Title

利用微生物與土壤添加物防治芥菜黃葉病

Translated Titles

Application of microorganism and soil amendment for controlling mustard Fusarium wilt

Authors

陳薏安

Key Words

芥菜 ; Fusarium oxysporum f. sp. conglutinans ; 芥菜黃葉病 ; 生物防治 ; Brevibacillus brevis ; 土壤添加物 ; 蝦蟹殼粉 ; mustard ; Fusarium oxysporum f. sp. conglutinans ; mustard Fusarium wilt ; biological control ; Brevibacillus brevis ; soil amendment ; shrimp and crab shell powder

PublicationName

中興大學植物病理學系所學位論文

Volume or Term/Year and Month of Publication

2016年

Academic Degree Category

碩士

Advisor

黃振文

Content Language

繁體中文

Chinese Abstract

芥菜黃葉病係由Fusarium oxysporum Schlecht. f. sp. conglutinans (Wollenw.) Snyder & H. N. Hansen所引起,可使芥菜植株矮化,葉片偏上生長、黃化及維管束褐變,嚴重時呈現萎凋,進而死亡,致使產量下降,造成農產經濟損失。目前用以防治黃葉病的策略有抗病品種、輪作、施用化學藥劑及生物防治等。近年來隨著社會大眾關心食安的議題,發展友善環境之防治方法確有其必要性。現今已有許多研究顯示生物防治菌具有效防治黃葉病的潛力,例如利用拮抗菌Trichoderma harzianum, Pseudomonas fluoresens及Bacillus subtilis皆曾被報導可有效防治作物黃葉病。施用土壤添加物是一種間接性的生物防治,其可藉由改善土壤物理及化學特性,進而改變土壤中的微生物相,達到降低作物土媒病原菌的功效。本研究之主要目的在於篩選生物防治菌,並配合土壤添加物,藉以研發有效防治芥菜黃葉病的方法。針對芥菜黃葉病菌Focn-05與Focn-38菌株之生理特性試驗結果,顯示病原菌菌絲於28℃生長最快速;厚膜孢子發芽率則於28℃最高。將芥菜種植於2×104 cfu/g soil 之病土中,兩週後可觀察到49-85%之罹病度。自台灣中南部芥菜田土分離68株芥菜黃葉病菌的拮抗微生物菌株,並於Nutrient agar平板進行對峙測試,發現其中有BG06、BG09、BG16、GL9-1、TKM01、TKM02、TKM03、YBJ13、YBJ15及YBJ19等10株菌株拮抗病原菌的能力最佳,進一步針對它們抑制病原菌分生孢子發芽百分率及是否促進植株生長進行測試,發現GL9-1菌株在Nutrient broth生長之發酵液可抑制87% Focn-05與81% Focn-38的分生孢子發芽率。此外,TKM03菌株可促進芥菜植株根系生長率達31.27%,至於YBJ13菌株則可提高20.3%的植株鮮重。將芥菜種子分別浸泡於YBJ13、TKM03及GL9-1三株拮抗細菌懸浮液後,播種於病菌土(2×104 cfu/g soil)中,經過兩週,GL9-1及YBJ13菌株可分別降低芥菜黃葉病罹病度達18.9-25.6%及10-20%。進一步,利用16S rDNA及16S-23S ITS序列分析鑑定三拮抗菌株,結果TKM03與YBJ13菌株鑑定為Bacillus amyloliquefaciens;GL9-1菌株鑑定為Brevibasillus brevis。評估土壤添加物對於植株生育之影響及抑制病害的試驗,發現蝦蟹殼粉可以顯著增加芥菜的株高及鮮重;同時蝦蟹殼粉、魚粉及牛血粉則分別可顯著降低芥菜黃葉病的罹病度。此外施用蝦蟹殼粉的處理亦可顯著降低病原菌的存活,其中將蝦蟹殼粉搭配B. brevis GL9-1細菌懸浮液更可顯著降低病原菌的存活率。此外,將蝦蟹殼粉與B. brevis GL9-1菌株混合後添加於病土中可使罹病度降低26.7-30.0%,證明可有效的防治芥菜黃葉病的發生。其中GL9-1菌株改以黃豆粉培養基發酵後再配合蝦蟹殼粉施用時,則防治效果更佳。

English Abstract

Fusarium oxysporum f. sp. conglutinans, the causal agent of mustard Fusarium wilt. The pathogen causes losses in the yield and quality of many economically cruciferous crops during warm growing seasons in the world. Current strategies for controlling mustard Fusarium wilt include resistant cultivars, crop rotation, soil fungicides or soil fumigants and use of biological agents. Due to safety and environmental concerns, developing a friendly control measure is needed. In past decade, several microorganisms including Trichoderma harzianum, Pseudomonas fluoresens and Bacillus subtilis have been reported to be effective in reducing crop disease severity caused by Fusarium wilt. In general, soil amendment could change soil physical and chemical properties and increase microbial activity, and control crop diseases. The purpose of this study is to use microbial agents and soil amendments for controlling Fusarium wilt of mustard. In this study, F. oxysporum f. sp. conglutinans isolates Focn-05 and Focn-38 grew well at 28℃ and caused 49-85% disease severity in infested soil (2×104 cfu/g soil). In total, 68 isolates of antagonistic bacteria were isolated from mustard seeds and soils where mustard plants were planted. Ten bacterial isolates (BG06, BG09, BG16, GL9-1, TKM01, TKM02, TKM03, YBJ13, YBJ15 and YBJ19) significantly inhibited mycelial growth of both Fusarium isolates tested. Two bacterial isolates, designated TKM03 and YBJ13 promoted plant growth while coated on mustard seeds, and isolate GL9-1displayed strong inhibitory effects on spore germination of the pathogen. According to the result of the sequences of partial 16S rDNA and 16S-23S internal transcribed spacer, TKM03 and YBJ13 were identified as Bacillus amyloliquefaciens and GL9-1was identified as Brevibacillus brevis. Mustard seeds were individually treated with TKM03, YBJ13 or GL9-1cell suspensions (108 cfu/ml) and sown in Fusarium infested soil. Results indicated that YBJ13 and GL9-1 could significantly reduce disease severity of mustard seedlings. Six soil amendments were evaluated for the efficacy on promoting mustard plant growth and controlling the disease, the result showed shrimp and crab shell powder was more effective in promoting the shoot length and plant weight of mustard plants. Moreover, shrimp and crab shell powder, fish meal or blood meal were effective in reducing the disease severity of mustard Fusarium wilt caused by F. oxysporum f. sp. conglutinans isolates Focn-05 and Focn-38. Furthermore, combination of shrimp and crab shell powder and Brevibacillus brevis GL9-1 could obviously reduce the survival of the pathogen in the soil. The combination was amended into the infested soil, and able to reduce 26.7-30.0 % of disease severity. In addition, the combination of shrimp and crab shell powder and B. brevis GL9-1 fermented liquid cultured in soybean meal broth was added into the infested soil. The results indicated that GL9-1 fermented liquid could markedly improve the efficacy of shrimp and crab shell powder for controlling mustard Fusarium wilt.

Topic Category 農業暨自然資源學院 > 植物病理學系所
生物農學 > 植物學
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