International Conference for Enhancing Scientific Research: New Horizons
Tanta Conference Hall 20-21 February 2008: PP 1-10
Differential Responses of Anabaena Variabilis and Nostoc Linckia to Salt Stress and Their Role in Improvement the Growth Conditions of Some Salt Stressed Plant Seedlings
Ahmed D. El-Gamal1, Nady A. E. Ghanem1, Eisha Y. El-Ayouty2 & Ehab F. El-Belely1.
1 Fac. Sci., Bot. Dept., Al-Azhar Univ. Nasr City, Cairo, Egypt. 2 Fac. Sci., Bot. Dept., Cairo Univ., Giza, Egypt
Two Species of cyanobacteria: namely, Anabaena variabilis and Nostoc linckia were isolated from Baltem and Sedi-Salem (saline-sodic soils), respectively (Kafr El-Sheikh Governorate, Egypt). A. variabilis was able to grow in salinity level between 4-6 g L-1 NaCl; above 6 g L-1 NaCl the growth rate was relatively constant till 10 g L-1NaCl. Over 10 g L-1 NaCl, the growth rate declined. Results indicated that A. variabilis was able to utilize NaCl between 1-10 g L-1. On the other hand, N. linckia had a wide range of salinity tolerance. Whereas, the growth rate increased gradually from 1- 4 g L-1 NaCl and more than 4 till 20 g L-1 NaCl the pattern of growth was nearly stable. N. linckia was able to withstand a wide range of salinity but it had no good ability to utilize NaCl. The results indicated that the addition of A. variabilis led to significant increase of shoot and root length as well as, significant decrease of proline content of bean, zea and barley seedlings grown in saline environment, respectively. Also, the presence of alga caused a significant increase in chlorophyll a content of zea seedlings as well as, the germination percentage of barley seedlings in as compared to control ones. N.linckia caused a significant decreasing in proline content of bean, zea and barley seedlings, while, it caused an increasing of both chlorophyll a content of zea and shoot length of barley seedlings as compared with control.
Salinity is one of the most serious factors limiting the productivity of many organisms (Rodríguez et al., 2006). Salinity affects organisms in estuaries both directly through inhibitory effects on physiology, and indirectly by affecting other environmental factors (Moisander et al., 2002). In accordance with FAO, approximately 7% of the land is affected by salt. Twelve percentage of this land is localized in the south and south east of Asia, where rice is the principal crop. Salted soils are extensive also in northern and southwestern Africa, including the Union of South Africa, Rhodesia, Egypt, Algeria, Morocco and Tunis.
Salt stress affects many aspects of algal metabolism and, as a result, growth rates of several cyanobacteria decrease under increasing salt concentrations, but the extent of growth inhibition can vary (Vonshak et al., 1988). Kirst (1990) stated that exposing some species to salinity stress results in a significant inhibition in their photosynthetic activity. On the other hand, certain algae, especially blue-greens, have the ability to adapt their physiological processes to very large fluctuations in salt concentrations in their environment (Borowitzka, 1986).
On the contrary, blue-green algae such as Aphanothece halophytica and Phormidium hypo-limneticum are known to be extremely halotolerant, growing at salinities in excess of 200 g L-1 (Dor and Ehrlic, 1987). Vonshak et al. (1988) demonstrated that Spirulina sp. have the ability of adapting to high sodium chloride concentrations.
Moisander et al. (2002) suggest that salinity may be a controlling factor for blooms of nitrogen fixing cyanobacteria in estuaries. They also found that Anabaenopsis sp. maintained similar growth rates in the full range of salinities from 2 to 20 g L-1 NaCl, while, Anabaena sp. grew at up to 15 g L-1 NaCl, but the maximum salinity 20 g L-1 NaCl was inhibitory. On the other hand, the upper limit for salinity tolerance of Cylindrospermopsis raciborskii was 4 g L-1, while Nodularia spp. also maintained similar growth rates in the full range of salinities from 0 to 20 g L-1 NaCl. Generally, previous studies indicated that, as a group of microorganisms, like cyanobacteria and green algae exhibit considerable salinity tolerance (Rao et al., 2007) and many species are adapted to hyper saline environments (Oren, 2000).
Also, several studies were carried out on the effect of salinity on the growth, metabolism and yield of the plants (Nandi et al., 1995; Moussa et al., 1996; Yildirm et al., 2006). It was found that high salt levels have a negative effect on plant growth and crop yield quality. Crop plants also differ in their tolerance to salinity.
Improvement of saline soil as well as salt-tolerant crop plants is needed to maintain the food output of the world. Therefore, there are many trials in this respect (Gurbaksh and Harpal, 1980; Soliman, 1984; Kamel et al., 1987; Ouda et al., 1991) showed that the employment of certain growth regulators such as gibberilic acid (GA3), decreases the injurious effect of salinity and improve the salt tolerance of the plants. Economically, growth regulators are approximately expensive and are non-practical especially, when applied in large amounts. Thus the idea to use other alternatives, such as, algae is very important aspects in order to play an economic role in soil reclamation, to increase soil fertility and to improve the plant conditioners under certain environmental factors (El-Gamal, 1998).
The role of cyanobacteria in the amelioration of salt affected soils was studied by Kaushik and Rajarao (1990). Apte and Thomas (1997) showed that amelioration of soil salinity by application of Anabaena torulosa during crop growth is possible, since it can also supplement the nitrogen requirements of the crop. El-Gamal (1998) investigated the effect of Anabaena variabilis (using extracts of the living or non living alga) on presoaked wheat grains subjected to various sodium chloride concentrations. The results revealed that the germination percentage was still stable for all the treated plants especially those which presoaked in both living or non living algae, while the germination percentage of the untreated plants reduced regularly and slowly, reaching 60 % at 3000 ppm of sodium chloride.
The aim of this work was to investigate the potentiality of Anabaena variabilis and Nostoc linckia to grow in saline media as well as to evaluate the effect of the selected algae on the growth and proline accumulation of salt stressed of bean, zea and barley seedlings.
MATERIALS & METHODS
Collection of soil samples: Five randomly-selected soil samples were obtained from each of the two study sites namely Baltem and Sedi-Salem (saline-sodic soils), Kafr El-Sheikh governorate, Egypt. After removing the uppermost cm of soil with a sterile spatula, about 10 cm diameter soil cores reaching 4-5 cm below the surface of the soil collected and placed in sterile plastic bags. Samples were transported to the laboratory within 24 hour of collection.
For isolation of the algae, all soil samples were crushed and passed through a 2 mm sieve (Shubert and Starks 1980).
Culturing and isolation of algae: Z and Allen's media were used for algal cultivation, as described by Staub (1961) and Allen (1968), respectively. Three methods were used for culturing and isolation of the two studied algae: semi-solid, liquid media (El-Ayouty and Ayyad, 1972) and filter paper method (Esmarch, 1914).
Purification of algae (es containing sterile meium patches filter paper through the pores. axenic culture): Algal sheath removal was done by the method described by Bradley and Pesano (1980). Algae were purified by using a commercial disinfectant known as TotilTM (Calgon corp). Also, washing with chlorine water was done by the method described by Fogg (1942).
Algal growth measurements: Growth measurement was carried out by determination of dry weight (El-Gamal, 1995), chlorophyll a content of algae (Nusch and Palme, 1975) and the optical density of the culture (Adhikary 1983).
Effect of salinity on the growth of investigated algae:
- Experimental design: Experiments of salinity were carried out in autoclaved 250 ml Erlenmeyer flasks with cotton plug. Cultures were inoculated with 2 mL of algal culture (15 to 20 day-old cultures) per 100 mL sterilized medium. One set of inoculant cultures was grown without salt as a control. Salinity treatments were 1, 2, 4, 6, 8, 10, 13, 15 and 20 g L-1 NaCl; with three replicates for each treatment. All flasks were incubated under suitable growth conditions of light and temperature, and swirled daily. Optical density, chlorophyll a content and the dry weight were used as a growth parameter in this study. Electrical conductivity of each culture medium filtrate was estimated at zero time and after 15 days of incubation period.
Effect of the investigated algae on the seedlings of some plants growing in saline environment:
- Experimental design: The experiment was carried out in plastic receptacles (9 cm length and 7 cm in diameter), each receptacle filled with 200 g of washed sterile soil. Then the soils were treated with different concentrations of NaCl (0.5, 1.0, 2.0, 3.0 and 4 gL-1), with three replicates for each concentration.
After that, each treated soil was inoculated with 50 ml of homogenized fresh highly biomass algal cultures. The receptacles were incubated under the light intensity of 2500 lux at 37°C for 1-2 weeks, and kept wetted. The receptacles were classified into the following categories:
-Set no. 1. Seeds/grains with non-treated soil (control).
- Set no. 2. Seeds/grains with salt-treated soil.
-Set no. 3. Seeds/grains with salt-treated soil and inoculated algal biomass.
For seedlings of bean, zea and barley, the following growth parameters were taken into consideration:
- Percentage of seeds and grains germination.
- Root and shoot length.
- Chlorophyll a content (Wettsteins, 1975).
- Determination of free proline (Bates et al., 1973).
Statistical analysis: In this respect, two-way ANOVA (analysis of variance) was used to examine whether an interaction effect existed between the presence of algae and salinity. Effect of algae on the germination percentage, root and shoot length, Chlorophyll a and proline content was tested using Duncan's Multiple Range Test, using three replicates. The results were analyzed at P < 0.05 (significant at 5 %) and at P < 0.01 (significant at 1 %).
The obtained results presented in Table (1) revealed that the maximum salinity level for growth of Anabaena variabilis was 4-6 g L-1 NaCl, from 6 to 10 g L-1 NaCl, the growth rate was relatively constant. Whereas, increasing the salinity over 10 g L-1 NaCl declined the growth rates. Also, results represented in Table (1) confirmed that A. variabilis was able to utilize the NaCl until high salinity levels (ranged from 1 to 10 g L-1 NaCl). Meanwhile, Nostoc linckia had a wide range of salinity tolerance, whereas the growth gradually increase from salinity 1 to 4 g L-1 NaCl, then the alga had relatively similar growth rate pattern from 4 to 20 g L-1 NaCl. Although N. linckia able to withstand along wide range of salinity, it had no significant capacity to utilize the NaCl salt.
Facing the great differentiations in values between all treatments, the significant data were sufficient to be restricted that the impacts of Anabaena variabilis, many parameters were affected by the presence of this alga. Where, a significant increasing in shoot and root length, and decreasing in proline content was recorded in bean seedlings (Table 2). Also, presence of the alga cause a significant increasing in root and shoot length, and chlorophyll a content, and decreasing in proline content in zea seedlings. Germination percentage, root and shoot length, and proline content of barley seedlings were also significantly affected by the alga (Table 2).
Existence of Nostoc linckia causes a significant decreasing in proline content in bean, zea and barley. Moreover, the presence of alga causes an increasing in chlorophyll a content in zea and shoot length in barley seedlings (Table 3).
Salinity is considered one of the barriers which stand as stumbling block in plant development. Therefore, many workers have thought about going beyond such problem that hinders the development process in developing countries. The potentiality of two selected blue-green algae was tested to grow in culture media supplemented with different NaCl concentrations. The results indicated their variation to grow in different NaCl concentration, as well as, their possible utilization of NaCl in culture media was also variable. Moisander et al. (2002) found that each of studied organisms is able to tolerate with NaCl differently. Also, Garcia-Pichel et al. (1999) observed a general pattern of decreasing photosynthesis and oxygen exchange capacity with increasing salinity in Cyanobacteria. NaCl stress is well known to suppress the growth of algae (Masojidek et al., 2000; Hagen et al., 2001). The effect of NaCl on present algae was tabulated in Table 1. The results indicated that the responses of two algae were different. Anabaena variabilis could grow under optimum salinity level at 4-6 g L-1 NaCl, but above 6 g L-1, the growth was relatively constant. On the other hand, Nostoc linckia showed a better growth when allowed to grow under a wide range of salinity, reaching the maximum at 8 g L-1 NaCl. Whereas, N. linckia able to withstand along a wide range of salinity. But it had no significant capacity to utilize NaCl.
Adaptation to high salinity levels includes the avoidance of internal toxic levels of inorganic ions, and synthesis and accumulation of osmoprotective compounds (Blumwald et al., 1983; Reed and Stewart, 1985). The present results showed that Anabaena variabilis had the power to allow NaCl to pass through cyanobacteria cells. In contrast, Nostoc linckia had another reaction, while the NaCl solution remains extracellular and incapable of entering the cyanobacterial cells; this was confirmed by measuring the electrical conductivity of culture media (ECm) at the beginning and after 15 days of the incubation period (Table 1).
It is seemed that the mucilaginous sheath had a role in an ameliorative effect on salt stress as more than 90 % of the cell-bound Na+ remained extracellulary trapped in the mucopolysaccharide sheath of cyanobacteria (Apte and Thomas, 1997).
The present data also indicated that the patterns of chlorophyll a content and dry weight of the algae are identical, i.e. embracing each other (in case of Anabaena variabilis).Whereas both patterns maintained similar, to some extent, in the full range of salinities from 4-20 g L-1 NaCl (in case of Nostoc linckia). Similar results were obtained by Moisander et al. (2002). Moreover, application of 0.3 M NaCl caused significant reduction in chlorophyll a content of N. linckia and A. subcylindrica as compared with the control after 15 days of inoculation (El-Naggar et al., 2004). On the other hand, an inverse relationship between the algal growth and NaCl concentration was observed (El-Sheekh and Omar, 2002; El-Naggar et al., 2004).
Concerning the chlorophyll a content of seedlings, the results indicate significant increase in chlorophyll a content in zea seedlings after Anabaena variabilis and Nostoc linckia treatments. In all cases Anabaena variabilis and Nostoc linckia significantly reduced the proline content in all tested crops. Proline accumulation under stress is proposed to be a part of the mechanisms of osmotic adjustment (Misra et al., 1996; Van Heerden and De Villier, 1996).
Generally, this study revealed that the members of cyanobacteria can alleviate the high concentrations of the ions. From agriculture point of view, soil salinity appears to be a major problem in agriculture and is becoming more serious from one year to another year. Sodium chloride is the most dominant salt in saline soil, although other salts are considered. Improving the fertility of saline soils is not adequate because initially saline soils are not suitable for crop production. The use of selected cyanobacteria would open the way for magnifying the role which could be played by algae on plant growth under saline condition. In General, cyanobacteria would be served as cheapest sources of biotechnology in natural habitats. However, a successful biotechnology requires the selection of suitable algal strain for proper plant but these were correlated by another factors rather than salinity status.
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