The Morphology of Contaminant Organism in Kappaphycus

alvarezii Tissue Culture

Ulfatus Zahroh

, Apri Arisandi

Biotechnology of fisheries and Marine Science, Universitas Airlangga

Departement of Marine Science, Universitas Trunojoyo Madura

Keywords: Kappaphycus alvarezii, Tissue culture, Cell morphology.

Abstract: The main problem in increasing the production of seaweed cultivation of Kappaphycus alvarezii is the

availability of quality seeds. One of the causes is because the seeds are susceptible to infectious diseases.

Tissue culture is one of the techniques to produce Specific Pathogen and Epiphyte Free /SPE

Nevertheless, the presence or absence of contamination needs to be analyzed to determine the cause of

contamination,, morphology of contaminating organisms, and changes of morphological cell of tissue

culture which can be used to prevent contamination during the next tissue culture and cultivation at the sea.

Based on the results, it could be revealed that the occurring contamination caused by bacteria and fungi as

well as caused by the less sterill culture process. Thallus morphology affected by the disease has slower

growth. There were also black spots, cotton-like substance as contaminated fungi (Saprolegnia sp and

phytopthora), and the fading of green and slimy pigment as bacteria contamination. In addition, the

morpology of ill seaweed cells has smaller cells and shrinked tissue compared to the healthy ones with their

bigger cells and no shrinkage.

1 INTRODUCTION

The species of seaweed widely cultivated in Madura

is Euchema Cottonii which is also known as

Kappaphycus alvarezii. It is the most important

seaweed and the largest on production volume in

Indonesia. This type contains Karaginan which is

useful as a Gelling agent, solidified agent, anda

fertilizer (Suryaningrum, 1998). The main problem

in increasing production of seaweed (Kappaphycus

Alvarezii) cultivation is the availability of good

seeds. One of the cause is that the seeds are

susceptible to the disease. Epiphytic attack caused

the decrease on seeds quality;, hence, the resistance

of seaweeds toward the disease is an indicator of

seaweed cultivation accomplishment. A widely

applied plain and low-cost technologyof seaweed

cultivation is not supported by the availability of

seeds which are unrestrained from disease and

epiphytic (specific pathogen and epiphyte free / spef).

Tissue culture is one of techniques to produce

quality seeds stock. Research that has been carried by

Parenrengi, et. a. in 2007; Hurtado and Biter in 2007;

Hurtado, et. al. 2009; and Yunque, et.al in 2010

revealed that seaweed K. alvarezii can be cultivated

by using tissue culture. However, those research did

not specifically examine occuring contamination and

identify the contaminant species. One of the

problems occurring in tissue culture is the

contamination. The condition of in vitro favored by

Eksplan contains sucrose and nutrient in high

concentration, high moisture, and suitable

temperature. These situations are also preferred by

microorganisms that grow more rapidly than eksplan.

A source of contaminating organisms can come from

unsterile environment, or they have existed in the cell

when eksplan of Kappaphycus alvarezii will be

cultivated through tissue culture. Therefore, it needs

to be controlled. The first step is by identifying the

types of contaminated microorganisms and by

acknowledging those symptoms.

2 LITERATURE REVIEW

2.1 Biology kappaphycus alvarezii

Kappaphycus alvarezii is a seaweed of class

Rhodophyceae. Based on the identification of the

karaginan fraction produced by Kappaphycus

alvarezii, kappa karaginan type, it is taxonomically

changed its name from Eucheuma alvarezii become

Zahroh, U. and Arisandi, A.

The Morphology of Contaminant Organism in Kappaphycus alvarezii Tissue Culture.

DOI: 10.5220/0007547605870594

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 587-594

ISBN: 978-989-758-348-3

587

Kappaphycus alvarezii (Patadjai 2007). The name

"alvarezii" given to Kappaphycus alvarezii comes

from the name of the Vicente (Vic) Alvarez. Vic is a

pioneer in cottonii cultivation method (Patadjai

2007). The world of seaweed trade is more familiar

with the name Eucheuma cottonii or cottonii only.

Meanwhile, seawater disease is defined as disruption

of structures and normal functions such as changes in

growth rate and appearance (color and shape) that

can affect productivity levels. Culture or tissue

culture in vitro is a method to isolate parts of the

plants grown on sterile artificial media, in sterile

culture bottles, and in aseptic conditions. Thus the

parts can proliferate and regenerate into a complete

plant. Tissue culture is a series of activities

undertaken to make plant parts (roots, buds, plant

growing tissues) grow into the whole (perfect)

condition in vitro (in glass) plants (Indrianto 2002).

The basic theories of tissue culture are: a. Cells of

a multicellular organism wherever it is located is

actually the same as a zygote cell because it

originates from one cell (omne cellula ex cellula). b.

The Cell Totipotency Theory by Schwann and

Schleiden (1898) states that the cell has Totipotency

nature that every living plant cell is equipped with

genetic information and complete physiological

devices to grow and develop into whole plants if the

conditions are appropriate. This theory believes that

every part of the plant can breed because all parts of

the plant are made up of living tissues. According on

Thorpe, (1981) , there are three main principles in

tissue culture:

a. Isolation of plant parts of whole plants (organs,

roots, leaves, stems);

b. Maintainance of the plant’s parts in the

appropriate environment and conditions of the

culture;

c. Maintenance under aseptic conditions.

3 METHOD

The implementation of tissue culture was carried out

in the Tissue Culture Laboratory Agroecotechnology

and research on contaminant organisms was

conducted at Marine Science Laboratory of

Trunojoyo University Madura. Tools used for tissue

culture process included Bottle Culture, Filter Paper,

Funnel, Glass Beaker 250 ml and 500 ml, Autoclave,

Laminar Flow, Microscope, Glass preparation +

cover glass and other tools.

Seaweed materials used were Kappaphycus

alvarezii cultivated by farmer groups in Aengdake

Village, Bluto District, Sumenep Regency. Conway

was utilized for tissue culture media and the seawater

was also sterilized.

The selected explant source had the following

criteria: (a) had many branches, dense and spiky

leaves, (b) no spots found and peeling (c) had specific

bright color (d) was live less 35 days, (e) weighed

between 50-100 grams per rumpon and was not

exposed to ice-ice disease. Tool sterilization used

70% alcohol and autoclave; while the material for

eksplan sterilization utilized 0,5% betadine and 25

ppt sterile sea water. The stages of tissue culture

implementation to explant observation was

conducted by methods from Hurtado and Biter

(2007) as mentioned below:

a. Preparation of tools and materials

1. Preparing the tool

▪ Washing the appliance with running water.

▪ Sterilizing equipment to be used with alcohol,

wet sterilization using autoclave with

temperature of 1210C at 1.5 atm pressure for 20

minutes

2. Preparing materials

▪ Filtering sea water into culture bottle ± 100ml

▪ Saving seawater that had been in autoclave in

the sterile room

▪ Filtering the media conway and sterilizing it

into an autoclave with a temperature of 121

C at

a pressure of 1.5 atm for 15 minutes.

Figure 3.1 Media Conway filter

b. Planting stage culture

Bottles containing media, as well as other

planting tools, were sprayed first with 70% alcohol.

Cut where Thallus was taken and put it into a

disinfectant solution I (1% betadine solution where

concentration 1 ml per 100ml seawater). Ten minutes

later, rinsed with sterile distilled water for subsequent

inclusion in a solution of disinfectant II (0.5%

betadine solution where 0.5 ml per 100ml seawater)

for 10 minutes. Then rinsed with sterile aquades for

3 times, aiming for no remnants of disinfectant

material stick on eksplan then drained in petridish.

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Implant planting was done in laminar air flow.

Eksplan should be planted in culture bottle

containing media; there was an eksplan planted on

each bottle by using tweezers clamp. This activity

was carried out in a laminar flow, placed in a room

temperature of 25 oC. The media was of 25 ppt

salinity, pH 7.5 and the explants were cultured in a

culture bottle amounted to 120 units. Then the bottle

was closed, removed, and arranged on a culture rack

in accordance with the placement plan, given 10

watts fluorescent lighting.

The location of the lamp was 30 cm on the top of

the shelf away from the culture bottle. The lighting

was programmed for 24 hours continuously.

c. Subculture stage

Every 5 days, eksplants grew in subculture to a

new culture bottle containing tissue culture media

with the same concentration and parameters with

previous culture media.

d. Maintenance stage

Maintenance is conducted on the culture room by

maintaining cleanliness and room temperature.

Culture bottles containing media and explants were

sprayed with 70% alcohol every day and

contaminated plants and media were immediately

removed from the room and observed.

e. Stage of data collection

Observation was carried out every day to see the

contamination of bacteria or fungi. Contaminated

media and explants were removed from the room and

their cells as well as their contaminants (Hayashi et

al. 2008) were observed.

The data taken include:

a. Percentage of contamination

The percentage of contamination is calculated

using the formula previously performed by

Amiluddin (2007) as listed below:

C (%) = A x 100%

description:

C: percentage of infected seaweed (%)

A: number of explants or infected seaweed

T: number of explants or bonded seaweed

points observed

b. Morphology cell

Cell of morphology was observed using the

microscope 100L Olympus with 100 times

magnification. It was photographed on size

1600 x 1200 with ISO 100 (Yulianto 1993).

c. Contaminant agent

The presence of epiphytic diseases and epiphytes

that infect the explants (seaweed) was observed

based on signs of morphological abnormalities.

Observed morphological abnormalities were

identified by looking at and comparing with the

images contained in the literature. The

morphology of contaminant organisms was

observed using the Olympus 100L microscope

with magnification 100 times. It was

photographed at the size of 1600 x 1200 with ISO

100 (Yulianto 1993).

d. Survival rate

The rate of seedling survival explants of K.

alvarezii. Based on Amiluddin (2007) was

calculated using the following formula:

SR (%) =

x 100

description: SR = Survival Rate (%)

S = Number of living seaweed

M = Number of dead seaweed

4 RESULTS

Epifit conditions on control treatment

Seaweed affected epiphytes in the sea

is characterized by the presence of

algae filament attached to the epidermis of the thallus

of appaphycus alvarezii as seen in Figure 4.1. Based

on the identification in the type and characteristics of

filament algae in accordance with Largo (2002), the

species identified are polysiphonia sp which is an

epiphytic competitor algae.

(a) (b)

(c)

Figure 4.1 Polysiphonia sp. on Kappaphycus alvarezii. a)

The state of Thallus the contaminated. b) morphology of

Polysiphonic attached to the epidermis thallus in a

magnification microscope 100x. c) Polysiphonia according

on Largo (2002).

The Morphology of Contaminant Organism in Kappaphycus alvarezii Tissue Culture

589

The existence of competitor algae can result in

disruption of the thallus in obtaining nutrients and

light. If the components needed in metabolism are

reduced; then over time, the seaweed Kappaphycus

alvarezii can become thin, flabby, pale and can cause

death.

The observation showed the presence of

abnormalities in tissue are characterized by: a

dangling lump in the epidermis, morphological

observations through a microscope found that

Polysiponia had afilament dangling and the base

through the part of the cell wall of Kappaphycus

alvarezii. According to Darmayati et. al. (2001),

Polyshiponia sp. is type of Rhodophyta-rhinoceros

and is a competitor algae in seaweed that can cause

disturbance in seaweed photosynthesis resulting

from the covering of thallus surface by

Polyshiponia.

Epiphytic organisms in tissue culture

Organisms that contaminate seaweeds resulting

from tissue culture which consists of fungi and

bacteria.

Contamination of fungus

Contaminated fungi consists of Saprolegnia sp.

and Phythopthora sp.,mushrooms of Oomycota class

that can only grow in environment with high

moisture or aqueous.

a. Saprolegnia sp.

The contaminated seaweed Saprolegnia sp. is

characterized by the whitening of thallus, mucus

covered by dirt like white flour, the peeled outer skin

or epidermis to reveal deep cell or medullaryin of the

tissue thallus, and the presence of white hifa on the

surface of the media.

Morphology Saprolegnia sp. as contained in Figure

4.3.

Saprolegnia sp. has a characteristic feature that

can grow at a temperature range of 0-35° C, with an

optimum growth interval of 15-30°C. It is a fungus

group of Oomycota also called a water fungus that

can live in aqueous / high humidity environment.

Saprolegnia sp. commonly attacks the injured part,

and may subsequently spread to other healthy tissues.

According to Wilfred et al. (1965) in Ningsih (2011),

the fungus of the family Saprolegniaceae can live in

freshwater and saltwater. Zoospora groups of these

fungi search for fertile substrate, then settle down and

start producing hypha. Mycelia grow over the

wounded tissue or the site of infection, and then

spread to normal tissue around the site of infection.

The fungal enzyme secreted by the fungus destroys

the surrounding tissue, kills cells, and progresses

mycelia. It is very dense and sticking out into the

water, making it look likes cotton.

(a)

(b)

Figure 4.3. Morphology of saprolegnia sp. a) Thallus

affected by fungi b) Endogenous of Saprolegnia sp. in

thallus tissue.

b. Phytophthora sp

Contaminated Thallus is pale green and has

specks of black spots. The morphological

observations of the tissues show the presence regions

of dark and rounded as shown in Figure 4.4 below:

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Figure 4.4 Contamination Phytophthora sp in thallus a)

Contaminated Morphology thallus, b) Morphology of

contaminated tissue Phytophthora sp at 100x

magnification microscope.

Diagnosis done in laboratories by taking mycelia

wasplaced on the surface of the glass slide and given

a little water for further observation under the

microscope. Mycelia cause saprolegniasis to have

branching with aseptate hypha structure.

The morphology of Phytophthora sp.,

sporangium, is oval to slightly rounded or pear-

shaped, whieh its spore has a whip feather (flagella)

that can move in water.

The pathogens may form in round

chlamydospores (Directorate of Holticulture

Protection 2011).

Contamination of bacteria

Contamination due to bacteria characterized by

exsplants showed symptoms of wilting and stingy

media smells. The observation of the thallus tissue

shows the presence of a ruptured cell due to a

bacterial infection. Observation of bacterial

morphology is done by first breeding bacteria

attached to wall of thallus in media agar and given

gram staining, then identified through electron

microscope. After doing bacterial culture in media

agar and identification under microscope, the result

showed that morphology of bacteria is single coccus,

paired, chained, and in the form of gram negative.

Thus, it indicated that the bacterium was

Streptococcuss sp.

The morphology is as shown in Figure 4.5 below:

(a)

(b)

Figure 4.5 Contamination on thallus by bacteria a)

morphology Thallus b) bacterial on a 100x magnification

through microscope.

Based on Figure 4.5, it was found that the

morphology and color of bacteria originating from

the culture room on the control are relatively the

same as the result of identified contaminant in the

contaminated explants. So, the contaminant

organism is indicated to have been present in the

source of the explants.

Changes in cell morphology

Cells observed in healthy thallus tissue appear more

clearly with scattered prevalence of staining (safranin)

throughout the surface of seaweed tissue. The vividly

dispersed safranin indicates that the liquid cell is

absorbed evenly by every space within the cell because

the state of the cell

wall is intact and no cytoplasm is

broken while the contaminated thallus tissue has an

incomplete cell shape and looks uneven due to the

damage on cell components or rupture of the

cytoplasm.

According to Juwono and Juniarto (2002) and

Lakitan 2011 in Arisandi (2011), the cell wall of the

plant has a main function as a protector of the cell

framework. When the cell wall is damaged by the

disease, it can lead to changes on shape and size of

the cell. Damage to the cell wall can interfere with

the absorption of nutrients into cells; it will also

disrupt metabolism and inhibit cell division. The

worsening damage causes the cell wall to burst,

leaving the fluid out and causing the cell to become

irregular and shrink (plasmolysis) subsequently as a

beginning to death (Musa and Wei 2008 in Arisandi

2011). The difference between healthy and

contaminated cells can be seen in Figure 4.6

The Morphology of Contaminant Organism in Kappaphycus alvarezii Tissue Culture

591

(a)

(b)

Figure 4.6. The difference between healthy and

contaminated cells in a 100x magnification. a) Healthy

cells, b) cells are contaminated

Contamination percentage

The percentage of contamination is the ratio of

thallus of infected seaweed to cultured seaweed,

calculated in percent. Based on the calculation of the

contamination, percentage on culture tissue activity

is 11.7% and the percentage of healthy seaweed is

88.3% with the amount of contamination per day is

as shown in Figure 4.7 below

Figure 4.7 Number of Contaminated Culture Bottles.

In the first week, contaminated grass sea culture

were in among 8 bottles; where the 6 bottles were

characterized by bleaching thallus, mucus covered by

dirt such as flour white, and the outer skin or

epidermis flaked. While in two other bottles, there

were black spots and brown line on the wall of the

thallus. Contamination in the first week was occurred

on day 3, day 4, and day 5; with the highest number

occurred on day 4. In the next week, the

characteristics of contamination were among in 6

bottles. Three bottles illustrated bleaching thallus

and three other bottles obtained black spots and

brown line on the wall of the thallus. Semangun

(2001) said that between the infection and the

seemingly occasional symptom, there is a long period

of time yet usually the symptoms of the disease will

appear after infection.

Surival rate

In the tissue culture activities conducted, the

survival rate was approximately 62.5%, which the

number of dead seaweed were as many as 45 bottles

and alive ones were 75 bottles as shown in Figure 4.8.

This indicates that the success rate of culture is

proven through the number of alive seaweed was

higher than the dead ones. Based on Perkasa (2011),

this survival rate is one of the determinants of success

in tissue culture activities. If the number of living

seaweed on the harvest is high and the number of

death is low, then the value of survival will be high.

Otherwise, if the number of the death is higher than

the other, the survival rate might be lower or below

as in the diagram:

Survival Rate

Figure 4.8 Survival rate level of tissue culture.

5 CONCLUSIONS

Based on the results obtained, it could be concluded

as follows:

1. There were two species of contaminants in

seawater tissue culture of Kappaphycus alvarezii:

fungus Saprolegnia sp., phythophthora

sp. and bacterium Streptococcus sp.

2. Fungal contamination was indicated that it is

already exist in prospective explant of seaweed to be

cultured, because the type of fungus is a water fungus

Not Survive

31,3 %

Survive

62,50%

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or Oomycota that can only grow in environments

with high moisture or watery levels.

3. Contaminated cells were smaller in size and

looked more shrunken than cells in tissue. A healthy

thallus looks bigger and has more sturdy cell walls

in support of its cell shape.

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