Pathogenicity test and the inhibition of bacterial isolates of Bacillus sp. against

Fusarium oxysporum causing wilt disease in plants Solanaceae

I Ketut Widnyana

Lecturer in the Department of Agrotechnology,

Faculty of Agriculture, University of Mahasaraswati Denpasar

Email: widnyanaketut@yahoo.com

Abstract

Bacterial species of the group Bacillus sp. can function optimally as fertilizer (biofertilizer) and as a

biological control agent (bioprotectan) so that the defect can control Fusarium wilt caused by

Fusarium oxysforum the Solanaceae plant, if developed without hope can suppress the use of synthetic

fertilizers and pesticides. Exploration to get Baccilus sp bacteria have been done on various rizosfere

Leguminoseae and solanaceae plants around the Bali island and followed by isolation Laboratory to

obtain pure isolates. Test capabilities as biopesticide Bacillus sp. beginning with the pathogenicity test

and test inhibition against Fusarium wilt pathogens in plants solanaceae performed to obtain isolates

with at least 50% inhibition of the growth of the fungus Fusarium.

The results of research to get 4 (four) isolates of Bacillus sp. which is able to inhibit the growth of

isolates of Fusarium oxysforum namely BS1, BS2, BS3, and BS4 are respectively 60.32%, 85.73%,

89.09%, and 54.99%. Fourth Bacillus sp isolates are also non-pathogenic so likely to be further

investigated as biological control agents against Fusarium wilt disease in the Solanaceae.

Keywords: Fusarium, Bacillus sp, antagonist, pathogenicity, solanaceae

1. Introduction

Fusarium wilt on Solanaceae especially on tomato plants caused by the fungus

Fusarium oxysporum, where this fungus Classification is as follows: Phylum Ascomycota

Class Sordariomycetes, Subclass Hypo- creomycetidae, Order Hypocreales, Family

Nectriaceae, GenusFusarium, Species F. oxysporum f.sp . lycopersici (WC Snyder & HN

Hansen, 1940) with its synonym F. bulbigenum var. lycopersici (Bruschi) Wollenw. &

Reinking, (1935), F. lycopersici Sacc., (1881) (Wong, M.Y., 2003)

Fusarium produces 3 asexual spores (conidia) that frequency and wide conidia

formation depends on the site and environmental conditions. The three types of conidia are

microconidium, makrokonidia, and chlamydospores. Microconidium have 1atau 2 cells, and

is the type most widely produced conidia both the phase and saprogenase patogenase.

Makrokonidia has a distinctive shape, consisting of 3-5 septa, curved like a crescent moon

and typically generated on the surface of infected plants seriously. Chlamydospores comprises

1-2 septa, thick-walled, produced at the end of the mycelium that had been old or in

makrokonidia, and the spores survive in unfavorable environments. At first, the mycelium is

white turbid (beige), then became pale yellow, pale pink to purplish (Agrios, 2005)

Land full of microscopic life forms including bacteria, fungi, actinomycetes, protozoa,

and algae. The different microorganisms, bacteria are by far the most common (ie, 95%). It

has been known for some time that the soil is hosts a large number of bacteria (approximately

108-109 cells per gram of soil) and the number of bacterial cells were cultured in the soil is

generally only about 1% of the total number of cells contained. Both the number and the type

of bacteria found in different soil is influenced by soil conditions including temperature,

humidity, and the presence of salts and other chemicals as well as the number and types of

plants. In addition, the concentration of bacteria found around the roots of plants (eg, in the

rhizosphere) is usually much larger than in the whole land. This is due to the presence of

nutrients, including sugars, amino acids, organic acids, and other small molecules from plant

root exudates (Glick, BR, 2012).

Although understanding the interaction PGPB (Plant Growth Promothing Bacteria) -

plants is limited, a number of these bacteria still used commercially in addition to

commercialized farming practices are: Agrobacterium radiobacter, Azospirillum brasilense,

Azospirillum lipoferum, Azotobacter chroococcum, fimus Bacillus, Bacillus licheniformis,

Bacillus megaterium, Bacillus mucilaginous, Bacillus pumilus, Bacillus sp., Bacillus subtilis,

Bacillus subtilis var. amyloliquefaciens, Burkholderia cepacia, Delfitia acidovorans,

Paenobacillus macerans, Pantoea agglomerans, Pseudomonas aureofaciens, Pseudomonas

chlororaphis, Pseudomonas fluorescens, Pseudomonas solanacearum, Pseudomonas sp.,

Pseudomonas syringae, Serratia entomophilia, Streptomyces griseoviridis, Streptomyces sp.,

Streptomyces sp lydicus and various Rhizobia. However, inoculation PGPB in plants represent

only a fraction of the current agricultural practices worldwide (Lucy, M., et al., 2004).

Bacillus sp. is one group of gram-positive bacteria are often used as a biological

control root diseases. Members of this genus have advantages, because the bacteria form

spores that are easily stored, has a long life durability, and relatively easily inoculated into the

ground. Bacillus sp. has been shown to have potential as biological control is good, for

example against pathogens such as R. solanacearum. Bacillus sp. can produce phytohormones

potential to develop sustainable agricultural systems. Phytohormones produced these soil

bacteria can affect the growth of plants, either directly or indirectly. Phytohormones indirectly

inhibits the activity of bacterial pathogens in plants, while the direct effect of these

phytohormones is increasing plant growth and can act as a facilitator in the absorption of

some nutrients from the environment (Greenlite, 2009).

Baccillus sp. role as biological control, have the ability to form a crystal protein toxin

that is capable of infecting a host of deadly insects. However, the effectiveness of the crystal

protein toxin can be reduced or even lost altogether if exposed to extreme conditions such as

the degree of acidity (pH) is too alkaline. Approximately 50 offspring of B. thuringiensis have

been found and separated from insects - insects and classified into 12 groups based on the

pattern - the pattern of esterase and according to the test - serological and biochemical tests.

Commercial utilization of these bacteria present are only two bacteria were manufactured and

registered for insect control in agriculture is Baccillus popilliae to control beetles in America,

B. thuringiensis to control pests of the nation lepidoptera (Silkworm) under the trade name

Florbac, Bactospen sp, and Thurycide (Haryanto, 2013).

2. Research Methods

2.1. Tools and materials

The tools used in this study, Petri dishes, test tubes, Erlenmeyer, autoclaving, plastic,

laminar air flow, sentrufuge, haemocytometer, handcounter, and EnCase. The materials used

are 70% alcohol, spirits, aluminum foil, PDA (Potato dextrose agar), PD Broth, NA (Nutrient

Agar), a solution of 0.1 M MgSO4.7H2O, rhizosphere various plants, and distilled water.

2.2. Isolation of the fungus F. oxysporum

F. oxysporum isolates obtained from the stems of tomato plants wilt disease. The plant

material has previously been disinfected with 70% alcohol and rinsed with sterile water, then

cut into pieces with a size of 1 cm and grown on PDA were placed in a Petri dish. Purification

occur if there are other microbes that grow along the target fungi. To ensure that the fungal

pathogen Fusarium wilt is then tested using the procedure described Koch's postulates.

Incubation was carried out at room temperature, within 3 days of pathogenic fungi spores

have grown and can be used as a source of inoculum.

Fusarium form white colonies on PDA and when it will be colored spore-forming

colonies grayish white. Observation under the microscope done, which characterize the

morphology is the microconidium spherical eggs and makrokonidia crescent with septa. If the

colony is quite old (about 7 days) are often formed spherical chlamydospores also complete

with a stalk.

2.3. Isolation of Bacillus sp. Isolation of Bacillus sp. started by taking soil samples taken from rizosphere legumes

and eggplants from the area of Bali. Soil samples were taken by 200 g of soil rizosphere

inserted into the filled plastic label with information about time and location of sampling, and

the type of plant. Soil samples were then stored in a protected place in direct sunlight until

rizobakteri isolation. Ten gram of soil sample was dissolved in 90 ml of sterile water and

shaken in a mechanical shaker for 30 minutes. Each sample was serially diluted from 10-2

to

10-6

(Widnyana, et al., 2013).

2.4. Pathogenicity test isolates of Bacillus sp. Pathogenicity test conducted in the greenhouse by using tobacco indicator plants aged

20 HST (days after planting). Crops planted in polybags with a mixture of soil and compost

(ratio 2: 5) which has been roasted to minimize contaminants. Colony density used in the

inoculation of indicator plants was 105 cfu / ml (Widnyana, 2001).

Pathogenicity test is done by inserting a bacterial suspension into the leaf tissue by

injecting a fine needle diameter of 0.4 mm between the two epidermis of leaves, then press to

enter the bacterial suspension injections so that the suspension of bacteria into the epidermis

of leaves without damaging the leaf epidermis. The existence of zoning (section looks

wetness) on leaf charged bacterial suspension means by entering the bacterial suspension is

correct. Furthermore, tobacco incubated for 24-48 hours. Dictated positive reaction when

formed necrotic symptoms on leaf tissue (in local zoning). While the leaf tissue did not

change significantly negative (Anonymous, 2008).

2.5. Test antagonistic Bacillus sp. against F. oxysporum.

Tests carried out at the Laboratory begins with the preparation of PDA media for the

isolation of the fungus F. oxysporum and for the isolation of Bacillus sp. Pure cultures of the

fungus Fusarium 4 days old on PDA taken with a diameter of 5 mm is placed face to face

with isolates of Bacillus sp. 2-day-old with the same size. Testing was conducted on PDA

with dual culture method to see rizobakteri inhibition against pathogens.

3. Results and Discussion

3.1. Isolation fungus F. oxysporum

Source of inoculum obtained in the village Luwus Baturiti Tabanan, with symptoms

suffered withering attack tomato plants, the leaves yellow, brown stem. Isolation fungus F.

oxysporum procedures performed by tissue planting method. Re-isolation was conducted by

streaking method to obtain pure isolates. Colonies fungus F. oxysporum isolation results are

presented in Fig.1

Figure 1. Colonies fungus Fusarium oxysporum on preparations age 6

days

Identification of Fusarium wilt pathogen begins with the observation under a

microscope as shown in Figure 2. Further testing is done by following the procedure of

Koch's postulates. Incubation was carried out at room temperature, within 3 days of

pathogenic fungi spores can already be used as a source of inoculum.

Figure 2. Morphology of the fungus F. oxysporum isolated from diseased tomato plants Luwus village

Baturiti Tabanan A. Vegetative growth. B. Growth generative

3.2. Isolation of Bacillus sp.

Isolation of Bacillus sp. rizoosfer begins with an exploration of various plants

solanaceae and Leguminosae. With the method of dilution up to 1 x 10-6

obtained various

isolates were further purified through repeated media NA so get Bacillus sp number 4 isolates

with power sufficient antagonist to be used in controlling Fusarium wilt. These four isolates is

presented in Figure 3. namely Bacillus sp. isolates BS1, BS2, BS3, BS4.

Figure 3. Bacillus sp. isolated on NA medium, left to right isolates BS1, BS2, BS3, and BS4.

3.3. Test of Bacillus sp. antagonistic to pathogenic F. oxysporum

Antagonistic test was conducted using a dual culture to see rizobakteri inhibition

against pathogens from the four isolates of Bacillus sp. BS1, BS2, BS3, and BS4. Antagonist

test results are presented in Figure 4 and Table 1.

A B C

Figure 4. Test antagonistic Bacillus sp. with the fungus F. oxysporum with A). Control, B). Bacillus

sp. isolates BS1, C). isolates BS2, D). isolates BS3, E) isolates of Bacillus sp. BS4

Table 1. Power rizobakteri Bacillus sp. inhibitory to the growth of the fungus F. oxysporum

No. isolates Code Mean extensive

fungal colony

(mm2)

wide zone

barriers (mm2)

inhibition

(%)

1 I0 (Kontrol) 4,310 - -

2 BS1 1.710 2,600 60.32

3 BS2 615 3,695 85.73

4 BS3 470 3,840 89.09

5 BS4 1.940 2,370 54.99 Note :

Data on average above the average yield antagonist test repetition as much as 10 times in each isolate

3.4. Test pathogenicity of Bacillus sp.

Pathogenicity test carried out on the four isolates of Bacillus sp namely BS1, BS2,

BS3, and BS4. Pathogenicity test results showed that all isolates of Bacillus sp. does not have

the power pathogenicity of the tobacco plant is a plant indicator. Test pathogenic bacterial

isolates can be seen in Figure 5 and Table 2.

Figure 5. Test the pathogenicity of Bacillus sp. the indicator plant (leaf tobacco) A). Initial

inoculation, B). Does not appear any symptoms on treatment

Table 2. Results of Bacillus sp. pathogenicity test on indicator plants (tobacco)

No Spesies bakteri Test results pathogenicity

1 Bacillus sp (BS1) Negatif

2 Bacillus sp (BS2) Negatif

3 Bacillus sp (BS3) Negatif

4 Bacillus sp (BS4) Negatif

Figure 5 shows that the three isolates pathogenicity test results showed a negative

reaction is evidenced by the absence of a rickshaw on the leaves after a given treatment

solution baakteri isolates respectively. This proves that the four isolates of Bacillus sp. BS1,

BS2, BS3, and BS4 is not a plant pathogen.

4. Conclusion

From the research that has been carried out, it can be concluded:

1. There are four isolates of Bacillus sp. which has the potential to suppress the

pathogen Fusarium wilt in pepper and tomato which isolates BS1, BS2, BS3, and

BS4

2. The four isolates of Bacillus sp. namely BS1, BS2, BS3, and BS4 able to inhibit the

growth of pathogenic fungi Fusarium respectively by 60.32%, 85.73%, 89.09%, and

54.99%.

2. Isolates of Bacillus sp (BS1, BS2, BS3, and BS4) is not pathogenic on plants

indicator means the isolates are not as pathogenic.

Acknowledgement

On this occasion the authors are grateful to the Directorate of Higher Education Kemendikbud

has funded research conducted by Kopertis 8 with contract number: 0991 / K8 / KM / 2014

dated May 2, 2014. Thanks also to the Rector of the University Mahasaraswati Denpasar,

Dean of the Faculty of Agriculture and friends who have helped this research.

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