Arbuscular Mycorrhizal Fungi and Soil Enzyme Activities in Different Fonio Millet ( Digitaria exilis Stapf.) Agroecosystems in Senegal

In plant roots, arbuscular mycorrhizal fungi (AMF) are the most prevalent microsymbionts, and thereby provide many key ecosystem services to natural and agricultural ecosystems. Despite AMF’s significance for the environment and the economy, little is known about the mycorrhizal inoculum potential and diversity of AMF associated with orphan African cereal crops, specially fonio millet ( Digitaria exilis stapf . ) under field conditions. We hypothesized that the type of fonio millet agroecosystem influences the AMF density and distribution in soils. We therefore, assessed the inoculum potential, density and diversity of AMF spores and soil enzyme activities in five fonio millet agroecosystems belonging to three climatic zones (Sudanian, Sudano-Sahelian and Sudano-Guinean). By combining AMF spore identification from field-collected soils and trap culture, 20 species belonging to 8 genera ( Acaulospora, Ambispora , Dendiscutata, Gigaspora, Glomus , Racocetra, Sclerocystis and Scutellospora ) were identified. Glomus was the most represented genus with 8 species, followed by Gigaspora (5 species) and Acaulospora (2 species); the remaining genera were each represented by one species. Except for Ambispora which was not found in the Sudanian area, all genera occurred in the three climatic zones. The abundance and diversity of AMF species and FDA-hydrolytic and phosphatase activities varied between fonio millet agroecosystems as well as between climatic zones. Soil pH and soil texture were the variables that best explained the density and distribution of AMF spores. Our results contribute to paving the way towards the development of microbial engineering approaches for agronomic improvement of fonio millet.


INTRODUCTION
Fonio millet (Digitaria exilis, stapf), also called "Acha", is one of the oldest cereal crops originated in West Africa. 1 It has very good prospects for semi-arid and upland areas as it tolerates poor soils and drought conditions, and matures very quickly (6-8 weeks). 2 Moreover, Fonio grains contain higher amounts of amino acids (e.g., methionine and cystine), 3 iron, potassium, calcium and phosphorus. 4,5owever, fonio consumption is still low, particularly in urban areas where it has long been considered as an orphan crop. 6,7][13] In the other hand, arbuscular mycorrhizal fungi (AMF) form the most prevalent microbial symbiotic association with the majority of terrestrial plant species. 14,15These beneficial soil microorganisms have a great potential for contributing to crop production and thereby helping to achieve sustainable global food security. 16,172][23] Hence, harnessing the potential of AMF is considered as a potential less costly solution to increase crop yields. 16,24,25Meanwhile, abiotic and biotic factors influence the effects of AMF taxa on plant development and production. 24,26,27n addition, it has been reported that AMF abundance and diversity varied depending on the ecological zone, 28,29 soil properties, [30][31][32] vegetation type 33 and agricultural management pratice. 34n Senegal, fonio millet is cultivated under various agricultural management practices across different climatic zones. 35However, little is known about the AMF density, diversity and distribution across the fonio millet agroecosystems.We hypothesized that the type of fonio millet agroecosystem and pedoclimatic conditions might influence the AMF density and distribution in soils.We therefore, assessed in this study the inoculum potential, density and diversity of AMF spores, and soil enzyme activities in five fonio millet agroecosystems belonging to three climatic zones (Sudanian, Sudano-Sahelian and Sudano-Guinean).
In each fonio millet agroecosystem, a sampling area of 100 × 100 m was delimited, soil was collected from 6 points at a depth of 0 to 25 cm and then the soil samples were pooled together in plastic bags and brought to the lab.The soil samples were sieved to <4 mm and kept at 4°C.

Characterization of soil properties
Physical (sand, silt, clay) and chemical (C, N, P, P 2 O 5 , C/N) characteristics of the five soils were analyzed at the Laboratory of Soil, Water and Plant of ISRA/CNRA at Bambey (Senegal) using standard methods.The soil physical characterization was carried out as described in Disale et al. 36 The soil samples were placed in a mechanical shaker and sieved for 5 min through a series of sieves to determine the size of different soil particles.The combustion system Thermo-Finnigan Flash EA 1112 (Thermo Finnigan, France) was used to quantify the total amount of soil carbon and nitrogen. 37The amount of soil total and available phosphorus was evaluated as described by Bibi and colleagues. 38Soil organic matter (OM) was determined from organic carbon as follows: OM (%) = organic carbon (%) × 1724.Soil pH was measured in soil-water (1:2,5) suspensions. 37

Determination of mycorrhizal inoculum potential
The mycorrhizal inoculum potential in each soil sample was evaluated by the dilution technique. 39Briefly, a quarter-fold dilution series (1, 1/4, 1/16, 1/64, 1/256 and 1/1024) was prepared by thoroughly mixing defined proportions of nonsterilized and sterilized soil.Then, 50 g of each diluted soil sample were placed in 5 pots, and 3 seeds of Zea mays L. (a highly mycotrophic plant) were sown per pot.The seedlings were thinned to one per pot and all plants were kept in glasshouse and watered with demineralized water.After 45 days, roots of all plants from the dilution ratios were harvested and stained with Trypan blue as described in Founoune-Mboup et al. 40 The presence of mycorrhizal infection in stained root segments was observed by light microscope at a magnification of 100X.The most probable number (MPN) of AMF propagules that can colonize plant roots was calculated as follows: Log MPN = (x log a)-K, where x represents mean of mycorrhized plants for all dilution ratios, a (factor of dilution) = 4, K=constant given by the table of Fisher & Yates. 41,42

Identification and enumeration of AMF spores from field-collected soils
The extraction of AMF spores from each field-collected soil was carried out using the wet sieving and decanting method. 43Briefly, 100 g of soil were mixed with 1 L of water and decanted in a series of 400-50 µm sieves.Then, the material of 200, 100 and 50 µm pore-sieves was re-suspended in water and collected in tubes.Two solutions of sucrose at 20% and 60% were successively added and centrifugation was done at 3000 g/min for 3 minutes.After that, the supernatant containing AMF spores was poured in a 50 µm mesh and rinsed with tap water.AMF spores were grouped and counted according to their morphological characters and using a dissecting microscope.The International Culture Collection of Arbuscular and Vesicular-Mycorrhizal Fungi was used for AM fungi description (https://invam.wvu.edu/methods/spores/enumeration-of-spores).The AMF spore density and abundance of each AMF species were expressed per 100 g of soil.Three replicates were made for each composite soil from each sampled site.

Determination of AMF species composition from trap culture
The trap culture method allows to confirm AMF spore identification (spores are sometimes damaged) from the field-collected soils.On the other hand, this method induces emergence of AMF that would not naturally sporulate. 44The trap culture was performed with field-collected soils using mays (Zea mays L.) for 3 months under glasshouse conditions.For this purpose, each field-collected soil was mixed with an autoclaved nutrient-poor sandy soil from Sangalkam (1:2 v/v) to serve as culture substrate.For each agroecosystem, 9 pots of 1 kg were filled with the culture substrate and 3 fonio seeds were sown per pot (9 replicates x 5 soil sites).Plants were watered every two days for three months.At the end of experiment, plants were harvested and AMF spores were isolated from soils, enumerated and identified as previously described. 43

Soil enzymatic activities
Enzymatic activities were determined from field-collected soils as described in Ndoye et al. 28,45 The activity of FDA (3'.6'-diacetylfluorescein) hydrolysis was measured according to Patle et al. 46 For this test, to 1 g of the soil, 15 ml of 60 mM potassium phosphate buffer (pH 7.6) and 0.2 ml of 1000 µg FDA ml 1 were added (with 3 replicates per soil origin).A blank enzyme without FDA and a blank substrate without soil were included.After 1h of shaking on an orbital incubator at 30°C, the flasks were removed and 1 ml of each suspension were transferred into Eppendorf tube and mixed with 1 ml of acetone to stop the reaction.After centrifugation (10000 rpm/min for 5 min), 1 ml of the supernatant was measured at 490 nm on a spectrophotometer (Ultrospec 3000 Pharmacia Biotech).The concentration of fluorescein was calculated using the calibration graph standard and expressed as µg FDA/g of soil/h.Acid and alkaline phosphatase activities were quantified using a colorimetric determination of p-nitrophenol released after soil incubation with p-nitrophenyl phosphate as substrate (pNPP, 5 mM). 47,48Briefly, 25 mg of soil sample was mixed with 400 µl of buffered sodium p-nitrophenyl phosphate solution (pH 6 and pH 11 for acid and alkaline phosphatase, respectively) and 100 µl pNPP (p-Nitrophenyl Phosphate, 5 mM).A blank enzyme without pNPP and a blank substrate without soil were included.After incubation at 37°C for 1 h, the reaction was complexed with 100 µl of CaCl 2 (0,5 M) then stopped by adding 400 µl of NaOH (0,5 M) solution.After centrifugation (10000 rpm/min for 5 min), 1 ml of the supernatant was measured at 400 nm on a spectrophotometer (Ultrospec 3000 Pharmacia Biotech).The amount of released p-nitrophenol was determined at 400 nm and expressed as µg pNPP/g of soil/h.

Statistical analysis
The Shapiro-Wilk and Levene tests were used to checked the normality and homogeneity of variance, respectively.So, comparisons of means were performed by Kruskal-Wallis test instead of one-way ANOVA when the test was significant.Statistical analyses were performed using rcompanion, FSA, TH.data, pgirmess packages in R software. 49,50The probability threshold "p value" was set at 0.05 in order to establish statistically significant differences between groups.
For each agroecosystem, AMF species richness, Shannon and Wiener diversity index (H') and Simpson Dominance index (D) were determined.The Shannon and Wiener diversity index was calculated as H' = -pi (ln pi) where pi represents the proportion of individuals found in the ith species, estimated as ni /N, ni being the number of individuals in the ith species and N, the total number of individuals.The inverse of the Simpson dominance index was evaluated using the following formula: where ni represents the number of the ith types and N the number of individuals in the population.
Pearson correlation coefficients were determined to investigate the relation between

Soil physicochemical characteristics
Our results showed that the sampled soils were sandy silt clay in Missirah and Mandina Findifeto and sandy clay silt in the other three sites with pH ranging from 5.84 to 6.98 (Table 1).For soil C, N, P, P 2 O 5 , and organic matter (OM) contents, the highest values were obtained in Togue, and the lowest values in Mandina Findifeto.On the contrary, soil pH follows an opposite trend, showing the lowest value in Togue and the highest in Mandina Findifeto.Considering the climatic zones, the Sudanian zone had the lowest C, N, P, P 2 O 5 and OM contents in soil and the highest value of soil pH, whereas the Sudano-Sahelian zone had intermediate values, as compared to the Sudano-Guinean zone (Table 1).

Inoculum potential, AMF species diversity and spore density in field-collected soils
Mycorrhizal soil infectivity of the 5 fieldcollected soils ranged from 5 to 71 propagules in 50 g of dry soil (Table 2).Mandina Findifeto soil showed higher MPN value (70.90 propagules per 50 g of soil) as compared to those of other field-collected soils (ranging from 5.20 to 12.60 propagules per 50 g of soil).Those latter soils did not differ significantly in terms of MPN values.The lowest MPN was obtained in Sare Yoba located in the same climatic zone with Mandina Findifeto.
The spore density of AMF species varied depending on soil origin (Table 3).For instance, Glomus sp.2 and Glomus sp.3 displayed their highest density in Koumbidia Soce (1017.33 and 770.67 spores/100 g of soil, respectively), while Scutellospora sp.aff.dipurpurascens and Dendiscutata sp.aff.heterogama had their highest density in Mandina Findifeto (97.67 and 920.67The total spore density of AMF also differed significantly between the five sites, ranging from 957 to 3166 spores per 100 g of dry soil (Table 3).The density of AMF spores was significantly higher in Mandina Findifeto (3165.33 spores/100 g of soil) than in other sites.It was followed by those of Koumbidia Soce, then Togue (2320.33 and 1379.67 spores/100 g of soil, respectively).The lowest AMF density was recorded in Sare Yoba (957.67 spores/100 g of soil).
Shannon index ranged from 1.63 to 1.29, while Simpson index varied from 0.77 to 0.67 and Hill index from 0.75 to 0.59.The highest diversity indices were observed in Sare Yoba in the Sudanian zone, whereas the soil from Koumbidia Soce in the Sudano-Sahelian zone showed the lowest diversity indices (Table 3).

Composition of AMF species from trap culture
A total of 20 AMF species belonging to 8 genera (Racocetra, Dendiscutata, Scutellospora, Gigaspora, Acaulospora, Ambispora, Glomus and Sclerocystis) were recorded from trap culture (Table 4).Of the 20 AMF species, 17 were found in Koumbidia Soce, 16 in Missirah, 15 in Togue, 15 in Sare Yoba and 11 in Mandina Findifeto.Only 10 out of the 20 AMF species were shared by the 5 sites, while two AMF species, Glomus sp.5 and Glomus sp.6, were recorded exclusively in Sare Yoba.Besides, 8 of the 20 AMF species revealed by trap culture were not detected by spore identification from field-collected soils (Tables 3 & 4).

Soil enzyme activities
Soil FDA-hydrolytic activity in Koumbidia Soce (0.53 µg FDA/g of soil/h) was significantly higher than those in Togue, Sare Yoba and Mandina Findifeto.The lowest FDA-hydrolytic activity was obtained in the Mandina Findifeto soil with 0.27 µg FDA/g of soil/h (Figure 5A).The activity of acid phosphatase was significantly higher in soils from Koumbidia Soce (188.98 µg pNPP/g of soil/h) and Missirah (187.14 µg pNPP /g of soil/h), the two sites located in the Sudano-Sahelian zone, as compared to other sites (Figure 5B).There were no statistically significant differences in acid phosphatase activity between soils collected from Togue, Sare Yoba and Mandina Findifeto.However, the greatest alkaline phosphatase activity was obtained in soil collected from Togue (276.67 µg pNPP /g of soil/h), followed by that from Missirah,  Sare Yoba, Mandina Findifeto and Koumbidia Soce (Figure 5C).

Correlation matrix between density and diversity of AMF, soil physicochemical properties and soil enzyme activities
Soil N, P, P 2 O 5 , C and OM had significant positive correlations between them (Figure 6).Soil pH was strongly positively correlated with AMF spore density (r 2 = 0.650, P-value = 0.235) and soil mycorrhizal potential (r 2 = 0.820, P-value = 0.089); and negatively correlated with the diversity of AMF from field-collected soil (r 2 = -0.419,P-value = 0.235) and the diversity of AMF from trap culture (r 2 = -0.810,P-value = 0.096).Soil C, N, P and available P were negatively correlated with soil MPN, spore density and the diversity of AMF from field-collected soils; and positively correlated with the diversity of AMF from trap culture although those correlations were not significant.

DISCUSSION
][56] Here, we analyzed the density and diversity of AMF; and the enzyme activities in soils from 5 fonio millet agroecosystems in Senegal.Our results revealed that agroecological conditions influence AMF spore density and diversity.These findings might be partially explained by differences in physicochemical characteristics of soils and rainfall.Previous study from Ndoye et al. 28 reported the influence of environmental factors on soil AMF spore density.Also, a significant difference in AMF spore density between three agroecological zones of the Central African Republic was observed by Djasbe and colleagues. 57This is consistent with the results of Maffo and coworkers 58 obtained from two agroecological zones in Cameroon.
On the other hand, it has been reported that AMF inoculum potential has a major influence on mycorrhizal effectiveness and early root infection. 59In this study, the observed high AMF spore density and mycorrhizal inoculum potential in Mandina Findifeto might be partially attributed to its lower clay, nutrient and organic matter contents; and higher pH and silt content as compared to other sites.Similarly, Swarnalatha and colleagues 60 had obtained a higher AMF spore density in a silty sandy loam soil compared to a silty clay loam soil.Moreover, the presence of clay might reduce the production of AMF spores. 60hese findings indicate the influence of soil type on AMF density.
One of the objectives of the present work was to determine the AMF diversity in soils from five fonio millet agrosystems.A total of 12 species from field-collected soils and 20 species from trap culture was recorded with differences between sites.Those site effects could be linked to soil physicochemical properties and environmental conditions. 60,61The negative correlations obtained between soil nutrient contents and AMF spore density and diversity collaborate other findings. 61,62In fact, it is reported that soil mineral nutrients, specially P might influence AMF diversity and density. 63For example, in North China, study of Lang and colleagues 64 in a long-term field experiment revealed that AMF alpha diversity gradually decreased as the P fertilizer rate increased.On the other hand, Delroy and colleagues 51 found that the diversity of AMF tends to expand at optimal P.However, evidence points out that P supply does not necessarily have a detrimental effect on AMF diversity. 65Those results suggested that besides nutrient contents, other parameters (organic matter, humidity, pH, etc) might influence soil AMF parameters. 639][70] In this respect, Zhao and colleagues 71 reported that increase in temperature and precipitation can promote mycelia and spore development by allowing the plant to supply more photosynthetic products to AMF.
3][74] Congruently, our results as well as those obtained from other agroecosystems in Senegal 75,76 showed site effects on AMF diversity.In the present work, AM fungi species richness in field-collected soils and trap culture was higher in Koumbidia Soce site which contains higher amounts of N, P, C, OM, sand and clay than Mandina Findifeto site.It is apparent that abiotic factors, particularly soil chemical properties, can influence the AMF community structure and abundance. 76,77Results of Song et al. 78 on Sephora flavescens Ait in China also supports the hypothesis that soil chemicals exert a selective effect of soil AMF population.
On the other hand, due to their effects on several ecosystem processes such as soil geochemical cycles, plant diversity and productivity, and soil composition, Glomeromycota communities have a wide environmental impact. 1976]79 This might be related to their greater environmental adaptability and capacity to colonize plant roots more widely because of their efficient production of mycelia and spores. 80Moreover, Glomus species have been reported to promote fonio growth and yield under glasshouse conditions. 9In the present study, the low spore density and diversity of Scutellospora and Racocetra might be explained by their huge spores which take longer to mature than small spores 81 and/or by the ability to grow only from an intact mycelium or with live spores. 82,83n addition, Glomus, Dendiscutata and Scutellospora dominated in Mandina Findifeto site which contained the lowest amounts of nutrient contents and OM compared to other sites.In contrast, Acaulospora has greater abundance in Togue site and lower abundance in Mandina Findifeto.Songachan and Kayang 83 noted that Glomus species dominated in natural sites and Acaulospora species in cultivated ones, due probably to the failure of hyphal network disturbance in environments that might have benefited Glomus species.
In this work, some AMF species (belonging to Ambispora, Glomus and Gigaspora) revealed by trap culture were not detected by spore identification from field-collected soils.Similar findings were reported by Leal et al., 84 Chairul et al. 85 and Rodriguez-Morelos et al. 86 This demonstrated that cryptic AMF spores that are invisible during sampling or in field conditions can be encouraged to germinate through trap culture. 44,87This shows the importance of the combination of spore identification from trap culture and field-collected soils in AMF analysis. 86urthermore, it was shown that soil pH might affect directly or indirectly AMF community composition by impacting P availability. 80,88Our results revealed positive relationships between soil mycorrhizal inoculum potential, AMF density and soil pH even if that was not significant.Bainard et colleagues 77 found a negative correlation between some AMF species and phosphate concentrations in the soil.Thus, the main factors influencing the spatial variation in the AMF community across the site appeared to be soil pH or pH-driven changes in soil chemistry and Electrical conductivity. 70,89he lower AMF diversity observed in Mandina Findifeto compared to Togue and other sites might partially be related to soil OM and carbon contents as reported by Zhang et al. 90 Moreover, many studies have shown correlations between soil nutrients and enzymatic activities, 91,92 as confirmed by our study.Also, we found that soil FDA activity was positively and significantly correlated with AMF diversity and negatively with soil MPN and soil pH.This is consistent with the study of Cheng and coworkers 93 showing a positive correlation between AMF diversity and soil enzyme activities.However, the correlation between soil alkaline phosphatase and soil nutrient contents was positive except for soil pH.In this way, Moradi et al. 94 observed a positive correlation between acid and alkaline phosphatase activity, soil OM and N.

CONCLUSION
Our study shows an appreciable AMF density and diversity in the five tropical soils in Senegal.The results in field-collected and trap culture samples, respectively, revealed 12 and 20 species of AMF belonging to 8 genera and 4 families from 5 fonio millet agroecosystems into three climatic zones (Sudanian, Sudano-Sahelian and Sudano-Guinean).The AMF diversity increases with the trap culture.In both field-collected and trap culture soils, Glomus was the dominant genus in term of spore density and diversity in the five agroecosystems.The abundance and diversity of AMF species and FDA-hydrolytic and phosphatase activities varied between fonio agroecosystems as well as between climatic zones.Thus, abiotic factors like soil physicochemical properties might influence AMF spore density and diversity.Furthermore, Soil pH and texture were the variables that best explained the distribution of AMF spores.
T h i s w o r k c o n t r i b u t e s t o o u r understanding of diversity and ecology of AMF in fonio millet agroecosystems.It therefore contributes to paving the way towards the development of microbial engineering approaches for agronomic improvement of fonio millet.However, more studies are necessary to better identify and explain the main driving factors of AMF community at different locations.

Figure 4 .
Figure 4. Relative abundance of AMF genera found in the rhizosphere of Digitaria exilis stapf

Figure 5 .
Figure 5. Enzyme activities of field-collected soils (A) FDA (Fluorescein diacetate.μg FDA/g of soil/h).(B) PHA and (C) PHB (Phospatasis acid and Basis.μg pNPP/g of soil/h) Boxes followed by the same letter are not significantly different according to the Kruskal-Wallis test (P < 0.05)

Figure 6 .
Figure 6.Correlation matrix of the different variables (AMF density and diversity.soil physicochemical properties and soil enzyme activities) Positive correlations are displayed in blue and negative correlations in red color.Color intensity and size of circle are proportional to correlation coefficients.In the right side of the correlogram.the legend color shows the correlation coefficients and the corresponding colors Ndoye et al | J Pure Appl Microbiol.2024;18(3):1866-1882. https://doi.org/10.22207/JPAM.18.3.37 AMF spore diversity and density, soil enzyme activities, and soil physicochemical characteristics.All statistical analyses were conducted in R v4.3.1.

Table 3 .
Density of arbuscular mycorrhizal fungal spores in the field-collected soils In row, values followed by the same letter are not significantly different according to the Kruskal-Wallis test (P < 0.05)