Phosphate solubilization by Trichoderma koningiopsis (NBRI-PR5) under abiotic stress conditions

2.1 Isolation, screening and growth media

Endophytic Trichoderma were isolated from rice plants collected from fields in Lucknow and Barabanki as described earlier by Rubini et al. (2005). Standard culture, Trichoderma harzianum NBRI-1055 (MTCC3841) was used as the reference strain at the time of screening (Singh et al., 2007). Trichoderma isolates and pathogenic fungi were grown and maintained on potato dextrose agar (PDA) at 28 °C and at 4 °C respectively. Standard Trichoderma strain and pathogenic fungi Sclerotium rolfsii, Sclerotinia sclerotiorum, Fusarium oxysporum and Alternaria solani were obtained from culture collection of Plant Pathology lab of CSIR-NBRI, Lucknow.

2.2 Screening for biocontrol activity

Biocontrol efficacy of NBRI-PR5 was determined on PDA plates inoculated against different soil-borne fungal plant pathogens following dual culture technique (Dennis and Webster, 1971). Per cent inhibition = [(C-T)/T] * 100, where C and T are radial growth of pathogen in control and dual culture plate resp.

2.3 Phosphate solubilization

All P solubilization experiments were carried out in broth modified from Trichoderma selective media (TSM) (Elad et al., 1981) and NBRIP-BPB broth (Mehta and Nautiyal, 2001), and named as NBRI-Trichoderma selective broth (NBRI-MTSB). NBRI-MTSB contained (per liter): glucose, 4 g; MgSO₄·7H₂O, 0.2 g; K₂HPO₄ 1.0 g; KCl, 0.15 g; (NH₄)NO₃, 1.0 g; Ca₃ (PO₄)₂, 5 g. For qualitative screening 0.025 g bromo phenol blue BPB dye was added to NBRI-MTSB broth (Mehta and Nautiyal (2001). An 8 mm bit of Trichoderma isolates were inoculated in 50 ml NBRI-MTSB-BPB broth and incubated at 30 °C with 150 rpm shaking on rotary shaker for qualitative screening. Change in color of the dye indicated lowering of pH and was recorded daily for 7 days. For quantitative P solubilization estimations NBRI-PR5 spore suspension adjusted to 6 log₁₀ units was inoculated at 1% in 50 ml NBRI-MTSB and flasks were incubated at 30 °C at 150 rpm on rotary shaker. Phosphate in culture supernatant was estimated on 3rd, 5th, 7th, 10th, and 15th day of inoculation and values were expressed as equivalent phosphate (µg ml⁻¹) (Fiske and Subbarow, 1925). For stress experiments the pH of the NBRI-MTSB was adjusted with 1 M NaOH before autoclaving to create alkaline conditions. For water stress NBRI-MTSB broth was supplemented with PEG-6000 at 2.5, 5.0, 10, 15 and 20% to induce matric potential (Steuter et al., 1981) and glycerol at 2, 5, 10, 15 and 20% to induce osmotic effect (Dallyn and Fox, 1980). The water potential (ψ) of one set of NBRI-MTSB with different treatments was determined after autoclaving the media at 25 °C using Wescor’s Vapour Pressure Osmometer model no. 5520. Interfering absorbance due to the additives, PEG and glycerol were nullified by substracting their respective blank readings taken against plain NBRI-MTSB broth. The P solubilization was calculated against a standard curve prepared with KH₂PO₄ (10–100 μg ml⁻¹ concentration). The experiments were conducted in triplicates and values are expressed as their means.

2.4 Morphological and molecular characterization of NBRI-PR5

Selected isolate NBRI-PR5 was morphologically characterized and identified by sequencing rDNA Internal transcribed spacer (ITS) and translation-elongation factor 1 alpha (tef1). Morphological and microscopic characterization of NBRI-PR5 cultures was carried out from cultures grown on PDA, corn meal dextrose agar (CMD) and synthetic low-nutrient Agar (SNA) as described earlier by Samuels et al. (2002). For mycoparasitic interaction study of NBRI-PR5 and Rhizoctonia solani, mycelia from the interaction zone of a dual culture plate was stained with cotton blue dye. Photographs were taken on an Olympus microscope at 10 and 40X magnification. Nessiers staining of the NBRI-PR5 mycelia harvested from the P solubilization experiments at different stress conditions was carried out using Modified Neisser’s metachromatic stain kit from Hi-media following the manufacturer’s instructions.

For ITS based identification PCR amplifications were done in a PTC200 thermocycler (MJ Research, Waltham, MA, USA) with primers ITS1 and ITS4 as described by White et al., (1990). Conditions for PCR amplification were initial denaturation of 1 cycle at 94 °C for 3 min; 30 cycles composed of denaturation at 95 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min; final extension at 72 °C for 10 min. The amplified products were sequenced with same primer pairs by Big Dye sequencing kit (Applied Biosystems, Foster City, USA). Two independent sequencing reactions generated data were examined on the web based molecular biology software Bioedit® (http://www.mbio.ncsu.edu/BioEdit/bioedit/). A final set of ITS sequence data and among vouched sequence data of Trichoderma was subjected to global search in BLASTn in TrichoBLAST for closest homology. The strain was further reconfirmed through the tef1 sequencing which can differentiate the members of Trichoderma koningii aggregate species using the primers EF1-728F and TEF1 rev and PCR conditions as described by Samuels et al. (2006).

2.5 Alkaline phosphatase assay

For determination of intracellular alkaline phosphatase enzyme activity, fungal mats were grown in NBRI-P medium at different pH and drought conditions as described in ‘Phosphate solubilization’ section. Fungal mat was washed at least 10 times with ice-cold distilled water to remove traces of extracellular enzymes and weighed after lightly pressing them between sheets of filter paper. A fine powder of the mat was prepared by crushing in liquid nitrogen and suspended in ice-cold sterilized distilled water. The extract obtained was centrifuged at 12,000 rpm for 20 min to settle the fungal debris. A clear supernatant containing the intracellular enzymes was obtained and made up to a known volume and used for assaying the intracellular alkaline phosphatase and total protein following the method of Tabatabai and Bremner (1969). Total protein in the enzyme extract was determined using method of Bradfords (1976) to express specific enzyme activity.

2.6 Organic acid analysis in broth

Crude culture filtrates were analyzed directly for the presence of organic acids after filtering through 0.22 μm nylon filters as described earlier, Cawthray (2003). All HPLC analyses were conducted by reversed phase chromatography on a 250 mm × 4.6 mm i.d., 5 µm particle C-18 Coloumn, having a UV detector from Cyberlab (Salo Terracce, Millsbury, USA) UV detector. All the chemicals and standards used were of HPLC grade. The mobile phase consisted of 25 mM KH₂PO₄ adjusted to pH 2.5 with concentrated orthophosphoric acid, and methanol (7%). The flow rate of the mobile phase was 0.7 ml min⁻¹ for all the chromatographic separations. The volume injected was 20 μl of the prepared sample or standard solution.

2.7 Data analysis

Data analyses were carried out using the descriptive statistical analysis tools of windows office excel 2007.

3.1 Isolation and screening

Total 33 endophytic Trichoderma isolates were obtained from surface sterilized tissues of rice plants and were named as NBRI-PR1-NBRI-PR33. Based on qualitative method for phosphate solubilization 22 isolates were selected which were further quantified for P solubilization and antagonistic activity against phytopathogens. Results for antagonistic activity, P solubilization, broth pH and mycelia dry weight 7 days post inoculation (dpi) is given in Table 1. Maximum P solubilization (70.79 µg ml⁻¹) was observed by Trichoderma isolate NBRI-PR5 which also showed 100% inhibition of various phyto-pathogens viz S. rolfsii, S. sclerotiorum, F. oxysporum, and A. solani Table 1. Based on the two screening results NBRI-PR5 was selected for further study and characterization. Microscopic study shows mycoparasitic colonization of Rhizoctonia solani by NBRI-PR5 as the mode of biocontrol activity (Fig. 1A).

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Fig. 1

Biocontrol and morphological characterization of T. koningiopsis-NBRI-PR5; Mycoparasitism against Rhizoctonia solani (A); Aerial growth with conidiation pattern on PDA (B), CMD (C) and SNA (D); Hemispherical to spherical pustules (E-G); Gross morphology with primary branch arrangement (H), intercalary phialide (I), developing conidiophores (J), phialides arise in whorls (K); conidia morphology (L-M). ITS based MP tree of T. koningiopsis NBRI-PR5 (N). tef1 based NJ tree of T. koningiopsis NBRI-PR5 (O) Pictures of pustules were taken using Stereomicroscope from Leica (E-G); Pictures of branching arrangement, phialide structure and spores were taken using Phase contrast microscope form Nikon (H-M).

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3.2 Morphological characterization and ITS sequencing of Trichoderma isolate NBRI-PR5

Puffy aerial growth with conidiation was observed on PDA after 96 h with 12 h dark and light conditions at 30 °C (Fig. 1B). Appressed aerial growth with conidiation in 2–3 concentric rings having light sweet, coconut odour was observed on CMD (Fig. 1C). Little aerial mycilium, conidia aggregated at margins was observed on SNA (Fig. 1D). Radial growth of 67.5 ± 3.5 mm on PDA 51 ± 4.2 mm on CMD 47 ± 2.8 mm on SNA in dark conditions was observed. No pigmentation diffusing in the agar was observed. Hemispherical to spherical pustules with rough surface less than 1 mm size on CMD was observed (Fig. 1E–G). The primary branch arrangement is pachybasium like that arise at acute angles. The phialides are laginiform mainly with pointed neck, rarely ampuliform and sinuous forms are seen. The phialides arise in the whorls of 3–5 directly on main axis and on supporting cell on primary branches (Fig. 1H, J–K). Despite T. koningi like colony morphology and conidiophore ornamentation, intercalary phialides as diagnostic characteristics of T. koningiopsis were observed (Fig. 1I). Light green conidia elliptical to ellipsoid, rarely sub-globose with smooth surface are present (Fig. 1L–M).

The ITS and tef1 region were nearly 600 and 900 bp single amplicon respectively and any other non specific amplifications were not detected on gel. The BLASTn and TrichoBLAST results on ITS sequence showed 99–100% homology to T. koningiopsis accessions and in particular to vouchered culture T. koningiopsis GenBank accession DQ379015. The tef1 showed 99–100% homology to T. koningiopsis accession DQ109549. Therefore the isolate under present study was designated as Trichoderma koningiopsis NBRI-PR5. The GenBank accession for ITS sequence of T. koningiopsis NBRI-PR5 is JN375992. ITS and tef1 based MP tree of T. koningiopsis NBRI-PR5 is given in supplementry Fig. 1N and O respectively.

3.3 Effect of pH and water stress on P solubilization ability of NBRI-PR5

Under ambient conditions (pH 7.0) NBRI-PR5 was found to solubilize TCP 40 times more efficiently than the standard culture NBRI-1055 (MTCC-3841) (Table 1). P solubilization under stress conditions was carried out in-vitro by using stress inducers such as salt, PEG and glycerol. The osmotic potential (OP) of the NBRIP broth in different stress treatments ranged from −0.78 ± 0.01 Mpa at control conditions to −11.91 ± 0.03 Mpa at 20% glycerol. Addition of PEG in the medium increased the osmotic potential to −1.88 ± 0.02 at 20% concentration while 20% glycerol increased the OP to −11.91 MPa ± 0.03 (Table 2). NBRI-PR5 optimally solubilized P at the pH range of 7 to 9. Decrease of 44% P solubilization was observed at pH 10.0 (47.64 μg ml⁻¹) which sharply declined to 0.37 μg ml⁻¹ at pH 11.0 (Fig. 2) Maximum P solubilization was observed at pH 8.0 which was 17% higher than in control (pH 7.0) (Fig. 2A). The P solubilization by NBRI-PR5 reduced with the increasing concentration of PEG and glycerol (Fig. 2A). Based on the osmotic potential developed by addition of PEG and glycerol, PEG was found to be more potent inhibitor for P solubilization. Correlation of P solubilization with mycelial dry mass (r² = 0.99) in alkaline condition showed a direct effect of pH on mycelial growth and P solubilization (Fig. 2B). On the contrary, although a progressive decrease in P solubilization was observed with increasing concentration of PEG, fungal biomass remained nearly constant in all the concentrations of PEG with a correlation coefficient of r² = 0.49 (Fig. 2B). Similarly in presence of lower concentrations of glycerol (2.5 and 5%) P solubilization was considerably reduced while the biomass remained close to the control. These results suggest that different mechanisms of P solubilization may be involved in different stress conditions in the fungi. Under drought stress conditions production of alkaline phosphatase enzyme was observed (Fig. 2A). This is further supported by the correlation observations of alkaline phosphatase activity with P solubilization in alkaline and drought stress conditions where a correlation of the enzyme with P solubilization is observed in presence of PEG (r² = 0.87) and glycerol (r² = 0.98) as compared to alkaline condition where r² = −0.07 (Fig. 2B). These observations are further strengthened from frequency distribution of P solubilization with respect to final pH of the broth, Fig. 3. The Fig. 3A shows the frequency distribution of the P solubized and its corresponding final pH (initial pH 7–11). The hightest P solubilization point (94 µg ml⁻¹) coincides with the lowest pH (3.5 pH) with a correlation coefficient of r² = 0.968 showing that in alkaline conditions the P solubilization is mainly achieved through acidification of the medium. However, under water stress conditions acidification of the medium does not show any correlation with the soluble P (r² = 0.543) (Fig. 3B). Besides, the final broth pH is 1–2 units higher in drought stress than in the alkaline stress.

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Fig. 2

Effect of pH and water stress on Phosphate solubilization (■), fungal biomass (Δ) and alkaline phosphatase enzyme (ο) (A). Correlation coefficient of P solubilized with different parameters of phosphate solubilization in broth conditions (B).

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Fig. 3

Role of pH in P solubilization under different stress conditions. Frequency distribution of P solubilized with respect to final pH of the broth at an initial pH range of 7.0–11.0 (◆) (A); Distribution of P solubilized with respect to final pH of the broth at a pH range of 7.0–11.0 (◆), PEG concentration 0–30% (×) and glycerol concentration 0–30% (●) (B).Dark vertical strip in A and B shows lower final pH (2.5–4.5) achieved for P solubilization under alkaline conditions. Light vertical strip in B shows higher pH (4.5–6.0) achieved for P solubilization under drought stress indicating presence of alternative mechanisms in alkaline and drought stress for equivalent amounts of soluble P.

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3.4 Effect of pH and water stress on organic acid and alkaline phosphatase enzyme production by NBRI-PR5 in broth

HPLC analysis of the organic acids produced by NBRI-PR5 in NBRIP broth showed qualitative and quantitative effects of alkaline conditions. Fig. 4 show chromatograms of blank media, control without TCP (-TCP), control with TCP (+TCP) (pH 7) (blue) and different treatments of pH (8–11), PEG (2.5–20%) and glycerol (2.5–20). Peaks corresponding to oxalic acid (RT 2.70) at pH 8 and two unknown peaks at RT 9.75 and 11.33 at pH 9 were observed which were not detected in other treatments. Organic acid production in presence of PEG and glycerol were highly reduced (Fig. 4B-C). The results show that role of organic acid production in P solubilization is more prominent in alkaline stress than in drought stress.

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Fig. 4

Comparative HPLC chromatograms of organic acids produced by T. koningiopsis NBRI-PR5 at different pH (A), PEG (B) and glycerol (C) treatments. Chromatograms of standard organic acids D. Concentration of standards: 100 mM of oxalic, formic, acetic, citric and succinic acid and 10 mM of d and l malic acid).

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Further on, a microscopic examination of the Neisser’s stained mycelia grown in presence of the stress conditions show polyphosphate accumulation in the mycelia (Fig. 5). Neisser’s staining differentially stains hyphae with accumulated polyphosphates in blue-black and the negative hyphae as brown or red. Positive staining was observed with blue to blue-black regions in hyphae in presence of TCP at various pH (inset Fig. 5B-D) and drought conditions (Fig. 5E-K). No staining in the blank (-TCP) treatment (Fig. 5A), and negative staining in presence of high concentrations of glycerol (Fig. 5L) was observed. Encrustation of NBRI-PR5 hyphae with salts was also observed under alkaline conditions (Fig. 5B-D). The P solubilization mechanisms in stress conditions, as infered from the present study in NBRI-PR5 is summurized in graphical abstract.

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Fig. 5

Polyphosphate granules (dark blue stains in hyphae) are absent in the negative control (-TCP) having P starvation conditions (A). Formation of polyphosphate granules was observed in presence of TCP at pH 7–9 (inset of B-D), glycerol 2.5% (E), 5% (F), 10% (G) and PEG 2.5% (I), 5% (J), 15% (K), 25% (K). Polyphosphate accumulation was absent in glycerol concentrations above 15% (H) showing brown staining all the treatments except at higher concentrations of glycerol (H). Encrustation of TCP granules was observed on the mycelia at pH 7.0, pH 8.0 and at pH 9.0 (B-D). Formation of chlamydospore was prominent in the presence of glycerol. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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