Taxonomy and species diversity of Ganoderma species in the Garden Route National Park of South Africa inferred from morphology and multilocus phylogenies.

Ganoderma is a cosmopolitan genus that encompasses species with cultural, economic, and pathogenic importance. Despite the importance of this genus, knowledge pertaining to the species diversity of Ganoderma in South Africa is limited. This study aimed at elucidating the identity and phylogenetic placements of Ganoderma samples obtained during a survey of wood-rotting fungi in the Garden Route National Park (GRNP) of South Africa, supplemented with isolates obtained from other localities across the country. Identification was achieved by means of multilocus phylogenetic inference combined with morphological evaluation. In total, eight distinct species of Ganoderma were recovered from different hosts and localities across the country. Of these, Ganoderma cf. cupreum and Ganoderma cf. resinaceum represent possible new records for South Africa. Two novel species are described, namely, G. eickeri. and G. knysnamense. Ganoderma eickeri, sp. nov., is characterized by a triquetrous and broadly attached basidiome, a sulcate or zonate yellowish brown to brown pilear surface, and ovoid to ellipsoid basidiospores. Ganoderma knysnamense is distinguished by an applanate to ungulate, sometimes convex, dimidiate to broadly attached basidiome, a chocolate-brown pilear surface covered with a hard woody-like crust and ellipsoid, broadly ellipsoid to ovoid basidiospores. The discovery of two new Ganoderma species in this study raises the known Ganoderma species in South Africa to 13.

Genetic variability of Ganoderma zonatum infecting palms in Florida.

Ganoderma zonatum is a lethal pathogen of palms (Arecaceae) in Florida (USA) because it degrades the wood of the lowest section of the palm trunk. This fungus is widespread throughout Florida, where it has been observed on over 60 species of palms. The authors examined the genetic variability of 25 isolates of G. zonatum obtained in Florida from 12 different palm species and representing 17 unique property locations in eight counties to determine if G. zonatum represents a species complex. The three genomic regions examined were the nuc rDNA ITS1-5.8S-ITS2 region (ITS), the coding region for RNA polymerase II subunit 2 (rpb2) domains 6 and 7, and the partial gene for translation elongation factor 1α (tef1α). The results indicated that variability among these three genomic regions was minimal, and the variability observed was not related to palm host or geographic region within Florida. Thus, in the geographic region surveyed, G. zonatum does not appear to represent a species complex.

About Ganoderma boninense in oil palm plantations of Sumatra and peninsular Malaysia: Ancient population expansion, extensive gene flow and large scale dispersion ability.

Wood rot fungi form one of the main classes of phytopathogenic fungus. The group includes many species, but has remained poorly studied. Many species belonging to the Ganoderma genus are well known for causing decay in a wide range of tree species around the world. Ganoderma boninense, causal agent of oil palm basal stem rot, is responsible for considerable yield losses in Southeast Asian oil palm plantations. In a large-scale sampling operation, 357 sporophores were collected from oil palm plantations spread over peninsular Malaysia and Sumatra and genotyped using 11 SSR markers. The genotyping of these samples made it possible to investigate the population structure and demographic history of G. boninense across the oldest known area of interaction between oil palm and G. boninense. Results show that G. boninense possesses a high degree of genetic diversity and no detectable genetic structure at the scale of Sumatra and peninsular Malaysia. The fact that few duplicate genotypes were found in several studies including this one supports the hypothesis of spore dispersal in the spread of G. boninense. Meanwhile, spatial autocorrelation analysis shows that G. boninense is able to disperse across both short and long distances. These results bring new insight into mechanisms by which G. boninense spreads in oil palm plantations. Finally, the use of approximate Bayesian computation (ABC) modelling indicates that G. boninense has undergone a demographic expansion in the past, probably before the oil palm was introduced into Southeast Asia.

Antitubercular Activity of Mycelium-Associated Ganoderma Lanostanoids.

In a continuation of our research into antitubercular lanostane triterpenoids from submerged cultures of Ganoderma species, three strains, Ganoderma orbiforme BCC 22325, Ganoderma sp. BCC 60695, and Ganoderma australe BCC 22314, have been investigated. Fourteen new lanostane triterpenoids, together with 35 known compounds, were isolated. Antitubercular activities of these mycelium-associated Ganoderma lanostanoids against Mycobacterium tuberculosis H37Ra were evaluated. Taken together with the assay data of previously isolated compounds, structure-activity relationships of the antitubercular activity are proposed. Most importantly, 3ß- and 15α-acetoxy groups were shown to be critical for antimycobacterial activity. The most potent compound was (24E)-3ß,15α-diacetoxylanosta-7,9(11),24-trien-26-oic acid (35).

Seasonal variation of Ganoderma spore concentrations in urban and suburban districts of the city of Szczecin, Poland.

According to recent studies, Ganoderma may be the third genus, after Alternaria and Cladosporium, the spores of which cause symptoms of allergy, and concentration is related to meteorological factors. The aerobiology of Ganoderma spores in Szczecin in urban and suburban districts was examined using Lanzoni Volumetric Spore Traps in 2008-2010. Ganoderma spores were present in the atmosphere on more than 90% of the days from June through September with peak concentrations in June, July and September. The number of days with spores was lower in the suburban district, while the total number of spores collected was higher there than in the urban district. Correlation and multiple regression analyses revealed weak relationships between Ganoderma and meteorological conditions, while testing the significance of differences between the districts showed that urban development did not have a clear impact on the values of meteorological parameters. A significantly higher abundance of spores in the suburbs of Szczecin seemed to be conditioned by the closeness of potential area sources. This study indicates that a single measuring site in the city centre insufficiently reflected the dynamics and level of Ganoderma spore concentration in peripheral districts.

Mycelial growth rate and macro- and micromorphological characteristics of medicinal species of genus Ganoderma (higher Basidiomycetes) from Iran.

Mycelial growth rate is a distinguishing quality that demonstrates continuous variation in different isolates collected from various hosts and locations. The objectives of this research were (1) to reinvestigate the previous identification of Iranian species, and (2) to recognize the best native isolate(s) for cultivation of different Ganoderma species. Of 78 samples collected from different hosts and sites, only 43 mycelia could be purified and examined for further study. Growth rate (GR; Δd/Δt) and growth coefficient (GC; dgh/t) were analyzed by growing isolate culture on 2% malt-extract agar medium (pH 5.5) incubated at 25°C. Macro- and micromorphological studies on mycelia and fruiting bodies such as basidiospore and cutis microcharacters as well as fruiting body quality were used for precise identification. Results revealed that samples belonged to 4 species: G. lucidum, G. applanatum, G. resinaceum, and G. australe. Among all samples, the isolate morphologically identified as G. applanatum showed the best GR (12 mm/day) and good GC (128 mm/day), followed by the 2 other isolates identified as G. resinaceum (GRs and GCs of 11 and 55 mm/day and 10.9 and 43.6 mm/day, respectively).

Rapid detection of Ganoderma-infected oil palms by microwave ergosterol extraction with HPLC and TLC.

Detection of basal stem rot (BSR) by Ganoderma of oil palms was based on foliar symptoms and production of basidiomata. Enzyme-Linked Immunosorbent Assays-Polyclonal Antibody (ELISA-PAB) and PCR have been proposed as early detection methods for the disease. These techniques are complex, time consuming and have accuracy limitations. An ergosterol method was developed which correlated well with the degree of infection in oil palms, including samples growing in plantations. However, the method was capable of being optimised. This current study was designed to develop a simpler, more rapid and efficient ergosterol method with utility in the field that involved the use of microwave extraction. The optimised procedure involved extracting a small amount of Ganoderma, or Ganoderma-infected oil palm suspended in low volumes of solvent followed by irradiation in a conventional microwave oven at 70°C and medium high power for 30s, resulting in simultaneous extraction and saponification. Ergosterol was detected by thin layer chromatography (TLC) and quantified using high performance liquid chromatography with diode array detection. The TLC method was novel and provided a simple, inexpensive method with utility in the field. The new method was particularly effective at extracting high yields of ergosterol from infected oil palm and enables rapid analysis of field samples on site, allowing infected oil palms to be treated or culled very rapidly. Some limitations of the method are discussed herein. The procedures lend themselves to controlling the disease more effectively and allowing more effective use of land currently employed to grow oil palms, thereby reducing pressure to develop new plantations.

Efficacy of Ganoderma sp. JAS4 in bioremediation of chlorpyrifos and its hydrolyzing metabolite TCP from agricultural soil.

A novel fungal strain JAS4 was isolated from agricultural soil and was found to be highly effective in degrading chlorpyrifos and its major degradation product 3,5,6-trichloro-2-pyridinol (TCP). The molecular characterization based on 18S rRNA sequence analysis, revealed strain JAS4 as Ganoderma sp. which could able to degrade chlorpyrifos and its metabolite in an aqueous medium with rate constant of 0.8460 day(-1), following first order rate kinetics, and the time in which the initial insecticide concentration was reduced by 50% (DT(50)) was 0.81 days. Studies on biodegradation in soil with nutrients showed that JAS4 strain exhibited efficient degradation of insecticide with a rate constant of 0.9 day(-1), and DT(50) was 0.73 day. In contrast, degradation of insecticide in soil without nutrients was characterized by a rate constant of 0.7576 day(-1) and the DT(50) was 0.91 day.

Ergosterol from the soilborne fungus Ganoderma boninense.

Ergosterol is the main component of the fungal membrane and is not found in plants or other microbial cells. Therefore, it can be a useful biomarker for the quantification of fungal biomass. We are now reporting the first isolation and characterisation of ergosterol from the mycelium of G. boninense. The ergosterol structure was detected by Thin Liquid Chromatography (TLC) and Ultra Performance Liquid Chromatography (UPLC) and confirmed with Gas Chromatography coupled with Mass Spectrometry (GCMS) and Nuclear Magnetic Resonance (NMR) analysis.

Skin test reactivity of allergic subjects to basidiomycetes' crude extracts in a tropical environment.

Fungal allergies can be detected by the skin prick test with extracts of the organisms, but not all fungi, including the basidiomycetes, are being examined. We determined the level of sensitization to basidiomycetes in allergic subjects and compared their reactivity to commercial extracts commonly used to detect allergies. Crude spore extracts of the basidiomycetes Ganoderma applanatum, Chlorophyllum molybdites, and Pleurotus ostreatus, which are known to release numerous spores, were examined along with commercial extracts on 33 subjects with asthma, allergic or non-allergic rhinitis. Overall, affected subjects showed the highest reactivity to mites (36%), followed by Ganoderma applanatum (30%), grass (27%) Chlorophyllum molybdites (12%) and Pleurotus ostreatus (12%). Allergic rhinitis patients were most reactive to mites (58%), grass (42%), Ganoderma applanatum (25%), Penicillium spp. (25%), and cat (17%). Those with asthma primarily responded to mites (44%), Ganoderma applanatum (44%), grass (33%), and Pleurotus ostreatus (22%). IgE levels correlated with positive basidiomycetes extracts. This finding, coupled with higher reactivity to basidiospores as compared to mitospores, and the similar sensitivities of patients to G. applanatum and mites, suggest that basidiomycetes are important allergen sources in the tropics.

The effects of meteorological factors on the occurrence of Ganoderma sp. spores in the air.

Ganoderma sp. is an airborne fungal spore type known to trigger respiratory allergy symptoms in sensitive patients. Aiming to reduce the risk for allergic individuals, we analysed fungal spore circulation in Szczecin, Poland, and its dependence on meteorological conditions. Statistical models for the airborne spore concentrations of Ganoderma sp.-one of the most abundant fungal taxa in the area-were developed. Aerobiological sampling was conducted over 2004-2008 using a volumetric Lanzoni trap. Simultaneously, the following meteorological parameters were recorded: daily level of precipitation, maximum and average wind speed, relative humidity and maximum, minimum, average and dew point temperatures. These data were used as the explaining variables. Due to the non-linearity and non-normality of the data set, the applied modelling techniques were artificial neural networks (ANN) and mutlivariate regression trees (MRT). The obtained classification and MRT models predicted threshold conditions above which Ganoderma sp. appeared in the air. It turned out that dew point temperature was the main factor influencing the presence or absence of Ganoderma sp. spores. Further analysis of spore seasons revealed that the airborne fungal spore concentration depended only slightly on meteorological factors.

Hourly predictive artificial neural network and multivariate regression trees models of Ganoderma spore concentrations in Rzeszów and Szczecin (Poland).

Ganoderma spores are one of the most airspora abundant taxa in many regions of the world, and are considered to be important allergens. The aerobiology of Ganoderma basidiospores in two cities in Poland was examined using the volumetric method, (Burkard and Lanzonii Spore Traps), from selected days in 2004, 2005 and 2006. Spores of Ganoderma were present in the atmosphere from June to November, with peak concentrations generally occurring from late July to mid-October. ANN (artificial neural network) and MRT (multivariate regression trees), models indicated that atmospheric phenomenon, hour and relative humidity were the most important variables influencing spore content. The remaining variables (air temperature, dew point, air pressure, wind speed and wind direction), also contributed to the high network performance, (ratio above 1), but their impact was less distinct. Those results are consistent with the Spearman's rank correlation analysis.

Evaluation of oil-palm fungal disease infestation with canopy hyperspectral reflectance data.

Fungal disease detection in perennial crops is a major issue in estate management and production. However, nowadays such diagnostics are long and difficult when only made from visual symptom observation, and very expensive and damaging when based on root or stem tissue chemical analysis. As an alternative, we propose in this study to evaluate the potential of hyperspectral reflectance data to help detecting the disease efficiently without destruction of tissues. This study focuses on the calibration of a statistical model of discrimination between several stages of Ganoderma attack on oil palm trees, based on field hyperspectral measurements at tree scale. Field protocol and measurements are first described. Then, combinations of pre-processing, partial least square regression and linear discriminant analysis are tested on about hundred samples to prove the efficiency of canopy reflectance in providing information about the plant sanitary status. A robust algorithm is thus derived, allowing classifying oil-palm in a 4-level typology, based on disease severity from healthy to critically sick stages, with a global performance close to 94%. Moreover, this model discriminates sick from healthy trees with a confidence level of almost 98%. Applications and further improvements of this experiment are finally discussed.

Ganoderma species discrimination by dual-mode chromatographic fingerprinting: a study on stationary phase effects in hydrophilic interaction chromatography and reduction of sample misclassification rate by additional use of reversed-phase chromatography.

Acetonitrile-water extracts of several Ganoderma species - a mushroom being used in Traditional Chinese Medicine - were analysed by liquid chromatography-UV detection in hydrophilic interaction chromatography (HILIC) and reversed-phase (RP) elution modes. A set of six polar stationary phases was used for HILIC runs. These columns had remarkably different separation properties under binary gradient conditions as evinced by hierarchical cluster analysis on retention patterns of seven test compounds. Complementary measurements of RP chromatograms were carried out on a C(18) packing. Injection precision (n=5) and intra-day precision (n=5) were each <2.0% RSD (HILIC) and <0.7% RSD (RP) for relative retention times of main characteristic peaks of a sample extract while for relative peak areas RSD values were max. 6.8%. Repetitive analysis (n=7) of a processed sample stored in the autosampler tray for 48h was used to confirm within-sequence sample stability. Eleven Ganoderma lucidum samples served as training set for the construction of column-specific simulated mean chromatograms. Validation with twelve samples comprising G. lucidum, Ganoderma sinense, Ganoderma atrum, and Ganoderma tsugae by correlation coefficient based similarity evaluation of peak patterns showed that a discrimination of G. lucidum from other Ganoderma species by means of chromatographic fingerprints is conceptually possible on all columns, except of a bare silica packing. The importance of the combined use of RP and HILIC fingerprints to improve the rate of correct sample classification was demonstrated by the fact that each one G. sinense specimen was wrongly assigned being G. lucidum by all HILIC fingerprints but not the RP fingerprint and vice versa. The present data revealed that (i) the analysis of complex biological materials by quasi orthogonal chromatographic modes such as HILIC and RP may deliver more discriminative information than single-mode approaches which strengthens the reliability of fingerprint-based sample classification and (ii) different retention and selectivity characteristics of polar bonded silica packings in the HILIC elution mode may only have a minor impact on chemometric sample discrimination capabilities in such kind of pattern-oriented metabolomics separation problems.

Molecular biology of Ganoderma pathogenicity and diagnosis in coconut seedlings.

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10-13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.

Ganoderma australe from southern India.

The genetic diversity of Ganoderma australe (Fr.) Pat. from southern India was investigated by using ITS1/2 rDNA. The phylogenetic analysis showed that six isolates clustered into two groups viz. biological species I and II. The four strains of BS I (YER03, MYC5, MYC2 and KE) clustered with G. australe TAI-01 and the two other strains of BS II (KMK3 and K39) were grouped with G. australe TAI-05 from Taiwan. The two strains namely TAI-01 and TAI-05 were described as G. australe intersterile Group 1 and 2 from Taiwan, respectively. The higher level nucleotide divergence among BS I and BS II and the high bootstrapping support clearly represent the presence of two biological species of G. australe in southern India which are genetically isolated.

Solid-state fermentation for enhanced production of laccase using indigenously isolated Ganoderma sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25 degrees C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.

Screening of fungal isolates and properties of Ganoderma applanatum intended for olive mill wastewater decolourization and dephenolization.

AIMS: To investigate different autochthonous isolates of wood-rotting fungi for the removal of both colour and phenolic compounds from olive mill wastewaters (OMW). METHODS AND RESULTS: The isolates Bjerkandera adusta Ba-100, Fomes fomentarius Ff-106, Ganoderma applanatum Ga-20, Irpex lacteus Il-3, Trametes versicolor Tv-101 and Tv-103 were preliminarily screened for their OMW-decolourizing potential on potato dextrose agar supplemented with different OMW concentrations. A further screening of batch cultures under different agitation speeds, to test the effect of shear stress, resulted in the selection of isolate G. applanatum Ga-20. Batch cultures grown in OMW-based medium exhibited strong laccase induction and significant decrease in the values of phenols, colour and chemical oxygen demand. Concomitant onset of laccase activity and colour removal was observed, and apart from laccase, neither lignin peroxidase nor manganese-dependent peroxidase activities were detected. Moreover, the depletion of aromatic compounds with high and low apparent molecular mass was observed by chromatographic analysis. CONCLUSIONS: Isolate G. applanatum Ga-20 exhibited interesting properties for its use in bioremediation of OMW, namely high removal of recalcitrant phenolic compounds and strong colour abatement. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time, the white-rot fungus G. applanatum proves to be effective for the decolourization and dephenolization of OMW.

Enhanced production of exopolysaccharides by fed-batch culture of Ganoderma resinaceum DG-6556.

The objectives of this study were to optimize submerged culture conditions of a new fungal isolate, Ganorderma resinaceum, and to enhance the production of bioactive mycelial biomass and exopolysaccharides (EPS) by fed-batch culture. The maximum mycelial growth and EPS production in batch culture were achieved in a medium containing 10 g/l glucose, 8 g/l soy peptone, and 5 mM MnCl(2) at an initial pH 6.0 and temperature 31 degrees C. After optimization of culture medium and environmental conditions in batch cultures, a fed-batch culture strategy was employed to enhance production of mycelial biomass and EPS. Five different EPS with molecular weights ranging from 53,000 to 5,257,000 g/mole were obtained from either top or bottom fractions of ethanol precipitate of culture filtrate. A fed-batch culture of G. resinaceum led to enhanced production of both mycelial biomass and EPS. The maximum concentrations of mycelial biomass (42.2 g/l) and EPS (4.6 g/l) were obtained when 50 g/l of glucose was fed at day 6 into an initial 10 g/l of glucose medium. It may be worth attempting with other mushroom fermentation processes for enhanced production of mushroom polysaccharides, particularly those with industrial potential.

Derivatization does not influence antimicrobial and antifungal activities of applanoxidic acids and sterols from Ganoderma spp.

Applanoxidic acids and sterols, isolated from Ganoderma spp., were acetylated and/or methylated. The antibacterial activity against Escherichia coli and Staphylococcus aureus and the antifungal activity against Candida albicans and Trichophyton mentagrophytes of the derivatives were investigated by a microdilution method, and compared with those of the natural products. Both natural and modified compounds exhibited comparable antibacterial and antifungal activities in a range of 1.0 to > 2.0 mg/ml minimal inhibitory concentration.