R. R. M. Paterson

A practical approach for identifications based on mycotoxin characters of Penicillium

The taxonomy of the penicillia is unstable particularly in the important antibiotic and mycotoxin-producing subgenus Penicillium. There are difficulties relating identifications to mycotoxin production. Also, the validity of dual nomenclature for pleomorphic fungi is under discussion increasingly. Patulin is an important mycotoxin produced by various fungi and has strict limits in the European Union. The mycotoxin and/or the isoepoxydon dehydrogenase (IDH) gene of the metabolic pathway have been assessed in 318 strains predominately of subgenus Penicillium. These data were used to classify the isolates. Subgenus Penicillium contained most of the IDH and patulin positives. The species and varieties in subgenus Penicillium which were associated with patulin detection can be reduced to one name, viz. Penicillium Pen p+ (p = patulin). This has been extended to other mycotoxin producing penicillia to indicate the scope of the scheme. The classification will lead to the number of taxa being reduced, while avoiding species names and hence dual nomenclature. Culture independent analysis of environmental samples is mentioned. The scheme could be used with advantage for other fungi.

Interaction with Penicillium expansum enhances Botrytis cinerea growth in grape juice medium and prevents patulin accumulation in vitro.

Interactions between fungi occur when they grow on the same host plant. This is the case of Botrytis cinerea and Penicillium expansum on grape. P. expansum is also responsible for production of the mycotoxin patulin. In this study, the influence of the interaction between both fungi on fungal growth parameters was studied as well as the effect on the accumulation of patulin by P. expansum. For that purpose, spores of B. cinerea and P. expansum were inoculated together (mixed inoculum), and the parameters growth rate, time for growth and patulin accumulation were assessed. The presence of P. expansum conidia shortened the time for growth of mixed inoculum colonies which, at the end of incubation, were B. cinerea‐like. Although some P. expansum growth was observed in mixed inoculum colonies, very low levels of patulin were observed. In assays carried out in patulin‐spiked medium, B. cinerea was capable to metabolize the mycotoxin. The capabilities of B. cinerea to shorten time for growth and prevent patulin accumulation are competing abilities that facilitate grape colonization.

Alternative patulin pathway unproven

We read with interest in your journal that Penicillium expansum strains S3, S31 and S87 (Rharmitt et al., 2016) were considered negative for possession of the isoepoxydon dehydrogenase (idh) gene for patulin (PAT) production and positive for PAT production. This led to speculation that an alternative pathway was involved in patulin production. However, the authors did not provide the diode array (DA) UV spectra of the HPLC peaks assigned to PAT, which assist in confirming its identification. UV spectra could usefully be provided in papers when DA analysis has been performed especially for unusual results such as these: At least a statement to say whether the spectra were identical, or not, to the PAT standard is required. One of the current authors (RRMP) reported for the first time the PCR test to assess mycotoxigenic fungi using the idh gene coupled with PAT detection by (a) TLC (Paterson et al., 2000; Paterson et al., 2003) and (b) HPLC UV (Paterson et al., 2003). idh negative and PAT positive strains were not detected. Similarly, Luque et al. (2011) did not detected this combination using micellar electrokinetic capillary electrophoresis, and HPLC-MS for PAT detection. The principle was employed in reverse transcription real-time PCR for idh (De Clercq et al., 2016). The current authors found that a compound which has (a) the same retention time as PAT, but (b) a different UV spectrum using Ultra HPLC DA detection (UHPLC DAD) in a Penicillium strain (Fig. 1), during recent screening work to detect patulin from penicillia isolated from Tunisian apples. Hence, the strain was negative for PAT detection. This was confirmed by the strain being idh negative. Incidentally, the chromatogram for P. expansum (S13) in Rharmitt et al. (2016) had a co-eluting metabolite with PAT as deduced by the peak having a distinct shoulder. The UV spectrum of this sample would probably be different from pure PAT although this was unreported. Perrone et al. (2017) recommended MS and NMR to establish correct ochratoxin A (OTA) production in species not associated conventionally with this mycotoxin. The questionable data were from HPLC-FLD (fluorescence detection) where other fluorescent compounds with the same retention time could be misidentified as OTA. Few laboratories may be able to afford MS and NMR and all methods need assessing on a cost-benefit basis to indicate what is suitable for most laboratories (Paterson et al., 2018). Rharmitt et al. (2016) employed HPLC DAD but the analytical potential of the technique was unfulfilled. Furthermore, Rharmitt et al. (2016) suggested the primers they employed may not have bound to the target DNA hence the negative idh result, which also undermines their premise of a different pathway for PAT.

Cordyceps – A traditional Chinese medicine and another fungal therapeutic biofactory?

Abstract Traditional Chinese medicines (TCM) are growing in popularity. However, are they effective? Cordyceps is not studied as systematically for bioactivity as another TCM, Ganoderma. Cordyceps is fascinating per se, especially because of the pathogenic lifestyle on Lepidopteron insects. The combination of the fungus and dead insect has been used as a TCM for centuries. However, the natural fungus has been harvested to the extent that it is an endangered species. The effectiveness has been attributed to the Chinese philosophical concept of Yin and Yang and can this be compatible with scientific philosophy? A vast literature exists, some of which is scientific, although others are popular myth, and even hype. Cordyceps sinensis is the most explored species followed by Cordyceps militaris. However, taxonomic concepts were confused until a recent revision, with undefined material being used that cannot be verified. Holomorphism is relevant and contamination might account for some of the activity. The role of the insect has been ignored. Some of the analytical methodologies are poor. Data on the “old” compound cordycepin are still being published: ergosterol and related compounds are reported despite being universal to fungi. There is too much work on crude extracts rather than pure compounds with water and methanol solvents being over-represented in this respect (although methanol is an effective solvent). Excessive speculation exists as to the curative properties. However, there are some excellent pharmacological data and relating to apoptosis. For example, some preparations are active against cancers or diabetes which should be fully investigated. Polysaccharides and secondary metabolites are of particular interest. The use of genuine anamorphic forms in bioreactors is encouraged.

Filamentous fungal characterizations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix‐assisted laser desorption/ionization time‐of‐flight intact cell mass spectrometry (MALDI‐TOF ICMS) is coming of age for the identification and characterization of fungi. The procedure has been used extensively with bacteria. UV‐absorbing matrices function as energy mediators that transfer the absorbed photoenergy from an irradiation source to the surrounding sample molecules, resulting in minimum fragmentation. A surprisingly high number of fungal groups have been studied: (i) the terverticillate penicillia, (ii) aflatoxigenic, black and other aspergilli, (iii) Fusarium, (iv) Trichoderma, (iv) wood rotting fungi (e.g. Serpula lacrymans) and (v) dermatophytes. The technique has been suggested for optimizing quality control of fungal Chinese medicines (e.g. Cordyceps). MALDI‐TOF ICMS offers advantages over PCR. The method is now used in taxonomic assessments (e.g. Trichoderma) as distinct from only strain characterization. Low and high molecular mass natural products (e.g. peptaibols) can be analysed. The procedure is rapid and requires minimal pretreatment. However, issues of reproducibility need to be addressed further in terms of strains of species tested and between run variability. More studies into the capabilities of MALDI‐TOF ICMS to identify fungi are required.

Biodegradation of ochratoxin a for food and feed decontamination.

Ochratoxin A (OTA) is one of the most important mycotoxins that is found in food and feed products. It has proven toxic properties, being primarily known for its nephrotoxicity and carcinogenicity to certain animal species. OTA is produced by several species of Aspergillus and Penicillium that can be found in a wide variety of agricultural products, which makes the presence of OTA in these products common. Many countries have statutory limits for OTA, and concentrations need to be reduced to as low as technologically possible in food and feed. The most important measures to be taken to control OTA are preventive in order to avoid fungal growth and OTA production. However, these measures are difficult to implement in all cases with the consequence of OTA remaining in agricultural commodities. Remediation processes are often used to eliminate, reduce or avoid the toxic effects of OTA. Biological methods have been considered increasingly as an alternative to physical and chemical treatments. However, examples of practical applications are infrequent. This review will focus on the (i) known microorganisms and enzymes that are able to biodegrade OTA; (ii) mode of action of biodegradation and (iii) current applications. A critical discussion about the technical applicability of these strategies is presented.

Filamentous fungi in drinking water, particularly in relation to biofilm formation

The presence of filamentous fungi in drinking water has become an area worthy of investigation with various studies now being published. The problems associated with fungi include blockage of water pipes, organoleptic deterioration, pathogenic fungi and mycotoxins. Fungal biofilm formation is a less developed field of study. This paper updates the topic and introduces novel methods on fungal biofilm analysis, particularly from work based in Brazil. Further recommendations for standard methodology are provided.

Internal amplification controls have not been employed in fungal PCR hence potential false negative results

Polymerase chain reaction (PCR) is subject to false negative results. Samples of fungi with the genes of interest (e.g. a disease or mycotoxin) may be categorized as negative and safe as a consequence. Fungi are eukaryotic organisms that are involved in many fields of human activity such as antibiotic, toxin and food production. Certain taxa are implicated in human, animal and plant diseases. However, fungi are difficult to identify and PCR techniques have been proposed increasingly for this purpose. Internal amplification controls (IACs) will ameliorate the situation and need to become mandatory. These are nucleic acids that posses a sequence which will provide a PCR product (i) using the same primers employed for the target gene, and (ii) that will not coincide on the gel with the product of the target gene. Only one group of workers employed an IAC, to respond to potential inhibition, which was reported in 1995 from this present assessment of numerous reports. Inhibitors in cultures need to be minimized, and secondary metabolites are an obvious source. The fields reviewed herein include medical mycology, mycotoxicology, environmental mycology and plant mycology. The conclusion is that previous reports are compromised because IACs have not been employed in fungal PCR; future research must include this control at an early stage.

Laccase activity from the fungus Trametes hirsuta using an air-lift bioreactor

Aim:  To produce high laccase activities from the white‐rot fungus Trametes hirsuta in an in‐house air‐lift bioreactor (ALB).

Survey of aflatoxins in chillies from Pakistan produced in rural, semi-rural and urban environments

Chilli peppers from Pakistan are consumed locally and also exported. Their quality is compromised by aflatoxins (AF) contamination. AF in chillies from rural, semi-rural and urban areas of the Punjab region of Pakistan were determined. Twenty-three (52.3%), 22 (50%) and 29 (65.9%) samples from rural, semi-rural and urban areas respectively, contained levels of aflatoxins which exceeded the European Union limits of >5 µg kg−1 for AFB1 and >10 µg kg−1 for total AF that apply to spices. Mean values for AFB1 in ground samples were 23.8, 14.8 and 14.0 µg kg−1 for rural, semi-rural and urban areas, respectively. Mean total AF in ground samples were 27.7, 17.7 and 16.2 µg kg−1 from equivalent locations. Eleven (50%), 12 (54.5%) and 14 (63.6%) whole samples from rural, semi-rural and urban areas, respectively, contained total levels of AF that exceeded European Union limits. The data indicate that individual localities have particular problems. In conclusion, the concentrations were often greater than the statutory limits set by the European Union.