Symbiotic N2-Fixation Efficiency of Lupine Plant under

Fungal and Bacterial Inoculation

Soad Y. El-Sayed, Hend, M. El-Egami and El-Tahlawy, Y.A.
Agric. Microbio. Res. Dept., Soils, Water and Environ. Res. Inst. (SWERI), Agric. Res. Center (ARC), Giza, Egypt.

Abstract
Plant growth promoting fungi (PGPF) and Plant growth promoting bacteria (PGPB) rep-
resent the highest portion of microorganisms (PGP-microorganisms) that inhabit the rhizosphere
(PGP-Microorganisms). So, Trichoderma harzianum and Serratia marscences and Bradyrhizo-
bium sp. were examined in vitro for achieving the traits that being related to plant growth pro-
moting effects such as, indol acetic acid (IAA) phytohormome production and phosphate solubil-
ization. Furthermore, a field experiment was conducted to study the co-inoculation effect of T.
harzianum, S. marscences and their mixture on the symbiotic N2-Fixation, some plant growth
characters and yield of lupine plant. The in vitro, all tested microorganisms were apparently able
to trigger PGP-properties. However, T. harzianum appeared to be superior in P-solubilization
and IAA production regarding to the other tested microorganisms. The available phosphorus
measured was (104.61, 96.72 and 83.72 ppm) for T. harzianum, S. marscences and Bradyrhizo-
bium, respectively. While the amounts of IAA produced were (105.84, 79.03 and 60.5 µg ml-1)
by T. harzianum, Bradyrhizobium and S. marscences, respectively. At the vegetative stage, the
results of field trail displayed that the co-inoculation of Bradyrhizobium with T. harzianum, S.
marscences and their mixture had significant effect on nodulation status (nodule number, nodule
dry weight and nitrogenase activity) comparing to control treatment of sole Bradyrhizobium in-
oculation. However, the treatment of T. harzianum was surpassed the other treatments. The same
trend was recorded at the harvest stage, where T. harzianum conducted with Bradyrhizobium,
which exhibited superiority for approve the lupine yield and its components (biological and seed
yield, 100 seed weight, harvest index and seed crude protein).
Keywords: PGP-Microorganisms; PGPF; PGPB; Symbiotic N2-Fixation; Trichoderma harzi-
anum; Serratia marscences; lupine.
Introduction
The symbioses between Rhizobium or Bradyrhizobium and legumes are representing a
renewable, cheaper and usually more effective agronomic practice source of N for agriculture
Zahran (1999). Many publications have indicated the necessity of legume inoculation with ef-
fective and efficient rhizobial strains, especially when the soil void of the specific Rhizobium
agents (Verma et al., 2010; Badawi et al., 2011). White lupine (Lupinus termis L.) is one of the
legume plants which grow as a traditional human food and animal feed since ancient times and
extensively cultivated in Egypt as well as other Mediterrean countries as a component sustaina-
ble farming system (Hefny, 2013).

1 
 
 Traditional nutrient management for preserving high crop productivity has been mainly
based on external fertilizer inputs. However, in the last decades, crop yield has not increased
proportionally with increasing fertilizer inputs, leading to low nutrient use efficiency and en-
hanced environmental risks (Zhang et al., 2010). Thus the aim of international research is to
make better use of natural microbial resources in a sustainable form of agriculture, in order to
reduce the input of fertilizers and pesticides and thus pollution of soils and ground water, as well
as to decrease energetic costs and the environmental pollution connected with the production of
agrochemical (Hoflich et al., 1994). Therefore, the overcoming of this challenge implies the im-
provement of crop nutrient use efficiency by exploiting the intrinsic biological potential of rhizo-
sphere processes (Pii et al., 2015). In this concern they added that soil microorganisms could
represent a promising method to improve plant use efficiency of nutrients, already present in soil
or supplied by fertilizers. Biofertilizers are considered as an alternative or complement to chemi-
cal fertilization to increase the production of crops in low input agricultural systems. Different
bacterial genera are involved in various biotic activities of the soil ecosystem making it dynamic
for nutrient turn over the sustainable for crop production (Ahemad and Khan, 2010).
Two paradigms that have emerged so far from the study of PGPR is that many of the best
strains are multifunctional, and secondly, that PGPR traits are commonly distributed among
many different species and genera of microoraganisms, many of which are indigenous numbers
of the soil microbial community (Viveros et al., 2010). Plant growth –promoting bacteria
(PGPB) which are belonging to a beneficial and heterogeneous group of microorganisms that can
be found in the rhizosphere, on the root surface or associated to it, are capable of enhancing the
growth of plants and protecting them from disease and a biotic stress through a wide variety of
mechanisms. The mechanisms by which PGPB enhancing the growth of plants, that involve soil
structure formation, decomposition of organic matter, recycling of essential elements, solubiliza-
tion of mineral nutrients, producing numerous plant growth regulators, degrading organic pollu-
tants, stimulation of root growth, crucial for soil fertility, biocontrol of soil and seed born plant
pathogens and in promoting changes in vegetation (Sivasakhti et al., 2014; Rocheli et al.,
2015). PGPR tested as co-inoculants with rhizobia include Bacillus sutilis, Bacillus thurigiensis,
S. proteomaculamts and Pseudomonas aureofaciens, Bradyrhizobium japonicum (Montano et
al., 2014).
Beside soil-borne bacteria, which are able to colonize plant roots and have beneficial ef-
fects on the plant, also fungi are considered as plant growth promoting agents. Plant growth
promoting fungi (PGPF) are class of non- pathogenic soil- born filamentous fungi that have ben-
eficial effects on plants. Several PGPF have been reported thus far, such as species belonging to
the genera Trichoderma, Fusarium, Penicillium and Phoma (Kleifeld and Chet, 1992;
Masunaka et al., 2011).
Trichoderma is a genus of fungus found associated with many root ecosystems (Garcia
et al., 2015) Species of Trichoderma have divers beneficial effects on plant growth and devel-
opment, they increase the proliferation of secondary roots, leaf area, shoot length, dry weight and

2 
 
crop yield. That is may be due to providing plant with hormones (IAA) and phosphorus to plant
through P- solubilizing (Hermosa et al., 2013; Mukherjee et al., 2013).
Co-inoculation studies with rhizobia and PGPR are becoming a frequent practice in the
development of sustainable agriculture, these studies are focused on the improvement of legumes
yield production by increasing the nitrogen fixed by Rhizobia (Montano et al., 2014). Some
PGPRs are able to modify nodule formation and biological nitrogen fixation (BNF) when they
are co-inoculated with rhizobia (Garcia et al., 2004).
The aim of the present study is mainly designed to study the effect of some PGPB and
PGPF for their plant growth promotion PGP-related properties in vitro and in field trail. As well
as their effect when co-inoculated with Bradyrhizobium on the efficiency of lupine-
Bradyrhizobium symbiosis under sandy soil condition.
MATERIALS and METHODS
Plant materials:
Seeds of lupine (Lupinus termis L.) c.v. (Giza 1) were kindly supplied from Legume
Crops Research Department; Field Crops Research Institute, ARC, Giza, Egypt.
Microorganisms:
Bradyrhizobium sp. (Lupinus) was used for inoculating of lupine seeds. Serratia
marscences used as plant growth promoting bacteria (PGPB) and Trichoderma harzianum used
as plant growth promoting fungi (PGPF). The mentioned microorganisms are kindly provided
from the Biofertilizers Production Unit of Agric. Microbiol. Res. Dept., (SWERI), (ARC), Giza,
Egypt. Bradyrhizobium sp. was grown on yeast extract mannitol broth medium (Vincent, 1970),
while peptone glycerol broth medium was prepared for S. marscences cultivation (Grimont and
Girmont, 1984) and T. harzianum was grown on a potato dextrose agar (PDA) (ATCC, 1992)
and incubated at 28oC for 4 days on rotary shaker. All microorganisms were loaded onto a pow-
dered and sterilized carrier of vermiculite supplemented with 10% Irish peat at the rate that satis-
fy 60% of the maximal water holding capacity.
Evaluation of plant growth promoting activities in vitro:
Estimation IAA production:
Indole acetic acid production was quantitatively measured by the method of Gordon and
Weber (1951). Microbial cultures were grown in a Luria-Bertani broth amended with L-
tryptophan (5mM) for 3-4 days. Cultures were centrifuged at 10,000 rpm for 20 min. Two ml of
supernatant was mixed with 4 ml of Salkowski reagent. Tubes were incubated at room tempera-
ture for 25 minutes. The intensity of the developed pink color was read at 530 nm spectrophoto-
metrically (Jenway 67-series spectrophotometer) and the amount of IAA produced was extrapo-
lated from the standard curve.

Estimation of phosphate solubilization

Phosphate solubilization was quantitatively measured according to Pikovskaya (1948).
Fungal and bacterial cultures were grown in Pikovskaya broth medium (containing insoluble
3 
 
tricalcium phosphate 2.5 g, glucose 13 g, (NH4)SO4 0.5 g, NaCl 0.2 g, MgSO4.7H2O 0.1 g , KCl
0.2g, yeast extract 0.5g, MnSO4 trace, FeSO4.7H2O trace, agar 20 g, pH adjusted to 7.2 and 1000
ml distilled water) and incubated at 28oC with continuous shaking. After 6 days, 10 ml sample of
each broth culture was centrifuged for 15 min. at 8000 rpm and phosphorus content was deter-
mined according to Watanabe and Olson (1965).
Field Experiment:
A field experiment was conducted at Ismailia Agric. Res. Station, Ismailia Governorate,
Egypt, during winter 2015/2016, using sprinkler irrigation system to evaluate the effect of inocu-
lation with Bradyrhizobium sp. either individually, as a control treatment, or co-inoculated with
the tested PGP-microorganisms (S. marscences, T. harzianum or both in a mixture) on the
growth and yield of lupine plant under sandy soil conditions. The following treatments were
conducted:
T1 - Inoculation with Bradyrhizobium sp. only (control).
T2 - Inoculation with Bradyrhizobium + S. marscences.
T3 - Inoculation with Bradyrhizobium + T. harzianum.
T4 - Inoculation with Bradyrhizobium and a mixture of Serratia and Trichoderma.
Compost, was applied as organic fertilizer at rate 5 ton/fed, kindly supplied by SWERI, ARC,
Giza, Egypt. All treatments received ammonium sulphate (20.5%N) at a rate of 20 Kg N/fed. as
an activation dose. The main characters of used soil and compost are shown in Table (1) and Ta-
ble (2), respectively. Two samples were taken at vegetative (after 60 days of sowing) and harvest
stages (120 days).
Table 1. Some chemical, physical and biological properties of experimental soil
Property Soil
Sand (%) 89.79
Silt (%) 2.48
Clay (%) 7.73
Texture Sandy
pH 7.32
-1 o
E.C (dS m at 25 C) 0.25
Organic matter (%) 0.25
Organic carbon% 0.15
Total nitrogen (%) 0.021
C/N ratio 7.14
Dehydrogenase activity* 264
CO2-evolution rate** 13.24
*µg TPF/100g dry soil/24h. **mg CO2/100g soil/24 h.
   

4 
 
Table 2. Some chemical, physical and biological properties of experimental compost
Property Compost
Sand (%) -
Silt (%) -
Clay (%) -
Texture -
pH 7.21
o
E.C (dS m-1at 25 C) 4.57
Organic matter (%) 36.67
Organic carbon% 21.32
Total nitrogen (%) 1.31
C/N ratio 16.30
Dehydrogenase activity* 689
CO2¬-evolution rate** 16.82
*µg TPF/100g dry soil/24h. **mg CO2/100g soil/24 h.

Plant growth analysis:

A non-destructive phenotyping of various parameters, including the root surface area,
leaf area and chlorophyll content, ware assayed depending on digital image analysis technique
(Dutta Gupta et al., 2013). For each treatment, nine fresh leaves of the same maturity stage
(third leave from the top) as well as nine roots were sampled. The images were captured with
300 dot per inch (dpi) resolution using flatbed scanner (HP Scanjet G2710) and stored as a TIFF
(tagged image file format) file for subsequent processing. The image processing as well as pho-
tometric values of red (R), green (G), blue (B) from the histogram and scale measurement were
extracted using Adobe Photoshop CS6 ver. 13-Extended. The leaf area and root surface area
were estimated according to Kaur et al. (2014) as the following:

6.45
Total chlorophyll content was calculated estimated according to Yadav et al. (2010) and
calculated according to Kawashima and Nakatani (1998) as the following:
⁄ 0.952 1.76

The plant height as well as shoot and root dry weight were measured. Furthermore, the
nodulation status was evaluated via number and dry weight of nodules. Nitrogenase enzyme ac-
tivity in fresh roots was estimated using acetylene reduction assay as described by Hardy et al.
(1973) as an indicator for the symbiotic nitrogen fixation. Also, total kjeldahl-N, P and K was
determined according to Page et al. (1982). Seed crude protein percentage was calculated by
multiplying N% by 6.25 (AOAC, 1990).

5 
 
Statistical analysis:
The experiment was set up in a completely randomized design. Each treatment was repli-
cated three times. Statistical analysis was done by means of the Analysis of Variance (ANOVA).
Multiple comparisons of treatment means were done by Fisher’s L.S.D. method at a 5% proba-
bility level. The analysis was performed using CoStat ver. 6.4 - CoHort Software.
RESULTS and DISCCUSION
Evaluation of plant growth promoting activities in vitro:
Phytohormome IAA and dissolved phosphate were quantitatively assayed as PGP-related
properties of microorganisms in vitro as shown in Table (2). Regarding to IAA production,
which determined by supplementation of 1000 µgmL-1 L-tryptophan as precursor of IAA synthe-
sis, the highest amount was produced by T. harzianum followed by those produced by Bradyrhi-
zobium and Serratia marscences. The corresponding amounts were 105.84, 79.03 and 60.50
µgmL-1 , respectively. This finding has been supported by Yadav et al. (2011) and Mara et al.
(2014) who reported that Trichoderma isolates are strong in IAA producing microorganisms and
the use of L-tryptophan precursor provided a positive effect as an inducer for the synthesis of this
phytohormome. Also, Badawi et al. (2011) reported that a high proportion of rhizo-
microorganisms is able to produce plant growth hormone (IAA), which acts as stimulator to root
growth as well as more branching and larger surface area.
Data in Table (3) show the ability of the tested microorganisms to solubilize tricalcium
phosphate supplemented to Pikovskaya broth medium. Results revealed that T. harzianum
showed strong phosphate solubilization as the amounts of available phosphorus measured was
(104.61 ppm) followed by Serratia (96.72 ppm) and Bradyrhizobium (83.72 ppm).
Table 3. Some PGP-related properties of Bradyrhizobium and the tested microorganisms in vitro.
Bacterial strains Amount of IAA (µg ml-1) Dissolved phosphorus (ppm)
Bradyrhizobium sp. 79.03 83.72
S. marscences 60.50 96.72
T. harzianum 105.84 104.61

The abovementioned results concluded that T. harzianum show a great phosphate solubil-
ization efficiency as compared to the other tested bacteria. This agreed with Yadav et al., 2011
who detected that fungi possess greater ability to solubilize phosphate than bacteria.
Solubilization of phosphate may due to the production of various organic acids by the
tested rhizo-microorganisms. This is in accordance with Vargas et al. (2009) who reported that
efficiency of rhizo-microorganisms to solubilize phosphate is due to the production of various
organic acids which have affinity to chelate the calcium ions.
 

6 
 
Growth aspects:
The response of lupine plants growth to co-inoculation with Bradyrhizobium sp., T. har-
zianum, S. marscences and their mixture after 60 days of planting is represented by some growth
aspects namely plant height, shoot and root dry weight, root and leaf surface area and chloro-
phyll content (Table, 4).
Statistical analysis showed that the co-inoculation with Bradyrhizobium sp. and the other
tested rhizo-microorganims for lupine plants significantly increased all growth parameters. How-
ever, co-inoculation with T. harzianum was always the superior for increasing all features of
plant vigor, T. harzianum stimulate plant growth and induced 17.23% increase in shoot dry
weight followed by mixture of tested PGP-microorganisms and S. marscences that gave increas-
es of 9.99 and 6.42%, respectively, above sole inoculation with rhizobia. The corresponding in-
creases of root dry weight were 30.47, 15.00 and 11.22% for T. harzianum, mixture and S.
marscences over sole rhizobial inoculation.
Regarding to root and leaf surface area, there were significant increases in both root and
leaf surface area due to co-inoculation with T. harzianum, S. marscences and their mixture as
compared to single inoculation with rhizobia.
Table 4. Effect of rhizo-microorganisms co-inoculated with Bradyrhizobium on some growth
aspects and nodulation status of lupine plant after 60 days of planting
Parameter Plant Shoot Root Root Leaf Nodule Nodule Nitroge Total
height D wt. D.Wt. Surfac Surfac Numbe Dry nase Chloro
Treatments (cm) (g/pl.) (g /pl.) e Area e Area r/plant Wt. (μmole phyll
(cm2) (cm2) (mg/pl. C2H4 /g (mg/cm
2
) nod/hr) )
T1 37.03 12.97 3.40 67.65 18.92 44.56 273.67 5.99 6.93
T2 40.63 13.86 3.83 80.00 21.45 41.33 301.44 6.84 6.96
T3 45.13 15.67 4.89 101.77 24.52 48.00 386.56 7.99 7.30
T4 40.83 14.41 4.00 88.33 21.69 43.89 341.33 7.80 7.40
LSD0.05 2.25 0.77 0.32 6.64 0.85 3.76 25.16 0.44 0.28
T1 - Inoculation with Bradyrhizobium sp. only (control).
T2 - Inoculation with Bradyrhizobium + S. marscences.
T3 - Inoculation with Bradyrhizobium + T. harzianum.
T4 - Inoculation with Bradyrhizobium with mixture of Serratia and Trichoderma.

Data in Table (4) revealed that the maximum values of root and leaf surface area of lu-
pine plant as affected by co- inoculation with T. harzianum were (101.77 and 24.52 cm2) respec-
tively, followed by those given by mixture of T.  harzianum  and  S.  marscences (88.3 and 21.69
cm2) PGP-microorganisms, while S. marscences recorded (80.00 and 21.45 cm2) for root and
leaf surface area, respectively. The same trend was observed in chlorophyll content as compare
to control (sole inoculation with rhizobia).
Indeed, the plant vigor depends mainly on the strength of strong root system architecture.
This is true and it is reflected by the salient flourishing of root mass and its surface area, which
gained as a functional of rhizo-microorganisms co-inoculation, particularly T. harzianum. Many
workers confirmed the beneficial effects of Trichderma sp. inoculation with T. harzianum T203

7 
 
to green potting mix increased leaf area, plant dry weight, general plant vigor and chlorophyll
content, in cucumber, compared with uninoculated controls (Inbar et al., 1994). Furthermore, T.
harzianum modifies root architecture, increased the total absorptive surface, facilites nutrient up-
take and the translocation of nutrients in the shoots, resulting in increased plant biomass through
an efficient use of N, P, K and micronutrients. They also have a wide abilities range to solubilize
plant nutrients such as phosphorus and micronutrients that become available (Samolski et al.,
2012; Hermosa et al., 2013). Regarding the phytohormones effect, PGPR also can alter root ar-
chitecture and promote plant development due to their ability to synthesize and secrete plant
hormones like indole-3-acetic acid (IAA) as reported by Boiero et al. (2007). The stimulatory
effect of the PGPRs comes from a manipulation of the complex and balanced network of plant
hormones that directly are involved in growth or stimulation of the root formation through bio-
synthesis of IAA (Sofo et al., 2011 and Montano et al., 2014).
The current findings revealed the role of the co-inoculation with Bradyrhizobium and the
mixtures of tested PGP microorganisms, which showed a significant increases in all growth as-
pects. This trend may due to the multiple mechanisms such as phosphate solubilization, nitrogen
fixation and IAA production that are responsible for improvement of plant growth. In this con-
text, Rudresh et al. (2005) reported that when a combination of Rhizobium, Bacillus and
Trichoderma was applied to chickpea, the mixture gave significantly greater effects than any of
the individual isolate with increases in germination, plant height, number of branches and total
biomass.
Nodulation Status:
The co-inoculation effect on lupine with Bradyrhizobium and tested PGP microorganisms
(S. marscences, T. harzianum and their mixture) on nodulation status represented by nodules
number, dry weight and nitrogenase activity are represented in Table (4).
Results demonstrated that rhizo-microorganisms lead to significant increase in nodulation
status of lupine plants in comparison to sole inoculation with rhizobia. However, co-inoculation
with Bradyrhizobium and T. harzianum was surpassing the other treatments for nitrogenase ac-
tivity, nodule number and nodule dry weight, which recorded 7.99 µmole C2H4/g nod/hr, 48.00
nodule/plant and 386.56 mg/plant, respectively.
The corresponding values of bradyrhizobia co-inoculated with a mixture of tested rhizo-
microorganisms and S. marscences were 7.80 and 6.84 µmole C2H4/g nod/hr for nitrogenase ac-
tivity are 3.89 and 41.33 nodule/plant for nodule number and 341.33 and 301.44 mg/plant for
nodule dry weight, respectively.
The results suggested that co-inoculation of lupine with Bradyrhizobium and tested rhizo-
microorganisms tended to improve nodulation status. However, T. harzianum surpassed the other
treatments followed by the mixture of tested rhizo-microorganisms then S. marscences as com-
pared to sole rhizobial inoculation.
Also these results indicated that co-inoculation of lupine seeds with Bradyrhizobium and
T. harzianum, S. marscences and their mixture in sandy soil may be the good tool to enhance
nodulation process. These results elicited also that these rhizo- microorganisms act to promote
8 
 
the initiation of nodules (its number and dry weight) and their performance efficiency (nitrogen-
ase activity), indicating that these co- inoculation has a vital role in boosting the nodulation pro-
cess along its duration. This may be accomplished by providing the rhizobia and lupine with
some promoting substances such as auxin. In this context, Garcia et al. (2004) found that com-
mon mechanisms used by rhizobacteria to alter nodule formation or biological nitrogen fixation
include a release of phytohormones such as auxins. They added that the effects of some phyto-
hormones are indirect, as they stimulate root growth, providing further sites for infection and
nodulation. Moreover, Yadav et al. (2011) detected that T. harzianum showed more significant
IAA production, which has a vital role in improvement of the nodulation status.
Hence, the tested rhizo-microorganisms proved that they are phosphate dissolving micro-
organisms as shown in Table (4), so they improve P concentration in lupine plant, consequently,
enhance nodulation status and N2 fixation. This is in agreement with Barea et al. (2005); Das et
al. (2014) who reported that inoculation with phosphate- solubilizing bacteria enhanced nodula-
tion and N2 fixation for alfalfa plants, in parallel with an increase in the P content of plant tis-
sues. It is therefore thought that an improvement in P nutrition of plant resulting from the pres-
ence of phosphate dissolving bacteria (PSB) was responsible for increasing nodulation and N2
fixation, as it is well-Known that these processes are (P-dependent). Also, Yadav et al. (2011)
tested T. harzianum for P-solubilization and found that it can be exploited for the solubilization
of phosphataes that fixed in the soil.
Nutrient status:
The total contents of nitrogen, phosphorus and potassium accumulated in the tissue of in-
oculated legumes may be considered an excellent indication of symbiotic efficiency and nutrient
uptake. Hence, accumulation of N, P and K in tissues of lupine plants as influenced by rhizo-
microorganisms co-inoculation was measured after 60 days of sowing and presented in Table (5).
Results revealed that the shoot N, P and K contents increased in treated plants by PGP-
microorganisms co-inoculated with Bradyrhizobium over sole rhizobial inoculation. The highest
shoot N (443.97 mg/plant) was recorded statically significant in plants treated with T. harzianum
followed by mixture treatment (396.87 mg/plant) and S. marscences (341.5 mg/plant) over con-
trol (single inoculation of Rhizobium).
Also, significant increases in shoot P and K contents were observed in response to PGP-
microorganisms. Similarly, co-inoculation of Bradyrhizobium and T. harzianum showed the
highest values in comparison to other treatments, where the values of shoot P and K contents in
response to T. harzianum inoculation were (42.28 and 82.67 mg/plant), respectively. While the
corresponding values in response to mixture of tested rhizo-microorganisms and S. marscences
were (38.77 and 37.35 mg/plant) for shoot P content and (77.51 and 73.30 mg/plant) for shoot K
content, respectively.
 

9 
 
Table 5. Nutrient constitutes of lupine plant as affected by co- inoculation of Bradyrhizobium
and some rhizo-microorganisms
Parameter Shoot content Root content
(mg/plant) (mg/plant)
Treatments Nitrogen Phosphorus Potassium Nitrogen Phosphorus Potassium
T1 283.03 29.56 66.96 23.99 5.33 18.47
T2 341.50 37.35 73.30 24.85 7.55 21.53
T3 443.97 42.28 82.85 32.75 9.32 30.88
T4 396.87 38.77 77.51 26.43 8.93 22.80
LSD0.05 27.69 3.91 7.59 2.78 2.77 2.64

Concerning the root N, P and K contents, data in Table (5) elicited a significant increase
due to co-inoculation of PGP-microorganisms and Bradyrhizobium in comparison to sole rhizo-
bial inoculation.
Inoculation with Bradurhizobium as main inoculants for lupine in conjunction with any
rhizo-microorganisms or their mixture resulted in marked increase in the N, P and K contents of
root. The values of root N, P, and K contents reached a peak with co-inoculant contained
Brdyrhizobium and T. harzianum as compared to other co-inoculants used (32.75, 9.32 and 30.88
mg/plant for N, P, K contents, respectively).
Based on the above mentioned results, it has been elicited that the salient enhancement in
the total N, P and K contents accumulated in the tissues of lupine plants was clearly emphasized
the distinct ability of PGP-microorganisms to increase the availability and uptake of the nutrients
in the rhizosphere, particularly for co-inoculated leguminous plants. This positive impression on
the nutrient status of lupine plants due to rhizo-microorganisms co-inoculation may be elucidated
by the key role of such tested rhizo-microorganisms in improvement of the nodulation and nitro-
gen fixation performance. This trend resulting in accumulating more fixed nitrogen in lupine tis-
sues. Additionally, these PGP-microorganisms have potential to enhance P content through its
ability to increase solubility of unavailable phosphate in the rhizosphere. This is in agreement
with Harman (2000) who reported that the improvement role of Trichoderma spp. in the im-
provement of plant growth occurs because of their ability to transform nutrients from unavailable
to available forms. Thus plant growth promotion correlates with improved availability of phos-
phorus and nitrogen as well. Yadav et al. (2011) reported that T. harzianum is the most signifi-
cant phosphate solubilizing fungus.
Plant growth promoting rhizobacteria have direct mechanisms that facilitate nutrient up-
take or increase nutrient availability by nitrogen fixation, solubilization of mineral nutrients,
mineralize organic compounds and production of phytohormones (Arora et al., 2012; Bhardwaj
et al., 2014; Gupta et al., 2015). Also Rhizobium and Serratia have been recorded as phosphate
solubilizing bacteria (Bhattacharyya and Jha, 2012).

Yield and other related components:

Biological yield, seed yield, 100 seeds weight, harvest index and seed crude protein of
lupine plant as affected by co-inoculation with Bradyrhizobium alone or in combination with T.
harzianum, S. marscences and their mixture are given in Table (6).

10 
 
 Generally, data in Table (6) revealed that lupine yield and its components responded
positively to addition of Bradyrhizobium in combination with the tested PGP-microorganisms.
Regarding to biological yield, seed yield, 100 seeds weight and harvest index appeared signifi-
cant increases due to co-inoculation of PGP-microorganisms with bradyrhizobia as compared to
sole inoculation of bradyrhizobia. Nevertheless, combination of T. harzianum and Bradyrhizobi-
um produced highest biological yield (7.152 ton/ha), (2.37 ton/ha) for seed yield, (40.45g) for
100 seeds weight and harvest index which recorded (33%), followed by mixture of T. harzianum
and S. marscences co-inoculated with bradyrhizobia (6.84 ton/ha) for biological yield, (2.23
ton/ha) for seed yield (37.02 g) for 100 seeds weight and (33%) for harvest index; and Serratia
(6.63, 2.15 ton/ha, 34.36 g and 32%) for biological and seed yield, 100 seeds weight and harvest
index, respectively.
Table 6. Effect of rhizo-microorganisms co-inoculated with Bradyrhizobium on lupine yield and
its components
Parameter
Biological yield Seed yield Harvest in- Seed crud pro- Seed 100weight
(ton/ha) (ton/ha) dex% tein% gm
Treatment
T1 6.73 2.05 30 21.01 29.5
T2 6.63 2.15 32 22.87 34.36
T3 7.152 2.37 33 25.08 40.45
T4 6.84 2.23 33 24.80 37.02
LSD0.05 0.13 0.04 0.07 1.82 3.48

Concerning the protein content concentration of lupine seeds, data in table (6) displayed
the positively response seed crude protein to co-inoculation of Bradyrhizobium and the tested
rhizo-microorganisms, in which, the percentage increase in the crude protein content accumulat-
ed in lupine seeds in response to conjunctive inoculation of Bradyrhizobium and T. harzianum, S.
marscences and their mixture were 16.22, 8.13 and 15.28, respectively over sole rhizobial inocu-
lation.
Those results clearly exhibited the promotive effect of rhizo- microorganisms on increas-
ing yield quantity, particularly in case of T. harzianum. Those findings may be interpreted by the
ability of such PGP-microorganisms to hasten the biological N2-fixation and nitrogen assimila-
tion and their translocation to yield constituents. This was generally agreed with Masunaka et
al. (2011) who reported that the main effect of PGPF on plant growth is therefore considered to
be the solubilization of minerals in the soil to help the plant to derive necessary nutrients in an
easily used form, thus enhancing plant yield. Ipstia and Singh (2014) reported that, conjunctive
use of Rhizobium with PGPR revealed synergistic effect on the symbiotic parameters and grain
yield of mung bean. They added that, significant increase in seed protein content was observed
due to bacterial inoculation supporting the hypothesis that biological nitrogen fixation by the
Rhizobium and PGPR to root associations could be responsible for the observed higher N uptake
of inoculated plants, which is cause significantly increased grain N uptake consequently in-
creased in grain protein. Plant growth promoting traits do not work indeptently of each other but

11 
 
additively as it was suggested in the “additive hypothesis” that multiple mechanisms such as
phosphate solubilization, dinitrogen fixation and IAA biosynthesis etc. are responsible for the
plant growth promotion and increased yield (Nehra and Choudhary, 2015).
It could be concluded that, the obtained results show a potential use of combination of
PGP-microorganisms (T. harzianum, S. marscences) and Bradyrhizobium in improve the effi-
ciency of lupine-Bradyrhizobium symbiosis through improvement of nodulation status, growth
characters and lupine yield under sustainable agriculture.
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‫ﻛﻔﺎءﺓ ﺗﺜﺒﻴﺖ ﺍﻟﻨﻴﺘﺮﻭﺟﻴﻦ ﺗﻜﺎﻓﻠﻴﺎ ﻟﻠﻨﺒﺎﺗﺎﺕ ﺍﻟﺘﺮﻣﺲ ﺑﺎﻟﺘﻠﻘﻴﺢ ﺍﻟﻔﻄﺮﻱ ﻭ ﺍﻟﺒﻜﺘﻴﺮﻱ‬

‫ﺳﻌﺎﺩ ﻳﻭﺳﻑ ﺳﺭﻱ‪ ،‬ﻫﻧﺩ ﻣﺻﻁﻔﻰ ﺍﻟﻌﺟﻳﻣﻲ‪  ،‬ﻳﺎﺳﺭ ﻋﺭﺑﻲ ﺍﻟﻁﺣﻼﻭﻱ‬
‫ﻗﺳﻡ ﺑﺣﻭﺙ ﺍﻟﻣﻳﻛﺭﻭﺑﻳﻭﻟﻭﺟﻳﺎ ﺍﻟﺯﺭﺍﻋﻳﺔ – ﻣﻌﻬﺩ ﺑﺣﻭﺙ ﺍﻷﺭﺍﺿﻲ ﻭ ﺍﻟﻣﻳﺎﻩ ﻭ ﺍﻟﺑﻳﺋﺔ – ﻣﺭﻛﺯ ﺍﻟﺑﺣﻭﺙ ﺍﻟﺯﺭﺍﻋﻳﺔ – ﺍﻟﺟﻳﺯﺓ ﻣﺻﺭ‬

‫ﺍﻟﻣﻠﺧﺹ ﺍﻟﻌﺭﺑﻲ‬
‫ﺗﻣﺛﻝ ﺍﻟﻔﻁﺭﻳﺎﺕ )‪ (PGPF‬ﻭ ﺍﻟﺑﻛﺗﺭﻳﺎ )‪ (PGPB‬ﺍﻟﻣﺷﺟﻌﺔ ﻟﻧﻣﻭ ﺍﻟﻧﺑﺎﺕ ﺍﻟﻣﻛﻭﻥ ﺍﻷﻋﻠﻰ ﻣﻥ ﺍﻟﻛﺎﺋﻧﺎﺕ ﺍﻟﺩﻗﻳﻘﺔ ﻭ ﺍﻟﺗﻲ ﺗﻌﻳﺵ ﺣﻭﻝ‬
‫ﺟﺫﻭﺭ ﺍﻟﻧﺑﺎﺕ‪ .‬ﻭ ﺑﻧﺎء ﻋﻠﻰ ﻫﺫﺍ ﺗﻡ ﺗﻘﻳﻳﻡ ﻓﻁﺭ ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ‪ Trichoderma harzianum‬ﻭ ﺑﻛﺗﺭﻳﺎ ﺍﻟﺳﻳﺭﺗﻳﺎ ‪Serratia marscences ‬‬
‫ﻣﻌﻣﻠﻳﺎ ﻛﻣﻳﻛﺭﻭﺑﺎﺕ ﺗﻣﺗﻠﻙ ﺑﻌﺽ ﺍﻟﺧﻭﺍﺹ ﺍﻟﻣﺷﺟﻌﺔ ﻟﻠﻧﻣﻭ ﻣﺛﻝ ﺍﻧﺗﺎﺝ ﺍﻧﺩﻭﻝ ﺣﺎﻣﺽ ﺍﻟﺧﻠﻳﻙ ‪ IAA‬ﻭ ﺍﻟﻘﺩﺭﺓ ﻋﻠﻰ ﺍﺫﺍﺑﺔ ﺍﻟﻔﻭﺳﻔﺎﺕ‪ .‬ﺑﺎﻹﺿﺎﻓﺔ‬
‫ﺍﻟﻰ ﺫﻟﻙ ﺗﻡ ﺍﺟﺭﺍء ﺗﺟﺭﺑﺔ ﺣﻘﻠﻳﺔ ﻟﺩﺭﺍﺳﺔ ﺗﺎﺛﻳﺭ ﻓﻁﺭ ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ‪ T. harzianum‬ﻭ ﺑﻛﺗﺭﻳﺎ ﺍﻟﺳﻳﺭﺍﺗﻳﺎ ‪ S. marscences‬ﻭ ﺍﻟﺧﻠﻳﻁ ﻣﻧﻬﻣﺎ‬
‫ﻋﻠﻰ ﻛﻔﺎءﺓ ﺗﺛﺑﻳﺕ ﺍﻟﻧﻳﺗﺭﻭﺟﻳﻥ ﺗﻛﺎﻓﻠﻳﺎ ﻭ ﺑﻌﺽ ﺻﻔﺎﺕ ﺍﻟﻧﻣﻭ ﻭ ﺍﻟﻣﺣﺻﻭﻝ ﻓﻲ ﻧﺑﺎﺕ ﺍﻟﺗﺭﻣﺱ‪ .‬ﺃﻅﻬﺭﺕ ﺍﻟﻧﺗﺎﺋﺞ ﺍﻟﻣﻌﻣﻠﻳﺔ ﺗﻔﻭﻕ ﻗﺩﺭﺓ ﻓﻁﺭ‬
‫ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ﻓﻲ ﺍﻧﺗﺎﺝ ‪ IAA‬ﻭ ﺍﺫﺍﺑﺔ ﺍﻟﻔﻭﺳﻔﺎﺕ‪ .‬ﻭﻗﺩ ﺃﻅﻬﺭﺕ ﺍﻟﻧﺗﺎﺋﺞ ﺍﻟﺗﺎﻟﻰ‪ 104.61 -:‬ﻭ ‪ 96.72‬ﻭ ‪) 83.72‬ﺟﺯء ﻓﻲ ﺍﻟﻣﻠﻳﻭﻥ( ﻟﻛﻝ ﻣﻥ‬
‫ﻓﻁﺭ ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ﻭ ﺑﻛﺗﺭﻳﺎ ﺍﻟﺳﻳﺭﺍﺗﻳﺎ ﻭ ﺍﻟﺑﺭﺍﺩﻱ ﺭﻳﺯﻭﺑﻳﺎ‪ ,‬ﻋﻠﻰ ﺍﻟﺗﻭﺍﻟﻲ‪ ,‬ﻓﻲ ﺍﺫﺍﺑﺔ ﺍﻟﻔﻭﺳﻔﻭﺭ ﺑﻳﻧﻣﺎ ﻛﺎﻧﺕ ﻛﻣﻳﺎﺕ ﺍﻻﻧﺩﻭﻝ ﺍﻟﻣﻧﺗﺟﺔ ﻛﺎﻟﺗﺎﻟﻰ‪-:‬‬
‫‪ 105.84‬ﻭ ‪ 79.03‬ﻭ ‪ 60.50‬ﻣﻳﻛﺭﻭﺟﺭﺍﻡ‪/‬ﻣﻝ ﻟﻛﻝ ﻣﻥ ﻓﻁﺭ ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ﻭ ﺍﻟﺑﺭﺍﺩﻱ ﺭﻳﺯﻭﺑﻳﺎ ﻭ ﺑﻛﺗﺭﻳﺎ ﺍﻟﺳﻳﺭﺍﺗﻳﺎ‪ ,‬ﻋﻠﻰ ﺍﻟﻭﺍﻟﻲ‪ .‬ﺃﻅﻬﺭﺕ‬
‫ﻧﺗﺎﺋﺞ ﺍﻟﺗﺟﺭﺑﺔ ﺍﻟﺣﻘﻠﻳﺔ ﻓﻲ ﺍﻟﻣﺭﺣﻠﺔ ﺍﻟﺧﺿﺭﻳﺔ ﺍﻟﺗﺄﺛﻳﺭ ﺍﻻﻳﺟﺎﺑﻰ ﻟﻠﺗﻠﻘﻳﺢ ﺍﻟﻣﺷﺗﺭﻙ ﻋﻠﻰ ﻋﻣﻠﻳﺔ ﺗﻛﻭﻳﻥ ﺍﻟﻌﻘﺩ ﺍﻟﺟﺫﺭﻳﺔ ﻣﺛﻝ ﻋﺩﺩ ﺍﻟﻌﻘﺩ ﻭ ﺍﻟﻭﺯﻥ‬
‫ﺍﻟﺟﺎﻑ ﻟﻠﻌﻘﺩ ﻭ ﺃﻳﺿﺎ ﻧﺷﺎﻁ ﺍﻧﺯﻳﻡ ﺍﻟﻧﻳﺗﺭﻭﺟﻳﻧﻳﺯ ﻣﻊ ﺗﻔﻭﻕ ﻣﻌﺎﻣﻠﺔ ﺍﻟﺗﺭﻳﻛﻭﺩﻳﺭﻣﺎ‪ .‬ﻛﻣﺎ ﺗﻔﻭﻕ ﺍﻟﺗﻠﻘﻳﺢ ﺍﻟﻣﺷﺗﺭﻙ ﺑﻔﻁﺭ ﺍﻟﺗﺭﺍﻳﻛﻭﺩﻳﺭﻣﺎ ﻓﻲ ﺗﺎﺛﻳﺭﻩ‬
‫ﺧﻼﻝ ﻣﺭﺣﻠﺔ ﺍﻟﺣﺻﺎﺩ ﻣﻥ ﺧﻼﻝ ﺍﻟﻣﺣﺻﻭﻝ ﺍﻟﺑﻳﻭﻟﻭﺟﻲ ﻭ ﻣﺣﺻﻭﻝ ﺍﻟﺑﺫﻭﺭ ﻭ ﻭﺯﻥ ‪ 100‬ﺑﺫﺭﺓ ﻭ ﻣﺣﺗﻭﻯ ﺍﻟﺑﺫﻭﺭ ﻣﻥ ﺍﻟﺑﺭﻭﺗﻳﻥ‪  .‬‬

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