One-vector CRISPR/Cas9 genome engineering of the industrial fungus Ashbya gossypii

The filamentous fungus Ashbya gossypii is currently used for the industrial production of vitamin B2. Furthermore, the ability of A. gossypii to grow using low-cost substrates together with the inexpensive downstream processing makes this fungus an attractive biotechnological chassis. Indeed, the production in A. gossypii of other high-added value compounds such as folic acid, nucleosides and biolipids has been described. Hence, the development of new methods to expand the molecular toolkit for A. gossypii genomic manipulation constitutes an important issue for the biotechnology of this fungus. In this work, we present a one-vector CRISPR/Cas9 system for genomic engineering of A. gossypii. We demonstrate the efficiency of the system as a marker-less approach for nucleotide deletions and substitutions both with visible and invisible phenotypes. Particularly, the system has been validated for three types of genomic editions: gene inactivation, the genomic erasure of loxP scars and the introduction of point mutations. We anticipate that the use of the CRISPR/Cas9 system for A. gossypii will largely contribute to facilitate the genomic manipulations of this industrial fungus in a marker-less manner.     

Growth substrates control the ability of Fusarium oxysporum to solubilize low-rank coal

The ability of the fungus Fusarium oxysporum to solubilize lignite was found to depend on the presence of a specific carbon source. When grown on glucose or another carbohydrate, the fungus is unable to solubilize coal but it produces the red dye bikaverin. In the coal-solubilizing state, which can be induced by cultivation in the presence of glutamate or gluconate, the fungus does not produce bikaverin. The presence or absence of the pigment can therefore be taken as an indicator of the ability of the fungus to solubilize coal. Addition of extracted and purified bikaverin to F. oxysporum growing on glutamate or gluconate inhibits coal solubilization. Hence, F. oxysporum offers a suitable system for investigating the mechanism of microbial coal degradation by comparing the two growth-substrate-controlled physiological states.     

Towards the biocontrol of bindweeds with a mycoherbicide

Within the framework of the European COST Action 816, afive-year collaboration between scientists from five Europeancountries has made an important contribution to biologicalcontrol of field and hedge bindweeds (Convolvulus arvensis andCalystegia sepium, respectively). A fungus Stagonosporaconvolvuli strain LA39, able to infect both field and hedgebindweed, was found in the UK and its biocontrol efficacyimproved by optimising mass production, formulation and storagetechniques. This fungus controlled bindweeds in both a cemeteryand in maize crops. Its use fits best in an integrated pestmanagement system where a green cover controls most of the weedsexcept the bindweeds. DNA marker analyses indicate that thefungus reproduces sexually, which could be used to furtherimprove this mycoherbicide. In addition, the insect Melanagromyzaalbocilia, which itself exhibits biocontrol potential againstbindweeds, may be used in combination with LA39 to improve theability of the fungus to penetrate the stem of bindweeds.Overall, the results suggest that S. convolvuli LA39 haspromising potential as a bioherbicide for control of field andhedge bindweed.     

Extracellular ligninase of Phanerochaete chrysosporium Burdsall has no role in the degradation of DDT

Summary The white rot fungus Phanerochaete chrysosporium Burdsall degraded DDT [1,1-bis(4-chlorophenyl)-2, 2,2-trichloroethane] in submerged agitated cultures. The ability of the fungus to metabolize this persistent environmental pollutant is not dependent on the formation of its extracellular lignin-degrading enzyme system.Dedicated to Professor E.-E. Bruchmann on the occasion of his 65th birthday     

Fungal colonization of the rooting system of the early land plant Asteroxylon mackiei from the 407‐Myr‐old Rhynie Chert (Scotland,UK)

Associations between plants and fungi were an important and varied feature of early terrestrial ecosystems, but in most instances their biological functions remain poorly understood. We document a new species of fungus colonizing the rooting system of the early lycopod Asteroxylon mackiei, based on exceptionally well‐preserved fossils from the Rhynie Chert. We investigated historical petrographic thin sections using standard optical microscopy and confocal laser scanning microscopy. P alaeozoosporites renaultii gen. nov., sp. nov. colonized the inner cortex of the plant rooting system. The fungus had an aseptate thallus with isotomous or sympodial branching. The mycelium bore distinctive porate, globose to elongated structures that we interpret as zoosporangia and resting sporangia. Doubts remain over the precise systematic affinity of P. renaultii, which closely resembles chytrids. Whereas most of the Rhynie Chert plants developed symbiotic associations of the mycorrhizal type, it seems that this was not the case for Asteroxylon mackiei, which possessed the most evolved rooting system among the Rhynie Chert plants. We argue that the new root‐borne fungus was probably parasitic. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 201–213.     

Identification of host plants and description of sclerotia of the truffle <Emphasis Type="Italic">Mattirolomyces terfezioides</Emphasis>

The truffle, Mattirolomyces terfezioides, is a hypogeous ascomycete with uncertain host relationships. The fungus has been regularly collected on sandy soils in the Carpathian Basin. During the study of the natural host plants of the fungus, strange, amorphous, belowground hyphal aggregates incorporating soil and sand particles have been found attached to the surface of the roots. The fruitbodies of M. terfezioides develop from these hyphal aggregates. This structure, similar to that formed by morels, could be interpreted as a sclerotium. Sclerotia were found both on roots of woody and herbaceous plants. To detect the roots colonized by M. terfezioides, a species-specific polymerase chain reaction was developed. Seven natural hosts of the fungus were identified by this method. No specificity regarding taxa or life form of the plants was found. The colonization of the roots by the septate hyphae of M. terfezioides was weak, particularly compared to the colonization by arbuscular–mycorrhizal fungi. This suggests that this fungus is not the dominant fungal partner of these plants. Therefore, using M. terfezioides as the only inoculum may be inappropriate in truffle cultivation experiments. Nevertheless, further in vitro experiments are needed to develop reliable knowledge on the still ambiguous symbiotic strategy of this fungus.     

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei,the hyper-cellulolytic filamentous fungus

The filamentous fungus Trichoderma reesei is a potent cellulase producer and the best-studied cellulolytic fungus. A lot of investigations not only on glycoside hydrolases produced by T. reesei, but also on the machinery controlling gene expression of these enzyme have made this fungus a model organism for cellulolytic fungi. We have investigated the T. reesei strain including mutants developed in Japan in detail to understand the molecular mechanisms that control the cellulase gene expression, the biochemical and morphological aspects that could favor this phenotype, and have attempted to generate novel strains that may be appropriate for industrial use. Subsequently, we developed recombinant strains by combination of these insights and the heterologous-efficient saccharifing enzymes. Resulting enzyme preparations were highly effective for saccharification of various biomass. In this review, we present some of the salient findings from the recent biochemical, morphological, and molecular analyses of this remarkable cellulase hyper-producing fungus.     

Aecidium dispori is the aecial anamorph ofPuccinia albispora, sp. nov. (Uredinales)

Aecidium dispori forms spermogonium and aecium onDisporum sessile andD. smilacinum, which are distributed in East Asia. TheAecidium species is found to be an aecial anamorph of aPuccinia fungus, with its uredinial-telial stage being formed onCarex conica, C. dolichostachya subsp.multifolia, C. pisiformis subsp.alterniflora andC. rugata. Urediniospores of this fungus are large, colorless, thick-walled with 4–5 equatorial germ pores. The morphological characteristics of urediniospores and the spermogonial-aecial host do not fit to any set of circumscribing characters of previously described species. We consider the fungus to be a new species and propose a new name,Puccinia albispora, for the fungus.     

Monitoring of white-rot fungus during bioremediation of polychlorinated dioxin-contaminated fly ash

Bioremediation is a low-cost treatment alternative for the cleanup of polychlorinated-dioxin-contaminated soils and fly ash when pollution spread is wide-ranging. An interesting fungus, Ceriporia sp. MZ-340, with a high ability to degrade dioxin, was isolated from white rotten wood of a broadleaf tree from Kyushu Island in Japan. We have attempted to use the fungus for bioremediation of polychlorinated-dioxin-contaminated soil on site. However, we have to consider that this trial has the potential problem of introducing a biohazard to a natural ecosystem if this organism is naturalized. We have therefore developed a monitoring system for the introduced fungus as a part of the examination and evaluation of bioremediation in our laboratory. We have also developed a PCR-based assay to reliably detect the fungus at the bioremediation site. DNA isolated from the site was amplified by PCR using a specific primer derived from internal transcribed spacer region (ITS: ITS1, 5.8S rDNA and ITS2) sequences of Ceriporia sp. MZ-340. We successfully monitored Ceriporia sp. MZ-340 down to 100 fg/µl DNA and down to 2 mg/g mycelium. We also successfully monitored the fungus specifically at the bioremediation site. The polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofuran content was observed to decrease in response to treatment with the fungus. The species-specific PCR technique developed in the present work is useful in evaluating the possibility of on-site bioremediation using the fungus Ceriporia sp. MZ-340.     

On the Mechanisms of the Excretion and Uptake of the Alkaloid Aurantioclavine during the Growth of the Fungus Penicillium nalgiovense VKM F-229

The biphasic dynamics of the alkaloid aurantioclavine in the culture liquid of P. nalgiovense VKM F-229 is shown to be due to the diauxic growth of the fungus on two carbon sources, succinate and mannitol. In the phase of active growth on succinate, the fungus synthesizes aurantioclavine and excretes it into the medium in an energy-independent manner, as a result of which the concentration of the alkaloid in the culture liquid rises. During the phase of metabolic adaptation to the other carbon source, mannitol, the concentration of aurantioclavine in the culture liquid falls, probably due to the energy-dependent uptake of the alkaloid by fungal cells. The reversible excretion of aurantioclavine in P. nalgiovense indicates that this process is regulatory and depends on the growth parameters and the physiological state of the fungus.     

Formulation and application of the entomopathogenic fungus: Zoophthora radicans (Brefeld) Batko (Zygomycetes: Entomophthorales)

Aims: To isolate and formulate a native strain of Zoophthora radicans naturally infecting larvae of diamondback moth, Plutella xylostella, existing in South Australia and to provide evidence that formulation of the fungus is effective against P. xylostella larvae, and therefore, it could be used as a tool in pest management of this insect. Methods and Results: Dose–response bioassays using formulated and unformulated forms of the fungus strain were carried out against third instar larvae of P. xylostella. Results obtained have indicated a significant increase in the larval mortality when higher concentrations of a formulated form of the fungus strain were applied compared to the treatments with the unformulated form (85·0 vs 57·5% of larval mortality, respectively, at the top concentration of 10⁷ conidia/ml). The median lethal concentration (LC50) for a formulated form was 100 times less than that of the unformulated form when they were applied against the third instar larvae of P. xylostella. In addition, the formulation used in the present bioassays has preserved the viability of introduced fungus conidia for longer time in comparison with the unformulated conidia. Conclusions: The effective application of a formulated fungus strain against P. xylostella larvae constitutes the first step towards its use in pest management of this insect. Significance and Impact of the Study: The formulated fungus in inverted emulsion could be used as an alternative tool to insecticides in pest management of P. xylostella larvae because of the development of resistance to insecticides in the treated larvae.     

Identification of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis Makino (Triuridaceae)

The identity of a mycorrhizal fungus in the roots of achlorophyllous Sciaphila tosaensis was investigated by DNA analysis and examination of the morphology of the association. The morphological features of the mycorrhizal fungus, i. e. aseptate hyphal coils, vesicles, arbuscule-like branching, and degenerate coils were similar to those previously reported for other achlorophyllous plants. Spore-like propagules identified asa glomalean fungus were propagated from root pieces of S. tosaensis in pot culture using alfalfa as the host trap plant. A PCR product was obtained from colonized root of S. tosaensis using the taxon-specific primers, VANS1 and VAGLO. Sequence analysis of the DNA fragment showed it to be almost identical to other Glomus species. Although it has been reported many times that the morphology of mycorrhizal fungi in achlorophyllous plants is quite similar to that of arbuscular mycorrhizal fungi, this is the first report of the isolation and identification of such a fungus itself.     

Decolorization of effluent from the bagasse-based pulp mills by white-rot fungus,Schizophyllum commune

Summary The effluent from bagasse-based pulp and paper mills can be decolorized with the white-rot fungus Schizophyllum commune. The influence of pH, nutrients and aeration on the decolorizing efficiency of this fungus has been determined. It was found that it could not degrade lignin unless a more easily metabolized carbon source was made available simultaneously. The addition of carbon and nitrogen not only improved the decolorizing efficiency of the fungus, but also resulted in reduction of the biological oxygen demand (BOD) and chemical oxygen demand (COD) of effluent. A 2-day incubation period was sufficient for lignin breakdown by S. commune. The efficiency of treatment of effluent with this fungus was highest at pH 4–5 and was further improved by intermittent aeration.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Survey of Verticillium nonalfalfae (Phyllachorales) on tree-of-heaven in the southeastern USA

Ailanthus altissima (Mill.) Swingle commonly known as tree-of-heaven is an invasive tree species that has spread through much of the USA. Significant mortality of A. altissima by a highly host-specific strain of the fungus, Verticillium nonalfalfae Inderb. (formerly Verticillium albo-atrum Reinke and Bethold), has been recently documented in Pennsylvania. To detect the regional distribution of this biological control agent in the Southeast, a windshield survey was conducted in Virginia, North Carolina and South Carolina to locate V. nonalfalfae-infected A. altissima stands. The survey covered 26,500 km of interstate highways and primary and secondary roads. There were 90 potential sites located throughout the three states. Of those, six were found to be infested with the fungus, all in the mountain regions of Virginia. Inoculation tests suggest no difference in virulence among the six V. nonalfalfae isolates on A. altissima, which resulted in 100% mortality in 9–11 weeks post-inoculation. Tree ratings revealed differences in health among sites, but all sites had an increase in disease incidence from 2011 to 2012 (mean = 4.7 ± 3.8%), suggesting this fungus is dispersing. This survey provides evidence that biological control using this fungus may be possible in Virginia.     

Association of the coprophilous fungus <Emphasis Type="Italic">Pirella circinans</Emphasis> with an indigenous beetle on the sub-Antarctic Bird Island

The zygomycete fungus Pirella circinans was isolated from cadavers of the beetle Hydromedion sparsutum from diverse sites on the sub-Antarctic Bird Island and was found to be the dominant or sole fungal coloniser during the primary degradation of cadavers. The fungus was observed to grow and colonise cadavers from discrete areas of the beetle carapace, some of which were not affected by alcohol surface sterilisation. The fungus is commonly reported from the dung of rodents and other small mammals, both of which are absent from Bird Island. Recovery of P. circinans as the only fungus from beetle cadavers is unusual and may indicate a close association between the beetle and the fungus.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Cerrena unicolor isolated from the mycangia of a horntail,Tremex longicollis,in Kochi Prefecture,Japan

A fungus was found to be stored in the mycangia of a horntail,Tremex longicollis, as hyphal fragments. All fungal isolates from the mycangia of 31 adult females of the horntail produced the same colonies on PDA. Basidiocarps ofCerrena unicolor occurred near the emergence hole of the horntail on a dead hackberry tree (Celtis sinensis). The cultures of this fungus were similar to those from the mycangia of the horntail in cultural characteristics. Mating between single-basidiospore mycelia ofC. unicolor and single-arthrospore mycelia from the mycangia of the horntail showed that they were compatible. These results revealed that the fungus isolated from the mycangia ofT. longicollis wasC. unicolor.