Diagnosing deep-seated mycoses continues to be a major challenge for a clinician. Non-culture-dependent laboratory assays with high sensitivity and specificity are needed for rapid diagnosis of deep-seated mycoses. In the future, clinical mycology laboratories will increasingly utilize nucleic acid-based methods for the recognition of pathogenic fungi in patient specimens and the identification of fungal isolates. Over the last 20 years, increasing numbers of papers have documented many molecular biological methods feasible for the detection of fungus-specific DNA sequences in clinical specimens. Polymerase chain reaction(PCR) and internal probes are central to these procedures. More recently, a non-isotopic in situ technique has been applied for the detection of pathogenic fungi. These methods have the potential to improve diagnostic accuracy, thereby accelerating the administration of appropriate antifungal therapy. This article will review recent advances in in situ molecular diagnosis of fungal infections.

The ecology and host differentiation of two heteroecious rust species, Melampsora chelidonii-pierotii and M. yezoensis, which parasitize Salix spp. and Papaveraceae, were demonstrated by field surveys of 12 riparian vegetative areas, parks, and botanical gardens, as well as inoculation experiments with aeciospores and urediniospores. Papaveraceae grow in or near willow forests, and they are used as spermogonial-aecial hosts in most fields. Melampsora chelidonii-pierotii has a narrow host range, and it contains two strains that have a differentiated host range in the uredinial-telial state, i.e. one is parasitic on S. chaenomeloides while the other parasitizes S. eriocarpa and S. pierotii. Both strains use Corydalis incisa as a spermogonialaecial host. On the other hand, M. yezoensis has a wider host range in both the spermogonial-aecial and uredinial-telial states. Melampsora yezoensis uses not only Corydalis incisa and Chelidonium majus var. asiaticum but also other Corydalis spp. as spermogonial-aecial hosts. This species use S. jessoensis and S. serissaefolia as natural hosts in the uredinial-telial state. Salix eriocarpa, S. pierotii, S. yoshinoi, and S. babylonica forma rokkaku were shown to act as hosts of M. yezoensis after artificial inoculation. These two Melampsora species appeared to be phylogenetically similar, although they had different survival strategies.

The biodiversity of soil-inhabiting mucoralean and mortierellalean fungi was surveyed using a baiting method. Two different substrates, dried petals of hibiscus flowers and spotted shrimps, were used as baits. Soil samples were collected from 18 temperate and subtropical regions in Japan. Eighteen species of six genera belonging to Mucorales and Mortierellales were isolated. The composition of the species isolated from hibiscus and shrimp baits were different. The results suggest that the baiting method is useful for isolating phylogenetically diverse mucoralean and mortierellalean fungi from soil.

This is the second part of the article that reviews the basic concepts, current status, and potential applications of genomics in mycology. It also provides brief introductions to the technology that is available for experimental and bioinformatics approach. With the advent of genome sequencing technology, many genome sequences of filamentous fungi have been deciphered, thereby enhancing the effectiveness of functional and comparative genomics. In over 50 fungal genera, the genome sequences of more than two species have been revealed. In addition to the comparison of static genome sequences, comparative functional genomics has been developed, which combines a number of genomic techniques. The phenotypes of a variety of eukaryotic microorganisms involve many genes, thereby making functional genomics data indispensable. The functional genomics data of several species, including model organisms, is accumulated in public databases. A comparison of these functional genomics data should facilitate the identification of genetic factors associated with the superior characteristics of fungi species. In the near future, genomics analysis will be incorporated into various fields of mycology. A variety of methodologies and disciplines will be applied to the design and construction of microorganisms with novel biological functions. This article also reviews recent technological advancements related to synthetic biology.

This review introduces three novel fungal cultivation and application systems, i.e., liquid-surface immobilization (LSI) , liquid-liquid interface bioreactor (L-L IBR) , and extractive liquid-surface immobilization (Ext-LSI) systems. In all systems, ballooned microsphere particles play a key role in immobilizing of fungal cells onto the surface of a liquid medium. Among the three systems, Ext-LSI facilitates the production of lipophilic secondary metabolites such as fungicidal substances at very high concentrations. Furthermore, unfavorable phenomena such as catabolite repression and proteolytic digestion of targeted enzymes can be effectively alleviated in the interfacial system. A novel fungal screening system was developed with Ext-LSI to enable the identification of novel antibacterial and antifungal agents. This system facilitates the high throughput and conventional screening of many fungi that produce antibacterial or antifungal agents with minimal medium (5 ml) and no shaking. Moreover, complex procedures such as solvent extraction and centrifugation are not needed. It is expected that this unique screening system will help identify many fungi having a high potential of producing valuable substances.

We propose a method to enhance the development of natural product drug discovery. Japan is located in a cool-temperate to subtropical zone where 12,300 fungal species have been recorded. At least 188,500 species are expected to be discovered in future. Of these fungal taxa, Hypocreales is one of the most attractive groups for the discovery of secondary metabolites. During the past ten years, we have established a diverse fungal collection mainly from Japan, containing 668 genera, 1,122 species, and 15,069 strains. We used these fungi to produce over 30,000 samples in a cultured extract library ready for assay. Previous successes in natural product drug discovery indicate that most lead candidates have a weak antifungal activity. Thus, it would be useful to prescreen a huge number of extracts against highly susceptible yeasts or fungi, to identify new active metabolites. A collection of these active compounds could then readily be evaluated by pharmaceutical companies.