FUNGI DIVERSITY IN THE RHIZOSPHERE OF Aspilia pruliseta Schweif. ext Schweif IN THE SEMI-ARID EASTERN KENYA

Semi-arid eastern Kenya is a fragile ecosystem with continuous cultivation of dryland pulses and grains. Farmers use artificial fertilizers most of which are deleterious to the environment. Previous studies have shown that soil microbes in the rhizosphere could be used to sustainably enhance levels of soil mineral nutrients and soil health. However, few studies have examined fungal diversity in the rhizosphere of wild and native Aspilia pruliseta shrub. In this study, amplicons of Internal Transcribed Spacer (ITS) region on Total Community DNA using Illumina sequencing were used to explore the fungal community composition within the rhizosphere. Operational taxonomic units (OTUs) were analyzed using QIIME 1.8.0, taxonomy assigned via BLASTn against SILVA 119 database. Hierarchical clustering was done using R programming software. 72,093, 50,539 and 43,506 sequence reads were obtained from samples MC1a, MC2a and MC3a respectively representing rhizosphere depth 0-20 cm, 21-40 cm and 41-60 cm. A total of 373 OTUs were realized at 3% genetic distance. Taxonomic analysis revealed that the genera Glomus was most prevalent in all soil depths with 85.60 % of the OTUs in depth 0-20 cm, 69.04 % in depth 21-40 cm and 48.45 % in depth 41-60 cm. The results revealed high levels of obligate arbuscular mycorrhiza fungi that if commercially cultured could enhance phosphates uptake in crops.


Introduction
vegetation of interest, the selected areas of the survey for each of the three sites had to have three 122 soil textural types (sandy loam, silt loam and silty clay). In each site, a quadrant measuring one 123 metre by one metre was thrown at random in each sub-site (a sub-site consisted of an area within 124 the site with one soil textural type). In case the quadrant contained more than one Aspilia pruliseta 125 plant, the one closest to the centre of the quadrant was chosen for collection of rhizosphere soil 126 fungal spores. The quadrant was thrown five times in each sub-site and soil was sampled at depth 1 , 127 0-20 cm; depth 2, 21-40 cm, depth 3 , 41-60 cm using a soil auger with a scooping capacity of 40cm 3 128 of soil. The sampled soil was put together for each rhizosphere depth from each sub-site and 129 homogenously mixed. 100 g of the mixture was put into khaki paper bags for soil and root DNA 130 analysis in the laboratory. was weighed using an electronic balance. The weighed soil was put into 100 ml beaker and about 135 50 ml tap water was added. The mixture was placed and mixed on an electronic stirrer overnight. 136 After 12 hours the mixture was washed several times by passing it through a 710 µ sieve placed 137 on top of a 45 µ sieve (14). The 710 µ sieve collected the roots and course debris while the 45 µ 138 prevented the spores from passing through. The roots and course debris from the 710 µ sieve were 139 put into a mortar and air-dried in a hood while the process of sieving continued by collecting sieved 140 water and soil mixture in a 1-litre cylinder (14) . The washing and decanting process was done 141 several times until near-clear water was obtained. This was followed by filling the centrifuge tubes 142 with the sieved content. Centrifugation was done for 5 minutes at 1500 revolutions per minute 143 (rpm) and the filtrate was poured off while the supernatant remained at the bottom of the tube. 48 % sucrose solution was added to the supernatant at equal volumes (50ml) and centrifuged for 1 145 minute at 1500 (rpm) (14). The filtrate was collected on the 45 µ sieve while the supernatant was 146 disposed off. The filtrate was then washed with slowly flowing tap water to wash off the sucrose.

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The washed content was then collected in a 50ml plastic cylinder and the contents poured into a 148 filter paper. Using a fine pair of forceps, the contents were picked and transferred to eppendorf

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Soil pH for the three studied depths was slightly acidic with a range from 5.9-6.1 (Table 1a, 1b &   214   1c). The middle depth (21-40 cm, Table 1b) was more acidic with a pH of 5.9 compared to 6.1 and 215 6.0 in the first and third soil depth respectively. There was more organic matter content in depth 216 two at 9.1% compared to 8.8% and 8.6% for depth one and depth three respectively. Soil      Analysis of similarity and distance based redundancy analysis at class (Figure 4)  proliferation of rhizosphere fungal growth (Table 1a, b &c). The level of soil organic matter was higher in the second rhizosphere layer (21-40 cm) but fungal microbe population was not 316 correspondingly high agreeing with the principal findings of (31) that soil has diverse elements 317 that contribute to its productivity and the proper balance between those elements is what actually 318 matters.

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The high sensitivity of Illumina sequencing enabled detection of rare species, thus providing more 320 detailed information on fungal diversity in the rhizosphere of Aspilia pruliseta plant. The phylum,

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Glomeromycota was more frequently identified in the plant's rhizosphere than those of