Enzyme Activity And Expression Of Key Genes Related To Phenolic Synthesis In Pecans After Botryosphaeria Dothidea Infection

Botryosphaeria dothidea is the causative fungus of pecan dry rot,infecting the branches and stems,causing the death of pecans,leading to serious economic losses.With the development of disease-resistant varieties,it was found that grafted seedlings with Hunan pecan and American pecan as rootstocks and Zhejiang pecan as scion were good in resistance and high in fruit yield.At present,they have been widely planted,but the research on the mechanism of disease resistance is still lacking.Early studies found that phenolic substances and related enzymes(PAL,PPO,POD)in severely diseased pecans are lower than those inanti-disease cultivars.These indicators play an important role in the identification of disease resistance.Therefore,we need to carry out further research on the synthesis of phenolic substances in the resistance reaction of pecans to B.dothidea infection.In this study,HG,AG and ZJ were used as plant materials to analyze the susceptibility and infection status of different varieties of pecans after inoculation.Secondly,the contents of total phenol and total flavonoids in different varieties were measured,and the enzyme activities related to disease resistance(PAL,PPO,POD,CAD,cell wall hydrolase)were analyzed.Finally,several key genes(PAL,C4 H,4CL,and CHS)of the phenylpropane metabolism pathway in the hickory defense pathway were cloned and analyzed,and the expression dynamics at different infection periods were studied.The main results of this study are as follows:1.After artificial inoculation of B.dothidea,ZJ is the most susceptible and HG is the most resistant.The susceptibility sequence of pecans is: ZJ> AG> HG.Scanning electron microscopy observation showed that infection of B.dothidea mycelium started from where it was inoculated,and the early hyphae attached to the host surface;in the middle stage,the hyphae invaded the epidermal structure and gradually expanded to form a network;later,the mycelium entered the tissue structure,wraped and destroied the tissue shape.However,the symptoms of susceptible varieties were more serious than that of resistant ones during the same period.2.After infection by B.dothidea,the total phenol and flavonoid content of HG was significantly higher than that of ZJ,while the content of AG was slightly lower than that of HG.PAL,a key enzyme in the metabolic pathway of phenylpropane,increased and then decreased in all three varieties,but the accumulation of enzyme activity in the resistant varieties was significantly higher than that in the susceptible varieties after inoculation.After inoculation,the activity of POD and PPO of HG showed an increase,while the ZJ showed no obvious fluctuation.The ?-1,3 glucanase in HG and AG showed an upward trend after infection,on the contrary,ZJ showed a decreasing response.After inoculation,the chitinase showed fluctuating changes in all varieties,the activity between the varieties was not much different,and the resistance behavior was not obvious.3.The complete sequences of the PAL,C4 H,4CL and CHS genes of pecans were obtained by PCR and RACE experiments.The results showed that the open reading frame of the pecan PAL gene is 2160 bp,which encodes 720 amino acids.The C4 H gene contains 1521 bp ORF and encodes 507 amino acids.The 4CL gene contains 1638 bp ORF,which encodes 545 amino acids.The CHS gene contains 1170 bp ORF that encodes 389 amino acids.The results of homology comparison analysis show that the nucleotide sequences of these four genes are more than 97% similar among the three varieties,and the homology was about 90% when compared with the sequences of walnut genus from the walnut family,and the homologyrange from 70% to 85% when compared with other genera.Phylogenetic tree analysis showed that the pecan gene sequence and Juglanaceae were clustered together,and the others from the same family were clustered together.4.The expression dynamics of key genes(PAL,C4 H,4CL and CHS)in phenylpropane metabolic pathway of different pecans infected by B.dothidea were determined by real-time fluorescence quantitative PCR.The expression of PAL gene was up-regulated in the early stage of inoculation,and the stronger the resistance of pecan was,the greater and earlier the expression of PAL gene was.Pathogen infection rapidly upregulated the expression of C4 H gene.The AG and HG reached their peaks on the 3rd day,which were 9.3 times and 9 times that of ZJ,respectively.After inoculation,there are differences in the expression level of 4CL gene in different varieties of pecans,and the relative expression level of ZJ is low.The better the resistance was,the higher the expression level of 4CL gene was,and the expression level of this gene was maintained at a higher level after 5 days of inoculation.The CHS gene is a key gene for the synthesis of flavonoids.The expression of HG among the three varieties is significantly higher than that of the other two varieties.