應用螢光假單胞菌Pseudomonas putida YLFP14防治

Abstract

由對Xanthomonas屬數種病原細菌具拮抗能力之葉表螢光假單胞菌菌株中，選取具抗銅性之Pseudomonas putida YLFP14菌株用於防治由Xanthomonas axonopodis pv. vesicatoria (Xav) 所引起的甜椒細菌性斑點病。P. putida YLFP14在含糖蜜、酵母粉及黃豆粉之MYS基質中，於30℃醱酵槽內經30小時培養後，其菌量可達到2×109 cfu/ml，且於4℃下保存10個月後，菌量仍可維持約108 cfu/ml以上並具防治效果。500倍稀釋之MYS-YLFP14為有效降低甜椒細菌性斑點病發生之最佳稀釋倍數，並可有效降低包括：翠美星、女王星、Early Calwonder及PBC137等不同品種甜椒細菌性斑點病之發生。處理MYS-YLFP14後72小時，再接種甜椒細菌性斑點病菌，或先接種病原菌24小時內再處理MYS-YLFP14，可明顯降低甜椒細菌性斑點病的發生。MYS-YLFP14可降低由抗銅及不抗銅之甜椒細菌性斑點病菌菌株所引起之甜椒細菌性斑點病，且MYS-YLFP14亦可明顯降低田間抗銅及不抗銅菌株所引起之甜椒細菌性斑點病的發生，與低量銅劑混合施用時效果更為穩定。研究中利用跳躍子Tn5突變P. putida YLFP14，獲得單一跳躍子插入所形成之抗生能力缺失突變菌株A378及鉗鐵物質缺失突變菌株F2，所獲得之突變菌株之生長曲線及在甜椒葉片上之存活能力與野生菌株並無明顯差異。突變菌株A378在PDA平板上對所測試之植物病原細菌菌株失去抑制能力，但突變株菌F2在PDA平板上對所測試之植物病原細菌菌株的抑制能力與YLFP14野生菌株相同。另外，A378菌株對甜椒葉片上Xav抑制能力明顯降低。而突變菌株A378對甜椒細菌性斑點病仍具防病能力，然其防病效果較野生菌株為低，顯示YLFP14產生之抗生物質在防治甜椒細菌性斑點病上佔重要角色；而突變菌株F2對甜椒細菌性斑點病之防治效果與YLFP14野生菌株並無明顯差異。YLFP14所產生之抗生物質經初步測試結果得知，此物質為熱穩定性、低分子量且於酸性環境始具抗生活性。YLFP14於PDA上始有拮抗Xav的能力，且添加葡萄糖於NA培養基上能增加其拮抗能力，以添加0.1~0.2%為最佳，當培養基pH值為6或9時及培養環境溫度於24℃時亦有較好之拮抗能力。利用平板菌量計數方式從葉表回收螢光假單胞菌發現，在高濕的環境有利於菌量的維持，低濕的環境24小時後便無法從葉片上分離到螢光假單胞菌。在本研究中另利用具表現螢光蛋白能力之載體pBREGFPexp以電轉型法導入螢光假單胞菌P. putida YLFP14及茄科細菌性斑點病菌X. axonopodis pv. vesicatoria XVT40，獲得能穩定表現螢光蛋白之轉型菌株YLFP14EG及XVT40EG，且轉型菌株之生長曲線與生理生化測試皆與野生型菌株相同。以掃描式電子顯微鏡可觀察到螢光假單胞菌P. putida YLFP14在高濕及低濕環境皆可存活於甜椒葉表上，而以雷射共軛焦顯微鏡觀察轉型菌株YLFP14EG發現，這些存在於葉表之螢光假單胞菌是具有活性的；混合YLFP14後使得XVT40EG菌體不易增殖而呈現較散佈的情形。

Pseudomonas putida YLFP14 isolated from the leaf surface of tomato inhibits the growth of several xanthomonads and is copper resistance. This strain has been shown to be a potential biocontrol agent for bacterial spot of sweet pepper caused by Xanthomonas axonopodis pv. vesicatoria (Xav). This study aimed to evaluate the efficacy of this strain for controlling the bacterial spot on leaves of sweet pepper in growth chamber and field trials, to study the role of antibiosis in the disease control and to study the colonization and interaction of this strain and Xav on the leaf surface of sweet pepper. A fermented liquid product, named MYS-YLFP14, prepared by growing P. putida YLFP14 in a fermentor using molasses, yeast extract and soybean powder (MYS) as substrates at 30℃ for 30 hrs was used in the disease control experiments. In growth chamber tests, treatment of intact leaves by dipping in mixture of MYS-YLFP14 (500-fold dilution) and Xav reduced significantly the disease severity. The disease was also suppressed by MYS-YLFP14 when applied on leaves 72 hrs before or within 24 hrs after inoculation with Xav. MYS-YLFP14 was effective for controlling the disease caused by copper-resistant and –sensitive strains of Xav. The freshly prepared MYS-YLFP14 contained about 2×109 cfu/ml of YLFP14, and after 10 months storage at 4℃, it still contained a high number of YLFP14 (about 108 cfu/ml) and remained effective in the disease control. In the field trials conducted in summer and winter seasons of two consecutive years also demonstrated the effectiveness of MYS-YLFP14 on the control of the disease. MYS-YLFP14 supplemented with low dosage of cupric hydroxide was more effective than MYS-YLFP14 alone. To study the role of antibiosis and siderophores in the disease control, two Tn5-induced mutants of YLFP14, A378 defective in antagonistic activity in vitro and F2 defective in producing siderophores, were isolated. The growth curves in nutrient broth and colonization ability on leaves of the two mutants were not different from their wild type strain. Inoculation tests revealed that both mutants still reduced the disease severity, but when applied at lower concentration (107 cfu/ml), the control efficiency of mutant A378 was significantly decreased as compared with the wild type and mutant F2. The ability for suppression of Xav population levels on the leaves was also less by A378 than by its wild type. The results suggest that the siderophore production may not be involved and that antibiotic mechanism of disease control by YLFP14. The crude extract prepared from the PDA culture of YLFP14 by extracting with acetone was heat stable and low molecular weight, and expressed the inhibitory activity against Xav at an acidic environment. The colonization study by the plating method showed that the population of YLFP14 increased gradually on the leaves at high relative humidity (100%) in a 5-day test period, but decreased rapidly at low relative humidity (70~85%). The increase in population levels of a Xav strain on the leaves was less in the presence of YLFP14. To visualize the bacterial cells on the leaf surface, YLFP14 and Xav XVT40 were marked by a green fluorescent protein (GFP). GFP-expressing vector pBREGFPexp was introduced into YLFP14 and XVT40 by electroporation, resulting in derivatives YLFP14EG and XVT40EG, respectively. These derivatives expressed egfp stably in vitro and their growth and carbon sources utilization were the same as their wild types. The Confocal laser scanning microscopic observation revealed that gfp in the marked cells was active. An examination of the leaves 24hrs after spraying showed that cells of YLFP14EG were mostly dispersed, but a few of large cell aggregates were also observed on leaves under high relative humidity, whereas, the cells were mostly in aggregates and the aggregates were small on leaves under relative humidity. When the leaves were sprayed with cells of XVT40EG and maintained at the high relative humidity condition, many large aggregates consisting of numerous cells of XVT40EG were observed, but when the leaves were sprayed with the mixture of XVT40EG and YLFP14, the cells of XVT40EG were fewer in number and were mostly dispersed.