Identification of two phthalide derivatives and an oxindole compound isolated from the edible mushroom Pleurotus ostreatus and their inhibitory activities against plant pathogenic microorganisms.

The excessive use of chemical pesticides in agricultural fields for controlling plant pathogenic microorganisms harms human health, the environment, and other beneficial microorganisms in the soil and plants. To address this challenge, it is essential to isolate and discover bioactive compounds from biological resources that could inhibit plant pathogenic microorganisms. In this study, the culture filtrate of the edible mushroom Pleurotus ostreatus was subjected to bioassay-guided isolation, and two phthalide derivatives-4,6-dimethoxyphthalide (1) and 5,7-dimethoxyphthalide (2)-were identified, along with an oxindole compound-3-hydroxy-3-methyloxindole (3). The inhibitory activities of the three compounds were evaluated against four fungal and five bacterial pathogens. Remarkably, 1 and 2 exhibited the lowest IC50 values against the conidial germination and germ tube elongation of the rice blast fungus Pyricularia oryzae. However, their effectiveness against bacterial pathogens was relatively low. The (S) and (R)-enantiomers of 3-hydroxy-3-methyloxindole showed different activities against plant fungal pathogens and bacterial plant pathogens.

Comparative Diversity and Functional Traits of Fungal Endophytes in Response to Elevated Mineral Content in a Mangrove Ecosystem.

This study investigates the impact of water quality, specifically elevated phosphate and zinc content, on the diversity and functional properties of mangrove fungal endophytes in two distinct mangrove forests. Mangrove plant performance is directly related to the presence of fungal leaf endophytes as these fungi could enhance plant health, resilience, and adaptability under stressed environmental conditions. Two distinct mangrove forest sites, one non-disturbed (ND) and one disturbed by aquaculture practices (D), were assessed for differences in water quality parameters. We further analyzed the fungal endophyte diversity associated with the leaves of a target host mangrove, Rhizophora mucronata Lamk., with the aim to elucidate whether fungal diversity and functional traits are linked to disturbances brought about by aquaculture practices and to characterize functional traits of selected fungal isolates with respect to phosphate (PO4) and zinc (Zn) solubilization. Contrary to expectations, the disturbed site exhibited a higher fungal diversity, challenging assumptions about the relationship between contamination and fungal community dynamics. Water quality, as determined by nutrient and mineral levels, emerged as a crucial factor in shaping both microbial community compositions in the phyllosphere of mangroves. From both sites, we isolated 188 fungal endophytes, with the ND site hosting a higher number of isolates and a greater colonization rate. While taxonomic diversity marginally differed (ND: 28 species, D: 29 species), the Shannon (H' = 3.19) and FAI (FA = 20.86) indices revealed a statistically significant increase in species diversity for fungal endophytes in the disturbed mangrove site as compared to the non-disturbed area (H' = 3.10, FAI = 13.08). Our chosen mangrove fungal endophytes exhibited remarkable phosphate solubilization capabilities even at elevated concentrations, particularly those derived from the disturbed site. Despite their proficiency in solubilizing zinc across a wide range of concentrations, a significant impact on their mycelial growth was noted, underscoring a crucial aspect of their functional dynamics. Our findings revealed a nuanced trade-off between mycelial growth and enzymatic production in fungal endophytes from ostensibly less contaminated sites, highlighting the relationship between nutrient availability and microbial activities. These insights provide a foundation for understanding the impact of anthropogenic pressures, specifically nutrient pollution, on mangrove-associated fungal endophytes.

Comparative Study on the Antimicrobial Activities and Metabolic Profiles of Five Usnea Species from the Philippines.

The rapid emergence of resistant bacteria is occurring worldwide, endangering the efficacy of antibiotics. Hence, there is a need to search for new sources of antibiotics that either exhibit novel structures or express a new mechanism of action. The lichen Usnea, with its wide range of unique, biologically potent secondary metabolites, may solve this problem. In this study, Usnea species were collected in the Northern Philippines, identified through combined morphological and biochemical characterization, and tested for antimicrobial activities against the multidrug-resistant ESKAPE pathogens, i.e., Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae, two standard antibiotic-sensitive test bacteria, and a yeast. A total of 46 lichen specimens were collected and later identified as Usnea baileyi (10), U. diffracta (10), U. glabrata (12), U. longissima (4), and U. rubicunda (10). The results show that the crude extracts of the Usnea species exhibited promising in vitro inhibitory activities against standard antibiotic-sensitive (E. faecalis ATCC 29212) and multidrug-resistant (methicillin-resistant S. aureus and E. faecalis) Gram-positive bacteria. Additionally, lichen compounds of representative specimens per species were identified and profiled using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The detection of lichen acids (LA) via HPLC showed the presence of 24 peaks of lichen acids. TLC-bioautography identified the bioactive lichen acids as alectronic acid, connorstictic acid, consalazinic acid, diffractaic acid, echinocarpic acid, erythrin acid, galbinic acid, hypoconstictic acid, hyposalazinic acid, hypostictic acid, lobaric acid, menegazzaic acid, micareic acid, pannarin, salazinic acid, stictic acid, and usnic acid. Our study highlighted the wide spectrum of opportunities for using lichens for the discovery of potential antimicrobial agents.

Isolation, structure elucidation, and biological activities of sesquiterpenes and phthalides from two edible mushrooms Pleurotus species.

Antimicrobial compounds were purified from culture filtrates from 2 edible Pleurotus species. Using a bioassay-guided fractionation of the culture filtrate extracts, 3 compounds (1-3) were obtained from Pleurotus ostreatus, and another compound (4) was obtained from Pleurotus pulmonarius. Spectroscopic analysis revealed that 1-3 was identified as 5,7-dimethoxyphthalide, 4,6-dimethoxyphthalide, and cheimonophyllon E, respectively, while 4 were identified as pleuroton A. The minimum inhibitory concentration and minimum bactericidal concentration of these compounds were determined against 6 pathogenic bacterial species, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. Compounds 2 and 4 were inhibitory against all tested bacteria, while 1 and 4 were inhibitory against 3 and 2 species, respectively. In addition, 1-4 inhibited tyrosinase, with IC50 values of 0.10-0.30 mg/mL, and α-glucosidase, with IC50 values of 0.12-0.54 mg/mL. However, their antioxidant capacities were marginal.

Monitoring of an Applied Beneficial Trichoderma Strain in Root-Associated Soil of Field-Grown Maize by MALDI-TOF MS.

The persistence of beneficial microorganisms in the rhizosphere or surrounding soil following their application is a prerequisite for the effective interaction with the plant or indigenous microbial communities in the respective habitats. The goal of the study was to analyze the establishment and persistence of the applied beneficial Trichoderma harzianum (OMG16) strain in the maize root-associated soil depending on agricultural practice (soil management practice, N-fertilizer intensity) in a field experiment. A rapid identification of the inoculated strain OMG16 is essential for its monitoring. We used a culture-based approach coupled to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis for the rapid identification of the inoculated Trichoderma strain as part of the beneficial microbe consortium (BMc). We isolated 428 fungal isolates from eight treatments of the field experiment. Forty eight percent of the isolated fungi equivalent to 205 fungal isolates were identified as Trichoderma, of which 87% (=179 isolates) were obtained from the fields inoculated with BMc. Gene sequence analysis showed a high similarity of the MALDI-TOF MS-identified Trichoderma, with that of the inoculated Trichoderma harzianum OMG16 confirming the re-isolation of the added beneficial fungus. This study highlighted the use of MALDI-TOF MS analysis as a quick, cost-effective detection and efficient monitoring tool for microbial-based bioinoculants in the field.

Isolation and Characterization of Nickel-Tolerant Trichoderma Strains from Marine and Terrestrial Environments.

Nickel contamination is a serious environmental issue that requires immediate action. In this study, 23 strains of Trichoderma were isolated from terrestrial and marine environments and identified using a polyphasic approach of morphological characterization and ITS gene sequence analysis. The Trichoderma strains were tested for their tolerance and biosorption of nickel. Our results showed the growth of all Trichoderma strains on Trichoderma Selective Medium (TSM) with 50-1200-ppm nickel, indicating their tolerance of this heavy metal even at a relatively high concentration. Six Trichoderma strains (three isolated from terrestrial substrates and three from marine substates) had the highest radial growth on TSM with 50-ppm Ni. Among these fungal isolates, Trichoderma asperellum (S03) isolated from soil exhibited the best growth after 2 days of incubation. For the biosorption of nickel, the accumulation or uptake efficiency by the six selected Trichoderma was determined in Potato Dextrose Broth (PDB) supplemented with 50-ppm Ni using a Flame Atomic Absorption Spectrophotometer (AAS). The percent uptake efficiency of the three strains of T. asperellum (S03, S08, and LL14) was computed to be up to 66%, while Trichoderma virens (SG18 and SF22) and Trichoderma inhamatum (MW25) achieved up to 68% uptake efficiency. Observation of the Trichoderma strains with Scanning Electron Microscopy (SEM) before and after the absorption of nickel showed very minimal damage on the hyphal and conidial surface morphology, but changes in the colonial characteristics were observed. Our study highlighted the potential of terrestrial and marine strains of Trichoderma for the bioremediation of nickel pollution.

Diversity, Phylogeny and Antagonistic Activity of Fungal Endophytes Associated with Endemic Species of Cycas (Cycadales) in China.

The culture-based approach was used to characterize the fungal endophytes associated with the coralloid roots of the endemic Cycas debaoensis and Cycas fairylakea from various population sites in China. We aim to determine if the assemblages of fungal endophytes inside these endemic plant hosts are distinct and could be explored for bioprospecting. The isolation method yielded a total of 284 culturable fungal strains. Identification based on the analysis of the internal transcribed spacer (ITS) rDNA showed that they belonged to two phyla, five classes, eight orders and 22 families. At least 33 known genera and 62 different species were confirmed based on >97% ITS sequence similarity. The most frequent and observed core taxa in the two host species regardless of their population origin were Talaromyces, Penicillium, Fusarium, Pochonia and Gliocladiopsis. Seventy percent was a rare component of the fungal communities with only one or two recorded isolates. Contrary to common notions, diversity and fungal richness were significantly higher in C. debaoensis and C. fairylakea collected from a botanical garden, while the lowest was observed in C. debaoensis from a natural habitat; this provides evidence that garden management, and to a minor extent, ex-situ conservation practice, could influence fungal endophyte communities. We further selected nineteen fungal isolates and screened for their antagonistic activities via a co-cultivation approach against the phytopathogens, Diaporthe sp. and Colletotrichum sp. Among these, five isolates with high ITS similarity matches with Hypoxylon vinosupulvinatum (GD019, 99.61%), Penicillium sp. (BD022, 100%), Penicillifer diparietisporus (GD008, 99.46%), Clonostachys rogersoniana (BF024, 99.46%) and C. rosea (BF011, 99.1%), which showed exceptional antagonistic activities against the phytopathogenic fungi with a significant inhibition rate of 70-80%. Taken together, our data presented the first and most comprehensive molecular work on culturable fungal endophytes associated with the coralloid roots of cycads. Our study also demonstrated that about 5% of fungal endophytes were not detected by the high-throughput sequencing approach, implying the equal importance of a culture-dependent approach to study fungal communities of cycads. We further highlighted the potential role of endemic and rare plants to discover and isolate unique plant-associated fungal taxa with excellent biocontrol properties.

Biodiscovery of Potential Antibacterial Diagnostic Metabolites from the Endolichenic Fungus Xylaria venustula Using LC-MS-Based Metabolomics.

Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC-MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for "dual" bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.

Full-Length 16S rRNA and ITS Gene Sequencing Revealed Rich Microbial Flora in Roots of Cycas spp. in China.

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

Protists as catalyzers of microbial litter breakdown and carbon cycling at different temperature regimes.

Soil bacteria and fungi are key drivers of carbon released from soils to the atmosphere through decomposition of plant-derived organic carbon sources. This process has important consequences for the global climate. While global change factors, such as increased temperature, are known to affect bacterial- and fungal-mediated decomposition rates, the role of trophic interactions in affecting decomposition remains largely unknown. We designed synthetic microbial communities consisting of eight bacterial and eight fungal species and tested the influence of predation by a model protist, Physarum polycephalum, on litter breakdown at 17 and 21 °C. Protists increased CO2 release and litter mass loss by ~35% at 17 °C lower temperatures, while they only had minor effects on microbial-driven CO2 release and mass loss at 21 °C. We found species-specific differences in predator-prey interactions, which may affect microbial community composition and functioning and thus underlie the impact of protists on litter breakdown. Our findings suggest that microbial predation by fast-growing protists is of under-appreciated functional importance, as it affects decomposition and, as such, may influence global carbon dynamics. Our results indicate that we need to better understand the role of trophic interactions within the microbiome in controlling decomposition processes and carbon cycling.

Biodiscovery of antibacterial constituents from the endolichenic fungi isolated from Parmotrema rampoddense.

A total of nine endolichenic fungi were isolated from the foliose lichen Parmotrema rampoddense (Nyl.) Hale. Of the nine endolichenic fungi, three taxa (Fusarium proliferatum, Nemania primolutea, Daldinia eschsholtzii) showed antibacterial activities as determined by the disk diffusion assay against ESKAPE bacterial pathogens. Fusarium proliferatum gave the most active fungal extract with zone of inhibition values of 15 mm and 19 mm against E. faecalis and S. aureus, respectively. Further chromatographic purification of the F. proliferatum ethyl acetate extract led to the isolation and identification of bis(2-ethylhexyl)terephthalate (1), acetyl tributyl citrate (2), and fusarubin (3). Acetyl tributyl citrate (2) exhibited moderate antibacterial activity against Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus.

Communicating Science through Editorial Cartoons in Microbiology Classrooms.

The use of graphical illustration in lecture presentations can make a seemingly boring lesson more attractive and enticing to students. Creating science-themed illustrations and science-based narratives can also lead to creative and critical thinking among students. We used writing editorials and creating editorial cartoons as a learning activity to promote critical thinking and creative skills that are essential in communicating scientific information. This activity can be used with a range of audiences, at various educational levels and in basic to advanced courses.

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps.

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70-650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.

Bioactive metabolites of Diaporthe sp. P133, an endophytic fungus isolated from Pandanus amaryllifolius.

Two new benzopyranones, diaportheone A (1) and B (2), were obtained via bioassay-guided isolation of the secondary metabolites from the endophytic fungus Diaporthe sp. P133 isolated from Pandanus amaryllifolius leaves. Their structures were determined from spectral analysis including mass spectrometry, 1D and 2D nuclear magnetic resonance. Both 1 and 2 inhibited the growth of the virulent strain of Mycobacterium tuberculosis H(37)Rv with minimum inhibitory concentrations of 100.9 and 3.5 µM, respectively.

Carbon source utilization by the marine Dendryphiella species D. arenaria and D. salina.

Carbon utilization by the marine Dendryphiella species, D. arenaria and D. salina, was investigated to detect differences in utilization and traits associated with their adaptation to the marine habitat. Fifty-four strains were isolated world-wide and tested for the utilization of various carbon sources using BIOLOG phenotype MicroArray (PM) and for the production of extracellular enzymes on solid culture media and on API ZYM assay strips. PM analysis showed that the fastest growth occurred on several monosaccharides and amino acids, 2-keto-d-gluconic acid, succinamide and turanose. Some polyols were poor carbon sources. However, the two species differed in their utilization rates of carbon sources, forming three major clusters: two separate clusters for D. arenaria and D. salina and a third cluster in which strains of the two species formed separate subclades that correlated with geographic origin. Several carbon sources were also found useful in differentiating the two speices. Dendryphiella salina did not utilize xylitol and quinic acid, whereas D. arenaria grew well on these substrates. The latter failed to grow on sorbitol and grew slowly on mannitol, both were good substrates for the former. There were also no qualitative differences between the extracellular enzymes produced, although laccase and peroxidase activities were confined only to some strains. The physiological similarities exhibited by the two species support the close relationship between D. arenaria and D. salina.