Вы находитесь на странице: 1из 72

›   ›

›
 ›     
    
  
  6  

 

   

ANDRIA AGUSTA1, YULIASRI JAMAL, PRAPTIWI AND AHMAD FATHONI

Laboratorium Bioscience, Botany Division, Research Center for Biology,


The Indonesian Institute of Sciences. Jl. Raya Bogor Km. 46, Cibinong 16911 West Java
*Alamat korespondensi, E-mail: bislunatin@yahoo.com

›


Totaly eighteen kinds of endophytic fungi have been isolated from the young
stems of kayu kuning [Archangelisia flava (L.) Merr.]collected from Bogor Botanic
Garden. All of isolated fungi have been screened for their capability to transform the host
plant alkaloid berberine in two kinds of liquid mediums, potato dextrose broth (PDB) and
glucose-yeast extract-peptone (GYP) respectively. Biotransformation reactions were
monitored and analyzed by a normal phase thin layer chromatography technique. The
screening process revealed that the fungus Coelomycetes AFKR-1 could be able to
transform berberine into a biotransformed product in PDB medium. Scaling-up
biotransformation reactions (5 x 200 ml medium in 500 ml size Erlenmeyer) which were
incubated at room temperature with agitation at 120 rpm yielded 20 % conversion rate of
substrate into a product in 48 hrs. The chemical structure of biotransformed product was
proposed as a 7-N-oxide-berberine based on their spectroscopic data.

  !"Archangelisia flava, endophytic fungi, Coelomycetes AFKR-1, berberine,


biotransformation.



 

Microorganisms have been paid attention by many researchers since they possess
a peculiar metabolic capacity, and shows an increasing demand on chemical and
pharmaceutical companies. The Merck Research Laboratory, USA screened more than
500 kinds of microbes for their capability to transform anticancer drugs, taxol and
cephalomannine in order to find out more water soluble derivatives (Chen et al., 2001).
From those biotransformation process, two hidroxy taxol derivatives and one hidroxy
cephalomannine derivative were achieved. The use of microbes was also widely applied
to produce an interesting derivatives from many kinds of steroidal drugs (Faramarzi et al.,
2008). Endophytic fungus  laria sp. isolated from the plant Cinchona pubescence
(Rubiaceae) reported can transform Cinchona alkaloids (quinine, quinidine, cinchonine,

c    


     u
cinchonidine) into 1-N-oxide derivatives and showing an anti-malarial activity with very
weak toxicity compared with the typical Cinchona alkaloid quinine (Shibuya et al.,
2003). Endophytic fungus Diaporthe sp. isolated from a tea plant Camelia sinensis also
reported can biotransform tea catechins into C-4 hydorxy derivatives, and could not react
with non tea catechins (Agusta et al., 2005). Recently, the endophytic fungi isolated from
Thitonia diversifolia, Viguiera arenaria and Viguiera robusta showed their capability to
transform a neuroleptic drug, thioridazine through regio- and stereoselective reactions
(Borges et al., 2008).


  
Ôlant Materials
The youg stems of kayu kuning [Archangelisia flava (L.) Merr.] were collected
from Bogor Botanical Garden in 2007 andidentified at Herbarium Bogoriensis, Botany
Division, Research center for Biology, Indonsian Institute of Sciences.

Isolation of Endoph tic Fungi


Young stems of kayu kuning were washed by tap water and then cut intopieces
with the length of 1 cm, followed by sterilization by soaking the stems in the 70%
alcohol for 2 minutes, then soaked in the NaOCl for 5 minutes and soaked again in 70%
alcohol for 30 second.The sterilized stems were sliced with sterile knife and then put on
Corn Meal Malt Agar (CMMC) media that had been added with 0.05 mg/ml
chloramphenicol followed by incubation in the room temperature for 1 week. Every
colony of endophitic fungi were serially transfered onto Potato Dextrose Agar (PDA)
media untill pure colony were obtained. The obtained endophytic fungi were preserve in -
80 oC at LIPI-MC.

Screening for Biotransformation of Berberine


Each of isolated endophytic fungi were cultivated in 20 ml of two kinds of liquid
medium (in 50 ml size Erlenmeyer), Potato dextrose broth (PDB) and glucose-yest
extract-peptone (GYP). After incubated at room temperature (26 ± 28 oC) on a reciprocal
shaker (100 rpm)for 5 days, 2 ml (1 mg/ml in methanol) of berberine was added into the

c    


     *
medium, and continue incubate on identical conditions. The biotransformation reactions
were monitored every days (until one week) throught a series of thin layer
chromatography (TLC) analysis of reaction extracts on a TLC plate GF254 (Merck) with
developing sovent CHCl3 ± MeOH ± HAc ( 6: 1 : 1 drop). The TLC chromatogram
patterns were visualized under expossure of UV light at 254 and 355 nm, and dragendorf
reagent.

Biotransformation of Berberine b the Endoph tic Fungus Coelom cetes AFKR-1


The endophytic fungus AFKR-1 was inoculated into the glucose±yeast extract±
peptone medium (200 ml) and cultivated for 5 d under shaking at 90 rpm at 26-28 °C. A
solution of berberine (20 mg) in MeOH (20 ml) was added to the cultivation medium and
shaking continued for 1 d. The reaction mixture was filtrated to remove the fungus
bodies. The filtrate was extracted with EtOAc and concentrated under reduced pressure to
give a product (25 mg), which was purified by Sephadex LH-20 column chromatography
(MeOH) and a preparative TLC (silica gel 60 F254, CHCl3-MeOH, 3:1) to afford a
biotransformed product (20 mg, 20%).
The chemical structure of biotransformed product was deduce based on their
spectroscopic data [NMR (500 MHz) and TOF-MS data].

  


Eighteen kinds of endophytic fungi isolates from young stems of kayu kuning
plant [Arcghangelisia flava (L.) Merr.]collected in Bogor Botanical Garden were
obtained in our laboratory. Through a set of screening test, it was found one of
endophytic filamentous fungus Coelomycetes AFKR-1 could biotransform berberine into
a transformed product in GYP medium at 26 ± 28 oC and 120 rpm. Scaling-up reaction to
be 1 L (5 x 200 ml in 500 ml size Erlenmeyer) with addition 100 mg (20 mg each) of
substrate berberine could achieved 20 mg (20 %) of a biotransformed product (Fig. 1).
The 1H-NMR and 13
C-NMR spectra of the biotransformed product showed an
identical signal patterns to those of berberine (Tabel 1.). The above 1D-NMR spectra
revealed there is no change in chemical environment of proton and carbon atoms in
berberine molecule and NMR analysis could not resolve the chemical structure of the
biotransformed product. On the other hand, both of biotransformed product and berberine

c    


     
gave a different spots on a TLC plate (Silica gel GF254, Merck) and different peaks on
an HPLC analysis (reverse phase C18 HPLC, 90 % acetonitrile, 40 oC, 340 nm).
Furthermore, the biotransformed product was subjected into a high resolution TOF-MS
analysis. The TOF-MS spectrum of biotransformed product shows the ion peak at [M+]
353, 16 amu.bigger than the ion peak of berberine (Fig 2.). These result revealed that the
biotransformation reaction of berberine by the endophytic fungus Coelomycetes AFKR-1
could placed one additional oxygen atom (mass 16 amu.) into the berberine molecule.
Based on the above data, we assume that chemical structure of the biotransformed
product is a new berberine 7-N-oxide derivative (submitted for publication). In addition,
the biological activity of biotransformed product berberine 7-N-oxide is in under
progress.
The biotransformation reaction of berberine into 7-N-oxide derivative is quite
similar with biotransformation reaction of Cinchona alkaloids by endophytic fungus
 laria sp. which was also reported produce N-oxide derivatives (Shibuya et al., 2003).

O
d d
 
 

 *  
 

  d



Fig 1.
#$%  13C-NMR and 1H-NMR data for biotransformed product and berberine in
DMSO-d6.

No. Atoms Biotransformed Product Berberine
13 1 13 1
C-NMR H-NMR C-NMR H-NMR
1 105.4 7.80 (1H, s) 105.4 7.80 (1H, s)
2 147.7 147.7
3 149.8 149.8
4 108.4 7.09 (1H, s) 108.4 7.09 (1H, s)
4a 130.7 130.7
5 26.3 3.20 (2H, dd, £l6.2 Hz) 26.3 3.20 (2H, dd, £l6.2 Hz)
6 55.2 4.93 (2H, dd, £l6.2 Hz) 55.2 4.93 (2H, dd, £l6.2 Hz)
8 145.5 9.90 (1H, s) 145.5 9.90 (1H, s)
8a 121.4 121.4
9 143.6 143.6
10 150.4 150.4

c    


     
11 126.7 8.20 (1H, d, £l9.1 Hz) 126.7 8.20 (1H, d, £l9.1 Hz)
12 123.5 8.01 (1H, d, £l9.1 Hz) 123.6 8.01 (1H, d, £l9.1 Hz)
12a 133.0 133.0
13 120.2 8.94 (1H, s) 120.2 8.95 (1H, s)
13a 137.5 137.5
13b 120.5 120.5
9-OCH3 61.9 4.10 (3H, s) 61.9 4.09 (3H, s)
10-OCH3 57.0 4.07 (3H, s) 57.1 4.07 (3H, s)
O-CH2-O 102.1 6.17 (2H, s) 102.1 6.17 (2H, s)






&'(

)   

This works were partially supported by IFS grant (No. F/4613-1) and LIPI
Internal research fund (DIPA).


 

Agusta, A., S. Maehara, K. Ohashi, P. Simanjuntak and H. Shibuya, 2005, Stereo


selective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp.
isolated from a tea plant, Chem. Ôharm. Bull., 53 (12), 1565-1569.
Borges KB., WDS Borges, MT Pupo, PS Bonato. 2008. Stereoselective analysis of
thioridazine-2-sulfoxide and thioridazine-5-sulfoxide: An investigation of rac-
thioridazine biotransformation by some endophytic fungi, £. Ôharm. Biomed.
Anal., 46, 945-952
ChenTS., Xiaoh Li, Dan Bollag, Yeuh-Chuen Liu and Ching-jer Chang. 2001.
Biotransformation of taxol. Tetrahedron Letters, 42, 3787-3789.

c    


     
Faramarzi MA., M Aghelnekad, MT Yadzi, M Amini, N Hajarolasvadi. 2008.
Metabolism of andros-4-en-3,17-dione by filamentous fungus Neurospora crassa,
Steroids, 73, 13-18.
Ping Xu, Dongliang Hua, and Cuiqing Ma, 2007, Microbial transformation of
propenylbenzenes for natural flavour production. TRENDS in Biotechnolog , 25,
571-576.
Shibuya H., C Kitamura, S Maehara, M Nagahata, H Winarno, P Simanjuntak, HS Kim,
Y Wataya and K Ohashi. 2003. Transformation of Chincona alkaloids into 1-N-
oxide derivatives by endophytic  laria sp. isolated from Chincona pubescence.
Chem. Ôharm. Bull., 51, 71-74

c    


     -
    
  

   


)  ›
 
  

ANDRY PRATAMA, RUHYAT KARTASUDJANA1, TUTI WIDJASTUTI1


1)
Lecturer of Faculty of Animals Husbandry, Padjadjaran University


›


An experiment was conducted to examine the effect of early feed restriction on
the compensatory growth in broiler chickens. One hundred and fifty broilers were
distributed to six treatment groups, with 30 broilers for each group. Each treatment group
was represented by five replication of five broiler each. Broiler were fed ad libitum
through out the experiment as the control, and other five group were fed restriction R1
(20% feed restricted of ad libitum), R2 (25% feed restricted of ad libitum), R3(30% feed
restricted of ad libitum), R4 (35% feed restricted of ad libitum), R5 (40% feed restricted
of ad libitum). Broiler were fed ad libitum conventional broiler diets from 1 to 7 and 15
to 42 days of age. Feed restriction was applied to broiler from 8 to 14 day of age so as to
reduce their weight. The present result showed that broilers showed compensatory growth
when they were restrict-refed. There is not decreased in finally body weight in broiler
chickens which have restricted feed and have a similar feed conversion at all treatments.

 !"Restricted Feeding, Growth, Broiler Chickens


 

Poultry businesses in Indonesia at this time continues progress very rapidly, it can
be seen from the number of livestock population of broiler chickens in Indonesia. As can
be seen in data released by the Department of Animal Husbandry of West Java Province,
an increase of 16 percent from 2007, amounting 58.084.470 to 69.562.266 head in 2008.
The increase was due to broiler farm felt quite profitable. Government support in the
form of livestock regulations contained in the government rules number 15 of 1977
which set the rules and regulations set up poultry farms. Government participate and
provide make good impact on the progress of broiler chicken farm it self so it would
increase the passion broiler breeder chickens in Indonesia. In addition, public awareness
of the nutritional needs of especially those derived from animal protein also indirectly
supports the increase in population of these broiler chickens.
On the other hand, the increase of population is not always followed by an
increase in welfare of farmers, because it is still dependent of imported feed ingredients,

c    


     †
so the feed ration price will be high and not balanced with the selling price of products on
the market. Classical problems such as these an obstacle for broiler breeders themselves
because results in lower profits. We need a solution to deliver optimal results and
efficient for the farmer, such as by improving efficiency in managing maintenance.
One way method for giving diets to chickens is usually giving ad libitum diets
(always available) to grow rapidly. This method causes the diet rations of broiler
chickens will consume 10-20 percent more than it needs, which in will increase the cost
incurred for diet rations. In addition, excess feed consumption caused diet ration energy
consumed is not always converted into flesh but will be stockpiled in the form of the final
product is achieved body broilers with high fattening. This fatty will begin to occur at the
beginning of growth and if this fatty has been formed in the early days, it will give
unfavorable effects of the fatty greater when harvested.
One effort that can be done to improve production efficiency in broiler chickens is
by limiting the amount of diet rations given in the early phase of growth between the ages
of 8-14 days (age 1-7 days and after 14 days were given ad libitum ration). This
restriction aims to inhibit the growth hypertrophy happened on broiler chickens aged 7-14
days that resulted in slowing growth or enlargement of the cell network. This restriction
will not change the total number of cells (hyperplasia) but only inhibits cell enlargement
(hypertrophy) so that when given diet rations ad libitum again, the growth of both
hyperplasia and hypertrophy network will return to normal (Hood and Allen, 1977; Jones
and Farell, 1992) .
The diet of 70 percent of total ad libitum feed intake of broiler chickens were
obtained with the same final body weight in chickens given feed ad libitum, reduced
mortality and a more efficient in feed conversion (Wijtten., et al, 2008). Reduced feed
rations by 30 percent during the age of 8-14 days of ad libitum broilers led slows growth,
this is due to the available protein in the diet reduced so that the growth hypertrophy
(enlarged cells) is inhibited, but after the restriction period stops then will be
compensated by chicken with consume more feed were given normal diet (ad libitum)
again, so the chicken will eventually reach the same body weight of chickens fed ad
libitum rations.

c    


     o

  

This research was conducted in Laboratory Poultry Husbandry Faculty,
Padjadjaran University. The method used is an experiment with Completely Randomized
Design (CRD). The material used is one day broiler chickens with Cobb strain, vita
chick, therapy, ND-B1, IBD vaccine. The tools used include plots cage (1 x 1 m), feeder,
drinking water, sprayer, scales, heating lamps, needles, syringes of blood storage.
150 chicks were divided into 6 treatment groups. R0 l the diet ad libitum, R1 l
The diet ration of 80% of ad libitum, R2 l The diet ration of 75% of ad libitum, R3 l The
diet ration of 70% of ad libitum, R4 l The diet ration of 65% of ad libitum, and R5 l The
diet ration of 60% of ad libitum).
The parameters observed were the final body weight, blood hematology, and
FCR. Data were analyzed with ANOVA. If there is a difference of treatment followed by
a test of Duncan¶s Multiple Range Test (Gasper,2006).

  



&*#%›!)&'+,

Table 1.The average body weight of the End of Each Treatment Research

Treatment
Repeated
R0 R1 R2 R3 R4 R5
«««««««««««««...gram««..««««««««««««
1 2915,0 2685,0 2812,0 2865,0 2752,0 2844,0
2 3095,0 2785,0 2816,0 2700,0 2847,5,0 2892,0
3 2780,0 2700,0 2754,0 2820,0 2880,0 2686,0
4 2965,0 2897,5 2699,0 2612,0 2762,5 2555,0
5 2468,0 2708,0 2648,0 2746,0 2936,0 2708,0
Total 14223,0 13775,5 13729,0 13743,0 14178,0 13685,0
Average 2844,6 2755,1 2745,8 2748,6 2835,6 2737,0

Based on the variance analysis showed that the rations in the early growth
restriction did not significantly affect the final body weight (P <0.05). These results
indicate that the chicken had left to pursue growth in the reduction diet ration (at age 8-14
days). This can be explained because the chicken can compensate for the lagging growth
in the next phase by consuming more feed, so the chickens get more protein for growth.
This is consistent with the results of Wijtten., (2008) that restricting the diet at the age of
7-14 days to get the same final body weight with ad libitum feeding. These result same

c    


     D
with described by Leeson and Zubair (1996), when the chickens are treated ration
restriction, it will cause disruption of growth, but when the chickens get back normal
intake of nutrients the growth will come back normal again.
This phenomenon can be explained because the chicken consuming protein and
energy of diet rations less than their needs. Treatment R0 consume 12,74 grams of
protein/day, whereas treatment of R1, R2, R3, R4 and R5 consume 10,19; 9,55; 8,91;
8,28; and 7,64 grams of protein. The amount is not sufficient for weight gain in chickens.
Lack of this protein causes chicken experience delays in network growth. Protein
consumed just enough for basic living needs and growth of hair.
During feed restriction, body weight gain of broiler chickens in the second week
decreased by 9,09 to 27,67% when compared with the R0 treatment (ad libitum ration).
However on the fourth week, weight gain greater than weight gain R0 treatment (ad
libitum), this can be seen in the curve of weight gain. At the five week, each treatment
consumes almost the same protein that is equal to 31,78 grams of protein, but what sets it
apart is the use of these proteins. R0 consume protein to meet the needs of his hunger
while others consume ration treatment to fulfill the requirement of protein for tissue
growth is inhibited during treatment restriction. This is consistent with described by
Leeson and Zubair (1996), that the chickens are given preferential treatment in the early
ration restriction will try to restore its growth on the diet phase back to normal. This
means that the phase compensation actually be used to pursue growth in a previously
disadvantaged.
-#,%'›%!

able 2. The average value of Hematology Blood from Each Treatment Research On
Day-14

Parameters
Treatment Erythrocytes (per ml) Hb (g/%) Hematocrit (%)
Days-7 Days-14 Days-7 Days-14 Days-7 Days-14
R0 1,337 x105 1,531 x105 6,00 6,65 19,43 26,15
R1 1,130 x105 1,638 x105 4,75 7,00 19,83 27,15
5
R2 1,370 x10 1,569 x105 5,67 6,05 24,00 25,60
5
R3 1,277 x10 1,348 x105 5,99 6,45 20,17 23,95
5
R4 1,017 x10 1,490 x105 4,67 7,85 20,13 29,65
5
R5 1,183 x10 1,362 x105 5,33 7,05 22,50 27,50


c    


     u
Based on statistical analysis showed that diet restriction had no significant effect
(p <0.05) in the amount of blood erythrocytes. Described by Koch (1973) and Sturkie
(1986) the number of red blood cells (erythrocytes) in the immature birds are more
variable and it ranged between 3-4 million or an average of 3.5 million per ml of blood.
So the number of blood cells in this study are still well below the normal amount of R0 to
R5. Nevertheless, in Table 2.seen a decline in the number of blood erythrocytes in the
treatment diet restrictions when compared to R0 treatment (ad libitum feed ration). This
is due to diet restriction treatment that has been done which caused the decrease in
erythrocyte formation process. As a result of the reduced number of red blood cells then
caused decrease supply of oxygen (O2) from the lungs to the tissues and carbon dioxide
(CO2) from tissues to the lungs carrier metabolism waste products, so that the formation
of ATP is reduced.
This means that the number of red blood cells and hemoglobin in the normal
range will be followed by an increase in growth (body weight). When the number of red
blood cells and hemoglobin low it will have an impact on low body weight. This result is
in accordance with the mechanism of "compensatory growth" that described by Leeson
and Zubair (1996), which states ration restriction will cause poor growth, in this case
hyperplasia relation to the growth of blood cells.
So do with blood hematocrit. Siswani (2006), report that blood hematocrit values
of maximum growth is at a certain value. This means that when the blood hematocrit
value is low, there will be an impact of low growth, but on the contrary if the hematocrit
value is high then the growth will also be low. This is because if the high hematocrit
value then causes the blood viscosity will increase, so that transport nutrients through the
blood becomes low.

!*. "&*

Table 3.Average Conversion Rations of Each Treatment Research In Week-six

Time Treatment
(weeks) R0 R1 R2 R3 R4 R5
1 1,47 1,64 1,78 1,53 1,59 1,69
2 1,60 1,44 1,32 1,38 1,42 1,33
3 1,42 1,40 1,37 1,37 1,19 1,27
4 1,47 1,48 1,47 1,42 1,36 1,49
5 1,69 1,86 1,59 1,87 1,77 1,81

c    


     uu
6 2,24 2,40 2,49 2,57 2,30 2,53

Based on the results of variance analyzes showed that diet restriction had no significant
effect (P <0.05) to feed conversion values. These results are in accordance with the
results obtained Wijtten., (2008) and Yagoub and Babiker (2008) which describes the
influence of diet restriction treatment did not significantly affect the value of feed
conversion. This can be explained, when the time of giving back to normal rations, the
chickens will consume more feed. This is because chickens need more nutrition to restore
or pursue growth retardation that has occurred during the diet restriction. This is
consistent with the opinion Yagoub and Babiker (2008) that the chickens will consume
more feed to meet the needs of the protein. This makes no difference in feed conversion
value at the end of the study.

 
Based on the results and discussion of this research, it can be taken to a
conclusion as follows:
1. Restricted feeding on early growth (8-14 days) can reduce the weight of chickens in
the second week but did not lead to lower final body weight in broiler chickens
reared for 42 days.
2. 30 percent restriction (70 percent of diet ad libitum) is the best level in the system
restriction feeding on broiler chickens reared 42 days.



 
Gaspersz, V. 2006.Teknik Analisis Dalam Ôenelitian Ôercobaan. Penerbit Tarsoto.
Bandung.
Hood, R.L. and Allen, C.E. 1977. Cellularit of Adipose Tissue: Effect of Growth and
Adiposit . £ournal of Lipid Research. 18: 275-284.
Jones, G.P.D. and Farrell, D.J. 1992. Earl -life Food Restriction of Chicken. II. Effect of
Food Restriction on the Development of Fat Tissue. British Poultry Science 33:
589-601.
Koch, T., 1973.Anatomi of The Chicken and Domenstic Birds. The Iowa State University
Press. Ames. Iowa.
Leeson. S., and A. K. Zubair.. 1996. Compensator growth in the broiler chicken : a
review. Worlds Poult. Sci. J. 52:189-201.

c    


     u*
Siswani.2006. Gambaran Darah Merah dan Ôertumbuhan Mandalung (Mule Duck) ang
Disuplementas Vitamin C. Fakultas Kedokteran Hewan.Institut Pertanian Bogor.
Sturkie, P.D., P. Griminger. 1986. Blood : Ôh sical Characteristics, Formed, Elements,
Hemoglobin, and Coagulation in Avian Ôh siolog . Springer Verlag. New York.
Wijtten, P. J. A., Hangoor, E., Perdok, H. B., and Sparla, J. K. W. M. 2008. The Effect of
Earl Life Feed Restriction on Ôerformance and Mortilit of Male Broilers.
World Poult.Congress.
Yagoub, M. Yagoub dan Babiker, S.A. 2008.Effect of compensatory Growth on The
Performance Carcass Charasteristics of The Broiler Chicks. Pakistan Journal of
Nutrition 7 (3) : 497-499.
 

c    


     u
›
           /
  ›
 
      


 
     

 

   0 

NESTI F. SIANIPAR1, GUSTAAF A. WATTIMENA2, MAGGY THENAWIJAYA S3.


HAJRIAL ASWIDINNOOR2, NURITA TORUAN-MATHIUS4 , GALE GINTING5

1
Department of Biology, Faculty of Science and Mathematics, Universitas Pelita Harapan,
Tangerang
2
Department of Agronomy, Bogor Agricultural University, Bogor
3
Department of Food Science and Technology, Bogor Agricultural University, Bogor4SEAMEO
Biotrop, Bogor
5
Oil PalmResearchCenter of Indonesia, Medan

›


Abnormality of somatic embryo (SE) can be resulted from genetic or epigenetic
changes. Thus, genetic changes can be analyzed by Random Amplified Ôol morphism
DNA(RAPD) and Randoml Amplified DNA Fingerprinting (RAF) techniques. The
objective of this research was to study the genetic changes of SE at several growth stages
and their plantlets using RAPD and RAF. Through RAPD technique the SE normal and
abnormal cotyledon could be distinguished using five primers (OPE-14,OPC-9, W-15,
AP-20 and SC10-19) at clone 638, while the SE normal and abnormal cotyledon at clone
558 could be distinguished using three primers (OPE-14, W-15 and AP-20).Among these
five primers pre-mentioned above, three primers, such as OPE-14, W-15 and AP-20 were
differentin the SE normal and abnormal cotyledon at clone 638, at a specific band of
1750 bp. Furthermore, the RAF technique detected the changes of DNA genome at 90 ±
358 bp. There were three amongstsix primers (AO-12, BB-18, W-15) genomic DNA
sequences were detected at 150 bp. Moreover, the primer AB16 could detect the
sequence DNA change of SE normal and abnormal cotyledon until one base pair at 95 bp.
Consequently, RAPD and RAF technique with the primers W 15 and AP 20 could
distinguish normal and abnormal SE cotyledon, plantlet and normal mother plant.

  !": somatic-embr oabnormalit , oil-palm, RAÔD, Randoml Amplified DNA
Fingerprinting (RAF).


 
Oil Palm (Elaeis guineensis) is a kind of cross breeding plants which its offspring
produced heterozygous. However, derived plants of oil palm can be produced
homozygous by such technique of tissue culture. There is a series of advantages of tissue
culture method, such as produceshigh similarity between the derivatives and mother plant

c    


     u
in a period of short time and in a large amount. Propagation of oil palm seedlings
through tissue culture can be done through the somatic embryo formation. However,
some of the resulted derivativessometimes show a certain high somaclonal variation
which can be investigated through an abnormality in cytology, mutation genotype,
karyotype changes and changes in DNA sequence(Duncan 1997; Kaeppler et al.,
2000).In addition to that, according to Deambrogio & Dale (1980) somaclonal variation
can be inducedby addition of a high concentration of 2,4 D during the process.
Somaclonal variability is the genetic diversity of plants produced during tissue
culture (Larkin & Scowcroft 1981).Genetic diversity occurs in tissue culture is caused by
doubling the number of chromosomes (fusion, endomitosis), chromosome structure
changes, gene changes and cytoplasm changes (Griffith et al. 1993; Kumar 1995).
Furthermore, according to van Harten (1998), somaclonal variation may be caused by
mitotic irregular, the role of chromosome instability and gene deletion.
There are several opinions about the occurrence of a genetic abnormality (Rao
and Daugh, 1990), such as disruption of gene expression caused by phytohormone (Jones
1991; Paranjothy et.al. 1993), callus structure is in compact calli, nodular calli or crumbs
calli with its the rapid growth produces 5 - 10 % and 100 % abnormal plants (Pannetier
et al. 1981; Duran et al. 1993).According Ginting and Fatmawati (1996)which their study
corresponded with Paranjothy et al. (1993), abnormalities have relation with the length
and age of callus subculture.Eeuwens et al. (2002) suggested that mantled flowers can be
occurred during culture condition in the embryo multiplication. Abnormalities can also
occur in male and female flowers that will be developed intoparthenorcarpy fruit or
mantled fruit (Corley et al. 1986).
DNA analysis is one of the molecular biologyapproaches to identify a genotype.
The main advantage of genotype analysis is DNA level changes with genetic distance
between individual with another can be investigated(Serret et al. 1997). Recently, the
development of a new technology has been able to develop the DNA polymorphism
analysis for genetic mapping, MAS, genomic fingerprinting. These technologies include
RFLP, RAPD, AFLP and microsatellite (SSR).Randomly Amplified DNA fingerprinting
(RAF) is the primary amplification technique based on arbitrary (Waldron et al. 2002)
RAF (Randomly Amplified DNA fingerprinting) is based on the technique of
amplification of arbitraryprimers(Waldron et al. 2002). RAF technique is similar to the

c    


     u
DAF. Advantages of RAF over previous protocols are its robustness and reliability, no
requirement for highly-purified DNA template, relatively requires few steps,the
opportunity for sensitive detection via-radio-labeling or fluorescent tagging, more
markers being simultaneously detected, and the ability to identify codominant loci
(Waldron et al. 2002).
Randomly amplified polymorphic DNA (RAPD) technique is one of the most
frequently techniques used for molecular method. RAPD technique has been widely used
to assist activities of plant breeding, including analysis of genetic variation and similarity
in various organisms. The application of molecular markers has been developed to
overcome problems such as morphological markers, cytology, histology and
biochemistry (Akagi et al. 1996; Ayers et al. 1997).
Hence, the objective of this research was to study the sequence changes of DNA
genome from somatic embryo at several growth stages and their plantlets using RAPD
and RAF.


  

%#*,#, &#%: Normal mother plantand somatic embryo of oil palm clone MK 558 and
MK 638 were obtained from Oil Palm Research of Indonesia (PPKS), Medan.
1, #2,&*: DNAs for RAPD and RAF analysis were extracted from young leaves
of normal mother plant and somatic embryo of oil palm using modified method of
Doyle& Doyle (1990).

 ""#DNA of each sample is used for PCR analysis, respectively by employing


10 primer10-mer with the composition of sequences shown on Table 1. Amplification
reaction wascarried outat volume of 25 ul. Composition of PCR analysis is as follows:
5.0 ul DNA template (5 ng/ul) ,0.2 ul dNTP, 2.5 ul PCR buffer (10X) + Mg Cl2, 1.0 ul
primer (10 pmal/ul), 0.2 ul Taq Polymerase (5U/ul), and 16.1 ul ddH2O.The
amplification was performed using aThermal Cycler Gene PCR (ABI 9700).The mixture
was pre-denaturated at 94°C for 4 min. The PCR conditions were as follows: 45 cycles at
94oC for 1 min for denaturation, 36oC for 1 min for annealing, 72oC for 2 min for
extension and 72oC for 4 min for post-extension.

c    


     u-
Amplified products were analyzed by electrophoresis. PCR results could be then
fractionated by using agarose gel 1.4% (w/v) in 40 ml 1X TAE solution. Electrophoresis
wascarried out at 75 volts for 1.5 h. Results of electrophoresis were documented with
Kodak Logic Dott Software.

 ""# The sampleswere somatic embryo and normal mother plant of clone MK
638. The RAF technique was performed according to the general steps as described by
Waldron et al., (2002). Each reaction volume of 10 uL cointained 1x PCR buffer (10mM
Tris pH 8.0, 10mM KCl, 5 mM MgCl2), 20uM dNTPs, 1,5 units AmpliTaqI Stoffel
Fagment DNA polymerase, 1 uCi Į-labelled 33P-dATP, and 5 uM single oligonucleotides
(Operon Technologies Inc.A kits) and 50 ng of genomic DNA template. PCR was
performed with a hot start (85oC), an initial denaturation at 94 oC for 5 min, followed by
30 amplification cycles of : 94 oC for 30s, 60s each at 57 oC,56oC, 55oC,54oC,53oC and
final extension step at 72 oC for 5 min. Eight primers were initially tested.
Furthermore, DNA analyzer (3130 DNA Analyser - Applied Biosystems) was
used to find DNA fragments. GeneScanTM-500LIZwas used as size Standard. As much as
2 ul sample of PCR aliquot result was then used and added with 0,2 ul GeneScanTM-
500LIZ and 7.8 uL HiDi formamide, denatured at 95oC for 5 min and finally cooled on
ice.
Table 1.Primer sequences from Operon Technologies Inc.
No Primer Primer sequence (5¶ ± 3¶)
1. AB-16 5¶-CCCGGATGGT-3¶
2. AE-11 5¶-AAGACCGGGA-3¶
3. AO-12 5¶-TCCCGGTCTC-3
4. AP-20 5¶-CCCGGATACA-3¶
5. BB-18 5¶-CAACCGGTCT-3¶
6. W-15 5¶-ACACCGGAAC-3¶
7. OPB-06 5¶-TGCTCTGCCC-3¶
8. OPC-02 5¶-GTGAGGCCTC-3¶
9. OPC-08 5¶-TGGACCGGTG-3¶
10. OPC-09 5¶-CTCACCGTCC-3¶
11. OPE-14 5¶-TGCGGCTGAG-3¶
12. SC-10-19 5¶-CGTCCGTCAG-3¶

  


V
 ""#

c    


     u†
The results of RAPD analysis on clone MK 638 and MK 558 produced polymorphic
pattern band that could differentiatenormal SE cotyledon and abnormal SE cotyledon.
Specifically on clone MK 638, both normal and abnormal SE cotyledon could be
distinguished using Primer OPE-14 with DNA fragment were at 3500 bp, 2250 bp, 1750
bp, 1400 bp and 850 bp. Similarity between primer OPE-14, W-15, AP-20 was that they
could distinguish either normal SE cotyledon or abnormal Se cotyledon at DNA fragment
of 1750 bp. Moreover, Primer SC-10-19 could distinguish normal and abnormal SE
globular at DNA fragment of 5000 bp, 4000bp, 3000 bp and 2250 bp.
However, as compared to clone MK 638, on clone MK558 differentiation between
normal and abnormal SE cotyledon using primers pre-mentioned above occurred at
different DNA fragments. For instance, OPE-14 could distinguish both normal and
abnormal SE cotyledon at the DNA fragment f 2500 bp, 3000 bp. Whilst primer AP-20
produced DNA fragment at 3000bp, 2000bp and 1750 bp and primer W-15 produced
DNA fragment DNA at 5000 bp and 2500 bp (Table 1 and Figure 1 and 2).
Consequently, using primers OPE-14, OPC-09, AP-20 and SC-10-19 could
producepolymorphic fragments of DNA for either plantlet or mother plantleaves on clone
MK 638 and MK 558.
According to Grattapaglia et al.,(1992), the numberof polymorphic DNAbands shows
the state of the plant genome, whereas the differences in the number and polymorphic
fragment DNA generated by every primer show the complexity of plant
genomes.Moreover, Phillips et al. (1990) suggested plants regenerated from callus and
relatively non-differentiated causes the possibility of a large number ofgenetic changes.
This study corresponds with those, which could be explained from the results in which
Primer OPE-14 and SC-10-19 produced mostly polymorphic bands on the SE cotyledon.
M Gn Gab Kn Kab P Dn Kn Kab Gn Gab P

10.000

3.000
2.000

1.000

500 
   
c    uo
Fig. 1. Primer W-18. M (marker 1kb), Clone 558 (lane 2-7) ; Normal globular somatic
embrio (Gn), Abnormal Globular (Gab), Normal cotyledon (Kn), Abnormal
cotyledon (Kab), Planlet (P), Leaves of mother plant (Dn). Clone 638 (lane 8 -12)
; Normal cotyledon somatic embryo (Kn), Abnormal cotyledon (Kab), Normal
Globular (Gn), Abnormal Globular (Gab), Planlet (P).

M Kn Kab Gn Gab P Kn Kab Gab P Gn Dn

10.000
3.000
1.500
1.000

250

Figure 2. Primer of SC 10-19. M (marker 1kb), Clone 558 (lane 2-6) ; Normal globular
somatic embrio (Gn), Abnormal Globular (Gab), Normal cotyledon (Kn),
Abnormal cotyledon (Kab), Planlet (P). Clone 638 (Lane 7 -12) ; Normal
cotyledon somatic embryo (Kn), Abnormal cotyledon (Kab), Abnormal SE
Globular (Gn), Planlet (P), Normal SE globular and Leaves of mother plant
(Dn).

Jones (1991) and Paranjothy et al. (1993) statedthe abnormality is occurredon oil
palm clonethat caused by gene expression changes. Abnormality is found on flowering of
oil palm clone that is usually caused by2.4- D as a plant growth regulator. The aim of
addition of 2,4 -Dinto the media is to induce callus and embryonic formation and has
been reported by Gintinget al.(1991) at Marihat Research Institute that indicates the
propagation of oil palm clones with addition of 2.4 D.Aside from addition of 2,4 -Dinto
the media, Edwin (1993) reportedsomaclonal variation causedbyabnormalities could also
occur due to utilization of mercury chloride as solvent/solution for the explants
sterilization.High concentration of mercury chloride could cause amino acid
compositions of plant tissue change and resulting in disruption of protein synthesis.
Tabel 1. Fragment polymorphisms by RAPD technique onMK638 dan MK 558 clone.

c    


     uD
DNA fragment Clone 638 Clone 558
pb OPE14 (5¶-TGCGGCTGAG-3¶) OPE14 (5¶-TGCGGCTGAG-3¶)
G+ G- K+ K- D DN G+ G- K+ K- D DN

1. 3500 + + '- + + + + '+ +


2. 3000) + + + + + + + + '+ +
3. - + + + + + + + + + + + +
4. 2250 + + ' + + + + + + + +
5. 1750 + + ' + - + + + + + -
6. 1400 - - - ' - + - - - - - +
7. + + + + + + + + + + + +
8. 850 - - ' - - - - - - - -
9. 750 - - - - - + - - - - - -
DNA W-15 (5¶-ACACCGGAAC-3¶) W-15 (5¶-ACACCGGAAC-3¶)
fragmentpb G+ G- K+ K- D DN G+ G- K+ K- D DN

1. 5000 + + - - + - + + - + + -
2. 2500 - - - - + - + + ' + -
3. + + + + + - + + + + + -
4. - - - - - + + + + + + +
5. - - + + + - + + + + + -
6. 1750 - - + - - - + + + + + -
7. 1500 + + + - + - + + + + + -
8. - - - - - + - - - - + +
9. - - + + - + - - - - - +
10. + + - - + - - - - - - -
DNA fragment OPC-09(5¶CTCAACGTCC-3¶) OPC-09(5¶CTCAACGTCC-3¶)
pb G+ G- K+ K- D DN G+ G- K+ K- D DN

c    


     *
1.3250 - - ' - - + + + + + -
2. + + + + - - + + + + + -
3.3000 + + + + + + + + + + + +
4. 2000 - - + - + + + + + + +
5. + + + + - - + + + + + +
6. - - - - - - - - - - - +
7. + + + + - - + + + + + -
8. - - - - - - - - - - - +
DNA AP-20(5¶-CCCGGATACA-3¶) AP-20(5¶-CCCGGATACA-3¶)
fragmentpb G+ G- K+ K- D DN G+ G- K+ K- D DN

1. 3000 - - ' - - - - ' - -


2. 2000 - - -+ - - - - + - - -
3. 1750 - - -+ - - - - +- - -
4. + + + + + + + + - ' + +
5. + + + + + + + + ' + +
6. 750 + + + + + - + + + + + -
7. + + + + + + + + + + + +
DNA SC10-19 (5¶-SGTCCGTCAG-3¶) SC10-19 (5¶-SGTCCGTCAG-3¶)
fragmentpb G+ G- K+ K- D DN G+ G- K+ K- D DN

1. 5000 - + ' -' - + + + + + -


2. 4000 - + + -+ - + + + + + -
3. 3000 - + + - + + + + + + + +
4. 2250 - + + -' + + + + + -
5. 2000 + - + + - - + + + + + -
6. - - + + - - + + + + + -

d 
: Band (+), No band (-), Normal Globular (G+), Abnormal Globular (G-), Normal
Cotyledon (K+), Cotyledon(K-), Plantlet leaves (D), Motherplant leaves (DN).

›
""#

c    


     *u
Through RAF analysis, there were six DNA genomes were amplified using
primers. Those could be amplified by Primer AB-16 (5'-CCCGGATGGT-3 '), whereas
using primer AO12 (5'-TCCCGGTCTC-3') produced at least one fragment DNA.
Furthermore, most polymorphic fragments could be amplified with primer AP-20 (5¶-
CCCGGATACA-3¶) (Picture 3 dan Tabel 2).
Normal and abnormal SE cotyledoncould be distinguished due to a change in
sequence of DNA genome. DNA genome of normal and abnormal SE cotyledon,
plantletleaves and mother plantleaveswere amplified using primers AB-16, AE-11, AP-
20, AO-12, BB-18 and W-15. SE cotyledon could produce 1-7 fragments from each
primer with total of 24 fragments and size of 90 -358 bp. Changes in the DNA sequences
of normal and abnormal cotyledon SE could be detected at approximately 90 bp, 91bp
and 146 bp using Primer AB-16. The primer AO-12 produced one polymorphic fragment
at 150 bp, while others, such as primer AE-11, AP-20, BB-18 and W-15 produced two
polymorphic fragments(Table 2). Moreover, the Primer AO-12, W-15 and BB-20 could
distinguish both normal and abnormal SE cotyledons at a specific fragment of about 150
bp (Table 2).













c    


     **
Fig.3. Fluorecence detection technique of RAF with primer AB-16 (5¶-CCCGGATGGT-
3¶).Labeled by FAM (6-carboxy-fluorecein) on oil palm plant clone 638. The
normal mother plant DNA genome (EG), Normal SE globular (2G+), Abnormal
SE globular (2G-),Normal SE cotyledon (2K+), Abnormal SE cotyledon (2K-)
and Plantlet leaves (2D).

Form the results, both RAF and RAPD techniquesemployed the same primers (W-
15 and AP-20) to produce polymorphic amplification. The results of RAF analysis could
be detected at around 100-400 bp and until at level of single-nucleotide base difference
using electrograph (Fig.3), whilst RAPD analysis produced polymorphic bands at 1500-
5000 bp (Fig. 1 and 2). The results of RAPD and RAF showed a change in normal and
abnormal SE cotyledon of plantlet and mother plantleaves (Fig 1 and 2). The Results
RAF using primers W-15 produced a change in DNA sequence at 150 bp, whereas the
RAPD analysis using primer W-15 produced a change DNA sequence at 1750 bp (Table
1 and 2).The RAF technique could detect DNA sequence changes until 1-
10bp.Thechanges of DNA sequence that could detect the location until one base called
mutation. Conclusively, the morphology change of normal SE cotyledon became
abnormal cotyledon could possibly be caused by changes in DNA sequence at specific
sites (Table 2). 
Table 2. Fragment polymorphisms by RAF technique on MK638 clone

Fragmen AB- 16 (5¶-CCCGkasiGATGGT-3¶) AO-12 (5¶-TCCCGGTCTC-3¶)


DNA 2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN
(pb)
90 - + - - - + 150 - + - - - +
91 + - + + + - 160 + - + + + -
138 + + + + + -
146 - + - - - +
248 + + + + + -
258 + + + + + -
375 + + + + + -
378 - - - - - +
380 + + - - - -

c    


     *
Fragmen AE-11(5¶-AAGACCGGGA-3¶) BB- (5¶-CAACCGGTCT-3¶)
DNA 18
(pb) 2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN
152 - + - - - + 90 - + - - - +
158 + - + + + - 150 - + - - - +
369 - - - - + - 158 + - + + + -
372 + + + + + +
Fragmen AP-20 (5¶-CCCGGATACA-3¶) W-15(5¶-ACACCGGAAC-3¶)
DNA (pb) 2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN
115 - + - - - - 150 - + - - - +
155 - + - - - + 214 + + + + + -
238 + + + + + - 217 + + + + + -
355 + - + + + - 255 - - - - - +
358 + - + + + - 258 - - - - - +
Keterangan : Fragment (+), Fragment (-), Normal Globular (G+), Abnormal Globular
(G-), Normal Cotiledon (K+), Abnormal Cotiledon (K-), Planlet Leaves
(D), Mother plant leaves (DN).
 
 In conclusion that RAPD technique showed similarity between primer OPE-14,
W-15, AP-20 was that they could distinguish either normal SE cotyledon or abnormal SE
cotyledon at DNA fragment of 1750 bp on clone MK638. The RAF technique detected
the changes of DNA genome at 90 ± 358 bp. The Primer AO-12, W-15 and BB-20 could
distinguish both normal and abnormal SE cotyledons at a specific fragment of about 150
bp by using RAF technique.There were three amongstsix primers (AO-12, BB-18, W-15)
genomic DNA sequences were detected at 150 bp. Consequently, RAPD and RAF
technique with the primers W 15 and AP 20 could distinguish normal and abnormal SE
cotyledon, plantlet and normal mother plant.

)  


The authors would like to thank the Oil Palm Research Center of Indonesia
(PPKS) for providing somatic embryo and mother plant samples. We also would like to
thank Dr. Rudy Lukman for his permission to work with RAF technique in the
laboratory of the Biotechnology Department at PT. BISI International Tbk.

c    
     *


 
Akagi H, Yokozeki Y, Inagaki A, Fujimura T. 1996. Microsatellite DNA markers for rice
chromosomes. Theor.Appl.Genet. 93 : 1071-1077.
Ayers NM et al. 1997. Microsatellites and single ± nukleotide polymorphism differentiate
apparent amylose classes in an extended pedigree of US rice germ plasm. Theor
Appl. Genet. 94 : 775 -780.
Corley RHV, Lee CH, Law LH, Wong CY. 1986. Abnormal flower development in oil
palm clones. Ôlanter (Kuala lumpur) 62 : 233-240.
Deambrogio E, Dale PI. 1980. Effect of 2,4 D on the frequency of regenerated plant in
barley (Hordeum vulgare) cultivar 'Akka' and on genetic variability between Them.
Cereal Res. Commun 8 : 417 - 424.
Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fersh
leaf tissue. Ôh tochem Bull. 19 : 11 -15.
Duncan RR. 1997. Tissue culture-induced variation and crop improvement. Adv. Agron.
58 : 201- 240.
Edwin FG. 1993. Plant Propagation by Tissue Culture. Part 1: 2nd Edition. Exegetics Ltd.
Edington, Wilts-England. p.420-425.
Ginting G, Fatmawati. 1997. Suspensi sel pada kelapa sawit (Elaeis guineensis Jacq.). £.
Ôen. Kelapa Sawit 5 (3) : 153 -160.
Ginting G, Lubis RA, AU Lubis. 1991. Kultur jaringan kelapa sawit (Elaeis guineensis)
di Pusat Penelitian Perkebunan Marihat. Seminar Bioteknologi Perkebunan dan
Lokakarya Biopolimer PAU-IPB, Bogor. P.115-119.
Grattapaglia D et al. 1992. Mapping in Woody Plant with RAPD Marker: Application to
Breeding in Forestry nd horticulture. P 37 -40. In : Aplication of RAPD
Technology to Plant Breeding. Joint Plant Breeding Symposia Series
CSSA/ASHS/AGA, Minneapolis, MN.
Grifftihs AJF, Suzuki J, Miller H, Lewontin RC. 1993. An Introduction to genetic
Analysis. Fifth edition. W.H. freeman and Co. 840p.
Jones LH. 1991. Endogenous cytokinin in oil palm (Elaeis guineensis Jacq.) callus,
embryoids and regenerant plants measured by radioimmunoassay. Ôlant Cell Tiss.
Org. Cult 20 : 201 - 209.
Kaeppler SM, Kaeppler HF, Rhee Y. 2000. Epigenetic aspects of somaclonal variation in
plants. Ôlant Mol Biol 43 : 179 -188.
Kumar A. 1995. Somaklonal variation and molecular genetics department. Scottish Crop
Research Institute, Invergrowrie Dundee. Canada. p. 197-212.
Larkin PJ, Scowcroft WR. 1981. Somaclonal variation-a novel source of variability from
cell culture for plant improvement. Theor.Appl.gen. 60 : 197 -214.
Paranjothy K, Othman R, Tan CC, Wang G, Soh AC.1993. Incidence of abnormalities in
relation to in vitro protocols. Di dalam : Rao V. et al, editor. Recent Devin Oil
Ôalm Tissue Cult & Biotechnol. Kuala Lumpur, p. 131 -141.

c    


     *
Pannetier C, Arthius P, LievouxDE, 1981. Neoformation de jeuneus planlets dÈElaeis
guineensis a partier de eals primaries obttenus sur fragments foliaires in vitro.
Oleagineux 36: 119 - 122.
Phillips RL, Plunkett DJ, Kaeppler SM. 1990. Do we understand somaclonal variation?
Di dalam : H.J.J. Nijkamp et al. editor. Progress in Plant Cellular and Molecular
Biology. Proc. 7th Internat. Cong. Plant Tiss. Cell Cult., p. 131 ± 14.
Serret ND, Udupa SM, and Weigand F. 1997. Assement of genetic diversity of
cultivated chickpea using microsatellite-derivate RFLP markers implication for
origin. Ôlant Breed. 116 : 573-578.
Van Harten AM. 1998. Mutation Breeding. Theory and Practikal Applications.
CambribgeUniv. Press. Wong, G., S.P. Chong, C.C tan, and A.C. Soh. 1999. Liquid
Suspension culture-A potential technique for mass production of oil palm clones.
Palm oil Res. Inst. Of Malaysia. P: 3-10.
Waldron J, at al. 2002. Randomly amplified DNA fingerprinting : a culmination of DNA
marker technologies based on arbitrarily-primed PCR amplifcation. £ Biomed and
Biotech 2 (3) : 141-150.

c    


     *-
 3           
 
›)   
  

NOOR ARIDA FAUZANA1), RATU SAFITRI2), SUKAYA SASTRAWIBAWA3)

1) Fisheries Faculty, Lambung Mangkurat University, Banjarbaru
2) Departement of Biology. FMIPA, Padjadjaran University, Bandung
3) Fisheries and Marine Science Faculty, Padjadjaran University, Bandung

›


The aims of this reesearch was to determine the type of fungus that can grow and
degrade cellulose has the potential banana waste. The research conducted by carrying out
isolation and identification and selection of cellulolytic fungi from banana waste. Banana
waste has a fairly complete nutritional content is to be a potential raw material for fish
feed. However, it is containinghigh cellulose that are not easily digested by fish, it is
necessary to need the microorganisms in the process. The research was conducted using
the descriptive method. Isolation of fungi with dilutionseries method on PDA and SDA
medium and identification with the moist chamber method. Cellulolytic fungi selection
through iodine test by looking at the diameter ratio of transparent zone and colony
diameter.
The result showed that total of 6 fungi were isolated from banana waste :
Ôenicillium nalgiovense Laxa, Aspergillus ochraceus Wilhelm, Aspergillus nidulans,
Aspergillus terreus, Aspergillus niger, andAspergillus or ae.Aspergillus terreus
andAspergillus or aemore potential degrade cellulose banana waste than other fungi that
found.

 !"cellulol tic fungi, banana waste





 
Banana waste is a potential as source of vegetable protein fish food because it
contains a fairly complete nutrition. The result of proximate analysis at the Laboratory of
Livestock Ruminant Nutrition and Food Chemistry Faculty of Animal Husbandry,
Universitas Padjadjaran (2009) content of nutrients and energy of a banana waste is as
follows: protein 10.09%, 18.01% crude fiber, crude fat 5.17 %, BETN 55.59%, Calcium
0.36%, 0.10% phosphorus and gross energy 3727 kcal / kg. Based on the analysis of van
Soest banana waste contains 27.36% ADF, NDF 39.27%, lignin20.21%, cellulose 7,15%
and hemicellulose 9.91%. Banana waste also contains tannin 0.0967%. The presence of
mineral and protein content is high enough waste illustrates the potential banana waste
can be used as an alternative feed ingredient for fish. The main problem using banana

c    


     *†
waste as a feed ingredient is content the high crude fiber; so it is difficult to digest.
Efforts to increase the value of the benefits of banana waste can be done through a
service known microbial fermentation process. Fungus have a greater ability to
degrading cellulose, it is necessary for reasons of isolation and selection of fungi from
banana waste.
The aims of this research was to determine 1) type of fungus that can grow on a
banana waste, 2) isolates fungus that have the potential to degrade cellulose of banana
waste, and 3) isolates the most potentialfungus to degrading cellulose of banana waste.


  

#, &#%"#*! 45&6-*,

The materials used in this research is the banana waste , distilled water, iodine
solution, physiological NaCl 0.9%, distilled water, PDA (potatoes dextrose agar), SDA
(Sabourad Dextrose Agar) methylated, and tetracycline. The tools used in this research
include autoclave, glass beaker, Bunsen, Petri dishes, measuring cups, incubator, needle
inoculation, labels, cotton, filter paper, analytical balance, erlenmeyer, ose, test tube rack,
test tube, microscope, mikropipet, pipettes, and a spatula.
2!5 
Isolation of Fungi
The research method begins with sampling of the banana waste that already retted,
whereas to isolate the fungus is through Dilution Series, it is expected fungus contained
in the sample can be grown, separated, isolated and subsequently on PDA and SDA
medium.
Identification of Fungi
After the isolates obtained, to identify the fungus through the moist chamber method.
Fungi identification is then performed by observing colony characteristics and
morphology of fungi with the help of fungi identification book.
Selection of cellulol tic fungi
Each isolate of fungi was inoculated on PDA medium, and incubated at room
temperature for 24 hours. At the end of the incubation medium surface drops iodine

c    


     *o
solution. If the fungus has the ability cellulolytic it will show the transparent zone around
the colony isolates of the fungus.




  


T pes of Fungi
There are 6 types of fungi isolates were isolated from banana waste, screening fungus in
PDA and SDA medium, (Figure 1 dan Figure 2).


Dilution 10-3 Dilution 10-4 Dilution 10-5



Picture 1. Screening Fungus in PDA medium


Dilution 10-3 Dilution 10-4 Dilution10-5

Picture 2. Screening Fungus in SDA medium

Six isolates of the fungus are as follows:


Isolate 1
This Isolate were identified as Ôenicillium nalgiovense Laxa , with the characteristics:
colony diameter from 2.5 to 3.5 cm for 5 to 7 days. Colonies are white and sometimes
gradually become pale green. Having a yellow-green conidiophores. Conidiophores have
two to three or even more branches, have hyalin. Conidia globose to subglobose, smooth,
hyalin, 3-4 ȝm.

c    


     *D
Isolate 2
Fungi isolate 2 were identified as Aspergillus ochraceus Wilhelm, with the
characteristics: colony diameter from 2.5 to 3.5 cm in 5-7 days. Colonies brown-black
with white side. Konidiophor solid yellow to brown. Head of young konidiophor globose
shaped and spread to two or more groups of columns. Konidiphor height 1.5 mm, yellow
to brown and rough-walled. Vesicles, globosa, with hyalin measuring 15-20 x ȝm.
Conidia globosa-subglobosa, hyalin-walled with rough or slippery.
Isolate 3
Fungi isolate 3 identified as Aspergillus nidulans, with the characteristics: colonies have
a white edge, having grown in 5 days time. Conidiophore brown. Head conidia compact
and columnar shaped.
Isolate 4
Fungi isolate 4 were identified as Aspergillus terreus, with the characteristics: colony
diameter from 3.5 to 5.0 cm in 5 to 7 days is green, generally consists of conidiophore
solid yellow brown, dark-colored growing increasingly dark. Head conidia compact,
high-columna generally 150-500 x 30-50 ȝm. Conidiophore hyalin, smooth-walled,
subglobose vesicles 10-20 ȝm. Phialide derived from metule 5-7 x 2.0 to 2.5 ȝm. Metulae
5.5 x 1.5 to 2.0. Conidia globose to ellipsodial 1.5 to 2.5 ȝm, yellow slippery hyalin.
Isolate 5
Fungi isolate 5 identified as Aspergillus niger , with the characteristics: colony reached a
diameter of 4-5cm in 5 to 7 days and consists of a compact basal layer of white to yellow
and a wide layer of conidiophores old ciklat colored to black. Head-colored conidia
Hiam, round, and tend to split into the fields in the colony of old age. Stipe of
conidiophores smooth-walled, colorless hyaline, but can also be brown. Spherical
vesicles up to semibulat, and a diameter of 50-100 ȝm. Phialid formed in metula, and size
(7.0 to 9.5) x (3-4) ȝm. Metula hyaline to brown in color, often bersepta, and size (15-25)
x (1.4 to 6.0) ȝm. Conidia spherical to oval, measuring 3.5 to 5.0 ȝm, brown, have
ornamentation in the form of bumps and spikes that are not uniform.
Isolate 6
Fungi isolat 6 identified as Aspergillus or ae , with the characteristics: colony diameter
between 4-5 cm in 5 days, generally consisting of the top with a long conodiophore.
Conidia heads radiate, yellow-green to brown. Conidiophore Hyalin measuring 4-5 mm,

c    


     
subglobose vesicles with a size of 40-80 ȝm. Phialide sometimes comes directly from
vesicles or metulae, usually measuring 8-12 x 4-5 ȝm. Conidia initially shaped ellipsodial
but the older the age-globosa fungi to shape subglobosa with size from 4.5 to 8 ȝm.
 

c    


     u

Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate6

Picture 3. Isolate of Fungus

Isolate 1 Isolate 2 Isolate 3


Ô
  #1# 6  
  )&%+%- 6  


Isolate 4 Species 5 Species 6

6  
 
Aspergillus niger 6  


Cellulol tic Fungi

Picture 4. Species of Fungus

Based on a comparison between the transparent zone and the colony diameter
formed by each isolate, it obtained two isolates of fungi that have a greater ability to
degrade cellulose, namely Aspergillus terreus and Aspergillus or ae. As for the
comparison between the transparentlear zone and colony diameter can be seen at Table 1.

c    


     *
Table 1. Comparison between Transparent Zone and Colony Diameter
Fungi Transparent Colony Diameter Comparison TZ Number of
Zone (mm) (mm) dan CD (mm) Fungi
Candidate
Isolate 1 8 7 1.14 5
Isolate 2 9 6 1.5 3
Isolate 3 27 22 1.22 4
Isolate 4 14 7 2.00 2
Isolate 5 5 5 1,00 6
Isolate 6 17 6 2.83 1

Isolates 1, 2, and 3

Before After

Isolates 4,5 dan 6

Picture 5. Iodium Test

The potential fungus to degdrade cellulose is Isolate 6. The greater the ratio of the
diameter of transparent zone and colony diameter, the fungus more potential degrade
cellulose (Kader, Omar, 1998). Fungi can degrade cellulose because it can form and
secrete the enzyme cellulase. The greater the amount of cellulase enzymes that are
secreted in the faster degradation of cellulose occur (Bagga and Sandhu, 1987 in
Zumrotiningrum, et al 2004).

 

c    


     
Retrieved 6 types of isolates fungus that grows on a banana waste, namely:
Ôenicillium nalgiovense, Laxa, Aspergillus ochraceus Wilhelm, Aspergillus nidulans,
Aspergillus terreus, Aspergillus niger, Aspergillus or ae. Aspergillus terreus and
Aspergillus or aewere the best candidates with the highest capability in cellulose
degradation.


 
Kader, A.J and O.Omar, 1998. Isolation of cellulol tic Fungi from Sa ap-Kinabalu Ôark,
Sabah. Article ASEAN Review of Biodiversity and Environmental Conservation
(ARBEC) 2:1-5.
Riadi, Lieke 2007. Teknologi Fermentasi. Graha Ilmu. Yogyakarta
Rismunandar. 1990. Bertanam Ôisang. C.V. Sinar Baru. Bandung
Samsons, Robert A, Ellen S.Hoekstra and Connie a N.Van Oorschot, 1981. Introduction
to Food Borne Fungi. Centraalbureau voor schimmelcultures, BAARN.
Zumrotiningrum, B.D, Ari Susilowati dan Wiryanto., 2004. Seleksi dan Identifikasi
Isolat Cendawan Selulotik dan Lignoselulolitik dari Limbah Ôen ulingan daun Ka u
Ôutih (Melaleuca leucadendron L) dari KÔH Gundih, Kabupaten Grobogan. Jurnal
Biofarmasi 2 (1): 24-28, Pebruari 2004.

c    


     
  
›  ›  
 
   
7  
  6  
 





PRAPTIWI, YULIASRI JAMAL, AHMAD FATHONI DAN ANDRIA AGUSTA1

Bioscience Laboratory, Botany Division, Research Center for Biology, The Indonesian Institute
of Sciences, Jl. Raya Bogor Km. 46, Cibinong 16911.
*Author forcorrespondence, E-mail: bislunatin@yahoo.com

›


The fungus AFK-8 is one of endophytic filamentous fungus isolated from young
stemsof kayu kuning[Archangelesia flava (L.) Merr.]. The ethyl acetate extract derived
from fungus culture shows their antibiotic activity tested against bacteria and fungi on a
disc diffusion method. Scaling up cultivation of endophytic fungus AFK-8 on 1L PDA
for 3 weeks yielded 143.3 mg of chloroform extract. Fractionation guided assay of these
extract showed that the fraction 6 is the most active fraction against tested microbes with
MIC values 8, 32, 4 and 64 ëg/ml against B. subtilis, M. luteus, S. aureus danE. coli
respectively. This fraction is also active against C. albicans, R. minuta and A. niger with
MIC value 32 mg/ml. The proposed chemical structure of active metabolit in fraction 6 is
1,2-diamino-9,10-anthracenedione which was determined through a GC-MS analysis.

Key words : Arcangelisia flava,endophytic fungi, metabolites, antimicrobial activity.


 
Microbial endophyte is a microbial colony which resident inside of healthy plant
tissues. Endophytic microbes have been recognize as a chemical producer with a broad
range biological activity (Tan and Zou, 2001). Some of endophytes could have ability to
mimicking their plant resindent chemical constituents such as Taxom ces
andreanaefromTaxus brevifoliathat also produce taxol (Stierle et al., 1993), an endophyte
fungus from the plantNotapod tes foetidathat produce camptotecin (Puriet al., 2005) in
laboratory. Some of Ôhomopsisfungi species associate with the plant ofSalix gracilost la
var. melanostach s(Horn et al., 1995), the fungusCr ptosporiopsis cf. quercinaassociate
with Tripterigium wilfordii(Strobel et al., 1999; Li et al., 2000), the fungusC tospora sp.
CR 200 andDiaporthe sp. CR 146 (Brady et al., 2000) have been reported produce
various secondary metabolites that shows an antibacterial activity.

c    


     
Kayu kuning [Arcangelisia flava (L.) Merr.]is a climbing plant belongs to
Menispermaceae plant family used extensively as a medicinal plant. The biologically
active chemical compounds of kayu kuning are protoberberin alkaloids i.e. berberine,
palmatine and jarorrhizine. According to Arrogo and Sibel (2009) berberinecan be used
to prevent metabolic illness, related to cardiacdisorder, also has antiinflammatory and
antiproliferation properties. Based on their traditional used as a medicinal plant, we could
interest to explore the bioprospecting of the endophytic fungi associated with kayu
kuning.


   
%#*,#, &#%"
The youg stems of kayu kuning (Archangelisia flava) were collected from
Sambas, West Kalimantan in 2007 and identified by DR. Sudarmono, Bogor Botanical
Garden.

"%#,&*8 *!6+,&25*'&
Young stems of kayu kuning were washed by tap water and then cut with the
length of 1-5 cm, followed by sterilization by soaking the stems in the 70% alcohol for 2
minutes, then soaked in the NaOCl for 5 minutes and soaked again in 70% alcohol for 30
second.The sterilized stems were sliced with sterile knife and then put on Corn Meal Malt
Agar (CMMC) media that had been added with 0.05 mg/ml chloramphenicol followed by
incubation in the room temperature for 1 week. Every colony of endophitic fungi were
serially transfered onto Potato Dextrose Agar (PDA) media untill pure colony were
obtained. The obtained endophytic fungi were preserve in -80 oC at LIPI-MC.

2 *&*'82*!# ,#$%&," !52,&*#*!*,&$#2, &#%""#
All of isolated endophytic fungi were cultivated in 20 mL PDA, Potato Dextrose
Broth(PDB) media and Glucose-Yeast extract-Pepton (GYP)in 100 mL test tube, then
incubated at room temperature for 3 weeks. After 3 weeks of incubation, fungi cultures
and its biomass were extracted twice with ethyl acetate : methanol (10 : 1 ), shaken with
vortex and allowed to form two layer. The upper layer was taken and concentrated with
rotary evaporator. The extracts were then analysed by Thin Layer Chromatography

c    


     -
(TLC) and eluted dichloromethane : methanol : acetic acid (6 : 1 : 1 drop). Then, the TLC
chromatograms were monitored by UV light at the wavelength of 254 nm dan 365 nm,
then sprayed with Ce(SO4)2.
Extract from each endophytic fungi were diluted in acetone and tested for its
antibacterial activity by diffusion method at the concentration of 10 mg/mL. Bacteria
isolates used for antibacterial activity were Bacillus subtilis, Micrococcus luteus,
Staphilococcus aureus andEschericia coli,inoculated in Nutrient Broth media and
incubated for 18 hours at room temperature. After that the tested bacteria was taken with
sterile micropippette(100 RL)and cultured on Mueller Hinton Agar (MHA) media. 10 RL
of extract was dropped on sterile paperdiscthen put on MHA media that had been
inoculated with bacteria isolate followed by incubation at 37oC for 24 hours.The growth
inhibition zone around paperdisc was then measured.

2#%&*'565%,&.#,&*85*'5"7
The endophytic fungus AFK-8 was cultivated onto 2 x 500 ml of PDA (in
Erlenmeyer 5 L) and incubated at room temperature. After 3 weeks, all ofmedium and
biomass were homogenazied and extracted with ethyl acetate-methanol (10:1, 3 times in
equal volumes) and concentrated under reduce pressure at 30 oC to achieved 143.3 mg of
yellowish extract.

"%#,&*82,&.,#$%&,"
The whole fungus extract (143,3 mg)was subjected into a Sephadex LH-20
column chromatography and eluted with methanol to achieved 6 fractions. All of
fractions were then subjected into TLC analysis and a disc diffusion antibacterial assay.
The fraction was disolved in aceton and tested for its antibacterial activity by
diffusion method at the concentration of 10 mg/mL,while bacteria isolates used were B.
subtilis, M. luteus, S. aureus andE. coli.Bacteria isolate was cultured on MHA media. The
sterile paper disc that had been added with the 10 RL extract was then put onMHA media
that had been inoculated with bacteria isolate, followed by incubation for 24 hoursat 37
o
C.The growth inhibitoin zone was the clear zone around the paper disc and the diameter
of clear zone was measured.

c    


     †

, -&*#,&*8 9#%58 #2,&*:
The MIC values were determined through a microdillution assay (Rodriguez-
Tudela , 2002). Determination of MIC value was done by preparing of fraction 6 stock
solution in DMSO at the concentration of 512 ug/ml. The medium for antifungi was
Saburaud Broth (SB), while for antibacterial test was Mueller Hinton Broth (MHB).
Fungi isolates used for the test were C. albicans, R. minuta, A. niger, A. flavus andS.
cerevisiae, while bacteria isolates used were M. luteus, E.coli, S. aureus, B. subtilis. In
each 96 microwell titer was added as : well 1 added with growth media 2x concentration
(100 ul) and stock solution (100 ul). Well 2 ± 14 added with growth media 1x
concentration (100 ul). Well 1 homogenized with micropippette, then pippette 100 ul and
put on well 2. The same method was done untill well 14. After that in each well was
added with bacteria inoculum (100 ul). Well 15 was positive control ( 100 uL growth
media and 100 uL inoculum), while well 16 was negative control (200 uL growth media).
The 96 microwell titer was then incubated in the shaker incubator for 24 hours at 35-
37oC. The MIC was observed visually.

*#%"&"8 #2,&*:
The chemical constituents of fraction 6 from the fungus AFK 8 extract cultivated
on PDA was analysed by an ion trap GC-MS (Varian, Saturn 2000) with the capillary
column VM 17 (0.25 mm x 30 m, varian). Conditions of analysis were : injector
temperature l 230 oC, column temperature was programmed from 80 oC (isothermal for 3
minutes) to 250 oC with temperature rate 5 oC/minute.Column pressure was 10,7 psi,
carrier gas flowwas 1,3 Ml/minute (Helium). Interfase temperature l 250 oC, trap
temperature l 150oC. The sample (in aceton solution) was injected 5 RL.



  
The isolation of endophytic fungi from kayu kuning (Arcangelisia flava (L.) Merr.
stem resulted in 9 endophytic fungi named AFK -1 ~ AFK-9. The TLC analysis results
showed that the TLC chromatograms oatterns of ethyl acetate-methanol extracts of fungi

c    


     o
AFK-1, AFK-2, AFK-3, AFK-5, AFK-6 and AFK-7 are different each other. On the other
hand, the TLC chromatogram pattern of AFK-4 is identical to AFK-2, while AFK-8 and
AFK-9 are identical to those of AFK-7. The above result revealed that the fungus AFK-2
is chemotaxonomically identical species with fungus AFK-4, and fungi AFK-7, AFK-8
and AFK-9 are also chemotaxonomically identical species.
From the antibacterial screening tests of ethyl acetate-methanol extract of all the
above endophytic fungi, it was found fungus AFKR-8 possess an antibacterial activity
againts all tested bacteria B. Subtilis, E. coli, M. luteus and S.aureus. In order to isolate
and characterize principle antibacterial metabolite(s) produce by the endophytic fungus
AFK-8, the fungus was then cultivated on 2 x 500 ml of PDA (in 5 L size Erlenmeyer),
and then extracted with ethyl acetate-methanol to achieve a yellowish extract.
Fractioation of AFK 8 extract with Sephadex LH-20 column chromatography yielded
6 fractions, i.e. F1 (55.1 mg), F2 (2.7 mg), F3 (25.5 mg), F4 (7.6 mg), F5 (34.8 mg) and
F6 (8.1 mg) respectively.
The antibacteria activity assay on a disc diffusion method showed that the F6
(fraction 6) had hihger antibacterial activity compare to other fractions. The diameter of
growth inhibition for fraction 6 was : M. luteus (17 mm), B. subtilis (17 mm), S. aureus

M. luteus B. subtilis S. aureus

Figure 1 Growth inhibition of fraction 6 of AFK 8 extract to several bacteria isolates

(14 mm) at the concentration of 100 ëg/10 ël, but failed to show their activity against E.
coli at tested concentration (100 ëg/10 ël).
Furthermore, the F6 was subjected into a subceptible antibacterial assay against 4
tested bacteria 5 tested fungi (Tabel 1) in a microdillution method. The results showed
that fraction 6 (MICl 4 mg/L) had higher activity compared to chloramphenicol (MICl
16) against S. aureus, and its activity was similar to chloramphenicol against B. subtilis

c    


     D
(MIC l 8 mg/L). Fraction 6 also had higher activity against R. minuta (MIC l 32
mg/L)dan S. cerevisiae (MIC l 32 mg/L) and similar to C. albicans (MIC l 32
mg/L)compared to nystatin and cabisidin. Based on the result, it was concluded that
fraction 6 of AFK-8 extract capable of inhibiting the growth of tested bacteria and fungi,
moreover its activity was better than comersial antibiotic against several bacteria and
fungi.
GC-MS analysis result of F6 (Fig. 2) showed a single main peak on its
chromatogram, indicate that F6 was almost pure. The MS spectrum of F6 shows base
peak and ion molecule at m/z 238.0. Comparison of mass spectrum (Fig. 3) of F6 with
known chemical mass spectra in NIST Librar and Wile , showed that the mass spectra of
F6 is identical with 1,2-diamino-9,10-antrasenediona. This compound was classified to
anthraquinone. According to Anonim (2010) antrasenediona has the ability as anticancer
and known as anticancer antibiotic. Based on the GC-MS analysis , the possibility of
molecule formula of fraction 6 AFK 8 PDA was C14H10N2O2 .
#$% MIC value of F6 (fraction 6) against tested bacteria and fungi.

MIC (mg/L)
No Microbe
isolate F6 chloramphenicol erythromycin nystatin Cabisidin
1 B. subtilis 8 8 0,03 nt nt
2 M. luteus 32 16 0,06 nt nt
3 m
 
 : ;( nt nt
4 E. coli 64 16 16 nt nt
5 C. albicans 32 nt nt 32 32
6 ©

;( nt nt : :
7 A. niger 32 nt nt 16 64
8 A. flavus 32 nt nt 16 32
9 m
   
;( nt nt : :

nt : not tested

c    


     
Figure 2 GC chromarogram of fraction 6

(a)

(b)

Figure 3. (a) MS spectrum of F6, (b) MS spectrum of 1,2-diamino-9,10-Antrasenediona


derived from NIST Library.

c    


     u
  
 Isolation ofendophytic fungi from kayu kuning stem [A. flava (L) Merr.] from
Sambas (West Kalimantan) yielded 9 endophytic fungi isolates which were
chemotaxonomically classified into into six group. Fraction 6 (F6) of fungus AFK-8
extract had the ability to inhibit the growth of several bacteria and fungi isolates better
than comersial antibiotic. The molecular structure of main constituent of fraction 6 was
proposed as 1,2-diamino-9,10-antrasenediona with a molecular formula of
C14H10N2O2.Further studyto determine its absolut chemical structure need to be done by
other spectroscopic analysis.


)   

This work was supported by IFS grant (No. F/4613-1) and LIPI Internal research
fund (DIPA). The authors would like to thank to DR. Sudarmono from Bogor Botanical
garden for collection and identification of plant materials.




Agusta A., Ohashi K and Shibuya H. 2006. Bisanthraquinone Metabolites Ôroduced


b the Endoph tic Fungus Diaporthe sp. Chem. Pharm. Bull., (4), 579.
Agusta A. 2009. Biologi dan Kimia £amur Endofit. Institut Teknologi Bandung.
Anonim.2010. Anthracenedione http://www.meb.uni-
bonn.de/cancer.gov/GlossaryTerm/CDR0000045594html
Arrigo FC&Sibel E. 2009.Metabolic and cardiovascular effects of berberine: from
preclinical evidences to clinical trial results. Clinical Lipidology vol. 4(5): 553-
563. http://www.futuremedicine.com/doi/abs/10.2217/clp.09.41
Brady SF., Singh MP., Janso JE., and Clardy J. 2000. C tosk rins A and B, new
BIAActive Bisanthroquinones Isolated from an Endoph tic Fungus.Organic
Letters ((25). 4047.
George C. 1988. Fungal Endoph tes in Stems and Leaves : From Latent Ôathogen
toMutualistic S mbiont. Ecological Societ of America, :< (1).
Geris dos Santos RM and Rodrigues-Filho E. 2003. Structures of Meroterpenes
Producedby Penicillium sp, an Endophytic Fungus Found Associated with Melia
Aedarach. £. Bra. Chem. Soc.., (5), 722.
Hossain SM.. 2005. Isolation and Structure Elucidation of Secondary Metabolites
fromEndophytic Fungi and the Plant Ôrismatomeris tetrandra and Synthesis of

c    


     *
(+)-Ochromycinone.
Hyde, KD and Soytong K. (2008). The fungal endophyte dilemma. Fungal
Diversit ;;,163 ± 173.
Janos B. 2005. Bioactive Microbial Metabolites. £. Antibiot.,7(1). 13
Keawpradub N., Dej-adisai S. and Yuenyongsawad S. 2005. Antioxidant and
c totoxicactivities of Thai medicinal plants named
Khaminkhruea:Arcangelisiaflava, Cosciniumblumeanum
andFibraureatinctoria.£. Sci. Technol.,(=( 2), 455-467.
Radji M. 2005. Ôeranan Bioteknologi dan Mikroba Endofit dalam Ôengembangan
ObatHerbal. Majalah Ilmu Kefarmasian, (3), 113-126.
Rodriguez-Tudela JL., Brchiesi F., Bille J., Chryssanthou E., Cuenca-Estrella
M.,Denning D., J. P. Donnelly, B. Dupont, W. Fegeler, C. Moore, M.
Richardson, P.

E. Verweij. (2002). Method for the determination of minimum inhibitor concentration


(MIC) b broth dilution of fermentative easts.Eucast Discussion -Document E.
Dis 7.1. 1.

Sofia L. 2006. Isolasi dan Uji Bioaktifitas Kandungan Kimia Utama Ôuding
merahdengan Metoda Uji Brine Shrimp. USU Repositor . 11-12.

Tan, R.X. and W.X. Zou. 2001. Endoph tes: A rich source of functional metabolites. Nat.
Ôrod. Rep., 18: 448²459

Tsai PL and Tsai TH. (2002). Simultaneous determination of berberine in rat blood,
liverand bile using microdil sis coupled to high-performance liquid
chromatograph .£ournal of Chromatograph 63 (961), 125-
130.ipcodeoo.com/Ôlants/A/Arcangelisia_flava/03 Mei 2009.

Verpoorte R., Siwon J., van Essen GFA., Tieken M. and Svendsen AB. 1982. Studies
onIndonesian Medicinal Ôlants VII Alkaloids ofArcangelisiaflava. £ournal
ofNatural Ôroducts,5).

Yang X, Strobel G, Stierle A, Hess WM., Lee J, Clardy J. 1994. A fungal endophyte-
treerelationship : Ôhoma sp. in Taxus wallachiana. Ôlant Science. 102.

c    


     
›    #% ›  
) 

 
  9 


PRIYANTI1, AZRI KUSUMA DEWI2, EFRILIA NURJANAH1

3
 # &8 &!##,5%%#+/#># ,#3(›
%#-#,6 &#*,&?5&*@#+2-


›


The Influence of NaCl and IBA at the MS media for the growth of rice mutant in
vitro was conducted in the tissue culture laboratory and green house plant¶s breeding
group (PATIR BATAN). The aims of this research was to know the concentrate variation
of NaCl, IBA, and MS media combination for growing percentage, height and root length
in planlet Diah Suci variety and three rice mutant lines. The experiment was arranged in
Completely Randomized Design, with three factors, fourty eight treatments and five
replications. The first factor was four concentration of NaCl (0%, 0.5%, 1% and 1.5%).
The second factor was three concentration of IBA (0%, 2.5% and 5%). The third factor
was Diah Suci variety as plant control and three rice mutant lines (Obs 1700, Obs 1701,
and Obs 1704. The result of four weeks observation was show that the added of NaCl up
to 1.5%, so the growth percentage, height and root length of plantlet would be to descend.
The concentration of IBA until 5% influenced to the growth percentage, height and root
length of planlet. The combination of three parameter refered to Obs 1704 better than
control and the others (Obs 1700 and Obs 1701) with the growth percentage was 80%,
planlet height 19.84 cm and root length 5.62 cm in combination of NaCl 1% and IBA 5%
on four weeks observation.

 !" in vitro, mutant lines, rice, NaCl and IBA

›


 Rice is the staple food of Indonesian people. The need is also increasing every
year but the production tends to decline. One factor is the cause is a narrowing of fertile
agricultural land and the transfer function of the land to non-agricultural activities such as
industry, housing, roads, and others. The farmers find alternative to large areas of land
used as rice cultivation, the vast land around the beach area. Problems that arise are
notall rice varieties resistant to soil with high salinity.
Salinity is either an accumulation of dissolved salts in the soil and become one of
the problems often faced in agriculture in the lowlands. Salinity stress on food crops can
cause plants can not grow. One of the efforts undertaken to overcome salinity stress is
through tissue culture. Through the selection of tissue culture can be improved salinity

c    


     
tolerance, drought, temperature, and disease in plant breeding programs (Nahlony et al.,
1997).

Variety Holy Diah (DS) is the rice varieties released by Nasinal Atomic Energy
Agency (BATAN) as a result of mutation breeding with gamma rays. Varieties of this
Holy Diah as high yielding varieties have been released nationally by the Ministry of
Agriculture in 2003. The advantages possessed by the Holy Diah varieties is the potential
for dry grain production high gilingnya namely 9.4 tons / ha, the area distribution and
planting a larger area compared with other neutron output of rice varieties, rice fluffier
texture, can adapt in Rice fields in the plains lower to a height of 650 m above sea level
atasa. Phenotypes of rice varieties Holy Diah still high enough to encourage pemulianya
to restore these varieties without changing other properties by re-irradiating with gamma
rays to obtain new and better varieties. The result of re-radiation of the Holy Diah rice
varieties with gamma rays is a mutant strain of observation (Obs) 1700, Obs Obs 1701
and 1704 at this time have entered the stage of yield trial. Salinity test in vitro the mutant
strains of rice varieties Holy Diah is expected to provide additional information about
mutant strains which are tolerant to abiotic stresses such as salinity. Mutant lines are
expected to be planted in areas with high salinity and can adapt well to other
environmental stress factors (ICRP, 2003).

 
   
#, &#%"#*! 45&6-*,
 The main material used is rice varieties explants Holy Diah sebgai mutannya
control and three lines of Obs 1700, Obs 17 001, and Obs 1704. Other materials are bacto
agar, sugar, aquades, MS culture media, NaCl, IBA, chlorox, Tween (for Saponification
in sterilization of explants), spirits, and alcohol 96%.
The instrument used is an autoclave, laminar air flow cabinet (LAFC), hot plate
and magnetic stirrer + spin bar, oven, microwave oven, analytical balance, pH meter
digitas, fridge, bottle plant, aluminum foil, Erlenmeyer, rubber bracelets, glass Backer ,
tweezers, pipette volume, measuring flask, rack culture, the culture bottles, dissection
tools, Petri dishes, Bunsen, plastic buckets, markers, paper labels, stroller, and equipment
cleaning glass tools.

c    


     
2!5 
V , &%&A#,&* 45&6-*,
Sterilization equipment in laboratory tissue culture BATAN there are two kinds of
concave and sterilization sterilization wet. Dry sterilization using the oven, the tools are
sterilized dry dissection tools, petri dish, Erlenmeyer and culture bottles. Wet sterilization
using an autoclave, the tools are sterilized moist culture media that have been made and
contaminated culture bottles.
(V 6# #,&*85%,5 !&#
Media dasar yang digunakan adalah media MS untuk memudahkan pembuatan media
maka Basic media used were MS media to facilitate media preparation is necessary first
made stock solution of 1 to 6. 1-3 stock solution of 10 ml, 4-6 stock solution of 5 ml.
NaCl concentration 0%, 0.5%, 1%, 1.5% and 0% IBA, 2.5%, 5% added to 1000 ml glass
Backer then added white sugar by 20 g and diluted with aquades as much as 1000 ml and
stirred with a magnetic stirrer + spin After the bar so homogeneous and homogeneous
medium pH measurements were taken using a digital pH meter, pH of media 5.8. The
next step is to enter into the media above 250 ml Erlenmeyer containing the bacto for as
much as 2 g. Erlenmeyer then covered with aluminum foil and tied with rubber. The
media was then sterilized using an autoclave for 15 minutes, 1 atm pressure, and
temperature of 121o C. Media that has been sterilized and then stored in the culture room.

;V *!52,&*
&2
Induction of rice conducted in LAFC. Each explant Holy Diah rice varieties as a control
and three mutant lines of rice (Obs 1700, 1701, 1704 Obs) peeled gabahnya chlorox then
sterilized in a solution of tween 40% and the solution is homogenized for 25 minutes.
Rice seed is cut into two parts, namely endosperm and embryo using dissection tools.
Part endosperm removed, bagia rice embryos grown in culture medium MS with
variakombinasi NaCl and IBA, in each culture bottle containing three embryos from the
Holy Diah varieties and three embryos from mutant strains Obs 1700, Obs 1701, Obs

c    


     -
1704. The culture bottles were incubated in culture room with temperature 26-28o C.
Observations were made at one week after planting (MST) to four MST.


$" .#,&* # #-, 
 Observations made at the age of one to four MST MST based on morphologic
characters. Observations were conducted one to two times each week. Observations were
a. Percentage of plantlets growing power
Plantlets are alive when plantlets grow, experiencing the inhibition or cessation of
growth but not dead. Plantlets growing starts counting from the beginning to the end of
observation (four MST).
b. High plantlets
High plantlets were measured at the age of two to four MST. Measurement-old
plantlets of two to three MST by using a ruler attached to the walls of culture bottles
because of plantlets is not removed from the culture bottles. The measurement starts from
the boundary until the surface of the upper main stem of the plant. Measurement of
plantlets at the age of four MST planet way out of the bottle culture, plantlets were
washed first with flowing ait then laid on a ruler and measured height from the main
trunk line to the end of the longest plant.
c. Root length
Root length measurement performed at the age of four MST. Plants that have
been removed from the culture bottle and were washed with running water and then
spread its roots to the ruler, measurement of boundary main stem up to the longest root
hairs.

16 &-*,#%"&'*
The experimental design using a completely randomized design (CRD) with three
factors of four concentrations of NaCl (0%, 0.5%, 1%, 1.5%), three concentrations of
IBA (0%, 2.5%, 5%) and four explants of one variety of rice Holy Diah (as control) and
three mutant lines of rice (Obs 1700, 1701, 1704 Obs.) The combination of these three
factors resulted in 48 treatments. The experiment was repeated five times.

c    


     †
#,#*#%"&"
 Data obtained statistically tested using ANOVA test. If significantly different
Duncan's test at 5% test level.



  



  2*,#'86%#*,%,"' &*'6 
 Data percentage of power from plants grown plantlets of control and three mutant
strains age of four MST shown in table 1.
Table 1. Percentage of Age Four Plantlets Growing Power MST

At NaCl concentrations of 0% and 0% IBA, the mutant strains grew Obs Obs
1700 and 1701 reached 100%. The percentage of mutant strains Obs Obs 1700 and 1701
higher than the DS (control) and Obs 1704. At NaCl concentration of 1.5% and 0% IBA
mutant lines Obs 1700 percentage growth of the power is still far better than the DS
(control), Obs Obs 1701 and 1704.
At NaCl concentrations of 0% and 5% IBA, the percentage of power grows
between the DS (control), Obs 1700, Obs 1701, and Obs 1704 showed no significant
differences ranged between 80-90%. At NaCl concentration of 1% and 5% IBA, 1700
Obs mutant strain has an ability to grow better (100%) compared with DS (control) that is
90% and Obs Obs 1701 and 1704 (80%). This indicates that the mutant lines Obs hidaup
1700 has a better ability than the control plants and other mutant lines (Obs Obs 1701 and
1704).

c    


     o
1% and 5% only mutant strains that have a high 1701 Obs better plantlets of 5.3 cm
compared with control plants and the two other mutant lines. At NaCl concentration of
1.5% and 5% higher IBA plantlets best seen in the mutant lines Obs 1701 (1.00 cm)
while the control plant height, Obs Obs 1700 and 1704 is less than 0.6 cm (Table 2).

Table 2. Interaction NaCl, IBA, High Against Mutant Lines Plantlets (cm) Rice In vitro
In the Age of Two MST

Description: Value averaging is followed by the same letter in same column indicates no
significant difference (Į l 5%)

Provision of NaCl concentration 0% and 5% in three IBA MST showed that


plantlets of both plant height control and mutant lines are not much different, high
plantlets ranged from 18.76-21.17 cm. At NaCl concentration of 1% and 5% higher IBA
plantlets between control plants with mutant strains Obs 1701 is not too much different
from the 16:06 and 16:15 cm. High Obs plantlets in 1700 (14.74 cm) and Obs in 1704
(10.71 cm) is much lower compared with control plants and mutant lines Obs 170.

c    


     D
Granting of NaCl 1.5% and 5% IBA addition, there are still high plantlet. High plantlets
in 1700 Obs mutant lines (1.70 cm) and Obs 1701 (1:22 cm) was still higher than the
control plant height (0.60 cm) and mutant strains Obs 1704 (12:17 cm) (Table 3).
At the age of four MST, NaCl concentration of 0% and 5% IBA showed no
significant difference between plant height control with a strain of mutant Obs 1700, Obs
1701, and 1704 Obs ranging from 27,25-28.64 cm. Giving NaCl 1% and 5% showed high
IBA best plantlet shown by control plants (23:40 cm). High plantlets Obs mutant strains
1701 (21.92) is still far better than the other mutant strains of high (<20 cm). At NaCl
concentration of 1.5% and 5% IBA, there is no height at the age of four plantlets of MST
(Table 4).
Table 3. Interaction NaCl, IBA, High Against Mutant Lines Plantlets (cm) Rice In vitro
In the Age of Three MST

Description: Value averaging is followed by the same letter in same column indicates
no significant difference (Į l 5%)

c    


     
Table 4. Interaction NaCl, IBA, High Against Mutant Lines Plantlets (cm) Rice In vitro
At Age Four MST

Description: Value averaging is followed by the same letter in same column indicates no
significant difference (Į l 5%)

According Suwarno (1985) that administration of NaCl at different concentrations


can increase leaf damage, reduce the number of tillers, plant height, dry weight of
canopy, roots and total plants. Damage to plant roots will cause difficulty in absorbing
the nutrient elements of the media and damage to the leaves causing the leaves are not
able to produce food through photosynthesis. The higher the salinity, the concentration of
Na, Mg, Ca and Mn in growing media while the solubility of P will decrease. NaCl
concentration of 5% can suppress the growth of plantlets of rice because of nutrient
imbalance absorption elements, elements kekahatan P, disruption of protein synthesis in
plants and poisoning Na and Cl (Dinata, 1985).

;
,*',+

At NaCl concentrations of IBA concentration 0% and 5% root length Obs 1700
mutant lines have a better appearance than control plants, mutant strains Obs Obs 1701
and 1704 is 4:50 cm. Provision of NaCl concentration of 1% and 5% IBA mutant lines
Obs 1704 has a better root length compared with control plants and the two other mutant
strains of 5.62 cm. At the age of four MST, 1701 Obs root mutant lines still have a long

c    


     u
accretion of about 0.70 cm compared with control plants and the two other mutant lines.
At NaCl concentration of 1.5% and 5% IBA color and texture more grim roots of fragile
roots and root hairs are few in number.
The results in line with the results of the research performed by Ishak (1994) on
rice roots Pelita I / I are given 1% NaCl concentration in MS media has lowered the
growth of roots and leaves as much as 30%, about 47% of control while the long growth
Atomita rice root length-2 root growth decreased by approximately 9% and leaf drop
about 38% of the control and rice roots Atomita-1 approximately 19% and 37% leaf. The
use of 1.5% NaCl concentration causes a decrease in rice root length growth padab Pelita
I / I almost 69% and 68% leaf, in rice Atomita length-2 root and leaf growth decreased by
74% and 70%, while in Atomita-1 root length growth and leaves decreased by 77% and
67%.
The high concentration of NaCl in the medium of plant growth will result in
growth suppression and disruption of root length on the formation of new roots that will
reduce the cruising range to reach the roots in nutrient elements. The amount of IBA
concentration is 5%, also can damage the plants that were injured (Sriwidodo, 1985 in
Dinata, 1985).
ZPT is not too high given its concentration on root cuttings to encourage the
growth of roots and shoots (Abidin, 1980). Plants already have a hormone in the body
itself but because jumahnya little needs to be done so that the addition of synthetic
hormone, plant growth is expected to be faster than before (Wudianto, 1989 in Harsanti
and Mugiono, 2001).

 

‡ The High Concentration of NaCl (1.5%), the percentage of power grows, plantlet height
and root length decreased.
‡ IBA concentration of 5% effect on the growth, plantlet height and root length.
‡ After 4 MST 1704 Obs mutant lines better than the control and the two other mutant
lines grew 80% the percentage of power, plantlet height 19.84 cm, 5.62 cm long roots in
NaCl concentration of 1% and 5% IBA

c    


     *
 The combination of IBA NaCl 1% and 5% the percentage of power grew, height and
root length of plantlets was still good.

c    


     


 

Abidin, Z. 1980.Knowledge Base about the growth regulator substances. Angkasa.
Bandung
BATAN. 2003. Superior Variety Rice Technology The combination of radiation and
Marriage Cross Movement. Applications of Radiation in the Field of Agriculture.
BATAN. Jakarta.
Dinata, K.K. 1985. Effect of Salinity on Growth and Production of Rice Varieties
Atomita II and IR 32.Thesis. IPB Bogor.
Ishak. 1994. Analysis of Amino Acid Content of Mutant Rice (Oryza sativa L.) CV
Atomita Atomita-1 and-2 and Its Relation to the Tolerance to Salinity. Zuriat Vol.5
No.2:56-64.
Nahlohy, Karmana, Darsa, dan Widiyanto. 1997. Variations Somaklon Rice Callus
induction and subculture results in Media with and without NaCl.Zuriat 8 (2): 64-67
Suwarno. 1985. Dissertation Summary: Inheritance and Physiological Properties Against
Salinity tolerance in Rice. IPB. Bogor


   
›  9  
9Ô 
 

  
)
     
› 

ELISA FRISKA ROMASI1*,ADOLF J. N. PARHUSIP1, YUNIWATY2


1)
Lecturer of Universitas Pelita Harapan, UPH Tower, Jl. MH Thamrin Boulevart 00-00, Lippo Karawaci,
Tangerang 15811, Indonesia,
2)
Alumnus from Food Technology Department of Universitas Pelita Harapan, UPH Tower, Jl. MH
Thamrin Boulevart 00-00, Lippo Karawaci, Tangerang 15811, Indonesia.
*
Coresponding author address :
UPHTower, Jl. MH Thamrin Boulevart 00-00, Lippo Karawaci, Tangerang15811, Indonesia, phone : (021)
5460901, Fax : (021) 5460910, e-mail : elisa_friska@yahoo.com

›


Guava leaves have been utilized traditionally as medicine and known as an
antimicrobial agent as well. In this research, guava leaves were extracted using
maceration method. The solvents used in this research were water, ethyl acetate, and
hexane. Guava leaves extracts were tested towards Escherichia coli, Staph lococcus
aureus, Bacillus cereus, and Ôenicillium sp. by the agar diffusion method. The objectives
of this research were to (1) determine the MIC and MBC of guava leaves extracts
towards tested microbes, (2) determine the active compound in guava leaves extract, (3)
observe the influence of certain pH, sugar concentration, salt concentration, and heat
treatment on the antimicrobial activity of guava leaves extract. The result showed that
ethyl acetate extract could inhibit all the tested bacteria excluding Ôenicillium sp. The
MIC and MBC for Escherichia coli, Staph lococcus aureus, and Bacillus
cereuswas0.017% and 0.067%, 1.177% and 4.707%,0.126% and 0.504%, respectively.
The active compounds found in guava leaves were alkaloid, saponin, tannin, phenol,

c    


     
flavonoid, triterpenoid, and steroid. The results indicate ethyl acetate extract was
influenced by pH and effective at pH 4. Sugar addition could increase the antimicrobial
activity. Furthermore, low concentration of salt could decrease the antimicrobial activity
towards B. cereus as well as that by heat. Moreover, the results also indicate ethyl
acetate extract could inhibit the growth of B. cereus spores.

 !" : guava leaf, antimicrobial activit


 
Guava plant (Ôsidium guajava L.) is a tropical plant that is easily found in
Indonesia. Many parts of this plant are utilized by human, especially its fruits and leaves.
Particularly, its fruit is commonly consumed as fresh fruit or processed food. Guava fruit
contains tryptophan lysine, pectin, calcium, phosphor, minerals and vitamin. Currently,
its fruit is also used to treat diabetes mellitus patient and people who have high level
blood cholesterol.
Besides its fruit prospective, other part of this plant is utilized for medicinal
purpose as well. Its root has potential utilizations, to stop dysentery, its young branch is
used to treat leucorrhea patient and its leaf is used to cure diarrhea, stomatitis, and
stomach-ache. Leaves of guava are reported to have antibacterial activity. Morton (2006)
reported about essential oil found in its leaves have, such as dendrene aromatic, -
selinen, nerolidiol, caryophyllene oxide, triterpenoids and -sitosterol.
Hence, this research was carried out to observe the antibacterial activity of guava
leaves extract against pathogenic microbe and consequently would increase the
economical applications of guava leaves.

   
The guava leaves used in this research were obtained from Muara Karang. All the
microbes were from PAU, Bogor Agriculture University and most of the chemicals were
purchased from Merck. The guava leaves were washed, freeze dried, then blended to
become powder. The powder was macerated with three kind of solvent, i.e. : water, ethyl
acetate, and hexane. The maceration process took 24 hour at room temperature. The
mixture then filtrated, condensed at 45oC with oven (for water as the solvent) or vacuum
evaporator (for ethyl acetate and hexane as the solvent) to obtained the extracts. The three

c    


     
kind of extracts then analyzed by Harborne method (Harborne, 1996) to determine the
active compound.
The antibacterial activities of all the extracts were tested by using agar diffusion
method. Four kinds of microbes, Escherichia coli, Staph lococcus aureus, Bacillus
cereus, and Ôenicillium sp. were used to test the antimicrobial activity of those extracts.
Every extract that were obtained from every solvent were tested in five concentrations
10%, 20%, 30%, 40%, and 50% and the solvent were used as control. The test was done
in 37oC. After 24 hour the diameter of inhibition zones were measured and the extract
that gave the highest diametrical inhibition with minimal concentration were chosen to be
used in the next analysis. Bloomfield method (1991) was used to determine the MIC and
MBC of the extracts. To observe the influence of pH, the chosen extracts were tested in
five kinds of pH value, 4, 5, 6, 7, and 8. The extract also tested in four kind of sugar
concentration : 10%, 20%, 30%, and 40%, four kind of salt concentrations : 1, 2, 3, and
4%, and also in two kind of temperatures : 80oC and 100oC for 5, 10, and 15 minutes.
The extract also tested against the Bacillus cereus spore for 24 hours in 37oC.

  


The water extracts did not inhibit all the microbes tested, in contrast the ethyl
acetate could inhibit all the bacteria tested but not Ôenicillium. The diameter of inhibition
of ethyl acetate extracts was between 6.17 mm ± 12.95 mm. Furthermore, hexane extract
could only inhibit B. cereus and the diameter of inhibition was 0.00 mm ± 6.79 mm.
(Table 1). For next analysis Ôencillium was not used as tested microbes.

Table 1. Diameter of Inhibition Zone of Guava Leaves Extract


Diameter of Inhibition Zone (mm)
Kind of Bacteria
E.coli S. aureus B. cereus Ôenicillium
0% 0.00 0.00 0.00 0.00
10% 0.00 0.00 0.00 0.00
s water 20% 0.00 0.00 0.00 0.00
30% 0.00 0.00 0.00 0.00
o 40% 0.00 0.00 0.00 0.00
50% 0.00 0.00 0.00 0.00
l  0% 0.00 0.00 0.00 0.00
 BC <; =<< := 0.00
v ,+%  20% 9.28 8.58 6.32 0.00

c    


     -
#2,#, 30% 9.49 9.52 7.17 0.00
e 40% 9.73 11.81 7.25 0.00
50% 10.06 12.95 7.51 0.00
n 0% 0.00 0.00 0.00 0.00
10% 0.00 0.00 0.00 0.00
t hexane 20% 0.00 0.00 5.04 0.00
30% 0.00 0.00 5.81 0.00
40% 0.00 0.00 5.79 0.00
50% 0.00 0.00 6.79 0.00

The MIC and MBC was determined for ethyl-acetate extract only. The
Bloomfield method was used and the result is in Table 2. The MIC and MBC for
Escherichia coli, Staph lococcus aureus, and Bacillus cereuswere0.017% and 0.067%,
1.177% and 4.707%,0.126% and 0.504% respectively.
Table 2. The MIC and MBC against tested Bacteria
Kind of Bacteria
E.coli S. aureus B. cereus
MIC MBC MIC MBC MIC MBC
0.017 % 0.067% 1.177% 4.707% 0.126% 0.504%

For ethyl ± acetate 10% extract could inhibit the tested bacteria with no
significant differences with the next higher concentration; the inhibition test was done
with the lower concentration, i.e. 2, 4, 6, 8, and 10% and the result shown in Table 3.
Base on the result, ethyl ± acetate 4% was chosen for next analysis to inhibit E. coli and
S. aureus, and ethyl acetate 6% was chosen to inhibit B. cereus.
Table 3. Diameter of Inhibition Zone of Ethyl ± acetate extract
Diameter of Inhibition Zone (mm)
Kind of Bacteria
E.coli S. aureus B. cereus
0% 0.00 0.00 0.00
2% 9.06 7.45 6.49
Concentration 4% <;; 7< 7.29
6% 9.59 8.36 7
8% 9.81 8.43 8.49
10 % 10.84 8.49 9.04

c    


     †
There were a lot of active compound in guava leaves. The active compounds in
guava leaves were alkaloid, saponin, tannin, phenol, flavonoid, triterpenoid, and steroid
(Table 4).
Table 4. The Active Compaound Found in Guava Leaves Extract
Kind of Extract
Active Compound Water Ethyl - acetate Hexane
Alkaloid + + +
Saponin + + -
Tannin + + +
Phenol + + -
Flavonoid + + +
Triterpenoid - + +
Steroid + + +

*8%5*286 * 1, #2,2,&.&,


It was found that ethyl acetate extract was effective under acid condition. It could
inhibit all the tested bacteria at pH 4, but at pH 5 it could not inhibit S. aureus, moreover
it could not inhibit all the tested bacteria at pH 6, 7, and 8 (Figure 1).
 
  


A
  


 
  
 

A AA     A AA A AA A AA
 A AA A AA A AA A AA A AA A AA A AA A AA A AA
A

    
^ 

        

Figure 1. Diameter of Inhibition Zone of Guava Leaves Extract in Several pH


Value

Most of the extract components were weak acid. At low pH, weak acids were not
dissociated. Non dissociated form weak acid would easy to diffuse inside the cell, then

c    


     o
the cell would react to maintain its pH. The cell reaction needs more energy, then the
energy to grow would decrease.

*8%5*285'# * 1, #2,2,&.&,


The result in Figure 2 shown that there was sugar concentration influence on the
antibacterial activity of the extract. The diameter inhibition range was 2.78 ± 9.70 mm.
The higher the sugar concentration, the higher the diameter inhibition. The sugar
concentration influenced the Aw value (water activity). At sugar concentration 10 ± 30%,
the water activity was 0.978, and at sugar concentration 40%, the water activity was
0.973. Not all water in the solution can be used by the bacterial for its growth. The water
that can be used by bacteria is stated as water activity, the water activity restrict the
growth of the bacteria.
  V
V   V


 A  
A
 V 

 

     

         

 
A AA
 A AA A AA
A


A A A A
½ V  V 

     
      

Figure 2. Diameter of Inhibition Zone of Guava Leaves Extract in Several Sugar


Concentration

*8%5*28#%,* 1, #2,2,&.&,
The data in Figure 3 shown that different kind of bacteria showed different result.
The diameter of inhibition zone were 4.52 ± 5.08 mm for E. coli 7.53 ± 8.06 mm for S.
aureus, and 3.98 ± 6.82 mm for B. cereus The extract activity could be influenced in
inhibiting B. cereus dissimilar with in inhibiting E. coli and S. aureus.
The salt will reduce the water activity value (Aw). Generally pathogen bacterial
can be inhibited at Aw (water activity) less than 0.92 that is the same with 13% (w/v) salt
concentration. The highest salt solution in this experiment was 4% (w/v). This

c    


     D
concentration was chosen for those were usually used for food. This salt concentration
was not sufficient to inhibit the bacterial growth. This data strengthen that the inhibition
was obtained by the extract activity, not by the salt.
   V
V   V


A  
 V 

 A     

     A
       

A AA
 A AA A AA
A

    
mV V 

  
       

Figure 3. Diameter of Inhibition Zone of Guava Leaves Extract in Several Salt
Concentration

*8%5*28 #,&*'* 1, #2,2,&.&,


The data in Figure 4 ± 6 shown that the ability of the antibacterial to inhibit the
bacterial growth will decrease when the heating temperature and time increase. The
diameter of inhibition zones were 5.24 ± 7.29 mm for E. coli (Fig. 4), 3.28 ± 5.15 mm for
S. aureus (Fig. 5), and 5.89 ± 8.04 (Fig. 6). The higher the heating temperature and the
longer the heating time, the less the active compound and the less the volatile component
of the extract (Ardiansyah, 2002).
  V
V   V


A A A A
A
 V 

   A


A    



A
A  A 
 V V 

A AA

Figure 4. Diameter of Inhibition Zone of Guava Leaves Extract in Several Heating Time
towards E. coli

c    


     -


V
 A
  

 
  
  
   
 
    

 V V
V
       
 
 
 
A
A  A 
  
×   V V   ½

A
AA


Figure 5. Diameter of Inhibition Zone of Guava Leaves Extract in Several Heating Time
towards S. aureus


V

 A
      "
! 
 

 !  ! 
   
   "
 

 V V
V

 
 
 
 
A
A  A 
  
×   V V   ½

A
AA

Figure 6. Diameter of Inhibition Zone of Guava Leaves Extract in Several Heating Time
towards B.cereus

1, #2,2,&.&,,# !"›
  6 
Figure 7 shown that the inhibition zone of vegetative cell of B. cereus was 8.94
mm in diameter and the inhibition zone of B. cereus spore was 8.67 mm in diameter.
Bacterial spore is more complex in structure than vegetative cell (Madigan et al., 2006).
Bacterial spore is resistant to heat, drying, radiation, acid, and disinfectant. This result
showed that the extract could inhibit bacterial spore, even though the spore was more
resistant than the vegetative cell.

c    


     -u

      

o  o 
o






 
î  

Figure 7. Diameter of Inhibition Zone of Guava Leaves Extract towards B. cereus Spore

 
From the entire experiment, it can be concluded that guava leaves have
antibacterial activity. The activity was influenced by pH, sugar, salt, and by heating
process. Moreover the antibacterial activity was strong enough to inhibit B. cereus
spores. This research indicates that guava leaves have potential natural antibacterial
compound and can be applied for certain food such as sour food, sugar added food, food
with no heating process or short heating process. Further research is suggested to study
the application of antibacterial activity of guava leaves.


 
Ardiansyah. Kajian Aktivitas Antimikroba Ekstrak Daun Beluntas (Ôlucea indica L.).
Program Pascasarjana IPB, 2002.
Bloomfield, S.F. Assesing Antimicrobial Activit . Oxford: Blackwell Scientific
Publication, 1991.
Harborne, J.B. Metode Fitokimia: Ôenuntun Cara Modern Menganalisis Tumbuhan.
Bandung: Penerbit ITB, 1996.
Madigan, M.T., H.M. Martinko, dan J. Parker. Brock Biolog of Microorganisms.
Southern Illinois : Prentice Hall, 2006.
Morton, J. F. ³Guava´. Home on-line. Available from
http://www.hort.purdue.edu/newcrop/morton/Description; Internet; accessed 5
August 2006.

c    


     -*
*,#'*&",&2 ,*28›#2, &# "%#,!8 -2#%&2  '#*&"-8#D#6

   #'#&*,"#-6&*'88&"#"©  


5+*#*!
+&  882,* ,+8
&2

Hersanti and Indah Fatmawati
Department of Plant Pests and Diseases, Faculty of Agriculture
Universitas Padjadjaran
Hersanti :hersanti09@gmail.com

6›m©6


Damping offdisease, caused by Rhioctonia solani Kuhn, is a major disease on rice. The
use of antagonistic bacteria as bio-control agents is an alternative control method toward
damping off disease which is friendly to environment.
The purpose of research was to evaluate the abilities of antagonistic bacterial isolate from
maja fruit to suppressing R. solani. The experiments were carried out in the
Phytopathology Laboratory and Glasshouse of the Department of Plant Pests and
Diseases, Faculty of Agriculture, Universitas Padjadjaran from June 2010 to September
2010.
There were two experiments i.e.: test of antagonistic ability of the isolates in dual culture
method and test of ability of the bacterial isolate to increase growth of rice var IR64 in
the glasshouse. Those experiments were arranged in the completely randomized design
consisted of 8 treatments and 3 replications.
Eight of bacterial isolates from maja fruit showed antagonistic abilityagainstR. solani in
vitro. The highest suppression was shown by BM4, BM5 and BM6 isolate, with
percentage inhibition 81,6% , 88,6% and 86,9% respectively. All bacterial isolates had be
ability to increase the growth of rice.

Keyword :Rhioctonia solani, Mol of maja fruit, rice, Antagonistic bacteria




c    


     -

 
Damping off diseasecaused by Rhioctonia solani is a major disease of rice (Agrios,
2004). This disease is also an important disease on rice in Indonesia; found in almost all
rice field, esspecially in wet season, with disease intensity of 50%(Semangun, 2004).
Organic plantation system with SRI (S stem of Rice Intensification) uses a mixture of
natural materials found in the area around rice plantation as the effort to control
destructive organisms to plant. The materials used are banana stump, coconut water,
water, rice, maja fruit, bamboo sprout, papaya, banana, sugar cane, chayote young
shoots, golden snail, and vegetables. Material¶s composition is adjusted to the available
source materials, and then fermented. Results of fermentation are known as the Local
Microorganisms or MOL (Ekamaida, 2008; Hersanti & Djaya, 2008). MOL has been
used by rice farmers, especially rice farmers using SRI planting methods (Hersanti &
Djaya, 2008). According to Uphoff (2004), organic rice intensification system does not
require chemical fertilizers and other chemicals, but will use the materials available in
nature.
Hersanti & Djaya (2008) obtained 19 (nineteen) bacterial isolates from different types of
Mol which were cebreng leaves, young shoots of chayote, maja fruit, bamboo sprouts,
banana stump. The dual culture of 19 (nineteen) bacterial isolates resulted in 6 isolates
which showed antagonistic ability against fungus Rhioctonia or ae and 4 isolates
showed antagonistic ability against fungus Cercospora or ae.
Antagonistic ability of the bacteria isolated from maja fruit MOL against rice diseases is
not known. Therefore, the research on utilizing the antagonists isolated from maja fruit
MOL needs to be done, so it can be used as biocontrol agent in controlling the damping
off disease on rice.

  
The experiments were conducted in the Laboratory of Phytopathology and Greenhouse of
Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran.


*.&, ",

c    


     -
The test of antagonistic potential of the bacteria isolated from maja fruit mol to prevent
the growth of fungi R. solani was done using the dual culture method,and arranged in the
Completely Randomized Design. There were six bacterial isolates obtained from the maja
fruit mol.


*.&. Test
The test to evaluate the ability of the bacterial isolates to increase the growth of rice
seedlings were done using the completely randomized design. The treatments were based
on the number of candidates of in vitro antagonistic bacteria, which were 6 isolates of
antagonistic bacteria and a control (R. solani). Each treatment was repeated three times.
The data were analyzed statistically using the SPSS version (16.0). Differences between
treatments were tested using the Duncan Multiple Range Test in the 5% significant..



  
*.&, ,",
From the maja fruit MOL, it was obtained 8 bacterial isolates. The isolates were
differentiated according to the color, shape, surface, and the edge of the colonies on
nutrient agar (Leung & Liu, 2002; Johnston, 2007). The colony characteristics of the
eight bacterial isolates were described in Table 1.

Table 1. Colony Morphology of bacteria isolated from Maja Fruit mol on the Nutrient
Agar medium
%* 6+%'
 "%#,"
%5  +#6 5 8#2 !'
1 BM1 White Circular Raised Entire
2 BM2 white Circular Convex Entire

c    


     -
3 BM3 yellow Circular Umbonate Entire
4 BM4 white Circular Raised Undulate
5 BM5 yellow Circular Convex Entire
6 BM6 white Circular Flat Entire
7 BM7 cream Circular Convex Entire
8 BM8 cream Irregular Flat Lobate
BM : Maja Fruit

Table 2. Level of inhibition caused by bacterial isolates of Maja Fruit MOL against R.
solani on the 3rd day.
 #*"8
#!&#%' ,+ .%8
 #,-*, 8©

2- &*+&$&,&*C
 ›EF
"%#*& 3772 :72
( ›(EF
"%#*& 372 :<72
; ›EF
"%#*& (3(! ::(!
 ›:EF
"%#*& 3$ =($
 ›=EF
"%#*& 372 :<(2
: ›7EF
"%#*& 37=2 :7=B2
= 2*, %", &% :3BB
#,  B
7 2*, %'8%&25  B3BB# BB#
Description: The average value which followed by the same letter in the same column is
not different
significantly according to Duncan Multiple Range Test in 5% significant

In dual culture test, most of the isolates caused inhibition zone, indicating the production
of metabolites toxic to the pathogen. Many antagonistic bacteria produce antibiotics,
siderofor, and other secondary metabolites toxic to or inhibit the growth of other
microorganisms (Fravel 1988, in Hasanuddin, 2003). For example Ôseudomonas

c    


     --
fluorescens was reported to produce antibiotic effectively inhibit the growth of
Rhioctonia solani, and also produce pyoluteorin antibiotic that inhibited Ô thium
ultimum growth (Howell and Stipanovic, 1979 in Hasanuddin, 2003). Some bacteria have
been widely used as biocontrol agents through the emphasis of antibiotics production to
suppress the development of pathogen or disease (Mukerji & Garg, 1988 in Yulia et al.
2008). Microbes that produce antibiotics are considered more appropriate to use as
biocontrol agents compared to the other mechanisms of antagonism such as competition
and parasitism (Yulia et al. 2008).
Other mechanism of the biocontrol agents is the competition of food source or certain
elements, so that constrain the growth room of other microorganisms (Weller 1988 in
Hasanuddin 2003; Brock, 1966 in Sudadi, 2005).

BM8 X R. BM1 XR. BM7 XR. BM2 X R. solani


solani solani solani solani sssolanisolanisol

BM4 X R. BM5 X R. solani BM8 X R. BM6 X R.


solani solani solani

kontrol
c    
     -†
Picture. Inhibition caused by bacterial isolates of maja fruit on the colony of R. solani on
potato dextrose agar, three days after inoculation

+882,8$#2, &#&"%#,!8 -#D# 5&,*,+' ,+8 &2

Result of the treatments of bacteria isolated from maja fruit mol on the seedling growth is
shown on Table 3. On 10 days after planting, the seed germination percentage and
seedling height of those treated with isolates of BM2, BM6 and BM7 were significantly
higher than control. This result showed that the bacteria isolated from maja fruit mol are
capable in inducing seed germination and growth of rice seedlings.

Table 3.The effect of bacteria isolated from Maja Fruit mol on the growth of rice
seedlings
  #,-*,  -&*#,&*C &'+,8
"!%&*'"2-
1. BM1 75,0 0 b 5,00 c
2. BM2 78,33 ab 8,71 a
3. BM4 76,11 ab 5,11 c
4. BM6 85,00 a 8,95 a
5. BM7 83,89 ab 8,22 ab
6. BM8 81,11 ab 7,93 ab
7. Kontrol + 63,33 c 3,08 d
8. Kontrol ± 83,33 ab 7,16 b
Description: The average value which followed by the same letter in the same column is
not different

c    


     -o
significantly according to Duncan Multiple Range Test in 5% significant

 Potency of the bacterial isolates from Maja fruit mol in increasing the percentage
of seed germination and seedling growth were similar to the result of Wahyudi (2009),
that Ôseudomonas sp and Bacillus sp. induced plant growth and produced growth
hormone and antimicrobial compound, so they decreased plant disease severity.

 
 Eight of bacterial isolates from maja fruit showed antagonistic
abilityagainstRhioctonia solani in vitro. The highest suppression was shown by BM1,
BM2, BM4, BM6 and BM7 and BM8 isolate, with percentage inhibition 68,51%,
69,81%, 64,62%, 74,25%, 69,25%, and 68,70% respectively. The bacterial isolates
(BM2, BM6, BM7 and BM8) had the ability to increase the growth of rice seedling..

c    


     -D


 

Agrios, GN. 2004. Plant Pathology. 4th Ed. Academic Press. London.
Ekamaida. 2008. Pengelolaan lahan pertanian ramah lingkungan dengan sistem
intensifikasi tanaman padi melalui pemanfaatan mikroorganisme lokal dalam pembuatan
kompos (Studi kasus di Desa Sidodadi Kabupaten Deli Serdang). Available online at
http://library.usu.ac.id (Diakses tanggal 8 September 2008).
Hasanuddin. 2003. Peningkatan peranan mikroorganisme dalam sistem pengendalian
penyakit tumbuhan secara terpadu. Jurusan Hama dan Penyakit Tumbuhan Fakultas
Pertanian Universitas Sumatera Utara. Available online at http://library.usu.ac.id (diakses
tanggal 8 September 2008).
Hersanti dan L. Djaya. 2008. Antagonism of Bacterial Isolates from Local
Microorganisms against Rhioctonia solani and Their Effect on the Growth of Rice

c    


     †
Seedlings. Mini Symposium Biocontrol in conjuction with ICMNS. Sekolah Tinggi Ilmu
Hayati Institut Teknologi Bandung.
Koga, H. 2001. Cytological aspects of infection by the rice blast fungus p ricularia
or ae. In: sreenivasaprasad, s, and r. Johnson (eds.). Major fungal disease of rice recent
advances. Kluwer Academic Publishes. pp. 87.http://books.google.co.id/books. Diakses
tanggal 24 September 2008.
Johnston, D. 2007. Colony Characteristics.http://faculty.clinton.edu/faculty/
donald.johnston/. Diakses tanggal 22 November 2008
Soenarjo, Edi, Djoko, S. Damarjati, dan Mahyuddin Syam. 1991. Buku 3 Padi. Penelitian
dan Pengembangan Pertanian.Pusat Penelitian dan Pengembangan Tanaman Pangan
Bogor.167 hlm.
Sudadi. 2005. Interaksi Mineral Lempung, Bahan Organik, dan Mikrobia tanah; Pengaruh
Terhadap Antagonisme dan Pemanfaatannya Dalam Pengendalian Hayati Penyakit
Tanaman Asal Tanah. Jurusan Ilmu Tanah Fakultas Pertanian UNS Surakarta.Jurnal Ilmu
Tanah dan Lingkungan. 5 (1): 18-29.
Suganda, T., E. Rismawati, E. Yulia, dan C. Nasahi, 2002.Pengujian kemampuan
beberapa bahan kimia dan air perasan daun tumbuhan dalam mengiduksi resistensi
tanaman padi terhadap penyakit bercak daun Cercospora. Jurnal Bionatura 4(1) : 17-28.
Suparyono., JLA Catindig, NP Castila, and FA Elazequi. 2003. Narrow Brown Leaf Spot
Available online at: http://www.knowledgebank.irri.org/
RiceDoctor/Fact_Sheets/Diseases/Narrow_Leaf_Brown_Spot.htm. (Diakses tanggal 4
Juni 2009).
Uphoff, N. 2004. System of rice intensification responds to 21st century needs. Grain of
truth.42 p.
Yulia, E., dan F. Widiantini. 2007. Potensi Bakteri Filoplen Asal Tanaman Padi dalam
Menekan Perkembangan Penyakit Busuk Batang (Helminthosporium sigmoideum Cav.)
pada tanaman Padi. Jurnal Agrikultura. 18 (3) : 190-196.
Yulia, E., F. Widiantini, R. Firmansyah, dan A. Karuniawan. 2008. Kemampuan Ekstrak
dan Bakteri Inhabitan Mucuna pruriens Linn.dalam Menekan Penyakit Bercak Daun
Cercospora dan Meningkatkan Pertumbuhan Tanaman Kacang Tanah. Jurnal Agrikultura.
19 (1) : 50-59.

c    


     †u
Yuliar. 2008. Skrining bioantagonistik bakteri untuk agens biokontrol Rhioctonia solani
dan kemampuannya dalam menghasilkan surfaktin. Biodiversitas 9(2):83-86.

c    


     †*