The survey, identification and etiology studies of rice root-knot nematode, Meloidogyne graminicola, in Taiwan

禾本科糧食作物水稻(Oryzae sativa L.)為東南亞地區人們主要的糧食作物。全世界的水稻栽培系統有四種，包含高地(upland)、雨澆低地(rain-fed lowland)、灌溉低地(Irrigated lowland)和深水或浮水(deep water or floating)水稻，水稻根瘤線蟲Meloidogyne graminicola被報導存在於各栽培系統中，可造成植株高度減少31-48%，產量損失5.3-73%。本研究經調查後，顯示水稻根瘤線蟲存在於台灣15個縣市的水稻田中，其中在苗栗縣和台中市的危害最為嚴重，田間罹病率達100%，在花蓮縣的田間罹病率最低，為52.94%。積溫試驗的結果可知曉水稻根瘤線蟲在20、24、28、30℃下，完成一次世代的時間依序分別為24、24、21、20天，以線性積溫方程式得出門檻溫度(t)為-13.5度，有效累積溫度(K)為833.3，推測造成門檻溫度為負值的原因為，該方程式於生物的最適生長範圍外的預測值造成的誤差以及實驗處理組數量不足間接造成的誤差。在田間監測淹水高度與水稻根瘤線蟲侵染和族群量的結果顯示，插秧後至曬田期的32天期間，田區為淹水狀態，在水稻根系內並未觀察到水稻根瘤線蟲的侵入與發育，而在插秧38天後，田區排水後再淹水，在水稻根系與土壤中可發現水稻根瘤線蟲。以淹水試驗模擬田間的結果顯示，在淹水狀態下線蟲會延遲入侵根系而延長水稻根瘤線蟲完成一次世代的時間，結果不同於田間的原因可能為實驗室的土壤環境含氧量較高，而可讓水稻根瘤線蟲入侵水稻根系；試驗的結果也證明水稻根瘤線蟲入侵水稻根系後，不會受淹水而影響其生長。本研究從病田採集的樣本中建立了54個單卵族群，經陰門膜紋、粒線體DNA中的CoII和核糖體DNA中的D2D3序列比對，再以酯酶圖譜佐證，其中13個單卵族群CL-2、ZD-2、SS-2、DYW、WF-1、PX-2、SiG-2、LiJ-1、MN-1、KD-2、FeL-2、FuL-3和CS-1為M. graminicola。各單卵族群的CoII序列屬於劉氏2013年報導中的haplotype 1，D2D3序列則出現haplotype 1、2和3型，每個單卵族群各具有1到3種haplotype。將其中12個族群CL-2、ZD-2、SS-2、DYW、PX-2、SiG-2、LiJ-1、MN-1、KD-2、FeL-2、FuL-3和CS-1與劉氏2013年鑑定為水稻根瘤線蟲的YM-Tn11和LBF族群共14個族群進行寄主範圍測試，測試7種本土作物的結果與前人文獻相符，即高麗菜、花椰菜、菜豆、毛豆、豌豆和番茄為寄主，甜椒為非寄主。但14個單卵族群在對本土品種的胡瓜、西瓜、玉米和黃秋葵四個作物上測試出6種不同的寄主反應，分別稱為玉米型、胡瓜型、葫蘆科型、黃秋葵型、胡瓜秋葵中間型和溫和型共6種水稻根瘤線蟲致病型。推測這些單卵族群各採集點受到冬季輪作作物相產生的寄主篩選壓力，而形成不同的致病型。本土北蔥(Allium fistulosum)和空心菜(Ipomoea aquatica)先前並無相關報導，本研究的實驗結果顯示，北蔥應為水稻根瘤線蟲的寄主，空心菜為抗性或耐性寄主。根系誘引率試驗結果顯示誘引率與寄主反應並無正相關，非寄主根系也可以誘引二齡線蟲。侵入和寄生試驗結果顯示，5種非寄主和13個單卵族群的組合中，27組單卵族群的二齡幼蟲不能入侵非寄主根系，YM-Tn11和LBF族群則可侵入玉米和胡瓜根系，但無法在其根系內建立與維持取食點。現今台灣並未有水稻根瘤線蟲危害嚴重的報導。然而未來面臨氣候變遷和缺水危機，無法利用淹水延後水稻根瘤線蟲的侵入或其二次感染源的散佈，因此水稻根瘤線蟲造成全球水稻產量的損失可能日趨嚴重。Rice (Oryza sativa) is a major food crop in South Asia. The rice cropping systems include upland, rain-fed lowland, irrigated lowland and deep water or floating rice. The rice root-knot nematodes (Meloidogyne graminicola) are reported occurring in all rice cropping systems. It could cause 31-48% decreased plant height, and 5.3-73% yield losses in different rice cropping systems. In this study, the rice root-knot nematodes were found to occurr in rice fields of 15 cities and counties in Taiwan. The infection rates of Miaoli county and Taichung city were both 100%, 52.94% of Hualien county was the lowest in all surveyed sities. When rice root-knot nematodes were cultured under four different sets of temperatures, the length of the life cycle was between 20 to 24 days. The threshold temperature (t) and thermal constant (K) were -13.5℃ and 833.33, respectively. The significant lower threshold temperature (t) might be due to the direct error of predicting the lower temperature with degree day linear equation or the indirect error of the insufficient temperature sets. The data of the monitored field indicated that under flooding conditions, rice plants were free of rice root-knot nematodes. However, the rice root-knot nematodes were discovered in rice roots and soil samples after the field was drained. The results of flooding test under laboratory conditions showed that flooding did not affect the development of rice root-knot nematode inside the roots, but if the flooding lasted till the penetration stage, it might delay the penetration and prolong the life cycle of rice root-knot nematodes. The oxygen content might affect the penetration of rice root-knot nematode. In this study, fifty-four single female lines were established. The mitochondiral DNA CoII sequence, 28S rDNA D2D3 sequence and the esterase profile were obtained from 13 of them. The isolates CL-2、ZD-2、SS-2、DYW、WF-1、PX-2、SiG-2、LiJ-1、MN-1、KD-2、FeL-2、FuL-3 and CS-1 were identified as M. graminicola。Twelve single female lines, CL-2、ZD-2、SS-2、DYW、PX-2、SiG-2、LiJ-1、MN-1、KD-2、FeL-2、FuL-3 and CS-1, in this study and two lines YM-Tn11 and LBF identified by Liu in 2013, were used in the host range test. Cabbage, cauliflower, common bean, soybean, garden pea and tomato were hosts of the rice root-knot nematodes, and pepper was non-host in our study. However, on cucumber, watermelon, corn and okra, the 14 tested isolates had 6 different responses and were classified as corn, cucumber, cucurbit, okra, cucumber okra intermediate and mild pathotype. The different pathotypes of rice root-knot nematodes might be due to the diversity of winter rotation crops. Bunching onion(Allium fistulosum) and water convolvulus (Ipomoea aquatica) were tested for their host status using rice root-knot nematodes for the first time. Our results showed that burching onion was a susceptible host and water spinach might be a tolerant or resistant host. The results of root tip chemotaxis tests showed that there was no positive correlation between host responses and attraction rates. The root tip of non-host also attraced 2nd stage juveniles. In invasion and parasitization tests, the 2nd stage juveniles of YM-Tn11 and LBF were found to invade the corn and cucumber roots but fail to establish the feeding sites. The other single female lines did not invade the roots of non-hosts. Significant yield losses caused by rice root-knot nematodes has not been found in Taiwan. However, the climate changes and the water shortage crisis in the future might accelerate the establishment of rice root-knot nematodes. Because field flooding could delay the invasion and spread of rice root-knot nematodes, the shortage of water might lead the rice root-knot nematodes to cause significant rice yield losses worldwide in the future.1. 前言 1 2. 材料與方法 7 一、台灣地區水稻根瘤線蟲之調查 7 二、水稻根瘤線蟲之病因學探討 7 (一) 積溫試驗 7 (二) 田間淹水與水稻根瘤線蟲族群量之關係 8 (三) 淹水試驗 8 三、建立台灣各地區水稻根瘤線蟲之單卵並鑑定 9 (一) 建立台灣各地區之單卵族群 9 (二) 陰門膜紋型態鑑定 9 (三) 分子生物方法鑑定 9 (四) 同功異構酶表型分析 10 四、十四個單卵族群之寄主反應試驗 11 (一) 本土作物寄主範圍 11 (二) 作物根尖誘引二齡幼蟲 12 (三) 評估非寄主作物對抗水稻根瘤線蟲的機制 13 3. 結果 14 一、台灣地區水稻根瘤線蟲之調查 14 二、水稻根瘤線蟲之病因學探討 15 (一) 積溫試驗 15 (二) 田間淹水與水稻根瘤線蟲族群量之關係 15 (三) 淹水試驗 16 三、建立台灣各地區水稻根瘤線蟲之單卵並鑑定 16 (一) 建立台灣各地區之單卵族群 16 (二) 陰門膜紋型態鑑定 17 (三) 分子生物方法鑑定 17 (四) 同功異構酶表型分析 18 四、十四個單卵族群之寄主反應試驗 18 (一) 本土作物寄主反應 18 (二) 作物根尖誘引二齡幼蟲 19 (三) 評估非寄主作物對抗水稻根瘤線蟲的機制 20 4. 討論 21 5. 參考文獻 29 6. 圖表 40 7. 附錄 6