**3. Production of arbuscular mycorrhizal fungi bio-fertilizers**

The commercial history of bio-fertilizers began with the launch of 'Nitragin' by Nobbe and Hiltner, a laboratory culture of Rhizobia in 1895, followed by the discovery of Azotobacter and then the blue green algae (BGA) and a host of other micro-organisms. Azospirillum and Arbuscular mycorrhizal fungi are fairly new discoveries [6]. Industrial manufacturing of AMF as crop inoculants is relatively new and, despite the practical demonstrations of their efficiency, their adoption by crop producers has been slow, most likely due to the quality and efficiency of marketed products [11]. In fact, production of previously selected AMF for their use as bio-fertilizers began in the decade of 1990 in a large part of the world [25]. Few companies throughout the world have manufactured and commercialized AMF inoculant using either a single AMF species or mixtures of AMF species that may include plant growth promoting rhizobacteria (PGPR) or other symbiotic and/or biocontrol fungi [11].

The production of inoculum differs from fungi family to another. Arbuscular mycorrhizal fungi are strict obligatory symbionts, they dependent on the presence of a host plant to accomplish their development and their multiplication. The inoculum producer is then required to co-cultivate the "fungus-host plant" complex. Without the use of host plants it would be impossible to complete the mycorrhizal life cycle until the production of new propagules/spores [7]. In addition to this monosporic inoculant, it is possible to produce inoculant with different native species with greater ease and speed [7]. In comparison with the commercial inoculant, it has a low cost, higher taxonomic diversity, and the use of locally adapted species [25, 26], which increases the chances of positive effects on the plant and avoid the introduction of exotic species [27]. The use of AMF inoculant produced from the forest soil is the most reliable and recommended method because of its high species diversity, the potential to accelerate the ecological restoration of the soil environment and to promote the germination and growth of the plants [9, 25–27].

#### **3.1 Conventional method of AMF bio-fertilizers production**

This method consists on AMF multiplication on pot culture with selective host plant under controlled conditions in a greenhouse or in a grow room [7].

#### *3.1.1 Mixture of AMF species bio-fertilizers production*

Native soil sampled from different plots of same natural sites must be mixed together to create one composite sample. The obtained mixture was distributed into pots (500 mL disposable cups) which were sown with trapping plant aiming to multiply and restore infective structures of the AMF species present in the trap cultures [27], and then kept in a greenhouse for four month. Two plant species are commonly used for trapping culture: cover "*Trifolum repens*" and leek "*Allium porrum*" [7, 27], But use of other legumes is also permitted such as Alfalfa "*Medicago sativa* [13] *Brachiaria* sp. [27]. Once the four month over, the areal part of plants is catted and the soils are mixed with roots for preparing a new plantation for other four months. Simultaneously, at each month, one pot of each plot was taken for analysis, using 50 g of soil for AMF spore isolation and identification and the roots for evaluation of the mycorrhizal colonization rate [26, 27]. A minimum of 12 months is required to obtain a good product, but the ideal is 24 months [13, 20]. Therefore, the obtained inoculums consist of different types of propagules: spores, fungal mycelium and fragments of mycorrhizal roots [7]. Multi-species products are closer to natural conditions because in ecosystems it is rare to encounter only one species of mycorrhizal fungus. The presence of several fungal species allows the inoculum to respond to a greater diversity of culture conditions.

#### *3.1.2 Monospecific AMF species bio-fertilizers production*

Production of monospecific AMF bio-fertilizer is based on the use of one selective AMF spore species isolated from natural soil using wet saving method [28], and cultivated on trapping culture with appropriate plant species on pot culture of 15 cm. Three months are needs to obtain AMF multiplication. Verification of AMF sporulation must be done each 20 days to one month. At same sanitary tests can also be performed to ensure that no contamination by parasitic fungi or sporulation of other AMF species has occurred. After four months, monospecific spores are ready for inoculation on seedlings of desired crops. In fact, [29] reported that Rhodes grass (*Chloris gayana*) is the best host for mass multiplication of *Glomus fasciculatum*. Bahia grass (*Paspalum notatum*) was used for multiplication of *Glomus deserticola* [27].

#### **3.2 In vitro technic of AMF bio-fertilizers production**

In vitro technic is an aseptic multiplication of AMF on roots cultivated on synthetic medium under sterile conditions. However, this technic started with the early work of [30], and subsequent development by [31, 32] and just after by [33], these authors developed the monoxenic cultivation system to produce contaminant-free AMF, allowing the realization of large-scale production under strictly controlled conditions [34]. The In vitro production of AMF bio-fertilizers consist on the extraction of potential viable propagules from soils with surface sterilization before optimization of growth conditions for germination under aseptic conditions. This aseptic technic consists on cultivation of number of AMF species in association with transformed host roots on synthetic growth medium [33]. Chabot et al. [35] established cultures from surface sterilized spores as starter material and produced 750 spores in 30 ml medium after a period of 4 months of growth in a mono-compartmental petri plate system. This is followed by the association of the propagules with a suitable excised host root for propagule production and recovery. Another system of in vitro AMF multiplication was developed by St Arnaud et al. [36], they used a bi-compartmental Petri plate system and obtained 15,000 spores in 3–4 months. Douds [37] improved this bi-compartmental system by replacing the medium in the distal compartment by fresh medium at regular intervals and obtained 65,000 spores in the distal side of the bi-compartment in a period of 7 months [34]. This technic of bi-compartment petri plate permitted to produce more than 250,000 propagules in 10 ml of medium, which made this technology attractive for industry. However, many process controls must be done to reduce the level of contamination, what should not exceed 3–5% [7, 34]. Once the AMF product is obtained; mass-produced propagules are then formulated in an utilizable form and stored before application to the target plant [7–34, 37]. This technic facilitates the efficient utilization of space and energy in the production system, using solid-state fermentation. Since the technology is more dependent on personnel, it lowers the number of mandays and achieves higher productivity [34].

The use of this technology remains useful for in vitro laboratory tests, but the mycorrhizal inoculum thus obtained (artificial environment on genetically modified roots) is not suited to applications in the agricultural field, providing overall unsatisfactory results [4].

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**Table 2.**

**Inoculum production technic**

Conventional method

Conventional method

Conventional method

Conventional method

On-farm method

*Native Arbuscular Mycorrhizal Fungi and Agro-Industries in Arid Lands…*

**Specific abiotic stress/zone**

Drought stress/Saoudian areas

Calcareous/Tunisian areas

Morocco

Draught/ Tafilalet- Morocco

Heavy metal Polluted soil/Oran- Algeria

In vitro method Draught/Tafilalet

*AMF bio-fertilizers produced in arid lands.*

**3.3 Production of arbuscular mycorrhizal fungi bio-fertilizers in arid lands**

Both conventional method and in vitro methods are practiced in arid areas to produce AMF bio-fertilizers. Several researches was focused on the increasing of plant production in arid land using AMF inoculum (**Table 2**), conventional method with AMF mixture was the most important technic of production adopted. Nevertheless, in vitro technic was also practiced such as by the energy and resource institute of India (TERI) [34]. They based on the faculty of Glomus genus to provide the possibility of using colonized roots as inoculum material with up to 80% of root colonization attained at 4 and 12 weeks [34, 39]. Despite, arid lands are often localized in underdeveloped country with low economical budget who cannot afford to allot enormous amounts in order to produce bio-fertilizers, so the conventional method remains the most appropriate technique under these conditions. In addition ecological conditions of arid lands give them specific characteristics that are not accommodating with all AMF strain. For that production of native AMF bio-fertilizers adapted to local conditions and to specific abiotic stress is essential [13]. Labidi et al. [39] developed a native AMF bio-fertilizer adapted to calcareous arid Tunisians soils. Abdelsalam et al. [38] produced AMF inoculum of desert saoudian areas using *Sorghum halepense* as trapping plant. Bencherif et al. [13] developed a specific AMF bio-fertilizer for arid saline soils. It is noted that in

**Propagule richness Infection** 

20 g of Sudan grass rhizosphere with 950mycorrhizal spores and 0.5 g of colonized roots

*Septoglomus constrictum*, *Funneliformis geosporum*, *Glomus fuegianum*, *Rhizophagus irregularis* et *Glomus* sp

> Rhizophagus irregularis

Glomus sp., Sclerocystis sp., Acaulospora sp

Acaulospora sp., Archaeospora sp., Glomus sp., Claroideoglomus sp., Ambispora sp., Diversispora sp

and *Claroideoglomus etunicatum*

Drought *Rhizophagus clarus*

**level**

**References**

et al. [38]

et al. [39]

et al. [5]

Meddich et al. [5]

Sidhoum and Fortas [40]

et al. [26]

78,5% Abdelsalam

90% Labidi

100% Meddich

80% Moreira

15, 9, 1, spores, /gr of soil

<50%, <20%, <5%,<5%, <5%/g of soil

*DOI: http://dx.doi.org/10.5772/intechopen.94084*

*Native Arbuscular Mycorrhizal Fungi and Agro-Industries in Arid Lands… DOI: http://dx.doi.org/10.5772/intechopen.94084*

*Mycorrhizal Fungi - Utilization in Agriculture and Forestry*

to respond to a greater diversity of culture conditions.

*3.1.2 Monospecific AMF species bio-fertilizers production*

**3.2 In vitro technic of AMF bio-fertilizers production**

days and achieves higher productivity [34].

unsatisfactory results [4].

*deserticola* [27].

to natural conditions because in ecosystems it is rare to encounter only one species of mycorrhizal fungus. The presence of several fungal species allows the inoculum

Production of monospecific AMF bio-fertilizer is based on the use of one selective AMF spore species isolated from natural soil using wet saving method [28], and cultivated on trapping culture with appropriate plant species on pot culture of 15 cm. Three months are needs to obtain AMF multiplication. Verification of AMF sporulation must be done each 20 days to one month. At same sanitary tests can also be performed to ensure that no contamination by parasitic fungi or sporulation of other AMF species has occurred. After four months, monospecific spores are ready for inoculation on seedlings of desired crops. In fact, [29] reported that Rhodes grass (*Chloris gayana*) is the best host for mass multiplication of *Glomus fasciculatum*. Bahia grass (*Paspalum notatum*) was used for multiplication of *Glomus* 

In vitro technic is an aseptic multiplication of AMF on roots cultivated on synthetic medium under sterile conditions. However, this technic started with the early work of [30], and subsequent development by [31, 32] and just after by [33], these authors developed the monoxenic cultivation system to produce contaminant-free AMF, allowing the realization of large-scale production under strictly controlled conditions [34]. The In vitro production of AMF bio-fertilizers consist on the extraction of potential viable propagules from soils with surface sterilization before optimization of growth conditions for germination under aseptic conditions. This aseptic technic consists on cultivation of number of AMF species in association with transformed host roots on synthetic growth medium [33]. Chabot et al. [35] established cultures from surface sterilized spores as starter material and produced 750 spores in 30 ml medium after a period of 4 months of growth in a mono-compartmental petri plate system. This is followed by the association of the propagules with a suitable excised host root for propagule production and recovery. Another system of in vitro AMF multiplication was developed by St Arnaud et al. [36], they used a bi-compartmental Petri plate system and obtained 15,000 spores in 3–4 months. Douds [37] improved this bi-compartmental system by replacing the medium in the distal compartment by fresh medium at regular intervals and obtained 65,000 spores in the distal side of the bi-compartment in a period of 7 months [34]. This technic of bi-compartment petri plate permitted to produce more than 250,000 propagules in 10 ml of medium, which made this technology attractive for industry. However, many process controls must be done to reduce the level of contamination, what should not exceed 3–5% [7, 34]. Once the AMF product is obtained; mass-produced propagules are then formulated in an utilizable form and stored before application to the target plant [7–34, 37]. This technic facilitates the efficient utilization of space and energy in the production system, using solid-state fermentation. Since the technology is more dependent on personnel, it lowers the number of man-

The use of this technology remains useful for in vitro laboratory tests, but the mycorrhizal inoculum thus obtained (artificial environment on genetically modified roots) is not suited to applications in the agricultural field, providing overall

**82**
