Impacts of Microbial Inoculants on the Growth and Yield of Maize Plant

2.1. Plant Material

Seeds of maize cultivars (Aflatoxin SYN 4W, TZB-SR, Aflatoxin R SYN-2Y, Aflatoxin SYN 3W and Aflatoxin SYN-2Y) used in this investigation were obtained from the International Institute of Tropical Agriculture, Ibadan, Nigeria. The seeds were sterilized with 70% ethanol for 2 minutes and washed with sterile distilled water.

2.2. Microbial Inoculant Preparation

P. kilonensis and P. protegens isolates selected for this study were obtained from the Microbial Ecology laboratory (C. Prigent-Combaret). The bacterial isolates were cultured for 48 hrs in Luria-Bertani Broth (LB) medium, and then cells were harvested by centrifugation at 12000g for 10 minutes. Each of the P.kilonensis and P. protegens was suspended in sterile distilled water and the concentration adjusted to give 10⁸cells/ml [15].

Ten grams of pectin were suspended in 1 L each of P. kilonensis and P. protegens suspensions, which were shaken for 10 min on a magnetic stirrer plate. Maize seeds were imbibed at the ratio of 500 g/L microbial solutions for 16 hrs.

The bio-inoculated seeds were then air-dried on filter paper for 1 hr before sowing. Another group of surface-sterilized maize seeds (70% ethanol for 2 min) were prepared as control treatments [16].

2.3. Collection of Soil Samples

Soil samples were collected randomly from Mafikeng and Itsoseng areas in South Africa. Mafikeng is located at 25°50'S, 25°38'E. The soil was grey loamy sand with 84% sand, 3% clay and 13% classified as Hutton and Itsoseng is located at 26°08'S, 25°86'E [17]. The soil was red sandy loam with 6.5% silt, 9.8% clay 83.6% sand classified as Ferallic Arenosols [17]. Using the random sampling method, auger samples (5kg) were collected from each sampling units at 0-15 cm. The soil samples collected from each of the sampling sites were bulked and transported to the laboratory in well labelled polyethylene bags. The core samples were then air dried for 3 days and passed through 2 mm sieve in the preparation for analysis.

2.4. Determination of Physical and Chemical Properties of Soil of Study Sites.

The physical and chemical properties measured include: pH using Kent pH metre model 7020, organic matter content using the wet oxidation method as described by [18]. The hydrometer method of Gee and Or. was employed in the determination of particle size [19]. Total nitrogen was estimated by the macro Kjeldahl method [20], available phosphorus (P) was determined by Bray-1 extraction method [21]. To determine E.C.E.C. the method of Chapman was employed [22]. These are shown in Table 1.

2.5. Plant Growth Promotion Studies Under Screen House

A pot experiment was laid in a Randomized Complete Block Design (RCBD) split plot arrangement. The main plot was Pseudomonas spp. (P. kilonensis, P. protegens CHAO and non-inoculated control) applied as seed treatment while the sub plots were maize genotypes (AFLATOXIN SYN 4W, TZB-SR, AFLATOXIN R SYN 2Y, AFLATOXIN SYN 3W and AFLATOXIN SYN-2Y 4W, TZB, AR, 3Wand 2Y) and 2 soil types (Itsoseng soil and Mafikeng soil). Bacterialized seeds were sown (2 seeds/pot) in 35cm-diameter pots filled with 12 Kg of sterilized soil. Soil was wet sterilized in an autoclave at 121ºC for 20 minutes. This is to ensure that only the inoculant introduced influence the sown seed. Only one plant per pot was maintained after germination. All treatments were in triplicates. Plants were irrigated daily at 6.00 hr and 18.00 hr using a watering can. Neither inorganic fertilizer nor pesticide was applied. Physical and chemical properties of the studied soils are shown in Table 1. While the phenotype characteristics of seeds used are recorded in Table 2.

2.6. Growth Parameter Data Collection

Growth parameters (plant height (cm) using a measuring tape, leaf length (cm) using a measuring tape, plant girth (cm) using a thin thread whose length was measured on a ruler, root length (cm) i.e length of the longest root using a measuring tape and root dry weight (g) per plant) were recorded at 12 weeks after planting. To obtain the root dry weight, the below ground portion of the plant was cut and washed to remove attached soil particles. The root was then air dried to a constant weight and the weight was recorded.

2.7. Assessing Plant Growth Promotion of Pseudomonas Spp. Under Field Condition

A field experiment was conducted between September, 2015 and February 2016 at North-West University, Mafikeng, (25°50'S, 25°38'E) South Africa. The Pseudomonas spp. tested were P kilonensis F113 and P protegens CHA0. Maize genotypes used include Aflatoxin SYN 4W, TZB-SR, Aflatoxin R SYN 2Y, Aflatoxin SYN 3W and Aflatoxin SYN-2Y.The soil of the study site was a loamy sand soil, slightly acidic with organic matter value of 1.66%, available phosphorus 1.39PPM, total nitrogen was 0.11 and effective cation exchange capacity was 10.84 (Table 1).

2.8. Experimental Layout

The experimental design was a randomized complete block (RCBD) split plot arrangement with three replications. The main plot was Pseudomonas spp. (P. kilonensisF113, P. protegens CHA0 and non-inoculated control) applied as seed treatment, while the sub plots were maize genotypes (Aflatoxinsyn 4w, TZB-SR, Aflatoxin R SYN 2y, Aflatoxin SYN 3W and Aflatoxin syn-2y). Plot size was 2.2 m². Interplant and row distance were 50 cm X 50 cm respectively. This gave a plant population of 8 plants per plot which is equivalent to 40,000 plants per hectare. Maize was sown at 3 seeds per hole. This was thinned to two per stand after germination and allowed to grow to maturity. Irrigation was solely by rainfall. Mafikeng had an average annual rainfall of 300 to 700 mm and temperatures range between 22ºC and 34ºC. No fertilizer was applied. Weeding was carried out manually using hoe at 4weeks, 8 weeks and 12 weeks after planting. Pest was controlled by the microbial inoculant. No chemical pesticide was applied. The maize varieties matured at different date however harvesting was carried out same day as maize were left on the stalk in the field to dry.

2.9. Growth Parameter and Yield Data Collection

Growth parameters (plant heights (cm) using a measuring tape, leaf length (cm) using a measuring tape, stem girth (cm) using a thin thread whose length was measured on a ruler were recorded at maturity. Dry maize cobs from each treatment were harvested threshed and the threshed maize seeds were weighed on a weighing scale and recorded as yield (tonnes/ha).

2.10. Statistical Analysis

Data collected from field and pot experiments were subjected to two way Analysis of Variance (ANOVA) using IBM SPSS statistical package 21. Means were compared using Duncan Multiple Range Test (p<0.05).

3.1. Effects of Microbial Inoculants, Soil Types and Maize Genotype on the Growth of Maize Planted in Pots

The results in Table 3 show that plant height, leaf length and plant girth on Itsoseng soil were not significantly different from those on Mafikeng soil. Both P. kilonensisF113 and P. protegens CHAO significantly increased the plant height. P. kilonensis F113 significantly increased plant girth. However, P. protegens CHAO decreased leaf length significantly. Also the growth parameters measurement significantly differed according to maize genotypes (P<0.05).

Combined effect of soil type × maize genotype and microbial inoculant × maize genotype increased significantly the plant height, leaf length and plant girth, while soil type × inoculant × maize genotype significantly increased the plant girth. However, combined effect of soil type × microbial inoculant was not significant on plant height, leaf length and plant girth as reported in Table 3.

Means followed by the same letter along the column for a factor are not significantly different at 5% level of probability using Duncan Multiple Range Test (DMRT).

3.2. Effects of Microbial Inoculants, Soil Types and Maize Genotype on Root Weight and Root Length of Maize Planted in Pots.

The effects of microbial inoculants, maize genotypes and soil type on root weight and root length of maize planted in pot was shown in Table 4. Maize plants inoculated with P. kilonensis F113 significantly increased root weight and root length. Soil from Mafikeng produced a significantly heavier and longer root system than soil from Blaawbank. Maize genotype Aflatoxin SYN 4W produced a significantly higher root weight while maize genotype Aflatoxin R SYN 2Y had a significantly higher root length compared to the other genotypes. Interactions of inoculant × maize genotype, and soil type × inoculant × maize genotypes had significant effect on the both root weight and root length of maize while soil type × inoculant and soil type × maize genotype had significant effect on root length but not on the root weight. Microbial inoculation thus resulted into elongation of stems and roots (Fig. 1).

3.3. Effects of Microbial Inoculants on the Growth and Yield of Maize Planted on the Field

Table 5 shows the effects of Pseudomonas inoculants on the growth and yield of maize planted on the field. Microbial inoculants had very high significant improved effect on growth and yield performance of maize plant. Both P. kilonensis F113 and P. protegens CHA0 significantly increased the plant height, leaf length and the yield of maize (Fig. 2 and Table 5). However, plants inoculated with P. kilonensis F113 had a higher height than those inoculated with P. protegens CHA0. This experiment evidence that the maize genotypes were not significantly different in growth but Aflatoxin SYN 4W and Aflatoxin SYN -2Y produced significantly higher yield followed by TZB SR and Aflatoxin SYN 3W while Aflatoxin R SYN -2Y has the least yield. There was no significant difference between maize genotypes for both growth and yield of maize. Similarly, the effect of maize genotype × inoculant on the growth and yield of maize is not significant. The growth and yield of all the genotypes tested were increased by the inoculants (P=0.05).