IJERD – International Journal of Environmental and Rural Development (2014) 5-1

Research article
erd

Effects of Pesticide on Phenotypic Characteristics of Plant
Growth - Promoting Rhizobacteria (PGPR) in Cassava
Production Systems
PIMUPSORN PANOMKHUM
Faculty of Agriculture, Khon Kaen University, Thailand

RATTIYAPON RUNGTHONG
Faculty of Agriculture, Khon Kaen University, Thailand

TIPPAYA NITTAYACHIT
Faculty of Agriculture, Khon Kaen University, Thailand

CHULEEMAS BOONTHAI IWAI
Faculty of Agriculture, Khon Kaen University, Thailand

MONGKON TA-OUN
Faculty of Agriculture, Khon Kaen University, Thailand

PHRUEKSA LAWONGSA*
Agricultural Biotechnology Research Center for Sustainable Economy: (ABRCSE),
Faculty of Agriculture, Khon Kaen University, Thailand
Email: phrula@kku.ac.th
Received 12 October 2013

Accepted 1 March 2014(*Corresponding Author)

Abstract Cassava (Manihot esculenta Crantz) is an important economic crop in Thailand.
Nowadays, insect pest is the major problem that causes severe damage to cassava leading
to considerable yield losses. Since, using chemical pesticide is always presence ofchemical residues on soil and may affect the population and diversity of soil
microorganism. Therefore, the aim of the study was to determine the effect ofthiamethoxam on plant growth promoting rhizobacteria (PGPR) population in cassava
production system. A total of 400 bacteria were isolated from 4 sites, including 1 (cassava
production system without thiamethoxam and fertilizer application 2 (cassava production
system with thiamethoxam application 3 (cassava production system with thiamethoxam
and organic fertilizer application 4 (cassava production system with thiamethoxam andchemical fertilizer application. These isolates were screened for their plant growthpromoting factors like production of indole-3-acetic acid (IAA), phosphate solubilizing
activity and their ability to grow in N-free medium. In addition, their biocontrol activity
like protease and chitinase enzyme production and siderophore production as well as
antagonistic activity against Fusarium sp. were investigated. The findings of this study
indicated that the application of thiamethoxam in cassava production system can affect
PGPR population. In this study, numbers of bacterial isolates demonstrate that they are apotential source to be used as microbial inoculant for crop production system.
Keywords Plant growth promoting rhizobacteria (PGPR), thiamethoxam, cassava

INTRODUCTION
Cassava (Manihot esculenta Crantz) is one of the most important economic crops in Thailand.
Cassava can be used for various food products, animal feed, raw material to produce ethanol etc. In
addition, an increasing demand for alternative energy products will increase the market value of
cassava in the near future.
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Nowadays, insect pest is the major problem that caused damage of cassava production. In
particular, the most of farmers prefer to use of chemical pesticide such as thiamethoxam,
insecticide in the group of neonicotinoids which is one of the most widely used, is resulting inchemical residue in soil. This may disturb the soil environment by affecting microorganisms in soil,
and also the physical -chemical properties of soil leading to infertility of soil. Biological control isthus being considered as an alternative or a supplemental way of reducing the use of chemicals inagriculture.
The using of introduced microorganisms with induced systemic resistance against plantdisease and stimulate plant growth has been extensively studied (Compant et al., 2005; Welbaum etal., 2004). The introduction of plant growth promoting rhizobacteria (PGPR) offers a promisingalternative to manage plant pathogen and enhance plant growth. PGPR can directly benefit plant
growth by increasing nitrogen and phosphorus uptake (Rodriguez et al., 1999) and indirectly byincreasing resistance to pathogen. PGPR may suppress plant pathogens by producing antimicrobial
metabolism (Duffy, 2004) and also by producing enzymes and/or fungicidal compounds(Bloemberg and Lugtenberg, 2001; Haas and Défago, 2005). This study was designed toinvestigate how chemical pesticide shaped the bacterial populations as well as plant growthpromoting activities for successful application of PGPR in cassava production systems to reduce
the use of chemical pesticide and soil fertility could be maintained.
OBJECTIVE
The purpose of this study was to investigate the influence of chemical pesticide on the populationand phenotypic characteristics of PGPR in cassava production system.
METHODOLOGY
Study Sites and Soil Sampling
Study sites used in this study were selected from Khon Kean province and Kalasin province (N 16°
28' 43", E 102' 49 and 37" N 16° 38' 3", E 103° 15' 15", respectively). Soil samples were collectedfrom rhizosphere of cassava cultivar Rayong 11 with three replications of each treatment wascollected at 0-15 cm depth. Pesticide used in this study for cassava stake priming wasthiamethoxam 25 WG. Four treatments used in this study, including 1) cassava production system
without thiamethoxam and fertilizer application 2) cassava production system with Thiamethoxam
application 3) cassava production system with thiamethoxam and organic fertilizer application,
1000 kg/1,600 Square meter 4) cassava production system with thiamethoxam and chemicalfertilizer application 15-7-18, 100 kg/1,600 Square meter. The rhizosphere samples were placed inplastic bags and stored at 4 °C for further microbial analysis. In addition, soil samples werecollected from 0-15 cm depth for the examination of physical and chemical properties of soil.
Isolation of PGPR from Cassava Rhizosphere
Bacterial strains were isolated from cassava rhizosphere by serial dilution plate technique on
nutrient agar medium (NAM) (Dubey and Maheshwari, 2002). The bacterial colonies were isolated
and maintained on NAM slants at 4 °C. One hundred isolates obtained from serial dilution platetechnique of each treatment were screened for their plant growth promoting factors like productionof indole-3-acetic acid (IAA), phosphate solubilizing activity and their ability to grow in N-free
medium. In addition, their biocontrol activity like protease and chitinase enzyme production andsiderophore production as well as antagonistic activity against Fusarium sp. were investigated.

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Phenotypic Characterization of PGPR
Indole-3-acetic acid (IAA) production: IAA production was determined using the method describedby Lawongsa (2008) with slight modification. Bacteria isolates were cultured in Tris-TMRT (Dmannitol 10 g, yeast extract 0.2 g, CaCl2.2H2O 0.2 g, MgSO4.7H2O 0.25 g, Tris-base 1.21 g, pH
6.8) supplemented with tryptophan 0.5 mM for 48 h. The measurement of IAA was done by adding
2 ml of 0.01 M FeCl3 in 35 % HClO4 into 1 ml of Tris-TMRT culture broth. The mixture wasincubated in the dark at 30 °C for 30 min. The detection of IAA was determined by thedevelopment of pink color.
Phosphate solubilizing assay: Solubilization of tricalcium phosphate was detected in national
botanical research institute’s phosphate growth medium (NBRIP) agar plate supplemented with
1.5% (w/v) agar (Nautiyal, 1999). Five microliters of each bacterial culture was dropped on NBRIP
agar plates. The development of halo zone around the bacterial colony indicated phosphatesolubilizing activity.
Nitrogenase activity: For rapid determination, nitrogenase activity was assayed after bacterial
strains were streaked onto N-free minimal medium supplemented with 1.5% (w/v) agar andincubated at 28 ± 0 °C for 3 days (Desnoues et al., 2003). Bacterial growth indicated nitrogenase
activity.
Protease assay: Bacteria were isolated for protease enzyme (casein degradation) using a method
described by Sjödahl et al. (2002). Samples were inoculated on LB agar plates containing skim
milk (20 %), then incubated at 28 ± 2 °C for two days (Uyar et al., 2011). The development of clear
zone around the bacterial colony indicated protease enzyme activity.
Chitinase assay: Chitinase activity was determined using colloidal chitin (Cattelan et al, 1999).
Five microliters of each bacteria culture were dropped on colloidal chitin agar (Colloidal Chitin 10
g, K2HPO4 0.5 g, MgSO4 7H2O 0.5 g, Na2HPO4 0.5 g, NaNO3 3.0 g, Yeast extract 1 g, Agar 20 g,
pH 7) and incubator at 28 °C for 7 days. The development of halo zone around the bacterial colony
indicated chitinase activity.
Siderophore assay: Siderophore was determined by chromazurol sulphonate agar (CAS) using themethod described by Clark and Bavoil, 1994. Bacterial inoculum was spotted into the center of a
CAS agar plate. After incubation at 28 °C for 5 days, siderophore production was assayed by clear
zone formation around the cell.
Antagonistic activity: An inhibition of phytopathogen by bacterial strains on potato dextrose agar
(PDA) plates was performed as detailed in previously study (Keel et al., 1996) with slight
modification. Briefly, bacteria was grown overnight in NB (Nutrient broth) medium, and 5 µl ofeach culture was spotted 2 cm from the edge of the plate (four spots per plate) and 0.3 cm squareplug from a culture of Fusarium sp. was placed at the center of the plate. The results were assessed
after 5 days by measuring the distance between the edges of the bacterial colony and the fungal
mycelium.
Statistical analysis: The data collected was analyzed statistically using Analysis of variance(ANOVA) along with least significant differences (LSD) by analytical software STATISTIC 8.
RESULT AND DISCUSSION
Soil Analysis
The data of physical and chemical properties of soil are shown in Table 1. The results showed thatsoil samples of all treatment were sandy soil and contained low fertility.
Effect on Bacterial Population of Rhizospheric Soil of Cassava
Total count of bacteria was found to decrease with the application of thiamethoxam (Table 2).
Interestingly, when the experimental plot received organic fertilizer as well as chemical fertilizer,Ⓒ ISERD

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there was slight increase in the bacterial count as compared to cassava production system without
any fertilizer.
Table 1 Physical and chemical properties of soil
Treatment
12
34
Organic matter; OM (%)
0.227 c0.360 b0.432 a0.368 b
Total N (%)
0.014 a0.009 b0.013 a0.009 bAvailable P (mg/kg)
7.230 c16.793 bc 51.890 a
26.330 b
Exchangeable K (mg/kg)
31.470 ab 24.037 b
33.057 ab43.870 a
Organic carbon (%)
0.132 c0.209 b0.250 a0.213 bSoil pH
4.411 a3.553 b4.556 a3.450 bElectrical conductivity (dS/m)
0.045 a0.026 b0.029 b0.032 bCation exchange capacity (cmol/kg)
1.725 b5.433 a3.333 ab6.000 aBulk density (g/cm3)
1.504 a1.411 a1.353 a1.442 aSoil moisture (%)
3.372 a2.935 a3.396 a3.009 aSoil texture
sand
sand
sand
sand
Treatment: 1) cassava production system without thiamethoxam and fertilizer application. 2) cassava production systemwith Thiamethoxam application. 3) cassava production system with thiamethoxam and organic fertilizer application,
1000 kg/1,600 Square meter. 4) cassava production system with thiamethoxam and chemical fertilizer application 15-718, 100 kg/1,600 Square meter. Means values followed by different letters are significantly different (ANOVA; LSD test,
P<0.05)

Table 2 Effect of pesticide application on bacterial population
Treatment Bacterial count (cfu/g soil)
1
5.94 x 1011 a
24.51 x 1010 b
35.42 x 1010 b
44.78 x 1010 b
Treatment: 1) cassava production system without thiamethoxam and fertilizer application. 2) cassava production systemwith Thiamethoxam application. 3) cassava production system with thiamethoxam and organic fertilizer application,
1000 kg/1,600 Square meter. 4) cassava production system with thiamethoxam and chemical fertilizer application 15-718, 100 kg/1,600 Square meter. Means values followed by different letters are significantly different (ANOVA; LSD test,
P<0.05)

Phenotypic Characterization of Bacterial Isolates for Plant Promotion and Biocontrol Traits
A total of 100 cultivable bacterial isolates of each treatment obtained after serial dilutions andplating onto NAM were screened for plant growth promoting traits through growth promoter assays
and biological control assays.
The bacterial ability to produce IAA in presence of L-tryptophan as precursor was tested. A
total of 305 isolates showed the ability to produce IAA, 60%, 64%, 89% and 92% of isolates thathave ability to produce IAA were obtained from treatment 1, 2, 3 and 4, respectively (Fig. 1).
Phosphate solubilizer produce clear zone around the bacterial colonies on media containing
insoluble mineral phosphate. A total of 245 isolates showed the ability to solubilize tricalcium
phosphate, 60%, 64%, 77% and 44% of isolates that have ability to solubilize phosphate wereobtained from treatment 1, 2, 3 and 4, respectively (Fig. 1).
A total of 310 isolates showed the ability to grow in nitrogen-free medium, 55%, 75%, 98%
and 82% of isolates that have ability to grow in nitrogen-free medium were obtained fromtreatment 1, 2, 3 and 4, respectively (Fig. 1).
Production of fungal cell wall degrading enzymes (protease and chitinase enzymes), an
important mechanism of fungal inhibition, was analyzed. A total of 294 isolates could produce halozone on skim milk agar that showed protease activity, 65%, 86%, 67% and 76% of isolates thathave ability to produce protease were obtained from treatment 1, 2, 3 and 4, respectively. A total of
285 isolates could produce halo zones on colloidal chitin agar that showed chitinase activity, 65%,77%, 84% and 59% were obtained from treatment 1, 2, 3 and 4, respectively (Fig. 1).
Bacterial isolates which have the ability to produce siderophore and showed clear zone on
CAS agar were considered as positive isolate. A total of 75 isolates could produce clear zone thatⒸ ISERD

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showed siderophore production, 5%, 22%, 35% and 13% were obtained from treatment 1, 2, 3 and4, respectively (Fig. 1).
In vitro antagonistic activity against Fusarium sp. was investigated. The result revealed that
only 16 isolates showed the ability to control Fusarium sp. growth among 400 isolates, 5%, 3%,
5% and 3% of isolates were obtained from treatment 1, 2, 3 and 4, respectively (Fig. 1).
From this present study, the results showed that bacteria showed variation in their growthpromoter and biocontrol characteristics. Numbers of bacteria still showed the ability to promote
plant growth directly and indirectly way even in the presence of pesticide. This could be certified to
the fact that certain soil bacteria can degrade pesticides (Sethunathan, 1973). In addition, certain
soil bacteria might have utilized pesticide as energy sources (Ahemad and Khan, 2011).
Furthermore, the results showed that the application of organic fertilizer can promote plant growthpromoting activities of bacteria.
100

Percentage of phenotypic sharacteristic

90
80
70
60

1

50

2

40

3

30

4

20
10
0
IAA
production

Phosphate Grouth in N- Proteasesolubilizing free medium
enzymeactivity
production

Chitinas
enzymeprodution

Siderophore Antagonistic
activity

Treatment: 1) cassava production system without thiamethoxam and fertilizer application 2) cassava production systemwith Thiamethoxam application 3) cassava production system with thiamethoxam and organic fertilizer application, 1000
kg/1,600 Square meter 4) cassava production system with thiamethoxam and chemical fertilizer application 15-7-18, 100kg/1,600 Square meter.

Fig. 1 Number of bacterial isolates showing phenotypic characteristic at each sampling site
displayed as a percentage of the total site number
CONCLUSION
This study has shown that thiamethoxam not only affect the bacterial population but also have an
impact on their plant growth promoting activities. In addition, the use of organic fertilizer canslight increase the number of bacteria that exert the positive effects on plant growth promoting via
direct and indirect mechanisms. The higher numbers of bacterial isolates which have the ability to
produce IAA, solubilize phosphate, grow in nitrogen-free medium, produce chitinase enzyme andproduce siderophore were found in cassava production system with thiamethoxam and organic
fertilizer addition when compared to control with thiamethoxam. Therefore, it can be concluded
that the use of pesticides should be applied with organic fertilizer, to maintain the biodiversity ofsoil microorganisms. Further study should be carried out with such efficient PGPR isolates toachieve the successful cassava management.
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ACKNOWLEDGEMENTS
This work was supported by a grant from National Science and Technology Development Agency
and Agricultural Biotechnology Research Center for Sustainable Economy, Khon Kaen University.
REFERENCES
Ahemad, M. and Khan, M.S. 2011. Assessment of plant growth promoting activities of rhizobacterium
Pseudomonas putida under insecticide-stress. Microbiol. J., 1, 54-64.
Bloemberg, G.V. and Lugtenberg, B.J.J. 2001. Molecular basis of plant growth promotion and biocontrol byrhizobacteria. Curr. Opin. Biotechnol., 4, 343-350.
Cattelan, M.E., Hartel, P.G. and Fuhrmann, J.J. 1999. Screening of plant growth-promoting rhizobacteria topromote early soybean growth. Soil Sci. Soc. Am. J., 63, 1670-1680.
Clark, V.L. and Bavoil, P.M. 1994. Methods in enzymology. Academic Press. London, UK.
Compant, S., Duffy, B., Nowak, J., Clement, C. and Barka, E.A. 2005. Use of plant growth-promoting
bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects. Appl.
Environ. Microbiol., 71, 4951-4959.
Desnoues, N., Lin, M., Guo, X., Ma, L., Carreno-Lopez, R. and Elmerich, C. 2003. Nitrogen fixation
genetics and regulation in a Pseudomonas stutzeri strain associated with rice. Microbiol., 149, 2262.
Dubey, R.C. and Maheshwari, D.K. 2002. Practical microbiology. S. Chand and Co., New Delhi, India.
Duffy, B., Keel, C. and Defago, G. 2004. Potential role of pathogen signaling in multitrophic plant-microbe
interactions involved in disease protection. Appl. Environ. Microbiol., 70, 1836-1842.
Haas, D. and Defago, G. 2005. Biological control of soil-borne pathogens by fluorescent pseudomonads.
Nature Rev. Microbiol., 3, 307-319.
Keel, C., Weller, D.M., Natsch, A., Defago, G., Cook, R.J. and Thomashow, L.S. 1996. Conservation of the
2,4-diacetylphloroglucinol biosynthesis locus among fluorescent Pseudomonas strains from diverse
geographic locations. Appl. Environ. Microbiol., 62, 552-563.
Lawongsa, P., Boonkerd, N., Wongkaew, S., O’Gara, F. and Teaumroong, N. 2008. Molecular andphenotypic characterization of potential plant growth-promoting Pseudomonas from rice and maizerhizospheres. World J. Microbiol. Biotechnol., 24, 1877-1884.
Nautiyal, C.S. 1999. An efficient microbiological growth medium for screening phosphate solubilizing
microorganisms. FEMS Microbiol. Lett., 170, 265-270.
Rodriguez, H. and Fraga, R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion.
Biotech. Adv., 17, 319-339.
Sethunathan, N. 1973. Microbial degradation of insecticides in flooded soil and in anaerobic cultures. Res.
Rev., 47, 143-165.
Sjödahl, J., Emmer, Å., Vincent, J. and Roeraade, J. 2002. Characterization of proteinases from antarctic krill(Euphausia superba). Protein Express. Purific., 26, 153-161.
Uyar, F., Porsuk, I., Kizil, G. and Yilmaz, E.I. 2011. Optimal conditions for production of extracellular
protease from newly isolated Bacillus cereus strain CA15. EurAsian J. BioSci., 5, 1-9.
Welbaum, G., Sturz, A.V., Dong, Z. and Nowak, J. 2004. Fertilizing soil microorganisms to improve
productivity of agroecosystems. Crit. Rev. Plant Sci., 23, 175-193.

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