Scholarly article on topic 'Response of Prosopis chilensis to biofertilization under calcareous soil of RasSudr 1-Vegetative growth'

Response of Prosopis chilensis to biofertilization under calcareous soil of RasSudr 1-Vegetative growth Academic research paper on "Agriculture, forestry, and fisheries"

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Abstract of research paper on Agriculture, forestry, and fisheries, author of scientific article — Fatma M.K. Faramawy

Abstract A field experiment was carried out during two successive seasons, 2006 and 2007 on Prosopis chilensis at six month old transplants. Experiment was conducted in RasSudr Research Station, Desert Research Center, at South Sinai Governorate, Egypt. Aiming to study the effect of adding biofertilizers Bradyrhizobium spp., Azotobacter chroococcum, Bacillus megaterium and vesicular arbuscular mycorrhizae (VAM) as well as their interaction, viz. control (without microbial inoculation) on soil microbials counts, plant growth parameters, total chlorophyll and some chemical contents of leaves and branches. Results revealed that different biofertilizer treatments increased the microbial counts (total microbial counts, azotobacter, azospirilla, phosphate dissolving bacteria (PDB) and VAM), the growth parameters (plant height, stem diameter, initiative branching point, number of branches, fresh weight and dry weight) and some chemical constituents such as total chlorophyll, crude protein, crude fiber and ash% compared to untreated plants. Concerning to inoculate plants with biofertilizers interaction treatments, a mixture of bradyrhizobium, azotobacter, PDB and VAM was the most effective in raising the productivity of prosopis plants followed by triple inoculated treatments, then double inoculation treatments and finally single inoculation treatments compared to control treatment.

Academic research paper on topic "Response of Prosopis chilensis to biofertilization under calcareous soil of RasSudr 1-Vegetative growth"

Annals of Agricultural Science (2014) 59(2), 253-262

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ELSEVIER

Faculty of Agriculture, Ain Shams University Annals of Agricultural Science

www.elsevier.com/locate/aoas

Response of Prosopis chilensis to biofertilization qma

under calcareous soil of RasSudr 1-Vegetative

growth

Fatma M.K. Faramawy *

Soil Fertility and Microbiology Dept., Desert Research Center, El Matarey, Cairo, Egypt

Received 18 August 2014; accepted 10 September 2014 Available online 12 December 2014

KEYWORDS

Prosopis chilensis;

Bio-fertilizers;

N-fixers;

Chemical constituents

Abstract A field experiment was carried out during two successive seasons, 2006 and 2007 on Prosopis chilensis at six month old transplants. Experiment was conducted in RasSudr Research Station, Desert Research Center, at South Sinai Governorate, Egypt. Aiming to study the effect of adding biofertilizers Bradyrhizobium spp., Azotobacter chroococcum, Bacillus megaterium and vesicular arbuscular mycorrhizae (VAM) as well as their interaction, viz. control (without microbial inoculation) on soil microbials counts, plant growth parameters, total chlorophyll and some chemical contents of leaves and branches. Results revealed that different biofertilizer treatments increased the microbial counts (total microbial counts, azotobacter, azospirilla, phosphate dissolving bacteria (PDB) and VAM), the growth parameters (plant height, stem diameter, initiative branching point, number of branches, fresh weight and dry weight) and some chemical constituents such as total chlorophyll, crude protein, crude fiber and ash% compared to untreated plants. Concerning to inoculate plants with biofertilizers interaction treatments, a mixture of bradyrhizobium, azotobacter, PDB and VAM was the most effective in raising the productivity of prosopis plants followed by triple inoculated treatments, then double inoculation treatments and finally single inoculation treatments compared to control treatment.

© 2014 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams

University. Open access under CC BY-NC-ND license.

Introduction

The Chilean mesquite (Prosopis chilensis (Molina) Stuntz) is a small to medium-sized legume tree belongs to Fabaceae up to 12 m in height and 1 m in diameter. It has a shallow and

* Tel.: +20 01121447985. E-mail address: f.faramawy62@yahoo.com. Peer review under responsibility of Faculty of Agriculture, Ain-Shams University.

spreading root system. It branches freely and its wood is hard and reddish, with brown and fissured bark. Its leaves are

4-7.5 cm long, compound, each with numerous leaflets along several pairs of pinnae. The flowers are greenish-white to yellow, about 5 mm long, abundant and occur in spike-like

5-10 cm long racemes. The pods are slender, slightly curved or straight, flat at maturity, 10-20 cm long, yellow when ripe, borne in drooping clusters. Seeds are bean-shaped, oblong,

6-7 mm, light brown, each in 4-angled case (Orwa et al., 2009). Prosopis chilensis flowers regularly in spring and

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sometimes sporadically again in late summer (Orwa et al., 2009).

Prosopis chilensis wood gives good charcoal, fiber, and a relatively dense timber valued for furniture and floors. The ground pods are eaten by native people in Northern Argentina. The leaves are not very palatable to animals but the sugary pods are eaten by livestock and the seeds are sometimes ground in a feed concentrate and have been observed to grow in seawater salinity (Gohl, 1982; Orwa et al., 2009). Native to Central America, Prosopis chilensis is common in Peru, Chile, Argentina and Uruguay. It is naturalized in many African countries as well as in the USA. Prosopis chilensis is found in the arid and semi-arid regions with ground water of between 3 and 10 m below the surface, such as drainage channels along ground water sinks. It grows between mean annual temperatures of 12-45 0C, under average annual rainfall of 350400 mm (Orwa et al., 2009). It is found in sandy, alkaline soils.

The main feed product provided by Prosopis chilensis is its pods. The pods are rather poor in protein (9-13% DM) and rich in fiber (crude fiber) (20-26% DM). (Feedipedia, 2011; Tran, 2013). Like other Prosopis pods, the ripe pods contain much sugar and can be a valuable source of energy (Gohl, 1982). The seeds are particularly rich in protein (32.5%). Reported values for leaves are rather high, with protein content ranging from 14.8 (Gabar, 1988) to 18.3% DM (for fresh loppings in India, Khirwar et al., 2003) and 22.5% DM.

This study aims to evaluate the effect of inoculation with biofertilizers on the growth of Prosopis chilensis and overcoming some stress conditions such as calcareous soil and saline irrigated water.

Material and methods

Field experiment was carried out on Prosopis chilensis at six month old transplants. Experiment was conducted in RasSudr Research Station, Desert Research Center, at South Sinai Governorate, Egypt, during two successive years (2006 and 2007). The soil of the location was highly calcareous. Analysis of the experimental soil and irrigation water was carried out at the Central Lab. at Desert Research Center (Tables 1 and 2).

1. Microorganisms used.

Active strains of Azotobacter chroococcum (as nitrogen fixing bacteria), Bacillus megaterium (as phosphate dissolving

bacteria), Bradyrhizobium japonicum (as symbiotic nitrogen fixer bacteria) and vesicular arbuscular mycorrhizal fungi (as phosphate solubilizer symbiont) having the capability to withstand the stressed desert conditions were provided by Unit of Soil Microbiology, Desert Research Center, Cairo.

1.1. Inoculum preparation.

Heavy cell suspension of A. chroococcum or B. megaterium or Bradyrhizobium spp. cells containing about 108 cells mP1 and VAM spore suspension (50 spore mP1) was used as standard inocula.

Mixtures of each strain were prepared just before inoculation by adding equal portion of the culture of each strain to inoculate Prosopis chilensis seedlings.

1.2. Procedure of inoculation.

Seedlings of P. chilensis were successively washed and immersed for 30 min in heavy cell suspensions of Azotobacter chroococcum or Bacillus megaterium or bradyrhizobium japonicum (108 cells mP1) or VAM spores (50 spore mP1) and a mixture of the culture of strain at the ratio of 1:1 carboxy-methyl cellulose solution 0.5% was used as an adhesive agent. Seedlings of control treatments were treated in the same manner but using N - deficient medium instead of bacterial culture. The inoculated seedlings were air dried at room temperature for 2 h before planting.

2. The experimental design was split plot in four replicates. The experimental treatments under the investigation were as follows:

a. control

b. B. japonicum

c. A. chroococcum

d. B. megaterium

e. VA mycorrhizae

f. B. japonicum + A. chroococcum

g. B. japonicum + B. megaterium

h. B. japonicum + VA

i. A. chroococcum + B. megaterium j. A. chroococcum + VA

k. VA + B. megaterium

l. B. japonicum + A. chroococcum + B. megaterium m. B. japonicum + A. chroococcum + VA n. B. japonicum + B. megaterium + VA o. A. chroococcum + B. megaterium + VA

Table 1 Soil analysis of the experimental soil. Depth (cm) Coarse sand% Fine sand% (1-0.5) (mm) (0.25-0.1) (mm) Total sand% Silt% (0.05 -0.002) (mm) Clay% (mm) <(0.002) Class texture

Mechanical properties 0-30 54.51 30-60 25.49 25.88 61.12 80.39 86.61 8.46 7.14 11.15 6.25 Sandy loam Sandy loam

Depth (cm) pH EC (ds/m2) CaCO3 O.M.% Saturation soluble extract

Soluble : anions (meq/L) Soluble cation (meq/L)

C03 HC03 S03 CI3 Ca+ + Mg+ + Na+ K+

Chemical properties 0-30 7.7 30-60 7.40 4.77 4.16 55.85 51.21 0.60 0.46 0.00 0.00 6.00 10.50 3.00 16.10 31.20 22.50 24.00 16.83 11.00 6.00 10.52 2.18 17.80 1.10

Table 2 Chemical analysis of irrigated water.

Well salinity (ppm) pH E C ds/m2 Soluble anions (meq/L) Soluble cation (meq/L)

CO3 HCO3 SO4 CI4 Ca++ Mg+ + Na + K +

45,000 8.60 9.23 0.00 2.50 16.22 81.28 23.65 19.18 57.17 0.51

p. B.japonicum + A. chroococcum + B.megaterium + VA

Densities of plants were 12 plants per each experimental unit (6 x 12 m).

The experimental site was irrigated just after transplanting by saline water pumped from a well of 45,000 ppm salinity, then irrigated each 7 days until the end of the experiment.

The experiment received 20 m3/feddan organic farm manure during soil preparation before transplanting, 150 kg calcium super phosphate/feddan (15.5% P205), 100 kg/fed of potassium sulfate (48% k2O) and ammonium sulfate (20.5% N), 60 kg/fed.

Four plants were randomly cut at the end of each season and the last four plants were left till pod production to evaluate the following characteristics:

3. Microbial counts

3.1. Rhizosphere soil samples were collected at harvesting stage of prosopis plants and analyzed for total microbial count according to Bunt and Rovira (1965). For counting and growing phosphate dissolving bacteria the same medium was used after adding 5 ml of 10% K2HPO4 as a sterile solution followed by adding 10 ml of sterile solution of 10% CaCl2 to each 100 ml of the medium (Abd-El Hafez, 1966), Azotobacter nitrogen deficient medium (Abd-El Malek and Isac, 1968); Azospirilla on Dobereiner's medium (Dobereiner, 1978).

3.2. Extraction of VA mycorrhizal spores

3.2.1. Spores were collected from rhizosphere and soil samples by wet sieving and decanting technique (Gerdemann and Nicolson, 1963).

3.2.2. Estimation of VA mycorrhizal colonization by using the method described by Trouvelot et al. (1986).

4. Growth characters

4.1. The studied growth parameters included (number of branches/plant, fresh and dry weight g/plant).

4.2. Estimation of total photosynthetic pigments' content by using Minolta chlorophyll meter (SPAD-502).

4.3. Ash content, according to method described by A.O.A.C. (1990).

4.4. Crude fiber (CF) content, according to method described by A.O.A.C. (1990).

4.5. Crude protein% (CP), total nitrogen was determined by modified microkjeldahl method described by Jackson (1958) according to Peach and Tracey (1956) and multiplied by 6.25.

4.6. Digestive protein content, by using the following formula: DP% = 0.9596 CP% - 3.55 as determined by Bredon et al. (1963).

4.7. Total digestive nutrients (TDN), estimated by using the equation TDN% = 74.43 + 0.35 CP% - 0.73 CF% according to Adams et al. (1964).

4.8. Total carbohydrate%, according to method described by A.O.A.C. (1990).

Results and discussion

Data in Table 3 clearly show that there are high variations of total counts and PDB counts between all treatments in prosopis rhizosphere in both two successive seasons. In case of monoinoculations azotobacter treatment shows the best results being 15.8 and 21.4 cfu x 107g-1 dry soil in case of total counts, while PDB treatments gave the highest densities of PDB (35.0 and 50.5 dry cfu x 103 g-1 soil), during the two successive seasons. In dual inoculations azotobacter + VAM show the best results followed by bradyrhizobium + azotobacter while bradyrhizobium + VAM treatment gave the lowest results in case of total count, but in case of PDB counts the highest results were recorded in azotobacter + PDB (65.5 and 78.2 dry cfu x 103g-1 soil) while all inoculants with VAM gave lower PDB counts.

The best results of total microbial densities (80.0 and 88.6 cfu x 107g-1 soil) and PDB counts (81.2 and 88.0 cfu x 103 g-1 soil) were obtained in quarto inoculation (Brady + Azoto + PDB + VAM) in the two successive seasons.

Data presented in Table 4 show the densities of azotobacter and azospirilla originated from the rhizosphere of prosopis plants. The counts of azotobacter ranged from 20.4 to 170.0 x 104g-1 dry soil (for the first season) and from 22.0 to 177.0 x 104 g-1 dry soil (for the second season). Adding azo-tobacter led to large increase in azotobacter numbers in both seasons. In case of azospirilla counts, all biofertilizers additions increased azospirilla counts compared with control. Quarto application gave the best results (41.50 and 42.2 x 104g-1 dry soil) in both seasons. These followed by triple application, which show little increase in counts compared with double inoculation. Finally monoinoculations gave the lowest azospi-rilla numbers in inoculated treatments.

The root colonization of prosopis plants and number of spores/100 g soil in the rhizosphere soil were affected by microbial inoculation (Figs. 1 and 2). The percent of root colonization was higher in the Prosopis chilensis inoculated with VAM (58.5 and 62.0 in the two seasons) compared to non-inoculated plants (6.0 and 7.5). However, adding other biofertilizers ( bradyrhizobium, azotobacter and PDB) increased coloniza-tion% and number of spores. It is noticed that adding azotobacter to VAM in all mixed treatments gave higher colonization% in prosopis roots and spores production in

Table 3 Densities of total microbial counts and phosphate dissolving bacteria in rhizosphere of Prosopis chilensis as influenced by

different biofertilizers in two successive seasons.

Treatments Total microbial count cfu • 107g" 1 dry soil Densities of PDB cfu x 103 g"1 dry soil

First season Second season First season Second season

Control 2.4 5.2 11.0 12.4

Bradyrhizobium 13.4 18.6 12.8 13.7

Azotobacter 15.8 21.4 18.5 20.2

PDB 12.5 17.4 35.0 50.5

VAM 13.6 19.1 12.5 14.4

Brady + Azotobact 31.3 35.9 22.1 12.9

Brady + PDB 28.7 32.8 48.8 59.3

Brady + VAM 30.9 37.1 20.2 22.1

Azotobact + PDB 30.8 35.7 65.5 78.2

Azotobact + VAM 32.2 40.0 26.8 33.0

PDB + VAM 29.6 33.5 42.3 51.6

Brady + Azoto + PDB 72.2 80.3 70.4 84.9

Brady + Azoto + VAM 77.8 84.3 26.7 27.6

Brady + PDB + VAM 72.0 77.6 77.2 85.7

Azoto + PDB + VAM 70.3 75.4 73.0 87.6

Brady + Azoto + PDB + VAM 80.0 88.6 81.2 88.0

L.S.D. at 0.05 3.18 2.79 1.55 1.12

Table 4 Densities of N2 fixers densities seasons. in Prosopis chilensis rhizosphere as influenced by different biofertilizers in two successive

Treatments Azotobacters x 104 g 1 dry soil Azospirilla x 104 g 1 dry soil

First season Second season First season Second season

Control 20.4 22.0 18.2 22.0

Bradyrhizobium 31.2 33.8 22.5 23.0

Azotobacter 110.0 122.0 28.4 28.8

PDB 27.5 30.7 21.0 24.2

VAM 33.3 38.6 21.8 25.6

Brady + Azotobact 120.0 144.0 30.1 33.1

Brady + PDB 41.7 43.0 33.0 34.4

Brady + VAM 44.0 47.7 30.8 32.0

Azotobact + PDB 111.0 129.0 32.4 33.8

Azotobact + VAM 118.0 136.0 33.7 34.5

PDB + VAM 32.0 33.8 32.1 32.8

Brady + Azoto + PDB 141.0 160.0 34.2 35.0

Brady + Azoto + VAM 144.0 166.0 37.5 39.0

Brady + PDB + VAM 48.0 52.0 35.4 35.9

Azoto + PDB + VAM 150.0 168.0 38.6 39.0

Brady + Azoto + PDB + VAM 170.0 177.0 41.5 42.2

L.S.D. at 0.05 3.01 4.22 1.01 1.21

rhizosphere regions followed by bradyrhizobium compared to control treatment. This finding is in accordance with observation of Morone-Fortunato et al. (2005) and Copetta et al. (2006). In contrast, adding PDB had negative effect on spore production and % of colonization, many researchers found that the increase of soil phosphorus availability which is caused by PDB action causes decrease in VAM propagation (Yousefi et al., 2012). Among triple inoculations VAM + azotobacter + bradyrhizobium gave the best results, resulted in 320.0 and 360.0 spores/100 g soil and 72.0% and 77.0% of root colonization. Non-significantly increased was observed between VAM + azotobacter treatment and VAM + azotobacter + bradyrhizobium + PDB treatment in both seasons.

Moreover the positive effect of mixed inoculation on the increase of root colonization% and numbers of VAM spores was recorded by Bahadori et al. (2013). These results are in conformity with the earlier finding of Garbaye (1994), who postulated that bacteria such as those of genus bacillus producing phytohormones and cohabiting in the rhizosphere with VAM fungi, could play a helping role in the plant-fungus interaction.

Prosopis total chlorophyll was increased by biofertilizers application in all treatments when compared to control (Fig. 3). Co-inoculation treatments gave better results than single ones; triple inoculation treatments gave higher results than double inoculation treatments in the two studied years.

Fig. 1 Densities of VAM spores of Prosopis chilensis as influenced by different biofertilizers in two successive seasons.

3 2.5 2 1.5

I First season ■ Second season

d fiill

Fig. 3 Total chlorophyll content of Prosopis chilensis as influenced by different biofertilizer treatments in two successive years.

However, quarto inoculation treatment gave the highest chlorophyll value, the second year had higher chlorophyll contents in all treatments, and this was due to the continuous plant growth. This was in agreement with the results obtained by Selvakumar et al., 2012.

These results may be due to the effect of microorganisms in biofertilizer or the role of N2 nutrition in producing growth promoting substances resulting in more efficient absorption of nutrients, which were the main components of photosyn-thetic pigments and consequently the chlorophyll content was increased (Gomaa and Abou-Aly, 2001).

The applied biofertilizer treatments significantly affected both fresh and dry forage weight/plant during the two studied seasons with variable magnitudes as recorded in Table 5. Results indicated that any of the applied biofertilizer treatments causes significant higher fresh and dry weight as compared with control.

Meanwhile, either of the bio-mixed treatments (double, triple and quatre) produced significantly higher fresh and dry weight than the single biofertilizer treatments. Among the single treatments azotobacter gave the highest values 691.3 and 998.3 g/plant for fresh weight and 230.10 and 346.63 g/plant for dry weight, then bradyrhizobium followed by VAM and finally PDB treatment. Data also showed that double inoculation treatments gave better results than single ones. No significant difference was recognized in the fresh and dry weights/ plant between treatments which received triple inoculation. Data also showed that brady + azoto + PDB + VAM treatment produced the highest fresh (818.0 and 1271.3 g) and dry (291.40 and 414.27 g) weight/plants.

The recorded data for the response of fresh and dry forage weight/plant could ensure the beneficial identities of exerting the effect of biofertilizers in their action of creating better soil condition in respect of physical, chemical and microfloral status of the soil as well as eliminating the environmental pollution and hazards when completely relying on chemical fertilizers. These were in agreement with Oyeyiola (2010) on vetiver and Himanni et al. (2013) on Prosopis juliflora.

The applied biofertilizer treatments caused slight significant differences on Crude Protein content of leaves and branches of prosopis in the two growing years (Table 6). Any of the applied biofertilizer treatments either single or mixed produced significant increase in CP percentage either in the leaves or in the branches as compared with control. These results were noticed in the two growing years.

Among the applied 15 biofertilizer treatments, brady + azoto + PDB + VAM treatment produced the highest CP percentage for leaves (16.20 and 15.67) and branches (14.54 and 13.62) in the two successive seasons, followed by (brady + azoto + VAM) treatment, which show no significant difference with the following one (azoto + PDB + VAM). Results reveal that mixing fertilizer produced the highest protein percentage (Table 8). Priority of mixing biofertilizer, may be that in these treatments supplied sufficient nitrogen by (azotobacter and bradyrhizobia) for protein synthesis by plant. Priority of quatro biofertilizer treatment, was probably because of presence of phosphate sol-ubilizing bacteria and VAM that caused the gradual and balanced supply of phosphorus, part of the energy needed for nitrogen fixation (IrajZarei et al., 2012). However, the first year showed better results than the second one in CP for both

Table 5 Effect of biofertilizer treatments on Prosopis chilensis fresh and dry weights (g)/plant during two successive seasons.

Treatments Fresh weight (g)/plant Dry weight (g)/plant

First season Second season First season Second season

Control 650.5 941.40 216.80 326.71

Bradyrhizobium 682.4 990.6 227.46 349.58

Azotobacter 691.3 998.3 230.10 346.63

PDB 671.5 982.4 222.06 337.19

VAM 677.6 991.6 225.2 344.30

Brady + Azotobact 729.3 1116.7 241.11 387.74

Brady + PDB 722.5 1109.6 239.08 385.27

Brady + VAM 725.6 1110.2 240.86 385.49

Azotobact + PDB 727.6 1118.2 241.90 388.26

Azotobact + VAM 731.4 1120.9 243.03 389.20

PDB + VAM 736.8 1116.1 242.80 387.53

Brady + Azoto + PDB 764.2 1184.3 252.73 404.82

Brady + Azoto + VAM 766.6 1192.6 253.43 411.61

Brady + PDB + VAM 761.4 1186.8 253.2 407.08

Azoto + PDB + VAM 760.9 1190.4 253.07 407.33

Brady + Azoto + PDB + VAM 818.0 1271.3 291.40 414.27

L.S.D. at 0.05% 6.54 8.48 1.78 6.48

Table 6 Effect of biofertilizer treatments on Prosopis chilensis crude protein% of leaves and branches during two successive seasons.

Treatments CP% of leaves CP% of branches

First season Second season First season Second season

Control 14.22 13.21 12.42 11.87

Bradyrhizobium 15.42 13.48 12.82 12.02

Azotobacter 15.68 13.56 12.91 12.52

PDB 15.36 13.41 12.70 12.00

VAM 15.55 13.53 12.77 12.45

Brady + Azotobact 15.80 14.11 13.12 12.90

Brady + PDB 15.77 14.00 13.10 12.88

Brady + VAM 15.85 14.23 13.22 12.90

Azotobact + PDB 15.87 14.25 13.23 12.80

Azotobact + VAM 15.88 14.33 13.35 12.84

PDB + VAM 15.71 14.27 13.15 12.92

Brady + Azoto + PDB 15.92 14.73 13.81 13.22

Brady + Azoto + VAM 15.96 14.78 13.85 13.23

Brady + PDB + VAM 15.91 14.82 13.76 13.11

Azoto + PDB + VAM 15.95 14.70 13.83 13.21

Brady + Azoto + PDB + VAM 16.20 15.67 14.54 13.62

L.S.D. at 0.05% 0.04 0.08 0.03 0.02

leaves and branches, this referred to the continuous consuming of protein in plant growth (Selvakumar et al., 2012).

Data in Table 7 show that, the highest carbohydrate% was obtained when using mixture of Bradyrhizobium, azotobacter, PDB and VAM being 44.88 and 46.30 for leaves and 39.89 and 41.13 for branches in the two successive years. Moreover each of the triple mixed biofertilizer treatments produced significantly higher carbohydrate% than the double inoculation, with superiority to Bradyrhizobium, Azotobacter and VAM treatment. Meanwhile, there were significant differences in carbohydrate percentage between each of the 15 biofertilizer treatments than control.

El-Quesni et al. (2013) reported that chlorophyll a, b and carotenoids were increased with mixed biofertilizers application. Total carbohydrates content significantly increased in leaves and roots of Jatropha seedlings treated with phospho-rien, microbien. Such increment in photosynthetic pigments,

which reflect in photosynthesis processes and led to increase in carbohydrate contents.

Results presented in Table 8 showed significant effect of biofertilizer treatments on total digestible nutrient of leaves and branches of prosopis plants in the two growing seasons. Any of the applied biofertilizers either single or mixed produced significant increase in total T D N either in leaves or in branches as compared with the control (without biofertiliz-ers). These results were noticed in the two growing seasons.

Meanwhile, the mixed biofertilizer treatment (Brady + Azoto + PDB + VAM) was more effective in producing the highest TDN than the other mixed treatments. This result was true for the two studied seasons. Here again such results insure the effect of biofertilizers in enhancing nutrients uptake which reflected on the growth and development of plants for better components in total digestible nutrients. The applied biofertilizers cause slight significant increase in DP percentage

Table 7 Effect of biofertilizer treatments on total carbohydrate % of leaves and branches of Prosopis chilensis during two successive seasons.

Treatments Total carbohydrate % of leaves Total carbohydrate % of branches

First season Second season First season Second season

Control 42.01 44.02 37.12 38.84

Bradyrhizobium 42.63 44.38 37.63 39.33

Azotobacter 42.86 44.85 37.84 39.56

PDB 42.36 44.47 37.56 40.01

VAM 42.52 44.63 37.60 40.09

Brady + Azotobact 42.87 44.89 38.36 40.55

Brady + PDB 42.91 44.64 37.98 40.16

Brady + VAM 42.78 44.91 38.22 40.44

Azotobact + PDB 42.98 45.09 38.41 40.52

Azotobact + VAM 43.09 45.13 38.52 40.35

PDB + VAM 43.00 44.68 38.48 40.11

Brady + Azoto + PDB 43.66 45.53 38.76 40.72

Brady + Azoto + VAM 44.34 45.87 39.61 40.65

Brady + PDB + VAM 44.11 45.48 39.23 40.82

Azoto + PDB + VAM 44.23 45.77 39.58 40.88

Brady + Azoto + PDB + VAM 44.88 46.30 39.89 41.13

LSD. at 0.0 5% 0.20 0.13 0.09 0.07

Table 8 Effect of biofertilizer treatments on total digestible nutrient (TDN) % of leaves and branches during two successive seasons

of Prosopis chilensis.

Treatments TDN % of leaves TDN % of branches

First season Second season First season Second season

Control 61.01 59.52 55.48 54.63

Bradyrhizobium 61.58 59.99 55.73 54.80

Azotobacter 61.70 60.09 55.89 55.01

PDB 61.16 59.74 55.66 54.83

VAM 61.53 59.82 55.70 55.05

Brady + Azotobact 61.91 60.03 55.94 55.26

Brady + PDB 61.72 60.14 55.95 55.24

Brady + VAM 61.83 60.25 56.00 55.21

Azotobact + PDB 61.93 60.28 55.67 55.18

Azotobact + VAM 61.96 60.35 56.17 55.21

PDB + VAM 61.18 61.04 56.10 55.12

Brady + Azoto + PDB 62.01 60.72 56.47 55.46

Brady + Azoto + VAM 62.06 60.96 56.50 55.53

Brady + PDB + VAM 62.10 60.89 56.50 55.64

Azoto + PDB + VAM 61.92 60.88 56.58 55.23

Brady + Azoto + PDB + VAM 62.051 61.33 57.00 55.95

L.S.D. at 0.05% 0.05 0.06 0.08 0.05

of leaves and branches of prosopis plants as shown in Table 9. Any of the applied biofertilizer treatments either single or in mixture produced slightly significant increase in D P percentage of prosopis plants either in leaves or in branches as compared with the control. These results were noticed in the two growing seasons. The best results were obtained when prosopis plants received quarto biofertilizers in both leaves and branches, this followed by triple inoculation treatments and then double inoculation, finally single inoculation gave the lowest values in inoculated treatments.

Results in Table 10 represent the effect of each of the applied biofertilizer treatments on ash contents of leaves and branches for prosopis plants during two successive years. The applied

biofertilizers caused significant increase in ash contents. Any of the applied biofertilizer treatments either single or mixed produced slightly significant increase in ash content either in leaves or in branches as compared with control. However, ash contents gradually increased from single inoculation passing through double and triple inoculation giving the highest values in quarto inoculation (Brady + Azoto + PDB + VAM) which gave 18.44% and 16.87% for leaves and 13.05% and 12.99% for branches, during the two successive years.

The effect of the mixed applied biofertilizer treatments on ash accumulation of prosopis plants may represent its benefits in providing plants with enough minerals and nutrients (Ahmed et al., 2013).

Table 9 Effect of biofertilizer treatments on total digestible protein (DP)% of leaves and branches during two successive seasons of Prosopis chilensis.

Treatments DP% in leaves DP% in branches

First season Second season First season Second season

Control 10.10 9.13 8.37 7.84

Bradyrhizobium 11.25 9.39 8.75 7.98

Azotobacter 11.50 9.46 8.84 8.46

PDB 11.45 9.32 8.64 7.97

VAM 11.37 9.43 8.70 8.38

Brady + Azotobact 11.61 9.99 9.04 8.83

Brady + PDB 11.58 9.88 9.02 8.81

Brady + VAM 11.69 10.11 9.14 8.83

Azotobact + PDB 11.67 10.12 9.15 8.73

Azotobact + VAM 11.68 10.20 9.26 8.77

PDB + VAM 11.53 10.14 9.07 8.85

Brady + Azoto + PDB 11.73 10.58 9.70 9.14

Brady + Azoto + VAM 11.77 10.63 9.74 9.15

Brady + PDB + VAM 11.72 10.67 9.65 9.03

Azoto + PDB + VAM 11.28 10.56 9.72 9.13

Brady + Azoto + PDB + VAM 12.00 11.39 10.40 9.52

L.S.D at 0.05% 0.04 0.02 0.02 0.02

Table 10 Effect of biofertilizer treatments on Prosopis chilensis ash% of leaves and branches during two successive seasons.

Treatments Ash% of leaves Ash% of branches

First season Second season First season Second season

Control 17.20 16.01 12.41 12.04

Bradyrhizobium 17.54 16.15 12.66 12.12

Azotobacter 17.44 16.33 12.71 12.29

PDB 17.78 16.28 12.54 12.19

VAM 17.63 16.28 12.20 12.60

Brady + Azotobact 17.87 16.62 12.81 12.31

Brady + PDB 17.89 16.49 12.77 12.35

Brady + VAM 17.79 16.40 12.75 12.33

Azotobact + PDB 17.88 16.56 12.76 12.37

Azotobact + VAM 17.91 16.62 12.80 12.41

PDB + VAM 17.94 16.55 12.74 12.29

Brady + Azoto + PDB 18.04 16.61 12.88 12.56

Brady + Azoto + VAM 18.11 16.77 12.96 12.74

Brady + PDB + VAM 18.09 16.85 12.92 12.68

Azoto + PDB + VAM 18.13 16.80 12.94 12.71

Brady + Azoto + PDB + VAM 18.44 16.87 13.05 12.99

L.S.D. at 0.05% N.S 0.06 0.07 0.05

General discussion

The growth and yield parameters of Prosopis chilensis such as, plant branching, fresh and dry weight, total chlorophyll, crude protein content, crude fiber and ash% were significantly increased by biofertilizers application in all applications when compared to control. Utilization of biological fertilizer increased plant growth due to increasing other nutrient absorption, Zahir et al. (1998).

Many investigators studied the effect of different sources of bio-fertilizers on different plant species. Hoshang et al. (2011) and AsadRokhzadi et al. (2008) indicated that inoculation of maize and chick pea with biofertilizers containing Azotobacter increased plant height, leaf number per plant, fruit mean weight and yield as compared with control (without biofertiliz-

er). Azotobacter fixed N from the atmosphere and released plant available N forms to soil, resulting in increased uptake and plant yield. Hassan et al. (2012) reported that bio-fertilizer treatments, enhanced plant height, branch number/plant, plant dry weight, Pods number/plant, Pods dry weight, seed index, seed number/plant and seed yield/plant and/feddan, nitrogen, phosphorus, potassium, protein percentage and Alkaloids percentage and Alkaloids content/plant(g). The biofertilizers stimulate the growth, yield and chemical constituents. These results in harmony with those obtained by Swaefy et al. (2007) on peppermint plant

Said Al Ahl (2005) reported that plant height, number of branches, plant fresh and dry weights, umbels number and fruits yield increased with bio-fertilizer treatment in Anethum graveolens, with the mixed biofertilizer treatment producing

the highest values, El-Shafie et al. (2010) on khella plants and Abd-El-Salam (2007) on roselle plants, Amin (1997) on coriander, fennel and caraway plants showed that, the growth characters were positively influenced by seed inoculation (Azotobacter sp. and Azospirillum sp.). The ability of Azoto-bacter to produce growth substances and antifungal substances in addition to fixed nitrogen made available to plants was probably the reason of higher yields. Nitrogen stimulates the meristematic activity for producing more tissues and organs. Abd-El-Fattah and Sorial (2000) ensured that increasing nitrogen levels increased the cytokinins and gibberellins which enhance cell division and cell enlargement and thus increased vegetative growth. Meanwhile, (Subb-Roa, 1984) stated that, the favorable effect of biofertilizers on growth parameters might be ascribed to its important role in fixing atmospheric N as well as increasing the secretion of natural hormones, namely IAA, GA3 and cytokinins, antibiotics and possibly raising the availability of various nutrients. Thus, it can be concluded that treating prosopis plants with bio-fertilizer increased the formation of branches. Yield of prosopis (Chauhan et al., 1996). In conclusion the increment in plant fresh weight may be attributed to a greater proliferation of root biomass resulting in the higher absorption of nutrients and water from the soil leading to production of higher vegetative biomass (Ahmed et al., 2013 and Abdel-Kader et al., 2012). The increase in plant fresh weight may be due to the increase of N in the root zone as a result of nitrogen application and fixed N by bacteria. Also, the solubilization of mineral nutrient synthesis of vitamins, amino acids and gibberellins, which stimulate growth and yield. The stimulation effects of nitrogen on vegetative growth characters may be due attributed to the well-known functions of nitrogen in plant life, being a part of protein; it is an important constituent of protoplasm. Also, enzymes, the biological catalytic agents, which speed up life processes, have N as their major constituents. Moreover, nitrogen involves in many organic compounds of plant system. A sufficient supply of various nitrogenous compounds is therefore, required in each plant cell for its proper functioning (Selvakumar et al., 2012). The superiority of bio-fertilizers alone or together for stimulating plant dry weight exhibited the same trend owing to the favorable effect of mixed bio-fertilizers on plant growth and yield attributes might be due to the improved nutrition and production of growth promoting substances by micro-organisms (Himani Bhatia et al., 2013 and El Gendy et al., 2013). The necessity of N, as a plant nutrient is emphasized by the fact that it is a main constituent of many organic compounds in plant (Daldoum and Musa, 2012 and Ghilavizadeh et al., 2013) The increase in chlorophyll content may be due to available nitrogen but also the increase in trace elements in the soil caused by the organic acids produced by microorganisms leading to a decrease in the pH of the soil (Subb-Roa, 1981).

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