南京农业大学学报  2017, Vol. 40 Issue (2): 251-257   PDF    
http://dx.doi.org/10.7685/jnau.201605017
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文章信息

韩杨会, 罗光华, 张月亮, 王利华, 方继朝
HAN Yanghui, LUO Guanghua, ZHANG Yueliang, WANG Lihua, FANG Jichao
杀菌剂处理稻苗后对水稻以及取食褐飞虱种群的影响
Changes of rice seedlings after fungicides treatment and effects of the brown rice planthopper feeding on the treatment rice seedlings
南京农业大学学报, 2017, 40(2): 251-257
Journal of Nanjing Agricultural University, 2017, 40(2): 251-257.
http://dx.doi.org/10.7685/jnau.201605017

文章历史

收稿日期: 2016-05-12
杀菌剂处理稻苗后对水稻以及取食褐飞虱种群的影响
韩杨会1,2, 罗光华2, 张月亮2, 王利华2, 方继朝1,2    
1. 南京农业大学植物保护学院, 江苏 南京 210095;
2. 江苏省农业科学院植物保护研究所, 江苏 南京 210014
摘要[目的]探究3种杀菌剂高剂量处理的稻苗对水稻及取食褐飞虱种群的影响。[方法]通过测定杀菌剂处理后稻苗体内草酸、还原糖以及可溶性蛋白的含量,并构建种群生命表探讨经杀菌剂处理的稻苗对取食褐飞虱种群生命参数的影响。[结果]糯稻经3种杀菌剂高剂量处理后,稻苗体内还原糖含量显著降低,碳氮比(C/N)有降低的趋势,草酸和可溶性蛋白的含量无显著变化。粳稻经3种杀菌剂高剂量处理后,C/N有升高的趋势,但草酸、还原糖和可溶性蛋白含量无显著变化。褐飞虱取食杀菌剂高剂量处理的糯稻后,5龄若虫历期、个体总发育历期以及雌成虫寿命显著延长,种群加倍时间、平均世代历期、种群趋势指数以及相对适合度增大;取食杀菌剂高剂量处理的粳稻后,褐飞虱种群趋势指数和种群适合度降低。此外,取食粳稻稻苗的褐飞虱种群趋势指数大于取食糯稻的褐飞虱。[结论]高剂量杀菌剂处理稻苗后能影响稻苗体内相关物质含量的变化,且不同水稻品种所受到的影响存在差异;褐飞虱取食经高剂量杀菌剂处理的稻苗后其部分种群生命参数会发生变化,而且处理的水稻品种不同对取食的褐飞虱影响也不同。
关键词褐飞虱   杀菌剂   生理生化物质   生命参数   
Changes of rice seedlings after fungicides treatment and effects of the brown rice planthopper feeding on the treatment rice seedlings
HAN Yanghui1,2, LUO Guanghua2, ZHANG Yueliang2, WANG Lihua2, FANG Jichao1,2    
1. College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China;
2. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
Abstract: [Objectives] This study aims to determine the effects on rice plant and the population dynamics of the brown planthopper Nilaparvata lugens(BPH). [Methods] The contents of oxalic acid, reducing sugars and soluble proteins in the rice seedlings were determined. And, the population life table of BPH was constructed. [Results] The results showed that the contents of reducing sugar in glutinous rice seedlings treated with 3 fungicides with high doses was significantly lower than the control; the C/N ratio also decreased while had no significantly difference; the contents of oxalic acid and soluble protein also had no significantly changes. On the contrary, the C/N ratio in japonica rice seedlings treated with fungicides increased to some extent. While, the contents of oxalic acid, reducing sugar and soluble protein had no significantly changes. When BPH feeding was applied on the treated glutinous rice seedlings, the development period of the 5 instar nymphs, the full life development duration and the life span of female were all extended, and the population doubling time, the average generation time, the index of population trend and the relative fitness were all increased. When BPH feeding was applied on the treated japonica rice seedlings, the index of population trend and the relative fitness were decreased. It is noteworthy that the index of population trend feeding on japonica rice seedlings was always higher than that feeding on glutinous rice seedlings. [Conclusions] High dose of three fungicides treatment could not only alter the plant physiological and biochemical indexes but also had different effects on the contents of related substances in different varieties of rice seedlings, which could indirectly affect the developmental dynamics of BPH population.
Key words: Nilaparvata lugens    fungicide    physiological and biochemical substances    life parameters   

褐飞虱[Nilaparvata lugens Stål (Hemiptera:Delphacidae)]是我国水稻上的重要害虫之一, 其主要通过刺吸取食水稻韧皮部营养物质和传播病毒病危害水稻[1-2]。褐飞虱属于r-对策昆虫, 极易暴发成灾, 近年来由于异常天气频发, 导致褐飞虱更加猖獗, 使我国水稻生产面临很大威胁[3-4]。研究发现用于水稻病害防治的杀菌剂同样能影响褐飞虱生长。如1/4推荐剂量的异稻瘟净, 4倍推荐剂量的叶枯灵、井冈霉素、三环唑和叶青双以及推荐剂量的甲基立枯磷等杀菌剂对褐飞虱若虫具有直接致死作用[5-6]。井冈霉素除了能够直接杀伤褐飞虱的若虫, 还能刺激褐飞虱产卵, 导致褐飞虱体内与产卵量相关的蛋白含量及卵黄原蛋白基因表达量升高, 造成了褐飞虱猖獗[7-9]。此外, 化学药剂的使用能够导致水稻植株一些生理生化物质含量的改变, 从而间接影响取食害虫的生长、发育以及繁殖[10-12]

纹枯病是我国水稻上的重要病害, 目前由于缺少优良的抗纹枯病水稻品种, 对纹枯病的防治主要以化学药剂防治为主[13]。己唑醇、噻呋酰胺以及嘧菌酯是目前我国水稻生产上比较常用的3种內吸性杀菌剂, 对水稻纹枯病具有良好的防治效果[14-16]。在农业生产中, 杀菌剂过量及频繁使用是造成病菌产生抗药性的重要原因[17], 伴随着抗性的逐渐升高, 若没有替代药剂可选择, 则药剂的使用量必定越来越高。田间己唑醇、噻呋酰胺和嘧菌酯用量增多, 对水稻植株有何影响, 以及对取食水稻的褐飞虱种群有何影响都不清楚。

目前, 杀菌剂对褐飞虱的间接影响研究较少, 且现有研究大多仅考虑单一杀菌剂对水稻生理生化指标或害虫的影响, 忽略了大田水稻病害防控通常是多种杀菌剂并用的现状。为了能更真实反映生产实际情况, 本研究模拟了生产上多种杀菌剂并用的情况, 进而探究这种生产条件下稻苗自身相关物质含量的变化以及对褐飞虱种群的影响, 以期了解多种杀菌剂大量使用条件下褐飞虱种群的变化, 为田间合理施药以及稻田病虫害综合防控提供理论参考。

1 材料与方法 1.1 材料

供试褐飞虱:室内连续传代饲养的敏感品系; 饲养条件:温度 (27±1)℃, 相对湿度为85%, 光照周期为14 h/10 h (光/暗), 期间不接触任何药剂。

供试水稻品种:糯稻为‘香糯833’, 粳稻为‘武运粳7号’。

供试药剂:95%己唑醇, 田间推荐用量75 g·hm-2; 96%噻呋酰胺, 田间推荐用量250 g·hm-2; 98%嘧菌酯, 田间推荐用量100 g·hm-2

1.2 稻苗处理

选取颗粒饱满的水稻种子, 清水洗净后浸种、催芽, 至大部分种子露白, 选取发育一致的水稻种子种植于水稻育苗盘上 (长29 cm, 宽17 cm), 每穴2粒, 保持充足的水肥, 保证水稻植株的正常发育。稻苗出土48 h喷洒4倍田间推荐用量的己唑醇, 隔36 h后喷洒4倍推荐用量的噻呋酰胺, 再隔36 h后喷洒4倍推荐用量的嘧菌酯。试验于喷洒嘧菌酯36 h后取样, 取样前喷洒清水洗净稻株表面的药剂。稻苗用吸水纸包裹根部保湿, 放入玻璃指形管内 (玻璃管直径3 cm、长20 cm), 每管3株苗期植物。

1.3 水稻体内草酸、还原糖和可溶性蛋白的测定

参考张继民等[18]的三氯化钛显色法测定稻苗体内草酸含量。取1 g新鲜稻苗样品, 用液氮研磨后加入10 mL的超纯水成匀浆后, 加入活性炭脱色30 min, 然后3 000 r·min-1离心15 min分离活性炭, 溶液呈无色或呈乳白色后, 取3 mL上清液加入1%三氯化钛溶液0.15 mL显色, 在UV2000分光光度计 (美国尤尼柯) 上于400 nm下测定吸光值。试验重复4次。用草酸制作标准曲线测定计算水稻体内草酸含量。

采用蒽酮比色法测定稻苗体内还原糖的含量。称取0.5 g水稻鲜样置于研钵中, 加液氮研磨, 用10 mL超纯水分次洗涤, 洗液转入试管中沸水浴10 min, 冷却后过滤, 摇匀备用。蒽酮试剂配制:称取0.5 g蒽酮, 用乙酸乙酯溶解定容至25 mL。还原糖含量的测定:取1 mL提取液加1 mL超纯水和0.5 mL蒽酮试剂, 再缓慢加入5 mL浓H2SO4, 沸水浴10 min (空白用1 mL的超纯水代替1 mL的提取液)。冷却至室温后, 在波长620 nm下测定吸光值 (UV2000分光光度计)。利用葡萄糖制作标准曲线计算稻苗体内还原糖含量[19]。对照和处理各重复测定4次。

可溶性蛋白含量的测定采用Bradford (G-250) 比色法[20]。考马斯亮蓝试剂:称取10 mg考马斯亮蓝G-250, 溶于5 mL的90%乙醇中, 加入85%(体积分数) 的磷酸10 mL, 用超纯水定容到100 mL, 棕色瓶避光放置。样品的提取:称取水稻鲜样0.2 g, 用液氮研磨后加5 mL的磷酸缓冲液匀浆, 3 000 r·min-1离心15 min, 上清液备用。样品测定:吸取样品提取液10 μL (将样品蛋白质含量稀释, 使蛋白质的浓度在分光光度计可测定的范围), 加入200 μL考马斯亮蓝试剂混匀, 放入酶标仪 (Elx808, 美国BioTek) 于595 nm下比色测定吸光值, 利用牛血清白蛋白制作标准曲线计算稻苗体内蛋白质的含量[19]。对照和处理各重复测定4次。

1.4 褐飞虱种群生命表的构建

参照刘泽文等[21]的方法构建褐飞虱种群生命表, 选取24 h内孵化的若虫置于玻璃指形管中单头饲养, 每天观察记录若虫的存活数与蜕皮情况 (褐飞虱的蜕皮及时用毛笔取出), 直至羽化为成虫。对照和处理各重复4次, 每个重复30头虫。羽化后的成虫, 1头雌虫和1头雄虫于玻璃管中单独配对, 观察并记录褐飞虱的单雌产卵量、单雌初孵若虫数、孵化率、交配率、雌雄成虫寿命, 据此计算下一代初孵若虫数和种群生命参数。对照和处理各重复3次, 每个重复20对。褐飞虱种群生命表构建:平均世代历期 (Td)=∑x·lx·mx/∑lx·mx(式中:x为特定的时期; lxx期种群存活率; mxx期每雌产雌率); 内禀增长率 (rm)=ln (∑lx·mx)/Td; 周限增长率 (λ)=erm; 种群加倍时间 (td)=ln2/rm[22]; 种群趋势指数 (I)=Nn+1/Nn(Nn为当代个体数; Nn+1为下一代个体数); 相对适合度 (Rf)=I处理/I对照[21]

1.5 数据处理

数据采用SPSS 17.0软件进行分析, 采用t检验法分析处理和对照间差异显著性。

2 结果与分析 2.1 药剂对稻苗的安全性评价

药剂处理糯稻和粳稻后均未出现茎叶失绿、黄化, 茎叶扭曲变形, 新叶伸出缓慢或停止, 植株矮化等药害症状[23], 因此这3种杀菌剂在4倍推荐剂量范围内对稻苗无明显药害。

2.2 杀菌剂处理后的稻苗草酸、可溶性糖以及可溶性蛋白含量的变化

杀菌剂处理后糯稻稻苗还原糖含量显著降低, C/N有降低的趋势, 但与对照相比差异不显著, 草酸和可溶性蛋白含量与对照相比差异也不显著; 粳稻稻苗3种生理生化物质含量以及C/N与对照相比差异不显著, 其中C/N有增加的趋势 (表 1)。

表 1 杀菌剂处理的水稻体内草酸、还原糖、可溶性蛋白的含量 Table 1 Contents of oxalic acid, reducing sugar and soluble protein in rice seedlings after treated with fungicides
生理生化物质
Physiological and biochemical substance
糯稻Glutinous rice 粳稻Japonica rice
对照Control 处理Treatment 对照Control 处理Treatment
草酸含量/(mg·g-1) Oxalic acid content 3.48±0.08a 3.53±0.08a 3.17±0.10a 3.35±0.08a
还原糖含量/(mg·g-1) Reducing sugar content 10.74±0.27a 9.51±0.40b 14.05±0.35a 16.02±0.75a
可溶性蛋白含量/(mg·g-1) Soluble protein content 19.24±0.21a 18.80±0.20a 17.21±0.15a 17.72±0.44a
碳氮比C/N ratio 0.56±0.01a 0.51±0.02a 0.83±0.02a 0.87±0.04a
注:同一水稻品种上标不同小写字母表示处理与对照间差异显著 (P < 0.05)。
Different lowercase letters indicate significant difference at 0.05 level of probability in the same rice variety. The same as follows.
2.3 杀菌剂处理的稻苗对取食褐飞虱若虫存活率的影响

褐飞虱取食杀菌剂处理的稻苗后其各龄若虫存活率以及总的存活率与各自对照相比差异都不显著 (表 2)。

表 2 取食杀菌剂处理的水稻苗后褐飞虱若虫的存活率 Table 2 The survival rate of nymphs of Nilaparvata lugens feeding on rice seedlings treated with fungicides
指标
Index
糯稻Glutinous rice 粳稻Japonica rice
对照Control 处理Treatment 对照Control 处理Treatment
初孵若虫数No.of newly-hatched individuals 120 120 120 120
1龄若虫存活率/% Survival rate of 1st instar larvae 98.00±1.15a 96.00±0.00a 96.00±2.83a 98.00±1.15a
2龄若虫存活率/% Survival rate of 2nd instar larvae 95.96±3.27a 98.96±2.08a 97.96±1.18a 97.96±1.18a
3龄若虫存活率/% Survival rate of 3rd instar larvae 98.91±1.09a 100.00±0.00a 98.86±1.14a 99.00±1.00a
4龄若虫存活率/% Survival rate of 4th instar larvae 100.00±0.00a 98.96±1.04a 100.00±0.00a 97.87±1.23a
5龄若虫存活率/% Survival rate of 5th instar larvae 97.87±1.23a 98.91±1.09a 94.58±2.13a 98.96±1.04a
初孵若虫至5龄若虫的存活率/%
Survival rate from newly-hatched to 5th instar larvae
91.00±1.91a 93.00±1.91a 88.00±4.00a 92.00±1.63a
2.4 杀菌剂处理的稻苗对褐飞虱若虫发育历期以及成虫寿命的影响

褐飞虱取食杀菌剂处理的糯稻稻苗后其5龄若虫以及总发育历期显著延长, 分别比对照延长0.42 d和0.75 d。取食杀菌剂处理的粳稻稻苗后, 褐飞虱的发育历期与对照相比差异不大 (表 3)。褐飞虱取食杀菌剂处理的糯稻稻苗后其雌成虫寿命显著延长3.82 d, 雄成虫寿命有延长的趋势, 但与对照相比差异不显著; 取食杀菌剂处理的粳稻稻苗后其雌、雄成虫寿命与对照相比差异不大 (表 4)。

表 3 取食杀菌剂处理的水稻苗后褐飞虱若虫的发育历期 Table 3 The developmental duration of Nilaparvata lugens feeding on rice seedlings treated with fungicides
指标
Index
糯稻Glutinous rice 粳稻Japonica rice
对照Control 处理Treatment 对照Control 处理Treatment
初孵若虫数No.of newly-hatched individuals 120 120 120 120
1龄若虫期/d Larva duration of 1st instar larvae 2.74±0.14a 2.96±0.02a 2.37±0.06a 2.50±0.07a
2龄若虫期/d Larva duration of 2nd instar larvae 2.07±0.03a 2.15±0.03a 2.04±0.00a 2.00±0.04a
3龄若虫期/d Larva duration of 3rd instar larvae 2.10±0.04a 2.15±0.04a 1.98±0.03a 2.03±0.03a
4龄若虫期/d Larva duration of 4th instar larvae 2.16±0.02a 2.34±0.05a 2.27±0.06a 2.32±0.05a
5龄若虫期/d Larva duration of 5th instar larvae 3.17±0.02a 3.59±0.14b 3.33±0.03a 3.32±0.09a
1~5龄发育历期/d Duration from 1st to 5th instar 12.24±0.06A 12.99±0.03B 11.99±0.02a 12.24±0.15a
注:同一水稻品种上标不同大写字母表示处理与对照差异极显著 (P < 0.01)。
Different uppercase letters indicate significant difference at 0.01 level of probability in the same rice variety.
表 4 取食杀菌剂处理的水稻苗后褐飞虱的雌、雄成虫寿命 Table 4 Longevity of female and male adults of Nilaparvata lugens feeding on rice seedlingstreated with fungicidesd
d
性别
Sex
每组试虫数
No.of individuals in each group
糯稻Glutinous rice 粳稻Japonica rice
对照Control 处理Treatment 对照Control 处理Treatment
雌虫Female 60 17.03±1.02a 20.85±1.55b 21.90±0.39a 22.42±0.78a
雄虫Male 60 29.73±0.69a 31.47±2.65a 26.73±1.07a 27.85±0.81a
2.5 杀菌剂处理的稻苗对褐飞虱繁殖力和种群适合度的影响

取食杀菌剂处理的糯稻稻苗后褐飞虱的产卵量、孵化若虫数都有增加的趋势; 而取食杀菌剂处理的粳稻后, 褐飞虱的产卵量、孵化若虫数都有降低的趋势, 但与对照之间都不存在显著性差异。取食杀菌剂处理的2种稻苗后褐飞虱的卵孵化率都有降低的趋势, 但与对照相比差异也不显著。杀菌剂处理的糯稻稻苗导致褐飞虱种群加倍时间、平均世代历期、种群趋势指数以及种群适合度都增大; 而杀菌剂处理的粳稻稻苗对褐飞虱种群加倍时间、平均世代历期影响不大, 但降低褐飞虱种群趋势指数和种群适合度, 且粳稻稻苗上取食的褐飞虱的种群趋势指数总大于糯稻稻苗上取食的褐飞虱; 取食杀菌剂处理的2种稻苗后褐飞虱种群内禀增长率和周限增长率几乎没有变化 (表 5)。

表 5 取食杀菌剂处理的稻苗后褐飞虱的繁殖力和种群适合度 Table 5 The fecundity and population fitness of Nilaparvata lugens feeding on rice seedlings treated with fungicides
生命参数
Life parameter
糯稻Glutinous rice 粳稻Japonica rice
对照Control 处理Treatment 对照Control 处理Treatment
产卵量Fecundity 288.90±14.45a 374.17±32.63a 459.63±24.37a 415.28±40.73a
孵化率/% Hatching rate 71.85±2.16a 68.15±3.11a 88.25±1.86a 84.10±2.89a
孵化若虫数No.of nymphs 203.60±7.09a 250.65±28.87a 398.18±27.48a 328.75±30.74a
内禀增长率/% Intrinsic rate of increase 0.14 0.13 0.14 0.14
周限增长率/% Finite rate of increase 1.15 1.14 1.15 1.15
种群加倍时间/d Population doubling time 4.88 5.14 4.85 4.86
平均世代历期/d The average generation time 19.60 21.48 19.53 19.89
种群趋势指数Index of population trend 86.77 106.99 169.85 140.32
种群相对适合度Relative fitness 1.00 1.23 1.00 0.83
3 讨论

本试验通过利用高剂量的己唑醇、噻呋酰胺和嘧菌酯依次处理水稻苗, 分析稻苗自身生理生化物质含量的变化以及用处理的稻苗喂食褐飞虱, 研究其对褐飞虱种群的影响。结果显示:稻苗自身生理生化物质含量的变化因水稻品种的不同所产生的变化有差异; 同时, 褐飞虱取食经处理的不同水稻品种后, 在种群参数上也表现出差异。这可能与农药品种的作用机制及水稻自身对不同农药的胁迫有不同反应所致[24]。此外, 当使用药剂的田间推荐剂量处理稻苗后, 糯稻稻苗体内的还原糖含量以及其上取食褐飞虱的发育历期和雌成虫寿命并没有出现显著性差异 (数据未列出)。这暗示随着田间致病菌抗性的形成与发展, 杀菌剂用量的增大, 将逐渐改变水稻体内相关物质的含量并影响田间褐飞虱种群的变化。本研究设置高剂量杀菌剂处理稻苗开展试验, 主要目的是为了探讨今后田间相应杀菌剂大剂量使用时产生的潜在影响, 可为相应药剂的使用策略提供一些理论指导。此外, 对于为什么水稻经不同剂量的杀菌剂处理后自身有不同的反应且对褐飞虱种群也有不一样的影响, 这值得更深入的研究。

农药喷施会对作物植株产生影响。吴进才等[24]研究了扑虱灵、吡虫啉、井冈霉素、杀虫双和三唑磷对水稻3个品种的生理生化影响, 发现这5种农药均能导致水稻草酸含量不同程度的下降。草酸是水稻叶鞘中强效抑制褐飞虱取食的物质, 稻株草酸含量高不利于褐飞虱的取食[25]。本试验结果显示高剂量杀菌剂处理的糯稻和粳稻, 它们的草酸含量均无显著性变化, 这可能是因为农药种类不同。Shi等[26]研究发现三唑磷和氯虫苯甲酰胺处理转基因水稻品种‘T2A-1’及其母本‘MH63’后会导致‘MH63’体内草酸含量显著高于‘T2A-1’, 这再次表明水稻自身对农药的胁迫反应有较大的差异。Wu等[11]发现井冈霉素处理水稻后会导致水稻体内C/N降低, 而水稻体内C/N降低能够刺激褐飞虱的取食。本研究显示高剂量杀菌剂处理糯稻后C/N有降低趋势, 处理粳稻后C/N有增加趋势。这暗示杀菌剂高剂量处理糯稻稻苗后对褐飞虱的抗性有降低的风险, 而杀菌剂高剂量处理的粳稻对褐飞虱的抗性有增强的趋势。

很多研究表明农药能够影响非靶标害虫。例如褐飞虱 (N.lugens) 取食经200 mg·L-1井冈霉素叶面喷雾处理的稻株能导致其产卵量显著增加; 家蚕 (Bombyx mori) 取食80%代森锰锌、70%丙森锌以及22.5%啶氧菌酯2倍推荐剂量处理3 d的桑叶后其4龄和5龄若虫龄期延长0.5~1.5 d; 柑桔红蜘蛛 (Panonychus citri) 受70%甲基托布津1 500倍液、50%多菌灵800倍液影响后其孵化率降低[7, 27-30]。上述相关研究都只考虑了单一药剂对非靶标害虫的影响。本研究模拟田间多种杀菌剂并用的情况, 由此探讨对非靶标害虫褐飞虱的影响。本研究基于构建褐飞虱种群生命表发现, 褐飞虱取食经高剂量杀菌剂处理的不同品种水稻后相关种群参数的变化有一定差异, 这与水稻品种不同有关。程家安等[31]研究发现, 在不同水稻品种上取食的褐飞虱种群内禀增长率、净增值率、平均世代历期和种群加倍时间差异较大; 丁识伯等[32]发现在‘青两优916’上取食的褐飞虱内禀增长率、净增值率以及种群趋势指数较高。这再次表明水稻品种、药剂种类、药剂用量等对非靶标害虫的影响是多种因素相互作用所形成的。因此, 如何避免某种药剂对非靶标生物的影响需要更深入细致的研究。

褐飞虱取食杀菌剂处理的稻苗后若虫的存活率与对照相比无差异, 推测是杀菌剂处理稻苗后对取食的褐飞虱没有直接的杀伤作用, 具体原因尚需要深入研究。

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