الفهرس 
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Abstract
The use of biofertilizers in medicinal plants production, particularly in Egypt, becomes unavoidable to minimize the nonstop addition of high doses of chemical fertilizers in which enormous amounts of deleterious heavy metal and other environmental pollutions might be present, as well as to lower their production costs. On the other hand, the application of some biofertilizers as biological control agents is considered to be of extreme importance due to their positive dual effect on plant growth. Therefore, the present work has been carried out to investigate the variations in soil, rhizoplane and rhizosphere microflora under the cultivation of medicinal plants and the effect of the root exudates of such plants on different isolates of N2-fixing bacteria. The influence of using locally isolated nitrogen fixing bacteria ( biofertilizers ) as biocontrol agents were also investigated, as well as the response of medicinal plants to biofertilizers.
5-1 The plan of the work consisted of six main topics:
5-1-1 Soil, rhizosphere and rhizoplane microflora as affected by the cultivation of some medicinal plants.
5-1-2 Effect of some medicinal plants root exudates on some isolates of N2-fixing bacteria.
5-1-3 Interaction between some medicinal plants and effectiveness of nitrogen fixing bacteria ( biofertilizers ).
5-1-4 Effect of N2-fixing bacteria on some phytopathogenic fungi in vitro.
5-1-5 Application of N2-fixing bacteria ( biofertilizer ) as natural agents for controlling soil born plant pathogens ( in vivo ).
5-1-6 Effect of mineral nitrogen and biofertilizers as well as combination between them ( 1:1 ),on growth plant characteristics of tested medicinal plants.
5-1-1 Soil, rhizosphere and rhizoplane microflora as affected by the cultivation of some medicinal plants:-
A pot experiment was carried out to correlate the effect of root exudates of the medicinal plants under investigation, toward different groups of microflora in rhizosphere and edaphosphere. This experiment was conducted in clay loam and sandy soil.
The following results were obtained:-
The rhizosphere of medicinal plants under investigation showed various effects toward the different groups of microorganisms under investigation. In some plants, the microbial content of the rhizosphere was more than that of non-rhizospheric soil giving positive rhizosphere effect, while the total counts of fungi in rhizosphere were less than those of non-rhizospheric soil giving negative rhizosphere effect. On the other hand, Nigella sativa, Trigonella foenum- graecum and Petroselinum sativum rhizosphere showed a more positive rhizosphere effect on Azotobacter and Azospirillum than those of non-rhizosphere soil.
Moreover, in this part, one hundred and twenty representative isolates of either Azotobacter and Azospirillum ( Sixty from Azotobacter and the other from Azospirillum ) were isolated from the rhizosphere and rhizoplane of black-cumin, fenugreek and parsley plants grown in clay loam or sandy soil. These isolates were tested toward their efficiency for nitrogen fixation by estimating the nitrogenase activity using the acetylene reduction technique. The obtained results could be summarized as follows: Six isolates of Azotobacter and six isolates of Azospirillum were screened for their activity in N2-fixation.
5-1-2 Effect of some medicinal plants root exudates on some isolates of N2-fixing bacteria:-
This experiment aims at determining the biological effect of the tested medicinal plants root exudates ( Nigella, Trigonella and Petroselinum ) on some isolates of Azotobacter and Azospirillum which were chosen according to their highly active on the N2-fixing.
Seeds of the tested medicinal plants were grown aseptically in large tubes containing glass beads ( 0.5 cm in diameter ) and 30 ml of distilled water to obtain root exudates. Seedling exudates were aseptically collected after one, two and three weeks from planting. These exudates were studied for their effects on twelve active strains of either Azotobacter and Azospirillum by using filter paper disks.
The following results were obtained:-
5.1.2.1 Effect of medicinal plants root exudates on active isolates of Azotobacter:-
The most stimulatory effect on Azotobacter was given by Nigella and Trigonella root exudates, respectively on Azotobacter isolate
( Az.Np7 ). The stimulative effect of Nigella root exudates was more pronounced than that of Trigonella. However, Trigonella and Petroselinum stimulated the growth of Azotobacter isolate ( Az.PR1 ). On the other hand, the most inhibitory effect against Azotobacter was given by parsley root exudates in all ages and dilutions.
5.1.2.2 Effect of medicinal plants root exudates on active isolates of Azospirillum:-
The most stimulatory effect on Azospirillum was given by Trigonella root exudates. Moreover, Nigella, Trigonella and Petroselinum root exudates stimulated the growth of the tested Azospirillum isolate ( As.Pp1 ). The stimulative effect of Petroselinum root exudates on Azospirillum isolate ( As.Pp1 ) was more pronounced than that of Nigella and Trigonella root exudates in all ages and dilutions.
5-1-3 Interaction between some medicinal plants and effectiveness of nitrogen fixing bacteria ( biofertilizers ):-
A pot experiment was carried out to correlate the effect of cultivation of tested medicinal plants inoculation with isolates of Azotobacter and Azospirillum on the growth of Azotobacter and Azospirillum counts ( or/ the ability of biofertilizer on withstand environmental condition ), and were also tested for their activity in N2-fixing and effect on plant growth under environmental conditions. This experiment was conducted in clay loam and sandy soil.
The following results were obtained:-
5.1.3.1 Effect of cultivation of tested medicinal plants inoculated with isolates of Azotobacter and Azospirillum on the growth of Azotobacter and Azospirillum counts:-
5.1.3.1.1 Rhizosphere of Nigella sativa stimulated growth of Azotobacter counts, while the stimulative effect of Nigella was more pronounced in case of inoculation with Azotobacter isolate
( Az.Np7 ).
5.1.3.1.2 The numbers of Azotobacter were sharply higher in the rhizosphere of Trigonella and Petroselinum plants inoculated with Azotobacter isolate ( Az.PR1 ).
5.1.3.1.3 The numbers of Azospirillum were sharply higher in rhizosphere of studied medicinal plants in case of inoculation with Azospirillum isolate ( As.Pp1 ).
5.1.3.2 Effect of inoculation with free nitrogen fixers on medicinal plants growth:-
5.1.3.2.1 Inoculation with Azotobacter isolates generally increased the amount of dry weight, N % and uptake of tested medicinal plants. But, the increase in these parameters was more pronounced in case of Nigella sativa plants inoculation with Azotobacter isolate ( Az.NP7 ). On the other hand, the increase in these parameters was more pronounced in Trigonella and Petroselinum plants inoculation with Azotobacter isolate
( Az.PR1 ).
5.1.3.2.2 Inoculation with Azospirillum isolates generally increased the amount of dry weight, N % and uptake of tested medicinal plants. The increase in these parameters was more pronounced at used Azospirillum isolate ( As.Pp1 ) in inoculation of the tested medicinal plants as compared with the other isolates.
5-1-4 Effect of N2-fixing bacteria on some phytopathogenic fungi in vitro:-
N2-fixing isolates caused different antagonistic effects against Fusarium solani and Aspergillus niger in vitro as indicated by the development of inhibition zones of different diameters.
The obtained results could be summarized as follows: Azotobacter isolates ( Az.NP7 and Az.PR1 ) inhibited the growth of tested fungi. On the other hand, strains of Azospirillum inhibited the growth of tested fungi, but the inhibitory effect of Azospirillum strain ( As.Pp1 ) was more pronounced than that of other strains.
The most active antagonistic, N2-fixing isolates and ability on withstand environmental conditions such as root exudates were selected. The following results were obtained:-Two isolates of Azotobacter out of 60 isolates, these isolates were ( Az.NP7- Az.PR1 ), and one isolate of Azospirillum out of 60 isolates, this isolate was ( As.Pp1 ).
5-1-5 Application of N2-fixing bacteria ( biofertilizer ) as natural agents for controlling soil born plant pathogens ( in vivo ).
A greenhouse experiment was conducted to evaluate the role of N-fixing bacteria as a natural agent for controlling soil born plant pathogens.
The following results were obtained:-
5.1.5.1 Seed inoculation of Nigella sativa with Azotobacter strain
( Az.NP7 ) and seed inoculation of Trigonella and Petroselinum with Azotobacter strain ( Az.PR1 ) increased emergence. Disease severity was reduced by these isolates.
5.1.5.2 Seed inoculation of Nigella, Trigonella and Petroselinum with Azospirillum isolate ( As.Pp1 ) increased emergence. Disease severity was reduced by this isolate.
5-1-6 Effect of mineral and biofertilizers as well as combination between them ( 1:1 ), on growth plant characteristic of tested medicinal plants:-
A field experiment was designed to study the effect of inoculation with N2-fixing bacteria ( biofertilizer ) on growth of plant parameters of tested medicinal plants. Inoculation was conjugated with the application of four doses of mineral N, i.e. 0, 50%, 75% and one full dose ( 100% ) on growth plant characteristics after 90 days of seed cultivation was also studied .
The following results were obtained:-
5.1.6.1 Inoculation with N2-fixer in the presence of a half dose of chemical N-fertilizer increased plant height, fresh and dry weight, N % and uptake as well as number of capsules/plant and yield of Nigella sativa plant.
5.1.6.2 Inoculation with N2-fixer without mineral N fertilizer enhanced plant height, fresh and dry weight, N % and uptake as well as number of pods/plant and seed yield of Trigonella foenum plant.
5.1.6.3 Inoculation with N2-fixer in the presence of a full dose of chemical N-fertilizer increased vegetative growth parameters, i.e. plant height, fresh and dry weight as well as N % and uptake of Petroselinum sativum plant.
As a general recommendation the application of biofertilizers as biocontrol agents are considered to be of extreme effect on medicinal plants growth and plant protection.