Controlled rate cooling of fungi using a stirling cycle freezer.

BACKGROUND: The use of a Stirling cycle freezer for cryopreservation is considered to have significant advantages over traditional methodologies including N2 free operation, application of low cooling rates, reduction of sample contamination risks and control of ice nucleation. OBJECTIVE: The study assesses the suitability of an 'N2-free' Stirling Cycle controlled rate freezer for fungi cryopreservation. METHODS: In total, 77 fungi representing a broad taxonomic coverage were cooled using the N2 free cooler following a cooling rate of -1 degrees C min(-1). Of these, 15 strains were also cryopreserved using a traditional 'N2 gas chamber' controlled rate cooler and a comparison of culture morphology and genomic stability against non-cryopreserved starter cultures was undertaken. RESULTS: In total of 75 fungi survived cryopreservation, only a recalcitrant Basidiomycete and filamentous Chromist failed to survive. No changes were detected in genomic profile after preservation, suggesting that genomic function is not adversely compromised as a result of using 'N2 free' cooling. CONCLUSION: The results demonstrate the potential of 'N2-free' cooling for the routine cryopreservation of fungi in Biological Resource Centres.

Using DNA barcodes to test the association of sexes and morphs in Calodesma spp. (Lepidoptera, Erebidae, Arctiinae, Arctiini, Pericopina) of Trinidad, West Indies, with an overview of the genus, taxonomic changes and a new species.

Phalaena militta Stoll, [1781], currently in the combination Thyrgis militta, is transferred to the new combination Calodesma militta. Phalaena militta is the type species of Thyrgis Walker, 1854, and so Thyrgis is a junior synonym of Calodesma Hübner, [1820]. The reinstated genus Seileria Dognin, 1923 is the next available name for the genus previously known as Thyrgis, and the remaining eight species and their subspecies currently in Thyrgis are transferred to new combinations as species of Seileria: S. angustifascia (Hering, 1925), S. basipunctata (Hering, 1926), S. constrictifascia (Dognin, 1919), S. flavonigra (Dognin, 1910), S. investigatorum (Toulgoët, 1988), S. marginata (Butler, 1875), S. meres (Druce, 1911), S. phlegon (Druce, 1885), S. phlegon ruscia (Druce, 1895), S. tenuifascia (Hering, 1930) and S. tenuifascia daguana (Hering, 1930). Eucyanoides Toulgoët, 1988, currently a synonym of Thyrgis, is made a new subjective synonym of Seileria. Based on DNA barcodes, we recognise three very similar, sexually dimorphic and in two cases polymorphic South American species of Calodesma with some phenotypes in common but very similar male genitalia: C. militta (BOLD:AAK1660), C. sp. cf. collaris (BOLD:ABZ2392) and C. pseudocollaris Cock new species (BOLD:AEI2170). Calodesma militta is widespread in South America, with two male morphs (collaris and dioptis) and two female morphs with variable markings (white and orange morphs). Centronia plorator Kaye, [1923] and Thyrgis lacryma Dognin, 1919 are variants of the white female morph and are new synonyms of Calodesma militta. A third female morph with red markings was not sequenced and could not be allocated to a species. Calodesma sp. cf. collaris (BOLD:ABZ2392) occurs in southern South America with both male morphs but only a white female morph. Calodesma pseudocollaris new species (BOLD:AEI2170) is only known from Trinidad, with one male morph (collaris) and the white female morph. Although more than ten morphs relating to this complex have been described as species, they cannot be synonymised without more data on distribution of the different species or DNA barcodes from the type specimens. Collated life history information indicates species of this group are split between Malpighiaceae feeders and Bromeliaceae feeders, but more work is needed to define these differences. The morphism patterns observed are discussed in terms of Müllerian mimicry and mimicry rings, and we suggest that in Trinidad (and elsewhere) there is a loose mimicry ring of diurnal black species with white spots or transparent patches on the wings which are most conspicuous and frequently observed when feeding on white Asteraceae flowers.

Tapping Culture Collections for Fungal Endophytes: First Genome Assemblies for Three Genera and Five Species in the Ascomycota.

The Ascomycota form the largest phylum in the fungal kingdom and show a wide diversity of lifestyles, some involving associations with plants. Genomic data are available for many ascomycetes that are pathogenic to plants, but endophytes, which are asymptomatic inhabitants of plants, are relatively understudied. Here, using short- and long-read technologies, we have sequenced and assembled genomes for 15 endophytic ascomycete strains from CABI's culture collections. We used phylogenetic analysis to refine the classification of taxa, which revealed that 7 of our 15 genome assemblies are the first for the genus and/or species. We also demonstrated that cytometric genome size estimates can act as a valuable metric for assessing assembly "completeness", which can easily be overestimated when using BUSCOs alone and has broader implications for genome assembly initiatives. In producing these new genome resources, we emphasise the value of mining existing culture collections to produce data that can help to address major research questions relating to plant-fungal interactions.

Description of Xenorhabdus magdalenensis sp. nov., the symbiotic bacterium associated with Steinernema australe.

A symbiotic bacterium, strain IMI 397775(T), was isolated from the insect-pathogenic nematode Steinernema australe. On the basis of 16S rRNA gene sequence similarity, this bacterial isolate was shown to belong to the genus Xenorhabdus, in agreement with the genus of its nematode host. The accurate phylogenetic position of this new isolate was defined using a multigene approach and showed that isolate IMI 397775(T) shares a common ancestor with Xenorhabdus doucetiae FRM16(T) and Xenorhabdus romanii PR06-A(T), the symbiotic bacteria associated with Steinernema diaprepesi and Steinernema puertoricense, respectively. The nucleotide identity (less than 97%) between isolate IMI 397775(T), X. doucetiae FRM16(T) and X. romanii PR06-A(T) calculated for the concatenated sequences of five gene fragments encompassing 4275 nt, several phenotypic traits and the difference between the upper temperatures that limit growth of these three bacteria allowed genetic and phenotypic differentiation of isolate IMI 397775(T) from the two closely related species. Strain IMI 397775(T) therefore represents a novel species, for which the name Xenorhabdus magdalenensis sp. nov. is proposed, with the type strain IMI 397775(T) ( = DSM 24915(T)).

The effect of cryopreservation on genomic stability in strains of the fungus Trichoderma.

Within Biological Resource Collections, the successful long-term storage of fungal cultures is essential because of their scientific and potential commercial value. Preservation procedures such as cryopreservation have traditionally been used to ensure genomic stability due to the suspension of metabolic activity at ultra-low temperatures. Genomic integrity is important with regards to conservation, as changes in the genome may compromise production of a desired metabolite, enzyme or activity. To evaluate cryopreservation as a conservation protocol, genomic integrity was assessed in five strains of the economically important fungus Trichoderma. Two polymerase chain reaction (PCR) fingerprinting techniques commonly used for molecular studies of fungi were applied. Three genetic polymorphisms were detected amongst replicates post preservation, indicating that even robust, standardised preservation cryopreservation methodologies can sometimes induce genomic change. However, the low number of polymorphisms suggests that cryopreservation is a reliable method for organism storage over long periods of time.

Occurrence of natural enemies of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) in Nigeria.

Fall armyworm (FAW; Spodoptera frugiperda), an exotic moth which recently invaded Africa, is a highly destructive pest of cereals especially maize a highly valued staple crop in Nigeria. The use of natural enemies such as predators or parasitoids for FAW control is more economically viable and environmentally safer than currently recommended synthetic insecticides. Natural enemies to combat the pest have not yet been reported in Nigeria. An exploration for the pests' natural enemies was undertaken by collecting FAW eggs and larvae from maize fields. These were reared in the laboratory for emergence, identification and efficacy as natural enemies. This yielded Euplectrus laphygmae (Hymenoptera: Eulophidae); Telenomus remus (Hymenoptera: Platygastridae) and Trombidium sp. (Acari.: Trombidiidae). Cotesia or Apanteles spp. were inferred to occur since Stictopisthus sp. (Hym.: Ichneumonidae), a secondary parasitoid, that attacks cocoons of Microgasterinae (e.g. Cotesia, Apanteles etc.) also emerged. Species of yet-to-be identified predators were also observed in various niches of maize plants. A positive relationship was found between FAW instar and the number of E. laphygmae eggs/instar ranging, on average, from 1.5 on second instar to 5.5 on fourth instars hosts. Parasitism rate of T. remus on FAW eggs was 100%. Parasitic mite infestation resulted in increasing paleness, reduced feeding, growth and movement as well as death of FAW 1st instars. Thus, the occurrence of FAW natural enemies in Nigeria calls for advocacy campaign to incorporate their use into integrated pest management strategies that attract and allow natural enemies to thrive for FAW management.

Parasitoid Complex of Fall Armyworm, Spodoptera frugiperda, in Ghana and Benin.

Keywords: biological control; Chelonus bifoveolatus; Coccygidum luteum; Telenomus remus; Trichogramma; West Africa.

Spectral Grouping of Nominally Aspergillus versicolor Microbial-Collection Deposits by MALDI-TOF MS.

Historical microbial collections often contain samples that have been deposited over extended time periods, during which accepted taxonomic classification (and also available methods for taxonomic assignment) may have changed considerably. Deposited samples can, therefore, have historical taxonomic assignments (HTAs) that may now be in need of revision, and subdivisions of previously-accepted taxa may also be possible with the aid of current methodologies. One such methodology is matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). Motivated by the high discriminating power of MALDI-TOF MS coupled with the speed and low cost of the method, we have investigated the use of MALDI-TOF MS for spectral grouping of past deposits made to the Centre for Agriculture and Bioscience International (CABI) Genetic Resource Collection under the HTA Aspergillus versicolor, a common ascomycete fungus frequently associated with soil and plant material, food spoilage, and damp indoor environments. Despite their common HTA, the 40 deposits analyzed in this study fall into six clear spectral-linkage groups (containing nine, four, four, four, four, and two members, respectively), along with a group of ten spectrally-unique samples. This study demonstrates the clear resolving power of MALDI-TOF MS when applied to samples deposited in historical microbial collections.

A Polyphasic Approach Aids Early Detection of Potentially Toxigenic Aspergilli in Soil.

Key chili and maize growing areas of Pakistan were selected for a focused baseline study of the levels of Aspergillus spp. Investigations were undertaken using a combination of molecular and culture-based techniques. Samples investigated included soil samples, one-year-old corn cobs, and fresh chili from selected locations. Aspergillus strains obtained from corn cobs were screened using coconut milk agar, resulting in one strain that was positive for aflatoxin production. Whole genome sequencing (WGS) with low coverage techniques were employed to screen the isolates for differences in the ribosomal RNA gene cluster and mitochondrial genome, with the aflatoxigenic strain proving to have a distinctive profile. Finally, strains were subjected to matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry (MALDI-ToF-MS) in order to obtain a proteomic 'fingerprint' which was used to distinguish the aflatoxigenic strain from the other isolates. The next generation sequencing (NGS) study was broadened to incorporate metabarcoding with ITS rRNA for determining the microbial biodiversity of the soil samples and presumptive screening for the presence of aflatoxigenic strains. Using information gleaned from the WGS results, a putative aflatoxigenic operational taxonomic unit (OTU) was observed in four of the 15 soil samples screened by metabarcoding. This method may have beneficial applications in early detection and surveillance programs in agricultural soils and commodities.

Telenomus Remus, a Candidate Parasitoid for the Biological Control of Spodoptera Frugiperda in Africa, is already Present on the Continent.

The fall armyworm, Spodoptera frugiperda, a moth originating from tropical and subtropical America, has recently become a serious pest of cereals in sub-Saharan Africa. Biological control offers an economically and environmentally safer alternative to synthetic insecticides that are being used for the management of this pest. Consequently, various biological control options are being considered, including the introduction of Telenomus remus, the main egg parasitoid of S. frugiperda in the Americas, where it is already used in augmentative biological control programmes. During surveys in South, West, and East Africa, parasitized egg masses of S. frugiperda were collected, and the emerged parasitoids were identified through morphological observations and molecular analyses as T. remus. The presence of T. remus in Africa in at least five countries provides a great opportunity to develop augmentative biological control methods and register the parasitoid against S. frugiperda. Surveys should be carried out throughout Africa to assess the present distribution of T. remus on the continent, and the parasitoid could be re-distributed in the regions where it is absent, following national and international regulations. Classical biological control should focus on the importation of larval parasitoids from the Americas.

A simple and inexpensive method for practical storage of field-sample proteins for subsequent MALDI-TOF MS analysis.

BACKGROUND: Protein-containing samples can readily be characterised and/or identified using matrix-assisted laser-desorption and ionisation time-of-flight mass spectrometry (MALDI-TOF MS). This technique however requires relatively-fresh biological material that contains proteins that have not yet undergone significant degradation. For field-work collection of samples, problems are often encountered due to delays between collection and sample processing, sample storage (possibly at elevated temperature and/or humidity in some climates), quarantine/regulatory restrictions on the transfer of living biological materials across national borders, and the potential to transfer unwanted microorganisms via non-living biological materials. RESULTS: In an attempt to overcome the above difficulties, we have developed a simple and inexpensive method for practical storage of field-sample proteins, for subsequent MALDI-TOF MS analysis, in which biological material is crushed onto filter paper and dried. The dried and protein-impregnated filter paper can then be soaked in an alcoholic solution suitable for the inactivation of microorganisms of concern and again dried for storage. After subsequent dry storage, the proteins may be eluted from the paper using a solution containing acetonitrile, trifluoroacetic acid, water, and MALDI-TOF MS matrix near to saturation. The extracted proteins are then pipetted onto the MALDI-TOF MS sample plate for subsequent analysis. Using this method, spectra of comparable quality to fresh-material controls have been obtained for acid-soluble proteins from Fallopia japonica and Impatiens glandulifera leaf material. Unlike untreated leaf material, high-quality spectra can be obtained with and without alcohol treatment even after storage for one month at up to 40 °C. CONCLUSIONS: We have developed a simple and inexpensive method for practical storage of field-sample proteins for subsequent MALDI-TOF MS analysis. Key benefits of this approach are a reduction in sample degradation, and consequent conservation of taxon-discriminatory spectral profiles, whilst minimising the potential for carryover of viable microorganisms.

A highly-simplified and inexpensive MALDI-TOF mass spectrometry sample-preparation method with broad applicability to microorganisms, plants, and insects.

Matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry prepares proteins intact in the gas phase with predominantly a single positive charge. The times-of-flight of charged proteins along a tube held at high vacuum after acceleration in an electrical field are proportional to the square root of the mass-over-charge ratios for the proteins, thereby allowing a mass spectrum to be generated, which can then be used to characterize or identify a protein-containing sample. Several sample-preparation methods are currently available but not all of these are applicable to some forms of fungal biomass and few of these are well suited to the analysis of plant or insect material. We have therefore developed a simplified method that: lyses cells, selectively solubilizes basic proteins, dissolves matrix to a suitable concentration, generates spectra with good intensity and peak richness, costs no more (and generally less) than current methods, and is not constrained in terms of throughput by the availability of centrifuges. Using this method, and a reagent formulation comprising α-cyano-4-hydroxycinnamic acid matrix close to saturation in 60%-65% (v/v) acetonitrile in water containing 2.5% (v/v) trifluoroacetic acid, we have been able to differentiate between strains for a representative subset of aflatoxin-producing and aflatoxin-non-producing strains of Aspergillus fungi, to differentiate between Indian and Pakistani strains of Himalayan balsam rust, to differentiate between closely-related Crassula spp. and regional biotypes of Crassula helmsii, and to differentiate between rubbervine introduced into Australia and Brazil. We have also analyzed fall armyworm and stem-borer samples stored in 70% (v/v) ethanol and old dried insect specimens.

Molecular methods to detect Spodoptera frugiperda in Ghana, and implications for monitoring the spread of invasive species in developing countries.

Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest indigenous throughout the Americas, which recently appeared in Africa, first reported from São Tomé, Nigeria, Bénin and Togo in 2016, and which we now report from Ghana. This species is recognised to comprise two morphologically identical but genetically distinct strains or species in the Americas, and we found both to be present in Ghana. We discuss possible routes of entry to Africa, of which the likeliest is adults and/or egg masses transported on direct commercial flights between the Americas and West Africa, followed by dispersal by adult flight within Africa. Identification of Lepidoptera is normally based on the markings and morphology of adults, and not on the larvae which actually cause the damage, and therefore larvae have to be reared through to adult for authoritative identification. We confirmed that the use of DNA barcoding allowed unequivocal identification of this new pest from Ghana based on the larvae alone. As authenticated barcodes for vouchered specimens of more pests become available, this approach has the potential to become a valuable in-country tool to support national capability in rapid and reliable pest diagnosis and identification.

Fungi in bottled water: a case study of a production plant.

A one-year fungal survey of a water bottling plant was conducted in order to evaluate the incidence and fluctuations of the mycobiota. The dominant fungal genera in order of highest numbers isolated were Penicillium, Cladosporium and Trichoderma followed by Aspergillus, Paecilomyces, and others. As expected, the highest number of isolates were collected during the warmer months, particularly May and June. Indeed during these two months there were more fungi present in the water, indicating that during those times of the year when fungal contamination is high, 0.4 mm filters should be changed on a more regular basis. In order to assess whether contamination was single or multi-loci, molecular methods based on the PCR were used for Penicillium brevicompactum. Overall, fungal contamination arose from multiple sources. Some P. brevicompactum strains were very "alike" and were detected during different sampling times, indicating that they were endemic to the plant. There was no evidence to suggest that fungi detected in the source water passed through to other parts of the plant. However, there was evidence that fungal strains isolated from the water filter were detected elsewhere in the factory, confirming the need to change filters more regularly during periods of high fungal contamination. In order to improve quality control a HACCP programme was implemented and Best Practice Guidelines introduced.

Comments on "Mycobiota and Mycotoxins in Traditional Medicinal Seeds from China. Toxins 2015, 7, 3858-3875"- in Attributing Ochratoxin A Biosynthesis Within the Genus Penicillium Occurring on Natural Agricultural Produce.

The unusual attribution of trace amounts of ochratoxin A in some Chinese food commodities to Penicillium polonicum is questioned by European experience in searches for ochratoxinogenic food-spoilage Penicillia, where mistaken attribution is now known to have been due to cryptic Penicillium verrucosum contamination. Consequently, selection of single-spore isolates is recommended as pre-requisite for attributing mycotoxin biosynthetic potential to fungi.