Plasmid profile of bacterial isolates from fertilizer amended tropical agro soils

1 Department of Microbiology, University of Uyo, P. M. B. 1017, Uyo, Akwa Ibom State, Nigeria. 2 Department of Soil and Environmental Science, Akwa Ibom State University, P. M. B. 1167, Uyo, Akwa Ibom State, Nigeria. 3 Department of Microbiology, University of Port Harcourt, P. M. B. 5323,Choba, Rivers State, Nigeria. 4 Molecular Biology and Biotechnology Division, Nigerian Institute for Medical Research, 6 Edmond Crescent, P. M. B. 2013, Yaba, Lagos, Nigeria.


INTRODUCTION
Arable lands or soil considered fit for growing crops (agro soils) contain natural nutrients.However, these nutrients are not readily available to the plants since they are being released in small portions through soil microbial activities (Girvan et al., 2003).Fertilizer amendments are added to improve or balance the soil nutrients, enhance soil quality and sustainability of the ecosystem.Soil microorganisms act as catalyst in biochemical transformations in soil, playing vital roles in maintaining soil fertility and plant yields.They exert critical effects on ecological stability *Corresponding author.E-mail: uduaknma@yahoo.com,Tel: +234 803 543 1746.and biological productivity of many fields, forest and grassland ecosystems (Havlin et al., 2009).Recently, research interest are focused on the introduction of sustainable management practices such as cropping systems, fertilizer application, cultivation practices, soil organic amendments and pesticide application in agriculture to maintain soil quality and productivity while minimizing the negative effects on the environment and soil resources (Sayali et al., 2012;Weiss et al., 2012;Ndubuisi-Nnaji et al., 2010).Several studies (Igwo-Ezikpe et al., 2010;Okoro et al., 2009;Coral and Karagoz 2005;Janniere et al., 1993;Okpokwasili et al., 1986) have associated plasmids with the metabolism and degradation of organic compounds like Poly Aromatic Hydrocarbons, Polychloro-biphenyl and pollutants or chemicals like pesticides, ammonium, Nitrous oxide, Phosphates and Carbamates.Although, most genes involved in these degradations have been shown to be plasmid-mediated, strains of Rhodococcus and Pseudomonas in which all the genes necessary for catabolism are chromosomallyborne have been reported (Takizawa et al., 1994;Bosch et al., 2000).
Plasmids generally carry genes that confer a selective advantage to their host in a specific environment; they increase access to the horizontal gene pool for adaptive traits that may be important in the overall physiology and survival of many bacteria (Obayori and Salam, 2010).Degradative plasmids, can mediate in the utilization of fertilizer amendments as they carry genes that can degrade chemical compounds and fix nitrogen (Okoro et al., 2009;Lipps, 2008).Some ecological studies (Igwo-Ezikpe et al., 2010;Obayori and Salam, 2010;Zhao and Zhou, 2005) have determined the incidence of plasmids in natural populations of terrestrial bacteria but few reports have dealt with the involvement of plasmids in degradation by bacterial populations of an agro ecosystem.This study was therefore aimed at evaluating the plasmid incidence, profiles and carriage frequencies of bacterial isolates from organic swine waste (SW) and inorganic (NPK) fertilizer amended tropical agro soils.

Experimental site
The experimental plot was established in the University of Uyo Commercial and Research farm in use Offot, Uyo Local Government Area of Akwa Ibom State, South-South Nigeria.The geographical position of the study site is at 5.0167°N latitude and 7.9667°E longitude and situated at an altitude of 38.1m above Sea level.The climate is tropical and the rainy season starts from April and continue till late October while the dry season is from November to March.The soil of the experimental site was sandy loam (72.60%) with moderate permeability, silt (9.04%) and clay (18.36%).The soil pH values ranged from 4.3 to 4.5.

Experimental design
Field and laboratory studies were carried out to evaluate the plasmid profile of bacterial isolates from fertilizer amended tropical agro soils.The experimental layout was a randomized complete block design (RCBD) of Anderson and Ingram (1993) with slight modifications.It comprised of four experimental blocks or replicates with four treatments namely: Control (None), Organic fertilizer (SW), Inorganic fertilizer (NPK) and combined organic and inorganic fertilizer (SW+NPK).The experimental blocks were located in areas with good drainage and topography and measured 15 × 15 m each.The dimensions of the individual plots within each block were 7 × 7 m with 1 to 2m line spacing between rows.

Sample sources and properties
The commercial grade of NPK 15:15:15 (inorganic fertilizer) used for the study was obtained from the Fertilizer Procurement Unit of the Akwa Ibom State Ministry of Agriculture, Uyo while the Swine waste SW (organic fertilizer) was collected from the Piggery Unit, Crop Science and Animal Production Department, University of Uyo Commercial and Research farm, Use Offot, Uyo, Akwa Ibom State, Nigeria.The swine waste was air dried before use.The rate of application per plot was calculated based on the recommended rates of field application per hectare (Havlin et al., 2009;Edem, 2007).

Field sampling and processing
Following the amendment of the soils with the different fertilizer treatment, soil samples were collected aseptically using a soil auger (sterilized by washing and swabbing with an alcohol-soaked cotton wool) at monthly intervals for a period of one year.On each sampling date, about 4 to 5 representative soil samples were taken randomly from the surface soil (0 to 30 cm depth) of each treatment plot, mixed and bulked into a composite sample.The soil samples were placed in polyethylene bags and labeled according to the fertilizer treatment for each block as follows: NONE, NPK, SW and SW + NPK respectively.The labeled samples were taken to the laboratory in ice cold packs for microbiological assays.Where the samples were not analyzed same day, they were stored at 4-5°C in a refrigerator and used subsequently.

Isolation and enumeration of culturable heterotrophic bacteria
The total heterotrophic soil bacteria was enumerated after surfaceplating on nutrient agar, supplemented with Nystatin to inhibit fungal growth according to the methods of Parham et al. (2003).Discrete colonies were further purified by sub-culturing on Nutrient agar and Mac Conkey agar respectively and incubated at 37°C for 18 to 24 h.They were identified using morphological and biochemical studies such as Gram's reaction, slide agglutination, coagulase, catalase, oxidase, citrate utilization and sugar fermentation tests were performed.Further identification of the isolates was done according to the methods of Holt et al. (1994), Barrow and Felthanm (2003) and Etok et al. (2004).To screen for their ability to utilize the fertilizer amendments, isolates were cultured on sterilized solid mineral salt agar (MSM) plates containing swine waste and NPK fertilizers (0.01% w / w ) respectively as sole carbon and energy source (Lalfakzuala et al., 2008).Culture plates and control plates (devoid of fertilizers) were incubated at room temperarture for 5 to 7days and observed for growth.

Plasmid DNA isolation and profiling
Plasmid studies was carried out at molecular biology and biotechnology laboratory, Nigerian Institute for Medical Research (NIMR) Yaba, Lagos.Seventy bacterial isolates that showed pronounced growth and good colonial morphology on mineral salt-fertilizer media were further screened for the presence or absence of plasmids to ascertain the involvement of plasmids in the utilization of fertilizer by the isolates.The plasmid DNA extraction was by the TENS Mini Prep method modified by Zhou et al. (1990).
Plasmid profiling was done by agarose gel electrophoresis.A DNA molecular weight marker (Hind III digest of λ -DNA and/or Ori kb -Promega, USA) was used as a standard.Gel electrophoresis was carried out in a horizontal tank at a constant voltage of 60V (Thermo EC machine, CBS scientific, USA) for 90 min.Plasmid DNA bands were viewed by fluorescence of bound ethidium bromide under a short wave ultraviolet (UV) light with a photo documentation system (Clinix, Japan) and photographed.The DNA bands were matched with those for λ DNA Hind III digest and/or Ori kb molecular weight maker.The approximate molecular weight of each plasmid was obtained by extrapolation on graphical plots of molecular weight of marker against the distance traveled by the respective band.

Statistical analysis
Statistical inferences were used to assess the plasmid carriage frequencies in the isolates and the relationship between plasmid carriage in the bacterial isolates and the fertilizer treatments.Study data was analyzed using SPSS statistical analysis package version 17.0.ANOVA was performed for bacterial population data, treatment options and their interactions.Chi square (χ 2 ) test of independence was performed to ascertain the relationship between plasmid carriage in bacterial isolates and the different fertilizer treatments.Result were considered significant at P < 0.05 (Ubom, 2004)

RESULTS AND DISCUSSION
The results from this study revealed the prevalence of culturable heterotrophic bacterial isolates in the fertilizeramended soils and the control.Diverse species of microorganisms namely; Bacillus, Acinetobacter, Rhodococcus, Pseudomonas, Staphylococcus, Micrococcus and Corynebacterium were isolated from both the fertilizer-amended and non-amended tropical agro soils.The abundance of a variety of microorganisms in these soils which are continuously cultivated thus suggests the adaptive abilities of these organisms to the environmental stress and agricultural inputs.Such mechanism of genetic adaptation by microorganisms usually acquired by evolution of metabolic/degradative ability is facilitated by the presence of plasmids (Top and Springeal, 2003).Plasmids are important in the overall physiology and survival of many bacteria as they carry genes that confer a selective advantage to their host in a specific environment.However, their genetic information is not essential to the host as cells that lack them usually function normally (Obayori and Salam, 2010).
Plasmid analysis of the seventy fertilizer utilizing bacterial isolates, revealed the presence of plasmids of different sizes (Table 1) with molecular weights ranging from 4.0 to 40.1 kb.Only twenty two (31%) of the isolates carried at least one kind of plasmid and forty eight (69%) of the isolates possessed no plasmids.Plasmid profiles revealed that isolates in lanes 1 to 11 (Figure 1) carried one plasmid each of size approximately 23.1kb except for isolates in lanes 8, 9 and 11 whose plasmid size was 25.2kb.Isolates in lanes 14, 15 and 16 (Figure 2) also carried one plasmid each of size approximately 35.7kb.Five of the isolates in lanes 12, 13, 17, 19 and 20 (Figure 2) carried two plasmids each with larger sizes/molecular weight.The largest plasmid size of 40.1kb was carried by Bacillus megaterium and Bacillus subtilis.These observations were similar to the reports of Kunnimalaiyan and Vary (2005) that many strains of B. megaterium carried 4 to 10 plasmids of sizes ranging from 5.4kb to over 165kb.Ohtani et al. (2008) also reported the presence of a 65kb conjugal plasmid pLS20 in B. subtilis.
Manure and other organic amendments have been shown to have numerous gross effects on soil microbiology.This study revealed that, the frequency of plasmid-carriage in bacterial isolates was higher in the organic fertilizer-amended soils (SW) (Table 3) than in the control/non-amended (NONE), inorganic (NPK) or combined (SW+NPK) fertilizer-amended treatment plots.Although Chi square (χ 2 ) test of independence indicated that there exist no relationship between the plasmid carriage in the isolates and the type of fertilizer treatments applied to the soil, Parham et al. (2003) reported that both dairy and swine manures generally increased bacterial population and activity.It was also observed that bacterial isolates taken from the swine waste-amended plots (Table 2), showed the highest plasmid incidence (38%) while isolates from the control or non-amended plots showed the lowest plasmid incidence (25%).The positive effect of organic fertilization accounted for these observations.
Although plasmid carriage in the bacterial isolates was independent of fertilizer treatments applied to soil, the ubiquity of bacterial plasmids in the heterotrophic population of tropical agro soils is indicative of the role of plasmids in the adaptation of these bacterial isolates to life in continuously cultivated and fertilizer amended soils.Identification of the plasmid-carrying isolates revealed approximately 75% of them to be Gram positive rods, particularly of the Bacillus genera.This corroborates earlier observations (Kunnimalaiyan et al., 2001;Igwilo-Ezikpe et al., 2010)

Conclusion
Plasmid incidence in about 31% of these bacterial isolates illustrates their adaptability to environmental stress induced by possible overuse or misuse of soil amendments.It also underscores the critical effects these microorganisms could have on the ecological stability and biological productivity of the agro ecosystem, as well as their potentials for use in bioremediation and eco restoration in order to ensure environmental sustainability and national development.

Table 1 .
Molecular weight of plasmid DNA from screened bacterial isolates.
ISN = Isolate Code

Table 2 .
Percentage of plasmid carrying bacterial isolates from each fertilizer-amended soil and control.

of isolates Screened No. of isolates with plasmids % of isolates with plasmids
No fertilizer amendment (control); SW = Farmyard Manure/Organic Fertilizer amendment; NPK = Inorganic Fertilizer amendment SW + NPK = Combined Organic and Inorganic Fertilizer amendment

Table 3 .
Frequency of plasmid carriage in bacteria isolated from different treatments.

Treatments No. of Isolates carrying Plasmids No. of Isolates not carrying Plasmids Total No. from each Treatment
NONE = No fertilizer amendment (control); SW = Organic Fertilizer amendment; NPK = Inorganic Fertilizer amendment; SW +NPK = Combined Organic and Inorganic Fertilizer amendment.