基于多组学量化松材线虫入侵对寄主马尾松的影响

徐清华; 郭芷晴; 贾佳宇; 苏军

doi:10.19303/j.issn.1008-0384.2024.06.011

基于多组学量化松材线虫入侵对寄主马尾松的影响

doi: 10.19303/j.issn.1008-0384.2024.06.011

1.
先正达（中国）投资有限公司，上海　200126
2.
福建农林大学金山学院，福建　福州　350002
3.
福建农林大学未来技术学院，福建　福州　350002

基金项目: 福建省林业科技项目（LZKG-202205）

详细信息

作者简介:
徐清华（1981 — ），女，硕士，高级工程师，主要从事花卉病虫害及林业有害生物防治技术研究，E-mail：qinghua.xu@syngentagroup.cn

通讯作者:
苏军（1986 — ），男，博士，副教授，主要从事森林保护研究，E-mail: junsu@fafu.edu.cn

中图分类号: S763.7
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出版历程
- 收稿日期: 2024-01-19
- 修回日期: 2024-04-01
- 网络出版日期: 2024-07-10
- 刊出日期: 2024-06-28

Multiomically Quantified Effects of Nematode Invasion on Pinus massoniana

XU Qinghua^1
,,
GUO Zhiqing²,
JIA Jiayu³,
SU Jun^{3
, ,}

1.
Syngenta (China) Investment Co., Ltd., Shanghai　200126, China
2.
College of Jinshan, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China
3.
College of Future Technology , Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的探究松材线虫入侵对寄主马尾松的影响。方法以4年生马尾松（Pinus massoniana）为试材，以皮接法接入5 000条松材线虫的马尾松为处理组[PWN（+）组]，以接入无菌水的马尾松为对照组[PWN（−）组]，基于多组学（表型组学、转录组学、宏基因组学、代谢组学）量化松材线虫入侵马尾松14 d后的变化。结果与PWN（−）组相比，PWN（+）组活性氧（ROS）含量和H₂O₂含量分别显著上升3.2倍和1.7倍（P＜0.05）；编码应激反应通路的c60547.graph_c0、c82953.graph_c0在PWN（+）组中的表达水平显著高于PWN（−）组（P＜0.05），萜类生物合成途径通路的c64867.graph_c0、c68789.graph_c0及合胞体形成通路的c81022.graph_c0在PWN（+）组中的表达水平显著低于PWN（−）组（P＜0.05）；PWN（+）组的微生物多样性显著低于PWN（−）组（P＜0.05），肉座菌目（Hypocreales）是其体内的优势微生物，在生物信息数据库KEGG的功能注释和丰度信息主要集中在复制修复通路、DNA复制通路（PATH:ko03030）、DNA复制蛋白通路（BR:ko03032）；代谢组上、下调的差异代谢物分别有365个和351个，PWN（+）组中根皮素、熊去氧胆酸、羧苄青霉素等物质的含量会增加以抵御松材线虫的侵染，差异显著的代谢物显著富集于ABC转运蛋白通路、花生四烯酸代谢通路、类黄酮生物合成通路、甘油磷脂代谢通路。结论当松材线虫入侵马尾松时，寄主会启动一系列复杂的防御反应。这些反应并非孤立存在，而是通过多种机制协同作用，共同应对松材线虫的侵染。上述结果有助于从多组学角度阐明松材线虫入侵对马尾松的影响，并为松材线虫病害诱导的森林衰退和寄主植物马尾松之间的相互作用提供依据。
- 松材线虫 /
- 马尾松 /
- 活性氧 /
- 转录组 /
- 宏基因组 /
- 代谢组
Abstract: Objective Effects of pine wood nematode (PWN) invasion on Pinus massoniana were quantified based on multiomics. Method Among 4-year-old Masson pine plants, a treatment, PWN(+), by inoculating 5 000 Bursaphelenchus xylophilus and a control, PWN(−), with the injection of sterile distilled water into the tree trunks were subjected to a 14 d post-inoculation followed by a quantified study including phenomics, transcriptome, microbiome, and metabolome. Result The pine trees in the PWN(+) group significantly increased over control on the contents of the reactive oxygen species (ROS) by 3.2-fold and hydrogen peroxide (H₂O₂) by 1.7-fold (P＜0.05), on the expressions of c60547.graph_c0 and c82953.graph_c0 in the unigenes encoding stress responsive pathways (P＜0.05), as well as on the expressions of c64867.graph_c0 and c68789.graph_c0 in the terpenoid biosynthesis pathways; and significantly lower on the c81022.graph_c0 in syncytium formation (P＜0.05) and the microbial diversity (P＜0.05). Hypocreales was the dominant order of microorganisms in the PWN(+) group. The functional annotations and abundance information from the KEGG database were mainly related to the replication and repair pathways, DNA replication pathway (PATH:ko03030), and DNA replication protein pathway (BR:ko03032). There were 365 upregulated and 351 downregulated differentially regulated metabolites in the metabolome. Metabolites like phloretin, ursodiol, and carbenpenicillin were notably enriched in the ABC transporters pathway, arachidonic acid metabolism, flavonoid biosynthesis, and glycerophospholipid metabolism in the PWN-infected pine wood to resist the invasion. Conclusion After PWN was inoculated in Masson pine, the host responded with a series of complex defensive reactions initiated by various mechanisms. Through multiomics, some such interactions between the species were unveiled providing clues for combating forestry declination by the infestation.
- Pine wood nematode (Bursaphelenchus xylophilus) /
- Pinus massoniana /
- reactive oxygen species /
- transcriptome /
- metagenome /
- metabolome

HTML全文

图 1 松材线虫接种效果的验证

以PWN作为阳性对照，以ddH₂O作为阴性对照。

Figure 1. Validation of PWN inoculation efficiency

PWN was used as a positive control and ddH₂O was used as a negative control.

下载: 全尺寸图片幻灯片

图 2 有无接种松材线虫对寄主马尾松ROS（a）和H₂O₂（b）含量的影响

不同小写字母代表差异显著（P＜0.05）。

Figure 2. Effects of PWN inoculation on ROS (a) and H₂O₂ (b) contents in P. massoniana

Different lowercase letters represent significant difference at P＜0.05.

下载: 全尺寸图片幻灯片

图 3 松材线虫入侵前后马尾松体内抗性基因的变化

Figure 3. Changes of resistance genes in P. massoniana before and after PWN inoculation

下载: 全尺寸图片幻灯片

图 4 不同处理条件下马尾松微生物群的多样性和结构功能

不同处理组微生物群落的多样性Shannon（a）、Simpson（b）、Invsimpson（c）指数差异，不同小写字母代表差异显著（P＜0.05）；（d）代表样本在目水平上的聚类柱状图；（e）样本微生物在KEGG level3水平上具有显著性差异的功能预测。

Figure 4. Diversity and structural functions of microflora in P. massoniana under varied conditions

Diversity of microbial communities in different sample groups differed in Shannon (a), Simpson (b), and Invsimpson (c) indexes; different lowercase letters represented significant differences at P＜0.05; d: clustering histogram of samples at order level; e: functional prediction of microorganisms at KEGG level 3.

下载: 全尺寸图片幻灯片

图 5 松材线虫入侵前后诱导的马尾松代谢物组成

（a）不同处理的OPLS-DA图；（b）不同处理间差异代谢物筛选火山图；（c）不同处理的差异代谢物VIP散点图。显示基于VIP值的前20种差异代谢物；(d)不同处理的代谢通路分析气泡图。（e）不同处理的所有差异代谢物的层次聚类分析热图。

Figure 5. Metabolites in P. massoniana before and after PWN inoculation

(a) OPLS-DA plots for different treatments; (b) differential metabolite screening volcano plot between different groups; (c)VIP scatter plots of differentially differentiated metabolites in different groups; top 20 differential metabolites based on VIP values are displayed; (d)Bubble map of metabolic pathway analysis in different groups;(e) Hierarchical cluster analysis heat map of all differential metabolites of groups.

下载: 全尺寸图片幻灯片

表 1 特异引物

Table 1. Specific primers

引物名称 Primer name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）
内对照 BxPP2A	AACGTCATTTCTAGCCGCCA	TCAGCCCTACAAACCCCTCT
Bx-cathepsin	TTGCATTCTACGGCCAGTCC	ACTGACTTTCGATGGCTCCG
c60547.graph_c0	TAAATTCCAAGTGCCCCGCA	ACCGTGATACACATTTCAGA
c64867.graph_c0	AAATCGTGTGTGTCCCTGCA	GGTTGCAATGATAACGGCCC
c68789.graph_c0	CGCCCGAATCTCTGCACTTA	TCGATGGTCTTGGTGATGGC
c81022.graph_c0	TTGGCTGTACAGATTCCCGT	ACCTATGGATGTCTGCTCCA
c82953.graph_c0	ACTGTTAACCTGGCTCACGG	CTACGCAAATTCACCGCCAC

下载: 导出CSV

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