Masenya Adegboye Babalola 2013 ALS SUPPL 9 129 147

ISSN 0117-3375

ASIA LIFE SCIENCESThe Asian International Journal of Life Sciences

Beyond Excellence©

SUPPLEMENT 9 AUGUST 2013CONTRIBUTIONS FROM AFRICA Series 2

©Rushing Water Publishers Ltd., 2013 Printed in the Philippinese-mails: [email protected] [email protected]

Printed in the Philippines

ASIA LIFE SCIENCES The Asian International Journal of Life Sciences Beyond Excellence©

©Rushing Water Publishers Ltd. 2013

ASIA LIFE SCIENCES - The Asian International Journal of Life Sciences (ISSN 0117-3375) is a non-profit, non-stock refereed scientific journal devoted to the publication of original research in the Life Sciences and related disciplines. Articles originating from anywhere in the world are most welcome. Two issues a year make a volume

BOARD OF EDITORS - Asia Life Sciences Supplement 9, 2013Chairman & Chief Editor: Dr. William Sm. Gruèzo, Institute of Biological

Sciences, College of Arts & Sciences (CAS), University of the Philippines Los Baños (UPLB), College 4031, Laguna, Philippines.

Members: Dr. Liding Chen, State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian, Beijing 100085, China. Dr. Leonila A. Corpuz-Raros, Crop Protection Cluster, College of Agriculture, UPLB, College 4031, Laguna. Dr. Maribel L. Dionisio-Sese, Plant Biology Division, Institute of Biological Sciences, CAS-UPLB, College, Laguna. Dr. Irineo J. Dogma Jr., Graduate School, University of Santo Tomas, España St., Manila. Dr. Agustine I. Doronila, School of Chemistry, University of Melbourne, Victoria 3010, Australia. Dr. Victor P. Gapud, Crop Protection Cluster, College of Agriculture, UPLB, College 4031, Laguna. Dr. Krishlex G. Gruèzo MD, 81 Gov. F.T. San Luis Avenue, Masaya, Bay 4033, Laguna. Dr. Rafael D. Guerrero III, National Academy of Science and Technology, Level 3, Science Heritage Building, Deparment of Science and Technology Complex, Bicutan, Taguig City, MetroManila. Michael G. Price, P.O. Box 468, Mi Ctr, Michigan 49254, USA. Dr. Xu Wu MD/PhD, 1924 Heritage Park Drive, Apt. 211, Oklahoma City, OK 73120, USA.

Technical Production Manager: Ydred Harriss G. GruezoDeadlines for submission of manuscripts. First issue-01 June; Second issue-01 January.

Please contact the Chairman, ALS Board of Editors concerning information for contributors (see addresses below).

Subscription Prices. Foreign: Institutional - US$750; Individual - US$500 (including Volumes 1-22,1992-2013+ 8 Supplements). Local: Institutional - PhP7500; Individual - PhP5000 (including Volumes 1-22, 1992-2013 + Supplements). Prepayment of order/back order is required. All issues are to be sent by air mail. Back orders will have an additional packing-handling and postage cost.

Send manuscripts, subscription orders and correspondence to: Dr. William Sm. Gruèzo, ASIA LIFE SCIENCES, The Asian International Journal of Life Sciences, 81 Gov. F.T. San Luis Avenue, Masaya, Bay 4033, Laguna, Philippines. Mobile phone no.(63) 915-360-4660; Telephone no. (63)(49) 501-2957. e-mails: [email protected] [email protected] website: http://journals.uplb.edu.ph/index.php/ALS

ASIA LIFE SCIENCES The Asian International Journal of Life Sciences

ISSN 0117-3375Supplement 9 August 2013

CONTENTS1 Determinants of smallholder cocoa farmers’ adaptation to climate change in Ile-oluji/Okeigbo Local

Government Area of Ondo State, Nigeria G.O. Ogunsola & A.S. Oyekale11 Flavonoids, carbohydrates, proanthocyanidins and

other phenolics in the leaves of selected Acacia species in Mankweng, Limpopo Province, South Africa H.K. Mokoboki & A. Sebola21 Impact of AIDS care and level of burnout among nurses

in selected hospitals in Limpopo Province, South Africa J.O. Igumbor & M. Davhana-Maselesele

33 Effect of phosphorus fertilizer and leaf cutting technique on biomass yield and crude protein content of two African indigenous leafy vegetables M. Seeiso & S.A. Materechera51 Examining a national agricultural extension and advisory system: A case study of the North West and South West regions of Cameroon G.N. Nyambi, G.C. Shinn & G.E. BriersCont. on Next Page

Reviewers for this Issue: Dr. N.S. Aggangan, Dr. R.T. Bagarinao, Dr. M.R. Delos Reyes, Dr. A.I. Doronila, Dr. M.L. Dionisio-Sese, Prof. R.K.B. Gallegos, Dr. V.P. Gapud, Dr. Wm.Sm. Gruèzo, Dr. J. Hu, Dr. K.M. Kasiotis, Dr. R.L. Lapitan, Dr. J. Ma, Dr. S.T. Meissner, Dr. M.L.T. Munarriz, Dr. F.P. Paras Jr., Dr. L.L. Pintor, Dr. C.L. Rapera, Dr. A.A. Rayos, Dr. W.L. Rivera, Dr. M.A.T. Tavanlar, Dr. A.T. Valerio, Dr. J. Wan, Dr. R. Wang & Dr. Y. Wang.

©Rushing Water Publishers Ltd. 2013 Printed in the Philippines

Contents 65 Implications of soil nutrient management practices for greenhouse gas mitigation in Africa O.I. Oladele & A.K. Braimoh79 Knowledge of and attitudes toward emergency cintraception among students at a South African university J.O. Igumbor, D.R. Phetlhu, N. Zulwayo, T. Badimo & P. Molefi 91 Strategy, operation and effects of the Nguni Cattle Project in the North-West Province, South Africa M.A. Antwi & O.I. Oladele 103 The use of polyethylene glycol in the diagnosis of polyphenolics in the foliage of Acacia species H.K. Mokoboki115 A critical review of South Africa’s policy response

to climate change R.L. Kenny & T.M. Ruhiiga 129 Identification of native rhizosphere community

composition of bacteria in Mahikeng soil, South Africa K. Masenya, M.F. Adegboye & O.O. Babalola149 Factors affecting adoption intensity of organic

agricultural practices in southwest Nigeria: Minimum tillage and fire S.A. Adebayo & O.I. Oladele

157 A review of efficiency measures in South Africa’s municipal solid waste management

N.V. Mudau, T.M. Ruhiiga & P.W. Malan173 Urban households’ willingness to consume vegetables

grown with urine: A case study in Ibadan, Oyo State, Nigeria A.A. Taiwo & A.S. Oyekale

185 Climate change mitigation potential of tree crop and alley farming practices in Africa O.I. Oladele & A.K. Braimoh

Contents199 1-Aminocyclopropane-1-carboxylate deaminase activity as a marker for identifying plant-growth promoting rhizobacteria in cultivated soil of South Africa M. Khantsi, M.F. Adegboye & O.O. Babalola213 Water management practices and carbon sequestration for climate change mitigation in Africa O.I. Oladele & A.K. Braimoh223 Nature and extent of inter-professional collaboration between traditional health practitioners and community nurses in a primary health care setting in South Africa M.G. Montshioa & U. Useh 233 The effects of breed, season, time of the day and period of browsing on woody plant species selection of goats C.K. Lebopa, E.A. Boomker, M. Chimonyo & S.D. Mulugeta245 Monitoring of urban sprawl using minimum distance supervised classification algorithm in Rustenburg, South Africa S.K. Bett, L.G. Palamuleni & T.M. Ruhiiga263 Development of a low-cost data acquisition system for total solar insolation and temperature monitoring S.R. Katashaya & R.O. Ocaya275 Seedpod traits and chemical composition of Acacia tortilis, A. robusta and A. hebeclada in Mafikeng, North West Province, South Africa

H.K. Mokoboki & C.K. Lebopa 283 Households’ access to improved drinking water and water treatment behavior in urban and rural Nigeria A.S. Oyekale301 Assessment of climate change vulnerability, housing quality and health status of cocoa farming households in Osun State, Nigeria A.A. Mufutau & A.S. Oyekale

Contents

315 Validation and shortening of the World Health Organisation Quality of Life instrument for people living with HIV (WHOQOL-HIV) in Limpopo Province, South Africa J.O. Igumbor & U. Useh329 Determinants of profitability of broiler production in the Ngaka Modiri Molema District, North West Province, South Africa R.A. Adem & M.A. Antwi345 Factors affecting adoption intensity of organic agricultural practices in southwest Nigeria: Crop rotation and intercropping S.A. Adebayo &

O.I. Oladele355 Land tenure pattern, farm investment decision and farming income in Egbeda Local Government Area of Ibadan, Nigeria T.A. Akintayo & A.S. Oyekale367 Information-seeking behavior of organic vegetable farmers in southwest Nigeria S.A. Adebayo &

O.I. Oladele379 Beneficiaries’ assessment of Comprehensive Agricultural Support Program on livelihoods in Ngaka Modiri Molema District, North West Province, South Africa M.A. Antwi & N. Nkwe395 Parental adaptation to children with disabilities: A consideration for rehabilitation timing in family-centred care U. Useh403 Isolation, characterization and antibacterial activity of Streptomycetes from rhizosphere soils in North West Province, South Africa M.F. Adegboye & O.O. Babalola423 Reviewers-Asia Life Sciences Supplement 9, 2013428 Board of Editors-Asia Life Sciences Supplement 9, 2013 ASIA LIFE SCIENCES Beyond Excellence ©


ASIA LIFE SCIENCES Supplement 9: 129-147, 2013The Asian International Journal of Life Sciences

Received 06 July 2013; Accepted 15 August 2013©Rushing Water Publishers Ltd. 2013

Identification of native rhizosphere community composition of bacteria in Mahikeng soil, South Africa

KEDIBONE MASENYA1, MOBOLAJI FELICIA ADEGBOYE1 and OLUBUKOLA OLURANTI BABALOLA1*

Soil is a complex environment and a hotspot of microbial diversity with millions of different bacterial species in a 1-g sample. In this study, bacteria from rhizosphere soil in Mahikeng were isolated and characterized using both conventional and molecular techniques. Most of the soil samples have colony forming units (cfu) ranging from 1.6×106 to 2.1×106 cfu/ml, which indicated that the samples were rich in microbial loads. The bacterial isolates were characterized by cultural, morphological, physiological and biochemical studies. Besides, 16S rRNA genes from genomic DNA were amplified by PCR and the amplicons were partially sequenced. Using 16S rRNA analysis, the majority of the 16S rDNA showed close similarity to those of Bacillus (11 species), and one each to Paenibacillus sp., Ensifer adhaerens, Aquamicrobium sp., Lactobacillus sp., Alcaligenes sp., Brevibacillus sp. and Proteus vulgaris. This analysis revealed that Bacillus sp. was the dominant population in all the rhizosphere soil samples collected from Mahikeng. These bacterial isolates exhibit plant growth promoting activities, which can enhance plant productivity and soil health.

Keywords: characterization, isolation, microbial diversity, rhizosphere, Mahikeng, South Africa

1Department of Biological Sciences, School of Environmental and Health Sciences, Faculty of Agriculture, Science and Technology, North West University, Mahikeng Campus, Private Bag X2046, South Africa

*Corresponding author: e-mail - [email protected] Tel:+27183892568, Fax:+27183892134

130

Masenya, Adegboye & Babalola 2013

Asia Life Sciences Supplement 9 2013

INTRODUCTIONMicrobial diversity is a vast frontier and a potential goldmine for the

biotechnology industry because it offers a number of new genes and biochemical pathways to probe for enzymes, antibiotics and other useful molecules (Gurung et al. 2010). The assessment of the structure within the microbial communities is one of the fascinating aspects of microbiology and the number of prokaryotic species of microorganisms described so far is remarkably low (van Elsas et al. 2006). Discovering novel microbes and characterizing their functions are the main goals in the study of microbial diversity. Historically, this was achieved through cultivation and subsequent characterization of strains (Rappe & Giovannoni 2003). These techniques can be used to study the structure and activity of soil microbial communities because the majority of organisms in natural soil have not yet been grown in culture and characterized. To appreciate their true functional diversity and activities they express in situ in soil in the response to different environmental constraints, it is necessary to develop new experimental approaches adapted to these microorganisms. One such approach developed in the recent years is molecular techniques (Bailly et al. 2007). The culture-independent methods have provided new tools to study the microbial world (Rappe & Giovannoni 2003).

Molecular characterization of bacteria has been shown to be effective as compared to traditional cultivation techniques such as plate counting methods, which have been increasingly considered inadequate, thus new and more sophisticated techniques have been developed for the isolation of bacteria from complex microbial habitats (Sait et al. 2002). Examples of bacteria found in soil are: decomposers, nitrogen fixers, nitrifying bacteria, actinomycetes, green blue agar bacteria, disease suppressors, sulphur oxidizers, decomposers/denitrifying bacteria include strains Bacillus subtilis and Pseudomonas fluorescens and Clostridium; these kinds of bacteria consume simple carbon compounds such as root exudates, and convert energy in soil organic matter which is useful to the rest of other organisms in soil (Waldrop et al. 2000). Nitrogen fixers include Rhizobium, Azotobacter, Frankia, Azospirillum and Agrobacterium. These kinds of bacteria are also called mutualists where both populations benefit for instance, Rhizobium bacteria, can be inoculated onto legume seeds to fix nitrogen in the soil (Green et al. 2010). They extract nitrogen gas from air and convert it to the form that plants can use. Efficient strains of nitrogen-fixing bacteria can save enormous means being spent on the nitrogen fertilizers and also prevent the degradation of the environment besides improving the yield (Bohlool et al. 1992).

Nitrifying bacteria includes Nitrosomonas, Nitrobacter and Nitrospira; these kind of bacteria add free nitrogen occurring in the form of nitrates in soil in the process of nitrification. In this process, they change ammonia to nitrite and nitrite is further changed to nitrate, which is leached more easily from the soil (Pratscher et al. 2011). Sulfur oxidizer includes Thiobacillus bacteria which can convert sulfides into sulfates and form sulfur which can be utilized by plants (Berthelin 2010). Actinomycetes helps to slowly breakdown humic acids; they are also able to degrade many complex substances such as cellulose and chitin (Hayakawa 2008).

131

Native rhizosphere bacteria in South Africa


This study was designed to isolate bacteria from rhizosphere soils collected from Mahikeng, identify and characterize the bacteria isolates using standard methods and assess the composition of the soil bacterial community using molecular techniques. The study sought also to clearly understand the bacterial community structure in soil as the extent of the diversity of microorganisms in soil is seen to be critical to the maintenance of soil health and quality. It helped in addressing problems of Mahikeng soil health and quality, for increased crop production since a wide range of bacteria is involved in biogeochemical cycles and as plant growth promoters. Thus, this study aimed at contributing new data on the occurrence of bacteria in soil for maintenance of soil health and quality.

MATERIALS AND METHODSSampling area. Rhizosphere soil samples were collected from different locations in Mahikeng. Mahikeng is located in the North-West Province (25.8500° S, 25.6333° E), Republic of South Africa. This region has an average annual precipitation of 559 mm, annual average temperature of 18.3°C and annual rainfall of 539 mm.Sample collection. Four crop studies were selected viz., onion, maize, spinach and cabbage grown in Mahikeng. Table 1 shows the location, sample code and pH of the soil samples. Five rhizosphere soil samples (100-500 g) were collected aseptically in sterile polythene bags to avoid contamination. Three samples were collected from Makogoe and the other two were collected from Madibe Makgabane by uprooting the root system and placing it in a cool box for transport to the laboratory. In some cases, samples were stored at 4°C when not used immediately.

Table 1. Areas in Mahikeng (North-West Province, South Africa) where the soil samples were collected, the sample isolate names and relevant parameters measured.

Location Crop Sample code

Isolate name pH1 pH2 Average pH

Mokogoe (25°53. 275’)

Cabbage MC MR1* MR2* MR18 7.61 8.44 8.03

Madibe Makgabane (25°5. 318’)

Maize MA MR4* MR5* MR13* MR16

8.29 8.35 8.32

Madibe Makgabane (25°53. 296’)

Spinach MS MR12* MR17* MR18* MR14

8.13 8.28 8.23

Mokogoe (25°56. 946’)

Maize MB MR3* MR11* MR15 8.2 7.79 7.99

Mokogoe (25°56. 657’)

Onion MO MR6* MR7* MR8* MR9* MR10

7.62 7.45 7.54

Culture methods. One gram of each soil sample was placed in 9 ml sterile distilled water in a test tube and subsequently, centrifuged. Serial dilutions were performed aseptically for each soil sample, and 0.1 ml of each diluted sample was spread on

132



nutrient agar (Merck) using spread plate method. Plates were incubated at 37°C for 24 hr.Biochemical characterization. Various biochemical tests were performed for identification of rhizosphere bacteria viz., catalase, oxidase, citrate utilization, ammonia production, glucose, lactose, sucrose, H2S production, starch hydrolysis, ammonia production, aesculin production and methyl red according to standard methods.DNA extraction from bacterial isolates. Genomic DNA bacterial isolates were conducted using ZR Soil Microbe DNA MiniPrep™ (Zymo Research, USA) extraction kit according to the manufacturer’s instructions. 16S rDNA gene amplification. PCR amplification of the 16S rDNA gene of the isolates was conducted using universal primers fD1 (5’-AGA GTT TGA TCC TGG CTC AG-3’) and rD1 (5’-TGA CTG ACT GAG GCT ACC TTG TTA CGA-3’) (Weisburg et al. 1991). It was performed in thermocycler, in a total volume of 50 μl containing 30-50 ng DNA, 100 mM of each primer, 0.05 U/µl Taq DNA polymerase, 4 mM MgCl2, and 0.4 mM of each dNTP (Integrated DNA Technology). The amplification reaction was performed with a DNA Engine DYAD Peltier thermal cycler (BioRad, USA). The thermal cycling conditions were: 5 min at 94°C for initial denaturation, 30 cycles of 30s at 95°C, 1 min at 54°C, 2 min at 72°C, and final extension for 5 min at 72°C. For each reaction, a negative control lacking DNA template was included. The PCR amplicons were analysed by electrophoresis in 1% (w/v) agarose gel. The gel containing Ethidium bromide (10 μg ml-1) was viewed under Syngene Ingenius Bioimager (UK) to confirm the expected size of the product. The remaining mixture was purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Germany). Nucleotide sequence determination. PCR purified products of the 16S rDNA of the strains were analysed for nucleotide sequence determination by using ABI PRISM® 3500XL DNA Sequencer (Applied Biosystems) at Inqaba Biotechnical Industrial (Pty) Ltd, Pretoria, South Africa. Molecular taxonomy determined by sequences and phylogenetic analysis. Phylogenetic and molecular evolutionary analyses were conducted using softwares where a sequence was carried to process raw data into high quality sequences. Nucleotide sequences were analyzed and edited by using BioEdit software (Hall 1999). After this initial analysis, sequences were compared to the basic local alignment search tool database of sequences deposited at the National Centre for Biotechnology (NCBI) using BLASTN website http://www.ncbi.nlm.nih.gov (Altschul et al. 1990). The partial 16S rDNA gene sequences were used to search the GenBank database with the BlastN algorithm to determine relative phylogenetic positions (Altschul et al. 1990). Multiple alignments of the sequences were carried out by Mafft program 6.864 (Katoh & Toh 2010) against corresponding nucleotide sequences retrieved from GenBank. Evolutionary distance matrices were generated as described by Jukes and Cantor (1969). Phylogenetic analyses were conducted using MEGA version 5.22 (Tamura et al. 2011) and Neighbor-joining (Fitch 1986) trees were constructed. The robustness of the tree topology was evaluated by

133



bootstrap analysis (Felsenstein 1985) based on 1,000 re-samplings. Manipulation and tree editing were carried out using TreeView (Page 1996).Nucleotide sequence accession numbers. The 16S rDNA gene sequences obtained in this study have been submitted to the GenBank database and were assigned accession numbers (indicated in parentheses). These are as follows: MR1 (JX971518), MR2 (JX971519), MR3 (JX971520), MR4 (JX971521), MR5 (JX971522), MR6 (JX971523), MR7 (JX971524), MR8 (JX971525), MR9 (JX971526), MR10 (JX971527), MR11 (JX971528), MR12 (JX971529), MR13 (JX971530), MR14 (JX971531), MR15 (JX971532), MR16 (JX971532), MR17 (JX971533) and MR18 (JX971534). Statistical analyses. Data were statistically analyzed by analysis of variance (ANOVA) using SPSS software version 10 for Window (SPSS, Chicago, IL, USA). Each treatment was replicated five times.

RESULTSIn this study, 18 isolates were chosen based on differences in the colony

morphology and sequencing results. Physical characteristics affecting the soil samples were measured (Table 1). The average pH of the soil sample which ranged from 7.54-8.23 was neutral to slightly alkaline. Onion crop soil sample (MO) indicated a high yield of bacteria, such as Bacillus thuringiensis MR6, Bacillus sp. MR7, B. mojavensis MR8, B. methylotrophicus MR9 and Lactobacillus sp. MR10. This was followed by Maize (MA) with Brevibacillus sp. MR4, B. vallismortis MR5, and B. amyloliquefaciens MR13, Paenibacillus sp. MR16). The low yield of bacterial population was obtained in cabbage (MC) composed of Bacillus subtilis MR1, B. clausii MR18) and Ensifer adhaerens MR2, Spinach (MS) yielded Proteus vulgaris MR1, Bacillus aerius MR1, B. cereus MR14) and Maize (MB) with Bacillus pumilus MR3, Aquamicrobium sp. MR11 and Alcaligenes sp. MR15) both showed the same number of bacterial yield.

The CFU for the total bacterial population was carried out using nutrient agar. Bacterial counts of soil samples obtained from five different sites were conducted. It was found that MO, MB and MC were significantly different from MA. MS was not statistically significant to MC. MA had the highest population with ± 2.1×106 cfu/ml, followed by MB (±1.7×106 cfu/ml) and the least being MC and MA with ±1.6×106 cfu/ml (Figure 1). Morphological, biochemical and phylogenetic analyses. Cultural characterization was carried out on all isolates and features such as elevation, color, margin, size, optical density, etc. of each isolate were recorded. The elevation is largely convex, color ranged from cream to red, optical density for all the isolates was opaque and the margin was largely smooth or entire. Gram reaction was conducted on all 18 isolates and 12 of the 18 isolates were found to be Gram positive. Both Gram negative and Gram positive were subjected to biochemical tests for genus level identification. On the basis of morphological, cultural and biochemical characteristics (Table 2), the bacterial isolates were identified as members of genus Bacillus, based on the rod structure and strictly aerobic nature of Gram positive bacteria; followed by Proteus spp, Pseudomonas spp. and Rhizobium spp. according to Bergey (1934).

134



Among these, Bacillus was predominant. Out of the 18 isolates 17 possessed catalase enzyme, 10 were oxidase negative, 11 were able to utilize citrate as their sole carbon source while 2 were able to liquefy gelatin, 13 were able to hydrolyze starch, while 16 reduced nitrate. For triple sugar fermentation, 6 produced glucose, 5 produced sucrose and only 2 of 18 isolates produced lactose, 3 produced H2S and 15 produced aesculin (Table 2). Phylogenetic analysis was also conducted and one representative of the various isolates with a high percentage similarity with each variety was selected for phylogenetic characterization on the basis of 16S rDNA (Table 2). The isolates indicated a high percentage identity of 80 to 100% with the sequences from the GenBank.

Figure 1. Bacterial colony forming units (CFU) for the total bacterial population of soil samples obtained from five different sites using nutrient agar. Bars represent standard error of the mean of five replicates. The bars with similar letters are not significantly different at p ≥ 0.05.

Plant growth promoting bacteria. Various plant growth promoting bacterial activities were conducted (Table 3) and the results of qualitative test HCN production showed that a low percent of isolated bacteria was capable of producing HCN. Bacillus subtilis, Proteus vulgaris and Pseudomonas sp. indicated high cyanogenic potential due to the yellow color change of the filter paper to dark brown to red when compared to other strains and was scored as positive indicating higher level of HCN production. Further tests indicated that the HCN producing isolates belonged to the genus Bacillus. However, other bacterial strains indicated no production of HCN based on the color development. Ammonia production was positive for all bacterial isolates. Bacillus amyloquefaciens and Aquamicrobium aerolatum showed antifungal activity over Fusarium oxysporum. Bacillus vallismortis isolate showed the solubilisation of the insoluble phosphate and three isolates, i.e. Paenibacillus sp., B. aerius and P. vulgaris showed the production of indole acetic acid. ACC deaminase production was observed in B. vallismortis, B. pumilus, B. subtilis and Alcaligenes sp.

135



Table 2. Species identification using biochemical characterization and molecular methods based on the basis of 16S rRNA gene sequences, centered on similarity of BLAST results returned from GenBank databases of rhizobacteria isolated from crop soil samples of cabbage, onion, maize and spinach.

Isol

ate

code

Gra

m re

actio

n

Cat

alas

e te

st

Oxi

dase

test

Citra

te u

tiliz

atio

n

Star

ch h

ydro

lysi

sG

elat

in h

ydro

lysi

s

Mot

ility

Nitr

ate

redu

ctio

n

Glu

cose

test

Lact

ose

test

Sucr

ose

test

H2S

pro

duct

ion

MR

test

v.pr

oska

uer t

est

MR1 + + - - + - - + - - + - + -

MR2 - + - + + - + + + - - - + -

MR3 + - - + - - + + + + - - + -

MR4 + + + + - + - - - - - + - +

MR5 + + - - - - - + - - - + - +

MR6 + + - - - - V + + - - - - +

MR7 + + - - - - + + + - - - + -

MR8 + + + + - - + + - - - - - +

MR9 + + + + + - + + - - - - + -

MR10 - + + + + - - + - - - - + -

MR11 - + + + + - + + - - - - + -

MR12 + + + + + - + + - - - - + +

MR13 + + + + + - + + + - - - + -

MR14 - + - + + - + + - + + - + -

MR15 - + - - + - - + - - + - + -

MR16 + + + + - + + + + - + - + -

MR17 + + - - + + - - - - + + + -

MR18 + + - - + - - + - - - + - -

136



Indo

le te

st

Aes

culin

test

Ass

igne

d A

cces

sion

no

Sequ

ence

Len

gth

(bp)

Clo

sest

rela

ted

spec

ies

Acc

essio

n no

% si

mila

rity

MR1 - + JX971518 1500 Bacillus subtilis JX094283 100

MR2 - - JX971519 1442Ensifer adhaerens EU221356 99

MR3 - - JX971520 1410 Bacillus pumilus JX293286 99

MR4 - + JX971521 1259 Brevibacillus sp. HQ01884 99

MR5 - - JX971522 1428 B. vallismortis JX144955 99

MR6 - - JX971523 1397Bacillus thuringiensis JQ988062 99

MR7 - + JX971524 1513 Bacillus sp. AF406633 99

MR8 - + JX971525 1470 B. mojavensis JQ236831 99

MR9 - + JX971526 1418B. methylotrophicus JX094955 99

MR10 - + JX971527 1501 Lactobacillus sp JN987182 99

MR11 - + JX971528 1346Aquamicrobium sp FM210786 96

MR12 - + JX971529 1466 Bacillus aerius JX009139 99

MR13 - + JX971530 1424 Paenibacillus sp HM352396 99

MR14 - + JX971531 1447 Proteus vulgaris JN409462 99

MR15 - + JX971535 1502 Alcaligenes sp AB118220 80

MR16 - + JX971532 1466Bacillus myloliquefaciens FJ889051 100

MR17 - + JX971533 1396 B. cereus JX293290 99MR18 + + JX971534 1519 B. clausii EU117277 89

Key: + = Positive; - =Negative; V = Variable

Table 3. Plant growth promoting activities of bacterial isolates from the rhizosphere soil of onion, maize, cabbage and spinach.

Isolate code

Ammonia production

ACC deaminase

Anti-fungal

Phosphate solubilization

Hydrogen Cyanide

Indole Acetic Acid

MR1 + + - + + -

MR2 + - - - -

MR3 + + - + - -

MR4 + - - + - -

137



MR5 + + - - - -

MR6 + - - - - -

MR7 + - - - - -

MR8 + - - - - -

MR9 + - - - - -

MR10 + - - - - -

MR11 + + - - - -

MR12 + - - - - +

MR13 + - - + - +

MR14 + - - - + +

MR15 + + + - - -

MR16 + + + - - -

MR17 + - - + - -

MR18 + - - - - -Key; + = Positive; - = Negative

16S rDNA gene amplification. Results presented in Figure 2 indicate that high molecular weight DNA recovered from the 15 bacterial isolates was successfully amplified using the universal primers. PCR amplification of the genomic DNA with universal primers resulted in approximately 1500 bp amplicons

Figure 2. Ethidium bromide stained agarose gel (1%) showing PCR amplification

of 16S rDNA of bacterial isolates obtained from the farming sites in Mahikeng, North-West Province, South Africa. M = DNA marker (1.5 Kb); PCR amplification of the 16S rDNA gene fragments from isolates obtained from soil samples in the rhizosphere of cabbage (Lanes 1-3), maize (Lanes 4-8), onion (Lanes 9-10) and spinach (Lanes 10-15), respectively.

Lactobacillus murinus (JN987182)

138



Phylogenetic analyses. Evolutionary analyses by Neighbor-Joining method (Figure 3) revealed the presence of groups that fell in several of the established bacteria. Evolutionary relationships of taxa derived from analysis of the 16S rDNA sequences of strains MR1-MR18 using more than one related sequences obtained from the NCBI GenBank was conducted. This lead to the discovery of evolutionary relationship among groups of organisms belonging to the phylum Firmicutes as well as Proteobacteria and gamma Proteobacteria. Seventy five percent of isolates were affiliated with the Firmicutes, while 25% were with the family Proteobacteria. The phylum Firmicutes was closely related to the members of named and characterized genus Bacillus, while 5% were closely related with Paenibacillus sp. and 25% were closely related to genus Proteus. Majority of the sequences were 5-20% different from those found in the database. The results suggest that the nucleotide sequences are highly diverse.

Clonopsis felicitatis was selected as an outgroup which is a sequence not contained within the group under study but is closely related to the Bacillus species. About 89 to 100% sequence identity was observed between the 16s rDNA sequences. Isolation of Pseudomonas and Bacillus also has been reported earlier from various crops plants (Vessey 2003, Minkwitz & Berg 2001, Chan et al. 1994). All isolates could be attributed to only 2 classes, namely: Proteobacteria and Firmicutes. The 16S rDNA were mostly related to the cloned 16S rDNA gene sequences obtained from the GenBank. MR16 was closely related to B. amyloquefaciens, MR7 isolate was closely related to Bacillus spp., MR2 closely related to Ensifer adhaerens, MR14 closely related to P. vulgaris, suggesting that the isolate had the same sequence as that from the GenBank.

Figure 3. Phylogenetic tree inferred using the Neighbor-Joining method. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method; evolutionary analyses were conducted in MEGA 5.22.

DISCUSSIONAnalysis of the bacterial communities present in the rhizosphere of onion,

maize, cabbage, and spinach was addressed by both culturing and molecular methods. pH value of the soil samples measured was found to be neutral to slightly alkaline which correlated with literature (Rousk et al. 2010) that the pH neutral to slightly alkaline is good for microbial existence and microbial activities like enzymatic activities. pH affects ionization and the binding and interaction of molecular processes. It further includes very basic things such as affecting the solubility of many substances the bacteria need. Though the pH of the cell contents growing in alkaline/acidic environments is neutral, the strains have transport mechanisms to keep a normal physiological hydrogen ion concentration inside the cell (Slonczewski et al. 2009). Furthermore, it has been reported that

139



140



soil properties such as pH and organic matter can affect rhizosphere community directly or indirectly (Garbeva et al. 2004). However, soil pH has been reported to not only determine H+ ion concentration but also influence the concentration of cations (Ashman & Puri 2002). According to Ashman and Puri (2002), a soil with pH range of 6.5-7.5 will have high concentrations of Ca2+, Mg2+ and K+ compared to an acidic soil (4.0-5.5), which they reported as being characteristic of agricultural soils. It was explained that agricultural soils are prone to gradual acidification after planting because cations which balance excess acidity are, on the other hand, enable soil microorganisms to modify the physico-chemical properties of the rhizosphere (Garbaye 1994) through their metabolic activities.

CFU results indicate that the samples were rich in microbial loads. It is known that organic matter introduced to soil stimulate microbial population and soil biological activity. Therefore, it was suspected that fertilizers added to the soil increased the soil bacterial load (Brady & Weil 1999) and also because the soil samples were taken from a rhizosphere, increased microbial activity is expected as there is a high concentration of microorganisms than in a soil distant from the plant and the increase is more pronounced with bacteria. The rhizosphere is considered to be a hotspot of bacterial diversity, it harbors bacterial flora whose diversity is mainly expressed in terms of functions adapted to root presence and in particular, to favor plant growth (Rawat et al. 2011). Phosphate deficiency also directly affects the growth of culturable bacteria. This will result in less pronounced colony forming units (Zhong & Cai 2007), therefore, it is concluded that the soil is fit for agricultural practices. Onion crop soil sample (MO) indicated a high yield of bacteria, with 100% bacteria belonging to Bacillus species. The high yield of bacteria from an onion crop sample was due to the release of root exudates which has been postulated to be a key factor influencing the diversity of microorganisms in the rhizosphere (Singh & Mukerji 2006). However, there is no direct evidence to support this hypothesis. Onion (MO) was followed by maize (MA) still Bacillus species were more predominant; however, Brevibacillus was also present. The low yield of bacterial population was indicated in cabbage (MC). In spinach (MS), P. vulgaris and Bacillus species were predominant while maize (MB) had bacterial yield of Alcaligenes sp. and Aquamicrobium sp. The poor yield of the soil samples from cabbage, spinach and maize is due to the inability of bacterial cells to move toward roots in response to root exudates (Dennis et al. 2010). The similarity of the crop soil samples is that all samples yielded Bacillus species. This is due to their endospore producing ability; thus, they survive harsh conditions.

The availability of pure culture isolates greatly simplifies investigations of the physiology and roles of bacteria, and initial biochemical studies in the laboratory (Sait et al. 2002). This study of diversity of bacteria isolated from Mahikeng soil resulted in the high predominance of Gram positive rod-shaped, aerobic spore-forming members of the genus Bacillus which are most widely represented in the soil. This is due to their ability to form spores and withstand a range of variable environmental conditions such as low nutrient availability, desiccation and chemical disinfections (Parvathi et al. 2009, Babalola & Akindolire 2011).

141



Plant rhizosphere is a preferential niche for various types of microorganisms in the soil. In the present investigation, 18 bacterial isolates were screened in vitro for their plant growth promoting (PGP) ability. Plant growth promoting rhizobacteria are considered to promote plant growth directly or indirectly. Their common traits include production of plant growth regulators, such as indole acetic acid (IAA) which is one of the physiologically active auxins. IAA is a common product of tryptophan metabolism by several microorganisms including plant growth promoting rhizobacteria (Xie et al. 1996). Bacillus aerius, Paenibacillus sp. and P. vulgaris from different crops appeared to have a great potential to synthesize IAA.

Phosphorus is one of the most limiting factors in crop production in many kinds of soils and in different geographical regions, as a result of high phosphorus fixation. Phosphorus deficiency is widespread and phosphorus fertilizers are required to maintain crop production. When it is added to the soil in the form of phosphate fertilizer, only a small portion is utilized by plants; the rest is converted into insoluble phosphates (Ryu et al. 2003). Soil microorganisms play an important role in making the phosphorus available to plants by mineralizing the organic phosphorus in the soil. Different bacteria i.e. B. subtilis, B. pumilus, B. cereus, B. brevis, Paenibacillus sp. and E. adhaerens showed the ability to solubilize phosphate. Rhizosphere phosphate solubilizing bacteria is capable of solubilizing inorganic phosphates to soluble organic forms (Khan et al. 2001). However, in the present study, the B. vallismortis strain either produced IAA or solubilized phosphorus. Earlier reports showed that some strains of B. subtilis and B. amyloliquefaciens produced certain volatile compounds such as 2-3, butanediol and acetoin that stimulated plant growth (Ryu et al. 2003)

Ammonium and nitrate are believed to be the principal sources of nitrogen for plant growth in agricultural and most natural environments. They are required in greater amounts than any other mineral nutrient (Howitt & Udvardi 2000). All the bacterial strains were found to be able to synthesize ammonia. This is explained by symbiotic nitrogen fixing bacteria species which are able to specifically interact with host plants and convert atmospheric nitrogen to ammonia (Dusha et al. 1987). Bacillus pumilus, B. subtilis and Pseudomonas sp. indicated a high production of ACC deaminase. ACC deaminase reduces the potential inhibitory effects of higher ethylene concentrations (Glick et al. 1998), which is of extreme importance when plants are exposed to stressful conditions such as heavy metals contamination of the soil (Grichko et al. 2000). According to the report of authors such as Shaharoona et al. (2006), it seems that this influence is real for other plant and bacterial combinations.

Fusarium oxysporum is a well-known soil-borne fungus and some strains of F. oxysporum are pathogenic to plants and are difficult to control; however, biological methods may be a reliable alternative to chemical methods for controlling soil-borne fungal growth. For applications in agriculture, the Bacillus species are considered important biological control agents. Bacillus amyloliquefaciens isolated from the rhizosphere soil, was found to efficiently antagonise F. oxysporum. Bacterial isolate B. amyloliquefaciens produces volatile compounds (acetylaldehyde, methanethiol,

142



ethanol and phenyl isothiocyanate) that inhibited the growth and spore germination of F. oxysporum. The antagonist strains exhibiting strong inhibitory activity against plant pathogens, have received much attention, (Xu et al. 2009, Mayak et al. 1999). The release of volatile compounds by soil microbes has been described to promote plant growth (Ryu et al. 2003); Bacillus isolates were found to be highly inhibitory of F. oxysporum growth whereas others showed mild activity or no activity at all. This suggests that the mode of action exerted and the type of anti-fungal metabolites produced by the isolates vary (Williams & Asher 1996).

Reduction of fungal growth by certain PGPR and formation of inhibition zones were presumably due to the compounds (antifungal substances and/or cell wall degrading enzymes) released by the bacteria into the culture medium. It has been reported that application of mixture of isolates inhibits pathogen growth more efficiently than a single isolate (Marjan et al. 2003). The reason why application of a single isolate does not control disease in better way might be related to insufficient root colonization. Therefore, the mechanisms of applying a mixture of isolates lead to more effective or at least more reliable biocontrol of root rot of wheat (Fatima et al. 2009).

From the analyses of 18 amplicons, bacterial soil samples were found to be phylogenetically diverse, with representatives from 2 different groups. The majority of the amplicons had sequences with 90-100% similarity with the sequences obtained from the current GenBank database. This suggests that these amplicons had really diverse phylogeny. Different diversity and phylogenetic analyses supported the hypothesis that bacterial communities associated with the rhizosphere of soils are different but share some genera like Bacillus. BLAST analysis of partial 16S rDNA gene sequences allowed the identification of several bacterial populations in the soil belonging to the following genera: for Bacillus eleven species were identified, and one species each to Paenibacillus, Ensifer (E. adhaerens), Aquamicrobium, Lactobacillus, Alcaligenes, Brevibacillus and Proteus (P.vulgaris).

A higher percentage of bacteria affiliated with the Firmicutes indicates that Firmicutes are considered to be a dominant bacterial family in the soil as shown by molecular methods. Firmicutes are characterized by heat resistant and desiccation ability and this explains the lower percentage of Proteobacteria in this study. Microbial diversity has been analysed using sequence based method by Borneman et al. (1996) using Wisconsin soil. Results of the present study are very different from the results of Borneman et al. (1996), because the majority of the sequences obtained were from Firmicutes group, followed by the Proteobacteria. In contrast to the Winconsin soil, most of the phylotypes in the soil samples were members of Proteobacteria (60.5%) and Fibrobacter (16%). Such differences can most likely be explained by differences in temperature, moisture, pH and vegetation.

All the sequences obtained were aligned with each other to determine genetic diversity amongst the root nodule bacteria. A consensus tree was drawn from the aligned sequences using MEGA version 5.22 on the basis of 16S rDNA sequence homology. Homology tree based on sequence alignment of the 16S rDNA bacteria isolates permitted rapid phylogenetic analysis; however, strains isolated from different geographic locations shared similar DNA homology. Phylogenetic analysis

143



on the basis of 16S rDNA sequence provided better understanding in the evaluation of genetic diversity of bacteria. Phylogenetic analysis of 1500 bp of 16S rDNA has been found to show the existence of large bacterial diversity (Clarridge III 2004)

The partial 16S rDNA sequences (1,500 bp) of these strains revealed 80-100% similarity with Bacillus, Proteus, Rhizobium and Pseudomonas species 16S rDNA sequences in the GenBank. Thus, a combination of conventional test and genetic analysis enabled identification of rhizosphere bacteria leading to a significantly better understanding of community structure, phylogeny and function of rhizosphere bacteria from Mahikeng soil.

Both biochemical characterization and molecular methods were carried out in this study to determine whether there was conformity between these two methods. The methods were reliable; however, distinguishing Bacillus species from each other using the classical biochemical test was difficult because of the fact that most Bacillus species only differ by one biochemical property thus, making the biochemical identification at the species level difficult. For instance, B subtilis and B pumilus are only distinguished by their ability to hydrolyse starch (Thompson et al. 1998) and this can alter the identification of the isolate. Molecular methods were found in this study to be an excellent alternative to classical biochemical identification procedures. The dominance of Gram positive spore-forming Bacillus species was observed using both methods; however, a shift towards the use of molecular methods is needed in order to provide a more robust classification and differentiation (McCartney 2002).

CONCLUSIONThe use of both molecular techniques and culturing methods has enabled to

identify bacterial community structure in Mahikeng soil. The bacterial isolates obtained from roots of various rhizosphere soils were found similar to the Firmicutes and Proteobacteria according to phylogenetic analysis. These bacterial isolates were essential for plant growth and nutrition as they are plant growth promoting thizobacteria (PGPR). The strategy of utilizing PGPR is relevant for achieving sustainable agriculture, as one of the goals for PGPR is to make reliable and accessible products for farmers such as biofertilizers and biocontrol agents. Consequently, continued research is needed to develop new approaches to improve efficiency of rhizobacteria and also to further understand their biochemical, genetic and ecological relationships. These rhizobacteria can be further used for the isolation of genes for biotechnological application. Their plant growth promoting abilities were correlated with IAA, HCN, ACC-deaminase activity and ammonia production of the isolates. Such isolates might have potential in future field applications as plant growth promoters. Bacillus species constituted key components of rhizosphere bacterial communities owing to their diverse and flexible physiological properties. Bacillus species from the rhizosphere are important as they perform defined roles in the soil such as ammonia oxidation. Using molecular techniques, it is now possible to identify bacteria up to the species level thus, enabling the detection of the presence and dominance of bacteria that perform specific functions.

144



ACKNOWLEDGMENTS The authors acknowledge the financial support received for this work from the

National Research Foundation and the North-West University, South Africa.

LITERATURE CITEDAltschul, S.F., W. Gish, W. Miller, E.W. Myers and D.J. Lipman. 1990. Basic local alignment

search tool. Molecular Biology 215: 403-410.Ashman, M.R. and G. Puri. 2002. Essential Soil Science: A Clear and Concise Introduction

to Soil Science. Blackwell Science Limited, United Kingdom, 198 p.Atlas, R.M., A. Horowitz, M. Krichevsky and A.K. Bej. 1991. Response of microbial

populations to environmental disturbance. Microbial Ecology 22(1): 249-256.Babalola, O.O. and A.M. Akindolire. 2011. Identification of native rhizobacteria peculiar to

selected food crops in Mmabatho Municipality of South Africa. Biological Agriculture and Horticulture 27: 294-309.

Bailly, J., L. Fraissinet-Tachet, M.C. Verner, J.C. Debaud, M. Lemaire, M. Wésolowski-Louvel and R. Marmeisse. 2007. Soil eukaryotic functional diversity, a metatranscriptomic approach. The ISME Journal VOLUME NUMBER?: 632-642.

Bergey, D.H. 1934. Bergey’s Manual of Determinative Bacteriology. The American Journal of the Medical Sciences 188, 282 p.

Berthelin, J. 2010. Soil Microorganism-mineral-organic Matter Interactions and the Impact on Metal Mobility. Molecular Environmental Soil Science at the Interfaces in the Earth’s Critical Zone, pp. 49-51.

Brady, N.C and R.R. Weil. 1999. The Nature and Properties of Soils, 12th edition, Prentice Hall, Upper Saddle River, New Jersey, USA.

Bohlool, B., J. Ladha, D. Garrity and T. George. 1992. Biological nitrogen fixation for sustainable agriculture: A perspective. Plant and Soil 141: 1-11.

Borneman, J., P.W Skroch, K.M. O’ Sullivan, J.A Palus, N.G. Rumjanek, J.L. Jansen, J. Nienhuis and E.W. Triplett. 1996. Molecular microbial diversity of an agricultural soil in Wiconsin. Application Environmental Microbiology 62: 1935-1943.

Chan, Y.K, W.L. Barraquio and R. Knowles. 1994. N2-fixing pseudomonads and related soil bacteria. FEMS Microbiology 13: 95-118.

Clarridge III, J.E. 2004. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious disease. Clinical Microbiology Reviews 17: 840-862.

Dennis, P.G., A.J. Miller and P.R Hirsch. 2010. Are root exudates more important than other sources of rhizodeposits in structuring rhizosphere bacterial communities?. Federation of European Microbiological Societies 72: 313-327.

Dusha, P., S. Kovalenko, Z. Banflavi and Z. Kondorosi. 1987. Rhizobium melilitic insertion element ISRm2 and its use for identification of the Fix X gene. Journal of Bacteriology 169: 1403-1409.

Ehrenfeld, J.G., B. Ravit and K. Elgersma. 2005. Feedback in the plant-soil system. Annual Review of Environment Resource 30(1): 75-115.

Fatima, Z., M. Saleemi, M. Zia, T. Sultan, M. Aslam, R. Rehman and M.F Chaudhary. 2009. Antifungal activity of plant growth-promoting rhizobacteria isolates against Rhizoctonia solani in wheat. African Journal of Biotechnology 8: 2-10.

Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39: 783-791.

Fitch, W.M. 1986. An estimation of the number of invariable sites is necessary for the accurate estimation of the number of nucleotide substitutions since a common ancestor. Progress in Clinical and Biological Research 218: 149-15_?.

145



Garbaye, J. 1994. Helper bacteria: A new dimension to the mycorrhizal symbiosis. New Phytopathology 128: 197-210.

Garbeva, P., J. Postma, J. Van Veen and J. Van Elsas. 2006. Effect of aboveground plant species on soil microbial community structure and its impact on suppression of Rhizoctonia solani AG3. Environmental Microbiology 82: 233-246.

Glick, B.R., D.M. Penrose and J. Li. 1998. A model for the lowering of plant ethylene concentrations by plant growth promoting bacteria. Journal of Theoretical Biology 190: 63-68.

Green, S.J., O. Prakash, T.M. Gihring, D.M. Akob, P. Jasrotia, P.M. Jardine, D.B. Watson, S.D. Brown, A.V. Palumbo and J.E. Kostka. 2010. Denitrifying bacteria isolated from terrestrial subsurface sediments exposed to mixed-waste contamination. Applied Environmental Microbiology 76: 32-44.

Grichko, V.P., B. Filby and B.R. Glick. 2000. Increased ability of transgenic plants expressing the bacterial enzyme ACC-deaminase to accumulate Cd, Co, Cu, Ni, Pb and Zn. Journal of Biotechnology 81: 45-53.

Gurung, T.D., C. Sherpa, V.P. Agrawal and B. Lekhak. 2010. Isolation and characterization of antibacterial Actinomycetes from soil samples of Kalapatthar, Mount Everest region. Nepal Journal of Science and Technology 10: 173-182.

Hayakawa, M. 2008. Studies on the isolation and distribution of rare actinomycetes in soil. Actinomycetologica 22: 12-19.

Hall, T.A. 1999. A user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.

Howitt, S.M and M.K. Udvardi. 2000. Structure, function and regulation of ammonium transporters in plants. Biochimica et Biophysica Acta (BBA)-Biomembranes 1465: 152-170.

Jukes, T.H. and C.R. Cantor. 1969. Evolution of protein molecules, pp. 21-132. In: Munro, H.N. (Ed.). Mammalian Protein Metabolism. Academic Press, New York, USA.

Katoh, K. and H. Toh. 2010. Parallelization of the MAFFT multiples sequence alignment program. Bioinformatics 26: 1899-1900.

Khan, A.A., G. Jilani, M.S. Akhtar, S.M.S. Naqvi and M. Rasheed. 2009. Phosphorus solubilizing bacteria: Occurrence, mechanisms and their role in crop production. Journal of Agricultural and Biological Science 1: 48-58.

Marjan, D.B., B. Peter, K. Frod, J.B. Joost, I. van der Sluis, L.C. van Loon and P.A.H.M. Bakker. 2003. Control of Fusarium wilt of raddish by combining Pseudomonas putida strains that have different disease suppressive mechanisms. Phytopathology 93: 626-632.

Mayak, S., T. Tivosh and B.R. Glick. 1999. Effect of wild type and mutant plant growth promoting rhizobacteria on the rooting of mungbean cuttings. Journal of Plant Growth Regulation 18: 49-53.

McCartney, A.L. 2002. Application of molecular biological methods for studying probiotics and the gut flora. British Journal of Nutrition 88(1): 29-37.

Minkwitz, A. and G. Berg. 2001. Comparison of antifungal activities and 16S ribosomal DNA sequences of clinical and environmental isolates of Stenotrophomonas maltophilia. Journal of Clinical Microbiology 39: 139-145.

Page, R.D.M. 1996. TREEVIEW, an application to display phylogenetic trees on personal computers. Computer Applications Bioscience 12: 357-358.

Pappas Jr, G.J., K. Benabdellan, B. Zingales and A.A. Gonzalez. 2005. Expressed sequence tags from the plant trypanosomatid Phytomonas serpens. Molecular Biochemistry and Parasitology 142: 149-157.

146



Parvathi, A., K. Krishna, J. Jose, N. Joseph and S. Nair. 2009. Biochemical and molecular characterization of Bacillus pumilus isolated from coastal environment in Cochin, India. Brazilian Journal of Microbiology 40: 269-275.

Pratscher, J., M.G. Dumont and R. Conrad. 2011. Ammonia oxidation coupled to CO2 fixation by archaea and bacteria in an agricultural soil. Proceedings of the National Academy of Sciences 108: 4170.

Rappé, M.S. and S.J. Giovannoni. 2003. The uncultured microbial majority. Annual Review of Microbiology 57: 369-394.

Rawat, S., A. Izhari and A. Khan. 2011. Bacterial diversity in wheat rhizosphere and their characterization. Advances in Applied Science Research Pelagia Research Library 2: 351-356.

Rousk, J., E. Bååth, P.C. Brookes, C.L. Lauber, C. Lozupone, J.G. Caporaso, R. Knight and N. Fierer. 2010. Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME Journal 4: 1340-1351.

Ryu, C.M., M.A. Farag, C.H. Hu, M.S. Reddy, H.X. Wei, P.W. Pare and J.W. Kloepper. 2003. Plant growth-promoting activities of Bacillus subtilis mbi 600 and its compatibility with commonly used fungicides in rice sheath blight management. International Journal of Microbiology 3: 120-130.

Sait, M., P. Hugenholtz and P.H. Janssen. 2002. Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environmental Microbiology 4: 654-666.

Shaharoona, B.M., Z.A. Arshad, A. Zahir and A. Khalid. 2006. Performance of Pseudomonas spp. containing ACC-deaminase for improving growth and yield of maize (Zea mays L.) in the presence of nitrogenous fertilizer. Soil Biology and Biochemistry 38: 2971-2975.

Singh, G. and K.G. Mukerji. 2006. Root exudates as determinant of rhizospheric microbial biodiversity. Microbial Activity in the Rhizosphere, Springer 1: 39-53.

Slonczewski, J.L., M. Fujisawa, M. Dopson and T.A. Krulwich. 2009. Cytoplasmic pH measurement and homeostasis in bacteria and archaea. Advances in Microbial Physiology 55: 1-79.

Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar. 2011. MEGA 5.0, Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 20: 2731-2739.

Thompson, J.M., W.M. Waites and C.E.R. Dodd.1998. Detection of rope spoilage in bread caused by Bacillus species. Journal of Applied Microbiology 85: 481-486.

van Elsas, J.D., J.K. Jansson and J.T. Trevors. 2006. Modern Soil Microbiology. CRC Press, New York, USA, pp. 84-105.

Vessey, J.K. 2003. Plant growth promoting rhizobacteria as biofertilizers. Plant and Soil 255: 571-586.

Waldrop, M., T. Balser and M. Firestone. 2003. Linking microbial community composition to function in a tropical soil. Soil Biology and Biochemistry 2: 1837-1846.

Weisburg, W.G., S.M. Barns, D.A. Pelletier and D.J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology 173: 697-703.

Williams, G.E. and M.J.C. Asher. 1996. Selection of rhizobacteria for the control of Pythium ultimum and Aphanomyces cochlioides on sugar beet seedlings. Journal of Crop Protection 15: 479-486.

Xie, H., J.J. Pastrenak and B.R. Glick. 1996. Isolation and characterization of mutants of the plant growth promoting rhizobacterium, Pseudomonas putida GR12-2 that over produce indole acetic acid. Current Microbiology 32: 67-71.

147



Xu, Z., C. Chen, J. He and J. Liu. 2009. Trends and challenges in soil research 2009, linking global climate change to local long-term forest productivity. Journal of Soils and Sediments 9: 83-88.

Zhong, W. and Z. Cai. 2007. Long-term effects of inorganic fertilizers on microbial biomass and community functional diversity in a paddy soil derived from quaternary red clay. Applied Soil Ecology 36: 84-91.

Beyond Excellence©

81 Governor F.T. San Luis Avenue, Masaya, Bay 4033Laguna, Philippines

Celfone nos. (063) (049) 0915-360-4660; 0921-374-9752Landline telephone no. (063)(049) 501-2957

e-mails: [email protected] [email protected]://journals.uplb.edu.ph/index.php/ALS

©Rushing Water Publishers Ltd., Philippines 2013

The papers published in Asia Life Sciences are indexed in theBiological Abstracts, CAB Abstracts, CAB Global Health,

Zoological Record, SciSearch®/Science Citation IndexExpanded, Journal Citation Reports/Science Edition, BIOSIS

Previews, ISI Web of Science®, ISI Web of Knowledge®and are covered by the

Thomson Reuters-Institute for Scientific Information (ISI), USAand CABI, Wallingford, Oxon, UK.

Asia Life Sciences has an Impact Factor of 0.259.

Asia Life Sciences is a recipient of the Journal AccreditationAward of the Commission on Higher Education (CHED),

Republic of the Philippines (2010-2013).

Printed on acid-free papers

148



Beyond Excellence©

81 Governor F.T. San Luis Avenue, Masaya, Bay 4033Laguna, Philippines

Celfone nos. (063) (049) 0915-360-4660; 0921-374-9752Landline telephone no. (063)(049) 501-2957

e-mails: [email protected]@gmail.com

http://journals.uplb.edu.ph/index.php/ALS


The papers published in Asia Life Sciences are indexed in theBiological Abstracts, CAB Abstracts, CAB Global Health,

Zoological Record, SciSearch®/Science Citation IndexExpanded, Journal Citation Reports/Science Edition, BIOSIS

Previews, ISI Web of Science®, ISI Web of Knowledge®and are covered by the

Thomson Reuters-Institute for Scientific Information (ISI), USAand CABI, Wallingford, Oxon, UK.

Asia Life Sciences has an Impact Factor of 0.259.

Asia Life Sciences is a recipient of the Journal AccreditationAward of the Commission on Higher Education (CHED),

Republic of the Philippines (2010-2013).

Printed on acid-free papers


ASIA LIFE SCIENCES Supplement 9: 423-427, 2013The Asian International Journal of Life Sciences

Actual Date of Publication - 31 August 2013 ALS Supplement 9 2013©Rushing Water Publishers Ltd. 2013

REVIEWERS - ASIA LIFE SCIENCES Supplement 9, 2013 - Contributions from Africa Series 2*

Dr. Nelly S. AgganganUniversity ResearcherNational Institute of Molecular Biology and Biotechnology (BIOTECH)University of the Philippines Los BañosCollege 4031, Laguna, Philippines

Dr. Ricardo T. Bagarinao Associate ProfessorEnvironmental Science (Restoration and Landscape Ecology)UP Open UniversityLos Baños, Laguna, Philippines

Dr. Mario R. Delos ReyesProfessor & DeanSchool of Urban and Regional PlanningUniversity of the PhilippinesE. Jacinto St., Diliman, Quezon City 1101Philippines

*Disclaimer: The use of trade names in this publication does not imply endorsement or criticism of the products named.

424



Dr. Agustine Ignatius Doronila Research FellowSchool of Chemistry University of Melbourne Victoria 3010 Australia

Dr. Maribel L. Dionisio-SeseProfessor 11Plant Biology DivisionInstitute of Biological SciencesCollege of Arts & SciencesUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines.

Dr. Marison R. DyAssociate Professor Department of Human and Family Development StudiesCollege of Human EcologyUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines.

Prof. Ralph Kristoffer B GallegosAssistant ProfessorAgricultural Machinery DivisionInstitute of Agricultural EngineeringCollege of Engineering and Agro-Industrial TechnologyUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines

Dr. Victor P. GapudProfessor EmeritusCrop Protection ClusterCollege of AgricultureUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines..Dr. William Sm. GruèzoProfessor 12Plant Biology DivisionInstitute of Biological SciencesCollege of Arts & SciencesUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines.

425

Reviewers - Asia Life Sciences Supplement 9 2013


Dr. Jianjun Hu3 Blackfan Circle, CLS-628-I6 BIDMC-Hospital, Harvard Medical SchoolBoston, MA 02115, USA

Dr. Konstantinos M. KasiotisChemist (Ph.D), Research AssistantBenaki Phytopathological InstituteDepartment of Pesticides Control and Phytopharmacy Laboratory of Toxicological Control of Pesticides Kifissia, Athens, Greece,

Dr. Renato L. LapitanAssociate ProfessorInstitute of Renewal Natural ResourcesCollege of Forestry and Natural ResourcesUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines

Dr. Jinxia MaResearch FellowJohns Hopkins Bloomberg School of Public Health615 N Wolfe St.BaltimoreMaryland 21205 USA

Dr. Scott T. MeissnerVisiting ProfessorInstitute of Biological SciencesCollege of Arts & SciencesUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines.

Dr. Ma. Lourdes T. MunarrizAssociate ProfessorSchool of Urban and Regional PlanningUniversity of the Philippines E. Jacinto St., Diliman, Quezon City 1101Philippines

426



Dr. Fernando O. Paras, Jr. Associate Professor Agricultural Machinery Division (AMD)Institute of Agricultural Engineering (IAE)College of Engineering and Agro-Industrial TechnologyUniversity of the Philippines Los BañosAMTEC Building, Pili DriveCollege 4031, Laguna, Philippines

Dr. Lynlei L. PintorEcosystem Research and Development BureauDepartment of Environment and Natural ResourcesCollege 4031, Laguna, Philippines

Dr. Corazon L. RaperaAssociate ProfessorCollege of Economics and ManagementUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines

Dr. Antonio A. Rayos, Sr.ProfessorAnimal and Dairy Science ClusterCollege of AgricultureUniversity of the Philippines Los BañosCollege 4031, Laguna, Philippines

Dr. Windell L. Rivera FACTM, FPAMDirector, Natural Science Research Institute andAssociate Professor of Microbiology,Institute of Biology, College of ScienceUniversity of the Philippines Diliman, Quezon City 1101Philippines

Dr. Mary Ann T. TavanlarUniversity ResearcherNational Institute of Molecular Biology and Biotechnology (BIOTECH)University of the Philippines Los BañosCollege 4031, Laguna, Philippines

427

Reviewers - Asia Life Sciences Supplement 9 2013


Dr. Alice T. ValerioProfessorAllied Business DepartmentDela Salle University-DasmariñasDasmariñas, Cavite, Philippines

Dr. Jin WanResearch FellowBSRB 5628, 109 Zina Pitcher PlaceMolecular and Behavior Neuroscience InstituteThe University of MichiganAnn Arbor, Michigan 48109-220, USA

Dr. Rui Wang.Department of BiologyUniversity of Utah257S 1400E Salt Lake City 84112 Utah, USA

Dr. Yanyang WangResearch FellowDepartment of SurgeryMassachusetts General Hospital and Harvard Medical School55 Fruit Street, Boston MA 02114 USA

Beyond Excellence©81 Governor F.T. San Luis Avenue, Masaya, Bay 4033, Laguna, Philippines

Celfone nos. (063) (049) 0915-360-4660; 0921-374-9752; 0916-526-0164Landline telephone no. (063)(049) 501-2957

e-mails: [email protected] [email protected]://journals.uplb.edu.ph/index.php/ALS


428



ASIA LIFE SCIENCES - The Asian International Journal of Life Sciences (ISSN 0117-3375) is a non-profit, non-stock refereed scientific journal devoted to the publication of original research in the Life Sciences and related disciplines. Articles originating from anywhere in the world are most welcome. Two issues a year make a volume

BOARD OF EDITORS - Asia Life Sciences Supplement 9, 2013Chairman & Chief Editor: Dr. William Sm. Gruèzo, Institute of Biological

Sciences, College of Arts & Sciences (CAS), University of the Philippines Los Baños (UPLB), College 4031, Laguna, Philippines.

Members: Dr. Liding Chen, State Key Laboratory of Urban and Regional Ecology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Shuangqing Road 18, Haidian, Beijing 100085, China. Dr. Leonila A. Corpuz-Raros, Crop Protection Cluster, College of Agriculture, UPLB, College 4031, Laguna. Dr. Maribel L. Dionisio-Sese, Plant Biology Division, Institute of Biological Sciences, CAS-UPLB, College, Laguna. Dr. Irineo J. Dogma Jr., Graduate School, University of Santo Tomas, España St., Manila. Dr. Agustine I. Doronila, School of Chemistry, University of Melbourne, Victoria 3010, Australia. Dr. Victor P. Gapud, Crop Protection Cluster, College of Agriculture, UPLB, College 4031, Laguna. Dr. Krishlex G. Gruèzo MD, 81 Gov. F.T. San Luis Avenue, Masaya, Bay 4033, Laguna. Dr. Rafael D. Guerrero III, National Academy of Science and Technology, Level 3, Science Heritage Building, Deparment of Science and Technology Complex, Bicutan, Taguig City, MetroManila. Michael G. Price, P.O. Box 468, Mi Ctr, Michigan 49254, USA. Dr. Xu Wu MD/PhD, 1924 Heritage Park Drive, Apt. 211, Oklahoma City, OK 73120, USA.

Technical Production Manager: Ydred Harriss G. Gruèzo

Deadlines for submission of manuscripts. First issue - 01 June; Second issue - 01 January. Please contact the Chairman, ALS Board of Editors concerning information for contributors (see addresses below).

Subscription Prices. Foreign: Institutional - US$750; Individual - US$500 (including Volumes 1-22,1992-2013+Supplements). Local: Institutional - PhP7500; Individual - PhP5000 (including Volumes 1-22, 1992-2013 + Supplements). Prepayment of order/back order is required. All issues are to be sent by air mail. Back orders will have an additional packing-handling and postage cost.

Send manuscripts, subscription orders and correspondence to: Dr. William Sm. Gruèzo, ASIA LIFE SCIENCES, The Asian International Journal of Life Sciences, 81 Gov. F.T. San Luis Avenue, Masaya, Bay 4033, Laguna, Philippines. Mobile phone no.(63) 915-360-4660; Telephone no. (63)(49) 501-2957. e-mails: [email protected] [email protected] website: http://journals.uplb.edu.ph/index.php/ALS

Front Cover (Clockwise): Irrigation system; Beef animal production; Free range poultry, and Lucerne farm; all in North West Province, South Africa. Credit – Prof. O.I. Oladele, North West University, Mafikeng Campus, Mmabatho 2735, South Africa.

ASIA LIFE SCIENCES The Asian International Journal of Life Sciences

ISSN 0117-3375 Supplement 9 August 2013

CONTENTS1 Determinants of smallholder cocoa farmers’ adaptation to climate change in Ile-oluji/Okeigbo Local Government Area of Ondo State, Nigeria G.O. Ogunsola & A.S. Oyekale11 Flavonoids, carbohydrates, proanthocyanidins and other phenolics in the leaves of selected Acacia species in Mankweng, Limpopo Province, South Africa H.K. Mokoboki & A. Sebola21 Impact of AIDS care and level of burnout among nurses in selected hospitals in Limpopo Province, South Africa J.O. Igumbor & M. Davhana-Maselesele 33 Effect of phosphorus fertilizer and leaf cutting technique on biomass yield and crude protein content of two African indigenous leafy vegetables M. Seeiso & S.A. Materechera51 Examining a national agricultural extension and advisory system: A case study of the North West and South West regions of Cameroon G.N. Nyambi, G.C. Shinn & G.E. BriersCont. on Inside Back Cover

Reviewers for this Issue: Dr. N.S. Aggangan, Dr. R.T. Bagarinao, Dr. M.R. Delos Reyes, Dr. A.I. Doronila, Dr. M.L. Dionisio-Sese, Prof. R.K.B. Gallegos, Dr. V.P. Gapud, Dr. Wm.Sm. Gruèzo, Dr. J. Hu, Dr. K.M. Kasiotis, Dr. R.L. Lapitan, Dr. J. Ma, Dr. S.T. Meissner, Dr. M.L.T. Munarriz, Dr. F.P. Paras Jr., Dr. L.L. Pintor, Dr. C.L. Rapera, Dr. A.A. Rayos, Dr. W.L. Rivera, Dr. M.A.T. Tavanlar, Dr. A.T. Valerio, Dr. J. Wan, Dr. R. Wang & Dr. Y. Wang.

©Rushing Water Publishers Ltd. 2013 Printed in the Philippines

Documents

Masenya Adegboye Babalola 2013 ALS SUPPL 9 129 147