3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

Response of Rhizobium on Nitrogen Accumulation In Leaf

by Jackbean Plant (Canavalia ensiformis l.)
P. Laksitarahmi and T. Surtiningsih
Department of Biology
Airlangga University, Surabaya, Indonesia
e-mail: laksitarahmi@yahoo.co.id

b chlorophyll and carotenoids which are also

contained yellow-orange pigment (Salisbury and
Ross, 1995) The rate of photosynthesis per unit
plant can be determined by leaf area. Leaf area is
one of the measurement parameters for considering
the function of leaf growth as a receiver of light and
photosynthesis instrument. Increased leaf area
allows large increase in catchment areas of light,
causing the leaves become thinner because the cells
palisade consists of only one or two layers. Leaf
area and the high amount of chlorophyll which will
cause the process of photosynthesis going well. The
larger the leaf area of plants then the acceptance
will be greater sunlight. With a high leaf area, then
the light will be more readily accepted by the leaves
as well (Fahrudin, 2009). Therefore the presence of
the element nitrogen can increase the rate of
photosynthesis of plants so as to stimulate
vegetative growth. Increasing the amount of
nitrogen in the soil produce proteins in large
quantities in plants, thereby increasing the growth
of plant tissues (Mapegan, 2007).

Abstract
The aim of this study was to know the response of
Rhizobium bacteria on the leaf area, chlorophyll
content a and b, N content in leaf of Jackbean
(Canavalia ensiformis L.). This research is an
experimental designed which is used Completely
Randomize Design (CRD). The treatment in this
experiment was giving the Rhizobium bacteria
dosage 0, 10, 20, and 30 ml/plant, it was inoculated
on planting hole of Jackbean plant. Data were
analyzed by ANOVA, with Duncan test at level 5
%. The results of this research showed that the
treatment of the Rhizobium bacteria dosage gave a
significant difference (p<0.05) for leaf area and
chlorophyll content a of Jackbean plant. The
highest leaf area (31,673,56 cm2/plant) and
chlorophyll content a (10,690,66 mg/g) gave by
Rhizobium bacteria dosage 20 ml/plant, but the best
N content (0,89%) gave by Rhizobium bacteria
dose 10 ml/plan in lea of Jackbean plant.
Key words : Canavalia ensiformis L., Rhizobium,
leaf, chlorophyll content a and b, nitrogen
1

Nitrogen fertilizer can be obtained either from

chemical fertilizers (urea) and biological fertilizers.
Biofertilizer is a gift that is useful for microbial
plant into the ground. One of the microbes included
in the fastening of microbial biofertilizer is N.
Nitrogen-fixing microbes there are 2 types of
microbe symbiosis with the roots of such plants
(Rhizobium) and there is no symbiosis is living
freely in the soil such as (Bacillus, Pseudomonas,
Azospirillum) (Akhtar and Siddiqui, 2009). While
the cultivation of legumes such as Jackbean plant,
required role of Rhizobium bacteria to attach the
element N in air into the elements available to
plants and helps to reproduce the formation of root
nodules. Rhizobium nodule that grows in
leguminoceae is a bacterial symbiosis with plant
roots, bacteria are able to take nitrogen from the air
direct to the joint activity of plant cells and bacteria.
Rhizobium has the ability to bind free nitrogen from
the air and turn it into a compound of ammonia
with the help of nitrogenase enzymes that are easily
absorbed by plants (Khalequzzaman and Hossain,

Introduction

Nitrogen is one of the macro elements needed by

plants. The element nitrogen is essential for
vegetative growth of plants because this element is
most needed plants to synthesize chlorophyll in the
plant functioned process photosynthesis . Leaf is a
place to synthesize food for the plant needs as well
as reserve food. Leaves have chlorophyll a role in
photosynthesis. Where chlorophyll or capture
function to receive the required light plant to treat
water (H2O) and carbon dioxide (CO2) into
carbohydrates become the primary food source for
the plant that can be used for the activity of
photosynthesis and other metabolic (Patreze and
Lazara, 2004). Chlorophyll is a pigment that is
very important in fotosintes, ranked second
chlorophyll pigments found on thylakoid membrane
is composed of green chlorophyll a and chlorophyll
B020

P. Laksitarahmi, Response of Rhizobium on Nitrogen Accumulation In Leaf By Jackbean Plant (Canavalia

ensiformis l.)

2008). Legume crops continues to increase its

nitrogen content and N uptake in legume plants
derived from the available N in the soil. While the
nitrogen fixed by legume crops have three
possibilities that nitrogen is used by the host plant
itself, the nitrogen excreted from the nodules into
the soil and used by other plants growing nearby
(Sutejo, 1999; Mapegan, 2007).
Jackbean plant is one of the legume family are
considered able to replace the role of soybean as
nutritional content is comparable with the soybeans.
To improve the cultivation of Jackbean plant can be
done with fertilizer. However, the current excessive
use of chemical fertilizers can harm agriculture, the
balance of nutrients in the soil to be disturbed,
decreased quality of agricultural products, the
presence of pests and diseases, and cause
environmental pollution. One attempts to restore
the fertility of agricultural land, get the production
of healthy food and avoid harmful chemicals is to
use biofertilizer with the addition of Rhizobium
that can reduce the use of chemical fertilizers.
Therefore, the purpose of this research was to
determine the response of Rhizobium bacteria on
the accumulation of nitrogen on leaf area,
chlorophyll content a and b.

Chlorophyll a = (13,7 x OD665) (5,76 x OD649)

Chlorophyll b = (25,8 x OD649) (7,7 x OD665)
Where, A665 and A649 represent the optical
density (OD) values at the respective wavelengths
Measuring levels of N, generally by using the
Kjeldahl method (Greenberg et al. 1989). The
analysis of leaf using Sudarmaji (1989) method.
Data were analyzed using ANOVA test and
correlation test, if significant will be followed by
Duncan's test of 5%.
3
Tabel 1:

Treatment
R0R0+
R10
R20
R30

2 Methodology
The research was conducted in two places is the
Laboratory of Microbiology, Department of
Biology, Faculty of Science and Technology,
Airlangga University Surabaya and Agricultural
Land in Surabaya. The research start from
September 2010 until February 2011. Jackbean
plant from the Laboratory of Food Biotechnology,
Center for Genetic Resources of Agricultural
Bogor. Isolate bacteria Rhizobium sp. from the
Microbiology Laboratory, Department of Biology,
Faculty of Science and Technology University
Press. This research is experimental using
Completely Randomized Design (CRD) with 5
replications per treatment. The treatment in this
study were Rhizobium bacteria at doses of 0, 10,
20, and 30 mL / plants and plants inoculated at the
hole the Jackbean plant. Plants allowed to grow
until the age of four months.The parameters
observed were leaf area growth, chlorophyll
content, leaf N content. The analysis of leaf
chlorophyll content using a spectrophotometer
(Winstermans and Mots, 1965 at Purnobasuki,
2010).

Result

Effect of Microbial Consortium on

vegetative growth in leaves of
Jackbean (C. enisiformis)

Leaf
area
(cm2)
23,20
2,77 a
24,53
0,38 a
28,98
2,93 b
31,673,
56 c
27,142,
97 b

Chlorophyll
content a
(mg/g)
0,15960,46
a
0,20130,32
ab
0,20420,36
ab
0,21370,53
b
0,18960,49
ab

Chlorophyll
content b
(mg/g)
0,13750,98
a
0,1777
0,14a
0,18330,98a

N content
in leaf
(%)
0,71

0,18080,11
a
0,17850,12
a

0,59

0,68
0,89

0,70

Description: R0-: non inoculated with Rhizobium,

R10: inoculated with Rhizobium 10 mL/plant, R20:
inoculated with Rhizobium 20 mL/plant, R30:
inoculated with Rhizobium 30 mL/plant and the R0
+ (control negative) chemically fertilized control
plant (5 g/plant). Different letters in the same line
significant different at <0.05
3.1
Leaf area
Table 1 shows that the results of statistical analysis
ANAVA test ( <0.05) of Rhizobium significantly
influenced leaf area ( = 0.00). For leaf area
parameters, giving the Rhizobium bacteria with a
dose of 20 mL showed a higher yield of 31.67 cm2,
compared to negative control treatment (23.20 cm2)
and positive control (24.53 cm2). This is in
accordance with the parameters of chlorophyll
content a in plant that showed that the addition of
20 mL of Rhizobium bacteria (R20) also showed the
highest (10.69 mg/g) when compared with the
negative control treatment that is only 7.98 mg / g
and a positive control 10.06 mg / g. This is the
optimal dose in affecting leaf area and chlotophyl
content a, due to the higher doses decreased. Based
on a treatment table R20 aligned with increasing
leaf area and levels of chlorophyll a, chlorophyll b,
while in harmony with higher levels of N. This is
presumably because the elements contained more
Mg accumulates on the leaves because it is the only
metal elements that make up the molecules of

Pigments from leaves of the developing seedlings

were extracted with 80% acetone and the amounts
of chlorophyll a and chlorophyll b were determined
as described (Winstermans and Mots, 1965 at
Purnobasuki, 2010).

B020

3rd International Conferences and Workshops on Basic and Applied Sciences 2010

ISBN: 978-979-19096-1-7

Rosemeyer, 2006; Elkoca et al, 2008. For N levels

in leaves of chlorophyll b in tune with, it is
assumed that the chlorophyll b has tasks as collect
light for photosynthesis. In the treatment of R20
leaf N content should be aligned with the high leaf
area and chlorophyll a and b. But in this study is not
the case, it is assumed at the time of sampling for
leaf N content is not homogeneous so as to produce
results that are not maximum. Not effect between
levels of N in leaves on the R10 treatment with leaf
area and levels of chlorophyll a. This is assumed
because of Rhizobium optimal dose of 10 g over the
fix nitrogen so as to affect the levels of N in the
leaves, so leaf area and chlorophyll a levels
increased. Nitrogen is a constituent of chlorophyll
dar 50-70% of total leaf N association with the
chloroplast and reflects the presence of enzymes
robulose bisphosphate carboxylase oxygenase
(rubisco). Both components play a role in
photosynthesis together with the light. Most of
photosynthesis of plant biomass accumulated and
their products (Paracer, S. and Ahmadjian, 2000)
Levels of nitrogen in leaves is not directly play a
role in photosynthesis, thus nutrient nitrogen
although this is a constituent of chlorophyll that has
been known as a raw material in the process of
photosynthesis (Sangeetha and Thevanathan, 2010).
Besides a role as a constituent of chlorophyll,
nitrogen also plays a role in the translocation of
carbohydrates from leaves to other plant organs. So
that nitrogen would overhaul the carbohydrate to
protein so as to speed up loading (phloem loading)
and unloading (phloem unloading) in the process
traslokasi carbohydrates. In such conditions no
accumulation of carbohydrates in the leaves,
thereby increasing the absorption of light energy
and photosynthetic rate also increased (Mapegan,
2007; Mogel, and Camle, 2011).

chlorophyll and the one that plays a role in

photosynthesis, so the nitrogen in the leaves also
increased (Rostini et al, 2003). The increating of
leaf area between treatment because there was
enough nutrients, especially nitrogen to be absorbed
quite a lot of plants that can affect plant growth,
especially for leaf formation. Leaf area also affects
the process of photosynthesis (Taiz and Zeiger,
1991; Sangeetha, V and Thevanathan, R. 2010).
Therefore, increased leaf area also increased the
chlorophyll content. So that the process of
photosynthesis will increase plant growth and
productivity.
3.2
Chlorophyll Content
Based on the results of analysis using ANAVA test
( <0.05) showed that administration of Rhizobium
bacteria significantly affect levels of chlorophyll a
( = 0.00) and b ( = 0.00). The treatment
Rhizobium bacteria is very effective in increasing
levels of chlorophyll a, which shows that the
magnitude chlorophyll content a in the treatment of
Rhizobium bacteria is higher than the negative
control and positive control. While to chlorophyll b
R10 highest yield on treatment of 0,1833 mg/g.
Between chlorophyll a and b in this study are not
aligned, but when seen from the overall average
increase between treatments. And for the average
chlorophyll a higher than chlorophyll b. This is
because in chlorophyll b has a function
asphotosynthetic antenna tasked with collecting the
light and then transferred to the reaction center and
the reaction center is composed of chlorophyll a, so
that the chlorophyll a were higher. These findings
are consistent with research by Selvakumar et al,
2009; Mogle dan Chamle, 2011).
3.3
Leaf Nitrogen Accumulation
The results of N uptake by the highest leaf (0.89%)
is achieved by Rhizobium treatment 10 ml/plant. N
uptake by the leaf was higher when compared with
negative control treatments 0.71% and the positive
control which were given 5 g NPK/plant 0.68%.
Dose of 20 mL of Rhizobium bacteria is more
effective in enhancing the growth of leaf area and
chlorophyll content. Leaf N content could
strengthen the evidence whether there is a process
belay from nitrogen atmosphere by Rhizobium in
the nodule or not. Based on table 1, the percentage
of leaf nitrogen content of Jackbean plant are
treated with Rhizobium inoculation of plants that
are not inoculated, not show positive effect, leaf N
content ranged from 0.59 to 0.89%. Increased
nutrient uptake by plant roots Jackbean not be
separated from the role of Rhizobium bacteria
inoculated into rhizosfir. In this result, the addition
of Rhizobium bacteria showed not the effect of leaf
area, chlorophyll b, but associated with levels of
chlorophyll a. This finding is in line with research

3.4
Correlation of leaf area, chlorophyll
content, N content in Leaf
Tabel 2:

The correlation between leaf area,

levels of chlorophyll a, chlorophyll b,
the levels of N leaves with a
Rhizobium bakteria

Treatment

Leaf
area

Chlorophyll a

Chlorophyll b

Leaf area
Chloropyll
a
Chlorophyll
b
N content
in leaf

1
0,292

0,292
1

0,609**
0,655**

N
content
in leaf
- 0,91
- 0,205

0,609

0,655**

- 0,208

0,91

- 0,205

- 0,208

Note: **) correlation positive is significant at the

0,01 level (2-tailed).

B020

P. Laksitarahmi, Response of Rhizobium on Nitrogen Accumulation In Leaf By Jackbean Plant (Canavalia

ensiformis l.)

soil, including nitrogen element. Nitrogen hooks

closely with the synthesis of chlorophyll and
synthesis of proteins and enzymes. Enzyme
(rubisco) acts as a catalyst in the fixation of CO2
that plants need for photosynthesis. Decreased
levels of nitrogen affect the photosynthesis of
plants is also good over the content of chlorophyll
and photosynthetic enzymes fotosintat is formed,
will further reduce the dried weight Jackbean plants
(Salisbury and Ross, 1992; Djukri and Bambang,
2003).

Based on table 2, the correlation coefficient is

indicated by a number between -1 to 1. If the value
of the correlation coefficient of 0 indicates no linear
relationship between two variables. If there are
asterisks (**) indicates a positive correlation
relationship. It turns out that showed a positive
correlation relationship of leaf area were positively
correlated to the levels of chlorophyll b and
chlorophyll a correlated positively to chlorophyll b.
Leaf area correlated with chlorophyll b, this is
presumably because the leaf area as one of the
parameters of leaf growth measurement serves as a
receiver of light and photosynthesis instrument.
Increasing leaf area will allow an increase in broad
areas of light capture. Increased leaf area of plants
in an effort to streamline the capture of light energy
for photosynthesis normally at low light intensity
conditions. Plant leaf surface area also affects the
process of photosynthesis. This is because the leaf
area is an attempt to capture light energy to
photosynthesis under conditions of low light
intensity (Taiz and Zeiger, 1991; Zeiny, 2007).
Plant growth are closely related to the absorbed
nutrients from the soil, including elements of N.
Where in the chlorophyll b as a light receiver which
will be ditrasfer to the reaction center. Therefore
between chlorophyll a and chlorophyll b are
correlated, this is because the chlorophyll b tasked
to transfer the light on the reaction center and the
reaction center chlorophyll is contained in a, where
the green pigment chlorophyll as the leaves serves
to absorb energy from sunlight and used in the
process of photosynthesis, causing the plant
vegetative growth lentils better sword. Elevated
levels of chlorophyll a and b is a testament to the
ability of plants to grow lentils kondisis under low
light (Talaat and Abdallah. 2008), this also agrees
with research Djukri and Bambang (2003). On leaf
area and the high amount of chlorophyll which will
cause the process of photosynthesis going well. The
larger the leaf area of plants is receiving sunlight
will also larger, therefore the leaf area correlated
with higher levels of chlorophyll. With a high leaf
area, then the light will be more readily accepted by
the leaves well. Thus, the leaf is an important organ
of plants that play a role in the process of
photosynthesis because the chlorophyll content in
it. Chlorophyll has a role as a catcher device
sunlight energy in photosynthesis to produce ATP
and NADPH and energy sources used to perform
formation fotosintat is light (Tambekar et al, 2009;
Fahrudin, 2009). With the increasing levels of
chlorophyll can be a sensitive indicator of plant
physiological conditions that affect plant growth
lentils sword. This is because more and more
ammonia in the leaves of N2 conversion by
microbes so the more chlorophyll is formed
(Sangeetha and Thevanathan, 2010). Closely
related to plant growth nutrients absorbed from the

Conclusions

From the results of this study, it can be concluded

that giving a bacterial Rhizobium in bacterial
concentration 20 mL gave the highest results for the
leaf area (31,673,56 ) dan chlorophyll content b
(10,690,66 mg/g ). Between leaf area, chlorophyll
a, and chlorophyll b showed that positive
correlation, except N content in leaf did not differ
significantly.
References
[1] Akhtar, M.S., dan Z.A. Siddiqui, Effects of
Phosphate Solubilizing Microorganisme and
Rhizobium sp. On The Growth, Nodulation,
Yield and Root-Rot Disese complex of
Chickpea Under Field Condition. Afican
Journal of biotechnology Vol 8(15): 34893496, 2009.
[2] Djukri dan Bambang S.P., Pengaruh Naungan
Paranet terhadap Sifat Toleransi Tanaman
Talas (Colocasia esculenta (l.) Schott). Jurnal
Ilmu Pertanian Vol 10(2): 17-25, 2003.
[3] Elkoca, E., F. Kantar, dan F. Sahin, Influence
of Nitrogen Fixing and Phosphorus
Solubilizing Bacteria on The Nodulation,
Plant Growth, and Yield of Chickpea. Journal
of Plant Nutrition 31: 157-1712008.
[4] Fachrudin, F., Budidaya Caisim (Brassica juncea
L.)Menggunakajan Ekstrak The dan Pupuk
Kacsing. Skripsi.
Fakultas Pertanian.
Universitas Sebelas Maret Surakarta, 2009.
[5] Greenberg, A.E., E. W. Rice, S. Clesceri, dan
A.D. Eaton. Standard Methods for The
Examination of Water and Wastewwater 21st
Edition. American Public Health Association
800 I Street. NW Washington, 2005.
[6] Khalequzzaman, K.M., dan I Hossain, Effect
of Rhizobium Starins and Biofertilizers on
Foot, Root Rot, and Yield of Bush Bean in
Sclerotinia Sclerotium Infested Soil. J. biosci. 16:73-78, 2008.

B020

3rd International Conferences and Workshops on Basic and Applied Sciences 2010

[7] Mapegan, Pengaruh Pupuk Nitrogen Terhadap

Pertumbuhan dan Hasil Tanaman Kacang
Hijau (Mungbean). Agripura 3 (2), 2007.

ISBN: 978-979-19096-1-7

Journal of Applied Sciences Research 4 (9):

1092-1102, 2008.
[20] Taiz L., and Zeiger E., Plant Physiology.
Tokyo. The Benyamin/Cumming Publishing
Company Inc. pp: 219-247, 1991.

[8] Mogle, U.P., dan D.R. Chamle, Evaluation of

Biofertilizers and Parthenium Vemicompost
on Tomato Crop. Journal of Ecobiotechnology
3(2): 11-13, 2011.

[21] Tambekar, D.H., S.R., Gulhane, D.O.,

Somkuwar, K.B., Ingle, S.P., and Kanchalwar.
Potential
Rhizobium
and
Phosphate
Solubilizers as a Biofertilizer from Saline Belt
of Akola and Buldhana District (India).
Research Journal of Agriculture and
Biological Sciences 5(4): 578-5, 2009.

[9] Rosemeyer, M., Biological Nitrogen Fixation:

Supplying Nitrogen Needs Of Sustainable a
Agriculture. Ecological Agricultur. New
Jersey, 2006.
[10] Salisbury, F.B., dan Ross, C.W., Fisiologi
Tumbuhan III edisi ke-4. Penerjemaah
Lukman, D.R. dan Sumaryini. ITB. Bandung,
1995.

[22] Zeiny, El., Effect Of Biofertilizer and Root

Exudates of Two Weeds as a Source of
Natural Growth Regulators on Growth and
Productivity of Bean Plants (Phaseolus
vulgaris). Research Journal of Agriculture
and Biological Sciences 3(5): 440-446, 2007.

[11] Sangeetha, V., and Thevanathan, R.,

Biofertilizer Potential of Traditional and
Pachagavya Amended with Seaweed Extract.
The Journal of American Science 6(2):61-67,
2010.
[12] Selvakumar, G, M. Lenin, P. Thamihiniyan,
and T. Ravimycin, Respone of Biofertilizer on
The Growth and Yield of Blackgram (Vigna
Mungo L.) Recent Research in Science and
Technology 1(4): 169-175, 2009.
[13] Sudarmaji, S. Haryono, and B. Suhardi.
Prosedur Analisis Bahan Makanan dan
Pertanian. Edisi Ketiga. Liberty. Yogyakarta,
1984.
[14] Sutejo,
Mulyani.
Pupuk
dan
Cara
Pemupukan. Rineka Cipta, Jakarta, 1999.
[15] Paracer, S. and Ahmadjian. Symbiosis: an
Introduction to Biological Associations. 2nd
edition. Oxford University Press Inc. New
York, 2000.
[16] Patreze, C.M., and Lazara Cordeiro, Nitrogen
Fixing and Vesicular Arbuscular Mycorrhizal
Symbioses in Some Tropical Legume Trees of
Tribe Mimoseae. Ecology and Management
196: 275-285, 2004.
[17] Purnobasuki, H., Panduan Praktikum
Fisiologi Tumbuhan. Departemen Biologi,
Universitas Airlangga. Surabaya, 2006.
[18] Rostini, Neni, A. Baihaki, Ridwan
Setiamihardja, and Giat Suryatmana, Korelasi
Kandungan Klorofil dan Beberapa Karakter
Daun Dengan Hasil Pada Tanaman Kedelai.
Jurnal Zuriat: 14 ( 2): 52, 2003.
[19] Talaat, N.B., dan A.M. Abdallah, Respone of
Faba Bean (Vicia faba L.) to Dual Inoculation
With Rhizobium and Mycorrhiza Under
Different Levels of N and P Fertilization.

B020