Application of arbuscular mycorrhizal fungi in combination with nitrogen fixing bacteria and other potential soil microbes as biofertilizer for soybean plant

The use of mycorrhizae as phosphate solubilizing fungus has been known since decade. Mycorrhizae could promote the growth of almost all species of plants. Biofertilizer product based on mycorrhizae fungus has been produced commercialization and easily found in the market. This study was aimed to understand the positive effect of mycorrhizae inoculation when it mixed with other microbes especially nitrogen fixing bacteria and growth hormone production bacteria. An experiment was set up at green house to run the activities of microbes inoculation on soybean plants. Soybean was mainly used for testing the potential of soil microbes since the target of the experiment is increasing the production of soybean seed and decreasing the used of chemical fertilizer. The result of this study showed that multi strains inoculation was better that single inoculation. Single mycorrhizae inoculation produced slightly higher number of pods. However when the mycorrhizae was combining with other potential microbes the number of pods produced by the plants even significantly higher than only single inoculation. Updating the quality of biofertilizer is needed by including the biodiversity of soil microbes to gain the promotion of green farming program.


Introduction
Soybean is the most promise crops for food after rice. Since several years ago the Government of Indonesia has been announced the program of self sufficient of soybean. Many programs have been launched to develop the soybean production including their agriculture management. The used of biofertilizer is recommended to reduce the requirement of chemical fertilizer which mainly caused soil pollution. Beside of that, the used of high doses of chemical fertilizer may caused the unhealthy quality of food. Therefore the used of potential microbes as biofertilizer is recommended as it will maintain the environment and confirmed the sustainable of friendly agriculture system.
Mycorrhizae is one of the potential soil fungus which could be use as biofertilizer. This fungus has potential on fixing and solubilize the phosphorus available in the soil and it could be fulfill the requirement of phosphorous beside others plant nutritions such as micronutrition, minerals and water. Fakuara in 1988 reported that mycorrhizae could increase the absorption of nitrogen, kalium, calsium, magnesium, cupper and mangan including zink also produced the plant growth hormones. Mycorrhizae are very important fungal symbiont in many plants. They act as natural barriers to the soil borne pathogens. By the hyphae that cover the surface of root plant, the mycorrhizae could become the buffer to protect the root plants from the microbial root diseases. The protein of pathogen The assessment of the beneficial of mycorrhizae has been study widely especially on plantation trees also crops. Kabirun [2] reported that mycorrhizae inoculation on upland rice could increase the rice growth, phosphate solubilizing and even increase the production of rice. Inoculation of mycorrhizae could increase the concentration of nitrogen up to 11.5 %, phosphate 14.9 % and kalium 12.2 % on upland rice [3].
The mechanism increasement of plant absorbtion by mycorrhizae was caused by the increase of metabolism system as a result of increasement of oksigen comsumption and phosphatase enzyme [4]. The mycorrhizae produced phosphatase enzyme which reguired for fixing the plant minerals to become available for the plants [5]. Dighton [6] reported that the mycorrhizae hypae was very effective for minerals absorption since the ability to penetrate the soil aggregate is high since the diameter of hyphae is very tiny so it provide more possibility to absorb the minerals.
Legumes plants such as soybean is the most capable plant which could develop the symbiotic living with Rhizobium. This plant was recognize as an important sources of protein for mankind. Rhizobium sp has widely known as beneficial bacteria especially on the ability on processing the nitrogen fixing from the air. The legumes-Rhizobium symbiosis is the most efficient system for Nitrogen fixation, the bacteria will interact with leguminous plant in the host specific manner and form N-fixing root bacteria [7]. Study of the beneficial of Rhizobium has been done widely following by the collection of its biodiversity strains to be conserve as culture collection. Development of this bacteria also has been investigated and tested as biofertilizer for plants and crops. At this stage, the production of biofertilizer based on rhizobium and the application into the plants has been socialize continuously to the farmer.
Pseudomonas sp. bacteria was known as phosphate solubilizing bacteria which found associate on the rhizosplane of root plants. The bacteria was confirmed to be able to mobilized phosphate to become available for the plants. Combination with other bacteria, Bacillus sp. could support the growth of soybean in term of full canopy covered and greenish color of leaf. Further more it could be improved the growth of plant which performed by the height of plants and its was comparable with the height of plants which fertilized with SP-36 for 200 kg per hectar [8]. The production of soybean in acid soil (pH 5.5) inoculated with Pseudomonas sp. significantly increased from 2.06 ton per hectar to 2.20 ton per hectar [9].
Other microbes such as Azospirillum has been known as beneficial microbes since they has potential on nitrogen fixing as well as growth hormone production. Azospirillum known as a rhizosphere bacteria and normally attach with non leguminous plants such as rice, corn etc. However positive effect of combined inoculation with Azospirillum and Rhizobium have been reported for different legumes. Volpin and Kalpunik [7] reported that Glycine max, Pisum sativum, Madicago sativa, Cicer arietinum, Psophocarpus tetragonolobus inoculated with Azospirillum and Rhizobium showed enhancement in early nodulation, increases nodule on main root, total nodule number, nodule weight and nodule specific activity of nitrogen fixation. In this case Azospirillum stimulated efficiency of Rhizobium to nodulate plants root. So, combination of Rhizobium and Azospirillum inoculation was recommended to be applied on growing food crops.
Agrobacterium species from the family of Rhizobiaceae (update scientific name is Rhizobium radiobacter) was known as a phytopatogenic bacteria which is the casual agent of crown gall diseases and Plant Growth Hormone, Rhizobiaceae (PGPR) bacteria beside commonly used as a model bacteria for molecular study, especially used for inserting the foreign gene into plant cell and to produced transgenic plants [9],[10]. Gram negative bacteria has a big size of plasmid namely T1plasmid which contain the gene factor for virulency of bacteria cause plant diseases. Through the gene in T-DNA, production of auxin and indole-3-acetic acid were encoded. In this case the DNA transmission capabilities of Agrobacterium have been vastly explores to insert foreign gene into plant [11]. Arbuscular Mycorrhizal Fungi (AMF) live symbiotically with almost all families of trees, therefore AMF could be develop as biofertilizer for many plants and crops. Soybean is one of target crops for the study. The effectiveness of mycorrhizae inoculation and combination of AMF with other potential microbes especially nitrogen fixation bacteria hopefully will promote the growth of soybean and its production. Endophytes microbes was isolated from forest trees (Shorea selanica) and it was selected for their potential for plant growth hormone production, indole acetic acid (IAA). Isolation of endophytes bacteria from Shorea selanica was done using MP media : Meat extract 5 g, Peptone 10 g, NaCl 5 g Nystatin 100 mg, Distilled water 1 liter [12].

Microorganisms
BIOVAM, arbuscular mycorrhizal fungi was produced in pilot scale production unit located in Cicurug, Sukabumi, West Java, Indonesia by the Sari Daun Farmer community.The production of BIOVAM was conducted in the green house using specific soil collected from mountain area near Sukabumi. The product actually consist of mycorrhizae root infected which could be spread out when its inoculated to the early germinates seed. At a present time the farmer continuously produce the BIOVAM for their own needed. [13]; K2HPO4 0.5 g, MgSO4.7H2O 0.2 g NaCl, 0.1 g, Mannitol 10 g, Yeast Extract 0.5 g, Aquadest 1 liter. Mass production of Bradyrhizobium sp. and Rhizobium sp. were done in YEM Broth and shake at 150 rpm for 4 days incubation periods.

Growth media Bradyrhizobium sp and Rhizobium (DCM) were grown in Yeast Extract Mannitol Broth
Pseudomonas sp. (BPF) was grown on Pikovskaya media (Hi Media) ; Ca3(PO4)2 5 g, NaCl, 0.2 g, KCl, 0.2 g, MgSO4.7H2O 0.1 g, NH4SO4 0.5 g, Glucosa 10 g, Yeast Extract, 0.5 g MnSO47H2O 0,0025 g, FeSO47H2O 0.0025 g , Agar 20 g, Aquadest 1 liter. This media used to screen the phosphate solubilizing bacteria base on their ability to used calcium phosphate for their energy sources. Pikovskaya Broth was used for cultivation of phosphate solubilizing microorganism [14].The inoculated plates were incubated 32 o C for 9 -10 days. Mass production was done by growing the bacteria in Pikovskaya broth media with shake in 150 rpm.

Green house experiments
The experiment was conducted at green house condition in Cibinong Treatment s were arranged using soil media in polybag. Each microbes used in this experiment were prepared ahead before treatment. The bacteria were grown in the broth media normally used for mass production and incubated by shaking according to the propose age. The fungus used in this experiment is a product of arbuscular mycorrhizae biofertilizer (BIOVAM) which have packed as commercial LIPI product.
Seed of soybean was inserted by nitrogen fixing bacteria (Bradyrhizobium sp.) using vacuum technology. Planting activities was done by mixing the soybean seed with the mycorrhizae until the fungus covered the outher part of seed. The other bacteria (endophytic, phosphate solubilizing bacteria and other rhizobium bacteria were inoculated directly in a hole of soil then incolated seed then were put inside the hole. Observation of the plant growth were done starting at early stages (1 month) and continue further from two months old plants until the plant is 3 months old. All parameter were observed as prepared

Results and Discussion
Biofertilizer are the product consisting selected and beneficial living microbes which are added to soil as microbial inoculants. They are gaining importance because of ecofriendly, non-hazardous and non toxic nature. Several organisms such as Rhizobium, Pseudomonas, phosphate solubilizing microorganism, Mycorrhizae, endophytic diazotroph and other soil potential microbes are presently being used as biofertilizer.
A greater part of soil phosphorous, approximately 95 to 99 % is present in the from of insoluble phosphate and hence cannot be utilized by plants [15]. Great proportion of phosphorus in chemical fertilizer becomes unavailable to the plants after its application in the soil [16].
Plant growth -promoting bacteria (PGPB) play an important role in supplementing phosphorous to the plant, allowing a sustainable use of phosphate fertilizer. Microorganism are involved in a range of process that effect the transformation of soil phophorus (P). Phosphate-Solubilizing Bacteria (PSB) is slowly emerging as important organism for soil improvement [17].
The important of vascular arbuscular mycorrhizae as a tool for improving the growth and productivity in diverse grop of plants was recognized [12]. Mycorrhizae is one of soil fungus which have potential on fixing phosphate to be available to the plants beside of that it help on inhibit the soil borne pathogen to infect the root since the hyphae of mycorrhizae cover the surface of roots [18]. Mycorrhizae known since decade as a potential microbes for biofertilizer. There are numbers of mycorrhizae base product found in the market and the quality of them are varies. The quality of biofertilizer become very important to confirm the successfully of the application on supporting the growth of plants. In case of BIOVAM product, the indicator needed to be confirmed was root infected percentage of inoculums before used it. Therefore in every step of production process, the percentage of root infection was analysed.  normally showed after 3 months inoculation. It was observed that control plant did not gave high percentage of infection rate which explained that the growth media used for mass production did not very much consist of mycorrhizae. The indigenous of mycorrhizae present in the soil, sometimes infected the root but often the potential on fixing phosphate was not significant. Method of application the mycorrhizae is simple, by inoculate the product content of chopped infected root. The hyphae of mycorrhizae fungus will then infected the new root development of host plant and further more established the symbiosis living with host plant.
Phosphorus deficiency leads to formation of small leaves, weak stem and slow development. Several mechanism like low soil pH, ion chelation and exchange reaction in the growth environmental have been reported to play a role in phosphate solubilizing by PSMs [17]. By inoculate the BIOVAM, the deficiency of phosphorus will not happened and the growth of plants were supported.
Root nodules of leguminous plants reported could be the host of other endophytes bacteria of diverse genera and species which are unrelated to rhizobia symbiotic nitrogen fixing bacteria [19]. These non-rhizobia nodule endophytes improve plant growth and nodulation when co-inoculated with rhizobium compare to inoculation with rhizobium alone [20]. .  Height of plants indicated the healthies of plants during growing periods. Figure 2 showed the data of soybean plants height at 1 to 2 months old plants. It is observed that the combination of some potential microbes especially combination between mycorrhizae, Rhizobium (DCM) and endophytes for growth hormone production (treatment no. 11) similarly with the combination of two strains of Rhizobium and two strains phosphorus solubilizing bacteria (treatment no 18) provide the best results on supporting the growth of plants compared to control plant (CO) and control nitrogen (CN). The inoculation with five differents microbes resulted approximately 120 cm height of plants, however the normal height of plant is about 50 -75 cm. This positif effect of inoculation open the possibility to develop new composisition of microbes.