Effects of postharvest oligochitosan treatment on fungal diseases and defence responses in Dongxue peach fruit.

In this study, the effects of oligochitosan treatment on controlling postharvest diseases in Dongxue peach ( Prunus Persica L. Batsch, cv Dongxuemi) were examined and the possible underlying mechanisms were discussed. Results showed that the disease incidence and lesion area in peach fruit inoculated with Monilinia fructicola and Penicillium expansum were all remarkably reduced by oligochitosan treatment. Oligochitosan treatment inhibited spore germination and mycelial growth of the two fungi in vitro. Oligochitosan treatment also induced upregulation of the salicylic acid signalling pathway-related genes (NPR1, PR1 and phenylalanine ammonia lyase) and enhanced the levels of total phenolics, flavonoids and lignin in peach. Meanwhile, enzymatic activities of superoxide dismutase, catalase, polyphenoloxidase, ascorbate peroxidase and phenylalanine ammonia lyase also increased. These findings suggest that the effects of oligochitosan on the disease control of peach fruit may be associated with its direct antimicrobial effects as well as increasing antioxidant, phenylpropanoid metabolism and accumulating antifungal compounds by activating the salicylic acid-dependent pathway.

Characterization of Paracoccidioides brasiliensis by FT-IR spectroscopy and nanotechnology.

Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, is a dimorphic fungus existing as mycelia in the environment (or at 25°C in vitro) and as yeast cells in the human host (or at 37°C in vitro). Because mycological examination of lesions in patients frequently is unable to show the presence of the fungus and serological tests can misdiagnose the disease with other mycosis, the development of new approach's for molecular identification of P. brasiliensis spurges is needed. This study describes the use of a gold nanoprobe of a known gene sequence of P. brasiliensis as a molecular tool to identify P. brasiliensis by regular polymerase chain reaction (PCR) associated with a colorimetric methods. This approach is suitable for testing in remote areas because it does not require any further step than gene amplification, being safer and cheaper than electrophoresis methods. The proposed test showed a color change of the PCR reaction mixture from red to blue in negative samples, whereas the solution remains red in positive samples. We also performed a Fourier Transform Infrared (FT-IR) Spectroscopy analysis to characterize and compare the chemical composition between yeast and mycelia forms, which revealed biochemical differences between these two forms. The analysis of the spectra showed that differences were distributed in chemical bonds of proteins, lipids and carbohydrates. The most prominent difference between both forms was vibration modes related to 1,3-ß-glucan usually found in mycelia and 1,3-α-glucan found in yeasts and also chitin forms. In this work, we introduce FT-IR as a new method suitable to reveal overall differences that biochemically distinguish each form of P. brasiliensis that could be additionally used to discriminate biochemical differences among a single form under distinct environmental conditions.

Isolation of the Entomopathogenic Fungal Strain Cod-MK1201 from a Cicada Nymph and Assessment of Its Antibacterial Activities.

The entomopathogenic fungus Cod-MK1201 was isolated from a dead cicada nymph. Three regions of ribosomal nuclear DNA, the internal transcribed spacers of nuclear ribosomal DNA repeats (ITS), the partial small subunit of rDNA (nrSSU) , and the partial large subunit of rDNA (nrLSU), and two protein-coding regions, the elongation factor 1α (EF-1α), and the largest subunit of the RNA polymerase II (rpb1) gene, were sequenced and used for fungal identification. The phylogenetic analysis of the ITS and the combined data set of the five genes indicated that the fungal isolate Cod-MK1201 is a new strain of Cordyceps sp. that is closely related to Cordyceps nipponica and C. kanzashiana. Crude extracts of mycelium-cultured Cod-MK1201 were obtained using distilled water and 50% (v/v) ethanol, and the antibacterial activity of each was determined. Both extracts had activity against Gram-positive and Gram-negative bacteria, but the ethanol extract was the more potent of the two. The antibacterial activity of the protein fractions of these extracts was also determined. The protein fraction from the ethanol extract was more antibacterial than the protein fraction from the aqueous extract. Three antibacterial constituents including adenosine, the total phenolic content (TPC), and the total flavonoid content (TFC) was also determined. The results showed that the adenosine content, the TPC, and the TFC of the ethanol extract were more active than those of the aqueous extract. Moreover, synergism was detected between these antibacterial constituents. In conclusion, the entomopathogenic fungal isolate Cod-MK1201 represents a natural source of antibacterial agents.

Immune augmentation and Dalton's Lymphoma tumor inhibition by glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus.

With the increase in cancer progression, alternatives in the medicinal field with minimal side effects need to be ascertained. In this context, for the first time novel glucans/glycans isolated from the mycelia and fruit body of Pleurotus ostreatus have been compared for their exquisite property as immunoceuticals. Glucans from both the sources displayed immunological functions which include lymphocyte proliferation, macrophage activation (nitric oxide production, ROS generation, phagocytosis, TNF-α production) as well as macrophage and NK cell mediated cytotoxicity. In vivo studies with Dalton's Lymphoma mice tumor model further enumerated the immune enhancing and tumor regression potential of the two glucan molecules. Highest tumor inhibition of about 75% and 71.4% were observed at 20mg/kg of mycelia and fruit body glucan/glycan treatments. A concomitant increase in the survival period of glucan treated tumor bearing mice was found to be primarily associated with immune boosting and apoptosis of cancerous cells. Both the glucan molecules exhibited similar degree of immune response at the systemic level with only subtle amount of differences in two dimensional in vitro cultures. Efficacy of glucans/glycans as immunomodulators may thereby provide decisive leads in strengthening the immune system along with other therapies.

Efficiency of Artemisia nilagirica (Clarke) Pamp. essential oil as a mycotoxicant against postharvest mycobiota of table grapes.

BACKGROUND: In order to get a potent botanical fungicide for the management of fungal decay of table grapes, an experiment was conducted in which 20 essential oils of higher plants were screened at 0.33 µL mL(-1) against dominant fungi causing decay of table grapes, including Aspergillus flavus, A. niger and A. ochraceus. Furthermore, the minimum inhibitory/fungicidal concentration, fungitoxic spectrum and mycotoxin inhibition activity of the most potent oil were determined. The efficacy of the most potent oil in preservation of table grapes, along with organoleptic evaluation, was also carried out by storing 1 kg of grapes in the oil vapour. RESULTS: Artemisia nilagirica oil was found to be most toxic, exhibiting 100% mycelia inhibition of all test fungi. Moreover, 0.29 µL mL(-1) A. nilagirica oil was fungistatic and 0.58 µL mL(-1) was fungicidal for all tested species of Aspergillus. The oil exhibited a broad range of fungitoxicity against other grape berry-rotting fungi. Artemisia nilagirica oil completely suppressed the growth and mycotoxin (AFB1 and OTA) secretion of aflatoxigenic and ochratoxigenic strains of Aspergillus at 1.6 µL mL(-1) . During the in vivo experiment, fumigation of 1 kg of table grapes with 200 and 300 µL dosage of A. nilagirica oil enhanced the shelf life for up to 9 days. The oil did not show any phytotoxic effect. Besides, oil application did not substantively change the sensory properties of the fruits. CONCLUSION: Artemisia nilagirica oil can be used as an alternative botanical fungicide for the control of fruit-rotting fungi of stored grapes.

Optimizing a culture medium for biomass and phenolic compounds production using Ganoderma lucidum

The present work was aimed at optimizing a culture medium for biomass production and phenolic compounds by using Ganoderma lucidum. The culture was optimized in two stages; a Plackett-Burman design was used in the first one for identifying key components in the medium and a central composite design was used in the second one for optimizing their concentration. Both responses (biomass and phenolic compounds) were simultaneously optimized by the latter methodology regarding desirability, and the optimal concentrations obtained were 50.00 g/L sucrose, 13.29 g/L yeast extract and 2.99 g/L olive oil. Maximum biomass production identified in these optimal conditions was 9.5 g/L and that for phenolic compounds was 0.0452 g/L, this being 100% better than that obtained in the media usually used in the laboratory. Similar patterns regarding chemical characterization and biological activity towards Aspergillus sp., from both fruiting body and mycelium-derived secondary metabolites and extracts obtained in the proposed medium were observed. It was shown that such statistical methodologies are useful for optimizing fermentation and, in the specific case of G. lucidum, optimizing processes for its production and its metabolites in submerged culture as an alternative to traditional culture.

Characterization of a cassiicolin-encoding gene from Corynespora cassiicola, pathogen of rubber tree (Hevea brasiliensis).

Corynespora Leaf Fall (CLF) is a major disease of rubber tree (Hevea brasiliensis) caused by the Ascomycota Corynespora cassiicola. Here we describe the cloning and characterization of a gene encoding cassiicolin (Cas), a glycosylated cystein-rich small secreted protein (SSP) identified as a potential CLF disease effector in rubber tree. Three isolates with contrasted levels of aggressiveness were analyzed comparatively. The cassiicolin gene was detected - and the toxin successfully purified - from the isolates with high and medium aggressiveness (CCP and CCAM3 respectively) but not from the isolate with the lowest aggressiveness (CCAM1), suggesting the existence of a different disease effector in the later. CCP and CCAM3 carried strictly identical cassiicolin genes and produced toxins of identical mass, as evidence by mass spectrometry analysis, thus suggesting conserved post-translational modifications in addition to sequence identity. The differences in aggressiveness between CCP and CCAM3 may be attributed to differences in cassiicolin transcript levels rather than qualitative variations in cassiicolin structure. Cassiicolin may play an important role in the early phase of infection since a peak of cassiicolin transcripts occurred in 1 or 2 days after inoculation (before the occurrence of the first symptoms), in both the tolerant and the susceptible cultivars.

Tolerance to and accumulation of cadmium by the mycelium of the fungi Scleroderma citrinum and Pisolithus tinctorius.

The behavior of ectomycorrhizal (ECM) fungi on exposure to cadmium dependent upon isolation remains a poorly understood phenomenon. The in vitro growth, tolerance, and accumulation of Cd were studied in three strains of ECM fungi exposed to six Cd concentrations (0-10 mg L(-1)). The fungi studied were a strain of Scleroderma citrinum Persoon (Sc) isolated from a tailings heap containing 5 mg kg(-1) available Cd, and two strains of Pisolithus tinctorius (Pers.) Coker and Couch from unpolluted sites (Pt1 and Pt2), both common ECM fungi used for remediation. The growth kinetic (36 days) of Sc was not affected by Cd concentration. By contrast, the ED(50) in Pt1 and Pt2 occurred at 4.8 and 6.9 mg L(-1) of Cd, respectively. The biomass of the three fungi exposed to the highest Cd concentration (10 mg L(-1)) was significantly different. Sc presented the highest biomass, while this was strongly reduced for Pt1 and Pt2. The tolerance index for Sc ranged from 78% to 95% at all Cd concentrations tested, while for Pt1 it was 49% and 31%, and for Pt2 it was 62% and 35% at 5 and 10 mg of Cd L(-1), respectively. The mycelium of both Pt strains accumulated more Cd than the Sc mycelium. At the highest Cd concentration, Pt1 and Pt2 accumulated 1.9 and 1.7 times more Cd than Sc. This study suggests that regardless of the differences in tolerance to Cd by the three ECM fungi, they could have biotechnological applications for soil remediation. However, Sc has greater possibilities of being used successfully when high concentrations of Cd prevail in the environment.

Viabilidade de agaricus blazei em diferentes condições de preservação / Viabilidad de agaricus blazei en diferentes condiciones de preservación / Viability of agaricus blazei under different preservation conditions

A técnica de transferências periódicas de fragmentos de micélio para novo meio de cultura é a mais utilizada para a preservação de Agaricus blazei. Entretanto, esta técnica apresenta maior risco de contaminação, degeneração genética e perda de caracteristicas biológicas. O desenvolvimento de técnicas de preservação que permitam a manutenção da viabilidade da espécie por mais tempo e a um menor custo é de interesse biotecnológico. Desse modo, o objetivo deste trabalho foi avaliar a viabilidade de A. blazei crescido em dois meios de cultivo e preservado à +4 ºC ou -20 ºC em diferentes recipientes de contenção. O fungo foi crescido em meio de ágar-extrato de malte ou ágar-grão de trigo moído e preservado à +4 ºC ou -20 ºC em diferentes recipientes de contenção, simples ou duplos, com adição de soluções aquosas de glicerol, sacarose, glicose, água ultrapura ou sem adição de crioprotetor. Após 1 ou 12 meses o micélio preservado foi transferido para ágar-extrato de malte para avaliação da viabilidade micelial. Os crioprotetores glicerol, sacarose e glicose, associados com o meio de cultura ágar-extrato de malte ou ágar-grão de trigo moído, em recipiente de contenção simples ou duplo são efetivos para preservação à +4 ºC por períodos curtos, um mês, mas não são efetivos para períodos longos, 12 meses. Os crioprotetores, meios de cultivo e recipientes de contenção simples ou duplos não são efetivos para criopreservação do fungo à -20 ºC. Os recipientes simples são tão eficientes quanto os recipientes duplos para evitar contaminações e preservar o fungo.

Continuous mycelial subculturing is frequently used for the preservation of Agaricus blazei. However, this technique has a higher risk of contamination, genetic degeneration and loss of biological characteristics. The development of preservation techniques that allow maintaining the viability of this species longer and at lower costs is of biotechnological interest. Thus, the objective of this study was to evaluate the viability of A. blazei grown in two culture media and preserved at +4 ºC or -20 ºC in different containment vessels. The fungus was grown on malt extract agar or grounded wheat grain agar culture medium and preserved at +4 ºC or -20 ºC in different containment vessels, single or double ones, with the addition of aqueous solutions of glycerol, saccharose, glucose, ultrapure water or without addition of cryoprotectant. After 1 or 12 months, the preserved mycelium was transferred to malt extract agar for assessment of mycelial viability. Glycerol, saccharose and glucose associated with malt extract agar or grounded wheat grain agar culture medium, in single or double containment vessels, are effective for preservation at +4 ºC for a short period, one month, but they are not effective for a longer period, 12 months. Cryoprotectants, culture media and single or double containment vessels are not effective for fungus cryopreservation at -20 ºC. Simple containment vessels are as efficient as double ones to prevent contamination and to preserve the fungus.

La técnica de transferencias periódicas de fragmentos de micelio para nuevo medio de cultura es la más utilizada para la preservación de Agaricus blazei. Sin embargo, esta técnica presenta mayor riesgo de contaminación, degeneraciones genéticas y pérdidas de características biológicas. El desarrollo de técnicas de preservación que permitan la manutención y viabilidad de la especie por más tiempo y con un costo más bajo es de interés biotecnológico. De esta manera, el objetivo de este estudio fue evaluar la viabilidad de A. blazei sembrado en dos medios de cultivo y preservados en +4 ºC o -20 ºC en diferentes recipientes de contención. El hongo se cultivó en medio de extracto de agar de malta o agar de grano de trigo molido y preservado en +4 ºC o -20 ºC en diferentes recipientes de contención simple o doble, con adición de soluciones acuosas de glicerol, sacarosa, glucosa, agua ultra pura o sin adición de crioprotector. Después de 1 o 12 meses, el micelio preservado fue transferido para extracto de agar de malta para evaluación de la viabilidad del micelio. Los crioprotectores glicerol, sacarosa y glucosa, asociados con el medio de cultura extracto de agar de malta o de agar de grano de trigo molido, en recipiente de contención simple o doble son eficaces para la preservación a +4 ºC por períodos cortos, un mes, pero no son eficaces por períodos largos, como 12 meses. Los crioprotectores medios de cultivo y recipientes de contención simples o dobles no son eficaces para la criopreservación del hongo a -20 ºC. Recipientes simples son tan eficaces como los dobles para evitar contaminaciones y preservar el hongo.

Growth inhibition and morphological alterations of Trichophyton rubrum induced by essential oil from Cymbopogon winterianus Jowitt ex Bor

Trichophyton rubrum is one of the most common fungi causer of dermatophytosis, mycosis that affect humans and animals around the world. Researches aiming new products with antifungal activity become necessary to overcome difficulties on treatment of these infections. Accordingly, this study aimed to investigate the antifungal activity of essential oil from Cymbopogon winterianus against the dermatophyte T. rubrum. The antifungal screening was performed by solid medium diffusion method with 16 T. rubrum strains, minimum inhibitory concentration (MIC) and minimum fungicide concentration (MFC) were determined using the microdilution method. The effects on mycelial dry weight and morphology were also observed. Screening showed essential oil in natura inhibited all the tested strains, with inhibition zones between 24-28 mm diameter. MIC50 and MIC90 values of the essential oil were 312 µg/mL for nearly all the essayed strains (93.75 percent) while the MFC50 and MFC90 values were about eight times higher than MIC for all tested strains. All tested essential oil concentrations managed to inhibit strongly the mycelium development. Main morphological changes on the fungal strains observed under light microscopy, which were provided by the essential oil include loss of conidiation, alterations concerning form and pigmentation of hyphae. In the oil presence, colonies showed folds, cream color and slightly darker than the control, pigment production was absent on the reverse and with evident folds. It is concluded that C. winterianus essential oil showed activity against T. rubrum. Therefore, it could be known as potential antifungal compound especially for protection against dermatophytosis.

Vegetative compatibility and heterokaryon formation between different isolates of Colletotrichum lindemuthianum by using the nit mutant system

Colletotrichum lindemuthianum, the causative agent of bean anthracnose, is one of the most common pathogens leading to expressive damage to plants beyond presenting noticeable variability. The knowledge on vegetative compatibility groups (VCGs) is of particular interest in asexual fungi as they subdivide the population in groups that can exchange genetic information via heterokaryosis and the parasexual cycle. Among the techniques used in studies about vegetative compatibility groups, the obtainment of nit mutants is apparent. This paper is aimed at obtaining heterokaryons between different isolates of C. lindemuthianum, grouping them in VCGs and evaluating their genetic variability by using the nit mutants system. Nit mutants were obtained from 20 single spore isolates. The mutants were phenotypically classified and paired for complementation and formation of heterokaryons so as to group them in VCGs. Seventeen mutants from the different phenotypic-rates were recovered: nit1, nit2, nit3 and nitM. At the same time, 10 mutants were selected for pairing and division of the anastomosis groups. Nine heterokaryons were obtained and the isolates were divided into 9 vegetative compatibility groups. In the combinations for the formation of anastomosis, 31 compatible combinations and 24 incompatible combinations were observed. It was concluded that the methodology used to select nit mutants in C. lindemuthianum made it possible to determine the vegetative compatibility groups and that such a technique was adequate to prove genetic variability.

Kinetic analysis of batch ethanol acetylation in isothermal non-stationary multiphase systems by lyophilized mycelium of Aspergillus oryzae

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.

New hosts of Myrothecium spp. in Brazil and a preliminary in vitro assay of fungicides

Myrothecium roridum and M. verrucaria are two plant pathogenic species causing foliar spots in a large number of cultivated plants. This paper aims to study the causal agents of foliar spots in vegetable crops (sweet pepper, tomato and cucumber), ornamental plants (Spathiphyllum wallisii, Solidago canadensis, Anthurium andreanum, Dieffenbachia amoena) and a solanaceous weed plant (Nicandra physaloides). Most of the isolates were identified as M. roridum; only the isolate 'Myr-02' from S. canadensis was identified as M. verrucaria. All the isolates were pathogenic to their original plant hosts and also to some other plants. Some fungicides were tested in vitro against an isolate of M. roridum and the mycelial growth recorded after seven days. Fungicides with quartenary ammonium, tebuconazole and copper were highly effective in inhibiting the mycelial growth of M. roridum. This paper confirms the first record of M. roridum causing leaf spots in sweet pepper, tomato, Spathiphyllum, Anthurium, Dieffenbachia and N. physaloides in Brazil. We also report M. roridum as causal agent of cucumber fruit rot and M. verrucaria as a pathogen of tango plants.

Comparison of methodologies for conidia production by Alternaria alternata from citrus / Comparação de metodologias para produção de conídios por Alternaria alternata do citros

Conidia production is a problem in the study of Alternaria alternata from citrus. Thus, this study aimed to compare existing methodologies for conidial production of A. alternata isolated from Ponkan tangerine (2 isolates), Cravo lemon (1 isolate), Pêra orange (2 isolates) and Murcott tangor (1 isolate). The methodologies used were conidia production with 12 and 24 hours under white fluorescent light, evaluation with 24 and 48 hours after applying fungal mycelium stress technique, cold stress followed by injury of mycelium and evaluation with 24 hours, using healthy vegetable tissue and the use of black fluorescent near ultraviolet (NUV) lamp. Satisfactory result was obtained with A. alternata isolate from Murcott tangor, with the production of 2.8 x 10(5) conidia mL-1, when fungal mycelium was stressed (Petri dish with 66.66 percent of fungi growth) and subsequently 24 h of growth. The use of white light (24 h) and black fluorescent NUV lamp also induced expressive conidia production by one isolate of Ponkan tangerine, which produced 17.2 x 10(5) and 10.1 x 10(5) conidia mL-1 and another of Murcott tangor, which produced 13.9 x 10(5) and 10.1 x 10(5) conidia mL-1, respectively. The remaining methodologies analyzed in this study were not able to induce conidia production in satisfactory quantity. The use of both mycelium stress technique and white light (24 h) and black fluorescent NUV lamp allowed the production of enough quantities of conidia to be used in vitro (detection of fungitoxic substances)and in vivo (pathogenicity test)assays, respectively.

A produção de conídios consiste em problema no estudo de Alternaria alternata do citros. Assim, este estudo objetivou comparar metodologias existentes para a produção de conídios de A. alternata por dois isolados de tangerina Ponkan, um de limão Cravo, dois de laranja Pêra e um de tangor Murcott. As metodologias empregadas foram a produção de conídios com 12 e 24 horas sob luz branca, avaliação com 24 e 48 horas após estressamento do micélio do fungo, choque térmico com imediato estressamento do micélio e avaliação com 24 horas, produção de conídios pelo emprego de tecido vegetal sadio e o emprego de luz negra ultravioleta. Produção satisfatória de conídios foi obtida com o isolado de A. alternata de tangor Murcott, a qual foi de 2,8 x 10(5) conídios mL-1, mediante emprego da técnica de estressamento da colônia e cultivo do fungo por 24 horas. Os empregos de luz branca (24 h) e negra ultravioleta promoveram expressiva produção de conídios por um isolado de tangerina Ponkan, a qual foi de 17,2 x 10(5) e 10,1 x 10(5) conidios mL-1 e por outro de tangor Murcott, a qual foi de 13,9 x 10(5) e 10,1 x 10(5) conídios mL-1, respectivamente. As outras metodologias analisadas neste estudo não foram capazes de induzir a produção de conídios em quantidade satisfatória. Com o emprego das técnicas de estressamento do micélio e a utilização de luz branca (24 h) e negra ultravioleta, tornou-se possível obter quantidades de conídios suficientes para serem utilizadas em testes in vitro (detecção de substâncias fungitóxicas)e in vivo (testes de patogenicidade), respectivamente.

Fourier transform infrared microscopy and imaging: detection of fungi in wood.

FTIR microscopy was used to detect and discriminate the two wood decaying fungi Trametes versicolor and Schizophyllum commune in experimentally infected beech wood blocks. The distribution of fungal mycelium in wood was locally resolved and semiquantitatively recorded using FTIR microscopy combined with a focal plane array detector and image analysis. Cluster analysis revealed major differences between FTIR spectra recorded from wood fibers and empty vessel lumina and spectra from mycelium of both fungal species, irrespective of whether the fungi were grown on the surface of wood or inside vessel lumina. Species-specific clustering of spectra of fungal mycelium grown on the wood surface and inside vessel lumina demonstrated the potential of FTIR microscopy to discriminate among fungal species decaying wood.

Biochemistry of ginseng root tissues affected by rusty root symptoms.

Ginseng rusty root, a disorder of unknown cause (s), in which reddish-brown to orange-brown areas develop on the surface of field-grown roots, was studied at the cellular and biochemical levels. Using light microscopy, the affected areas were shown to comprise of the epidermis and underlying 6-8 cell layers of the cortical tissues. Rusty root areas ranged from small clusters of 3-4 cells to larger expanding areas of >80 cells. These cells appeared golden-brown and stained a bluish-green with Toluidine Blue indicating the presence of phenolic compounds. Energy-dispersive X-ray spectroscopy and atomic emission spectrometry of affected epidermal cells revealed a significant accumulation of Fe, Al, Si, Mg and other cations when compared to adjoining healthy cells. The concentrations of the six most common ginsenosides found in ginseng roots (Rg(1), Re, Rb(1), Rc, Rb(2), and Rd) were reduced by 40-50% in rusty root-affected epidermal and cortical tissues when compared to adjacent healthy tissues. Total phenolic compounds were increased by up to threefold in affected tissues and HPLC analysis revealed significantly higher levels of quercetin, cinnamic acid, vanillic acid, p-coumaric acid, benzoic acid, chlorogenic acid and catechin. In vitro phenolic-metal binding assays confirmed that phenolic compounds were able to sequester positively-charged metal ions, in particular Fe, to form a phenolic-metal ion complex. In ginseng callus cultures, accumulation of phenolic compounds was increased threefold within 12 h of treatment with chitosan (1%), and to a lesser extent by wounding. Specific defense enzymes, namely phenylalanine ammonia-lyase (PAL, E.C. 4. 3. 1. 5.), polyphenoloxidase (PPO, E.C. 1. 10. 3. 1.) and peroxidase (POD, E.C. 1. 11. 1. 7.), were also significantly enhanced in treated callus tissues and in rusty root tissues. On field-grown ginseng roots, application of chitosan induced symptoms similar to rusty root, whereas wounding and ethylene treatments did not. Based on these results, rusty root symptoms on ginseng are proposed to result from an induction of host defense responses, especially phenolic production, in epidermal and underlying cortical cells. This induction is likely due to attempted invasion by as-yet uncharacterized chitin-containing soil fungi, which were observed in many of the affected cells. Subsequent oxidation of phenolic compounds and sequestration of metal ions, in particular Fe, appear to be largely responsible for the symptoms observed.

Mycelial extract of Cordyceps ophioglossoides prevents neuronal cell death and ameliorates beta-amyloid peptide-induced memory deficits in rats.

Ameliorating effects of methanol extracts of Basidiomycetes against in vitro and in vivo model of Alzheimer's disease were investigated. The extracts of Cordyceps ophioglossoides and Hypocrea citrina var. citrina prevented the beta-amyloid((25-35)) (Abeta((25-35)))-induced cell death in SK-N-SH neuronal cells. However, in rat model of Alzheimer's disease, 30-d intraperitoneal administration with only the extract of Cordyceps ophioglossoides significantly prevented spatial memory loss by intracranial injection of Abeta((25-35)), which was assessed in water maze task. Interestingly, the scavenging activity of the reactive oxygen species (ROS) generated in Abeta((25-35))-treated cells was also found in the extract of Cordyceps ophioglossoides, but not in the extract of Hypocrea citrina var. citrina. These results suggest that the extract of Cordyceps ophioglossoides may protect the Abeta-induced neuronal cell death and memory loss through free radical scavenging activity. These results further suggest that Cordyceps ophioglossoides mycelium may be valuable for the protection from Alzheimer's dementia.

Supplementation of sugarcane bagasse with rice bran and sugarcane molasses for shiitake (Lentinula edodes) spawn production

The purpose of this study was to assess the myceliation rate, mycelial vigor and "estimated biomass" of Lentinula edodes (Berk.) Pegler, grown on a sugarcane bagasse substrate enriched with rice bran and sugarcane molasses for spawn production. The proportions of rice bran used were 0, 10, 15, 20, 25, 30 and 40 percent (dry weight/dry weight of bagasse) and the sugarcane molasses concentrations tested were 0, 10, 20, 30, 40, 50 and 60 g/kg (dry weight/dry weight of bagasse plus rice bran). The myceliation rate was decreased by the addition of the higher quantities of rice bran. The 25 and 30 percent rice bran proportions induced the highest stimulation of mycelial vigor. The addition of sugarcane molasses did not change myceliation rate or mycelial vigor. The "estimated biomass" values were similar when intermediate rice bran proportions were used and for all sugarcane molasses concentrations. Based on response surface obtained for the "estimated biomass" data, higher values were obtained with substrates containing 20 to 25 percent rice bran combined with 10 to 30 g sugarcane molasses, although the latter supplement was not considered to stimulate L. edodes growth.

In situ hybridization for the identification of filamentous fungi in tissue section.

Identification of fungi in tissue sections can be difficult because of limited biopsy tissue with only a few organisms present, or mycelial elements may be the only forms present, rendering common organism types indistinguishable from one another. In situ hybridization may assist in the rapid and accurate identification of such fungi. In this study, DNA probes were directed against the 5S or 18S ribosomal RNA sequences of three groups of fungi with a high degree of specificity for each. Two of the three, Aspergillus and Zygomycetes species, are usually seen in tissue purely in their hyphal forms. The third, Candida species is seen less commonly as predominantly mycelial elements. Probes were tested on 61 formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these organisms (Candida species, n = 21; Aspergillus species, n = 27; Zygomycetes, n = 13). Accuracy of both in situ hybridization (ISH) and morphology, based on the examination of Grocott methanamine silver (GMS)- and periodic acid-Schiff (PAS)-stained slides, was compared with culture. The results showed that morphologic examination (GMS and PAS) showed a slightly greater sensitivity in detecting the presence of fungi (98%) compared with in situ hybridization (95%). DNA probes, however, were more accurate in correctly identifying those organisms present. Although ISH specific probes showed 97% positive predictive value (PPV), examination of GMS-and PAS-stained slides had an 86% PPV when compared with culture-based identification methods. These results show that ISH, directed against ribosomal RNA, provides a rapid and accurate technique for the identification of mycelial fungal organisms in histologic tissue sections. Its primary use lies in the ability to accurately distinguish between organisms that have similar or identical morphologic features by light microscopy.