A LAMP-Based Toolbox Developed for Detecting the Major Pathogens Affecting the Production and Quality of the Chinese Medicinal Crop Aconitum carmichaelii.

Aconitum carmichaelii Debeaux is a traditional Chinese medicinal herb that has been utilized for approximately 2,000 years. However, as cultivation has increased, there have been more reports of A. carmichaelii infections caused by four major pathogenic fungal species, Fusarium oxysporum, F. solani, Mucor circinelloides, and Sclerotium rolfsii, resulting in increased disease incidences and limited production and quality. To detect these infections, we developed a LAMP-based toolbox in this study. The cytochrome c oxidase subunit 1 (cox1) gene, translation elongation factor-1α (EF-1α), internal transcribed spacer (ITS) regions of rDNA, and alcohol dehydrogenase 1 (ADH1) gene, respectively, were used to design species-specific LAMP primer sets for F. oxysporum, F. solani, S. rolfsii, and M. circinelloides. The results showed that the LAMP-based toolbox was effective at detecting pathogens in soil and plant materials. We also used this toolbox to investigate pathogen infection in the main planting regions of A. carmichaelii. Before harvesting, F. oxysporum, M. circinelloides, and S. rolfsii were commonly found in the planting fields and in infected A. carmichaelii plants. Therefore, the toolbox we developed will be useful for tracking these infections, as well as for disease control in A. carmichaelii.

Identification and Characterization of Sclerotium delphinii Causing Southern Blight on Aconitum kusnezoffii in Northeast China.

Aconitum kusnezoffii is a perennial medicinal plant that belongs to the Ranunculaceae family and is distributed mainly in Northeast and North China. In July 2018, a typical southern blight disease of A. kusnezoffii was observed in commercial fields of Qingyuan County, Fushun City, Liaoning Province, China. The fungus mainly infected stem base and tuberous roots of the plant by wrapping the hyphae and absorbing nutrition, resulting in tuberous root wilted or whole plant death. Morphological characteristics of colony and sclerotia of three representative strains isolated from the diseased plants differed from those of Sclerotium rolfsii isolated from A. carmichaelii. Sclerotia were large (0.8 to 5.1 mm), reddish-brown, and irregular and had pitted surfaces, and the hyphae were white, compact, or fluffy, with a growth rate ranging from 8.0 to 10.1 mm/day. Phylogenetic analysis of the internal transcribed spacer and the large subunit sequences of Akln6, Akln9, and Akln15 showed that three strains isolated from A. kusnezoffii formed a unique and well-supported clade that groups with the reference isolates of S. delphinii. Based on phylogenetic analysis and cultural and morphological characteristics, the three isolates of A. kusnezoffii were identified as S. delphinii. The optimum temperature for mycelial growth of the three tested isolates was 30°C, and sclerotia formed and matured more easily at 20°C. Light promoted the growth of mycelial, whereas dark was beneficial to the formation and maturation of sclerotia. The pathogenicity of S. delphinii showed stronger than S. rolfsii at low temperature (20°C). This is the first report of S. delphinii causing southern blight on A. kusnezoffii in China, and this finding provides a basis for disease-accurate diagnosis and the development of effective management strategies.

Soil fungal biodiversity and pathogen identification of rotten disease in Aconitum carmichaelii (Fuzi) roots.

Aconitum carmichaelii, commonly known as Fuzi, is a typical traditional Chinese medicine (TCM) herb that has been grown for more than one thousand years in China. Although root rot disease has been seriously threatening this crop in recent years, few studies have investigated root rot disease in Fuzi, and no pathogens have been identified. In this study, fungal libraries from rhizosphere soils were constructed by internal transcribed spacer (ITS) sequencing using the HiSeq 2500 high-throughput platform. A total of 948,843 tags were obtained from 17 soil samples, and these corresponded to 195,583,495 nt. At 97% identity, the libraries yielded 12,266 operational taxonomic units (OTUs), of which 97.5% could be annotated. In sick soils, Athelia, Mucor and Mortierella were the dominant fungi, comprising 10.3%, 10.1% and 7.7% of the fungal community, respectively. These fungi showed 2.6-, 1.53- to 6.31- and 1.38- to 2.65-fold higher enrichment in sick soils compared with healthy soils, and their high densities reduced the fungal richness in the areas surrounding the rotted Fuzi roots. An abundance analysis suggested that A. rolfsii and Mucor racemosus, as the dominant pathogens, might play important roles in the invading Fuzi tissue, and Phoma adonidicola could be another pathogenic fungus of root rot. In contrast, Mortierella chlamydospora, Penicillium simplicissimum, Epicoccum nigrum, Cyberlindnera saturnus and Rhodotorula ingeniosa might antagonize root rot pathogens in sick soils. In addition, A. rolfsii was further verified as a main pathogen of Fuzi root rot disease through hypha purification, morphological observation, molecular identification and an infection test. These results provide theoretical guidance for the prevention and treatment of Fuzi root rot disease.

Antiviral anthraquinones and azaphilones produced by an endophytic fungus Nigrospora sp. from Aconitum carmichaeli.

A new hydroanthraquinone derivative, 6-O-demethyl-4-dehydroxyaltersolanol A (1), and two new azaphilones, 8,11-didehydrochermesinone B (6) and (7S)-7-hydroxy-3,7-dimethyl-isochromene-6,8-dione (8), along with five known analogues (2-5 and 7), were isolated from the culture broth of Nigrospora sp. YE3033, an endophytic fungus obtained from Aconitum carmichaeli. Their structures were elucidated on the basis of spectroscopic analyses. Biological activity test indicated that compounds 1-3, and 7 exhibited the inhibitory effects on influenza viral strain of A/Puerto Rico/8/34 (H1N1) with the IC50 values of 2.59, 8.35, 7.82, and 0.80µg/mL, respectively, while the low cytotoxicity of 7 with the CC50 value of 184.75µg/mL, displaying a promising potential of 7 in the development of anti-influenza A virus drugs.

Nonomuraea flavida sp. nov., a novel species of soil actinomycete isolated from Aconitum napellus rhizosphere.

A novel actinomycete strain, YN-5-1T, isolated from the rhizosphere soil of a medicinal plant, Aconitum napellus, was characterized by a polyphasic approach to determine its taxonomic position. The strain showed highest 16S rRNA gene sequence similarities of 97.3, 97.2 and 97.1 % to Nonomuraea turkmeniaca DSM 43926T, Nonomuraea ferruginea DSM 43553T and Nonomuraea candida DSM 45086T, respectively. A wide range of genotypic and phenotypic characteristics, as well as levels of DNA-DNA relatedness between strain YN-5-1T and N. turkmeniaca DSM 43926T (57.46 %), N. ferruginea DSM 43553T (53.50 %) and N. candida DSM 45086T (48.80 %), distinguished the novel isolate from its closest phylogenetic neighbours. The morphological characteristics of strain YN-5-1T were typical of the genus Nonomuraea. Chemotaxonomic characteristics, such as diagnostic diamino acid of the peptidoglycan, whole-cell sugars, phospholipid type, major menaquinone and major fatty acids, further supported the assignment of strain YN-5-1T to the genus Nonomuraea. The G+C content of the genomic DNA was 72.1 mol%. Based on the above data, strain YN-5-1T is considered to represent a novel species of the genus Nonomuraea, for which the name Nonomuraea flavida sp. nov. is proposed. The type strain is YN-5-1T ( = CCTCC AB 2012909T = KCTC 29143T).