476
2.4 Determination of Percentage Mycorrhizal Colonization
of the Maize Genotypes
Fine roots were washed in running tap water and thereafter cleared with 10% KOH
at 80 °C water baths. The cleared roots were rinsed thoroughly with distilled water,
acidified with 2% HCl, followed by staining with 0.05% (w/v) trypan blue in lactic
acid (Liu et al. 2016). The samples were then washed with 10% lactic acid and
stored at 4 °C in a refrigerator until analysis. From each sample, random subsets of
about 100 root segments were selected for AMF colonization assessment. The percentage of root length colonized by AMF was determined by observing the stained
roots for arbuscules, vesicles and intraradical hyphae under a dissecting microscope
using the gridline intersect method (Giovannetti and Mosse 1980).
2.5 Soil Analysis
A pH meter was used to determine soil pH in a 1:2.5 soil/water suspension. Soil
organic matter was analysed via the wet combustion Walkley and Black oxidation
method. Macro-Kjeldahl digestion, titration and distillation method was used to
determine total nitrogen (Okalebo et al. 2002). Available P was determined using
the Olsen extraction method (Okalebo et al. 2002). The soils were leached using
ammonium acetate and the amounts of exchangeable cations (Ca
2+
and Mg
2+
) in the
leachate analysed using atomic absorption spectroscopy. Flame emission spectroscopy was used to measure K
+
and Na
+
concentrations.
2.6 Statistical Analyses
Percentage data on maize root AMF colonization were arcsine transformed before
analysis to fulfil the assumptions of ANOVA. The results were then analysed using
two-way analysis of variance, considering the source of soil and crop genotypes as
independent factors. Wherever feasible, post hoc testing was performed using
Tukey’s HSD test. For cowpea genotypes, data on the number of nodules and nodule
dry weight were analysed using two-way analysis of variance (ANOVA). Means
were separated by Tukey’s HSD test at 5% probability level. All ANOVAs and post
hoc tests were done using SAS software version 9.2. The bacteria isolate groups
were clustered based on their morphological characteristics applying the Euclidean
distance similarity and single linkage (nearest-neighbour) procedures using PAST
software version 3.20. Redundancy analysis (RDA) to relate soil characteristics
with plant growth characteristics (shoot and root dry weights), cowpea nodulation
and maize AMF colonization potential was done using Canoco software version 5.
RDA is a multivariate analysis that is utilized to determine the relationship between
two different sets of data.
E. M. Njeru et al.
2.4 Determination of Percentage Mycorrhizal Colonization
of the Maize Genotypes
Fine roots were washed in running tap water and thereafter cleared with 10% KOH
at 80 °C water baths. The cleared roots were rinsed thoroughly with distilled water,
acidified with 2% HCl, followed by staining with 0.05% (w/v) trypan blue in lactic
acid (Liu et al. 2016). The samples were then washed with 10% lactic acid and
stored at 4 °C in a refrigerator until analysis. From each sample, random subsets of
about 100 root segments were selected for AMF colonization assessment. The percentage of root length colonized by AMF was determined by observing the stained
roots for arbuscules, vesicles and intraradical hyphae under a dissecting microscope
using the gridline intersect method (Giovannetti and Mosse 1980).
2.5 Soil Analysis
A pH meter was used to determine soil pH in a 1:2.5 soil/water suspension. Soil
organic matter was analysed via the wet combustion Walkley and Black oxidation
method. Macro-Kjeldahl digestion, titration and distillation method was used to
determine total nitrogen (Okalebo et al. 2002). Available P was determined using
the Olsen extraction method (Okalebo et al. 2002). The soils were leached using
ammonium acetate and the amounts of exchangeable cations (Ca
2+
and Mg
2+
) in the
leachate analysed using atomic absorption spectroscopy. Flame emission spectroscopy was used to measure K
+
and Na
+
concentrations.
2.6 Statistical Analyses
Percentage data on maize root AMF colonization were arcsine transformed before
analysis to fulfil the assumptions of ANOVA. The results were then analysed using
two-way analysis of variance, considering the source of soil and crop genotypes as
independent factors. Wherever feasible, post hoc testing was performed using
Tukey’s HSD test. For cowpea genotypes, data on the number of nodules and nodule
dry weight were analysed using two-way analysis of variance (ANOVA). Means
were separated by Tukey’s HSD test at 5% probability level. All ANOVAs and post
hoc tests were done using SAS software version 9.2. The bacteria isolate groups
were clustered based on their morphological characteristics applying the Euclidean
distance similarity and single linkage (nearest-neighbour) procedures using PAST
software version 3.20. Redundancy analysis (RDA) to relate soil characteristics
with plant growth characteristics (shoot and root dry weights), cowpea nodulation
and maize AMF colonization potential was done using Canoco software version 5.
RDA is a multivariate analysis that is utilized to determine the relationship between
two different sets of data.
E. M. Njeru et al.