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Effect of acid phosphatase produced by Trichoderma asperellum Q1 on growth of Arabidopsis under salt stress
Journal of Integrative Agriculture 2017, 16(6): 1341–1346 Available online at www.sciencedirect.com
ScienceDirect
RESEARCH ARTICLE
Effect of acid phosphatase produced by Trichoderma asperellum Q1 on growth of Arabidopsis under salt stress ZHAO Lei1, 2*, LIU Qun2*, ZHANG Ya-qing2*, CUI Qing-yu2*, LIANG Yuan-cun3 1 2 3
Institute of Environment and Ecology, Shandong Normal University, Jinan 250014, P.R.China College of Life Science, Shandong Normal University, Jinan 250014, P.R.China College of Plant Protection, Shandong Agricultural University, Tai’an 271018, P.R.China
Abstract Salt stress is a major environmental factor that inhibits crop growth. Trichoderma spp. are the most efficient biocontrol fungi and some of the strains can stimulate plant growth. Phosphate solubilization is known as one of the main mechanisms in promoting plant growth, but the underlying mechanisms of phosphate solubilization in the salinity still need to be explored. The Trichoderma asperellum Q1 isolated and identified in our lab is a beneficial rhizosphere biocontrol fungus with a high phosphate solubilization activity. It could produce acid and alkaline phosphatases when using insoluble organic phosphorus as the sole phosphorus source, the salt stress increased the phosphorus-solubilization ability of the strain and the activities of the two enzymes. Furthermore, an acid phosphatase was purified from the fermentation broth by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. Its molecular weight was 55 kDa as determined by SDSPAGE. The purified acid phosphatase was used to investigate growth performance of Arabidopsis thaliana by plate assay and the result showed that it contributed to Arabidopsis growth by transforming organic phosphate into a soluble inorganic form under salt stress. To our knowledge, this is the first report on acid phosphatase purification from T. asperellum and its function in regulation of plant growth under salt stress. Keywords: Trichoderma asperellum, Arabidopsis thaliana, acid phosphatase, plant-growth promotion, salt stress
Schinner 1992). Many soils throughout the world are P
1. Introduction Phosphorus (P) is one of the most essential macronutrients required for plant growth and development (Illmer and
deficient because the free P concentration (the form available to plant) is low (Rodrı́guez et al. 1999). Even in fertile soils, it is not sufficient for plants due to the high reactivity of soluble P with calcium, iron, or aluminum, which leads to P precipitation (Rodrı́guez et al. 1999; von Wandruszka 2006). In order to overcome the defects, phosphate fertilizers are frequently added into the soil in order to obtain
Received 11 May, 2016 Accepted 21 September, 2016 Correspondence ZHAO Lei, Tel: +86-531-86188195, Fax: +86531-86180107, E-mail: [email protected]; LIANG Yuan-cun, Tel: +86-538-8242301, E-mail: [email protected] * These authors contributed equally to this study. © 2017, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61490-9
good productivity (Pradhan and Sukla 2009). Moreover, the production of traditional P fertilizers is a costly chemical processing and the overuse of them may results in soil salinization (Vassilev et al. 2006). It is known that some microorganisms are involved in the solubilization of insoluble phosphate into the soluble
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form (Son et al. 2006). These P-solubilizing microbes excrete inorganic acids, organic acids, and phosphatase to dissolve phosphorus (Zhong et al. 2014). However, the mechanisms of P-solubilization not only differ among fungal isolates but also depend on the applied P sources (de Oliveira Mendes et al. 2014). Trichoderma is a highly desirable bioagent with dual potentials for biocontrol and plant growth promotion. Certain effects of Trichoderma spp. on plant development have been studied, such as production of 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase (Viterbo et al. 2010), siderophore (Qi and Zhao 2013), and solubilization of insoluble minor nutrients or phosphate (Altomare et al. 1999). However, the underlying mechanisms of the phosphate-solubilizing ability in promoting plant stress tolerance have not been fully elucidated. The previous study has confirmed that there were positive correlation between hydrolyzed organic P compounds and the secretion of phosphatases or phytase in Trichoderma harzianum and Trichoderma viride (Aseri et al. 2009). However, the effect of acid phosphatase (ACPase) on P-solubilization and plant growth promotion needs to be proved. Therefore, we investigated the solubilization of insoluble phosphorus and phytin of T. asperellum Q1 and the effect of its ACPase on Arabidopsis growth-promoting under salt stress.
2. Materials and methods 2.1. Strain T. asperellum Q1 was isolated and identified previously from the rhizosphere of cucumber in a greenhouse in Shandong Province, China (Qi and Zhao 2013). The strain was maintained on potato dextrose agar slants at 4°C.
2.2. Quantitative assay of phosphate solubilizing activity In order to determine the differences in the phosphate solubilization with or without salt stress, the following experiments were conducted. 1 mL spore suspension (1×107 spores mL–1) of T. asperellum Q1 was used to inoculate 250 mL Erlenmeyer flasks containing 100 mL medium, and then incubated at 28°C on a rotary shaker at 180 r min–1. The media which contained tricalcium phosphate (TCP), dibasic calcium phosphate (DCP) or phytin individually without salt stress were used as the control. After 12, 24, 36, 48, 60, and 72 h of incubation, each culture supernatant was obtained by centrifugation and the available phosphorus was analyzed using the ammonium vanadate-molybdate spectrophotometry method (Kang et al. 2005).
2.3. Phosphatase activity assay Pikovskaya’s broth (Phytin was supplied as the sole P-source) was used to measure the phosphatase activity by employing the p-nitrophenyl phosphate method (Żyla et al. 1989). The inoculation and culture conditions were the same as the described above. After 1–5 days incubation, the supernatant of each culture broth was obtained by centrifugation and then analyzed. Acid phosphatase activity was determined in 0.1 mol L–1 citrate buffer (pH 5.0), and alkaline phosphatase activity was determined in 0.1 mol L–1 carbonate buffer (pH 9.7). One unit of acid or alkaline phosphatase activity was defined as the amount of enzyme that produced 1 μmol L–1 of p-nitrophenol from p-nitrophenyl phosphate in one minute under standard conditions (pH 5.0 or 9.7, 37°C).
2.4. Preparation of crude enzyme T. asperellum Q1 was fermented in a medium containing 0.75% (w/v) bran, 0.5% (w/v) peptone, 0.125% (w/v) NaCl, 0.02% (w/v) MgSO4·7H2O, 0.5% (w/v) KNO3, and 0.005% (w/v) FeSO4·7H2O in distilled water (pH 7.0–7.5). The inoculation and culture conditions were the same as the described above. After 3 days, the supernatant containing crude enzyme was obtained by centrifugation at 4 000 r min–1 for 10 min.
2.5. Purification of ACPase The crude enzyme was concentrated by (NH4)2SO4 fractionation and was first brought to 20% saturation with (NH4)2SO4. The mixture was stirred for 4 h at 4°C and then centrifuged at 12 000 r min–1 for 20 min. The supernatant was then brought to 80% saturation with (NH4)2SO4. The precipitate was suspended in 30 mL of 50 mmol L–1 Tris-HCl (pH 5.8), and was dialysed overnight at 4°C against 50 mmol L–1 Tris-HCl (pH 5.8). The enzyme was further purified by ion exchange chromatography with DEAE-Sepharose Fast Flow (Sigma, USA). The column (1.0 cm×10 cm) was equilibrated with 50 mmol L–1 sodium acetate buffer (pH 5.8) in advance. After loading the enzyme sample, the column was washed with the equilibration buffer until the absorbance of the effluent at 280 nm reached baseline levels, and a linear gradient elution was conducted with 100 mL of 50 mmol L–1 sodium acetate buffer (pH 5.8) and 100 mL of the same buffer containing 1 mol L–1 NaCl. The flow rate was 3 mL min–1 and the fractions were collected in each tube per minute and tested for ACPase activity. The active fractions were pooled for further purification. The concentrated enzyme solution was loaded into the Sephadex G-100 column (3.0 cm
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2.7. Effects of acid phosphatase on Arabidopsis seedlings growth Arabidopsis seeds were surface-sterilized with 75% (v/v) ethanol for 5 min and 20% (v/v) bleach for 7 min. After five washes in distilled water, the seeds were germinated and grown on 1/2 MS medium plates supplemented with 60 mmol L–1 NaCl. Plates were placed vertically at an angle of 65°C to allow root growth along the agar surface as well as unimpeded aerial growth of the hypocotyls. The plants were placed in a plant growth chamber with a photoperiod of 16 h of light/8 h of darkness, a light intensity of 300 μmol m2 s–1, and a temperature of 22°C (Contreras-Conejo et al. 2009). The treatments and corresponding media were shown in Table 1.
2.8. Statistical analysis Data were presented as the means±standard deviations, and the statistical significance was determined by Duncan’s multiple range test with P-values<0.05.
3. Results 3.1. The P-solubilization ability of T. asperellum Q1 under salt stress Generally, insoluble phosphates in soils include organic and inorganic forms. Therefore, the solubilization ability of T. asperellum Q1 for TCP, DCP, and phytin under salt stress was examined. As shown in Fig. 1, salt stress stimulated P-solubilization ability compared to controls after inoculation of T. asperellum Q1 at 36 h. Solubilization rates of TCP and DCP were increased by 16.5 and 29.4%, respectively, compared to controls after 36 h of inoculation under salt stress (Fig. 1-A and B). Solubilization of phytin achieved a maximum level of 28.40 mg L–1 after 60 h of incubation, which was 46.8% higher than that of the control (Fig. 1-C).
Treatments1) Medium components2) CK 20 mL 1/2 MS+1 mL buffer+60 mmol L–1 NaCl E 20 mL 1/2 MS+3.3×10–3 U enzyme+60 mmol L–1 NaCl P 20 mL 1/2 MS+0.1 g phytin+1 mL buffer+60 mmol L–1 NaCl E+P 20 mL 1/2 MS+0.1 g phytin+3.3×10–3 U enzyme+60 mmol L–1 NaCl 1)
CK, treated without acid phosphatase and phytin; E, treated with acid phosphatase and without phytin; P, treated with phytin but without acid phosphatase; E+P, treated with acid phosphatase and phytin. 2) Buffer, 50 mmol L–1 sodium acetate buffer (pH 5.8). Each treatment was repeated five times. 10–20 seeds were used in each treatment.
CK
A TCP solubilization(mg L–1)
SDS-PAGE was used to determine ACPase purity and its molecular weight using a 12% (w/v) acrylamide gel. High-sensitivity silver staining was used for detecting proteins (Blum et al. 1987).
Table 1 The design of plate experiments
B DCP solubilization (mg L–1)
2.6. Determination of purity and molecular weight of ACPase
This indicated that salt stress can improve P-solubilization ability of T. asperellum Q1. Additionally, the increasing degree of solubilization of insoluble organic phosphorus was
C Phytin solubilization (mg L–1)
×100 cm, Sigma, USA) that had been equilibrated with 50 mmol L–1 sodium acetate buffer (pH 5.8). The column was washed at a flow rate of 0.3 mL min–1 with the same buffer, and fractions were collected in each tube every 20 min. The fractions with ACPase activity were then pooled for subsequent analysis.
Salt stress
250 200 150 100 50 0 180 160 140 120 100 80 60 40 20 0 45 40 35 30 25 20 15 10 5 0
0
12
24
36
48
60
72
0
12
24
36
48
60
72
0
12
24 36 48 Incubation time (h)
60
72
Fig. 1 Spectrophotometric determination of available phosphorus concentration by Trichoderma asperellum Q1. A, tricalcium phosphate (TCP). B, dibasic calcium phosphate (DCP). C, phytin. Liquid fermentation medium without salt stress was used as the controls. Error bars indicate the standard error (SE) of the means. The experiments were repeated three times.
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much higher than that of insoluble inorganic phosphorus (Fig. 1-A, B and C).
3.3. Purification of ACPase
3.2. Phosphatase activity assay Previously, we have demonstrated that 200 mmol L–1 of salt concentration had inhibitory effect on the growth of T. asperellum Q1 (data not shown). T. asperellum Q1 was cultivated in liquid medium containing phytin as the sole P source, which acted as the main substrate of phosphatase. As shown in Fig. 2, activities of ACPase and alkaline phosphatase reached peaks at 2 days after incubation and then decreased gradually until 5 days. Furthermore, activities of ACPase and alkaline phosphatase in culture supernatant of T. asperellum Q1 were significantly increased in salt stressed media compared with the controls. The highest activity of ACPase was 9.61×10–3 U mL–1 at 2 days after inoculation of T. asperellum Q1 (Fig. 2-A). Additionally, the maximum activity of alkaline phosphatase was merely 1.24×10–3 U mL–1, which was lesser than the ACPase activity (Fig. 2-B). Both of the acid and alkaline phosphatase activities of T. asperellum Q1 were increased by 30 and
CK
Acid phosphatase activity (×10–3 U mL–1)
A
Alkaline phosphatase activity (×10–3 U mL–1)
B
12 10 8 6 4 2 0
0
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
1
2
20% respectively at 2 days after inoculation (Fig. 2-A and B).
Salt stress
3
4
5
The ACPase secreted by T. asperellum Q1 was purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. All the purification steps were summarized in Table 2. The final result was 9.94-fold purification with an overall yield of 18.9%.
3.4. Determination of purity and molecular weight of ACPase After purification, the sample with ACPase activity showed a single band with molecular weight of 55 kDa on SDS-PAGE stained with silver nitrate (Fig. 3).
3.5. Effects of ACPase on Arabidopsis seedlings growth To reveal the effect of ACPase on plant growth-promoting, the Arabidopsis seedlings were grown on 1/2 MS plates with 4 different treatments according to Table 1. The result showed that the treatment containing both ACPase and phytin (E+P) resulted in the highest root length compared with the control (Fig. 4-A) and the other two treatments (Fig. 4-B and C). Meanwhile, there was an obvious hydrolyzation circle on the plates because of the insoluble phosphorus has been hydrolyzed by ACPase (Fig. 4-D). All the Arabidopsis seedlings in each treatment were investigated under a stereoscope for measurement. The seedling height and root length in E+P treatment increased by 33.1 and 34.3%, respectively, compared with the control. These results showed that ACPase from T. asperellum Q1 plays an important role in regulation of Arabidopsis growth under salt stress.
4. Discussion 0
1
2 3 Incubation time (h)
4
5
Fig. 2 Effect of salt stress on the phosphatase activity in the culture supernatant of Trichoderma asperellum Q1. A, acid phosphatase activity. B, alkaline phosphatase activity. Error bars indicate the standard error (SE) of the means. The experiments were repeated three times.
It has been reported that Na+ is an activator of many enzymes including phytase and phosphatase. For example, Na+ can stimulate extracellular phytase isolated from the marine yeast Kodamaea ohmeri BG3 (Li et al. 2008). Moreover, Na+ could enhance the activity of alkaline phosphatase from Aspergillus caespitosus by 51% (Guimarães et al.
Table 2 The purification table for acid phosphatase from Trichoderma asperellum Q1 Purification step Crude enzyme (NH4)2SO4 DEAE-sepharose fast flow Gel filtration
Protein (mg) 2 258.9 550.4 58.5 42.7
Total activity (×10–3 U) 1 462.5 728.2 339.0 275.8
Specific activity (×10–3 U mg–1) 0.65 1.32 5.79 6.46
Purification (fold) 1.0 2.03 8.91 9.94
Yield (%) 100 49.8 23.2 18.9
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MW (kDa)
1
A
B
170 130 100 70 55 40
E
CK C
D
35 25 E+P
P
15 10
Fig. 3 Silver nitrate-stained SDS-PAGE analysis of purified acid phosphatase (ACPase) from Trichoderma asperellum Q1. MW, molecular weight standards; lane 1, purified ACPase.
2007). So this may be similar to the P-solubilization ability of T. asperellum Q1 which could be improved by salinity. The P-solubilization mechanisms of Trichoderma spp. may differ from the different P sources added into the medium. Both inorganic and organic acids can release PO43– from phosphatic deposit while phosphatase mainly hydrolyzes organic P compounds (de Oliveira Mendes et al. 2014). ACPase could hydrolyze phosphomonoester and converted insoluble organic P into a soluble form. But its role in P-solubilization differs from different kinds of microorganisms. Shen et al. (2011) demonstrated that most plant growth-promoting rhizo-bacteria can excrete ACPase, and some studies have also been conducted on the purification and characterization of ACPase from fungi (Shimizu 1993; Guimarães et al. 2004; Leitão et al. 2010). An ACPase from T. harzianum was purified in a single step using a phenyl-sepharose chromatography column. Its molecular weight was approximately 57.8 kDa, and the optimum pH and temperature was 4.8 and 55°C, respectively (Leitão et al. 2010). In this study, the ACPase from T. asperellum Q1 was purified by a combination of ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The purified enzyme showed as a single band on SDS-PAGE and the apparent molecular weight was generally consistent with that of T. harzianum (Leitão et al. 2010). However, it was obviously less than that from A. caespitosus (186 and 190 kDa) (Guimarães et al. 2004) and greater than that from A. fumigatus (18 kDa) (Bernard et al. 2002). Although we have verified that the fermentation broth of T. asperellum Q1 that contains ACPase could promote plant growth by P-solubilization (Zhao and Zhang 2015), the effects of other components cannot be ruled out.
Plant height and root length (cm)
E 3.2
Plant height
2.8
Root length
2.4 2.0 1.6 1.2 0.8 0.4 0.0
CK
E P Treatment
E+P
Fig. 4 Effect of acid phosphatase (ACPase) produced by Trichoderma asperellum Q1 on height and root length of Arabidopsis under salt stress. A, CK, treated without ACPase and phytin. B, E, treated with ACPase and without phytin. C, P, treated with phytin and without ACPase. D, E+P, treated with ACPase and phytin. E, plant height and root length. All plates were supplemented with 60 mmol L–1 NaCl in 1/2 MS medium. Means of five replicates (±SE) followed by the same letter are not significantly different at P<0.05.
T. asperellum Q1 has the ability to produce siderophore and organic acids, which have been testified in our lab (Zhao et al. 2014). Some researchers have also discovered that the siderophore produced by arsenic-resistant bacteria could solubilizing P from inorganic minerals, phosphate rock and organic phytate (Ghosh et al. 2015). However, our study revealed the effect of the ACPase purified from T. asperellum Q1 on plant growth promotion by P-solubilization under salt stress. So the significance of this study shed light on the function of ACPase from T. asperellum Q1 in promoting plant growth by P-solubilization under salt stress.
5. Conclusion This study revealed that the ACPase produced by T. asperellum Q1 showed significant effect on growth of Arabidopsis under salt stress. This will represent an
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important step forward in understanding the mechanisms of plant-growth promoting under salt stress and may improve Trichoderma to become a candidate for phosphate solubilizing biofertilizer.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31171806).
Reference Aseri G K, Jain N, Tarafdar J C. 2009. Hydrolysis of organic phosphate forms by phosphatases and phytase producing fungi of arid and semi-arid soils of India. American-Eurasian Jouranl of Agricultural Environmental Science, 5, 564–570. Altomare C, Norvell W A, Björkman T, Harman G E. 1999. Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22. Applied and Environmental Microbiology, 65, 926–933. Bernard M, Mouyna I, Dubreucq G, Debeaupuis J P, Fontaine T, Vorgias C, Latge J P. 2002. Characterization of a cellwall acid phosphatase (PhoAp) in Aspergillus fumigatus. Microbiology, 148, 2819–2829. Blum H, Beier H, Gross H J. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, 8, 93–99. Contreras-Conejo H A, Macias-Rodriguez L, Cortés-Penagos C, López-Bucio J. 2009. Trichoderma virens, a plant beneficial fungus, enhances biomass production and promotes lateral root growth through an auxin-dependent mechanism in Arabidopsis. Plant Physiology, 149, 1579–1592. Ghosh P, Rathinasabapathi B, Ma L Q. 2015. Phosphorus solubilization and plant growth enhancement by arsenicresistant bacteria. Chemosphere, 134, 1–6. Guimarães L H S, Júnior A B, Jorge J A, Terenzi H F, Polizeli M L T M. 2007. Purification and biochemical characterization of a mycelial alkaline phosphatase without DNAase activity produced by Aspergillus caespitosus. Folia Microbiologica, 52, 231–236. Guimarães L H S, Terenzi H F, Jorge J A, Leone F A, Polizeli M D L. 2004. Characterization and properties of acid phosphatases with phytase activity produced by Aspergillus caespitosus. Biotechnology and Applied Biochemistry, 40, 201–207. Illmer P, Schinner F. 1992. Solubilization of inorganic phosphates by microorganisms isolated from forest soils. Soil Biology and Biochemistry, 24, 389–395. Kang Y, Hu J, Shan J, He F, Piao Z, Yin S. 2005. Solubilization capacity of insoluble phosphates and it’s mechanism by two phosphate solubilizing fungi (PSF). Microbiology China, 33, 22–27. (in Chinese) Leitão V O, de Melo Lima R C, Vainstein M H, Ulhoa C J. 2010. Purification and characterization of an acid phosphatase from Trichoderma harzianum. Biotechnology Letters, 32,
1083–1088. Li X Y, Chi Z M, Liu Z Q, Li J, Wang X H, Hirimuthugoda N Y. 2008. Purification and characterization of extracellular phytase from a marine yeast Kodamaea ohmeri BG3. Marine Biotechnology, 10, 190–197. de Oliveira Mendes G, de Freitas A L M, Pereira O L, da Silva I R, Vassilev N B, Costa M D. 2014. Mechanisms of phosphate solubilization by fungal isolates when exposed to different P sources. Annals of Microbiology, 64, 239–249. Pradhan N, Sukla L B. 2009. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. African Journal of Biotechnology, 5, 850–854. Qi W Z, Zhao L. 2013. Study of the siderophore-producing Trichoderma asperellum Q1 on cucumber growth promotion under salt stress. Journal of Basic Microbiology, 53, 355–364. Rodrıg ́ uez H, Fraga R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances, 17, 319–339. Shen J B, Yuan L X, Zhang J L, Li H G, Bai Z H, Chen X P, Zhang W F, Zhang F S. 2011. Phosphorus dynamics: from soil to plant. Plant Physiology, 156, 997–1005. Shimizu M. 1993. Purification and characterization of phytase and acid phosphatase produced by Aspergillus oryzae K1. Bioscience, Biotechnology, and Biochemistry, 57, 1364–1365. Son H J, Park G T, Cha M S, Heo M S. 2006. Solubilization of insoluble inorganic phosphates by a novel salt- and pHtolerant Pantoea agglomerans R-42 isolated from soybean rhizosphere. Bioresource Technology, 97, 204–210. Vassilev N, Vassileva M, Nikolaeva I. 2006. Simultaneous P-solubilizing and biocontrol activity of microorganisms: potentials and future trends. Applied Microbiology and Biotechnology, 71, 137–144. Viterbo A, Landau U, Kim S, Chernin L, Chet I. 2010. Characterization of ACC deaminase from the biocontrol and plant growth-promoting agent Trichoderma asperellum T203. FEMS Microbiology Letters, 305, 42–48. von Wandruszka R. 2006. Phosphorus retention in calcareous soils and the effect of organic matter on its mobility. Geochemical Transactions, 7, 1–8. Zhao L, Wang F, Zhang Y Q, Zhang J J. 2014. Involvement of Trichoderma asperellum strain T6 in regulating iron acquisition in plants. Journal of Basic Microbiology, 54, 115–124. Zhao L, Zhang Y Q. 2015. Effects of phosphate solubilization and phytohormone production of Trichoderma asperellum Q1 on promoting cucumber growth under salt stress. Journal of Integrative Agriculture, 14, 1588–1597. Zhong C Q, Cao G X, Huang W Y, Luan X S, Yang Y F. 2014. Dissolving mechanism of strain P17 on insoluble phosphorus of yellow-brown soil. Brazilian Journal of Microbiology, 45, 937–943. Żyla K, Kujawski M, Koreleski J. 1989. Dephosphorylation of phytate compounds by means of acid phosphatase from Aspergillus niger. Journal of the Science of Food and Agriculture, 49, 315–324. (Managing editor ZHANG Juan)
ScienceDirect
RESEARCH ARTICLE
Effect of acid phosphatase produced by Trichoderma asperellum Q1 on growth of Arabidopsis under salt stress ZHAO Lei1, 2*, LIU Qun2*, ZHANG Ya-qing2*, CUI Qing-yu2*, LIANG Yuan-cun3 1 2 3
Institute of Environment and Ecology, Shandong Normal University, Jinan 250014, P.R.China College of Life Science, Shandong Normal University, Jinan 250014, P.R.China College of Plant Protection, Shandong Agricultural University, Tai’an 271018, P.R.China
Abstract Salt stress is a major environmental factor that inhibits crop growth. Trichoderma spp. are the most efficient biocontrol fungi and some of the strains can stimulate plant growth. Phosphate solubilization is known as one of the main mechanisms in promoting plant growth, but the underlying mechanisms of phosphate solubilization in the salinity still need to be explored. The Trichoderma asperellum Q1 isolated and identified in our lab is a beneficial rhizosphere biocontrol fungus with a high phosphate solubilization activity. It could produce acid and alkaline phosphatases when using insoluble organic phosphorus as the sole phosphorus source, the salt stress increased the phosphorus-solubilization ability of the strain and the activities of the two enzymes. Furthermore, an acid phosphatase was purified from the fermentation broth by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. Its molecular weight was 55 kDa as determined by SDSPAGE. The purified acid phosphatase was used to investigate growth performance of Arabidopsis thaliana by plate assay and the result showed that it contributed to Arabidopsis growth by transforming organic phosphate into a soluble inorganic form under salt stress. To our knowledge, this is the first report on acid phosphatase purification from T. asperellum and its function in regulation of plant growth under salt stress. Keywords: Trichoderma asperellum, Arabidopsis thaliana, acid phosphatase, plant-growth promotion, salt stress
Schinner 1992). Many soils throughout the world are P
1. Introduction Phosphorus (P) is one of the most essential macronutrients required for plant growth and development (Illmer and
deficient because the free P concentration (the form available to plant) is low (Rodrı́guez et al. 1999). Even in fertile soils, it is not sufficient for plants due to the high reactivity of soluble P with calcium, iron, or aluminum, which leads to P precipitation (Rodrı́guez et al. 1999; von Wandruszka 2006). In order to overcome the defects, phosphate fertilizers are frequently added into the soil in order to obtain
Received 11 May, 2016 Accepted 21 September, 2016 Correspondence ZHAO Lei, Tel: +86-531-86188195, Fax: +86531-86180107, E-mail: [email protected]; LIANG Yuan-cun, Tel: +86-538-8242301, E-mail: [email protected] * These authors contributed equally to this study. © 2017, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(16)61490-9
good productivity (Pradhan and Sukla 2009). Moreover, the production of traditional P fertilizers is a costly chemical processing and the overuse of them may results in soil salinization (Vassilev et al. 2006). It is known that some microorganisms are involved in the solubilization of insoluble phosphate into the soluble
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form (Son et al. 2006). These P-solubilizing microbes excrete inorganic acids, organic acids, and phosphatase to dissolve phosphorus (Zhong et al. 2014). However, the mechanisms of P-solubilization not only differ among fungal isolates but also depend on the applied P sources (de Oliveira Mendes et al. 2014). Trichoderma is a highly desirable bioagent with dual potentials for biocontrol and plant growth promotion. Certain effects of Trichoderma spp. on plant development have been studied, such as production of 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase (Viterbo et al. 2010), siderophore (Qi and Zhao 2013), and solubilization of insoluble minor nutrients or phosphate (Altomare et al. 1999). However, the underlying mechanisms of the phosphate-solubilizing ability in promoting plant stress tolerance have not been fully elucidated. The previous study has confirmed that there were positive correlation between hydrolyzed organic P compounds and the secretion of phosphatases or phytase in Trichoderma harzianum and Trichoderma viride (Aseri et al. 2009). However, the effect of acid phosphatase (ACPase) on P-solubilization and plant growth promotion needs to be proved. Therefore, we investigated the solubilization of insoluble phosphorus and phytin of T. asperellum Q1 and the effect of its ACPase on Arabidopsis growth-promoting under salt stress.
2. Materials and methods 2.1. Strain T. asperellum Q1 was isolated and identified previously from the rhizosphere of cucumber in a greenhouse in Shandong Province, China (Qi and Zhao 2013). The strain was maintained on potato dextrose agar slants at 4°C.
2.2. Quantitative assay of phosphate solubilizing activity In order to determine the differences in the phosphate solubilization with or without salt stress, the following experiments were conducted. 1 mL spore suspension (1×107 spores mL–1) of T. asperellum Q1 was used to inoculate 250 mL Erlenmeyer flasks containing 100 mL medium, and then incubated at 28°C on a rotary shaker at 180 r min–1. The media which contained tricalcium phosphate (TCP), dibasic calcium phosphate (DCP) or phytin individually without salt stress were used as the control. After 12, 24, 36, 48, 60, and 72 h of incubation, each culture supernatant was obtained by centrifugation and the available phosphorus was analyzed using the ammonium vanadate-molybdate spectrophotometry method (Kang et al. 2005).
2.3. Phosphatase activity assay Pikovskaya’s broth (Phytin was supplied as the sole P-source) was used to measure the phosphatase activity by employing the p-nitrophenyl phosphate method (Żyla et al. 1989). The inoculation and culture conditions were the same as the described above. After 1–5 days incubation, the supernatant of each culture broth was obtained by centrifugation and then analyzed. Acid phosphatase activity was determined in 0.1 mol L–1 citrate buffer (pH 5.0), and alkaline phosphatase activity was determined in 0.1 mol L–1 carbonate buffer (pH 9.7). One unit of acid or alkaline phosphatase activity was defined as the amount of enzyme that produced 1 μmol L–1 of p-nitrophenol from p-nitrophenyl phosphate in one minute under standard conditions (pH 5.0 or 9.7, 37°C).
2.4. Preparation of crude enzyme T. asperellum Q1 was fermented in a medium containing 0.75% (w/v) bran, 0.5% (w/v) peptone, 0.125% (w/v) NaCl, 0.02% (w/v) MgSO4·7H2O, 0.5% (w/v) KNO3, and 0.005% (w/v) FeSO4·7H2O in distilled water (pH 7.0–7.5). The inoculation and culture conditions were the same as the described above. After 3 days, the supernatant containing crude enzyme was obtained by centrifugation at 4 000 r min–1 for 10 min.
2.5. Purification of ACPase The crude enzyme was concentrated by (NH4)2SO4 fractionation and was first brought to 20% saturation with (NH4)2SO4. The mixture was stirred for 4 h at 4°C and then centrifuged at 12 000 r min–1 for 20 min. The supernatant was then brought to 80% saturation with (NH4)2SO4. The precipitate was suspended in 30 mL of 50 mmol L–1 Tris-HCl (pH 5.8), and was dialysed overnight at 4°C against 50 mmol L–1 Tris-HCl (pH 5.8). The enzyme was further purified by ion exchange chromatography with DEAE-Sepharose Fast Flow (Sigma, USA). The column (1.0 cm×10 cm) was equilibrated with 50 mmol L–1 sodium acetate buffer (pH 5.8) in advance. After loading the enzyme sample, the column was washed with the equilibration buffer until the absorbance of the effluent at 280 nm reached baseline levels, and a linear gradient elution was conducted with 100 mL of 50 mmol L–1 sodium acetate buffer (pH 5.8) and 100 mL of the same buffer containing 1 mol L–1 NaCl. The flow rate was 3 mL min–1 and the fractions were collected in each tube per minute and tested for ACPase activity. The active fractions were pooled for further purification. The concentrated enzyme solution was loaded into the Sephadex G-100 column (3.0 cm
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2.7. Effects of acid phosphatase on Arabidopsis seedlings growth Arabidopsis seeds were surface-sterilized with 75% (v/v) ethanol for 5 min and 20% (v/v) bleach for 7 min. After five washes in distilled water, the seeds were germinated and grown on 1/2 MS medium plates supplemented with 60 mmol L–1 NaCl. Plates were placed vertically at an angle of 65°C to allow root growth along the agar surface as well as unimpeded aerial growth of the hypocotyls. The plants were placed in a plant growth chamber with a photoperiod of 16 h of light/8 h of darkness, a light intensity of 300 μmol m2 s–1, and a temperature of 22°C (Contreras-Conejo et al. 2009). The treatments and corresponding media were shown in Table 1.
2.8. Statistical analysis Data were presented as the means±standard deviations, and the statistical significance was determined by Duncan’s multiple range test with P-values<0.05.
3. Results 3.1. The P-solubilization ability of T. asperellum Q1 under salt stress Generally, insoluble phosphates in soils include organic and inorganic forms. Therefore, the solubilization ability of T. asperellum Q1 for TCP, DCP, and phytin under salt stress was examined. As shown in Fig. 1, salt stress stimulated P-solubilization ability compared to controls after inoculation of T. asperellum Q1 at 36 h. Solubilization rates of TCP and DCP were increased by 16.5 and 29.4%, respectively, compared to controls after 36 h of inoculation under salt stress (Fig. 1-A and B). Solubilization of phytin achieved a maximum level of 28.40 mg L–1 after 60 h of incubation, which was 46.8% higher than that of the control (Fig. 1-C).
Treatments1) Medium components2) CK 20 mL 1/2 MS+1 mL buffer+60 mmol L–1 NaCl E 20 mL 1/2 MS+3.3×10–3 U enzyme+60 mmol L–1 NaCl P 20 mL 1/2 MS+0.1 g phytin+1 mL buffer+60 mmol L–1 NaCl E+P 20 mL 1/2 MS+0.1 g phytin+3.3×10–3 U enzyme+60 mmol L–1 NaCl 1)
CK, treated without acid phosphatase and phytin; E, treated with acid phosphatase and without phytin; P, treated with phytin but without acid phosphatase; E+P, treated with acid phosphatase and phytin. 2) Buffer, 50 mmol L–1 sodium acetate buffer (pH 5.8). Each treatment was repeated five times. 10–20 seeds were used in each treatment.
CK
A TCP solubilization(mg L–1)
SDS-PAGE was used to determine ACPase purity and its molecular weight using a 12% (w/v) acrylamide gel. High-sensitivity silver staining was used for detecting proteins (Blum et al. 1987).
Table 1 The design of plate experiments
B DCP solubilization (mg L–1)
2.6. Determination of purity and molecular weight of ACPase
This indicated that salt stress can improve P-solubilization ability of T. asperellum Q1. Additionally, the increasing degree of solubilization of insoluble organic phosphorus was
C Phytin solubilization (mg L–1)
×100 cm, Sigma, USA) that had been equilibrated with 50 mmol L–1 sodium acetate buffer (pH 5.8). The column was washed at a flow rate of 0.3 mL min–1 with the same buffer, and fractions were collected in each tube every 20 min. The fractions with ACPase activity were then pooled for subsequent analysis.
Salt stress
250 200 150 100 50 0 180 160 140 120 100 80 60 40 20 0 45 40 35 30 25 20 15 10 5 0
0
12
24
36
48
60
72
0
12
24
36
48
60
72
0
12
24 36 48 Incubation time (h)
60
72
Fig. 1 Spectrophotometric determination of available phosphorus concentration by Trichoderma asperellum Q1. A, tricalcium phosphate (TCP). B, dibasic calcium phosphate (DCP). C, phytin. Liquid fermentation medium without salt stress was used as the controls. Error bars indicate the standard error (SE) of the means. The experiments were repeated three times.
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much higher than that of insoluble inorganic phosphorus (Fig. 1-A, B and C).
3.3. Purification of ACPase
3.2. Phosphatase activity assay Previously, we have demonstrated that 200 mmol L–1 of salt concentration had inhibitory effect on the growth of T. asperellum Q1 (data not shown). T. asperellum Q1 was cultivated in liquid medium containing phytin as the sole P source, which acted as the main substrate of phosphatase. As shown in Fig. 2, activities of ACPase and alkaline phosphatase reached peaks at 2 days after incubation and then decreased gradually until 5 days. Furthermore, activities of ACPase and alkaline phosphatase in culture supernatant of T. asperellum Q1 were significantly increased in salt stressed media compared with the controls. The highest activity of ACPase was 9.61×10–3 U mL–1 at 2 days after inoculation of T. asperellum Q1 (Fig. 2-A). Additionally, the maximum activity of alkaline phosphatase was merely 1.24×10–3 U mL–1, which was lesser than the ACPase activity (Fig. 2-B). Both of the acid and alkaline phosphatase activities of T. asperellum Q1 were increased by 30 and
CK
Acid phosphatase activity (×10–3 U mL–1)
A
Alkaline phosphatase activity (×10–3 U mL–1)
B
12 10 8 6 4 2 0
0
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0
1
2
20% respectively at 2 days after inoculation (Fig. 2-A and B).
Salt stress
3
4
5
The ACPase secreted by T. asperellum Q1 was purified by ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. All the purification steps were summarized in Table 2. The final result was 9.94-fold purification with an overall yield of 18.9%.
3.4. Determination of purity and molecular weight of ACPase After purification, the sample with ACPase activity showed a single band with molecular weight of 55 kDa on SDS-PAGE stained with silver nitrate (Fig. 3).
3.5. Effects of ACPase on Arabidopsis seedlings growth To reveal the effect of ACPase on plant growth-promoting, the Arabidopsis seedlings were grown on 1/2 MS plates with 4 different treatments according to Table 1. The result showed that the treatment containing both ACPase and phytin (E+P) resulted in the highest root length compared with the control (Fig. 4-A) and the other two treatments (Fig. 4-B and C). Meanwhile, there was an obvious hydrolyzation circle on the plates because of the insoluble phosphorus has been hydrolyzed by ACPase (Fig. 4-D). All the Arabidopsis seedlings in each treatment were investigated under a stereoscope for measurement. The seedling height and root length in E+P treatment increased by 33.1 and 34.3%, respectively, compared with the control. These results showed that ACPase from T. asperellum Q1 plays an important role in regulation of Arabidopsis growth under salt stress.
4. Discussion 0
1
2 3 Incubation time (h)
4
5
Fig. 2 Effect of salt stress on the phosphatase activity in the culture supernatant of Trichoderma asperellum Q1. A, acid phosphatase activity. B, alkaline phosphatase activity. Error bars indicate the standard error (SE) of the means. The experiments were repeated three times.
It has been reported that Na+ is an activator of many enzymes including phytase and phosphatase. For example, Na+ can stimulate extracellular phytase isolated from the marine yeast Kodamaea ohmeri BG3 (Li et al. 2008). Moreover, Na+ could enhance the activity of alkaline phosphatase from Aspergillus caespitosus by 51% (Guimarães et al.
Table 2 The purification table for acid phosphatase from Trichoderma asperellum Q1 Purification step Crude enzyme (NH4)2SO4 DEAE-sepharose fast flow Gel filtration
Protein (mg) 2 258.9 550.4 58.5 42.7
Total activity (×10–3 U) 1 462.5 728.2 339.0 275.8
Specific activity (×10–3 U mg–1) 0.65 1.32 5.79 6.46
Purification (fold) 1.0 2.03 8.91 9.94
Yield (%) 100 49.8 23.2 18.9
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MW (kDa)
1
A
B
170 130 100 70 55 40
E
CK C
D
35 25 E+P
P
15 10
Fig. 3 Silver nitrate-stained SDS-PAGE analysis of purified acid phosphatase (ACPase) from Trichoderma asperellum Q1. MW, molecular weight standards; lane 1, purified ACPase.
2007). So this may be similar to the P-solubilization ability of T. asperellum Q1 which could be improved by salinity. The P-solubilization mechanisms of Trichoderma spp. may differ from the different P sources added into the medium. Both inorganic and organic acids can release PO43– from phosphatic deposit while phosphatase mainly hydrolyzes organic P compounds (de Oliveira Mendes et al. 2014). ACPase could hydrolyze phosphomonoester and converted insoluble organic P into a soluble form. But its role in P-solubilization differs from different kinds of microorganisms. Shen et al. (2011) demonstrated that most plant growth-promoting rhizo-bacteria can excrete ACPase, and some studies have also been conducted on the purification and characterization of ACPase from fungi (Shimizu 1993; Guimarães et al. 2004; Leitão et al. 2010). An ACPase from T. harzianum was purified in a single step using a phenyl-sepharose chromatography column. Its molecular weight was approximately 57.8 kDa, and the optimum pH and temperature was 4.8 and 55°C, respectively (Leitão et al. 2010). In this study, the ACPase from T. asperellum Q1 was purified by a combination of ammonium sulphate precipitation, ion-exchange, and gel filtration chromatography. The purified enzyme showed as a single band on SDS-PAGE and the apparent molecular weight was generally consistent with that of T. harzianum (Leitão et al. 2010). However, it was obviously less than that from A. caespitosus (186 and 190 kDa) (Guimarães et al. 2004) and greater than that from A. fumigatus (18 kDa) (Bernard et al. 2002). Although we have verified that the fermentation broth of T. asperellum Q1 that contains ACPase could promote plant growth by P-solubilization (Zhao and Zhang 2015), the effects of other components cannot be ruled out.
Plant height and root length (cm)
E 3.2
Plant height
2.8
Root length
2.4 2.0 1.6 1.2 0.8 0.4 0.0
CK
E P Treatment
E+P
Fig. 4 Effect of acid phosphatase (ACPase) produced by Trichoderma asperellum Q1 on height and root length of Arabidopsis under salt stress. A, CK, treated without ACPase and phytin. B, E, treated with ACPase and without phytin. C, P, treated with phytin and without ACPase. D, E+P, treated with ACPase and phytin. E, plant height and root length. All plates were supplemented with 60 mmol L–1 NaCl in 1/2 MS medium. Means of five replicates (±SE) followed by the same letter are not significantly different at P<0.05.
T. asperellum Q1 has the ability to produce siderophore and organic acids, which have been testified in our lab (Zhao et al. 2014). Some researchers have also discovered that the siderophore produced by arsenic-resistant bacteria could solubilizing P from inorganic minerals, phosphate rock and organic phytate (Ghosh et al. 2015). However, our study revealed the effect of the ACPase purified from T. asperellum Q1 on plant growth promotion by P-solubilization under salt stress. So the significance of this study shed light on the function of ACPase from T. asperellum Q1 in promoting plant growth by P-solubilization under salt stress.
5. Conclusion This study revealed that the ACPase produced by T. asperellum Q1 showed significant effect on growth of Arabidopsis under salt stress. This will represent an
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important step forward in understanding the mechanisms of plant-growth promoting under salt stress and may improve Trichoderma to become a candidate for phosphate solubilizing biofertilizer.
Acknowledgements This work was supported by the National Natural Science Foundation of China (31171806).
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