Pathogenicity of monokaryotic and dikaryotic mycelia of Ganoderma boninense revealed via LC-MS-based metabolomics.

This study compared the pathogenicity of monokaryotic (monokaryon) and dikaryotic (dikaryon) mycelia of the oil palm pathogen Ganoderma boninense via metabolomics approach. Ethyl acetate crude extracts of monokaryon and dikaryon were analysed by liquid chromatography quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) coupled with multivariate data analysis using MetaboAnalyst. The mummichog algorithm was also used to identify the functional activities of monokaryon and dikaryon without a priori identification of all their secondary metabolites. Results revealed that monokaryon produced lesser fungal metabolites than dikaryon, suggesting that monokaryon had a lower possibility of inducing plant infection. These findings were further supported by the identified functional activities. Monokaryon exhibits tyrosine, phenylalanine, and tryptophan metabolism, which are important for fungal growth and development and to produce toxin precursors. In contrast, dikaryon exhibits the metabolism of cysteine and methionine, arginine and proline, and phenylalanine, which are important for fungal growth, development, virulence, and pathogenicity. As such, monokaryon is rendered non-pathogenic as it produces growth metabolites and toxin precursors, whereas dikaryon is pathogenic as it produces metabolites that are involved in fungal growth and pathogenicity. The LC-MS-based metabolomics approach contributes significantly to our understanding of the pathogenesis of Ganoderma boninense, which is essential for disease management in oil palm plantations.

Antioxidant and L-asparaginase activities of culturable endophytic fungi from ornamental Dendrobium orchids.

This study reports the antioxidant potential and L-asparaginase production of culturable fungal endophytes from Dendrobium orchids in Malaysia. Twenty-nine isolates were screened using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay to determine their free radical scavenging activities and antioxidant capacity (IC50 and AEAC). L-asparaginase production of fungal endophytes was detected by the qualitative plate assay, and the enzyme activities estimated via the Nesslerization method. All 29 endophytic isolates exhibited various degrees of radical scavenging activities (35.37%-77.23%), with Fusarium fujikuroi (D1) identified as having the highest antioxidant capacity (IC50 6.097 mg/mL) and the highest AEAC value (11.55  mg/g). For L-asparaginase production, the majority of the isolates (89.66%) showed positive results, especially among the culturable species of Fusarium, Trichoderma, and Daldinia. Most Fusarium spp. were able to produce L-asparaginase (80.77%), but the highest L-asparaginase activity was detected in Daldinia eschscholtzii (D14) with 2.128 units/mL. Results from this study highlighted the potential of endophytic fungi from medicinal orchids (Dendrobium sp.) as natural sources of bioactive compounds to be developed into novel antioxidants and anticancer drugs.

Characterization and modeling of diffusion kinetics of rosemary oleoresin extract from gellan gum-based film.

Fresh food products are highly prone to oxidation and microbial attack, rendering them unsuitable for consumption. Thus, active food packaging was developed to protect and prolong food shelf-life. Zein/gellan gum (GG) based active film is developed by incorporating rosemary oleoresin extract (ROE) (0-20%). The films were characterized by their barrier and antioxidant properties. The release behavior of ROE in fatty and hydrophilic food stimulants was investigated via mathematical modeling. The active films incorporated with 20% ROE have significantly higher oxygen barrier and oxygen transfer is reduced by 20% compared to the control. A tortuous path is created with ROE, which impedes oxygen movement across the film. ROE addition improved water resistance performance by reducing the active film swelling ratio by 31%. This improvement is attributed to the hydrophobic nature of ROE. FTIR shows that the interaction between ROE and the active film is primarily hydrogen bonding and electrostatic interactions. Active film exhibits excellent antioxidant activity, with high TPC, DPPH scavenging activity, and FRAP. Mathematical modeling revealed a higher diffusivity (D) of ROE in fatty food stimulants at 24 °C, attributed to high polarity and solubility in fatty food stimulants. Overall, this active film has an excellent antioxidant effect and could potentially be used as food packaging for high-fat food products to prevent oxidation.

Bioprocessing and purification of extracellular L-asparaginase produced by endophytic Colletotrichum gloeosporioides and its anticancer activity.

L-asparaginase is an enzyme commonly used to treat acute lymphoblastic leukemia. Commercialized bacterial L-asparaginase has been reported to cause several life-threatening complications during treatment, hence the need to seek alternative sources of L-asparaginase. In this study, the novelty of upstream and downstream bioprocessing of L-asparaginase from a fungal endophyte, Colletotrichum gloeosporioides, and the cytotoxicity evaluation was demonstrated. Six variables (carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate) known to influence L-asparaginase production were studied using One-Factor-At-A-Time (OFAT) approach, with four significant variables further optimized using Response Surface Methodology (RSM). The crude extract produced using optimized condition was purified, characterized and examined for its anticancer effect. Purification of fungal L-asparaginase was performed via ultrafiltration and size exclusion chromatography, which are less common techniques. The protein profile and monomeric weight of L-asparaginase were determined using SDS-PAGE and Western blot. Cytotoxicity of purified L-asparaginase on leukemic Jurkat E6 and oral carcinoma cells were studied using MTS assay for 24 h and 48 h. OFAT results from optimization showed that glucose and L-asparagine concentrations, incubation period and temperature, were significant factors affecting L-asparaginase production by C. gloeosporioides. RSM analysis further evidence the significant interaction between glucose and L-asparagine concentrations in inducing L-asparaginase production. Purified L-asparaginase was profiled with specific activity of 255.02 IU/mg protein, purification fold of 6.12, and 34.63% of enzyme recovery. SDS and Western blot revealed that the purified L-asparaginase might be a tetramer with monomeric units of 25 kDa. Purified L-asparaginase was discovered to be more efficient against Jurkat leukemic cells than against H103 oral carcinoma cells, as lower IC50 value was observed for Jurkat cell lines (46 .36 ± 1.52 µg/mL for Jurkat and 125.56 ± 7.28 µg/mL for H103). In short, purified L-asparaginase derived from endophytic C. gloeosporioides showed high purity and significant anticancer effect toward cancer cells. This study therefore demonstrated the potential of fungal L-asparaginase as alternative chemotherapy drug in the future.

Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation.

L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.

Bacterial and eukaryotic microbial communities in urban water systems profiled via Illumina MiSeq platform.

Microbial communities from a lake and river flowing through a highly dense urbanized township in Malaysia were profiled by sequencing amplicons of the 16S V3-V4 and 18S V9 hypervariable rRNA gene regions via Illumina MiSeq. Results revealed that Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the dominant prokaryotic phyla; whereas, eukaryotic communities were predominantly of the SAR clade and Opisthokonta. The abundance of Pseudomonas and Flavobacterium in all sites suggested the possible presence of pathogens in the urban water systems, supported by the most probable number (MPN) values of more than 1600 per 100 mL. Urbanization could have impacted the microbial communities as transient communities (clinical, water-borne and opportunistic pathogens) coexisted with common indigenous aquatic communities (Cyanobacteria). It was concluded that in urban water systems, microbial communities vary in their abundance of microbial phyla detected along the water systems. The influences of urban land use and anthropogenic activities influenced the physicochemical properties and the microbial dynamics in the water systems. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-020-02617-3.

Determining Soil Microbial Communities and Their Influence on Ganoderma Disease Incidences in Oil Palm (Elaeis guineensis) via High-Throughput Sequencing.

Basal stem rot (BSR), caused by Ganoderma boninense, is the most devastating oil palm disease in South East Asia, costing US$500 million annually. Various soil physicochemical parameters have been associated with an increase in BSR incidences. However, very little attention has been directed to understanding the relationship between soil microbiome and BSR incidence in oil palm fields. The prokaryotic and eukaryotic microbial diversities of two coastal soils, Blenheim soil (Typic Quartzipsamment-calcareous shell deposits, light texture) with low disease incidence (1.9%) and Bernam soil (Typic Endoaquept-non-acid sulfate) with high disease incidence (33.1%), were determined using the 16S (V3-V4 region) and 18S (V9 region) rRNA amplicon sequencing. Soil physicochemical properties (pH, electrical conductivity, soil organic matter, nitrogen, phosphorus, cation exchange capacity, exchangeable cations, micronutrients, and soil physical parameters) were also analyzed for the two coastal soils. Results revealed that Blenheim soil comprises higher prokaryotic and eukaryotic diversities, accompanied by higher pH and calcium content. Blenheim soil was observed to have a higher relative abundance of bacterial taxa associated with disease suppression such as Calditrichaeota, Zixibacteria, GAL15, Omnitrophicaeota, Rokubacteria, AKYG587 (Planctomycetes), JdFR-76 (Calditrichaeota), and Rubrobacter (Actinobacteria). In contrast, Bernam soil had a higher proportion of other bacterial taxa, Chloroflexi and Acidothermus (Actinobacteria). Cercomonas (Cercozoa) and Calcarisporiella (Ascomycota) were eukaryotes that are abundant in Blenheim soil, while Uronema (Ciliophora) and mammals were present in higher abundance in Bernam soil. Some of the bacterial taxa have been reported previously in disease-suppressive and -conducive soils as potential disease-suppressive or disease-inducible bacteria. Furthermore, Cercomonas was reported previously as potential bacterivorous flagellates involved in the selection of highly toxic biocontrol bacteria, which might contribute to disease suppression indirectly. The results from this study may provide valuable information related to soil microbial community structures and their association with soil characteristics and soil susceptibility to Ganoderma.

A novel study based on adaptive metal tolerance behavior in fungi and SEM-EDX analysis.

Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.

Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction and ergosterol assay.

This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL-1) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via high performance liquid chromatography, HPLC) confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.