以高頻微波消除甘藍種子之黑腐病菌之研究

Study on eliminating Xanthomonas campestris pv. campestris from cabbage seeds by high frequency microwave

廖證翔(Cheng-Hsiang Liao)

指導教授 ： 張雅君

共同指導教授 ： 柯俊成(Chiun-Cheng Ko)

國立臺灣大學/生物資源暨農學院/植物醫學碩士學位學程/碩士(2019年)

https://doi.org/10.6342/NTU201903663

摘要

十字花科蔬菜黑腐病是由黑腐病菌 (Xanthomonas campestris pv. campestris，簡稱XCC) 所引起的世界性嚴重病害，在臺灣地區影響甘藍菜的產量甚鉅，而防治上最有效的方法便是使用無帶菌的健康種子，減少初次接種源，以降低田間發病率。目前雖已經有許多種子消毒方法被開發出來，但多具有無法完全殺菌、降低種子發芽率或處理時間太長等缺點。因此本研究欲利用臺大物理系所開發之24 GHz高頻微波系統，建立一套快速且能有效消除十字花科蔬菜種子黑腐病菌之技術，期望能提供種子生產業者全新的種子處理選項。首先以300 W高頻微波照射 XCC 1-1菌株之1 ml懸浮液，結果顯示8.91秒便能將XCC 1-1完全消除，說明了以高頻微波進行種子消毒的可能性。於是將甘藍種子浸泡於XCC 1-1懸浮液，風乾1天後作為人工接菌種子，平均每粒帶菌種子約有6萬CFU (colony forming unit) 的菌量。當直接以人工接菌的乾種子進行高頻微波的照射55秒，種子平均溫度達90℃，雖可以降低種子帶菌量至52%，但發芽率亦下降為25%。為提升高頻微波的殺菌效果，嘗試將種子進行噴霧處理後再照射高頻微波，結果顯示以該方法將種子加熱至平均溫度94℃時，能降低種子帶菌量至24%，同時發芽率提高為58%，較直接以乾種子照射高頻微波效果更佳，顯示水分可能為提升殺菌效果的關鍵。為了確認水在殺菌過程中的角色，進一步比較乾燥 (dry)、噴霧 (misting) 及浸水 [包含種子浸水於倒立離心管 (water-soaking in inverted tube) 及種子浸水 (water-soaking) 於正立離心管兩種處理] 等四種種子處理，經200 W高頻微波照射後，種子溫度達80℃左右之細菌族群量及發芽率。結果顯示種子表面所覆蓋的水分越多，殺菌效果越好，在兩種種子浸水的處理中，皆能將XCC 1-1從種子上完全消除；而在發芽率試驗中，只有種子浸水 (water-soaking) 的處理在高頻微波照射後，發芽率仍可維持100%。於是種子浸水處理配合高頻微波成為最佳種子消毒方式。為進一步探討種子浸水 (water-soaking) 後照射高頻微波，種子達80℃高溫下仍然得以存活的關鍵，於是藉由調控高頻微波功率和照射時間，將甘藍種子加熱至不同溫度，以找出XCC 1-1死亡而種子得以存活的溫度區間。由實驗結果發現以600 W照射0.6~1秒的快速加熱下，甘藍種子最高存活溫度為91℃，同時種子與XCC 1-1的致死溫度差可達13℃以上；相對在20 W照射35秒的慢速加熱下，甘藍種子最高僅能承受63℃，種子與XCC 1-1的致死溫差亦降為11℃；顯示高功率高頻微波的快速加熱是種子得以在高溫存活的主要原因，並且加熱速度越快越有利於種子消毒。綜合以上結果，本研究成功開發出以甘藍種子浸水 (water-soaking) 方式配合照射600 W高頻微波1秒鐘，作為高頻微波的標準種子消毒方法。此外，本研究亦證實了高頻微波的照射不會影響甘藍種子發芽及植株的生長，且能有效將幼苗黑腐病之發病率由15%下降為0%，於是本研究成為世界首度成功利用微波進行種子消毒的案例。基於人工接菌種子的成功，本研究進一步嘗試利用該技術消除種子內部的XCC。為獲得自然感染的帶菌種子，將XCC 1-1懸浮液噴灑於油菜花上，待種子成熟後採收果莢，經表面消毒後，證實每粒種子內部帶菌量約為3,500~16,000 CFU。將經表面消毒之種內帶菌油菜種子以種子浸水 (water-soaking) 的方式照射高頻微波，可以消除99%的種內細菌量；在進一步嘗試後發現，事先將種子浸水10分鐘，再以種子浸水方式處理高頻微波，便能將種子內部XCC全數殺死，但種子也無法存活。此外，本研究欲瞭解高頻微波如何於有水環境下將XCC 1-1殺死，於是初步收集種子浸水後經高頻微波照射之XCC 1-1，其中一部分細菌經propidium iodide染色後以共軛焦顯微鏡觀察，發現XCC 1-1細胞均無螢光產生，表示細胞膜並未受到破壞；另一部分細菌塗佈於SYG-X培養基後，完全無法在培養基生長，代表XCC 1-1已死亡，且死亡原因應與細胞膜損害無關。同時在掃描式及穿透式電子顯微鏡的觀察中，發現XCC 1-1經高頻微波照射後會有細胞縮短及細胞質產生均勻分布的電子緻密小體，推論這些效應可能與XCC致死的機制有關，但仍有待後續實驗證實。

關鍵字

高頻微波 ； 十字花科黑腐病菌 ； 甘藍 ； 種子消毒 ； 致死溫度

並列摘要

Black rot of crucifers caused by Xanthomonas campestris pv. campestris (XCC) is one of the most important diseases in the world, and it affects the yield of cabbage especially in Taiwan. The most effective method for prevention and control of black rot disease is the use of healthy pathogen-free seeds to reduce the initial inoculum and thus to decrease the disease incidence in the field. Though many seed disinfection methodshavebeen developed, but most of these methods cannot eliminate pathogenic bacteria completely, affect seed germination, or require long processing time. Therefore, this study intends to use the 24 GHz high-frequency microwave system developed by Department of Physics at National Taiwan University to establish a technology which can quickly and effectively eliminate XCC from vegetable seeds. Therefore, a new seed disinfection option can be provided for seed producers. At first, 1 ml of XCC 1-1 strain suspension was irradiated with 300 W high frequency microwave, and the result showed that XCC 1-1 was completely eliminated in 8.91 seconds, indicating the possibility of seed sterilization by high frequency microwave. The cabbage seeds were immersed in XCC 1-1 suspension, air-dried for 1 day and used as the artificially inoculated seeds, and the average bacterial population per seed was about 60,000 CFU (colony forming unit). While these dry inoculated seeds were directly irradiated with high frequency microwave for 55 seconds and the average seed temperature reach 90℃, XCC 1-1 population decreased to 52%, and seed germination rate was also reduced to 25%. In order to improve the sterilization effect of high frequency microwave, the seeds were misting-treated and then irradiated with high frequency microwave.. The results indicated when the misting-treated seeds were heated to the average temperature of 94℃, bacterial population per seed was reduced to 24%, and germination rate increased to 58%. Because the bacteria elimination of misting-treated seeds were better than dry seeds after irradiation with microwave, indicating that water might be the key factor of improving sterilization effect. To confirm the role of water in sterilization process, bacterial population and germination rate of dry, misting, and water-treated (including water-soaking in inverted tube and in upright tube) seeds were analyzed after irradiated with 200 W high frequency microwave, at about 80℃. The results revealed that the more water seed surface was covered by, the better bactericidal effect it had. In both water-treated seed treatments, XCC 1-1 could be eliminated from seeds completely; but only seeds of water-soaking treatment (water-soaking in upright tube)could maintain 100% germination rate. Consequently, seed with water-soaking treatment and high frequency microwave irradiation is the best disinfection method. In order to further explore the reason seed after water-soaking microwave treatement can still survive at high temperature of 80℃, the cabbage seeds were heated to different temperatures by adjusting the high frequency microwave power and irradiation times, and then tried to find out the temperature range in which XCC 1-1 die but seeds survive. According to the experimental results, it was found that under rapid heating of 600 W microwave irradiation for 0.6-1 seconds, the highest survival temperature of cabbage seeds was 91℃, and the lethal temperature difference between seed and XCC 1-1 reached above 13℃. Whereas under slow heating of 20 W irradiation for 35 seconds, the highest survival temperature of cabbage seed was 63℃, and the lethal temperature between seed and XCC 1-1 was reduced to 11℃. It can be concluded that rapid heating of high power high frequency microwave is the main reason why seed can survive at high temperature, and fast heating rate, is benificial to seed disinfection. Based on the above results, this study successfully developed a standard seed disinfection protocol for high frequency microwave as following water-soaking seed were irradiated with 600 W high frequency microwave for 1 seconds. In addition, our study also demonstrated that high frequency microwave irradiation did not affect seed germination and plant growth, also it could effectively reduce the black rot disease incidence of seedlings from 15% to 0%. So this study became the world’s first successful case of seed disinfection using microwave. Because of the success on artificially inoculated seeds, we further attempted to use the same technology to eliminate XCC inside the seeds. To obtain naturally infected seeds, the XCC 1-1 suspension was sprayed on the rape flowers, and the seed pods were harvested after seed matured. These seeds were surface sterilized, and the internal bacterial population was determined as 3,500-16,000 CFU per seed. Surface-sterilized rape seeds treated with water-soaking microwave irradiation method could eliminate 99% of XCC inside the seed; in advance, it was found that seeds pre-soaking in water for 10 minutes then treated with water-soaking microwave irradiation method could eliminate XCC inside the seed completely, but seed cannot survive. Further more, to understand how XCC was eliminated by high-frequency microwave in a watery environment, XCC 1-1 was collected after water-soaking microwave irradiation treatment, and part of the bacteria were stained with propidium iodide and observed by confocal microscope. Beacuse there had no fluorescence in XCC 1-1 cells, it suggested that bacterial cell membranes were not damaged. Another part of irradiated XCC 1-1 was cultured on medium, but no colony was observed, indicating these bacteria were dead and cell membrane destruction seemed not the causal reason. According to the observations of scanning and transmission electron microscopy, it was found that XCC 1-1 cells became shortend and electron-dense objects appeared in cytoplasm after high frequency microwave irradiation. It is inferred that these effects may be related to the mechanism of XCC 1-1 death, but it still needs to be confirmed.

並列關鍵字

high frequency microwave ； Xanthomonas campestris pv. campestris ； cabbage ； seed disinfection ； lethal temperature

參考文獻

Aladjadjiyan A. 2010. Effect of microwave irradiation on seeds of lentils (Lens culinaris, Med.). Rom. J. Biophys. 20: 213-221.

Araújo SDS, Paparella S, Dondi D, Bentivoglio A, Carbonera D, Balestrazzi A. 2016. Physical methods for seed invigoration: advantages and challenges in seed technology. Front. Plant Sci. 7: 646.

Banik S, Bandyopadhyay S, Ganguly S. 2003. Bioeffects of microwave–a brief review. Bioresour. Technol. 87: 155-159.

Bhaskara Reddy MV, Raghavan GSV, Kushalappa AC, Paulitz TC. 1998. Effect of microwave treatment on quality of wheat seeds infected with Fusarium graminearum. J. Agric. Eng. Res. 71: 113-117.

Bhat NA, Syeed N, Bhat KA, Mir SA. 2010. Pathogenicity and host range of Xanthomonas campestris pv. campestris-incitant of black rot of crucifers. J. Phyto. 2: 01-05.