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定量评价南方根结线虫趋性方法的建立及应用

花匠小妙招
2025-01-05 14:51

摘要: 为了能够定量评价南方根结线虫Meloidogyne incognita 2龄幼虫的趋化性，本研究以Pluronic F-127胶体为载体，建立了一种能够借助体式显微镜相机及ImageJ灰度值统计法对根结线虫移动轨迹进行量化检测，从而判断根结线虫2龄幼虫趋性的测试方法，并用10种已报道的具有趋性活性的化合物验证了方法的可靠性。另外，采用该方法测定了40种化合物对南方根结线虫的趋性活性。研究结果表明，10 μg/mL质量浓度下，22种测试化合物具有吸引活性、11种化合物具有驱避活性，其中对茴香胺和间苯三酚分别具有较强的吸引和驱避线虫活性。进一步研究表明，对茴香胺对南方根结线虫2龄幼虫具有低浓度吸引和高浓度驱避的趋性活性，而间苯三酚对该幼虫的驱避活性随浓度增高而增强。该研究为快速定量评价化合物对南方根结线虫2龄幼虫的趋性及发现高活性根结线虫驱避剂提供了一种可靠的方法。

Abstract: To quantitatively evaluate the chemotaxis of the second-stage juveniles (J2s) of Meloidogyne incongnita, an experimental method using stereomicroscope camera and ImageJ grayscale statistics method to detect the chemotaxis of J2s by analyzing the movement trajectory of the root-knot nematode in Pluronic F-127 gel was established, and verified the reliability of the method with 10 reported compounds to induce chemotaxis. In addition, the method was used to determine the chemotaxis of 40 compounds against M. incongnita. The results showed that 22 compounds had attractive activity and 11 compounds had repellent activity at the concentration of 10 μg/mL, with p-anisidine and phloroglucinol being the most attractive and repellent to nematodes, respectively. Further studies showed that p-anisidine exhibited attraction activity at low concentrations and repellent activity against J2s of M. incongnita at high concentrations, while the repellent activity of phloroglucinol increased with higher concentrations. This study provides a reliable method for rapid and quantitative evaluation of the chemotaxis of compounds against M. incongnita and for the discovery of highly active root-knot nematode repellents.

图 1 模型设计示意图

A为待测化合物侧，B为溶剂侧，中央白色部分为空白缓冲区，红色圆圈为线虫注射区。

Figure 1. Model design diagram

A represents the test compound side and B represents the solvent side, white part represents blank buffer zone, red circle represents nematode injection zone.

图 2 10 μg/mL十三烷处理侧与溶剂侧对比：A为十三烷侧，B为溶剂 (95%乙醇) 侧

Figure 2. Comparison between 10 μg/mL tridecane and solvent treatment: A represents tridecane treatment, B represents solvent (95% ethanol) treatment

图 3 50 μg/mL苯并噻唑处理侧与溶剂侧对比：A为苯并噻唑侧，B为溶剂 (95%乙醇) 侧

Figure 3. Comparison between 50 μg/mL benzothiazole and solvent treatment: A represents benzothiazole treatment, B represents solvent (95% ethanol) treatment

图 4 10 μg/mL对茴香胺处理侧与溶剂侧对比：A为对茴香胺侧，B为溶剂 (95%乙醇) 侧

Figure 4. Comparison between 10 μg/mL p-anisidine and solvent treatment: A represents p-anisidine treatment, B represents solvent (95% ethanol) treatment

图 5 10 μg/mL间苯三酚处理侧与溶剂侧对比：A为间苯三酚侧，B为溶剂 (95%乙醇) 侧

Figure 5. Comparison between 10 μg/mL phloroglucinol and solvent treatment: A represents phloroglucinol treatment, B represents solvent (95% ethanol) treatment

图 6 梯度浓度的对茴香胺(A)和间苯三酚(B)对南方根结线虫的趋性活性

Figure 6. Chemotaxis activity of p-anisidine (A) and phloroglucinol (B) with gradient concentration against M. incognita

表 1 验证化合物对南方根结线虫的趋性活性

Table 1 Validation of chemotaxis activity of compounds against M. incognita

化合物　　　
Compound　　　文献报道
趋性
Reported
chemotaxis测试质量浓度
Test
concentration/
(μg/mL)趋性指数
Chemotaxis
index,
CI 硝酸钾
Potassium nitrate 驱避[8]
Repellent 1000 0.54 ± 0.06 百里酚
Thymol 驱避[7]
Repellent 50 0.58 ± 0.04 苯并噻唑
Benzothiazole 驱避[9]
Repellent 50 0.62 ± 0.04 2-辛酮
2-Octanone 驱避[10]
Repellent 50 0.77 ± 0.03 二苯并呋喃
Dibenzofuran 吸引[9]
Attractant 10 1.18 ± 0.03 3-甲氧基苯甲酸
m-Anisic acid 吸引[13]
Attractant 10 1.26 ± 0.03 水杨酸甲酯
Methyl salicylate 吸引[11]
Attractant 1 1.31 ± 0.05 1,5-戊二胺
1,5-Pentanediamine 吸引[17]
Attractant 10 1.36 ± 0.07 1,4-丁二胺
1,4-Butanediamine 吸引[17]
Attractant 10 1.46 ± 0.04 十三烷
Tridecane 吸引[7]
Attractant 10 1.53 ± 0.07

表 2 测试化合物对南方根结线虫的趋性活性

Table 2 Chemotaxis activity of test compounds against M. incognita

化合物　　　　　
Compound (10 μg/mL)　　　　　趋性指数
CI化合物　　　　　
Compound (10 μg/mL)　　　　　趋性指数
CI 对茴香胺 p-Anisidine 1.53 ± 0.09 N-乙基苄胺 N-Ethylbenzylamine 1.06 ± 0.06 4-羟基-5,6-二甲基吡喃-2-酮
4-Hydroxy-5,6-dimethylpyran-2-one 1.45 ± 0.11 2,4-二氯苯乙酮 1-(2,4-dichlorophenyl)ethanone 1.06 ± 0.05 混旋樟脑磺酸 DL-Camphor sulfonic acid 1.45 ± 0.08 桂皮醛 Cinnamaldehyde 1.05 ± 0.08 四水合酒石酸钾钠 Potassium sodium tartrate tetrahydrate 1.44 ± 0.11 1-辛醛 Octyl aldehyde 1.03 ± 0.04 对正丁基苯胺 4-n-Butyaniline 1.41 ± 0.13 α-紫罗兰酮 α-Ionone 1.01 ± 0.03 苯甲酸 Benzoic acid 1.39 ± 0.10 2,6-二氯苯胺 2,6-Dichloroaniline 0.99 ± 0.04 香芹酮 (–)-Carvone 1.30 ± 0.13 2,6-二氯-4-氨基苯酚 4-Amino-2,6-dichlorophenol 0.99 ± 0.03 苯甲酸乙酯 Ethyl benzoate 1.30 ± 0.10 水杨羟肟酸 Salicylhydroxamic acid 0.97 ± 0.03 2-氨基苯并咪唑 2-Aminobenzimidazole 1.29 ± 0.08 对羟基苯丙酸甲酯
Methyl 3-(4-hydroxyphenyl)propionate 0.96 ± 0.11 三苯基膦 Triphenylphosphine 1.27 ± 0.12 1-四氢萘酮 1-Tetralone 0.93 ± 0.12 1-氯-4-硝基苯 4-Nitrochlorobenzene 1.26 ± 0.09 邻氨基苯酚 2-Aminophenol 0.93 ± 0.06 对羟基苯甲醛 4-Hydroxybenzaldehyde 1.24 ± 0.05 碳酸丙烯酯 Propylene carbonate 0.93 ± 0.02 4-羟基-6-甲基-2-吡喃酮 4-Hydroxy-6-methyl-2-pyrone 1.23 ± 0.08 2-甲基间苯二酚 2-Methylresorcin 0.92 ± 0.05 苯甲腈 Benzonitrile 1.17 ± 0.08 β-香茅醇 (–)-β-Citronellol 0.91 ± 0.04 乙嘧酚 Ethirimol 1.17 ± 0.04 2,3-二甲氧基苯酚 2,3-Dimethoxyphenol 0.90 ± 0.05 2-溴吡啶 2-Bromopyridine 1.16 ± 0.07 噻吩-2-羧酸甲酯 Thiophenate methyl 0.82 ± 0.09 吡啶 Pyridine 1.09 ± 0.10 4-乙基苯胺 4-Ethylaniline 0.82 ± 0.06 3,5-二氯-4-甲基苯胺 3,5-Dichloro-4-methylaniline 1.08 ± 0.03 香叶醇 Geraniol 0.82 ± 0.04 2,6-二氯苯硼酸 (2,6-Dichlorophenyl)boronicacid 1.07 ± 0.04 亚磷酸二乙酯 Diphenyl ethylhexyl phosphite 0.81 ± 0.05 1-溴辛烷 1-Bromooctane 1.06 ± 0.09 间苯三酚 Phloroglucinol 0.69 ± 0.06

表 3 对茴香胺和间苯三酚的浓度梯度对南方根结线虫的趋性活性相关性分析

Table 3 Correlation analysis of concentration chemotaxis activity of p-anisidine and phloroglucinol against M. incognita

供试药剂
Test compound回归方程 (y = )
Regression equation相关系数
Correlation coefficient, r 对茴香胺
p-Anisidine −0.5873logx + 1.775 0.7742 间苯三酚
Phloroglucinol −0.1786logx + 0.8698 0.9797 [1]

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