| American ginseng is a perennial medicinal plant with anti-inflammatory,anti-tumor and immunity enhancement effects,and is widely cultivated throughout China,especially in Jilin,Liaoning,Shandong.With the expansion of American ginseng cultivation area and the increase of planting years,diseases have become a major obstacle to its yield and quality.Root rot is the most serious underground disease,while gray mold and black spot mainly affect the above-ground parts of American ginseng.Plant nutritional status is an important factor affecting the occurrence of diseases,and reducing disease occurrence through nutritional control is an important part of green control technology.The research found that calcium,as an essential nutrient for plants,is second only to nitrogen and potassium in the growth of American ginseng,and calcium deficiency significantly reduces the growth of American ginseng.The laboratory investigated 22 planting plots in Wendeng,Weihai City,Shandong Province,and found that the available calcium content of soil varied greatly,yet there was almost a gap in research on the calcium requirements of American ginseng and the role of calcium on the occurrence of disease in American ginseng.In this paper,we studied the calcium nutritional requirements of American ginseng and the effect of calcium on the main diseases of American ginseng,aiming to provide a reference for the rational application of calcium fertilizer and disease control of American ginseng.1.Effect of exogenous calcium on the growth and quality of American ginsengThe 2-year-old American ginsengs were cultivated in sand culture and field pot.During cultivation,different amounts of CaCl2 solution(0、0.56、1.11、2.22、3.33 g·L-1)or calcium oxide(0、0.5、1.5、4.5 g·kg-1)are added,and the height,leaf area,biomass,saponin content and nutrient uptake of American ginseng were measured.The results of the sand culture experiment showed that calcium treatment mainly affected the growth of above-ground parts of American ginseng,and the calcium content of underground and aboveground parts of American ginseng showed an increasing trend with increasing calcium concentration,where the 3.33 g-L-1group showed an increase of 42.63%and 148.65%compared to the control(p<0.05).Moreover the aboveground part has a higher calcium content than the underground part.The plant height and above-ground fresh weight had the maximum in the 0.56 g·L-1 treatment.The results of field pots showed that the height and leaf area were greatly affected by the calcium treatment.The 0.5 g·kg-1 treatment showed 22.73%and 44.93%increase in plant height and leaf area at week 6 compared to the control(p<0.05),and the total saponin content in the roots had a maximum after 80 days of calcium treatment(p>0.05).2.Effect of exogenous calcium on root rot of American ginsengIn order to reduce the use of pesticides,a pot experiment was conducted to investigate the effects of calcium application and inoculation separately two pathogens(Fusarium solani and Ilyonectria mors-panacis)and possible physiological and ecological mechanisms of calcium in root rot disease control.During the ginseng cultivation,different amounts of calcium oxide(0、0.5、1.5、4.5 g·kg-1)was applied to the soil.The root biomass,disease incidence,root nutrient content,soil chemical properties and soil microbial community were measured.The results showed that low calcium additions increased root rot disease index,however high calcium additions decreased root rot disease index.Compared with the control,adding 4.5 g·kg-1 calcium oxide decreased root rot disease index of I.mors-panacis by 87.69%(p<0.05),meanwhile,the root rot disease index of F.solani was reduced by 66.67%(p>0.05).The calcium content in American ginseng roots of the two inoculation groups were increased by 101.28%and 61.50%(p<0.05),respectively.Nitrogen and phosphorus content in the roots were positively correlated with the root rot disease index of the F.solani group.On the other hand,the structure and composition of soil microbial communities inoculated with two pathogens differed significantly.Ca treatment mainly affected soil fungal diversity and community composition,and had little effect on bacterial community,moreover,4.5 g·kg-1 calcium oxide treatment had an inhibition on the relative abundance of Didymella and Phoma.Overall,the potential mechanism for high dose calcium to reduce root rot disease of American ginseng by more than 65%is related to increasing calcium content in roots,reducing nitrogen and phosphorus content in roots,and decreasing the relative abundance of potential pathogens in soil.The results of this study provide a theoretical basis for reducing root rot disease of American ginseng through rational application of calcium fertilizer.Tests of 0%,0.1%,0.2%,0.4%,0.8%,1.6%and 3.2%CaCl2 on the growth of F.solani and I.mors-panacis were designed,and the results showed that high concentrations of CaCl2(>3.2%)had inhibitory effect on F.solani and promoted I.mors-panacis.In order to explore the effect of exogenous calcium on the expression of American ginseng disease resistance related genes,in this study,the water/calcium treated American ginseng roots were inoculated with F.Solani and I.mors-panacis,and the expression of PRs genes(PR4,PR6,PR10)and jasmonic acid pathway-related genes(AOS 1,AOC1,JAR1)in the roots of American ginseng at 6 h,12 h,24 h and 72 h after inoculation were measured.The results showed that the root rot caused by F.solani were reduced in the 0.4%Ca treatment compared with the control.The expression of three PR genes began to be significantly upregulated at 72 h of inoculation,but only PR4 was higher than that of the control group.In addition,the content of jasmonic acid in ginseng roots in the Ca treatment group was significantly higher than that in the control group(p<0.05)6 h after inoculation,but the three genes related to the jasmonic acid synthesis pathway in the treatment group were not higher than those in the control group.The relative expression of AOS 1 was higher in the Ca+F.solani group compared to the F.solani group from 12 h to 72 h after inoculation,where it was 4.76 times higher at 12 h(p<0.05);At 72 h,the relative expressions of PR4,PR6 and PR10 in the Ca treatment group were lower than those in the control group,and the expression of PR6 was significantly reduced(p<0.05),while the relative expression of the three genes AOS1,AOC1 and JAR1 related to jasmonic acid synthesis was higher than that of the control group at 72 h after inoculation,but there was no significant difference(p>0.05).3.Effects of exogenous calcium on American ginseng leaf diseasesIn the CaCl2 concentration range of 0.1%～3.2%,the inhibitory effect of 0.4%CaCl2 on Botrytis cinerea was 6.11%(p<0.05).In order to investigate the mechanism of exogenous calcium on the gray mold of American ginseng,in this study,water/calcium-treated American ginseng leaves were inoculated with B.cinerea,and a total of 12 samples were analyzed by transcriptome sequencing at 36 h and 72 h after inoculation.The results showed that the symptoms of gray mold were reduced in the 0.4%Ca treatment,and the concentration could significantly inhibit the diameter of the pathogen(p<0.05).Compared to the control,the Ca treatment had 44 differential genes at 36 h and 142 differential genes at 72 h.In addition,KEGG pathway analysis showed that 36 h after inoculation,differentially expressed genes were mainly involved in glutathione metabolism,cysteine and methionine metabolism.At 72 h,differentially expressed genes were mainly involved in phenylpropane biosynthesis,plant hormone signal transduction and plant-pathogen interaction.Gene expression in these pathways may play an important role in calcium-mediated resistance to B.cinerea infestation in American ginseng leaves.The effects of different CaCl2 concentrations(0.1%～3.2%CaCl2)on the growth of two strains of Alternaria panax(5747,Q and 6197,R)showed that the promotion effect of 0.4%calcium concentration on the two pathogens was 22.00%and 27.80%(p<0.05),respectively.In order to explore the mechanism of exogenous calcium on black spot,in this study,the leaves of American ginseng treated with water/calcium were inoculated with Q and R,and a total of 24 samples were analyzed by transcriptome sequencing at 36 h and 72 h after inoculation.The results showed that at 72 h,there was no significant difference in the symptoms caused by the two pathogens between the Ca treatment group and the control group.For Q,the Ca treatment had 89 unique differentially expressed genes at 36 h and 276 unique differentially expressed genes at 72 h.Moreover,KEGG pathway analysis revealed that differentially expressed genes in the flavonoid biosynthesis pathway in Ca treatment were more abundant at 36 h in response to Q,and the plant hormone singal transduction had the highest number of differentially expressed genes at 72 h,genes involved in the plant-pathogen interaction pathway were higher at both time points.For R,the Ca treatment had 580 unique differentially expressed genes at 36 h and 312 unique differentially expressed genes at 72 h.Ca treatment had the largest number of differentially expressed genes involved in phenylpropane biosynthesis at 36 h,and the most differentially expressed genes in the plant-pathogen interaction pathway were found at 72 h,differential genes involved in plant hormone singal transduction were more abundant at both time points.In contrast,Ca-mediated resistance-related genes in American ginseng leaves responded more to the R strain than to the Q strain. |
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