World J Microbiol Biotechnol (2012) 28:31973206 DOI 10.

1007/s11274-012-1130-2

ORIGINAL PAPER

Isolation of Oxalic acid tolerating fungi and decipherization of its potential to control Sclerotinia sclerotiorum through oxalate oxidase like protein
Shivani Yadav Alok K. Srivastava Dhanajay P. Singh Dilip K. Arora

Received: 11 February 2012 / Accepted: 11 July 2012 / Published online: 4 August 2012 Springer Science+Business Media B.V. 2012

Abstract Oxalic acid plays major role in the pathogenesis by Sclerotinia sclerotiorum; it lowers the pH of nearby environment and creates the favorable condition for the infection. In this study we examined the degradation of oxalic acid through oxalate oxidase and biocontrol of Sclerotinia sclerotiorum. A survey was conducted to collect the rhizospheric soil samples from Indo-Gangetic Plains of India to isolate the efcient fungal strains able to tolerate oxalic acid. A total of 120 fungal strains were isolated from root adhering soils of different vegetable crops. Out of 120 strains a total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration. Then we examined the antagonistic activity of the 15 isolates against Sclerotinia sclerotiorum. These strains potentially inhibit the growth of the test pathogen. A total of three potential strains and two standard cultures of fungi were tested for the oxalate oxidase activity. Strains S7 showed the maximum degradation of oxalic acid (23 %) after 60 min of incubation with fungal extract having oxalate oxidase activity. Microscopic observation and ITS (internally transcribed spacers) sequencing categorized the potential fungal strains into the Aspergillus, Fusarium and Trichoderma. Trichoderma sp. are well studied biocontrol agent and interestingly we also found the oxalate oxidase type activity in these strains which further strengthens the potentiality of these biocontrol agents. Keywords Biocontrol ITS Oxalate oxidase Oxalic acid
S. Yadav (&) A. K. Srivastava D. P. Singh D. K. Arora National Bureau of Agriculturally Important Microorganisms, Mau Nath Bhanjan, Uttar Pradesh 275101, India e-mail: shivaniyadav09@gmail.com

Introduction Oxalic acid is an organic compound with the formula H2C2O4. It occurs ubiquitously in nature, sometimes as a free acid, but more commonly as soluble potassium or sodium oxalate or as insoluble calcium oxalate. Oxalic acid and oxalates are produced by many fungi, bacteria, actinomycetes and plants and whereas it occurs naturally in animals. It has been suggested that it inhibit the growth of fungi that are more sensitive to acidity or oxalate, thus having an impact on competition between fungal species. Role of oxalic acid has been reported by the many researchers in invasion of host tissue by phytopathogenic fungi (Bateman and Beer 1965; Maxwell and Bateman 1968). Oxalic acid produced by Sclerotinia sclerotiorum, during pathogenesis acts synergistically with endopolygalacturonase, lowering the pH of the infected tissues to a level optimal for the activity of this enzyme. Oxalic acid strongly chelates the calcium present in the structural pectates as a result plant tissues becomes more susceptible to invasion by Sclerotinia sclerotiorum (Punja and Jenkins 1984). Three different oxalate-degrading enzymes have been reported in fungi, bacteria and plants: oxalate decarboxylases (ODC, EC 4.1.1.2), oxalate oxidases (OXO, EC 1.2.3.4) and oxalyl-CoA decarboxylases (OXC, EC 4.1.1.8) (Svedruzic et al. 2005)). Oxalate decorboxylase is a Mncontaining enzyme that decomposes oxalic acid to formic acid and CO2 in a reaction that requires O2 (Reinhardt et al. 2003). Oxalate oxidase have a greater extent of protein sequence similarity with oxalate decoroxylase is accordingly at rst oxidized by O2. However, upon catalysis, OXO cleaves oxalic acid to two CO2 molecules together with generation of H2O2. Oxalate oxidase activity has extensively studied in plants, and as an intracellular enzyme. On the other hand, it has been also studied in two

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white-rot basidiomycetous species i.e. Ceriporiopsis subvermispora and Abortiporus biennis (Aguiar et al. 2006; Gra z et al. 2009). In fact, Ceriporiopsis subvermispora is the rst fungal species in which both ODC and OXO activities were reported (Aguiar et al. 2006; Watanabe et al. 2005). The third oxalate-cleaving enzyme, oxalylCoA decarboxylase, is a bacterial enzyme, which converts activated oxalyl-CoA to formyl-CoA and CO2 employing thiamine pyrophosphate as cofactor. Oxalate oxidase have advantage over ODC, H2O2-generating OXO can evoke defence responses and confer disease resistance in plants. In the present study, fungi were isolated from soil and screened for oxalate oxidase activity.

concentration were subsequently tested against Sclerotinia sclerotiorum. Genomic DNA isolation and ITS amplication The subsequent genomic DNA was extracted by CTAB method. PCR reactions were performed on a Biorad thermal cycler (MJ MiniTM personal thermal cycler Biorad, Hercules, USA). Primers ITS1: TCC GTA GGT GAA CCT GCG G and ITS2: GCT GCG TTC TTC ATC GAT GC were used for the amplication of internal transcribed spacer (White et al. 1990). The amplication was carried out in a 50 ll volume by mixing 5090 ng template DNA with the polymerase reaction buffer (109); 10 mM dNTPs; primers pA and pH (100 ng each) and 1.0 U Taq polymerase (Banglore Genei P.Ltd., Banglore, India). The amplication conditions were as follow: initial denaturation of 5 min at 94 C, followed by 30 cycles of 50 s at 94 C, 60 s at 55 C and 1 min 30 s at 72 C and a nal extension period of 10 min at 72 C. After amplication the PCR product was resolved by horizontal electrophoresis in 1.2 % agarose gel in 19TAE buffer. Gels were stained with ethidium bromide (10 mg ml-1) and visualized on a gel documentation system (AlphaImager Mini System, Bucher Biotec, Basel, Switzerland) and gel images were digitalized. Diversity indices The indices of fungal diversity, species richness and species evenness were calculated using the number of isolates belonging to each group based on ITS sequences. Diversity P was calculated using the Shannon index: H0 = - S {(n1/n) ln (n1/n)}, where n1 is number of isolates in each group and n is number of isolates in all groups (Shannon and Weaver 1949). ITS sequencing The PCR amplied internal transcribed spacer was puried with a Quaquick purication kit (QIAGEN India Pvt. Ltd., New Delhi- India). The DNA sequence was double checked by sequencing both strands using primers ITS1 and ITS2 for forward and reverse reaction, respectively. The nucleotide sequences were di-deoxy cycle sequenced with uorescent terminators (Big Dye, Applied Biosystems) and run in 3130xl Applied Biosystems ABI prism automated DNA sequencer. The ITS sequences were approximately 650 bb in length. Accession number The sequences were submitted to GenBank and accession numbers were assigned for nine isolates from JQ436556 to JQ436564.

Materials and methods Sampling site A total of 30 root adhering soils were collected from vegetable (Brinjal, Chilli) from different part of the eastern region of the Indo-Gangetic plains of India in December 2010. The sampling sites were identied in different locations viz., Kanpur, Allahabad, Lucknow, Bahraich, Ballia, Mau and Rampur (coordinates 26270 3900 N 80200 0000 E). Ten composite samples were made from 30 samples, collected using a soil auger along zigzag paths (zigzag sampling) to achieve randomness. Isolation The populations of rhizospheric fungal members in the soil samples were enumerated by the standard serial dilution plating technique and results were expressed per gram of soil on a moisture free basis. One gram of soil sample was added to 9 ml of sterile distilled water. The samples were diluted and appropriate dilutions were spread on potato dextrose agar and malt extract agar plate. The plates were incubated at 25 C, fungal species and the population was enumerated after 7 days of incubation. All the fungal strains were preserved for long term by lyophilisation method at our culture collection facility. Screening of oxalic acid tolerant isolates Oxalic acid tolerant fungi were screened on potato dextrose agar (PDA) and malt extract agar amended with 10, 20, 30, 40 and 50 mM of oxalic acid were used to isolate fungi. The fungal strains were isolated from soils obtained from 10 composite samples. After 710 days of incubation, 120 fungal colonies were randomly picked and were transferred to a fresh medium at least three times for purication. Fungal strains which were able to tolerate the oxalic acid

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3199

Multiple sequence alignment and phylogenetic analysis ITS sequences of the strains were compared with ITS sequences available by the BLAST search (Altschul et al. 1997). Multiple sequence alignments were performed using Clustal W (Thompson et al. 1994). Minor modications were done manually on the basis of conserved domains and columns containing more than 50 % gaps were removed. The phylogenetic tree was constructed on the aligned datasets using neighbor joining (NJ) (Saitou and Nei 1987) method using the program MEGA 4.0.2 (Tamura et al. 2007). Bootstrap analysis was performed as described by Felsenstein (1985) on 1,000 random samples taken from the multiple alignments. Preparation of mycelium crude extract and enzymatic assays After removing the fungal colonies from the agar plates by peeling the biomass from the dialysis membrane, the mycelia were homogenized in an ice-chilled motor-driven Potters homogenizer in 5 mM phosphate buffer pH 7. The homogenates were then centrifuged (10 min, 10,0009g, 4 C) and the supernatant fractions were collected and analyzed for enzymes activities and for the level of proteins. The protein concentration was determined using the Bradford method and bovine serum albumin as a standard (Bradford 1976). Oxalate oxidase (OXO) activity was determined by the formation of H2O2 by oxalate oxidase was coupled to the horseradish peroxidase-mediated oxidation of 2,20 -azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The assays were monitored by an increase in absorbance at 650 nm at 22 C (e 10,000 M-1 cm-1 for the ABTS radical product under these conditions). Assay mixtures contained 50 mM sodium succinate buffer, pH 4.0, with horseradish peroxidase (24 units; as dened by supplier), ABTS (5 mM), potassium oxalate (20 mM) and oxalate oxidase, which was added last, in a total volume of 1 ml. The enzyme was equally active in citrate and succinate buffers. The pH optimum of the enzyme was determined to be 4.0 using citrate buffers with an oxygen electrode, according to Requena and Bornemann (1999). One unit of enzyme activity (U) was dened as the amount of enzyme producing 1 mol of H2O2 per minute, and the specic activity was expressed in U mg-1 of proteins. Degradation of oxalic acid through oxalate oxidase HPLC High-performance liquid chromatography (HPLC) was performed for each sample according to a protocol by Singh et al. (2003). The HPLC system (Waters, Massachusetts

USA) was equipped with two Waters 515 reciprocating pumps, a photo diode array detector (Waters 2996) and a system controller with Waters Empower software for data integration and analysis. Reverse phase chromatographic analysis was carried out under isocratic conditions (C-18 100 A, 5 mm, 250 4.6 mm i.d.; solvent system0.5 mM K2HPO4: acetonitrile, 10:90 V/V; ow rate, 1.0 ml min) with 10 ll injection volume and detection at 214 nm. Samples were ltered through membrane lter (0.45 lm) prior to injection. Oxalic acid (1 mg/ml) was used as internal and external standards. Degradation of oxalic acid by oxalate oxidase was identied by comparing the retention time of standard compounds and by co-injection. Quantitative analysis of oxalic acid degradation was carried out by comparing peak areas of reference (oxalic acid) with those of the sample run under the same elution condition. Antagonistic activity in broth Sclerotinia sclerotiorum were grown in the potato dextrose broth (50 ml), after 3 days when mycelia were completely formed without sclerotia then fungal extract of isolates positive for oxalate oxidase activity (conrmed by HPLC) were inoculated (20 ml) and for control we added only sterile water.

Result Isolation A total 120 fungal strains were randomly selected from the potato dextrose and malt extract agar from 10 composite samples. The plates were incubated at 25 C; the fungal species were enumerated after 7 days of incubation. All the fungal strains were observed in the compound microscope for the initial identication. All the 120 strains were tentatively categorised into four different genera i.e. Fusarium, Trichoderma, Aspergillus, and Penicillium respectively. Screening of oxalic acid tolerant isolates All the fungal isolates isolated from potato dextrose agar and malt extract agar was screened for the ability to tolerate or degrade oxalic acid. A total of 80 isolates were able to grow at 10 mM of oxalic acid whereas only 15 isolates were grow at 50 mM of oxalic acid concentration (Table 1, Fig. 1). ITS sequencing and phylogenetic analysis of its data A total of nine potential isolates were selected for the ITS sequencing based on it tolerance at 50 mM of oxalic acid. ITS sequencing and phylogenetic analysis revealed that all the

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3200 Table 1 Table showing the number of fungal strains which are tolerating different concentration of oxalic (mM) Concentration of oxalic acid (mM) 10 20 30 40 50 No of fungal strains 80

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100 100

S5 Fusarium udum S6
100

100

Fusarium lycopersici

S4
100 100 100

Fusarium oxysporum S9 Trichoderma koningii

65 43 28 15
65 100 100

S8 S7 Trichoderma viride Trichoderma harzianum

isolates showed 99100 % similarity with the sequences within the GenBank. Isolates belonged to Fusarium udum, Fusarium lycopersici, Fusarium oxysporum, Trichoderma koningii, Trichoderma viride, Trichoderma harzianum, Aspergillus terreus, Aspergillus avus and Penicillium sp. (Fig. 2). Diversity indices Two typing methods (ITS sequencing and identication through microscopic examination) adopted for examining the diversity of fungal strains from rhizospheric soils of vegetable crops, have given the diverse resolution. To quantify the diversity, the data were subjected to statistical analysis of diversity indices. Shannon index of diversity (H0 ); species richness (R), and species evenness (E) (Shannon and Weaver (1949), calculated for isolates
0.1

100

S1 Aspergillus terreus (HQ449678) S2 Aspergillus flavus (HQ607957)

100

100

97 100

S3 Penicillium sp.(HE608809) Coniothyrium minitans(ATCC32870 TM) Abortiporus biennis

Fig. 2 Unrooted phylogenetic tree based on ITS gene sequences of potential strains of fungi isolated from Indo-Gangetic plains of India. The tree was created by the neighbour-joining method. The numbers on the tree indicates the percentages of bootstrap sampling derived from 1,000 replications. Bar inferred nucleotide substitutions per nucleotides

obtained from three different soil samples. The diversity index was 1.756, species richness was 9.000 and species evenness was recorded to 0.936.

Fig. 1 Some of the representative isolates growing on the Potato Dextrose Agar amended with 50 mM concentration of oxalic acid

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World J Microbiol Biotechnol (2012) 28:31973206 Table 2 Oxalate oxidase (OXO) activity after 7 days of cultivation of different fungal strains isolated from Indo-Gangetic Plains of India and standard reference strains of fungi Fungal Strains Enzyme activity (U/mg) 4.80 4.17 4.98 1.27 1.22 6.72 7.56 2.13 1.85 0.31 0.77 0.49 Fungal Strains Enzyme activity (U/mg) 0.74 0.59 0.79 0.86 0.76 0.48 0.67 0.16 0.22 0.24 4.50 1.06

3201

Antagonistic activity in potato dextrose broth Oxalate oxidase extract completely inhibit the formation of sclerotia of Sclerotinia sclerotiorum in the potato dextrose broth whereas in the positive control Sclerotinia sclerotiorum formed the sclerotia after 7 days (Fig. 5).

S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12

S13 S14 S15 S16 S17 S18 S19 S20 S21 S22 C. minitans (ATCC 32870) A. biennis (MTCC 1176)

Discussion Oxalic acid is one of the most potent toxic organic compounds secreted by the lamentous fungi. It chelates the calcium ion which results in the destabilization of the pectate polymers and so that different pectolytic enzymes secreted by the pathogen easily degrades the cell wall. Researchers have already reported that the production of oxalic acid is one of the important factors for sclerotial development and pathogenecity in Sclerotinia sclerotiorum (Donaldson et al. 2001; Godoy et al. 1990; Kesarwani et al. 2000; Rollins and Dickman 2001; Zhou and Boland 1999). Production of oxalic acid acidies the environment which favours the activity of pathogenecity related enzymes such as polygalacturonase and acidic protease produced by Sclerotinia sclerotiorum (Marciano et al. 1983; Cotton et al. 2003). However, oxalate oxidase protects the different vegetable crops by catabolising the oxalate to hydrogen peroxide and carbon dioxide (Donaldson et al. 2001; Burke and Rieseberg 2003; Kesarwani et al. 2000), whereas in grain plants like barley oxalate oxidase protects from pathogen by increasing defence mechanisms (Dumas et al. 1995). Several researchers have reported the oxalate oxidase activity in fungi, bacteria and plants (Escutia et al. 2006; Graz et al. 2009; Ren et al. 2007; Franceschi and Nakata 2005; Libert and Franceschi 1987; Hamel et al. 1999; Nakata and He 2010). The Indo-Gangetic Plain (IGP) is the most important region of India in case of the economic point of view. Topographically the plain is homogeneous, with only

Assay of oxalate oxidase The oxalic acid degrading enzyme (oxalate oxidase) was found in a total of 22 isolates ranging from 0.16 to 7.56 U mg-1 (Table 2, Fig. 3). We further selected 5 isolates having enzyme activity more than 4.0 U mg-1 to examine the oxalic acid degradation. Degradation of oxalic acid through oxalate oxidase A total of 3 potential fungal isolates were checked for the oxalic acid degradation at different time interval ranging from 10 to 60 min (Fig. 4, Table 3). Strain Fusarium oxysporum and Trichoderma viride showed highest activity among the 3 isolates (Table 3).

Fig. 3 Some of the representative isolates showing the positive activity for oxalate oxidase

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W2996 PDA 254.0 nm at 1.2

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Fig. 4 Peak area showing the degradation of oxalic acid by oxalate oxidase after 60 min of incubation period by standard cultures and fungal isolates from Indo-Gangetic plains of India

oodplain bluffs and other related features of river erosion and changes in river channels forming important natural features. Different types of vegetable crop are subjected in this region e.g. brinjal, chilli and oilseeds, like sunower and pulses, like chick pea. Sclerotinia sclerotiorum is one of the important fungal pathogen which causes disease in the economically important crops. Viewing these importances a study was conducted to search some potential fungal strains which can prevent the disease caused by the pathogen. In this study we found that a total of 80 fungal strains out of 120 were able to grow at the 10 mM of oxalic acid.

However, only 15 strains were able to grow on the 50 mM concentration of oxalic acid. In another study, it is reported that at the 16 mM concentration of oxalic mycelia biomass was 193 mg whereas the mycelia biomass was severely decreased at the higher concentration (Ren et al. 2009). The strains which were able to grow at 50 mM were further selected for the oxalate oxidase activity. We selected Coniothyrium minitans as the reference strains for the oxalic acid degradation, which has been reported by Ren et al. (2007) as the efcient degraders of oxalic acid. In contrast we found the oxalate oxidase in our study in the strain.

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Fig. 4 continued

Earlier some researchers have reported that oxalate degrading bacteria effectively suppress the infection of plant tissue by Sclerotinia sclerotiorum. It has been reported that metals toxicity is prevented by the production of oxalic acid, which forms the oxalate metal complex (Jarosz-Wilkoazka and Graz 2006), oxalic acid concentration is precisely controlled by the action of two different enzymes i.e. oxalate oxidase and oxalate decarboxylase (Svedruzic et al. 2005). In this study we found that the oxalate oxidase activity was ranging from 0.16 to 7.56 U mg-1. Although the molecular mass of oxalate oxidase from Ceriporiopsis subvermispora showing more resemblance from oxalate decarboxylase of bacterial and fungal strains whereas it has less resemblance from plant oxalate oxidase (Escutia et al. 2005). Oxalate oxidase activity is only

reported in two fungi i.e. Ceriporiopsis subvermispora, a wood rot fungi (9.3 U/mg) (Escutia et al. 2005) and Abortiporus beinnis (6.0 U/mg) (Gra z et al. 2009). Interestingly, in this study we identied the two isolates as Trichoderma viride and Fusarium sp. based phenotypic and molecular analysis showing the oxalate oxidase activity. In another study it has reported that oxalate oxidase from Ceriporiopsis subvermispora unexpectedly shared the greatest similarity to microbial bicupin oxalate decarboxylases rather than monocupin plant oxalate oxidases (also known as germins) and this study they have also showed that two fungal isoforms, one of which was heterologously expressed in Escherichia coli, are indeed oxalate oxidases that possess \0.2 % oxalate decarboxylase activity and that the organism is capable of rapidly degrading exogenously supplied oxalate (Escutia et al. 2005). It has been already

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3204 Table 3 Degradation of oxalic acid after different time interval by potential fungal strains isolated from Indo-Gangetic Plains of India and standard culture Fungal strains C. minitans Time (min) 0 15 30 45 60 S7 0 15 30 45 60 S6 0 15 30 45 60 S3 0 15 30 45 60 Abortiporus beinnis 0 15 30 45 60 Percentage of degradation (oxalic acid) 0.0 2.5 12.6 18.4 22.8 0.0 11.4 12.6 18.8 23.1 0.0 3.1 7.0 12.8 21.0 0.0 1.9 2.3 11.6 17.3 0.0 1.3 2.1 7.6 19.7

World J Microbiol Biotechnol (2012) 28:31973206

proved the oxalate oxidase activity in plant with oxalate (OxO, EC 1.2.3.4) prepared from wheat bran, based on specic oxidation of oxalate to produce H2O2 and the

H2O2 formation has been colorimetrically determined using horseradish peroxidase-catalyzed oxidation of 4-aminoantipyrine and N,N-dimethylaniline by H2O2 and this method has been tested on rice, buckwheat, soybean and oxalis leaves showing it is precise, sensitive, inexpensive, highly reproducible and simple to perform and good agreement has been obtained between this method and the HPLC (Liu et al. 2009). In this study we also conrmed the activity by this method and we also found that the fungal extract prevent the formation of sclerotia in the potato dextrose broth (Fig. 5). In a very recent study a DNA fragment was found in Sclerotinia rolfsii genome which has 32 % sequence similarity with oxalate oxidase gene of barley (Schmid et al. 2010). From this study researchers suggested that the oxalate oxidase reaction is the likely oxalate degradation route in S. rolfsii (Schmid et al. 2010). In our study we also found the fungi having the positive result for oxalate oxidase activity, so it could be assumed that our isolates have oxalate oxidase type protein which catalysing the conversion of oxalate to hydrogen peroxide and carbon dioxide. Fungi identied in this study have already extensively studied by many researchers but there is no available report on the oxalate oxidase production. However, Trichoderma sp. are well studied biocontrol agent, so we assumed that our strains have some protein with oxalate oxidase like activity. Further characterisation is under progress to clearly identify the protein sequences for better understanding of the oxalate oxidase activity. In this study we found that the Indo-Gangetic Plains have the potential fungal strains having oxalate oxidase type activity which is benecial to check the growth of plant pathogenic fungi like Sclerotinia sclerotiorum. In contrast these strains could be used in the medical purposes in the form of oxalate oxidase production, like in treatment of kidney-urinary tract stones etc. and for different biotechnological applicability.

Fig. 5 a Sclerotinia sclerotiorum without fungal extract (control); b Sclerotinia sclerotiorum with fungal extract containing oxalate oxidase enzyme

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World J Microbiol Biotechnol (2012) 28:31973206 Acknowledgments This work was supported by Indian Council of Agricultural Research, India, Network Project on Application of Microorganisms in Agriculture and Allied Sectors.

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