| Objective:This study provides scientific basis for field management on branch blight disease and quality evaluation on honeysuckle by investigating the occurrence regularity of branch blight disease in the field and exploring its effect on the quality of honeysuckle.Methods:The occurrence rule of branch blight disease of honeysuckle was studied by field investigation.The fungi were isolated from the diseased tissues and healthy tissues.The genus and species of the strains were determined by morphological characteristics and rDNA-ITS sequence identification.Specific primers were designed based on the Ceratobasidium theobromae ITS sequence and five specific gene sequences to detect molecular pathogens and determine the sensitivity of the primers.To test the sensitivity of primers by using the genomic DNA of healthy leaves,healthy branches,diseased leaves and diseased branches and establish the detection system of branch blight.To investigate the yield of honeysuckle on different disease rating of honeysuckle plants.The contents of chlorogenic acid and luteolin-7-O-glucosid in honeysuckle flower samples and loganin and chlorogenic acid in honeysuckle stem samples from all diseased grades are measured according to method illustrated in Pharmacopoeia of People's Republic of China?2015?.Result:1.The law of branch blight disease of honeysuckleBranch blight occurs throughout the entire growth cycle.Its high-incidence season continues from July to September every year.In the germination period,shoots of honeysuckle endangered by the branch blight do not germinate,and the number of shoots is less than that of healthy plants during the germination period in late March and early April of the following year,and the growth of the whole tree is also relatively weak.In the flowering period,the budding time of diseased plants is 7 to 9 days later than that of healthy plants,and the yield of flowers will be affected accordingly.The high-temperature and high-humidity cultures of branches collected from the disease were found to started to produce mycelium in May of that year and continue until December.In the hot and humid environment after the rain,a large number of hyphae were found in the cut branches of the field branches,wounds on the leaves,and deciduous leaves.At the beginning,the mycelium was white and fluffy,and then slowly gathered into light yellow lump.The chlorosis of the diseased shoots and the yellowish dryness generally develop from the lower end to the upper end of the shoots.The leaf mesophyll turns green and yellow,and the veins appear green.The pattern is fishbone pattern and gradually fades from the tips of the leaves.Each layer of leaves from the chlorosis to dry off about 27 days,a layer of leaves chlorosis yellow to the next layer of the leaf began to show symptoms about 7 to 9 days.The main branches of leaves from the onset of the disease to all dry off about 50 to 72 days,collateral from the27th day began to show symptoms of chlorosis,about the 80th day all leaves off.2.Isolation and identification of strains of honeysuckle plant tissue13 strains were isolated from healthy branches.16 strains were isolated from healthy leaves.28 strains were isolated from diseased branches.57 strains were isolated from diseased leaves.The strains were classified according to the morphology and microscopic characteristics,a total of 12 genera and 113 strains.Using the primers ITS1/ITS4,the ITS sequence of the representative strains of each genus were amplified by PCR and cloned and sequenced.A total of 15 fungi were identified,including Choanephora cucurbitarum,Rhizopus oryzae,Fusarium incarnatum.,Colletotrichum gloeosporioides,Botryosphaeria dothidea,Mucor ellipsoideus,Fusarium oxysporum,Curvularia spicifera,Fusarium chlamydosporum,Colletotrichum gloeosporioides,Botryosphaeria dothidea,Mucor ellipsoideus,Fusarium oxysporum,Curvularia spicifera,Fusarium chlamydosporum Phomopsis capsici,Diaporthe phaseolorum,Alternaria tenuissima,Sordaria fimicola,Fusarium verticillioides,Ceratobasidium theobromae.3.Design and screening of specific primers for Ceratobasidium theobromaeIn this study,we verified the reportorial and the self-designed 13 pairs of primers.The results showed the five pairs of primer,Th-t02.0406F/R?Th-t07.0104F/R?Th-t09.0300F/R?Th-t80.0268F/R?Th-t02.0204F/R,were specific through optimization of the reaction conditions,and the size of specific amplification products were 587bp,645bp,813bp,1271bp,and 253bp,respectively.4.The detection of sensitivity of primersThe specific primers of Th-t02.0406F/R,Th-t07.0104F/R,Th-t09.0300F/R,Th-t80.0268F/R,and Th-t02.0204F/R were used to detect the DNA sensitivity of Ceratobasidium theobromae and plant tissue.The primers of Th-t80.0268F/R and Th-t02.0204F/R had the highest sensitivity to Ceratobasidium theobromae DNA and the smallest detectable DNA concentration was 9.765×10-3 ng/?L.The minimum DNA of Ceratobasidium theobromae detected by the primer of Th-t09.0300F/R was 9.765×10-2ng/?L,the primer of Th-t02.0406F/R was 9.765×10-1 ng/?L,the primer of Th-t07.0104F/R was 9.765×10-2 ng/?L.The primer of Th-t02.0204F/R had the lowest detectable mycelium content of 150 mg/g from leaves and stems.The primer of Th-t80.0268F/R had the lowest detectable mycelium content of 150 mg/g in stems and 500 mg/g of mycelia in leaves.The primer of Th-t02.0204F/R can be used as a specific primer for the detection of Ceratobasidium theobromae.5.The effect of different grades of branch blight of honeysuckle on production of honeysuckle flowerThe difference in the budding amount of honeysuckle flower existed in each disease level is extremely significant.The average budding amount per plant of the grade 0 was the highest,with 216 flowers,and the average budding per plant of the grade 7 was the lowest,with only 68 flowers.The budding amount of the plant decrease with the increase of the incidence level,the more severe the disease,the smaller the amount of budding.The average plant yields of the grade 1,3,5,and 7 plants were significantly lower than those of the 0plants.The average yield per plant of the grade 0 plant was the highest,and the dry weight reached 34.19 g.The average yield per plant of the grade 7 plant was the lowest,only 6.13 g,which was about 1/6 of the yield of the grade 0 plant,and the difference was extremely significant.6.The quality detection of honeysuckle flower and honeysuckle stemThe content of active components including chlorogenic acid and luteolin-7-O-glucosid in honeysuckle flower samples and chlorogenic acid and loganin in the honeysuckle stem from all diseased grades was in accord with the Pharmacopoeia of People's Republic of China?2015?.The content of chlorogenic acid in all honeysuckle flower samples was ranged from 2.31%to 3.46%,among which the content of chlorogenic acid in the honeysuckle flower sample from grade 1 of diseased tree was the highest,while that from grade 0 was the lowest.The content of chlorogenic acid in the samples from diseased tree was significantly higher than that from he1thy plants?P<0.01?.The content of luteolin-7-O-glucosid in honeysuckle flower samples was ranged from 0.050%to 0.065%,among which the content of luteolin-7-O-glucosid from the grade 0 honeysuckle flower sample was the lowest.The content of luteolin-7-O-glucosid in healthy plants was significantly different from other disease grades?P<0.01?.The content of chlorogenic acid in honeysuckle stem was 0.43%to0.54%,and the content of chlorogenic acid in healthy plants was higher than other disease-grade plants?P<0.01?.The content of loganin in honeysuckle stem was 0.82%to1.58%,and the content of loganin in all diseased sample plants of L.japonica caulis was significantly lower than that of healthy plants.Conclusion:1.Through the field investigation,we have mastered the pathogenicity of branch blight,which can provide guidance for the prevention and control of branch blight disease.2.Through the specific primer screening and sensitivity detection,the PCR detection system of Ceratobasidium theobromae was established.3.The occurrence of branch blight of L.japonica is beneficial to the accumulation of chlorogenic acid and luteolin in honeysuckle,but it will significantly reduce the yield of honeysuckle and the content of chlorogenic acid and loganin in L.japonica Caulis,resulting in a decline in the quality of L.japonica Caulis.It is necessary to pay attention to the prevention and treatment of branch blight.The content of active components including chlorogenic acid and luteolin-7-O-glucosid in honeysuckle flower samples and chlorogenic acid and loganin in the honeysuckle stem from all diseased grades was in accord with the Pharmacopoeia of People's Republic of China?2015?. |
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