Chapter 3 Isolation, screening, morphological and biochemical characterization of fungal isolates

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1 Chapter 3 Isolation, screening, morphological and biochemical characterization of fungal isolates

2 3.1 Introduction Phosphorus is one of the major nutrients, second only to nitrogen in requirement for plants. A greater part of soil phosphorus, approximately % is present in the form of insoluble phosphates and cannot be utilize by the plants (Vassileva et al., 2001). To increase the availability of phosphorus for plants, large amounts of fertilizers are being applied to soil. However, a large proportion of fertilizer phosphorus after application is quickly transformed into the insoluble form. Therefore, very little percentage of the applied phosphorus is available to plants. Phosphorus deficiencies are widespread on soil throughout the world and phosphorus fertilizers represent major cost for agricultural production. The phosphate solubilizing fungi (PSFs) play an important role in supplementing phosphorus to the plants, allowing a sustainable use of phosphate fertilizers. It has been reported that fungi possess greater ability to solubilize insoluble phosphate than bacteria (Nahas, 1996). Species of Aspergillus, Penicillium and Trichoderma have been widely mentioned as efficient strains of phosphate solubilizers. In the present study fungal strains having potential to solubilize, insoluble phosphates have been isolated, screened and characterized on their morphological and biochemical means. These PSFs were checked for the ability to solubilize insoluble phosphates on plate assay and their comparative analysis has been performed. 3.2 Materials and Methods Collection of soil sample The soil samples were collected from different locations of the Kukrail forest, Lucknow, Lawns of Integral University, Lucknow and Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow. The soil samples were taken from two zones viz., upper zone, which is cm deep and lower zone, which is cm in depth Isolation and screening of phosphate solubilizing fungi The soil rhizospheric samples were screened for the presence of phosphate solubilizing fungi (PSF) Media preparation for isolation of fungi

3 The fungi were grown on potato dextrose agar (PDA) medium. For this PDA medium comprising of potato 20 %, dextrose 2 % was prepared and ph adjusted to 7.0. This medium was complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify Serial dilutions of soil samples Soil rhizospheric samples from various locations were taken and serial dilutions were made. For this 1 g sample was taken and added in tube containing distilled water and mixed thoroughly. This represented 10-1 dilution. Under aseptic conditions, 10-2 to 10-9 dilutions of samples were prepared Screening of phosphate solubilizing fungi The isolates were screened on the basis of plate assay. Two media viz., Pikovskaya s media (Pikovskaya, 1948) and bromophenol blue media were used for screening. The Pikovskaya s medium consisted of yeast extract 0.50 (g/l), dextrose (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate (g/l), ferrous sulphate (g/l). Bromophenol media consist of yeast extract 0.50 (g/l), dextrose (g/l), calcium phosphate 5.00 (g/l), ammonium sulphate 0.50 (g/l), potassium chloride 0.20 (g/l), magnesium sulphate 0.10 (g/l), manganese sulphate (g/l), ferrous sulphate (g/l) and 0.5 % of bromophenol blue dye. Both the media were prepared and ph adjusted to 7.0. This medium was complemented with agar 1.5 % and autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify. Fungal colonies were inoculated on petriplates containing medium for plate assay and the plates were incubated in inverted position in incubator for up to 72 h at 28 C. Positive cultures were screened by observing transparent halo zones in Pikovskaya s medium and yellow halo zone on bromophenol blue medium Pure cultures of phosphate solubilizing fungal species Positive fungal colonies were subculture, on fresh petriplates containing medium for plate assay. Fungal cultures were isolated by incubating the plates in inverted position in incubator for up to 72 h at 28 C. Positive cultures were screened by observing transparent halo zones in

4 Pikovskaya s medium, which is due to the solubilization of insoluble tricalcium phosphate into the soluble form and yellow halo zones appears on bromophenol blue medium which is due to the production of organic acids leading to lowering of ph within the medium Morphological characterization of fungal isolates by lactophenol staining The identification of the isolated phosphate solubilizing fungi was done by its staining procedure. A fungal colony was first grown on the Sabouraud agar medium and its morphology was studied using standard cover-slip technique and lactophenol cotton blue staining procedure. The cover slip was inserted in tilted position in the petriplate itself and the culture was allowed to grow for some time. Then the cover slip was taken out with the help of forceps and put inverted on slide containing a drop of lactophenol cotton blue stain and visualized under microscope at 40 X magnification. Thin mycelia of fungal isolates were also spread on the glass slide and teased with needles followed by addition of a drop of lactophenol stain. The stained and air-dried slides were further examined under microscope at 40 X magnification. The fungi were identified on the basis of mycelial and spore characteristics Biochemical characterization of phosphate solubilizing fungi Solubilization index based on colony diameter and halo zone for each PSF indicate the efficiency of solubilization of insoluble phosphate into the soluble one thus forming a transparent halo zone around the colony. Hence, the positive fungal isolates were also analyzed qualitatively for their ph change, dry weight and acid phosphatase enzyme. Beside it the starch hydrolysis test and cellulose hydrolysis test were also performed Solubilization index (SI) 200 mg-500 mg ml of each PSF culture preserved in sterile distilled water was placed on Pikovskaya s agar (Pikovskaya, 1948) plates and incubated for seven days. Solubilization Index was measured using following formula (Edi-Premono et al., 1996). SI = Colony diameter + halo zone diameter Colony diameter

5 ph change 200 mg-500 mg of five days old culture of fungus was added to sterile 100 ml Pikovskaya s broth (PB) medium and kept on shaker for seven days at 28 C. Sterile uninoculated medium served as control. Initial ph and change in ph was recorded on 3 rd, 5 th and 7 th day by digital ph meter Dry weight determination of fungus 200 mg-500 mg of 5 days old culture of fungus was added to sterile 100 ml Pikovskaya s broth (PB) medium and kept on shaker for seven days at 28 C. During this period, the fungal cultures reached the maximal level of their biomass in the medium. This was determined by measuring dry weight of cultures for which mycelia were taken and dried at 40 C and their respective weights were measured Starch hydrolysis test Starch agar medium (starch 20.0 g/l, peptone 5.0g/l, yeast extract 3.0 g/l, agar 15.0 g/l; ph 7.0) was inoculated with isolated fungal cultures. The plates were incubated at 25 C in inverted position for 5 to 7 days. The surface of the plates was flooded with iodine solution for 30 sec. Examined the disappearance of starch from the starch agar media plates by observing the disappearance of clear zones around the fungal growth Cellulose hydrolysis test The Czapek-mineral salt agar medium consisted of KCl 0.5 (g/l), K 2 HPO (g/l), NaNO (g/l), MgSO 4.7H 2 O 0.5 (g/l), peptone 2.0 (g/l), carboxymethyl cellulose (CMC) 5.0 (g/l). This medium was complemented with agar 2 % and autoclaved at 15 psi for 15 min. Autoclaved medium was poured in sterile petriplates (25 ml/plate) under laminar flow hood and allowed to solidify. The plates were allowed to inoculate with isolated fungal cultures. The plates were incubated at 35 C in inverted position for 5 days. The surface of the plates was flooded with 1 % aqueous solution of hexadecyltrimethyl ammonium bromide for 30 sec. The plates were observed for the formation of a clear zone around the fungal growth.

6 Analysis of acid phosphatase enzyme activity from various isolates Medium preparation and fungal culture. All the fungal isolates tested positive in plate assay were subjected to analyses of activity of acid phosphatase enzyme. Fungal colonies tested positive in plate assay were inoculated in Pikovskaya s broth, poured in test tubes (20 ml/tube) and autoclaved at 15 psi for 15 min. The tubes were incubated in incubator shaker at 120 rpm, 28 C for h. 10 ml of above grown fungal culture was taken and filtered through Whatman no. 1 filter paper. This was considered as enzyme or protein sample. The enzyme acid phosphatase was assayed using para nitrophenyl phosphate (PNP-P) as a substrate. The reaction mixture contained 2.5 ml (0.1 M) sodium acetate buffer (ph 5.8), 1 ml (1 mm) magnesium chloride, 0.5 ml 1 % PNP-P and 0.5 ml of a suitable dilution of enzyme preparation. One ml of the reaction mixture was transferred to 2 ml of 0.2 M sodium hydroxide before and after 15 min incubation at 37 C to stop the reaction. The sodium hydroxide solution added before incubation acts as a control sample for each analysis. The amount of para nitro phenol (PNP) liberated was measured by recording the absorbance at 420 nm using an appropriate calibration curve. Activity is expressed as nmol PNP liberated min -1. The blank was run in a similar manner using distilled water. Reaction showing acid phosphatase enzyme activity with para nitrophenyl phosphate substrate

7 Determination of protein content by Lowry s method 500 l of fungal culture was taken in microfuge tube and protein was precipitated with equal volume of ice-cold 20 % trichloroacetic acid (TCA) and kept at 4 C overnight. The pellet was recovered by centrifuging at 12,000 rpm for 5 min at room temperature and decanting the supernatant. The pellet was washed with 0.1 ml ice-cold 10 % TCA and ice-cold acetone. Depending on the pellet size, it was dissolved in ml of 0.1 N NaOH. The solution was subjected to heating for 5 min in boiling water bath and vortexed vigorously. The protein content was determined by Lowry s method (Lowry et al., 1951). For protein content determination, 0.5 ml of protein solution was taken in test tube and 2.5 ml of alkaline solution [prepared by mixing 2 % Na 2 CO 3 solution (in NaOH), 2 % sodium potassium tartrate and 1 % CuSO 4.5H 2 O in 100:1:1. was added. The contents were mixed well and the tubes were incubated at room temperature for 10 min. This was followed by addition of 0.25 ml of 1.0 N Folin s reagent. The contents in the tube were mixed thoroughly and after 10 min, absorbance at 660 nm against reagent blank was determined spectrophotometrically using bovine serum albumin fraction V as standard. 3.3 Results and Discussion Isolation and screening of phosphate solubilizing fungi Based on serial dilution thirty-two fungal isolates from different soil samples were isolated, from which 20 fungal isolates having potential phosphate solubilizing ability were screened by plate assay. Various fungi, which were isolated from soil, were preserved on the potato dextrose medium (PDA). The shape, structure and type of colony were analyzed on the Sabouraud agar medium and further the lactophenol staining was performed. The tricalcium phosphate present in Pikovskaya s medium in insoluble form was converted into the soluble form by the phosphate solubilizing fungi (PSF), thereby giving a clear zone around a positive colony (Fig. 3.1).

8 Figure 3.1 Fungi showing phosphate solubilization leading to formation of clear zone in Pikovskaya's medium In bromophenol blue medium (Fig. 3.2) all the positive fungal isolates were capable of producing organic acids, which led to change in ph and thereby color change from blue to yellow. Figure 3.2 Fungi showing yellowing of bromophenol blue medium Fungal isolates were characterized by analyzing their shape and the structure of colonies, presence of amylase and cellulase/cellobiose enzyme. The isolates were examined and the results are given in Table 3.1. Variations were observed among the isolates in their colony morphology Morphological characterization of PSFs by microscopic examination

9 All the twenty fungal isolates were characterized by microscopic analysis using lactophenol cotton blue staining procedure. Wide variations were observed among the fungal isolates in their colony morphology (Table 3.1). Table 3.1 Morphological characterization of phosphate solubilizing fungal isolates by colony morphology and microscopic analysis Fungal isolates Colony morphology analysis on Sabouraud agar medium Microscopic analysis Fungus identified FNP 1 Aspergillus niger FUK 29 Bipolaris tetramera FNC 27 Alternaria brassicae

10 FUC 5 Verticillium FSK 12 Rhizoctonia FNK 18 Fusarium oxysporum FSI 33 Gliocladium FNK 2 Schizophyllum commune

11 FNI 10 Alternaria SPWF166 FUI 16 Trichoderma FUK 17 Phoma FNC 11 Aspergillus flavus

12 FNU 4 Aspergillus paecelomyces FSC 15 Ascobolus FUK 6 Alternaria azaubiae FSK 14 Gliocladium

13 FNK 20 Amauroderma FNI 7 Helminthosporium FUC 13 Botrytis FNK 19 Aspergillus sulphuracea Biochemical characterization of fungal isolates Solubilization index (SI)

14 All the 20 isolates were able to solubilize tricalcium phosphate (TCP) in Pikovskaya s agar medium and the diameter of the zones of solubilization indicated wide variations among the isolates. The results are shown in Table 3.2. Fungal isolates FUK 29, FNK 18, FNK 2, FNI 10, FNK 20 and FNC 11 have shown higher solubilization zones (Fig. 3.3) indicating a high level of phosphate solubilization while others have showed less solubilization index. Table 3.2 Comparative analysis of solubilization indices of fungal isolates Fungal isolates Solubilization Index (SI) FNP 1 2±0.15 FUK ±0.12 FNC ±0.13 FUC 5 2.7±0.09 FSK ±0.11 FNK ±0.12 FSI ±0.13 FNK 2 3.7±0.129 FNI ±0.1 FUI ±0.13 FUK ±0.14 FNC ±0.1 FNU 4 2.4±0.137 FSC ±0.12 FUK 6 2.5±0.15 FSK ±0.12 FNK ±0.12 FNI 7 2.2±0.13 FUC ±0.18 FNK ±0.19

15 SI(cm) FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI 33 FNK 2 FNI 10 FUI 16 FUK 17 FNC 11 FNU 4 FSC 15 FUK 6 FSK 14 FNK 20 FNI 7 FUC 13 FNK 19 Fungal isolates Figure 3.3 Histogram showing solubilization indices of fungal isolates ph change All the 20 isolates were allowed to grow on Pikovskaya s broth supplemented with TCP (0.5 % w/v). Decrease in ph was observed among fungal isolates with time up to 7 days. Minimum ph was observed on the 7 th day. The ph lowered down due to the liberation of organic acids in broth media. The maximum decrease of ph was exhibited by FUK 29 i.e., 2.9 after 7 th day of growth. Even the ph of FNK 20, FNI 10, FNK 18, FNC 11 and FNK 2 showed decreasing trend in ph, which was observed as 3, 3.1, 3.2, 3.3 and 3, respectively (Table 3.3) thus confirmed of exhibiting the phosphate solubilizing ability. However, no significant decrease in ph were found in two isolates FSK 14 and FSC 15 viz., even after 7 th day which was observed as 5.9 and 5.4, respectively (Fig. 3.4).

16 Table 3.3 Comparative analysis of ph change on 3 rd, 5 th and 7 th day Fungal isolates ph 3 rd day ph 5th day FNP FUK FNC FUC FSK FNK FSI FNK FNI FUI FUK FNC FNU FSC FUK FSK FNK FNI FUC FNK ph 7th day

17 ph FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI 33 FNK 2 FNI 10 FUI 16 FUK 17 FNC 11 FNU 4 FSC 15 FUK 6 FSK 14 FNK 20 FNI 7 FUC 13 FNK 19 Fungal isloates Figure 3.4 Histogram showing comparative analysis of ph on different days of growth Dry weight determination weight of fungus After 1 week of incubation, the fungal mycelia were filtered and dried at 45 C followed by measuring their dry weight. Thus, fungal isolate FNP 1, FUI 16, FNC 11, FNU 4 and FNU 4 exhibited an increase in the dry weight, which was 2.031, 2.494, 2.102, and 2.234, respectively (Table 3.4).

18 Table 3.4 Dry weight determination of fungal isolates Fungal isolates Dry weight (g) FNP ±0.05 FUK ±0.06 FNC ±0.07 FUC ±0.09 FSK ±0.16 FNK ±0.09 FSI ±0.06 FNK ±0.06 FNI ±0.04 FUI ±0.07 FUK ±0.04 FNC ±0.03 FNU ±0.01 FSC ±0.012 FUK ±0.092 FSK ±0.07 FNK ±0.09 FNI ±0.07 FUC ±0.09 FNK ±0.08

19 FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI 33 FNK 2 FNI 10 FUI 16 FUK 17 FNC 11 FNU 4 FSC 15 FUK 6 FSK 14 FNK 20 FNI 7 FUC 13 FNK 19 Dry weight (g) Fungal isolates Figure 3.5 Histogram representing the dry weight of fungal isolates Starch hydrolysis test This test was used to determine the ability of an organism to hydrolyze starch, by enzymatic action. Starch is a mixture of two polyglucose polysaccharide molecules: linear amylase and branched amylopectin. Iodine combines with the amylase starch fraction to form an intense, deep blue colour complex. If starch is hydrolyzed, this network disintegrates and test fails to maintain the blue colour. Here some PSF gave positive results and some were tested negative. The colony, which showed the disappearance of blue color from the starch agar media plates due to utilization of starch, was a positive colony for starch test (Table 3.5) Cellulose hydrolysis test Cellulose is a polysaccharide comprising of long linear chain of glucose units linked by β-1, 4 glycosidic bonds. Fungi, bacteria and actinomycetes bring about degradation of cellulose by the secretion of extracellular enzyme, cellulose. It is a complex enzyme composed of at least three components viz., endoglucanase, exoglucanase and β-glucosidase. The cooperative action of these three enzymes is required for the complete hydrolysis of cellulose to glucose. Evidence for

20 the microbial utilization of cellulose can be detected using hexadecyltrimethyl ammonium bromide. This reagent precipitates intact carboxymethyl cellulose (CMC) in the medium and thus clear zones around the colony in an otherwise opaque medium indicating degradation of CMC. In the cellulose hydrolysis test, all fungal isolates gave positive results (Table 3.5). Table 3.5 Starch hydrolysis and cellulose hydrolysis tests of fungal isolates Fungal isolates Starch hydrolysis test Cellulose hydrolysis test FNP 1 Positive Positive FUK 29 Negative Positive FNC 27 Negative Negative FUC 5 Positive Positive FSK 12 Positive Positive FNK 18 Positive Negative FSI 33 Negative Positive FNK 2 Positive Positive FNI 10 Negative Negative FUI 16 Negative Negative FUK 17 Positive Positive FNC 11 Positive Positive FNU 4 Positive Positive FSC 15 Positive Positive FUK 6 Positive Negative FSK 14 Negative Positive FNK 20 Positive Positive FNI 7 Positive Positive FUC 13 Negative Positive FNK 19 Positive Positive

21 Quantitative analysis of acid phosphatase enzyme activities The selected PSFs were grown on Pikovskaya s broth containing 0.5 % TCP and their acid phosphatase activities were measured. The phosphatase activity was estimated in the supernatant of broth taken after centrifugation at 10,000 X g at 4 C. The main mechanism for the solubilization of insoluble organic and inorganic phosphate was due to production of an enzyme acid phosphatase, which catalyzes hydrolysis of phosphate to liberate inorganic phosphorus (Pi). Thus, the isolates were evaluated for their acid phosphatase producing ability by measuring Pi liberated. Among all the positive isolates, six fungi exhibited significantly higher amount of acid phosphatase enzyme activity, including fungi FUK 29, FNK 20, FNI 10, FNK 18, FNC 11 and FNK 2 (Fig. 3.6). Moreover, these isolates also showed relatively higher solubilization index. Thus, the solubilization index and the acid phosphatase enzyme activity are directly proportional to each other indicating that high enzymatic activity results in the formation of large halo zone. The acid phosphates activities of all the isolates are presented in Table 3.6. Table 3.6 Acid phosphatase enzyme activity of fungal isolates Fungal isolates Acid phosphatase enzyme activity (nmole/ml) FNP ± ±0.015 FUK ± ±0.035 FNC ± ±0.023 FUC ± ±0.043 FSK ± ±0.032 FNK ± ±0.029 FSI ± ±0.017 FNK ± ±0.023 FNI ± ±0.015 FUI ± ±0.025 FUK ± ±0.018 FNC ± ±0.034 Acid phosphatase enzyme specific activity (Activity/mg protein)

22 nmoles of Pi/ml FNU ± ±0.075 FSC ± ±0.051 FUK ± ±0.025 FSK ± ±0.037 FNK ± ±0.015 FNI ± ±0.035 FUC ± ±0.025 FNK ± ± FNP 1 FUK 29 FNC 27 FUC 5 FSK 12 FNK 18 FSI FNK 2 FNI FUI 16 FUK 17 FNC 11 FNU 4 Fungal isolates FSC FUK 6 FSK FNK 20 FNI 7 FUC 13 FNK 19 Figure 3.6 Histogram indicating acid phosphatase activity of fungal isolates 3.4 Conclusion The results indicated existence of variation among the fungal isolates in their morphology. Some of fungi have black conidiophores, sickle shaped spores, some of them having brown colony while others formed light green, brown and orange colonies. All the fungal isolates showed halo zones on Pikovskaya s agar media indicating there phosphate solubilizing ability. Based on diameter of transparent halo zone, the solubilization index of the isolates was measured, Further, dry weight determination, change in ph of media at different days and acid phosphatase activities were also determined. The isolated strains were identified on the basis of microscopic analysis using lactophenol cotton blue stain which includes Schizophyllum commune,

23 Trichoderma, Aspergillus sulphuracea, Botrytis, Helminthosporium, Amauroderma, Gliocladium, Alternaria azaubiae, Ascobolus, Aspergillus paecelomyces, Aspergillus flavus, Alternaria sp., Gliocladium, Fusarium oxysporum, Aspergillus niger, Bipolaris tetramera, Alternaria brassicae, Verticillium, Rhizoctonia and Phoma. Prominent halo zones were found in case of positive PSF isolates on Pikovskaya s agar. Based on transparent halo zone the isolates that exhibited higher SI also exhibited higher acid phosphatase activity. Beside it, the starch and cellulose hydrolysis test were also studied. However, morphological and biochemical characterization of isolates did not give complete information about isolates. Hence, polyphasic approach involving molecular marker analyses along with phenotypic evaluation is essential for identification of fungal isolates and determination of genetic variation. Thus, all fungal isolates were further subjected to diversity analysis at molecular level.