| Panaxginseng C.A.Meyer is a perennial medicinal plant with high medicinal and economic value.It is a valuable Chinese herbal medicine.The ginseng can not be repeated within 10-20 years.Otherwise,there will be problems such as“burning,rotting roots,frequent disease,and reduced yield”.In this study,the phenolic acids in the root exudates were isolated and identified by high performance liquid chromatography in ginseng continuous cropping.The effects of genomic DNA sequencing on the fungal diversity and structure of ginseng rhizosphere soil were determined by macrogenomic sequencing.Trichoderma harzianum Tri41 was used to inhibit ginseng rust rot and reduce phenolic acid in ginseng root exudates.It was confirmed by gene labeling that Tri41 could colonized in soil and organic fertilizer mixed matrix.A set of disease prevention and control technology of"sunlight rotary tillage+chemical killing+biological pesticide-fertilizer recovery"was proposed,which aims to provide a theoretical basis for the removal of ginseng continuous cropping obstacles.1.Five kinds of autotoxic phenolic acids:salicylic acid,gallic acid,benzoicacid,3-phenyl propionate and cinnamic acids were identified as the main allelopathy of root secretion in the continuous cropping.In this study,the species and content of five phenolic acids,such as salicylic acid,gallic acid,benzoic acid,3-phenyl propionate acid and cinnamic acid,were collected and identified in the rhizosphere soil of ginseng for six years.It was confirmed that these phenolic acids could inhibit the germination of ginseng seeds and the growth of ginseng seedlings,and found that the five phenolic acids had influence on the mycelium growth and spore germination of Cylindrocarpon destructans,and the severity of this effect on the incidence of C.destructans was determined by the concentration of these phenolic acids in soil.Both indoor and potted experiments showed that salicylic acid,gallic acids and benzoic acid were at 0.5mmol L-1 concentration,3-phenyl-propionic acid and cinnamic acid at0.05mmol L-1 concentration.The inhibitory effect of phenolic acid on the germination and seedling growth of ginseng was the strongest,meanwhile the promoted effect of the pathogenic fungal was the strongest.The presence of these five kinds of phenolic acids will increase the incidence of C.destructans t,affect the quality of ginseng,and aggravate the obstacle of continuous cultivation of ginseng.2.This study used the metagenomic molecular sequencing method to find out that the autotoxic phenolic acid produced by ginseng roots can change the fungal structure and diversity in ginseng rhizosphere soil.Five kinds of autotoxic phenolic acids were extracted from the roots of ginseng,and they were treated with ginseng root soil samples（except cinnamic acid）,and the treated soil samples were analyzed.The results showed that the number of sexual and asexual OTUs of C.destructans was significantly increased in the treated soil.This indicates that phenolic acids in the rhizosphere of ginseng can promote the growth of ginseng rust pathogens.The MiSeq high-throughput sequencing analysis of the treated soil samples showed that the number of OTUs of some beneficial fungal（Mortierella and Humicola）increased after treatment with five phenolic acids（except cinnamic acid）.At the same time,the number of OTUs of soil-borne diseases（C.destructans,and Fusarium）increased,and the increase was greater than that of beneficial fungal.This indicates that the harmful effects of pathogenic function are greater than the antagonism of beneficial function.This may be an important reason for the seriousness of soil-borne diseases in ginseng continuous cropping soil.PCA analysis showed that the fungal content of these three soils was farther than that of other soil samples,which were the soil of new forest land without ginseng,continuous ginseng soil,and continuous ginseng soil treated with cinnamic acid.At the same time,the spatial distribution of the three soil fungal contents is also far from each other.This shows that ginseng continuous cropping and phenolic acid treatment can change the structure and diversity of soil fungi.The results of field experiments showed that after ginseng was planted in ginseng treated with phenolic acid,the growth index of ginseng,the incidence of ginseng rust and the severity of disease were lower than those of untreated.According to the results of soil fungal phylogenetic analysis and the results of outdoor experiments,it can be concluded that the soil samples after cinnamic acid treated are similar to those untreated However,the incidence and severity of C.destructans,were not significantly different from those of the other four phenolic materials.It is speculated that cinnamic acid in phenolic acid may not be the main cause of ginseng continuous cropping obstacle.3.Trichoderma harzianum 41 can effectively inhibit the growth of the main disease（C.destructans）in ginseng continuous cropping and at the same time reduce the autotoxic phenolic acid present in the soil.Tri41 inhibits the growth of ginseng rust by spatial competition and nutrient competition.On the ninth day of growth of Tri41,the inhibition rate of C.destructans was 72.32%at 20°C;the inhibition rate of C.destructans was 79.53%at25°C.By co-cultivating Tri41 and autotoxic phenolic acid,it was found that Tri41 can significantly reduce the content of autotoxic phenolic acid.Through the outdoor pot experiment,which is to determine the important defense enzyme activities in the roots of three kinds of ginseng,proves that five phenolic acids can reduce the activity of ginseng root defense enzyme,while the biocontrol Tri41 reduces the phenolic acid in the soil.Strengthening the activity of the root defense enzymes in life.The experiment of inoculation of C.destructans can prove that the addition of phenolic acid to the soil can aggravate the main soil-borne diseases of ginseng continuous cropping obstacles,mainly in the increase of morbidity and the severity of disease.After adding Tri41,the content of five phenolic acids was reduced,and the incidence and severity of C.destructans were reduced at same time.4.This experiment compared the indicators Tri41 and transformant Tri41-g,and determined that Tri41-g can be colonized in the soil-fertilizer mixture for a long time.The Tri41-g was obtained by protoplast introduction.Compared with the wild strain Tri41,not obvious phenomenon was observed:the genetic stability of Tri41-g was stronger,the growth rate of mycelium was slower,and the number of spores was less produced.The biological characteristics of Tri41-g were not significantly different,and Tri41-g had the same inhibitory ability against pathogens as Tri41.The mycelial growth and sporulation of Tri41-g did not change the optimum conditions for the environment.The colonization experiment was carried out on the transformant Tri41-g,and the colonization of Tri41-g in different treatment groups was analyzed and explored.The ginseng was planted in three mediums:organic fertilizer,new forest soil and soil-fertilizer mixture.Tri41-g was then colonized in the above six treatment groups,and each treatment group was monitored by green fluorescent gene labeling technology.The results showed that the number of colonies and colonization time of Tri41-g in the soil-fertilizer treatment group was significantly higher than that of other treatment groups.At the same time,the cultivation of ginseng was conducive to the colonization of Tri41-g.5.This study proposes a set of disease prevention and control technology of“physical disinfection+chemical elimination+biological fertilizer recovery”.It has been proved that this technology can effectively improve soil microorganism,reduce the incidence of ginseng diseases,and improve the yield and quality of ginseng.In this study,in cooperation with the planting bases in the main producing areas of ginseng,the two chemical agents,which are most commonly used in production and have little impact on the environment,were used for composting.At the same time,the soil was rotated and turned under the sun.The effects of improved methods on soil microbial flora,soil physical and chemical properties,soil enzyme activity,ginseng main growth index and incidence of ginseng rust rot were studied.The results showed that the soil with the highest content of soil fungi,the soil acidification was the worst,the soil enzyme activity was the lowest,the ginseng growth index was the lowest,and the incidence of ginseng rust was the most serious.Both chemical treatment and physical treatment can reduce the number of fungi in ginseng continuous cropping soil and the incidence of C.destructans,increase soil pH and increase soil enzyme activity.Compared with the corn rotation treatment group and the defrosting treatment group,the pesticide-fertilizer treatment group has better improvement effect,especially the cotton long-mix fertilizer treatment,which can effectively mitigate the ginseng continuous cropping obstacle. |
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