Characterization of cultural traits and fungicidal activity of strains belonging to the fungal genus Chaetomium

Compare and characterize Chaetomium strains with special regard to their potentialities as biocontrol agents.


Introduction
Members of the fungal genus Chaetomium are generally cosmopolitan and reside in soil on cellulose-rich materials or on dung (Ahmed et al. 2016). Chaetomium species are, however, also present in many other compartments of the environment. They are, for example, found associated with living plants, for example, as endophytes in Ginkgo biloba and Pinus sp. (Skolko and Groves 1953;Qin et al. 2009;Mart ınez-Alvarez et al. 2016), on waterdamaged building-materials (Wang et al. 2016a), on ancient works of arts (Coronado-Ruiz et al. 2018), and in extreme habitats such as deserts or salt lakes (Murgia et al. 2018;Abdel-Azeem 2020). Since the establishment of the genus more than 300 species have been described (Asgari and Zare 2011). In 2019, the Index Fungorum (www.indexfungorum.org) listed 273 accepted species names (Abdel-Azeem 2020). Both a high degree of competitiveness towards other micro-organisms (Chang and Kommendhal 1968;Hubbard et al. 1982) and the ability to form a large number of bioactive metabolites, including some with fungicidal activity, indicate that certain species of the genus may be interesting candidates for the biological control of plant pathogens (Madbouly and Abdel-Wareth 2020).
Indeed, suppression of plant pathogens by spore suspensions, filtrates, biochemical fractions or formulated materials from different Chaetomium species has been recorded not only in vitro, but also on detached leaves, in the greenhouse as well as in the field. For example, an isolate of C. cochliodes controlled Fusarium blight of oat seedlings when added to seeds or to the soil, both in greenhouse and field experiments (Tveit and Wood 1955). Strains of C. globosum reduced the development of tan spot (causal agent Pyrenophora tritici-repentis; Istifadah and McGee 2006; Larran et al. 2016) and spot blotch (Bipolaris sorokiniana) on potted wheat, and of Northern Corn Leaf Blight (Setosphaeria turcica) on detached maize leaves (Zhang et al. 2013). A commercially available product based on pellet and powder formulations of both C. cupreum and C. globosum suppressed a number of pathogens on different crops (Soytong et al. 2001). Under greenhouse conditions, application of spores and methanol extracts of Chaetomium globosum, Chaetomium lucknowense and Chaetomium cupreum to pomelo seedlings inoculated with Phytophthora nicotianae reduced the extent of root rot and increased plant weight (Hung et al. 2015a), and a bioformulation of C. elatum controlled Fusarium wilt of tomato (Soytong 2015).
Parasitism and antibiosis were identified as causative for the suppression of Cochliobolus sativum (= B. sorokiniana) by C. globosum (Aggarwall et al. 2004). Phytophthora nicotianae was inhibited by three different species of Chaetomium (Hung et al. 2015b) although no evidence of direct parasitism was recorded for C. globosum suppressing Botrytis aclada (K€ ohl et al. 1997). Chaetomium species are known to produce more than 300 secondary metabolites with enormous structural variation (Hamed et al. 2020) and several of these have been implicated in the biocontrol of plant pests and pathogens (Madbouly and Abdel-Wareth 2020). Among this variety of metabolites several types of bioactive compounds have been identified, such as the chaetoglobins (Ge et al. 2008), xanthenone (Pontius et al. 2008), orsellides (Schl€ orke and Zeeck 2006), polyhydroxylated steroids (Zhang et al. 2009), armochaetoglobins (Chen et al. 2015) and azaphilones (Yamada et al. 2011). Some of these natural products possess not only antifungal activity, but also anti-inflammatory, cytotoxic and enzyme inhibiting properties (Li et al. 2016). In addition to bioactive secondary metabolites, extracellular enzymes produced by Chaetomium species are suspected to contribute to the antagonism towards plant pathogens (Sun et al. 2006;Raguchander et al. 2014). More information on secondary metabolites of Chaetomium species and compounds suspected to be involved in the interaction with plant pathogens are presented in recent research papers and reviews (e.g. Yan et al. 2018Yan et al. , 2019Abdel-Azeem 2020).
For the legal registration of biocontrol agents, information on the biological properties of their active ingredients must be supplied. This includes the potential toxicity and pathogenicity towards humans and animals. Both may be critical especially in the case of Chaetomium, since some of its metabolites are potentially harmful (Ohtsubo et al. 1978;Sekita et al. 1981), and several species of the Chaetomiaceae have been reported to cause serious opportunistic infections in immunocompromised patients. Species able to grow at body temperature may break the thermal exclusionary zone of the human body and emerge as potential pathogens in immunocompromised humans (Ahmed et al. 2016). Furthermore, biocontrol agents based on living organisms can only function under appropriate temperature conditions. For this reason, candidate biocontrol strains should be checked for their temperature requirements early in the development process.
In this study, 12 strains of Chaetomium isolated from different habitats and environments were evaluated. The strains were taxonomically identified and characterized regarding cultural characteristics and temperature requirements for mycelial growth. The potential as biocontrol agents was assessed based on the inhibition of phytopathogens by culture filtrates of the Chaetomium strains. Ethyl acetate extracts from culture filtrates were separated by semi-preparative HPLC and the antifungal activity of the resulting fractions was determined in bioassays. The UV spectra of fractions with antifungal activity were analysed for the presence of known natural products.

Fungal isolates
Twelve strains of Chaetomium were tested (Table 1). Of these, six were supplied by the JKI Fungal Culture Collection (maintained by JKI Institute for Epidemiology and Pathogen Diagnostics, Braunschweig). They were originally isolated from different substrates and deposited in the collection under the species names listed (Table 1). The other six Chaetomium strains were isolated in the context of this analysis from plant material after surfacedisinfection and placement on PDA. The disinfection procedure included immersion in 96% EtOH for 1 min, followed by 1% NaOCl for 5 min (potato slices, leaves) or 10 min (wheat kernels) and several washes in sterile distilled water. All Chaetomium strains were maintained at room temperature on potato dextrose agar (PDA; Sigma Aldrich, Darmstadt, Germany

Molecular species determination of Chaetomium sp. isolates
Fungal isolates of Chaetomium sp. were grown on 2% (w/v) PDA at 24°C in darkness for 14 days. Fungal mycelia were scraped from the Petri dishes and ground with 0Á25 mm glass beads in 2 ml tubes in the Precellys24 (Peqlab, Erlangen, Germany), followed by DNA extraction using the DNeasy Plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration was determined photometrically using the Nanodrop (Peqlab). For differentiating Chaetomium sp., three barcodes were used, based on the results of Wang et al. (2016a). In detail, b-tubulin (primers T1 and T22;O'Donnell and Cigelnik 1997) and RNA polymerase II second largest subunit (primers RPB2AM-1bf and RPB2AM-7R; Miller and Huhndorf 2005) were used. Additionally, the internal transcribed spacer region was included with the primers ITS-1 and ITS-4 as described in White et al. (1990). PCR conditions were set as follows: 5 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 55°C and 45 s at 72°C, followed by a final extension of 5 min at 72°C. PCR fragment size and single product amplification was checked by gel electrophoresis. PCR products were cleaned up with the ExoSAP-IT PCR Product Cleanup Reagent (Thermo Fisher, Berlin, Germany) and sent for sequencing to LGC Genomics (Berlin, Germany). For tree construction and alignment purposes each PCR reaction was done in three independent replicates. After removal of sequence parts of minor quality, a consensus sequence was determined that was used for further analyses. For genotyping, one PCR reaction of good quality was sufficient that was sequenced in both directions. The highest BLAST hit was taken for species determination. In case more than one hit showed equal high probability, all most likely results were used, as shown in the results section (Table 2).

Microscopy
Ascospores of the Chaetomium strains were scraped from sporulating cultures, mounted in water and viewed under differential interference contrast optics of a Zeiss Axioscope 2 light microscope. Ascospores were photographed with a Zeiss Axiocam 105 color digital camera. The width and length of 120 ascospores per strain were determined using the camera software.

Determination of effect of temperature on mycelial growth
Mycelial plugs (7 mm diameter) were cut from 7-to 14day-old actively growing colonies of each of the Chaetomium strains on PDA using a cork borer and placed in the centre of fresh PDA plates. The inoculated plates were incubated in darkness in microbiological incubators set at 15, 20, 25, 30 or 35°C. Three plates were evaluated per strain-temperature combination. Every other day, colony diameters were recorded along two perpendicular lines until the mycelium reached the edge of the plate, or for the more slowly growing colonies for a duration of maximal 14 days. For each plate, mean colony diameters (minus the diameter of the inoculum plug) were calculated and used to compute the means and standard deviation for the three plates. The experiments on effect of temperature on mycelial growth as well as all other experiments described below were performed at least twice.

Dual cultures
Four mycelial plugs (7 mm diameter) from actively growing colonies on PDA of P. graminea or B. sorokiniana respectively were placed equidistantly on three plates each with synthetic nutrient poor agar (SNA) (Nirenberg and O'Donnell 1998), about 2 cm from the edge of the plates. One mycelial plug of each of the Chaetomium strains was placed in the centre of the plates. The plates were incubated in the dark at room temperature and photographs were taken after 5 days (P. graminea) or 10 days (B. sorokiniana) after inoculation.

Production of culture filtrates and assessment of antifungal activities
Culture filtrates were produced on a rotary shaker in potato dextrose broth (PDB; Roth, Karlsruhe, Germany). Erlenmeyer flasks (300 ml) containing 50 ml autoclaved PDB were inoculated with three mycelial plugs from sporulating Chaetomium plates and placed for 14 days on a rotary shaker at 25°C and 150 rev min À1 . Following separation of the culture filtrate from the mycelium with a B€ uchner funnel using paper filter and vacuum suction, the filtrates were sterilized by passing through Rotilabo â syringe filters (0Á45 µm pore size) (Roth) and added to molten PDA shortly before solidification to obtain a final concentration of 10%. Three plates each were inoculated in the centre with mycelial plugs from actively growing cultures of the phytopathogenic fungi listed in the section 'Fungal isolates'. The controls were PDA plates not amended with culture filtrate. The inoculated plates were incubated at 20°C, and mycelial diameters were determined at regular intervals as described above. Inhibitory activity (%) of the culture filtrates was computed in relation to the mycelial diameters on the control plates using the formula: (diameter on control plateÀdiameter on amended plate)/diameter on control plate 9 100%. Additionally, fungicidal activity of the culture filtrates was assessed in microplates at concentrations of 10 and 30%. Conidia suspensions from B. cinerea, B. fabae, B. sorokiniana or F. culmorum for inoculation of the microplates were obtained from sporulating cultures on PDA. About 5 ml of PDB were pipetted onto the cultures, followed by rubbing of the surface with a spatula. The resulting conidial suspensions were passed through gauze (MUllro â ; Hartmann) and the concentration of conidia was adjusted to 5 9 10 4 ml À1 with a haemocytometer. In the case of P. infestans, suspensions of sporangia were obtained from cultures on rye agar and also adjusted to 5 9 10 4 ml À1 . For preparation of rye agar 100 g of rye seeds were autoclaved, left to soak overnight and passed through cheesecloth. The mucilage was resuspended in water to 500 ml, and 2Á5 g glucose and 6 g agar were added. The medium was autoclaved and poured into Petri dishes. Filtrates of 14-day-old Chaetomium cultures were diluted in a ratio 3 : 7 with PDB containing conidia of the respective pathogens to obtain a final concentration of 30% culture filtrate. Filtrate concentrations of 10% were prepared by adding to concentrated culture filtrates PDB (P. infestans: pea-juice medium) and PDB containing conidia (P. infestans: pea juice medium containing sporangia) in a ratio 1 : 2 : 7. Aliquots of 200 µl were pipetted into three wells each of 96-well microplates.
The controls were wells with 200 µl PDB or pea-juice medium containing fungal conidia or sporangia of P. infestans; respectively without culture filtrate added. The microwell plates were incubated for 2-7 days at room temperature before fungal growth was rated visually or under a dissecting microscope according to the following differentiation: no inhibition (as in controls); amount of mycelium in wells increasingly reduced; conidia (P. infestans: sporangia) ungerminated or with only short mycelial strands.

Determination of fungicidal activity of ethyl-acetate extracts in multi-well plates
Cultures of the strains C. globosum 1, C. cochliodes 3, C. aureum, C. nozdrenkoae, C. ramosissimum and C. elatum were raised by adding 12 agar plugs from freshly grown plates to 200 ml PDB in 1-l flasks and passed through a B€ uchner funnel as described above. Using a separating funnel, the filtrates were extracted three times with ethylacetate (volumes: 150, 100, 100 ml). The ethyl-acetate fractions were combined and evaporated to dryness with a rotary evaporator. The dried crude extracts thus obtained (corresponding to 200 ml liquid fungal culture) were dissolved in 4 ml 99% ethanol (stock solution). Aliquots from stock solutions were diluted with ethanol in a ratio 1 : 4, and 4 µl each were pipetted into three wells each of 96-well microtiter plates (well volume 300 µl), and 296 µl PDB or pea juice medium containing fungal conidia or sporangia of P. infestans respectively were added to each well. After incubation for 2-7 days at room temperature, fungal growth was assessed as described above.

HPLC fractionation and profiling of crude extracts
The dried crude extracts of the fermentation of C. globosum 1, C. cochliodes 3, C. aureum and C. nozdrenkoae were dissolved in acetonitrile and analysed by HPLC (Agilent 1100 Series) equipped with a LiChrospher RP 18 column (4 9 125 mm; 5 lm; Merck, Darmstadt, Germany). The column was used at 40°C with a flow rate of 1 ml min À1 . An elution gradient was used composed of H 2 O and acetonitrile, starting from 99% H 2 O to 100% acetonitrile over a period of 20 min and then the 100% acetonitrile-flow was continued for further 4 min. The compounds were detected via a diode array detector. For separation of the crude extracts, the same method was used and a fraction collector (Agilent 1100 Series) was used to collect eluted samples. The use of 96-well plates for collecting the flow through resulted in 92 different fractions of 250 µl from each extract. The solvent of each fraction was then evaporated. In the bioassays, 6 µl methanol (P. infestans: 4 µl ethanol) were pipetted into each well and 194 µl PDB containing fungal conidia (P. infestans: 196 µl pea juice medium containing sporangia) were added. Incubation of the plates and assessment of inhibitions were as described above.
The molecular weight of the selected peaks was determined using a HPLC-MS (Agilent 1260 Series LC and 6130 Series Quadrupole MS System; Agilent, Santa Clara, CA). The mass spectra were recorded using atmospheric pressure chemical ionization with positive and negative polarization. A Superspher RP 18 (125 9 2 mm; 4 lm; Merck) column was used at 40°C. For every run, 1 ll of a sample at a concentration of 1 mg ml À1 was injected. The elution was performed with a gradient of H 2 O and acetonitrile and a flow rate of 0Á45 ml min À1 .

Molecular species determination and biology of the Chaetomium isolates
Molecular species determination of Chaetomium sp. was done with three different genotyping markers. When BLAST results named several species with a similar high probability, all hits were listed (Table 2). Except for the C. globosum isolates, species identification with the ITSmarker was rather unspecific, leading to up to twelve equally high hits in BLAST. The primer pairs for the tubulin and rpb-2 (RNA polymerase II second largest subunit) loci were much more specific and mostly resulted in only one or two distinct most likely hits in BLAST. Combining the results obtained with the three primer pairs allowed us to assign the strains to the species C. globosum, C. cochliodes, C. aureum, C. nozdrenkoae and C. ramosissimum.
The ascospores of C. globosum and C. cochliodes were very similar in shape and size (C. globosum: minimal width 7Á3 µm, maximal length 10Á0 µm; C. cochliodes: Based on their morphology, the ascospores of the single isolates of C. aureum, C. ramosissimum and C. elatum were distinguishable from each other as well as from those of C. globosum and C. cochliodes. The ascospores of C. globosum, C. cochliodes, C. elatum and C. ramosissimum were limoniform and bilaterally flattened, while those of C. aureum were rather spindle shaped (Fig. S1).
The isolate of C. nozdrenkoae failed to produce perithecia and ascospores on PDA, malt extract agar and a number of other agar media tested (not shown).
On PDA plates (Figs S2a-e and S3a), as well as in shake culture in PDB (Fig. 2) all isolates of C. globosum produced a typical brown pigment. C. aureum produced a red pigment that stained PDA plates red (Figs S2i and S3b) and made the culture filtrate appear dark red to almost black. The culture filtrates of C. cochliodes, C. nozdrenkoae and C. ramosissium were more or less yellow, while the filtrate from C. elatum appeared translucent with no pigmentation (Fig. 2).
Temperature affected the growth of Chaetomium strains on PDA differently ( Fig. 3 and Fig. S3). For C. ramosissimum the speed of mycelial growth increased with incubation temperature, accordingly the slowest and fastest growth was at 15 and 35°C respectively (Fig. 3a). The growth pattern of C. nozdrenkoae was similar to that of C. elatum, but the maximal growth rate was recorded at 25/30°C (Fig. 3b). C. elatum ( Fig. 3c and Fig. S3d) and strains C. cochliodes 1-3 (not shown) were unable to grow at 35°C and reached their maximal speed of growth at 20/25°C (C. elatum, C. cochliodes 1) or at 25°C (C. cochliodes 2 and 3). All five isolates of C. globosum had their slowest mycelial growth at 35°C (Fig. S3a). Mycelial growth was fastest at 30°C (C. globosum 1; Fig. 3d) or at 20°C (C. globosum 2-4; Fig. 3e). C. aureum was unique in that it grew comparatively slow at all incubation temperatures (Fig. 3f).

The isolates produce biological active natural products
To determine the ability of the different Chaetomium strains to produce antifungal compounds, filtrates from shake cultures were added to PDA in Petri dishes. The growth of different phytopathogenic fungi on these media was then compared to growth on unamended plates (Fig. 4). For the filtrates from C. globosum, a clear inhibitory activity (>50%) was observed towards B. cinerea and P. graminea. Inhibition of B. sorokiniana was below 20% and around 30% in the case of F. culmorum and P. infestans (Fig. 4a). This pattern was strikingly similar for all five isolates of C. globosum tested. As an example, inhibition by the filtrates from isolate C. globosum 1 is shown in Fig. 4a. The effect of the culture filtrates was also similar among the three isolates of C. cochliodes that all caused a moderate inhibition of the growth of P. infestans by around 35-40%, as exemplified by isolate C. cochliodes 3 (Fig. 4b). Growth of the other fungi was either not affected or the degree of inhibition was below 15%. The culture filtrates from C. aureum had a similar broad antifungal spectrum as those from C. globosum. However, the activity was strongest against P. infestans but only moderate against B. cinerea and D. graminea. In addition, the filtrates from C. aureum caused a considerable inhibition of growth of F. culmorum and F. oxysporum (Fig. 4c). Interestingly, F. solani was always less affected than F. culmorum and F. oxysporum. The filtrates from C. nodzdrenkoae showed activity only against the mycelial growth of P. infestans (Fig. 4d), whereas those from C. ramosissimum failed to inhibit the growth of any of the fungi tested (Fig. 4e). In the tests with the filtrates from C. elatum mycelial growth of P. infestans was inhibited by around 30% (Fig. 4f). Inhibition of the other tested fungi was below 20% and a high variability was observed in the case of F. culmorum. Inhibition of the mycelial growth of P. graminea by C. globosum was also seen in dual culture plates. In these tests, the inhibitory activity of all five strains of C. globosum was very similar (Fig. S4a-e). The moderate inhibition of D. graminea by C. aureum observed on filtrateamended agar plates was not apparent in dual cultures (Fig. S4i). Bipolaris sorokiniana was strongly inhibited in dual culture with C. globosum. Bipolaris sorokiniana was equally strongly affected by all five isolates of C. globosum (exemplified by isolate C. globosum 1 in Fig. S4).
In further tests performed in 96-well plates, culture filtrates at concentrations of 30 or 10% were inoculated with conidia of B. fabae, B. cinerea, F. culmorum and B. sorokiniana (first set of experiments; Table 3) or sporangia of P. infestans (second set of experiments; Table 4). In these tests, filtrate of C. globosum 1 served as an example of the C. globosum strains and the filtrates of C. cochliodes 1 and 3 respectively were used as representatives of the C. cochliodes strains. In the first set of experiments, a moderate to strong effect on B. fabae, B. cinerea, F. culmorum and D. graminea was recorded for C. globosum 1. The filtrate from C. aureum caused a clear inhibition of F. culmorum and D. graminea, but had just a mild effect on B. fabae and B. cinerea. A mild effect on F. culmorum was also recorded for the filtrate from C. cochliodes. Generally, the inhibitory effects observed in these experiments were dose-dependent. No or only very weak effects were observed with all other filtrates (Table 3). Dose dependency of the effects of the culture filtrates was also observed with P. infestans. All culture filtrates tested caused a moderate to strong inhibition of P. infestans. Autoclaving of the filtrates tended to reduce the inhibitory activity (Table 4).
Ethyl acetate extracts of C. globosum 1 caused a moderate to strong inhibition of B. cinerea and P. infestans and a low inhibition of F. culmorum (Table 5). The C. cochliodes extract showed a high activity only against P. infestans, while the extract from culture filtrate from C. aureum inhibited the growth of all the three test organisms. The extracts from C. nozdrenkoae, C. ramosissimum and C. elatum were inactive against B. cinerea and F. culmorum and had only a very low inhibitory effect on P. infestans.

Fractionation of biological active extracts
Ethyl acetate extracts were separated by HPLC and the fractions were deposited into 92 wells of micro plates. Four wells served as controls. In the bioassays with the HPLC fractions from C. globosum, inhibition of the growth of B. cinerea, B. sorokiniana, F. culmorum and P. infestans was recorded within wells D1-D2 and E4-E6 (Figs 5a,c and 8a). The strongest and highly reproducible activity was found by inhibition of B. sorokiniana and B. cinerea and in wells E4-6 (Fig. 5a,c). The HPLC signals corresponding to the wells D1-D2 and E4-E6 were found to be related to the group of the chaetoglobosines and these are responsible for the antifungal activity in the crude extracts of C. globosum (Fig. 5b). The assumption that the chaetoglobosines are the biologically active compounds within the wells D1-D2 and E4-E6 from the fractionated extract was supported by the 3D-chromatogram (Fig. 5d). Apart from the UV-spectra of the chaetoglobosin class of compounds, there were no other signals detectable in the range between 200 and 600 nm wavelength at the respective retention times and thus in the wells D1-D2 and E4-E6. The fractions from C. cochliodes 3 caused a strong inhibition of P. infestans in wells E1-10 and inhibited F. culmorum, B. sorokiniana and B. cinerea within wells E3-E9 (Fig. 8b). In the bioassays with fractionated extracts from C. aureum, the test organism P. infestans was strongly inhibited in the wells A9-A12 and B10-B12 (Figs 6a and 8c). The signals and UV-spectra, which were found to correspond to the compounds responsible for the biological activity, did not match to any of the natural products in our libraries (Fig. 6b,c).

(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l)
Thus, the compounds could not yet be identified. The reddish-brown colour on the well bottom suggested that the dark red pigment of C. aureum, reported previously as oosporein (Taniguchi et al. 1984), was deposited in these wells. The UV-spectra indicated the presence of oosporeine, but due to smearing and interference with signals from other compounds an unequivocal identification of oosporein was not possible. The involvement of oosporein in the observed inhibition of P. infestans appears nevertheless likely. In case of the bioassay with the fractionated extracts from C. aureum against F. culmorum the inhibition was more or less weak. The sporulation of F. culmorum was reduced whereas mycelial growth was little affected, extended over several wells and was only visible under a dissecting microscope at the beginning of the incubation period, which made interpretation of the results difficult (Figs 7a and 8c). Identification of the compounds potentially responsible for the biological activity was not possible since the signals and UV-spectra in the corresponding wells did not match with any natural product in our library (Fig. 7b,c). Furthermore, in the bioassays with fractions of the extract from C. nozdrenkoae and B. cinerea, F. culmorum and P. infestans, fungicidal activity was not observed, which fits to the results obtained in the bioassays with

c i n e r e a P . g r a m i n e a B . s o r o k i n i a n a F . o x y s p o r u m F . c u l m o r u m F . s o l a n i P . i n f e s t a n s B . c i n e r e a P . g r a m i n e a B . s o r o k i n i a n a F . o x y s p o r u m F . c u l m o r u m F . s o l a n i P . i n f e s t a n s B . c i n e r e a P . g r a m i n e a B . s o r o k i n i a n a F . o x y s p o r u m F . c u l m o r u m F . s o l a n i P . i n f e s t a n s B . c i n e r e a P . g r a m i n e a B . s o r o k i n i a n a F . o x y s p o r u m F . c u l m o r u m
F . s o l a n i P . i n f e s t a n s  Table 5.

Discussion
Molecular identification of the 12 Chaetomium strains was achieved by PCR and sequencing of the amplicons using primers targeting the ITS, tubulin and rpb2 genes (Wang et al. 2016a). The specificity differed among the primers, where the ITS-primer is the least specific. Taken together, the use of three primer pairs based on three different loci was necessary and sufficient to resolve the identity of the different Chaetomium strains down to the species level. The results of the molecular identification were supported by microscopic observations on the size and shape of the ascospores. The morphological characteristics match with descriptions in the taxonomic literature (Arx et al. 1986;Wang et al. 2016b). For five of the isolates obtained from the JKI Culture Collection, the previous identification based on morphology could be confirmed. However, the strain identified as C. ramosissimum in the present study was deposited in the collection in 1975 under the name C. indicum. This apparent discrepancy can be explained by taxonomic changes, since Wang et al. (2014) recognised C. ramosissimum as separate species within the C. indicum group. As optimum and maximum temperatures for growth, these authors state 24 and 30°C for C. indicum and 30 and 41°C for C. ramosissimum respectively. This is in agreement with the observation that growth of our C. ramosissimum strain was substantially faster at 30 and 35°C than at 25°C. Also, the limoniform shape of the ascospores and their size correspond well to the data given by Wang C. globosum 1 ++/+++ ++ ++/+++ ++ +/++ +/++ +++ ++ C. cochliodes 3 AE À AE À + À AE AE C. aureum + + + + +++ ++ +++ + C. nozdrenkoae À À À À À À À À C. ramosissimum AE À À À AE À AE À C. elatum À À À À AE À AE À À no inhibition, as in controls; AE, +, ++ amount of mycelium in wells increasingly reduced; +++ conidia nongerminated or with only short mycelial strands.
et al. (2014) and indicated C. ramosissimum as the correct taxon.
The formation of extracellular pigments with various biological functions is known from many fungi (Caro et al. 2016) and has also been reported for strains and species of Chaetomium (Brewer et al. 1968;Cooke and Collins 1970;Rodr ıguez et al. 2002). In our study, the culture filtrates from C. aureum and from all five strains of C. globosum were stained deep red and brown respectively. Formation of a number of red acidic metabolites on PDA has also been reported for C. aureum strain MF-91 . According to Taniguchi et al. (1984), the red pigment produced by C. aureum is oosporein. The nature of the brown pigment from C. globosum is as yet unknown. Hu et al. (2012) reported interruption of pigment production of C. globosum by a specific inhibitor of the biosynthetic pathway for melanin.
Most Chaetomium species are mesophilic, with an optimal temperature range of 25-28°C (Prokhorov and Linnik 2011). Among the incubation temperatures tested, 35°C was the optimum for C. ramosissimum. The ability of a Chaetomium species to grow near to the temperature of 37°C is used as an indicator for their potential to infect humans (Wang et al. 2014). From the toxicological point of view, the use as biocontrol agents of such strains must be evaluated critically. In contrast to C. ramosissimum, C. elatum and C. cochliodes ceased their growth at 35°C. For C. elatum this observation is in agreement with previous studies (Asgari and Zare 2011;Prokhorov and Linnik 2011). The isolates of C. globosum were able to grow at 35°C in our study, but growth was strongly restricted compared to the other temperatures employed. Based on the observation of a low radial growth, morphological changes in ascocarp development and absence of mature ascocarps, Prokhorov and Linnik (2011) concluded that temperatures of 33°C or higher were unfavourable for growth of C. globosum. With this in mind, the species C. globosum has been assigned to risk group I, the lowest of four groups categorizing the risk of C. aureum grew comparatively slow at all temperatures in our study, with maximal growth at 25°C. This is in contrast to C. aureum MF-91 that was reported to grow rapidly on PDA with the maximum at 35°C .
On agar medium amended with culture filtrate, an inhibition of P. infestans was recorded for all Chaetomium strains except for C. elatum, whereas in microplates the inhibition was observed also for the filtrate from C. elatum. Components with activity against P. infestans, B. cinerea and F. culmorum could be extracted with ethyl acetate from the culture filtrates of C. globosum, C. cochliodes and C. aureum, whereas no or only low antifungal activity was present in the extracts from C. nozdrenkoae, C. ramosissimum and C. elatum. Based on these observations, the latter three strains were not or only partially included in the experiments with fractions of extracts separated by HPLC.
In our study, a broad spectrum of phytopathogenic fungi was inhibited on agar media amended with culture filtrates of C. globosum and C. aureum. For C. globosum, our results fit well with the literature stating antifungal activity against several phytopathogens including oomycetes (Aggarwall 2015). Among the most named compounds reported to be involved in the biological activity are the chaetoglobosins, a family of macrocyclic polyketide alkaloids belonging to the class of cytochalasans. Unfortunately, some of them are highly cytotoxic and irreversibly bind to actin (Kretz et al. 2019), which may pose a hurdle for the use of Chaetomium species in biocontrol. In the bioassays of our study with the fractionated extracts from C. globosum, the HPLC and MS analysis of the active compounds identified the chaetoglobosins. Chaetoglobosins were found to be the main antifungal compounds for the activity of C. globosum and other Chaetomium species against Bipolaris sorokiniana (Biswas et al. 2012), Saetosphaeria turcica (Zhang et al. 2013, Fusarium oxysporum (Soytong 2015), Sclerotinia sclerotiorum (Zhao et al. 2017) and F. sporotrichoides (Jiang et al. 2017). Chaetoglobosins A and C, isolated from culture filtrates of a strain of C. globosum, inhibited the maize pathogen Setosphaeria turcica in vitro, and Chaetoglobosin A Another hazardous compound shown to be present in extracts of Chaetomium is sterigmatocystin (Rank et al. 2011), which is biosynthetically related to the aflatoxins. However, by HPLC and MS analysis with pure sterigmacystin as standard we could not detect this compound in our extracts. We were also not able to detect flavipin, a metabolite from C. globosum reported to inhibit the growth of several plant pathogenic fungi, especially F. graminearum (Xiao et al. 2013).
Compared to C. globosum, much less work has been done on the potential of C. aureum and C. cochliodes as biocontrol agents. Both showed a strong activity against P. infestans, C. aureum inhibited also F. culmorum. For C. aureum, in vitro fungicidal activity and biocontrol activity have been reported against Pythium aphanidermatum (Pornsuriya et al. 2010), Fusarium circinatum (Mart ınez-Alvarez et al. 2016), the rice blast pathogen Magnaporthe grisea and the sheath blight pathogen Rhizoctonia solani . C. aureum produces a number of bioactive secondary metabolites, including oosporein (Taniguchi et al. 1984) that was shown to have antifungal activity against R. solani, Botrytis cinerea and Pythium ultimum (Mao et al. 2010). In previous studies crude extracts from C. cochliodes showed activity in vitro against Phytophthora sp. (Rujira et al. 2017) and Drechslera oryzae (syn. Bipolaris oryzae) (Soytong 2014). Chaetoglobosin A was isolated from culture filtrates of C. cochliodes and suggested to be responsible for the inhibition of P. ultimum and other pathogens (Kang et al. 1999).
In our bioassays with the fractionated extracts from C. aureum and C. cochliodes it was possible to determine active components, but the compounds are as yet unknown (Figs 6 and 7).
Overall, our results regarding the identification of antifungal metabolites produced by the Chaetomium strains are still preliminary, given the large number of biologically active compounds reported for the genus (Zhang et al. 2012). Structure elucidation was not accomplished since the amount of the highly active components was insufficient. In further studies the aim will be to find a way (optimization of fermentation conditions and/or genetic manipulation) to increase the production and to isolate enough pure compounds for  elucidation. This will include testing of media other than the standard laboratory medium PDB used here. Our present study can nevertheless serve as starting point for further elaboration of the compounds produced by our strains. With regard to the safety of potential biocontrol products based on Chaetomium the antifungal versus the cytotoxic properties of the compounds could be of specific interest.

Authors' contributions
A.L. performed molecular analyses of Chaetomium sp., S.J. carried out HPLC fractionation and profiling. P.Z. and M.M. determined antagonistic potential and temperature behaviour of the fungal isolates. W.M. maintained and provided the fungal strains and contributed to the molecular analyses. E.K. carried out microscopic analyses.