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Isolation of Indigenous Soil Microbes and Evaluation of Storage Effects to Develop a Viable Soil-Microbial Inoculum

Authors:

Abstract

Rhizosphere microorganisms (RMs) are important in crop production but, they have been badly affected by agrochemicals. Hence, restoration of beneficial RMs is required for soil rehabilitation. This study was designed to isolate indigenous soil microbes (ISM) and tested their survival in a nutrient-enriched broth culture medium (BCM). BCM was prepared by using ingredients i.e., sugarcane molasses, rice bran and water. A virgin soil sample was collected from Udawalawe, Dry zone of Sri Lanka and it was introduced to BCM and incubated at ambient conditions for six months. After that, BCM was filtered and stored in amber colour containers. The standard dilution plating method with specific culture mediums was followed to identify RMs present in BCM and recorded their colony-forming units (CFU). Accordingly, BCM was tested for storage conditions i.e., ambient condition, refrigerator (4°C), sunny and darker. In vitro results indicated that ISM viz. Anaerobic nitrogen-fixing bacteria, Herbaspirillum spp., Root nodule-Rhizobium spp., Azospirillum spp. and Phosphate solubilizing bacteria can be survived in the developed BCM for more than six months with the average CFU/ml counts of 7.00×10 58 , 8.75×10 58 , 7.50×10 58 , 15.00×10 58 and 57.00×10 58 respectively. The direct sunlight exposure reduced the population of ISM in BCM. In darker conditions, microbes were survived successfully. Under the refrigerator condition, ISM was sustained well. The ambient condition was better for the prolonged survival of ISM in a cost-effective way. For future advancements, in vitro morphological and species-level characterisation studies and field-level studies on effectiveness as an SMI are needed.
B.R. Kulasekara*, A. Maralanda, H.A.S. Weerasinghe and U.W.L.M. Kumarasiri (*kulasekaraya@gmail.com)
Rhizosphere microorganisms (RMs) are important in crop production but, they have been badly affected by agrochemicals. Hence, restoration of beneficial RMs is required for soil rehabilitation. This study was
designed to isolate indigenous soil microbes (ISM) and tested their survival in a nutrient-enriched broth culture medium (BCM). BCM was prepared by using ingredients i.e., sugarcane molasses, rice bran and water. A
virgin soil sample was collected from Udawalawe, Dry zone of Sri Lanka and it was introduced to BCM and incubated at ambient conditions for six months. After that, BCM was filtered and stored in amber colour
containers. The standard dilution plating method with specific culture mediums was followed to identify RMs present in BCM and recorded their colony-forming units (CFU). Accordingly, BCM was tested for storage
conditions i.e., ambient condition, refrigerator (4°C), sunny and darker. In vitro results indicated that ISM
viz.
Anaerobic nitrogen-fixing bacteria,
Herbaspirillum
spp., Root nodule-
Rhizobium
spp.,
Azospirillum
spp.
and Phosphate solubilizing bacteria can be survived in the developed BCM for more than six months with the average CFU/ml counts of 7.00×1058, 8.75×1058, 7.50×1058,15.00×1058 and 57.00×1058 respectively.
The direct sunlight exposure reduced the population of ISM in BCM. In darker conditions, microbes were survived successfully. Under the refrigerator condition, ISM was sustained well. The ambient condition was
better for the prolonged survival of ISM in a cost-effective way. For future advancements, in vitro morphological and species-level characterisation studies and field-level studies on effectiveness as an SMI are needed.
Keywords:
Indigenous soil microbes, inoculum, rhizosphere
Sugarcane Research Institute of Sri Lanka
RMs have the capacity to significantly influence plant growth by improving the bioavailability of
soil-borne nutrients and they are capable to produce plant growth-promoting secretions.
RMs have been badly affected by intensive crop cultivation and agrochemicals.
Restoration of beneficial RMs is required for soil rehabilitation and sustainable crop cultivation.
However, the isolation of indigenous soil microbes and preservation up to a viable soil microbial
inoculum is challenging.
This study was designed to isolate ISM and to evaluate the storage effects on microbes in a
nutrient-enriched broth culture medium.
An undisturbed virgin floor soil sample, primarily organic horizon was collected from Uda
Walawe, (6°24'N, 80°49'E and 72 m above sea level) Dry zone of Sri Lanka to isolate ISM for
the evaluation.
Dry zone - mean annual rainfall (<1,750 mm), mean annual temperature (32.1 °C), mean
annual bright sunshine duration (4.3 to 9.3 hours per day)
The collected soil belonged to the Reddish Brown Earths local soil category
(USDA - Order: Alfisols, Suborder: Ustalfs, Great group: Haplustalfs)
Ingredients of BCM: sugarcane molasses, rice bran and water
Storage conditions : ambient , refrigerator (4°C) , sunny and darker
conditions for three months
Screened by standard dilution plating method by using six specific
culture media and recorded colony forming units (CFU)/ ml of BCM
Azotobactor Medium : to screen
Azotobacter
spp.
Clostridium Medium : to screen anaerobic N-fixing microorganisms
JNFb Medium : to screen
Herbaspirillum
spp
.
Yeast-Mannitol Agar (YMA) Medium containing Congo red :
to screen root nodule bacteria-
Rhizobium
spp
.
Roderquez Caceras (RC) Medium : to screen
Azospirillum
spp
.
Picovaskaya’s (PVK) Medium : to screen the phosphate-solubilising bacteria
Preparation of
BCM
Introduction of
ISM
Incubation at
ambient condition
for 6 months
Screening
Storing
Filtering the
BCM
Allen, E.K., and Allen, O.N., (
1950
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Olivares, F.L., Baldani, V.L.D., Reis, V.M.
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(1996). Occurrence of the endophytic diazotrophs
Herbaspirillum
spp.in roots, stems, and leaves,
predominantly of Gramineae.
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Onyeze Rosemary, C., Onah Gloria, T., and Igbonekwu Cecilia, C., (2013). Isolation and Characterization of Nitrogen-Fixing Bacteria in the Soil.
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No.3,pp.438-445.
Pikovskaya, R.I., (1948). Mobilization of phosphorus in soil in connection with the vital activity of some microbial species.
Microbiology
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According to the in vitro screening, there were five different types of RMs in the enriched BCM.
Azotobactor
spp. were not found in vitro condition.
Anaerobic N
-fixing microorganisms
(ANFB)
Transforming atmospheric nitrogen into fixed nitrogen
Living non-symbiotic and fix N in the absence of oxygen
Herbaspirillum
spp.
Promoting plant growth by biological nitrogen fixation
and phytohormone production.
Coloniesing in roots and aerial plant tissue endophytic N
-
fixing bacteria
Root nodule
Rhizobium
spp.
Forming a symbiotic relationship with Legume plants by
making nodules on the plant roots
onverting atmospheric nitrogen into ammonium form
Producing of plant hormonal substances, notably
gibberellins
Azospirillum
spp.
Stimulating plant growth through phytohormones.
Phosphate solubilising bacteria
(PSB)
Hydrolyzing organic and inorganic insoluble P
compounds into soluble forms
Types
of RMs
Initial
CFU /ml
(BCM in
ambient
condition)
CFU /ml in different storage conditions
after 3 months
6 months after
CFU /ml
(BCM in
ambient
condition)
Ambient
condition
Refrigerator
condition (
4°C
)
Sunny
condition
Darker
condition
ANFB
6.00×10
59
12.50×10
58
5.50×10
58
2.00×10
0.50×10
58
7.00×10
58
Herbaspirillum
spp. 3.17×10
59
3.75×10
58
5.00×10
58
1.50×10
3.00×10
58
8.75×10
58
Root nodule
Rhizobium
spp. 3.77×10
59
1.00×10
58
2.37×10
59
1.25×10
58.50×10
58
7.50×10
58
Azospirillum
spp. 4.52×10
59
19.25×10
58
1.90×10
59
1.25×10
56.50×10
58
15.00×10
58
PSB
4.17 ×10
59
64.25×10
58
1.50×10
59
5.00×10
21.75×10
58
57.00×10
58
The isolated ISM can be survived in the artificially enriched BCM successfully and the storage
conditions of BCM are directly affected to the average CFU counts of ISM.
In vitro morphological and species-level characterization studies on ISM are required for future
advancements.
Field-level studies are needed for the identification of the effectiveness of the isolated ISM as a soil
microbial inoculum.
Filtered BCM
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