Determination of endoglucanase activity of paper decaying fungi from an old library at the ancient Medina of Fez

Paper from an ancient library of the cultural city of Fez (Morocco) is exposed to rapid deterioration by variety of microorganisms, especially cellulolytic fungi. For this, ten isolates fungi previously isolated from historical biodeteriorated paper were screened for their ability to produce endoglucanase (CMCase), amylase, polygalacturonase and ligninase enzymes. The CMCase activity of cellulolytic strains was essayed in liquid media at 25°C for 10 days. Influence of temperature and pH were assessed for the production of CMCase by all the fungus isolated from decaying paper. The research findings from the present study demonstrate that all the tested isolates had cellulase, amylase, pectinase and ligninase activities. It was found that Mucor racemosus PF15, Aspergillus niger, and Aspergillus oryzae exhibited the maximum endoglucanase activity in liquid medium (0.256, 0.236, and 0.216 UI/mL in descending order) for six days. Temperature profiling revealed optimum endoglucanase activity at 25 and 30°C. Maximum activity was observed at pH 5 and pH 6.     

Identification, phylogenetic analysis and characterization of obligate halophilic fungi isolated from a man-made solar saltern in Phetchaburi province, Thailand

A total of 17 species from 43 isolates were obtained through serial dilutions of soil samples isolated from one of the man-made solar salterns located in Ban Laem district of Phetchaburi province, Thailand. Soil analysis of the sample revealed high salinity and moisture content, slight alkalinity and low amounts of nitrogen, total organic carbon and organic matter in the habitat. Morphological analysis was performed on all isolates, and molecular identification and phylogenetic analysis were carried out only on the halophilic fungi isolated. Six halophilic fungi, belonging to four species, were identified among the isolates, including five strains of Aspergillus genus [Aspergillus flavus, A. gracilis, A. penicillioides (2 strains) and A. restrictus]. One species was found to be a yeast, namely, Sterigmatomyces halophilus, which was the most frequent isolate found among the halophilic fungi. All other isolates were halotolerant fungi. Characterization of the halophilic fungal isolates showed that they were best adapted to conditions of 10–15 % NaCl (w/v), slight alkalinity (pH 7.0–7.5) and a temperature range of 30–35 °C.     

Bioaerosol assessment in the library of Istanbul University and fungal flora associated with paper deterioration

Health problems in people who are in indoor environments with poor ventilation have resulted in an increase in the number of studies regarding air quality. Microorganisms and inferior indoor air-climatic conditions not only affect human health but also cause decay of invaluable materials present in libraries. Therefore, this study aimed to assess the culturable bioaerosol composition and concentration in the library of Istanbul University. The culturable fungal flora, a biodeterioration agent, of the damaged archival materials was also examined. The air was sampled for a year, as were the surfaces of 207 biologically damaged books. The fungal colonies’ range was between 235 and 1850 CFU/m³, and the bacterial colonies range between 270 and 1920 CFU/m³. This is the first volumetric record in Turkey. Although the microbial contamination was not very high, molds such as Aspergillus versicolor, Cladosporium sphaerospermum, Penicillium chrysogenum, Alternaria alternata, Chaetomium globosum, Aspergillus flavus, and Aspergillus ochraceus might cause harm to human beings or to books as biodeterioration agents. The highest amount of fungus was determined in Archive 3, which contained damaged books. The fungal species isolated from the air and books were essentially the same. Therefore, it is important to determine not only the numeric values but also the microbiological composition of fungal colonies because the variety of fungal species is indicative of a deterioration process in effect over a long period. The deterioration of books must be remedied.     

High diversity of root-associated fungi isolated from three epiphytic orchids in southern Ecuador

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum, Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens. Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens. No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi.     

Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra

This multidisciplinary research combines knowledge in molecular biology with fungal morphology, aiming at the identification of infecting fungi from historical documents on the Archive of the University of Coimbra. The identification of infecting fungi on several bibliographic documents and support materials was based both on ribosomal DNA loci amplification and sequencing, and morphological identification, using macro- and microscopical traits. A high fungal diversity was found in all types of support: parchment, laid-paper and wood-pulp paper. Fourteen fungal genera were isolated, identified, and kept in culture. The most frequent were Cladosporium, Penicillium and Aspergillus, and other less frequent genera, such as Alternaria, Botrytis, Chaetomium, Chromelosporium, Epicoccum, Phlebiopsys and Toxicocladosporium were also present. Within these genera, 20 different species were identified, from which 15 were found only in a single support type. Cladosporium cladosporioides and Penicillium chrysogenum were the only species present in all support types.     

Bird feather fungi from Svalbard Arctic

Despite feather fungi being an important component of the Arctic fungal flora, their ecological role and diversity are not fully known. In the current study, fungal cultures were isolated from feathers (barnacle goose, common eider, and glaucous gull) collected in the Ny-Ålesund region, Svalbard. Isolates were identified by ITS region sequences, which include the ITS1, ITS2, and 5.8S rRNA. The result showed culturable yeast and filamentous fungi belonging to three classes: Ascomycota (Pyrenochaetopsis pratorum, Cladosporium herbarum, Thelebolus microsporus, Aspergillus versicolor, Penicillium commune, and Venturia sp.), Basidiomycota (Mrakia blollopis and Rhodotorula mucilaginosa), and Zygomycota (Mucor flavus). Most of the fungal isolates appeared to be cold-tolerant, and about 60 % of the isolates showed keratinase activity. The reasonably low fungal diversity colonizing feathers indicates that the birds of Svalbard are casual carriers of fungi which may result in a negligible impact on their health. To the best of our knowledge, this is the first record of fungal communities present on the feathers of birds in the high Arctic.     

Diverse Deep-Sea Fungi from the South China Sea and Their Antimicrobial Activity

We investigated the diversity of fungal communities in nine different deep-sea sediment samples of the South China Sea by culture-dependent methods followed by analysis of fungal internal transcribed spacer (ITS) sequences. Although 14 out of 27 identified species were reported in a previous study, 13 species were isolated from sediments of deep-sea environments for the first report. Moreover, these ITS sequences of six isolates shared 84–92 % similarity with their closest matches in GenBank, which suggested that they might be novel phylotypes of genera Ajellomyces, Podosordaria, Torula, and Xylaria. The antimicrobial activities of these fungal isolates were explored using a double-layer technique. A relatively high proportion (56 %) of fungal isolates exhibited antimicrobial activity against at least one pathogenic bacterium or fungus among four marine pathogenic microbes (Micrococcus luteus, Pseudoaltermonas piscida, Aspergerillus versicolor, and A. sydowii). Out of these antimicrobial fungi, the genera Arthrinium, Aspergillus, and Penicillium exhibited antibacterial and antifungal activities, while genus Aureobasidium displayed only antibacterial activity, and genera Acremonium, Cladosporium, Geomyces, and Phaeosphaeriopsis displayed only antifungal activity. To our knowledge, this is the first report to investigate the diversity and antimicrobial activity of culturable deep-sea-derived fungi in the South China Sea. These results suggest that diverse deep-sea fungi from the South China Sea are a potential source for antibiotics’ discovery and further increase the pool of fungi available for natural bioactive product screening.     

The effects of book disinfection to the airborne microbiological community in a library environment

The management of fungal contaminants inside libraries and archives has become a big challenge for librarians, restorers and scientists. Several disinfection treatments have been developed in recent years, using both chemical and physical approaches on book collections and indoor environments. However, there is a lack of knowledge about the temporal efficiency of these cleanings, especially in relation with the preservation environments. The aim of this study was to determine the long-term effect of a chemical disinfection that interested a previous-contaminated book collection inside a University library. The monitoring after 6 months and 1 year from the cleaning confirmed any fungal growth on the disinfected books and the reduction of 90% of the airborne fungal load, highlighting anyway the presence of high fungal diversity. Sixty-eight different airborne fungal entities were isolated, in particular Aspergillus vitricola, Bulleromyces albus, Cladosporium cladosporioides, Cladosporium pseudocladosporioides, Cladosporium sphaerospermum, Penicillium brevicompactum, Rodothorula mugillaginosa and Sporodiobolus pararoseus. Several fungal species were sampled from the disinfected books, in particular Aspergillus penicillioides and Penicillium chrysogenum. The presence of these fungi both as airborne and as settled particles highlights the importance to maintain clean the preservation environments in order to prevent further microbial contaminations.     

Mycoflora and mycotoxin contamination of Roundup Ready soybean harvested in the Pampean Region, Argentina

A total of 89 freshly harvested soybean seed samples (Roundup Ready [transgenic] soybean cultivars) from the 2010/2011 crop season were collected from five locations in the Northern Pampean Region II, Argentina. These samples were analyzed for internal mycoflora, toxin production of isolated fungi, and for a range of mycotoxins. Mycotoxin analysis of aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs) and ochratoxin A (OTA) was done by HPLC-FLD (high performance liquid chromatography with postcolumn fluorescence derivatization), alternariol and alternariol monomethyl ether with HPLC-UV (HPLC with UV detection), trichothecenes (deoxynivalenol, nivalenol, T-2 toxin, HT-2 toxin, fusarenon X, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were analyzed by GC-ECD (gas chromatography with electron capture detector). Fungal colonization was more frequently found for samples from América, Saladillo and Trenque Lauquen than for samples from General Villegas and Trenel; a total of 1,401 fungal isolates were obtained from the soybean seeds. The most commonly identified fungal genera were Alternaria, Sclerotinia, Chaetomium, Cladosporium, Aspergillus, Penicillium, Phomopsis and Fusarium. Alternaria alternata, A.tenuissima, Aspergillus flavus, Penicillium citrinum, Fusarium verticillioides and F.semitectum were the predominant toxigenic fungal species. Mycotoxin production was confirmed for several isolates of toxigenic species, including Aspergillus flavus, A. parasiticus, Alternaria alternata, A.tenuissima, Fusarium graminearum, F semitectum and F. verticillioides. In particular, the percentage of mycotoxigenic Alternaria alternata (100 %), A.tenuissima (95 %) and aflatoxigenic strains of A. flavus (57 %) were remarkably high. Although none of the mycotoxins, AFs, ZEA, FBs, trichothecenes and OTA, were directly detected in samples of soybean seeds, the frequent presence of toxigenic fungal species indicates the risk of multiple mycotoxin contamination.     

Fungal endophytes from cactus Cereus jamacaru in Brazilian tropical dry forest: a first study

Endophytic fungi live within the healthy tissues of plants and can promote host species tolerance to different environmental stresses. However, most studies have been of plants in humid environments, and there are few reports of the benefits of such associations for plants of extreme environments. Our aims were to identify endophytic fungi using morphological taxonomy, to explore richness and estimate species frequency in a cactus, C. jamacaru, in Brazilian tropical dry forest (Caatinga). We thus identified 59 taxa, corresponding to 69.7 % of the total number of isolates; the other 30.3 % were sterile mycelia. Cladosporium cladosporioides and Fusarium oxysporum were the species most commonly isolated, followed by Acremonium implicatum, Aureobasidium pullulans, Trichoderma viride, Chrysonilia sitophila, and Aspergillus flavus. Forty-seven species are recorded for the first time as endophytic fungi of cacti, and 18 others as endophytes for Brazil; this suggests that C. jamacaru harbors a highly diverse fungal community as measured by a diversity index. However, species accumulation curves suggest that our study still underestimates endophyte diversity because it does not provide an exhaustive sample. To our knowledge, this is the first report of endophytic fungi from C. jamacaru in tropical dry forests.     

Nimesulide inhibits pathogenic fungi: PGE2-dependent mechanisms

Certain non-steroidal anti-inflammatory drugs can inhibit fungal growth, fungal prostaglandin E2 production, and enzyme activation. This study aims to investigate the antifungal effect of nimesulide against pathogenic filamentous fungi and yeast. The experiments detailed below were also designed to investigate whether the action is dependent on E2 fungal prostaglandins. Our data showed that nimesulide exhibited potent antifungal activity, mainly against Trichophyton mentagrophytes (ATCC 9533) and Cryptococcus neoformans with MIC values of 2 and 62 μg/mL, respectively. This drug was also able to inhibit the growth of clinic isolates of filamentous fungi, such as Aspergillus fumigatus, and dermatophytes, such as T. rubrum, T. mentagrophytes, Epidermophyton floccosum, Microsporum canis, and M. gypseum, with MIC values ranging from 112 to 770 μg/mL. Our data also showed that the inhibition of fungal growth by nimesulide was mediated by a mechanism dependent on PGE2, which led to the inhibition of essential fungal enzymes. Thus, we concluded that nimesulide exerts a fungicidal effect against pathogenic filamentous fungi and yeast, involving the inhibition of fungal prostaglandins and fungal enzymes important to the fungal growth and colonization.     

Molecular identification of isolated fungi from stored apples in Riyadh,Saudi Arabia

Fungi causes most plant disease. When fruits are stored at suboptimal conditions, fungi grows, and some produce mycotoxin which can be dangerous for human consumption. Studies have shown that the Penicillium and Monilinia species commonly cause spoilage of fruits, especially apples. Several other genera and species were reported to grow to spoil fruits. This study was conducted to isolate and identify fruit spoilage by fungi on apples collected in Riyadh, Saudi Arabia and conduct a molecular identification of the fungal isolates. Thus, we collected 30 samples of red delicious and Granny Smith apples with obvious spoilage from different supermarkets between February and March of 2012 in Riyadh, Saudi Arabia. Each apple was placed in a sterile plastic bag in room temperature (25–30 °C) for six days or until fungal growth was evident all over the sample. Growth of fungal colonies on PDA was counted and sent for molecular confirmation by PCR. Six fruit spoilage fungi were isolated, including Penicillium chrysogenum, Penicillium adametzii, Penicillium chrysogenum, Penicillium steckii, Penicillium chrysogenum, and Aspergillus oryzae. P. chrysogenum was the most frequent isolate which was seen in 14 of a total of 34 isolates (41.2%), followed by P. adametzii and A. oryzae with seven isolates each (20.6%) and the least was P. steckii with six isolates (17.6%). Penicillium species comprised 27 of the total 34 (79.4%) isolates. Sequence analysis of the ITS regions of the nuclear encoded rDNA showed significant alignments for P. chrysogenum, P. adametzii and A. oryzae. Most of these fungal isolates are useful and are rarely pathogenic; however they can still produce severe illness in immune-compromised individuals, and sometimes otherwise healthy people may also become infected. It is therefore necessary to evaluate the possible production of mycotoxins by these fungi to determine a potential danger and to establish its epidemiology in order to develop adequate methods of control.     

Monitoring of the bacterial and fungal biodiversity and dynamics during Massa Medicata Fermentata fermentation

The microbial community dynamics play an important role during Massa Medicata Fermentata (MMF) fermentation. In this study, bacterial and fungal communities were investigated based on the culture-dependent method and polymerase chain reaction-denaturing gradient gel electrophoresis analysis. Meanwhile the dynamic changes of digestive enzyme activities were also examined. Plating results showed that MMF fermentation comprised two stages: pre-fermentation stage (0–4 days) was dominated by bacterial community and post-fermentation stage (5–9 days) was dominated by fungal community. The amount of bacteria reached the highest copy number 1.2?×?10¹⁰ CFU/g at day 2, but the fungi counts reached 6.3?×?10⁵ CFU/g at day 9. A total of 170 isolates were closely related to genera Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Mucor, Saccharomyces, Rhodotorula, and Amylomyces. DGGE analysis showed a clear reduction of bacterial and fungal diversity during fermentation, and the dominant microbes belonged to genera Enterobacter, Pediococcus, Pseudomonas, Mucor, and Saccharomyces. Digestive enzyme assay showed filter paper activity; the activities of amylase, carboxymethyl cellulase, and lipase reached a peak at day 4; and the protease activity constantly increased until the end of the fermentation. In this study, we carried out a detailed and comprehensive analysis of microbial communities as well as four digestive enzymes' activities during MMF fermentation process. The monitoring of bacterial and fungal biodiversity and dynamics during MMF fermentation has significant potential for controlling the fermentation process.     

A preliminary survey on the occurrence of mycotoxigenic fungi and mycotoxins contaminating red rice at consumer level in Selangor, Malaysia

Red rice is a fermented product of Monascus spp. It is widely consumed by Malaysian Chinese who believe in its pharmacological properties. The traditional method of red rice preparation disregards safety regulation and renders red rice susceptible to fungal infestation and mycotoxin contamination. A preliminary study was undertaken aiming to determine the occurrence of mycotoxigenic fungi and mycotoxins contamination on red rice at consumer level in Selangor, Malaysia. Fifty red rice samples were obtained and subjected to fungal isolation, enumeration, and identification. Citrinin, aflatoxin, and ochratoxin-A were quantitated by ELISA based on the presence of predominant causal fungi. Fungal loads of 1.4?×?10⁴ to 2.1?×?10⁶?CFU/g exceeded Malaysian limits. Monascus spp. as starter fungi were present in 50 samples (100 %), followed by Penicillium chrysogenum (62 %), Aspergillus niger (54 %), and Aspergillus flavus (44 %). Citrinin was present in 100 % samples (0.23–20.65 mg/kg), aflatoxin in 92 % samples (0.61–77.33 μg/kg) and Ochratoxin-A in 100 % samples (0.23–2.48 μg/kg); 100 % citrinin and 76.09 % aflatoxin exceeded Malaysian limits. The presence of mycotoxigenic fungi served as an indicator of mycotoxins contamination and might imply improper production, handling, transportation, and storage of red rice. Further confirmatory analysis (e.g., HPLC) is required to verify the mycotoxins level in red rice samples and to validate the safety status of red rice.     

Diversity of cultivable fungi associated with Antarctic marine sponges and screening for their antimicrobial, antitumoral and antioxidant potential

The diversity of sponge-associated fungi has been poorly investigated in remote geographical areas like Antarctica. In this study, 101 phenotypically different fungal isolates were obtained from 11 sponge samples collected in King George Island, Antarctica. The analysis of ITS sequences revealed that they belong to the phylum Ascomycota. Sixty-five isolates belong to the genera Geomyces, Penicillium, Epicoccum, Pseudeurotium, Thelebolus, Cladosporium, Aspergillus, Aureobasidium, Phoma, and Trichocladium but 36 isolates could not be identified at genus level. In order to estimate the potential of these isolates as producers of interesting bioactivities, antimicrobial, antitumoral and antioxidant activities of fungal culture extracts were assayed. Around 51 % of the extracts, mainly from the genus Geomyces and non identified relatives, showed antimicrobial activity against some of the bacteria tested. On the other hand, around 42 % of the extracts showed potent antitumoral activity, Geomyces sp. having the best performance. Finally, the potential of the isolated fungi as producers of antioxidant activity seems to be moderate. Our results suggest that fungi associated with Antarctic sponges, particularly Geomyces, would be valuable sources of antimicrobial and antitumoral compounds. To our knowledge, this is the first report describing the biodiversity and the metabolic potential of fungi associated with Antarctic marine sponges.     

A comparative study of siderophore production by fungi from marine and terrestrial habitats

Siderophore producing potential of 20 fungal isolates (same 10 species from each marine and terrestrial habitat) were examined and compared. Except marine Aspergillus flavus, all isolates produced siderophores as evidenced by positive reaction in FeCl₃ test, CAS assay and CAS agar plate test. The results indicated widespread occurrence of siderophores in both the habitats. Examination of the chemical nature of siderophores revealed that mucoraceous fungi produced carboxylate, while others produced hydroxamate siderophores. Thus, the nature of siderophore was found to be independent of habitat. Among all the isolates, Cunninghamella elegans (marine form) was maximum siderophore producer (1987.5 μg/ml) followed by terrestrial form of C. elegans (1248.75 μg/ml). There was no marked variation in siderophore concentration of Penicillium funiculosum strains. Comparison of quantification of siderophore production between marine and terrestrial revealed that four terrestrial isolates (Aspergillus niger, Aspergillus ochraceous, Penicillium chrysogenum, Penicillium citrinum) were ahead in siderophore production, while, the other four marine isolates (Aspergillus versicolor, C. elegans, Rhizopus sp., Syncephalastrum racemosum) were found to be more potent siderophore producers, indicating that they were equally competent.     

Preliminary Study of the Fungal Ecology at the Haematology and Medical-Oncology Ward in Bamako,Mali

Data on fungal epidemiology in sub-Saharan African countries are scarce. This exploratory study aimed to characterize the fungal flora at the Onco-Haematology ward of the National Teaching Hospital of Point G in Bamako, Mali. A cross-sectional survey was conducted in the dry and in the rainy seasons. Nasal swab and sputum samples were collected from the hospitalized patients while airborne fungal spores were collected using electrostatic dust-fall collectors. Fungi were identified by their morphological characteristics and MALDI-TOF mass spectrometry. Candida albicans was the most frequent yeast species colonizing patients; Aspergillus species were isolated in 86 % of the patients and were the main airborne environmental contaminants. Overall, airborne fungal contamination rates increased from 33.8 % in the dry to 66.2 % in the rainy season (p < 0.001). The most frequent Aspergillus species were Aspergillus niger (36.6 %) and Aspergillus flavus (32.92 %). In contrast, Aspergillus fumigatus (5.43 %) was relatively rare. This high level of fungal exposure raises concern regarding the management of at-risk patients in this Onco-Haematology ward and stresses the need for strengthening the mycological diagnostic capacities to accompany the implementation of adapted fungal infection prevention and management policies.     

Isolation and characterization of endophytic fungi from Camptotheca acuminata

In this study, a total of 161 endophytic fungal isolates from Camptotheca acuminata were obtained and classified to 16 taxa according to morphological and molecular analysis. These taxa were composed of 2 frequent genera (Botryosphaeria and Fusarium) and 14 infrequent groups such as Xylaria, Diaporthe, Rhizopus, Epicoccum, and Preussia, demonstrating that fungal endophytes in C. acuminata were highly abundant and diverse. Antimicrobial activity screening using filter-paper diffusion method showed that 47.6 % of the tested isolates had antimicrobial activity against at least one of the test microorganisms. Screening of fungal endophyte-derived camptothecin analogues by TLC and LC–MS/MS³ demonstrated that a strain Botryosphaeria dothidea, X-4 could produce 9-methoxycamptothecin (9-MCPT) when cultured in Sabouraud’s dextrose broth for 12 days under shake flask and bench-scale fermention conditions. This work showed that the fungal endophytes from C. acuminata could be an alternative source for the production of 9-MCPT and other natural antimicrobial compounds.     

Cryopreservation of ectomycorrhizal fungi has minor effects on root colonization of Pinus sylvestris plantlets and their subsequent nutrient uptake capacity

The use of ectomycorrhizal (ECM) fungi for afforestation, bioremediation, and timber production requires their maintenance over long periods under conditions that preserve their genetic, phenotypic, and physiological stability. Cryopreservation is nowadays considered as the most suitable method to maintain the phenotypic and genetic stability of a large number of filamentous fungi including the ECM fungi. Here, we compared the ability of eight ECM fungal isolates to colonize Pinus sylvestris roots and to transport inorganic phosphate (Pi) and NH₄ ⁺ from the substrate to the plant after cryopreservation for 6 months at ?130 °C or after storage at 4 °C. Overall, the mode of preservation had no significant effect on the colonization rates of P. sylvestris, the concentrations of ergosterol in the roots and substrate, and the uptake of Pi and NH₄ ⁺. Comparing the isolates, differences were sometimes observed with one or the other method of preservation. Suillus bovinus exhibited a reduced ability to form mycorrhizas and to take up Pi following cryopreservation, while one Suillus luteus isolate exhibited a decreased ability to take up NH₄ ⁺. Conversely, Hebeloma crustuliniforme, Laccaria bicolor, Paxillus involutus, and Pisolithus tinctorius exhibited a reduced ability to form mycorrhizas after storage at 4 °C, although this did not result in a reduced uptake of Pi and NH₄ ⁺. Cryopreservation appeared as a reliable method to maintain important phenotypic characteristics (i.e., root colonization and nutrient acquisition) of most of the ECM fungal isolates studied. For 50 % of the ECM fungal isolates, the colonization rate was even higher with the cultures cryopreserved at ?130 °C as compared to those stored at 4 °C.     

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development.