放線菌Streptomyces plicatus B4-7拮抗青椒疫病菌之機制與綜合防治

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放線菌Streptomyces plicatus B4-7拮抗青椒疫病菌之機制與綜合防治

The biocontrol mechanism and integrated pest management of Streptomyces plicatus B4-7 on controlling Phytophthora capsici

陳盈宇 , Ph.D　　Advisor：蔡東纂　　Co-advisor ：陳珮臻

英文

Streptomyces plicatus B4-7 ； Streptomyces saraceticus strain 31 ； Phytophthora capsici ； Meloidogyne incognita ；生物防治；超寄生；抗生素； borrelidin ；農藥；作物病蟲害綜合防治； Streptomyces plicatus B4-7 ； Streptomyces saraceticus strain 31 ； Phytophthora capsici ； Meloidogyne incognita ； biocontrol ； hyperparasitism ； antibiotic ； borrelidin ； pesticide ； copper hydroxide ； etridiazole ； integrated pest management

Abstract │ Reference (139) │ Altmetrics

Abstract 〈TOP〉

自新竹縣芎林鄉柑橘園竹葉上分離純化的放線菌菌株B4-7，經生理生化測試與16S rDNA鑑定為Streptomyces plicatus。對峙試驗中，B4-7可抑制75%以上多種疫病菌菌絲的生長，包含Phytophthora capsici、P. cinnamomi、P. palmivora與P. parasitica。利用電子顯微鏡觀察超寄生時，可見B4-7纏繞於青椒疫病菌P. capsici菌絲上，並分泌酵素破壞細胞壁與入侵菌絲與孢子囊，證實B4-7具有超寄生能力。將B4-7搖瓶培養於幾丁質馬鈴薯蔗糖培養液 (chitin-potato sucrose broth；CP)、馬鈴薯蔗糖培養液 (potato sucrose broth；PS)、米糠培養液 (rice bran broth；RB) 與燕麥培養液 (oatmeal broth；OM) 六天後，四種B4-7培養濾液對P. capsici可表現32.5 ~ 55.0%菌絲生長抑制率。測試四種B4-7培養濾液對P. capsici游走孢子發芽率的影響，發現處理24小時後， RB與OM培養濾液可完全抑制P. capsici游走孢子發芽，CP培養濾液處理可達82.9%游走孢子發芽抑制率，而PS培養濾液處理完全無法抑制游走孢子發芽；然以顯微鏡觀察B4-7 CP與PS培養濾液處理後之P. capsici游走孢子發芽情況，游走孢子的發芽管皆呈現畸形與不規則分岐膨大。將四種B4-7培養濾液分別與P. capsici游走孢子一同接種於青椒葉片上，三天後觀察其發病狀態，結果顯示四種B4-7培養濾液的疫病發病抑制率皆為100%。在青椒根系接種P. capsici游走孢子三小時後，澆灌B4-7 OM培養液並觀察莖基部發病情況，結果顯示B4-7 OM培養液可減少75%的發病率。為純化分離B4-7的抗疫病菌物質，B4-7 OM培養液經乙酸乙酯 (ethyl acetate, EA) 初步萃取後，將具有抗疫病菌物質的有機層產物濃縮並進行管柱層析，以矽膠為靜相，不同比例EA與Hexan調配為動相。經管柱層析純化與儀器分析後，B4-7的抗疫病菌物質確認為borrelidin，這是首次發現S. plicatus能產生此抗種生素。將純化自B4-7的borrelidin稀釋為1、5與10 mg/L三種濃度並直接滴在P. capsici 菌絲塊上測試抑制效果，24小時後菌絲生長抑制率分別為50.0%、92.9%與100%。以上實驗結果顯示S. plicatus B4-7具有超寄生與產生抗生物質等抗病機制，相當有潛力運用在作物病蟲害綜合防治(Integrated pest management)上。為了瞭解生物防治放線菌在田間與它種農藥的關係，將用於防治南方根瘤線蟲的Streptomyces saraceticus strain 31(SS31)與防治青椒疫病的S. plicatus B4-7分別與73支與12支常用農業藥劑進行親和性試驗。在培養基實驗中，當放線菌與藥劑同時處理時，結果顯示73支藥劑中，有40支對SS31無抑制效果；12支藥劑中，有4支對B4-7無抑制效果。當放線菌先在培養基上生長24小時後再處理藥劑，結果顯示分別有55支與8支藥劑對SS31與B4-7無抑制效果。在溫室試驗中，施用SS31可有效降低根瘤線蟲造成的根瘤指數與減少土中二齡幼蟲的數量，而在SS31與藥劑共同施用的處理中，結果顯示四氯異苯腈、氫氧化銅、普伏松與加保扶不會影響SS31防治線蟲的能力，甚至有加成的效果。當B4-7分別與依得利和氫氧化銅混合施用來防治青椒疫病，可有良好的防治效果，且當減少農藥劑量時，B4-7與依得利混合施用仍可完全抑制青椒疫病發生，顯示適當的運用生物防治菌可降低農藥施用量。

Parallel Abstract 〈TOP〉

A Streptomyces plicatus B4-7 was obtained from the bamboo leaf in a citrus farm (Qionglin Township, Hsinchu County, Taiwan) and identified by 16S rDNA methods. B4-7 suppressed up to 75% mycelial growth of Phytophthora capsici, P. cinnamomi, P. palmivora and P. parasitica in the dual culture test on PSA (potato sucrose agar) plates. The hyperparasitism of B4-7 against P. capsici was confirmed under the scanning electron microscope. The cultural filtrates of B4-7 in chitin-potato sucrose broth, potato sucrose broth, rice bran broth and oatmeal broth can inhibited the mycelial growth and the zoospore germination of P. capsici in the in vitro tests. Abnormal formation of P. capsici zoospore germ tubes were observed after treated with B4-7 cultural filtrates 24 h. When the B4-7 cultural filtrates and P. capsici zoospore were inoculated simultaneously on bell pepper leaves, 100% disease suppression was observed. In the pot test, the B4-7 OM cultural broth when added 3 h post inoculation (HPI) of P. capsici showed 75% crown rot disease reduction. The OM cultural broth was extract with ethyl acetate, and the EA supernatant was purified by column chromatography eluted with ethyl acetate/hexane as the developing solvent system. A compound was isolated from the anti-Phytophthora fraction of B4-7 OM broth, and was identified as borrelidin according to the spectral analysis. This study is the first report of S. plicatus capable of producing this antibiotic. Borrelidin isolated from B4-7 showed 50%, 92.86% and 100% inhibitory to the mycelial growth of P. capsici when treated 1, 5 and 10 mg/L for 24 h, respectively. The results indicated B4-7 could be a good biocontrol agent candidate for the integrated pest management (IPM) program to control Phytophthora blight. To evaluate the potential of Streptomyces spp. applied in an integrated pest management system, commonly used pesticides from different pesticide categories were tested in vitro and in vivo for the compatibility. Two biocontrol actinomycetes, Streptomyces saraceticus strain 31 and S. plicatus B4-7, were used to control Meloidogyne incognita and Phytophthora capsici, respectively. In the in vitro test, 73 pesticides were tested with SS31, and 12 with B4-7. When the pesticides applied with Streptomyces spp. at the same time in vitro (S1), SS31 was found to be compatible with 40 out of 73 pesticides tested, and 4 out of 12 for B4-7. The in vitro test results indicated that when SS31 and B4-7 were established 24 h before the pesticide application (S2), the inhibition effects of the pesticides were reduced. Under the S2 treatments, 55 pesticides were compatible with SS31, and 8 with B4-7. SS31 could lower both the M. incognita galling index and J2 population in greenhouse trials, and was compatible with chlorothalonil, copper hydroxide, ethoprop and carbofuran regardless of the pesticide application sequences. Application of B4-7 mixed with etridiazole or copper hydroxide on controlling P. capsici in vivo showed the pesticides could achieve the same control efficacies at lower dosage. Both SS31 and B4-7 compatible with the majority pesticides when applied in the field with an appropriate time interval are suitable BCAs for the IPM.

Reference ( 139 ) 〈TOP〉

Ref. of Introduction
連結：
Manulis, S., Shafrir, H., Epstein, E., Lichter, A., Barash, I., 1994. Biosynthesis of indole-3-acetic acid via the indole-3-acetamide pathway in Streptomyces spp. Microbiology 140, 1045-1050.
連結：
Salla, T.D., Silva, T.R., Astarita, L.V., Santarem, E.R., 2014. Streptomyces rhizobacteria modulate the secondary metabolism of Eucalyptus plants. Plant Physiol. Bioch. 85, 14-20.
連結：
Abd-Allah, E.F., 2001. Streptomyces plicatus as a model biocontrol agent. Folia Microbiol. 46, 309¬-314.
連結：
Aghighi, S., Shahidi Bonjar, G.H., Saadoun, I., 2004. First report of antifungal properties of a new strain of Streptomyces plicatus (strain101) against four Iranian phytopathogenic isolates of Verticillium dahliae, a new horizon in biocontrol agents. Biotechnol. 3, 90-97.
連結：

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