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桿菌屬細菌表現系統之建立及應用─桿菌屬拮抗細菌之開發與改良(3/3)

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行 政 院 國 家 科 學 委 員 會 專 題 研 究 計 畫 成 果 報 告

桿 菌 屬 拮 抗 細 菌 之 開 發 與 改 良

D e v e l o p m e n t a n d m o d i f i c a t i o n o f B a c i l l u s a n t a g o n i s t 計畫編號: NSC 88-2316-B-002-016-B19 (3/3) 執行期間:87年8月1日 至 88年7月31日 計 畫 主 持 人 : 陳 昭 瑩 執 行 單 位 : 臺 灣 大 學 植 物 病 理 學 系 一 、 中 英 文 摘 要 已知許多細菌可產生幾丁質分解 酵素分解真菌細胞壁,抑制真菌的生 長,故被應用於植物真菌性病害的防 治上。本研究將 Bacillus circulans chiA 基因導入拮抗細菌 Bacillus subtilis 細 胞中,測試其對拮抗菌抑制病原真菌 生長的影響。首先將含起動子的 chiA 基 因 次 選 殖 於 shuttle vector pHY300PLK 上,以原生質體轉形法送 入 B. subtilis 細胞中。可於 B. subtilis 的培養上清液中測得幾丁質分解酵素 的活性,顯示 ChiA1 蛋白質可分泌於 B. subtilis 的細胞外。B. subtilis 於 LB 中培養 24 小時後可以測得幾丁質分解 酵素的活性;在幾丁質培養基中則需 培養較長時間才可測得幾丁質分解酵 素的活性。在大腸桿菌細胞中,ChiA1 蛋白質主要分泌於細胞間質中。當培 養時間延長時,可於幾丁質培養基上 菌落外緣出現透明圈。將 B. circulans chiA 基因 表現於無抑 菌作用的變 異 株 , 可 以 觀 察 到 ChiA 對 Botrytis elliptica 的生長具有抑制效果;但幾丁 質分解酵素 ChiA1 並不能穩定地促進 B. subtilis 野生菌株的抑菌效果。

Several chitinase - producing bacteria are known to degrade the fungal cell wall and are able to inhibit the growth of fungi. Applications of chitinase-producing bacteria in plant disease control have been reported. In this study, we transferred the chiA gene of chitinase-producing bacterium, Bacillus circulans, into the antagonist, Bacillus subtilis and examined the effect of ChiA1 protein on the antagonistic activity of B. subtilis. Firstly, the chiA gene was subcloned with its own promoter into the shuttle vector, pHY300PLK, and transferred into the bacterial cells of Bacillus subtilis by protoplast transformation. The expression of ChiA1 protein was detected in the supernatant of B. subtilis culture, indicating that the ChiA1

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protein is secreted out of the Bacillus cell. The ChiA1 protein was detected 24 hr after cultivation of Bacillus in LB broth and in a later period when incubated in chitin medium. In Escherichia coli cells, the ChiA1 protein is translocated into the periplasmic space. The chitinase is released from E. coli cells after prolonged incubation. The expression of chiA gene in a non-antagonistic mutant of B. subtilis could display inhibition effect on the growth of Botrytis elliptica. However, when the chiA gene was expressed in the antagonistic strain of B. subtilis, the enhancing effect of ChiA1 on the antagonistic activity of B. subtilis was not readily observed.

二、計畫緣由與目的

The antagonistic activity of biocontrol agent could be due to the antabiosis, competition of nutrition, and parasitism. Some antagonistic bacteria are capable of producing chitinases which are involved in the biocontrol of fungal pathogens (Inbar and Chet, 1991; Kobayashi et al., 1995; Ordentlich et al. 1988; Pleban et al., 1997; Sneh, 1981). Chitinases are able to degrade the chitin components in the cell wall of fungi (Horikoshi and Iida, 1959). Chitin, a polymer of N-acetyl-D-glucosamin in beta- 1,4 -linkage, is one of the components of cell wall of pathogenic fungi in Ascomycetes, Basidiomycetes,

and Deuteromycetes. Antifungal activity of chitinase from chitinolytic bacteria is reported in many cases (Chernin et al., 1995; Mavingui and Heulin, 1994). Chitinolytic bacteria or chitinase has been used with antagonistic organism to enhance its toxicity, such that the ChiA1 protein of Bacillus circulans could enhance the toxicity of Bacillus thuringiensis subsp. kurstaki toward diamondback moth larvaes (Wiwat et al., 1996). Endochitinase ChiAII of Serratia marcescens has a synergistic effect on the activity of CryIC endotoxin of

Bacillus thuringiensis toward

Spodoptera littoralis larves (Regev et al., 1996).

In the genus Bacillus, B. circulans (Tanaka and Watanabe, 1995; Watanabe et al., 1990), B. cereus (Pleban et al., 1997), and B. polymyxa (Mavingui et al., 1994) are well known to produce chitinase. Most of Bacillus subtilis are not chitinase-producers, many are able to produce antibiotics and are good biocontrol agent for fungal diseases of plants (Aldrich and Bader, 1970, Asaka and Shoda, 1996; Baker and Stavely, 1985, Berger et al., 1996, Tchen, 1987; Podile et al. 1985). In this study, we examined the effect of chitinase on the antagonistic activity of B. subtilis with the chiA gene of B. circulans.

二、 結果與討論

The 2.4-kb chiA gene of B. circulans was cloned into the vector

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pCR2.1 and subcloned into the shuttle vector, pHY300PLK (4.8 kb). The clone was designated pNTU111. Clear zone was observed around the colony of E.

coli DH5α(pNTU111) on chitin medium

after 7-day incubation. E. coli DH5α(pHY300PLK) hardly grew on chitin medium. In SDS-PAGE analysis, the activity of chitinase was detected in the periplasmic fraction of E. coil cells.

The activity of chitinase was detected in the supernatant of B. subtilis 28-6(pNTU111) which incubated in LB, nHA and chitin medium. In LB broth, the chitinase was detected significantly 24 hr after inoculation and the activity still could be detected 48, and 96 hr after inoculation. During these incubation periods, chitinase activity was not detected in the culture of B. subtilis 28-6 及 B. subtilis 28-6(pHY300PLK). The chitinase activity appeared about one day delayed when B. subtilis was cultured in nHA medium and two days delayed in chitin medium. In addition, the chitinase collected from the supernatant of B. subtilis 28-6(pNTU111) could significantly release the fluorescence compound, MU, from 4-MU-(GlcNAc)3.

The chiA fragment amplified in this study includes its own promoter and a 2,097-bp open reading frame for a protein product, ChiA1 (Watanabeet al., 1990). Although the expression of chiA is inducible in B. circulans (Watanabe et al., 1990), it is consistently expressed in both E. coli and B. subtilis when chiA

was constructed in the shuttle vector, pHY300PLK. The constitutive expression of chiA in B. subtilis may be explained by the possibilities that a repression control in B. circulans does not present in B. subtilis and E. coli or another upstream promoter on the vector may drive the expression of chiA gene.

By using nHA medium for the growth of target fungi and antagonist, B.

subtilis 28-6 (pNTU111) showed

stronger inhibition effect on the growth

of Rhizoctonia solani AG4 than B.

subtilis 28-6(pHY300PLK); however, this effect could not reproduced regularly. This inconsistency might be due to the states for the production of antibiotic and chitinase were not well controlled. The instability of pNTU111 in B. subtilis 28-6 was observed under a non-selective condition. Thus, a test was performed in the medium containing tetracycline for selection of plasmid. This phenomenon will become a problem in pot assay. An improvement on this point will be considered. Plasmid pHY300PLK have been reported stable in Bacillus thuringiensis and still a stable construct with a chitinase gene of Aeromonas sp. (Wiwat et al., 1996). Although plasmid pHY300PLK was also stable in B. subtilis, the construct was not as stable with the chiA gene of B. circulans in a non-selective condition.

To clarify the effect of chitinase expressed in Bacillus subtilis on the growth of target fungi, the chiA gene was introduced into a B. subtilis mutant

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fenE which could not inhibit the growth of Rhizoctonia. By using weak malt extract agar for the growth of target fungi and chitin medium for the growth of antagonist, B. subtilis fenE (pNTU111) showed stronger inhibition effect on the growth of B. elliptica than B. subtilis fenE (pHY300PLK), indicating that chitinase ChiA1 could inhibit the growth of B. elliptica. Similar effect was not observed on B. cinerea, Rhizoctonia solani AG-1, and Fusarium oxysporum. The reason is not clear.

四、成果自評

We showed that chiA gene of B. circulans could inhibit some pathogenic fungi when the gene was introduced into a B. subtilis strain. The enhancing effect of ChiA protein on the antagonistic activity of B. subtilis had been observed; but not consistently displayed. The interactions of ChiA1 protein of B. circulans and antibiotics of B. subtilis may exist. The expression of chiA gene and antibiotic synthetase genes within same Bacillus subtilis cell may be counteracted. A better way to use chitinase gene in plant disease control will be put in deep consideration.

五、參考文獻

1. Aldrich, J. and Bader, R. 1970. Biological control of Fusarium roseum

f. sp. dianthi by Bacillus subtilis. Plant Dis. Report. 54:446-448.

2. Asaka, O. and Shoda, M. 1996.

Biocontrol of Rhizoctonia solani

damping-off of tomato with Bacillus

subtilis RB14. Appl. Environ.

Microbiol. 62:4081-4085.

3. Baker, C. J. and Stavely, J. R. 1985. Biocontrol of bean rust by Bacillus subtilis under field conditions. Plant Dis. 69:770-772.

4. Berger, F., Li, H., Frazer, D. W., and Leifert, C. 1996. Effect of pathogen inoculum, antagonist density, and plant species on biological control of phytophthora and pythium damping-off by Bacillus subtilis Cot1 in high-humidity fogging glasshouses. Phytopathology 86:428-433.

5. Chernin, L., Ismailov, Z., Haran, S., and Chet, I. 1995. Chitinolytic

Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Environ. Microbiol. 61:1720-1726. 6. Horikoshi, K. and Iika, S. 1959. Effect

of lytic enzyme from Bacillus circulans

and chitinase from Streptomyces sp. on

Aspergillus oryzae. Nature 183:186-187.

7. Inbar, J. and Chet, I. 1991. Evidence that chitinase produced by Aeromonas caviae is involved in the biological control of soil-borne plant pathogens by this bacterium. Soil. Biol. Biochem. 23:973-978.

8. Kobayashi, D. Y., Guglielmoni, M., and Clarke, B. B. 1995. Isolation of the chitinolytic bacteria Xanthomonas maltophilia and Serratia marcescens as biological control agents for summer patch disease of turfgrass. Soil. Biol.

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Biochem. 27:1479-1487.

9. Mavingui, P. and Heulin, T. 1994. In vitro chitinase and antifungal activity of a soil, rhizosphere and rhizoplane population of Bacillus polymyxa. Soil. Biol. Biochem. 26:801-803.

10. Ordentlich, A., Elad, Y., and Chet, I. 1988. The role of chitinase of Serratia marcescens in biocontrol of Sclerotium rolfsii. Phytopathology 78:84-88. 11. Pleban, S., Chernin, L., and Chet, I.

1997. Chitinolytic activity of an endophytic strain of Bacillus cereus.

Lett. Appl. Microbiol. 25:284-288. 12. Podile, A. R., Prasad, G. S., and Dube,

H. C. 1985. Bacillus subtilis as antagonist to vascular wilt pathogens. Curr. Sci. 54:865-865.

13. Regev, A., Keller, M., Strizhov, N., Sneh, B., Prudovsky, E., Chet, I., Ginzberg, I., Koncz-Kalman, Z., Koncz, C., Schell, J., and Zilberstein, A. 1996. Synergistic activity of a Bacillus

thuringiensis δ-Endotoxin and a bacterial endochitinase against

Spodoptera littoralis larvae. Appl. Environ. Microbiol. 62:3581-3586. 14. Sneh, B. 1981. Use of rhizosphere

chitinolytic bacteria for biological control of Fusarium oxysporum f. sp.

diantibi in carnation. Phytopath. Z. 100:251-256.

15. Tschen, J. S. M. 1987. Control of

Rhizoctonia solani by Bacillus subtilis. Trans. Mycol. Soc. Japan 28:483-493. 16. Wiwat, C., Lertcanawanichakul, M.,

Siwayapram, P., Pantuwatana, S., and Bhumiratana, A. 1996. Expression of chitinase-encoding genes from

Aeromonas hydrophila and

Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis. Gene 179:119-126.

參考文獻

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