The study of Egyptian algal flora is still a non-depletion source, although numerous studies were carried out in such field

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DISCUSSION

The study of Egyptian algal flora is still a non-depletion source, although numerous studies were carried out in such field. In Egypt, the role of algae is still at the bottom or, in other words, being tested or tried, therefore the scientific life needs intensive studies concerning algae that may help to bridge the gaps between science and reality, or in other words to solve the crossword puzzle in people's endurance. For that reason, the present study was performed to construct the first attempt to the survey the soil algal flora of Kafr El-Sheikh governorate, in addition to study the role of algae which can be played for withstanding the high levels of salinities during the growth of some salinity stressed plants.

In the present work, it has been tried to isolate algae from ten possible locations in Kafr El-Sheikh, all this locations were chosen on the basis of their physiological and chemical characteristics, as well as on their geographic locations. The results of mechanical analysis of soil samples showed that some soils, to some extent, were similar in their texture, while the others were different. It was found that soil "1" had no stones, while fine gravels were represented by the lowest fraction, on the contrary soil "1, 2, 3, 5, 6 and 9" had the highest fractions of very fine sand. Generally, the variations of soil textures from one region to the next may have a role in algal populations and give the habitat a stimulator role in algal selection, but this role might be also attributed to the non-biotic factors and cultivation status of soil under study, this may clear the distribution frequency of intended algae under study.
The present data reflect the results of maximum water holding capacities, gravitational, hygroscopic and capillary water, where the data revealed that soil "1 and 10" had the lowest levels of their water holding capacity, on contrary the results showed that soil "5 and 6" had the highest levels of maximum holding capacities. On the other hand, the data in Table (3) also reflect the reversal relationship between the results of maximum water holding capacity and gravitational water, also the results in Table (3) and Fig. (6) clearly revealed that soil "5 and 6" appear to be the highest percentages of capillary water, while soils "1 and 10" had the lowest percentages. On the other hand, soil "3" shows the highest percentage of hygroscopic water, while the lowest percentage observed only in soil "1". It was evident that maximum water holding capacity includes both hygroscopic and capillary water and the both play a noticeable role in predominance of all forms of algal life, whether vegetative or resting forms.
The highest values of both total soluble salts and electrical conductivity (5.28 % and 68.13 dS/m in soil paste extract, respectively) were recorded in soil "2", on contrary soil "3" showed the lowest values of both (0.35 % and 1.58 dS/m in soil paste extract, respectively). Generally, all soils which collected from waste lands (1, 2 and 5) could be described as saline soils, while the rests appeared to be non-saline ones.
The determination of pH values of paste soil extract indicated that all soils had alkaline nature. The determination of pH in 1:5 (w/v) soil extract showed a little lowering in pH values.
The maximum content of organic carbon was observed in soil "6", parallel, the same soil had the highest percentage of carbonate. Concerning, the bicarbonate content, the highest percentage was recorded in soil "9".
The assessment of chlorides, Ca⁺⁺, Mg⁺⁺, Na⁺ and K⁺ ions indicated that soil "2", the non-cultivated, had the highest values of these ions, the values were (3.9 %, 840, 519.3, 4946.5 and 2877.6 ppm, respectively). The concentrations of ions were arranged from higher to the lower following order: Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺, only in the case of soils "1, 2, 5 and 9", however, in all cases Mg⁺⁺ was the lowest concentration.
In this respect Fathi and Zaki (2003) demonstrated that the higher ions of soils were recorded at site (IV) which was non-cultivated. Monovalent and divalent cations play an important role in the productivity of soils (Starks et al., 1981). The present results showed pronounced fluctuations of monovalent and divalent cations from site to site. Generally, the results give at first sight an impression for the importance of cations content for the growth and abundance of algae, as well as, their productivity should go at all time paralleled to the change of soil cations parameters (Fathi and Zaki, 2003).
Stanier et al., (1971) demonstrated that it is possible to make such taxonomic distinction, as well as physiological studies, only by investigations with organisms in pure culture. Generally, under most conditions, algae grow as mixed communities with other organisms, so it is necessary to obtaining such algae in a culture containing a single species of algae (axenic culture).
Algal species used in our work were grown generally in artificial isolation from other species because it is difficult otherwise to attribute a particular feature or response to the organism under investigation. In this respect, many techniques were applied to obtain axenic cultures, the success of particular technique depends largely on the algae under investigation and the contaminating bacteria and/or fungi witch were present. The applied techniques to obtaining unialgal culture include Streak plating (which was more suitable for obtaining of unicellular algae) and Pour plate method. On the other hand, trials for purified the unialgal cultures (especially algae with gelatinous sheath) from bacteria were very difficult because the presence of epiphytic bacteria which usually become embedded in the mucilaginous algal sheaths for this reason, Algal sheath removal technique (Bradley and Pesano, 1980) was more effective in the purification of algae which have a gelatinous sheath. However, other techniques such as washing with diluted chlorine water, treatment with Totil^TM detergent and with antibiotics were also effective in purification of the algal isolates. Many antibiotics inhibit the growth of both blue-green algae and bacteria (due to their susceptibility to antibiotics), for this reason, using of antibiotics, such as penicillin and streptomycin, were difficult to employ with blue-green algae, but it were more effective when used with green algae. During our work, it needs to suggest that, successive subculturing several times is useful to confirm the purity.
The present study cleared that out of 40 identified species, 25 belonging to Cyanophyta and 15 belonging to Chlorophyta. The oscillatoroid species of Cyanophyta represented the majority of most isolates during this work. The dominance of Cyanophyta as compared with other types of soil algae is a matter of tolerance and adaptability (Ahmed, 1994; Hifney, 1998 and El-Attar, 1999). Whitton, (1992) stated that, numerous forms of cyanobacteria have been recorded from environments with fluctuating salinities. In this respect, results of isolation also reflected the wide distribution of soil algae in the investigated area, especially in saline soils. According to Richards (1954) and depending on determination of electrical conductivity, soils "2 and 5" are classified as very strong saline soils, while soil "1" classified as strong saline soil, nevertheless, 11 species of blue-green algae were isolated and about 5 species of green algae, including Gloeocystis major, Chlorococcum humicola, Characium acuminatum, Protococcus viridis and Closterium parvulum, were also isolated from the saline soils. Isolation of Chlorococcum humicola from saline environment was also performed by El-Gamal (1995), in addition to Prescott (1973), who described the dominance of alga as common on beach soils.

In a comparison between the soil samples, concerning the distribution and number of algal species, soil "6" exhibited the highest number of species (13 algal species), representing 32.5 % of the total number of isolated species, on the other hand, soil "10" showed the lowest number of isolates (3 species) representing 7.5 % of the total isolated species.

As regards to the Chlorophyta the coccoid, unicellular and colonial forms were the predominant in all investigated soils, such Chlamydomonas globosa, Pandorina sp., Chlorella vulgaris, Scenedesmus dimorphus etc., such results clearly indicated the role which can be played by organic matter and cations of soils in modifying algal population and activities in various soils. These results are in accordance with that of El-Attar (1999), and Fathi and Zaki (2003).
It is well known that a proper identification of algae can be possible after careful examination of cultures. The significance of algal cultivation lies in the fact that they allow to detect the important diagnostic features especially the mode of reproduction and morphological variability. From Table (7), it can be seen, the different distribution and frequency of algal isolates from one area to another. The application of different culture media proved all favorable conditions for growth of algae and gave the possibility to detect all morphological variability as well as the reproduction modes of algae. The present data agree with those of Starks et al., (1981) as they mentioned that the current knowledge of soil algae is based mainly upon enrichment culture studies, which may have caused bias in the resulting species lists.
The identification of algae was carried out using the following recommended criteria: thallus color, thallus morphology, dimension and sizes of heterocysts, vegetative and reproductive cells. The description of isolated algae was accompanied by colored photographs. The identification of algae is still problematic, so the identification was carried out by using intended literatures, as well as the description of algae was reflecting the all possible criteria which were recorded to solve the identification circumstance. For that reason, not all algal genera were identified to species rank because the characters of organisms are slightly deviated from the type species.
Concerning the effect of different culture media on the algal growth, the results proved that Z-medium was apparent to be most suitable culturing media for the growth of the most investigated algae, although other media were proper for the growth of others. In this respect, El-Gamal (1995) obtained the same results as Z-medium proved the favorable conditions for the growth of algae either blue-greens or coccoid green algae (84 % of the total species isolated).

Carbon is essential element for living organisms, the except- ional position of carbon among other elements is based on the ability of its atoms to link manifoldly together to form a huge variety of compounds which are essential to the development of living things (Zehnder, 1982 and Vymazal, 1995). Results dealing the effect of different carbon sources on the growth and biomass production revealed that xylose was preferable for most investigated blue-greens, while the investigated green algae gave the highest biomass production when were allowed to grow in inorganic carbon sources.

Nitrogen acts as a main constituent of protein and genetic materials. Nitrogen also is of a major importance in amino acids, purines, pyrimidines, amino sugars or amines. Algae utilize nitrate, ammonium salts and organic nitrogen as the main source for nitrogen, although ammonium ion appears to be preferentially absorbed over nitrate (Chapman and Chapman, 1973_b). With respect to the effect of different nitrogen sources on the growth and biomass production, the results indicated that the most investigated algae utilized inorganic nitrogen sources better than the organic ones, this results also were suggested by Fogg et al., (1973) who found that, in laboratory culture most algae grow less well on organic nitrogen than on inorganic nitrogen. Generally, the assimilation of inorganic nitrogen (especially nitrate-nitrogen, ammonium-nitrogen and elemental nitrogen) salts are differed due to different algal species, and also depends on various factors such as the levels and types of combined nitrogen supplied (Fogg et al., 1973). Salah El-Din et al., (2004) noticed that Nostoc humifusum grown in Z-medium supplemented with 2 % NaCl reached the maximum growth at the best nitrogen source as (NH₄)₂CO₃. Also, (NH₄)₂NO₃, NaNO₂ and NH₄Cl stimulated N. humifusum to high growth similarly. However, KNO₃, a nitrogen source had similar effect as the control on N. humifusum. On the other hand, N. humifusum grew with lower growth at both (NH₄)₂SO₄ and urea as compared to the control. On the contrary, all the investigated nitrogen sources did not increase the growth of Oscillatoria earlei but more likely were inhibitory in compared to the control in the following order: (NH₄)₂NO₃, peptone, NaNO₂, urea, (NH₄)₂CO₃, (NH₄)₂SO₄, NH₄Cl, L-Proline and KNO₃.
Basic environmental factors such as pH, temperature and light are essential factors which affect the growth and decline mass population of terrestrial algae (Nicklisch, 1992).
Hydrogen ion concentration (pH) is an important factor which directly or indirectly affects the growth, distribution and abundance of terrestrial algae. However data in Table (11 a and b) showed that the optimum pH values for the growth of the investigated species differ one to the other, generally, blue-green algae prefer neutral to alkaline substrata for their development, while green algae favor neutral and slightly acidic media for their growth. These results are in reasonable agreement with those obtained by Gerloff et al., (1950_b), Kratz and Myers (1955), Shields and Durrell (1964), Fogg et al., (1973), and Starks et al., (1981) who accepted that neutral and alkaline soils are more favorable to the development of blue-green algae, while the Chlorophyta were better represented in acidic soils (pH 6-4.6). The effect of pH may be represented in influences the metabolism of algae and so determines the solubility of carbon dioxide and minerals in the medium (Becker, 1993), for example at high pH levels the poor growth may be due to soluble iron being precipitated out of solution as ferric hydroxide which thus becomes largely unavailable, on the other hand, low pH levels could lead to an unavailability of molybdenum which is essential for nitrogenase and nitrate reductase (Fogg et al.,1973).
Concerning the effect of temperature on investigated algal growth, the data indicated that the optimum temperature ranged from 25-30˚C for all investigated blue-green algae, while the optimum temperature was 25˚C for all investigated green algae. The present results are in conformity with the findings of Kratz and Myers (1955), Fogg et al., (1973), Hoffmann (1989) and El-Gamal (1995). Generally the lower or higher temperatures inhibited the algal growth. Fogg et al., (1973) stated that low temperature may be exert at least two effects: physical damage produced by ice crystals and a slowing down of the chemical reaction, on the other hand, at high temperatures cause an imbalance of metabolism results from the differential acceleration of compound reactions as well as, from a denaturation of proteins. In this connection, the terrestrial algae are resistant to low as well as to high temperature and live in cold and hot desert soils. Fogg et al., (1973), also stated that blue-green algae are well known for their ability to withstand and to grow at extremes of temperature.
Light intensity is a factor which appears to exert different effects in the field and in the laboratory. The effect of different light intensities on investigated alga; growth can be summarized as follows: the investigated blue-greens were able to grow within a range between 2000-5000 lux, while this range decreased between 1000-3000 lux in the case of investigated green algae under study. In addition, many algal species comply with variations in light intensity by varying their cellular content of chlorophyll a and or altering the activities or concentration of photosynthetic enzymes (Soeder and Stengel, 1974). It was obvious that, continuous illumination for the algal cultures give better yields than intermittent illumination (16 h light : 8 h dark), this finding is supp- orted by the same observation of Fogg et al., (1973).
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 four 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 was 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 and Hagen et al., 2001). The effect of NaCl on our present was tabulated in Table (15 a and b), the results indicated that the responses of four algae were different as the growth of Oscillatoria sp.(1) increased with increasing the salinity level until 6 g L^-1, while Anabaena variabilis could grow under optimum salinity level at 4-6 g L^-1, but above 6, the growth was relatively constant. Nostoc linckia showed a better growth when allowed to grow under a wide range of salinity, reaching the maximum at 8 g L^-1, although N. linckia able to withstand along a wide range of salinity but it had no significant capacity to utilize NaCl. Tolypothrix tenuis appeared to be much sensitive to NaCl treatment when comparing with control. The utilization of NaCl in media was not so obvious.
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 and Reed and Stewart, 1985). The present results showed that both Oscillatoria sp. (1) and Anabaena variabilis had the power to allow NaCl to pass through cyanobacteria cells, while Nostoc linckia and Tolypothrix tenuis had another reaction, as the NaCl solution remains extracellular incapable of entering the cyanobacterial cells, this was confirmed by measuring the electrical conductivity of culture media (EC_m) at the beginning and after 15 days of the incubation period (Table 15 a and b, and Figs. 75, 77, 79 and 81). 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 mucopolysacch-aride sheath of cyanobacteria (Apte and Thomas, 1997).
The data also indicated that the patterns of chlorophyll a content and dry weight of the algae were been identical, i.e. embracing each other (in case of Oscillatoria sp. (1) and Anabaena variabilis), where as the both patterns maintained similar, to some extent, in the full range of salinities from 4-20 g L^-1 (in case of Nostoc linckia), or the patterns of chlorophyll a content and dry weight were not identical as in Tolypothrix tenuis. Similar results were obtained by Moisander et al., (2002). Moreover, application of NaCl caused significant reduction in chlorophyll a content as compared with the control (El-Naggar et al., 2004). On the other hand, an inverse relationship between the algal growth and NaCl concentration was observed by many investigators (Reynose and De-Gamboa, 1982; Rai and Tiwari, 1999; Masojidek et al., 2000; El-Sheekh and Omar, 2002 and El-Naggar et al., 2004).
The present study was conducted to investigate the potentiality of some isolated cyanobacteria in salt affected soils through studying the interactive effects of NaCl and algae on the growth and proline accumulation in seedlings of bean, durra and barley. The results show that the response of plants to salinity and algal treatment varied remarkably from one alga to another. It is seemed that, the interactive effects of NaCl and algae on the growth of plant seedlings were organism dependent. Although, the effects of algae on plant growth were variable, however the results show more or less significant increase in root or shoot length of the test crops. On the other hand, Anabaena variabilis and Tolypothrix tenuis induced significantly the germination percentage of barley seedlings.
Concerning the chlorophyll a content of seedlings, the results indicate significant increase in chlorophyll a content in durra seedlings after Anabaena variabilis, Nostoc linckia and Tolypothrix tenuis treatments. In all cases Oscillatoria sp. (1), Anabaena variabilis and Nostoc linckia reduced significantly the proline content in all test crops. While, Tolypothrix tenuis had no significant effect on investigated plants. It seemed that the relative growth of plants respond very differently to NaCl and algal treatments. Proline accumulation under stress is proposed to be a part of the mechanisms of osmotic adjustment (Ahmad et al., 1981; Roy et al.,1992; Misra et al., 1996, and Van Heerden and De Villier, 1996).
Generally, this study revealed that the members of cyanob-acteria 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 every year. Sodium chloride is the dominant salt in saline soil, although another salts considered. Improving the fertility of saline soils is not adequate as saline soils are not suitable for crop production. The use of selected cyanobacteria would open the way for magnifying the role could be played by algae on plant growth under saline condition. Generally, cyanobacteria would be served as cheapest sources of biotechnology in natural habitats. However, a successful biotechnology requires the selection of algae strain for proper plant and that was correlated by another factors rather than salinity status.