探討紅冠腐菌對大豆根瘤產生氧化亞氮 (N2O) 之影響

Abstract

大豆 (Glycine max) 是世界上重要的糧食作物之一，其根系在種植過程中能與根瘤菌共生形成根瘤，並藉由根瘤菌的固氮酶 (nitrogenase) 將大氣中的氮氣 (N2) 固定成氨以供植物利用。除了固氮作用外，有些根瘤菌如 Bradyrhizobium diazoefficiens USDA110還能進行反硝化作用，將硝酸鹽依序還原成亞硝酸鹽、一氧化氮、氧化亞氮 (N2O) 及 N2。然而有些根瘤菌如 Bradyrhizobium japonicum USDA6因缺乏反硝化作用最後一步的 N2O 還原酶 (N2O reductase)，導致最終產物為 N2O。N2O 是一種強溫室作用氣體，其溫室效應比二氧化碳強約300倍，因此若缺乏 N2O 還原酶、或乘載 N2O 還原酶的 nosZ 基因表現下降時，皆會造成較多 N2O 產生並逸散至大氣中。許多研究已證實環境因子會影響 N2O 還原酶活性或 nos 基因表現，然而紅冠腐菌 (Calonectria ilicicola) 是否會影響 nos 基因表現而增加 N2O 逸散的風險目前尚未被研究。本研究將 B. diazoefficiens 及 B. japonicum 分別培養在添加 KNO3 的液態培養基中，透過 N2O 微電極器檢測發現，缺乏 nosZ 基因的 B. japonicum 確實產生較多 N2O。後續在與 C. ilicicola 分生孢子共同培養時，結果發現不論是具有 nosZ 基因的 B. diazoefficiens 或缺乏 nosZ 基因的 B. japonicum ，其 N2O 產量皆無顯著差異，而 B. diazoefficiens 的 nosZ 基因表現亦未受到影響。盆栽試驗結果顯示大豆接種 B. japonicum 產生的 N2O 約為接種 B. diazoefficiens 的6.5倍，另外大豆受到 C. ilicicola 感染會降低 B. diazoefficiens 的 nosZ 基因表現量而導致 N2O 的產量增加。另計算根罹病度、根瘤重量、根瘤數量及根瘤大小，發現罹病大豆根瘤的健康狀況較對照組差，因此推測根瘤的健康可能會影響 B. diazoefficiens 的nosZ 基因表現下降，故而提高 N2O 產量。鑑於缺乏 nosZ 基因可造成約6.5倍的 N2O 產量，本研究進一步調查臺灣大豆田的根瘤菌組成及 nosZ 基因普及率，結果發現 B. diazoefficiens、Bradyrhizobium elkanii 及 Bradyrhizobium liaoningense 在花蓮及桃園是優勢菌種且根瘤樣本內皆可測得 nosZ 基因；然而主要產毛豆的高屏地區發現 B. elkanii 及 B. liaoningense 為優勢菌種但普遍缺乏 nosZ 基因。綜合上述，本研究建立檢測大豆根瘤內 N2O 產量的試驗系統，證實選擇具有 nosZ 基因的根瘤菌種至關重要，後可配合防治病原菌以降低 N2O 產量，為大豆種植在淨零碳排的願景下提供理論基礎。

Soybean (Glycine max) is one of the most important crops worldwide, and soybean roots can form nodules with symbiotic rhizobia to fix nitrogen (N2) into ammonia as extra nutrients. In addition to N2 fixation, some rhizobia can also perform denitrification. For example, Bradyrhizobium diazoefficiens USDA110 can sequentially reduce nitrate to nitrite, nitric oxide, nitrous oxide (N2O), and N2. Another species, Bradyrhizobium japonicum USDA6, which lacks N2O reductase, produces N2O as the final product. N2O is a greenhouse gas with global warming potential 300 times stronger than CO2. Therefore, the absence of N2O reductase or the decreased expression of the N2O reductase-coding gene nosZ can lead to more N2O production. Many studies have shown that environmental factors can affect the activity of N2O reductase or the expression of the nosZ gene. However, whether Calonectria ilicicola could affect nosZ gene expression or N2O emission has not been studied. This study used N2O microsensor to detect B. diazoefficiens and B. japonicum culture broth supplemented with KNO3, and it was found that B. japonicum indeed produced detectable N2O. Then co-culturing of B. diazoefficiens and B. japonicum with C. ilicicola conidia revealed that N2O emission was not affected by C. ilicicola, and the expression of the B. diazoefficiens nosZ gene was unaffected either. Pot experiments showed that soybeans inoculated with B. japonicum produced approximately 6.5 times N2O more than those inoculated with B. diazoefficiens. Additionally, soybeans infected with C. ilicicola reduced the expression of the B. diazoefficiens nosZ gene, leading to more N2O production. The results of root disease severity, nodule weight, nodule number, and nodule size showed that C. ilicicola affects nodules health, which may reduce the expression of the nosZ gene and consequently increase N2O production. Considering the importance of having nosZ gene in soybean nodules to minimize N2O production, our field survey revealed that B. diazoefficiens, Bradyrhizobium elkanii, and Bradyrhizobium liaoningense are dominant rhizobia species in Taoyuan city and Hualien county, and the nosZ gene was detected in most soybean nodules from these regions. However, Kaohsiung city and Pingtung county, which are the main regions for edamame production, showed that B. elkanii and B. liaoningense are dominant species, and low detection rate of nosZ gene. In summary, this study establishes a system for detecting N2O production in soybean nodules, and the results confirm the importance of nosZ gene in the rhizobia species, following by the pathogen control to reduce N2O emissions for soybean industry under the net-zero vision.