| Pine wilt disease, induced by pine wood nematode(Bursaphelenchus xylophilus), is a kind of catastrophic forest diseases. It would endanger forest ecological security and cause huge economic losses. Pinus massonianaLamb. is the most widely distributed native pine species and suffer the most serious damage from pine wood nematodes in China. Resistant trees exist among P. massoniana population. Use of resistant trees instead of susceptible trees is the most economic and effective way to control pine wood nematodes. However, the resistant mechanism remains poorly understood. The plant materials in this study were different resistant P. massoniana clones that had been selected by a serious of inoculating test. Some physiological and biochemical responses were measured and anatomical structure characteristic in the stem was observed in the clones post inoculated. The high throughput sequencing was used for selecting the differentially expressed genes between high resistant and susceptible genotypes post inoculation at transcriptome level.These research were explored the molecular mechanism of resin as the defense when the plant was invaded by pine wood nematode. These would be the theoretical basis for evaluation and selecting high resistant clones in the early age. The results showed as below.(1) The resin yield and component content were measured in different resistant clones. 19 components were detected in P. massoniana. The percentage of monoterpenes, sesquiterpene and diterpene was 12.08%, 5.16% and 79.53%. There was non-significant difference in content of α-pinene, β-phellandrene, 4-carene, sandaracopimaric acid and neoabietic acid between high resistant and susceptible clones. The other 14 components content between high resistant and susceptible clones were statistically significant difference. There was significantly positively correlated between resistance degree and resin yield, and the correlation coefficient was 0.169. The resistance degree was also significantly positively correlated with the content of camphene, β-pinene, β-Myrcene and D-limonene. The correlation coefficient was from 0.507 to 0.622. There was significantly negative correlation between resistance degree andlongifolene content, and the correlation coefficient was-0.420. These results indicted that the resistant of P. massoniana could be preliminarily evaluated by resin yield and component content. When the plants were not attacked by nematodes, there was non-significant difference in content of α-pinene between high resistant and susceptible clones. The average of α-pinene was distributed from 178 to 198 μg·mL-1. The percentage of β-pinene and D-limonene in different resistant clones was significant difference. The content of these two components in high resistant clones was 2.11 times and 2.42 times more than susceptible ones. The longifolene content in susceptible clones were 1.92 times higher than the high resistant ones. There was significant difference in content of α-pinene, β-pinene, D-limonene and longifolene between different resistant clones post inoculated nematode. The α-pinene content in high resistant ones was lower than susceptible ones at 1 day post inoculated, and higher than susceptible ones from 7d to 15 d. There was significant difference in content of β-pinene between different resistant clones at 1d and 7d. The percentage of D-limonene in high resistant clones was 1.79 times higher than susceptible ones.The longifolene content in susceptible ones was 13.32% higher than high resistant ones. The results demonstated α-pinene, β-pinene, D-limonene and longifolene were closely related with resistant. When the P. massoniana invaded by pine wood nematode, terpene percentage of the resin was changed for defense.(2) As the structre of secreting and transporting, resin canal was observed and there was significant difference between different resistant clones. The resin area in high resistant clones was 1.4 times higher than susceptible ones post inoculation. Also the resin canal in diferent resin yield clones was surveyed. The canal area in the low yielder clones was 10.83% lower than the high yielders. The resin canal area amplification in high yielders was 2.02 times higher than low ones. The similar trend indicated defense of respond to stimulate in different resistant clones may resemble the different yield clones. These verified the significant positive correlation between resistant degree and resin yield from anatomical structure.(3) The dynamic variation of signal molecular including H2O2, O2·-, NO and Ca2+was detected in needles of different resistant clones post inoculation. The H2O2 content was significant difference between different resistant clones during the whole nematode invading time. The H2O2 content in high resistant ones was 14.22% higher than susceptible ones at 1d. But at the other day, the H2O2 content in high resistant ones was lower. The content in susceptible ones was 1.7 times higher than high resistant ones at 15 d. The O2·- content was significant difference between different resistant clones at 3d~30d. The content in high resistant ones was 21.27%, 26.36%, 16.43% higher than susceptible ones at 3d, 7d, 15 d. And the content in susceptible ones was 43.35% higher than high resistant ones at 30 d. The NO content was significant difference between different resistant clones except 7d. The variation trend of Ca2+content was similar between different resistant clones post inoculation, but there was significant difference at every time points.The content in high resistant ones was 15.83% higher than susceptible ones at 1d. And the content in susceptible ones was 27.69%, 1.86 times higher than high resistant ones at 3d, 7d. The content in high resistant ones was 23.85% higher at 15 d and 41.74% lower than susceptible ones at 30 d. All of the results showed that signal molecular transmited the stimulus message and involved in systemic defense when P. massoniana was infected by nematode. The signal molecular played an important role in resistant mechansim of P. massoniana.(4) The content of plant endogenous hormones including salicylic acid(SA) and jasmonic acid(JA) was significant difference between different resistant clones post inoculation. SA content was significant difference between different resistant clones at different time points. The content in high resistant ones was 2.88 times and 2.36 times at 1d and 30 d and was 1.14~1.68 times higher susceptible ones at other time point. The JA content was significant difference between different resistant clones except 7d. The content in high resistant ones was 3.52 times, 1.93 times, 1.56 times higher than susceptible ones at 1d, 3d, 30 d. However, the content in high resistant ones was 67.74% lower than susceptible ones at 15 d. The results indicated plant endogenous hormones had been involved in defense against invading by nematode. They could regulate the process of resin biosynthesis.(5) The transcriptome information was obtained from P. massoniana stem RNA at 1d, 15 d and 30 d post inoculation by RNA-seq. 80 340 unigenes were assembled and average length was 675 nt. Among of them, 50 227(62.5%)unigenes obtained functional annotation. U2 af, β-TUB, EF and CAC were selected as reference genes by geNorm, NormFinderand BestKeeper. Both of them could express stably in different genotypes at different time points. Differentially expressed genes(DEGs) were confirmed at different time points and the number of DEGs in susceptible genotypes was higher than the high resistant ones. Most of the DEGs were down-regulation. These results indicated that physiological and biochemical responses were acute in susceptible genotypes post inoculation. The high resistant and susceptible genotypes had 371 and 2 179 DEGs at 15 d, which were the highest number of DEGs presented. The number was lowest at 30 d. The expression of some terpene biosynthesis-related DEGs including DXS, HDS, MECPS, HMGR,(-)-α-pinene synthase,(-)-β-pinene synthase,(+)-α-pinene synthase,(-)-α/β-pinene synthase,(-)-limonene synthase, longifolene synthase,Δ-selinene synthase and CYP720 B changed significantly. There was significant difference between different resistant genotypes at expression of DXS, HMGR,(-)-α-pinene synthase,(-)-β-pinene synthase,(+)-α-pinene synthase,(-)-limonene synthase, longifolene synthaseand CYP720 B, which indicted these genes may related to the resistant. Additionally, the expression of the pathogenesis-related genes, cell wall-related genes, reactive oxygen species(ROS) responsive genes and other resistant-related genes had changed in high resistant and susceptible genotypes post inoculation. The genes encoding PR-3 and defensin-1 between two genotypeswere significant difference. The expression of these unigenes indicated by qRT-PCR agreed well with transcriptome analysis. The expression of DEGs was matched with dynamic variation of resin component and signal molecular, indicted systemic defense was activated when P. massoniana was infected by nematode. Multiple resistance mechanisms were activated together to resist the infection of nematode. |
PDF Full Text Request