First report of soft rot of Aconitum carmichaelii caused by Pectobacterium brasiliense in China

Aconitum carmichaelii Debx. is a Chinese traditional medicine herb, and is widely planted in China. The processed lateral roots of A. carmichaelii is known as Fuzi, and is used for the treatment of pain and inflammation in the joints (Zhou et al., 2015). In July 2019, a high incidence (approximately 50-100%) of soft rot of A. carmichaelii was observed in several commercial fields in Jiangyou County of Szechuan Province of China. Soft rot brownish lesions developed on infected stems, leading to collapse and wilting of entire plants. From symptomatic plants, the margins between the diseased and healthy areas were cut into pieces (5 × 5 mm), which were surface sterilized using 75% ethanol for 30 s and 2% NaOCl for 1 min, followed by three rinses with sterile water. The sterilized sections were macerated in drops of sterile water, and the extract was streaked onto King’s B (KB) agar medium and incubated for 48 h at 30°C. Single colonies that are round, convex and creamy on the plates after 2 days were streaked on KB agar plates. Ten bacterial strains were isolated, and the strain Fuzi915 was chosen for further analyses. The 16S rDNA gene sequence (GenBank accession MZ881946) amplified by primer pair 27F/1492R (Monciardini et al., 2002) showed 99.85% identity to the sequence of Pectobacterium brasiliense (syn. Pectobacterium carotovorum subsp. brasiliense, Pcb) strain HNP201736 (MN393938.1) and P. carotovorum subsp. carotovorum strain PJP201706 (MN394020.1), respectively, and also showed 99.78% identity to P. brasiliense strain SX309 (CP020350.1). To further identify the Fuzi915 strain, the PCR assay was carried out using primer pairs Y1/Y2, EXPCCF/EXPCCR and BR1f/L1r (De Boer and Ward, 1995; kang et al., 2003; Duarte et al., 2004), specific to P. carotovorum, P. carotovorum subsp. carotovorum and P. carotovorum subsp. brasiliense (Pcb), respectively. Specific fragments of 434 bp and 322 bp were amplified by the Y1/Y2 and BR1f/L1r primer sets, receptively, but there was no amplification by the EXPCCF/EXPCCR primer set, indicating that the Fuzi915 strain belongs to Pcb (Onkendi and Moleleki, 2014). Additional phylogenetic trees based on two housekeeping genes mdh (MZ892962) and gapA (MZ892963) were constructed using Maximum-likelihood method with 1000 bootstraps. The Fuzi915 strain clustered with all P. brasiliense strains including type strain P. brasiliense BC1. Further, a pathogenicity test was conducted on healthy A. carmichaelii roots and seedlings maintained in a growth chamber at 25°C and 95% humidity. Root inoculation was followed by drenching 107 CFU/ml of the cell suspension of Fuzi915 strain in soil surrounding the A. carmichaelii roots. Ten roots were inoculated with cell suspension while 10 roots were drenching inoculated with sterile water as negative control. Stem inoculation was followed by injecting 103 CFU/ml of the cell suspension in the stem of 10 A. carmichaelii seedlings, while 10 were injected with sterile water as negative control. After 5 days, Pcb-inoculated roots became brown and soft, and Pcb-inoculated seedlings became wilted and water soaked and started to collapse, similar to symptoms observed in the field. No symptoms were observed on the control plants inoculated with sterile water. The strain was re-isolated successfully from symptomatic A. carmichaelii and was identified as P. brasiliense by using PCR with the same primers to complete Koch’s postulates. To our knowledge, this is the first report of the soft rot of A. carmichaelii caused by P. brasiliense in China.

The Aconitum carmichaelii F3′5′H Gene Overexpression Increases Flavonoid Accumulation in Transgenic Tobacco Plants

Aconitum carmichaelii Debx. is a herbal species that contains many precious bioactive substances, which are alkaloids, flavonoids, steroids, and glycosides. Flavonoids, which are major secondary compounds, play an important role in maintaining redox balance in the cells of the plant body. Many flavonoids have antibacterial, antioxidant, and anticancer properties. However, studies have mainly focused on aconitine, which is a highly toxic group A poison belonging to the alkaloid group, but with little mention of flavonoids. The flavonoids in A. carmichaelii are a group of substances with high content, concentrated in leaves and flowers, including quercetin and kaempferol. F3′5′H (Flavonoid 3′5′-hydroxylase) has been identified as the key enzyme involved in the final steps of flavonoid biosynthesis in plants in general and in A. carmichaelii specifically. This study offers the first report, and demonstrates that the overexpression of the F3′5′H gene from a herbal plant, A. carmichaelii, increases flavonoid content in genetically modified tobacco plants. The A. carmichaelii gene was transformed into tobacco leaf tissue to create transgenic tobacco plants. The AcF3′5′H gene was incorporated into the tobacco genome and was expressed in four transgenic tobacco lines (T01, T03, T05, and T014). The F3′5′H content increased from 20.33% to 32.00% compared with that in non-transformed plants (P < 0.001). Therefore, the flavonoid content of four transgenic tobacco lines increased compared to the WT, from 69.23% to 122.23% (P < 0.001). The results of the successful expression of the AcF3′5′H gene in model tobacco plants are the basis for using the AcF3′5′H gene for improving flavonoid content in other medicinal plants. Thus, the AcF3′5′H gene considered in this work could be a candidate for gene technology to enhance flavonoid accumulation in plants.

Characteristics of Isolates of Pseudomonas aeruginosa and Serratia marcescens Associated With Post-harvest Fuzi (Aconitum carmichaelii) Rot and Their Novel Loop-Mediated Isothermal Amplification Detection Methods

Fuzi (the lateral root of Aconitum carmichaelii Debx.) is a traditional Chinese medicine that is cultivated in more than eight provinces in China. However, it can be easily devastated by post-harvest rot, causing huge losses. Therefore, it is extremely important that the primary causal pathogens of post-harvest Fuzi rot are identified and appropriate detection methods for them are developed to prevent and control losses. In this study, two bacterial strains (X1 and X2) were isolated from rotten post-harvest Fuzi. Based on their morphological, physiological, and biochemical characteristics, housekeeping gene homologies, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) results, these isolates were identified as Pseudomonas aeruginosa and Serratia marcescens. The pathogenicities of these isolates were confirmed by fulfilling Koch’s postulates demonstrating that they were post-harvest Fuzi rot pathogens. Two loop-mediated isothermal amplification (LAMP) methods targeting the gyrase B subunit (gyrB) gene of P. aeruginosa and the phosphatidylinositol glycan C (pigC) gene of S. marcescens were successfully developed, and it was found that the target genes were highly specific to the two pathogens. These LAMP methods were used to detect P. aeruginosa and S. marcescens in 46 naturally occurring Fuzi and their associated rhizosphere soil samples of unknown etiology. The two bacterial assays were positive in some healthy and rotten samples and could be accomplished within 1 h at 65°C without the need for complicated, expensive instruments. To our knowledge, this is the first report of P. aeruginosa and S. marcescens causing post-harvest Fuzi rot. The newly developed methods are expected to have applications in point-of-care testing for the two pathogens under different Fuzi planting procedures and will significantly contribute to the control and prevention of Fuzi rot.

Transcriptome Analysis of Lateral Roots Development in Aconitum Carmichaelii

Background: Fuzi is a processed product of the lateral root of Aconitum carmichaelii Debx, a plant of the Ranunculaceae, and has been used to treat various diseases. This study used Illumina Hiseq High-throughput platform to sequence, assemble and annotate, screen development related genes, transcription pathways and functional enrichment in true root, lateral roots and “bridge”, and analyzed their correlations with the formation and development of lateral roots of A.carmichaelii, which can reveal the process and regulation mechanism of lateral roots growth and maturation of A. carmichaelii. Results: By sequencing, a total of 66.13Gb clean data and 28,982 unigenes with function annotation were finally obtained, with N50 of 1,627 bp, and 12,833 genes were assigned to 130 specific metabolic pathways by Kyoto Encyclopedia of Genes and Genomes (KEGG), then 2,599 were significantly differentially expressed. The KEGG analysis of the DEGs revealed that it was mainly enriched in starch and sucrose metabolism, ribosome, carbon metabolism, plant hormone signal transduction which play an important role in the expansion of lateral roots. The DEGs and pathways indicated that there was little differences between true root and “bridge”, while a big difference between them and lateral roots. The DEGs of auxin, cytokinin and other pathways may be conducive to the formation of lateral roots, which explained the development mechanism of lateral roots from a molecular point of view. Conclusions: This study provides a reference for the study of cultivation and management of lateral roots of A. carmichaelii.

Components and Pharmacodynamical Mechanism of Yinfupian Based on Liquid Chromatography-Mass Spectrometry and Proteomics Analyses

Objective: According to the treatment records of Yang deficiency syndrome (YDS) with characteristic decoction pieces of lateral root of Aconitum carmichaelii—Yinfupian (YF) in traditional Chinese medicine prepare school, known as “Jianchangbang”. The aim of this study was to investigate differences in the composition and therapeutic mechanism of the unprocessed lateral root of Aconitum carmichaelii (ULRA) and its processed product (YF).Methods: Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and orthogonal partial least squares discriminant analysis method were used to determine and screen the main components of ULRA and YF. Changes in the histological structure and morphology of gonads in rats were observed using hematoxylin-eosin. Enzyme-linked immunosorbent assay was used to determine the contents of serum cyclic adenosine monophosphate and cyclic guanosine monophosphate in YDS rats treated with ULRA and YF. Tandem mass tag proteomics analysis was used to identify the differentially expressed proteins in YDS rats treated with ULRA and YF.Results: Both ULRA and YF exerted certain therapeutic effects on rats with YDS. They improved the gonadal morphology and increased the contents of serum cyclic adenosine monophosphate and cyclic guanosine monophosphate. After processing of ULRA into YF, the content of C19-diester-diterpenoid alkaloids decreased (converted into C19-monoester-diterpenoid alkaloids and C19-alkylol amine-diterpenoid alkaloids), whereas that of C20-diterpene alkaloids increased. Proteomics analysis showed that cytochrome P450 and aldehyde oxidase 3 (AOX3) were downregulated, whereas cathepsin G (CTSG) was upregulated in rats with YDS. Treatment with ULRA mainly downregulated the expression of α-actinin, fast skeletal troponin, creatine kinase, and myosin. Treatment with YF mainly upregulated the expression of mitochondrial ribosomal protein and mitochondrial inner membrane protein.Conclusion: ULRA and YF exerted good therapeutic effects on YDS; the main difference in components between these preparations was in C19-diterpenoid alkaloids. ULRA mainly acts on the muscle contraction-related proteins and is closely related to inflammation and myocardial injury. YF mainly acts on the mitochondrial proteins and is closely related to adenosine triphosphate energy metabolism.