Diversity of Arbuscular Mycorrhizal Fungi Species Associated with Soybean ( Glycine max L. Merill) in Benin

Arbuscular Mycorrhizal Fungi (AMFs) could be used to sustainably improve crop yields. The present study evaluated the diversity of AMF species associated with soybean (Glycine max L. Merill) in main soybean-producing For the mycorrhization parameters, the mycorhization frequencies did not vary from one production areas to another, unlike the intensities, which significantly varied from one production areas to another (2.31% to 24.62%). Finally, this study revealed that the physico-chemical parameters of the soils had an influence on the other parameters studied. Moreover, there were an abundance and a significant diversification of AMFs associated with soybean in the different production areas, which are influenced by certain physi-co-chemical soil parameters.


Introduction
In Africa, one of the main constraints in agriculture is the constant decline in the level of soil fertility [1] which leads to crop yield losses. Farmers often rely on the use of chemical fertilizers whether registered or not, so as to increase crop productivity. This increased application of chemical fertilizer leads to increased crop yields, while having negative environmental impacts [2] and constitutes a major concern [3].
An alternative to application of chemical fertilizer could be the use of legumes as they are able to symbiose with nodule bacteria (rhizobia) present in most, if not all, tropical soils [4] and these rhizobia possess the nitrogenase complex, an enzyme capable of reducing atmospheric nitrogen (N2) into compounds assimilable by the host plant [4]. This attribute then fertilizes the soil allowing adequate yield in N-deficient soils where non-nodulated crops such as cereals fail.
Maximizing N2 fixation is an economical way to cope with the shortage of expensive nitrogenous fertilizers in tropical countries and this biological nitrogen fixation by legume crops is even beneficial to subsequent cereal crops in rotation or association with the [4].
As a food legume with many agronomic and nutritional benefits [5], soybean (Glycine max L. Merill) is, sometimes considered as a miracle plant [6] and has progressively been adopted by several farmers, especially in Benin. The majority of the soybean farmers do not use fertilizers to improve the soil fertility level claiming that the crop does not need fertilizers [7]. This situation, despite the attribute of soybean plant able to fertilize soils, leads to deficiency in nitrogen, phosphorus and potassium in the soybean-producing soils, reducing the yield worryingly [8] [9].
In Benin, the importance of soybean cultivation is thus reflected in the different sectors of the economy and it appears beneficial to the whole society [10].
However, yields remain low and fluctuate around 1100 kg/ha [11] compared to the potential yield of 4000 kg per hectare [12]. This is said to result from poor agricultural practices and soil erosion leading to deficiency of nitrogen and as-  [7]. It is then crucial to identify the best practices able to increase soybean productivity without degrading soil and environment. One of these practices could be application of arbuscular mycorrhizal fungi (AMF) known to increase crop capacity in soil minerals absorption in general, and phosphorus in particular [13]. [14] Indicated that AMF increase crop absorption of phosphorus in two ways: first, by mineralizing organic phosphorus through activity of phosphatases located in mature arbuscular and intercellular hyphae, and second, by solubilizing insoluble phosphorus (tricalcium phosphate, rock powder) through acid production.
Mycorrhizal fungi are microorganisms naturally inhabiting crop rhizosphere, but the species richness depends on the crop species. The objective of the current study was to evaluate the diversity and density of arbuscular mycorrhizal fungi associated with soybean (Glycine max L. Merrill) cultivation in the major production areas in Benin.

Study Area
The study was carried out in the major soybean production areas of Benin. It covered 13 production areas, falling into 4 agroecological zones. Soil sampling was carried out during the rainy season in September 2017. Four sites were selected in each production areas resulting in a total of 52 samples for the study.
Survey was conducted at each site in the soybean rhizosphere considering producers with at least 1 ha of cultivated area, farming experience (time) and land cultivation history ( Figure 1).

Plant Material
The plant material used was the soybean variety TGX 1910-14F developed by the International Institute of Tropical Agriculture (IITA). This variety has good agronomic and morphological characteristics [7].

Trapping Device of AMF
In order to reveal the diversity of AMF in the sampled soils, a trapping device was set up using the trapping method of [15]. It consisted of growing soybean in pots containing the sampled soils. There were four replicates per soil sample. A total of 52 pots with 2 kg soil were displayed. The trial was daily watered until five days before harvest to create water stress and facilitate the multiplication of mycorrhizal fungi.

Evaluation of Mycorrhization Parameters
Roots were sampled, carefully washed and stained using modified method [16].
The youngest roots were sampled and cut into 1 -2 cm length, immersed into a KOH solution (10%) and heated in a 90˚C water bath for about 45 -60 min. The roots were then rinsed with water and immersed into a 0.5% Trypan Blue solution.  The contents were placed in the water bath for 45 min at 90˚C again. The frequency and intensity of mycorrhization were assessed under a light microscope as described [17].

Spores Extraction
Spores were extracted by the wet sieving method described [18]. Dry soil sample of 100 g was suspended in 500 ml of tap water and stirred. After 30 seconds, the suspension was poured onto a series of four superimposed sieves of decreasing mesh size (500-200-100-50 μm). This was repeated three times in order to re-American Journal of Plant Sciences cover maximum number of spores. For each sieve, the resulting mixture was suspended in tap water and then distributed into centrifuge tubes (Corex tubes).
A viscosity gradient is created by carefully injecting at the bottom of each tube, using a pipette, firstly 5 ml of a 20% sucrose solution and secondly 5 ml of a 60% sucrose solution. The corex tubes were then centrifuged for 3 min at 3000 rpm and at 4˚C. The spores were finally recovered with a Pasteur pipette, rinsed with water through a 50-μm sieve and deposited in a petri dish for observation and enumeration.

Spores Enumeration and Identification
Spores abundance was assessed under a binocular magnifying glass using a gridded Petri dish (5 cm diameter). Spores were counted and classified into different morphotypes according to their size, color, shape, cluster shape and mode of attachment of the suspensory hyphae. They were then photographed using a binocular magnifying glass with a camera connected to a computer. Spore identification was carried out based on their morpho-anatomical characteristics. Biological diversity indices were then calculated. For microscopic identification, healthy spores were mounted on glass slides and stained with polyvinyl lactic acid glycerol (PVLG) mixed with Melzer's reagent (1:1 vol/vol) as reported [19]. They were then left for 72 h in the dark. The spores were examined under a binocular microscope (G x 40) and photographed. Spore identification was done using INVAM (International culture collection of vesicular/arbuscular mycorrhizal fungi) database according to their morphological characteristics.

Biological Diversity Indexes
Different diversity indexes were used to assess the structure of the mycorrhizal fungi population referring or not to a concrete spatio-temporal framework.

The Shannon Diversity Index (H')
The [20] diversity index is the most commonly used in the literature. It is de-

Simpson's Diversity Index (1-D)
The Simpson index measures the probability that two randomly selected individuals belong to the same species [21]. It is defined as:

Hill's Diversity Index (1-Hill)
It is a measure of proportional abundance, allowing the Shannon-Weaver and Simpson indices to be combined. It is defined by:

Analysis of Soil Parameters
The physico-chemical analyses of the collected soil samples were carried out at the Laboratory of Soil Microbiology and Microbial Ecology (LMSEM) of the Faculty of Agricultural Sciences of the University of Abomey-Calavi in Benin.
Granulometry was determined with Robinson pipette method; organic carbon was measured using [22]; available phosphorus was determined with [23] whilst pH water and pHKCl were determined by potentiometric method and Cationic Exchange Capacity (CEC) [23].

Statistical Analysis
Statistical analyses were performed under R software version 4.1.0. After checking the normality and equality of variances, collected data and calculated parameters were subjected to an analysis of variance (ANOVA) at a threshold of 5%.
The physical and chemical parameters of soil were used to assess the relationship among them and the diversity of AMFs using correlation tests (Pearson). Factorial Component Analysis (FCA) was carried out to obtain the characterization of the AMF genera in the different study sites.

Density and Diversity of AMF Spores AMF Spore Density in Soils
The results showed that the AMFs spore density significantly (p < 0.0001) varied from one site to another. Thus, spore densities are higher in Copargo soils (3584 spores/100g soil) and lower in Dassa soils (1146 spores/100g soil) ( Figure 2).

Biological Diversity Indices of AMFs in Sampled Soils
There was a significant difference (p < 0.05) among the biological diversity in-  diversity indexes whereas the production areas of Zakpota recorded the lowest.
As for the Hill index, it varied from 0.70 to 0.4. The diversity of AMFs therefore differs significantly from one production areas to another (Table 1).

Distribution of Morphotypes Inventoried in the Sampled Soils
Eight species were identified in all the soils sampled. The identified species were totally eight with two belonging to Glomus genus, three to Gigaspora genus, two to Acaulospora genus and one species to Diversispora genus. The results of the spore enumeration are as shown in Table 2. The eight (08) species identified were found in all the sampled soils with the exception of the species "Scutellospora savannicola" absent in the soils sampled in the production areas of Agbangnizoun.

Relative Abundance of Different Spores
All identified species were recorded in all sampled soils except for "Scutellospora savannicola" species absent in the soils sampled in the production areas of Agbangnizoun. The species with high dominance in all soils were "Rhizophagus partial" followed by "Acaulospora denticulate". The species with low dominance in sampled soils were "Scutellospora savannicola" and "Racocetra crispa".

Typology of Studied Communities According to AMF Species
The first two components present 80% of the information that is taken into account. The results of the data analysis can be validly exploited as they exceed 50% (Figure 3).
The results of the Factorial Component Analysis showed that the production areas of Dassa, Djougou, Savè, Bohicon, Zakpota and Bassila are negatively correlated by the dominance of Rhizophagus partial. Diversispora sp and Scutellospora savanicola are very well represented on axis 2 and characterize the production areas of Nikki. The production areas of Zangnanado and Glazoué are characterized by the species Acaulospora denticulate and Paraglomus occultum.
As for the production areas of N'dali, Parakou and Copargo, they are positively H. B. Houngnandan et al.

Evaluation of the Relationship of the Studied Parameters
In general, in the study areas, there was a significant (p < 0.05) correlation among available phosphorus and Cationic Exchange Capacity; organic carbon and sodium; sodium, calcium, Shannon index and Hill index; potassium and magnesium; mycorrhization intensity and magnesium; sodium and Shannon intensity, Hill intensity. There was also a highly significant correlation (p < 0.01) between sodium and potassium. Significant differences (p < 0.01) are noted between calcium and magnesium; mycorrhization frequency and mycorrhization intensity and finally among all biological diversity indices (Shannon, Simpson and Hill) ( Table 3).

Discussion
The results of the current study reveal a high level of spore density for all the studied production areas (approximately 23,980 spores/100g soil  [24] under maize (Zea mays L.) (6260 spores per 100 g soil) but higher than the results reported by [25] under cowpea (Vigna unguiculata (L.) Walp.) (202 ± 42 per 100 g soil in different agro-ecological zones of Benin) [26], under cashew (Anacardium occidentale L.) plantation in central Benin [27], in the Wari-Maro classified forest in northern Benin under Isoberlinia doka (237 to 258 spores per 100 g soil). This difference in spore density levels could be due to the plant species itself. Indeed, a plant species can directly influence the abundance and composition of mycorrhizal fungi spores [28] [29]. According to [30], the presence and natural distribution of glomerales is a function of floristic composition as well as environmental conditions. [31] showed that legumes grow better on poor soils, partly because of the symbiotic microorganisms that colonize their root system such as mycorrhizal fungi and rhizobia. They have the ability to promote the development of fungal propagules (mycelial hyphae, spores) by releasing exudates into their rhizosphere that promote the development of microorganisms, including mycorrhizal fungi.
In early studies, [25] showed no significant difference between the different Agro-Ecological Zones of Benin (AEZ) regarding the spore density of Glomeromycota associated with cowpea cropping. These results are contrary to those from the current study showing a significant difference among production areas in terms of number of spores recorded with Copargo showing the highest. It should be noted that this production areas is located in one of the cotton growing areas and food crops and therefore in perpetual use, which favors the development of microorganisms. In addition, the fields constitute an environment of continuous crop rotation which, in the long term, favors high abundance of spores [27]. In the current study, the soil samples were taken at full bloom, but also under water stress. It is said that period from sowing to flowering associated with favorable environmental conditions (moisture and presence of plant roots) would have allowed an activation and multiplication of spores and this could, indeed, explain the high densities recorded. According to [32] [33], the number of spores is higher in the soil after being subjected to water stress conditions. This is consistent with the results from this current study.
The results in the current study showed that the diversity of fungal spores varies from one production areas to another with a dominance of the genus Glomus in almost all the production areas studied. Indeed, this dominance of Glomus has also been reported in AMF morphotypes in various tropical soils [27] [36] and in agricultural soils in temperate zones [28] [37]. According to [38], the predominance of species of the genus Glomus in most ecosystems suggests a better adaptation of this genus either to the most hostile conditions such as drought, salinity and other environmental stresses, or to a wide range of ecological niches [27]. Furthermore, according to [39] the genera Glomus would spread much more by spores which are forms of resistance of AMF to harsh conditions while the genera Gigaspora and Scutellospora would spread more with other types of propagules such as hyphae, extra root mycelial fragments. Determination of the indices of biological diversity indicates that there are significant differences between production areas on all indices (Shannon-Weiner, Simpson and Hill). The AMF community is very diverse in all communities.
The results from this study show positive and negative correlations among different parameters evaluated. Indeed, there is a positive correlation between the assimilable phosphorus and the biological diversity indices (case of the Savè production areas). The major role of AMFs is in the mobilization for the plant nutrients that are not very mobile in the soil, mainly phosphorus [24] [40]. Depending on the soil pH, this element is mostly trapped by iron, aluminum or calcium in forms that are difficult for plants to mobilize [41]. Moreover, according to the studies by [25] [42] [43] [44], there is a broad and diverse influ-ence of soil properties on AMFs.
In general in the study communities, no significant correlation was found between physico-chemical parameters namely pH and mycorhization frequencies. These results are contrary to those [27], who reported a negative correlation between soil phosphorus, nitrogen and carbon with spore density, but also between mycorrhizal frequency and spore density. Subramanian et al. (2006) also showed that phosphorus application can positively or negatively influence spore production. According [45], phosphorus can be a limiting factor for spore abundance, either at too high or too low concentrations [46]. This could also be explained by the fact that sampling was not carried out in the same area and under the same conditions, but also that the specific diversity of the different samples is not the same and varies according to the production areas. It is therefore important to note that in order to optimize soybean production, it will be necessary to determine the right concentration of mycorrhizal inoculum to apply to get good yield.

Conclusion
The overall objective of this study was to evaluate the diversity of arbuscular mycorrhizal fungi (AMF) associated with soybean cultivation in the main soybean production areas of Benin. This study showed that there are mycorrhizal flora of significant spore density associated with soybean and which differs from one production areas to another. From the results obtained, it appeared that soil parameters had an influence on the diversity of AMF. Variability in spore densities was also observed from one production areas to another but with high levels of diversity not necessarily stipulating a strong mycorrhizal symbiosis. A total of eight species belonging to three genera and three families were obtained in the studied production areas. The genera are Glomus, Acaulospora and Gigaspora in all the production areas with an abundance of the genus Glomus. These species belong to the following families: Diversisporales, Glomérales and Paraglomérales. Finally, the current study revealed that there was an abundance and diversity of AMF associated with soybean cultivation in the different soybean-producing production areas in the Republic of Benin. It will be necessary to produce inoculums based on these local species and test their response on soybean crop to determine the best way to achieve good soybean productivity in Benin.