An account of Colletotrichum species associated with anthracnose of Atractylodes ovata in South Korea based on morphology and molecular data.

Ovate-leaf atractylodes (OLA) (Atractylodes ovata) is a well-known medicinal plant in Korea; its dried rhizome and root extracts are used in herbal medicine. However, anthracnose is a great challenge to the OLA cultivation in South Korea. Colletotrichum spp. is a major group of plant pathogens responsible for anthracnose on a range of economically important hosts. Its occurrence on OLA remains unresolved. To investigate the diversity, morphology, phylogeny, and biology of Colletotrichum spp., 32 fungal isolates were obtained from 30 OLA-affected leaves collected from five different farms, in two regions in South Korea, Mungyeong and Sangju. The phylogenetic analysis with four or five gene loci (ITS, TUB2, ACT, GAPDH, and CHS-1) along with morphology of 26 representative isolates delineated six previously known Colletotrichum species including C. fructicola, C. gloeosporioides sensu stricto (s.s), C. cigarro, C. plurivorum, C. siamense and C. sojae, and one new species, described here as C. ovataense. Amongst these species, C. gloeosporioides s.s. and C. plurivorum were the most prevalent species. A pathogenicity test on the detached leaves revealed that different Colletotrichum species presented a distinct degree of virulence, confirming Koch's postulates. In this study, C. fructicola, C. cigarro, C. plurivorum, C. siamense, and C. sojae were reported from A. ovata for the first time, as the causal agent of ovate-leaf atractylodes anthracnose. Understanding the diversity and biology of the Colletotrichum species population will help in managing this disease.

Endophytic Pseudomonas induces metabolic flux changes that enhance medicinal sesquiterpenoid accumulation in Atractylodes lancea.

The bacterial endophyte Pseudomonas fluorescens ALEB7B significantly enhances photosynthate accumulations in Atractylodes lancea. These carbohydrates are preferentially used by the host plant to synthesize secondary metabolites, rather than to increase plant biomass accumulation. Mechanisms underlying the allocation of endophyte-increased carbohydrate in different plant metabolic processes are largely unknown. We have studied how P. fluorescens ALEB7B enhances photosynthate accumulation and how bacterial elicitors regulate metabolic flux and increase medicinal sesquiterpenoid formation in A. lancea using the sterile tissue culture plantlets. P. fluorescens ALEB7B enhances plant photosynthate accumulation by synthesizing and secreting indole-3-acetic acid, which has been demonstrated using high-performance liquid chromatography analysis. The increased endogenous indole-3-acetic acid promotes plant root development and then assimilation. Increased carbohydrates provide the material basis for the formations of terpenoid hydrocarbon scaffolds, which has been proved using gas chromatography analysis. Further, protein and polysaccharide elicitors secreted by P. fluorescens ALEB7B have been separated and purified from the bacterial fermentation broth, which have been applied to A. lancea plantlets. Both elicitors can stimulate the conversions of terpenoid hydrocarbon scaffolds to oxygenous sesquiterpenoids, the active medicinal ingredients in A. lancea, by triggering the oxidative burst in planta. Moreover, this study separates an ABC transporter substrate-binding protein from protein elicitors secreted by P. fluorescens ALEB7B with an ÄKTA Prime Plus Purifier System and firstly shows that this protein is essential to induce oxygenous sesquiterpenoid accumulation in A. lancea. This study provides new perspectives for mechanisms of medicinal oxygenous terpenoid synthesis, which has important reference values to the cultivation of medicinal plants that have terpenoids as their active ingredients, such as Artemisia annua and Taxus chinensis.

[Identification of endophytic bacteria BZJN1 and research on biological control of root rot of Atractylodes macrocephala].

In this study， an endophytic bacteria strain BZJN1 was isolated from Atractylodes macrocephala， and identified as Bacillus subtilis by physiological and biochemical tests and molecular identification. Strain BZJN1 could inhibit the growth of mycelia of Ceratobasidium sp. significantly， and the inhibition rate was more than 70%. The mycelium growth deformity with bulge as spherical and partially exhaustible in apex or central with microscopic observation. The inhibitory rates under 3% and 6% concentrations of the cell free fermentation were 22.7% and 38.7% expectively. The field test proved that the control efficacy of treatment of 1×108 cfu·mL⁻¹ is 75.27% and 72.37% after 10 and 20 days. All the treatments of strain BZJN1 was able to promote the growth of A. macrocephala， the treatment of 1×108 cfu·mL⁻¹ could able to increase the yield to 14.1%.

Reactive oxygen species and hormone signaling cascades in endophytic bacterium induced essential oil accumulation in Atractylodes lancea.

MAIN CONCLUSION: Pseudomonas fluorescens induces gibberellin and ethylene signaling via hydrogen peroxide in planta . Ethylene activates abscisic acid signaling. Hormones increase sesquiterpenoid biosynthesis gene expression and enzyme activity, inducing essential oil accumulation. Atractylodes lancea is a famous Chinese medicinal plant, whose main active components are essential oils. Wild A. lancea has become endangered due to habitat destruction and over-exploitation. Although cultivation can ensure production of the medicinal material, the essential oil content in cultivated A. lancea is significantly lower than that in the wild herb. The application of microbes as elicitors has become an effective strategy to increase essential oil accumulation in cultivated A. lancea. Our previous study identified an endophytic bacterium, Pseudomonas fluorescens ALEB7B, which can increase essential oil accumulation in A. lancea more efficiently than other endophytes; however, the underlying mechanisms remain unknown (Physiol Plantarum 153:30-42, 2015; Appl Environ Microb 82:1577-1585, 2016). This study demonstrates that P. fluorescens ALEB7B firstly induces hydrogen peroxide (H2O2) signaling in A. lancea, which then simultaneously activates gibberellin (GA) and ethylene (ET) signaling. Subsequently, ET activates abscisic acid (ABA) signaling. GA and ABA signaling increase expression of HMGR and DXR, which encode key enzymes involved in sesquiterpenoid biosynthesis, leading to increased levels of the corresponding enzymes and then an accumulation of essential oils. Specific reactive oxygen species and hormone signaling cascades induced by P. fluorescens ALEB7B may contribute to high-efficiency essential oil accumulation in A. lancea. Illustrating the regulation mechanisms underlying P. fluorescens ALEB7B-induced essential oil accumulation not only provides the theoretical basis for the inducible synthesis of terpenoids in many medicinal plants, but also further reveals the complex and diverse interactions among different plants and their endophytes.

Volatiles released by endophytic Pseudomonas fluorescens promoting the growth and volatile oil accumulation in Atractylodes lancea.

Atractylodes lancea is a well-known, but endangered, Chinese medicinal plant whose volatile oils are its main active components. As the volatile oil content in cultivated A. lancea is much lower than that in the wild herb, the application of microbes or related elicitors to promote growth and volatile oil accumulation in the cultivated herb is an important area of research. This study demonstrates that the endophytic bacterium Pseudomonas fluorescens ALEB7B isolated from the geo-authentic A. lancea can release several nitrogenous volatiles, such as formamide and N,N-dimethyl-formamide, which significantly promote the growth of non-infected A. lancea. Moreover, the main bacterial volatile benzaldehyde significantly promotes volatile oil accumulation in non-infected A. lancea via activating plant defense responses. Notably, the bacterial nitrogenous volatiles cannot be detected in the A. lancea - Pseudomonas fluorescens symbiont while the benzaldehyde can be detected, indicating the nitrogenous volatiles or their precursors may have been consumed by the host plant. This study firstly demonstrates that the interaction between plant and endophytic bacterium is not limited to the commonly known physical contact, extending the ecological functions of endophyte in the phytosphere and deepening the understandings about the symbiotic interaction.

[Pathogen identification, regularity of development and control measures of diseases on Atractylodes lancea].

Based on current progress and field investigations,this review summarized the symptoms,epidemiology and control methods of 11 diseases on Atractylodes lancea, including the most severely harmed root diseases such as root rot and southern blight, as well as the sclerotinia rot that was newly happened. This review aims to demonstrate the progress of studies on diseases of A. lancea, providing guidance for field production. Sclerotonia disease and leaf spot disease are new diseases,suggesting the awareness of this disease on plant quarantine.

Involvement of abscisic acid and salicylic acid in signal cascade regulating bacterial endophyte-induced volatile oil biosynthesis in plantlets of Atractylodes lancea.

The enormous biological diversity of endophytes, coupled with their potential to enhance the production of bioactive metabolites in plants, has driven research efforts focusing on endophytes. However, limited information is available on the impacts of bacterial endophytes on plant secondary metabolism and signaling pathways involved. This work showed that an endophytic Acinetobacter sp. ALEB16, capable of activating accumulation of plant volatile oils, also induced abscisic acid (ABA) and salicylic acid (SA) production in Atractylodes lancea. Pre-treatment of plantlets with biosynthetic inhibitors of ABA or SA blocked the bacterium-induced volatile production. ABA inhibitors suppressed not only the bacterium-induced volatile accumulation but also the induced ABA and SA generation; nevertheless, SA inhibitors did not significantly inhibit the induced ABA biosynthesis, implying that SA acted downstream of ABA production. These results were confirmed by observations that exogenous ABA and SA reversed the inhibition of bacterium-induced volatile accumulation by inhibitors. Transcriptional activities of genes in sesquiterpenoid biosynthesis also increased significantly with bacterium, ABA and SA treatments. Mevalonate pathway proved to be the main source of isopentenyldiphosphate for bacterium-induced sesquiterpenoids, as assessed in experiments using specific terpene biosynthesis inhibitors. These results suggest that Acinetobacter sp. acts as an endophytic elicitor to stimulate volatile biosynthesis of A. lancea via an ABA/SA-dependent pathway, thereby yielding additional insight into the interconnection between ABA and SA in biosynthesis-related signaling pathways.

[Screening and identification of an endophytic fungus from Atractylodes lancea which utilizes volatile oil selectively].

In order to transform main active ingredient of volatile oil, endophytic fungi were screened from the root of Atractylodes lancea. Transformation method was used in vitro. The changes of volatile oil were traced by gas chromatography. One endophytic fungus (strain ALG-13) which could uitilize volatile oil selectively was screened. Single factor experiment were conducted for exploring the effects of various factors that including kinds of carbon source, speed, liquid volume, pH and concentration of plant tissue on degradation by this strain. Subsequently, the main affecting factors carbon source, speed, pH and liquid volume were optimized using orthogonal array design. Results showed that endophytic fungus ALG-13 selectively used the volatile oil, change the relative percentage of the main components of volatile oil, Atractylon and Atractydin were increased, While, beta-eudesmol and Atractylol decreased. After selectively degradation by fungus, volatile oil components percentage were closer to the geo-herbs. Strain ALG-13 was identified as Bionectria ochroleuca according to its morphological characteristics and systematic analysis of ITS sequence. The optimal conditions were as follows: sucrose used as carbon source, rotating speed was 200 r x min(-1), initial pH for medium was 4.5, 50 mL liquid was added in 250 mL flask. The endophytic fungus ALG-13 could degrade the volatile oil selectively, which was benefit for forming geoherbs A. lancea volatile oil composition.

[Effect of endophytic fungal elicitors on growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea].

OBJECTIVE: To investigate the effects of endophytic fungal elicitors on the growth and atractylodin accumulation of cell suspension cultures of Atractylodes lancea. METHOD: The endophytic fungal elicitors were added to the medium with different concentrations and culture period. Their effects on biomass, atractylodin content and relevant enzyme activities in suspension cultured cells were studied. RESULT: The cell growth was not affected by elicitors at low concentration and obviously inhibited at high concentration. Inhibition rate reached 46.7% by 100 mg L(-1) elicitor. In addition, six strains from A. lancea, among which Rhizoctonia SP1 activity was higher, had distinctly promoted the accumulation of atractylodin. Atractylodin biosynthesis was notably promoted by 20-60 mg L(-1) Rhizoctonia SP1 elicitor. When 40 mg L(-1) Rhizoctonia SP1 elicitor was added in the medium at the 12 day, the maximum content of atractylodin was 28.06 microg L(-1) at the 21 day with 48.3% higher than that of the control and PPO, POD and CAT activities remarkably increased. CONCLUSION: Adding the endophytic elicitors to the medium is able to be effective approaches to enhance atractylodin yield in the suspension culture cell of A. lancea.

[Effects of Arbuscular Mycorrhizae on growth and essential oil of Atractylodes lancea].

OBJECTIVE: To study the effects of Arbuscular Mycorrhizae on cultivated Atractylodes lancea. METHOD: Pot experiment of A. lancea, with (code as AM) or without (code as CK) Glomus mosseae (GM) was conducted 5 times respectively, then the biomass, essential oil, and soil nutrition, soil organism, soil microbial were detected after A. lancea were harvested. RESULT: (1) Mycorrhizal dependency of A. lancea was 245%, and height of individuals, numbers of leaves, leaf area, biomass of A. lancea were all higher in AM than in CK (P < 0.05). (2) GC-MS analysis with cluster analysis and principal components analysis showed that there were no differences in essential oil of A. lancea between AM and CK. (T3) Total N, available N, available P and available K in AM soil were all lower than in CK soil. (4) GC-MS analysis showed organic matters changed differenly in AM soil and CK soil, components 5,6 in AM soil were higher than that in CK soil, but component 9, 10, 11 were lower in AM soil than that in CK soil. (5) Biolog detect showed AWCD of AM soil microbe were higher than that of CK soil throughout the incubation, and AWCD of the former was 0.66, and the later was 0.46 after 192 h incubation. and t-test showed, Shannon seven indices and McIntosh'seven indices were same both at 72 h and 192 h, and diversity indices of Shannon and McIntosh were also same at 72 h, but AM soil microbe were higher than CK soil microbe at 168 h (P < 0.05). CONCLUSION: AM could promote nutrition uptake, improve the function diversity and activity of microbe in rhizosphere of A. lancea, influence the composition of the organic matter, that lead the growth of A. lancea, but not to the quality.