Study On The Pathogeny Of Cyclocarya Paliurus Anthracnose And Its Resistance Mechanism

Cyclocarya paliurus（Batal.）Iljinskaja belongs to Juglaruiaceae and Cyclocarya.It is an important endangered and rare plant in China.It has high medicinal,wood and ornamental value.At present,the research on C.paliurus focuses on artificial breeding,chemical active ingredients and its medicinal and health care functions,but there are few reports on its disease and its disease resistance mechanism.In recent years,during the disease investigation,it was found that the anthracnose of C.paliurus occurred in many plantations in Jiangsu,with the characteristics of large range,wide distribution,and rapid spread.The food industry has caused serious economic losses.Based on this,the present study collected anthracnose diseased leaves from C.paliurus plantations in three cities of Jiangsu Province,isolated and identified the pathogen,and clarified the species composition,distribution range and hazard characteristics of C.paliurus anthracnose,in order to provide scientific basis for targeted prevention and control in the future.In addition,from the physiological,biochemical,transcriptional and metabolic levels,the defense strategies of C.paliurus in response to the dominant pathogen C.fructicola infection were systematically studied,in order to clarify the disease resistance mechanism of C.paliurus.In addition,the defense strategies of C.paliurus against the dominant pathogen C.fructicola infection were systematically studied from the physiological,biochemical,transcriptional and metabolic levels,so as to clarify the disease resistance mechanism of C.paliurus against anthracnose infection,in order to provide an important theoretical basis for further mining the disease resistance genes of C.paliurus and the breeding of disease resistant varieties of C.paliurus in the future.In addition,the antifungal effect of secondary metabolites of C.paliurus on plant pathogenic fungi was clarified for the first time,and its mechanism was preliminarily revealed,which laid a foundation for the development of new control agents.The main research results are as follows:1.Forty-four single spore strains were obtained from the leaves of C.paliurus planted in three cities of Jiangsu Province（Nanjing,Yancheng and Changzhou）.By Bayesian/Maximum Likelihood multigene phylogenetic analysis and morphological identification（morphological characteristics such as colony,conidia,appressorium,ascospore and ascus）,6 strains were identified as C.aenigma;10 strains were identified as C.gloeosporioides;28 strains were identified as C.fructicola,so C.fructicola was the pathogenic dominant species of C.paliurus anthracnose.The virulence determination results of 14 representative strains of 3 species showed that there were significant differences in virulence among species.The virulence of C.gloeosporioides and C.fructicola was relatively strong,while the virulence of C.aenigma is the weakest;the inoculation symptoms of each strain is basically the same as those in the field.The results of the optimal culture temperature measurement showed that the 14 representative strains all showed similar growth patterns on PDA medium:the optimal growth temperature of the three species was 25℃-30℃,and none of them were observed mycelial growth at 5℃;isolates of C.aenigma and C.fructicola are more sensitive to high temperature,grow very slowly（or fail to grow）at 40°C,and their average growth rate is slower than that of C.gloeosporioides isolates.2.Using the dominant pathogen of Cyclocarya paliurus anthracnose as the inoculation material,the resistance of C.paliurus provenances from 6 different provinces/autonomous regions was identified.In vivo inoculation results showed that the incidence of anthracnose was between 42 and 97.3%,and the disease index was between 16.5 and 83.4;the incidence and disease index of Wufeng provenance were the lowest among the six provenances,and there is an obvious"chlorosis"phenomenon around the lesions,which is the most disease-resistant.After inoculation of fruit anthracnose from the Jinggangshan Provenance,the lesions were large and connected into pieces.The incidence rate and disease index were the highest among the six provenances,and they were the most susceptible.The results of the in vitro inoculation test showed that the C.paliurus of Wufeng provenance produced an obvious"chlorotic circle"around the lesions 72 hours after inoculation,and the lesions could not continue to expand from 72 hours to 7 days.The lesions of C.paliurus from Jinggangshan provenance continued to expand from 24h to 7 d after inoculation.Therefore,Wufeng provenance was identified as disease-resistant provenance,and Jinggangshan provenance was identified as susceptible provenance.3.After inoculation with C.fructicola,there were significant differences in the physiological and biochemical indexes of the resistant and susceptible provenances of C.paliurus.The results of hormone determination showed that the content of SA in the resistant provenance C.paliurus increased first and then decreased after inoculation,and was significantly higher than that in the susceptible provenance at 24 hpi;the change trend of JA content was opposite to that of SA;after inoculation,the resistant and susceptible varieties The ABA content of the source showed a downward trend,and the resistant provenance was lower than the susceptible provenance.The results of defense enzyme activity assay showed that after inoculation,the enzyme activities of PPO,PAL,POD and CAT were all increased in resistant provenances,and their enzyme activities were higher than those in susceptible provenances.related to resistance.The change of H₂O₂content was consistent with the change trend of CAT enzyme activity,and the H₂O₂ content of resistant provenance was significantly higher than that of susceptible provenance 24 h after inoculation.Histochemical staining test showed that the accumulation of both H₂O₂ and O₂ in the resistant provenance was higher than that in the susceptible provenance 24 h after inoculation.To sum up,it can be considered that the disease-resistant provenance has a stronger ROS burst and scavenging ability.4.Screen DEGs with|log2Fold Change|>1 and FDR<0.05 as the criteria,set up four comparison groups of S＿0 h vs S＿24 hpi,S＿0 h vs S＿72 hpi,R＿0 h vs R＿24 hpi and R＿0 h vs R＿72 hpi,in order to study the type and function annotations of DEGs at different time points before and after inoculation of C.fructicola with resistant and susceptible provenances of C.paliurus.Venn diagram screened out 352 common DEGs in resistant and susceptible provenances.Trend analysis showed that most of these DEGs were up-regulated after inoculation,and in the resistant provenances,the expression duration was longer;GO enrichment analysis showed that the common DEGs were mostly related to redox components are related.It is speculated that after inoculation of fruit anthracnose,ROS bursts and the disease-resistant provenance response is stronger,which also confirms the physiological results from the molecular level.At the same time,Venn diagram screened 2204 DEGs specific to disease-resistant provenance,and trend analysis showed that 1369 DEGs were up-regulated,and826 DEGs were down-regulated.GO enrichment analysis showed that the disease-resistant provenance-specific DEGs were mainly enriched in membrane and redox process-related functional pathways,which may be related to programmed cell death（PCD）of C.paliurus.KEGG enrichment analysis showed that the disease-resistant provenance-specific DEGs were significantly enriched in plant-pathogen interaction,plant hormone signal transduction,phenylalanine metabolism,plant MAPK signaling pathway,and peroxisome pathways.According to heatmap visualization analysis and gene function annotation,DEGs in important disease resistance signaling pathways such as R genes（NLRs and PRRs）,ROS,SA,Ca²⁺,and MAPK signaling pathways are strongly expressed in disease resistance provenances.and the expression was continuously up-regulated from 0 h to 72 hpi,suggesting the effectiveness of these DEGs in the disease resistance mechanism of C.paliurus.5.Through the broad-target metabolome analysis technology,365 secondary metabolites were detected in 18 samples before and after inoculation of C.paliurus,and 145 of them were annotated in the database.The differentially accumulated metabolites（DAMs）of the resistant and susceptible provenances were screened at different time points before and after inoculation of C.fructicola with VIP>1 and differential multiples FC>2 or FC<0.5 as the criteria.KEGG enrichment analysis showed that the biosynthetic pathways of flavonoids and flavonols,flavonoid biosynthetic pathways,metabolic pathways and tryptophan metabolic pathways were differentially enriched in the comparison groups of resistant and susceptible provenances at different time points;especially the biosynthetic pathway of flavonoids and flavonols.With the prolongation of the inoculation time,the significance of differential enrichment gradually increased,and the differential substances gradually accumulated from the flavonoid biosynthesis pathway to the downstream biosynthesis pathway of flavonoids and flavonols,indicating that different flavonoids in the biosynthetic pathway of flavonols and flavonols may play a key role in the disease resistance of C.paliurus.Combined analysis of transcriptome and metabolome showed that in resistant provenances,the expression of flavonoid biosynthesis pathway genes increased sharply with the extension of inoculation time;while in susceptible provenances,the expression levels of these genes had little or no significant difference compared with those before inoculation,and even showed down-regulated expression,Moreover,DAMs were also significantly enriched in the flavonoid and flavonol biosynthesis pathways downstream of the flavonoid synthesis pathway;in addition,correlation analysis also showed that multiple differential metabolites of this pathway were strongly correlated with differentially expressed genes in the transcriptome.It is speculated that the disease-resistant provenance of C.paliurus can inhibit the further infection of C.fructicola by reprogramming the flavonoid biosynthesis pathway,regulating the biosynthesis of downstream flavonoids and flavonols,and increasing the accumulation of phytoalexins.6.Extract the total flavonoids of C.paliurus after inoculation of C.fructicola at 72 hpi with resistant and susceptible provenances,to evaluate the antifungal effect and reveal the underlying mechanism.The results showed that the total flavonoids of the resistant and susceptible provenances had significant inhibitory effects on the mycelial growth and biomass of C.fructicola,and the total flavonoids of the resistant provenances were stronger.The total flavonoids from both resistant and susceptible provenances of C.paliurus could not inhibit the conidial germination of C.fructicola,but they could significantly inhibit the formation of appressorium.The results of q PCR showed that after treatment with total flavonoids from the resistant and susceptible provenances,the genes related to the formation of appressorium（Cf Lac,Cf End,Cf Gdh,Cf C₂H₂）were significantly down-regulated,and the inhibitory effect of total flavonoids from the resistant provenances was better.In addition,it was found that the C.fructicola hyphae had the phenomenon of internode swelling and swelling after the total flavonoids treatment.It was speculated that the total flavonoids affected the fruiting anthracnose mycelium cell membrane permeability.The results of the determination of the physiological and metabolic indexes of the C.fructicola after treatment with the total flavonoids of C.paliurus showed that the total flavonoids of the resistant and susceptible provenances of C.paliurus lead to the increase of the content of malondialdehyde in C.fructicola,and the extravasation of nucleic acid,protein and soluble sugar and other contents.Therefore,it can be proved that one of the antifungal mechanisms of the total flavonoids of C.fructicola is to destroy the cell membrane of C.fructicola,resulting in the extravasation of its contents.In this study,C.fructicola,C.gloeosporioides and C.aenigma were identified as the pathogens of Cyclocarya paliurus anthracnose,and the morphological and pathogenic differences and phylogenetic relationship of the three pathogens were analyzed;It was clear that C.fructicola was the dominant pathogen of Cyclocarya paliurus anthracnose.The resistant（Wufeng）and susceptible（Jinggangshan）provenances were selected through resistance identification.The phenotypic,physiological,biochemical and transcriptional differences of resistant and susceptible provenances after inoculation with dominant pathogen C.fructicola point to ROS burst and hypersensitive response（HR）play an important role in the disease resistance mechanism of C.paliurus.The combined analysis of transcriptome and metabolome revealed that C.paliurus regulated the reprogramming of flavonoid biosynthesis and promoted the synthesis of downstream products and phytoalexin in response to the infection of C.fructicola.Total flavonoids of C.paliurus can inhibit the growth of mycelium,biomass and the formation of appressorium of C.fructicola,and destroy the cell membrane of mycelium and lead to the gushing of contents.