, P. cecidicola and P. rubicundum are clustered with Talaromyces purpureus, T. stipitatus and Sagenomonella bohenica. In second, strains of Talaromyces purpurogenum and T. austrocalifornicus formed sub-cluster and externally linked by Talaromyces udagawa and Penicillium diversum. And the third sub-cluster comprised Penicillium purpurogenum, P. minioluteum, P. samsonii, and Talaromyces minioloteum.

LSU sequences based identification and phylogeny

Total 23 representative sequence of LSU rDNA region having > 97% similarity in BLAST analysis, comprises of 14 sequences of Talaromyces species and nine sequences of related species of Penicillium, Thermoascus and Rasamana were retrieved from NCBI (http://ncbi.nlm.nih.gov) nucleotide sequence database. Phylogenetic tree (supplementary Fig. 4) based on LSU regions of 23 taxa and of T. trachyspermus 4014 (Accession number: KX668277) was generated. Tree is divided into three clades, where cluster-1 comprises of two strains of Talaromyces flavus, two strains of T. verruculosus, T. pinophilus, T. aculeatus, T. purpurogenus, T. viridis, T. helices, T. funiculosus and T. trachyspermus strain 4014; cluster-2 comprises of Talaromyces related species as, Byssochlamys spectabilis (Paecilomyces variotii), Penicillium limosum, P.citrinum, P. simplicissimum, P. oxalicum, and P. solitum and cluster-3 comprises of three Talaromyces species as, T. bacillisporus, T. variabilis, and T. rugulosus. These may be separated from other Talaromyces species due to sequences length variation.

β–tubulin gene sequences based identification and phylogeny

The BLASTN analysis of β-tubulin gene (MF509777) partial sequence against nucleotide sequence database of NCBI showed very few hits having above 95% similarity in local alignment. The 14 sequences were retrieved and aligned including our β–tubulin gene sequence by ClustalW program of MEGA6 software. Phylogenetic tree is shown in Fig. 5 (supplementary file), which is divided into three main clusters but distantly related. Samson et al. (2014, 2016) studied the phylogenetic relationships of various Talaromyces species and derived the four sections under genus Talaromyces. Beta tubulin gene sequences were used by various researchers to derive the identity and phylogenetic relationships of Talaromyces species as, T. purpurogenus (Yilmaz et al. 2012); T. atroroseus (Frisvad et al. 2013); T. cellulolyticus (Fujii et al. 2013); T. trachyspermus (Lee et al. 2005); T. amazonensis, T. francoae, T. purgamentorum and T. columbiensis (Houbraken et al. 2016) etc. In this study Fig. 5 shows clustering of T. trachyspermus (MF509777) with T. assiutensis with 100% replication and 0.0 unit distance, which is linked to cluster of three strains of T. trachyspermus and T. subinflatus clustered with 100% similarity of replicates and 0.05 unit distance. The isolated T. trachyspermus clustered with T. assiutensis, may be due to partial beta-tubulin gene sequence of isolated T. trachyspermus (W. somnifera), but the local alignment of conserved regions are important in functional assignment to sequences and also in deriving phylogenetic relationship. The closely relatedness of the T. trachyspermus, T. assiutensis and T. gossypii have been discussed in various studies (Frisvad et al. 2013; Samson et al. 2014, 2016) and form monoclade. Other strains of Talaromyces species are grouped into two clades.

Conclusion

The prominent endophytes of W. somnifera as, Penicillium sp, Aspergillus sp., Trametes sp, Sarocladium sp., Penicillium sp, Colletotrichum sp., Ceratobasidium sp. and Hypocrea sp. were detected by various researchers. The secondary metabolite, withanolide producing endophyte, Talaromyces pinophilus reported by Sathiyabama and Parthasarathy (2018), is the only report on Talaromyces species from W. somnifera plant. The present study is the first report on T. trachyspermus an endophyte isolated from leaves of W. somnifera plant, which has shown both the antagonistic and plant growth promoting properties. On the whole, results of the present study are promising and point out that the fungal isolate may be used for biofertilization. Therefore, it would be valuable to perform further studies on the isolate in pot and field trials. Their successful application in the agriculture may diminish the use of chemicals and protect the biological resources and environment which is important for a sustainable agricultural system. Further the genome sequencing and annotation may reveal the characteristics of genome to help in understanding the important secondary metabolite synthesis pathway and gene involved in symbiotic relationship.

Notes

Acknowledgments

The first author is financially supported by DBT-Builder Programme Barkatullah University Bhopal (M.P.) The authors are grateful to Dr. S. K. Singh, Coordinator, National Fungal Culture Collection of India (NFCCI) Agharkar Research Institute Pune, for identification of fungi.

Conflict of interest

The author shows no conflict of interest.

Supplementary material

42398_2019_45_MOESM1_ESM.docx (38 kb)
Supplementary material 1 (DOCX 38 kb)

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© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Department of MicrobiologyBarkatullah UniversityBhopalIndia
  2. 2.Department of Biotechnology and Bioinformatics Center (SubDIC)Barkatullah UniversityBhopalIndia

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