Prosiding Seminar Ilmiah dan Pertemuan Tahunan PEI dan PFI XVI Komda Sul-Sel, 2005 ISBN : 979-95025-6-7

EFFECT OF TOXIN FROM Pyricularia grisea ON RICE, CORN, MUNGBEAN, AND STRINGBEAN SEEDLINGS Amran Muis Balai Penelitian Tanaman Serealia

ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh toxin dari cendawan Pyricularia grisea pada biji padi, jagung, kacang hijau, dan kacang panjang. Penelitian ini dilakukan di Jurusan Penyakit Tanaman University of the Philippines Los Baňnos yang berlangsung dari bulan Juni hingga September 2003. Penelitian meliputi dua kegiatan yakni: pengaruh toxin terhadap panjang akar dari beberapa benih, serta pengaruh toxin terhadap daun padi yang sehat dan luka. Toxin dihasilkan dengan cara menumbuhkan cendawan P. grisea pada potato dextrose broth selama 7-14 hari kemudian disaring. Hasil penelitian menunjukkan bahwa pertumbuhan akar tanaman-tanaman yang diberi perlakuan dengan toxin murni (crude filtrate) lebih pendek dibanding akar tanaman yang diberi perlakuan lain. Kata kunci: Toxin, Pyricularia grisea.

INTRODUCTION The ability to withstand toxic compounds in the natural environment is an important adaptation for all particular, micromicroorganisms. In organisms often encounter an array of preformed and inducible chemical barriers during their attempts to infect plants. To defend themselves against fungal pathogens, plants produce an array of antifungal proteins (Dixon et al., 1994) as well as specialized antibiotics called phytoalexins. Only pathogens which can evade these plant defense responses during early infection stages are able to survive and cause disease (Urban et al., 1999). Rice blast caused by Pyricularia grisea is one of the most important diseases of rice and is distributed worldwide. The disease is particularly severe where rice is irrigated or receives high amounts of rainfall. Yield losses can range from 50-90% in severe epidemics. P. grisea is the most important pathogen of rice worldwide. As rice is the most important starch source, rice blast is arguably the most important plant pathogen of those considered as anticrop weapons. This pathogen produces toxins

(pyricularin, alpha-picolinic acid, etc.) that appear to contribute to the development of the disease (Agrios, 1997). Pyricularin is one of the non-hostspecific toxins. It is toxic not only to higher plants but also to a variety of microorganisms (Goodman, et al., 1986). Nevertheless, P. grisea can produce large quantities of pyricularin in vitro. This experiment was design to (1) determine the effect of toxin on root elongation of rice, corn, mungbean, and stringbean; (2) determine its effect on detached and intact rice leaves; (3) test the activity of toxin at several levels of dilutions; (4) test the effect of heat on toxin activity. MATERIAL AND METHODS The experiment was conducted in laboratory of Plant Pathology Dept., UPLB from June to September 2003. Seed materials used in this experiment were; rice, corn, mungbean, and stringbean. The pathogen Pyricularia grisea was obtained from infected rice leaves and was transferred into potato dextrose agar (PDA) plates then grown in vitro at room temperature.

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There were two activities on this experiment: (1) effect of toxin on root elongation of several crops, (2) effect of toxin on intact and wounded rice leaves. For the first activity the experiment was arranged in factorial completely randomized design with 3 replications. Factor I was incubation time of the fungal culture, consist of two levels (7 and 14 days old culture), factor II was the treatment of toxin (filtrate). The treatments consist of: 1. Crude filtrate 2. Boiled filtrate for 10 minutes. 3. 1:10 dilution of filtrate 4. 1:30 dilution of filtrate 5. 1:50 dilution of filtrate 6. Distilled water. Observation conducted three days after treatment by measuring the root length of each seedling from each treatment. For the rice leaves, observation was addressed toward the necrosis symptoms using + and – codes, where + indicate necrotic symptom appear and – indicate no necrotic appear. Toxin was produced by growing the fungus for 7 or 14 days (depend on the treatment) in potato dextrose broth at room temperature. 125-ml erlenmeyer flasks containing 25 ml of fluid are suitable.

Then, the culture was filtered through Seitz filter with 5 layers of filter paper. For bioassay test, the seeds of rice, corn, mungbean, and stingbean were germinated at room temperature for 24 to 48 hrs in Petri plates containing moist filter paper. Then dilution of the cell-free filtrate was prepared using boiled filtrate, crude,1:10, 1:20, and 1:50. Diluents was sterile distilled water. In each 3 Petri plates, 2 ml of dilution was placed and 10 selected, uniform, germinated susceptible seeds were placed in each of the plates. This was repeated three times. The same solution was also given to the detached and intact rice leaves. RESULT AND DISCUSSION Analysis of variance for the effect of toxin on root elongation of rice showed that there was significantly different on treatments and incubation of culture due to the root elongation and there was an interaction between treatments and incubation. On the 7 days old culture there was not significantly different between crude and boiled filtrate, but they were significantly different with other treatments (Table 1). Visually, rice seedling’s roots treated with crude filtrate were shorter than others (Fig. 1).

Table 1. Effect of toxin from 7 and 14 days old culture of P. grisea on root elongation of rice Treatments Crude filtrate Boiled filtrate 1:10 dilution 1:20 dilution 1:50 dilution Distilled water

Root length (cm) 7 d old culture 14 d old culture 0.4 a 0.9 a 0.2 a 1.1 a 0.9 b 2.1 b 2.0 c 2.2 b 2.8 d 2.2 b 3.1 d 2.5 b

Mean (cm) 0.7 0.6 1.5 2.1 2.5 2.8

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

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Prosiding Seminar Ilmiah dan Pertemuan Tahunan PEI dan PFI XVI Komda Sul-Sel, 2005 ISBN : 979-95025-6-7

Figure 1. Effect of toxin from 7 days old culture of P. grisea on root elongation of rice at 3 days after inoculation.

On the 14 days old culture there was not significantly different between crude and boiled filtrate, but they were significantly different with other treatments. The longest roots were found in seedlings treated with distilled water and not

significantly different with treatment 1:50 dilution (Table 1). Visually, seedling’s roots treated with crude and boiled filtrate were shorter than others and some of the roots on treatment 1:10 dilution still shorter than those on treatment distilled water (Fig. 2).

Figure 2. Effect of toxin from 14 days old culture of P. grisea on root elongation of rice at 3 days after inoculation

Analysis of variance for the effect of toxin on root elongation of corn showed that there was significantly different on treatments but there was not significantly

different on incubation of culture due to the root elongation and there was an interaction between treatments and incubation. On the 7 days old culture the

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shortest root length was shown by boiled filtrate treatment, but not significantly different with other treatments and the longest root length was shown by treatment distilled water (Table 2). Visually, root length of corn seedlings treated with crude filtrate, boiled filtrate, dilution 1:20, and dilution 1:50 were almost the same. And normal seedling growth was shown by treatment distilled water (Fig. 3). On the 14 days old culture crude filtrate and boiled filtrate gave a same

result but they were not significantly different with treatments 1:10 dilution, 1:20 dilution, and 1:50 dilution. The longest roots elongation were found in treatments distilled water that significantly different with other treatments (Table 2 and Fig. 4). Visually, corn seedlings treated with distilled water showed normal growth unlike those that treated with toxin filtrate (Fig. 4).

Table 2. Effect of toxin from 7 and 14 days old culture of P. grisea on root elongation of corn Treatments Crude filtrate Boiled filtrate 1:10 dilution 1:20 dilution 1:50 dilution Distilled water

Root length (cm) 7 d old culture 14 d old culture 0.6 a 1.4 a 0.6 a 1.2 a 1.4 a 1.5 a 1.5 a 1.5 a 1.6 a 1.6 a 4.9 b 2.1 a

Mean 1.0 0.9 1.5 1.5 1.6 3.5

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Figure 3. Effect of toxin from 7 days old culture of P. grisea on root elongation of corn at 3 days after inoculation

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Prosiding Seminar Ilmiah dan Pertemuan Tahunan PEI dan PFI XVI Komda Sul-Sel, 2005 ISBN : 979-95025-6-7

Figure 4. Effect of toxin from 14 days old culture of P. grisea on root elongation of corn at 3 days after inoculation

Analysis of variance for the effect of toxin on root elongation of mungbean showed that there was significantly different on treatments and incubation of culture due to the root elongation and there was an interaction between treatments and incubation although, there was no significantly different among the treatments on the 7 days old culture. On the 7 days old culture the shortest root length was shown by crude filtrate treatment followed by boiled filtrate, but

there was not significantly different with other treatments (Table 3 and Fig. 5). On the 14 days old culture there was not significantly different between crude and boiled filtrate, but they were significantly different with other treatments. And the shortest root elongation was shown by crude filtrate treatment (Table 3 and Fig. 6). There was no significantly different between treatment 1:10 dilution and 1:20 dilution but it was significantly different with treatment distilled water.

Table 3. Effect of toxin from 7 and 14 days old culture of P. grisea on root elongation of mungbean Treatments Crude filtrate Boiled filtrate 1:10 dilution 1:20 dilution 1:50 dilution Distilled water

Root length (cm) 7 d old culture 14 d old culture 2.3 a 1.1 a 2.6 a 1.2 a 6.0 c 1.5 a 6.0 c 1.4 a 5.7 bc 1.8 a 5.1 b 1.6 a

Mean 1.7 1.9 3.7 3.7 3.8 3.4

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

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Figure 5. Effect of toxin from 7 days old culture of P. grisea on root elongation of mungbean at 3 days after inoculation

Figure 6. Effect of toxin from 14 days old culture of P. grisea on root elongation of mungbean at 3 days after inoculation

Analysis of variance for the effect of toxin on root elongation of stringbean showed that there was significantly different on treatments and incubation of culture due to the root elongation and there was an interaction between treatments and incubation. On the 7 days old culture the shortest root length was shown by crude

filtrate treatment and not significantly different with treatments boiled filtrate, 1:10 dilution, 1:20 dilution, and 1:50 dilution but they were significantly different with treatment distilled water which showed the longest roots (Table 4 and Fig. 7).

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Prosiding Seminar Ilmiah dan Pertemuan Tahunan PEI dan PFI XVI Komda Sul-Sel, 2005 ISBN : 979-95025-6-7

Table 4. Effect of toxin from 7 and 14 days old culture of P. grisea on root elongation of stringbean Treatments Crude filtrate Boiled filtrate 1:10 dilution 1:20 dilution 1:50 dilution Distilled water

Root length (cm) 7 d old culture 14 d old culture 2.5 a 2.3 a 2.5 a 2.5 a 3.0 b 2.6 a 3.7 d 2.7 a 3.5 cd 2.6 a 3.2 bc 3.1 b

Mean 2.4 2.5 2.8 3.2 3.0 3.2

In a column, means followed by a common letter are not significantly different at 5% level by DMRT.

Stringbean

Figure 7. Effect of toxin from 7 days old culture of P. grisea on root elongation of stringbean at 3 days after inoculation

On the 14 days old culture crude and boiled filtrate gave the same result in inhibiting the roots of stringbean and significantly different with other treatments (Table 4 and Fig. 8). The longest root

elongation was shown on treatment 1:50 dilution which was not significantly different with treatment distilled water (Table 4).

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Figure 8. Effect of toxin from 14 days old culture of P. grisea on root elongation of stringbean at 3 days after inoculation

The effect of toxin from the 7 days and 14 days old culture on wounded (detached) and intact rice leaves are shown on Table 5. The symptom was resulted from both 7 and 14 days old culture incubation. Although on the 14 days old culture only slight necrotic symptom appeared. Since the filtrate toxin also impregnated into the moist paper tissue, it is systemically also entered into the tissue through cut-end intact leaves or through wounded

(detached) leaves, and produce systemicyellowing tissue. According to Callow (1983) toxins can produce many of the commonly observed symptoms of disease by interfering with amino acid synthesis, by inhibiting photosynthetic ATP formation, or by inhibiting amino acid formation. Until two days after inoculation, leaves inoculated with sterile distilled water remain fresh.

Table 5. Effect of toxin from 7 and 14 days old culture of P. grisea on wounded and intact rice leaves at 2 days after inoculation Treatments

Crude filtrate Boiled filtrate 1:10 dilution 1:20 dilution 1:50 dilution Distilled water

Reaction of toxin on rice leaves 7 days old culture 14 days old culture Wounded Intact Wounded Intact + + + + + + + + + + + + + + + + + + + + -

+ necrotic symptom - no symptom

The overall result of observation showed that crude filtrate gave the best result to inhibit root elongation. No effect of

boiling the filtrate due to its effectiveness to inhibit elongation of the roots (Fig. 9 and Fig. 10). Since these toxins are heat-

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Prosiding Seminar Ilmiah dan Pertemuan Tahunan PEI dan PFI XVI Komda Sul-Sel, 2005 ISBN : 979-95025-6-7

stable, ordinary cooking and procedures for heat processing do not substantially reduce toxin levels. The result also indicated that among the concentration, diluting the filtrate lessen the effect of the toxin against root elongation. The variation of diluting effect among the crops indicates the different response of the crops.

Regarding the incubation time of the culture, it is seems that the 7 days old culture of P. grisea is more effective in inhibiting the roots compared to the 14 days old culture. The toxin inhibited the roots of all crops tested. It is proof that the toxin produced by P. grisea is non-host specific.

3.5

Rice

Root elongation (cm)

3.0

Corn Mungbean

2.5

Stringbean

2.0 1.5 1.0 0.5 0.0 Crude Boiled

1:10

1:20

1:50

dH2O

Treatments

Figure 11. Effect of toxin from 7 days old culture of P. grisea on root elongation of rice, corn, mungbean, and stringbean at 3 days after inoculation.

7.0

Rice

Root elongation (cm)

6.0

Corn Mungbean

5.0

Stringbean

4.0 3.0 2.0 1.0 0.0 Crude Boiled

1:10

1:20

1:50

dH2O

Treatments

Figure 12. Effect of toxin from 14 days old culture of P. grisea on root elongation of rice, corn, mungbean, and stringbean at 3 days after inoculation.

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CONCLUSION •

• • • •

Toxin produced by Pyricularia grisea inhibited the root growth of rice, corn, mungbean, and stringbean seedlings. Toxin did not affected by heating. Diluting the filtrate lessen the effect of toxin against root elongation. The highest root inhibition resulted from exposing the seedlings on crude filtrate. The filtrates including crude, boiled, and diluted showing necrotic symptoms on rice leaves.

REFERENCES Agrios, G.N. 1997. Plant Pathology. 4th ed. Academic Press. 635 p.

Callow, J.A. 1983. Biochemical Plant Pathology. John Wiley & Sons.484 p. Dixon, R.A., M.J. Harrison and C.J. Lamb. 1994. Early events in the activation of plant defense responses. Annu. Rev. Phytopathol. 32:479-501. Goodman, R.N., Z. Kiraly, and K.R. Wood. 1986. The Biochemistry and Physiology of Plant Disease. University of Missouri Press, Columbia. 433 p. Urban, M., T. Bhargava, and J.E. Hamer. 1999. An ATP-driven efflux pump is a novel pathogenicity factor in rice blast disease. The EMBO Journal Vol. 18:512-521.

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