day 1-fullpaper poster

BIOOXIDATION OF BERBERINE BY THE ENDOPHYTIC FUNGUS COELOMYCETES AFKR-1 ISOLATED FROM KAYU KUNING [Archangelisia

flava (L.) MERR.: Menispermaceae]

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: [email protected]

ABSTRACT

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.

Key words: Archangelisia flava, endophytic fungi, Coelomycetes AFKR-1, berberine, biotransformation.

INTRODUCTION

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 Xylaria sp. isolated from the plant Cinchona pubescence

(Rubiaceae) reported can transform Cinchona alkaloids (quinine, quinidine, cinchonine,

International Seminar Biotechnology P1

mailto:[email protected]

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).

MATERIALS AND METHODS

Plant 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 Endophytic 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


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 by the Endophytic Fungus Coelomycetes 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].

RESULTS AND DISCUSSION

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 13C-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

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

Xylaria sp. which was also reported produce N-oxide derivatives (Shibuya et al., 2003).

Fig 1.

Tabel 1. 13C-NMR and 1H-NMR data for biotransformed product and berberine in DMSO-d6.

No. Atoms Biotransformed Product Berberine13C-NMR 1H-NMR 13C-NMR 1H-NMR

12344a5688a

105.4147.7149.8108.4130.726.355.2145.5121.4

7.80 (1H, s)

7.09 (1H, s)

3.20 (2H, dd, J=6.2 Hz)4.93 (2H, dd, J=6.2 Hz)9.90 (1H, s)

105.4147.7149.8108.4130.726.355.2145.5121.4

7.80 (1H, s)

7.09 (1H, s)

3.20 (2H, dd, J=6.2 Hz)4.93 (2H, dd, J=6.2 Hz)9.90 (1H, s)


910111212a1313a13b9-OCH310-OCH3O-CH2-O

143.6150.4126.7123.5133.0120.2137.5120.561.957.0102.1

8.20 (1H, d, J=9.1 Hz)8.01 (1H, d, J=9.1 Hz)

8.94 (1H, s)

4.10 (3H, s)4.07 (3H, s)6.17 (2H, s)

143.6150.4126.7123.6133.0120.2137.5120.561.957.1102.1

8.20 (1H, d, J=9.1 Hz)8.01 (1H, d, J=9.1 Hz)

8.95 (1H, s)

4.09 (3H, s)4.07 (3H, s)6.17 (2H, s)

Fig. 2.

ACKNOWLEDGEMENT

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

Internal research fund (DIPA).

REFERENCE

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. Pharm. 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, J. Pharm. 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.

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 Biotechnology, 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 Xylaria sp. isolated from Chincona pubescence. Chem. Pharm. Bull., 51, 71-74


THE EFFECT OF EARLY FEED RESTRICTION ON THE COMPENSATORY GROWTH IN BROILER CHICKENS

ANDRY PRATAMA, RUHYAT KARTASUDJANA1, TUTI WIDJASTUTI11)Lecturer of Faculty of Animals Husbandry, Padjadjaran University

ABSTRACT

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.

Key words : Restricted Feeding, Growth, Broiler Chickens

INTRODUCTION

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,

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.


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 = the diet ad libitum, R1 =

The diet ration of 80% of ad libitum, R2 = The diet ration of 75% of ad libitum, R3 = The

diet ration of 70% of ad libitum, R4 = The diet ration of 65% of ad libitum, and R5 = 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).


Final Body Weight

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

RepeatedTreatment

R0 R1 R2 R3 R4 R5…………………………………...gram……..………………………………

1 2915,0 2685,0 2812,0 2865,0 2752,0 2844,02 3095,0 2785,0 2816,0 2700,0 2847,5,0 2892,03 2780,0 2700,0 2754,0 2820,0 2880,0 2686,04 2965,0 2897,5 2699,0 2612,0 2762,5 2555,05 2468,0 2708,0 2648,0 2746,0 2936,0 2708,0

Total 14223,0 13775,5 13729,0 13743,0 14178,0 13685,0Average 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

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.

Hematology Blood

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

TreatmentParameters

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 26,15R1 1,130 x105 1,638 x105 4,75 7,00 19,83 27,15


R2 1,370 x105 1,569 x105 5,67 6,05 24,00 25,60R3 1,277 x105 1,348 x105 5,99 6,45 20,17 23,95R4 1,017 x105 1,490 x105 4,67 7,85 20,13 29,65R5 1,183 x105 1,362 x105 5,33 7,05 22,50 27,50

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.

Feed Conversion

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

Time Treatment


(weeks) R0 R1 R2 R3 R4 R51 1,47 1,64 1,78 1,53 1,59 1,692 1,60 1,44 1,32 1,38 1,42 1,333 1,42 1,40 1,37 1,37 1,19 1,274 1,47 1,48 1,47 1,42 1,36 1,495 1,69 1,86 1,59 1,87 1,77 1,816 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.

CONCLUSIONBased 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.

REFERENCESGaspersz, V. 2006. Teknik Analisis Dalam Penelitian Percobaan. Penerbit Tarsoto.

Bandung.

Hood, R.L. and Allen, C.E. 1977. Cellularity of Adipose Tissue: Effect of Growth and Adiposity. Journal of Lipid Research. 18: 275-284.

Jones, G.P.D. and Farrell, D.J. 1992. Early-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. Compensatory growth in the broiler chicken : a review. Worlds Poult. Sci. J. 52:189-201.

Siswani. 2006. Gambaran Darah Merah dan Pertumbuhan Mandalung (Mule Duck) yang Disuplementas Vitamin C. Fakultas Kedokteran Hewan. Institut Pertanian Bogor.

Sturkie, P.D., P. Griminger. 1986. Blood : Physical Characteristics, Formed, Elements, Hemoglobin, and Coagulation in Avian Physiology. Springer Verlag. New York.

Wijtten, P. J. A., Hangoor, E., Perdok, H. B., and Sparla, J. K. W. M. 2008. The Effect of Early Life Feed Restriction on Performance and Mortility 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.


ABNORMALITY ANALYSIS OF OIL PALM (ELAEIS GUINEENSIS JACG) SOMATIC EMBRYO USING RANDOMLY AMPLIFIED POLYMORPHISM

DNA (RAPD) AND RANDOMLY AMPLIFIED DNA FINGERPRINTING (RAF) TECHNIQUES

NESTI F. SIANIPAR1, GUSTAAF A. WATTIMENA2, MAGGY THENAWIJAYA S3. HAJRIAL ASWIDINNOOR2, NURITA TORUAN-MATHIUS4 , GALE GINTING5

1Department of Biology, Faculty of Science and Mathematics, Universitas Pelita Harapan, Tangerang

2Department of Agronomy, Bogor Agricultural University, Bogor3Department of Food Science and Technology, Bogor Agricultural University, Bogor4SEAMEO

Biotrop, Bogor5Oil PalmResearchCenter of Indonesia, Medan

ABSTRACT

Abnormality of somatic embryo (SE) can be resulted from genetic or epigenetic changes. Thus, genetic changes can be analyzed by Random Amplified Polymorphism DNA(RAPD) and Randomly 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.

Key words: somatic-embryoabnormality, oil-palm, RAPD, Randomly Amplified DNA Fingerprinting (RAF).

INTRODUCTION

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


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

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.


Plant Material: Normal mother plantand somatic embryo of oil palm clone MK 558 and

MK 638 were obtained from Oil Palm Research of Indonesia (PPKS), Medan.

DNA extraction: 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).

RAPD Assay: 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.

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.

RAF Assay: 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 α-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’


3.000

500

1.000

2.000

10.000


A. RAPD Assay

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


3.000

250

1.0001.500

10.000

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

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.

DNA fragment

pb

Clone 638 Clone 558

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

fragmentpb

W-15 (5’-ACACCGGAAC-3’) W-15 (5’-ACACCGGAAC-3’)


1. 5000

2. 2500

3.

4.

5.

6. 1750

7. 1500

8.

9.

10.

+ + - - + -

- - - - + -

+ + + + + -

- - - - - +

- - + + + -

- - + - - -

+ + + - + -

- - - - - +

- - + + - +

+ + - - + -

+ + - + + -

+ + - + + -

+ + + + + -

+ + + + + +

+ + + + + -

+ + + + + -

+ + + + + -

- - - - + +

- - - - - +

- - - - - -

DNA fragment

pb

OPC-09(5’CTCAACGTCC-3’) OPC-09(5’CTCAACGTCC-3’)



1.3250

2.

3.3000

4. 2000

5.

6.

7.

8.

- - + - - -

+ + + + - -

+ + + + + +

- - + - - +

+ + + + - -

- - - - - -

+ + + + - -

- - - - - -

+ + + + + -

+ + + + + -

+ + + + + +

+ + + + + +

+ + + + + +

- - - - - +

+ + + + + -

- - - - - +

DNA

fragmentpb

AP-20(5’-CCCGGATACA-3’) AP-20(5’-CCCGGATACA-3’)


1. 3000

2. 2000

3. 1750

4.

5.

6. 750

7.

- - - + - -

- - -+ - -

- - -+ - -

+ + + + + +

+ + + + + +

+ + + + + -

+ + + + + +

- - + - - -

- - + - - -

- - + - - -

+ + - + + +

+ + - + + +

+ + + + + -

+ + + + + +

DNA

fragmentpb

SC10-19 (5’-SGTCCGTCAG-3’) SC10-19 (5’-SGTCCGTCAG-3’)


1. 5000

2. 4000

3. 3000

4. 2250

5. 2000

6.

- + + -+ -

- + + -+ -

- + + - + +

- + + -+ -

+ - + + - -

- - + + - -

+ + + + + -

+ + + + + -

+ + + + + +

+ + + + + -

+ + + + + -

+ + + + + -

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

B. RAF Assay


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).


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

DNA

(pb)

AB- 16 (5’-CCCGkasiGATGGT-3’) AO-12 (5’-TCCCGGTCTC-3’)

2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN

90

91

138

146

248

258

375

378

380

- + - - - +

+ - + + + -

+ + + + + -

- + - - - +

+ + + + + -

+ + + + + -

+ + + + + -

- - - - - +

+ + - - - -

150

160

- + - - - +

+ - + + + -

Fragmen AE-11(5’-AAGACCGGGA-3’) BB- (5’-CAACCGGTCT-3’)


DNA

(pb)

18

2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN

152

158

369

372

- + - - - +

+ - + + + -

- - - - + -

+ + + + + +

90

150

158

- + - - - +

- + - - - +

+ - + + + -

Fragmen

DNA (pb)

AP-20 (5’-CCCGGATACA-3’) W-15(5’-ACACCGGAAC-3’)

2K+ 2K- 2G+ 2G- 2D DN 2K+ 2K- 2G+ 2G- 2D DN

115

155

238

355

358

- + - - - -

- + - - - +

+ + + + + -

+ - + + + -

+ - + + + -

150

214

217

255

258

- + - - - +

+ + + + + -

+ + + + + -

- - - - - +

- - - - - +

Keterangan : Fragment (+), Fragment (-), Normal Globular (G+), Abnormal Globular (G-), Normal Cotiledon (K+), Abnormal Cotiledon (K-), Planlet Leaves (D), Mother plant leaves (DN).

CONCLUSION

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.

ACKNOWLEDGMENTS

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.


REFERENCES

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. Planter (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. Phytochem 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.). J. Pen. 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. Plant Cell Tiss. Org. Cult 20 : 201 - 209.

Kaeppler SM, Kaeppler HF, Rhee Y. 2000. Epigenetic aspects of somaclonal variation in plants. Plant 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 Palm Tissue Cult & Biotechnol. Kuala Lumpur, p. 131 -141.


Pannetier C, Arthius P, LievouxDE, 1981. Neoformation de jeuneus planlets d'Elaeis 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. Plant 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. J Biomed and Biotech 2 (3) : 141-150.


ISOLATION, IDENTIFICATION AND SELECTION OF CELLULOLITIC FUNGI FROM BANANA WASTE (Musa paradisiaca)

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

1) Fisheries Faculty, Lambung Mangkurat University, Banjarbaru2) Departement of Biology. FMIPA, Padjadjaran University, Bandung

3) Fisheries and Marine Science Faculty, Padjadjaran University, Bandung

ABSTRACT

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 containing high 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 dilution series 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 : Penicillium nalgiovense Laxa, Aspergillus ochraceus Wilhelm, Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, and Aspergillus oryzae. Aspergillus terreus and Aspergillus oryzae more potential degrade cellulose banana waste than other fungi that found.

Keywords : cellulolytic fungi, banana waste

INTRODUCTION

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%, lignin 20.21%, 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 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 potential fungus to degrading cellulose of banana waste.


Materials and Equipment

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.

Procedur

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 cellulolytic 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


solution. If the fungus has the ability cellulolytic it will show the transparent zone around

the colony isolates of the fungus.


Types 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 Penicillium 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 μm.


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 μm.

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 μm. Conidiophore hyalin, smooth-walled,

subglobose vesicles 10-20 μm. Phialide derived from metule 5-7 x 2.0 to 2.5 μm. Metulae

5.5 x 1.5 to 2.0. Conidia globose to ellipsodial 1.5 to 2.5 μm, 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 μm. Phialid formed in metula, and size

(7.0 to 9.5) x (3-4) μm. Metula hyaline to brown in color, often bersepta, and size (15-25)

x (1.4 to 6.0) μm. Conidia spherical to oval, measuring 3.5 to 5.0 μm, brown, have

ornamentation in the form of bumps and spikes that are not uniform.

Isolate 6

Fungi isolat 6 identified as Aspergillus oryzae , 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,

subglobose vesicles with a size of 40-80 μm. Phialide sometimes comes directly from

vesicles or metulae, usually measuring 8-12 x 4-5 μm. Conidia initially shaped ellipsodial

but the older the age-globosa fungi to shape subglobosa with size from 4.5 to 8 μm.


Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate6

Picture 3. Isolate of Fungus

Cellulolytic 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 oryzae. As for the

comparison between the transparent lear zone and colony diameter can be seen at Table

1.


Isolate 1 Penicillium nalgiovense Laxa

Isolate 3 Aspergillus nidulans

Isolate 4Aspergillus terreus

Species 5 Aspergillus niger

Species 6 Aspergillus oryzae

Isolate 2 Aspergillus ochraceus Wilhelm

Table 1. Comparison between Transparent Zone and Colony DiameterFungi Transparent

Zone (mm)Colony Diameter

(mm)Comparison TZ

dan CD (mm)Number of

Fungi Candidate

Isolate 1 8 7 1.14 5Isolate 2 9 6 1.5 3Isolate 3 27 22 1.22 4Isolate 4 14 7 2.00 2Isolate 5 5 5 1,00 6Isolate 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).

CONCLUSION


Retrieved 6 types of isolates fungus that grows on a banana waste, namely:

Penicillium nalgiovense, Laxa, Aspergillus ochraceus Wilhelm, Aspergillus nidulans,

Aspergillus terreus, Aspergillus niger, Aspergillus oryzae. Aspergillus terreus and

Aspergillus oryzae were the best candidates with the highest capability in cellulose

degradation.

REFERENCES

Kader, A.J and O.Omar, 1998. Isolation of cellulolytic Fungi from Sayap-Kinabalu Park, Sabah. Article ASEAN Review of Biodiversity and Environmental Conservation (ARBEC) 2:1-5.

Riadi, Lieke 2007. Teknologi Fermentasi. Graha Ilmu. Yogyakarta

Rismunandar. 1990. Bertanam Pisang. 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 Penyulingan daun Kayu Putih (Melaleuca leucadendron L) dari KPH Gundih, Kabupaten Grobogan. Jurnal Biofarmasi 2 (1): 24-28, Pebruari 2004.


ANTIMICROBIAL METABOLITE FROM THE CULTURE OF ENDOPHYTIC

FUNGUS AFK-8 ISOLATED FROM KAYU KUNING[Archangelisia flava

(L.)MERR.]

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: [email protected]

ABSTRACT

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 g/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.

INTRODUCTION

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 Taxomyces

andreanaefromTaxus brevifoliathat also produce taxol (Stierle et al., 1993), an endophyte

fungus from the plantNotapodytes foetidathat produce camptotecin (Puriet al., 2005) in

laboratory. Some of Phomopsisfungi species associate with the plant ofSalix gracilostyla

var. melanostachys(Horn et al., 1995), the fungusCryptosporiopsis cf. quercinaassociate

with Tripterigium wilfordii(Strobel et al., 1999; Li et al., 2000), the fungusCytospora sp.

CR 200 andDiaporthe sp. CR 146 (Brady et al., 2000) have been reported produce

various secondary metabolites that shows an antibacterial activity.


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.

MATERIAL AND METHODE

Plant Materials

The youg stems of kayu kuning (Archangelisia flava) were collected from

Sambas, West Kalimantan in 2007 and identified by DR. Sudarmono, Bogor Botanical

Garden.

Isolation of Endophytic Fungi

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.

Screening of Secondary MetabolitesProduction and Antibacterial Assay

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


(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 µL)and cultured on Mueller Hinton Agar (MHA) media. 10 µL

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.

Scaling up Cultivation of Fungus AFK-8

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.

Isolation of Active Metabolites

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 µL extract was then put onMHA media

that had been inoculated with bacteria isolate, followed by incubation for 24 hoursat 37 oC.The growth inhibitoin zone was the clear zone around the paper disc and the diameter

of clear zone was measured.


Determination of MIC Value of Fraction 6

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.

GC-MS Analysis of Fraction 6

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 = 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 = 250 oC, trap

temperature = 150oC. The sample (in aceton solution) was injected 5 µL.

RESULT AND DISCUSSION

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


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 g/10 l, but failed to show their activity against E.

coli at tested concentration (100 g/10 l).

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 (MIC= 4 mg/L) had higher activity compared to chloramphenicol (MIC=

16) against S. aureus, and its activity was similar to chloramphenicol against B. subtilis


(MIC = 8 mg/L). Fraction 6 also had higher activity against R. minuta (MIC = 32

mg/L)dan S. cerevisiae (MIC = 32 mg/L) and similar to C. albicans (MIC = 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 Library and Wiley, 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 .

Table 1. MIC value of F6 (fraction 6) against tested bacteria and fungi.

No Microbe isolate

MIC (mg/L)

F6 chloramphenicol erythromycin nystatin Cabisidin

1 B. subtilis 8 8 0,03 nt nt

2 M. luteus 32 16 0,06 nt nt

3 S. aureus 4 16 32 nt nt

4 E. coli 64 16 16 nt nt

5 C. albicans 32 nt nt 32 32

6 R. minuta 32 nt nt 64 64

7 A. niger 32 nt nt 16 64

8 A. flavus 32 nt nt 16 32

9 S. cerevisiae 32 nt nt 64 64

nt : not tested


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.


CONCLUSION

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.

ACKNOWLEDGEMENT

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.

REFERENCE

Agusta A., Ohashi K and Shibuya H. 2006. Bisanthraquinone Metabolites Produced bythe Endophytic Fungus Diaporthe sp. Chem. Pharm. Bull.,54 (4), 579.

Agusta A. 2009. Biologi dan Kimia Jamur 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. Cytoskyrins A and B, new BIAActive Bisanthroquinones Isolated from an Endophytic Fungus.Organic Letters 2(25). 4047.

George C. 1988. Fungal Endophytes in Stems and Leaves : From Latent Pathogen toMutualistic Symbiont. Ecological Society of America, 69 (1).

Geris dos Santos RM and Rodrigues-Filho E. 2003. Structures of Meroterpenes Producedby Penicillium sp, an Endophytic Fungus Found Associated with Melia Azedarach. J. Braz. Chem. Soc..,14 (5), 722.

Hossain SM.. 2005. Isolation and Structure Elucidation of Secondary Metabolites fromEndophytic Fungi and the Plant Prismatomeris tetrandra and Synthesis of


http://www.futuremedicine.com/doi/abs/10.2217/clp.09.41

http://www.meb.uni-bonn.de/cancer.gov/GlossaryTerm/CDR0000045594html

(+)-Ochromycinone.

Hyde, KD and Soytong K. (2008). The fungal endophyte dilemma. Fungal Diversity33,163 – 173.

Janos B. 2005. Bioactive Microbial Metabolites. J. Antibiot., 58(1). 13

Keawpradub N., Dej-adisai S. and Yuenyongsawad S. 2005. Antioxidant and cytotoxicactivities of Thai medicinal plants named Khaminkhruea:Arcangelisiaflava , Cosciniumblumeanum andFibraureatinctoria.J. Sci. Technol.,27( 2), 455-467.

Radji M. 2005. Peranan Bioteknologi dan Mikroba Endofit dalam Pengembangan ObatHerbal. Majalah Ilmu Kefarmasian, II (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 inhibitoryconcentration (MIC) by broth dilution of fermentative yeasts.Eucast Discussion -Document E. Dis 7.1. 1.

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

Tan, R.X. and W.X. Zou. 2001. Endophytes: A rich source of functional metabolites. Nat. Prod. Rep., 18: 448—459

Tsai PL and Tsai TH. (2002). Simultaneous determination of berberine in rat blood, liverand bile using microdilysis coupled to high-performance liquid chromatography.Journal of Chromatography A, (961), 125-130.zipcodezoo.com/Plants/A/Arcangelisia_flava/03 Mei 2009.

Verpoorte R., Siwon J., van Essen GFA., Tieken M. and Svendsen AB. 1982. Studies onIndonesian Medicinal Plants VII Alkaloids ofArcangelisiaflava. Journal ofNatural Products,45 (5).

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


COMBINED EFFECT OF NaCl AND IBA ON MS MEDIA ON GROWTH OF RICE MUTANT STRAIN IN VITRO

PRIYANTI1, AZRI KUSUMA DEWI2, EFRILIA NURJANAH1

1, 1 UIN Syarif Hidayatullah Jakarta, 2 BATAN Alamat: [email protected]

ABSTRACT

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.

Key words: in vitro, mutant lines, rice, NaCl and IBA

BACKGROUND

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


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).

RESEARCH METHOD

Materials and Equipment

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.

Procedure

1. Sterilization Equipment

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.

2. Preparation of Culture Media

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.

3. Induction Rice

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

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.

Observation Parameter

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.

Experimental Design

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.

Data Analysis

Data obtained statistically tested using ANOVA test. If significantly different

Duncan's test at 5% test level.


1. Percentage of plantlets growing power

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).

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 (α = 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.

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 indicatesno significant difference (α = 5%)


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 (α = 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).

3.Root Length


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

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).

CONCLUSION

• 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

The combination of IBA NaCl 1% and 5% the percentage of power grew, height and

root length of plantlets was still good.


REFERENCES

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

STUDY OF ANTIMICROBIAL ACTIVITY FROM GUAVA (Psidium guajava L.) LEAF EXTRACT TOWARDS PATHOGENIC MICROBES

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 :UPH Tower, Jl. MH Thamrin Boulevart 00-00, Lippo Karawaci, Tangerang 15811, Indonesia, phone :

(021) 5460901, Fax : (021) 5460910, e-mail : [email protected]

ABSTRACT

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, Staphylococcus aureus, Bacillus cereus, and Penicillium 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 Penicillium sp. The MIC and MBC for Escherichia coli, Staphylococcus aureus, and Bacillus cereus was 0.017% 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, 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.

Keywords : guava leaf, antimicrobial activity

INTRODUCTION

Guava plant (Psidium 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.

METHODE

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


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, Staphylococcus aureus, Bacillus

cereus, and Penicillium 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.

RESULT AND DISCUSSION

The water extracts did not inhibit all the microbes tested, in contrast the ethyl

acetate could inhibit all the bacteria tested but not Penicillium. 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 Pencillium 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 Penicillium

s

o

l

water

0% 0.00 0.00 0.00 0.0010% 0.00 0.00 0.00 0.0020% 0.00 0.00 0.00 0.0030% 0.00 0.00 0.00 0.0040% 0.00 0.00 0.00 0.0050% 0.00 0.00 0.00 0.000% 0.00 0.00 0.00 0.00

10% 9.34 7.99 6.17 0.00


v

e

n

t

ethyl - acetate

20% 9.28 8.58 6.32 0.0030% 9.49 9.52 7.17 0.0040% 9.73 11.81 7.25 0.0050% 10.06 12.95 7.51 0.00

hexane

0% 0.00 0.00 0.00 0.0010% 0.00 0.00 0.00 0.0020% 0.00 0.00 5.04 0.0030% 0.00 0.00 5.81 0.0040% 0.00 0.00 5.79 0.0050% 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, Staphylococcus aureus, and Bacillus cereus were 0.017% 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

Concentration

0 % 0.00 0.00 0.002 % 9.06 7.45 6.494 % 9.33 8.19 7.296 % 9.59 8.36 8.448 % 9.81 8.43 8.4910 % 10.84 8.49 9.04


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 + + +

Influence of pH on Extract Activity

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).

0.00 0.00 0.00 0.000.00 0.00 0.00 0.001.62

4.17

0.00

6.16

0.00 0.00 0.000.00

4.73

1.68

0

2

46

8

10

12

Control 4 5 6 7 8

pH value

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)

E. coli S. aureus B. cereus

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


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

energy to grow would decrease.

Influence of Sugar on Extract Activity

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.

0.00

7.59

9.70 9.43

0.00

4.123.993.342.78

3.92

0.00

5.434.834.844.55

0

2

46

8

10

12

Control 10 20 30 40

sugar concentration (%)

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)


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

Concentration

Influence of Salt on Extract Activity

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


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.

0.00 0.00

4.65 4.794.52 5.08

0.00

8.06 7.81 7.53 7.84

4.35

6.82

3.984.92

0

2

46

8

10

12

control 1 2 3 4

Salt concentration (%)

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)


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

Influence of Heating on Extract Activity

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).

7.29

5.58

6.80

10.09

5.246.036.83

10.09

0

2

46

8

10

12

0 5 10 15

Heating Time (minute)

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)

80oC 100oC

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


3.28 3.364.48

5.15

8.43

3.334.85

8.43

0

2

4

6

8

10

0 5 10 15

Heating Time (minutes)

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)

80oC 100oC

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

7.286.83

8.948.447.83 7.14

5.89

8.44

0

2

4

6

8

10

0 5 10 15

Heating Time (minutes)

Dia

met

er o

f In

hib

itio

n

Zo

ne

(mm

)

80oC 100oC

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

Extract Activity towards B. cereus Spore

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.


8.94 8.67

0

2

4

6

8

10

Vegetative Spora

Cell typesD

iam

eter

of

Inh

ibit

ion

Zo

ne

(mm

)

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

CONCLUSION

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.

REFERENCES

Ardiansyah. Kajian Aktivitas Antimikroba Ekstrak Daun Beluntas (Plucea indica L.). Program Pascasarjana IPB, 2002.

Bloomfield, S.F. Assesing Antimicrobial Activity. Oxford: Blackwell Scientific Publication, 1991.

Harborne, J.B. Metode Fitokimia: Penuntun Cara Modern Menganalisis Tumbuhan. Bandung: Penerbit ITB, 1996.

Madigan, M.T., H.M. Martinko, dan J. Parker. Brock Biology of Microorganisms. Southern Illinois : Prentice Hall, 2006.

Morton, J. F. “Guava”. Home on-line. Available from http://www.hort.purdue.edu/newcrop/morton/Description; Internet; accessed 5 August 2006.


http://www.hort.purdue.edu/newcrop/morton/Description

Antagonistic Potency of Bacteria Isolated from Local Microorganism of Maja (Aegle

marmelos L. Correa) Againts Damping Off Disease (Rhizoctonia solani Kuhn.) and

Their Effect on Growth of Rice

Hersanti and Indah Fatmawati

Department of Plant Pests and Diseases, Faculty of Agriculture

Universitas Padjadjaran

Hersanti : [email protected]

ABSTRACT

Damping off disease, caused by Rhizoctonia 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 ability against R. 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 : Rhizoctonia solani, Mol of maja fruit, rice, Antagonistic bacteria


mailto:[email protected]

INTRODUCTION

Damping off disease caused by Rhizoctonia 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 (System 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 Rhizoctonia oryzae and 4 isolates

showed antagonistic ability against fungus Cercospora oryzae.

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.

METHODS

The experiments were conducted in the Laboratory of Phytopathology and Greenhouse of

Department of Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran.


In-vitro Test

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.

In-vivo 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..


In-vitro test

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

No IsolatesColony Morphology

Colour Shape Surface Edge

1 BM1 White Circular Raised Entire


2 BM2 white Circular Convex Entire

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.

No.

Treatment

Means of Radial growth

of R. solani (cm)

Level of

inhibition (%)

1 BM 1 >< R. solani 1,88 c 68.51 c

2 BM 2 >< R. solani 1,81 c 69.81 c

3 BM 4 >< R. solani 2,12 d 64.62 d

4 BM 6 >< R. solani 1,54 b 74.25 b

5 BM 7 >< R. solani 1,84 c 69.25 c

6 BM 8 >< R. solani 1,87 c 68.70 c

7 control (-) sterile

water

6,00 e

0 e

8 control (+) folicur 0,00 a 100 a

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 Pseudomonas


fluorescens was reported to produce antibiotic effectively inhibit the growth of

Rhizoctonia solani, and also produce pyoluteorin antibiotic that inhibited Pythium

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).


BM7 X R. solaniBM8 X R. solani solani

BM1 X R. solani BM2 X R. solani sssolanisolanisola

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

kontrol

Picture. Inhibition caused by bacterial isolates of maja fruit on the colony of R. solani on

potato dextrose agar, three days after inoculation

The effect of bacteria isolated from Maja Fruit MOL on the growth of rice

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

No. Treatmen

t

Germination (%) Height of

seedlings (cm)

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

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 Pseudomonas sp and Bacillus sp. induced plant growth and produced growth

hormone and antimicrobial compound, so they decreased plant disease severity.

CONCLUSION

Eight of bacterial isolates from maja fruit showed antagonistic ability against

Rhizoctonia 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..


REFERENCES

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 Rhizoctonia solani and Their Effect on the Growth of Rice


http://library.usu.ac.id/

http://library.usu.ac.id/

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 pyricularia

oryzae. 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.


http://www.knowledgebank.irri.org/RiceDoctor/Fact_Sheets/Diseases/Narrow_Leaf_Brown_Spot.htm

http://www.knowledgebank.irri.org/RiceDoctor/Fact_Sheets/Diseases/Narrow_Leaf_Brown_Spot.htm

http://faculty.clinton.edu/faculty/%20donald.johnston/

http://faculty.clinton.edu/faculty/%20donald.johnston/

http://books.google.co.id/books

Yuliar. 2008. Skrining bioantagonistik bakteri untuk agens biokontrol Rhizoctonia solani

dan kemampuannya dalam menghasilkan surfaktin. Biodiversitas 9(2):83-86.


day 1-fullpaper poster

Documents