TW201938787A

TW201938787A - Streptomyces misionesis KHY26, cultivation method for increasing KHY26 and use for controlling plant pathogens

Info

Publication number: TW201938787A
Application number: TW107108324A
Authority: TW
Inventors: 陳泰元
Original assignee: 行政院農業委員會高雄區農業改良場
Priority date: 2018-03-12
Filing date: 2018-03-12
Publication date: 2019-10-01
Also published as: TWI638046B

Abstract

The present invention discloses a Streptomyces misionesis KHY26, a cultivation method for increasing the KHY26 and a use for controlling plant pathogens. The cultivation method comprises incubating the KHY26 in a predetermined medium having a specific composition for cultivation under a specific condition for a specific time period to obtain a fermented product of the KHY26 having a high concentration. The KHY26 has an antibacterial activity to inhibit growth of many plant pathogens including Lasiodiplodia theobromae, Colletotrichum gloeosporioides, and Meloidogyne spp. that exists in Cucurbitaceae, Solanaceae or other plants. Accordingly, the KHY26 can be used as a biological pesticide for preventing plant pathogens including Meloidogyne spp. in Cucurbitaceae, Solanaceae or other plants.

Description

鏈黴菌KHY26菌株及其增量培養方法與防治植物病原微生物之用途Streptomyces KHY26 strain and its incremental culture method and use for controlling plant pathogenic microorganisms

本發明係關於一種具有防治多種植物病原微生物(含植物寄生性線蟲)功效之鏈黴菌(Streptomyces misionesis )KHY26菌株及其增量培養方法與用途。The invention relates to a Streptomyces misionesis KHY26 strain with the effect of controlling various plant pathogenic microorganisms (including plant parasitic nematodes), and an incremental culture method and application thereof.

作物的種植過程中常常會遭遇植物病原菌或害蟲的侵害，目前常見防治害蟲的方法包含，化學防治、物理防治、生物防治等等。化學防治法雖然使用上較為方便，但若使用不當時很容易造成使用者中毒、藥劑殘留以及環境污染等等缺失，反觀，生物防治法，例如使用生物農藥，則對於環境較為友善。生物農藥包含具有植物保護功效的天然物質，例如源自植物的除蟲菊精或魚藤精，或是應用於微生物防治的蘇力菌，又或是用於防治害蟲的昆蟲性費洛蒙等等。During planting of crops, plant pathogens or pests are often encountered. At present, common methods for controlling pests include chemical control, physical control, and biological control. Although the chemical control method is more convenient to use, if it is not used properly, it can easily cause user poisoning, drug residues, and environmental pollution. On the other hand, biological control methods, such as the use of biological pesticides, are more friendly to the environment. Biological pesticides contain natural substances with plant protection effects, such as plant-derived pyrethrum or roe deer, or selenium used for microbial control, or insect pheromones used to control pests, etc. Wait.

由於微生物具有能大量複製且對環境較為友善之優點，因此不少研究係將微生物應用於生物農藥的製備；例如中華明國專利第TW 201439035(A)號專利公開案係一種農用微生物製劑粉劑配方及其製作方法，係將微生物菌粉、賦形劑與增量劑混合，以製備微生物製劑；其中的微生物為菌粉可為芽孢桿菌、假單孢桿菌、固氮菌、酵母菌、枯草桿菌、木霉菌、真菌、溶磷菌或藻類。又，中華明國專利第TW 201438582(A)號專利公開案係為使用細黃鏈黴菌(Streptomyces microflavus)株及使用彼等來控制植物疾病和害蟲之方法，係將細黃鏈黴菌突變株應用於防治植食性害蟲與對植物有害之真菌。Because microorganisms have the advantages of being able to reproduce in large quantities and being more environmentally friendly, many researches have applied microorganisms to the preparation of biological pesticides; for example, China Mingguo Patent No. TW 201439035 (A) Patent Publication is an agricultural microbial preparation powder formulation The preparation method is to mix microbial fungus powder, excipients and extenders to prepare a microbial preparation; the microorganisms may be bacterial powder, which may be Bacillus, Pseudomonas, nitrogen-fixing bacteria, yeast, Bacillus subtilis, Trichoderma, fungi, phosphate-soluble bacteria or algae. In addition, the China Mingguo Patent No. TW 201438582 (A) discloses a method for using Streptomyces microflavus strains and using them to control plant diseases and pests, and applies a mutant strain of Streptomyces microflavus To control plant-eating pests and fungi that are harmful to plants.

今，發明人乃一本孜孜不倦之精神，並藉由其豐富專業知識及多年之實務經驗所輔佐，據此研創出本發明之具有防治多種植物病原微生物(含植物寄生性線蟲)功效之鏈黴菌KHY26菌株(以下簡稱KHY26)以及大量生產KHY26之培養方法，並將KHY26用於防治包含植物寄生性線蟲在內之多種植物病原微生物。Today, the inventor is a tireless spirit, supplemented by his rich professional knowledge and many years of practical experience, and based on this, he has created the Streptomyces that has the effect of controlling various plant pathogenic microorganisms (including plant parasitic nematodes). KHY26 strain (hereinafter referred to as KHY26) and a culture method for mass production of KHY26, and KHY26 is used to control various plant pathogenic microorganisms including plant parasitic nematodes.

本發明係一種鏈黴菌(Streptomyces misionesis )KHY26菌株(以下簡稱KHY26)，以及含有KHY26之生物農藥。鏈黴菌KHY26菌株寄存於財團法人食品工業發展研究所，寄存編號為BCRC 910799，其中KHY26係具有防治多種植物病原微生物(含植物寄生性線蟲)之功效。The invention relates to a Streptomyces misionesis KHY26 strain (hereinafter referred to as KHY26) and a biological pesticide containing KHY26. The KHY26 strain of Streptomyces is deposited with the Institute of Food Industry Development, and the deposit number is BCRC 910799. Among them, KHY26 has the function of controlling various plant pathogenic microorganisms (including plant parasitic nematodes).

本發明亦提供培養鏈黴菌(Streptomyces misionesis )KHY26菌株(以下簡稱KHY26)的方法，包含：將KHY26培養於一特定培養基，並於25~35℃培養一特定時間，以獲得含有大量KHY26活菌體之發酵產物。The present invention also provides a method for culturing Streptomyces misionesis KHY26 strain (hereinafter referred to as KHY26), which comprises: culturing KHY26 in a specific medium and culturing at 25-35 ° C for a specific time to obtain a large amount of KHY26 viable cells Of fermentation products.

本發明進一步提供一種鏈黴菌(Streptomyces misionesis )KHY26菌株(以下簡稱KHY26)用於製備生物農藥之用途，係以一有效劑量之KHY26，製作成液劑或粉劑，施予一植物以防治包含植物寄生性線蟲在內之多種植物病原微生物。The invention further provides a strain of Streptomyces misionesis KHY26 (hereinafter referred to as KHY26) for the preparation of biological pesticides, which is prepared as a liquid or powder with an effective dose of KHY26 and applied to a plant to prevent and control plant parasites. Sexual nematodes include a variety of plant pathogenic microorganisms.

於本發明之一實施例中，植物病原微生物可為木瓜疫病菌(Phytophthora palmimora )、木瓜根腐病菌(Pythium aphanidermatum )、蓮霧黑腐病菌(Lasiodiplodia theobromae )、蓮霧果腐病菌(Pestalotiopsis euginae )、炭疽病菌(Colletotrichum gloeosporioides )、芒果畸形病菌(Fusarium mangiferae )、番石柳黑星病菌(Phyllosticta psidiicola )、番石榴瘡痂病菌(Pestalotiopsis psidii )、稻熱病菌(Magnaporthe grisea )以及瓜類根瘤線蟲(Meloidogyne spp.)與茄科根瘤線蟲(Meloidogyne spp.)。Embodiment, papaya plant pathogenic microorganisms may blight (Phytophthora palmimora), papaya root rot (Pythium aphanidermatum) was inoculated to one embodiment of the present invention, wax black rot pathogen (Lasiodiplodia theobromae), wax fruit rot pathogen (Pestalotiopsis euginae) , anthracnose (Colletotrichum gloeosporioides), mango malformation pathogen (Fusarium mangiferae), Liu fan stone Venturia inaequalis (Phyllosticta psidiicola), guava scab fungus (Pestalotiopsis psidii), rice germs heat (Magnaporthe grisea) and melons root-knot nematode (Meloidogyne spp.) and Meloidogyne spp.

於本發明之一實施例中，其一培養基為液態培養基，包含1~10 wt%黃豆漿、0.1~1 wt%酵母抽出物、0.05~0.5 wt%磷酸二氫鉀(KH₂ PO₄ )、0.05~0.5 wt%磷酸氫二鉀(K₂ HPO₄ )、0.01~0.1 wt%硫酸鎂(MgSO₄ ·7H₂ O))、0.05~0.5 wt%幾丁聚醣(Chitoson)與剩餘百分比之水，且培養之特定時間為3~5天。In one embodiment of the present invention, one of the culture media is a liquid culture medium, which contains 1 to 10 wt% soybean milk, 0.1 to 1 wt% yeast extract, 0.05 to 0.5 wt% potassium dihydrogen phosphate (KH ₂ PO ₄ ), 0.05 ~ 0.5 wt% dipotassium hydrogen phosphate (K ₂ HPO ₄ ), 0.01 ~ 0.1 wt% magnesium sulfate (MgSO ₄ · 7H ₂ O)), 0.05 ~ 0.5 wt% Chitoson and the remaining percentage of water , And the specific time of cultivation is 3 ~ 5 days.

於本發明之一實施例中，另一培養基為固態培養基包含一固體部分與一液體部分，固體部分以重量比100%計算包含5~35 wt%燕麥粒、5~35 wt%大麥片、10~20 wt%玉米粉、5~30 wt%稻殼、5~15 wt%蚵殼粉，液體部分為該固體部分總重量之40~60 wt%的水，且培養之特定時間為10~18天。In one embodiment of the present invention, the other medium is a solid medium comprising a solid portion and a liquid portion, and the solid portion comprises 5 to 35 wt% oat grains, 5 to 35 wt% barley flakes, 10% by weight based on 100% by weight. ~ 20 wt% corn flour, 5 ~ 30 wt% rice husk, 5 ~ 15 wt% coriander powder, the liquid part is 40 ~ 60 wt% water of the total weight of the solid part, and the specific cultivation time is 10 ~ 18 day.

於本發明之一實施例中，生物農藥為液劑或粉劑。In one embodiment of the present invention, the biological pesticide is a liquid or a powder.

於本發明之一實施例中，生物農藥係為液態發酵產物所製成之液劑或粉劑，且液劑之有效劑量為濃度1 x 10⁵ ~1 x 10¹⁰ cfu/mL之KHY26菌株，粉劑之有效劑量為濃度1 x 10⁷ ~1 x 10¹⁰ cfu/g之KHY26菌株，且粉劑包含鏈黴菌KHY26菌粉與麥芽糊精。In one embodiment of the present invention, the biological pesticide is a liquid or powder made of a liquid fermentation product, and the effective dose of the liquid is a KHY26 strain with a concentration of 1 x 10 ⁵ to 1 x 10 ¹⁰ cfu / mL, a powder. The effective dose is KHY26 strain with a concentration of 1 x 10 ⁷ ~ 1 x 10 ¹⁰ cfu / g, and the powder contains Streptomyces KHY26 bacterial powder and maltodextrin.

於本發明之一實施例中，生物農藥為固態發酵產物所製成之粉劑，且粉劑之有效劑量係為濃度1 x 10⁸ ~1 x 10¹¹ cfu/g之KHY26菌株。In one embodiment of the present invention, the biological pesticide is a powder made from a solid fermentation product, and the effective dose of the powder is a KHY26 strain with a concentration of 1 x 10 ⁸ to 1 x 10 ¹¹ cfu / g.

藉此，本發明鏈黴菌(Streptomyces misionesis )KHY26菌株對根瘤線蟲及多種植物病原菌具抗生活性，因此可用於作為生物農藥或是果實保護組成物。Therefore, the KHY26 strain of Streptomyces misionesis of the present invention is resistant to rhizobial nematodes and various plant pathogens, and therefore can be used as a biological pesticide or a fruit protection composition.

本發明之目的及其結構功能上的優點，將依據以下圖面所示之結構，配合具體實施例予以說明，俾使審查委員能對本發明有更深入且具體之瞭解。The purpose of the present invention and its structural and functional advantages will be explained based on the structure shown in the following drawings, in conjunction with specific embodiments, so that the reviewing committee can have a deeper and more specific understanding of the present invention.

本發明為一種鏈黴菌(Streptomyces misionesis )KHY26菌株(以下簡稱KHY26)，寄存於財團法人食品工業發展研究所，寄存編號為BCRC 910799。鏈黴菌KHY26可用於製備生物農藥，並可抑制多種植物病原微生物如：木瓜疫病菌(Phytophthora palmimora )、木瓜根腐病菌(Pythium aphanidermatum )、蓮霧黑腐病菌(Lasiodiplodia theobromae )、蓮霧果腐病菌(Pestalotiopsis euginae )、炭疽病菌(Colletotrichum gloeosporioides )、芒果畸形病菌(Fusarium mangiferae )、番石柳黑星病菌(Phyllosticta psidiicola )、番石榴瘡痂病菌(Pestalotiopsis psidii )、稻熱病菌(Magnaporthe grisea )以及瓜類根瘤線蟲(Meloidogyne spp.)與茄科根瘤線蟲(Meloidogyne spp.)。以鏈黴菌KHY26所製備之生物農藥可為液劑或粉劑，粉劑可包含鏈黴菌KHY26與其固態發酵培養基質，並可以一有效劑量，抑制根瘤線蟲等植物寄生性線蟲之生長與繁殖。The present invention is a Streptomyces misionesis KHY26 strain (hereinafter referred to as KHY26), which is deposited in the Institute of Food Industry Development, and the deposit number is BCRC 910799. Streptomyces KHY26 be used to prepare biological pesticides, and can inhibit a variety of phytopathogenic microorganisms, such as: papaya blight (Phytophthora palmimora), papaya root rot (Pythium aphanidermatum), black rot pathogen wax (Lasiodiplodia theobromae), fruit rot pathogen wax ( Pestalotiopsis euginae ), Colletotrichum gloeosporioides , Fusarium mangiferae , Phyllosticta psidiicola , Pestalotiopsis psidii , and Magnaporthe grina Meloidogyne spp. And Meloidogyne spp. The biological pesticide prepared with Streptomyces KHY26 can be a liquid or a powder. The powder can contain Streptomyces KHY26 and its solid fermentation medium, and can inhibit the growth and reproduction of plant parasitic nematodes such as Rhizobium nematodes at an effective dose.

本發明亦提供增量培養鏈黴菌KHY26菌株之方法，包含將鏈黴菌KHY26菌株培養於一培養基中，並於25~35℃培養一特定時間。其中該培養基可為液態培養基，包含1~10 wt%黃豆漿、0.1~1 wt%酵母抽出物、0.05~0.5 wt%磷酸二氫鉀(KH₂ PO₄ )、0.05~0.5 wt%磷酸氫二鉀(K₂ HPO₄ )、0.01~0.1 wt%硫酸鎂(MgSO₄ ·7H₂ O)、0.05~0.5 wt%幾丁聚醣(Chitoson)與水，且培養之特定時間為3~5天。此外，培養基亦可為固態培養基，固態培養基包含一固體部分與一液體部分，固體部分以重量比100%計算，包含5~35 wt%燕麥粒、5~35 wt%大麥片、10~20 wt%玉米粉、5~30 wt%稻殼與5~15 wt%蚵殼粉，以及液體部分為該固體部分總重量之40~60 wt%的水，且固態發酵培養之特定時間為10~18天。The present invention also provides a method for incrementally growing the KHY26 strain of Streptomyces, which comprises culturing the KHY26 strain of Streptomyces in a medium, and culturing the KHY26 strain at 25-35 ° C for a specific time. The medium may be a liquid medium, including 1 to 10 wt% soybean milk, 0.1 to 1 wt% yeast extract, 0.05 to 0.5 wt% potassium dihydrogen phosphate (KH ₂ PO ₄ ), and 0.05 to 0.5 wt% hydrogen phosphate dihydrogen. Potassium (K ₂ HPO ₄ ), 0.01 ~ 0.1 wt% magnesium sulfate (MgSO ₄ · 7H ₂ O), 0.05 ~ 0.5 wt% Chitoson and water, and the specific culture time is 3 ~ 5 days. In addition, the medium can also be a solid medium. The solid medium contains a solid portion and a liquid portion. The solid portion is calculated by 100% by weight, and contains 5 ~ 35 wt% oat grain, 5 ~ 35 wt% barley flakes, and 10 ~ 20 wt. % Corn flour, 5 ~ 30 wt% rice husk and 5 ~ 15 wt% husk flour, and the liquid part is 40 ~ 60 wt% water of the total weight of the solid part, and the specific time for solid fermentation culture is 10 ~ 18 day.

此外，藉由下述具體實施例，可進一步證明本發明可實際應用之範圍，但不意欲以任何形式限制本發明之範圍。In addition, through the following specific examples, the scope of the present invention can be further proved, but it is not intended to limit the scope of the present invention in any form.

實驗一、鏈黴菌KHY26菌株及其培養方法Experiment I. Streptomyces KHY26 strain and its culture method

(( 一One )) 鏈黴菌KHY26菌株之分離與鑑定Isolation and Identification of Streptomyces KHY26 Strain

自臺灣各地農田及惡地地形取得15處土壤樣本，經分離篩選後純培養，再以菌落外觀型態分類，共得到219株菌株；以菌株對於蓮霧黑腐病菌(Lasiodiplodia theobromae )及木瓜根腐病菌(Pythium aphanidermatum )的抗生活性作為菌株功效篩選指標，於馬鈴薯葡萄糖瓊脂培養基平板(potato dextrose agar，PDA)上，將分離之菌株分別與蓮霧黑腐病菌及木瓜根腐病菌進行對峙培養，試驗結果獲得對蓮霧黑腐病菌及木瓜根腐病菌皆具抗生活性之微生物，共16株菌株，且其中編號KHY26之菌株對前述兩種病原菌皆具有最佳之抗生活性。請參見第一圖(A)，為KHY26菌株於PDA培養基平板上生長之型態圖，係為邊緣呈現平滑狀之圓形菌落；另請見第一圖(B)，將KHY26培養於羊血培養基平板，並沒有產生溶血反應，表示KHY26具有生物安全性。Fifteen soil samples were obtained from farmlands and bad terrains in various parts of Taiwan. After isolation and screening, they were purely cultured, and then classified according to the appearance of the colonies. A total of 219 strains were obtained. The strains were Lasiodiplodia theobromae and papaya root. The life resistance of Pythium aphanidermatum was used as a screening index for strain efficacy. The isolated strains were cultured on potato dextrose agar (PDA) against the black rot fungus of lotus fog and the root rot fungus of papaya. As a result of the test, there were 16 strains of microorganisms that were resistant to the black rot of the lotus mist and the root rot of papaya. A total of 16 strains were obtained, and the strain No. KHY26 had the best resistance to the aforementioned two pathogens. Please refer to the first picture (A), which shows the growth pattern of KHY26 strain on the PDA culture plate, which is a round colony with smooth edges; see also the first picture (B), which is cultured in sheep blood The culture plate did not produce a hemolytic response, indicating that KHY26 was biologically safe.

將KHY26菌株進行16s rDNA定序，並委託財團法人食品工業研究所進行菌種鑑定，確認KHY26菌株為鏈黴菌(Streptomyces misionesis )(請參見附件1)；其16s rDNA之序列請參見序列表之SEQ ID NO:1，另，SEQ ID NO:2為KHY26菌株上，帶有的鏈黴菌特殊專一性片段。The KHY26 strain was sequenced with 16s rDNA, and the food industry research institute was entrusted to carry out strain identification, and the KHY26 strain was confirmed to be Streptomyces misionesis (see Annex 1); the sequence of its 16s rDNA is shown in SEQ of the Sequence Listing ID NO: 1, and SEQ ID NO: 2 is a special specific fragment of Streptomyces on KHY26 strain.

(( 二) 液態發酵之培養基成分對KHY26菌株生長的影響2) Effect of liquid fermentation medium components on the growth of KHY26 strain

下列實驗以搖瓶培養系統進行培養，以觀察培養基之不同穀物基質、酸鹼值及其他添加物對於本發明KHY26菌株生長之影響，其中下列所提及之基本培養液為黃豆漿培養基，成分包含3 wt%黃豆漿、0.3 wt%酵母抽出物、0.1 wt%磷酸二氫鉀(KH₂ PO₄ )、0.1 wt%磷酸氫二鉀(K₂ HPO₄ )與剩餘百分比的水。The following experiments were performed using a shake flask culture system to observe the effects of different cereal substrates, pH values and other additives on the growth of the KHY26 strain of the present invention. The basic culture solution mentioned below is a soybean milk culture medium, which contains 3 wt% soybean milk, 0.3 wt% yeast extract, 0.1 wt% potassium dihydrogen phosphate (KH ₂ PO ₄ ), 0.1 wt% dipotassium hydrogen phosphate (K ₂ HPO ₄ ), and the remaining percentage of water.

以下所有實驗中之菌量計算方法如下：於無菌環境中取1 mL待測菌液與9 mL 無菌水混合均勻，此即為10倍稀釋菌液，並以此方法繼續進行菌液的10倍序列稀釋；取0.1 mL之稀釋菌液滴至PDA平板，並以消毒過之三角玻璃棒將稀釋菌液均勻塗佈於PDA平板表面後，置於27℃下培養，待菌落生長至肉眼可見之大小時，計算各稀釋倍率之菌落數；每組實驗皆進行三重複，計算時係將三重複之菌落數加總後除以3以獲得一平均菌落數；平均菌落數再以下列計算公式推算原始菌數：The calculation method of the bacteria amount in all the following experiments is as follows: in a sterile environment, take 1 mL of the bacteria solution to be tested and mix with 9 mL of sterile water, which is 10 times the diluted bacteria solution, and continue to perform 10 times the bacteria solution by this method. Sequential dilution; take 0.1 mL of the diluted bacteria solution onto the PDA plate, and apply the diluted bacteria solution to the surface of the PDA plate with a sterilized triangular glass rod, and culture at 27 ° C until the colonies grow to the naked eye. For large hours, calculate the number of colonies at each dilution rate. Each group of experiments is performed in triplicate. The calculation is performed by adding the triplicate colonies and dividing by 3 to obtain an average colony number. The average colony number is then calculated by the following calculation formula. Number of original bacteria:

原始菌數(單位：cfu/mL)=平均菌落數 × 稀釋倍率之倒數 × 10Original bacterial count (unit: cfu / mL) = average colony count × reciprocal of dilution ratio × 10

菌量計算結果將以顯著性測試進行統計分析，若未達顯著水準則以費雪最小差異測試法(Fisher’s Least Significance Test, LSD)檢定，測定5%之顯著差異，若各組間具有顯著差異則會標記不同的字母，若各組間不具有顯著差異則會標記相同的字母。The bacterial count calculation results will be statistically analyzed using a significance test. If the significant water criterion is not met, the Fisher's Least Significance Test (LSD) test will be used to determine a significant difference of 5%. If there is a significant difference between groups Different letters will be marked, and the same letters will be marked if there are no significant differences between the groups.

(1)(1) 不同穀物基質對KHY26菌株生長的影響Effects of different grain substrates on the growth of KHY26 strain

將菌量濃度為10⁹ cfu/mL之KHY26菌體懸浮液，取1 mL菌體懸浮液作為接種源，加入400 mL之3種培養液中，於27℃，轉速150 rpm下，搖瓶培養5天，並於第5天測量菌量；3種培養液係分別為3 wt%黃豆漿培養液、3 wt%燕麥漿培養液以及3 wt%粉頭培養液，；其成分包含3 wt%之黃豆漿(或3 wt%燕麥漿、或3 wt%粉頭培養液)、0.3 wt%酵母抽出物、0.1 wt%磷酸二氫鉀(KH₂ PO₄ )、0.1 wt%磷酸氫二鉀(K₂ HPO₄ )與剩餘百分比的水；黃豆漿之製備方法為將市售黃豆以果汁機打成漿，燕麥漿之製備方法為將市售燕麥以果汁機打成漿；粉頭之製備方法為將市售粉頭以果汁機打成漿；實驗結果請參見表一，以3 wt%黃豆漿培養液培養所得到的菌量最多。The KHY26 bacterial cell suspension with a concentration of 10 ⁹ cfu / mL was taken, 1 mL of the bacterial cell suspension was taken as an inoculation source, and 400 mL of three kinds of culture liquid were added to the flask culture at 27 ° C and a rotation speed of 150 rpm. 5 days, and the amount of bacteria was measured on the 5th day; 3 kinds of culture liquid systems are 3 wt% soybean milk culture liquid, 3 wt% oatmeal culture liquid and 3 wt% flour culture liquid, and the composition contains 3 wt% Soymilk (or 3 wt% oatmeal, or 3 wt% flour culture), 0.3 wt% yeast extract, 0.1 wt% potassium dihydrogen phosphate (KH ₂ PO ₄ ), 0.1 wt% dipotassium hydrogen phosphate ( K ₂ HPO ₄ ) and the remaining percentage of water; the preparation method of soybean milk is to make commercially available soybeans into a fruit juice machine, and the preparation method of oat milk is to make commercially available oats into a fruit juice machine; In order to pulp the commercially available powder head with a fruit juice machine; the experimental results are shown in Table 1. The amount of bacteria obtained from the 3 wt% soymilk culture solution was the largest.

表一 Table I

(2)(2) 不同酸鹼值對KHY26菌株生長的影響Effects of different pH values on the growth of KHY26 strain

以含有3 wt%黃豆漿之培養液為基底，調整其酸鹼值以測試酸鹼值對於KHY26菌株生長之影響，培養條件為27℃、轉速150 rpm、搖瓶培養5天；結果請參見表二：試驗中，以pH=7.5培養液所得到的菌量最高為3.5 x 107 cfu/mL。Based on a culture solution containing 3 wt% soybean milk, adjust its pH value to test the effect of pH value on the growth of KHY26 strain. The culture conditions are 27 ° C, 150 rpm, and shake flask culture for 5 days; please refer to the table for the results. 2: In the test, the maximum bacterial amount obtained with the culture solution at pH = 7.5 is 3.5 x 107 cfu / mL.

表二 Table II

(3)(3) 硫酸鎂(MgSO4)對於KHY26菌株生長的影響Effect of magnesium sulfate (MgSO4) on the growth of KHY26 strain

以含有3 wt%黃豆漿、pH=7.5之培養液為基底，調整硫酸鎂(MgSO₄ ·7H₂ O)濃度以測試硫酸鎂與KHY26生長之關係，培養條件為27℃、轉速150 rpm、搖瓶培養5天；結果請見表三，以0.05 wt%硫酸鎂之培養液所得到的菌量最高，為3.1 x 10⁷ cfu/mL。Based on a culture solution containing 3 wt% soybean milk and pH = 7.5, the concentration of magnesium sulfate (MgSO ₄ · 7H ₂ O) was adjusted to test the relationship between magnesium sulfate and KHY26 growth. The culture conditions were 27 ° C, 150 rpm, and shaking. The flask was cultured for 5 days; the results are shown in Table 3. The 0.05% by weight magnesium sulphate broth gave the highest bacteria amount, 3.1 x 10 ⁷ cfu / mL.

表三 Table three

(4)(4) 幾丁聚醣Chitin (Chitosan)(Chitosan) 對於KHY26菌株生長的影響Effect on the growth of KHY26 strain

以含有3 wt%黃豆漿、pH=7.5之培養液為基底，添加1~5 wt%幾丁聚醣(Chitosan)並測試對KHY26菌株生長之影響，培養條件為27℃、轉速150 rpm、搖瓶培養5天；結果請見表四，以1 wt%幾丁聚醣之培養液所得到的菌量最高，為6.0 x 10⁷ cfu/mL。Based on a culture solution containing 3 wt% soybean milk and pH = 7.5, add 1 to 5 wt% chitosan and test the effect on the growth of KHY26 strain. The culture conditions are 27 ° C, 150 rpm, and shake. The flask was cultured for 5 days; the results are shown in Table 4. The highest bacterial amount was obtained with a 1 wt% chitosan culture solution, which was 6.0 x 10 ⁷ cfu / mL.

表四 Table four

(( 三) KHY26菌株之固態發酵培養方式C) KHY26 strain solid state fermentation culture method

此固態發酵所使用之固態培養基成分包含燕麥粒、大麥片、玉米粉、稻殼、蚵殼粉與甲殼素，將原料磨成細粉後充分混合均勻，再加入一定比例範圍之純水，以獲得使用之固態發酵培養基。本發明之固態發酵培養基包含一固體部分與一液體部分，固體部分若以重量比100%計算，包含5~35 wt%燕麥粒、5~35 wt%大麥片、10~20 wt%玉米粉、5~30 wt%稻殼與5~15 wt%蚵殼粉；液體部分為上述固態部分總重量40~60 wt%的水。其中固體部分的較佳比例為30 wt%燕麥粒、30 wt%大麥片、15%玉米粉、20%稻殼與5%蚵殼粉，液體部分的較佳添加量為固態部分總重量60 wt%之水。The solid medium components used in this solid fermentation include oat grains, barley flakes, corn flour, rice husks, husk flours and chitin. The raw materials are ground into fine powders and mixed thoroughly, and then a certain proportion of pure water is added. The solid fermentation medium used was obtained. The solid fermentation medium of the present invention includes a solid portion and a liquid portion. If the solid portion is calculated by 100% by weight, it contains 5 ~ 35 wt% oat grain, 5 ~ 35 wt% barley flakes, 10 ~ 20 wt% corn flour, 5 ~ 30 wt% rice husk and 5 ~ 15 wt% husk powder; the liquid part is water with a total weight of 40 ~ 60 wt%. Among them, the preferred proportion of the solid portion is 30 wt% oatmeal, 30 wt% barley flakes, 15% corn flour, 20% rice husk and 5% husk flour, and the preferred addition amount of the liquid portion is the total weight of the solid portion of 60 wt. % Of water.

固態發酵培養步驟如下：將菌量濃度為10⁹ cfu/mL之KHY26菌體懸浮液，取1 mL菌體懸浮液作為接種源，以稻殼、燕麥粒、大麥片、玉米粉及蚵殼混合之固態培養基於27℃培養14天，以獲得固態發酵增量培養菌體及培養基之混合固型物。將混合固型物以粉碎機充分混拌及粉碎，以獲得固態發酵菌粉，作為KHY26粉劑；在菌粉原始菌量為1.4 x 10¹⁰ cfu/g之狀況下，若粉碎後以80目之濾網過濾，製備得的KHY26粉劑菌量較高，為1.2 x 10¹⁰ cfu/g，而以100目濾網過濾者所得之粉劑菌量較低，為9.4 x 10⁹ cfu/g。The solid fermentation culture steps are as follows: KHY26 bacterial cell suspension with a concentration of 10 ⁹ cfu / mL, 1 mL of the bacterial cell suspension is used as the inoculation source, and rice husk, oat grain, barley flakes, corn flour and husk are mixed The solid medium was cultured at 27 ° C for 14 days to obtain a mixed solid of the solid fermentation fermentation culture body and the medium. The mixed solid is thoroughly mixed and pulverized with a pulverizer to obtain a solid fermented bacterial powder as a KHY26 powder; under the condition that the original bacterial amount of the bacterial powder is 1.4 x 10 ¹⁰ cfu / g, if it is pulverized, it is The strain of the KHY26 powder prepared by filtering with a filter is 1.2 x 10 ¹⁰ cfu / g, and the powder of the powder obtained by filtering with a 100-mesh filter is low, 9.4 x 10 ⁹ cfu / g.

(( 四) KHY26液劑及粉劑之儲存安定性測試4) Storage stability test of KHY26 liquid and powder

(1) KHY26(1) KHY26 液劑儲存安定性測試Liquid storage stability test

將依前述液態發酵配方及條件，搖瓶培養5天之KHY26菌液液劑儲存於4℃、16℃與27℃，並定期觀察其菌量之變化；實驗結果請參見表五，儲存時間為2個月時，三種儲存溫度之KHY26的菌量並無顯著變化；儲存至3個月時，KHY26菌量開始下降，並以儲存溫度為27℃者最為明顯，菌量僅剩下9.5 x 10⁵ cfu/mL，且於儲存6個月後菌量即低於10³ cfu/mL；而儲存於4℃與16℃者，至儲存6個月後，菌量仍可維持在10⁷ cfu/mL以上。Store the KHY26 bacteria liquid solution for 5 days in shake flask culture according to the aforementioned liquid fermentation formula and conditions at 4 ° C, 16 ° C, and 27 ° C, and periodically observe the changes in the amount of bacteria; please refer to Table 5 for the experimental results. The storage time is At 2 months, the bacterial count of KHY26 at three storage temperatures did not change significantly. When stored to 3 months, the bacterial count of KHY26 began to decrease, and it was most obvious at the storage temperature of 27 ° C. The bacterial count was only 9.5 x 10 ⁵ cfu / mL, and the bacteria volume after storage for 6 months is less than 10 ³ cfu / mL; and those stored at 4 ° C and 16 ° C, the bacteria volume can still be maintained at 10 ⁷ cfu / Above mL.

表五 Table five

此外，KHY26菌液液劑亦可進一步以乾燥機進行乾燥，以製備成粉劑使用。其製備方式有兩種，第一種製備方式為：將前述液態發酵小型量產之KHY26菌液，以低溫噴霧乾燥機於噴霧出口溫度50℃下進行噴霧乾燥處理，再將乾燥後之粉末，加入不同載體(玉米粉、小麥粉、乳糖及麥芽糊精)混合攪拌，以製成粉劑。第二種製備方式為：將前述液態發酵小型量產之KHY26菌液，以冷凍乾燥機進行乾燥處理，其步驟依序為(1)冷凍樣品階段，目的為將樣品冷凍，進行時令樣品溫度在1小時內下降至-40℃；(2)第一階段乾燥，目的為將冷凍後之樣品內的冰晶階段性昇華，進行方式為抽真空至真空度為100 mT，並設定溫度於90分鐘內上升至-30℃，再於下一個90分鐘內上升至-20℃，接著於360分鐘內上升至-10℃，最後於360分鐘內上升至0℃；(3)第二階段乾燥，目的為使樣品升溫至常溫以使樣品更加乾燥，進行方式為破真空至真空度為1 mT，並設定溫度於240分鐘內上升至10℃，再於240分鐘內上升至20℃，最後於360分鐘內上升至25℃；(4)乾燥結束，維持上述的溫度及真空度至冷凍乾燥機關閉為止，以獲得冷凍乾燥後的粉末。接著再將冷凍乾燥後之粉末，加入不同載體(玉米粉、小麥粉、乳糖及麥芽糊精)混合攪拌，以製成粉劑。In addition, the KHY26 bacteria liquid solution can also be dried with a dryer to prepare a powder. There are two ways to prepare it. The first way is to spray-dry the KHY26 bacteria liquid produced by the aforementioned small-scale liquid fermentation with a low-temperature spray dryer at a spray outlet temperature of 50 ° C, and then dry the powder. Add different carriers (corn flour, wheat flour, lactose and maltodextrin) and stir to make a powder. The second preparation method is: drying the KHY26 bacteria liquid produced by the aforementioned liquid fermentation small-scale mass production with a freeze dryer, and the steps are (1) the freezing sample stage in order to freeze the sample and perform the seasonal sample temperature. Decrease to -40 ° C within 1 hour; (2) The first stage of drying, the purpose is to sublimate the ice crystals in the frozen sample in a stepwise manner. The method is to evacuate to a vacuum of 100 mT and set the temperature to 90 minutes. The temperature rises to -30 ° C, then rises to -20 ° C in the next 90 minutes, then rises to -10 ° C in 360 minutes, and finally rises to 0 ° C in 360 minutes; (3) the second stage of drying, the purpose In order to make the sample warm to normal temperature to make the sample more dry, the method is to break the vacuum to a vacuum degree of 1 mT, and set the temperature to rise to 10 ° C in 240 minutes, then to 20 ° C in 240 minutes, and finally in 360 minutes. The temperature rises to 25 ° C; (4) After the drying is completed, the above-mentioned temperature and vacuum are maintained until the freeze dryer is turned off to obtain a freeze-dried powder. Then, the freeze-dried powder is added with different carriers (corn flour, wheat flour, lactose and maltodextrin) and mixed to make a powder.

(2)(2) 粉劑製作與儲存安定性測試Powder production and storage stability test

將前述固態發酵物粉碎並以80目篩網過篩後，所製備之KHY26粉劑儲存於27℃以量測其儲存安定性，結果請見表六，於儲存4個月之後KHY26之菌量雖略有下降，但仍可維持在10¹⁰ cfu/g，其儲存安定性頗佳。The solid fermented material was pulverized and sieved through an 80-mesh sieve. The prepared KHY26 powder was stored at 27 ° C to measure its storage stability. The results are shown in Table VI. Although the bacteria volume of KHY26 was 4 months after storage, Slightly decreased, but still maintained at 10 ¹⁰ cfu / g, and its storage stability is quite good.

表六 Table six

(( 五)Fives) 鏈黴菌KHY26菌株之生理生化測試Physiological and Biochemical Test of Streptomyces KHY26 Strain

將KHY26菌株(以下簡稱KHY26)培養於特定之篩選培養基上，如SMA培養基(skim milk agar)、YSA培養基(yeast extract-soluble starch agar)、Tween-80 agar與MRA培養基(Mandel-Reese agar)、EYA培養基(egg-yolk agar)與PCA培養基(powdered chitin agar)，等菌落長出後觀察培養基之狀態，以判定KHY26所具備的酵素活性；請參見第二圖，於Tween-80 agar上，KHY26菌株周圍會產生白色沉澱環，即KHY26具有脂肪分解酶(lipase)活性；EYA培養基上，KHY26菌株周圍產生一透明環，表示其具有磷脂質分解酵素(phospholipase)的活性；YSA培養基上，KHY26菌株周圍具有大透明圓環，表示KHY26亦具有澱粉分解酵素(amylase)活性；生長於SMA培養基之KHY26菌落周圍會產生透明圓環，表示KHY26具有蛋白分解酵素(protease)活性；MRA培養基上，KHY26菌株周圍產生橘黃色環，表示KHY26具有纖維素分解酵素(cellulase)活性；PCA培養基上，KHY26菌落周圍生成一透明環，表示KHY26具有幾丁質分解酵素(chitinase)活性KHY26 strain (hereinafter referred to as KHY26) is cultured on a specific screening medium, such as SMA medium (skim milk agar), YSA medium (yeast extract-soluble starch agar), Tween-80 agar and MRA medium (Mandel-Reese agar), EYA medium (egg-yolk agar) and PCA medium (powdered chitin agar). After the colonies grow, observe the state of the medium to determine the enzyme activity of KHY26; see the second picture on Tween-80 agar, KHY26 A white precipitation ring will be generated around the strain, that is, KHY26 has a lipolytic enzyme (lipase) activity; on EYA medium, a transparent ring is generated around the KHY26 strain, indicating that it has phospholipase activity; on YSA medium, the KHY26 strain There is a large transparent circle around, indicating that KHY26 also has amylase activity; a transparent circle will be generated around KHY26 colonies grown in SMA medium, indicating that KHY26 has protease activity; on MRA medium, KHY26 strain An orange-yellow ring is generated around it, indicating that KHY26 has cellulase activity; on PCA medium, KHY26 colonies are generated around A transparent ring, indicating that KHY26 has chitinase activity

(( 六) 鏈黴菌KHY26菌株對農藥之耐受性6) Tolerance of Streptomyces KHY26 Strains to Pesticides

利用農藥平板法以及農藥液混合法，檢測鏈黴菌KHY26菌株(以下簡稱KHY26)對農藥之耐受性。農藥平板法是將菌液塗佈於含有不同農藥之PDA培養基平板，培養後觀測細菌的生長情形；農藥液混合法是將菌液與農藥液均勻混合30分鐘後，再塗佈於PDA培養基平板上，並觀察細菌生長。請參見表七，為KHY26對於各種農藥耐受性測試結果，「O」代表具有耐受性(菌株生長良好)、「X」代表無耐受性(菌株無法生長)、「△」代表低耐受性(菌株可生長但生長狀態不佳)；在兩種測試方法中，KHY26對於甲基多保淨、亞托敏、免賴得、達特安、第滅達安、納乃得、益達胺、芬殺松與第滅寧皆具有耐受性。Pesticide plate method and pesticide solution mixing method were used to test the resistance of Streptomyces KHY26 strain (hereinafter referred to as KHY26) to pesticides. The pesticide plate method is to coat the bacterial solution on a PDA medium plate containing different pesticides, and observe the growth of bacteria after cultivation; the pesticide solution mixing method is to uniformly mix the bacterial solution and the pesticide solution for 30 minutes, and then apply it on the PDA medium plate Up and observe bacterial growth. Please refer to Table 7 for the results of KHY26's tolerance test for various pesticides. "O" represents tolerance (good growth of the strain), "X" represents no tolerance (strain cannot grow), and "△" represents low tolerance Susceptibility (strains can grow but the growth status is not good); in two test methods, KHY26 is safe for methyl polychloride, subtomin, raid Dadamin, fenfluxone, and diphenirin are all tolerated.

表七 Table seven

實驗二、鏈黴菌KHY26之抗生活性範圍評估Experiment 2: Evaluation of the Anti-Life Range of Streptomyces KHY26

為了大範圍評估KHY26菌量(以下簡稱KHY26)的抗生活性，係將KHY26於PDA培養基平板之一側劃線培養1日之後，再於KHY26對側接種欲觀測之植物病原真菌，並於27℃進行對峙培養，以觀察該病原菌之生長情形。請參見第三圖，於對峙培養的試驗中，KHY26能有效抑制木瓜疫病菌、木瓜根腐病菌、蓮霧黑腐病菌、蓮霧果腐病菌、芒果炭疽病菌、芒果畸形病菌、番石榴黑星病菌以及番石榴瘡痂病菌的生長；第四圖係使用分離自臺灣各地的稻熱病菌(Magnaporthe grisea )分離株與KHY26菌株進行對峙培養，結果顯示KHY26亦能有效抑制不同地區分離之稻熱病菌菌株的生長。In order to evaluate the resistance of KHY26 bacteria (hereinafter referred to as KHY26) on a large scale, KHY26 was streaked on one side of a PDA culture plate for 1 day, and then the opposite side of KHY26 was inoculated with the phytopathogenic fungi to be observed, and the temperature was 27 ° C Face-off culture was performed to observe the growth of the pathogenic bacteria. Please refer to the third figure. KHY26 can effectively inhibit papaya disease, papaya root rot, lotus fog black rot, lotus fog fruit rot, mango anthracnose, mango deformity, guava black star The growth of pathogens and guava scabs; the fourth picture shows the use of Magnaporthe grisea isolates from various parts of Taiwan and KHY26 strains for confrontation culture. The results show that KHY26 can also effectively inhibit rice fever strains isolated in different regions. Growth.

實驗三、鏈黴菌KHY26菌株對於果實之保護功效Experiment 3: Protective effect of Streptomyces KHY26 strain on fruits

本部分之實驗結果皆使用費雪最小差異測試法(Fisher’s Least Significance Test, LSD)檢定，標記不同英文字者代表該二組別之間具有5%顯著差異，標記相同英文字者代表不具有顯著差異。The experimental results in this section are all tested using Fisher's Least Significance Test (LSD). Markers with different English characters represent a 5% significant difference between the two groups, and those with the same English characters represent no significant difference. difference.

(( 一One )) 防治芒果炭疽病Prevent and cure mango anthracnose

採收露天種植的芒果，並浸泡水(對照組)或KHY26菌液，再以農友貫行之方式裝箱並於室溫下放置7天，以觀察芒果炭疽病之發病情形；結果顯示對照組的罹病率為88.9%，罹病度為59.3%；而KHY26菌液組的罹病率為22.2%，罹病度為11.1%，顯著低於對照組。The mangoes grown in the open air were harvested, soaked in water (control group) or KHY26 bacterial solution, and then boxed in a manner consistent with farmers and left at room temperature for 7 days to observe the incidence of mango anthracnose; the results showed that the control The attack rate of the group was 88.9%, and the attack rate was 59.3%. The attack rate of the KHY26 bacterial solution group was 22.2%, and the attack rate was 11.1%, which was significantly lower than that of the control group.

芒果炭疽病罹病度係藉由罹病指數求得，罹病指數分為0~4級，0級代表無任何病徵，1級為果實炭疽病斑面積占1~5%，2級為果實炭疽病斑面積占6~10%，3級為果實炭疽病斑面積占11~25%，4級為果實炭疽病斑面積26%以上；罹病度之公式如下：The disease severity of mango anthracnose was obtained from the disease index. The disease index was graded 0 ~ 4. Grade 0 represents no symptoms. Grade 1 is the area of fruit anthracnose lesions, and grade 2 is the fruit anthracnose spot. The area occupies 6 ~ 10%, level 3 is the area of fruit anthracnose spots accounting for 11 ~ 25%, level 4 is the area of fruit anthracnose spots above 26%; the formula for the degree of disease is as follows:

罹病度(%)＝[(Σ罹病指數 x 該指數果實數) /(4 x 總果實數)] x 100%Disease degree (%) = [(Σ disease index x number of fruits of this index) / (4 x total number of fruits)] x 100%

罹病率(%)＝(罹病果實數 /總果實數) x 100%Disease rate (%) = (number of diseased fruits / total number of fruits) x 100%

(( 二) 防治棗炭疽病(Colletotrichum gloeosporioides )2) Prevention and Treatment of Colletotrichum gloeosporioides

於印度棗(Zizyphus mauritiana )果實上製造20個傷口後，先噴霧處理水、濃度為2 x 10⁷ cfu/mL之KHY26菌液或是44.2%克收欣溶液(2000倍稀釋)；24小時之後接種濃度為1 x 10⁶ spore/mL炭疽病孢子懸浮液，並於室溫下培養7天觀察罹病度。參見表八，對照組的罹病度為63.35%，克收欣之罹病度為50.0%，而KHY26菌液組的罹病度為13.3%，顯著低於對照組。再參見第五圖，對照組與克收欣組的印度棗的果實比面上明顯可見白色菌絲的生長，但是KHY26菌液組的印度棗果實表面無明顯的白色菌絲，果實外觀亦相對完整美觀。After making 20 wounds on Zizyphus mauritiana fruit, first spray-treated with water, KHY26 bacteria solution with a concentration of 2 x 10 ⁷ cfu / mL or 44.2% gram solution (2000-fold dilution); after 24 hours The anthracnose spore suspension was inoculated at a concentration of 1 x 10 ⁶ spore / mL and cultured at room temperature for 7 days to observe the disease severity. See Table 8. The disease severity of the control group was 63.35%, the disease severity of Ke Shouxin was 50.0%, and the disease severity of the KHY26 bacteria solution group was 13.3%, which was significantly lower than that of the control group. Referring again to the fifth figure, the growth of white hyphae on the fruit surface of the control group and the Keshouxin group is obvious, but there is no obvious white hypha on the surface of the fruit of the group of KHY26 bacteria, and the appearance of the fruit is also relatively Complete and beautiful.

棗炭疽病罹病度係藉由罹病指數求得，罹病指數分為0~5級，0級代表無任何病徵，1級為果實炭疽病斑面積占1~5%，2級為果實炭疽病斑面積占6~10%，3級為果實炭疽病斑面積占11~25%，4級為果實炭疽病斑面積26~50%，5級為果實炭疽病斑面積51%以上；罹病度之公式如下：The disease severity of jujube anthracnose is obtained from the disease index. The disease index is divided into grades 0 ~ 5. Grade 0 represents no symptoms. Grade 1 is the fruit anthracnose spot area which accounts for 1 ~ 5%. Grade 2 is the fruit anthracnose spot. The area is 6 ~ 10%, the level 3 is the fruit anthracnose spot area 11-25%, the level 4 is the fruit anthracnose spot area 26-50%, the level 5 is the fruit anthracnose spot area 51% or more; the formula of the disease degree as follows:

罹病度(%)＝[(Σ罹病指數 x 該指數果實數) /(5 x 總果實數)] x 100%Disease degree (%) = [(Σ disease index x number of fruits of this index) / (5 x total number of fruits)] x 100%

表八 Table eight

(( 三three )) 防治蓮霧黑腐病菌Prevention and treatment of black rot of lotus fog (Lasiodiplodia theobromae )( Lasiodiplodia theobromae )

於蓮霧果實上製造20處傷口後，立即噴灑濃度約1 x 10⁶ spore/mL之蓮霧黑腐病菌，5分鐘後再噴灑水(對照組)、濃度約2 x 10⁷ cfu/mL之KHY26菌液、KHY26培養濾液或10%保粒黴素甲(1000倍稀釋)，將蓮霧果實於室溫下培養7天後觀察。請參見第六圖，對照組的每顆蓮霧果實上皆生長大量白色菌絲，噴灑保粒黴素甲的蓮霧果實上亦觀察到菌絲生長；噴灑KHY26培養濾液之果實則有少量菌絲，噴灑KHY26菌液之蓮霧果實觀察到白色菌絲量又少於噴灑KHY26濾液者。Immediately after making 20 wounds on the lotus mist fruit, spray the lotus mist black rot fungus at a concentration of about 1 x 10 ⁶ spore / mL, and then spray water (control group) at a concentration of about 2 x 10 ⁷ cfu / mL after 5 minutes. KHY26 bacterial solution, KHY26 culture filtrate, or 10% troomycin a (1000-fold dilution), the lotus mist fruit was cultured at room temperature for 7 days and observed. See Figure 6. A large number of white hyphae were grown on each lotus mist fruit in the control group. Mycelial growth was also observed on the lotus mist fruit sprayed with fulomycin A; the fruit sprayed with KHY26 culture filtrate had a small amount of bacteria. Silk, sprayed with KHY26 bacterial liquid on the lotus mist fruit observed that the amount of white mycelium was less than those sprayed with KHY26 filtrate.

進一步測試施予防治物時機與蓮霧黑腐病菌的防治功效：同時處理組係於病原菌接種後5分鐘噴灑KHY26菌液或保粒黴素甲；預處理組則是先施予蓮霧果實KHY26菌液或保粒黴素甲，24小時後再於蓮霧上製造傷口並接種病原菌；接種病原菌後於室溫培養7天，並觀察菌絲的生長且記錄；參見第七圖，KHY26菌液組的罹病度皆低於保粒黴素甲組，且菌絲生長之情形也較輕微；另，不論是給予KHY26菌液或是保粒黴素甲，預處理組的菌絲生長情形皆低於同時處理組。Further test the timing of application of the control substance and the control effect of the black rot fungus of lotus fog: Simultaneously, the treatment group was sprayed with KHY26 bacterial solution or fulomycin A 5 minutes after the pathogen was inoculated; the pretreatment group was first given the lotus fog fruit KHY26 bacterial solution Or granulomycin A, make wounds on lotus mist and inoculate pathogenic bacteria after 24 hours; incubate for 7 days at room temperature after inoculating pathogenic bacteria, and observe the growth of hyphae and record; see figure 7, KHY26 bacterial solution group The degree of disease was lower than that of the mycotoxin A group, and the mycelial growth was mild. In addition, the mycelial growth of the pretreatment group was lower than that of the pretreatment group, regardless of the KHY26 bacterial solution or the mycotoxin A group.

蓮霧黑腐病菌罹病度係藉由罹病指數求得，罹病指數分為0~5級，0級代表無任何病徵，1級為果實黑腐病斑面積占1~5%，2級為果實黑腐病斑面積占6~10%，3級為果實黑腐病斑面積占11~20%，4級為果實炭疽斑面積21~35%，5級為果實黑腐病斑面積36~50%以上，6級為果實黑腐病斑面積51%以上；罹病度之公式如下：The disease degree of the black rot fungus of lotus mist is obtained by the disease index. The disease index is divided into grades 0 to 5. Grade 0 represents no disease symptoms. Grade 1 is the fruit with black rot spots occupying 1 to 5%. Grade 2 is the fruit. The area of black rot spots accounts for 6 ~ 10%, the area of black rot spots for grade 3 accounts for 11 ~ 20%, the area of black rot spots for fruits ranks 21 ~ 35%, and the area for black rot spots of fruits ranks 36 ~ 50 Above 6%, the area of fruit black rot spots is more than 51%. The formula of disease severity is as follows:

罹病度(%)＝[(Σ罹病指數 x 該指數果實數) /(6 x 總果實數)] x 100%Disease degree (%) = [(Σ disease index x number of fruits of this index) / (6 x total number of fruits)] x 100%

(( 四)four) 防治木瓜疫病Control of Papaya Disease

將採收後之木瓜浸泡於水(對照組)、它牌枯草桿菌、2000倍稀釋的62.5%賽普護汰寧藥劑，或是菌量濃度為2 x 10⁵ cfu/mL之KHY26稀釋菌液，浸泡1分鐘後陰乾，放於27℃下放置於封口袋內，8天後觀察木瓜病害的發生情形。請參見表九與第八圖，浸泡賽普護汰寧藥劑與KHY26菌液之木瓜的罹病率與罹病度皆顯著低於對照組以及處理它牌枯草桿菌處理組，且KHY26菌液組的罹病率與罹病度又低於賽普護汰寧藥劑組；參見第八圖，處理KHY26菌液的木瓜果皮較完完整美觀，並無觀察到病斑或是菌絲。Soak the harvested papaya in water (control group), its brand Bacillus subtilis, a 62.5% Saipu Hutongning agent diluted 2000 times, or a KHY26 diluted bacteria solution with a concentration of 2 x 10 ⁵ cfu / mL After soaking for 1 minute, it was dried in the shade and placed in a sealed bag at 27 ° C. After 8 days, the occurrence of papaya disease was observed. Please refer to Tables 9 and 8. The disease rate and disease severity of papaya soaked with Sepp Hu Ting Ning and KHY26 bacterial solution were significantly lower than those of the control group and the B. subtilis treatment group, and the KHY26 bacterial solution group The rate and disease severity were lower than those of Sepp Hu Tie Ning group; see Figure 8, the papaya peel treated with KHY26 bacterial solution was more complete and beautiful, and no disease spots or hyphae were observed.

木瓜疫病罹病度係藉由罹病指數求得，罹病指數分為0~4級，0級代表無任何病徵，1級為果實疫病病斑面積占1~5%，2級為果實疫病病斑面積占6~10%，3級為果實疫病病斑面積占11~20%，4級為果實疫病病斑面積21%以上；罹病度之公式如下：The degree of papaya disease is calculated from the disease index. The disease index is divided into grades 0 to 4. Grade 0 represents no symptoms. Grade 1 is the disease blight area of fruit blaming 1 ~ 5%. Grade 2 is the fruit blight area. It accounts for 6 ~ 10%, level 3 is the fruit blight area of 11-20%, and level 4 is the blight area of fruit is more than 21%. The formula for the degree of disease is as follows:

表九 Table nine

另，將木瓜浸泡於水、2 x 10⁷ cfu/mL之KHY26菌液，與2000倍稀釋的62.5%賽普護汰寧藥劑1分鐘後，取出陰乾並放置於紙箱，以電石催熟1天，再於室溫下放置5天，並觀察木瓜病害之發生情形。請參見表十，於木瓜疫病的防護上，KHY26菌液與賽普護汰寧的罹病率與罹病度皆為0%，明顯低於對照組；而於木瓜蒂腐病的防護上，KHY26菌液與賽普護汰寧的罹病率與罹病度皆亦低於對照組。In addition, after soaking papaya in water, 2 x 10 ⁷ cfu / mL KHY26 bacterial solution, and 62.5% Saipu Hu Ting Ning medicament diluted 2000 times for 1 minute, take out the dry and put it in a cardboard box, and ripen with calcium carbide for 1 day , And then left at room temperature for 5 days, and observe the occurrence of papaya disease. Please refer to Table 10. In terms of protection against papaya disease, the incidence and severity of KHY26 bacteria solution and cyprotinin are both 0%, which are significantly lower than those in the control group. In terms of protection against papaya rot, KHY26 bacteria The attack rate and disease severity of the liquid and Saipu Hutongning were also lower than those of the control group.

木瓜疫病罹病度係藉由罹病指數求得，罹病指數分為0~4級，0級代表無任何病徵，1級為果實疫病病斑面積占1~5%，2級為果實疫病病斑面積占6~10%，3級為果實疫病病斑面積占11~20%，4級為果實疫病病斑面積21%以上。The degree of papaya disease is calculated from the disease index. The disease index is divided into grades 0 to 4. Grade 0 represents no symptoms. Grade 1 is the disease blight area of fruit blaming 1 ~ 5%. Grade 2 is the fruit blight area. It accounts for 6 ~ 10%, level 3 is the fruit blight area of 11-20%, and level 4 is the blight area of fruit more than 21%.

木瓜蒂腐病罹病度係藉由罹病指數求得，罹病指數分為0~4級，0級代表無任何病徵，1級為果實蒂腐病病斑面積占1~5%，2級為果實蒂腐病病斑面積占6~10%，3級為果實蒂腐病病斑面積占11~20%，4級為果實蒂腐病病斑面積21%以上。上述二種疾病罹病度之公式如下：The disease severity of papaya rot is calculated from the disease index. The disease index is divided into grades 0 to 4. Grade 0 represents no symptoms. Grade 1 is the fruit pediculosis lesion area which accounts for 1 to 5%. Grade 2 is the fruit. The area of pedicle rot lesions accounts for 6 ~ 10%, the area of pedicle rot lesions of grade 3 accounts for 11 ~ 20%, and the area of pedicle rot lesions of grade 3 accounts for more than 21%. The formula for the above two diseases is as follows:

表十 Table ten

(( 五) 防治木瓜根腐病(Pythium aphanidermatum ) 5) Prevention and Treatment of Papaya Root Rot ( Pythium aphanidermatum)

將生長45天大的木瓜移植含有木瓜根腐病菌(Pythium aphanidermatum )的土壤中，土染的病菌量為10² spore/g-土壤後，每株分別澆灌總體積為10 mL之水(對照組)及稀釋5~500倍、原始濃度為6 x 10⁷ cfu/mL)之KHY26菌液菌液，並於室溫培養7天以觀察病害的發生。請參見表十一，澆灌KHY26菌液各稀釋倍數皆能明顯抑制罹病之植株，其罹病率亦顯著低於對照組。The 45-day-old papaya was transplanted into the soil containing Pythium aphanidermatum , and the amount of soil-stained bacteria was 10 ² spore / g-soil, and each plant was irrigated with a total volume of 10 mL of water (control group) ) and diluted 5 to 500 times the original concentration of 6 x 10 ⁷ cfu / mL) of bacteria KHY26 broth, and incubated at room temperature for 7 days to observe the occurrence of disease. Please refer to Table 11. The dilution of KHY26 bacterial solution can significantly inhibit diseased plants, and the disease rate is significantly lower than that of the control group.

罹病率(%)＝(罹病株數 /總株數) x 100%Disease rate (%) = (number of diseased plants / total number of plants) x 100%

表十一 Table eleven

另，將生長60天大之木瓜穴盤苗，連同栽培介質分別以100~1000倍稀釋、原始濃度為6 x 10⁷ cfu/mL之KHY26種子菌液進行浸根處理，再移植到含有菌量為10² spore/g-土壤之木瓜根腐病病菌之培養土內，於室溫培養12天。請參見表十二，以浸根方式處理之木瓜植株，其罹病率可降低至10%，所能達到的防治效果優於以澆灌方式給予KHY26菌液之植株；且以浸根方式處理木瓜植株，僅需要使用1000倍稀釋的菌液就可以達到極佳的防護效果。In addition, the 60-day-old papaya plug seedlings, together with the cultivation medium, were diluted 100-1000 times with KHY26 seed bacterial solution at an original concentration of 6 x 10 ⁷ cfu / mL, soaked in roots, and then transplanted to the bacterial content. In the culture soil of 10 ² spore / g-soil of Papaya root rot fungus, it was cultured at room temperature for 12 days. Please refer to Table 12. The disease rate of papaya plants treated by root soaking can be reduced to 10%, and the control effect can be better than that of plants given KHY26 bacterial solution by watering; and the papaya plants are treated by root soaking , Only need to use 1000 times diluted bacterial solution to achieve excellent protection.

表十二 Table 12

另，於美濃的木瓜園進行田間試驗，自木瓜穴盤苗移植至田間起，每2周澆灌一次KHY26菌液，連續澆灌6次後；無澆灌KHY26菌液之對照組於颱風後發生根腐病的機率為44.4%，而KHY26菌液澆灌組別發生根腐病的機率僅為22.2%。In addition, field experiments were carried out in the papaya garden in Mino. From the transplantation of papaya plug seedlings to the field, KHY26 bacterial solution was irrigated every 2 weeks, and after continuous irrigation for 6 times; the control group without irrigation of KHY26 bacterial solution had root rot after typhoon. The incidence of disease was 44.4%, while the incidence of root rot in the KHY26 bacterial solution irrigation group was only 22.2%.

(( 六)six) 防治瓜類根瘤線蟲(Meloidogyne spp.)Control of Meloidogyne spp.

將欲種植甜瓜之植穴，於種植前一日分別處理500 mL水(對照組)、500 mL之100倍稀釋無菌培養液、500 mL之100倍稀釋、原始濃度為2x10⁷ cfu/ml之KHY26菌液及於植穴旁開溝條並施以 7.2g的歐殺滅，毆殺滅之施予比例約為 60 kg/公頃；其中種植於處理KHY26菌液植穴之組別，其植株會再以100倍稀釋之KHY26菌液進行浸根處理，再定植；定植後每週再分別以水、100倍稀釋之KHY26菌液(原始濃度為2x10⁷ cfu/ml)或100倍稀釋之無菌培養液進行澆灌處理，本實驗共進行5次澆灌處理。請參見表十三，為各組別之植株根瘤數目及土壤線蟲數，KHY26組別能有效減少植株的根瘤數目，以及土壤中的線蟲數；再請參閱第九圖，處理KHY26菌液處理組別的根瘤數明顯低於對照組以及無菌培養液組。The melon planting points to be planted were treated with 500 mL of water (control group), 500 mL of 100-fold diluted sterile culture solution, 500 mL of 100-fold diluted KHY26 at the original concentration of 2x10 ⁷ cfu / ml on the day before planting. The bacterial solution and the ditch strip next to the planting hole were applied with 7.2g of European killing. The application rate for the killing was about 60 kg / ha; of which, the plants that were planted in the group that processed KHY26 then 100-fold diluted bacterial suspension KHY26 root dip treatment, and then planting; after planting and then were water, diluted 100-fold KHY26 bacteria (the original concentration of 2x10 ⁷ cfu / ml) per week, or 100-fold diluted culture sterile The solution was irrigated, and this experiment was performed a total of 5 times. Please refer to Table 13. For the number of plant nodules and soil nematodes in each group, the KHY26 group can effectively reduce the number of plant nodules and the number of nematodes in the soil. Please refer to Figure 9 for the KHY26 bacterial solution treatment group. The number of other nodules was significantly lower than that of the control group and the sterile culture medium group.

表十三 Table 13

(( 七)Seven) 以KHY26液劑防治茄科根瘤線蟲(Meloidogyne spp.)KHY26 Solution for Meloidogyne spp.

將番茄穴盤苗於移植至田間前，連同穴盤栽培介質以50倍稀釋、原始濃度為2x10⁷ cfu/ml之KHY26液劑進行浸根處理，再移植至田間植穴中，於移植後，每14天，每株分別澆灌處理500 mL水(對照組)、500 mL之100倍、200倍、400倍稀釋KHY26液劑(原始濃度為2x10⁷ cfu/ml)，本實驗共進行7次澆灌處理。請參見表十四，為各組別之土壤線蟲數，KHY26液劑各濃度處理組皆能有效減少土壤中的線蟲總數，以及減少根瘤線蟲及其他植食性線蟲數目。Before transplanting tomato plug seedlings into the field, they were immersed in a 50-fold diluted KHY26 solution with an original concentration of 2x10 ⁷ cfu / ml together with the plug cultivation medium, and then transplanted into the field planting points. After the transplantation, Every 14 days, each plant was irrigated with 500 mL of water (control group), 500 mL of 100-fold, 200-fold, and 400-fold diluted KHY26 solution (the original concentration was 2x10 ⁷ cfu / ml). A total of 7 irrigations were performed in this experiment. deal with. Please refer to Table XIV. For the number of soil nematodes in each group, the KHY26 solution at each concentration can effectively reduce the total number of nematodes in the soil, and reduce the number of nodular nematodes and other plant-feeding nematodes.

表十四 Table 14

(( 八)Eight) 以KHY26粉劑防治茄科根瘤線蟲(Meloidogyne spp.)KHY26 Powder for Controlling Meloidogyne spp.

將番茄穴盤苗於移植至田間前，將不同處理量(1 g、2 g或4g)、原始菌量為1x10⁹ cfu/g之KHY26粉劑施灑於植穴中土壤1次，再將番茄苗移植至田間植穴中，於移植後，每14天，每株分別施灑1 g、2 g或4 g之KHY26粉劑於植株莖基處，無任何施灑者為對照組，每14天施灑於植株莖基部周遭土壤1次，並於每次施用後澆水，本實驗共進行7次施灑處理。請參見表十五，為各組別之土壤線蟲數，KHY26粉劑各施用量處理組皆能有效減少土壤中的線蟲總數，以及減少根瘤線蟲及其他植食性線蟲數目。Before transplanting tomato plug seedlings to the field, KHY26 powder with different treatment amount (1 g, 2 g, or 4 g) and the original amount of bacteria was 1x10 ⁹ cfu / g was applied to the soil in the planting hole once, and then the tomato The seedlings were transplanted into the field planting points. After transplantation, each plant was sprayed with 1 g, 2 g, or 4 g of KHY26 powder at the base of the plant every 14 days. No sprayer was used as the control group, every 14 days. It was applied to the soil around the base of the stem of the plant once, and watered after each application. This experiment was performed 7 times in total. Please refer to Table 15. For the number of soil nematodes in each group, the treatment group of each application amount of KHY26 powder can effectively reduce the total number of nematodes in the soil, and reduce the number of nodular nematodes and other plant-feeding nematodes.

表十五 Table 15

由上述之實施說明可知，本發明與現有技術相較之下，本發明具有以下優點：As can be seen from the foregoing implementation description, compared with the prior art, the present invention has the following advantages:

1. 本發明之鏈黴菌KHY26菌株對於多種植物病原菌具有抗生活性，並對根瘤線蟲等線蟲具有抑制效果，且因不具溶血特性故其安全性亦高；KHY26菌液噴灑生長中之植物、將植株進行浸根處理、或噴灑於採收後之果實皆能有效降低蓮霧黑腐病、炭疽病、木瓜疫病、木瓜根腐病、甜瓜根瘤線蟲之感染，可應用於製備廣效性生物農藥。1. The Streptomyces KHY26 strain of the present invention is resistant to various plant pathogens, and has an inhibitory effect on nematodes such as Nematode nematodes, and has high safety because it does not have hemolytic properties; KHY26 bacterial solution is sprayed on growing plants and plants Root soaking treatment or spraying on the harvested fruits can effectively reduce the infection of lotus fog black rot, anthracnose, papaya disease, papaya root rot, and melon nodule nematode infection, and can be used to prepare a broad-spectrum biological pesticide.

2. 本發明之鏈黴菌KHY26菌株能有效避免蓮霧、木瓜、印度棗或芒果果皮產生黑斑或腐壞，除了可延長水果的保存以外亦能持水果外觀的美觀程度，提高其經濟價值。此外且KHY26能有效防治瓜類及茄科作物之根瘤線蟲，使瓜類及茄科作物植株生長良好。2. The Streptomyces KHY26 strain of the present invention can effectively prevent dark spots or spoilage of lotus mist, papaya, Indian jujube or mango peel. Besides prolonging the preservation of the fruit, it can also maintain the aesthetic appearance of the fruit and improve its economic value. In addition, KHY26 can effectively control the nodular nematodes of melons and solanaceous crops, and make the plants of melons and solanaceous crops grow well.

3. 本發明提供鏈黴菌KHY26菌株之製備方法包含液態發酵法與固態發酵法，皆能獲得較高濃度之菌量，且製備成的粉劑亦具有優秀的儲存安定性。3. The invention provides a method for preparing Streptomyces KHY26 strain including liquid fermentation method and solid state fermentation method, both of which can obtain a higher concentration of bacteria, and the prepared powder has excellent storage stability.

綜上所述，本發明鏈黴菌KHY26及其培養方法與用途，的確能藉由上述所揭露之實施例，達到所預期之使用功效，且本發明亦未曾公開於申請前，誠已完全符合專利法之規定與要求。爰依法提出發明專利之申請，懇請惠予審查，並賜准專利，則實感德便。To sum up, the streptomyces KHY26 of the present invention, its culture method and use can indeed achieve the expected use effect through the above-disclosed embodiments, and the present invention has not been disclosed before the application, and it has fully met the patent Regulations and requirements. I filed an application for an invention patent in accordance with the law, and I urge you to examine it and grant the patent.

惟，上述所揭之說明，僅為本發明之較佳實施例，非為限定本發明之保護範圍；其；大凡熟悉該項技藝之人士，其所依本發明之特徵範疇，所作之其它等效變化或修飾，皆應視為不脫離本發明之設計範疇。However, the description disclosed above is only a preferred embodiment of the present invention, and is not intended to limit the scope of protection of the present invention; it; anyone who is familiar with the technology, the scope of features according to the present invention, etc. Effect changes or modifications should be regarded as not departing from the design scope of the present invention.

無no

第一圖：鏈黴菌KHY26菌株於PDA培養基上之菌落型態圖。The first picture: the colony pattern of Streptomyces KHY26 strain on PDA medium.

第二圖：鏈黴菌KHY26菌株之脂質分解酵素(lipase)、磷脂酶分解酵素(phosphplipase)、澱粉分解酵素(amylase)、蛋白質分解酵素(protease)、纖維分解酵素(cellulase)及幾丁質分解酵素(chitinase)活性分析圖。Second picture: Lipase, phospholipase, amylase, protease, cellulase and chitinase of Streptomyces KHY26 strain (chitinase) activity analysis chart.

第三圖：鏈黴菌KHY26菌株對不同植物病原真菌之抗生活性分析圖。Third figure: Analysis of the resistance of Streptomyces KHY26 strain to different plant pathogenic fungi.

第四圖：鏈黴菌KHY26菌株對不同地區稻熱病菌之抗生活性分析圖。Figure 4: Analysis of the resistance of Streptomyces KHY26 strain to rice fever in different regions.

第五圖：鏈黴菌KHY26菌液防治印度棗炭疽病分析圖。Fifth graph: An analysis of the prevention and cure of Indian jujube anthracnose by the bacterial solution of Streptomyces KHY26.

第六圖：鏈黴菌KHY26菌液及濾液防治蓮霧黑腐病菌分析圖。Figure 6: An analysis of the control of Streptomyces KHY26 bacterial liquid and filtrate against the black rot of lotus fog.

第七圖：鏈黴菌KHY26菌液之施用時機對防治蓮霧黑腐病影響分析圖。Figure 7: Analysis of the effect of the timing of application of Streptomyces KHY26 bacterial solution on the prevention and treatment of lotus rot black rot.

第八圖：鏈黴菌KHY26菌液防治木瓜疫病分析圖。Figure 8: Analysis of the control of papaya blight by Streptomyces KHY26.

第九圖：鏈黴菌KHY26菌液防治甜瓜根瘤線蟲分析圖。The ninth picture: An analysis of the control of Streptomyces spp.

附件1：鏈黴菌KHY26學名鑑定鑑定報告。Attachment 1: Identification report of scientific name of Streptomyces KHY26.

國內寄存資訊Domestic hosting information

財團法人食品工業發展研究所Food Industry Development Institute

民國106年10月19號October 19, 106

寄存編號：BCRC 910799Registration Number: BCRC 910799

＜110＞行政院農業委員會高雄農業改良場＜120＞鏈黴菌KHY26及其培養方法與用途＜160＞ 2 ＜170＞ PatentIn version 3.5 ＜210＞ 1 ＜211＞ 1287 ＜212＞ DNA ＜213＞ Streptomyces misionesis ＜400＞ 1 cgtgacgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcag caatgctgat 60 ctgcgattac tagcgactcc gacttcatgg ggtcgagttg cagaccccaa tccgaactga 120 gaccggcttt ttgagattcg ctccacctca cggtatcgca gctcattgta ccggccattg 180 tagcacgtgt gcagcccaag acataagggg catgatgact tgacgtcgtc cccaccttcc 240 tccgagttga ccccggcggt ctcccgtgag tccccagcac cacaagggcc tgctggcaac 300 acgggacaag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 360 gacagccatg caccacctgt acaccgacca caaggggggc actatctcta atgctttccg 420 gtgtatgtca agccttggta aggttcttcg cgttgcgtcg aattaagcca catgctccgc 480 cgcttgtgcg ggcccccgtc aattcctttg agttttagcc ttgcggccgt actccccagg 540 cggggcactt aatgcgttag ctgcggcacg gacaacgtgg aatgttgccc acacctagtg 600 cccaccgttt acggcgtgga ctaccagggt atctaatcct gttcgctccc cacgctttcg 660 ctcctcagcg tcagtatcgg cccagagatc cgccttcgcc accggtgttc ctcctgatat 720 ctgcgcattt caccgctaca ccaggaattc cgatctcccc taccgaactc tagcctgccc 780 gtatcgagct gcagacccgg ggttaagccc cgggctttca caaccgacgc gacaagccgc 840 tacgagctct ttacgcccaa taattccgga caacgcttgc gccctacgta ttaccgcggc 900 tgctggcacg tagttagccg gcgcttcttc tgcaggtacc gtcactctcg cttcttccct 960 gctgaaagag gtttacaacc cgaaggccgt catccctcac gcggcgtcgc tgcatcaggc 1020 tttcgcccat tgtgcaatat tccccactgc tgcctcccgt aggagtctgg gccgtgtctc 1080 agtcccagtg tggccggtcg ccctctcagg ccggctaccc gtcgtcgcct tggtgagccg 1140 ttacctcacc aacaagctga taggccgcgg gctcatcctg caccgccgga gctttacacc 1200 atcaaggatg cccaagatgg tcatatccgg tattagaccc cgtttccagg gcttgtccca 1260 gagtgcaggg cagattgccc acgtgtt 1287 ＜210＞ 2 ＜211＞ 889 ＜212＞ DNA ＜213＞ Streptomyces misionesis ＜400＞ 2 tgcaggacac ttcgtatcgc ccggccttta tggctagtcc tacggccaag accaggttgg 60 agcgctccgc ctcaagccat ccgagggcat cgctctgcct cctgaagtaa cgagaaggcg 120 tggggctagg cggcaggtgg gaggatgcgg catgtgcacc gtggacgtaa taatccagga 180 gccgacccaa tgcaaggtcg cgctgatcct cgacacgaaa ctctccgcct agattatcgg 240 catataggcg aatcaagtcg tggagccgcc acctgcccca ggtttctccc ggttcgacga 300 ggtgcatcct cgccagatct tcaagcaatc gatcagcagc cgactcttcg attgcggcca 360 gttgcgccgc agccctgctc gatatatctg cccctggatt gattgggatt aggcgaaaga 420 ggcgggcctg atcttcggtg agcctccgat aggaaacatc aaatgcggcc gtgaccgttc 480 ttgtgccgcg ctcaagatgt ccaagtcgcg tgtgtgcctg agagagggct tgtttcattg 540 aagtcagtgg gcgaccgggg aaatctgcca ggagagcgcc gcagatctgc aaggctattg 600 ggaggtttcc gcagagccga gcgatttctt tcgcggcatc gaggtcctcc tccacccgga 660 tgtcgtcgac accgttagta ttcttcaata cgcttcgcaa gaggttgacc gatgctgctg 720 atggcaagac ctcaaggctg tggcggcggg cattgatgtc caagctgtcg cgcgaggtaa 780 tcagaaccgc gttagccgtg tcactcggca gcagtggctg tacctgatca atcgacgaca 840 cattgtcgat aattagaagt attcgacgct tatgctttgc gtacgctgc 889＜ 110 ＞ Kaohsiung Agricultural Improvement Field of the Administrative Committee of the Executive Yuan ＜ 120 ＞ Streptomyces KHY26 and its cultivation method and use ＜ 160 ＞ 2 ＜ 170 ＞ PatentIn version 3.5 ＜ 210 ＞ 1 ＜ 211 ＞ 1287 ＜ 212 ＞ DNA ＜ 213 ＞ Streptomyces misionesis <400> 1 cgtgacgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcag caatgctgat 60 ctgcgattac tagcgactcc gacttcatgg ggtcgagttg cagaccccaa tccgaactga 120 gaccggcttt ttgagattcg ctccacctca cggtatcgca gctcattgta ccggccattg 180 tagcacgtgt gcagcccaag acataagggg catgatgact tgacgtcgtc cccaccttcc 240 tccgagttga ccccggcggt ctcccgtgag tccccagcac cacaagggcc tgctggcaac 300 acgggacaag ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac 360 gacagccatg caccacctgt acaccgacca caaggggggc actatctcta atgctttccg 420 gtgtatgtca agccttggta aggttcttcg cgttgcgtcg aattaagcca catgctccgc 480 cgcttgtgcg ggcccccgtc aattcctttg agttttagtag ttgcggccgt actccccaggact 540 cgggg tgcggcacg gacaacgtgg aatgttgccc acacctagtg 600 cccaccgttt acggcgtgga ctaccagggt atctaatcct gttcgctccc cacgctttcg 660 ctcctcagcg tcagtatcgg cccagagatc cgccttcgcc accggtgttc ctcctgatat 720 ctgcgcattt caccgctaca ccaggaattc cgatctcccc taccgaactc tagcctgccc 780 gtatcgagct gcagacccgg ggttaagccc cgggctttca caaccgacgc gacaagccgc 840 tacgagctct ttacgcccaa taattccgga caacgcttgc gccctacgta ttaccgcggc 900 tgctggcacg tagttagccg gcgcttcttc tgcaggtacc gtcactctcg cttcttccct 960 gctgaaagag gtttacaacc cgaaggccgt catccctcac gcggcgtcgc tgcatcaggc 1020 tttcgcccat tgtgcaatat tccccactgc tgcctcccgt aggagtctgg gccgtgtctc 1080 agtcccagtg tggccggtcg ccctctcagg ccggctaccc gtcgtcgcct tattagaccc cgtttccagg gcttgtccca 1260 gagtgcaggg cagattgccc acgtgt tggtgagccg 1140 ttacctcacc aacaagctga taggccgcgg gctcatcctg caccgccgga gctttacacc 1200 atcaaggatg cccaagatgg tcatatccgg t 1287 <210> 2 <211> 889 <212> DNA <213> Streptomyces misionesis <400> 2 tgcaggacac ttcgtatcgc ccggccttta tggctagtcc tacggccaag accaggttgg 60 agcgctccgc ctcaagccat ccgagggcat cgctctgcct cctgaagtaa cgagaaggcg 120 tggggctagg cggcaggtgg gaggatgcgg catgtgcacc gtggacgtaa taatccagga 180 gccgacccaa tgcaaggtcg cgctgatcct cgacacgaaa ctctccgcct agattatcgg 240 catataggcg aatcaagtcg tggagccgcc acctgcccca ggtttctccc ggttcgacga 300 ggtgcatcct cgccagatct tcaagcaatc gatcagcagc cgactcttcg attgcggcca 360 gttgcgccgc agccctgctc gatatatctg cccctggatt gattgggatt aggcgaaaga 420 ggcgggcctg atcttcggtg agcctccgat aggaaacatc aaatgcggcc gtgaccgttc 480 ttgtgccgcg ctcaagatgt ccaagtcgcg tgtgtgcctg agagagggct tgtttcattg 540 aagtcagtgg gcgaccgggg aaatctgcca ggagagcgcc gcagatctgc aaggctattg 600 ggaggtt tcc gcagagccga gcgatttctt tcgcggcatc gaggtcctcc tccacccgga 660 tgtcgtcgac accgttagta ttcttcaata cgcttcgcaa gaggttgacc gatgctgctg 720 atggcaagac ctcaaggctg tggcggcggg cattgatgtc caagctgtcg cgcgaggtaa 780 tcagaaccgc gttagccgtg tcactcggca gcagtggctg tacctgatca atcgacgaca 840 cattgtcgat aattagaagt attcgacgct tatgctttgc gtacgctgc 889

Claims

一種鏈黴菌(Streptomyces misionesis )KHY26菌株，係寄存於財團法人食品工業發展研究所，寄存編號為BCRC 910799，其中KHY26菌株係對植物病原微生物具抗生活性，該植物病原菌係包含木瓜疫病菌(Phytophthora palmimora )、木瓜根腐病菌(Pythium aphanidermatum )、蓮霧黑腐病菌(Lasiodiplodia theobromae )、蓮霧果腐病菌(Pestalotiopsis euginae )、炭疽病菌(Colletotrichum gloeosporioides )、芒果畸形病菌(Fusarium mangiferae )、番石柳黑星病菌(Phyllosticta psidiicola )、番石榴瘡痂病菌(Pestalotiopsis psidii )、稻熱病菌(Magnaporthe grisea )以及瓜類根瘤線蟲(Meloidogyne spp.)其中至少之一。A Streptomyces misionesis strain KHY26 is deposited with the Food Industry Development Institute of the Consortium, and the deposit number is BCRC 910799. The KHY26 strain is resistant to plant pathogenic microorganisms. The plant pathogenic strain includes Phytophthora palmimora ), Papaya root rot fungus ( Pythium aphanidermatum ), lotus fog black rot fungus ( Lasiodiplodia theobromae ), lotus fog fruit rot fungus ( Pestalotiopsis euginae ), anthracnose fungus ( Colletotrichum gloeosporioides ), Fusarium mangiferae black At least one of Phyllosticta psidiicola , Pestalotiopsis psidii , Magnaporthe grisea , and Meloidogyne spp.

一種培養高濃度鏈黴菌(Streptomyces misionesis )KHY26菌株之培養方法，包含：將鏈黴菌KHY26菌株接種於一培養基，並於25~35℃培養一特定時間，以獲得含有鏈黴菌KHY26之發酵產物。A method of culturing a strain KHY26 method of high concentration Streptomyces (Streptomyces misionesis) culture, comprising: KHY26 Streptomyces strain was inoculated in a culture medium, and incubated for a certain time to 25 ~ 35 ℃, to obtain a fermented product containing the Streptomyces KHY26.

如申請專利範圍第2項所述之培養方法，其中該培養基係為液態培養基，包含1~10 wt%黃豆漿、0.1~1 wt%酵母抽出物、0.05~0.5 wt%磷酸二氫鉀(KH₂ PO₄ )、0.05~0.5 wt%磷酸氫二鉀(K₂ HPO₄ )、0.01~0.1 wt%硫酸鎂(MgSO₄ ·7H₂ O))、0.05~0.5 wt%幾丁聚醣(Chitoson)與剩餘百分比之水，且該特定時間為3~5天，並將該鏈黴菌KHY26菌株以轉速100~300 rpm、溶氧量0.5~10 ppm之條件培養3~8日。The culture method according to item 2 of the scope of the patent application, wherein the medium is a liquid medium, which contains 1 to 10 wt% soybean milk, 0.1 to 1 wt% yeast extract, and 0.05 to 0.5 wt% potassium dihydrogen phosphate (KH _{_{2 PO 4), 0.05 ~ 0.5}} wt% dipotassium hydrogen phosphate _{_{(K 2 HPO 4), 0.01}} ~ 0.1 wt% of magnesium sulfate _{_{(MgSO 4 · 7H 2 O)}} ), 0.05 ~ 0.5 wt% chitosan (Chitoson) And the remaining percentage of water, and the specific time is 3 to 5 days, and the Streptomyces KHY26 strain is cultured at a speed of 100 to 300 rpm and a dissolved oxygen amount of 0.5 to 10 ppm for 3 to 8 days.

如申請專利範圍第2項所述之培養方法，其中該培養基係為固態培養基，包含一固體部分與一液體部分，該固體部分以重量比100%計算包含5~35 wt%燕麥粒、5~35 wt%大麥片、10~20 wt%玉米粉、5~30 wt%稻殼與5~15 wt%蚵殼粉，以及該液體部分為該固體部分總重量之40~60 wt%的水，且該特定時間為10~18天。The cultivation method according to item 2 of the scope of patent application, wherein the culture medium is a solid medium and includes a solid portion and a liquid portion, and the solid portion comprises 5 to 35 wt% oat grains, 35 wt% barley flakes, 10-20 wt% corn flour, 5-30 wt% rice husk and 5-15 wt% husk flour, and the liquid part is 40-60 wt% water of the total weight of the solid part, And the specific time is 10-18 days.

一種包含鏈黴菌(Streptomyces misionesis )KHY26菌株之生物農藥，係以一有效劑量之KHY26菌株施予一所需植物，以抑制植物病原微生物之入侵、生長及病徵表現，其中該植物病原菌係包含木瓜疫病菌(Phytophthora palmimora )、木瓜根腐病菌(Pythium aphanidermatum )、蓮霧黑腐病菌(Lasiodiplodia theobromae )、蓮霧果腐病菌(Pestalotiopsis euginae )、炭疽病菌(Colletotrichum gloeosporioides )、芒果畸形病菌(Fusarium mangiferae )、番石柳黑星病菌(Phyllosticta psidiicola )、番石榴瘡痂病菌(Pestalotiopsis psidii )、稻熱病菌(Magnaporthe grisea )以及根瘤線蟲(Meloidogyne spp.)其中至少之一。A biopesticide containing Streptomyces misionesis strain KHY26, which is applied to a desired plant with an effective dose of KHY26 strain to inhibit the invasion, growth, and symptoms of plant pathogenic microorganisms, wherein the plant pathogenic strain includes papaya disease fungus (Phytophthora palmimora), papaya root rot (Pythium aphanidermatum) bacteria, wax black rot pathogen (Lasiodiplodia theobromae), wax fruit rot pathogen (Pestalotiopsis euginae), anthracnose (Colletotrichum gloeosporioides), mango malformation pathogen (Fusarium mangiferae), At least one of Phyllosticta psidiicola , Pestalotiopsis psidii , Magnaporthe grisea , and Meloidogyne spp.

如申請專利範圍第5項所述之生物農藥，該生物農藥係為液態發酵所製成之液劑或粉劑，該液劑之有效劑量係為濃度1 x 10⁵ ~1 x 10¹⁰ cfu/mL之KHY26菌株，該粉劑之有效劑量係為濃度1 x 10⁷ ~1 x 10¹⁰ cfu/g之KHY26菌株，且該粉劑係包含鏈黴菌KHY26菌粉與麥芽糊精。According to the biological pesticide described in item 5 of the patent application scope, the biological pesticide is a liquid or powder made by liquid fermentation, and the effective dose of the liquid is 1 x 10 ⁵ ~ 1 x 10 ¹⁰ cfu / mL For KHY26 strain, the effective dose of the powder is KHY26 strain with a concentration of 1 x 10 ⁷ to 1 x 10 ¹⁰ cfu / g, and the powder contains Streptomyces KHY26 powder and maltodextrin.

如申請專利範圍第5項所述之生物農藥，其中該生物農藥係為固態發酵所製成之粉劑，該粉劑之有效劑量係為濃度1 x 10⁸ ~1 x 10¹¹ cfu/g之KHY26菌株。The biological pesticide according to item 5 of the scope of the patent application, wherein the biological pesticide is a powder made by solid state fermentation, and the effective dose of the powder is a KHY26 strain with a concentration of 1 x 10 ⁸ to 1 x 10 ¹¹ cfu / g .

一種鏈黴菌(Streptomyces misionesis )KHY26菌株用於製備生物農藥之用途，係以一有效劑量之鏈黴菌KHY26菌株施予一所需植物以防治植物病原微生物。A strain of Streptomyces misionesis KHY26 is used to prepare biological pesticides, which is to apply an effective dose of Streptomyces KHY26 strain to a desired plant to control plant pathogenic microorganisms.

如申請專利範圍第8項所述之用途，其中該生物農藥為KHY26菌株液態發酵所製成之液劑或粉劑，該液劑之有效劑量係為濃度1 x 10⁶ ~1 x 10¹⁰ cfu/mL之KHY26菌株，該粉劑之有效劑量係為濃度1 x 10⁷ ~1 x 10¹⁰ cfu/g之KHY26菌株，且該粉劑係包含鏈黴菌KHY26菌粉與麥芽糊精。The use as described in item 8 of the scope of patent application, wherein the biological pesticide is a liquid or powder made by liquid fermentation of KHY26 strain, and the effective dose of the liquid is 1 x 10 ⁶ ~ 1 x 10 ¹⁰ cfu / The effective dose of the KHY26 strain of mL is the KHY26 strain with a concentration of 1 x 10 ⁷ to 1 x 10 ¹⁰ cfu / g, and the powder contains Streptomyces KHY26 powder and maltodextrin.

如申請專利範圍第8項所述之用途，其中該生物農藥為KHY26菌株固態發酵所製成之粉劑，該粉劑之有效劑量係為濃度1 x 10⁸ ~1 x 10¹¹ cfu/g之KHY26菌株。The use as described in item 8 of the scope of patent application, wherein the biological pesticide is a powder made by solid fermentation of the KHY26 strain, and the effective dose of the powder is a KHY26 strain with a concentration of 1 x 10 ⁸ to 1 x 10 ¹¹ cfu / g .