Virulence of Fungi Isolated from Ambrosia Beetles to Acer amoenum Branches.

In Japan, no association between the ambrosia beetle and their fungal symbionts causing branch dieback or tree mortality on maple, Acer amoenum, has been reported. However, we identified dieback of several branches and numerous holes created by three species of ambrosia beetles, Euwallacea fornicatus, Euwallacea interjectus, and Platypus calamus, on Acer amoenum trees at the University of Tokyo Tanashi Forest, Tokyo Metropolis, Japan, in 2016. The high attack density of the beetles was observed on the weakened trees; however, the contribution of the associated fungi to the branch dieback was still unknown. We isolated fungi carried by these three beetles and inoculated them to Acer amoenum cut main trunks and sapling branches to determine whether the associated fungi caused the branch dieback. Fusarium euwallaceae was isolated from all Euwallacea fornicatus and Euwallacea interjectus, whereas Arthrinium phaeospermum, Raffaelea cyclorhipidia, and Epicoccum nigrum were isolated from P. calamus, with 35, 15, and 5% isolation frequencies, respectively. Inoculation with F. euwallaceae and R. cyclorhipidia induced statistically significantly wider sapwood discoloration (six and four times wider for F. euwallaceae and R. cyclorhipidia, respectively) than the controls, and larger water-conductance loss (2 and 1.7 times larger for F. euwallaceae and R. cyclorhipidia, respectively) than the controls. However, the observed lesions were not large enough to cause discoloration, and symptoms of dieback were not observed, even 13 months after the inoculation. Therefore, we concluded that the virulence of the four investigated fungi to Acer amoenum was very low and that these fungi were likely not the primary cause of the branch dieback.

From colony to rodlet: "A six meter long portrait of the xerophilic fungus Aspergillus restrictus decorates the hall of the Westerdijk institute."

The extreme xerophilic fungus Aspergillus restrictus is used as a model for a large artwork created out of five microscopic pictures in total measuring 80 cm by 624 cm. The artwork is printed on aluminium and located at the entrance of the Westerdijk Institute, Utrecht, The Netherlands. The first picture is made from a colony of the fungus, which has a dimension of 1 cm and the last picture shows details of ornamentation on conidia and phialides of the fungus. The first two pictures of the artwork are made using a unique method of light microscopy in which many hundreds of pictures are made at different focal depths resulting in high detail and resolution of the pictures. For three other pictures, cryo-electron scanning microscopy was used including both a conventional system for lower magnification and a field emission scanning electron microscope for high resolution micrographs. The range of magnification is, at real size, between 78 and 63,000 times. When the observer passes the artwork it acts like a virtual microscope, just by walking past it you zoom-in to the smallest possible details. This coherent increase of magnification of one fungus, with very high quality light- and electron microscopy micrographs, shows different layers of fungal organization and emergent properties. These include the occurrence of secondary outcrops of hyphae and conidiophores in a colony; the formation of a stipe on a thin aerial hyphae; the presence and formation of characteristic structures on stipes, vesicles and phialides and a continuous zone between the forming conidia and phialides.

High-Resolution Melting (HRM) Curve Assay for the Identification of Eight Fusarium Species Causing Ear Rot in Maize.

Maize plants are often infected with fungal pathogens of the genus Fusarium. Taxonomic characterization of these species by microscopic examination of pure cultures or assignment to mating populations is time-consuming and requires specific expertise. Reliable taxonomic assignment may be strengthened by the analysis of DNA sequences. Species-specific PCR assays are available for most Fusarium pathogens, but the number of species that infect maize increases the labor and costs required for analysis. In this work, a diagnostic assay for major Fusarium pathogens of maize based on the analysis of melting curves of PCR amplicons was established. Short segments of genes RPB2 and TEF-1α, which have been widely used in molecular taxonomy of Fusarium, were amplified with universal primers in a real-time thermocycler and high-resolution melting (HRM) curves of the products were recorded. Among major Fusarium pathogens of maize ears, F. cerealis, F. culmorum, F. graminearum, F. equiseti, F. poae, F. temperatum, F. tricinctum, and F. verticillioides, all species except for the pair F. culmorum/F. graminearum could be distinguished by HRM analysis of a 304 bp segment of the RPB2 gene. The latter two species could be differentiated by HRM analysis of a 247 bp segment of the TEF-1α gene. The assay was validated with DNA extracted from pure cultures of fungal strains, successfully applied to total DNA extracted from infected maize ears and also to fungal mycelium that was added directly to the PCR master mix ("colony PCR"). HRM analysis thus offers a cost-efficient method suitable for the diagnosis of multiple fungal pathogens.

A novel antifungal property for the Bacillus licheniformis ComX pheromone and its possible role in inter-kingdom cross-talk.

Quorum sensing molecules (QSMs) regulate, through a chemical communication process, multiple complex systems in bacterial and some fungal populations on the basis of cell density. The bacterial QSMs involved in inter-kingdom cross-talk may exhibit antagonistic activity against fungi. This provides an important opportunity for biocontrol of fungal invasion in plants. It has been shown that cultures of Bacillus spp. inhibit fungal growth. Here, we explore the inhibitory potential of the industrial workhorse Bacillus licheniformis NCIMB-8874 and its QSM (ComX pheromone) on the growth of Aspergillus flavus, a cereal, legume, and nut crop pathogen. Our studies show that ComX filtered extracts from cultures of B. licheniformis can cause a significant reduction in the growth of A. flavus NRRL 3357 and ESP 15 at a concentration as low as 13 µg ml-1. This work evidences, for the first time, the inter-kingdom utility of the bacterial quorum sensing ComX pheromone indicating potential antifungal food security against A. flavus.