Occurrence of arbuscular mycorrhizal fungi spores in soils of some legumes and their response to varying concentrations of phosphorus application

A potted experiment arranged in a 5 × 3 factorial in a randomized complete block design was undertaken to investigate the occurrence of arbuscular mycorrhizal fungi (AMF) in the soil of five leguminous plants: Cajanus cajan (L.) Huth, Centrosema pascuorum Martius ex Benth, Crotalaria ochroleuca G. Don, Lablab purpureus (L.) Sweet and Mucuna pruriens (L.) DC. The effects of varying phosphorus concentrations (P0) (0 kg/ha of single superphosphate), P1 (100 kg/ha of single superphosphate) and P2 (200 kg/ha of single superphosphate) on the population of AMF spores under these legumes were also carried out. The AMF spores in soil samples were extracted at 19 weeks after planting, using the wet sieving and decanting method, and enumerated with the aid of a stereoscopic microscope. Spores of different species of genera Glomus and Gigaspora were encountered in the soils of the five leguminous plants. Spores of Glomus species predominated while the spores of Gigaspora species were found in lower numbers. The total AMF population was significantly affected by legume species (p ≤ 0.05). The total AMF spore counts were higher in the soils of Mucuna pruriens and Crotolaria ochroleuca (p ≤ 0.05). The populations of Glomus mossae in soils decreased with increasing level of applied phosphorus (p ≤ 0.05). A positive correlation was recorded between the total AMF spores, the predominant AMF spores and soil pH, while the organic matter content and the available phosphorus were negatively correlated with both the total AM spores and the predominant AMF spores.


Introduction
The Leguminosae family is the second largest among worldwide food crops after cereals. Legumes are second to Poaceae (the grasses) in agricultural and economic importance (Curiel et al. 2015). Legumes have an important place in crop rotation, provide protein-rich food for man and livestock, are used in admixture with grasses in hay and pastures and are also used as cover crops and green manures. The growth of legumes on soils improves the nutrient status of soil by increasing the nitrogen content of soil. This also has a profound effect on soil characteristics, such as soil structure, soil texture and water content They protect crops from the soil-borne pathogens such as nematode (Cavallazzi et al. 2007) and weed (Rinaudo et al. 2010). They improve soil structure development and aggregate stabilization (Gianinazzi et al. 2010). AMF are essential for ecosystem sustainability, plant development and maintenance of biological diversity (Smith et al. 2011).
The major soils of Southern Guinea Savanna of Nigeria have coarse-textured surface and are low in organic matter and chemical fertility (Alori et al. 2012). The productivity of these soils is generally low in most of the existing farming systems. These soil problems have necessitated the intensification of research in this geographical zone. This includes research into biological nitrogen fixing systems as an alternative to chemically produced fertilizer. Certain  Mucuna pruriens (L.) DC were sown at 2 per pot, and this was thinned to 1 per pot 10 days after germination. Crops were irrigated to soil moisture capacity at 6.00 and 18.00 h daily using a watering can. These crops were allowed to grow to maturity (19 weeks). Soil samples were collected from the soil from these pots cropped with different legumes: C. cajan, C. pascuorum, C. ochroleuca, L. purpureus and M. pruriens with the aid of small hoe blade and transported to the laboratory in the well-labeled polythene bags for analysis.

Determination of some selected soil chemical properties
The chemical properties determined include pH using Kent pH meter model 7020, soil organic matter (SOM), using wet oxidation method as described by Shamshuddin et al. (1994). Available phosphorus in soil samples was determined using the modified No. 1 method of Bray and Kurtz (1945).

Extraction and morphological characterization of isolates of AMF spores in soils
The wet sieving and decanting technique for the extraction of AMF spores developed by Brundrett et al. (1996) was employed for the extraction of AMF spores. The isolated spores were then picked up with a needle under a dissecting microscope and were mounted in both polyvinyl alcohol lactic acid-glycerol (PVLG), Meltzer's reagent and PVLG mixed with Meltzer's reagent (1:1 v/v). All spores were examined using a compound microscope. The morphological properties of these spores were determined according to the key proposed by Trappe (1982). The characteristics used include shape, size, color, distinct wall layer, attached hyphae, sporocarps, bulbous attachment, clustering and surface ornamentation of spores. Characterization was made by using the description provided by the international collection of vesicular and AMF (INVAM 2001).

Statistical analysis
The percentage frequency of occurrence of AMF spores in each legume soil was calculated using the formula below: × No of an AMF sp Total no AMF spores 100 The correlation between the AMF spore counts and variables such as pH, organic matter content and available phosphorus in soil of each legume was analyzed. Data collected were subjected to analysis of variance using Genstat statistical package. Means were separated using Duncan's multiple range test.

The percentage frequencies of occurrence of the predominant AMF spores under different legumes
The percentage frequencies of occurrence of the predominant AMF spores under different legumes are shown in Figure 1. The spores of Glomus fasciculatus were predominant in soils of the legumes except M. pruriens in which the spores of Glomus macrocarpus var. macrocarpus were predominant. The spores of Glomus macrocarpus var. macrocarpus were the next in abundance to Glomus fasciculatus in soils of C. pascuorum, C. ochroleuca and L. purpurens, respectively. The spores of Gigaspora corralloidae, however, had lowest counts in all the five legumes soils.

Total AMF spore count and some chemical properties of soil under different leguminous plants
The total AMF spore count and some chemical properties of soil under different leguminous plants as reported in Table 1 show that most of the soils at different levels of applied phosphorus were slightly acidic, with the soils under C. ochroleuca at 200 kg/ha of applied phosphorous level having the lowest pH (5.0). The soils of C. cajan at 200 kh/ha level of applied phosphorus and M. puriens at zero (0 kg/ha) level of applied phosphorus were, however, neutral (7.0 and 7.1). There were also variations in the level of available phosphorus of the soils of the five legumes 19 weeks after planting. C. cajan at zero (0 kg/ha) level of applied phosphorus had the highest available phosphorus of 32 ppm, while M. pruriens at the same level of applied phosphorus had the lowest available phosphorus of 3.16 ppm.

Effect of the applied phosphorus levels
and different legumes on the total AMF spores Table 2 shows the effect of the applied phosphorus levels and legume types on the total AM spores. The population of AMF spores was significantly higher in the soils of M. pruriens and C. ochroleuca than the soils of other legumes. The lowest AM spore count was recorded in the soils of L. purpurens.
Higher AMF counts were recorded in the soil of M. pruriens when no phosphorus was applied than with applied phosphorus. A decline in the spore count was recorded with increase in the applied phosphorus. On the other hand, C. ochroleuca had lower AM counts in soils with no applied phosphorus, whereas an increase in AMF count was observed with increasing level of applied phosphorus.

Effects of different legumes and varying phosphorus levels on the population of Glomus mossae
The population of G. mossae spores was significantly higher in the soil of M. pruriens than in the soils of the other legumes. G. mossae spores were more numerous in soils with zero (0 kg/ha) level of applied phosphorus. The number of spores in soils when phosphorus was not applied was in fact significantly higher than the number of spores in soils of 100 and 200 kg/ha levels of applied phosphorus ( Table 3).

Effects of different legumes and varying phosphorus levels on the population of
Glomus macrocarpus var. geosporus and Glomus macrocarpus var. macrocarpus spores As revealed by Tables 4 and 5, the populations of G. macrocarpus var. geosporus and G. macrocarpus var. macrocarpus spores were significantly higher in the soil of M. pruriens than in the soils of the other legumes. However, the varying P levels have no significant effect on the spore population of both AMF species.    Table 6 reveal that the population of G. fasciculatus spores encountered at the different legume species was not significantly different from one another at p ≤ 0.05. However, the application of 0 kg/ha of P resulted in a significantly higher G. fasciculatus spore compared to the application of 200 kg/ha of P at p ≤ 0.05.

Effects of different legumes at varying P levels on Gigaspora coralloidea spore population
G. coralloidea spore count in soils under different legumes at varying levels of applied P in Table 7 shows that the population of G. corralloidae spores was not affected significantly either by the legume species or by the P level (p ≤ 0.05).

Correlations between some soil
properties and AMF spore population in soil Table 8 shows the correlations between some soil properties and AMF spore population in soil. Significant negative correlations were observed between AMF spore population and available P and between AMF spore population and SOM content.

The percentage frequencies of occurrence of the predominant AM spores under different legumes
The occurrence of Glomus spores reported in this research ( Figure 1) was also reported by Alori et al.    phosphates in response to low levels of available phosphorus. The total AMF spores count in the soils of M. pruriens was higher than that of the soils of C. cajan.
There are slight variations in the quantity of the organic matter of the soils studied. This probably had an effect on the level of available phosphorus in the soils. A soil with high organic matter content will likely have a higher microbial population. Immobilization of phosphorus by these organisms will cause reduction in the available phosphorus of that particular soil. The reduction in pH in soils under Crotalaria ochroleuca at 200 kg is most likely due to the extrusion of H + , following the uptake of ammonium by the fungal hyphae.

Effect of the applied phosphorus levels and different legumes on the total AMF spores
Variation in the abundance of AMF under different legumes and varying P levels could probably be due to the differences in morphological structures, growth characteristics and nitrogen-fixing ability of the legumes.

Effects of different legumes at varying P levels on Glomus fasciculatus spore population
The lack of significant difference in the spore population of G. fasciculatus under different legumes implies that exudate from the legumes studied favors the growth of G. fasciculatus. The significant decline in G. fasciculatus spore population observed with increasing level of the applied P is consistent with the report of the study by Johnson et al. (2013). Increased phosphorus supply has previously been reported to exert strong inhibition of arbuscular mycorrhizal development (Nouri et al. 2015).

Conclusion
This study has revealed that spores of Glomus sp. and Gigaspora sp. occur in the soils of Southern Guinea Savanna, ecological zone of Nigeria. However, spores of Glomus sp. are abundant than spores of Gigaspora sp. in soils of this zone. The occurrence of AMF spores in soils under leguminous plant -C. cajan, C. pascuorum, C. ochroleuca, L. purpureus, and M. pruriens was also established. M. pruriens soil was observed to have more AMF spores than the other legume soils. The use of M. pruriens in crop rotation could help to improve soil fertility in the study area. The mere presence of mycorrhizal spores does not necessarily reflect the abundance of mycorrhizal infection. There is, therefore, the need for further studies on the rate of infection of these legumes with the existing mycorrhizal spores.
This study has also shown that soil properties such as pH, organic matter content, and the available P do have effects on the AMF population. Further work is required to observe the correlation between the presence of AMF and the amount of P and N in tissues of different legumes. This information will enable the manipulation of AMF symbiosis for the benefit of a minimum input agricultural system in Southern Guinea Savanna Zone of Nigeria.