Penicillium hermansii, a new species causing smoky mould in white button mushroom production

Penicillium hermansii, a new species causing smoky mould in white button mushroom production Competing fungi in white button mushroom (Agaricus bisporus, champignon) cultivation causes significant episodic losses. One of these competing fungi is known as Bsmoky mould^. It owes its name to the production of high numbers of spores after the disturbance of compost, which resembles smoke. We investigated strains isolated from smoky mould cases in the Netherlands, UK and Canada and show that these outbreaks were caused by a new Penicillium species, named P. hermansii sp.nov.(type strain CBS 124296 ). Several Penicillium species are reported to cause smoky mould. However, we so far have no indications that smoky mould is caused by other Penicillium species than P. hermansii. This species belongs to section Exilicaulis and differs from other Penicillia by its slow growth rate on Czapek yeast agar (CYA) and malt extract agar (MEA) and its inability to grow on CYA supplemented with 5% salt and CYA and MEA incubated at 37 °C. . . . Keywords Agaricus Taxonomy Phylogeny Mycobiota Introduction last decade, several outbreaks occurred in the Netherlands (C. Hermans, pers. comm.). The first signal of infection by smoky A critical step during mushroom production is the colonisa- mould is an increase of temperature of the compost during tion of the pasteurised phase II compost by Agaricus bisporus. growth that is difficult to control even by lowering air temper- Competitive moulds from spawn-run compost can have dev- ature. Mushrooms grown on compost with a minor smoky astating effects on production levels. An important and well- mould infection are slightly paler, mature quicker and there known example is Trichoderma aggressivum and much re- is usually reduced pinning. In more severely infected com- search is performed on this species (Kosanovic et al. 2015; post, areas lacking growth and even complete empty mush- O’Brien et al. 2014;O’Brien et al. 2017; Radvanyi et al. room beds occur. Digging into these infested areas releases 2016). Another less commonly and poorly documented com- large clouds of spores resembling smoke, hence the name peting contaminant is known as ‘smoky mould’, which is smoky mould. capable of wiping out a complete crop (Fletcher and Gaze Various names, such as Penicillium chermesinum, P. 2008; Grogan and Harvey 1999; Grogan et al. 2000). In the citreonigrum, P. implicatum and P. fellutanum have been ap- plied to smoky mould outbreaks (Baars et al. 2011; Beyer 2002; Fletcher and Gaze 2008; Grogan et al. 2001). These This article is part of the BSpecial Issue on hyphomycete taxonomy and species are distantly related to each other and belong to dif- diversity in honour of Walter Gams who passed away in April 2017^. ferent Penicillium sections (Houbraken and Samson 2011; Section Editor: Hans-Josef Schroers and Marc Stadler Visagieetal. 2014). The use of these names in literature might suggest that smoky mould is caused by multiple species. * J. Houbraken However, in the past Penicillium, identification was primary j.houbraken@westerdijkinstitute.nl based on phenotypic and physiological characters. It is prob- able that only one fungus is involved and that some or all of Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 these identifications are incorrect. CT Utrecht, The Netherlands In this study, strains isolated from smoky mould out- Biodiversity (Mycology), Eastern Cereal and Oilseed Research breaks in the Netherlands, Canada and UK were studied Centre, Agriculture & Agri-Food Canada, 960 Carling Ave., using a polyphasic approach. Physiological, macro- and Ottawa, ON K1A 0C6, Canada Mycol Progress microscopical characteristics combined with partial calmodu- 5% NaCl (CYAS), malt extract agar (MEA, Oxoid), lin (CaM), β-tubulin (BenA) and RNA polymerase II second oatmeal agar (OA), creatine sucrose agar (CREA), largest subunit (regions 5–7) (RPB2) sequences demonstrate dichloran 18% glycerol agar (DG18), yeast extract su- that this set of strains represents a new species in Penicillium crose agar (YES) (Frisvad and Samson 2004). section Exilicaulis. Additional CYA and MEA plates were incubated at 15, 30 and 37 °C. Colony diameters and other macro- scopic colony characteristics (e.g. colour conidia, colony Material and methods texture) were recorded after 7 days of incubation at 25 °C. Mounts were made from the MEA medium in Strains lactic acid and microscopic features were studied by light microscopy using a Zeiss Axiokop 2 Plus. The An overview of examined strains is presented in Table 1.The temperature-growth response was investigated on CYA 16 investigated strains were isolated from compost or the air by recording diameters of three-point inoculated colo- of mushroom production farms, where Agaricus bisporus cul- nies of seven strains (CBS 122432, CBS 124296, CBS tivations were infected with smoky mould. The strains that 124297, DTO 032-A2, DTO 032-A3, DTO 173-C9 and were isolated in different years were from different outbreaks DTO 173-D1) after 14 days at 6–36 at 3 °C intervals and different production farms. No information is recorded at and 40 °C. Alphanumeric codes for conidial and re- farm level and strains isolated in the same year could be ob- verse colours refer to (Kornerup and Wanscher 1978). tained from more than one production location. Two of the Isolates were also examined for the production of alka- strains are legacy strains from previous outbreaks, HRI 1043- loids with Ehrlich reagent using the filter paper method D from the UK and the main strain mentioned in previous described by Lund (1995). Thegrowthrateoftwo P. literature (Fletcher et al. 1989;Grogan et al. 2000), and VM- hermansii strains (CBS 124296 and DTO 173-D4) and 2 from Canada. four other Penicillium species (P. brevicompactum DTO 099-D1; P. chrysogenum CBS 306.48; P. daleae DTO Phenotypic and physiologic characterisation 099-B8; P. glabrum, DTO 099-A6) was studied on an autoclaved compost based medium containing 250 g All pure cultured strains were inoculated at three points on Agaricus grown compost (phase III) and 15 g agar per Czapek yeast autolysate agar (CYA), CYA supplemented with litre. Table 1 Overview of P. hermansii isolates used in this study Culture collection number Substrate Location Year DTO 031-A3 = CBS 122432 = Mushroom compost (phase III) with smoky mould UK Around 1995 DAOM 242544 = HRI 1043-D = DC-283 DTO 194-C7 Mushroom compost with smoky mould The Netherlands 1997 DTO 194-C8 = CBS 132824 Mushroom compost with smoky mould The Netherlands 1997 DAOM 242545 = VM-2 Mushroom compost Nova Scotia, Canada 2000 DTO 032-A2 Mushroom compost with smoky mould The Netherlands 2006 DTO 032-A3 Mushroom compost with smoky mould The Netherlands 2006 DTO 079-D4 = IBT 29994 Mushroom compost with smoky mould The Netherlands 2008 DTO 079-D5 = CBS 124296 Mushroom compost with smoky mould, type The Netherlands 2008 DTO 079-D6 = CBS 124297 Air in mushroom growing room The Netherlands 2008 DTO 173-C9 Mushroom compost with smoky mould, second harvesting period The Netherlands 2011 DTO 173-D1 Mushroom compost with smoky mould, second harvesting period The Netherlands 2011 DTO 173-D2 Mushroom compost with smoky mould The Netherlands 2011 DTO 173-D3 Air in mushroom growing room The Netherlands 2011 DTO 173-D4 Air in mushroom growing room The Netherlands 2011 DTO 282-D3 Air in mushroom growing room The Netherlands 2013 DTO 282-D6 Air in mushroom growing room The Netherlands 2013 CBS, culture collection of the Westerdijk Institute; DC, disease collection at the Pennsylvania State University’s Mushroom Spawn Lab, USA; DTO, internal culture collection of department Applied and Industrial Mycology, housed at the Westerdijk Institute; HRI, culture collection of Warwick HRI, UK Mycol Progress DNA extraction, sequencing and phylogenetic Fig. 1. Nine smoky mould isolates were included in this anal- analysis ysis and those isolates form a unique clade within section Exilicaulis. Two well-supported clades are present in this sec- Strains were grown for 7–14 days at 25 °C on MEA tion and the smoky mould strains group in a cluster previously prior to DNA extraction. Genomic DNA was extracted named the Penicillium parvum-clade (Visagie et al. 2016). using the Ultraclean Microbial DNA Isolation Kit (MoBio Penicillium canis and P. striatisporum are basal to the clade Laboratories Inc., Carlsbad, USA) following the manufac- containing the smoky mould isolates; however, statistical sup- turer’s protocol. The ITS barcode and parts of the BenA, port is lacking. With the data available, it was not possible to CaM and RPB2 genes were amplified, sequenced and conclusively identify the phylogenetic sister species of the annotated according to the method described by Houbraken smoky mould isolates. and Samson (2011) and Houbraken et al. (2012). Newly gen- BenA, CaM and RPB2 gene sequences were generated to erated sequences are deposited in GenBank with accession confirm the phylogenetic coherence of the smoky mould numbers MG386210–MG386247 and MG333469– strains at the species level, and their relationship with other MG333479. species in the P. parvum-clade. The Tamura Nei (TN93) mod- Publically available BenA, CaM and RPB2 sequences of el using the gamma distribution (+G) was found to be optimal the type strains of species belonging to section Exilicaulis for the BenA data set (length 372 sites); and the TN93 were retrieved from GenBank and supplemented with the model using a discrete gamma distribution with invari- newly generated sequences. A concatenated dataset with ant sites (G+I) was optimal for the CaM (length 518 BenA, CaM and RPB2 sequences including nine smoky sites) and RPB2 (length 789 sites) datasets. The best- mould strains was generated and used to determine the phy- scoring ML trees are presented in Fig. 2 and show that logenetic relationship of the smoky mould strains with other the smoky mould strains form a coherent species level members of Penicillium section Exilicaulis.Singlegene clade, distinct from other members of the P. parvum- phylogenies were made by combining the generated se- clade. The smoky mould strains cluster together in trees quences from smoky mould isolates with sequences of based on all loci on a strongly supported branch (> 95% strains belonging to the P. parvum-clade (Visagie et al. bs, 1.00 pp). Most bootstrap percentages and posterior 2016)ofsect. Exilicaulis. The sequence data sets were probability (pp) values were low (< 70% bs, < 0.95 pp) in aligned using MAFFT (Katoh and Standley 2013). Prior these gene trees. to analysis, the optimal substitution model was deter- mined in MEGA v.6.06. (Tamura et al. 2013), utilising Morphology and physiology the Akaike information criterion (AIC). Maximum like- lihood (ML) analysis of the single gene datasets were Phenotypic characters of the smoky mould strains were re- analysed in MEGA 6.06 and the combined dataset in corded and compared with each other. All strains displayed RAxML-VI-HPC v. 7.0.3 (Stamatakis 2006)using the similar macro- and microscopical characters. Most strikingly, GTRGAMMA substitution model. The robustness of the isolates grew slow on CYA and MEA, even at an optimum the phylograms was evaluated by 1000 bootstrap (bs) growth temperature (27–30 °C). Furthermore, the conidio- runs. Bayesian inference (BI) was performed in MrBayes phores had short stipes (10–40 (− 120) μm) and the strains v.3.2.1 (Ronquist et al. 2012) using Markov chain did not grow on CYAS, and CYA and MEA incubated at Monte Carlo (MCMC) algorithm. The analysis stopped 37 °C. The optimum growth temperature on CYA was be- when the average standard deviation of split frequencies tween 27 and 30 °C, with a colony diameter of 15–18 mm reached 0.01. The sample frequency was set to 100; the after 14 days. The minimum growth temperature was first 25% of trees were removed as burnin. Convergence between 12 and 15 °C and the maximum growth tem- and ESS values of the runs were examined by Tracer 1.6 perature between 33 and 36 °C. Strains incubated at (Rambaut et al. 2014). 33 °C reached 11–14mm andnogrowthwas observed at 36 °C. The colony diameters of the smoky mould strains on autoclaved compost agar (CA) were similar Results to those on MEA (CA 9–12 vs MEA 9–12 mm). Also the growth density was similar on both media. The oth- Phylogeny er examined Penicillium species grew well on CA and their colony diameters were similar or slightly smaller The total length of the combined data set was 1729 characters than on MEA (P. brevicompactum,CA18–22 vs MEA (individual datasets: BenA 421; CaM,518; RPB2, 788 char- 20–24 mm; P. chrysogenum,CA40–43 vs MEA 30–35 mm; acters) and the proportion of gaps was 5.7%. The phylogram P. daleae,CA 24–27 vs MEA 34–37 mm and P. glabrum,CA was drawn to scale and the results of this analysis are shown in 38–40 vs MEA 41–45 mm). Mycol Progress Fig. 1 Best-scoring maximum likelihood tree using RAxML based on a combined dataset with partial BenA, CaM and RPB2 sequences, showing the relationship of species accommodated in section Exilicaulis. The phylogram is rooted with P. glabrum CBS 125543. Bootstrap percentages and posterior probability values are presented at the nodes. Values less than 70% bs or 0.95 pp are not shown and branches with more than 95% bs and 1.00 pp are thickened. The bar indicates the number of substitutions per site Taxonomy In: Penicillium subgenus Aspergilloides section Exilicaulis. Based on phylogenetic coherence of the smoky mould causing Etymology: Named after Con Hermans, who studied the isolates and their unique phenotypic and physiologic charac- occurrence of smoky mould and other competing fungi in ters, we describe them here as a new species named the Netherlands. Penicillium hermansii sp. nov. Diagnosis: Slow growth on CYA (5–10 mm) and MEA (8– 15 mm) at 25 °C after 7 days incubation; no growth on CYAS; Penicillium hermansii Houbraken, Seifert & Samson, sp. no growth on MEA and CYA incubated at 37 °C; conidio- nov. Mycobank MB823949 (Fig. 3). phores biverticillate and short-stiped, 10–40 (−120) μm. Mycol Progress Fig. 2 Maximum likelihood (ML) trees generated for BenA, CaM and presented at the nodes. Values less than 70% bs or 0.95 pp are not shown RPB2 with MEGA6. The phylograms are rooted with P. corylophilum and branches with more than 95% bs and 1.00 pp are thickened. The bar CBS 312.48. Bootstrap percentages and posterior probability values are indicates the number of substitutions per site Typus: the Netherlands, ex compost with Agaricus metulae, and sometimes subterminal or intercalary metulae bisporus, 2008, collected by C. Hermans, isolated by J. present; in degenerated strains monoverticillate conidiophores Houbraken (holotype CBS H-21028, culture ex-type CBS predominantly present. Terminal and subterminal metulae un- 124296 = DTO 079-D5). equal in length, 10–20 (− 25) × 2.0–3.5 μm, often with inflat- ITS barcode: ITS = MG333472 (alternative markers: ed apex, 3–5 μm wide. Phialides ampulliform, in verticils of BenA = MG386214, CaM = MG386229, RPB2 = 4–10, closely packed, 7–9×2–3.5 μm. Conidia globose to MG386242). All examined P. hermansii strains share identical subglobose, smooth-walled, 2.0–2.5 μm. Ascomata and scle- ITS, BenA, CaM and RPB2 sequences. The species can be rotia not observed. differentiated from other known Penicillium species by ITS sequences. Distribution and ecology: The Netherlands, United Discussion Kingdom, Canada. Isolated from compost colonised by Agaricus bisporus and from the air of white button mushroom Grogan et al. (2001) expressed uncertainty about the identity production farms. of the smoky mould fungus in mushroom cultivation because Colony diameter: 7 days, in mm, 25 °C (unless stated oth- taxonomic authorities that they consulted did not agree on a erwise): CYA 5–10; CYA15°C no growth or spore germina- common identification. Most identifications of smoky mould tion; CYA 30 °C 7–12; CYA 37 °C: no growth; MEA 8–15; isolates were performed before the era of DNA sequence- YES 5–9; DG18 3–7; CYAS no growth; OA 10–15; creatine based identification. Grogan et al. (2000) studied the identity agar no growth or spore germination. Optimum growth tem- of four smoky mould isolates (including isolate HRI 1043-D) perature on CYA 27–30 °C. using ITS sequences, but the exact identification of the species Colony characters: Sporulation on CYA absent to moder- involved in smoky mould problems remained uncertain. As ate; colonies entire, plane, velvety; mycelium white, sporula- demonstrated in this paper, the smoky mould isolates also turn tion grey-green to greyish-brown green (~ 5D3); exudate ab- out be a previously undescribed species. Although several sent; soluble pigments absent; colony reverse brown (5C3– Penicillium species (e.g. P. chermesinum, P. citreonigrum, P. E3). Soluble pigments on YES absent, sporulation light, implicatum and P. fellutanum) were named as causal agents of (pale) grey-green or inconspicuous; exudate absent, reverse smoky mould, we suggest that these outbreaks were probably brown. Colonies on MEA velvety, dull green or grey-green all caused by P. hermansii alone. Strains referred to as smoky (25–26E3), reverse brown (5D3). Ehrlich reaction negative. mould in the literature share a slow growth rate on agar media Micromorphology: Conidiophores borne from surface; and most were reported to be monoverticillate (Baars et al. slightly vesiculate; stipes short, 10–40 (− 120) × 2–3.5 μm, 2011;Beyer 2002; Fletcher and Gaze 2008). Penicillium with walls smooth or very finely roughened; conidiophores hermansii also grows slowly, but fresh isolates have terminal of freshly isolated strains bearing terminal verticils of 2–4 verticils of 2–4 metulae. Mycol Progress Mycol Progress Fig. 3 Penicillium hermansii, a 7 day-old cultures, 25 °C, left to right; Trichoderma aggressivum. This species grows quickly first row, all obverse, CYA, YES, DG18, MEA; second row, CYA on agar media and seems to compete with Agaricus reverse, YES reverse, OA obverse and CREA obverse. b–e for space and nutrients effectively. The association of Conidiophores. f Conidia. Scale bars = 10 μm T. aggressivum with Agaricus production is probably first based on chemical communication via extrolites, Phylogenetically, P. hermansii is most closely related to P. including volatiles, and/or extracellular enzymes. For canis, P. catenatum, P. erubescens, P. guttulosum, P. example, the mycelium of A. bisporus is required for menonorum, P. nepalense, P. papuanum, P. parvum, P. induction of intensive sporulation in T. aggressivum pimiteouiense, P. rubidurum, P. striatisporum and P. vinaceum (Krupke et al. 2003; Mamoun et al. 2000; Mumpuni et al. that all align to the P. parvum-clade of section Exilicaulis. 1998;Seaby 1987;Seaby 1996). Similarly, Beyer (2002)in- These species share a slow growth rate on CYA and MEA dicated an interaction between the mycelium of Agaricus and and produce short-stiped, monoverticillate conidiophores. smoky mould and suggested that the Agaricus is either Like the other members of this clade, P. hermansii also grows parasitised or repressed. More research is needed to under- slowly on agar media and along with P. nepalense and P. stand the biology of smoky mould in button mushroom catenatum is among the slowest growing species of the clade. cultivation. Morphologically, it differs from all other species of this clade Acknowledgements We thank Nancy Nickerson, Robert Davies and by the production of biverticillate conidiophores, with Leonard North for supplying the Canadian isolate of P. hermansii and monoverticillate conidiophores only seen in older, presum- Gerry Louis-Seize for early phylogenetic studies of that strain. ably degenerated strains. Penicillium hermansii also differs from most of the phylogenetically related species by its inabil- Compliance with ethical standards ity to grow at 37 °C, a character shared only with P. nepalense (Pitt 1980 [‘1979’]; Takada and Udagawa 1983). Using the Conflict of interest The authors declare that they have no conflict of dichotomous key published in the Penicillium monograph of interest. Pitt (1980 [‘1979’]), P. hermansii keys out as P. fellutanum. Open Access This article is distributed under the terms of the Creative That species most obviously differs from P. hermansii by its Commons Attribution 4.0 International License (http:// faster growth rate on CYA (5–10 vs 17–25 mm). creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- In addition to its distinctive phenotype, P. hermansii is also priate credit to the original author(s) and the source, provide a link to the unique in Penicillium for its association with Agaricus- Creative Commons license, and indicate if changes were made. colonised compost. Until now, this species has never been isolated from other habitats, despite extensive surveys of soil, food, indoor air and other substrates from all over the world. Strong associations between Penicillium species and particu- lar habitats have been known for a long time (Westerdijk References 1949). For example, P. italicum, P. ulaiense and P. digitatum are associated with rot of citrus fruits and P. allii is strongly Baars J, Rutjens J, Mumm R (2011) Can volatiles emitted by compost associated with rot of garlic. Generally, Penicillium species during spawn run be used to detect green mould infection early? In: with a specific association to certain substrates grow well on Savoie J-M, Foulongne-Oriol M, Largeteau M, Barroso, G. (eds) Proceedings of the 7th International Conference on Mushroom standard laboratory media and do not require special com- Biology and Mushroom Products (ICMBMP7), vol 1. INRA, pounds from its associated natural source, e.g. citrus peels or Villenave d’Ornon Cedex, pp 474–483 garlic (Frisvad and Samson 2004). In contrast, P. hermansii Beyer DM (2002) Pest species biology and control—weed and indicator grows slowly on general purpose agar media CYA, MEA and moulds. In: Mushroom integrated pest management handbook. 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Penicillium hermansii, a new species causing smoky mould in white button mushroom production

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Abstract

Competing fungi in white button mushroom (Agaricus bisporus, champignon) cultivation causes significant episodic losses. One of these competing fungi is known as Bsmoky mould^. It owes its name to the production of high numbers of spores after the disturbance of compost, which resembles smoke. We investigated strains isolated from smoky mould cases in the Netherlands, UK and Canada and show that these outbreaks were caused by a new Penicillium species, named P. hermansii sp.nov.(type strain CBS 124296 ). Several Penicillium species are reported to cause smoky mould. However, we so far have no indications that smoky mould is caused by other Penicillium species than P. hermansii. This species belongs to section Exilicaulis and differs from other Penicillia by its slow growth rate on Czapek yeast agar (CYA) and malt extract agar (MEA) and its inability to grow on CYA supplemented with 5% salt and CYA and MEA incubated at 37 °C. . . . Keywords Agaricus Taxonomy Phylogeny Mycobiota Introduction last decade, several outbreaks occurred in the Netherlands (C. Hermans, pers. comm.). The first signal of infection by smoky A critical step during mushroom production is the colonisa- mould is an increase of temperature of the compost during tion of the pasteurised phase II compost by Agaricus bisporus. growth that is difficult to control even by lowering air temper- Competitive moulds from spawn-run compost can have dev- ature. Mushrooms grown on compost with a minor smoky astating effects on production levels. An important and well- mould infection are slightly paler, mature quicker and there known example is Trichoderma aggressivum and much re- is usually reduced pinning. In more severely infected com- search is performed on this species (Kosanovic et al. 2015; post, areas lacking growth and even complete empty mush- O’Brien et al. 2014;O’Brien et al. 2017; Radvanyi et al. room beds occur. Digging into these infested areas releases 2016). Another less commonly and poorly documented com- large clouds of spores resembling smoke, hence the name peting contaminant is known as ‘smoky mould’, which is smoky mould. capable of wiping out a complete crop (Fletcher and Gaze Various names, such as Penicillium chermesinum, P. 2008; Grogan and Harvey 1999; Grogan et al. 2000). In the citreonigrum, P. implicatum and P. fellutanum have been ap- plied to smoky mould outbreaks (Baars et al. 2011; Beyer 2002; Fletcher and Gaze 2008; Grogan et al. 2001). These This article is part of the BSpecial Issue on hyphomycete taxonomy and species are distantly related to each other and belong to dif- diversity in honour of Walter Gams who passed away in April 2017^. ferent Penicillium sections (Houbraken and Samson 2011; Section Editor: Hans-Josef Schroers and Marc Stadler Visagieetal. 2014). The use of these names in literature might suggest that smoky mould is caused by multiple species. * J. Houbraken However, in the past Penicillium, identification was primary j.houbraken@westerdijkinstitute.nl based on phenotypic and physiological characters. It is prob- able that only one fungus is involved and that some or all of Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 these identifications are incorrect. CT Utrecht, The Netherlands In this study, strains isolated from smoky mould out- Biodiversity (Mycology), Eastern Cereal and Oilseed Research breaks in the Netherlands, Canada and UK were studied Centre, Agriculture & Agri-Food Canada, 960 Carling Ave., using a polyphasic approach. Physiological, macro- and Ottawa, ON K1A 0C6, Canada Mycol Progress microscopical characteristics combined with partial calmodu- 5% NaCl (CYAS), malt extract agar (MEA, Oxoid), lin (CaM), β-tubulin (BenA) and RNA polymerase II second oatmeal agar (OA), creatine sucrose agar (CREA), largest subunit (regions 5–7) (RPB2) sequences demonstrate dichloran 18% glycerol agar (DG18), yeast extract su- that this set of strains represents a new species in Penicillium crose agar (YES) (Frisvad and Samson 2004). section Exilicaulis. Additional CYA and MEA plates were incubated at 15, 30 and 37 °C. Colony diameters and other macro- scopic colony characteristics (e.g. colour conidia, colony Material and methods texture) were recorded after 7 days of incubation at 25 °C. Mounts were made from the MEA medium in Strains lactic acid and microscopic features were studied by light microscopy using a Zeiss Axiokop 2 Plus. The An overview of examined strains is presented in Table 1.The temperature-growth response was investigated on CYA 16 investigated strains were isolated from compost or the air by recording diameters of three-point inoculated colo- of mushroom production farms, where Agaricus bisporus cul- nies of seven strains (CBS 122432, CBS 124296, CBS tivations were infected with smoky mould. The strains that 124297, DTO 032-A2, DTO 032-A3, DTO 173-C9 and were isolated in different years were from different outbreaks DTO 173-D1) after 14 days at 6–36 at 3 °C intervals and different production farms. No information is recorded at and 40 °C. Alphanumeric codes for conidial and re- farm level and strains isolated in the same year could be ob- verse colours refer to (Kornerup and Wanscher 1978). tained from more than one production location. Two of the Isolates were also examined for the production of alka- strains are legacy strains from previous outbreaks, HRI 1043- loids with Ehrlich reagent using the filter paper method D from the UK and the main strain mentioned in previous described by Lund (1995). Thegrowthrateoftwo P. literature (Fletcher et al. 1989;Grogan et al. 2000), and VM- hermansii strains (CBS 124296 and DTO 173-D4) and 2 from Canada. four other Penicillium species (P. brevicompactum DTO 099-D1; P. chrysogenum CBS 306.48; P. daleae DTO Phenotypic and physiologic characterisation 099-B8; P. glabrum, DTO 099-A6) was studied on an autoclaved compost based medium containing 250 g All pure cultured strains were inoculated at three points on Agaricus grown compost (phase III) and 15 g agar per Czapek yeast autolysate agar (CYA), CYA supplemented with litre. Table 1 Overview of P. hermansii isolates used in this study Culture collection number Substrate Location Year DTO 031-A3 = CBS 122432 = Mushroom compost (phase III) with smoky mould UK Around 1995 DAOM 242544 = HRI 1043-D = DC-283 DTO 194-C7 Mushroom compost with smoky mould The Netherlands 1997 DTO 194-C8 = CBS 132824 Mushroom compost with smoky mould The Netherlands 1997 DAOM 242545 = VM-2 Mushroom compost Nova Scotia, Canada 2000 DTO 032-A2 Mushroom compost with smoky mould The Netherlands 2006 DTO 032-A3 Mushroom compost with smoky mould The Netherlands 2006 DTO 079-D4 = IBT 29994 Mushroom compost with smoky mould The Netherlands 2008 DTO 079-D5 = CBS 124296 Mushroom compost with smoky mould, type The Netherlands 2008 DTO 079-D6 = CBS 124297 Air in mushroom growing room The Netherlands 2008 DTO 173-C9 Mushroom compost with smoky mould, second harvesting period The Netherlands 2011 DTO 173-D1 Mushroom compost with smoky mould, second harvesting period The Netherlands 2011 DTO 173-D2 Mushroom compost with smoky mould The Netherlands 2011 DTO 173-D3 Air in mushroom growing room The Netherlands 2011 DTO 173-D4 Air in mushroom growing room The Netherlands 2011 DTO 282-D3 Air in mushroom growing room The Netherlands 2013 DTO 282-D6 Air in mushroom growing room The Netherlands 2013 CBS, culture collection of the Westerdijk Institute; DC, disease collection at the Pennsylvania State University’s Mushroom Spawn Lab, USA; DTO, internal culture collection of department Applied and Industrial Mycology, housed at the Westerdijk Institute; HRI, culture collection of Warwick HRI, UK Mycol Progress DNA extraction, sequencing and phylogenetic Fig. 1. Nine smoky mould isolates were included in this anal- analysis ysis and those isolates form a unique clade within section Exilicaulis. Two well-supported clades are present in this sec- Strains were grown for 7–14 days at 25 °C on MEA tion and the smoky mould strains group in a cluster previously prior to DNA extraction. Genomic DNA was extracted named the Penicillium parvum-clade (Visagie et al. 2016). using the Ultraclean Microbial DNA Isolation Kit (MoBio Penicillium canis and P. striatisporum are basal to the clade Laboratories Inc., Carlsbad, USA) following the manufac- containing the smoky mould isolates; however, statistical sup- turer’s protocol. The ITS barcode and parts of the BenA, port is lacking. With the data available, it was not possible to CaM and RPB2 genes were amplified, sequenced and conclusively identify the phylogenetic sister species of the annotated according to the method described by Houbraken smoky mould isolates. and Samson (2011) and Houbraken et al. (2012). Newly gen- BenA, CaM and RPB2 gene sequences were generated to erated sequences are deposited in GenBank with accession confirm the phylogenetic coherence of the smoky mould numbers MG386210–MG386247 and MG333469– strains at the species level, and their relationship with other MG333479. species in the P. parvum-clade. The Tamura Nei (TN93) mod- Publically available BenA, CaM and RPB2 sequences of el using the gamma distribution (+G) was found to be optimal the type strains of species belonging to section Exilicaulis for the BenA data set (length 372 sites); and the TN93 were retrieved from GenBank and supplemented with the model using a discrete gamma distribution with invari- newly generated sequences. A concatenated dataset with ant sites (G+I) was optimal for the CaM (length 518 BenA, CaM and RPB2 sequences including nine smoky sites) and RPB2 (length 789 sites) datasets. The best- mould strains was generated and used to determine the phy- scoring ML trees are presented in Fig. 2 and show that logenetic relationship of the smoky mould strains with other the smoky mould strains form a coherent species level members of Penicillium section Exilicaulis.Singlegene clade, distinct from other members of the P. parvum- phylogenies were made by combining the generated se- clade. The smoky mould strains cluster together in trees quences from smoky mould isolates with sequences of based on all loci on a strongly supported branch (> 95% strains belonging to the P. parvum-clade (Visagie et al. bs, 1.00 pp). Most bootstrap percentages and posterior 2016)ofsect. Exilicaulis. The sequence data sets were probability (pp) values were low (< 70% bs, < 0.95 pp) in aligned using MAFFT (Katoh and Standley 2013). Prior these gene trees. to analysis, the optimal substitution model was deter- mined in MEGA v.6.06. (Tamura et al. 2013), utilising Morphology and physiology the Akaike information criterion (AIC). Maximum like- lihood (ML) analysis of the single gene datasets were Phenotypic characters of the smoky mould strains were re- analysed in MEGA 6.06 and the combined dataset in corded and compared with each other. All strains displayed RAxML-VI-HPC v. 7.0.3 (Stamatakis 2006)using the similar macro- and microscopical characters. Most strikingly, GTRGAMMA substitution model. The robustness of the isolates grew slow on CYA and MEA, even at an optimum the phylograms was evaluated by 1000 bootstrap (bs) growth temperature (27–30 °C). Furthermore, the conidio- runs. Bayesian inference (BI) was performed in MrBayes phores had short stipes (10–40 (− 120) μm) and the strains v.3.2.1 (Ronquist et al. 2012) using Markov chain did not grow on CYAS, and CYA and MEA incubated at Monte Carlo (MCMC) algorithm. The analysis stopped 37 °C. The optimum growth temperature on CYA was be- when the average standard deviation of split frequencies tween 27 and 30 °C, with a colony diameter of 15–18 mm reached 0.01. The sample frequency was set to 100; the after 14 days. The minimum growth temperature was first 25% of trees were removed as burnin. Convergence between 12 and 15 °C and the maximum growth tem- and ESS values of the runs were examined by Tracer 1.6 perature between 33 and 36 °C. Strains incubated at (Rambaut et al. 2014). 33 °C reached 11–14mm andnogrowthwas observed at 36 °C. The colony diameters of the smoky mould strains on autoclaved compost agar (CA) were similar Results to those on MEA (CA 9–12 vs MEA 9–12 mm). Also the growth density was similar on both media. The oth- Phylogeny er examined Penicillium species grew well on CA and their colony diameters were similar or slightly smaller The total length of the combined data set was 1729 characters than on MEA (P. brevicompactum,CA18–22 vs MEA (individual datasets: BenA 421; CaM,518; RPB2, 788 char- 20–24 mm; P. chrysogenum,CA40–43 vs MEA 30–35 mm; acters) and the proportion of gaps was 5.7%. The phylogram P. daleae,CA 24–27 vs MEA 34–37 mm and P. glabrum,CA was drawn to scale and the results of this analysis are shown in 38–40 vs MEA 41–45 mm). Mycol Progress Fig. 1 Best-scoring maximum likelihood tree using RAxML based on a combined dataset with partial BenA, CaM and RPB2 sequences, showing the relationship of species accommodated in section Exilicaulis. The phylogram is rooted with P. glabrum CBS 125543. Bootstrap percentages and posterior probability values are presented at the nodes. Values less than 70% bs or 0.95 pp are not shown and branches with more than 95% bs and 1.00 pp are thickened. The bar indicates the number of substitutions per site Taxonomy In: Penicillium subgenus Aspergilloides section Exilicaulis. Based on phylogenetic coherence of the smoky mould causing Etymology: Named after Con Hermans, who studied the isolates and their unique phenotypic and physiologic charac- occurrence of smoky mould and other competing fungi in ters, we describe them here as a new species named the Netherlands. Penicillium hermansii sp. nov. Diagnosis: Slow growth on CYA (5–10 mm) and MEA (8– 15 mm) at 25 °C after 7 days incubation; no growth on CYAS; Penicillium hermansii Houbraken, Seifert & Samson, sp. no growth on MEA and CYA incubated at 37 °C; conidio- nov. Mycobank MB823949 (Fig. 3). phores biverticillate and short-stiped, 10–40 (−120) μm. Mycol Progress Fig. 2 Maximum likelihood (ML) trees generated for BenA, CaM and presented at the nodes. Values less than 70% bs or 0.95 pp are not shown RPB2 with MEGA6. The phylograms are rooted with P. corylophilum and branches with more than 95% bs and 1.00 pp are thickened. The bar CBS 312.48. Bootstrap percentages and posterior probability values are indicates the number of substitutions per site Typus: the Netherlands, ex compost with Agaricus metulae, and sometimes subterminal or intercalary metulae bisporus, 2008, collected by C. Hermans, isolated by J. present; in degenerated strains monoverticillate conidiophores Houbraken (holotype CBS H-21028, culture ex-type CBS predominantly present. Terminal and subterminal metulae un- 124296 = DTO 079-D5). equal in length, 10–20 (− 25) × 2.0–3.5 μm, often with inflat- ITS barcode: ITS = MG333472 (alternative markers: ed apex, 3–5 μm wide. Phialides ampulliform, in verticils of BenA = MG386214, CaM = MG386229, RPB2 = 4–10, closely packed, 7–9×2–3.5 μm. Conidia globose to MG386242). All examined P. hermansii strains share identical subglobose, smooth-walled, 2.0–2.5 μm. Ascomata and scle- ITS, BenA, CaM and RPB2 sequences. The species can be rotia not observed. differentiated from other known Penicillium species by ITS sequences. Distribution and ecology: The Netherlands, United Discussion Kingdom, Canada. Isolated from compost colonised by Agaricus bisporus and from the air of white button mushroom Grogan et al. (2001) expressed uncertainty about the identity production farms. of the smoky mould fungus in mushroom cultivation because Colony diameter: 7 days, in mm, 25 °C (unless stated oth- taxonomic authorities that they consulted did not agree on a erwise): CYA 5–10; CYA15°C no growth or spore germina- common identification. Most identifications of smoky mould tion; CYA 30 °C 7–12; CYA 37 °C: no growth; MEA 8–15; isolates were performed before the era of DNA sequence- YES 5–9; DG18 3–7; CYAS no growth; OA 10–15; creatine based identification. Grogan et al. (2000) studied the identity agar no growth or spore germination. Optimum growth tem- of four smoky mould isolates (including isolate HRI 1043-D) perature on CYA 27–30 °C. using ITS sequences, but the exact identification of the species Colony characters: Sporulation on CYA absent to moder- involved in smoky mould problems remained uncertain. As ate; colonies entire, plane, velvety; mycelium white, sporula- demonstrated in this paper, the smoky mould isolates also turn tion grey-green to greyish-brown green (~ 5D3); exudate ab- out be a previously undescribed species. Although several sent; soluble pigments absent; colony reverse brown (5C3– Penicillium species (e.g. P. chermesinum, P. citreonigrum, P. E3). Soluble pigments on YES absent, sporulation light, implicatum and P. fellutanum) were named as causal agents of (pale) grey-green or inconspicuous; exudate absent, reverse smoky mould, we suggest that these outbreaks were probably brown. Colonies on MEA velvety, dull green or grey-green all caused by P. hermansii alone. Strains referred to as smoky (25–26E3), reverse brown (5D3). Ehrlich reaction negative. mould in the literature share a slow growth rate on agar media Micromorphology: Conidiophores borne from surface; and most were reported to be monoverticillate (Baars et al. slightly vesiculate; stipes short, 10–40 (− 120) × 2–3.5 μm, 2011;Beyer 2002; Fletcher and Gaze 2008). Penicillium with walls smooth or very finely roughened; conidiophores hermansii also grows slowly, but fresh isolates have terminal of freshly isolated strains bearing terminal verticils of 2–4 verticils of 2–4 metulae. Mycol Progress Mycol Progress Fig. 3 Penicillium hermansii, a 7 day-old cultures, 25 °C, left to right; Trichoderma aggressivum. This species grows quickly first row, all obverse, CYA, YES, DG18, MEA; second row, CYA on agar media and seems to compete with Agaricus reverse, YES reverse, OA obverse and CREA obverse. b–e for space and nutrients effectively. The association of Conidiophores. f Conidia. Scale bars = 10 μm T. aggressivum with Agaricus production is probably first based on chemical communication via extrolites, Phylogenetically, P. hermansii is most closely related to P. including volatiles, and/or extracellular enzymes. For canis, P. catenatum, P. erubescens, P. guttulosum, P. example, the mycelium of A. bisporus is required for menonorum, P. nepalense, P. papuanum, P. parvum, P. induction of intensive sporulation in T. aggressivum pimiteouiense, P. rubidurum, P. striatisporum and P. vinaceum (Krupke et al. 2003; Mamoun et al. 2000; Mumpuni et al. that all align to the P. parvum-clade of section Exilicaulis. 1998;Seaby 1987;Seaby 1996). Similarly, Beyer (2002)in- These species share a slow growth rate on CYA and MEA dicated an interaction between the mycelium of Agaricus and and produce short-stiped, monoverticillate conidiophores. smoky mould and suggested that the Agaricus is either Like the other members of this clade, P. hermansii also grows parasitised or repressed. More research is needed to under- slowly on agar media and along with P. nepalense and P. stand the biology of smoky mould in button mushroom catenatum is among the slowest growing species of the clade. cultivation. Morphologically, it differs from all other species of this clade Acknowledgements We thank Nancy Nickerson, Robert Davies and by the production of biverticillate conidiophores, with Leonard North for supplying the Canadian isolate of P. hermansii and monoverticillate conidiophores only seen in older, presum- Gerry Louis-Seize for early phylogenetic studies of that strain. ably degenerated strains. Penicillium hermansii also differs from most of the phylogenetically related species by its inabil- Compliance with ethical standards ity to grow at 37 °C, a character shared only with P. nepalense (Pitt 1980 [‘1979’]; Takada and Udagawa 1983). Using the Conflict of interest The authors declare that they have no conflict of dichotomous key published in the Penicillium monograph of interest. Pitt (1980 [‘1979’]), P. hermansii keys out as P. fellutanum. Open Access This article is distributed under the terms of the Creative That species most obviously differs from P. hermansii by its Commons Attribution 4.0 International License (http:// faster growth rate on CYA (5–10 vs 17–25 mm). creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- In addition to its distinctive phenotype, P. hermansii is also priate credit to the original author(s) and the source, provide a link to the unique in Penicillium for its association with Agaricus- Creative Commons license, and indicate if changes were made. colonised compost. Until now, this species has never been isolated from other habitats, despite extensive surveys of soil, food, indoor air and other substrates from all over the world. Strong associations between Penicillium species and particu- lar habitats have been known for a long time (Westerdijk References 1949). For example, P. italicum, P. ulaiense and P. digitatum are associated with rot of citrus fruits and P. allii is strongly Baars J, Rutjens J, Mumm R (2011) Can volatiles emitted by compost associated with rot of garlic. Generally, Penicillium species during spawn run be used to detect green mould infection early? In: with a specific association to certain substrates grow well on Savoie J-M, Foulongne-Oriol M, Largeteau M, Barroso, G. (eds) Proceedings of the 7th International Conference on Mushroom standard laboratory media and do not require special com- Biology and Mushroom Products (ICMBMP7), vol 1. INRA, pounds from its associated natural source, e.g. citrus peels or Villenave d’Ornon Cedex, pp 474–483 garlic (Frisvad and Samson 2004). In contrast, P. hermansii Beyer DM (2002) Pest species biology and control—weed and indicator grows slowly on general purpose agar media CYA, MEA and moulds. In: Mushroom integrated pest management handbook. 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Stud Mycol:1–173 occur in compost and seem to have little or no effect on Grogan H, Harvey L (1999) The effects of compost moulds in mushroom Agaricus growth (Grogan et al. 2001). compost. Horticulture Research International, Warwick Very little work has been carried out on competition with Grogan HM, Scruby A, Harvey L (2000) Moulds in spawn-run compost Agaricus by P. hermansii and most research on compet- and their effect on mushroom production. Science and Cultivation of Edible Fungi, Vols 1 and 2:609–615 ing fungi in Agaricus production has focused on Mycol Progress Grogan H, Parker L, Scruby A (2001) Survey of compost moulds in O’Brien M, Kavanagh K, Grogan H (2017) Detection of Trichoderma aggressivum in bulk phase III substrate and the effect of T. traditional and bulk Phase III (spawn-run) compost. 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Journal

Mycological ProgressSpringer Journals

Published: May 30, 2018

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