Abstract
The toxic metals released from industrial production, mining, smelting and traffic contaminate the agricultural soils. This has raised concerns not only for crop quality but also for human health. Engineering and/or microbial based technologies are used to remove the toxic metals from contaminated soils. But these approaches are costly and less efficient in comparison to phytoremediation technique which has emerged as more efficient and cost effective method for decontamination of the toxic metal affected sites. There are highly specialized plants that have the ability to accumulate and tolerate high concentrations of toxic metals from soils and may provide the basis for remediation of heavy metal contaminated sites. Mustard has a key role in phytoremediation technology and is a potential candidates for the introduction of genes aimed at phytoremediation. For enhancing the biomass of this crop for phytoremediation, modern agricultural practices require the use of chemical fertilizers that pose environmental hazards of various kinds. A better alternative to the chemicals are the soil microbes that reside in the rhizosphere of plants and stimulate their growth. Rhizosphere of Brassica spp. is colonized by several beneficial microbes that can be isolated, mass cultured and utilized to maximize the biomass of this crop plant for use in phytoremediation technology.
Key words:
Mustard, Phytoremediation, Biomass, Trichoderma spp.
INTRODUCTION
Toxic metals such as cadmium, lead, zinc etc. released from industrial production, mining, smelting and traffic contaminate agricultural soils and threaten crop production by their toxicity to plants (Wang et al., 2009). Heavy metals have been found to accumulate in human body through ecological food chain and eventually pose serious health hazards (di Toppi and Gabbrielli, 1999). Pyhtoremediation i.e. use of plants to remove toxic metals from contaminated sites is generally viewed as more efficient and cost effective technique compared to other methods of decontamination such as engineering and/or microbial approaches (Vickers and Lemaux, 1998). With an extensive root system for absorption and transport, large amounts of heavy metals can be directed to the shoots and removed by biomass harvesting (Palmer et al., 2001). The metal-enriched plant material can then be removed from the site, the contaminants concentrated, disposed or if possible, the metal element recovered and valuable metal recycled (Palmer et al., 2001). Plants therefore, represent natural environmentally safe way to remediate contaminated sites (Palmer et al., 2001). The process of phytoremediation depends on the extent of root development, biomass production and adaptability for growth in a particular soil environment (Salt and Raskin, 1999). For enhancing the root development, biomass production and consequently phytoremediation potential of Brassica species, the modern agriculture requires the use of chemical fertilizers. But, these chemical fertilizers are expensive, hazardous to the environment and also are available in limited supply. Therefore, to explore the possibility of supplementing the inorganic chemical fertilizers with organic ones such as the biofertilizers of microbial origin is the need of the day. A reliable approach is the sustainable agricultural system which maintains and improves human health, benefits producers and consumers both economically and spiritually, protects the environment, and produces enough food for an increasing world population (Higa, 1991). Sustainableagriculture that integrates environmental health, economic profitability and social or/and economic equity, is based on substantial use of beneficial soil microorganisms that hold tremendous potential for use to enhance plant growth and yield at a low cost (Higa, 1991). Microbial inoculants are cheaper as compared to the chemical fertilizers, and are not hazardous to the environment. There are many species of beneficial soil microbes such as Trichoderma spp., Bacillus megaterium, Verrucomicrobium spinosum,Phyllobacterium brassicacearum, Variovorax paradoxus etc. flourishing in the rhizosphere of Brassica plants that, have immense potential to enhance plant growth by a plethora of mechanisms including phytohormone production (Mehnaz et al., 2001), complex substrate degradation and/or siderophore production (Masalha et al., 2000) etc. and are therefore, of agricultural importance.
Role of Mustard in phytoremediation
Mustard has been recognized as potentially useful candidate for phytoremediation (Palmer et al., 2001). It is able to produce significant amount of biomass, which is of definite advantage in phytoremediation (Palmer et al., 2001). With rapid growth potential of mustard, multiple cropping can be achieved during the growing season and since flowering and seed production is not essential, contaminant recycling by leaf fall can be avoided (Palmer et al., 2001). It exhibits superior heavy metal accumulation characteristics (Gleba et al., 1999) and improved lines have already been developed for Pb accumulation ability by conventional selection methods (Nanda Kumar et al., 1995). It has been found that Pb accumulation in these plants varies from 1416 to 18,812 µg-1 dry weight (Nanda Kumar et al., 1994).This plant has also been demonstrated to show substantial tolerance to zinc and copper (Ebbs and Kochian, 1997) and significant amounts of Zn have been found to accumulate in shoots of the plant grown in contaminated sites (Ebbs and Kochian, 1998). To be affective in phytoremediation and removel of specific elements, Brassica species must exhibit significant tolerance to a number of heavy metals or xenobiotics and have the ability to grow under varied growth conditions (Palmer et al., 2001).
Materials and Methods
Isolation Trichoderma spp. from rhizosphere
Mustard plants were collected from a field at Rajouri in Jammu and Kashmir, India. Care was taken to dig out, as far as possible, the whole root system with a sterilized spatula. The root systems were then brought to the laboratory in separate polyethylene bags. The roots were given gentle taping to loosen-off the lightly adhering soil, in order to have just the rhizosphere soil attached to the root system. Small pieces of roots (2 cm) of different diameter were cut with sterilized scissors under aseptic condition and 25 such root pieces for each sample were transferred to flasks (one for healthy and the other for diseased roots) containing 100 ml of sterilized distilled water. The flasks were shaken vigorously with the help of a shaker to get a homogenous suspension of the rhizosphere soil. Taking this as the stock solution, conventional soil dilution plate method (Warcup, 1950) was followed for isolation of the rhizosphere fungi. Dilutions of 1:100, 1:1000, and 1:10000 were prepared. Three replicates of sterilized Petri plates were inoculated with one ml aliquots from all the diluted suspensions. To this was added 20 ml melted and cooled (40 C) potato dextrose agar medium and the plates were rotated slowly in clock-wise and anti-clock wise directions to disperse the soil solution uniformly in the culture medium. All the inoculated plates were then incubated at 25±2 C. The plates were examined regularly and the colonies of fungi appearing on the medium were transferred into fresh sterilized Petri plates containing PDA medium to avoid over-running by the fast growing forms. The pure culture of the Trichoderma spp. was identified using standard literature and preserved on PDA slants at 4C.
Preparation of mass culture of the fungus
The mass culture of Trichoderma spp. was prepared on barley grains (Shivanna et al., 1994). Clean and intact barley grains were taken for this purpose. The grains were pre-wetted by boiling them in water for 20-30 min so as to raise the moisture content of the grains up to 40-50% and to make them soft enough for the profuse growth of the fungus. After boiling, the grains were spread on wire mesh so as to drain the excess of water. The grains were then mixed with gypsum (calcium sulphate 2%) and chalk powder (calcium carbonate 0.5%) on dry weight basis to check pH of the medium and prevent grains from sticking with each other. Clean glucose bottles were filled with such barley grains (100g each) which were then steam sterilized for 1-2 h. The bottles were then allowed to cool at room temp. and inoculated with five agar blocks (5 mm diam. each) cut from the margin of actively growing culture of the fungus. The bottles were incubated at 25 ± 2C for 10 d. The bottles were shaken once or twice daily for rapid and uniform colonization of the fungus. Barley grains colonized by the fungus were air dried and aseptically stored at 4C for further use.
Preparation of pots
The soil sample collected from the agricultural field was air dried at room temp. and ground to fine powder form with the help of pestle and mortar. The pure inoculum of the fungus, which was prepared on barley grains, was mixed separately with sterilized natural soil (1% w/w). The soil samples so prepared were separately filled in clay pots (15
´
25 cm). The pots were kept at room temp. for a week during which the fungus developed and colonized the soil particles. Soil supplemented with barley grains without inocula was used as control. The moisture level of the soil (25-30%) was maintained by watering the pots from time to time. Twenty surface sterilized seeds of mustard were sown in each pot 8 days after soil amendment with rhizosphere fungus. The experiments were set in replicates of three pots in a greenhouse. The observations for the effect of rhizosphere fungus on biomass of mustard plants were made on plant height and number of branches/plant at 150 days after sowing (DAS).
Results and Discussion
The results clearly indicated that Trichoderma spp. stimulated the biomass of mustard. In Trichoderma spp. amended soil, at 150 DAS, mustard plant height was recorded to be
36.1
±
0.05 cm
which is much higher than the control. Number of branches per plant under treatment was recorded to be 10 while in control it was only 4. This way the biomass of mustard was significantly promoted by the fungus which has direct impact on phytoremediation potential of the plant
(P < 0.05).
Numerous microorganisms indigenous in the rhizosphere of Brassica species such as Phyllobacterium brassicacearum, Serratia plymuthica, Comamonas terrigena, Stenotrophomonas maltophilia, Agromyces cerinus, Acinetobacter rhizosphaerae, Microbacterium oxydans, Paenibacillus lautus, Arthrobacter globiformis, Pseudomonas fluorescens, Variovorax paradoxus Trichoderma spp. etc. have been reported to influence the growth and phytoremediation potential of this crop plant (Larcher et al., 2008; Adams et al., 2007). Many studies have demonstrated that application of beneficial microorganisms colonizing the rhizosphere of plants considerably reduce heavy metal toxicity to plants and enhance the accumulation of heavy metals in the plants (Wang et al., 2009). Sheng and Xia (2006) reported that inoculation of cadmium resistant plant growth promoting bacteria enhance dry weight and Cd uptake of oilseed rape in Cd contaminated soils. Growth conditions, root surface area and ion exchange capacity of the roots have been advanced as the factors affecting uptake and removel of toxic metals by the plants (Salt et al., 1997). Biomass stimulation by the rhizosphere soil microbes can be attributed to their ability to suppress pathogenic soil microorganisms, produce growth promoting substances such as phytohormones and/or degrade complex substrates in the soil (Unyayar, 2000; Dubey et al. 2007). Cristescu et al. (2002) studied ethylene production by phytopathogenic fungus Botrytis cinerea causing post harvest rot of perishable plant products including tomato and found that this fungus has the ability to produce ethylene in vitro. Altmore et al. (1999) investigated the capability of Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize some insoluble or sparingly soluble minerals in vitro and reported that T-22 was able to solubilize MnO2, metallic zinc and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium. This phosphate solubilising activity of T. harzianum might be responsible for its plant growth promoting ability.
________________________________________________________________________
Table 1. Impact of soil amendment with Trichoderma spp. on its biomass potential
(in pots)
________________________________________________________________________
Treatment No. of days Plant height (cm) No. of branches/plant
Trichoderma
spp. 150 36.1
±
0.05 10
Control (without PGPF) 150 22.03
±
0.03 4 _____________________________________________________________________
Average of all three replicates; ±, Standard error of mean (SEM).
Data were statistically analyzed which were found to be significant (P < 0.05)
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