IJERD – International Journal of Environmental and Rural Development (2018) 9-1

Research article
erd

Effects of Legume Residues with and without
Allelochemicals on Biodiversity of Plant Growth Promoting
Bacteria in Long-Term Organic Residues Amendment
Systems
PHRUEKSA LAWONGSA*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: phrula@kku.ac.th

SAOWALAK SOMBOON
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

RATTIYAPON RUNGTHONG
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

PATCHAREE SAENJAN
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

PATMA VITYAKON
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Received 7 January 2018

Accepted 5 May 2018(*Corresponding Author)

Abstract Application of organic residues into infertile soil is one way to restore the fertility
and productivity of arable soils. However, organic residues have different qualities(especially chemical compositions including allelochemicals) that may influence theactivity and type of microorganisms present in the soil. Therefore, this study aims toinvestigate the effect of allelochemicals on plant growth promoting bacteria (PGPB)
biodiversity in 17 years long-term field experiment continuous application of different
quality residues, groundnut stover (Arachis hypogaea) (high quality organic residue; withallelochemicals) and tamarind (Tamarindus indica L.) (intermediate quality organic
residue; without allelochemicals), compare to control (no organic residue applied). PGPB
isolates were screened for their plant growth promoting factors such as production ofindole-3-acetic acid (IAA), phosphate solubilizing activity and their ability to grow innitrogen-free medium. In addition, the biocontrol activities which are protease production
and antagonistic activity against Fusarium sp. were investigated. PGPB biodiversity wasdetermined on a basis of amplified rDNA restriction analysis (ARDRA). The majority ofbacteria were found to belong to the genera of Bacillus, Staphylococcus, andBrevibacterium. In addition, at 52 weeks after residue application, PGPB richness oftamarind treatment was higher than groundnut treatment. The findings of this study also
indicated that long-term legume residues amendment with and without allelochemicals canaffect PGPB richness and biodiversity.
Keywords plant growth promoting bacteria (PGPB), groundnut stover, tamarind residue,
allelopathy

INTRODUCTION
Application of organic residues into infertile soil is one way to restore the fertility and productivityof arable soils. In addition, incorporating organic matter into soil affects on the biological, chemicaland physical properties of the soil and its overall health. However, organic residues have different
qualities (especially chemical compositions including allelochemicals) that influence the numberⒸ ISERD

57

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

and type of microorganisms present in the soil (Aciego Pietri and Brooks, 2009; Marschner et al.,2003). Among allelopathic plants, tamarind is well known for its allelopathic potential. There areseveral allelochemicals playing role as allelopathic substances were found in tamarind leaves suchas tannins, phenolic compounds, flavoniod and alkaloids (Bhadoriya et al., 2011; Lewis andNeelakantan, 1964). Total phenols such as gallic acid, Caffeic acid were observed in tamarind
leaves for 14 g/kg and tannic acid for 8 g/kg. These chemicals also showed antimicrobial activities(Lawongsa et al., 2016a) and leaf extract of tamarind which showed decreasing bacterial
population in sandy soil (Somboon et al., 2017). Moreover, applications of different quality oforganic residue also have a tendency to induce plant growth promoting bacteria (PGPB) in soil
(Rungthong et al., 2013). The PGPB represent numerous species of soil bacteria which, whengrown in association with a host plant, result in stimulation of growth of their host. PGPB are used
as inoculants for biofertilization, phytostimulation and biocontrol (Bloemberg and Lugtenberg,
2001). PGPB can directly benefit plant growth by fixing nitrogen, which can subsequently be usedby the plant, thereby improving plant growth when the amount of nitrogen in the soil is limiting(Vessey and Buss, 2002), produce phytohormone such as indole-3-acetic acid (IAA) (Ahemad andKhan, 2012; Sachdev et al., 2009) and phosphorus uptake (Rodriguez et al., 1999). Indirectly, byincrease resistance to pathogen and stimulate activation of induced systemic resistance (ISR) andsystemic acquired resistance (SAR) pathways (Duffy and Défago, 1999). The accumulation oflegume residue with allelochemicals into soils beyond certain threshold levels to overcome soil
degradation is may affect PGPB diversity.
OBJECTIVE
The purpose of this study was to investigate the effect of allelochemicals on plant growthpromoting bacteria (PGPB) biodiversity in 17 years long-term field experiment continuous
application of different quality of legume residues, groundnut stover (Arachis hypogaea) (highquality organic residue; without allelochemicals) and tamarind (Tamarindus indica L.)(intermediate quality organic residue; with allelochemicals), compare to control (no organic residue
applied).
METHODOLOGY
Study Sites and Soil Sampling
The long-term field experiments were established at the research station of the Agriculture and Cooperatives of Northeast at Tha Phra subdistrict, Khon Kaen province, Thailand (16°20´ N; 102°49´E) since 1995. This experiment has been designed as the randomized complete block design(RCBD). Three treatments apart from control soil (C; soil with no organic residues applied), there
were two legume residue treatments applied in early May every year including:, groundnut stover(Arachis hypogaea) as high quality organic residue (GN; C/N ratio: 17.1, Lignin: 67.6 g/kg;
Polyphenols: 12.9 g/kg) and tamarind (Tamarindus indica L.) leaves + petiole litter as intermediate
quality organic residue (TM; C/N ratio: 31.5, Lignin: 87.7 g/kg; Polyphenols: 31.5 g/kg) at the rateof 10 Mg/ha/year to bare soil plots. The organic materials were incorporated to a depth of 20 cm ina 4 x 4 m2 plot. The average of soil moisture content in the control field, GN field and TM field allyear were 6.24%, 6.26% and 6.6%, respectively. Weeds were controlled at approximately monthly
intervals. For the present study, soil samples were obtained in April 2012, 17 years after the field
experiment had started. Three random soil samples from each of three replicate plots of eachtreatment were collected at 0-15 cm depth for PGPB analysis at 0, 26, 52 weeks after residue
application. The soil in this study was characterized as a Khorat sandy loam (fine loamy siliceous
isohyperthermic Typic (Oxyaquic) Kandiustults (USDA, 2014). Soil texture of all plot sites were
sand. The proportion of sand in the topsoil (0–15 cm depth) was between 93.86 - 94.93 %. The pHof all soil samples were found to vary from 5.1 to 6.62 which indicated the slight acidity of soils.
The soil pH values of 5.10, 6.37 and 6.62 were obtained from C, GN and TM, respectively. TheⒸ ISERD

58

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

electrical conductivity (EC) values of all soil samples were found in the range of 33.66 to 64.66µS/cm indicated that all soil treatments were low in salinity. The soil samples were placed inplastic bags and stored at 4°C for further microbial analysis.
Isolation of PGPB from Soil Samples
Bacterial strains were isolated from soil samples by serial dilution plate technique on nutrient agar
medium (NAM). The bacterial colonies were isolated and maintained on NAM slants at 4°C. One
hundred isolates obtained from serial dilution plate technique of each treatment were screened fortheir plant growth promoting factors like production of indole-3-acetic acid (IAA), phosphatesolubilizing activity and their ability to grow in N-free medium. In addition, their biocontrol
activity like protease enzyme production as well as antagonistic activity against Fusarium sp. wasinvestigated.
Total Genomic DNA Isolation
PGPB isolates were grown in a nutrient broth at 28°C overnight and then were harvested bycentrifugation at 5,000xg for 5 min and washed twice in 500 µl of TEN buffer (50 mM Tris, 20
mM disodium EDTA, and 50 mM NaCl, pH 8.0). Cell lysates were prepared by mixing the cellpellet with 200 µl of 20% (w/v) sucrose in TEN buffer to this 20 µl of 2 mg/ml of lysozyme and 20µl of 10 mg/ml of RNase was added. Cell mixtures were incubated at 37°C for 60 min. Then 75 µlof 5 M NaCl and 100 µl of 10% SDS were added before gentle mixing. The solution was purified
twice by using phenol:chloroform:isoamyl-alcohol (25:24:1, by volume). The upper phase wascollected and precipitated by using isopropanol and 3 M sodium acetate. The DNA pellet was
resuspended in sterilized deionized-water and total genomic DNA was kept at -20°C before use(Sambrook and Russell, 2001).
Amplified rDNA Restriction Analysis (ARDRA)
The selected promising plant growth promoting traits were continued analyzed for biodiversity
analysis using ARDRA. The 16S rDNA universal primers fD1 (5’-AGA GTT TGA TCC TGG
CTC AG-3’) and rP2 (5’-AAG GAG GTG ATC CAG CC-3’) (Weisburg et al. 1991) were used toamplify a 1.5-kb internal region of the 16S rRNA gene. An initial denaturation at 95°C for 5 min
was followed by 35 cycles with denaturation at of 95°C (30 s), annealing at 58°C (1 min) andextension at 72°C (2 min), and a final extension at 72°C for 7min. Restriction analysis was
performed with 5 µl of amplified product and 10 µl of restriction buffer containing 2 U of either the
restriction enzymes AluI. After a 4 h digestion at the appropriate temperature, the enzyme wasinactivated by heating the preparations at 65°C for 20 min. For each isolate, PCR amplification andrestriction analysis were performed at least three times. Calculation of the pair-wise coefficients ofsimilarity was based on the presence or absence of bands. A cluster analysis with the UPGMA
algorithm was performed with the NTSYS-pc numerical taxonomy and multivariate analysis
system. Then, Representatives of each group were selected for cloning and partial 16S rRNA genesequencing to retrieve sequence similarity and bacterial identity from sequence databases.
Diversity Index Analysis
Based on these clusters, PGPB community richness and diversity index (Shannon) was calculated.
The formula is as follows:Ⓒ ISERD

59

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

where H’ is the species diversity index, s is the number of species, and pi is the proportion ofindividuals of each species belonging to the species of the total number of individuals (Nolan andCallahan, 2006).
RESULTS AND DISCUSSIONPGPB Richness
The PGPB diversity was estimated by Shannon’s index. Shannon value can reflect the species
diversity of the community, affected by both species richness and species evenness, that is the twovalues also consider the abundance of each species. The greatest PGPB richness was observed in
GN (0.14) and TM (0.14) at 26 weeks after residue application (Table 1). At 52 weeks after residue
application, PGPB richness of tamarind treatment was higher than groundnut treatment. This study
indicated that application of legume residue with allelochemicals like tamarind induced PGPB
richness overtime. In a relatively study showed that a large number of bacteria and fungi have theirability to degrade allelochemicals (Zhang et al., 2010; Chen et al., 2011). These microorganisms
could mineralize the allelochemicals as its sole source of carbon and energy. This could be certified
to the fact that the PGPB richness can increase eventually.
Table 1 PGPB richness from the three treatments
Treatments*
C
GN
TM

Shannon’s index (weeks after residue application)
0
26
52
0.02
0.10
0.07
0.02
0.14
0.10
0.02
0.14
0.12

*Treatments (C; no long-term application of organic residue in tropical sandy soil, GN; long-term applicationof groundnut strover in tropical sandy soil, TM; long-term application of tamarind in tropical sandy soil)

ARDRA Analysis and 16S rRNA Gene Partial Sequencing on PGPB Isolates
The cluster dendrogram of ARDRA analysis of PGPB isolates obtained from soil samples of 17
years long-term field experiment is illustrated in Fig. 1. Digestion of amplified 16S rDNA with
AluI revealed seven main clusters of ARDRA dendrogram. Cluster I, II, III and V of ARDRA
dendrogram showed representative of genera/species from all treatment. Cluster IV contained
representative of PGPB genera/species only from groundnut treatment while, cluster VI and VII
showed representative of PGPB genera/species only from tamarind treatment. Remarkably, Cluster
I, IV and V of ARDRA dendrogram showed representative of genera/species from all weeks after
residue application. No representative of PGPB genera/species isolated at 0 week after residue
application was observed in cluster II, III, VI and VII of ARDRA dendrogram. In addition, no
representative of PGPB genera/species isolated from 0 and 52 weeks after residue application wasobserved in cluster VI and VII of ARDRA dendrogram.
On the basis of the 16S rRNA gene sequence analysis, in excess of 1 kb fragments weresequenced for most of isolates, with similarities ranging between 88 and 99%. Thirteen isolates
were identified as Bacillus spp., Staphylococcus spp., Brevibacterium spp., Ralstonia spp.,
Pseudomonas spp., Brevundimonas spp., Sphingomonas spp., Stenotrophomonas spp.,
Paenibacillus sp., Burkholderia sp., Acinetobacter sp. and Lysinibacillus sp.
Further, Bacillus spp., Staphylococcus spp. and Brevibacterium spp. were observed in alltreatments. The PGPB in the genus of Brevundimonas was only found in groundnut treatment. In
addition, the Sphingomonas spp. was only found in tamarind treatment.
The previous study reported that long-term application of organic residues strongly affectedbacterial and fungal community structure and diversity (Lawongsa et al., 2016b; Kamolmanit et al.,2013). This study suggested that not only application of legume residue with or without
allelochemicals can alter bacterial and fungal diversity, but also PGPB diversity.
Ⓒ ISERD

60

IJERD – International Journal of Environmental and Rural Development (2018) 9-1* The different isolates were designated T followed by the weeks after residue application (0, 26, or 52), treatment name(G;groundnut or T; tamarind) and by progressive numbers of PGPB isolation.

Fig. 1 Dendrogram of PGPB isolates representing each ARDRA group
CONCLUSION
In this study, thirteen bacterial strains isolated from 17 years long-term field experiment continuous
application of different quality residues, groundnut stover (Arachis hypogaea) (high quality organic
residue) and tamarind (Tamarindus indica L.) (intermediate quality organic residue), compare tocontrol (no organic residue applied) were characterized. Most of the bacteria were member in thegenera of Bacillus spp., Staphylococcus spp., Brevibacterium spp., Ralstonia spp., Pseudomonas
spp., Brevundimonas spp., Sphingomonas spp., Stenotrophomonas spp., Paenibacillus sp.,
Burkholderia sp., Acinetobacter sp. and Lysinibacillus sp. The bacterial communities were
dominated by Bacillus spp. (42.86%), Staphylococcus spp. (11.90%) and Brevibacterium spp.
(9.52%). Interestingly, the richness of PGPB strains showed no different between groundnut
treatment and tamarind treatment at 0 and 26 weeks after residue application. However, therichness of PGPB strains of tamarind residue had increased at 52 weeks after residue application.
Notably, only the Brevundimonas spp. was only found in groundnut treatment. In addition, theSphingomonas spp. was only found in tamarind treatment. These findings suggested thatapplication of legume residue with allelochemicals like tamarind can alter PGPB richness anddiversity. The direction of the future research should thus consider, apart from studying PGPB, also
decomposing fungi and archaea in long term field experiment. This may include phylogenetic
studies and functional genes to identify microbial community directly involved in the degradationof organic materials in soils.
ACKNOWLEDGEMENT
This work was supported by a grant from Agricultural Biotechnology Research Center for
Sustainable Economy and Soil Organic Matter Management Research Group, Khon Kaen
University.

Ⓒ ISERD

61

IJERD – International Journal of Environmental and Rural Development (2018) 9-1

REFERENCES
Aciego Pietri, J.C. and Brookes, P.C. 2009. Substrate inputs and pH as factors controlling microbial biomass,
activity and community structure in an arable soil. Soil Biol. and Biochem., 41, 1396-1405.
Ahemad, M. and Khan, M.S. 2012. Productivity of green gram in tebuconazole-stressed soil by using atolerant and plant growthpromoting Bradyrhizobium sp. MRM6 strain. Acta Physiol. Plant., 34, 245-254.
Bhadoriya, S.S., Ganeshpurkar, A., Narwaria, J., Rai, G. and Jain, A.P. 2011. Tamarindus indica, Extent of
explored potential. Pharmacogn Rev., 9, 73-81.
Bloemberg, G.V. and Lugtenberg, B.J.J. 2001. Molecular basis of plant growth promotion and biocontrol byrhizobacteria. Curr. Opin. Biotechnol., 4, 343-350.
Chen, L., Yang, X., Raza, W., Li, J., Liu, Y., Qiu, M., Zhang, F. and Shen, Q. 2011. Trichoderma harzianum
SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers. Appl.
Microbiol. Biotechnol., 89, 1653-1663.
Duffy, B. and Défago, G. 1999. Environmental factors modulating antibiotic and siderophore biosynthesis by
Pseudomonas fluorescens biocontrol strains. Appl. Environ. Microbiol., 65, 2429-2438.
Kamolmanit, B., Vityakon, P., Kaewpradit, W., Cadisch, G. and Rasche, F. 2013. Soil fungal communities
and enzyme activities in a sandy, highly weathered tropical soil treated with biochemically contrastingorganic inputs. Biol. Fertil. Soils, 49, 905-917.
Lawongsa, P., Rungthong, R., Kamolmanit, B., Saenjan, P. and Vityakon, P. 2016a. Responses of soil
bacterial population to the appearance of allelopathic substances. Khon Kaen Agri. J., 44, Supplement 1,
984-990.
Lawongsa, P. and Vityakon, P. 2016b. Diversity and structure of soil bacterial communities under long-term
application of different quality of organic residues in tropical sandy soil. The 4th International
Symposium on Biological Engineering and Natural Sciences. July, 19-21. Sapporo, Japan. 941-952.
Lewis, Y.S. and Neelakantan, S. 1964. The chemistry, biochemistry and technology of tamarind. J. Sci. Ind.
Res., 23, 150-204.
Marschner, P., Kandeler, E. and Marschner, B. 2003. Structure and function of the soil microbial community
in a long-term fertilizer experiment. Soil Biol. Biochem., 35, 453-461.
Nolan, K.A. and Callahan, J.E. 2006. Beachcomber biology, The Shannon-Weiner species diversity index. In
Proceeding Tested Studies for Laboratory Teaching, Vol. 27, 27th Workshop/Conference of the
Association for Biology Laboratory Education (ABLE), 334-338, Virginia 21-25 June, USA.
Rodriguez, H. and Fraga, R. 1999. Phosphate solubilizing bacteria and their role in plant growth promotion.
Biotech. Adv., 17, 319-339.
Rungthong, R., Nittayachit, T., Pnomkhum, P., Vityakon, P., Saenjan, P. and Lawongsa, P. 2013. Screeningof bacterial isolates containing plant growth promoting traits in organic residue-treated soils. Khon Kaen
Agri. J., 41, Supplement 2, 113-120.
Sachdev, D.P., Chaudhari, H.G., Kasure, V.M., Dahavale, D.D. and Chopade, B.A. 2009. Isolation andcharacterization of indole acetic acid (IAA) producing Klebsiella pneumoniae strains from rhizosphere ofwheat (Triticum aestivum) and their effect on plant growth. Indian J. Exp. Biol., 47, 993-1000.
Sambrook, J. and Russell, D.W. 2001. Molecular cloning, A laboratory manual. Cold Spring Harbor
Laboratory, New York.
Somboon, S., Klinpratum, C., Tippawan, P., Sawatwor, K. and Lawongsa, P. 2017. The appearance ofallelopathic substances affecting the presence of Burkholderia pseudomallei. Khon Kaen Agri. J., 45,
Supplement 1, 1307-1314.
USDA. 2014. Keys to soil taxonomy, USDA-Natural Resources Conservation Service. Washington, USA.
Vessey, J.K. and Buss, T.J. 2002. Bacillus cereus UW85 inoculation effects on growth, nodulation, and N
accumulation in grain legumes. Controlled-Environment Studies. Can. J. Plant Sci., 82, 282-290.
Weisburg W.G., Barns S.M., Pelletier D.A. and Land D.J. 1991. 16S ribosomal DNA amplification forphylogenetic study. J. Bacteriol., 173, 697-703.
Zhang, Z.Y., Pan, L.P. and Li, H.H. 2010. Isolation, identification and characterization of soil microbes
which degrade phenolic allelochemicals. J. Appl. Microbiol., 108, 1839-1849.

Ⓒ ISERD

62