Abstract
High phosphate solubility is one of the most important factors for increasing plant growth. This study focused on the zinc phosphate (ZP) solubilizing capacity of thermo-halotolerant Aspergillus terreus, where the growth showed halo zones on Pikovskaya agar medium that appeared at high NaCl concentrations (up to 10%) and a wide range of temperatures (up to 45 °C). Acidification of the broth was assumed to be the major mechanism for ZP solubilization by A. terreus, where the growth was related to the pH decrease in the medium containing ZP. Under pot conditions, A. terreus increased the biomass and phosphorus content of Hordeum vulgare plants. A. terreus showed a phosphorus solubilization ability with a NaCl concentration of up to 10%; therefore, A. terreus can be of great benefit in maintaining the available phosphate levels for crops in saline soils. Finally, it was found that A. terreus with ZP can substitute chemical fertilizer and help improve crop production.
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Molecular Identification of Rhizospheric Thermo-halotolerant Aspergillus terreus and its Correlation to Sustainable Agriculture
Tarek M. Abdel-Ghany,a,b,* and Mohamed M. Alawlaqi a
High phosphate solubility is one of the most important factors for increasing plant growth. This study focused on the zinc phosphate (ZP) solubilizing capacity of thermo-halotolerant Aspergillus terreus, where the growth showed halo zones on Pikovskaya agar medium that appeared at high NaCl concentrations (up to 10%) and a wide range of temperatures (up to 45 °C). Acidification of the broth was assumed to be the major mechanism for ZP solubilization by A. terreus, where the growth was related to the pH decrease in the medium containing ZP. Under pot conditions, A. terreus increased the biomass and phosphorus content of Hordeum vulgare plants. A. terreus showed a phosphorus solubilization ability with a NaCl concentration of up to 10%; therefore, A. terreus can be of great benefit in maintaining the available phosphate levels for crops in saline soils. Finally, it was found that A. terreus with ZP can substitute chemical fertilizer and help improve crop production.
Keywords: Aspergillus terreus; Zinc phosphate; Solubilization; Biofertilizers
Contact information: a: Biology Department, Faculty of Science, Jazan University, Jazan, Saudi Arabia; b: Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Egypt;
* Corresponding author: tabdelghany@yahoo.com
INTRODUCTION
Phosphorus (P) is one of the most important macronutrients, after nitrogen, for the growth and development of plants (Hameeda et al. 2008; Sharma et al. 2013). The majority of soil P, approximately 95% to 99%, is in an insoluble form linked with metals, such as iron, zinc, aluminum, and calcium; therefore, it cannot be used by plants (Son et al. 2006) and must be converted into a soluble form. Phosphorus participates remarkably in plant metabolism, photosynthesis, energy and sugar production, and synthesis of nucleic acids, and it enhances nitrogen fixation (Saber et al. 2005).
Many microorganisms in agricultural soil plays an important role in sustainable agriculture (Abdel-Ghany et al. 2015, 2018a,b). Phosphate-solubilizing microorganisms (PSMs) can play a great role in dissolving both fertilizer P and bound P in the soil, which is environmentally friendly and sustainable (Khan et al. 2007).
These PSMs can solubilize and mineralize P from the inorganic and organic pools of the total soil P and may be utilized as inoculants to increase the availability of P to plants (Richardson 2001; Oliveira et al. 2009; Abdel-Ghany et al. 2013). Bhattacharyya and Jha (2012) stated that in the natural environment numerous microorganisms in the soil and rhizosphere are effective at releasing P from the total soil P through solubilization and mineralization. Numerous researchers have conducted investigations on PSMs in soils (Gupta et al. 2012), mangrove (Holguin et al. 2001), and the rhizosphere (Oliveira et al. 2009). Nahas (1996) reported that fungi possess a better ability to solubilize insoluble phosphate than bacteria. A broad range of soil fungi are reported to solubilize insoluble P, including Aspergillus niger and Penicillium species, which are the most common fungi capable of phosphate solubilization (Whitelaw et al. 1999; Nelofer et al. 2016).
Conflicting results have been reported for the effect of temperature on phosphorus solubilization by fungi and bacteria. Varsha et al. (2002) recorded 28 °C as the best temperature for P solubilization by Aspergillus aculeatus. A. awamori is capable of solubilizing insoluble phosphate over a wide range of high temperature (Walpola et al. 2012). On the other hand, Johri et al. (1999) reported solubilization at a low temperature of 10 °C.
Several mechanisms, including lowering the pH by acid production, ion chelation, and exchange reactions in the growth environment, have been reported to play a role in phosphate solubilization by soil fungi and bacteria (Abd-Alla 1994; Whitelaw 1999; Han et al. 2006). Penicillium and Aspergillus are able to change insoluble soil phosphates into soluble forms by secreting acids, such as formic, acetic, propionic, lactic, glycolic, fumaric, and succinic acids. These acids reduce the soil pH and bring about the dissolution of phosphate bonds (Bolan et al. 1997). Most of the reports in the literature suggested that tri-calcium phosphate is dissolved by acidification. Therefore, any microorganisms that acidify the external medium will show some level of phosphate-solubilizing activity (Chai et al. 2011; Sanjotha et al. 2011).
Inoculation with phosphate-solubilizing fungi (A. terreus and P. pinophilum) and the application of reactive phosphate rock improves the P uptake of plants, which results in an increased dry matter yield of sorghum under controlled conditions (Steiner et al. 2016). These PSMs have considerable synergistic effects on the growth and development of crops (Tallapragada and Gudimi 2011). Salt-tolerant or halophilic soil microorganisms that also exhibit the ability to solubilize insoluble P facilitate the development of saline-alkali soil-based agriculture (Zhu et al. 2011). The objective of this study was to select promising phosphate-solubilizing fungi under stressful salt and temperature conditions for the development of sustainable agriculture.
EXPERIMENTAL
Rhizosphere Sampling for Phosphate-solubilizing Fungi
Wild halophilic plants (Suaeda monoica) in Jizan, Saudi Arabia were uprooted from salt marsh soil (Fig. 1A) and gently shaken to remove the superfluous soil, and the rhizosphere soils were placed in clean sterilized polyethylene bags. A known weight of roots with soil was immersed in a flask containing sterilized distilled H2O. After being shaken, suitable dilutions were prepared. One milliliter of the suitable rhizosphere soil suspension was transferred to each sterilized petri plate and covered with melted, but cooled medium. The plates were incubated at 28 °C for 7 d to 10 d, and the developing fungi were counted and isolated.
Salt and Temperature Tolerance of the Fungal Isolates
The salt tolerance of the isolates was determined by inoculating the cultures in triplicate on Czapek medium amended with NaCl concentrations of up to 15% w/v. Rosebengal (1/15000, w/v) and chloramphenicol (50 ppm) were added to the growth medium to suppress bacterial growth. For temperature tolerance, the fungal isolates were cultivated on Czapek agar medium and incubated at different temperatures up to 50 °C. The fungal growth in terms of the colony diameter was recorded after 7 d of incubation. The fungus that was highly resistant to NaCl (halophilic/halotolerance) and temperature was purified.
Morphological and Molecular Identification of Fungal Isolate
The fungus that was highly resistant to NaCl (halophilic/halotolerance) and temperature was purified, identified morphologically and microscopically as A. terreus (Fig. 1B) according to established literature protocols (Raper and Fennell 1973; Pitt and Hocking 1997), and confirmed by molecular identification.
Fig. 1.(A) Hypersaline soil with Suaeda monoica plant for fungal isolation and (B) A. terreus colony growth
DNA Extraction and PCR Amplification
Fungal mycelia (0.2 g) of fungus grown on potato dextrose medium were harvested and placed in liquid nitrogen for 10 min, then vigorously homogenized to extract its genomic DNA. A DNA extraction buffer (500 µL) containing of 200 mM Tris-HCl, 240 mM NaCl, 25 mM EDTA, and 1% SDS at pH 8.0 has been mixed with the DNA extract, then shaken for 5 min and then centrifuged for 5 min at 10,000 rpm. Next, the supernatant has been mixed with an equal volume of phenol:chloroform (1:1 v/v) for 30 min and again centrifuged for 5 min at 12,000 rpm. The upper part has been gently outgoing and mixed with an equal volume of 3 M sodium acetate (pH 5.2) and 2 volumes of 96% ethanol for 60 min at -20 °C. After centrifugation, the collected DNA was washed with ethanol (70%), dried until removal of the ethanol, and re-suspended in 100 µL of distilled water.
Molecular identification of A. terreus was based on Internal transcribed spacer (ITS) rDNA sequence (18S- 28S rRNA), according to White et al. (1990). The sequences of the ITS1 and ITS4 primers were 5‘-TCCGTAGGTGAACCTGCGG-3‘and 5′- TCCTCCGCTTATTGATATGC-3′, respectively. Molecular evolutionary genetic analysis software MEGA6 (Germany) was used for phylogenetic analyses. The sequence was annotated and submitted to the NCBI Gen Bank database. A basic local alignment search tool (BLAST) homology search was performed to find the closest homology, and a sequence analysis was performed by comparison with ITS rDNA sequences of ten other fungal species obtained from the Gen Bank database. The evolutionary tree was reconstructed using the neighbor joining method.
Phosphate-solubilizing Ability Test
Aspergillus terreus was checked for a phosphate-solubilizing ability on Pikovskaya (PKV) agar medium that contained 0.5 g/L (NH4)2SO4, 0.1 g/L MgSO4·7H2O, 0.02 g/L NaCl, 0.02 g/LKCl, 0.003 g/L FeSO4·7H2O, 0.003 g/L MnSO4·H2O, 5 g/L Ca3(PO4)2, 10.0 g/L glucose, 0.5 g/L yeast extract, 15.0 g/L agar, and 1000 mL of distilled water (Pikovskaya 1948) containing zinc phosphate (ZP). The formation of a clear halo zone around the fungal growth after 5 d of incubation indicated a phosphate-solubilizing ability.
Zinc Phosphate Solubilization Efficiency
The PKV agar medium containing ZP was prepared and poured into sterilized petri plates. A. terreus (5-mm disc of fungal colony) was spotted on these plates and incubated for 7 days at different temperature ranged from 10 to 45 °C. Those that showed halo zones around the colonies were capable of phosphate solubilization. The solubilization index was calculated according to Eq. 1,
(1)
where SI is the solubilization index, C is the colony diameter (cm), and H is the halo zone diameter (cm).
Effect of the Salinity on Zinc Phosphate Solubilization
The effect of the salinity on the phosphate solubilization ability of A. terreus was determined in a PKV medium amended with NaCl (0% to 10%) over 5 d. The medium was inoculated with A. terreus and incubated at 30 °C. The ZP solubilization efficiency was determined using the method mentioned above.
Pot Experiment
Grains of Hordeum vulgare were surface sterilized, rinsed six times with sterile water, and dried. Spore suspensions of A. terreus at different levels (2 mL and 4 mL, each containing 2 × 106 spores) were added to autoclaved soils 48 h before sowing. The uninoculated seeds served as a control treatment for comparison. Zinc phosphate was added (0.5 g/kg) to the soil before seeding, except to the control. The plants were irrigated with sterilized water when required. The pots with different treatments were arranged in a randomized complete block design with eight replicates for each treatment. The treatments were as follows: control (soil without ZP and A. terreus), soil treated with ZP, soil treated with ZP and 2 mL of spore suspension, and soil treated with ZP and 4 mL of spore suspension. After 20 d, a random sample (six plants of H. vulgare) was taken from each treatment to determine some growth parameters, including the plant height (cm), dry weight/plant (g), nitrogen (N) (extracted from the plants with sulfuric acid and detected using the semimicro Kjeldahl method), and P (extracted by nitric perchloric acid digestion and detected using the vanadono-molybdo-phosphoric colorimetric method).
RESULTS AND DISCUSSION
The identification of the A. terreus fungus was confirmed using molecular characterization, which is based on ITS rDNA (Figs. 2Aand 2B). This method of molecular identification of fungi at the species level is primarily based on the variable nature of the ITS regions of DNA (Romanelli et al. 2010; Delgado-Serrano et al. 2016). The 18S rRNA sequence of the A. terreus isolate was searched for on a database (BLAST) using multiple sequence alignment (Fig. 1B).
Fig. 2.(A) Phylogenetic relationships between the A. terreus strain and the ITS sequences of closely related fungal strains retrieved from the NCBI GenBank database; and (B)cluster analysis of A. terreus (B)
From the alignment profile results, it was concluded that the A. terreus strain PKKS1 18S rRNA amplicon closely matched other A. terreus isolates (> 99%). The constructed phylogenetic relatedness (Fig.2B) of the whole sequence of A. terreus strain PKKS1 18S rRNA was compared with closely related strains from the BLAST database. This procedure revealed the molecular identity of the isolated fungus strain of this study. The highest growth, irrespective of the NaCl concentration, was recorded with the isolate A. terreus, which grew with up to 10% NaCl (Table 1).
The growth of A. terreus in general decreased with an increasing NaCl concentration, but the maximum growth was noted at 2% NaCl. This concentration appeared to be optimal for growth. Similar data were obtained by Nasmetova et al. (2010) with halophilic A. terreus that was isolated from hypersaline environments. Nasmetova et al. (2010) and Nazareth et al. (2012) reported that A. fumigatus and A. terreus can be isolated with a high frequency from salty soil and hypersaline water samples, with salinities ranging from 3% to 15% NaCl. Kanse et al. (2015) found that increasing the salinity had no effect on the phosphate solubilization ability of Talaromyces funiculosus. In the present study, A. terreus was able to grow at temperatures of up to 45 °C (Table 2), but the growth decreased at temperatures above 35 °C. This result confirmed the thermotolerance of A. terreus and agreed with the results of Ahirwar et al. (2017).
Table 1. Growth of A. terreus under Different NaCl Concentrations
SD*, ± standard deviation
Table 2. Growth of A. terreus at Different Temperatures
SD*, ± standard deviation
These results indicated that A. terreus may be a potential candidate for ZP solubilization at environmentally stressed temperatures (Fig. 3), as the phosphate solubilization efficiency of A. terreus at different temperatures up to 45 °C was clearly high from the appearance of clear halo zones around the fungal colonies. Similarly, other researchers (Gharieb et al. 1998; Sayer and Gadd 1998) have reported phosphate solubilization at different temperatures by Aspergillus sp. Halos were produced because of the solubilization of insoluble phosphates, which in turn were mediated via the production of organic acid in the surrounding medium (Gaur1990).
Fig. 3. Phosphate solubilizing ability (halo zone) of A. terreus at different temperatures: (A) 30 °C; (B) 25 °C; (C) 20 °C; (D) 35 °C; (E) 40 °C; and (F) 45 °C
The greatest decrease in the medium pH was observed with the ZP-amended medium and unamended medium during A. terreus growth after up to 10 d of incubation, particularly at a low concentration (500 ppm) (Table 3). The presence of insoluble ZP was not the factor necessary to stimulate acid production. Solubilization might have occurred because of the production of acids by A. terreus, as the pH of the culture broth shifted from neutral to highly acidic. Previous studies on the effects of the pH on the availability of phosphate have had contrasting results (Whitelaw 1999). However, the effects of the pH on the solubility and speciation of metals have been well documented (Gadd and Sayer 2000; Gadd 2001). Fankem et al. (2006) stated that phosphate solubilization is the result of the combined effects of pH decrease and organic acid production by microorganisms. Another explanation for phosphate solubilization was reported by Kim et al. (2005), who stated that organic acids may form soluble complexes with metal ions associated with insoluble P, and thus P is released.
Table 3. pH Detection at Different Incubation Periods and Different ZP Concentrations
SD*, ± standard deviation
A. terreus showed P solubilization with up to 10% NaCl present (Table 4). Similarly, Nautiyal (1999) reported solubilization in the presence of 10% NaCl, but there was a general decrease in solubilization with an increasing NaCl concentration. According to Srividya et al. (2009), the phosphate-solubilizing ability of A. niger was enhanced by the presence of NaCl. In addition, previous studies reported that solubilization of inorganic phosphates was improved in the presence of 1% NaCl (Kim et al. 1997; Kang et al. 2002). The strain A. terreus can thus be of great benefit in maintaining the available phosphate levels for crops in saline soils.
Table 4. Solubilizing Efficiency of the ZP at Different NaCl Concentrations
SD*, ± standard deviation
This is not the first study to use Aspergillus sp. for phosphate solubilization or as a biofertilizer (Jain et al. 2012; Xiao et al. 2013). The best growth of Hordeum vulgare was recorded for the plant treated with A. terreus at low and high spore suspensions with ZP, followed by the control with ZP and no fungal inoculation, when compared with that of the control without ZP and fungal inoculation (Fig. 4).
Fig. 4. Hordeum vulgare treated with phosphate solubilizing A. terreus: (A) control (soil without ZP and A. terreus); (B) soil treated with ZP; (C) soil treated with ZP and 2 mL of spore (2×106 ) suspension; and (D) soil treated with ZP and 4 mL of spores (2×106)
The control plant revealed that a deficiency of available phosphate retarded plant growth for various parameters, such as the shoot length and fresh weight, while increases in these parameters may have been because of the P released from ZP by fungal inoculation (Table 5).
Table 5. Effects of Phosphate Solubilizing A. terreus on Some Growth Parameters, P, and N Percentages in Hordeum vulgare Plants
SD*, ± standard deviation
Two mL** and 4 mL*** of distilled water, each containing 2×106 A. terreus spores
CONCLUSIONS
- Rhizosphere A. terreus appeared to have a promising effect on the solubilization of zinc phosphate (ZP) under stressful temperature and salinity conditions.
- The pot experiment studies revealed that A. terreus increased the P and nitrogen concentrations in H. vulgare plants.
ACKNOWLEDGEMENTS
The authors extend their appreciation to the Deanship of Scientific Research at Jazan University for funding this work through the research group project No. JUP8//000217.
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Article submitted: April 14, 2018; Peer review completed: August 17, 2018; Revised version received: August 26, 2018; Accepted: August 30, 2018; Published: September 7, 2018. DOI: 10.15376/biores.13.4.8012-8023