Phytoremedial Potential of Tagetes Erecta Under Mycorrhizal Inoculation in Heavy Metal Polluted Soil

Institute of Ecology and Environmental Studies, Obafemi Awolowo, University, Ile-Ife, Nigeria Department of Pure and Applied Chemistry, Ladoke Akintola University of Technology, P.M.B 4000, Ogbomoso, Oyo State. Nigeria Department of Industrial Chemistry, Federal University, Oye-Ekiti, Nigeria Department of Microbiology, Obafemi Awolowo, University, Ile-Ife, Nigeria Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P.M.B 4000, Ogbomoso, Oyo State. Nigeria


Introduction
In many parts of the world, the problem of environmental pollution has assumed an unprecedented proportion (Giuffré et al., 2012). Industrial revolution and increase in world population have inevitably been responsible for environmental pollution. Many events of coming decades already were predetermined with past and present activities of man on planet earth. Major pollution sources include point sources such as emissions, waste discharge from industries, vehicle exhaustion and nonpoint sources such as soluble salts (natural and artificial), insecticides/pesticides, disposal of industrial and municipal waste and excessive use of fertilizers (Olarenwaju et al., 2009). About 60% of these pollutants are referred to as heavy metals (Kamnev, 2003). Heavy metals are the most insidious pollutants because of their non-biodegradable nature and properties that affect all forms of ecological system (Saba et al., 2013). Despite the toxicity potentials of heavy metals, some are essential for normal healthy growth and reproduction at low but critical concentration. Unfortunately, developing countries of the world such as Nigeria could not afford the cost of remediation because of lack of adequate fund, misplacement of priorities, poor management strategy and low level of technology advancement to handle problems of pollution. To address these challenges, the application of a method that offers a cost effective and an environmentally friendly approach that utilizes bio-agents with appropriate techniques such as phytoextraction should be employed. Although, over 500 plants species had been identified as metalliferous or hyperaccumulators of metals from polluted sites, majority of which are exotic species with adaption to a species natural habitat (Krämer, 2010). Thus, the need to also access the phytoremedial potentials of some indigenous plant species or weeds whose potential are yet to be known, as hidden potentials of some commonly grown weeds needs to be unveiled for human benefit particularly as it relates to improving the quality of the environment.
Hence, this study investigate the ability of the weed plants as African Marigold (Tagetes erecta) to extract heavy metals from polluted soils.

Experimental Site
The greenhouse experiment was carried out at the Faculty of Agriculture, Obafemi Awolowo University (OAU).

Experimental Procedure
A total of 120 plastic pots with drainage holes at the bottom with 3 kg each of air-dried soil. The soil that was collected was air dried and sterilized at 121°C for 2 h using autoclave to eliminate native mycorrhiza fungi propagules as well as other microorganisms present in the soil and sieved using 2mm mesh. These concentrations were used to contaminate the soil in the pots. The soil was left for four days for equilibration before mycorrhiza treatments were applied. The mycorrhiza treatment consisted of 30 pots each of Glomus mosseae (mycorrhiza) and non-mycorrhiza respectively. Soil mycorrhiza inoculum of Glomus mosseae was applied at the rate of 12 g per pot of 100 spores. Five seeds of Tagetes erecta were planted per pot and were thinned to two stands per pot at two weeks after planting and replicated 3 times in a completely randomized design. The pots were maintained weed free and pots were watered regularly with deionized water to moisture capacity to maintain soil moisture at 70% of field water holding capacity.

Parameters Determined
At the end of twelve weeks after planting, plant growth, percentage of root colonization by Arbuscular Mycorrhizal fungi, soil analysis, N, K, Ca, Mg, Zn, Pb, Fe, Cd, Cu, K and Na were all determined based on series of methods used at the end of twelve weeks after planting.

Statistical Analyses
A statistical comparison of means was made with analysis of variance (ANOVA) and treatment means were separated using Duncan Multiple Range Test at p<0.05 available in the SPSS. 16 statistical packages version.

Results and Discussion
The results of the pre-experiment analysis conducted on the soil samples used for this study indicated that the soil was sandy loam (84.4% and 15.4%, sand and silt composition respectively) and were characterized by mean pH of 6.5, available P 12.8 mg kg 1 , 2.1 mg kg 1 Organic carbon, Nitrogen 0.22 mg kg 1 and Ca 2+ was 2.02 mg kg 1 . The Pb and Cu contents of the soil were 0.22 mg kg 1 and 0.74 mg kg 1 . The physical and chemical characteristics of soil used in the experiment is presented in the Table 1 above and Fig. 1 shows the weed plants of African Marigold (Tagetes erecta) planted on the soil.

Number of Leaves
The response of leaves of T. erecta to different levels of Cu concentration varied with Glomus mosseae inoculation. In an uncontaminated Cu (0 mg kg 1 ), the effect of Glomus mosseae inoculation on the number of leaves in T. erecta was evident at 6, 10 and 12 Weeks After Planting (WAP). However, at 125 mg kg 1 of Cu contamination, the effect of Glomus mosseae became evident at 12 weeks after planting on leaf development T. erecta.

Fig. 1: Tagetes erecta
With the increase in the level of Cu contamination to 250 mg kg 1 , the number of leaves in T. erecta was enhanced by Glomus mosseae inoculation at 4-12 WAP, (Fig. 2).
The response of leaf of T. erecta to different levels of Pb contamination under the influence of Glomus mosseae varied with the concentrations of Pb. In the 0 mg Pb kg 1 contaminated soil, Glomus mosseae inoculation significantly (p≤0.05) enhanced the number of leaf (Fig. 5). When the soil was contaminated with 25 mg kg 1 of Pb, Glomus mosseae improved leaf production T. erecta. At 100 mg Pb kg 1 , there was no effect of Glomus mosseae on the effect of Glomus mosseae on number of leaves.

Plant Height
In an uncontaminated Cu soil (0 mg kg 1 ), Glomus mosseae enhanced the plant height of T. erecta at 6 to 12 WAP. At 125 mg kg 1 of Cu contamination, plant height of T. erecta was enhanced by Glomus mosseae inoculation except at 12 weeks after planting in T. erecta, there was no marked effect of mycorrhiza inoculation on the plant height. Similar, trend was also observed for T. erecta 250 mg kg 1 Cu contamination.
When the Cu contamination was doubled (500 mg kg 1 ) the effect of Glomus mosseae significantly (p≤0.05) enhanced the plant height in both plants, particularly at 10 to 12 WAP ( Fig. 3d). At a higher concentration (1000 mg kg 1 ) the effects of mycorrhizal inoculation on the plant height of T. erecta significantly (p≤0.05) increase the plant height at early stage of development (2 to 4 weeks after planting) (Fig. 3).

Stem Girth
At 0 mg kg 1 of Cu soil contamination, mycorrhizal inoculation did not have appreciable effect on the stem girth of T. erecta except at 4 and 10 WAP, where Glomus mosseae was more pronounced (Fig. 4a). Similar trend was also observed at the level of 125 mg kg 1 of Cu contamination (Fig. 4b). Mycorrhizal inoculation increased the stem girth of T. erecta at 8 to 12 Weeks After Planting (WAP) under 500 mg kg 1 of Cu contaminated soil. At 1000 mg kg 1 concentration of Cu, Glomus mosseae inoculation significantly (p≤0.05) enhanced the stem girth of both T. erecta, at 8 to 12 Weeks After Planting (WAP) in T. erecta.

Number of Leaves
The response of leaf of T. erecta to different levels of Pb contamination under the influence of Glomus mosseae varied with the concentrations of Pb. In the 0 mg Pb kg 1 contaminated soil, Glomus mosseae inoculation significantly (p≤0.05) enhanced the number of leaf. When the soil was contaminated with 25 mg kg 1 of Pb, Glomus mosseae improved leaf production in both T. erecta. At 100 mg Pb kg 1 , there was no effect of Glomus mosseae on the effect of Glomus mosseae on number of leaves.
In an uncontaminated (0 mg kg 1 ) soil under Pb treatment experiment, the height of Glomus mosseae inoculated T. erecta was higher than those with noninoculated plants.

Plant Height
However, at 50 mg Pb kg 1 soil contamination, the influence of Glomus mosseae on plant height of T. erecta was reduced throughout the period of plant growth. The response of plant height of T. erecta to 75 mg Pb kg 1 contaminated soil was significantly (p≤0.05) enhanced by Glomus mosseae inoculation at 12 WAP. At a higher concentration of 100 mg kg 1 of Pb, the plant plants height did not follow a definite pattern with respect to mycorrhizal inoculation (Fig. 6).

Influence of Arbuscular Mycorrizal (AM) Fungi on the Nutrient Uptake of T. erecta in Cu and Pb in Contaminated Soil
Glomus mosseae inoculation enhanced the N uptake of T. erecta in Cu polluted soil, the highest N uptake of T. erecta in Cu polluted soils occurred at 250 mg Cu kg 1 contamination. The P uptake decreased as the concentration of contaminant increased in Glomus mosseae inoculated plants. There was a marked effect of Glomus mosseae inoculation on the P uptake of T. erecta compared with noninoculated plants. The Zn uptake decreased as the Cu concentration increased in Glomus mosseae inoculated plant. Glomus mosseae had significantly (p≤0.05) enhanced the uptake of Fe in T. erecta. Fe uptake by T. erecta decreased as the Cu concentration increases in the soil (Table 2).
In the uncontaminated soil, Glomus mosseae inoculation improved the N, P, K uptakes However, in the 25 and 50 Pb kg 1 contaminated soil, mycorrhizzal inoculation enhanced N, P and K contents of T. erecta. Mycorrhizal inoculation enhanced Zn uptake at 250 mg Pb kg 1 contamination. Fe uptake was significant at 50 mg Pb kg 1 under Glomus mosseae inoculation (Table 3).

Influence of Arbuscular Mycorrizal (AM) Fungi on the Plant Dry Weight, Cu and Pb uptakes in T. erecta
Glomus mosseae inoculation enhanced plant dry weight in Cu contaminated soil irrespective of the level of the concentrations except at 1000 mg kg 1 Cu. The uptake of Cu by T. erecta as influenced by Glomus mosseae is shown in Cu uptake increased with increasing level of soil contamination to a threshold level and then declined. Threshold level of Cu uptake with non-inoculated plants was at 250 mg kg 1 contamination, while with Glomus mosseae was at 500 mg kg 1 (Table 4).
Similar to Cu contaminated soil, the Glomus mosseae inoculation influenced the plant dry weight of T. erecta in Pb contaminated soils (Table 5). Glomus mosseae increased Pb uptake in T. erecta with increased level of contamination to a threshold before it declined. Plant optimum uptake for lead was at 75 mg kg 1 contamination level with Glomus mosseae. 04c Values in the same group followed by the same letter did not differ significantly at p≤0.05 using Duncan Multiple Range Test. Legend: Trt-Treatment, NM-Non-mycorrhiza; GM-Glomus mossea   2.47c 0.23e Values in the same group followed by the same letter did not differ significantly at p≤0.05 using Duncan Multiple Range Test. Legend: Trt-Treatment, NM-Non-mycorrhiza; GM-Glomus mosseae  Table 6 Inoculated and non-inoculated plants were both infected by AM fungi. Percentage root infection however was significantly higher at P<0.05 in plants inoculated with Glomus Mosseae (GM) than in the nonmycorrhizal (GM). Greater colonization occurred between 0-250 mg Cu kg 1 and 0-50 mg Pb kg 1 than at higher concentrations of contaminations.

Discussion
This study assessed the potential of indigenous plant species (Tagetes erecta) in removing Cu and Pb from contaminated soil using Glomus mosseae as an enhancer. In a view to achieve this, T. erecta was planted in greenhouse with Glomus mosseae inoculation under different concentration levels of Cu and Pb soil contamination. The knowledge of AM associations with T. erecta exposed to different levels of Cu and Pb contamination could provide a basis for alleviating the stress of heavy metal toxicity in weeds studied. Specific criteria had been put into cognizance when screening, selecting and identifying weed species with, tropical climate, primary goal of identifying potential hyperaccumulators high potential for phytoremediation of Cu and Pb.
The result revealed that the growth parameters (number of leaves, plant height and stem girth) were enhanced by Glomus mossea in T. erecta. Leaf production of T. erecta was sustained at highest level of (1000 mg kg 1 ) of Cu concentration, similar observation had been made on the effect of AM inoculation on Solenostemon monostachyus grown in Cu and Cd contaminated soil (Awotoye et al., 2012). This suggests that AM infection offers protection against heavy metal toxicity.
The study also observed the toxicity effect of Pb which was noticed on the leaf production of AM inoculated T. erecta at 50-100 mg kg 1 concentrations. This indicates that the effectiveness of AM inoculation in a polluted environment may be controlled primarily by the concentration level of the contaminant. This supports the findings of Awotoye et al. (2013) on mycorrhizal inoculation of Amaranthus spinosus, Synedralla nodiflora, Sida acuta and Euphorbia heterophylla where increase Pb concentration had pronounced negative effects on the plant growth parameters.
Arbuscular Mycorrizal fungi are able to enhance heavy metal uptake through their external mycelium, which support a wider exploration of soil volumes by spreading beyond root exploration zone (Malcova et al., 2003), thus providing access to greater volume of metals present in the rhizosphere. Another relevance of AM fungi symbiosis is that it can increase plant establishment and growth despite high levels of soil heavy metal (Enkhtuya et al., 2002), due to better nutrition (Fewtrell et al., 2003) and water availability (Augé, 2001) and soil aggregation properties (Rillig and Steingerg, 2002). It has been suggested that mycorrhizal status is related to environmental elements, especially contamination levels in soil (Smith and Read, 1997). Cu and Pb addition decreased the percent of root colonization in the present study and root colonization decreased with increasing soil Cu and Pb concentrations. This result demonstrates that high levels of soil Cu and Pb inhibited the growth of AMF and then inhibited the formation of mycorrhiza. Andrad et al. (2004) reported that the increase of Pb concentrations decreased the root colonization and the spore numbers, which is confirm insistent with the present study.

Conclusion
Arbuscular mycorrhiza fungi improved the growth of Tagetes erecta under high toxicity of Copper (1000 mg kg 1 ). AM fungi also improved the uptake of Cu and Pb Tagetes erecta. This study shows that AM fungi are beneficial to the phytoremediation process and that Tagetes erecta and Crotalaria juncea are suitable to grow and rehabilitate a Cu and Pb contaminated site.