Exploration Of Germplasm Resources And Action Of Microbial Communities In Continuous Cropping Obstacle Of Aconitum Carmichaelii

Aconiti lateralis radix praeparata.(Fuzi,FZ)is a processed root of Aconitum armichaelii Debeaux and the most extensive species in the Aconitum genus,with large-scale cultivation in provinces such as Sichuan,Shaanxi,Yunnan,and Guizhou.The plant has undergone variations in adaptation to different environments due to differences in geographical location,climate,and cultivation conditions.As a result,germplasm resources with distinct characteristics and rich genetics have been formed.Traditional farming practices for FZ have relied on experiential knowledge to selectively intercrop and breed multiple varieties,leading to confusion in germplasm sources,reduced yields,and increased disease and pest problems.Moreover,limited research has been conducted on the quality differences between cultivated germplasms,thereby hindering the promotion of agricultural "varieties".Consequently,germplasm issues have emerged as a bottleneck for developing the FZ industry.Despite the rapid growth in FZcultivation,limited land has forced growers to resort to long-term,high-intensity continuous cropping,resulting in obstacles such as soil acidification,nutrient imbalance,increased incidence of pests and diseases,reduced yields,and adverse economic consequences for growers.These challenges are hindering the industrial development of FZ.Hence,the chloroplast(cp)genome sequence of the germplasm of FZ was obtained using high-throughput sequencing technology,and its structure,sequence variation,and phylogenetic relationship was analyzed using bioinformatics techniques.The content of effective components and toxic components was determined by high-performance liquid chromatography,and the quality difference evaluation was carried out by combining cluster analysis(HCA),principal component analysis(PCA),orthogonal partial least squares discrimination(OPLS-DA)method,and quality markers were screened out.The physicochemical properties,microbial community structure,and diversity of FZ rhizosphere soils under continuous cropping and rotation modes were studied using soil nutrient rapid detection equipment,inductively coupled plasma optical emission spectroscopy(ICP-OES),and high-throughput sequencing technology.The main results are as follows:(1)Yunnan province possesses a relatively abundant germplasm resource of FZ,cultivated across a wide range of areas mainly concentrated in Heqing,Eryuan,Weixi,Yulong,Yunlong,and Binchuan counties,spanning longitude 99.13 ～ 100.68 °E,latitude 25.67 ～ 27.18 °N,and altitude 1930.88 ～ 2967.17 m.Based on phenotypic characteristics,27 samples can be categorized into four typical germplasm types: Yunfu 1 has artemisia-shaped leaves,elongated conical roots that possess numerous branches,small and inconspicuous buds,as well as white seed coats.Yunfu 2 is characterized by its pumpkin-shaped leaves,inverted conical roots that with few branches,large and upward-pointing buds,and black seed coats.It grows in Eryuan,Yunlong,Heqing,and Binchuan.Yunfu 3 displays leave resembling Artemisia in shape,while its roots assume an inverted conical form with limited branching,and its buds are relatively large and point upwards,with black seed coats,and grows in Yunlong and Heqing.Conversely,Yunfu 4 features leave akin to those of a pumpkin,long conical roots with numerous branches,inconspicuous tiny buds,and white seed coats,and are distributed in Eryuan and Heqing counties.The study results indicate that some excellent aconite germplasm resources have limited mobility and are confined to a few areas,and there has been a lack of good variety breeding efforts.(2)The complete chloroplast(cp)genome sequences of 27 samples varied in length from158,800 to 155,891 bp,containing 132 functional genes,including 87 protein-coding genes,37 t RNA genes,and 8 r RNA genes.Among these samples,Yunfu 1 had a length of 155,880 bp,consisting of 86,347 bp in the LSC region,16,947 bpi n the SSC region,and 53,586 bp in the IR region.In contrast,Yunfu 2 possessed a full-length sequence of 155,858 bp,with86,330 bp in the LSC region,16,944 bp in the SSC region,and 52,854 bp in the IR region.Meanwhile,Yunfu 3 and Yunfu 4 had a full-length sequence length of 155,891 bp,with86,359 bp in the LSC region,16,946 bp in the SSC region,and 52,586 bp in the IR region.These findings indicated that different aconite germplasms could be preliminarily differentiated based on the length of their chloroplast genome,and their IR,SSC,and LSC fragments.Maximum likelihood phylogenetic analysis revealed that the samples could be classified into three distinct clades,with Yunfu 3 and Yunfu 4 clustering together into a unique branch with 100% bootstrap support and separated from the other samples with the same level of support.On the other hand,Yunfu 1 and Yunfu 2 formed two separate branches,each with100% bootstrap support.These results demonstrated that different aconite germplasms were distinguishable from each other,with Yunfu 1 and 2 being closely related and Yunfu 3 and 4being more closely related.(3)The results of hierarchical clustering heatmap analysis demonstrated that the 27 samples could be divided into two distinct clusters.Interestingly,samples from the same cluster exhibited dispersed origins and germplasm,indicating a lack of clear regional or germplasm-specific characteristics.Specifically,S19,S02,S04,S14,S19,S26,and S27 were classified into Cluster I,while the remaining samples were grouped into Cluster II.The results of Principal component analysis(PCA)were inconclusive,as no clear grouping pattern emerged.In contrast,orthogonal partial least squares discriminant analysis(OPLS-DA)was able to correctly classify most samples into two categories based on germplasm type,with an accuracy rate of 88%.However,extremely high or low chemical content could not be grouped into either category.Furthermore,the linear discriminant analysis raw validation model exhibited an overall accuracy rate of 88.9%,whereas the cross-validation model displayed a lower accuracy rate of 59.3%.The function graph of group centroids revealed that different germplasms exhibited unique spatial distribution patterns.Germplasm 3 was relatively distant from the other,while germplasm 1,2,and 4 displayed varying degrees of overlap,reducing in discrimination accuracy.The results show that the chemical compositions of different genotypes are similar,and the quality differences are small,but they have their own characteristic compounds.(4)The continuous cultivation of FZ resulted in a significant increase in the content of available potassium(AK),total potassium(TK),and total phosphorus(TP),as well as lead(Pb),chromium(Cr),and mercury(Hg).On the other hand,it led to a decrease in the content of ammonium nitrogen(NH4+)and copper(Cu).The contents of arsenic(As)and cadmium(Cd)remained stable.Interestingly,maize-FZ rotation was found to effectively reduce the content of Pb and Hg,while rice-FZ rotation reduced the content of Cr and Cu.Both rotation modes decreased the content of AK,TK,and TP,which addressed soil fertility imbalances.(5)After continuous cropping of FZ,there was a decrease in bacterial community richness and diversity indices such as Richness,Shannon,and Chao1.However,when rice-FZ or maize-FZ crop rotation was applied,and the indices either increased or remained stable.On the other hand,both continuous cropping and crop rotation increased in fungal Richness and Chao1 values.Therefore,rotating rice and FZ can significantly improve the diversity and richness of both bacterial and fungal communities.Regarding community structure,continuous cropping led to a relative abundance increase of Deinococcus-Thermus,Basidiomycota,Bacteroidetes,and pathogenic Fusarium,while rice/maize-FZ rotation significantly decreased these bacterial groups.Since Fusarium is a common root rot pathogen in crops,rotation can reduce the risk of FZ disease.The bacterial co-linearity network results showed that the number of negative correlation edges,modularity,and critical groups increased in the bacterial co-linearity network after rotation,increasing the stability of the bacterial network structure.Additionally,continuous cropping weakened soil nitrogen fixation,affecting normal plant growth,while rotation enhanced nitrogen fixation and alleviated the harm of continuous cropping.To summarize,the rotation system can effectively alleviate FZ continuous cropping obstacles.This study provides a theoretical basis for identifying FZ germplasm resources,selecting and breeding excellent varieties and cultivation methods,and lays a research foundation for the high-quality,efficient,and sustainable development of FZ production.