CC BY-NC-ND
0Annals of Agricultural Science (2011) 56, 17-25
Faculty of Agriculture, Ain Shams University Annals of Agricultural Science
www.elsevier.com/locate/aoas
ORIGINAL ARTICLE
Peanut plant growth and yield as influenced by co-inoculation with Bradyrhizobium and some rhizo-microorganisms under sandy loam soil conditions
F.Sh.F. Badawi *, A.M.M. Biomy, A.H. Desoky
Agricultural Microbiology Department, Soils, Water and Environ. Res. Inst., ARC, Giza, Egypt
Received 24 March 2011; accepted 10 April 2011 Available online 10 August 2011
KEYWORDS
Peanut (Arachis hypogea L.); Bradyrhizobium spp.; Serratia marcescens; Trichoderma harzianum; PGP-related properties; Nodulation;
Peanut yield components
Abstract The ability of tested rhizomicrobial isolates (Serratia marcescens and Trichoderma harzianum) along with a strain of root nodule bacteria (Bradyrhizobium spp.) to exhibit some PGP-properties was evaluated in vitro conditions. The main PGP-properties, namely the ability to solubilize-P and production of IAA, as well as production of siderophores and HCN were examined. Additionally, field trials were conducted on sandy loam soil at El-Tahrir Province during two successive summer seasons to study the effect of co-inoculation with Bradyrhizobium either individually or together with S. marcescens and/or T. harzianum on nodulation, some plant growth characters, peanut yield and its yield components. The in vitro experiment revealed that all of the tested microorganisms were apparently able to trigger PGP-properties. Phosphate solubilization was the common feature of the employed microorganisms. However, T. harzianum appeared to be superior to other microorganisms, and Bradyrhizobium displayed the lowest capacity. The ability of the microorganisms to produce indole compounds showed that S. marcescens was more effective in IAA production and followed by Bradyrhizobium. Capacity of S. marcescens and T. harzianum to excrete ferric-specific ligands (siderophores) and HCN was detected, while Bradyrhizobium failed to produce such compounds. Results of field trials showed that the uninoculated peanut had the least nodulation status, N2-ase activity and all vegetative growth characters in both studied seasons. Bacterization of peanut seeds with bradyrhizobia exerted considerable improvement in number and mass of root nodules, increased the rate of acetylene reduction and all growth characters in
Corresponding author. Tel.: +20 2 1296 4444. E-mail addresses: farid-badawi@yahoo.com, journalaaru@yahoo. com (F.Sh.F. Badawi).
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doi:10.1016/j.aoas.2011.05.005
comparison to the uninoculated control. The synergy inoculation between bradyrhizobia and any of the tested microorganisms led to further increases of all mentioned characters and strengthened the stimulating effect of the bacterial inoculation. However, the promotive action on peanut nodula-tion, N2-fixation performance and vegetative characters was obvious with the dual inoculation with Bradyrhizobium plus S. marcescens. The other tested treatments, Bradyrhizobium conjugated with Trichoderma or with a mixture of the examined microorganisms, occupied the second rank. Additionally, peanut yield (pods and straw) and it's components (seed and straw protein contents, hundred seed weight, as well as pod and seed weight/plant and shelling%) along the two consecutive seasons followed a similar pattern to that of the vegetative growth stage.
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Peanut (Arachis hypogaea L.) is considered one of the most important edible oil crops in Egypt, which is due to its seeds' high nutritive value for humans, as well as the produced cake and the green leafy hay for feeding livestock, in addition to the seed oil's importance for industrial purposes. The main growing areas are located in the north of the country; they include reclaimed desert to the east and west of the Nile Delta. Peanut seeds are characterized by their high oil content (50%), which is utilized in different industries, besides they contain 26-28% protein, 20% carbohydrates and 5% fiber (Fageria et al., 1997).
One of the well known N2-fixing plant-microorganism interactions is the legume-rhizobia symbiosis, which is considered the most efficient and important process in crop production, so as to improve soil fertility and farming system flexibility (Mylona et al., 1995). Numerous publications have indicated the necessity of legume inoculation with effective and efficient rhizobial strains, especially when the soil is void of the specific Rhizobium agents (Jensen and Hauggaard, 2003; Kandil et al., 2008; Verma et al., 2010).
There is a currently considerable interest in the role of rhi-zosphere organisms in plant growth promotion and biological control of soil borne pathogens (Kloepper et al., 1989; Vargas et al., 2009). There are several PGPR commercialized, whose plants growth-promoting activities have been demonstrated in several ways, including production of iron-sequestering sid-erophores and antimicrobial compounds that hinder colonization of hosts by phytopathogens, induction of host systemic disease resistance, ability to produce ACC deaminase to reduce the level of ethylene in the roots of the developing plants, sol-ubilization of precipitated mineral nutrients, production of plant growth hormones, and/or improving the function of roots to absorb nutrients and water (Dey et al., 2004; Zahir et al., 2004; Verma et al., 2010).
Serratia marcescens is a species of Gram-negative bacterium. It has been widely known as a food-spoilage organism (Abdour, 2003) and an insect pathogen (Bahar and Demirbag, 2007). It is well known for its red pigmentation "prodigiosin" produced at temperatures below 30 0C. It has a unique ability to produce extracellular enzymes that have the ability to degrade chitin, a substance that mainly comprises fungal cell walls. These chitinolytic enzymes could have possible industrial and agricultural uses such as the introduction of these genes for chitin degrading enzymes into crops (Hejazi and Falkiner, 1997). It had also been reported to promote plant growth by the production of antagonistic substances (De Queiroz and De Melo, 2006), inducing resistance against plant pathogens (Tilak et al., 2006) and solubilization of phosphates (Tripura et al., 2007). In this respect, Khalid et al. (2004) and Verma et al. (2010) found that S.
marcescens was able to produce higher values of all plant growth promotion traits (solubilize phosphate and produce IAA besides producing siderophore and HCN) that enhance plant growth and its productivity.
Trichoderma are fungi that are present in almost all soils and other diverse habitats. Some strains of such fungus induce plants to ''turn on'' their native defense mechanisms against pathogenic fungi and nematodes (Siddiqui and Shaukat, 2004). Most biocontrol agents belonging to the species T. har-zianum and T. hamatum are used as fungicides. Trichoderma harzianum can also be used as a biofertilizer to reduce chemical inputs in the perspective of sustainable agriculture and conservation of natural resources (Harman, 2011). It is used for foliar application, seed treatment and/or soil treatment for suppression of various fungal diseases. The presence of Trichoderma inside the roots of treated plants made it a mycorrhizal organism (Neumann and Laing, 2006). A recent list of mechanisms has been suggested by many investigators, i.e. host-plant resistance, competition for space and nutrients, secretion of chitin-olytic enzymes, mycoparasitism, inactivation of the pathogen's enzymes, tolerance to stress through enhanced root and plant development, solubilization and sequestration of inorganic nutrients (Baker, 1989; Yedidia et al., 1999; Roco and Perez, 2001; Yadav et al., 2011). In this concern, Carvajal Liliana et al. (2009) and Harman (2011) reported that T. harzianum as a PGP-fungi had numerous speculative explanations of mechanisms involved in the increased growth and productivity including inhibition and alteration of normal root microflora; growth stimulating substances, nutrient availability or stimulation of nutrient uptake, and decreasing inhibitory substances to plant growth. Also, Trichoderma were highly efficient producers of many extracellular enzymes (T. resii is used for cellu-lase and hemicellulose and T. harzianum is used for chitinase).
Enhancement of nodulation and biological nitrogen fixation of legumes by co-inoculation with PGP microorganisms are becoming a practical way to improve nitrogen availability in sustainable agricultural production system (Bai et al., 2002; Abdel-Wahab et al., 2008). The most commonly implicated mode to stimulate legume-Rhizobium symbiosis is phytohor-mones inducing stimulation of root growth, to provide more sites for rhizobial infection and nodulation (Vessey and Buss, 2002). Also, PGPRs can promote legume nodulation and nitrogen fixation via producing flavonoid like compounds and/or stimulating the host legume to produce more flavonoid signal molecules (Parmar and Dadarwal, 1999; Bai et al., 2002). Potentiality for improving plant yield by combining rhi-zobacteria with rhizobia has been reported by many workers (Pan et al., 2002; Tilak et al., 2006; Verma et al., 2010).
The present investigation aimed at examining some microorganisms for their plant growth promotion (PGP)-related properties in both laboratory (in vitro) and field trials. The study was also concerned with the impacts of such microbial inocula on the peanut-Bradyrhizobium symbiosis, peanut plant growth, yield and some yield attributes in sandy loam soil of Egypt.
Materials and methods
Microorganisms used
Bardyrhizobium spp. (strain USDA 3456) and S. marcescens (EG 10) were kindly obtained from the Biofertilizers Production Unit, Agric. Microbiology. Dept., Soils, Water and Environ. Res. Inst. (SWERI) Agric. Res. Center (ARC), Giza, Egypt. T. harzianum was kindly provided by the Plant Pathology Res. Inst., ARC, Giza, Egypt.
Preparation of inocula
Bradyrhizobium was cultured in a yeast extract mannitol broth medium (Vincent, 1970) and Serratia was grown in King's medium B (Atlas, 1995). Cultures were incubated at 28 0C for three
days on a rotary shaker until early log phase to ensure population density of 109 cfu/ml culture. Trichoderma was grown on a potato dextrose agar (PDA) according to ATCC (1992) and incubated at 28 0C for 48 h on a rotary shaker.
Powdered vermiculite supplemented with 10% Irish peat (plus 10% wheat bran for fungi inoculant) was packed into polyethylene bags (200 g carrier per bag), then sealed and sterilized with gamma irradiation (5.0 x 106 rads). Each bacterial culture (120 ml of log phase growing culture) was injected into a sterilized carrier to satisfy 60% of the maximal water holding capacity, then mixed thoroughly and left for a week for curing. While, Trichoderma culture was injected into a sterilized carrier one day before sowing to guarantee the efficiency of peanut seeds inoculation with the fungi.
Assay of PGP-related properties in vitro
Ability of the tested rhizo-microorganisms to exhibit some PGP-properties was evaluated in in vitro conditions, through the determination of their efficiency to solubilize phosphate on DCP media (Frioni, 1990), production of indoleacetic acid (IAA) (Gordon and Waber, 1951), production of siderophores (Alexander and Zuberer, 1991), as well as cyanogenesis was examined as described by Bakker and Schippers (1987).
Field experiments
Two field experiments were conducted at El-Tahrir Province in the West Nile Delta, during two successive summer seasons of 2009 and 2010, using sprinkler irrigation system, to evaluate the effect of co-inoculation with Bradyrhizobium spp. (strain USDA 3456) either individually or interacting with the tested microorganisms (S. marcescens and/or T. harzianum) on peanut root nodulation, plant growth, yield and yield attributes, under sandy loam soil conditions.
Representative soil samples were collected from the top 20 cm layer of the experimental field, air-dried and sieved through a 2 mm screen. Physical, chemical and biological properties of the soil are shown in Table 1. Organic fertilizer
Table 1 Physical, chemical and biological properties of the experimental soil in both seasons of the study.
Property Value Season I Season II
Particle size distribution (%)
Sand 68.90 68.82
Silt 24.85 25.02
Clay 6.25 6.16
Texture grade Sandy loam Sandy loam
Saturation percent (S.P%) 41.0 40.8
pH (soil paste) 7.33 7.28
E.C (dS m_1, at 25 oc) 0.37 0.41
Soluble cations (meq/L)
Ca+ + 1.85 1.96
Mg+ + 0.97 1.02
Na+ 0.81 0.86
K + 0.28 0.31
Soluble anions (meq/L)
CO^ - -
HCO3 0.81 0.96
cr 0.79 0.82
SO4- 2.31 2.37
Organic matter (%) 0.62 0.65
Organic-C (%) 0.36 0.38
Total-N (%) 0.033 0.035
Total soluble-N (ppm) 34.0 40.0
Available-P (ppm) 7.20 8.00
Available-K (ppm) 164.6 182.4
DTPA-extractable (ppm)
Fe 3.52 3.80
Mn 3.14 3.41
Zn 1.25 1.12
Cu 0.31 0.40
Total count of bacteria (cfu/g) 6.9 x 105 6.3 x 105
Total count of fungi (cfu/g) 0.9 x 104 1.1 x 104
Total count of actinomycetes (cfu/g) 1.1 x 104 1.3 x 104
Dehydrogenase activity (ig TPF/g/day) 5.40 6.10
Table 2 The main traits of the used compost.
Property Value
pH (1:10 extract) 6.75
E.C (dSm41, at 25 oc) 5.80
O.C (%) 24.52
Total-N (%) 1.38
C/N ratio 17.77
Total-P (%) 0.89
Total-K (%) 1.46
Total soluble-N (ppm) 862.3
Available P (ppm) 269.5
Available K (ppm) 911.3
DTPA-extractable (ppm)
Fe 508.4
Mn 52.6
Zn 38.2
Cu 8.7
Total count of bacteria (cfu/g) 14 x 107
Total count of fungi (cfu/g) 12 x 106
Total count of actinomycetes (cfu/g) 2.3 x 106
Dehydrogenase activity (ig TPF/g/day) 141.5
Germination test of cress seeds (%) 89.0
(Compost) used was kindly supplied by Soil, Water and Environ. Res. Inst. (SWERI), ARC, Giza, Egypt. The main chemical and biological traits of the compost used are shown in Table 2.
The following co-inoculation treatments were conducted:
(1) Uninoculated (control).
(2) Inoculation with Bradyrhizobium only.
(3) Inoculation with Bradyrhizobium + S. marcescens.
(4) Inoculation with Bradyrhizobium + T. harzianum.
(5) Inoculation with Bradyrhizobium + S. marcescens + T. harzianum.
A randomized complete block design with four replicates was used with a plot area of 10.5 m2 (1/400 feddan). Compost at a rate of 10 m3/fed, superphosphate (15.5% P2O5) at a rate of 200 kg/fed and potassium sulfate (48% K2O) at a rate of 50 kg/fed were incorporated into the soil for all studied treatments 7 days before sowing. All treatments received ammonium sulfate (20.5% N) at a rate of 20 kg N/fed at planting time as an activator dose.
Peanut seeds (variety Giza 6) were provided by the Oil Crops Research Department, Field Crops Research Institute, ARC, Giza, Egypt. Seeds were inoculated with Gamma irradiated ver-miculite-based inocula for each microorganism used at a rate of 400 g/40 kg seeds, prior to sowing using 16% Arabic gum solution as an adhesive agent, then the inoculants were added and thoroughly mixed. Peanut seeds were sown into hills, at 50 cm between ridges and 10 cm between hills. After 15 days of sowing, plants were thinned to secure one plant per hill.
After 75 days of sowing, five plants were uprooted at random from the second row of each plot to evaluate the nodula-tion status, N2-ase activity, plant dry matter and its N-contents. At harvest, ten guarded plants were randomly taken from the second inner two rows of each experimental unit to determine yield components, namely weight of pods and seeds/plant, 100-seed weight, as well as crude protein and shelling percentages. Plants in the middle three ridges of each plot (3 m2) were harvested and their pods were air dried to calculate pod yield (ardab/feddan) and straw yield (ton/fed).
Methods of analysis
- Soil properties and compost traits were determined according to Piper (1950) and Page et al. (1982).
- Nitrogenase enzyme activity in fresh roots was measured
using acetylene reduction assay (ARA), as described by
Hardy et al. (1973).
The oven dried plant materials were wet digested using a mixture of pure HClO4 and H2SO4 at a ratio of 1:1, according to Jackson (1973). Total nitrogen content was assayed according to the methods of Page et al. (1982). Seed crude protein percentage was calculated by multiplying N% by 6.25 (AOAC, 1990). Shelling% was calculated according to the equation:
Shelling % =
Seed weight/plant Pod weight/plant
- The experimental data obtained were subjected to analysis of variance (ANOVA), according to the procedures outlined by Snedecor and Cochran (1980).
Results and discussion
Ability of the tested microorganisms to exhibit some PGPproperties in vitro condition
Data in Table 3 present some of PGP-related properties of the tested microorganisms. In general all of the tested microorganisms were apparently able to trigger PGP-properties in in vitro conditions.
Results showed that P-solubilization is the common feature of all of the tested microorganisms grown on synthetic media as expressed by halo clarification zone formed around their colonies (zone diameters ranged from 2.85 to 3.15 cm). However, T. harzianum appeared to be superior to the other microorganisms, as it produced 3.15 cm of clear zone and Bradyrhizobium sp. displayed the lowest capacity (2.85 cm). The clear zone caused by the tested microorganisms may indicate excretion of particular groups of organic acids, which have high affinity to chelate the calcium ions. The ability of bacteria and fungi to dissolve the precipitated phosphorus depends on its efficiency to produce inorganic and organic acids and/or CO2 (Antoun et al., 1998 and Vargas et al., 2009). In this concern, Altomare et al. (1999) and Neumann and Laing (2006) reported that the capability of the plant-growth-promoting and biocontrol fungus to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of che-lating metabolites, and redox activity. Verma et al. (2010) and Yadav et al. (2011) reported that fungi possess greater ability to solubilize rock-phosphate than bacteria and it is preferred to use fungal P-solubilizers arguing that bacterial strains can lose their ability to solubilize P after several cycles of
Table 3 Evaluation of the ability of the used microorganisms to exhibit some PGP-properties in in vitro conditions.
Microorganism P-solubilization IAA-production Siderphoresd production Cyanogens (HCN)
Zonea clarification Zone diameter (cm) Colorb intensity 1g/mlc color intensity color intensity
Bradyrhizobium spp. + + + 2.85 ++ 11.80 - -
Serratia marcescens + + + 2.92 +++ 19.60 ++ +
Trichoderma harzianum + + + 3.15 ++ 10.55 ++ +
—, Negative result; +, low; ++, moderate; + + +, high.
a Diameter of the clarification zone around colonies on DCP media plates. b,c Intensity of the pink to red color and the quantity of IAA produced (ig/ml) in liquid culture. d Intensity of the orange halo around colonies on the chrome azurol S agar plates.
Table 4 Effect of co-inoculation with the different rhizo- microorganisms on nodulation status of peanut roots after 75 days from
sowing.
Treatments Root nodules
Season I Season II
Number/plant Dry weight (mg/plant) Number/plant Dry weight (mg/plant)
Uninoculated 83.25 165.75 120.50 243.75
Bradyrhizobium spp. (Br.) 183.00 287.00 208.00 330.75
Br. + Serratia marcescens (S.) 246.5 461.00 264.00 493.75
Br. + Trichoderma harzianum (T.) 213.75 395.25 241.25 388.25
Br. + S. + T. 239.00 428.75 259.75 472.00
L.S.D. at 0.05 26.370 36.980 16.360 45.950
in vitro culture. Therefore, the unavailable forms of phosphorus can be partially dissolved and enhance its availability against the adverse conditions by the action of phosphate dissolving microorganisms naturally occurring or introduced into the soil (Dobbelaere et al., 2004 and Abdel-Wahab et al., 2008).
The results originated from both qualitative and quantitative assays of IAA reflected the ability of all tested microorganisms to produce indole compounds. The three tested microorganisms exhibited a pink to red color with a little variation in intensity. In the quantitative measurements, the highest value of auxin production was obtained by S. marcescens, followed by Bradyrhizobium as they produced 19.60 and 11.80 p,gml_1, respectively, while T. harzianum produced nearly lower amount of IAA being 10.55 p,gml_1. Indeed, a high proportion of rhizo-microorganisms are able to produce plant growth hormone, i.e. indole acetic acid, which acts to stimulate root growth and provides it with more branching and larger surface area. In fact, many investigators consider the indole secretion, by PGPRs, as a vital mechanism to clarify plant promotion (Glickman et al., 1998; Zahir et al., 2004; Ab-del-Wahab et al., 2008; Verma et al., 2010).
Another interesting trait of PGPR's is the ability to secrete ferric-specific ligands, which are generally termed as sidero-phores. Numerous PGPRs have the ability to produce biocide compounds, including cyanide that has poisonous phytopath-ogenic agents. Data in Table 3 declared that the tested microorganisms, except Bradyrhizobium, were able to excrete siderophores (which were indicated by moderate yellow-orange halo around their colonies due to removing the color from CAS dye-Fe III-complex) and low cyanide (expressed as a weak orange-red pigmentation). In fact, under iron-limiting conditions, some microorganisms produce a range of low-molecular weight compounds, namely siderophores which are able to acquire ferric iron. These iron chelators are thought to sequester the limited supply of iron available in the rhizo-sphere, thereby depriving pathogenic fungi of this essential element and consequently restricting their growth, as well as contribute to iron uptake and transport in the plant. Thus, cyanide and siderophores have an important role in the bio-control activity against soil borne phytopathogens, beside the essential function of siderophores in the improvement of iron nutrition. These results are in accordance with those obtained by Press et al. (2001), Zahir et al. (2004), Neumann and Laing (2006). While, Dowling and O'Gara (1994) reported that HCN production can reduce plant growth, but the net effect improved plant development leading to more growth, consequently increased the yield of agricultural crops.
Table 5 Effect of co-inoculation with the different rhizo-microorganisms on the activity of nitrogenase enzyme of peanut roots after 75 days from sowing.
Treatments N2-ase activity(imol
C2H4/g d.wt nodules/h)
Season I Season II
Uninoculated 9.74 10.59
Bradyrhizobium spp. (Br.) 19.07 18.22
Br. + Serratia marcescens (S.) 22.69 22.07
Br. + Trichoderma harzianum (T.) 20.94 21.34
Br. + S + T. 20.13 19.90
L.S.D. at 0.05 1.580 3.115
In view of the above results, it could be concluded that the microorganisms were able to exhibit PGP-properties, which may display several modes of beneficial action. This finding was emphasized by other investigators (Bertrand et al., 2000; Vargas et al., 2009; Verma et al., 2010).
Response of peanut plants to co-inoculation with Bradyrhizobia and rhizo-microorganisms
Root nodulation status
The nodulation features of the peanut plants as affected by inoculation with bradyrizobia and rhizo-microorganisms after 75-days from sowing are presented in Table 4. The data revealed that the uninoculated peanut had the least number of nodules, being 83.25 and 120.50 nodule/plant and recorded the least nodule dry weights, 165.75 and 243.75 mg/plant in both growth seasons. The results suggest that the presence of native rhizobia of peanut in the experimental soil is of inadequate number, having a low efficiency of nitrogen fixation. These results point out to the necessity of using effective strains of Bradyrhizobium. This observation was also reported by El-Sawy et al. (2006), Mekhemar et al. (2007). Inoculation of peanut with bradyrhizobia exerted a great improvement in nodulation status, which led to significant increases in number and dry weight of nodules in comparison to the uninoculated treatment. This could be observed from the striking differences between the inoculated and uninoculated treatments and emphasized the vital importance to continue inoculation of peanut seeds successively with effective strains. In this concern, Minamisowa et al. (1992), Vlassak and Vanderleyden (1997) mentioned that the problem of inoculated legumes in most countries is the occurrence in their soils of highly competitive
Table 6 Effect of co-inoculation with the different rhizo-microorganisms on the plant dry weights and N-content of peanut plants
after 75 days from sowing.
Treatments Dry weight (g/plant) N-content (mg/plant)
Shoot Root Shoot Root
Season I
Uninoculated 18.78 1.71 500.50 20.98
Bradyrhizobium spp. (Br.) 26.40 1.90 792.00 30.30
Br. + Serratia marcescens (S.) 30.65 2.05 832.75 33.73
Br. + Trichoderma harzianum (T.) 27.73 1.91 799.50 31.18
Br. + S. + T. 27.20 1.92 802.50 31.25
L.S.D. at 0.05 3.508 0.207 119.60 4.472
Season II
Uninoculated 21.22 1.76 557.93 26.98
Bradyrhizobium spp. (Br.) 28.69 2.13 804.10 39.47
Br. + Serratia marcescens (S.) 34.68 2.36 1161.88 46.05
Br. + Trichoderma harzianum (T.) 30.89 2.22 925.63 41.68
Br. + S. + T. 33.18 2.33 1020.55 44.18
L.S.D. at 0.05 2.472 0.097 85.980 1.774
indigenous populations of N2-fixing strains, which in many cases are less efficient than the inoculated strains.
Moreover, co-inoculation of peanut with bradyrhizobia and any of the tested microorganisms tended to remarkably improve nodule number and nodule dry weight, in both seasons. However, Serratia surpassed the other treatments as it gave 34.70% and 26.92% increase in nodule number and 60.63% and 49.28% in nodule dry weight, respectively, in both tested season, more than bradyrhizobia inoculated plants. The second rank of increases was recorded with the mixture treatment (Serratia + T. harzianum) giving 30.60 and 24.88% increases in nodule number and 49.39% and 42.71% increase in mass of nodular tissues, respectively, for the same above-mentioned order. Improvement of nodulation pattern may oc-curr by providing the peanut-bradyrhizobia system with some synergistic substances (Table 3), such as auxins, flavonoids-like compounds and siderophores, which enhance root proliferation and provide more infection sites occupied by rhizobia and in synchronism enhancing the survival and activity of microsymbionet in the peanut rhizosphere. A range of evidences have been reported by many investigators on PGPR stimulation of nodulation, as well as creation of more infection sites on the hairs and epidermis of the leguminous plant roots (Vessey and Buss, 2002; Kloepper, 2003; Abdel-Wahab et al., 2008; Verma et al., 2010).
Nitrogenase activity of peanut root nodules Nitrogen fixation by rhizobia is evidenced by the rate of acetylene reduction by root nodules, as well as the quantities of N-assimilation by the seedling shoots.
Nitrogenase activity of peanut root nodules as affected by co-inoculation with Bradirhizobium and tested PGP-microorganisms are presented in Table 5. The results revealed that, there were various values of nitrogenase activity among different inoculation treatments in both seasons of the study. The uninoculated plants showed low a N2-ase activity, i.e. 9.74 and 10.59 imol C2H4/g d.wt of nodules/h, respectively. Rhizo-bial inoculation increased the rate of acetylene reduction by root nodules as they recorded 19.07 and 18.22 imol C2H4/ g d.wt nodules/h, respectively, for the successive seasons.
The maximal rates of acetylene reduction in both seasons were 22.69 and 22.07 imol C2H4/g d.wt of nodules/h, for Bradyrhizobium conjugated with Serratia. The other tested treatments (Bradyrhizobium conjugated with Trichoderma or with a mixture of PGPR's) came at the second rank and achieved values of nitrogenase activity 20.94 and 20.13 imol C2H4/g d.wt of nodules/h in the first season and 21.34 and 19.90 imol C2H4/g d.wt of nodules/h in the second one, respectively. the presence of PGPR in the rhizosphere may enhance legume nodulation and nitrogen fixation by affecting signal exchange between the plants and rhizobia. These results are in accordance with those obtained by Zhang et al. (1996), Bai et al. (2002), Neumann and Laing (2006) who found that co-inoculation of some PGPR's along with effective rhizobia stimulated legumes growth, nodulation and nitrogen fixation.
Plant dry matter and its nitrogen content Plant dry matter and its nitrogen content as affected by Bradyrhizobium inoculation with different PGPR's are given in Table 6. Data exerted that bacterization of peanut seeds with bradyrhizobia significantly increased the plant dry matter as compared with the uninoculated plants. In both seasons, increases in shoot dry weights were 40.58% and 35.20% and those in roots were 11.11% and 21.02%, respectively, above the uninoculated plants. This could be due to the essential role of Bradyrhizobium in enhancing plant growth and N2-fixation as reported by Mekhemar et al. (2005).
The synergy inoculations between bradyrhizobia and any of the tested PGPR's exhibited increases of peanut shoots and roots, relatively to the untreated treatment or that inoculated with Rhizobium only. Treatments comprising Serratia, Tricho-derma or their mixture confirmed their synergistic interaction to stimulate plant growth and induced 63.21%, 47.65% and 44.83% increases in shoot dry weight in the first season and 63.43%, 45.57% and 56.36% in the second season, respectively, over the uninoculated control. The corresponding values of root dry weights were 19.88%, 11.69% and 12.28%, in the first season, and 34.09%, 26.14% and 32.39%, in the second season, respectively, for the same order mentioned above. The promotive impression on the plant growth was
Table 7 Effect of co-inoculation with the different rhizo-microorganisms on peanut yield and its components.
Treatments Pod yield Straw yield Seed Straw 100-seed Pod weight Seed weight Shelling (%)
(ardab/fed)a (ton/fed) protein (%) protein (%) weight(g) (g/plant) (g/plant)
Season I
Uninoculated 16.34 1.60 22.99 12.93 77.07 64.89 40.87 64.13
Bradyrhizobium (Br.) 23.02 2.05 26.46 14.12 80.77 72.26 49.66 68.79
Br. + S. marcescens (S.) 25.92 2.18 27.22 14.65 84.85 74.83 52.67 70.38
Br. + T. harzianum (T.) 25.35 2.01 26.54 13.91 80.87 72.47 49.90 69.06
Br. + S. + T. 24.11 1.96 26.21 13.75 80.93 71.91 49.61 68.99
L.S.D. at 0.05 0.709 0.097 0.618 0.529 1.415 0.614 0.581 1.626
Season II
Uninoculated 17.35 1.63 23.14 13.34 77.59 65.97 42.98 65.20
Bradyrhizobium (Br.) 23.67 2.18 26.65 14.46 83.33 71.97 50.13 69.65
Br. + S. marcescens (S. ) 27.00 2.32 27.80 14.92 86.89 74.54 52.03 69.81
Br. + T. harzianum (T.) 26.05 2.27 26.70 14.20 85.01 73.97 50.16 67.82
Br. + S. + T. 24.49 2.25 26.56 13.84 84.64 73.60 50.43 68.52
L.S.D. at 0.05 0.643 0.069 0.485 0.244 0.699 1.543 1.077 2.114
a One ardab = 75 kg.
obvious with S. marcescens followed by the mixture treatment, which may be attributed to one or more of PGP-related properties indicating an increase in root surface area and enhancement of nutrient uptake capacity. The promotion effect of PGPR's on plant vigor has been reported by many investigators (Asghar etal., 2002; Bai etal., 2003; Tilak etal., 2006; Ya-dav and Verma, 2009).
Efficiency of nitrogen fixation performance is expressed by nitrogen accumulation in the plant tissues. Data in Table 6 exhibited that the percentage increases in total N-content of peanut shoots and roots reached to 58.24% and 44.42% in the first season and 44.12% and 46.29% in the second season, due to the sole bradyrhizobial inoculation, then it was magnified as a result of co-inoculation with the various PGPRs. The percentage increases in the shoot N-content were 66.38%, 59.74% and 60.34%, in the first season and 108.25%, 65.90% and 82.92%, in the second season, due to co-inoculation with S. marcescens, T. harzianum and their mixture, respectively, above the uninoculated control. The corresponding increases in peanut roots were 60.77%, 48.62% and 48.95%, in the first season and 70.68%, 54.48% and 63.75%, in the second one, respectively. These results confirmed that co-inoculation of peanut with S. marcescens followed by the mixture treatment resulted in pronounced accumulation of nitrogen in shoot and root tissues. Hence, results emphasized the key role of PGPRs co-inoculation in the improvement of biological nitrogen fixation by peanut-brady-rhizobia system and greatly helped in increasing the root biomass and thus indirectly enhanced absorption of nutrients from surrounding environment. These results are in the same line with those obtained by Abdel-Wahab et al. (2008), Sad-aghiani et al. (2008), Verma et al. (2010).
Peanut yield and some yield attributes
Pod and straw yields, seed and straw protein contents, hundred seed weight as well as pod and seed weight/plant and shelling percentage of peanut crop along the two consecutive seasons, as affected by co-inoculation with bradyrhizobia alone or in combination with Serratia and/or Trichoderma are given in Table 7. Results elicited that the sole inoculation with Bradyrhizobium resulted in significant increases in pod
yield (40.88% and 36.43%) during the two growth seasons above the uninoculated treatment. Co-inoculation of peanut with bradyrhizobia and any of the tested PGPRs significantly magnified the peanut yield, as compared to the uninoculated control. However, peanut plants exerted high responses to the dual inoculation with Bradyrhizobium and Serratia, which surpassed the other co-inoculation treatments. For instance, S. marcescens, T. harzianum and their mixture attained increases in pod yield, i.e. 12.60%, 10.12% and 4.74%, in the first season and 14.07%, 10.05% and 3.46%, in the second one, respectively, above the Bradyrhizobium treatment. Data of straw yield behaved similarly to the pod yield and exhibited relatively high values (2.18 and 2.32 ton/fed) by using Serratia in combination with Bradyrhizobium, while the other inoculation treatments showed higher values, as compared with the uninoculated plants, with no significant differences between them. In fact, PGPRs have been shown to greatly improve the productivity and quality of many legumes, when they co-inoculated with rhizobia. This synergistic effect may be elucidated by their ability to enhance the N2-fixation performance, as well as nutrients availability and uptake from soil, which results in the production of substances like hormones, sidero-phores, phosphate solubilization and improvement of nutrients and water uptake. These results are in harmony with those obtained by Tilak et al. (2005), Abdel-Wahab et al. (2008), Yadav and Verma (2009) and Verma et al. (2010).
Additionally, data of seed and straw protein contents (Table 7) followed a similar pattern to that of pod and straw yields. Inoculation of peanut seeds with bradyrhizobia alone resulted in increasing seed and straw protein contents by 15.09% and 9.20%, in the first season and they were 15.17% and 8.40%, in the second one, respectively, above the uninoc-ulated treatment, such increases were improved to 14.01% to 18.40% and 6.34% to 13.30%, in the first season and 14.78% to 20.14% and 3.75% to 11.84%, in the second one, respectively, due to the co-inoculation with the tested microorganisms. These positive results of dually inoculated peanut plants could be ascribed to the promotive effects of PGPRs, which exerted their influence on early nodulation (increased number of nodules, higher N2-fixation rates) and a general improvement of root development and consequently nutrient
uptake (Luz, 2001; Kloepper, 2003; Zahir et al., 2004; Abdel-Wahab et al., 2008).
Regarding the effect of co-inoculation treatments on peanut yield components, data in Table 7 illustrated also that Bradyrhizobium inoculation individually or in combination with any of the tested microorganisms caused significant increases in all peanut yield components, as compared with the uninoculated treatments. This trend was true in both seasons of the study. The results again confirmed the superiority of co-inoculation with Bradyrhizobium and Serratia treatment in achieving the highest values of 100-seed weight, pod weight, seed weight and shelling (84.85 g, 74.83 g/plant, 52.67 g/plant and 70.38%) in the first season and (86.89 g, 74.54 g/plant, 52.03 g/plant and 69.81%) in the second one, respectively. Co-inoculation with Bradyrhizobium and Trichoderma treatment, generally, came in the second position giving 80.87 g, 72.47 g/plant, 49.90 g/plant and 69.06%, in the first season and 85.01 g, 73.97 g/plant, 50.16 g/plant and 67.82%, in the second season, respectively. These results may be attributed to the nature of root exudates, which act as suitable substrates for the associative microorganisms, that release plant promoting substances mainly indole acetic acid, gibbrellines and cyt-okinines. These promotive effects of PGPRs could stimulate plant growth, absorption of nutrients and their efficiency, as well as the metabolism of photosynthates. These results stand in accordance with those obtained by Kloepper (2003), Tilak et al. (2005), Verma et al. (2010).
From the mentioned results, it could be concluded that exploitation of co-inoculation with rhizobia and plant growth promoting rhizo-microorganisms is becoming an efficient strategy for enhancing the productivity of legumes, as well as to provide legume plants with natural bioprotection against phytopathogens under sustainable agriculture system (Luz, 2001; Bai et al., 2002; Zaidi and Khan, 2007; Verma et al., 2010).
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