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Article Navigation >  Journal of Zhejiang A&F University  > 2022  >  39(1): 153-158

LI Wenjie, CUI Lulu, MA Yunjiao, et al. Insect resistance and metabolome of four willow species[J]. Journal of Zhejiang A&F University, 2022, 39(1): 153-158. DOI: 10.11833/j.issn.2095-0756.20210166
Citation: LI Wenjie, CUI Lulu, MA Yunjiao, et al. Insect resistance and metabolome of four willow species[J]. Journal of Zhejiang A&F University, 2022, 39(1): 153-158. DOI: 10.11833/j.issn.2095-0756.20210166
shu

Insect resistance and metabolome of four willow species

DOI: 10.11833/j.issn.2095-0756.20210166

  • College of Forestry, Hebei Agricultural University, Baoding 071000, Hebei, China

  • Received Date: 2021-02-10
  • Rev Recd Date: 2021-06-20
    • Available Online: 2021-09-01
  • Publish Date: 2022-02-14
  •   Objective  This purpose is to explore the insect resistance of different willows (Salix spp.) to Plagiodera versicolora, and to provide reference for the breeding of willow cultivars with strong insect resistance.  Method  S. caprea, S. matsudana, S. babylonica and 3 clones of S. triandra were tested in laboratory for insect feeding, and the differential secondary metabolites between insect resistant and non insect resistant groups were determined and screened.  Result  The survival rate of indoor feeding was 98% for S. matsudana, 93% for S. babylonica, 90% for S. caprea and 0 for S. triandra. There were 23 main different metabolites between the non insect resistant group and the insect resistant group, among which 15 were up-regulated, including fer-agmatine, L-ascorbic acid, gallic acid, (−)-epigallocatechin, (+)-gallocatechin, myricitrin, L-(+)-tartaric acid, D-(−)-arabinose, hydroxy-methoxycinnamate, N-feruloyl agmatine, gluconic acid, 2 ′-Hydroxygenistein, N-Acetyl-DL-tryptophan, eupatilin 3-glucoside, and hexadecylsphingosine, and 8 were down-regulated, including quercetin 3-O-(6″-O-malonyl)-galactoside, hesperidin, rutin, D-galactopyranuronate, 2,5-dihydroxy benzoic acid O-hexside, quercetin O-acetylhexoside, 3-hydroxy-5-methylphenol-1-Oxy-β-D-glucose, and bioquercetin.  Conclusion  S. triandra has the best insect resistance to P. versicolora. There are significant differences in the main secondary metabolites between the non insect resistant group and the insect resistant group, which indicates that the secondary metabolites of S. triandra may be toxic to the larvae of P. versicolora, or lack of nutrients necessary for larvae growth and development, resulting in the death of the larvae. [Ch, 4 fig. 1 tab. 23 ref.]
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Insect resistance and metabolome of four willow species

doi: 10.11833/j.issn.2095-0756.20210166
  • College of Forestry, Hebei Agricultural University, Baoding 071000, Hebei, China
  • Received Date: 2021-02-10
  • Rev Recd Date: 2021-06-20
  • Available Online: 2021-09-01
  • Publish Date: 2022-02-14

Abstract:   Objective  This purpose is to explore the insect resistance of different willows (Salix spp.) to Plagiodera versicolora, and to provide reference for the breeding of willow cultivars with strong insect resistance.  Method  S. caprea, S. matsudana, S. babylonica and 3 clones of S. triandra were tested in laboratory for insect feeding, and the differential secondary metabolites between insect resistant and non insect resistant groups were determined and screened.  Result  The survival rate of indoor feeding was 98% for S. matsudana, 93% for S. babylonica, 90% for S. caprea and 0 for S. triandra. There were 23 main different metabolites between the non insect resistant group and the insect resistant group, among which 15 were up-regulated, including fer-agmatine, L-ascorbic acid, gallic acid, (−)-epigallocatechin, (+)-gallocatechin, myricitrin, L-(+)-tartaric acid, D-(−)-arabinose, hydroxy-methoxycinnamate, N-feruloyl agmatine, gluconic acid, 2 ′-Hydroxygenistein, N-Acetyl-DL-tryptophan, eupatilin 3-glucoside, and hexadecylsphingosine, and 8 were down-regulated, including quercetin 3-O-(6″-O-malonyl)-galactoside, hesperidin, rutin, D-galactopyranuronate, 2,5-dihydroxy benzoic acid O-hexside, quercetin O-acetylhexoside, 3-hydroxy-5-methylphenol-1-Oxy-β-D-glucose, and bioquercetin.  Conclusion  S. triandra has the best insect resistance to P. versicolora. There are significant differences in the main secondary metabolites between the non insect resistant group and the insect resistant group, which indicates that the secondary metabolites of S. triandra may be toxic to the larvae of P. versicolora, or lack of nutrients necessary for larvae growth and development, resulting in the death of the larvae. [Ch, 4 fig. 1 tab. 23 ref.]

LI Wenjie, CUI Lulu, MA Yunjiao, et al. Insect resistance and metabolome of four willow species[J]. Journal of Zhejiang A&F University, 2022, 39(1): 153-158. DOI: 10.11833/j.issn.2095-0756.20210166
Citation: LI Wenjie, CUI Lulu, MA Yunjiao, et al. Insect resistance and metabolome of four willow species[J]. Journal of Zhejiang A&F University, 2022, 39(1): 153-158. DOI: 10.11833/j.issn.2095-0756.20210166
shu

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  • 杨柳科Salicaceae柳属Salix植物种类繁多，有450~520种；分布广泛，主要分布在北半球[1]，在中国属于原生树种；适应性强，耐湿，抗旱，耐低温，抗风性好，又具有很高的观赏价值。柳蓝叶甲Plagiodera versicolora又名柳圆叶甲，属鞘翅目Coleoptera叶甲科Chrysomelidae，是柳树主要食叶害虫，易在柳树较多或集中的地方大量发生。目前柳蓝叶甲仍以化学防治为主，因此对环境污染较大[2]。如何安全有效地预防和控制柳蓝叶甲的危害成为亟待解决的问题。植物抗虫性是植物与昆虫共同进化过程中形成的遗传性状，具有专一性、累积性、持续性、与环境的协调性、与其他防治措施的兼容性等特点，提高植物抗性是害虫综合防治策略中的一项重要措施[3-5]。在促使植物具有抗虫性的诸多因素中，化学因素起着重要作用[6]。穆丹等[7]研究证明：植物的气味能够有效调节和诱导柳蓝叶甲的行为。MATSUDA等[8]研究发现：绿原酸对某些柳树甲虫具有拮抗作用。IKONEN等[9]研究表明：叶甲拒食的柳叶中有高含量的绿原酸和水杨酸。由于技术的限制，植物次生代谢物基因转录和调控机制仍然没有得到解决。代谢组学技术在理解代谢网络和转录基因功能方面起着关键作用[10-14]。随着质谱、核磁共振等相关技术的迅速发展，代谢组学与其他组学技术的结合成为代谢组学研究的热点[12-14]。本研究选取旱柳S. matsudana、 黄花柳S. caprea、 垂柳S. babylonica以及三蕊柳S. triandra的3个无性系进行抗虫性测定，根据结果将柳树分成抗虫组与非抗虫组，再通过代谢组学技术分析抗虫组与非抗虫组之间的差异次生代谢物，为选育抗虫性强的柳树品种提供依据。

1.   材料与方法

    1.1.   试验材料

  • 选取三蕊柳的3个无性系W10、W4、W21，对照包括旱柳K24，黄花柳HHL，垂柳BD3，共6个材料。

    • 1.2.   试验方法

    1.2.1.   抗虫性指标测定

  • 在养虫盒底部放入湿润的滤纸，在每个供试材料上摘取完整嫩叶，用浸湿的棉球包裹叶柄以防止叶片失水，把嫩叶放于湿润的滤纸上，接入相同龄期的30只柳蓝叶甲幼虫，每个材料均设3组重复，每组5片叶，7 d后观察幼虫存活数量并计算存活率。

    • 1.2.2.   代谢组学分析

  • 植物代谢组学是一种高通量、无偏倚的综合分析[15]。将供试样品根据抗虫性指标测定的结果分为非抗虫组和抗虫组，采集新鲜叶片，液氮速冻用箔纸包好，−80 ℃冰箱保存备用。基于UPLC-Q-TOF-MS和GC-MS的代谢组学分析方法，对不同样品的代谢谱进行分析[16]。OPLS-DA是代谢组学数据分析中常用的分析方法，是PLS-DA的扩展[17]，通过去除与分组无关的系统自身差异筛选代谢差异物，使分离达到最大化[18]，再通过差异聚类热图分析[19]对样品和指标进行分类。

    • 1.3.   数据分析

  • 使用Excel 2010对数据进行整理；使用SPSS 20.0对数据进行独立样本t检验以及相关性分析；使用Origin 8.0作图。用OPLS-DA、单维分析(t检验)和差异倍数筛选组间差异代谢物。筛选获得的差异代谢物用http://www.hmdb.ca/和https://metlin.scripps.edu/数据库进行定性。

2.   结果与分析

    2.1.   室内幼虫存活率

  • 由图1可见：喂食黄花柳、旱柳、垂柳的幼虫平均存活率分别为90%、98%、93%，其中，喂食旱柳的幼虫存活率最高。喂食三蕊柳3个无性系的幼虫存活率均为0，在接虫后的第4天全部死亡。因此，把柳树分类非抗虫组(旱柳K24、黄花柳HHL和垂柳BD3)以及抗虫组(三蕊柳的3个无性系W10、W4和W21)。

    Figure 1.  Survival rate of indoor larvae

    • 2.2.   正交偏最小二乘法判别分析

  • 由OPLS-DA得分图(图2)显示：不同抗性组合模型参数中R2X=0.615，说明该模型对自变量X的解释程度为61.5%；R2Y=0.997，表示对分类变量Y的解释程度为99.7%；Q2=0.844，表示该模型对样本变量的预测程度为84.4%，说明OPLS-DA模型稳定性良好且预测能力较强。OPLS-DA的得分图显示非抗虫组与抗虫组的数据点在OPLS1上完全区分，表明两者存在明显化学差异。

    Figure 2.  OPLS-DA score chart of non-insect resistant group and insect resistant group

    • 2.3.   差异代谢物火山图

  • 从图3可以看出：在指定阈值P＜0.01时，共筛选出23种显著差异代谢物。结果显示：抗虫组与非抗虫组比较，其中上调的差异代谢物数目为15种，下调的代谢物数目为8种。

    Figure 3.  Volcano map of differential metabolites

    • 2.4.   差异代谢物筛选

  • 不同柳树样本中共检测到651个物质信号，经过标准样品数据库和参考文献比对，对有生物学重复的，采取将差异倍数，t检验的P值和OPLS-DA模型的变量投影重要度(VIP)相结合的方法来筛选差异代谢物，筛选的标准为差异倍数FC＞1，t检验P＜0.01和VIP＞1。由表1可见：经筛选后，非抗虫组与抗虫组共筛选出23种主要的差异代谢产物。其中阿魏酰胍丁胺、抗坏血酸、没食子酸、绿茶儿茶酚、没食子儿茶素、杨梅苷、葡萄酸铜、D-(−)-阿拉伯糖、4-羟基-3-甲氧基肉桂酸乙酯、N-阿魏酰胍丁胺、葡糖酸、2′, 4′, 5, 7-四羟基异黄酮、N-乙酰-DL-色氨酸、异泽兰黄素-3-葡萄糖苷、十六聚甲烷基鞘氨醇等15种产物表现为上调，而槲皮素-3-O-(6″-O-乙酰基)-β-D-吡喃半乳糖苷、橙皮甙、芦丁、聚半乳糖醛酸、2, 5-二羟基苯甲酸、槲皮素O-乙酰己烯苷、3-羟基-5-甲基苯酚-1-氧化基-β-D-葡萄糖、槲皮素等8种产物表现为下调。并且其中异泽兰黄素-3-葡萄糖苷在非抗虫组中并未检测到，但其在抗虫组的含量较高。

    化合物CAS编号非抗虫组
    代谢物丰度    		抗虫组
    代谢物丰度    		FCPVIP调节变化
    阿魏酰胍丁胺 N/A 360 000 1 923 333 5.343 0.001 1.708 上调
    抗坏血酸 50-81-7 4 197 336 37 266 667 8.879 0.004 1.647 上调
    槲皮素-3-O-(6″-O-乙酰基)-β-D-吡喃半乳糖苷 N/A 59 033 333 16 443 333 0.279 0.005 1.643 下调
    没食子酸 149-91-7 74 367 501 333 6.741 0.002 1.697 上调
    橙皮甙 520-26-3 20 766 667 1 596 673 0.077 0.001 1.695 下调
    绿茶儿茶酚 970-74-1 1 613 333 17 633 333 10.930 0.007 1.708 上调
    没食子儿茶素 4233-96-9 10 050 000 32 900 000 3.274 0 1.707 上调
    杨梅苷 17912-87-7 2 901 667 39 633 333 13.659 0 1.708 上调
    芦丁 153-18-4 48 933 333 6 833 339 0.140 0.010 1.618 下调
    葡萄酸铜 815-82-7 122 700 521 000 4.246 0.003 1.661 上调
    聚半乳糖醛酸 25990-10-7 58 167 30 233 0.520 0.009 1.608 下调
    D-(−)-阿拉伯糖 10323-20-3 674 333 1 220 000 1.809 0.010 1.603 上调
    4-羟基-3-甲氧基肉桂酸乙酯 N/A 70 100 221 667 3.162 0.008 1.594 上调
    N-阿魏酰胍丁胺 N/A 265 000 1 373 333 5.182 0 1.721 上调
    2, 5−二羟基苯甲酸 490-79-9 34 966 667 12 733 333 0.364 0 1.692 下调
    槲皮素O-乙酰己烯苷 N/A 53 433 333 10 726 667 0.201 0.005 1.627 下调
    葡糖酸 133-42-6 526 333 2 393 333 4.547 0.001 1.689 上调
    2′, 4′ , 5, 7-四羟基异黄酮 1156-78-1 2 000 003 12 063 333 6.032 0.009 1.594 上调
    3-羟基-5-甲基苯酚-1-氧化基-β-D-葡萄糖 N/A 2 373 333 158 433 0.067 0.002 1.682 下调
    槲皮素 20364-84-5 52 666 667 6 833 339 0.130 0.007 1.626 下调
    N-乙酰-DL-色氨酸 87-32-1 24 970 264 333 10.586 0.007 1.617 上调
    异泽兰黄素 3-葡萄糖苷 N/A 9 31 866 667 3 540 740.740 0.007 1.694 上调
    十六聚甲烷基鞘氨醇 N/A 4 260 000 6 223 333 1.461 0.009 1.675 上调
    　　说明：CAS编号是美国化学文摘服务社(chemical abstracts service, CAS)为化学物质编制的登记号；N/A表示该物质并未查询到
    　　　　　CAS编号

    Table 1.  Differential metabolite screening

    • 2.5.   差异聚类热图

  • 筛选具有显著差异(P＜0.05)的代谢物进行聚类热图分析，进一步挖掘特征，并对筛选后的差异代谢物归一化处理。由图4显示：抗虫组样本丰度明显区别于非抗虫组样本，抗虫组样本组内也有差异但不明显，非抗虫组中黄花柳的样本成分分布丰度与垂柳和旱柳存在差异。可以看出：能明显划分成4个区域冷暖色差分异，在非抗虫组丰度高而在抗虫组中丰度低的代谢物，为8种下调代谢产物；在抗虫组中丰度高而在非抗虫组中丰度低的代谢物为15种上调代谢物。

    Figure 4.  Differential metabolism clustering heat map

3.   讨论与结论

  • 本研究对4种不同柳树无性系进行了室内饲虫，结果显示三蕊柳对柳蓝叶甲表现出了高抗性；通过应用代谢组学方法对非抗虫组与抗虫组的次生代谢产物进行测定，筛选并最终鉴定出23种主要的差异代谢产物。上调次生代谢物包括阿魏酰胍丁胺、抗坏血酸、没食子酸、绿茶儿茶酚、没食子儿茶素、杨梅苷、葡萄酸铜、D-(−)-阿拉伯糖、4-羟基-3-甲氧基肉桂酸乙酯、N-阿魏酰胍丁胺、葡糖酸、2′, 4′, 5, 7-四羟基异黄酮、N-乙酰-DL-色氨酸、异泽兰黄素-3-葡萄糖苷、十六聚甲烷基鞘氨醇等15种。下调次生代谢物包括槲皮素-3-O-(6″-O-乙酰基)-β-D-吡喃半乳糖苷、橙皮甙、芦丁、聚半乳糖醛酸、2, 5-二羟基苯甲酸、槲皮素O-乙酰己烯苷、3-羟基-5-甲基苯酚-1-氧化基-β-D-葡萄糖、槲皮素等8种。这些差异特定代谢物影响的机制尚待进一步研究。

    凌嘉昊[20]通过调查野外虫害等级和室内取食率来对比簸箕柳S. suchowensis和三蕊柳的抗虫性，发现三蕊柳表现出了高抗性，与本研究结果一致；该研究还发现三蕊柳的挥发物会使柳蓝叶甲产生趋避反应，并且鉴定分析显示22种挥发物组分在释放量方面有显著差异。而本研究发现：柳蓝叶甲的幼虫对三蕊柳存在取食行为，在接虫后的第4天全部死亡，表明三蕊柳除挥发物会对柳蓝叶甲产生趋避反应外，其次生代谢产物可能会对柳蓝叶甲幼虫产生毒害作用，或缺乏幼虫生长发育所必需的营养物质而导致其死亡。黄酮类物质是植物中一类重要的次生代谢物质，可以影响昆虫的行为和代谢，使之产生忌避、拒食等行为，破坏昆虫的正常代谢，严重时导致昆虫中毒甚至死亡，是对昆虫有毒的一类次生代谢产物[21]。生物碱是一大类含氮的碱性物质，对昆虫的防御作用主要表现在阻碍昆虫的取食以及引起昆虫中毒2个方面[22]。有研究表明：树木中的酚酸类物质含量越高，对天牛Cerambycidae的抗性就越强，但不同酚酸在不同无性系间的变化规律随其种类的不同而不同[23]。本研究可为培育抗虫性强、性状优良的柳树品种提供理论基础，三蕊柳的具体抗虫机制还有待进一步研究。

Reference (23)

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  • 1.   材料与方法

  • 1.1.   试验材料

  • 1.2.   试验方法

  • 1.2.1.   抗虫性指标测定
  • 1.2.2.   代谢组学分析

  • 1.3.   数据分析

  • 2.   结果与分析

  • 2.1.   室内幼虫存活率

  • 2.2.   正交偏最小二乘法判别分析

  • 2.3.   差异代谢物火山图

  • 2.4.   差异代谢物筛选

  • 2.5.   差异聚类热图

  • 3.   讨论与结论

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