Isolation and Screening of Antagonistic Diazotrophic Endophytic

Bacteria from Oil Palm Roots against Ganoderma boninense

Yurnaliza

, Meylisa Purba

, Dita Isnaini Rambe

, Listra Sarimunggu

, Isnaini

Nurwahyuni

and Sovia Lenn

Departement of Biology, Faculty Matematics and Natural Sciences, Jl. Bioteknologi No 1, Universitas Sumatera Utara,

Medan, 20155, Indonesia.

Departement of Chemistry, Faculty Matematics and Natural Sciences, Jl. Bioteknologi No 1, Universitas Sumatera Utara

, Medan, 20155, Indonesia.

Keywords: Diazotroph, bacterial endophyte, oil palm, Ganoderma boninense.

Abstract: Ganoderma boninense was pathogenic fungi affected rot disease on the basal stem of oil palm. Strategies to

control G.boninense should be increased to reduced disease incidence. The aims of the study were to obtain

isolates of bacterial endophyte diazotroph from oil palm roots and to examine their growth inhibiting

activity to G. boninense. Oil palm roots were collected from three locations of oil palm plantation, namely

Universitas Sumatera Utara (USU), PTPN IV Adolina, and community’s oil palm plantations at Desa

Bingkat, Serdang Bedagai. Bacterial endophytes was isolated from sterile roots and cultivated on Nutrien

Agar and Ashby's specific medium. Dual culture methods were used as antagonistic test to G.boninense.

Potential isolates were identified by Vitek test analyzer. The results showed that population of the bacteria

were abundant in non-commercial plantation (USU and community plantation). Screening of bacterial

isolates to inhibit G.boninense colony resulted 13 isolates of bacterial with various of percentage inhibition

growth ranged from 11 to 62.5%. One of potential isolate of bacteria performed red color and by Vitek

analyzer was identified as Seratia marcencens.

1 INTRODUCTION

Oil palm is most planted plant as producer of

vegetable oils in the word. The function of oil palm

crops is not only in the food products manufacture

(Murphy, 2014), but also in wood industry for

improving wood density in construction (Sulaiman

et al., 2012). For the reason, the government support

to keep the growth and productivity of oil palm. The

major current problem in oil palm plantation is the

high rate disease attack due to Ganoderma

boninense. The fungi cause diseases in all stage of

oil palm growth even when plant in nursery stage

(Ariffin et al., 2000).

Some intensive controlling efforts were

continued to be done, physically, chemically (Jee et

al., 2015) or used bio-control agent as fungi

(Yurnaliza et al., 2014) or bacteria (Bivi et al.,

2010). The controlling results are still needed to be

improved. The previous research about bio-control

activity of fungal endophytes to protect oil palm

against G.boninense infection showed that some

species of fungal endophytes were able to produced

antifungal, lytic enzymes (Yurnaliza et al., 2014)

and induced plant resistance (Yurnaliza, 2015) and

(Yurnaliza et al., 2017) to protected oil palm from

G.boninense infection.

This study is focused to search a new bacteria for

oil palm protection against G.boninense. Bacterial

antagonist are expected to have antifungal and lytic

enzyme activity, plant growth promoting compound

or the ability to induce their host resistance.

Diazotrophic bacteria were the potential candidate

bacteria that have ability as growth promoting of

plant and also as biological control agent

Diazotrophic are the nitrogen-fixing bacteria from

atmosphere that live symbiotically or free. This

bacteria live abundant in rhizosphere or as

endophytes in the tissues plant. The diazotrophic

endophytes are very important to improve of plant

growth, because of the plant will get converting

product of nitrogen by bacteria directly (Aryantha et

al., 2018). Besides that, some diazotroph bacteria

also produce indole-3-acetic acid for stimulating

Yurnaliza, ., Purba, M., Rambe, D., Sarimunggu, L., Nurwahyuni, I. and Lenny, S.

Isolation and Screening of Antagonistic Diazotrophic Endophytic Bacteria from Oil Palm Roots against Ganoderma boninense.

DOI: 10.5220/0010088700730077

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

73-77

ISBN: 978-989-758-449-7

plant growth (Zega et al., 2018), antifungal

compound or lytic enzymes to control pathogenic

fungi (Ji et al., 2014). Therefore, as a endophytes,

the diazotroph bacteria are promising to be expanded

as a bio-control agent candidate to control

G.boninense. This research is expected to obtain

diazotroph endophytic bacteria from oil palm root as

bio-control agent of G.boninense.

2 MATERIAL AND METHODS

2.1 Samples of Oil Palm Roots

The roots of oil palm were obtained from

Universitas Sumatera Utara (USU), state-owned

enterprise of PTPN IV Adolina, and small holder of

community oil palm plantations at Desa Bingkat,

Serdang Bedagai.

2.2 Isolation Bacteria

Isolation of bacterial endophyte from oil palm roots

were started from surface sterilization according to

procedure of Yurnaliza et al., (2014) with

modification. Oil palm roots (10 g) were washed

with running tap water, and surface sterilized

respectively using ethanol 70% v/v (2 min), sodium

hypochlorite 5.25% (5 min), ethanol 70% (30 sec)

and last rinsed with sterile distilled water (3 times

each). The sterile roots were homogenized by mortar

and pestle and dissolved with 10 ml of sterile

distilled water. The root suspension was diluted by

serial dilution until 10

-3

and the dilution suspension

were spread onto Ashby's and Nutrient Agar (NA)

medium. The culture was incubated for 24-48 hrs at

temperature ± 28ºC. The rising colony bacteria on

the medium were calculated the total colony and

sub-cultured to the new medium for purification.

The single culture as pure culture were characterized

the morphology of colony, Gram stain and several

biochemical characters.

2.3 Selection Antagonistic Bacterial

against Ganoderma boninense

Antagonistic activity of bacterial endophytes

diazotroph were selected by dual test assay. The

bacteria was co-cultured with G.boninense on the

extract potato medium (PDA, Merck®). The

G.boninense was cultured in medium at the center of

dish (9 cm in the diameter). The endophytic bacteria

were cultured besides of G.boninense at distance 3

cm from the center of inoculated fungus. The culture

plates were stored on dark room at room temperature

during 5 days.The percentage inhibition of colony

G.boninense by bacterial endophytes were

calculated as formulated (Yurnaliza et al., 2014)

%CGI = (r1-r2 ) x 100%

(1)

Noted: CGI is radial colony growth inhibited , r1and

r2 were respectively radius growth of G.boninense

colonies to control (no bacterial) and test bacterial.

2.4 Characterization of Bacteria

The bacteria were characterized morphology colony,

Gram stain type, motility and catalase activity. The

high potential bacteria was identified by Vitek

analyser (bioMerieux, UK). The procedure

identification is accomplished by biochemical

methods.

3 RESULTS AND DISCUSSION

3.1 Population of Bacterial

Endophytes Diazotoph

Total population of endophytic bacteria from sterile

roots of oil palm segments were dissimilar in each

sample location and type medium for cultivation. Oil

palm roots from Universitas Sumatera Utara had

more abundant bacteria than other locations.

Percentage of population bacterial diazotrophic

endophytes indicated that in un-commercial

locations (USU and community plantation) the

population dizotrophic bacteria were more higher

than PTPN IV. PTPN IV Adolina was a larger

plantation state-owned enterprise in agribusiness.

Normally, application of chemical fertilizers in

company management was a standard procedure to

improve productivity of plant growth. Presence of

diazotrophic bacteria as endophytes in oil palm roots

were affected by utilization of chemical fertilizer.

Communities of diazotroph and total bacteria were

effected by fertility management of the land (Orr et

al., 2012). Ayuni et al., (2015) said the population

diazotroph bacteria of Stenotrophomonas maltophila

and it’s nitrogenase enzyme were affected

significantly by urea-N treatment to rice plant.

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

Table 1. Population of bacterial endophytes from

roots of oil palm from 3 locations.

Location Total

bacteria

(10

)

Diazotroph

bacteria (10

)

Percentage of

diazotroph

bacteria/10g

oil palm root

(%)

University

Sumatera

Utara

(USU)

4.64 2.19 47,2

PTPN IV

Adolina

4.06 1.08 27

Community

lantation

1.08 0.075 6,9

Counting of total population bacterial

endophytes in oil palm roots using nutrient agar

indicated the total population generally, but specific

for diazotrophic bacteria were calculated on Ashby’s

medium. Ashby’s medium is a selective medium

without N-source for isolating and cultivating

diazotrophic bacteria (Stella et al., 2012). Only

bacteria that have nitrogenase enzyme will grow in

this medium. The highest percentage of diazotrophic

bacteria in 10 g root sample were in community

plantation (Table 1).

Based on morphological colony of bacterial growth

on Ashby’s medium were selected as much as 30

isolates. Next selected bacteria was based on their

activity to inhibited Ganoderma mycelium growth in

dual culture assay test.

3.2 Characterization Bacteria and

Antagonistic Activity Assay

Dual culture assays between 30 isolates of bacterial

endophytes diazotroph and G.boninense resulted 13

potential isolates with %CGI 11.1-62.5 %. Almost

all isolates were coccus Gram negative. Bacterial

endophytes from oil plam roots were isolated by

Ramli et al., (2016) and Bivi et al., (2010) also

dominated by Gram negative bacteria. The motility

tests showed several bacteria motile and all isolates

were unable to reduce hydrogen peroxide (catalase

negative) (Table 2). The colour of colony was

variable from white, yellow, yellowish, orange and

red. The red colour bacteria was usually Genus of

Serratia. One of the red colour bacteria (AP 35) had

the highest %CGI (62.5%) (Figure 1).

All of endophytic diazotroph bacterial isolates has

different abilities in inhibiting G.boninense. From

dual assay was resulted two model inhibition

mycelium type, inhibited and inhibited with

depletion of mycelium (Figure 1). Inhibited of radial

growth mycelium probably caused by antifungal

activity of bacteria.

The depletion of mycelium was occurred when

the fungal hypa was contact and close to bacterial

colony. It’s probably caused by lytic enzyme that

was produced by bacteria. The enzymes such as

chitinase and glucanase used chitin and glucan from

cell wall of G.boninense as a substrate. Chitin and

glucan components which are the major constituents

of the cell wallof the fungus. Bacterial endophyte

diazotroph in this research (AP 39) are possibly able

to produce bothenzyme. The activity of lytic

Table 2: Gram stain, cell type, morphology colony and percentage of colony growth inhibition (CGI) activity bacterial

endophytes diazotroph against G. boninense

Code

Gram

Cell

shape

Morphology of colony CGI

(%)

Motility Catalase

test

Shape elevation margin colour

AU 6 - Coccus iregular Raised Curled yellow 40.0

- -

AU 30 - Coccus Circular Flat Lobate white 11.1

+ -

AU 33 - Coccus Iregular Flat Lobate white 22.0

+ -

AP 23

Coccus Circular Flat entire white 44.4

- -

AP 24 + Coccus Circular Flat Entire white 48.8

- -

AP 25 + Coccus Iregular Raised Undulate yellowish 11.1

+ -

AP 32 - Coccus Circular Convex Undulate white 20.0

+ -

AP 33 + Coccus Iregular Convex Undulate Orange 22.2

- -

AP 34 - rod Iregular Raised Lobate red 22.2

+ -

AP 35 - rod Iregular Flat Lobate red 62.5

+ -

AP 36 - rod Circular Raised Undulate white 15.0

- -

AP 39 - Coccus Iregular Flat Curled pink 44.4

- -

AK 25 + rod Circular Flat entire white 16.6

- -

Isolation and Screening of Antagonistic Diazotrophic Endophytic Bacteria from Oil Palm Roots against Ganoderma boninense

enzyme made the colony thinning and the growth

inhibited.

Bacterila diazotroph not only be able to produce

chemical compound to induce plant growth, but also

produce antifungal (Weber et al., 2007) and fungal

mycelium lysis enzymes. Ji et al., (2014) showed

that the diazotroph endophytic bacteria were able to

inhibited mycelium of Fusarium oxysporum and

Rhizoctonia solani in dual culture test. According to

Bivi et al., (2010) in addition to enzyme activity, the

use of endophytic bacteria as a biocontrol agent of

G.boninense is an effective means. That's because

endophytic bacteria are indigenous bacteria that

colonize the oil palm root vascular system, so it has

better nutrition and space competitiveness than

G.boninense, so the growth of G.boninense will be

depressed.

Figure 1: Morphological characteristics of inhibited

colony G.boninense when antagonistic activity assay with

bacterial endophytes diazotroph (a). Inhibited growth and

(b) depletion mycelium, C (no bacteria)

Identification of the red one of diazotrophic

bacteria by Vitek analyzer resulted species of

Serratia marcencens with probability number was

99%. The biochemical detail in Table 3.

Table 3. Biochemical test results of isolate AP 35 using Gram Negative (GN) Card by Vitek analyzer

(bioMerieux, UK)

Well Test Results Well Test Results

2 Ala-Phe-Pro-Arylamidase - 33 Saccharose/Sucrose +

3 Adonitol + 34 D-Tagatose -

4 L-yrrolydonyl-Arylamidase + 35 D-Trehalose +

5 L-arabitol + 36 Citrat (sodium) +

7 D-cellobiose - 37 Malonate -

9 Beta Galactosidase - 39 5- Keto Gluconate -

10 H2S Production - 40 L-Lactate alkalinisation +

11 Beta-N-Acetyl-

Glucosaminidase

+ 41 Alpha Glucosidase -

12 Glutamyl Arylamidase pNA - 42 Succinate alkalinisation -

13 D-Glucose + 43 Beta-N-acetyl-Galactosaminidase -

14 Gamma-Glutamyl-Transferase + 44 Alpha Galactosidase -

15 Fermentation Glucose + 45 Phosphatase +

17 Beta-Glucosidase + 46 Glycine Arylamidase -

18 D-maltose + 47 Ornithine Decarboxylase +

19 D-Manitol + 48 Lysine Decarboxylase +

20 D-Mannose + 52 Decarboxylase Base -

21 Beta-Xylosidase - 53 L-Histidine assimilation -

22 Beta-Alanine arylamidase pNA - 56 Coumarate +

23 L-Proline Arylamidase + 57 Beta-Glucoronidase -

26 Lipase - 58 o/129 resistance [comp vibrio] +

27 Palatinose - 59 Glu-Gly-Arg-Arylamidase +

29 Tyrosine Arylamidase - 61 L-Malate assimilation -

31 Urease - 62 Ellman -

32 D-sorbitol + 64 L-Lactate assimilation -

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

4 CONCLUSIONS

The thirteen isolates of bacterial endophytes

diazotroph from oil pam roots inhibited of

G.boninense mycelium with percentage colony

growth inhibition was 11-62,5%.Diazotroph bacteria

were dominated by Gram negative bacteria with the

coccus cell shape. The potential isolate was a red

colony bacteria and identified as Serratia

marcencens by Vitek analyzer (bioMerieux, UK).

ACKNOWLEDGEMENTS

Author would like to say extremely thank you to

Indonesian Goverment for financial support of

Penelitian Dasar Unggulan Perguruan Tinggi

(PDUPT) Project No. 48/UN5.2.3.1 /PPM/KP-

DRPM/2018.

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Isolation and Screening of Antagonistic Diazotrophic Endophytic Bacteria from Oil Palm Roots against Ganoderma boninense