Antagonism of rhizosphere Trichoderma brevicompactum DTN19 against the pathogenic fungi causing corm rot in saffron (Crocus sativus L.) in vitro.

Introduction: Corm rot in saffron (Crocus sativus L.) significantly impacts yield and quality. Non-toxic fungi, particularly Trichoderma species, are valuable for biological control due to their production of diverse and biologically active secondary metabolites. Methods: This study aimed to isolate an effective antagonistic fungus against the pathogenic fungi causing corm rot in saffron. Four pathogenic fungi (Fusarium oxysporum, Fusarium solani, Penicillium citreosulfuratum, and Penicillium citrinum) were isolated from diseased saffron bulbs in Chongming. Initial screening through dual culture with these pathogens re-screening from rhizosphere soil samples of C. sativus based on its inhibitory effects through volatile, nonvolatile, and fermentation broth metabolites. The inhibitory effect of biocontrol fungi on pathogenic fungi in vitro was evaluated by morphological observation and molecular biology methods. Results: Antagonistic fungi were identified as Trichoderma brevicompactum DTN19. F. oxysporum was identified as the most severe pathogen. SEM (scanning electron microscope) and TEM (transmission electron microscope) observations revealed that T. brevicompactum DTN19 significantly inhibited the growth and development of F. oxysporum mycelium, disrupting its physiological structure and spore formation. Additionally, T. brevicompactum DTN19 demonstrated nitrogen fixation and production of cellulase, IAA (Indole acetic acid), and siderophores. Whole-genome sequencing of strain DTN19 revealed genes encoding protease, cellulase, chitinase, ß-glucosidase, siderophore, nitrogen cycle, and sulfate transporter-related proteins. Discussion: T. brevicompactum DTN19 may inhibit the propagation of pathogenic fungi by destroying their cell walls or producing antibiotics. It can also produce IAA and iron carriers, which have the potential to promote plant growth. Overall, T. brevicompactum DTN19 showed the development prospect of biological agents.

Feedback regulation of plant secondary metabolism: Applications and challenges.

Plant secondary metabolites offer resistance to invasion by herbivorous organisms, and are also useful in the chemical, pharmaceutical, cosmetic, and fragrance industries. There are numerous approaches to enhancing secondary metabolite yields. However, a growing number of studies has indicated that feedback regulation may be critical in regulating secondary metabolite biosynthesis. Here, we review examples of feedback regulation in secondary metabolite biosynthesis pathways, phytohormone signal transduction, and complex deposition sites associated with secondary metabolite biosynthesis. We propose a new strategy to enhance secondary metabolite production based on plant feedback regulation. We also discuss challenges in feedback regulation that must be overcome before its application to enhancing secondary metabolite yields. This review discusses recent advances in the field and highlights a strategy to overcome feedback regulation-related obstacles and obtain high secondary metabolite yields.

AaWRKY6 contributes to artemisinin accumulation during growth in Artemisia annua.

Artemisinin, which is extracted from the plant Artemisia annua L., is a crucial drug for curing malaria and has potential applications for treating cancer, diabetes, pulmonary tuberculosis, and other conditions. Demand for artemisinin is therefore high, and enhancing its yield is important. Artemisinin dynamics change during the growth cycle of A. annua; however, the regulatory networks underlying these changes are poorly understood. Here, we collected A. annua leaves at different growth stages and identified target genes from transcriptome data. We determined that WRKY6 binds to the promoters of the artemisinin biosynthesis gene artemisinic aldehyde Δ11(13) reductase (DBR2). In agreement, overexpression of WRKY6 in A. annua resulted in higher expression levels of genes in the artemisinin biosynthesis pathway and greater artemisinin contents than in the wild type. When expression of WRKY6 was down-regulated, artemisinin biosynthesis pathway genes were also down-regulated and the content of artemisinin was lower. WRKY6 mediates the transcriptional activation of artemisinin biosynthesis by binding to the promoter of DBR2, making it a key regulator for modulating the dynamics of artemisinin changes during the A. annua growth cycle.

Antagonism of Rhizosphere Streptomyces yangpuensis CM253 against the Pathogenic Fungi Causing Corm Rot in Saffron (Crocus sativus L.).

Plant diseases lead to a significant decline in the output and quality of Chinese herbal medicines. Actinomycetes play a vital role in the rhizosphere ecosystem. This is especially true for Streptomyces, which have become a valuable biological control resource because of their advantages in producing various secondary metabolites with novel structures and remarkable biological activities. The purpose of this study was to isolate an effective antagonistic actinomycete against the pathogen of corm rot in saffron. An antagonistic actinomycete, CM253, was screened from the rhizosphere soil samples of Crocus sativus, by plate co-culture with four pathogenic fungi (Fusarium oxysporum, Fusarium solani, Penicillium citreosulfuratum, and Penicillium citrinum). CM253 inhibited the growth and development of F. oxysporum hyphae by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Furthermore, by analyzing the degrading enzyme, the growth-promoting performance, and the whole genome of strain CM253, it was identified as Streptomyces yangpuensis, which produces NH3, protease, glucanase, cellulase, IAA, and ACC deaminase. In addition, 24 secondary metabolite synthesis gene clusters were predicted in antiSMASH. We identified genes encoding 2,3-butanediol; methionine; isoprene (metH, mmuM, ispEFH, gcpE, idi, and ilvABCDEH); biofilm formation; and colonization (upp, rfbBC, efp, aftA, pssA, pilD, fliA, and dhaM). Above all, S. yangpuensis CM253 showed the potential for future development as a biocontrol agent.

In Vitro Study of Biocontrol Potential of Rhizospheric Pseudomonas aeruginosa against Pathogenic Fungi of Saffron (Crocus sativus L.).

Plant rhizosphere soil contains a large number of plant-growth promoting rhizobacteria, which can not only resist the invasion of pathogenic microorganisms and protect plants from damage, but also promote the growth and development of plants. In this study, Pseudomonas aeruginosa strain YY322, isolated and screened from the rhizosphere soil of saffron (Crocus sativus L.), was found through a plate confrontation experiment to show highly effectual and obvious antagonistic activity against the pathogens of saffron, including Fusarium oxysporum, Fusarium solani, Penicillium citreosulfuratum, Penicillium citrinum and Stromatinia gladioli. In addition, the volatile organic compounds of strain YY322 had great antagonistic activity against these pathogens. Observation under a scanning electron microscope and transmission electron microscope reflected that strain YY322 had a significant effect on the hyphae and conidia of F. oxysporum and F. solani. Through the detection of degrading enzymes, it was found that P. aeruginosa can secrete protease and glucanase. The plant growth promoting performance was evaluated, finding that strain YY322 had the functions of dissolving phosphorus, fixing nitrogen, producing siderophore and producing NH3. In addition, whole genome sequencing analysis indicated that the YY322 genome is comprised of a 6,382,345-bp circular chromosome, containing 5809 protein-coding genes and 151 RNA genes. The P. aeruginosa YY322 genome encodes genes related to phenazine (phzABDEFGIMRS), hydrogen cyanide(HCN) (hcnABC), surfactin (srfAA), salicylate (pchA), biofilm formation (flgBCDEFGHIJKL, motAB, efp, hfq), and colonization (minCDE, yjbB, lysC). These results collectively indicated the role of P. aeruginosa YY322 in plant growth enhancement and biocontrol mechanisms. All in all, this study provides a theoretical basis for P. aeruginosa as the PGPR of saffron, paving the way for the subsequent development and utilization of microbial fertilizer.

First Report of Black Spot Caused by Penicillium citreosulfuratum on Saffron in Chongming Island, China.

Saffron (Crocus sativus L.) is a world-famous source of dye and spices and an important medicinal plant, which is cultivated on a large scale on Chongming Island (31.62° N, 121.39° E) in Shanghai, China. In August 2020, a survey of saffron was conducted in this area, and black spots were observed on about 10% of plants. Characteristics of the disease were: The bottom of the corm was darkened in the groove and scattered black spots could be observed after peeling off the membranous scale leaves. The junction of lesions and healthy parts was light brown. As the lesions expanded, approximately 80% of the surface of the corm became dark brown to bluish gray. The inside of the corm was also necrotic. In order to isolate the pathogen, ten diseased corms with typical symptoms were selected. All corms were first treated with 75% ethanol for 30 s, 0.1% HgCl2 for 5 min, and then rinsed with sterile water 5 times. Next, tissue pieces (5 mm × 5 mm) at the margin of lesions were cut out and placed on the potato dextrose agar (PDA) medium. After incubating at 28°C for 5 days, fungi were separated and purified by using the hyphal-tip technique. A total of six pure cultures with different colony morphologies were obtained, of which only the isolate MF3 was present in ten diseased corms. The isolate MF3 was inoculated on the PDA and cultured at 28°C for 10 days and characteristics of the fungus colonies were: colonies sub-circular, reaching 28 to 30 mm diam, from above rough, dense, fluffy, blue-gray, with some white spots and central point raised, and outer margin form an irregular, narrow, white ring; from below, yellow with light-green. The hyphae were slender, with many septa. The conidiophores were typically smooth walled, short, and slender and either monoverticillate with very short stipes or as irregularly biverticillate. Phialides were ampulliform, 5 to 10 per metula, 5 to 8 × 2 to 3 µm. Conidia were smooth and globose, and ranged in diameter from 1.4 to 1.7 µm (n=50). Molecular identification of the fungus was made by PCR amplification of the internal transcribed spacer (ITS) region of rDNA and ß-tubulin gene using primers ITS1/ITS4 (White et al. 1990), TUB2Fd/TUB4Rd (Aveskamp et al. 2009) respectively. The ITS (MW881446) and ß-tubulin (MW911464) sequences of the fungus were similar to the ITS (MN592912) and ß-tubulin (KY469126) sequences of the epitype of Penicillium citreosulfuratum with 99.81% and 99.56% identity, respectively. According to the morphological and molecular characterization, the isolate MF3 was identified as P. citreosulfuratum (Visagie et al. 2016). For pathogenicity testing, the fungus was grown on PDA and incubated at 28°C for 5 days. Then mycelial plugs (5 mm diam.) were inoculated on the scalpel incision square wounds of surface-disinfected corms and mock-inoculated corms received only PDA plugs. Corms were placed in sterile plastic bottles and observed after culturing at 28°C for 21 days. Each treatment had three replicates and the experiment was repeated twice. The results showed that corms inoculated with P. citreosulfuratum developed diseased with similar symptoms as in the field. No disease symptoms were observed on control corms. Re-isolations were performed from inoculated corms, and all re-isolated fungi were confirmed as P. citreosulfuratum, verifying the fungus as the pathogen based on Koch's postulates. To our knowledge, this is the first report of the pathogen causing black spot disease of saffron. Although the disease is not fatal to saffron, to a certain extent it will cause a reduction in the production of the crop. In addition, this pathogen has not been reported to be pathogentic to other plant species.

Active anti-acetylcholinesterase component of secondary metabolites produced by the endophytic fungi of Huperzia serrata

Background An endophytic fungus lives within a healthy plant during certain stages of, or throughout, its life cycle. Endophytic fungi do not always cause plant disease, and they include fungi that yield different effects, including mutual benefit, and neutral and pathogenic effects. Endophytic fungi promote plant growth, improve the host plant's resistance to biotic and abiotic stresses, and can produce the same or similar biologically active substances as the host. Thus, endophytic fungal products have important implications in drug development. Result Among the numerous endophytic fungi, we identified two strains, L10Q37 and LQ2F02, that have anti-acetylcholinesterase activity, but the active compound was not huperzine A. The aim of this study was to investigate the anti-acetylcholinesterase activity of secondary metabolites isolated from the endophytic fungi of Huperzia serrata. Microbial cultivation and fermentation were used to obtain secondary metabolites. Active components were then extracted from the secondary metabolites, and their activities were tracked. Two compounds that were isolated from endophytic fungi of H. serrata were identified and had acetylcholine inhibitory activities. In conclusion, endophytic fungal strains were found in H. serrata that had the same anti-acetylcholinesterase activity. Conclusion We isolated 4 compounds from the endophytic fungus L10Q37, among them S1 and S3 are new compounds. 6 compounds were isolated from LQ2F02, all 6 compounds are new compounds. After tested anti acetylcholinesterase activity, S5 has the best activity. Other compounds' anti acetylcholinesterase activity was not better compared with huperzine A.

Microbial biotransformation of gentiopicroside by the endophytic fungus Penicillium crustosum 2T01Y01.

Endophytic fungi are symbiotic with plants and possess multienzyme systems showing promising metabolite potency with region selectivity and stereoselectivity. The aim of this study was to use these special microorganisms as an in vitro model to mimic the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, compound 1). The fungi isolated from a medicinal plant, Dendrobium candidum Wall. ex Lindl., were screened for their biotransformation abilities with GPS as the substrate, and one strain with high converting potency was identified as Penicillium crustosum 2T01Y01 on the basis of the sequence of the internal transcribed spacer of the ribosomal DNA region. Upon the optimized incubation of P. crustosum 2T01Y01 with the substrate, seven deglycosylated metabolites were detected by ultraperformance liquid chromatography/quadrupole time of flight mass spectrometry (UPLC/Q-TOF MS). Preparative-scale biotransformation with whole cells of the endophytic fungus resulted in the production of five metabolites, including three novel ones, 5α-(hydroxymethyl)-6ß-methyl-3,4,5,6-tetrahydropyrano[3,4-c]pyran-1(8H)-one (compound 2), (Z)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 3), and (E)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (compound 4), along with two known ones, 5α-(hydroxymethyl)-6ß-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 5) and 5α-(hydroxymethyl)-6α-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (compound 6), aided by nuclear magnetic resonance and high-resolution mass spectral analyses. The other two metabolites were tentatively identified by online UPLC/Q-TOF MS as 5-hydroxymethyl-5,6-dihydroisochromen-1-one (compound 7) and 5-hydroxymethyl-3,4,5,6-tetrahydroisochromen-1-one (compound 8), and compound 8 is a new metabolite. To test the metabolic mechanism, the ß-glucosidase activity of the fungus P. crustosum 2T01Y01 was assayed with ρ-nitrophenyl-ß-d-glucopyranoside as a probe substrate, and the pathway of GPS biotransformation by strain 2T01Y01 is proposed. In addition, the hepatoprotective activities of GPS and metabolite compounds 2, 5, and 6 against human hepatocyte line HL-7702 injury induced by hydrogen peroxide were evaluated.

Evidence-based antioxidant activity of the essential oil from Fructus A. zerumbet on cultured human umbilical vein endothelial cells' injury induced by ox-LDL.

BACKGROUND: The essential oil from Fructus Alpiniae zerumbet (FAZ) is its principal bioactive ingredient, and is widely used in Miao folk herbs in Guizhou province for the treatment of gastrointestinal and cardiovascular diseases. Several studies have confirmed that FAZ ameliorates hyperlipidemia and atherosclerosis. Because endothelial dysfunction often accompanies cardiovascular diseases, especially hyperlipidemia and atherosclerosis, the present study concentrated on evaluating the endothelial protective effects of the essential oil from FAZ (EOFAZ) on oxidized low-density lipoprotein (ox-LDL)-induced injury of cultured human umbilical vein endothelial cells (HUVECs) and on the regulation of oxidative stress. METHODS: Cell viability was analyzed with the MTT assay and trypan blue exclusion staining (TBES). Cell injury was assessed by lactate dehydrogenase (LDH) release. Biochemical enzymatic methods were used to evaluate the oxidative stress, including the lipid peroxidation product, malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). RESULTS: The redox status of HUVECs was significantly exacerbated after exposure to ox-LDL. EOFAZ protected HUVECs against ox-LDL injury as assessed by the MTT assay, TBES and LDH release. Furthermore, EOFAZ ameliorated the oxidative stress by elevating the activities of SOD, CAT and GSH-Px, and increasing the GSH levels, in addition to attenuating the MDA contents. CONCLUSIONS: The present data provide the first experimental evidence that EOFAZ protects endothelial cells against ox-LDL-induced injury, and indicate that this protection involves ameliorating the redox status.

[Screening of endophytic fungi from Huperzia serrata for acetylcholinesterase inhibitory activity and its taxonomic identification].

OBJECTIVE: To screen out fungus strains with acetylcholinesterase inhibitory activity from Huperzia serrata. METHOD: Endophytic fungi fermentation products from 59 H. serrata strains were stained with acetylcholinesterase hydrolyzed alpha-naphthaleneacetic ethyl ester and fast blue B salt, and screened for acetylcholinesterase inhibitory activity with thin-layer chromatography-bioautography. Target strains were classified and identified through the sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics. RESULT: Fungus strain LQ2F01 from H. serrata showed positive color reaction in the screening for acetylcholinesterase inhibitory activity. The sequence analysis on 18s rDNA and 5.8s rDNA combined with morphological characteristics showed the strain LQ2F01 belonged to Acremonium. CONCLUSION: Endophytic Fungi LQ2F01 from H. serrata shows identical acetylcholinesterase inhibitory activity with the host plant, which is of great significance to the development of natural medicines and the studies on the relationship between the endophytic gungi and the host plant.