الفهرس 
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Abstract
It is well known that out of 87 million fed. arable area of Egypt, 2.6 million fed. are salt- affected. Most of these lands are lost annually to cultivation due to salinity. In view of vital role of some plant growth promoting rhizomicroorganisms (PGPR) which have positively influence plants vitality and the ability of the plants to cope with a biotic stress conditions such as salinity.
The aim of this study is to isolate and identified some salt-tolerant plant growth promoting rhizomicroorganisms (PGPR) from different salt-affected soils in Egypt to be used as a plant biostimulant under saline stress. As well, study the efficiency of those isolated PGPR in presence of soil conditioners (compost and humic acid) on tomato and pepper growth performance and productivity.
For this purpose, a series of laboratory and pot experiments were designed and carried out. Obtained results can be summarized as follows:
The first part
5.1. Isolation of PGPR strains
Two hundred and seven isolates of bacteria and actinobacteria were isolated from different salt-affected soils of Egypt namely Kafr El-Shikh (Sakha), El-Qalubia (Meet Kenana), El-Behira (El-Nubaria) and North Sinai (Sahl El-Tina) Governorates. 5.2. Screening of PGPR isolates
5.2.1. Primary screening of PGPR
Results indicated that 97% of total isolates were able to produce IAA in culture media. While, 3% of the isolates were negative for IAA production. Moreover, 11% of total isolates produced highly amounts of indole acetic acid. Therefore, these isolates were subjected again to secondary screening under saline stress. Regarding the inorganic phosphate solubilization by all investigated isolates, results indicated that 67.6 % of isolates were able to solubilize rock phosphate on PVK plates. Whereas, 32.4% were not able to solubilize phosphate on the same medium. Also, about 3.9% of the isolates gave the highest records of available phosphorus in the culture medium.
Concerning siderophores production by PGPR isolates, results indicated that 35.7% of isolates were able to grow on TSA medium and produce siderophores. Whereas, the other isolates showed negative results. In case of verifying the nature of the produced siderophores, 28.5% of the isolates were able to produce two types of siderophores (catecholate and citric acid), while 22.2% aren’t able to produce any siderophores types. Results also indicated that 40% of total isolates were isolated on nitrogen free media and fix atmospheric nitrogen, the highest ten isolates in N2-ase activity were chosen to secondary screening.
5.2.2. Secondary screening of PGPR
Results indicated that the isolates number 135 and 139 produced the highest amounts of IAA at different sodium chloride concentrations (1-5%). Also, the isolates number 23 and 159 able to solubilize inorganic insoluble phosphate at all tested sodium chloride concentrations. On the other hand, the nitrogenase activity of isolates number 178 and 207 were slightly affected under all tested sodium chloride concentrations. Therefore, the isolates number 23, 159, 135, 139, 178 and 207 were chosen and identified to be used in the further studies.
5.3. Identification of the more potent plant growth promoting rhizobacteria (PGPR) isolates
from the morphological characteristics, staining properties, spore formation and physiological properties, it was clear that the isolates could be identified as Paenibacillus alive D135, Bacillus pumilus D139, Bacillus megaterium D159, Pseudomonas fluorescence D23, Azospirillum lipoferumD178 and Azospirillum lipoferum D207.
5.4. Tolerance of identified PGPR strains for saline stress
In this experiment, NaCl concentrations (1-5%) and sea water at different dilutions (1:2-1:10) were used.
5.4.1. Nitrogenase activity (N2-ase) for PGPR under saline stress
It was obvious that two out of six identified PGPR strains (A. lipoferum D178 and A. lipoferum D207) were able to fix atmospheric nitrogen under saline stress. Obtained results clearly indicated that nitrogenase activity was higher under sodium chloride concentrations than those at different sea water dilutions. The highest N2-ase activity was observed by A. lipoferum D207 at 1% sodium chloride. On the other hand, the lowest nitrogenase activity was observed when A. lipoferum D178 was tested at (1:2) ratio of sea water.
5.4.2. Phosphate availability by the PGPR strains under saline stress
Results indicated that the six strains could solubilize phosphate in culture medium at all sodium chloride concentrations and sea water dilutions. Respecting the effect of saline stress on phosphate availability, results showed that B. megaterium D159 and Ps. fluorescence D23 were the highest in solubilizing phosphate. While, A. lipoferum D207 lost its ability to solubilize phosphate when sodium chloride was added to culture medium at a concentration of 5%.
5.4.3. Indole acetic acid production by PGPR strains under saline stress
Six strains produced IAA under all NaCl concentrations and sea water dilutions. But, IAA amounts decreased at 5% sodium chloride and (1:2) sea water dilution. Results emphasized that the highest produced amounts of IAA were observed by P. alive D135 and B. pumilus D139 when sea water was diluted at ratio of (1:10). On the other hand, the lowest amounts of IAA were produced by B. megaterium D159 and A. lipoferumD178 at 5% sodium chloride.
5.4.4. Gibberellins production by PGPR strains under saline stress
All investigated strains possess the ability to produce gibberellins under saline stress. But, gibberellins amounts were reduced at high NaCl concentrations and sea water dilutions. Paenibacillus alvie D135 was the highest producer of gibberellins under all sodium chloride and sea water concentrations followed by B. megaterium D159. Whereas, A. lipoferum D207 was the lowest producer of gibberellins.
5.4.5. Siderophores production by PGPR strains under saline stress
Respecting the effect of saline stress on siderophores production, results showed that B. megaterium D159 was the highest producer for catecholate-types at all applied sodium chloride concentrations followed by P. alive D135 then A. lipoferum D178. Results also showed that Ps. fluorescence D23 was the only investigated PGPR strain which was able to produce citric acid-types under all tested sodium chloride concentrations. Also, data revealed that B. pumilus D139 produced two types of siderophores at all applied sea water dilutions.
5.5. Colonization ability of PGPR strains for tomato and pepper roots
5.5.1. In vitro
Results revealed that when tomato and pepper cultivars were inoculated with the six identified PGPR strains, the highest colonization intensity in tomato cultivars root was observed with Super strain B when inoculated with Pseudomonas fluorescence D23, Paenibacillus alvie D135 and Azospirillum lipoferum D178. Whereas, the highest colonization intensity in pepper cultivars root was observed when Romy cultivar inoculated with Bacillus megaterium D159, Bacillus pumilus D139 and Azospirillum lipoferum D207.
5.5.2. Colonization experiment
5.5.2.1. Colonization intensity of tomato and pepper roots
Results revealed that after 15 and 30 days the colonization intensity with PGPR in tomato and pepper cultivated in washed sand only without any amendments not detected. While, at 30 days all soil treatments recorded higher colonization intensity than the control. The highest colonization intensity was recorded in tomato and pepper plants cultivated in soil amended with biostimulant combined with compost and humic acid.
5.5.2.2. Dehydrogenase, phosphatase and nitrogenase activity in soil
Results indicated that no activity was observed in control treatment with phosphatase and nitrogenase. The highest records of the determined enzymes were observed in case of soil inoculated with biostimulant combined with compost in presence of humic acid. This trend of results was observed with tomato and pepper.
5.5.2.3. Biometric observations of tomato and pepper
The lowest records of the growth characteristics i.e. leaves number, shoot size and dry weights of shoot and root were observed in sandy soil irrigated with sea water at ratio of 1:2, this evidence was observed in both tomato and pepper. On the other hand, the highest records of tomato and pepper plants growth characteristics were observed when cultivated in salinized sandy soil and inoculated with biostimulant in combination with compost and humic acid.
The second part
Growth performance and yield of tomato affected by biostimulant, humic acid and organic manure application under saline stress
This experiment was conducted in greenhouse conditions using a randomized complete block design (RCBD) with three replicates in the experimental farm station of Fac. Agric. at Moshtohor during 2011 season, in order to study the efficacy of biostimulant strains (Pseudomonas fluorescence D23, Bacillus pumilus D139 and Azospirillum lipoferum D178), humic acid and organic manure (compost) application on growth and yield of tomato.
5.6. Dehydrogenase activity (DHA)
Results indicated that rhizosphere of tomato cultivated in moderately saline soil with no amendments (control) gave the lowest dehydrogenase activity at all determination periods. Results also revealed that the highest significant increase of DHA was observed in case of tomato inoculated with biostimulant combined with humic acid and compost at 1½ dose.
5.7. Phosphatase activity
The obtained results revealed that no significant difference was observed between phosphatase activity in soil treated with biostimulant only and soil treated with chemical fertilizers at 30 days. Soil inoculated with biostimulant combined with humic acid in presence of compost at one and half dose gave the highest significant values of phosphatase activity at initial, 60 and 120 day.
5.8. Nitrogenase activity (N2-ase)
Higher records of N2-ase activity were observed in soil treated with compost than biostimulant each one singularly at 15 and 60 days. While, at 30 days no significant differences were observed between compost and biostimulant treatments. Also, rhizosphere of tomato which inoculated with salt-tolerant PGPR recorded higher values of N2-ase activity than rhizosphere of plants treated with chemical fertilization, but still less those treated with combination of PGPR and compost or humic acid.
5.9. Total, available nitrogen and phosphorus in tomato’s rhizosphere
Obtained results revealed that the lowest records of total and available nitrogen and phosphorus were observed in moderately saline soil without any amendments (control). Soil inoculated with biostimulant in presence of humic acid gave significant higher values of total nitrogen and available nitrogen and phosphorus than soil inoculated with biostimulant only. While, no significant differences in total phosphorus values were observed in these treatments.
5.10. Tomato growth characteristics
Dual treatment of tomato with biostimulant strains and compost gave higher records of root size than plants cultivated in soil treated with either biostimulant or compost only. The highest significant number of leaves was observed in case of tomato inoculation with biostimulant combined with compost at one and half dose in presence of humic acid.
5.11. Nitrogen, phosphorus and potassium uptake in tomato (N, P and K)
Nitrogen, phosphorus and potassium uptake in tomato shoots were positively affected by all investigated treatments compared to control. Results showed that the lowest values of nitrogen and phosphorus uptake were observed in moderately saline soil without any amendments (control). While, the highest records of nitrogen uptake by tomato shoots were observed when soil was amended with biostimulant combined with compost at 1½ dose in presence of humic acid.
5.12. Proline accumulation and oxidative enzymes in tomato leaves
Tomato inoculated with biostimulant combined with compost at one and half dose in presence of humic acid gave the lowest amounts of accumulated proline. Whereas, moderately saline soil without any amendments (control) gave higher amounts of proline in tomato plants.
Plant oxidative enzymes (nitrate reductase, peroxidase and polyphenol oxidase) were affected with soil amendments. Results clearly indicated that tomato inoculation with biostimulant in combination with compost at different doses in presence or absence of humic acid significantly increased the activities of nitrate reductase and peroxidase compared to either biostimulant or compost each one individually.
5.13. Photosynthetic pigments
The lowest values of chlorophyll A, B, total chlorophyll and carotenoids were observed in tomato cultivated in free amendments moderately saline soil. Results also indicated that no significant differences were found between chlorophyll A in tomato inoculated with biostimulant combined with compost at different doses. On contrast, soil amended with biostimulant combined with humic acid gave higher records than soil treated with compost at different doses in combination with biostimulant.
5.14. Phenols, sugars and amino acids in tomato leaves
The lowest values of total and free phenols were observed in control treatment. On the other hand, the highest values of total and conjugated phenols were observed when tomato inoculated with biostimulant in presence of compost at 1½ dose combined with humic acid. Tomato inoculated with biostimulant combined with compost at one and half dose in presence of humic acid gave the highest significant records of total sugars compared with tomato inoculated with biostimulant or compost each one solely. While, the lowest values of total and reduced sugars were observed in tomato cultivated in free amendments moderately saline soil (control). In addition, dual amendments of tomato with biostimulant combined with compost at different doses gave higher values of total amino acids than tomato inoculated with biostimulant or compost each one solely.
5.15. Titratable acidity, vitamin C and total soluble solids in tomato fruits
Tomato inoculated with biostimulant combined with compost at one and half dose in presence of humic acid gave the highest records of titratable acidity. Results also showed that the highest and the lowest records of vitamin C were observed in tomato inoculated with biostimulant combined with compost at one and half dose in presence of humic acid and free amendments moderately saline soil, respectively. On the other hand, tomato inoculated with biostimulant combined with compost at 1½ dose in presence of humic acid gave the highest records of T.S.S.
5.16. Yield and yield components of tomato
The lowest number of fruits/plant, weight of tomato fruits and plant yield were observed in tomato grown in moderately saline soil without any amendments (control). While, the highest weight of one tomato fruit and yield of tomato fruits were observed when tomato inoculated with biostimulant in combination with compost at one and half dose in presence of humic acid.
Growth performance and yield of pepper affected by biostimulant, humic acid and organic manure application under saline stress
This experiment was conducted in greenhouse conditions using a randomized complete block design (RCBD) with four replicates in the experimental farm station of Fac. Agric. at Moshtohor during 2011 season, in order to study the efficacy of biostimulant strains (Bacillus megaterium D159, Paenibacillus alvie D135 and Azospirillum lipoferum D207), humic acid and organic manure (compost) on growth and yield of pepper.
5.17. Dehydrogenase activity (DHA)
Results indicated that the lowest DHA values at initial and 30 days were observed in moderately saline soil without any amendments. On the other hand, dehydrogenase activity was gradually increased through the first 60 days and thereafter were decreased. Results also showed that at 60 and 120 days pepper inoculated with biostimulant combined with compost at different doses in presence of humic acid significantly increased the DHA values than other treatments.
5.18. Phosphatase activity
The moderately saline soil without any amendments (control) recorded the lowest significant values of phosphatase activity at 30, 60 and 120 days. Whereas, the highest significant values of phosphatase activity at 60 and 120 days were observed in soil inoculated with biostimulant strains combined with compost at 1½ dose and HA. Except of the control treatment, chemical fertilization of pepper gave lower values of phosphatase activity at all determination periods except at 15 days rather than various soil treatments.
5.19. Nitrogenase activity (N2-ase)
Results emphasized that lower nitrogenase activity was recorded in moderately saline soil without any amendments through the first 60 days. While, higher significant records of N2-ase activity were observed in soil inoculated with biostimulant than soil treated with compost only, this result was observed at 15 and 60.
5.20. Total, available nitrogen and phosphorus in pepper’s rhizosphere
Moderately saline soil inoculated with biostimulant strains combined with compost at one and half dose showed higher available phosphorus comparing with chemical fertilization. On contrast, the free amendments moderately saline soil showed the lowest values of available amounts of nitrogen and phosphorus.
5.21. Pepper growth characteristics
The lowest records of all determined growth characteristics i.e. leaves, branches and flowers number, length and dry weight of shoot and dry weight and size of root were observed in free amendments moderately saline soil. The addition of biostimulant combined with compost and HA to the moderately saline soil cultivated with pepper gave higher records of growth characteristics than biostimulant only.
5.22. Nitrogen, phosphorus and potassium uptake in pepper (N, P and K)
Results indicated that the highest significant records of N and K uptake were observed in soil inoculated with biostimulant combined with compost at one and half dose in presence of humic acid. Whereas, the highest uptake of P was observed in application of compost at full dose. On contrast, the lowest records of macronutrients uptake were observed in moderately saline soil without any amendments (control).
5.23. Proline accumulation and oxidative enzymes in pepper leaves
The obtained results emphasized that the highest amounts of proline were observed in pepper leaves grown in moderately saline soil without any amendments (control). While, the application of biostimulant in combination with compost at different doses and/or humic acid significantly decreased the amounts of accumulated proline in pepper leaves comparing with control. Results also indicated that the highest significant activities of nitrate reductase and peroxidase were observed when pepper inoculated with biostimulant combined with compost at 1½ dose with humic acid. On contrast, no significant differences were observed in polyphenol oxidase activity between all treatments except chemical fertilization and control treatments.
5.24. Photosynthetic pigments in pepper leaves
Results indicated that the highest values of chlorophyll A were observed in pepper grown in soil amended with biostimulant in combination with compost at one and half dose in presence of humic acid. Also, when pepper cultivated in free amendments moderately saline soil (control) the highest records of carotenoids and the lowest values of chlorophyll B were observed.
5.25. Phenols, sugars and amino acids in pepper leaves
Results indicated that the lowest values of total and free phenols were observed in soil treated with chemical fertilization. In addition, the dual inoculation of pepper with biostimulant and humic acid increased the total, free and conjugated phenols than the dual inoculation with biostimulant and compost at different doses. The highest significant amounts of total and non-reduced sugars were observed in soil amended with biostimulant, humic acid and compost at 1½ dose. Whereas, the lowest values of total and reduced sugars were observed in soil without any amendments. In addition, the highest records of total amino acids were observed in soil amended with biostimulant in combination with compost at 1½ dose in presence of humic acid.
5.26. Titratable acidity, vitamin C and total soluble solids in pepper fruits
The obtained results indicated that moderately saline soil without any amendments gave the lowest values of titratable acidity, vitamin C and total soluble solids of pepper fruits. On contrast, the highest records of vitamin C and T.S.S. were observed in pepper inoculated with biostimulant combined with humic acid in presence of compost at full dose.
5.27. Yield and yield components
The highest fruit yield of pepper was observed in soil amended with biostimulant combined with compost at one and half dose in presence of humic acid. On contrast, results revealed that the control treatment gave the lowest number of fruits per plant, weight of one fruit and fruit yield/plant.
CONCLUSION AND RECOMENDATION
In view of the obtained results which showed that the used soil in the current study characterized with high salts concentration, since the vegetable crops used in this study couldn’t grow well under these conditions without any soil conditioners addition.
Therefore, the Agric. gypsum was added at a rate of ½ ton/fed.; compost at a rate of 10 ton/fed. and humic acid at a rate of 4 kg/fed. Also, the course sand was added at a rate of 20 ton/fed. in order to soil texture improvement.
All these pre-treatments mentioned above lead to improvement of physical and chemical soil properties for increasing the used PGPR efficiency in improvement tomato and pepper growth and productivity under similar conditions.