An essay about a double inoculation of Arachis hypogaea (L.) by Glomus sp and isolated Bradyrhizobium sp of El Frin site (Wilaya of El Tarf, Northeastern Algeria).
Symbiosis is an association of mutual benefit between two living organisms.
In plants, some symbioses occur through the formation of specialized joint bodies such as nodules Fabaceae and mycorrhizae of most plants [29,31,12,14].
These symbioses have played a fundamental role in the evolution of species . About 400 million years ago, the colonization of land by plants has been associated with the formation of mycorrhizal symbioses, symbiosis between plant roots and soil fungi [9,27,15].
In this symbiosis, the fungus is generally unable to use the complex polysaccharides which are the main sources of carbon in the soil; on the contrary it uses carbohydrates that are excreted or produced by the plant cells. This production is also strongly stimulated by the auxins that the fungus releases [19,15].
The mycorrhizal plant benefits from the association by the increase in root and aerial systems due to the mycorrhizal fungi (MF) that allow it to better use some soil components which are difficult for the roots to access [2,32].
The usefulness of mycorrhizae is not limited only to the phosphorus absorption. Indeed, even the MF intervenes in the absorption and transfer of other elements such as water, copper, and zinc . and may play a protective role in relation to fungal diseases .
In some cases, the symbiotic phenomenon is not about a single symbiotic organism; there may be collaboration between, for example, mycorrhizal fungi and atmospheric nitrogen N2-fixing bacteria (AFB).
Indeed, although present to 80% in the earth's atmosphere, molecular nitrogen represents a major limiting factor in the growth of cultivated plants .
This paradox is due to the fact that the N2 nitrogen molecule is very stable and the higher organisms (eukaryotes) are unable to use it. Only symbiotic bacteria are able to reduce N2 to ammonia NH3 making it absorbable and its production may be higher than the industrial production .
This production is carried out under anaerobic conditions at the nodes' level . These two types of root endosymbioses i.e. mycorrhizal symbiosis and nitrogen-fixing symbiosis play a considerable role in the phosphate and nitrogen nutrition of plants .
It is in this context that we are interested in peanut (Arachis hypogaea) (L.) which is a plant of great alimentary, fodder and industrial interest. It is a herbaceous plant with a strong root system consisting of a primary taproot that sinks to a depth of 1m.
Several studies have shown that peanuts can be naturally nodulated and mycorrhized  and that controlled inoculation could enhance the growth and production of the plant.
The aim of the present work is to search for, isolate Rhizobia and arbuscular mycorrhizal fungi (AMF) from peanut roots that grow in El frin station (north eastern Algeria),then multiply them in a pure cultivation and inoculate them to seeds of peanuts in order to assess the contributions of microsymbionts on the growth and the yield of the plant.
MATERIALS AND METHODS
Presentation of the study site:
The study site is a farm located in El Frin, town of Ain Assel, wilaya of El-Tarf (Fig .1).
The farm was named after the Martyr Abdullah BOUTELLAH and covers an area of 17 hectares. It is bordered to the north by Djebel Boumerchen, to the west by Lake Oubeira, to the south by Kef Bouffa and Kef Rokaba and to the east by the national road No.44.
Crop production is centered on peanut farming which occupies an area of 15 hectares and the production of vegetables (tomato, cucumber) in an area of 1.5 hectares.
Preparation of inocula:
About 300g of soil, randomly selected from the study site, were collected in the first 20 cm in 3 different points. They were properly mixed so that to form a single sample from which 50g were removed to undergo a wet sieving according to Gerdemann and Trappe technique (1963), Daniels and Skipper (1982). It is to sift the soil sample through a series of sieves (50, 100, 150, 300 and 400 pm) superimposed and subjected to a powerful water jet until the water that comes out becomes clear. The selected sieves coresponding to each mesh were observed under a binocular microscope. It showed a great spore diversity which is not the purpose of the present study but from which an extremely abundant morphotype was kept in the 150 pm fraction.
This morphotype named Glomus sp has been the aim of a monospore culture then multiplied on high semi axenically peanut seedlings.
The colonized roots (up to 60%) by Glomus sp were divided into fragments of about 1 cm and used as fungal inoculum in the present study. As far as the rhizobial inoculum, there are two Bradyrhizobium strains that were previously isolated from nodules root of peanut that is grown in the station and multiplied in a pure culture.
They were selected in the exponential phase of growth and were used for coating the peanut germinated seeds.
To investigate the ability of the Peanut in forming rhizobial nodules, as well as its dependence on the AMF, we have conducted a controlled inoculation experiment with a strain of fungus Glomus sp1 (G) and two strains of Bradyrhizobium (Br1 and Br2).
Peanut seeds, previously disinfected with H2O2 to 10 volumes for 15 minutes and rinsed several times with sterile distilled water, were germinated in axenic conditions at 30 [degrees] C for 3 days in Petri dishes containing filter paper moistened with sterile water.
Healthy seeds sprouted and coated with Bradyrhizobium were transplanted into pots containing 5L of the station's soil and sterilized 3times at 100[degrees] for 1 hour.
While transplanting, 1g of mycorrhizal roots was mixed with the soil of each pot and around the hole that was intended to receive the seedlings which were previously coated in the rhizobial culture according to the following procedure:
Br1: Inoculation by Bradyrhizobium 1
Br2: Inoculation by Bradyrhizobium 2
G: Inoculation by Glomus sp
G+Br1: Inoculation by Glomus sp + Bradyrhizobium 1
G+Br2: Inoculation by Glomus sp + Bradyrhizobium 2
NI: Not inoculated
Each treatment was repeated 6 times at a rate of 3graines per pot.
The device was conducted under ambient conditions from May to September 2012 and watering was done with sterile water.
Main Measured Parameters:
the average height of the main stem, the weight of the fresh weight of the aerial part, average number of flowers per plant.
average number of pods and pod size.
--Parameters of symbiosis:
the length of the root system, the weight of the root system, the number and the fresh weight of nodules, mycorrhizal colonization after processing with potash and blue trypan staining according to Phillips and Hayman method (1970), the leaves nitrogen content using the spectrophotometry method (digestion with peroxodisulfate and 2,6-imethyl digestion: DIN EN ISO 11905-1H Determination. ISO 78901, DIN 38405 D9) and the phosphorus content of the leaves usig the spectrophotometry method (365.2 + 3; APHA 4500-PE; DIN EN ISO 6878-D11).
RESULTS AND DISCUSSION
The Br1et Br2 treatment allowed the formation of nodules, and equally the inoculation with Glomus sp enabled effective peanut root colonization. The absence of nodules and mycorrhizae in the non-inoculated processings shows that the device is free from contamination.
Height and fresh weight of air system per plant:
Measuring the height and fresh weight of the aerial system was carried out at harvest time. The average height and weight of the fresh material of all plants is highly significant compared to the control.
Note the superiority of processing with Glomus sp alone or in interaction with Bradyrhizobium.
Dual inoculation remains higher (highly significant) compared to all other processings.
Average number of flowers per plant based on the treatments:
Counting this parameter was carried out in full bloom, seven weeks after the implementation of the cultivation. It led to the values expressed in figure 4. The control treatment allowed the formation of an average of 5 flowers per plant. Br1, Br2 and G sp has doubled the number and the interaction tripled it.
The effects of single inoculation (Br 1, Br 2 and G) and that of the double inoculation (Br1 and Br2 + G + G) are highly significant compared to the control.
Similarly, the dual inoculation is very highly significant compared to the single one.
Number and weights of pods per plant:
The inoculation of the peanut by arbuscular fungi and isolated Bradyrhizobium induced an increase of yield both in pods and weight of their fresh material (Fig 5, 6).
These results are valued by the significant production of flowers at the association level "bacteria--host plant" and "fungus--host plant."
The clear superiority of the interaction Br1+ G and Br2 + G is again demonstrated by Figures 5 and 6. The differences are highly significant. The number and weight of pods are respectively doubled and tripled in these treatments compared to the control.
Compared together, Br1 and Br2 treatments are in favor of the first with a significant difference.
Average size of pods per plant:
Whatever the processing, the average size of pods per plant has improved compared to the control (Fig7 8). The differences are highly significant for Br1, significant to Br2 and G and very highly significant for the interaction Br + G.
Length and average weights of root system per plant:
The values obtained by the double inoculation (Br + G) with respect to the single inoculation (Br1, Br2 and G) and to the control, are highly significant for the length of the root system and very highly significant for its weight.
Many authors [22,6] have shown the effect of endosymbioses on root development. In fact, it is recognized that when preparing the symbiosis with Rhizobium or with Glomus, molecular signals between the plant and the microsymbiont partner take place and and has the effect of the proliferation of root system which would allow the root to achieve the microorganism in the soil and establish the symbiotic relationship with it
Number and average weights of nodules per plant:
The difference is significant between the two bacteria, with the advantage to (Br1).
The absence of nodulation for the witness and for G denotes the absence of contamination and confirms the good soil sterilization.
The leaves of the inoculated plants with the isolates are green to dark green. Non-inoculated plants have yellow to green leaves. This indicates that the inoculated bacteria are efficient as suggested Dommergues and Mangenot  who regard the green color as an appropriate fixation indicator
The amount of nitrogen in various simple treatments (Br 1, Br 2 and G) or in the double inoculation (Br1 + G and Br2 + G) is very highly significant compared to the control (Fig 13).
--The Results obtained through the double inoculation Br1 and Br2 + G + G with respect to the single inoculation Br1, Br2 and G and to the control proved to be very highly significant.
--The Difference between Br1 and Br2 is significant with the former being more effective than the latter.
As regards the comparison between the two bacteria and the fungus, the difference is significant in favor of the bacterial inoculum.
It is demonstrated in the present work that the peanuts can host simultaneously in its roots two types of symbiotic Glomus and Bradyrhizobium. As suggested by Smith and Read  the Rhizobium nodulation is greatly increased in legumes with the presence of endomycorrhizal fungi in the roots.
 stated that dual inoculation by Rhizobium and AMF increases the nitrogen fixation rate, which results in a better growth of the plant.
The numbers obtained by the double inoculation, compared to the single G inoculation Br1et Br2 and the control, are very highly significant for the phosphorus dose.
Compared with each other, both Br1 and Br2 bacteria express significant differences (in favor of Br1) (Fig 14).
Regarding the comparison between the two bacteria and the fungus, the difference is significant,as the last is more effective than Br1et Br2.
As for Parniske  the presence of mycorrhizae allows the absorption of soluble phosphorus both beyond the depletion zone and in sufficiently high amount for the plant, even if this element is not present in the soil only at very low concentrations. It also enables the rise of the mineralization of organic phosphorus in the soil .
Thus, the plants mycorrhized with G indicate higher phosphorus content than non-mycorrhized plants. This result is in accordance with those of Fraga-Beddiar and Le Tacon  according to whom the mycorrhizal symbiosis significantly improves the phosphate nutrition of mycorrhizal plants.
The main objective of our work was to carry out a controlled inoculation of Arachis hypogaea (L.) by two Rhizobium strains and one Glomus strain.
--Concerning the growth of the peanut, this present essay has shown that simple inoculation (Br1, Br2, G) has more than doubled the results compared to the control. The interaction (Br1 + G) and (Br2 + G) has tripled them.
--It Is the same for the parameters of symbiosis where double inoculation has more than tripled values compared to the control.
--Regarding the yield, we find that, whatever the processing is, yields in number and in weight of pods have significantly improved.
The positive effects of nutrients provided by the single or double inoculation on legume yields were observed in several studies. Like all crops, peanut needs nutrients which depending on their availability influence its yields.
These results are quite comparable to those obtained by Rusli et al. . In a literature review on symbiotic nitrogen fixation by tropical legumes, Peoples and Herridge . showed that most parts of research works locate peanut potential fixation from 30 to 130 kg N ha-1. In this literature review, Peoples and Herridge  concluded that without inoculation with selected strains of rhizobia, peanut fixes between 22-49% of its total nitrogen in the atmosphere. The inoculation of the soil by efficient strains can improve the nitrogen fixation potential in peanut which represents 47 to 78% of its nitrogen needs 
This work with encouraging results should be taken under natural conditions i.e. on unsterilized soil to see the effect of the natural soil microflora in interaction with the inoculated strains. It is also essential to identify bacterial and fungal strains used and characterize them the molecular tool.
Associated with mycorrhiza, rhizobial symbiosis open up promising prospects for the Algerian agriculture and at the same time enables it to avoid the massive use of fertilizers the fact that will protect our environment and allow significant budget savings accordingly.
The research goal was to enable us to assess the effect of various processing on the following parameters: increasing yields (and production consequently), decreasing the use and cost of inputs (fertilizers and pesticides), the improvement of product quality as well as the preservation of the environment through sustainable development where chemicals will be lightly used.
Received 28 December 2015; Accepted 28 January 2016; Available online 24 February 2016
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Wided Touil and Arifa Beddiar
Departement of Biology, Faculty of Natural and life Sciences, University of El taref, 36000 El Taref, Algeria.
Address For Correspondence:
Wided Touil, Departement of biology, Faculty of Natural and life Sciences, University of El taref, 36000 El Taref, Algeria.
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|Author:||Touil, Wided; Beddiar, Arifa|
|Publication:||Advances in Environmental Biology|
|Date:||Jan 1, 2016|
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