Resistance Mechanism Of Camellia Oleifera Cultivars To Colletotrichum Gloeosporioides

The mechanisms of Colletotrichum gloeosporioides resistance for different varieties of Camellia oleifera were studied on the basis of morphology,pHysiology and biochemistry,and molecular biology.The following results are obtained:1.Among the 8 varieties of Camellia oleifera investigated and tested,4 varieties,such as Camellia yuhsienensis,huizhoudahong and shuchengdahong,are Camellia gloeosporioides resistant ones,which incidence were less than 10%;two varieties (ShuChengDaQing and HuiZhouXiaoQing) are susceptible to the disease,which incidence were over 50%.The incidences of the disease for other two varieties were between 10% and 50%.2.The result from the relative swelling of peel demonstrated that the variety with great relative swelling was usually susceptible to the disease,such as ShuChengDaQing,HuiZhouXiaoQing and HuiZhouXiaoQing which relative swelling were all round 70%. The Colletotrichum gloeosporioides-resistant varieties were usually low relative swelling, such as Camellia yuhsienensis and HuiZhouXiaoHong which relative swelling were about 50%.In addition,this experiment showed that the Colletotrichum gloeosporioides-resistant varieties had high water content,such as HuiZhouDaHong which water content reached 76%.It is probably correlated to the chemical composition of peel,and the difference in fruit maturity because of fruit collection in the same time.3.The effects of peel extracts on spore germination and hypHa growth differed significantly among varieties.The extracts from high resistant variety had a strong restraint of spore germination and hypha growth;but had no or lower resistant of spore germination for QingGuo-type varieties,which spore germination reached over 80%,particularly 1000,'o for HuiZhouDaQing.This indicates that the extracts contained a lot of matter fitting for spore germination.The experiment of inoculation of spore in juice culture medium showed that there existed significant difference between Colletotrichum gloeosporioides-resistant and -susceptible varieties,which was similar to the result from the experiment of spore germination.4.Resistance to the leaves epidermal structure of different resistance levels had found that the structure of leaves skin has great influence on resistance.Waxy layer thickness of the leaves,the cuticle thickness,the palisade tissue thickness and cell density of palisade tissue were positive effect on resistance.5.Of the 6 pHenols in peel measured,the contents of catechol and nipa were closely correlated to the disease resistance.The contents of catechol and nipa in disease-resistant varieties were 5-10 times higher than those in disease-susceptible varieties.6.The relationship between disease-resistant and the clearance rate of leaves,the peel extract against DPPH was also researched.The results showed that in the 2.0 mg/mL concentration,both of leaves or the skin,the clearance rate of DPPH has reached more than 85 percent,the highest more than 90 percent.The free radical scavenging activity is greatly different in leaves and peel extract from different grades disease-resistant,but there is no linear correlation between them.It is linear correlation between the free radical scavenging activity and the concentrations of extractives or between the free radical scavenging activity and the holding time after been added with DPPH.7.Enzymatic of pHenylalanine(PAL),enzymatic of polypHenoloxidase(PPO), enzymatic of peroxidase(POD) of different disease-resistant varieties of Camellia were measured before and after inoculating with Colletotrichum gloeosporioides.The results showed that in the absence of flu cases,three kinds of enzymatic in different disease-resistant were not obvious.After the vaccination,the stronger the disease-resistant varieties have,the faster the increased speed and rate of change rise. PPO activity in healthy peel for disease-resistant varieties was 4 times as high as that for disease-susceptible ones.There existed great difference in PAL activity among varieties.PAL activity averaged 12096 OD/g for disease-resistant varieties,which was significantly higher than that for disease-susceptible varieties with an average of 8021 OD/g. 8.The result from molecular biological experiment revealed that the DNA concentration of Camellia oleifera was about 918ug/ml fulfilling the requirement of RAPD analysis.Moreover,UPGMA analysis demonstrated that the 17 DNA samples from the whole country could be divided into 5 groups,of which the disease-resistant and disease-susceptible groups were evident.This result suggests that there is similar DNA fragment in genes for the same group.9.High performance HPLC-MS analysis was employed to compare components difference of extracts from methanol and acylacetate mixture extracts from leaves of three resistive Camellia oleifera varieties,namely Dabieshan 1,Dabieshan 2 and Dabieshan 3.Results of HPLC-DAD-MS analysis showed that there were species and quantity difference between the two extracts.Analysis of the red fruit showed that Dabieshan 1 contained three unique molecules namely C₁₄H₁₄O₃,C₇H₁₂O₆ and C₂₉H₄₀O₁₄.These compounds may relate to the resistive property against anthracnose. Analysis of Dabieshan 2 and Dabieshan 3 showed that they shared the same characteristic molecules C₇H₁₂O₆ and C₂₉H₄₀O₁₄.These compounds may relate to the resistive property against anthracnose.Comparison of the compounds possibly related property of the three varieties against anthracnose revealed that there were multiple compounds related property of Camellia oleifera against anthracnose,and the most possible compound related resistive property was possibly C₂₉H₄₀O₁₄.10.High performance HPLC-MS analysis was employed to compare components difference of extracts from methanol and acylacetate mixture extracts from fruits of resistive and non-resistive Camellia oleifera varieties.Results of HPLC-DAD-MS analysis showed that there were species and quantity difference between the two kinds extracts.HPLC-MS analysis results showed the resistive variety had unique molecules C₂₇H₃₀O₁₄ and C₁₅H₁₄O₆,these were possibly related to the resistive properties.At the same time,the non-resistive variety had unique molecules C₇H₁₀O₅,C₂₄H₂₀O₁₁ and C₃₁H₂₄O₁₅.These compounds were possibly related to the properties of easier being infected by anthracnose of Camellia oleifera.11.High performance HPLC-MS analysis was employed to compare components difference of extracts from methanol and acylacetate mixture extracts from leaves of resistive and non-resistive Camellia oleifera varieties.Results of HPLC-DAD-MS analysis showed that there were species and quantity difference between the two kinds extracts.Analysis results showed on the HPLC chromatogram of the resistive variety had unique picks with retention time 7.4 and 14.0 min,while the non-resistive 8.5 min.On the negative ion flow of the resistive variety there was a unique ion with m/z 553 and a possible molecule formula C₃₄H₂₃O₇.This was the most possible compound related resistive property of Camellia oleifera.12.High performance HPLC-MS analysis was employed to compare components difference of extracts from red fuit and green fruit of Camellia oleifera Abel.Results of HPLC-DAD-MS analysis showed that there were species and quantity difference between the two extracts.Analysis of the red fruit showed that there were two unique ions with m/z 577 and 289 and corresponding molecules C₂₇H₃₀O₁₄ and C₁₅H₁₄O₆.Because of that red Camellia oleifera has usually more of resistant ability against anthracnose,it is deducable that this two compounds were related to the fastness.Investigation of the components of the green fruit revealed that there were unique ions with m/z 331,125,483,635 and 787,and corresponding molecules C₁₇H₁₆O₇,C₆H₆O₃,C₂₄H₂₀O₁₁,C₃₁H₂₄O₁₅ and C₃₈H₂₈O₁₉.These compounds may related to factors making Camellia oleifera easier being infacted for that green fruit species of Camellia oleifera are usually easier being infected by anthracnose.