Agriculture and Natural Resources 52 (2018) 309e316

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Agriculture and Natural Resources

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Original Article

Dynamics and diversity of cultivable rhizospheric and endophytic

bacteria during the growth stages of cilembu sweet potato
(Ipomoea batatas L. var. cilembu)
Agustina Monalisa Tangapo,a, b, 1 Dea Indriani Astuti,a, 1 Pingkan Aditiawatia, *
a
Microbial Biotechnology Research Group, Bandung Institute of Technology, Bandung, Indonesia
b
Department of Biology, Faculty of Mathematic and Natural Sciences, Sam Ratulangi University, Manado, Indonesia

a r t i c l e i n f o a b s t r a c t

Article history: Cultivable rhizospheric and endophytic bacteria were isolated from cilembu sweet potato during the 5 mth
Received 13 February 2018 period post planting to assess the diversity and dynamics of its bacterial community. The number of colony
Accepted 15 May 2018 forming units of rhizospheric bacteria was significantly higher than for the endophytic bacteria. The
Available online 18 October 2018
diversity and genera richness of the bacteria associated with cilembu sweet potato in the early stages of
growth were higher than in the last stages. The different cultivable bacteria were identified using 16S rRNA
Keywords:
gene sequencing as: Alphaproteobacteria (Methylobacterium, Sphingomonas, Paracoccus), Gammaproteo-
Cilembu sweet potato
bacteria (Klebsiella, Enterobacter, Pseudomonas, Serratia), Bacteroidetes (Chryseobacterium, Sphingobacte-
Endophyte
Ipomoea batatas
rium), Firmicutes (Exiguobacterium, Bacillus, Staphylococcus) and Actinobacteria (Streptomyces, Arthrobacter,
Plant growth-promoting (PGP) Kocuria, Microbacterium, Micrococcus). The nitrogen content in the soil may significantly affect the change of
Rhizosphere bacterial diversity in the rhizosphere during the growth of cilembu sweet potato. All isolates were capable of
producing plant growth-promoting traits, alone or in combination, such as indole acetic acid production,
phosphate solubilization, ammonia production, nitrogen fixation, cellulolytic and amylolytic activity.
Copyright © 2018, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction areas with similar environmental conditions has become an impor-

tant focus of research. Cilembu soil with its physico-chemical char-
Sweet potato (Ipomoea batatas L. Lam) has been cultivated in acteristics, organic and inorganic nutrients provides a unique habitat
many countries around the world and is used as staple food, as well as for bacteria (Subroto, 2010; Nedunchezhiyan et al., 2012). Under-
a raw material for animal feed and alcohol production, so that it ranks standing plant-associated bacteria is very important because they
sixth as the most important food crop after rice, wheat, potatoes, play a crucial ecological role in nutrients recycling and plant growth
maize and cassava (Food and Agriculture Organization, 2002). One of promotion (Berg and Smalla, 2009). Marasco et al. (2013) reported
the most popular sweet potato varieties in Indonesia is cilembu sweet the plant growth-promoting (PGP) potential of grapevine-associated
potato (I. batatas L. var. cilembu) from Cilembu village, West Java bacteria under three different agroclimatic conditions and the ma-
province. Cilembu sweet potato, also known as “Ubi Madu Cilembu” jority of isolates showed multiple PGP activities, which promoted
(cilembu honey sweet potato), has superiority in terms of taste, as it is plant growth directly, indirectly or synergistically.
sweeter than other sweet potatoes (Arifin, 2002; Solihin et al., 2016). The association between bacteria and roots is in the soil,
The high demand for cilembu sweet potato as an export has pushed rhizosphere and endorhiza. The rhizosphere is defined as the zone
the increase in its production, which has become difficult due to land of soil that is affected by exudates produced by living plant roots,
space limitations (Solihin et al., 2017). Several studies have shown the while the soil habitat is not directly influenced by exudates sup-
trademark cilembu sweet potato flavor and sweetness are hardly plied by plant roots (Mahaffee and Kloepper, 1997). Endophytes are
produced outside Cilembu village (Arifin, 2002; Solihin et al., 2018). defined as “all microorganisms which for all or part of their lifetime
Consequently, enhancing the growth of this commodity in other colonize internal plant tissues” (Hardoim et al., 2015). Some rhi-
zospheric bacteria can penetrate and colonize within root tissues
* Corresponding author.
as endophytic bacteria. These bacteria have the potential to
E-mail address: [email protected] (P. Aditiawati). maintain the biotic diversity in plant-associated bio-communities
1
Equal contribution. (Lodewycks et al., 2002; Hardoim et al., 2015).

https://doi.org/10.1016/j.anres.2018.10.003
2452-316X/Copyright © 2018, Kasetsart University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
creativecommons.org/licenses/by-nc-nd/4.0/).
310 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316

The study of the role of bacterial diversity in species variety and phosphate as inorganic P. For ammonia production, bacterial iso-
abundance is important for sustainable development (Singh and lates were grown in peptone water (peptone 5 g/L) and incubated
Varaprasad, 2008). However, there is no information on the dy- for 48 hr at room temperature, and then 0.5 mL Nessler's reagent
namics and diversity of the rhizospheric and endophytic bacteria of (Iwata et al., 2012) was added and development color indicated
cilembu sweet potato or the functions of these bacteria. Although NH3 production (Cappucino and Sherman, 1992). For nitrogen fix-
the number of studies on the productivity of cilembu sweet potato ation, nitrogen free Malate media containing bromothymol blue as
has increased, it is not easy to collect information about the bacterial an indicator was used (Gothwal et al., 2008). The amylolytic activity
communities associated with cilembu sweet potato. The objective of was determined using the method of Hankin and Anagnostakis
the current study was to isolate and characterize the bacteria in the (1975) in NA medium containing 1% soluble starch and after incu-
rhizosphere and inside the roots (endophyte) of cilembu sweet po- bation, 5 mL of 1% iodine solution was added to the plates; a clear
tato during its cultivation process, focusing on the cultivable bacteria zone around the colonies indicated amylase production. For cellu-
isolates and analyzing their abundance and diversity. The study also lolytic activity, the bacterial colonies were grown on carboxy-
aimed to analyze the relation between bacterial diversity and soil methylcellulose agar medium and incubated for 2 d. The plates
parameters, as well as examining the functional properties of the were flooded with Congo red solution (1 mg/mL) for 15 min. The
bacteria. All this information will be beneficial in identifying the role plates were destained using 1 M NaCl for 15 min and a clear zone of
of bacteria in the quality establishment of cilembu sweet potato. hydrolysis indicated cellulose degradation (Gupta et al., 2012).

Materials and methods Molecular identification

Sampling of plants and rhizosphere soil and isolation of bacteria For polymerase chain reaction (PCR) amplification, a loopful from
a bacterial colony was suspended in 30 mL of TE buffer (100 mM Tris
Plant and rhizosphere soil samples were collected from a sweet HCl, 10 mM ethylenediaminetetraacetic acid, pH 8.0), and heated in
potato var. cilembu cultivation site at Cilembu (geographical co- a boiling water bath for 5 min. One mL of cell extract was used on a
ordinates: 6
54‘24.500 S, 107
50‘46.300 E), Pamulihan, Sumedang, PCR template to amplify the 16S rRNA gene (Arturo et al., 1995) in a
West Java, Indonesia. The rhizospheric soil sampling was carried out 50 mL reaction volume with Kapa Taq Extra HotStart ReadyMix kit
each month for 5 mth after planting (R1-R5). At each sampling time, (Kapa Biosystem; Wilmington, MA, USA). The universal bacterial
five plants were randomly harvested and the soil loosely adhering to primers 27F (5‘- AGAGTTTGATC [A/C]TGGCTCAG-3‘) and 1492R (5‘-G
the roots was shaken, the resulting roots were pooled and then, the [C/T]TACCTTGTTACGACTT-3‘) were used in the reaction. PCR am-
rhizosphere soil was collected by brushing off the roots or tuberous plifications were performed using an T100 Thermal Cycler machine
roots per plant followed by homogenization by sieving (Marquez (Bio-Rad; Hercules, CA, USA) with the following program: 5 min for
et al., 2014). In addition, a soil sample was taken before planting initial denaturation at 95
C and then 35 cycles of 30 s min at 96
C,
(S0). The sample of soil (10 g) was suspended in 90 mL of sterile 30 s at 65
C, and 1 min at 72
C and finally 10 min at 72
C. The
phosphate-buffered saline and agitated at 150 revolutions per amplified genes were subjected to electrophoresis using 1.2%
minute for about 30 min, then serially diluted and spread on agarose gel with a size marker (1 kb DNA ladder).
nutrient agar (NA). The plates were incubated at room temperature The PCR products were sent to Macrogen Inc. (Seoul, Korea) for
for 24e72 hr in triplicate (Kumar et al., 2012; Marquez et al., 2014). nearly full-length 16S rRNA (~1500 bp) sequencing. The basic local
The total numbers of cultivable bacteria were determined as colony alignment tool (BLAST) at the National Center for Biotechnology
forming units (CFUs) on nutrient agar plates using the dilution plate Information (http://www.ncbi.nlm.nih.gov/) was used as to find
method with three replications each time. The cilembu sweet potato similar known sequences.
roots/tuberous roots were used for the isolation of endophytic
bacteria (EG1-EG5). The roots were first surface sterilized to remove Statistical analysis
bacteria from the surface. The roots were washed with tap water
and sterile distilled water followed by washing in 70% ethanol. The All calculation of diversity indices were performed using the
roots were then washed with sterile distilled water and incubated computer program PAST (Paleontological Statistics), version 3.14
for 10 min with 5% NaOCl. To confirm that the sterilization process (Hammer et al., 2001). The PAST software was adopted to measure the
0 P
was successful, the samples were plated on NA and incubated for Shannon-Wiener (H0 ) (formula: H ¼ pi ln pi) where H0 the di-
24 hr at 28
C. The samples without contamination were used for versity index and pi is the proportion of species i to the total number of
isolation of the endophytic bacteria. For isolation, the samples were species. Species richness (S) is the total number of different species in
homogenized under aseptic conditions, then serially diluted and each sample. The evenness index (E) provides information about the
spread on NA (Del Giudice et al., 2008; Kumar et al., 2012). The distribution of individual over species (Heip et al., 1998).
numbers of colonies obtained on all agar plates were counted. In- All enumerations of total numbers of CFU were analyzed using
dividual bacterial colonies were purified by sub-culturing on fresh Annova 5% and then followed with Tukey's Method. The correlation
media and preserved on basal media containing glycerol at 20
C. between the soil properties and microbial diversity was deter-
mined using principal component analysis (PCA) using the corre-
Functional characterization of bacterial isolates lation matrix to calculate eigen values and eigenvectors (using the
PAST program).
Plant growth-promoting activities were also screened, consist-
ing of: IAA (indole acetic acid) production, phosphate (P) solubili- Results and discussion
zation, ammonia productions and nitrogen fixation. The ability of
isolates to produce IAA was evaluated in nutrient broth (NB) culture Isolation of bacteria during the growth of cilembu sweet potato
(Atlas, 2010), the colorimetric method of Gordon and Weber (1951)
was used. The IAA production was observed as development of a The bacterial abundance of rhizospheric bacteria during the
pink-red color and the optical density was recorded at 530 nm. The growth of cilembu sweet potato substantially increased during the
mineral P-solubilizing ability of the isolates was determined on first 2 mth of growth. The abundance peaked in the second month
Pikovskaya's medium (Pikovskaya, 1984) amended with tricalcium of the growth phase (R2) with 13.07  106 CFU/g (Fig. 1). After the
 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316 311

Fig. 1. Changes in total abundance during growth stages of cilembu sweet potato of: (A) rhizospheric bacteria isolates; (B) endophytic bacteria isolates.

second month, this value continued to fall to 6.60  106 CFU/g in communities represent a subgroup of the rhizobacterial commu-
the third month (R3) and then to 5.80  106 CFU/g in the fourth nities, which has the ability to enter the root interior of the plant
month (R4). The bacterial density and number of isolates of rhi- and thus, there is a selection of the bacteria in the rhizosphere that
zospheric bacteria (R1-R5) were higher than in non-rhizospheric colonize the plant (Sessitsch et al., 2002).
bacteria (soil before planting; S0). In total, 40 morphologically
different bacteria were isolated from the rhizosphere of different- Diversity of bacteria during the growth of cilembu sweet potato
aged sweet potato variety cilembu. The highest species richness
was in the first month of growth (24 of 40 isolates), while the The diversity of isolates was calculated for each month based on
lowest number was in the soil before planting (S0) with nine iso- several indices. The Shannon index (H0 ) accounts for both the
lates. In the rhizosphere, the bacteria abundance in the early stages abundance and evenness, and may explain the diversity of bacteria
of growth was significantly higher than for the last stages. Plant (Stirling and Wilsey, 2001). H0 analysis indicated that the diversity
growth and development stages influence the abundance and of the bacteria in the rhizosphere was highest in the third month
diversity of rhizospheric bacteria and it has been reported that the sample (R3, H’ ¼ 2.83) and lowest in the fifth month sample (R5,
composition of root exudates changes according to plant growth H’ ¼ 2.06), whereas for the endophytic bacteria it was highest in the
and development stage (Chaparro et al., 2013). The concentrations first month sample (H’ ¼ 2.34) and lowest in the samples in the
of sugar and sugar alcohols in the root exudates were higher earlier third and fourth months (EG3 and EG4, H’ ¼ 2.01). These values
on and decreased with plant growth, while amino acids and phe- were higher than the H0 value from the soil before planting (S0,
nolics increased over time. Based on this information, it was H0 ¼ 1.68) as shown in Table 1. This result showed that there was a
assumed that in early of growth stages when roots were growing significant increase in bacterial diversity during the growth period
quickly, sugar was released as substrate for a diversity range of of cilembu sweet potato, which peaked in the third month of
bacteria in their early stages of development (Badri et al., 2013; growth for the rhizosphere and in the first month for endophytes.
Chaparro et al., 2013). Moreover, the range in the evenness index for the rhizosphere was
In the endophytes, a significantly higher abundance was also 0.85e0.98 and for the endophytes was 0.83e0.93, where a higher
observed in the mid-growth stages from the root/tuberous root value reflects a uniform distribution of the individuals in each
samples compared to the last 2 mth. The total numbers of bacteria species (Table 1). There was no dominant species in any sample.
from the root/tuberous root of cilembu sweet potato were between However, several isolates, (BR1, BR2, BR3, BR12 for rhizospheric
4.05  103 and 11.9  103 CFU/g of wet sample and reached the bacteria and BE6, BE8, BE11, BE12, BE13 (1), BE13 (3) for endophytic
highest abundance in the third month of growth (Fig. 1). The bacteria0 were always detected each sampling time (Fig. 1).
decrease in the bacterial population in the last stages could have Significant changes in bacterial diversity were found during the
been due to the unavailability of essential nutrients for bacteria growth of cilembu sweet potato perhaps as a result of many
during storage root growth (bulking) stages of cilembu sweet po- different factors such as plant species and cultivars, plant devel-
tato. Changes in plant physiology can lead to the development of a opmental stage, properties of soil and the season (Berg and Smalla,
distinct endophytic bacteria community (Hallman et al., 1997). The 2009). The presence of crops in the soil may change the soil
bacterial density of the rhizosphere was significantly greater than properties by producing plant exudates, and cause a rapid increase
of endophytes for all sampling times. Endophytic bacteria are in bacterial diversity and abundance in the rhizosphere as was seen
mostly derived from the rhizosphere as endophytic bacteria in the first month. The release of root exudates may become a
312 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316

Table 1
Diversity indices for bacterial communities from different ages of cilembu sweet potato.

Sample Age (mth) Species richness (S) Shannon index (H0 ) Evenness_index (E)

Soil before planting 0 (S0) 9 1.68 0.82

Rhizosphere 1 (R1) 24 2.61 0.85
2 (R2) 20 2.49 0.87
3 (R3) 23 2.83 0.97
4 (R4) 20 2.72 0.98
5 (R5) 11 2.06 0.92
Endophytes 1 (EG1) 15 2.34 0.87
2 (EG2) 12 2.08 0.83
3 (EG3) 12 2.01 0.86
4 (EG4) 10 2.01 0.91
5 (EG5) 11 2.20 0.93

source of carbon compounds for growth substrates or a signal for have been reported by Nasution et al. (2017). Soil pH in the rhizo-
root-associated microorganisms (Compant et al., 2010). Plant sphere soil was in the range 4.8e5.4 and was lower than in bulk soil
development stages may also influence the variety and amount of (5.9). The N values in the rhizosphere soil were in the range
exudates produced, which could serve as a selective pressure in the 0.16e0.29%.
rhizosphere. This includes the bacteria, which have an important Component 1 from the PCA explained 74.5% and component 2
role during plant growth. Cilembu sweet potato is known to start 12.3% of the total variation. The N content had a direct correlation
forming tubers during the second month of its growth (Van de with bacterial diversity (H0 ), while the C:N ratio had an inverse
Fliert and Braun, 1999), which may change the amount and type correlation (Fig. 2). These findings may suggest that the N content,
of exudates produced. This finding may explain the change in as a chemical property of soil, may significantly affect the change in
bacterial diversity and abundance in the second month, when the bacterial diversity in the rhizosphere during the growth of cilembu
species richness began to drop due to the selective pressure by the sweet potato. The lowest N content had the highest bacterial
produced exudates. As the number of species decreased, the diversity (sample R1). Based on Bobbink et al. (2010), N accumu-
abundance of the remaining bacteria increased, as was seen in the lation can cause a decline in microbial diversity by the expansion of
third month of growth. nitrophilous species and the competitive exclusion of others. The
current study also recorded a decrease in the soil pH with an in-
Soil characteristics: rhizospheric bacteria diversity relationship crease in N. The increase in N can induce soil acidification, exerting
deleterious effects on microbial growth (Wei et al., 2013). Some
The production of exudates by plants may cause some changes studies suggested that soil texture and pH are parameters that
in the chemical and physical properties of the soil including the pH, might affect the richness and diversity of bacterial communities in
total C, N, and available P and K contents (Nardi et al., 2000). These the soil (Sessitsch et al., 2002; Beneduzzi et al., 2013).
properties are known to affect the general composition of the
bacterial community. Changes in bacterial diversity and dynamics Molecular identification of bacterial isolates
in the rhizosphere during the development stages of plants are the
result of differences in the physical, chemical and biological prop- All isolates were characterized morphologically and identified
erties of the root-associated soil (Berg and Smalla, 2009). The PCA using PCR amplification with the 16S rRNA gene (Table 2). The
results showed the relationships between bacterial diversity (H0 ) identified bacterial genera were: Klebsiella, Enterobacter, Pseudo-
and soil parameters (Fig. 2). The soil parameters used in this study monas, Chryseobacterium, Sphingomonas, Micrococcus, Kocuria,

Fig. 2. Statistical correlation between soil composition (pH, C, N, P, K, C/N) and diversity index (H0 ) where nitrogen and H0 are located in the same quadrant, which indicates a
significant correlation.
 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316 313

Table 2
Identification of rhizospheric and endophytic bacteria of cilembu sweet potato using NCBI BLAST-N of 16S rRNA gene sequences.

Isolates Closest match/species identity Family Phylum

Rhizospheric bacteria
BR1 Klebsiella pneumoniae NR 041750 (99%) Enterobacteriaceae Proteobacteria
BR2 Enterobacter cloacae NR 118568 (99%) Enterobacteriaceae Proteobacteria
BR3 Pseudomonas nitroreducens NR 113601 (99%) Pseudomonadaceae Proteobacteria
BR4 Chryseobacterium indologenes NR112975 (98%) Flavobacteriaceae Bacteroidetes
BR5 Sphingomonas paucimobilis NR 114999 (99%) Sphingomonadaceae Proteobacteria
BR6 Micrococcus luteus NR 114673 (99%) Micrococcaceae Actinobacteria
BR7 Kocuria rhizophila NR 026452 (99%) Micrococcaceae Actinobacteria
BR8 Sphingomonas endophytica NR117869 (100%) Sphingomonadaceae Proteobacteria
BR9 Microbacterium laevaniformans NR 115540 (99%) Microbacteriaceae Actinobacteria
BR10 Exiguobacterium acetylicum NR 113585 (99%) Bacillaceae Firmicutes
BR11 Sphingobacterium multivorum NR 113076 (94%) Sphingobacteriaceae Bacteroidetes
BR12 Bacillus mycoides NR 036880 (99%) Bacillaceae Firmicutes
BR13A Bacillus mojavensis NR 024693 (99%) Bacillaceae Firmicutes
BR13B Bacillus pumilus NR 112637 (99%) Bacillaceae Firmicutes
BR14 Bacillus licheniformis NR 074923 (99%) Bacillaceae Firmicutes
BR15 Streptomyces albus NR 118467 (99%) Streptomycetaceae Actinobacteria
BR16 Streptomyces sp. NR 112305 (99%) Streptomycetaceae Actinobacteria
BR17 Streptomyces abikoensis NR 118286 (99%) Streptomycetaceae Actinobacteria
BR18 Streptomyces griseus NR 115669 (98%) Streptomycetaceae Actinobacteria
BR19P Methylobacterium platani NR 044211 (99%) Methylobacteriaceae Proteobacteria
BR19T Bacillus aquimaris NR 025241 (95%) Bacillaceae Firmicutes
BR20 Bacillus simplex NR 115603 (99%) Bacillaceae Firmicutes
BR21 Bacillus megaterium NR 117473 (97%) Bacillaceae Firmicutes
BR22 Bacillus aryabhattai NR 115953 (100%) Bacillaceae Firmicutes
BR23 Bacillus badius NR 112633 (98%) Bacillaceae Firmicutes
BR24A Bacillus subtilis NR 116017 (100%) Bacillaceae Firmicutes
BR24B Bacillus cereus NR 074540 (99%) Bacillaceae Firmicutes
BR24C Bacillus flexus NR 113800 (94%) Bacillaceae Firmicutes
BR25 Bacillus sp. MF 948374 (99%) Bacillaceae Firmicutes
BR26 Bacillus acidiceler NR 043774 (99%) Bacillaceae Firmicutes
BR27 Streptomyces sp.S000651623 (80%) Streptomycetaceae Actinobacteria
BR6B Arthrobacter ureafaciens NR 029281 (97%) Micrococcaceae Actinobacteria
BR29 Serratia marcencens NR 114043 (99%) Enterobacteriaceae Proteobacteria
BR30 Paracoccus sp. NR 149253 (97%) Rhodobacteraceae Proteobacteria
BR31 Bacillus sp. NR 113945 (95%) Bacillaceae Firmicutes
BR32 Staphylococcus saprophyticus NR 074999 (99%) Staphylococcaceae Firmicutes
BR33 Unidentified
BR34 Bacilllus amyloliquefaciens NR 041455 (99%) Bacillaceae Firmicutes
BR37 Bacillus methylotrophicus NR 116240 (99%) Bacillaceae Firmicutes
BR38 Bacillus luciferensis NR 025511 (95%) Bacillaceae Firmicutes
Endophytic bacteria
BE1 Bacillus mycoides NR 113990 (99%) Bacillaceae Firmicutes
BE2 Klebsiella pneumoniae NR 041750 (99%) Enterobacteriaceae Proteobacteria
BE3 Unidentified
BE4 Unidentified
BE5 Arthrobacter ureafaciens KT 380556 (99%) Micrococcaceae Actinobacteria
BE6 Pantoea NR 111998 (94%) Enterobacteriaceae Proteobacteria
BE7 Streptomyces abikoensisNR 118286 (99%) Streptomycetaceae Actinobacteria
BE8 Enterobacter cloacae NR 118568 (98%) Enterobacteriaceae Proteobacteria
BE9 Microbacterium laevaniformans NR 115540 (99%) Microbacteriaceae Actinobacteria
BE10 Paracoccus communis NR 133809 (97%) Rhodobacteraceae Proteobacteria
BE11 Unidentified
BE12 Bacillus subtilis NR 113265 (99%) Bacillaceae Firmicutes
BE13-1 Bacillus mojavensis NR 112725 (99%) Bacillaceae Firmicutes
BE13-3 Bacillus badius NR 112633 (98%) Bacillaceae Firmicutes
BE14 Bacillus megaterium NR 117473 (99%) Bacillaceae Firmicutes
BE15A Bacillus licheniformis NR 074923 (99%) Bacillaceae Firmicutes
BE15B Bacilllus amyloliquefaciens NR 041455 (99%) Bacillaceae Firmicutes
BE16 Bacillus safensis NR 113945 (95%) Bacillaceae Firmicutes
BE17 Sphingobacterium multivorum NR 113706 (94%) Sphingobacteriaceae Bacteroidetes
BE18 Staphylococcus haemolyticus NR 113345 (100%) Staphylococcaceae Firmicutes
BE19 Pseudomonas putida NR 113651 (98%) Pseudomonadaceae Proteobacteria
BE20 Bacillus pumilus NR 112637 (99%) Bacillaceae Firmicutes

Microbacterium, Sphinghobacterium, Exiguobacterium, Bacillus, Strep- Methylobacteriaceae (2.5%), Sphingobacteriaceae (2.5%), Rhodo-
tomyces, Paracoccus, Staphylococcus, Methylobacterium, Arthrobacter bacteraceae (2.5%) and Staphylococcaceae (2.5%). Furthermore, all
and Serratia. The results showed that all the classified isolates the classified isolates of endophytic bacteria belonged to nine fam-
of rhizospheric bacteria belonged to 12 families: the Bacillaceae ilies: the Bacillaceae (40.9%), Enterobacteriaceae (13.6%), Micro-
(47.5%), Streptomycetaceae (12.5%), Enterobacteriaceae (7.5%), coccaceae (4.5%), Streptomycetaceae (4.5%), Microbacteriaceae
Micrococcaceae (7.5%), Sphingomonadaceae (5%), Pseudomonada- (4.5%), Rhodobacteraceae (4.5%), Sphingobacteriaceae (4.5%), Staph-
ceae (2.5%), Flavobacteriaceae (2.5%), Microbacteriaceae (2.5%), ylococcaceae (4.5%) and Pseudomonadaceae (4.5%). All the isolates
314 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316

Fig. 3. Relative abundance each month during growth stages of: (A) rhizospheric bacteria; (B) endophytic bacteria.

could be attributed to four phyla (Proteobacteria, Bacteroidetes, sampling times. The analysis showed that isolate BR1 had the
Actinobacteria and Firmicutes), which have been known to be plant- highest sequence similarity to Klebsiella pneumonia subsp. ozaenae,
associated (Reinhold-Hurek et al., 2015). Molecular identification while isolate BR2 was similar to Enterobacter cloacae (Table 2). A
using 16S rRNA gene sequencing showed Bacillus was the dominant previous study showed that the Klebsiella strain was present in the
genus of rhizospheric and endophytic bacteria. This was consistent rhizosphere and showed PGP traits (Sachdev et al., 2009). Entero-
with the study in sweet potato rhizosphere by Marquez et al. (2014), bacter spp. were also found in the rhizosphere of sugarcane
where the genus Bacillus was enriched in the tuber rhizosphere (Mirza et al., 2001). The presence of Enterobacter was also detected
samples of all sweet potato genotypes studied, while other genera as endophytic bacteria (BE8).
showed a plant genotype-dependent abundance.
Species richness and estimation of the diversity of rhizospheric Functional characterization of rhizospheric and endophytic bacteria
bacteria provide information on their composition and roles in the
environment. The present study found that the presence of the All the rhizospheric and endophytic bacteria of the cilembu
Enterobacteriaceae, Pseudomonadaceae, Bacillaceae and Strepto- sweet potato were further screened for the presence of PGP traits in
mycetaceae was not influenced by plant age, as they were detected order to understand their role in their habitat. The PGP traits
on all sampling occasions. In contrast, the Methylobacteriaceae evaluated in this study were: IAA production, phosphate solubili-
were only present in the first month sample (R1) as shown in Fig. 3. zation, ammonia production, nitrogen fixation, cellulolytic and
The isolates BR1 and BR2 were the most abundant isolates in the amylolytic activity. The results showed that more than 95% of the
majority of the rhizosphere soil samples, and were also found at all rhizospheric and endophytic bacteria possessed more than one PGP

Fig. 4. Plant growth-promoting (PGP) potential of rhizospheric and endophytic bacteria of cilembu sweet potato: (A) percentage of isolates showing multiple PGP traits. Percentage
of isolates showing PGP traits during growth stages for IAA production (IAA), phosphate solubilization (PS), ammonia production (Amn), nitrogen fixation (NF), cellulolytic (Cel) and
amylolytic (Amy) activity of: (B) rhizospheric bacteria (R); (C) endophytic (EG) bacteria.
 A.M. Tangapo et al. / Agriculture and Natural Resources 52 (2018) 309e316 315

trait, which may promote plant growth synergistically. None of the Acknowledgements
rhizospheric bacteria and only 4.5% of the endophytic bacteria
showed only one or no activity, while about 86% of isolates from Authors thank the Institut Technologi Bandung (ITB) and the
both location showed more than two PGP traits (Fig. 4A). Only Indonesian Ministry of Higher Education and Research (Kemen-
12.5% (5 out of 40) of the rhizospheric bacteria were positive for IAA ristekdikti) for support and providing facilities.
production, phosphate solubilization, ammonia production, nitro-
gen fixation and cellulolytic and amylolytic activity (six PGP traits)
while 22.7% of the endophytic bacteria were positive for six PGP References
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