Diversity, antibacterial and phytotoxic activities of culturable endophytic fungi from Pinellia pedatisecta and Pinellia ternata.

BACKGROUND: Endophytic fungi of medicinal plants, as special microorganisms, are important sources of antibacterial compounds. However, the diversity and antibacterial activity of endophytic fungi from Pinellia Tenore have not been systematically studied. RESULTS: A total of 77 fungi were isolated from roots, stems, leaves, and tubers of Pinellia ternata and P. pedatisecta. All fungi were belonged to five classes and twenty-five different genera. Biological activities tests indicated that 21 extracts of endophytic fungi exhibited antibacterial activities against at least one of the tested bacteria, and 22 fermentation broth of endophytic fungi showed strong phytotoxic activity against Echinochloa crusgalli with the inhibition rate of 100%. Furthermore, four compounds, including alternariol monomethyl ether (1), alternariol (2), dehydroaltenusin (3) and altertoxin II (4), and three compounds, including terreic acid (5), terremutin (6), citrinin (7), were isolated from Alternaria angustiovoidea PT09 of P. ternata and Aspergillus floccosus PP39 of P. pedatisecta, respectively. Compound 5 exhibited strong antibacterial activities against Escherichia coli, Micrococcus tetragenus, Staphylococcus aureus, and Pseudomonas syringae pv. actinidiae with the inhibition zone diameter (IZD) of 36.0, 31.0, 33.7, 40.2 mm and minimum inhibitory concentration (MIC) values of 1.56, 3.13, 1.56, 1.56 µg/mL respectively, which were better than or equal to those of positive gentamicin sulfate. The metabolite 7 also exhibited strong antibacterial activity against P. syringae pv. actinidiae with the IZD of 26.0 mm and MIC value of 6.25 µg/mL. In addition, the compound 7 had potent phytotoxic activity against E. crusgalli with the inhibition rate of 73.4% at the concentration of 100 µg/mL. CONCLUSIONS: Hence, this study showed that endophytic fungi of P. ternata and P. pedatisecta held promise for the development of new antibiotic and herbicide resources.

Transcriptomic analysis of Pinellia ternata (Thunb.) Breit T2 plus line provides insights in host responses resist Pectobacterium carotovorum infection.

Transcriptome is used to determine the induction response of Pinellia ternata (Thunb.) Breit T2 plus line (abbreviated as PT2P line) infected with Pectobacterium carotovorum. The main objective of the study was to deal with the transcriptome database of PT2P line resistance to soft rot pathogens to provide a new perspective for identifying the resistance-related genes and understanding the molecular mechanism. Results indicated that water soaking and tissue collapse started at 20 h after PT2P line was infected by P. carotovorum. A total of 1360 and 5768 differentially expressed genes (DEGs) were identified at 0 h and 20 h, respectively. After 20 h of infection, growth and development-related pathways were inhibited. Meanwhile, DEGs were promoted the colonization of P. carotovorum pathogens in specific cell wall modification processes at the early infected stage. A shift to a defensive response was triggered at 0 h. A large number of DEGs were mainly up-controlled at 20 h and were substantially used in the pathogen recognition and the introduction of signal transformation cascades, secondary metabolites biosynthesis, pathogenic proteins activation, transcription aspects and numerous transporters. Furthermore, our data provided novel insights into the transcript reprogramming of PT2P line in response to P. carotovorum infestation.

Siderophore Production by Rhizosphere Biological Control Bacteria Brevibacillus brevis GZDF3 of Pinellia ternata and Its Antifungal Effects on Candida albicans.

Brevibacillus brevis GZDF3 is a gram-positive, plant growth-promoting rhizosphere bacterium (PGPR) isolated from the rhizosphere soil of Pinellia ternata (an important herb in traditional Chinese medicine). The GZDF3 strain produces certain active compounds, such as siderophores, which are the final metabolite products of non-ribosomal peptide synthetase (NRPS) and independent non-ribosomal peptide synthetase (NIS) activity. With the present study, we attempted to investigate the siderophore production characteristics and conditions of Bacillus sp. GZDF3. The antibacterial activity of the siderophores on pathogenic fungi was also investigated. Optimal conditions for the synthesis of siderophores were determined by single factor method, using sucrose 15 g/l, asparagine 2 g/l, 32°C, and 48 h. The optimized sucrose asparagine medium significantly increased the production of siderophores, from 27.09% to 54.99%. Moreover, the effects of different kinds of metal ions on siderophore production were explored here. We found that Fe3+ and Cu2+ significantly inhibited the synthesis of siderophores. The preliminary separation and purification of siderophores by immobilized-metal affinity chromatography (IMAC) provides strong antibacterial activity against Candida albicans. The synergistic effect of siderophores and amphotericin B was also demonstrated. Our results have shown that the GZDF3 strain could produce a large amount of siderophores with strong antagonistic activity, which is helpful in the development of new biological control agents.

Microbiota and Chemical Compounds in Fermented Pinelliae Rhizoma (Banxiaqu) from Different Areas in the Sichuan Province, China.

This study focused on the microbiota and chemical compounds of the fermented Pinelliae Rhizoma produced in Longchang (LC), Zizhong (ZZ) and Xindu (XD), in Sichuan Province (China). High-throughput sequencing was used to analyze the microbiota. GC-MS and LC-MS were used to detect the compounds produced during the three different Pinelliae Rhizoma fermentation processes. The bacteria and fungi of the three fermented Pinelliae Rhizoma differed substantially, with the bacterial content mainly composed of the Bacillus genus, while the common fungi were only included in four OTUs, which belong to three species of Eurotiomycetes and Aspergillus cibarius. 51 volatile compounds were detected; they varied between LC, XD, and ZZ fermented Pinelliae Rhizoma. C10 and C15 terpenes were most frequently detected, and only curcumene and ß-bisabolene were detected in the three fermented Pinelliae Rhizoma. 65 non-volatile compounds were detected by LC-MS, most were of C16, C18, C20, C21 and C22 structures. Cluster analysis showed more similarity between LC and XD fermented Pinelliae Rhizoma with regards to volatile compound content, but more similarity between the XD and ZZ fermented Pinelliae Rhizoma for non-volatiles. Moreover, no correlation between geographical distance and microflora or compounds of fermented Pinelliae Rhizoma was observed. These results showed that hundreds of compounds are produced by the natural mixed fermentation of Pinelliae Rhizoma, and may mostly relate to the microorganisms of five species.This study focused on the microbiota and chemical compounds of the fermented Pinelliae Rhizoma produced in Longchang (LC), Zizhong (ZZ) and Xindu (XD), in Sichuan Province (China). High-throughput sequencing was used to analyze the microbiota. GC-MS and LC-MS were used to detect the compounds produced during the three different Pinelliae Rhizoma fermentation processes. The bacteria and fungi of the three fermented Pinelliae Rhizoma differed substantially, with the bacterial content mainly composed of the Bacillus genus, while the common fungi were only included in four OTUs, which belong to three species of Eurotiomycetes and Aspergillus cibarius. 51 volatile compounds were detected; they varied between LC, XD, and ZZ fermented Pinelliae Rhizoma. C10 and C15 terpenes were most frequently detected, and only curcumene and ß-bisabolene were detected in the three fermented Pinelliae Rhizoma. 65 non-volatile compounds were detected by LC-MS, most were of C16, C18, C20, C21 and C22 structures. Cluster analysis showed more similarity between LC and XD fermented Pinelliae Rhizoma with regards to volatile compound content, but more similarity between the XD and ZZ fermented Pinelliae Rhizoma for non-volatiles. Moreover, no correlation between geographical distance and microflora or compounds of fermented Pinelliae Rhizoma was observed. These results showed that hundreds of compounds are produced by the natural mixed fermentation of Pinelliae Rhizoma, and may mostly relate to the microorganisms of five species.

Alteration of crop rotation in continuous Pinellia ternate cropping soils profiled via fungal ITS amplicon sequencing.

Pinellia ternata is a traditional Chinese herb that has been shown to suffer from continuous cropping. Deterioration in continuous P. ternata cropping soils and the effect of alteration of crop rotation on the soil were evaluated by using Illumina high-throughput sequencing technology. Soil physicochemical properties and fungal diversity were significantly lower in continuous P. ternate cropping soil compared with those of uncultivated soil. In contrast, significant increases in soil factors and fungal diversity were detected after the continuous cropping soil was subjected to rotational cropping treatments, indicating a remediation effect of crop rotation on continuous cropping soil. Moreover, with the extension of wheat cropping in the rotations, fungal richness and most physicochemical properties exhibited a significantly increasing trend, suggesting that the remediation effect of crop rotation could be enhanced by extending the cropping of break crops. Furthermore, compared with those of continuous cropping soils, microbes that were potentially beneficial to P. ternate were enriched in the rhizosphere by rotational cropping, whereas micro-organisms that supposedly lead to soil deterioration were suppressed. Redundancy analysis indicated that Podospora and Alternaria were the major microbes that were responsible for increasing soil physicochemical properties; by contrast, the deterioration in soil fertility was mainly attributed to Fusarium micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of deterioration in continuous Pinellia ternate cropping soils and how crop rotations alter the deteriorated soil is still limited, especially regarding the presence of fungi and soil fertility. This research found remediation effects of crop rotation on continuous P. ternate cropping soils and indicates that these effects could be enhanced by extending the cropping of break crops. Community shifts between the continuous and rotational cropping soils and the associated changes in soil fertility will have a considerable impact on the diagnosis of obstacles to continuous P. ternate cropping and enhancement of the remediation effects of crop rotation.

Cytotoxic seco-cytochalasins from an endophytic Aspergillus sp. harbored in Pinellia ternata tubers.

Six new seco-cytochalasins A-F (1-6), two new asperlactones G-H (7-8) along with three known cytochalasins (9-11) were isolated from the solid cultures of an endophytic fungus Aspergillus sp. Their structures were elucidated by comprehensive spectral analysis, and their absolute configurations were determined through Mo2(OCOCH3)4-induced electronic circular dichroism (ECD) spectra and Rh2(OCOCF3)4-induced ECD experiment. Compounds 5 and 6 were rare seco-cytochalasins possessing an α, ß-unsaturated furanone structure in their side-chains. These isolates exhibited cytotoxicity against human lung cancer A-549 cell line with IC50 values ranging from 7.8 to 70.2 µM. At the concentration of 16 µM, compound 4 also exerted a 3-fold enhancement of doxorubicin susceptibility on doxorubicin-resistant human breast cancer (MCF-7/DOX) cell line.

Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system.

Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.

New phenalenone derivatives from Pinellia ternata tubers derived Aspergillus sp.

Nine new phenalenone derivatives (1-9), along with two known analogues (10-11) have been isolated from the solid cultures of an endophytic fungus Aspergillus sp. which was obtained from Pinellia ternate. Their structures were established through interpretations of spectroscopic evidence, and some of their absolute configurations were determined by electronic circular dichroism (ECD) and Mo2(OCOCH3)4 induced ECD. All of the phenalenones are unusual acyclic diterpenoid adducts, which are diversely oxidized and partly epoxidized to form different heterocycles. In addition, compound 10 exhibited significant antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis with MIC50 values of 1.87, 2.77, and 4.80µg/mL, respectively.

Antimicrobial metabolites from the plant endophytic fungus Penicillium sp.

Five rare dichloro aromatic polyketides (1-5) were obtained from an endophytic fungus Penicillium sp., along with five known metabolites (6-10). Their structures were elucidated by extensive spectroscopic analysis, Mosher methods, as well as [Rh2(OCOCF3)4]-induced electronic circular dichroism (ECD) experiments. Compounds 2-4 and 6 structurally involved acyclic 1.3-diols, the uneasy configuration determinations of which were well carried out by double-derivation NMR methods. Compounds 1-10 were evaluated for their antibacterial and antifungal activities against five strains of human pathogenic microorganisms. Helvolic acid (7) showed potent inhibitory effects against Staphylococcus aureus and Pseudomonas aeruginosa with MIC (minimum inhibitory concentration) values of 5.8 and 4.6µg/mL, respectively.

[Identification of dominant microbes from traditional Chinese medicine Pinelliae Rhizoma Fermentata].

To investigate the microbial species, amount changes as well as the isolation and identification of domain strains at different fermentation time points of Pinelliae Rhizoma Fermentata, and provide basis for exploring the mechanism of Pinelliae Rhizoma Fermentata processing. Five samples were chosen at the time points (0, 18, 36, 54, 72 h) of Pinelliae Rhizoma Fermentata processing. Bacteria, mold and yeast from the samples were cultured; their colonies were counted, and the dominant strains were isolated and purified. The dominant bacteria and dominant fungi were identified by 16S rDNA and 26S rDNA sequencing respectively. The results showed that the bacteria count was low with slow and smooth changes in the fermentation process;while mold and yeast grew dramatically after 54 h culturing and reached 1×107 CFU•mL⁻¹ at the end of fermentation. Through the NCBI homology alignment and phylogenetic tree construction, the dominant bacteria were identified as Streptomyces sp., Bacillus pumilus, B. subtilis, B. aryabhattai and other Bacillus sp.; the dominant yeast was identified as Meyerozyma guilliermondii; the dominant mold were identified as Paecilomyces variotii, Byssochlamys spectabilis, and Aspergillus niger in the processing of Pinelliae Rhizoma Fermentata. The results indicated that multiple microbe species, especially yeast and mold, played a role in the fermentation processing of Pinelliae Rhizoma Fermentata. M. guilliermondii, P. variotii, P. variotii and A. niger and Bacillus sp. can be the crucial factors in the processing of Pinelliae Rhizoma Fermentata.

[Influence on AM fungi infection rate and medicine quality of Pinellia ternata in condition of three soil impact factors].

OBJECTIVE: To explore the influence on AM fungi infection rate and medicine quality of Pinellia ternate in the condition of three soil impact factors. METHOD: Set the orthogonal test of three factors and levels. Determinate the AM fungi infection rate in early stage of mature & stage of mature of P. ternata, and the water content, water soluble extract, butanedioic acid content and alkaloid content of P. ternata tuber that be harvested also had be determinated. RESULT AND CONCLUSION: With the P levels to 30 mg x kg(-1) and 90 mg x kg(-1), AM fungi infection was the best when mixed inoculated of EM. Microbial agent inoculated played a decisive role in P. ternata growth and physiological activity, secondary influenced factor was P concentration, and the water stress was the minimal impact. Mixed inoculated of AM fungi and EM treatment with the low P levels (30, 90 mg x kg(-1)) proved better effect on enhancing the water extract content, anedioic acid and alkaloid content, while decreasing the water contents of P. ternata tuber.

[Infection progress of arbuscular mycorrhizae on tissue-cultured plantlets of Pinellia ternata].

OBJECTIVE: To study the Arbuscular mycorrhizal (AM) formation progress and infection characteristics between tissue culture plantlets of Pinellia ternata and Glomus mosseae. METHOD: The tissue culture plantlets of P. ternata were inoculated with G. mosseae, the formation of AM were sampled and observed with microscopy by staining. RESULT AND CONCLUSION: The hyphae of G. mosseae began to penetrate the root epidermis after 10 days of inoculation. Lots of intracellular hyphae formed in cortex cells at the 15th day. Arbuscules started to form and there were some hyphae on the root at the 20th day. At the 25th day, many arbuscules formed and most as Arum type. Some arbuscles started to disintegrate at the 30th day, and a few of vesicles occurred. Lots of spores formed after 35 days. At the 40th day, some vesicles began to decline. The hand section showed that the intercellular hyphae gradually formed in intercellular space, and the hyphae branched in cortex cells and occupied most cell lumen finally. It is expounded that P. ternata and G. mosseae could recognize each other quickly and form a symbiont system.

[Pathogen identification of Pinellia ternata tuber disease and selection of fungicide].

The rotten tuber of Pinellia ternata was found as an important disease during the growing season in Tianshui production area. The isolated pathogens were tested following Koch's postulates and identified as Fusarium oxysporum. The suitable growth conditions for the F. oxysporum were 15-30 degrees C, pH 6-8, the optimal condition was 21.9 degrees C and pH 7.2. Some fungicides were demonstrated to be effective to inhibit the pathogen growth. 70% thiophanate-methyl and 58% metalaxyl MZ were most effective to inhibit the pathogen. The EC50 were 0.002 7, 0.066 2 g x L(-1), respectively.

Characterization of the endophytic antagonist pY11T-3-1 against bacterial soft rot of Pinellia ternata.

AIMS: Biocontrol is an emerging trend aimed at reducing chemical input while increasing plant fitness, productivity and resistance to diseases in sustainable agriculture. An antagonist, pY11T-3-1, was herein characterized for potential applications against soil-borne plant diseases. METHODS AND RESULTS: In vitro antagonistic assays, the antagonist pY11T-3-1 was demonstrated able to obviously reduce the occurrence of the soft rot disease on Pinellia ternata, potato, pepper, tomato, cucumber and eggplant tubers or fruits, with higher prevention (90%) on P. ternata. It showed a broad antagonistic spectrum against 23 tested bacterial and fungal phytopathogens, which were distributed in 14 genus and 17 species. However, it inhibited only two of the seven bacterial nonpathogens. Phenotypic characterizations showed that the antagonist pY11T-3-1 was similar to Pseudomonas aeruginosa. Its major fatty acids were 18:1 w7c (22.17%), 16:0 (20.21%), 12:0 2OH (12.45%), 16:1w7c/15 iso2OH (10.95%) and 10:0 3OH (10.79%), which is a different profile from that of Ps. aeruginosa. The 16S rRNA and gyr B gene sequences shared 100 and 99% similarity with Ps. aeruginosa, respectively. The phylogenetic trees showed that it was clustered with Ps. aeruginosa. CONCLUSIONS: The antagonist pY11T-3-1 was characterized as Ps. aeruginosa with a unique fatty acid profile. SIGNIFICANCE AND IMPACT OF THE STUDY: With broad antagonistic spectrum and host selectivity, the antagonist pY11T-3-1 may provide a more environmental and economical alternative to the control of soil-borne disease on P. ternata, which needs further investigation.

[Arbuscular mycorrhiza of cultivated and wild Pinellia ternata].

OBJECTIVE: To study the arbuscular mycorrhiza and arbuscular mycorrhizal fungi associated with cultivated and wild Pinellia ternata in Guizhou province. METHOD: Wild and cultivated P. ternata roots were observed through staining and microscopic examination, the arbuscular mycorrhizal fungi spores were isolated through wet thieving according to Gerdemann & Nicolson (1963), the spores were identified following the description of Schenck & Pérez (1988), and some previous publications. RESULT: The typical arbuscular mycorrhiza (AM) structure was showed according to a research of wild and cultivated P. ternata. In the survey of AM fungi species in the rhizosphere of wild and cultivated P. ternata, 3 genera and 21 species were found, 3 genera and 7 species were identified. 5 species of them belong to Glomus, 1 species belongs to Scutellospora, 1 species belongs to Gigaspora, including Glomus mosseae, G. intraradices, G. melanosporum, G. deserticola, G. aggregatum, Scutellospora castanea, Gigaspora albida, and one of them was a new record, i.e., Scutellospora castanea which was the dominant species in Bijie. CONCLUSION: The diversity of AM fungi between wild and cultivated Pinellia ternata was showed on this survey, the fungi associated with wild ones are different form the cultivated ones, such as Gigaspora albida only occurs in cultivated ones, Glomus melanosporum only occurs in wild ones, while Glomus mosseae and Glomus intraradices occur in both wild and cultivated ones, and there were specialization species in Bijie, all these can provide new though for solving degradation problem of cultivated Pinellia ternata.

[Study on influence of arbuscular mycorrhizal fungi Pinellia ternata yield and chemical composition].

OBJECTIVE: To study the influence of arbuscular mycorrhizal fungi to Pinellia ternata leaf index, propagation coefficient, tuber yield and chemical composition. METHOD: The Pinellia ternata leaf length, width, plant height and leafstalk diameter were measured during growing time till P. ternata loded. The leaf index was calculated and the tuber yield were measured after harvest. The contents of guanosine was determined by HPLC method, and the alkaloid contents were determined by spectrophotometric method. RESULT AND CONCLUSION: Inoculated AM fungi could increase P. ternata propagation coefficient and the yield of tuber. Inoculated AM fungi could increase P. ternata leaf index, made leaf tends to be more like bamboo leaf type. Inoculated AM fungi could delayed P. ternata lodging time; G. mosseae + G. intraradices treatment (early inolulation time) proved better influence on increasing fresh weight and dry weight of P. ternata tuber and the propagation coefficient. Inoculated AM fungi could increase guanosine and alkaloid contents of Pinellia ternata tuber, and the contents of guanosine and alkaloid in inoculated treatments were higher than those in cultivated P. ternata from Hezhang; G. mosseae + G. intraradices treatment (later inolulation time) proved good effect on increasing the contents of guanosine and alkaloid.

[Isolation and identification of endophytes from Pinellia].

OBJECTIVE: To investigate the varieties of entophytes in different parts of Pinellia ternata. METHOD: The solidified plates were applied for the isolation of the endophytes, and three methods were used for the identification of endophytic fungi. RESULT: Eighty four strains of the entophytes were isolated from the P. ternata collected from 3 habitations. Endophytic fungi were morphologically identified belonging to 15 genera, 4 families. CONCLUSION: It indicated that the entophytes in P. ternata were diversity and rich, and there were some differences at quantity and species in different organs of P. ternata.

Isolation and characterization of endophytic and rhizosphere bacterial antagonists of soft rot pathogen from Pinellia ternata.

Pinellia ternata, a traditional Chinese herb that has been used in China for over 1000 years, is susceptible to a soft rot disease, which may cause major loss of yield. The use of bacteria as potential antagonists against Pectobacterium carotovorum SXR1, the causal agent of the disease on P. ternata, was evaluated. Altogether, 1107 candidate bacteria were isolated from the rhizosphere and surface-sterilized plants of P. ternata. In Petri dish tests, 55 isolates inhibited the growth of strain SXR1, and 21 of these reduced the disease development on P. ternata slices by over 50%. Four selected antagonists significantly reduced the disease incidence on tissue culture seedlings, and also prevented the disease on the transplants. Agonist P-Y2-2 yielded a good prevention level of 81.9%. The four antagonists rapidly colonized the tissue culture seedlings and transplants, whereas greater populations of the antagonists (10(7)-10(9) CFU g(-1) fresh tissues) were observed in the seedlings and in the preinoculated transplants than in those inoculated during transplanting. The use of pathogen-free tissue culture seedlings pre-inoculated with antagonist may provide a strategy for production of P. ternata plantlets resistant to soft rot disease. This is the first report on the efficacy of biocontrol agents against pathogens on P. ternata.