First Report of Seedling Stem Rot on Jinxianlian (Anoectochilus roxburghii) Caused by Fusarium oxysporum in China

Anoectochilus roxburghii is an important Chinese herbal medicine plant belonging to Orchidaceae and known as Jinxianlian. This orchid is cultivated and mostly adopted to treat diabetes and hepatitis. About 2 billion artificially cultivated seedlings of Jinxianlian are required each year and approximately $600 million in fresh A. roxburghii seedlings is produced in China. From 2011, sporadic occurrence of stem rot on Jinxianlian have been observed in greenhouses in Jinhua City (N29°05′, E119°38′), Zhejiang Province. In 2018, nearly 30% of seedlings of Jinxianlian grown in greenhouse conditions were affected by stem rot in Jinhua City. Symptoms initially occurred in the stem at the soil line causing dark discoloration lesions, rotted tissues, wilting, and eventually leading to the death of the plants. A total of 23 diseased seedlings collected from seven different greenhouses were surface sterilized with 1.5% sodium hypochlorite for 3 min, then rinsed in water. Pieces of tissues disinfected from each sample were plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 5 days (Kirk et al. 2008). A total of 19 isolates were recovered. They developed colonies with purple mycelia and beige or orange colors after 7 days of incubation under 25°C on PDA and carnation leaf agar (CLA) media (Kirk et al. 2008; Zhang et al. 2016). Colonies on PDA had an average radial growth rate of 3.1 to 4.0 mm /d at 25°C. Colony surface was pale vinaceous, floccose with abundant aerial mycelium. On CLA, aerial mycelium was sparse with abundant bright orange sporodochia forming on the carnation leaves. Microconidia were hyaline and oval-ellipsoid to cylindrical (3.7 to 9.3 × 1.3 to 2.9 μm) (n=19). Macroconidia were 3 to 5 septate and fusoid-subulate with a pedicellate base (27.4 to 35.6 × 3.2 to 4.2 μm) (n=19). These morphological features were consistent with Fusarium oxysporum (Sun et al. 2008; Lombard et al., 2019). To confirm the identification based on these morphological features, the internal transcribed spacer region (ITS) and translation elongation factor1 (TEF) were amplified from the DNA of 3 out of 19 isolates chosen at random respectively using the set primer ITS1/ITS4 and EF1/ EF2 (Sun, S., et al. 2018; Lombard et al., 2019). BLAST analysis revealed that the ITS sequences (OK147619, OK147620, OK147621) had 99% identity to that of F. oxysporum isolate JJF2 (GenBank MN626452) and TEF sequence (OK155999, OK156000, OK156001) had 100% identity to that of F. oxysporum isolate gss100 (GenBank MH341210). A multilocus phylogenetic analysis by Bayesian inference (BI) and maximum likelihood (ML) trees based on ITS and TEF indicated that the pathogen grouped consistently with F. oxysporum. Three out of 19 isolates chosen at random were selected to evaluate pathogenicity. Uninfected healthy A. roxburghii seedlings about 40 day-old planted in sterilized substrates were sprayed with distilled water containing 2 x 106 conidia per ml suspensions as inoculums, and plants sprayed with distilled water alone served as controls. Plants were then incubated at 25°C and 85% relative humidity. Ten plants were inoculated for each isolate. After 10 days, all plants inoculated developed stem rot symptoms, while control plants remained healthy. Cultures of Fusarium spp. were re-isolated only from inoculated plants with the frequency of 100% and re-identified by morphological characteristics as F. oxysporum, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of F. oxysporum causing stem rot on A. roxburghii seedlings. As F. oxysporum is a devastating pathogenic fungus with a broad host range, measures should be taken in advance to manage stem rot of A. roxburghii.

Combination of Anoectochilus roxburghii Polysaccharide and Exercise Ameliorates Diet-Induced Metabolic Disorders in Obese Mice

Metabolic syndrome is a cluster of metabolic disorders that threatens public health. Nevertheless, its exact mechanism and relative intervention remain largely obscure. Accumulating evidence indicate that tither Anoectochilus roxburghii polysaccharide (ARP) or exercise (EX) exhibited the beneficial effects on metabolic health. However, the synergetic beneficial effects of ARP and EX as a combined intervention on obesity-induced metabolic disorders remain largely obscure. Male C57BL/6 mice were fed a high-fat diet (HFD) and intervened with ARP and EX for 12 continuous weeks. The results indicated that the ARP, EX, and ARP combined with EX treatment group regulated lipogenesis by suppressing the fatty acid pathway, dampening the system oxidative stress by stimulating Nrf2-mediated phase II enzyme system, and promoting the mitochondrial function by activating the mitochondrial complexes and PGC-1α in HFD mice. More importantly, the combination of ARP and EX showed an even greater beneficial effects relative to either ARP or EX alone, especially in decreasing reactive oxygen species (ROS) level and increasing adenosine triphosphate (ATP) content. Taken together, these findings further confirmed that ARP and EX could be effective interventions on obesity-induced metabolic abnormalities, and that the combination of ARP and EX exhibited the beneficial synergetic effects.

Dietary Supplement of Anoectochilus Roxburghii Polysaccharides Ameliorates Cognitive Dysfunction Induced by High Fat Diet via “Gut-Brain” Axis

Background：Anoectochilus roxburghii, a traditional Chinese herbal medicine, has been widely used for treating numerous metabolic diseases. Anoectochilus roxburghii polysaccharide (ARPs) is an important constituent of Anoectochilus roxburghii. This study tried to investigate whether ARPs could improve cognitive dysfunction in diet-induced obesity.Methods: C57BL/6J male mice were randomly divided into the normal chow diet group (CD), the high-fat diet group (HFD) and HFD supplemented with ARPs (HARPs). Morris water maze test and Y maze test were used to evaluate spatial learning and memory. Serum TNF-α and LPS concentrations were detected by ELISA. Genes involved in Neurotrophic factors, inflammation and Intestinal permeability were detected by RT-PCR. Immunohistochemistry was used to detect TNF-α and ZO-1 proteins. BDNF in hippocampus was detected by immunofluorescence staining. 16S rDNA sequencing was used to detect intestinal microbial diversity.Results: Dietary supplement of ARPs ameliorates cognitive dysfunction induced by HFD. Compared with HFD mice, body weight, plasma glucose, total cholesterol and triglyceride levels in HARPs group were decreased significantly. Moreover, the cognitive function and memory of HFD mice were decreased, while ARPs abolished these effects. In addition, Tau protein levels were decreased and BDNF increased significantly in hippocampus from HARPs mice. Furthermore, compared with HFD group, IL-6 and TNF-α levels in hippocampus and colon from HARPs mice were decreased. Moreover, ARPs increased the tight junction protein levels and intestinal microbiota diversity significantly.Conclusions: Diet supplemented with ARPs prevented mice from HFD-induced cognitive dysfunction, indicated that ARPs has a good effect on obesity-related neuropathy. Mechanically, these effects at least in part attribute to the “gut-brain” axis regulation, and which can provide reference for the development of ARPs as functional foods in the future.