ENDOPHYTES RHIZOBIA BACTERIA, ISOLATED FROM ACACIA MANGIUM, ITS
POTENSIAL TO SUPPORT THE GROWTH OF SOYBEAN.
Harmastini Sukiman
Research Center for Biotechnology, Indonesian Institute of Sciences
Jl. Raya Bogor KM 46 Cibinong, Bogor.
Telp: 021-8754587 Fax: 021-8754588
[email protected]
ABSTRACT
Acacia mangium is a legume tree species which is commonly used for the re-greening programme of
critical land. This tree can easily grow in many different conditions and adjustable to the dry condition. The
wood of acacia has economic value and most popular to be used for pulp industries. Isolation of endophytes
rhizobia bacteria has been successfully done from Acacia mangium, and based on 16SrRNA analysis, it was
identified that the bacteria belong to the Rhizobia tropici. The potential of the endophytes bacteria has been
studied on its ability to support the growth of soybean. Green house experiment showed that single inoculation
of endophtyes bacteria and rhizobia isolated from soybean alone gave better growth in term of plant height,
upper plant biomass and production of seed compared to control plant and chemical fertilizer treatment. Double
inoculation of bacteria did not give significant result of seed production. The ability of endophyte rhizobia to
face the stress is important for the application. The result of stress test showed that the bacteria could bebetter
adapted to acidity and salinity including to UV exposure and cold condition. However theendophytecould not
survive to the high temperature and oxidative condition. Aim of this research wasto study the possibility of
cross inoculation of rhizobia endophytes bacteria isolated from Acacia mangium to soybean plant and to find
the best combination of inoculants for development the quality of biofertilizer.
Keyword : endophytes, rhizobia, inoculants , acacia.
ISOLASI BAKTERI ENDOFITIK RHIZOBIA DARI ACACIA MANGIUM ,
POTENSINYA DALAM MENUNJANG PERTUMBUHAN KEDELAI
ABSTRAK
Acacia mangium adalah tanaman dari family Leguminosae yang banyak digunakan dalam programme
penghijauan lahan kritis. Tanaman ini merupakan tanaman yang dapat tumbuh di berbagai kondisi alam dan
mampu beradaptasi dengan kondisi tanah yang kering. Kayu akasia mempunyai nilai ekonomi tinggi dan
sangat popular untuk industri kayu lapis ( pulp). Isolasi bakteri penambat nitrogen rhizobia telah berhasil
dilakukan dari Acacia mangium dan berdasarkan analisis 16SrRNA diketahui bahwa salah satu isolat
berpotensi tersebut adalah Rhizobia tropici. Potensi dari bakteri endofitik tersebut telah diteliti khususnya
dalam kemampuannya menunjang pertumbuhan kedelai. Percobaan rumah kaca menunjukkan bahwa inokulasi
tunggal dari bakteri endofitik rhizobia dan rhizobia asal kedelai memberikan hasil yang lebih baik pada tinggi
tanaman, biomasa tanaman bagian atas dan juga produksi biji kedelai dibandingkan dengan tanaman kontrol
mautun tanaman yang diberi pupuk kimia. Inokulasi ganda antara rhizobia dan bakteri endofitik tidak
memberikan hasil yang signifikan pada produksi biji kedelai.Kemampuan bakteri endofitik dalam menghadapi
cekaman lingkungan merupakan hal yang penting untuk kesuksesan aplikasinya di tanaman. Bakteri endofitik
rhizobia menunjukkan kemampuan dalam beradaptasi pada kondisi cekaman asam, salinitas tinggi, penyinaran
sinar UV dan suhu rendah namun bakteri tersebut tidak tahan terhadap cekaman suhu tinggi dan kondisi
oksidatif.Tujuan dari penelitian ini mempelajari kemungkinan dilakukannya inokulasi silang antara rhizobia
asal tanaman tinggi, Acacia mangium , kepada tanaman pangan kedelai dan didapatnya kombinasi komposisi
antar mikroba berpotensi yang terbaik sebagai pengembangan kualitas inokulum.
Kata kunci : endofitik, rhizobia, inokulan dan akasia
INTRODUCTION
Acacia mangium is a species of leguminous plant tree which belongs to the family of
Fabaceae. This is a native trees species from Northern Eastern of Queensland in Australia.
Acacia mangium is a fast growing tree that produces lots of seeds and commonly used for
forestry and ecological restoration. In Indonesia, this tree is abundantly grown for the forest
timber estate programme[1].
Acacia mangium is a suitable species in humid tropical lowland area and it is successfully
used for regreening programme of critical land since the plant is vigorously extreme in growth
rate and highly tolerance to acid, low nutrient soils, and also resistant to diseases. In addition to
that, this tree could grow reasonably well where the competition is severe, for example with
Imperata cylindrica . The wood of Acacia mangium is suitable for wide range of uses especially
for pulp and paper industry.[2]
Nitrogen fixation of this plant has been known widely and it has beendetermined that Acacia
mangium could develop the living symbiosis with rhizobia through their specific nodulation.
Effective N2 fixing nodule of Acacia mangium has been confirmed to content rhizobia bacteria
with a restricted range of Bradyrhizobium spp. strain.[3]. Acacia rhizobia are monotrichously
flagellated and their slow growing also produces alkalinity without serum zone formation in
litmus milk. The result of a study showed that rhizobia acacia has ecological adaptation to high
temperature and arid condition.[4] Immunological identification of the Bradyrhizobium strains
present in the nodules confirmed the persistence of more efficient introduced strains up to 42
months after transfer of the inoculated trees to the field.[5]
Cross inoculation of rhizobia, which is originally isolated from nodule of acacia has not been
reported much. The possibility of cross inoculation is open as far as the rhizobia endophytes are
compatible with the crops. Diversity and adaptability of soybean in their compatibility with
number of rhizobia strain havebeen identified. Soybean and common bean nodulate with many
different species of rhizobia and the occurrence of both slow and fast growing strains in the
species of tropical legumes havebeen reported[6].
Previous report indicated that some rhizobial strains such as Rhizobia tropici could be
isolated from tree legumes. Rhizobium tropici is a novel species which commonly nodulates
Phaseoulus vulgaris L.beans and Leucena sp.tress. However these strains actually could also
nodulate other tree legumes, such as acacia. Inoculation with genetic diversity of rhizobia strains
recommended for soybean hasresulted in the establishment of rhizobia population in soil.[7]
Environmental conditions are limiting factors to growth and activity of the N2 fixing plants.
Typical environmental stresses found by the rhizobia bacteria in their legume nodules symbiosis
are acidity, alkalinity, salinity, temperature and some physical exposed such as UV incusing
chemicals. Population of Rhizobium bacteria species vary in their tolerance to major
environmental factors. Therefore it is essential to screen the bacteria through environmental
stresses and screening for tolerant strains is essential.[8]
Biological N2 fixation represents the major sources of N input in agriculture soils. The
behavior of those some N2-fixing bacteria under severe environmental conditions such as
acidity, salinity, alkalinity, physical condition.[9]
Inoculants development required the update combination of bacteria which could provide the
plant nutrition needed on supporting growth of soybean. Possible inoculants development could
be achieved by outsourcing the other nitrogen fixing bacteria which have potential on specific
condition and compatible to the host crops.
The aim of this research is to collect the biodiversity of endophytes microbes from specific
host plant and to open the possibility sources of rhizobia strains on supporting the growth of
soybean. In addition to that is to improve the quality of inoculants using better composition of
potential microbes.
MATERIAL AND METHOD
Isolation of rhizobia endophytes bacteria
Isolation of endophytes bacteria was done from root nodules of Acacia mangium. Sampling of
acacia root nodules was collected by digging the soil surrounding the plant tree and finding the
roots which have root nodules. Isolation of bacteria was done based on the method described by
Vincent.[10 ] Undamaged nodules was selected, cleaned and surface sterilization was performed
to remove the debris outside nodules and also contaminant microorganisms. Isolation was done
by crushed the nodules using flat dissect knife on sterile Petridish and take a loop of milky
suspension of bacteria to grow in Yeast Extract Mannitol Agar (YEMA) media. Purification was
done continuously by transferring a single colony to the fresh YEMA media with addition of
Congo Red as indicator. Colonies that did not absorb the Congo red were suspected as rhizobia.
Pure cultures were preserved in YEMA slant agar media to conserve and for further used
Molecular identificationof endophytes bacteria
Endophytes rhizobia bacteria isolated from Acacia mangium were identified by 16S rRNA gene
sequence. Single colony of bacterium was picked using sterile toothpicks and dipped it into PCR
tube. A 49 µl of PCR mixture was added into PCR tube and pipetted up and down to mix. The
PCR mixture was contained 25 µl of ready mix, 25 µl of dNTP, 2 µl of each oligonucleotide
primer 9F (5’ GAG TTT GAT CCT GGC TCAG 3’), 1541R (5’ AAG GAG GTG ATC CAG
CC 3’) and 20 µl of sterile distilled water. Denaturation was run at 96°C for 5 min. Thirty cycles
of amplification consisted of denaturation at 96°C for 30 sec, annealing at 55°C for 30 sec and
extension at 72°C for 1 min were run. An elongation phase was performed at 72°C for 7 min. 3
µl of amplified reaction mixture was analyzed by agarose (1 % w/v) gel electrophoresis in TAE
buffer. After run at 100 V for 30 min, the gel was stained with Atlas sight DNA for 30 min and
was observed by UV transilluminator. The 16S rRNA gene sequence obtained was compared to
BLAST analysis.
Stress test.
To determine the ability of endophytes bacteria to adapt with stress, the endophytes were
exposed to extreme condition namelyheat and cold shock, UV irradiation, osmotic and oxidative
stress. DCM 2.1.2, the nitrogen fixing potential of endophyte was used in this study. Cells were
grown aerobically at 30oC in NB media. After 24 hours, the cells were exposed to different stress
test. UV irradiation of cell suspensions (10 ml) was performed by exposing the cells to UV light
for 2 h. For osmotic shock, the cells were incubated with 3 M NaCl for up to 2 h at 30 oC. For
acid stress, cells were grown at pH 3.0 and 4.0 for 2 h at 30oC. For oxidative stress, cells were
treated with 3% H2O2 and grown for 2 h at 30oC. For heat shock, cells were exposed to 75oC for
5 min by immersion of cultures in a water bath. For cold treatment, cultures were incubated at
8oC for 6 days. After that 50 µl of the diluted treated cells were dropped on NA plates and
incubated at 30oC for 24 hr. The presence of colonies indicated cell tolerant to stress condition.
Cross inoculation of endophytes bacteria from acacia to soybean
Pot experiment was conducted to study the cross inoculation of endophytes bacteria from
tree legumes to soybean crop. Soybean was used as a model of plant since that crop is normally
fulfilling the nitrogen requirement from symbiotic living with rhizobia. Germinated seedling was
soaked into bacterial suspension with 107cells per ml density. Seedling was deep soaked for 30
minutes. After that, seedlings were transplanted into the 10 kg growth media ( soil : compost ) in
ratio 1:1 in polybag. Each polybag was planted by 2 seedlings. In addition to that, each hole was
added by 1 ml of bacterial suspension. The endophytes bacteria (DCM 2.1.2) was grown in YEM
broth for 3 days. Rhizobia (Rh B64 ) and mixer of Rh B64 and DCM 2.1.2 was used as
comparison and prepare by growing in YEM broth for 7 days. For control of nitrogen (CN)
soybean was fertilized by chemical fertilizer (NPK) 0.8 g per polybag and without fertilizer
(CO). During the soybean growth, inoculants treatments were fertilized with NPK (0.4g/polybag)
and applied twice at 15 and 45 days after planting. Inoculants treatments were fertilized with
NPK (0.4 g/polybag). Chemical fertilization was done 15 and 45 days after planting. Each
treatment was 3 replicates. Data collection was done by measuring the height of plants during 1
and 2 month old plants. Harvesting time was done after 3 months and biomass of plant was
measured by weighing the upper and lower plants. The upper and lower plants were dried in an
oven of 70o C for 24 hours and dry weight per plant was measured. Number of pod and seed
weight of each plant were also observed.
RESULT AND DISCUSSION
Rhizobium strain has diverse geographical origin of parents plant or hybrid plants.[11 12]
Almost all rhizobia strains isolated from parental legumes trees could be grouped into
Bradyrhizobium elkanii, while all strains isolated from hybrid plant were grouped together in a
clade close to Bradyrhizobium japonicum .[13 ] 16S rRNA analysis of endophyte from Acacia
mangium showed that the isolates belong to Rhizobium tropici with the value of query coverage
is 99 %. This result explained that the endophytes from tree legumes might have diverse species
of Rhizobia.
Table 1. BLAST analysis of DCM 2.1.2 based on 16S rRNA gene sequence
Description
Rhizobium tropici strain CAF-440 16 S ribosomal
Total
Query
E
Max
score
coverage
Value
ident
2608
99%
0.0
99%
2608
99%
0.0
99%
2608
99%
0.0
99%
2606
99%
0.0
99%
RNA gene, partial sequence
Rhizobium tropici strain CAF-225 16 S ribosomal
RNA gene, partial sequence
Rhizobium tropici CAF 46 16 S ribosomal RNA
gene, partial sequence
Rhizobium tropici CIAT 899, complete genome
Many forest trees are legumes which nodulate with the bacterium Rhizobium (fast growing) or
Bradyrhizobium (slow grower) and fix gaseous nitrogen thereby utilizing some of the 84,000
tones of nitrogen gas in the air above each hectare of land. There are more than 18,000 species of
legumes of which about 7.200 species are woody species. Only about 18 % of those woody
species have been examined for nodulation.
[1,14]
Nuswantara et.al 1997 and Christine Le Roux
et.al 2009 reported that phylogeny of acacia rhizobia has been actually confirmed by the partial
sequence of the rRNA operation and resulted that the bacteria could be grouped in
Bradyrhizobium japonicum and Bradyrhizobium alkanii.[9,
13,15]
Most of tested acacia rhizobia
isolates nodulated the trees werebelong to the fast growing bacteria when they were growen in
Yeast Extract Mannitol Agar. However, it did not perform nodulation in renodulation test to
confirm the infectiveness of strains in isolation host.[15]
Table 2. Resistance of isolate Rhizobium DCM 2.1.2 to various stress conditions
DCM 2.1.2 (cfu)
Normal condition
0.1 x109
UV exposure
5.5x107
Heat shock
-
Cold shock
0.4x108
pH 3
6.5x106
pH 4
1.95x106
NaCl (3 M)
4.5x106
H2O2 (3%)
-
The successful application of the endophytes rhizobia to crop plant very much depends to the
ability of bacteria facing the extreme environments. As we understand that environment in
surrounding the root system become the specific niche of condition for the bacteria to survive
and to grow well.
Result of stress test nitrogen fixing bacteria showed that DCM 2.1.2 survived enough to some
stress that were acidity ( pH 3.0 and pH 4.0) and salinity. The population of bacteria slightly
decreased from 109 to 106 cfu. UV exposed made the population of bacteria a bit decline from
109 to 107 cfu similarly with cold shock which the number of cells remain in 108 cfu. The
bacteria could not survive on the heat shock same as to the oxidative stress. Hamdi Husen
Zahran 1999 reported that soil acidity is a significant problem faced in agriculture area while
most leguminous plant require neutral soil for growth especially when they depend on symbiotic
N2 fixation. So, the failure of legume to nodulate under acid soil conditions is common,
especially in soil with pH of less than 5.0 [8] This study result may be now updating since it was
found that rhizobia acacia isolates actually could resistant on acid condition.
Considering the environmental conditions especially in relation with the programmes of
extensification of agriculture land which are more likely will be in the acid and unfertile soils,
these endophytes rhizobia have possibility to be used as a biofertilizer product together with
other bacteria used to compose biofertilizer.These bacteria could give specific benefit for the
plant.
month 1
Height of plants (cm)
80
60
57
49.33
60.33
44
75.67
month 2
66
60.67
46.67
47.33
45.33
40
20
0
CO
CN
DCM
RH
DCM+RH
Treatments
Graph 1. Height of 1 and 2 month old of soybean plants
Graph 1 shows the effect of inoculation on the growth of soybean plants. In the first month of
growing, the effect of inoculation bacteria, did not show significant different among treatments.
At the first month old plant,the growth of soybean approximately reached 50 cm in average,
while after 2 months old, the effect of inoculation treatment wasmore significant. Inoculation
with single strain of rhizobia (Rhizobium B-64) showed better result of plant height, with
anaverage of up to 80 cm, compared to other inoculations which were range from 65-70 cm.
Inoculation with endophyte rhizobia alone showedinsignificant result on plants height compared
to control and chemical fertilizer treatment. This condition mightbe caused by the soil condition
used for the experimentthat contained enough nutrients coming from organic matter applied in
the growth media [17 )[10,16]( Vincent, 1970,. Somasegaran and Hoben , 1994].
Upper plant
Dry weight (gram)
7
Lower plant
6.14
6
5.25
4.83
5
3.75
4.08
4
2.37
3
1.79
2
1.97
1.63
1.34
1
0
CO
CN
DCM
RH
RH + DCM
Treatments
Graph 2. Dry weight of upper and lower plants inoculated bacteria
In regard to
plant biomass, single inoculation that is rhizobia, endophyte rhizobia
isolated from acacia and combination among the two bacteria showed significant result on the
biomass of upper plant compared to control and chemical fertilizer treatments. However, the
biomass of lower plants in all treatments did not show significant different results. In conclusion,
rhizobia bacteria inoculation positively supported the growth of plant especially by increasing
the upper part of plant biomass.
It is reported that the bacteria most often used to inoculate forage crops is bacteria in the
genus Rhizobium. Furthermore it has been determined that different forage legumes require
different species or even different biovars within species of rhizobium for successful formation
of nodules.
Ʃ pods
fresh weight of pods
Seed weight
89.33
90
80
71.33
70
60
56.33
54.33
50
40
54.67
47.65
32.88
30
44.99
37.59
36.41
28.03
23.4
19.71
18.84
16.95
20
10
0
KO
KN
RH
Treatments
DCM
RH. DCM
Graph 3. Number of pods and seed weight per two plants
Graph 3 shows that soybean significantly responded to the single Rhizobium inoculation
in regard to the number of pods and seed production compared to the inoculation of rhizobia
endophyteisolated from Acacia mangium. However the combination between Rhizobium and
rhizobia endophytes from Acacia mangium gave high number of pod and seed weight compares
to control plant and chemical fertilizer. This result explained that single inoculation could be
applied even better than the double inoculations.
This experiment concluded that all treatments showed that potential microbes, rhizobia
and rhizobia endophyte, could give positive effect on the production of crop plants, number of
pods and seed production compared to control plant and chemical fertilizer treatment. However,
double inoculation of potential microbes did not always guarantee to stimulate the high
production of seed. The highest seed weight was performed by rhizobia inoculation followed by
double inoculation between rhizobia and endophytes. and single endophyte inoculation
performed the same production of seed as chemical fertilizer treatment.
CONCLUSION
The positive effect of inoculation especially on plant height could be seen after 2 months of
inoculation. The process was the formation of nodules that happened after the bacteria infected
the root plant and further more developed the symbiosis living with the host plant, then running
the nitrogen process to support
the growth of plant
(Graph 1). Single strain inoculation
wasfound better than double inoculation strains shown by significant results of upper plant
biomass, whereas the effect of double strain inoculation did not significantly respond tothe lower
plant biomass. ( Graph 2) . Single inoculation of rhizobia provided thebest result on the
production of soybean same as endophyte of acacia inoculation although it was not as good as
rhizobia. This experiment confirmed the possibility for cross inoculation of endophyte from tree
legumes such as Acacia to crops plant (soybean) . Inoculation of microbes from different host
trees are wide open to be used to crops plants.
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ACKNOWLEDGEMENT
I would like to express my gratitude to all of my colleages, Sylvia Lekatompessy, Tiwit
Widowati, Rumella Simarmata, Lisye Nurjanah, Nuriyanah and Adang. R who have given
valuable supports in conducting this experiment and analysis of the data. This research was
supported by the Indonesian Institute of Sciences Research Funding on the topic of National
Food Sustainability Programme.
APPENDIX
Fig 1. The growth of soybeans at one month old
Fig 2. The growth of soybeans at two months old