Antimicrobial Activity of Cultivable Endophytic and Rhizosphere Fungi Associated with "Mile-a-Minute," Mikania cordata (Asteraceae).

Endophytic and rhizosphere fungi are understood to be aiding the host plant to overcome a range of biotic and abiotic stresses (nutrition depletion, droughts, etc.) hence, they remain to be reservoirs of plethora of natural products with immense use. Consequently, this investigation of endophytic and rhizosphere fungi isolated from Mikania cordata (a perennial vine that is well established in Sri Lanka) for their antimicrobial properties was performed with the aim of future derivation of potential beneficial pharmaceutical products. Leaves, twigs, and roots of M. cordata were utilized to isolate a total of 9 endophytic fungi out of which the highest amount (44%) accounted was from the twigs. A sample of the immediate layer of soil adhering to the root of M. cordata was utilized to isolate 15 rhizosphere fungi. Fusarium equiseti and Phoma medicaginis were endophytes that were identified based on colony and molecular characteristics. The broad spectrum of antimicrobial activity depicted by F. equiseti (MK517551) was found to be significantly greater (p ≤ 0.05, inhibitory against Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 25853) than P. medicaginis (MK517550) (inhibitory against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 25853) as assessed using the Kirby-Bauer disk diffusion method. Trichoderma virens and Trichoderma asperellum were rhizospere fungi that exhibited remarkable antimicrobial properties against the test pathogens chosen for the study. T. asperellum indicated significantly greater bioactivity against all four bacterial pathogens and Candida albicans ATCC 10231 under study. The ranges of minimum inhibitory concentrations (MICs) of the fungi depicting antimicrobial properties were determined. The results obtained suggest that F. equiseti, P. medicaginis, T. asperellum, and T. virens of M. cordata harness bioprospective values as natural drug candidates. This is the first report on isolation and evaluation of the antimicrobial properties of endophytic and rhizosphere fungi of Mikania cordata.

Isolation, identification, and the growth promoting effects of two antagonistic actinomycete strains from the rhizosphere of Mikania micrantha Kunth.

Actinomycetes are an important group of gram-positive bacteria that play an essential role in the rhizosphere ecosystem. The confrontation culture and Oxford cup method were used to evaluate the antagonistic activities of strains, which were isolated from the rhizosphere soil of Mikania micrantha. The two isolates were identified using morphological and physiological tests combined with 16S rRNA-based molecular analysis, respectively. The type I polyketone synthase (PKS-I) was amplified. The constituents of fermentation metabolites were analyzed by gas chromatography mass spectrometry. The plant growth promoting effect was determined. Finally, the growth of wheat seedlings was assessed using the Petri dish method. Overall, of the isolated twelve strains, WZS1-1 and WZS2-1 could significantly inhibit target fungi. Isolate WZS1-1 was identified as Streptomyces rochei, and WZS2-1 was identified as Streptomyces sundarbansensis. In particular, Fusarium graminearum (FG) from wheat was inhibited by more than 80%, and the inhibitory bandwidths against FG were 31 ±â€¯0.3 mm and 19 ±â€¯0.5 mm, respectively. The genes PKS-I were successfully amplified, confirming that these strains are capable of producing biosynthetic secondary metabolites. Major component analysis revealed aliphatic ketones, carboxylic acids, and esters, with n-hexadecanoic acid being the most abundant compound. Plant growth promoting test indicated that both strains produced IAA, presented with orange loops on CAS plates, dissolved phosphorus and potassium, fixed nitrogen, but did not generate organic acids; both strains colonized in soil, while only WZS1-1 colonized in wheat roots. Additionally, the fermentation broth significantly promoted the growth of wheat.

Decontamination of Mikania glomerata Leaves by Gamma Irradiation: Coumarin Determination by HPLC-DAD, Microbiological Control and Genotoxicological Studies.

Gamma irradiation as a decontaminating physical agent could be an important tool in the production chain of herbal medicines by improving the microbiological quality of raw materials and the safety of final products. This study was undertaken to investigate the genotoxic potential and eventual chemical modifications of a batch of Mikania glomerata raw material decontaminated by different doses of gamma irradiation (2.0, 3.5, and 5.0 kGy), using a cesium-137 source. DNA damage was assessed in vitro by agarose gel electrophoresis in regard to double-chain breaks of plasmid pUC 9.1 DNA and in vivo by micronucleus test in bone marrow cells of Wistar rats. Cytotoxicity in bone marrows was assessed by scoring polychromatic and normochromatic erythrocytes ratio. An HPLC-DAD method was adapted and validated for the enhancement of coumarin selectivity from the other matrix constituents. The microbial load was satisfactorily reduced, leading to sterilization at the highest dose. Genotoxic and cytotoxic effects were not increased in the in vitro and in vivo models. The concentration of coumarin and the chromatographic profiles of the hydroalcoholic plant extracts (ethanol 70% v/v) were not modified after such process. Therefore, this work suggests that gamma irradiation of M. glomerata raw material is suitable and safe for microbiological control purposes at the employed doses.

Biotechnological prospecting of foliar endophytic fungi of guaco (Mikania glomerata Spreng.) with antibacterial and antagonistic activity against phytopathogens.

Mikania glomerata (Spreng.), popularly known as "guaco", is a plant from the Asteraceae family that has many therapeutic properties. The use of medicinal plants has been examined in studies on endophytic diversity and bioprospecting; endophytes inhabit the interior of plants without harming them. Microorganism-host complex interactions are related to the production of compounds that may confer resistance to pathogens or to production of bioactive compounds or growth regulators. In this study, we evaluated foliar endophytic fungi of M. glomerata to examine the control of plant pathogens, molecular identification, and production of compounds with antimicrobial activity. In the antagonism test, 6-mm diameter disks were placed equidistant from the endophyte and plant pathogen, and pathogen growth area was measured. The endophytic strains G-01, G-02, and G-03 were effective against Fusarium solani and Didymella bryoniae. The endophyte rDNA regions corresponding to internal transcribed spacer 1-5.8S-internal transcribed spacer 2 were sequenced, and the results were compared with sequences deposited in the NCBI database. The G-01, G-02, and G-03 strains were identified as Diaporthe citri. This identification was confirmed by phylogenetic analysis. The crude extract of the secondary metabolites of the G-01 strain was tested against Escherichia coli and Staphylococcus aureus; the metabolites showed antimicrobial activity against S. aureus. The endophytes tested in this study have potential for use in biotechnological applications.

Short-term parasite-infection alters already the biomass, activity and functional diversity of soil microbial communities.

Native parasitic plants may be used to infect and control invasive plants. We established microcosms with invasive Mikania micrantha and native Coix lacryma-jobi growing in mixture on native soils, with M. micrantha being infected by parasitic Cuscuta campestris at four intensity levels for seven weeks to estimate the top-down effects of plant parasitism on the biomass and functional diversity of soil microbial communities. Parasitism significantly decreased root biomass and altered soil microbial communities. Soil microbial biomass decreased, but soil respiration increased at the two higher infection levels, indicating a strong stimulation of soil microbial metabolic activity (+180%). Moreover, a Biolog assay showed that the infection resulted in a significant change in the functional diversity indices of soil microbial communities. Pearson correlation analysis indicated that microbial biomass declined significantly with decreasing root biomass, particularly of the invasive M. micrantha. Also, the functional diversity indices of soil microbial communities were positively correlated with soil microbial biomass. Therefore, the negative effects on the biomass, activity and functional diversity of soil microbial community by the seven week long plant parasitism was very likely caused by decreased root biomass and root exudation of the invasive M. micrantha.

Molecular cloning and characterization of two novel NAC genes from Mikania micrantha (Asteraceae).

NAC proteins, which are plant-specific transcription factors, have been identified to play important roles in plant response to stresses and in plant development. The full-length cDNAs that encode 2 putative NAC proteins, designated as MmATAF1 and MmNAP, respectively, were cloned from Mikania micrantha by rapid amplification of cDNA ends. The full-length cDNAs of MmATAF1 and MmNAP were 1329 and 1072 bp, respectively, and they encoded deduced proteins of 260- and 278-amino acid residues, respectively. The proteins MmATAF1 and MmNAP had a calculated molecular mass of 29.81 and 32.55 kDa and a theoretical isoelectric point of 7.08 and 9.00, respectively. Nucleotide sequence data indicated that both MmATAF1 and MmNAP contained 2 introns and 3 exons and that they shared a conserved genomic organization. Multiple sequence alignments showed that MmATAF1 showed high sequence identity with ATAF1 of Arabidopsis thaliana (61%) and that MmNAP showed high sequence identity with NAP of A. thaliana (67%) and CitNAC of Citrus sinensis Osbeck (62%). Phylogenetic analysis showed that the predicted MmATAF1 and MmNAP proteins were classified into the ATAF and NAP subgroups, respectively. Transient expression analysis of onion epidermal cells indicated nuclear localization of both MmATAF1-GFP and MmNAP-GFP fusion proteins. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that MmATAF1 was expressed in all the tissues tested, but in varying abundance, while MmNAP was specifically expressed in stems, petioles, shoots, and leaves, but not in roots. The transcript levels of MmATAF1 and MmNAP in shoots and in infected stems were induced and strengthened by wounding, exogenous ZnSO(4), abscisic acid, salicylic acid, and Cuscuta campestris infection on the basis of semi-quantitative RT-PCR and real-time PCR analyses, respectively. Collectively, these results indicated that MmATAF1 and MmNAP, besides having roles in M. micrantha adaptation to C. campestris infection and abiotic stresses, also integrated signals derived from both C. campestris infection and abiotic stresses.

The biology and taxonomy of rust fungi associated with the neotropical vine Mikania micrantha, a major invasive weed in Asia.

Three microcyclic rust species were collected during surveys of the perennial asteraceous vine Mikania micrantha (Eupatorieae: Asteraceae) throughout its native range in the Neotropics but were absent in its invasive range in Asia. The commonest species, Puccinia spegazzinii with brown telioid telia, occurred wherever M. micrantha was found in South and Central America including the Caribbean island of Trinidad. Dietelia portoricensis, with occasional vestigial spermogonia and grayish-white to pale yellow columnar aecioid telia, was collected only in Costa Rica; while D. mesoamericana sp. nov., apparently restricted to Mesoamerica, can be distinguished by its abundant, yellowish-orange, fertile spermogonia, yellow to pale brown telial columns, larger teliospores, and 4-spored rather than 2-spored metabasidia. The fact that all three species share a fundamentally similar symptomatology suggests a common origin.