effect of arbuscular mycorrhizal fungi inoculation...

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87 Philippine Journal of Science 142 (1): 87-96, June 2013 ISSN 0031 - 7683 Date Received: ?? Feb 20?? Key Words: alleviate, arbuscular mycorrhizal fungi, copper, Desmodium cinereum, phytoremediation, toxicity *Corresponding author: [email protected] 1 Department of Crop Protection, College of Agriculture, Central Luzon State University, Science City of Muñoz, Nueva Ecija 2 Forest Biological Sciences, College of Forestry and Natural Resources, University of the Philippines, Los Baños, College, Laguna 3 National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, College, Laguna Joden M. Adiova 1 , Nelson M. Pampolina 2 and Nelly S. Aggangan 3* The effect of arbuscular mycorrhizal fungi (AMF) inoculation on copper (Cu) uptake and toxicity of Desmodium cinereum (Kunth) D.C. was studied. This legume produces large amount of biomass that can serve as buffer in areas with high concentration of Cu. Pre-germinated seeds of D. cinereum inoculated and non-inoculated with AMF were grown in sand-soil mixture treated with increasing Cu concentration (0, 400, 800, 1200, 1600ppm Cu). Increasing Cu concentration gave a corresponding reduction on height, diameter, leaf area, and biomass of the plants. Root growth and nodule formation at 1200 and 1600ppm Cu level were inhibited (p<0.05 and p<0.01, respectively). Mycorrhizal inoculation increased plant height, biomass, and stem diameter at lower Cu level. Inoculation enhanced nodulation and also improved phosphorus concentration in the leaves, stem and roots at 0 and 400ppm Cu level. Increasing Cu concentration resulted to a greater Cu accumulation in the roots while Cu concentration on stem and leaves remained at a normal level. Inoculation with AMF increased Cu uptake of roots and stem at 800, 1200, and 1600ppm Cu levels. The ability of mycorrhizal fungi to improve Cu uptake, increase plant growth, increase phosphorus uptake, and promote growth of other beneficial microorganisms such as nitrogen fixing bacteria (as exemplified by the nodulation in the roots) for D. cinereum, make it an ideal tool for phytoremediation of Cu contaminated sites. Effect of Arbuscular Mycorrhizal Fungi Inoculation on Growth and Cu Uptake and Toxicity of Desmodium cinereum (Kunth) D.C. INTRODUCTION Copper (Cu) is the third most used commercial metal because of its availability and attractive properties (Fjällborg and Dave 2003). Likewise, it is also one of the most common and problematic heavy metal (HM) soil pollutants. Since Cu can be released both naturally and through human activity, it is very common in the environment. It is often found near mines, industrial settings, landfills and waste disposals causing problems to humans. Increasing widespread pollution has caused vast areas of land to become unproductive and hazardous for both wildlife and human population. Utilization of these lands for agricultural and development purposes require a safe and efficient decontamination. Reclamation of lands contaminated with heavy metals (HMs), however, is a very

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Page 1: Effect of Arbuscular Mycorrhizal Fungi Inoculation …philjournalsci.dost.gov.ph/images/pdf/pjs_pdf/vol142no1/...The role of arbuscular mycorrhizal fungi (AMF) in plant nutrition is

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Philippine Journal of Science142 (1): 87-96, June 2013ISSN 0031 - 7683Date Received: ?? Feb 20??

Key Words: alleviate, arbuscular mycorrhizal fungi, copper, Desmodium cinereum, phytoremediation, toxicity

*Corresponding author: [email protected]

1Department of Crop Protection, College of Agriculture, Central Luzon State University, Science City of Muñoz, Nueva Ecija

2Forest Biological Sciences, College of Forestry and Natural Resources, University of the Philippines, Los Baños, College, Laguna

3National Institute of Molecular Biology and Biotechnology, University of the Philippines Los Baños, College, Laguna

Joden M. Adiova1, Nelson M. Pampolina2 and Nelly S. Aggangan3*

The effect of arbuscular mycorrhizal fungi (AMF) inoculation on copper (Cu) uptake and toxicity of Desmodium cinereum (Kunth) D.C. was studied. This legume produces large amount of biomass that can serve as buffer in areas with high concentration of Cu. Pre-germinated seeds of D. cinereum inoculated and non-inoculated with AMF were grown in sand-soil mixture treated with increasing Cu concentration (0, 400, 800, 1200, 1600ppm Cu). Increasing Cu concentration gave a corresponding reduction on height, diameter, leaf area, and biomass of the plants. Root growth and nodule formation at 1200 and 1600ppm Cu level were inhibited (p<0.05 and p<0.01, respectively). Mycorrhizal inoculation increased plant height, biomass, and stem diameter at lower Cu level. Inoculation enhanced nodulation and also improved phosphorus concentration in the leaves, stem and roots at 0 and 400ppm Cu level. Increasing Cu concentration resulted to a greater Cu accumulation in the roots while Cu concentration on stem and leaves remained at a normal level. Inoculation with AMF increased Cu uptake of roots and stem at 800, 1200, and 1600ppm Cu levels. The ability of mycorrhizal fungi to improve Cu uptake, increase plant growth, increase phosphorus uptake, and promote growth of other beneficial microorganisms such as nitrogen fixing bacteria (as exemplified by the nodulation in the roots) for D. cinereum, make it an ideal tool for phytoremediation of Cu contaminated sites.

Effect of Arbuscular Mycorrhizal Fungi Inoculation on Growth and Cu Uptake and

Toxicity of Desmodium cinereum (Kunth) D.C.

INTRODUCTIONCopper (Cu) is the third most used commercial metal because of its availability and attractive properties (Fjällborg and Dave 2003). Likewise, it is also one of the most common and problematic heavy metal (HM) soil pollutants. Since Cu can be released both naturally

and through human activity, it is very common in the environment. It is often found near mines, industrial settings, landfills and waste disposals causing problems to humans. Increasing widespread pollution has caused vast areas of land to become unproductive and hazardous for both wildlife and human population. Utilization of these lands for agricultural and development purposes require a safe and efficient decontamination. Reclamation of lands contaminated with heavy metals (HMs), however, is a very

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difficult task because unlike any other contaminants, HMs cannot be degraded or destroyed and tend to accumulate in soils and sediments, thereby, posing great danger to living organisms (Khan 2006). One of the solutions that have been a subject of vast research is the use of plants and their associated rhizosphere microorganisms to remove, degrade, or contain chemical contaminants present in the soil, sediments, groundwater, surface water and even atmosphere. This promising technology is known as phytoremediation.

The role of arbuscular mycorrhizal fungi (AMF) in plant nutrition is well documented. AMF can facilitate uptake and transport of nutrients like phosphorus (P) (Thingstrup et al. 2000; Jakobsen et al. 2001), improve plant growth under water stress (Kaya et al. 2003), reduce pathogenic infections (Abdalla & Abdel-Fatta 2000; Elsen et al. 2008) and protect plants against stressed induced by fungicide (Campagnac et al. 2010). Arbuscular mycorrhizal fungi also improve micronutrient acquisition by increasing the surface area of soil explored by mycorrhizal roots and increasing the solubility of metals by producing metal-chelators (Szaniszlo et al. 1981). These important capabilities made AMF an ideal tool to aid plants in phytoremediation. However, the mechanism by which it contributes to HM tolerance is not clearly understood. This is reflected on different and sometimes contradicting results from various studies. Many studies reported that AMF protect plants from HM stresses by reducing uptake and translocation of HMs from the soil (Jentschke et al. 1998; Hildebrandt et al. 1999; Huang et al. 2002). On the other hand, a number of studies showed that AMF colonizationcan significantly increase HM accumulation of the plants. For example, Joner & Leyval (2001) found that AMF colonization enhanced total cadmium(Cd) uptake of Trifolium subterraneum while Ahonen-Jonnarth & Finlay (2001) reported that AMF could also enhance total uptake of cadmium (Cd) and nickel (Ni) by the host plant Pinus sylvestris. Even the mechanisms by which plants partition the accumulated HM in their tissues as influenced by AMF is also not fully understood. Some studies reported different results on where accumulated metals are stored in the plant tissues. Others showed that AMF increases accumulation of HMs in the shoots of the plants (Leung et al. 2006) while some reported that most of the HMs are stabilized in the roots preventing their translocation into the shoot (Chen et al. 2001, 2003). These conflicting results only show the complexity of mechanisms by which AMF affects HM accumulation in plants.

There have been no extensive studies conducted to explore the possibility of using AMF and legumes as its host for phytoremediation. Most of the studies conducted on phytoremediation using AMF focused

on agricultural crops and grasses, with a very little understanding on other higher plants particularly on woody-legumes such as Desmodium cinereum (Kunth) D.C. This forage legume can produce tremendous amount of shoot biomass and long deep penetrating roots and consequently may provide high organic matter. D. cinereum can provide a unique and more effective means of cleansing HM contaminated soils and offers better potential for phytoremediation. Thus, this study was conducted to assess the potential of mycorrhizal D. cinereum for phytoremediation, specifically, it aimed to: 1) determine the effect of AMF on plant growth and P uptake at different Cu concentration, 2) determine the effect of AMF inoculation on Cu uptake and distribution of Cu within the plant, 3) determine the effect of elevated metal concentration on AMF colonization in the roots, and 4) determine the effects of AMF inoculation and soil Cu concentration on nodulation.

MATERIALS AND METHODS

Experimental DesignThe experiment was established in a greenhouse of the National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños (UPLB), College, Laguna. The experiment was conducted following a two factor (mycorrhiza inoculation and Cu levels) in Randomized Complete Block Design with ten replicates.

Soil Collection and PreparationThe soil used in the experiment was collected from a grassland (0-20cm depth) in San Isidro, Cuenca, Batangas. The soil was brought to UPLB, air dried for one week, pulverized and sieved in a 2mm wire mesh and mixed with sand at 1:1(v/v) ratio. The soil is characterized as slightly acidic (pH 6.2, 1:1 water), sandy loam texture, water holding capacity of 52%, with 2.87% OM, 0.1% N, 72ppm P, 3.1 cmol(+)/kg K, CEC of 20.1 cmol(+)/kg and 11ppm Cu. Soil analyses was done at the Analytical Laboratory of the College of Agriculture, University of the Philippines. The soil and sand mixture was sterilized for 48 hours in a force draft oven set at 1000C in order to eliminate indigenous microbial population including arbuscular mycorrhizal propagules. Seven hundred fifty (750g) grams of the mixture was placed in 4” x 8” polyethylene plastic bags. Copper was applied on the soil as a dilute solution. Different concentrations were obtained by adding 200 mL of aqueous solutions of Cu sulfate pentahydrate (CuSO4∙5H2O) containing 0, 400, 800, 1200, and 1600ppm Cu. The medium was allowed to stabilize for 10 days before using.

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Selection of Host PlantReconnaisance survey was conducted along three ecosystems: grassland (500-700masl), secondary forest (800-1000masl), and primary forest (1200-1600masl) in Tampakan mountain ecosystem and vicinities of South Cotabato. Selection of host plant was based on their distribution on three chosen ecosystems, morphological characteristics and based on interview with local community. The morphological features considered for phytoremediation were adaptability to disturbed ecosystem, economic importance and symbiotic synergism with beneficial microorganisms. Based from the survey, D. cinereum was one of the top three plant species that matched the most number of favorable characteristics.

Preparation of Planting Materials and InoculationSeeds of D. cinereum were provided by the International Center for Tropical Agriculture (CIAT), International Rice Research Institute (IRRI), Los Baños, Laguna. The seeds were surface sterilized with 50% alcohol for two min. then rinsed three times in sterile distilled water. Plants were inoculated with AMF using MYKOVAMTM, a commercial mycorrhizal inoculant developed and solely produced at present by BIOTECH, UPLB, College, Laguna. The inoculant is a soil based containing spores of AMF Glomus etunicatum, G. macrocapum, and Gigaspora margarita and infected roots of bahia grass (as trap plant). Approximately 10 grams of MYKOVAM was applied at the middle part of pregerminating trays where seeds were sown. Five seeds were sown in each container and later thinned to one seedling per hole two days after germination. Seeds for the control treatment were sown in different trays to avoid cross contamination.

Pot Experiment and Measurement of ParametersTwenty five days after sowing, mycorrhizal inoculated and non-inoculated seedlings with similar height (5.5-6cm) were transplanted on polyethylene bags (no holes) treated with different concentration of Cu and later placed on steel benches. The seedlings were grown inside a screen house for eight weeks.

Height was measured weekly using a ruler starting from the third day after transplanting. It was measured one inch above the soil surface to the tip of the young shoot. Stem diameter, total biomass, root biomass, shoot biomass, root nodules, and total leaf area of the youngest fully expanded leaf (YFEL) were measured at harvest. The YFEL of each plant was taken and their total area was measured using computer softwares (Adobe Photoshop CS2 and MS Visio 2006). Shoots and roots were separated, washed with tap water to remove adhering debris, oven dried at 70O C for three days and then weighed separately. Total biomass

was obtained by adding thebiomass of shoots and roots. The partitioned plant parts were wrapped individually with tissue paper and placed inside brown envelops prior to oven drying.

Mycorrhizal Colonization and Tissue Analysis The extent of mycorrhizal colonization in the roots was evaluated after the roots were cleared and stained (Philipps & Hayman 1970) using the grid line intersect method of Giovanetti & Mosse (1980). Detached leaves, stem and roots were ground in a Wiley mill and then sent to Central Analytical Services Laboratory, BIOTECH, UPLB for analyses of P and Cu concentration. Phosphorus and Cu uptakes were computed as the product of plant weight and P and Cu concentration, respectively.

Statistical AnalysisAll data gathered were subjected to two-way analyses of variance (ANOVA) using the SAS (v8.01) program. Comparison of treatment means was done using Duncan’s Multiple Range Test at p<0.05.

RESULTS

Plant GrowthThe general appearance of uninoculated and AMF-inoculated D. cinereum treated with increasing Cu after eight weeks in a greenhouse is shown in Figure 1. Increasing Cu concentrations significantly reduced height (p<0.01), root biomass (p<0.05), total plant biomass (p<0.01), stem diameter (p<0.01) and leaf area (p<0.01) of the inoculated and non-inoculated plants (Table 1, Figures 2 & 3). More pronounced reduction on the different parameters were observed on plants grown inCu levels amended with 800ppm, 1200ppm, and 1600ppm Cu.

Mycorrhizal inoculation, significantly increased height at 0ppm, 400ppm and 800ppm, while root biomass, total biomass and stem diameter of mycorrhizal plants were highest at 0ppm (Figure 2). Leaf area were not affected by mycorrhizal inoculation (Figure 3A). By contrast, nodule dry weight was significantly increased by mycorrhizal inoculation at 0ppm, 400ppm and 800ppm Cu levels (Figure 3B). No nodules were observed at Cu levels beyond 800ppm.

Increasing soil Cu concentrations resulted to an increase in mycorrhizal colonization. Plants exposed to 1600ppm Cu level had the highest colonization rate while plants at the 0 and 400ppm Cu levels had the lowest mycorrhizal colonization rate (Figure 4).

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Phosphorus and Copper ConcentrationIncreasing Cu concentration resulted to significant reductions on phosphorus concentration on leaves, stem and roots of both inoculated and non-inoculated plants (Table 2). No significant differences, however, were observed at 1200 and 1600ppm Cu level in both inoculation treatments. Mycorrhizal inoculation on the other hand, significantly increased phosphorus uptake at 0 and 400ppm Cu levels. No further significant changes on P concentration were observed at the higher Cu levels.

Increasing Cu concentration on the soil resulted to increasing Cu concentration on stem and roots (Table 3). The significant increase was observed on roots at

800ppm, 1200ppm, and 1600ppm Cu levels and stem at 400-1600ppm Cu levels. Inoculation of mycorrhiza significantly increased root and stem Cu concentration with the highest increase being observed at the 1200 and 1600ppm Cu levels.Leaf Cu concentrations on the other hand were neither affected by mycorrhizal inoculation nor increasing soil Cu concentration.

Total Cu Uptake and Distribution on Leaves, Stem, and RootsMycorrhizal inoculation increased total Cu uptake of the plants (Figure 5). Total plant copper uptake was highest at 400 and 800ppm Cu level by both non-inoculated and inoculated plants (Figure 6). The

Table 1. Effects of mycorrhizal inoculation, copper levels, and their interaction on growth parameters, nodule biomass, phosphorus and copper concentration of D. cinereum.

Parameter Mycorrhizal inoculation (Factor A)

Cu level (Factor B) A x B

Height at harvest ** ** **

Stem diameter at harvest ** ** NS

Total biomass ** ** **

Root biomass ** * **

Leaf area NS ** NS

Nodule dry weight ** ** **

Leaf P concentration * ** NS

Stem P concentration NS ** NS

Root P concentration NS ** NS

Leaf Cu concentration NS NS NS

Stem Cu concentration ** * NS

Root Cu concentration ** ** NS

Total Cu uptake * * NS

NS,*,** = Not significant, significant at p<0.05 and p<0.01, respectively.

Figure 1. General appearance of uninoculated and arbuscular mycorrizal fungi-inoculated D. cinereum treated with increasing Cu after eight weeks in a greenhouse.

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Table 2. Phosphorus concentration (%) in the leaves, stem and roots of eight- week old Desmodium cinereum as affected by mycorrhizal inoculation and Cu levels. n= 3

Cu level (ppm)Leaves Stem Roots

Uninoc Inoc Uninoc Inoc Uninoc Inoc

0 0.257b 0.335a 0.23a 0.255a 0.236ab 0.252a

400 0.274b 0.323a 0.181b 0.231a 0.187c 0.219b

800 0.218c 0.215c 0.105c 0.109c 0.14de 0.164cd

1200 0.135e 0.171d 0.064d 0.069d 0.081g 0.121ef

1600 0.120f 0.172de 0.045e 0.078d 0.094g 0.101fg

Treatment means (in each plant part) with the same letter(s) are not significantly different from each other using DMRT at p<0.05.

Table 3. Copper concentration (ppm) in the leaves, stem and roots of eight- week old Desmodium cinereum as affected by mycorrhizal inoculation and Cu levels. n= 3

Cu level (ppm)Leaves Stem Roots

Uninoc Inoc Uninoc Inoc Uninoc Inoc

0 6d 8abcd 4fg 2g 38d 28d

400 5d 9abcd 7ef 11cd 204c 222c

800 7bcd 14a 7ef 13c 255c 365b

1200 8abcd 13ab 9de 18b 288bc 580a

1600 6dc 12abc 9de 23a 344b 583a

Treatment means (in each plant part) with the same letter(s) are not significantly different from each other using DMRT at p<0.05.

Figure 2. Height (A), stem diameter (B), total biomass (C) and root biomass (D) of uninoculated and mycorrhiza inoculated Desmodium cinereum at different Cu levels (0-1600ppm).

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Figure 4. Mycorrhizal colonization of inoculated Desmodium cinereum. Roots of non-inoculated plants were not infected with mycorrhiza.

Figure 3. Leaf area (A) and nodule dry weight (B) of uninoculated and inoculated Desmodium cinereum at different copper levels.

lowest Cu uptake was at the 0ppm Cu level in both inoculation treatments. Increasing Cu concentration on the soil resulted to greater partitioning of Cu to the roots. Roots took up 90 to 98% of the total Cu uptake by the plants from 400 to 1600ppm Cu levels. Whereas at 0ppm, only 72% and 59% of the total Cu uptake where retained in the roots of non-inoculated and inoculated plants, respectively.

DISCUSSIONCopper is an essential element for normal growth of plants as well as mycorrhizal fungi (Marschner 1995). The amount of Cu needed depends on the species of plants and mycorrhizal fungi. Moreover, different crops and microorganisms differ in their tolerance to Cu and other HMs (Taiz & Zeiger 2002). In this experiment, it was shown that increasing concentration of Cu in the soil resulted into significant reduction on plant height, plant biomass, stem diameter and total leaf area. Elevated Cu

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Table 4. Percentage distribution of Cu uptake in the leaves, stem and roots of eight- week old Desmodium cinereum as affected by mycorrhizal inoculation and Cu levels. n= 3

Cu level (ppm)(ppm)

Leaves Stem Roots

Uninoc Inoc Uninoc Inoc Uninoc Inoc

0 21% 35% 7% 5% 72% 59%

400 4% 6% 3% 4% 94% 90%

800 3% 5% 1% 3% 96% 92%

1200 2% 2% 1% 2% 96% 96%

1600 1% 1% 1% 2% 98% 97%

Figure 5. Mycorrhizal colonization of inoculated Desmodium cinereum. Roots of non-inoculated plants were not infected with mycorrhiza.

concentration also inhibited nodule formation in plant roots. Mycorrhizal inoculation on the other hand improved plant height, plant biomass and stem diameter. The ability of the mycorrhizal fungi to improve plant growth is well documented in different studies (Ortasi 2012; Poulton et al. 2002; Perner et al. 2006). In this study however, improvement on plant growth was only observed at lower Cu concentrations specifically at the 0, 400 and 800ppm Cu level. Interestingly, at the same Cu levels, improvement on nodulation as affected by mycorrhizal inoculation was also observed. The improved nodulation could have enhanced plant nutrition contributing to the overall increase in plant growth at lower Cu levels. Castillo (1993) reported that mycorrhizal inoculation could enhance nodulation especially in soils with low P content. Inoculated plants at 0ppm and 400ppm Cu levels have significantly higher P concentration than non-

inoculated plants. This observation conforms the findings of Jakobsen et al. (2001), that nodulation is directly related with the P supply in the plants which was improved by AMF inoculation.

Another interesting result of the study is that increasing Cu levels added into the soil significantly increased the rate of AMF colonization. Though other studies showed similar results (Weissenhorn et al. 1995; Hildebrandt et al. 1999), there is no enough proof to say that high Cu concentration stimulated better mycorrhizal colonization. The high colonization rate that was found on plant roots exposed to elevated Cu concentration could be attributed to the fact that root development at higher Cu levels were extremely inhibited. Root sampling for examination of mycorrhizal colonization rate was limited with these few and less developed roots compared with the more developed and extensive root system at lower Cu levels.

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Figure 6. Total Cu uptake and its distribution in roots, stem and leaves of non-inoculated and mycorrhizal inoculated Desmodium cinereum at increasing Cu levels.

So there are lesser roots to be sampled from the less developed root system resulting to higher chance of sampling mycorrhizal infected roots.

Mycorrhizal inoculation significantly increased Cu concentration of the roots and stem at elevated Cu levels. Surprisingly, leaf Cu content was still at the normal range of 5-30ppm regardless of the Cu concentration on the soil and roots in contrast with the study by Chen et al. (2005) where mycorrhizal inoculation increased metal concentration on the leaves. Leaf Cu concentration was independent with the soil Cu concentration unlike root Cu concentration which increases as soil Cu increases. Although mycorrhizal colonization significantly increased plant Cu concentration, HM was mostly retained on the roots. Some studies show that AMF increases translocation of HM to the shoot (Leung et al. 2006). The results of this study had shown otherwise. AMF helped retained HM on plant roots preventing its translocation into the shoot especially to the leaves. Similarly, Chen et al. (2001, 2003) reported increased retention of HM in the roots of mycorrhizal plants clover and maize. AMF could have immobilized large amount of Cu in the roots through passive adsorption to their fungal cell wall (Joner et al. 2000).

Plants that accumulated the highest biomass at treatments where Cu was added had the highest total Cu uptake. Plants grown at the 400ppm Cu level had the highest total Cu uptake despite having the lowest tissue Cu concentration among the four treatments where Cu was added. It was followed by the plants at 800ppm Cu level and the least Cu uptake was that of the plants with the highest tissue

Cu concentration. Mycorrhizal inoculation significantly increased total Cu uptake in all treatments where Cu was added. However, most of the Cu accumulated was retained in the roots while only a small amount was transported on the shoot. About 90 to 98 % of Cu accumulated at these Cu treatments were retained in the roots while only1 to 6 % being translocated to the leaves.

Phytoremediation is an ideal technology where plants are used to clean polluted air, water or land. However, success of this strategy lies on finding plants that can efficiently and cost effectively removed contaminants from the soil. Yang (2005), pointed out that an ideal plant for phytoremediation should be fast-growing, should have high biomass production, should have extensive root system, and can accumulate large amount of HM. Looking for a plant with these characteristics, however, is a challenge. Heavy metal accumulating plants that were identified so far have root penetration to only shallow depths and produces small plant biomass. This study showed the importance of biomass production in extracting HMs from the soil and that high biomass production could readily translate into high HM accumulation and thus, reduces the toxic effects of HM stress.

Desmodium cinereum is a leguminous fast growing and high biomass accumulating plant species. Because of its extensive root system, it is popularly used in contour hedgerows for alley cropping to control erosion which is a usual problem especially in degraded and polluted areas. It can also increase soil fertility thru its association with beneficial microorganisms like mycorrhiza and nitrogen

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fixing bacteria. These ideal characteristics make it a good candidate for phytoremediation. However, this plant is also used as an important source of animal feed and forage plants are not traditionally used in phytoremediation because of the issue of mammalian toxicity. Results of the study however, show that leaf Cu concentration is still at a normal level regardless of the soil and root Cu concentration making it safe for mammalian consumption.

SUMMARY AND CONCLUSIONIncreasing Cu concentrations reduced height, root biomass, total plant biomass, stem diameter and leaf area of the inoculated and non-inoculated plants. Nodulation was also affected by increasing Cu levels especially at 1200ppm and 1600ppm where almost zero nodules were observed in both inoculation treatments. Improvement on root biomass, total biomass, and stem diameter as a result of inoculation was only observed at 0ppm Cu level. Mycorrhizal inoculation significantly improved nodule formation at 0ppm, 400ppm, and 800ppm Cu levels. Interestingly, mycorrhizal colonization was not affected by different Cu treatments. Even at the highest Cu level, mycorrhiza was still able to colonize the root system infecting a considerable amount of roots. Increasing Cu concentration reduced phosphorus concentration on leaves, stem and roots of both inoculated and non-inoculated plants. Mycorrhizal inoculation on the other hand increased P concentration at 0ppm and 400ppm Cu levels. Copper concentration on stem and roots was significantly increased by the increasing Cu treatments. Leaf Cu concentration on the other hand, remained at a normal level (5-30ppm) regardless of the amount of Cu applied in the soil. Inoculation of mycorrhiza significantly increased root and stem Cu concentration with the highest increase observed at the 1200 and 1600ppm Cu levels. Mycorrhizal inoculation increased total Cu uptake of the plants at all treatments amended with Cu. The total Cu uptake was highest at 400ppm (0.272mg Cu/plant) and 800ppm (0.231mg Cu/plant) Cu levels. Roots retained 90 to 98% of the total Cu uptake by the plants from 400 to 1600ppm Cu levels. Whereas at 0ppm, only 72% and 59% of the total Cu uptake where retained in the roots of non-inoculated and inoculated plants respectively. It is concluded that mycorrhizal D. cinereum could extract considerable amount of Cu from the soil even up to 1600ppm Cu level making it favorable for phytoremediation purposes. In addition, leaf Cu concentration remained at normal range thus making it still safe for animal consumption. Their nitrogen-fixing capability and extensive root system makes them an ideal tool in controlling erosion and restoring nutrients which is mostly a problem in degraded lands.

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