Abstract: The Khewra salt mine area lies in the District Jhelum, Pakistan. The Precambrian Salt Range Formation is exposed in the foothills of southern Potwar. The Salt Range Formation consists of mainly halite (NaCl), sylvite (KCl) and gypsum (Ca So4) salts. The alkaline soil is the product of weathering of the Salt Range Formation. The soil on which the plants are growing is rich in Na+, K+ and Ca++ metals. The seeds of Acacia nilotica and Peganum harmala were collected from salt polluted soil out side of the Khewra salt mine and the non-polluted soil from Muzaffarabad, Azad Kashmir, Pakistan. These seeds were grown in different concentrations of Na+, K+ and Ca++ at room temperature (22 ± 2°C). The study of salt polluted seeds in presence or absence of fungus Rhizopus, shows that the germination rate and biomass increase with increasing concentration levels of Na+ and Ca++ up to 30 µg ml‾1 and K+ up to 400 µg ml‾1. However, the germination rate and biomass of non-polluted seeds in absence of fungus, decrease with increasing the concentration levels of Na+, K+ and Ca++. In contrast, no single non-polluted seed germinated in the presence of fungus. The present study shows that if seeds from A. nilotica and P. harmala plants are sown and grown in saline soils of Punjab and Indus plains, these will reduce the salinity of soil without fungal infection in such plants.
Introduction
In Khewra salt mine area, the Precambrian Salt Range Formation is exposed (Gee, 1945; Fatmi, 1973 and Shah, 1977) in southern Potwar. The Formation consists of red colour marl, rock salts and gypsum. The red colour marl includes clay, gypsum and dolomite layers. The main rock salts are NaCl, KCl and CaSo4. The weathering and erosion of the Salt Range Formation formed soil rich in Na+, K+ and Ca++ metals. The soil samples collected from out side of Khewra mine and Muzaffarabad were analyzed by the Department of Agriculture Muzaffarabad. The alkaline soil from Khewra mine area has pH 8.1, organic matter 0.00025 mg kg–1, NO3–N 13.57 mg kg–1, K+ 1000 mg kg–1, Ca++ 50 mg kg–1 and Na+ 392 mg kg–1. Whereas, the alkaline soil from Muzaffarabad has pH 7.38, K+ 256 mg kg–1, Ca++ 26/kg and Na+ 24 mg kg–1. These metals are major essential nutrients used by living organisms in high concentration.
The required amount of mineral nutrition is essential for the better growth of plants growing in metal contaminated soil. Plants absorb metals in different concentration based on external and internal factors. Heavy metals uptake, accumulation and tolerance have been studied in the British population of the metallophyte Thalaspi caerulescens by Baker et al. (1994). They showed that Zn++, Cd++, Mn++ and Ni++ were transported to the shoot as accumulators. Whereas, Al+++, Cr+++, Cu++, Fe++ and Pb++ were predominantly immobilized in the roots.
Baker (1981) described the pattern of metal up taking for accumulator and non-accumulator plants. In accumulators, there is a rapid increase of metal contents in the arial tissues until saturation is reached. For non-accumulators, restricted transport of metals from root to shoot occurs until a soil concentration is reached and there is unrestricted transport and metal toxicity ensures. Each organism, regardless of whether it lives in a metal enriched environment or not, has a certain ability to cope with non-essential metals or excessively available essential metals. However, there are limits in the occurrence of metal tolerance in the higher plants (Bradshaw and McNeilly, 1991).
Natural vegetation and crops are sensitive to salinity level both at germination and subsequent growth stage. High salinity in nature creates very poor vegetation of halophytic community and poor productivity of natural and cultivated crops. The plants growing in saline area modify themselves to resist salt.
The general vegetation in Khewra area gives a clear picture of subtropical zone due to the presence of Acacia modusta, A. nilotica, Dalbergia sisu etc. in tree layer, Calotropis procera, Dutura, Dodonea viscosa, Ziziphus oxyphylla etc. in shrub layer and Peganum harmala, Fumaria indica, Lathyrus aphaca, Poa annua, Themeda anathera, Sacharum spontaneum, Euphorbia prostrata, Sonchus asper etc. in herb layer. Major essential nutrients are required in high concentration for plants as well as fungi. These nutrients show toxic effect if they are present in higher concentration, for example, Na+ shows toxic effect in germination of seeds.
Fungi are, in general, easy to grow and produce high yield of biomass. The other kinds of biomass arising from industrial fermentation include Rhizopus arrhizus, Aspergillus terreus and Penicellium. Toxic effects are believed to affect fungal population by reducing abundance. However, the effect of toxic metals on microbial abundance in natural habitat varies with the metal species and other environmental factors (Duxbury, 1985). This study deals with the effect of alkaline metals on the growth rate of Acacia nilotica and Peganum hamala in the presence or absence of fungus Rhizopus sp.
Materials and Methods
The seeds of Acacia nilotica and Peganum harmala were collected from salt polluted soil of Khewra salt mine and non-polluted soil of Muzaffarabad in the month of June 2001. The seeds were brought and placed in cold room. The seeds were used after sterilizing with calcium hypochlorite for detailed experiments.
To observe the effect of metals on the germination and growth of seeds, Na+, K+ and Ca++ solutions were prepared from the salt NaCl, KCl and CaSo4 respectively. The seeds were sterilized by Millipore filtration. The metal solution was added to 20 ml of Rorison nutrient solution in 9cm diameter Petridishes having sterilized filter paper. The final metal concentrations were 0, 10, 20, 30, 40 and 50 Fg ml–1 of Na+ and Ca++ and 0, 200, 400, 600, 800 and 1000 Fg ml–1 of K+. These concentrations were chosen to reflect the range of concentrations likely to be found under field conditions. The Petridishes were rotated to mix the solution with the Rorison solution. Three replicates were prepared for each metal concentration. Ten sterilized seeds of each A. nilotica and P. harmala were placed in each Petridish. Two sets of Petridishes were prepared containing Na+, K+ and Ca++ solutions. Each Petridish in one set was inoculated centrally with an 8 mm disc cut from the margin of a three days old culture of fungus Rhizopus species. Whereas, the other set was remained without this fungus. After 7 days at room temperature (22±2°C), the percentage of seed germination was determined. The biomass (dry weight) of shoots of A. nilotica and P. harmala were calculated after 10 days.
Results
The germination rate of seeds and biomass of shoots of A. nilotica and P. harmala from salt polluted area increase with increasing the concentrations of Na+ and Ca++ up to the level of 30 Fg ml–1 with and without Rhizopus sp., whereas in case of K+ this growth rate increases up to the level of 600 Fg ml–1. The growth rate above these limits decreases in the presence of fungus (Tables 1-6). However, the germination rate is equal or greater even at the range of 40-50 Fg ml–1 Na+ and Ca++ than that of 0 Fg ml–1. The overall biomass in K+ and Ca++ concentrations is smaller than Na+, but the pattern of increasing or decreasing of growth rate is similar in all observed metal concentrations. The germination rate and biomass from non-polluted plant seeds decrease with increasing metal concentrations of Na+, K+ and Ca++ in the absence of fungus. No germination is found in any Petridish when inoculated with fungus (Tables 1-6). The germination rate of salt polluted seeds increases upto the level of 600 Fg K+ ml–1 and biomass of shoots is maximum at the level of 400 Fg K+ ml–1. The germination rate and biomass decrease above these limits. The growth rate of both plant seeds growing in non-polluted area, shows descending order in growth, during increase in concentrations of K+ in absence of fungus. The seed germination is not seen in any concentration of K+ when inoculated with fungus (Table 3- 6).
Discussion
This study deals with the comparison of the growth between the same plants growing in salt contaminated soil at Khewra salt mine area and non-contaminated soil at Muzaffarabad, in presence or absence of fungus Rhizopus, at different concentrations of Na+, K+ and Ca++. The result shows that the plants growing in Na+, K+ and Ca++ contaminated soil are not only have much better growth rate but also defend themselves against the parasitic attack. In contrast, the seeds growing in non-polluted soil are not resistant to high concentration of metals and they are totally susceptible against parasite or pathogens (Tables 1-6). This study supports the tolerant theory (Boyd and Marten, 1992) against higher concentration of metals or pollutants (Iqbal and Qadir, 1982) in plants growing in polluted area and defense theory (Reeves et al., 1981; Boyd and Morten, 1992) against the parasite or pathogens. It is clear from the results obtained during the present study that the seeds of plants from polluted area show highest germination rate (70-75%) at the level of 600 Fg K+ ml–1 concentration. In contrast, the non-polluted seeds show extremely low germination rate of 9-10% (Table 3).
| Table 1: | Effect of Na+ on germination of seeds of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (%/7 days; mean values) |
| Table 2: | Effect of Ca++ on germination of seeds of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (%/7 days; mean values) |
| Table 3: | Effect of K+ on germination of seeds of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (%/7 days; mean values) |
| Table 4: | Effect of Na+ on biomass of shoot of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (mg/10 days; mean values) |
| Table 5: | Effect of Ca++ on biomass of shoot of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (mg/10 days; mean values) |
| Table 6: | Effect of K+ on biomass of shoot of Acacia nilotica and Peganum harmala from salt polluted and non-polluted areas with and without Rhizopus (mg/10days; mean values) |
The biomass of the seedling from polluted seeds at the level of 400 Fg K+ ml–1 is 103 mg, whereas the biomass of seedling from non-polluted seeds contains only 1-3 mg (Table 6). In conclusion, this study confirms the results that the plants growing in polluted soils, defend themselves against the parasites and pathogens, due to the presence of high concentration of metals (Tables 1-6).
Natural vegetation and crops are sensitive to salinity both at germination and subsequent growth stages. High salinity in nature creates poor halophytic vegetation and poor productivity of natural and cultivated crops. Some plants modify their growth in saline environments. The natural modifications help them to survive in saline areas which decrease the salt pollution.
The fungi are sensitive to high concentration of essential elements. Polluted soil may be nutrient poor, variable pH and may contains additional toxicants (Gadd and Griffiths, 1978). These factors effect fungal populations. Rhizopus species was isolated from non-polluted soil of Muzaffarabad. This fungus do not resist in the high concentration of metals. Rhizopus stolonifer cause disease soft rot of sweat potatoes and leak of strawberries, raspberries, peaches etc. The stored non-polluted seeds or grains are spoiled when attacked by some species of Rhizopus. This study shows that the Rhizopus species do not effect the growth rate of the plants growing in salt polluted soils. Whereas, the seeds germination rate and biomass in the non-polluted soil are severely effected by the fungus.
It is concluded from the present findings that the plants growing in metals or salt polluted soils have the ability to tolerate and survive in high concentration of metals. If such plants are grown in salty and saline soils, the environmental pollution can be controlled to greater extent. Resultantly, on one hand, this will assist in increasing vegetation and purification of environment and on the other, pathogen resistant plant species can be introduced.