IMA Genome - F19 : A genome assembly and annotation guide to empower mycologists, including annotated draft genome sequences of Ceratocystis pirilliformis, Diaporthe australafricana, Fusarium ophioides, Paecilomyces lecythidis, and Sporothrix stenoceras.

The pace at which Next Generation Sequence data is being produced continues to accelerate as technology improves. As a result, such data are increasingly becoming accessible to biologists outside of the field of bioinformatics. In contrast, access to training in the methods of genome assembly and annotation are not growing at a similar rate. In this issue, we report on a Genome Assembly Workshop for Mycologists that was held at the Forestry and Agricultural Biotechnology Institute (FABI) at the University of Pretoria, South Africa and make available the 12 draft genome sequences emanating from the event. With the aim of making the process of genome assembly and annotation more accessible to biologists, we provide a step-by-step guide to both genome assembly and annotation, intended to encourage and empower mycologists to use genome data in their research.

Fungal Species and Mycotoxins Associated with Maize Ear Rots Collected from the Eastern Cape in South Africa.

Maize production in South Africa is concentrated in its central provinces. The Eastern Cape contributes less than 1% of total production, but is steadily increasing its production and has been identified as a priority region for future growth. In this study, we surveyed ear rots at maize farms in the Eastern Cape, and mycotoxins were determined to be present in collected samples. Fungal isolations were made from mouldy ears and species identified using morphology and DNA sequences. Cladosporium, Diplodia, Fusarium and Gibberella ear rots were observed during field work, and of these, we collected 78 samples and isolated 83 fungal strains. Fusarium was identified from Fusarium ear rot (FER) and Gibberella ear rot (GER) and Stenocarpella from Diplodia ear rot (DER) samples, respectively. Using LC-MS/MS multi-mycotoxin analysis, it was revealed that 83% of the collected samples contained mycotoxins, and 17% contained no mycotoxins. Fifty percent of samples contained multiple mycotoxins (deoxynivalenol, 15-acetyl-deoxynivalenol, diplodiatoxin and zearalenone) and 33% contained a single mycotoxin. Fusarium verticillioides was not isolated and fumonisins not detected during this survey. This study revealed that ear rots in the Eastern Cape are caused by a wide range of species that may produce various mycotoxins.

Efficacy of Commercially Available Entomopathogenic Agents against the Polyphagous Shot Hole Borer in South Africa.

The invasive ambrosia beetle, Euwallacea fornicatus, was first reported in South Africa in 2018. The beetle has now spread to eight provinces of the country and has had a devastating impact on both native and non-native tree species. This is especially true for trees located in urban and peri-urban environments. Recent predictions are that the South African E. fornicatus invasion will cost an estimated ZAR 275 billion (approx. USD 16 billion) if it continues to spread uncontrollably, justifying an urgent need for its effective management in the country. One option is biological control, which is preferred over the use of chemicals due to its lower environmental impact. We tested two broad-spectrum fungal entomopathogenic agents, Eco-Bb® and Bio-Insek, which are commercially available in South Africa, for efficacy against E. fornicatus. Initial laboratory assays yielded promising results. However, beetle infestation trials using treated pieces of woody castor bean stems showed little effect on beetle survival and reproduction.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Along the footpath of Penicillium discovery: Six new species from the Woodville Big Tree Forest Trail.

In this study, we studied the diversity of Penicillium occurring in soil collected along the Woodville Big Tree Forest Trail situated close to the coastal town of Wilderness in South Africa. Strains were accessioned into a collection and then identified to species based on ß-tubulin DNA sequences, which is the recommended DNA barcode for the genus. The 74 strains were found to represent 18 species, including six we consider undescribed. Here, we introduce them as Penicillium claroviride, P. kalander, P. mattheeae, P. outeniquaense, P. subfuscum, and P. umkhoba. Phylogenetic comparisons were made, and genealogical concordance was demonstrated for these new species using DNA sequences from nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode), ß-tubulin, calmodulin, and RNA polymerase II second largest subunit. Notes on morphological characters distinguishing the new species from their close relatives are provided.

Da Vinci's yeast: Blastobotrys davincii f.a., sp. nov.

A new species of the yeast genus Blastobotrys was discovered during a worldwide survey of culturable xerophilic fungi in house dust. Several culture-dependent and independent studies from around the world detected the same species from a wide range of substrates including indoor air, cave wall paintings, bats, mummies, and the iconic self-portrait of Leonardo da Vinci from ca 1512. However, none of these studies identified their strains, clones, or OTUs as Blastobotrys. We introduce the new species as Blastobotrys davincii f.a., sp. nov. (holotype CBS H-24879) and delineate it from other species using morphological, phylogenetic, and physiological characters. The new species of asexually (anamorphic) budding yeast is classified in Trichomonascaceae and forms a clade along with its associated sexual state genus Trichomonascus. Despite the decade-old requirement to use a single generic name for fungi, both names are still used. Selection of the preferred name awaits a formal nomenclatural proposal. We present arguments for adopting Blastobotrys over Trichomonascus and introduce four new combinations as Blastobotrys allociferrii (≡ Candida allociferrii), B. fungorum (≡ Sporothrix fungorum), B. mucifer (≡ Candida mucifera), and Blastobotrys vanleenenianus (≡ Trichomonascus vanleenenianus). We provide a nomenclatural review and an accepted species list for the 37 accepted species in the Blastobotrys/Trichomonascus clade. Finally, we discuss the identity of the DNA clones detected on the da Vinci portrait, and the importance of using appropriate media to isolate xerophilic or halophilic fungi.

Morphology and multigene phylogeny of Talaromycesamyrossmaniae, a new synnematous species belonging to the section Trachyspermi from India.

A new Talaromyces species, T.amyrossmaniae, isolated from decaying fruit and litter of Terminalia bellerica, is described and illustrated. On the natural substrate, the new species produces determinate synnemata, with a well-defined, vivid orange red to orange red cylindrical stipe, and a greyish green capitulum. Conidiophores are typically biverticillate, or sometimes have subterminal branches, with acerose phialides that produce globose to subglobose, smooth to slightly roughened conidia. Multigene phylogenetic analyses based on the internal transcribed spacer region (ITS), and partial sequences of ß-tubulin (BenA), calmodulin (CaM), and DNA directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization, revealed that these isolates are distinct and form a unique lineage of Talaromyces in section Trachyspermi, closely allied to T.aerius, T.albobiverticillius, T.heiheensis, T.erythromellis, and T.solicola. The new species T.amyrossmaniae is the first species in section Trachyspermi with determinate synnemata.

Taxonomic annotation of public fungal ITS sequences from the built environment - a report from an April 10-11, 2017 workshop (Aberdeen, UK).

Recent DNA-based studies have shown that the built environment is surprisingly rich in fungi. These indoor fungi - whether transient visitors or more persistent residents - may hold clues to the rising levels of human allergies and other medical and building-related health problems observed globally. The taxonomic identity of these fungi is crucial in such pursuits. Molecular identification of the built mycobiome is no trivial undertaking, however, given the large number of unidentified, misidentified, and technically compromised fungal sequences in public sequence databases. In addition, the sequence metadata required to make informed taxonomic decisions - such as country and host/substrate of collection - are often lacking even from reference and ex-type sequences. Here we report on a taxonomic annotation workshop (April 10-11, 2017) organized at the James Hutton Institute/University of Aberdeen (UK) to facilitate reproducible studies of the built mycobiome. The 32 participants went through public fungal ITS barcode sequences related to the built mycobiome for taxonomic and nomenclatural correctness, technical quality, and metadata availability. A total of 19,508 changes - including 4,783 name changes, 14,121 metadata annotations, and the removal of 99 technically compromised sequences - were implemented in the UNITE database for molecular identification of fungi (https://unite.ut.ee/) and shared with a range of other databases and downstream resources. Among the genera that saw the largest number of changes were Penicillium, Talaromyces, Cladosporium, Acremonium, and Alternaria, all of them of significant importance in both culture-based and culture-independent surveys of the built environment.

Indoor airborne fungal pollution in newborn units in Turkey.

Pathogenic and/or opportunistic fungal species are major causes of nosocomial infections, especially in controlled environments where immunocompromised patients are hospitalized. Indoor fungal contamination in hospital air is associated with a wide range of adverse health effects. Regular determination of fungal spore counts in controlled hospital environments may help reduce the risk of fungal infections. Because infants have inchoate immune systems, they are given immunocompromised patient status. The aim of the present study was to evaluate culturable airborne fungi in the air of hospital newborn units in the Thrace, Marmara, Aegean, and Central Anatolia regions of Turkey. A total of 108 air samples were collected seasonally from newborn units in July 2012, October 2012, January 2013, and April 2013 by using an air sampler and dichloran 18% glycerol agar (DG18) as isolation media. We obtained 2593 fungal colonies comprising 370 fungal isolates representing 109 species of 28 genera, which were identified through multi-loci gene sequencing. Penicillium, Aspergillus, Cladosporium, Talaromyces, and Alternaria were the most abundant genera identified (35.14, 25.40, 17.57, 2.70, and 6.22% of the total, respectively).

IMA Genome-F 6: Draft genome sequences of Armillaria fuscipes, Ceratocystiopsis minuta, Ceratocystis adiposa, Endoconidiophora laricicola, E. polonica and Penicillium freii DAOMC 242723.

The genomes of Armillaria fuscipes, Ceratocystiopsis minuta, Ceratocystis adiposa, Endoconidiophora laricicola, E. polonica, and Penicillium freii DAOMC 242723 are presented in this genome announcement. These six genomes are from plant pathogens and otherwise economically important fungal species. The genome sizes range from 21 Mb in the case of Ceratocystiopsis minuta to 58 Mb for the basidiomycete Armillaria fuscipes. These genomes include the first reports of genomes for the genus Endoconidiophora. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these pathogens cause disease.

Discovery of a sexual cycle in Talaromyces amestolkiae.

Talaromyces amestolkiae is a common cosmopolitan species that has been cultured from indoor house dust, sputum and lungs from cystic fibrosis patients, indoor air, wheat, soil, pineapple, sculptures and manure. It was described as an asexual Talaromyces species and was reported to produce black sclerotia. In this study we report on the induction of sexual reproductive structures in T. amestolkiae. The mating type of 18 T. amestolkiae strains was determined with MAT-specific primers. Subsequently opposite mating types were inoculated on oatmeal agar and malt-extract agar and incubated 6-20 wk at 25 and 30 C in darkness. After incubation single ascospore isolations were made and evidence of recombination in the offspring was examined by amplified fragment length polymorphism and pairwise homoplasy index test, which is implemented in Splitstree4. The offspring displayed clear evidence of recombination on a genetic level as shown in the variations observed between banding patterns in the amplified fragment length polymorphism. Also a net-like and reticulated NeighborNet was observed and the pairwise homoplasy index test for recombination supported the presence of recombination (P = 0.003372). The distribution of MAT1-1 and MAT1-2 genes in the progeny showed a close to 1:1 ratio. Talaromyces amestolkiae is only the second heterothallic Talaromyces species to produce ascomata and ascospores under laboratory conditions.

Talaromyces atroroseus, a new species efficiently producing industrially relevant red pigments.

Some species of Talaromyces secrete large amounts of red pigments. Literature has linked this character to species such as Talaromyces purpurogenus, T. albobiverticillius, T. marneffei, and T. minioluteus often under earlier Penicillium names. Isolates identified as T. purpurogenus have been reported to be interesting industrially and they can produce extracellular enzymes and red pigments, but they can also produce mycotoxins such as rubratoxin A and B and luteoskyrin. Production of mycotoxins limits the use of isolates of a particular species in biotechnology. Talaromyces atroroseus sp. nov., described in this study, produces the azaphilone biosynthetic families mitorubrins and Monascus pigments without any production of mycotoxins. Within the red pigment producing clade, T. atroroseus resolved in a distinct clade separate from all the other species in multigene phylogenies (ITS, ß-tubulin and RPB1), which confirm its unique nature. Talaromyces atroroseus resembles T. purpurogenus and T. albobiverticillius in producing red diffusible pigments, but differs from the latter two species by the production of glauconic acid, purpuride and ZG-1494α and by the dull to dark green, thick walled ellipsoidal conidia produced. The type strain of Talaromyces atroroseus is CBS 133442.

Effects of irradiation dose and O(2) and CO(2) concentrations in packages on foodborne pathogenic bacteria and quality of ready-to-cook seasoned ground beef product (meatball) during refrigerated storage.

Combined effects of gamma irradiation and concentrations of O(2) (0, 5, 21%) and CO(2) (0, 50%) on survival of Escherichia coli O157:H7, Salmonella enteritidis, Listeria monocytogenes, lipid oxidation, and color changes in ready-to-cook seasoned ground beef (meatball) during refrigerated storage were investigated. Ground beef seasoned with mixed spices was packaged in varying O(2) and CO(2) levels and irradiated at 2 and 4 kGy. Irradiation (4 kGy) caused about 6 Log inactivation of the inoculated pathogens. Inactivation of Salmonella was 0.9- and 0.4-Log lower in 0 and 5% O(2), respectively, compared to 21% O(2). Irradiation at 2 and 4 kGy increased thiobarbituric acid reactive substances in meatballs by 0.12 and 0.28 mg malondialdehyde kg(-1), respectively, compared to control. In reduced-O(2) packages, radiation-induced oxidation was lower, and the initial color of an irradiated sample was maintained. Packaging with 0% + 50% CO(2) or 5% O(2) + 50% CO(2) maintained the oxidative and the color quality of irradiated meatballs during 14-day refrigerated storage. MAP with 5%O(2) + 50% CO(2) combined with irradiation up to 4 kGy is suggested for refrigerated meatballs to reduce the foodborne pathogen risk and to maintain the quality.

Maintenance of safety and quality of refrigerated ready-to-cook seasoned ground beef product (meatball) by combining gamma irradiation with modified atmosphere packaging.

UNLABELLED: Meatballs were prepared by mixing ground beef and spices and inoculated with E. coli O157:H7, L. monocytogenes, and S. enteritidis before packaged in modified atmosphere (3% O2 + 50% CO2 + 47% N2) or aerobic conditions. The packaged samples were irradiated at 0.75, 1.5, and 3 kGy doses and stored at 4 °C for 21 d. Survival of the pathogens, total plate count, lipid oxidation, color change, and sensory quality were analyzed during storage. Irradiation at 3 kGy inactivated all the inoculated (approximately 106 CFU/g) S. enteritidis and L. monocytogenes cells in the samples. The inoculated (approximately 106 CFU/g) E. coli O157:H7 cells were totally inactivated by 1.5 kGy irradiation. D¹°-values for E. coli O157:H7, S. enteritidis, and L. monocytogenes were 0.24, 0.43, and 0.41 kGy in MAP and 0.22, 0.39, and 0.39 kGy in aerobic packages, respectively. Irradiation at 1.5 and 3 kGy resulted in 0.13 and 0.36 mg MDA/kg increase in 2-thiobarbituric acid-reactive substances (TBARS) reaching 1.02 and 1.49 MDA/kg, respectively, on day 1. Irradiation also caused significant loss of color and sensory quality in aerobic packages. However, MAP effectively inhibited the irradiation-induced quality degradations during 21-d storage. Thus, combining irradiation (3 kGy) and MAP (3% O2 + 50% CO2 + 47% N2) controlled the safety risk due to the potential pathogens and maintained qualities of meatballs during 21-d refrigerated storage. PRACTICAL APPLICATION: Combined use of gamma irradiation and modified atmosphere packaging (MAP) can maintain quality and safety of seasoned ground beef (meatball). Seasoned ground beef can be irradiated at 3 kGy and packaged in MAP with 3% O2 + 50% CO2 + 47% N2 gas mixture in a high barrier packaging materials. These treatments can significantly decrease risk due to potential pathogens including E. coli O157:H7, L. monocytogenes, and S. enteritidis in the product. The MAP would reduce the undesirable effects of irradiation on quality, and extend the shelf life of the product for up to 21 d at 3 °C.