EFFECTS OF ARBUSCULAR MYCORRHIZA FUNGI, ORGANIC FERTILIZER AND DIFFERENT MOISTURE REGIMES ON SOIL PROPERTIES AND YIELD OF AMARANTHUS CRUENTUS

: A pot experiment was conducted to assess the influence of two arbuscular mycorrhiza (AM) fungi and organic fertilizer (OF) on the growth and yield of Amaranthus cruentus under varying soil moisture regimes. This was done with a view to providing information on the crop adaptation to drought conditions and to also sustaining soil nutrient balance for increased crop yields. The experiment consisted of 36 treatments ( Glomus clarum , Glomus deserticola and no AM), organic fertilizer made from market wastes at different rates (0, 5 and 10 t ha - 1 ) and varied water regimes (25, 50, 75 and 100% field capacity [FC]). Each of the treatments was replicated thrice. The treatment combination, 10 t ha -1 OF and G. clarum produced the highest fresh vegetative yield of 48.82 t ha -1 which was not significantly (p > 0.05) different from only 45.78 t ha -1 fresh yield obtained with 5 t ha -1 OF and G. clarum when water levels were compared. The repeated experiment with only water addition gave lower and comparable yields of A. cruentus . We concluded that the addition of G. clarum in combination with 5 t ha -1 of organic fertilizer to soil optimally improved the growth and yield of A. cruentus in water stress conditions.


Introduction
Crops and soil nutrition are intrinsically linked because the soil houses and provides nutrients for crops, and as a result, soil nutrient decline could lead to low quality and quantity in crop production (Murrell et al., 2015). In addition to this, there are other abiotic stresses like drought and salinity that cause one third of global agricultural losses (Vandenberghe et al., 2017). Some soil nutrient deficiencies have indirectly enhanced human activities to impact negatively on the soil ecosystem through variable cultural farm operations and agricultural Effects of arbuscular mycorrhiza fungi on soil properties and yield of A. cruentus 149 (Zhigila et al., 2014). In Nigeria, it is commonly called 'tete' among the Yorubas, 'green' among the Igbos, and 'aleho' among the Hausas (Mshelmbula et al., 2017;Towolawi et al., 2017) and it is a good source of vitamins and dietary minerals (Cyril et al., 2014). This study therefore assessed the influence of two arbuscular mycorrhiza fungi (Glomus clarum, Glomus deserticola) and organic fertilizer (OF) on the growth and yield of Amaranthus cruentus under varying soil moisture regimes.

Materials and methods
Experimental location, design used and agronomic practices employed The experiment was conducted in the greenhouse of the Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria. Viable spores of AM fungi, namely: Glomus clarum and Glomus deserticola, obtained from the Department of Agronomy, University of Ibadan, Ibadan, Nigeria were propagated using the Zea mays plant for a period of three months. During the propagation period, maize plants were watered regularly and chopped leaves of Gliricidia sepium were used to nourish the previously sterilized sandy soil used on a weekly basis. At three months, water was withdrawn from maize plants to allow for multiplication of fungi spores for two weeks. The organic fertilizer made from market wastes was procured from a waste recycling firm in a local market in Ibadan, Nigeria. Viable seeds of Amaranthus cruentus were obtained from the National Horticultural Research Institute, Jericho, Ibadan, Nigeria.
Surface soil sample was collected from an infertile land, sieved and sterilized and three kilograms of the sterilized soil were filled into each of the polythene pots. The experiment consisted of 36 treatments, namely: ([10 g G. clarum, 10 g G. deserticola and no AM fungi], organic fertilizer at different rates [0, 5 and 10 t ha -1 ], varied water regimes [25,50,75] and 100% field capacity [FC]). Each of the treatments was replicated thrice and factorially arranged in a completely randomized design to give a total of 108 pots. Treatments were applied at sowing and plants in each pot were thinned to two stands at two weeks after sowing (WAS). The pots were maintained weed-free throughout the experimental period and they were watered appropriately.
Collection of data on growth performance commenced at 3 WAS and continued weekly thereafter until 6 WAS. Growth parameters assessed included plant height, number of leaves, stem girth and leaf area. Fresh biomass yield per pot was determined immediately after harvest using a weighing balance. The plants were then oven-dried at 70°C to constant weight using a binder FED 400 model to determine the dry biomass yield. There was a repeated experiment immediately after the harvest of the first set of the vegetable crop to test for residual effects of AM fungi and organic fertilizer, but with only different water regimes as treatment addition.
Propagation of arbuscular mycorrhiza fungi Soil inocula containing viable spores of AM fungi, namely G. clarum and G. deserticola were obtained from the Department of Agronomy, University of Ibadan, Ibadan, Nigeria. Fifty grams of each inoculum were weighed into 10 kg of sterilized coarse sand, and two seeds of maize (Zea mays) sown into the pots. The plants were regularly watered and chopped leaves of Gliricidia sepium were used to nourish the soil weekly. Three months after sowing, water was withdrawn to allow for multiplication of fungi spores. Two weeks after, maize shoots were cut and soil air-dried.

Extraction and counting of arbuscular mycorrhiza fungi spores
Pre-and post-planting of extraction and counting of AM fungi spores were determined using the wet sieving and decanting method of Habte and Osorio (2001). One hundred grams of air-dried soil were taken from the sterilized bulk soil sample and the AM treatment pots, and each was thoroughly mixed. Each was soaked for 30 minutes with 250 ml of distilled water in a beaker. Each sample was thereafter made with distilled water to 1000 ml suspension and agitated for 30 minutes to dislodge the fungal spores from the soil. The soil suspension was decanted over a stack of sieves (250, 75 and 53 µm) arranged in descending order of their sizes. Thereafter, materials left in the last two sieves (75 and 53 µm) were collected, suspended in 40% sucrose solution and centrifuged at 3000 rpm for 5 minutes. The spores were later examined and counted under a dissecting microscope.

Laboratory analyses
Pre-cropped soil and organic fertilizer (OF) samples were analysed for their properties using standard methods of Page et al. (1982). Soil pH was determined in 1:1 soil/water using a pH meter; total N of the soil and OF were determined using the macro-Kjedahl method, available P of the soil and OF were determined using the Bray P1 method, and organic C of the soil and OF were determined using the Walkley-Black wet oxidation method. Exchangeable bases (Ca 2+ , Mg 2+ , K + , and Na + ) of the soil and OF were extracted using 1 M ammonium acetate and their concentrations were read using the spectrophotometric method. The summation of the exchangeable bases, Ca 2+ + Mg 2+ + K + + Na + , gave cation exchange capacity (CEC) of the soil.

Statistical analyses
Data on the yield of A. cruentus were subjected to analysis of variance and their means were separated using the Duncan's multiple range test using SAS 9.2 statistical software at p < 0.05. Data on the growth parameters were plotted using GraphPad Prism 5.0 software at p < 0.05.

Results and Discussion
Mycorrhizal spore count Total spore counts of AM fungi: Glomus clarum and Glomus deserticola were 114.17 ± 35.12 and 58.44 ± 17.44 per 100 g of soil, respectively. Okon and Solomon (2014) earlier obtained AM fungi spore count range of 18-112 per 100 g of soil from varieties of crops they worked on.

Soil properties
The physical and chemical properties of the soil and organic fertilizer used in the experiment are presented in Table 1. The soil was of sandy loam texture with sand, silt and clay proportions 692.00, 154.00 and 154.00 g kg -1 respectively. The soil was slightly acidic with soil pH of 6.70. Organic carbon and total N in the soil were 0.77 and 0.07 g kg -1 respectively. The organic fertilizer had organic C content of 79.26 g kg -1 and total N of 6.97 g kg -1 , giving a C:N ratio of 11:1; an attribute that the organic fertilizer used has potential to decompose fast for enhanced soil fertility. The C:N ratio is a critical factor in the decomposition of organic material. The lower the C:N ratio the faster will be its rate of decomposition and nitrogen release in crop husbandry (Ge et al., 2013).
Soil water plays a significant role in organic matter decomposition, mineralization and nutrient availability during crop production. From 75% FC water regime, soil properties, particularly total N, organic C and CEC were significantly increased compared to when soil moisture regimes were much lower (Table 2). However, Rana et al. (2017) observed no significant influence of water regimes on soil organic carbon, a contrary report from our own. Variations from different studies could be attributed to differences in soil properties, chemistry of water used for wetting and water use efficiency by different crop species. These soil properties sustained the second A. cruentus cultivation without further soil amendment additions, but with a reduction in the yield of the test crop. Increased soil organic carbon and slightly acidic soil conditions were observed by Tits et al. (2014), Babajide and Olla (2014), and Okonji et al. (2018) in AMF treated soils.
The symbiotic association of AMF in plants that allows for more P as observed by Cundiff (2012) and Bhardwaj et al. (2014) was equally observed in this study (Table 2). Pots without AMF had significantly (p < 0.05) lower available P of 47.46 mg kg -1 when compared with 54.67 and 55.30 mg kg -1 of available P for G. deserticula and G. clarum fungal additions, respectively.

Agronomic growth of Amaranthus cruentus
Growth parameters of A. cruentus improved with the use of organic fertilizer and the AM fungi. Moyin-Jesu (2015) earlier observed significant improvement in similar growth parameters with organic fertilizer treatments when compared to the control. However, there were reduced growth parameters during the second planting (Figures 1 to 4). Growth parameters increased with increasing rates of water addition, but varied slightly during the repeated experiment, where plants with 75% FC treatments had comparable growth with 100% FC treatment plants. This could be the reasonable soil moisture that allows for availability of nutrients to plants. This agreed with the findings of Khalil and Yousef (2014) where growth parameters of the test plant were significantly lowered with decreasing rates of water availability.
The highest mean plant height of 64.83 ± 6.72 cm obtained with the treatment containing 10 t ha -1 OF and G. clarum was not significantly different from 61.83 ± 4.91 cm obtained with the treatment containing 5 t ha -1 OF and G. clarum at 100% FC ( Figure 1). Soil inoculation with G. clarum gave the best crop physiological growth parameters by Zuccarini and Savé (2016). At 75% FC, the highest mean plant height of 44.67 ± 5.36 cm was obtained with the treatment containing 10 t ha -1 OF and G. deserticola. The highest mean stem girth of 3.50 ± 0.00 cm was obtained with 10 t ha -1 OF and G. deserticola at 100% FC; while at 75% FC, the highest mean stem girth of 2.63 ± 0.07 cm was obtained with 10 t ha -1 OF ( Figure  3). The highest mean leaf area of 98.51 ± 7.61 cm 2 was also obtained with the treatment containing 10 t ha -1 OF and G. deserticola (Figure 4). Comparable results were obtained during the second planting, though with reduced values. These results agreed with Minaxi et al. (2013) and Cyril et al. (2014) whose work showed the highest plant height of A. cruentus with the synergistic use of manure and AM fungi when combined, which was significantly higher than when each of the treatments was singly applied.

Effects of arbuscular mycorrhiza fungi on soil properties and yield of
The repeated experiment without further application of AM fungi and OF treatments gave lower and comparable values. These results agreed with the findings of Cyril et al. (2014) where the application of manure in combination with AM fungi showed a significantly higher vegetative yield of A. cruentus than the control or the use of inorganic fertilizer. Salami and Osonubi (2003) similarly recorded the improved cassava yield with AM fungi inoculation, and Bona et al. (2016) equally showed that AM fungi were useful for sustainable agriculture.

Conclusion
The synergistic use of AM fungi and organic fertilizer improved the availability of total N, organic C and CEC of the soil. The growth and yield of A. cruentus cultivated under different soil moisture regimes when AM fungi and organic fertilizer were applied also varied. The addition of G. clarum fungi with 5 t ha -1 of organic fertilizer to soil optimally improved the growth and yield of A. cruentus in water stress conditions.