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Potential Antagonist Organisms against Poria hypolateritia of Red Root Disease in Tea Plantation.

Byline: K.P CHONG AND M.Y. NOOR FAZILA

ABSTRACT

A dual culture method was practiced in order to observe the interaction between Poria and the soil isolates. This method was slightly modified by allocating Poria in the middle and the soil isolate was inoculated 2 cm from the periphery of the plate; opposite to each other. The soil isolates was labeled as Noor Fazila (NF)-1, NF- 2, NF- 3, NF- 4, NF- 5, NF- 6, NF- 7, NF- 8, NF- 9, NF- 10, NF- 11 and NF- 12. After twelve days of interaction, two isolates; NF-1 and NF-5 were identified as potential antagonist organisms and suspected to be Cunninghamella sp. Other successfully isolated fungi are Trichoderma, Stylopage, Verticillium, Blastomyces, Ovulariopsis, Periconia and Ustilago. The rest of the fungi showed either agonism or mutualism interaction against Poria. (c) 2012 Friends Science Publishers

Key Words: Poria; Dual culture; Cunninghamella

INTRODUCTION

Pytopathologists have neglected for a long time the role of the microbial population of the soil, considering only the parasite and the disease. Many decades ago, researchers had begun the study of the relationship between soil microorganisms and the pathogen host complex (Chandniwala, 1995). However, the seminal work that crystallized biocontrol research into coherent discipline was 'Ecology of Soil-Borne Plant Pathogens: Prelude to Biological Control', based on the meeting in 1963 at Berkeley, California (Paulitz, 2000). The distribution of antagonist organisms varied widely according to the different samples of the soil. Among the many antagonistic bacteria were Bacillus subtilis and the most frequently occurring antagonistic fungi were species of Penicillium, Aspergillus, Trichoderma and Trichothecium (Chandiwala, 1995).

One of the species in Trichoderma; Trichoderma pseudokoningii found to be able to impede the spread over of Poria hypolateritia; the causal agent of red root disease in tea plantation. Aspergillus also can hamper this pathogen but merely as pre-pathogen (Cooray and Balasuriya, 2002). The infection of Poria on tea bushes not only means a gradual loss of yield and income during the period of infection, but also a loss of capital because of death of the entire bush (Fuchs, 1989). Recently, Sabah Tea Plantation found to be infected by this disease. Countless methods have been applied including planting Guatemala grass, uprooting, drenching and sanitation by burning the infected area. All the mentioned methods did not reach the target to fully eradicate the disease severity. In fact, methyl bromide had been sprayed over to eradicate Poria. But, this merely jeopardized Sabah Tea Plantation as one of the few organic tea producers. Therefore, application of methyl bromide has been discontinued.

Thus, biological control may be the best alternative to overcome this problem.

MATERIALS AND METHODS

Root and soil sampling: The infected tea root from Sabah Tea Plantations was uprooted and placed in a clean plastic bag together with the soil swiftly. Fifteen samples were taken representing the infected tea's roots. Whereas, 10 soil samples were dug surrounding the healthy tea root which is far from the infected tea bushes. The samples were kept in autoclaved bottles and transferred to Laboratory in University Malaysia Sabah.

Plate inoculation with infected root: The tea's root surface was washed properly before sterilization by immersing into 75% of alcohol for several minutes and rinsed in three changes of sterile distilled water. In a sterile dish, a small portion of infected root was transferred to a PDA plate.

Besides, another method described by Chong et al. (2004) also was applied to detect the existence of Poria colony. Preparation of serial dilution and plate inoculation: 1 g of soil sample was diluted in 10 mL of distilled water in a test tube. One mL of the diluted soil was dropped in another test tube for further dilutions (Tortora et al., 2004). The dilution factor was 1 x 10-5. Then, the sample was inoculated on three PDA plates using a pipette, streaked using a hockey stick rode. The steps were repeated for other soil samples.

Inoculation of isolated organisms: In vitro comparisons consisted of removing four mm diameter disks from the edge of expanding colonies grown. Both Poria and soil- borne fungus were placed in a new Petri dish; the soil-borne fungus colony was placed two cm from the periphery of the plate, whereas Poria was placed in the middle of the plate.

The paired culture was incubated at ambient room temperature (25+-2oC) for one week. All pairings were replicated three times (Bell et al., 1982).

Measurement of the radius: The calculation method was extracted from the text written by Kasturi (1999). The inoculated plates were observed and the radius of Poria colony towards the soilborne organisms was measured. The percent inhibition of radius growth (PIRG) was calculated using the followed equation:

PIRG = R1 - R2 x 100

R1

equation:

R1-Radius of Poria towards two cm from the periphery of the control plate.

R2-Radius of Poria towards the potential antagonism organism colony.

Determination of antagonist organisms: The plate with the highest PIRG value was sifted. The morphology of the colonies was observed through microscope. The morphology and the spores' structure were observed to identify the potential antagonist by referring to the fungi, which were identified to the method of Barnett and Hunter (1972).

RESULTS AND DISCUSSION

Table I showed the different diameter measured, morphology and color of the soil isolates on petri dishes after one week.

table

Interspecific interaction and PIRG measurement: From a serial of repeated experiments (at least three times) the interactions between soil isolates and Poria are summarized in Table II. Isolate NF 1 and Isolate NF 5 showed an antagonistic interaction against, whereas NF 3 is suspected to be agonist organism based on the condition observed after the interaction in dual culture (Fig. 1). Other soil isolates showed an interaction, which can be classified as either agonism or mutualism (Fig. 2). Mean of percentage of inhibition in radius growth (PIRG) of twelve soil isolates against Poria showing that only NF1 and NF5 are significant, which the mean values are 86.65% for NF 1 and 85.33% for NF 5, respectively. No PIRG observed in the other isolate.

Classification of isolated fungi: The structures of NF 1 (Fig. 3a and b) and other soil isolates were observed under the microscope to identify their genera based on the previous research and finally listed in Table III.

Table I: Traits of twelve fungi colonies isolated from the soil samples

###Diameter (mm)###A###M###E###Color###

NF 1###90###I###F###R###White

NF 2###45###I###E###F###White greenish

NF 3###10###I###E###X###Green brownish

NF 4###5###I###U###X###Grey

NF 5###45###I###F###R###White

NF 6###90###C###F###U###White yellowish

NF 7###90###C###F###R###White

NF 8###90###I###F###R###White

NF 9###90###C###F###U###white

NF 10###90###C###E###F###Yellow greenish

NF 11###90###C###E###F###White yellowish

NF 12###90###C###F###R###Green greyish

Table II: Interspecific interaction between Poria and soil isolates (NF 1-NF 12)

Soil isolates###Interspecific interaction

NF 1###Antagonism

NF 2###Agonism/mutualism

NF 3###Agonism

NF 4###Agonism/mutualism

NF 5###Antagonism

NF 6###Agonism/ mutualism

NF 7###Agonism/mutualism

NF 8###Agonism/mutualism

NF 9###Agonism/mutualism

NF 10###Agonism/mutualism

NF 11###Agonism/mutualism

NF 12###Agonism/mutualism

NF 1 is suspected to be Cunnighamella based on the morphology structures observes under the microscope. Cunninghamella, which belongs to class Zygomycetes is able in metabolizing chlorpromazine (CPZ) and methdilazine (MDZ) to produce essential anti-stress compounds for humans. Cunninghamella is able to secrete P-450 enzymes. These enzymes are also secreted in human body. Thus, it is suspected that this fungus has the capability to secrete complex enzymes to degrade Poria's mycelia (Zhang et al., 1996).

Besides Cunninghamella, NF 3, which is suspected to be Periconia, has a distinctive interaction against Poria compared to other isolates. Thus, Periconia can be pointed

Table III: Taxonomy of isolated soil fungi based on the microscopic features

Isolate###Classification###

###Phylum###Class###Order###Family###Genus

NF 1###Zygomycota###Zygomycetes###Mucorales###Cunninghamellaceae###Cunninghamella

NF 2###Deutromycota###Hyphomycetes###Moniliales###Moniliaceae###Verticilium

NF 3###Deutromycota###Hyphomycetes###Moniliales###Dematiaceae###Pericornia

NF 4###Zygomycota###Zygomycetes###Zoopagales###Zoopagaceae###Stylopage

NF 5###Zygomycota###Zygomycetes###Mucorales###Cunninghamellaceae###Cunninghamella

NF 6###Deutromycota###Hypomycetes###Moniliales###Moniliaceae###Trichoderma

NF 7###Ascomycota###Eauscomycetes###Onygenales###Onygenaceae###Blastomyces

NF 8###Ascomycota###Eauscomycetes###Onygenales###Onygenaceae###Blastomyces

NF 9###Ascomycota###Ascomycetes###Erysiphales###Erysiphaceae###Ovulariopsis

NF 10###Deutromycota###Hypomycetes###Moniliales###Moniliaceae###Trichoderma

NF 11###Deutromycota###Hypomycetes###Moniliales###Moniliaceae###Trichoderma

NF 12###Basidiomycota Basidiomycetes###Ustilaginales Ustilaginaceae###Ustilago

pathogenic fungus, which causes root and brown rot of the grain sorghum Sorghum bicolor (Macko et al., 1992).

The toxins are called peritoxins and periconins. These toxins are actually host specific toxins; solely excreted to cause disease in sorghum. But, the main concern is on the precursor of these compounds to be synthesized. The precursor is known as circinatin (Fig. 4). When the fungus was grown under suppressive conditions, it is suspected that circinatin acts as biosynthetic precursor of the unknown toxins (Macko et al., 1992). Thus, there is a potential for the circinatin to be the precursor to synthesize toxin, which can inhibit the growth of Poria.

CONCLUSION

The soil isolates were Cunninghamella (Isolates NF 1 and NF 5), Verticillium (Isolate NF 2), Periconia (Isolate NF 3), Stylopage (Isolate NF 4), Trichoderma (Isolates NF 6, NF 10 and NF 11), Blastomyces (Isolate NF 7 and NF 8), Ovulariopsis (Isolate NF 9) and Ustilago (Isolate NF 12). From twelve soil isolates, only one was interacted as antagonist organism, whereas the rest showed either agonism or mutualism interaction against Poria. Further insight into the potential antagonist of the soil isolate will aid in the knowledge for sourcing possible future of antifungal compounds.

Acknowledgement: We thank Sabah Tea Garden for the samples and explanation given during sampling was in progress.

REFERENCES

Barnett, H.L. and B.B. Hunter, 1976. Illustrated Genera of Imperfect Fungi, 3rd edition. Burgess Publishing Company, Minnesota, USA

Bell, D.K., H.D. Wells and C.R. Markham, 1982. In vitro antagonism of Trichoderma species against six fungal plant pathogen. Pytopathology, 72: 379-381

Chandniwala, K.M., 1995. Recent Advances in Plant Pathology, Vol. 1. Anmol Publications PVT LTD, New Delhi

Chong, K.P., A. Gassner, A. Markus, Md. Faisal, Md. Noor and Md. Khairi Abdullah, 2004. Identification of pathogen causal root disease of tea plant at Sabah Tea Plantation. In: Dayou, J., P.C. Lee, K. Saibeh, D. Gabda, P.Y. Moh and J.J. Silip (eds.), Proceedings of the Seminar on Science and Technology 2004, pp: 16-20. Universiti Malaysia Sabah, Kota Kinabalu, Sabah

Cooray, B.A.P. and A. Balasuriya, 2002. Antagonism of three naturally occurring fungi against major tea root disease of Sri Lanka and their sensitivity to recommended systemic fungicides. Proceedings of the 58th Annual Session, p: 39. 2-7 December 2002, Sri Lanka, 39

Fuchs, H.J., 1989. Tea Environments and Yield in Sri Lanka. Margraf Scientific Publishers, Germany

Kasturi, R.S., 1999. Biological Studies of Trichoderma Harzianum and its in Vitro Antagonicity against Three Root Pathogens. Dissertation Bachelor of Science, Universiti Putra Malaysia

Macko, V., M.B. Stimmel, T.J. Wolpert, L.D. Dunkle, W. Acklin, R. Banteli, B. Jaun and D. Arigoni, 1992. Structure of the host-specific toxins produced by the fungal pathogen. Periconia circinata.

Proceedings of the National Academy of Sciences of the United States of America, pp: 9574-9578. October 1992, USA

Paulitz, I.C., 2000. Population dynamics of biocontrol agents and pathogens in soils and rhizospheres, European J. Plant Pathol., 106: 401-413

Tortora, G.J., B.R. Funke and C.L. Case, 2004. Microbiology: An Introduction, 8th edition. Pearson Education, Inc, United State of America

Zhang, D., J.P. Freeman, J.B. Sutherland, A.E. Walker, Y. Yang and C.E.

Cerniglia, 1996. Biotransformation of chlorpromazine and methdilazine by Cunninghamella elegans. Appl. Environ. Microbiol., 62: 798-803

School of Science and Technology, University Malaysia Sabah, 88400, Kota Kinabalu, Sabah, Malaysia, 1Corresponding author's e-mail: chongkp@ums.edu.my, To cite this paper: Chong, K.P. and M.Y. Noor Fazila, 2012. Potential antagonist organisms against Poria hypolateritia of red root disease in tea plantation., Int. J. Agric. Biol., 14: 457-460
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Author:Chong, K.P.; Fazila, Noor
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9MALA
Date:Jun 30, 2012
Words:2024
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