Effects of plant growth promoting bacterial isolates from Kavango on the vegetative growth of Sorghum bicolor

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726 Afr. J. Microbiol. Res.
Table 1. Source and plant growth promoting trait profiles of bacterial isolates.
Bacterial isolate Site Plant rhizosphere PO3-4 solub. N2 fixation IAA Anti-fungal
S. maltophilia LCS2-11* Lukas 2 Sorghum - + + -
P. stutzeri ACM2-32 ATR Pearl millet + - + -
E. cloacae FCM2-50 Field E Pearl millet - + ++ -
B. subtilis ASM1-59 ATR Pearl millet - - ++ +
B. amyloliquefaciens LSM1-61 Lukas 2 Pearl millet - - ++ +
*Siderophore producing isolate; ATR coordinates - 17°53′49.75″ S 20°09′07.07″ E; Lukas 2 coordinates - 17°53′43.80″ S
20°14′05.26″ E; Field E coordinates - 17°54′04.40″ S 20°14′14.34″ E.
plant diseases can be fulfilled by using plant root
associated bacteria (Akhtar and Siddiqui, 2011; Glick,
2012). Various groups of bacteria found in the volume of
soil affected by the presence of plant roots (Uren, 2007),
also known as the rhizosphere, have been shown to be
beneficial for the growth, yield and crop quality of plants
(Orhan et al., 2006). These bacteria are able to colonize
the rhizosphere and in some instances enter the roots of
plants eventually inducing a beneficial effect on the host
plant (Kloepper et al., 1980). Mechanisms by the
bacteria aimed at inducing plant growth promotion
include production of antibiotics against pathogenic
microorganisms, production of antifungal compounds,
production of plant hormones, increasing the availability
of soluble phosphorus, increasing iron availability to
plants, nitrogen fixation and regulation of ethylene
concentration (Lucy et al., 2004).
The sorghum plant has the capacity to grow in
moderately poor semi-arid and sub-tropical conditions of
Africa (Taylor, 2003). Sorghum (Sorghum bicolor (L.)
Moench) is an important crop throughout Western,
Eastern and Southern Africa, that is used mainly for food
and beverage production. Sorghum meal is used for
making porridge and as an added substance to lower
consistency and increase supplement and caloric
thickness in porridge produced from other grains (Smith
and Frederiksen, 2000; Ohiokpehai, 2003). In beer
production, sorghum malt is used in the saccharification
of the starchy substrate prior to fermentation (Smith and
Frederiksen, 2000). Despite the genetic potential,
generally low sorghum grain yields are experienced as a
consequence of major constraints such as nutrient
deficiency, soil water deficiency and plant diseases
(Wortmann et al., 2009).
Commercial inorganic fertilizers may offer a short term
solution for crop production but the financial component
and long term soil fertility concerns labels them as an
unfavourable option (Namibia Resource Consultants and
Vigne and Associates Consultants, n.d.). For those
countries that do not manufacture fertilizers, as opposed
to need to import them, bacterial inoculants can be used
to produce local cereals and agricultural products at a
reasonably less expensive cost. The utilization of
rhizosphere related microorganisms offers an appealing
option to agrochemicals considering the fact that their
plant growth and crop yield enhancing capacities have
been shown over the recent decades (Saharan and
Nehra, 2011). This study was carried out to assess the
effects of treating S. bicolor seed with peat based plant
growth-promoting rhizobacterial suspensions.
MATERIALS AND METHODS
Bacterial isolates
Native bacterial isolates (Stenotrophomonas maltophilia LCS2-11
and Pseudomonas stutzeri ACM2-32, Enterobacter cloacae FCM2-
50, Bacillus subtilis ASM1-59 and Bacillus amyloliquefaciens LSM1-
61) exhibiting increasing nutrient availability, plant hormone
production and anti-fungal capabilities were obtained from The
Department of Biological Sciences, University of Namibia. The
bacteria were isolated from the rhizospheres of pearl millet and
sorghum plants that were grown in the fields of subsistence farmers
along the Kavango River (Table 1).
Preparation of treatments
Bacterial isolates were grown in VM–ethanol broth at 28 ±2°C for 3
days. The bacterial cell concentration was adjusted to OD660 = 0.9
in 50 ml VM–ethanol broth volume, washed with sterile distilled
water and resuspended in 50 ml of 0.85% NaCl. This procedure
was repeated for some bacterial isolates depending on the number
of treatments and number of replicates. Starke Ayres® palm peat
was prepared according to the manufacturer’s instructions and
dried overnight at 60°C. Approximately 50 g dry palm peat was
placed into separate aluminium foil containers and sterilized via
autoclaving. The palm peat was then aseptically transferred into
Ziploc® plastic bags, moistened with 5 ml sterile distilled water per
bag and kept at 4°C.
The application of phosphate solublizers alone or in combination
with nitrogen fixers is beneficial for the growth of cereal (Zaidi and
Khan, 2005). Therefore, combination treatments were made up of
one phosphate solubilizing isolate (P. stutzeri ACM2-32), a N2–fixer
and an isolate with antifungal capability. The inoculum treatments
were prepared according to Rose et al. (2011) with slight
modifications. Treatments (Table 2) consisted of 50 g palm peat
and 20 ml of bacteria solution, that is, 3 ml bacteria-0.85% NaCl
suspension + 17 ml sterile distilled water for single bacterial
treatments and 3 ml bacteria-0.85% NaCl suspension (×3 different
isolates) + 11 ml sterile distilled water for combination bacterial
treatments, whereas 3 ml 0.85% NaCl + 17 ml sterile distilled water
was the control. After transferring the bacteria solutions to the palm
peat enclosed in Ziploc® bags, the treatments were incubated for 3
days at 30°C before applying to soil. The non-inoculum control
treatments were a commercial fertilizer, Hygrotech Terra Nova

Haiyambo et al. 727
Table 2. Comparisons of treatments for root masses, plant masses and
root : shoot ratios.
Treatment Root mass (g) Plant mass (g) Root : shoot ratio
T1 0.09 ± 0.01c 0.31 ± 0.04b 0.39
T2 0.05 ± 0.01bc 0.22 ± 0.06b 0.31
T3 0.09 ± 0.03c 0.29 ± 0.02b 0.42
T4 0.07 ± 0.05bc 0.17 ± 0.16b 0.86
T5 0.09 ± 0.02c 0.39 ± 0.18b 0.33
T6 0.07 ± 0.01bc 0.23 ± 0.11b 0.50
T7 0.08 ± 0.00c 0.34 ± 0.08b 0.33
T8 0.08 ± 0.04c 0.34 ± 0.30b 0.39
T9 0.10 ± 0.03c 0.45 ± 0.04ab 0.28
T10 0.14 ± 0.07a 0.83 ± 0.16ac 0.20
T11 0.07 ± 0.09bc 0.18 ± 0.23b 0.58
T12 0.19 ± 0.05a0.39 ± 0.08
b
0.95
Data is presented as mean ±SD for root and plant masses and as decimal
form for root mass : shoot mass. a = mean difference between treatment and
peat + water is significant at the 0.05 level. b = mean difference between
treatment and fertilizer is significant at the 0.05 level. c = mean difference
between treatment and no peat is significant at the 0.05 level. T1 = LCS2-11
(Stenotrophomonas maltophilia); T2 = ACM2-32 (Pseudomonas stutzeri); T3 =
FCM2-50 (Enterobacter cloacae); T4 = ASM1-59 (Bacillus subtilis); T5 = LSM1-
61(Bacillus amyloliquefaciens); T6 = ASM1-59: LCS2-11: ACM2-32; T7 =
ASM1-59: FCM2-50: ACM2-32; T8 = LSM1-61: LCS2-11: ACM2-32; T9 =
LSM1-61: FCM2-50: ACM2-32; T10 = Fertilizer; T11 = peat + water; T12 = no
peat.
applied at 200 kg/hectare and a treatment with no peat.
Application of treatments and planting sorghum seeds
Plant pots (15 cm diameter x 12 cm depth) containing 1.6 kg of
unprocessed arenosol type soil collected from a field (17°53′57.90″
S; 20°14′04.39″ E) were used in this study. Using a sterile trowel,
treatments were transferred from the Ziploc® bags and mixed with
soil in the plant pots. S. bicolor seeds bought from Rundu Open
Market were surface sterilized by soaking in 70% ethanol for 5 min,
then in 1.5% sodium hypochlorite for 1 min and rinsed three times
in sterile distilled water. The seeds were dried for 2 h in sterile
conditions and planted into the pots containing treatments. There
were two replicates for each treatment with one seed planted per
pot. After 25 days, the dry mass was determined by drying plants in
an oven (50°C) until the weight remained constant; the length and
mass of shoots and roots were recorded.
Specifics for greenhouse pot experiments
The pot experiments were carried out at the University of Namibia
Main campus’ (Windhoek) greenhouse facility for 25 days. The
plant pots were arranged in a randomized block manner with two
blocks. The plants were watered every day with an average
atmospheric pressure of 1006.923 hPa, an average maximum
temperature of 34.1°C and an average 13 h 26 m 58 s daylight
length per day for the duration of the pot experiments.
Statistical analysis
SPSS statistics (SPSS, version 22.0.0.0, 2013) was used to analyse
the data. Analysis of variance (ANOVA)\Kruskal-Wallis one-way
analysis of variance procedure was performed followed by post hoc
Fisher’s least significant difference (LSD). All analyses were tested
at 5% level of significance.
RESULTS AND DISCUSSION
Three of the single inoculant treatments (T1, T3 and T5)
and three combination treatments (T7, T8 and T
9) had
comparatively similar growth effects on sorghum root
mass as the fertilizer treatment. Treatment T9 was able to
enhance sorghum plant growth significantly as compared
to the water control. Apart from inoculants T2 and T4, the
remaining peat based bacterial suspensions evoked a
valuable impact on the development of S. bicolor.
PGP and biocontrol bacteria inoculation effects on
sorghum
Three of the single inoculant treatments (T1, T3 and T5)
and three combination treatments (T7, T8 and T
9) had
comparatively similar growth effects on sorghum root
mass as the chemical fertilizer treatment. Treatment T9
was able to enhance sorghum plant growth significantly
as compared to the water control. Though two of the
single inoculants T2 and T4 did not bring about any
improved growth on the plants, it was determinable that
the peat based bacterial suspensions elicited a beneficial

728 Afr. J. Microbiol. Res.
effect on the growth of S. bicolor.
The results showed that single bacterial suspension
treatments consisting of K. cloacae FCM2-50 (p = 0.089),
S. maltophilia LCS2-11 (p = 0.089) and B.
amyloliquefaciens LSM1-61 (p = 0.122) enhanced root
growth of S. bicolor. The combination bacterial treat-
ments T7 (B. subtilis ASM1-59: K. cloacae FCM2-50: P.
stutzeri ACM2-32) and T8 (B. amyloliquefaciens LSM1-
61: S. maltophilia LCS2-11: P. stutzeri ACM2-32) also
produced enhanced root growth on S. bicolor. Treatment
T9 (B. amyloliquefaciens LSM1-61: K. cloacae FCM2-50:
P. stutzeri ACM2-32) enhanced both S. bicolor root
growth (p = 0.196) and whole plant biomass (p = 0.032).
Unsurprisingly, the difference in mean root dry mass
between the fertilizer and the water control was
statistically significant (p = 0.044). K. cloacae FCM2-50,
B. subtilis ASM1-59 and B. amyloliquefaciens LSM1-61
are described as high producers of IAA, thus enabling
root growth stimulation. Additionally, Kosakonia spp. are
known to promote seedling root elongation via ACC
deaminase activity (Li et al., 2000).
The water control and the no peat treatments were
significantly different (p = 0.003) with regard to sorghum
plant dry mass. Sorghum plants that grew in the no peat
control treatment often had greater and at times
statistically significant than most of the inoculant treat-
ments. However, the average root-shoot ratio of 0.95
(0.58 for water control) for sorghum plants in the no peat
treatment suggests that nitrogen availability was lower in
the no peat treatment as compared to the inoculation
treatments.
N2-fixing bacteria play a critical role in the accumulation
of plant biomass by providing an environment where the
plant acquires nitrogen for assimilation (Pilbeam, 2010).
The root-shoot ratio of the plant is also determined by
nitrogen availability. The average root : shoot ratio of T4
(0.86) and the no peat treatment (0.95) were greater than
that of the water control treatment (T11 = 0.58). The rest of
the treatments had a smaller average root : shoot ratio as
compared to the water control. A nitrogen deficiency
often causes the growth of an increased root fraction so
that the root system is allowed to increase nutrient
acquisition (Pilbeam, 2010). As compared to the water
control, the lower root-shoot ratios in plants treated with
inoculations suggests that there was more nitrogen
available as a result of the bacterial treatments.
We can conclude from our data that bacterial treat-
ments were able to enhance sorghum growth, compa-
rable to that of the commercial fertilizer in terms of root
biomass. K. cloacae FCM2-50: B. amyloliquefaciens
LSM1-61: P. stutzeri ACM2-32 enhanced sorghum plant
biomass. Enhancement of sorghum growth in terms of
root biomass comparable to the level of commercial
fertilizer was accomplished by single inoculants of S.
maltophilia LCS2-11, K. cloacae FCM2-50, and B.
amyloliquefaciens LSM1-61. Similarly, combination
inoculants of B. amyloliquefaciens LSM1-61: K. cloacae
FCM2-50: P. stutzeri ACM2-32, B. amyloliquefaciens
LSM1-61: S. maltophilia LCS2-11: P. stutzeri ACM2-32
and B. subtilis ASM1-59: K. cloacae FCM2-50: P. stutzeri
ACM2-32 promoted sorghum vegetative root growth.
Conclusion
From this study, it is concluded that PGP bacteria
inoculants improve the growth of sorghum seedlings to
level comparable to chemical fertilizers. These findings
show the possibility of using bacterial inoculants as an
inexpensive, effective and environmentally friendly alter-
native for increased agricultural crop productivity. An
added advantage is that these PGP bacteria are
ecologically adapted to the soils of this agro ecological
zone as they were originally isolated from there. The
eventual goal is to prove that the inoculants facilitate and
improve plant growth and increase grain seed yield in
sorghum. By developing inoculants consisting of native
PGPR and bio-control bacteria, we improve our potential
to alleviate challenges of heavily depending on importing
fertilizers in countries that do not have chemical fertilizer
manufacturing companies like Namibia or where
subsistence farmers do not afford the price of the
chemical fertilisers. The advancement of field trials at
multiple locations is a necessary step towards assuring
the accomplishments of effective bacterial inoculants.
These inoculants offer a cheap preferential option to
support current and future sorghum based industries.
Conflict of interests
The authors did not declare any conflict of interest.
ACKNOWLEDGEMENTS
This study was funded by The Future Okavango (TFO)
Project through the BMBF (Federal Ministry of Education
and Research, Germany) Research Framework
Programme, Research for Sustainable Development
(FONA). The University of Namibia, Department of
Biological Sciences is thanked for providing the research
facilities.
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