Water Air Soil Pollut (2011) 219:3–10 DOI 10.1007/s11270-010-0679-3
Dual Inoculation of Arbuscular Mycorrhizal and Phosphate Solubilizing Fungi Contributes in Sustainable Maintenance of Plant Health in Fly Ash Ponds A. Giridhar Babu & M. Sudhakara Reddy
Received: 26 April 2010 / Accepted: 20 October 2010 / Published online: 3 November 2010 # Springer Science+Business Media B.V. 2010
Abstract Fly ash is one of the residues produced during combustion of coal, and its disposal is a major environmental concern throughout coal-based powergenerated counties. Deficiencies of essential nutrients, low soil microbial activity, and high-soluble salt concentrations of trace elements are some of the concerns for reclamation of fly ash ponds. The effect of fly-ash-adapted arbuscular mycorrhizal (AM) fungi and phosphate solubilizing fungus Aspergillus tubingensis was studied on the growth, nutrient, and metal uptake of bamboo (Dendrocalamus strictus) plants grown in fly ash. Co-inoculation of these fungi significantly increased the P (150%), K (67%), Ca (106%), and Mg (180%) in shoot tissues compared control plants. The Al and Fe content were significantly reduced (50% and 60%, respectively) due to the presence of AM fungi and A. tubingensis. The physicochemical and biochemical properties of fly ash were improved compared to those of individual inoculation and control. The results showed that combination of AM fungi and A. tubingensis elicited a synergetic effect by increasing plant growth and uptake of nutrients with reducing metal translocation.
A. G. Babu : M. S. Reddy (*) Department of Biotechnology, Thapar University, Patiala 147004 Punjab, India e-mail:
[email protected]
Keywords Arbuscular mycorrhizal fungi . Reclamation . Aspergillus tubingensis . Glomus spp. . Dendrocalamus strictus
1 Introduction Fly ash is one of the residues generated in the combustion of coal and causes air and water pollution if proper management measures are not taken in time. Fly ash disposal is a major environmental concern throughout coal-based power-generated counties. In India alone, the annual production of fly ash is about 120 MT by 82 power plants and expected to rise to 150–170 MT per year by end of 2012 (MOEF 2007). Disposal of fly ash has significant impacts on terrestrial and aquatic ecosystems due to leaching of toxic substances from the ash into soil and ground water, as well as reduction in plant establishment and growth (Wong and Wong 1990). In many cases, reclamation practices have been ineffective due to high mortality of tree seedlings which might be due to deficiency of essential nutrients (usually N and P), low soil microbial activity, high soluble salt concentrations of trace elements, and the presence of compacted and cement layers on ash disposal sites (Selvam and Mahadevan 2002). However, successful rehabilitation must involve the development of microbially driven organic matter turnover and mineral nutrient cycling for the long-term provision of plant nutrients (Banning et al. 2010).
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Remediation of contaminated sites may be facilitated by selection of tolerant plant species as well as microorganisms which play a critical role to establish early ecosystem development, due to functional abilities such as nitrogen fixation, organic matter turnover, mycorrhizal symbiosis, and potential facilitation of plant establishment (Hodkinson et al. 2002; Walker et al. 2003). Among soil microorganisms, arbuscular mycorrhizal (AM) fungi play relevant roles for establishment, survival of plant species, and improved soil properties in stressed environments (Ortega-Larrocea et al. 2010). Arbuscular mycorrhizal fungi also alter the soil microbial communities in rhizosphere directly or indirectly through changes in root exudation patterns (Barea et al. 2005) and enhance the soil enzyme activities (Wang et al. 2006). The effects of selected isolates of AM fungi play an important role on the plant growth, nutrient uptake, and aggregation of fly ash (Enkhtuya et al. 2005; Wu et al. 2009). However, spontaneous selection of infective and effective AM fungi is a long process in fly ash ponds. It has also been demonstrated that the use of adapted AM fungal strains, in restoration and bioremediation studies, is more effective than applying non-adapted strains (Vivas et al. 2005). Oliveira et al. (2010) reported that AM fungi quickly lose their symbiotic efficacy when cultivated without edaphic stresses of the environment from where they are originally isolated. They also recommended that the inoculum should be produced with original edaphic stresses especially for AM isolates from extreme environments. We have isolated AM fungi from the plants growing in fly ash pond and multiplied in pots using fly ash to maintain its adaptation. Many fungi can survive and grow at high concentrations of toxic metals and the genus Aspergillus play multi-fascinated roles such as P solubilization, heavy metal bioleaching, plant growth promotion, and synergetic effects with mycorrhizal fungi (Medina et al. 2006; Yang et al. 2009). The selection of A. tubingensis in this study is based on its ability to solubilize the insoluble phosphates and improvement of growth of plants under stress conditions (Krishna et al. 2005). Bamboo plants (Dendrocalamus strictus) were selected because of its extensive underground root and rhizome system (having significant capacity to bind the top soil), accumulation of leaf mulch, abundant litter fall. It also serves as an efficient agent in rehabilitation of degraded land, preventing soil erosion, conserving
Water Air Soil Pollut (2011) 219:3–10
moisture, reinforcement of embankments, and drainage channels (Zhou et al. 2005). The present study was aimed to study the influence of fly-ash-adapted AM fungi along with co-inoculation of phosphate solubilizing fungus A. tubingensis on the growth, nutrient, and metal uptake of bamboo plants grown in fly ash. An improvement of physicochemical and biochemical properties of fly ash due to inoculation of these fungi is also reported.
2 Materials and Methods 2.1 Fly Ash Sample Collection Fly ash samples were collected (0–40 cm depth) randomly from a different location of fly ash sedimentation pond of National Aluminium Company Ltd. (NALCO), Damnjodi (18°46′22″ N 082°53′23″ E), Orissa, India. The samples were mixed thoroughly, and a portion of the fly ash was used to analyze various physicochemical characteristics. The physicochemical properties and elemental levels of fly ash were presented in Table 1. Table 1 Physicochemical properties of fly ash
Parameters
Units
pH
7.35
EC(mS/cm)
0.30
Organic Carbon (%) Olsen P (mg/kg) N(mg/kg)
0.30
Elemental analysis (mg/kg) Al2O3 Fe2O3
2.30 85.0
281,500 51,500
P2O5
4,500
SO3
13,500
CaO
8900
MgO
3800
Na2O
3700
MnO
500
K2O
800
ZnO
100
CuO
150
Water Air Soil Pollut (2011) 219:3–10
2.2 Isolation and Inoculum Production The roots and rhizosphere fly ash samples of dominant plants growing in fly ash pond were collected up to 30 cm depth using a stainless steel shovel. Three replicates were collected from each plant species. Samples were mixed thoroughly and placed in a plastic bag and stored at 4°C until their use. The dominant plants growing in this pond are Acacia pennata (Mimosaceae), Calotropis gigantea (Asclepiadaceae), Cassia occidentalis (Caesalpiniaceae), Cynodon dactylon (Poaceae), Lantana camara (Verbenaceae), and Jatropha gossypifolia (Euphorbiaceae). AM fungal spores were isolated from these samples by sieving and decanting technique (Gerdemann and Nicholson 1963). Isolated spores were propagated on three trap plants; maize, wheat, and sorghum (seeds surface sterilized with 0.01% HgCl2) grown separately in earthen pots containing the fly ash for two successive cycles, each cycle of 3 months long. No fertilizers were added to fly ash while multiplying the AM fungi. The inoculum was prepared with density of spores 90–100 spores per 10 g of fly ash. AM inoculum consisted of fly ash containing spores and AM-infected root pieces from a pot culture. The phosphate solubilizing fungus A. tubingensis (Reddy et al. 2002) was used as coinoculant. The fungus was grown on Pikovskaya’s agar (Pikovskaya 1948) plates at 28°C up to dense formation of spores. Spores were scraped and mixed with vermiculite with inoculum size of 1.7×108 spores/g vermiculite. Prior to inoculation, subsamples of spores isolated from pots and fly ash pond were identified following the current taxonomic criteria (Schenck and Perez 1990) and information of INVAM (http://www.invam. caf.wdu.edu/). The AM fungal isolates were identified as Glomus rosea, Glomus magnicaule, Glomus etunicatum, Glomus heterogama, Glomus maculosum, Glomus multicaule, Scutellospora nigra, and Scutellospora heterogama.
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plot was 60×60×40 cm3. Three replicates were maintained for each treatment. The experiment consisted of four treatments (1) fly ash, (2) fly ash and AM fungi, (3) fly ash and A. tubingensis, and (4) fly ash with AM fungi and A. tubingensis. In each treatment, nine bamboo plants (2 months old) obtained from the NALCO nursery were planted and inoculated with AM fungi (inoculum size 50 gm/plant with 90–100 spores/10 g) and 5 g/plant (1.7×108 spores) of A. tubingensis fungal spores. After 6 and 12 months of plantation, the shoot height and number of branches were measured. AM colonization was determined by collecting the root samples randomly from three places for each plot. The percentage of root length colonized by AM fungi was calculated by the gridline intersect method (Giovannetti and Mosse 1980) after staining with trypan blue (Phillips and Hayman 1970). The spores were isolated from rhizospheric soil samples of fly ash and were counted (Gerdemann and Nicholson 1963). To determine the uptake of minerals and metals by bamboo in different treatments, three randomly collected shoots from each plot were used. The shoot tissues were digested with nitric acid and perchloric acid (3:1) as described in Page et al. (1982). These samples were analyzed for mineral nutrients such as K, Ca Na, Mg, and heavy metals viz., Al, Fe, Zn, and Cu by inductively coupled plasma mass spectroscopy. Total P content in the shoot tissues was determined by colorimetric method as describes in Kitson and Mellon (1944). To determine the physicochemical changes of fly ash, the samples were harvested randomly from each treatment plot after 6 and 12 months. The organic carbon was estimated by the method as described in Walkley and Black (1934) and the total N by using TOC analyzer. Urease activity was determined by the method of McGarity and Myers (1967) and acid phosphatase by Tabatabai and Bremner (1969). Available P was estimated by Olsen et al. (1954) and Bray and Kurtz (1945) methods according to the pH of samples.
2.3 Nursery Experiment 2.4 Statistical Analysis The nursery experiment was conducted at NALCO nursery, Damanjodi, Orissa, India. Fly ash was used as substrate to grow the bamboo plants. Randomized blocks design was followed and size of each treatment
The data were analyzed by ANOVA, and means were compared by using Turkey’s tests at P
