# Effect of abiotic factors and culture media on the growth of cheese-associated Nectriaceae species

Océane Savary, Monika Coton, Jean-Luc Jany, Louis Coroller, Emmanuel Coton

► To cite this version:

Océane Savary, Monika Coton, Jean-Luc Jany, Louis Coroller, Emmanuel Coton. Effect of abiotic factors and culture media on the growth of cheese-associated Nectriaceae species. International Journal of Food Microbiology, 2022, 364, 10.3390/foods11081114. [https://doi.org/10.3390/foods11081179]

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Effect of abiotic factors and culture media on the growth of cheese-associated *Nectriaceae* species.

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Océane Savaryª, Monika Cotonª, Jean-Luc Janyª, Louis Corollerª and Emmanuel Cotonª#

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ªUniv Brest, Laboratoire Universitaire de Biodiversité et Écologie Microbienne, F-29280 Plouzané, France

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Running title: Physiology of cheese-associated *Nectriaceae* spp.

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#Corresponding author: Pr. Emmanuel COTON, emmanuel.coton@univ-brest.fr
17 ABSTRACT

18 *Nectriaceae* species have been described in various natural environments or as plant
19 or human pathogens. Within this family, the *Bisifusarium domesticum* species is of
20 particular interest for food mycologists as it is used for a technological function in
21 various cheese productions. Moreover, it has only been isolated from the cheese
22 environment so far and, until recently, was the only *Nectriaceae* species described in
23 this food product. Recently, four novel cheese-associated *Nectriaceae* species have
24 been described, including two associated to the *Bisifusarium* genus and two to a new
25 genus, *Longinectria* gen. nov.. These observations raise questions concerning the
26 potential adaptation of these species to the cheese environment. In this context, this
27 study first focused on determining the impact of abiotic factors on the growth of isolates
28 belonging to the five cheese-associated species (i.e. *B. allantoides* sp. nov., *B.
29 domesticum*, *B. penicilloides* sp. nov., *L. lagenoides* gen. nov. sp. nov. and *L.
30 verticilliforme* gen. nov. sp. nov.) but also included phylogenetically close species. To
31 do so, fungal growth kinetics in liquid medium (Potato Dextrose Broth) were
32 determined by laser nephelometry at different temperatures, pH and water activities
33 using NaCl as a depressor. Growth modeling was then performed to estimate cardinal
34 values for each abiotic factor. Secondly, fungal growth was also evaluated on Potato
35 Dextrose Agar (synthetic medium), cheese agar (cheese-mimicking medium) and
36 Raclette de Savoie cheese (actual cheese). Our results clearly highlighted
37 physiological differences in growth characteristics between the studied cheese-
38 associated *Nectriaceae* spp. and the "non-cheese" species which could suggest, for
39 the former, an adaptation to this food matrix. Indeed, regarding the impact of the tested
40 abiotic factors, statistical analyses confirmed this dichotomy, with for example the
41 lowest optimal temperatures being estimated for the cheese-associated species (*T*<sub>opt</sub>
42 19.1–23.1 °C) while the other *Bisifusarium* species exhibited the highest optimal 43 temperature (*T*<sub>opt</sub> 26.1–36.2 °C). As for the impact of the growth media, radial growth 44 measurements highlighted that *B. domesticum* was the least affected species by a 45 growth on Raclette de Savoie and even grew faster on cheese agar than on synthetic 46 medium confirming its strong adaptation to the cheese environment.

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48 Keywords: *Bisifusarium*, *Longinectria*, modeling, predictive mycology, cardinal values,
49 laser nephelometry.
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# 1. Introduction

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The *Nectriaceae* family encompasses a vast number of species including numerous important plant and human pathogens but also technologically relevant species.

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Among the latter, *Bisifusarium domesticum* is used by cheesemakers as an adjunct culture during semi-soft, washed rind cheese productions (e.g. Saint-Nectaire, Reblochon) (Ropars et al., 2012) as well as for its ability to prevent surface stickiness (hence its "Anticollanti" denomination) of some smear-ripened cheeses (e.g. Raclette, Gruyère, Appenzeller) (Bachmann et al., 2005, 2003). Noteworthy, the mechanisms associated with this "Anticollanti" property are still unknown and some questions remain unanswered regarding this species tolerance to washed rind cheese conditions (cheese rind regularly washed with brine during ripening, ripening cellar temperature ~8-15°C) that are usually rather unsuitable for filamentous fungal growth.

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In 2015, Lombard et al., introduced a new nomenclature for the *Nectriaceae* family, resulting in the inclusion of six new genera based on morphological observations and a ten-gene phylogeny. For example, this led to the reclassification of the *Fusarium dimerum* species complex into the *Bisifusarium* genus. Since then, the nomenclature of the *Nectriaceae* family, and more particularly of the *Fusarium* genus, has been highly debated (Geiser et al., 2021; O'Donnell et al., 2020 vs. Crous et al., 2021; Lombard et al., 2015). Regardless of the nomenclature debate, the *Bisifusarium* genus (i.e. *Fusarium dimerum* species complex sensu Geiser et al., 2021) includes to this date eight species (Lombard et al., 2015; Schroers et al., 2009; Sun et al., 2017). They correspond to *B. biseptatum* (Schroers, Summerbell & O'Donnell) L. Lombard & Crous, 2015, *B. delphinoides* (Schroers, Summerbell, O'Donnell & Lampr.) L. Lombard & Crous, 2015, *B. dimerum* (Penz.) L. Lombard & Crous, 2015, *B. domesticum* (Fr.)

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75 L. Lombard & Crous, 2015, *B. lunatum* (Ellis & Everh.) L. Lombard & Crous 2015, *B. nectrioides* (Wollenw.) L. Lombard & Crous, 2015 and *B. penzigii* (Schroers, Summerbell & O'Donnell) L. Lombard & Crous, 2015 (Lombard et al. 2015) and to *B. tonghuanum* (B.D. Sun, Y.G. Zhou & A.J. Chen, 2017). The latter was described as a new member of the genus *Bisifusarium* based on its phylogenetic positioning using four target sequences (ITS, LSU, *tef1* and *tub2*) and thus using Lombard et al. (2015) nomenclature (Sun et al., 2017). More recently, based on a ten-gene phylogeny, two novel *Bisifusarium* spp. associated with cheese were also described (Savary et al., 2021), namely *B. allantoides* (O. Savary, M. Coton, E. Coton and J-L. Jany) and *B. penicilloides* (O. Savary, M. Coton, E. Coton and J-L. Jany). Therefore, upon acceptance of the two latter species, this genus will gather ten species which are cosmopolitan saprotrophs as the different species have been observed in various habitats with highly contrasted intrinsic and extrinsic characteristics (soil, plants, human samples and cheese), some having been described as plant and/or human pathogens (Lombard et al., 2015; Schroers et al., 2009; Sun et al., 2017). More precisely, *B. biseptatum*, *B. nectrioides* and *B. penzigii* have been found to be associated with natural environments (soil) (Schroers et al., 2009) while *B. tonghuanum* was isolated from *Atriplex centralasiatica* and *Suaeda salsa* plants (Sun et al., 2017). As for *B. lunatum*, it has mainly been isolated from *Opuntia* or *Gymnocaycium cacti* (Schroers et al., 2009) and is considered to be a plant pathogen (Flores-Flores et al., 2013; Gryzenhout et al., 2017; Mongiardino Koch et al., 2015). *B. delphinoides* is also considered to be a plant pathogen, isolated from stem lesions of *Hoodia gordonii* and found in soil and plant materials (Kulkarni et al., 2011; Schroers et al., 2009). However, *B. penzigii* and *B. lunatum* are also considered as potential human pathogens. They were previously isolated from a corneal ulcer and human
sinus, respectively (do Carmo et al., 2016; Schroers et al., 2009). *B. delphinoides* was
reported in human eye or skin infections (Collado et al., 2013; Hassan et al., 2016;
Park et al., 2019; Salah et al., 2015; Schroers et al., 2009). As for *B. dimerum*, even if
it was first isolated from a citron (*Citrus medica* L.) (Schroers et al., 2009), it is
considered as a human pathogen causing several human mycoses as keratitis,
keratomycosis, onychomycosis and endogenous endophthalmitis (Hassan et al.,
2016; Ray et al., 2016; Salah et al., 2015; Schroers et al., 2009; Simon et al., 2018).
Finally, as previously mentioned, *B. domesticum* is particularly of interest for food
mycologists as this species is used in the dairy industry for cheese production and
has, so far, only been found on cheese surfaces (Bachmann et al., 2005, 2003;
Lombard et al., 2015; Ropars et al., 2012; Schroers et al., 2009). Until recently, it was
the only *Nectriaceae* member clearly associated with the cheese environment.
However, as previously stated, we recently isolated several fungal isolates from
different cheeses that we proposed to belong to four new *Nectriaceae* species based
on the ten-gene phylogeny used by Lombard et al. (2015) and morphological traits
(Savary et al., 2021). Beyond the mentioned *B. allantoides* and *B. penicilloides*
species, two others were proposed to pertain to a new genus *Longinectria* gen. nov.
(*L. lagenoides* O. Savary, M. Coton, E. Coton and J-L. Jany and *L. verticilliforme* O.
Savary, M. Coton, E. Coton and J-L. Jany) (Savary et al., 2021).
The existence of cheese-associated *Nectriaceae* and closely related species living in
habitats with contrasted environmental conditions raises questions concerning their
adaptation to their respective environment.
In the context of this study, we used a two-step approach to evaluate the potential
physiological adaptation of the cheese-associated *Nectriaceae* spp. to this matrix by
monitoring the impact, firstly, of abiotic factors (using laser nephelometry) and,
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secondly, of growth media (Potato dextrose agar, cheese agar and Raclette de Savoie) on fungal growth and comparing it to that of closely related species.

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## 2. Materials and methods

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### 2.1. Nectriaceae isolates

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Type strains of the cheese-associated *Nectriaceae* species (i.e. *B. allantoides*, *B. domesticum*, *B. penicilloides*, *L. lagenoides* and *L. verticilliforme*) and of the "non-cheese" *Bisifusarium* species (i.e. *B. biseptatum*, *B. delphinoides*, *B. dimerum*, *B. lunatum*, *B. nectrioides* and *B. penzigii*) were used for interspecific comparison.

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Additionally, supplementary isolates of *B. allantoides* (n=2), *B. domesticum* (n=4) *B. penicilloides* (n=2) and *L. lagenoides* (n=3), as well as *B. dimerum* (n=4, only for laser nephelometry), were analysed for intraspecific variability evaluation. The different isolates were obtained from various culture collections as listed in Table 1. Monospores were obtained in all cases prior to experimental use.

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### 2.2. Isolate selection

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For intraspecific variability analyses, isolate selection was done either based on isolation origins (*B. allantoides*, *B. penicilloides*, *L. lagenoides* and *B. dimerum*) or based on Random Amplified Polymorphic DNA (RAPD) profiles (*B. domesticum*, Figure S1). For *B. domesticum* RAPD profiling, 25 isolates (Table S1, identifications first confirmed with alpha elongation factor gene sequencing, data not shown) were subjected to amplification using the ari1 primer (5'CTGCTTGGCACAGTTGGCTTC3') (Geisen et al., 2001). The PCR mixture contained molecular biology grade water, PCR buffer (1X), 1.5 mM MgCl₂, 3.5 µM ari1 primer, 0.2 mM dNTPs, 0.05 U/µL GoTaq

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polymerase (Promega) and 40 ng of DNA. Amplifications were performed using the following program: initial denaturation at 94°C for three minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 48°C for 30 seconds and extension at 72°C for two minutes with a final extension at 72°C during ten minutes. Amplicons were analyzed on a 2% (w/v) agarose gel with a migration of ten µL of each PCR-product at 90 V during two hours in 1X Tris-acetate-EDTA buffer (molecular biology grade, Promega).

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## 2.3. Conidia production

For conidia production, each fungal isolate was pre-cultured for ten days at 25°C on Potato dextrose agar (PDA, Difco, Fisher Scientific) then two mL of Tween 80 (0.015 %, v/v) were added on the surface of each culture to collect a conidia suspension. Conidia were counted in Malassez cells and, for each isolate, the suspension concentration was adjusted to $10^6$ conidia/mL in Tween 80.

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## 2.4. Growth media

Potato dextrose broth (PDB, Difco, Fisher Scientific) was used for the nephelometric method with the following conditions. Temperature effect (5-45°C) was directly evaluated with PDB while for pH, PDB was adjusted with hydrochloric acid or sodium hydroxide to obtain the target pH values in pH 2-14 (± 0.1) range. For water activity ($a_w$), NaCl (Sigma-Aldrich, Germany) was used with concentrations ranging from 2.9-21 % (corresponding to theoretical values ± 0.05 $a_w$ 0.98-0.83). Except for pH modified media, which were filtered at 0.22 µm, all other preparations were sterilized at 121°C for 15 minutes and stored at 4°C until use.

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For radial growth, synthetic PDA medium (pH 5.8 ± 0.0), a cheese-mimicking medium, cheese agar, (pH 4.0 ± 0.0) prepared as described in Morin-Sardin et al., (2016) and a Raclette de Savoie cheese (pH 5.4 ± 0.2) were used. For Raclette de Savoie cheese, only the internal sections below the rind were used. To do so, a ten mm thick rind and cheese sample was removed using a sterile knife, then five mm thick slices were vertically cut using a sterile lyre and standardized to 70 mm diameter pieces which then were transferred into sterile Petri dishes. Finally, the cheese slices were exposed to UV light for 15 minutes and conserved at 4°C until used.

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## 2.5. Impact of abiotic factors on fungal growth determined by laser nephelometry

The tested liquid culture media (190 µL, except for the negative control, 200 µL), were distributed into sterile 96 well microplates (Thermo Scientific). Then 10 µL of inoculum at $10^6$ conidia/mL were added to each well. In parallel, fungal suspensions were inoculated onto PDA to insure growth and purity. Microplates were placed in plastic containers with sterile distilled water placed at the bottom to regulate humidity before incubation in darkness, in an external incubator (BF 240, Binder GmbH, Germany) at 25.0°C for pH and $a_w$ conditions or at the studied temperature (incubators BF 240 and KB 240 Binder GmbH, Germany; INB500, IPP110 and IPP500, Memmert GmbH, Germany; MIR-253 and MIR-154, Sanyo, Japan). The measurements were performed using a laser-based microplate nephelometer (NEPHELOstar<sup>plus</sup>, BMG Labtech) equipped with a laser diode at 635 nm. Before each measurement, a 20 second 500 rpm double-orbital agitation step was performed by the nephelometer microplate reader. All microplates were monitored two to three times daily until signal saturation (or up to one month when no signal increase was observed). Two or three biological

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replicates and a minimum of two technical replicates per biological replicate were systematically performed for each studied isolate.

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## 2.6. Impact of media on fungal growth using radial measurements

The impact of fungal growth kinetics on synthetic PDA media in comparison to cheese agar and Raclette de Savoie was assessed for multiple cheese related isolates, B. domesticum (n=5 isolates), B. allantoides (n=3), B. penicilloides (n=3), L. lagenoides (n=4) and L. verticilliforme (n=1), and "non-cheese" (B. biseptatum, B. delphinoides, B. dimerum, B. lunatum, B. nectrioides and B. penzigii) type strains (Table 1). To do so, five µL of conidia suspensions (10⁶ conidia/mL) were inoculated onto the center of each medium with at least two biological and two technical replicates. All media were incubated in darkness at 15.0°C and 25.0°C, then radial growth was monitored in two perpendicular directions, for 30 to 40 days or 15 to 28 days, respectively.

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## 2.7. Data analysis and modeling

### 2.7.1. Primary modeling

At the end of each experiment, growth kinetics obtained either by nephelometry or radial growth measurements (expressed as Relative Nephelometric Units -RNU- and diameters in millimeters, respectively) were obtained as a function of time. The obtained data was assembled with Microsoft Excel (after export from the Nephelostar Galaxy software for nephelometry).

Each kinetic (RNU or diameters in mm, times) was fitted by the modified Gompertz equation (Equation (1)) (Zwietering et al., 1990):

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$$y = a \times \exp \left[ - \exp \left[ \left( \frac{\mu m \times \exp (1)}{a} \right) (\lambda - t) + 1 \right] \right] \quad (1)$$
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where y is the RNU or diameter observed at the time t, a corresponds to maximal amplitude of signal or growth, µm is the maximal growth rate (RNU/hour or mm/day) and λ the lag time (hours or day), corresponding to the time before detectable fungal growth.

For the nephelometry method, as a signal was measurable in wells containing only culture medium, negative controls were performed in each microplate to check for potential well contamination, and to standardize the kinetics modeling between analyses. Each negative control kinetic was also fitted, then the fitted values were subtracted from the corresponding growth kinetics data obtained with the inoculated well in the same conditions.

### 2.7.2. Secondary modeling

The maximal growth rates were fitted by the cardinal model (Equations 2 & 3) (Rosso et al., 1993) which is used to study the impact of abiotic factors on microbial growth (Dagnas and Membré, 2013):

$$ \mu m = \mu_{opt} \times CMn(X) \quad (2) $$

$$ CMn(X) = \begin{cases} \frac{0}{(X - X_{max}) \cdot (X - X_{min})^n} & , X_{min} \le X < X_{max} \\ \frac{0}{(X_{opt} - X_{min})^{n-1} \cdot \{(X_{opt} - X_{min}) \cdot (X - X_{opt}) - (X_{opt} - X_{max}) \cdot [(n-1) \cdot X_{opt} + X_{min} - n \cdot X]\}} & , X_{min} < X < X_{max} \\ 0 & , X > X_{max} \end{cases} \quad (3) $$

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where µopt corresponds to the optimal growth rate (RNU/hour or mm/day), X represents the studied parameter (temperature, pH or a<sub>w</sub>) and X<sub>min</sub>, X<sub>max</sub> and X<sub>opt</sub> are respectively the minimum, maximum and optimal parameter values for which growth is possible. According to the literature, n=2 for temperature and a<sub>w</sub> and n=1 for pH (Pinon et al., 2004; Rosso et al., 1995). Moreover, for a<sub>w</sub> modeling, the a<sub>w max</sub> was fixed at 1 as done by Pinon et al. (2004) and a<sub>w opt</sub> at 0.99 (highest observed value).
As for radial growth measurements, for each solid media, the optimal growth rate was estimated fitting the maximal growth rates using the cardinal model with the cardinal values estimated during laser nephelometry method ($T_{min}$, $T_{opt}$ and $T_{max}$).

The primary and secondary models were fitted with these equations by minimizing the sum of squared error (nlinfit function, MATLAB R2018b software, Natick, Massachusetts: The MathWorks Inc.). Five percent confidence intervals were computed using the Jacobian matrix (nlparci function, MATLAB R2018b software, Natick, Massachusetts: The MathWorks Inc). As the sum of the squared error (SSE), the Root Mean Square Error (RMSE) were computed as fitting criterions.

### 2.7.3. Statistical analyses

A principal component analysis (PCA) was performed based on the estimated cardinal values to identify the main trends among the 26 isolates belonging to the eleven studied species. Then, a one-way analysis of variance (ANOVA) and a multiple comparison test were performed to compare the cardinal values between the two identified groups.

An additional ANOVA (*p* value < 0.05) was also done in order to compare the growth rates obtained among *Nectriaceae* isolates. All statistical analyses were done using the MATLAB software.

To specifically compare some cardinal values, the likelihood ratio test was also applied as done by Trunet et al., (2015) and Morin-Sardin et al., (2016). To do so, the secondary modeling was fitted with one parameter value fixed for all the isolates (constrained model, C), then the obtained SSE were compared to those obtained with the previous conditions (unconstrained model, U) applying Equation (4).
$$S_L = n \cdot \ln \ln \text{SSE}_C - n \cdot \ln \ln \text{SSE}_U \quad (4)$$

where *n* corresponds to the number of data, SSE<sub>C</sub> and SSE<sub>U</sub> to the Sum of the Squared Error for the constrained and unconstrained model, respectively. When *n* tends to infinity, the limiting distribution of *S*<sub>L</sub> is a Chi square test (Chi2) distributed with a degree of freedom defined as *p*<sub>U</sub> – *p*<sub>C</sub>, where *p*<sub>U</sub> and *p*<sub>C</sub> are the number of parameters in unconstrained and constrained models. If *S*<sub>L</sub> is lower than χ<sup>2</sup> (α = 0.05), the difference was considered as not significant, that is to say that the fixed parameter is equal to the determined parameter.

## 3. Results

### 3.1. Impact of abiotic factors on fungal growth

#### 3.1.1. Preliminary studies

For *B. domesticum* RAPD profiling, the obtained results (Figure S1) showed low genetic diversity among the 25 tested isolates but allowed selecting five representative strains with the most diverse profiles.

For growth studies, using nephelometry measurements, we also ensured that discontinuous measurements, for high throughput parallel assays, and microplate agitation before measurements did not impact growth kinetics (data not shown). Noteworthy, the *µm* estimated between technical replicates highlighted standard error inferior to 10.0 % (3.0 to 8.4 %) at a temperature close to the optimal (30.0°C).

#### 3.1.2. Interspecific comparative study

The interspecific study using laser nephelometry was performed on the eleven type strains of *Bisifusarium* spp. and *Longinectria* spp.. For temperature, different growth behaviors were observed among *Nectriaceae* spp. (Figure 1, Figure 2 and Table 2).
B. allantoides, B. domesticum, B. penicilloides, L. lagenoides and L. verticilliforme (cheese-associated species) but also B. biseptatum, B. lunatum, and B. penzigii ("non-cheese" species), type strains developed slowly at 5.0°C while no growth was observed for B. delphinoides, B. dimerum and B. nectrioides ("non-cheese" species).

Interestingly, for the cheese-associated species included in this study, no growth was observed at 37.0°C for B. allantoides, B. domesticum, L. lagenoides and L. verticilliforme and even at 30.0°C for B. penicilloides. Conversely, for "non-cheese" species, growth was observed up to 38.0°C, and even 40.0°C for B. dimerum. Based on the estimated cardinal values via secondary modeling (Table 2), the lowest optimal and maximal temperature values were observed for cheese-associated Nectriaceae spp. (i.e. Topt estimated 22.3 ± 0.4°C, 22.4 ± 0.2°C, 20.1 ± 0.6°C, 21.8 ± 0.2°C and 19.1 ± 0.5°C for B. allantoides, B. domesticum, B. penicilloides, L. lagenoides and L. verticilliforme, respectively) while all the other Bisifusarium species had significantly higher optimal (between 26.1°C and 36.2°C) and maximal (between 38.0°C and 41.2°C) temperature values (Figure 1, Figure 2 and Table 2).

Concerning pH impact, variations between pH 5.0 and 10.0 only had a slight impact on fungal growth, i.e. on both lag phase and growth rate (data not shown) for all type strains belonging to the eleven species. No growth was observed at pH 2.0 and pH 14.0, and even pH 3.0, for the five cheese-associated species. Based on the estimated cardinal values (Table 2), the highest minimum pH values were obtained for cheese-associated species with a pHmin = 3.7 ± 0.1 for B. domesticum (Table 2) or pHmin = 3.9 ± 0.2 for B. penicilloides (Table 2). For the other "non-cheese" species, this value was close to pH 2.9 (Table 2). However, it should be noted that this condition was really drastic for some Bisifusarium spp. (e.g. B. delphinoides, B. nectrioides and B. lunatum)
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as a significant decrease in growth rate between pH 4 and pH 3 was observed (p value
<0.001 for all species except *B. dimerum* p value = 0.004).

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Concerning sodium chloride used as an *a*<sub>w</sub> depressor, as expected, growth rates
decreased with decreased *a*<sub>w</sub> and several growth profiles were identified. The highest
halotolerance was observed for *B. allantoides* type strain which grew in presence of
~13.0 % NaCl with an estimated *a*<sub>wmin</sub> 0.87 (corresponding to ~17.0 % NaCl). Then,
*B. dimerum*, *B. delphinoides*, *B. penicilloides* and *B. penzigii* type strains grew in the
presence of approximately 11.0 % of salt, corresponding to *a*<sub>w</sub> 0.93, and their
estimated *a*<sub>wmin</sub> was between 0.90–0.88 (14.5–16.0 % NaCl). On the other hand, *B.
domesticum*, *B. nectrioides*, *L. lagenoides* and *L. verticilliforme* type strains only grew
at 6.0 % NaCl (*a*<sub>w</sub> 0.97) (*a*<sub>wmin</sub> = 0.96–0.97) while *B. lunatum* and *B. biseptatum* type
strains only grew at NaCl concentrations ≤ 3.0 % (*a*<sub>w</sub> 0.98) (*a*<sub>wmin</sub> = 0.97) (Figure 1,
Figure 2 and Table 2).

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3.1.3. Intraspecific comparative study

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Intraspecific growth behavior variability was evaluated among different isolates
belonging to four cheese-associated species (excluding *L. verticilliforme* as only one
isolate is currently available) and five *B. dimerum* isolates as a "non-cheese"
representative species. Isolates were selected according to their isolation origins,
except the five *B. domesticum* isolates which were selected, as previously stated,
according to their different RAPD profiles (§ 3.1.1, Figure S1). This was done to avoid
a representativity bias as some *B. domesticum* isolates are commonly used as
commercial adjunct cultures in cheese production. Distinct profiles between cheese-
associated isolates and the selected "non-cheese" strains could be noted (Table 3 and
Figure 3). As for cheese-associated species, *B. allantoides*, *B. domesticum* and *L*.
lagenoides isolates, fungal growth was observed in the 5.0-30.0°C temperature range, and even 5.0-25.0°C for B. penicilloides, with Topt estimated at 22.7 ± 0.4 °C; 20.9 ± 0.9 °C; 21.4 ± 1.2°C; and 21.9 ± 0.7 °C respectively. Among these species, one L. lagenoides isolate (UBOCC-A-120038) grew only up to 25.0°C and for one B. domesticum isolate (UBOCC-A-120004), growth was observed in the 10.0-25.0°C temperature range. In addition, for B. domesticum MNHN-RF-05627NT, a faster growth than for the other B. domesticum isolates was observed between 20.0 and 25.0 °C (Table 3 and Figure 3). For pH, no notable differences could be noted within each species, and as already observed for the type strains, no major impact on fungal growth between pH 5.0 and pH 10.0 and no growth at pH 3.0 were observed for all cheese-associated tested isolates. However, for B. domesticum, significantly slower growth was observed for one isolate (UBOCC-A-120001) at pH 4.0 (*p* value < 0.05) and pH 5.0 (*p* value < 0.01). Regarding NaCl impact, while for B. allantoides and B. penicilloides, similar results within a considered species were observed, few differences could be noted for B. domesticum and L. lagenoides. For the latter, UBOCC-A-120038 grew up to aw 0.98 vs. 0.97 for the other isolates. As for B. domesticum, the most tolerant isolate (UBOCC-A-120001) grew up to 10 % NaCl (estimated awmin 0.93) while the most sensitive isolate (UBOCC-A-120004) could only grow up to 3 % NaCl (awmin 0.97). The three other tested isolates exhibited intermediary behaviors (growth up to 9 % NaCl -awmin 0.96- for MNHN RF-05627NT and 6 % NaCl - awmin 0.96- for UBOCC-A-120002 and UBOCC-A-120003) (Table 3 and Figure 3). As for the selected "non-cheese" species, B. dimerum isolates grew faster than cheese-associated species (e.g. on average with temperature impact modeling, 1.04 x10⁵ RNU/hour vs. 6.14 x10⁴ RNU/hour for B. domesticum isolates, *p* value <0.001) and, different growth characteristics than those of cheese-associated species were noted.
B. dimerum isolates could not grow under 10°C in these experimental conditions but grew up to 40.0 °C with Tmax estimated at 41.1 ± 0.4 °C and Topt at 30.1 ± 1.1 °C for the five tested isolates. Finally, for pH impact, similar curves profiles were observed with no notable differences between the tested isolates. The same observation was done for NaCl impact, with a growth still observed at 13.0 % NaCl (aw 0.91) and awmin values estimated to range from 0.86-0.88. Overall, for each species, similar profiles to those obtained during the interspecific study (performed on type strains) were observed with little variations within each species.

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### 3.1.4. Principal component analysis

The principal component analysis performed on all studied isolates, confirmed the previous inter- and intra-specific observations separating the isolates into two clusters corresponding, on one hand, to the cheese-associated species and, on the other, to "non-cheese" ones (Figure 4). Moreover, it was observed that the "cheese" cluster exhibited significantly lower optimal growth rates (for temperature, pH and aw) than the "non-cheese" cluster (p values ≤ 0.001), but also a significantly lower Tmax and Topt (p values ≤ 0.001) while pHmin and pHopt were lower for the "non-cheese" cluster (p values ≤ 0.01).

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## 3.2. Impact of media on fungal growth

Growth of the cheese-associated Bisifusarium and Longinectria spp. (i.e. B. allantoides -n=3-, B. domesticum -n=5-, B. penicilloides -n=3-, L. lagenoides -n=4- and L. verticilliforme -n=1-), as well as of the "non-cheese" Bisifusarium species type strains (i.e. B. biseptatum, B. delphinoides, B. dimerum, B. lunatum, B. nectrioides and
B. penzigii), was monitored, at 15.0°C and 25.0°C, using synthetic media (PDA) and cheese matrices (cheese agar and Raclette de Savoie cheese). Noteworthy, growth was observed for all twenty-two studied isolates in all tested conditions and the secondary modeling accurately fit (low RMSE values ranged from 0.02 to 0.33 mm/day) the maximal growth rates estimated by the primary modeling at both temperatures for each media. Different growth characteristics were highlighted depending on the considered isolate, temperature and medium (Table 4, Figures 5 and 6). Regarding the growth on cheese agar of B. allantoides, L. lagenoides and L. verticilliforme isolates, compared to PDA, optimal growth rates slightly decreased between 15.6–18.8 %, 8.6–18.4 % and 8.8 % respectively. Among L. lagenoides isolates, it could be noted that one isolate (UBOCC-A-120038) appeared as less impacted (-8.6 %). For B. penicilloides, intraspecific variability was also noted. Indeed, for one B. penicilloides isolate, $\mu_{opt}$ decreased by 18.5 % (UBOCC-A-120034) while for the two other isolates, it was unchanged or even increased (UBOCC-A-120021NT and VTT-D-041022 - +16.1 % - respectively). Interestingly, for B. domesticum isolates, their $\mu_{opt}$ were actually higher on cheese agar than on PDA for the five tested isolates with high or moderate increase depending on the considered isolate. Indeed, for UBOCC-A-120002, UBOCC-A-120004 and MNHN-RF-05627NT, $\mu_{opt}$ increased by 3.8–9.7%, while for UBOCC-A-120001 and UBOCC-A-120003 the increase was more substantial (28.6 % and 25.0 %, respectively). As for Raclette de Savoie, B. allantoides growth was notably slower than on PDA (on average, decreasing by 43.6 ± 1.9 %) but B. penicilloides, L. lagenoides and L. verticilliforme $\mu_{opt}$ were negatively affected (between 25.8–55.6 %; 22.9–39.5 % and 35.3 %, respectively). Moreover, among B. penicilloides isolates, it could be noted that UBOCC-A-120034 growth was more affected than the other ones (-55.6 % vs. -25.8 % and -31.4 %). As for B. domesticum,
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UBOCC-A-120001 and UBOCC-A-120003 isolates could be considered as little or not impacted by growth on Raclette de Savoie compared to PDA as a very slight $\mu_{opt}$ decrease was noted (4.8 % and 10.0 %). For UBOCC-A-120002 and MNHN-RF-05627NT, $\mu_{opt}$ decreased by 19.2 % and 19.4 %, while for UBOCC-A-120004 the decrease reached 40.0 %.

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Comparing the cheese-associated species to the "non-cheese" species, it could be noted, first, that the latter grew faster (except *B. nectrioides*) on the three media (e.g. $\mu_{opt}$ = 12.8 ± 0.3 mm/day for *B. biseptatum* CBS 110311 vs 3.1 ± 0.1 mm/day for *B. domesticum* MNHN-RF-05627 on PDA) (Table 4, Figures 5 and 6). However, on cheese agar, compared to synthetic medium, important $\mu_{opt}$ decreases for *B. delphinoides*, *B. penzigii* (22.1 and 31.7 %, respectively) and a slight one for *B. biseptatum*, *B. dimerum* and *B. lunatum* (16.4 %; 19.6 % and 5.1 % respectively) were observed. Conversely, for *B. nectrioides*, the $\mu_{opt}$ increased by 31.4 % (4.6 mm/day on cheese agar vs. 3.5 mm/day on PDA). As for Raclette de Savoie cheese, compared to PDA medium, growth rates of *B. biseptatum*, *B. delphinoides*, *B. lunatum* and *B. penzigii* showed a 48.8 to 54.5 % decrease) while they were of 39.1 % and 22.9 %, affected for *B. dimerum* and *B. nectrioides*, respectively.

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## 4. Discussion

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Species pertaining to the *Nectriaceae* family are associated with a wide variety of biotopes. Indeed, while some species have been reported to be associated to natural environment (e.g. soil, plants), others are emerging or invasive species causing infections in immunocompromised patients but also immunocompetent humans, and are mainly associated with eye infections (Schroers et al., 2009). However, a small
number of species have also been associated with the cheese environment (Savary et al., 2021). Among the latter, *B. domesticum* has been used for decades for daily productions of some washed-rind cheeses due to its "anticollanti" ability that prevents cheese rind stickiness. Yet, despite its industrial use, information on this species is scarce. Other species, namely *B. allantoides* sp. nov., *B. penicilloides* sp. nov., *L. lagenoides* gen. nov. sp. nov. and *L. verticilliforme* gen. nov. sp. nov., were recently isolated from diverse cheeses (Savary et al., 2021) although their potential origin and role is not yet elucidated. As these five species have solely been isolated from cheeses to date, questions concerning their potential adaptation to this environment arise.

In this context, this study aimed at determining the impact of cheese-relevant abiotic factors and diverse media on fungal growth of these cheese-associated species. We also compared them to "non-cheese" but phylogenetically close species. Growth kinetics for ten *Bisifusarium* and two *Longinectria* species were thus determined.

Acquiring physiological data for fungal growth modeling can be done using different methods ranging from classical radial growth measurements on solid media, to more high throughput methods (Aunsbjerg et al., 2015; Joubert et al., 2010; Medina et al., 2012; Sardella et al., 2018). In the context of this study, we first applied a high throughput discontinued microplate laser nephelometry method, which has already been successfully used to monitor fungal growth (Joubert et al., 2010; Lecomte et al., 2012), to investigate the impact of cheese relevant abiotic factors corresponding also to those classically used to describe microbial growth, namely temperature, pH and $a_w$ (using NaCl as a depressor as notable concentrations are added for cheese production). Preliminary studies were performed to ensure that the nephelometric method could be applied under these experimental conditions to efficiently monitor fungal growth over time using discontinuous measurements (data not shown), thus
allowing for simultaneous monitoring of a much larger number of fungal strains in multiple conditions (data not shown). It should be noted that, while this method is much faster and more sensitive than classical radial growth measurements, it does have some limits. Laser nephelometry uses an arbitrary unit that could be considered difficult to link to radial growth, which is considered as the most biologically relevant criterion. Nevertheless, a linear relationship between conidiogenesis and the nephelometric signal was already described (Joubert et al., 2010) and it can be argued that the systematic use of the optimal growth rate as the reference parameter should preclude any bias. Moreover, it can be difficult or even not possible to use the substrate (or a mimicking-medium) from which a specimen was isolated as this method requires the use of a culture medium enabling light scattering measurements. This explains why in the framework of this study, in a second time, radial growth measurements were also used to compare growth on cheese matrices, namely a cheese-mimicking medium (cheese agar) and Raclette de Savoie core (a cheese in which *B. domesticum* is commonly used as a rind adjunct culture) in comparison to PDA medium.

For primary modeling, several models are frequently applied to model bacterial or fungal growth. They include the logistic function, asymmetric model, Baranyi model and modified Gompertz equation (Dagnas and Membré, 2013; Kosegarten et al., 2017; Tjørve and Tjørve, 2017). The latter has already been shown to be more robust (Declerck et al., 2001) than the others and, after a preliminary model comparison using our data, the modified Gompertz equation had the best fitting (data not shown) and was therefore applied.

Concerning the impact of abiotic factors on fungal growth, the obtained results revealed common intraspecific growth patterns and interspecific differences among
Bisifusarium spp. and Longinectria spp. The most contrasted behaviors were related to temperature. Indeed, B. allantoides, B. domesticum, B. penicilloides, L. lagenoides and L. verticilliforme, that are to this date the only known Nectriaceae species associated to cheese, were better adapted to low temperatures than the six "non-cheese" Bisifusarium spp.. Indeed, B. domesticum MNHN-RF-05627NT had the significantly lowest Topt (22.4 ± 0.2°C) and Tmax (30.6 ± 0.1°C) among the Bisifusarium genus and the same observation was done on the four other isolates. Regarding the other eleven isolates of cheese-associated Nectriaceae spp. nov., similar Topt were observed, in the 19.1-23.1°C range. Moreover, the performed statistical analyses highlighted that the Topt and Tmax of cheese-associated Nectriaceae spp. were highly significantly lower than those of "non-cheese" Bisifusarium spp. (p value ≤ 0.001), this can be linked to their better adaptation to a cheese environment. Interestingly, B. allantoides, B. domesticum, B. penicilloides, L. lagenoides and L. verticilliforme (all isolates studied) were the only species for which no growth was observed in the tested conditions at temperatures higher than 30.0°C and no growth was even observed at this temperature for all B. penicilloides isolates and L. lagenoides UBOCC-A-120038. This is of particular interest as the observed values are compatible with temperatures used during cheese making and ripening. On the other hand, B. biseptatum, B. delphinoides, B. lunatum, B. nectrioides and B. penzigii (type strains) grew at 37.0°C (with a slightly higher estimated Tmax between 38.0°C and 40.9°C, Table 2) and the five B. dimerum isolates even grew at 40.0°C (e.g. Tmax 41.9 ± 0.5°C for MNHN-RF-05625ET). This thermotolerance is compatible with a higher potential to colonize the human body and can explain why these species are observed to be human opportunistic pathogens. Interestingly, the obtained data were highly concordant with those described by Schroers et al. (2009) based on radial growth measurements on
malt extract agar media. Indeed, *T*<sub>opt</sub> between 21.0°C and 24.0°C were estimated for *B. domesticum*, and between 24.0°C and 30.0°C for *B. nectrioides* while it was between 27.0°C and 30.0°C for all other *Bisifusarium* spp.. The only discordance between the two studies concerns *B. lunatum* CBS 632.76 for which, our data led to a higher estimated optimum value (36.2 ± 0.4°C on PDB vs. 27.0–30.0°C on malt extract agar). Similarly, the impact of temperature on *B. dimerum* growth was also investigated in another previous study using optical density and the optimal temperature was determined to be between 35.0°C and 37.0°C using minimal medium (Steinberg et al., 2015). Moreover, observed growth limits for *B. allantoides*, *B. penicilloides*, *L. lagenoides* and *L. verticilliforme* (Table 2, Table 3) were concordant with those reported on PDA by Savary et al., (2021). The similarity of the obtained results between radial growth and nephelometry confirmed the interest of this high throughput method to determine fungal growth cardinal values. Concerning the minimal temperature, *B. allantoides*, *B. domesticum*, *B. penicilloides*, *L. lagenoides* and *L. verticilliforme* isolates but also *B. biseptatum* CBS 110311, *B. lunatum* CBS 632.76 and *B. penzigii* CBS 317.34, were able to grow at 5.0°C in contrast to *B. delphinoides* CBS 120718, *B. dimerum* MNHN-RF-05625 and *B. nectrioides* CBS 176.31 that no longer grew in these experimental conditions, even after one month. This led to wider confidence intervals for estimated *T*<sub>min</sub>. In fact, the difficulty to study low temperatures and the absence of intermediate values in the experimental design between detectable and undetectable growth conditions is a limit of secondary modeling, because zero values cannot be appended to the dataset although some species are really sensitive to low temperature variations. The same problem was observed for the estimated *T*<sub>max</sub> for the *B. delphinoides* CBS 120718<sup>HT</sup>, *B. domesticum* UBOCC-A-120004 and *B. penicilloides* isolates which were probably overestimated
and explained by the lack of intermediate values between good growth and absence of growth. Therefore, one of the limits that can be classically encountered in modeling is based on the experimental design adopted which can induce for some isolates, very distant values not allowing for an optimal model fit. As a result, the model cannot always be used to infer the distal values of the curve.

Regarding the impact of pH on *Nectriaceae* spp. growth, no major differences were observed between species with nevertheless a significantly higher *pH*min and *pH*opt for the cheese-associated *Bisifusarium* and *Longinectria* spp. (e.g. *pH*min 3.7 ± 0.1 and pH 6.7 ± 0.4, respectively for *B. domesticum* MNHN-RF-05627NT) compared to the “non-cheese” species (*p* values ≤ 0.001 and 0.01 respectively). The latter six *Bisifusarium* spp. type strains were able to grow in a wide range of pH values (pH 3.0–12.0) with, overall, only a minor impact on growth rates between pH 5.0 and pH 10.0. Noteworthy, as expected, *B. domesticum* and other cheese-associates species growth at pH values classically measured in brines or on cheeses (pH 5.0–6.0) (Guinee and Fox, 2017; Mounier et al., 2017) was confirmed.

Water activity is a factor with high impacts on fungal growth (Pitt and Hocking, 2009). In this study, *a*<sub>w</sub> was modified by sodium chloride as it is commonly used as a cheese ingredient not only for its flavor enhancing ability, but also for food preservation (Guinee, 2004; Nguyen Van Long et al., 2017). As already demonstrated on several filamentous fungi, this study highlighted that a decrease in *a*<sub>w</sub> induced a significant decrease in *Bisifusarium* and *Longinectria* spp. growth rates with different effects depending on the considered species. The “non-cheese” species presented the lowest estimated *a*<sub>wmin</sub> values (slightly significant, *p* value ≤ 0.01) although *B. allantoides*, *B. dimerum*, *B. penzigii* and *B. delphinoides* presented the highest halotolerance (*a*<sub>wmin</sub> 0.86–0.89, corresponding to minimum ~15.0 % NaCl). For *B. domesticum*, although
highly associated with cheese, the growth limit was estimated at ~7.0 % NaCl ($a_{wmin}$ 0.96) except for *B. domesticum* UBOCC-A-120001 for which $a_{wmin}$ was estimated at 0.93 (~8.5 %) and UBOCC-A-120004 which only grew up to ~3.0 % NaCl, with an estimated $a_{wmin}$ at 0.97 (~6.0 % NaCl). Similar halotolerance was observed for *Longinectria* spp. isolates with $a_{wmin}$ estimated between 0.96 and 0.97. Finally, *B. penicilloides* spp. isolates presented higher halotolerance with $a_{wmin}$ 0.91–0.90 (corresponding to minimum ~13.0 % NaCl). Although *B. allantoides* seems more adapted to high salt concentration, overall, the cheese-associated species were not the most halotolerant, but their growth range in the tested conditions is compatible with NaCl concentrations encountered during cheese making (overall 1.1–2.5 % in ripening conditions) (Guinee and Fox, 2017; Mounier et al., 2017). Noteworthy, for the same water activity values, using glycerol as an $a_w$ depressor, a lower impact on growth was observed for all *B. biseptatum*, *B. delphinoides*, *B. dimerum*, *B. domesticum*, *B. lunatum*, *B. nectrioides* and *B. penzigii* type strains (data not shown). This has been regularly observed for other fungi (Morin-Sardin et al., 2016; Nguyen Van Long et al., 2017) and is linked to the toxicity of Na+ and Cl⁻ ions, and to the ionic strength increase which can alter enzyme activities (Bois et al., 2006; Morita et al., 2004).

The second part of this study was performed on cheese matrices and investigated growth on cheese-mimicking medium (cheese agar) and actual Raclette de Savoie cheese for *Bisifusarium* and *Longinectria* species compared to growth on PDA. This was performed for the cheese-associated species (n=16 isolates) and the type strains of the “non-cheese” *Bisifusarium* species (n=6) at 15.0°C and 25.0°C. Overall, it should be noted that all tested species and isolates, including the “non-cheese” ones, were able to grow on the tested media. On cheese agar, *B. allantoides*, *L. lagenoides* and *L. verticilliforme* grew slightly slower than on PDA ($\mu_{opt}$ decrease between 8.6 and
18.8 %), while for *B. penicilloides* isolates, the optimal growth rate decreased for one isolate or was unchanged or increased for the two others. Interestingly, for *B. domesticum* isolates, the μopt were considerably higher on this medium, in particular for two isolates (UBOCC-A-120001 and UBOCC-A-120003; +28.6 and +25.0 %, respectively). As for the "non-cheese" species, a decrease was noted between 5.1 % and 31.7 % (overall, slightly higher negative impact than for cheese-associated species), except for *B. nectrioides* which grew better on cheese agar than on PDA (μopt +31.4 %). As for Raclette de Savoie, *B. allantoides*, *B. penicilloides*, *L. lagenoides* and *L. verticilliforme* had an important μopt decrease (22.9-55.6 %). *B. biseptatum*, *B. delphinoides*, *B. dimerum*, *B. lunatum* and *B. penzigii* grew faster than other *Nectriaceae* species on cheese as well as on the two other media, but despite that, comparing growth on Raclette de Savoie to that on PDA, they seemed less adapted to this matrix (μopt decreased by 22.9 to 54.5 %). Interestingly, for *B. domesticum*, although a slower growth was observed on cheese compared to PDA, members of this species appeared less impacted by a growth on Raclette de Savoie (μopt decreased by only 4.8 to 19.4 %), except for one isolate (UBOCC-A-120004, -40.0 %). A recurrent problem associated with modeling approaches is that they often rely on monitoring growth of a single strain per species. This clearly raises questions about the representativeness of the selected strain and therefore of the obtained results. Thereby, the use of several strains with different isolation origins and/or genetic profiles for a given species is crucial to obtain robust and reliable results that can be considered as representative of a species physiology. For this reason, we performed, when possible, the analyses on various isolates for the cheese-associated species (and for *B. dimerum* as an outgroup, for nephelometry method only). As previously stated, analysis of the secondary models and cardinal values confirmed similar profiles
within each species, with low variability regardless of the approach used (laser nephelometry in liquid medium or radial growth on solid medium). Noteworthy, among the *B. penicillioides* isolates, VTT-D-041022 was the only isolate not isolated from cheese but from the outer surface of an Abbey in Scotland (Suihko et al., 2007), and despite that, it presented the same growth profiles as the two other *B. penicilloides* isolates for the nephelometry study and was the least impacted isolate within the species by a growth on cheese agar and Raclette de Savoie.

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In conclusion, the obtained results provide new physiological insights on cheese-associated *Nectriaceae* spp. but also closely related *Bisifusarium* spp. for which knowledge is still scarce. For these former, growth characteristics were concordant with cheese making and ripening conditions. Noteworthy, *B. domesticum* seemed better adapted to the cheese matrix. Beyond new knowledge, the obtained data could be useful for optimizing *B. domesticum* use as a cheese adjunct or even of the novel *Nectriaceae* spp.. In the future, it would also be of interest to evaluate the cheese-associated species functional diversity and impact on cheese matrices (e.g. flavor compounds, proteolytic and lipolytic activities).

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640 Acknowledgments

We are grateful to Dr. Jeanne Ropars (Laboratoire Écologie, Systématique et Évolution, Université Paris-Saclay) for providing isolates as well as all the people who kindly provided the various cheeses samples to implement the *B. domesticum* isolate collection used for RAPD analyses. We would also like to thank members of the French Dairy Interbranch Organization (CNIEL), in particular, Laurence Abraham from
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Entremont and the Lactalis R&D team for kindly providing Raclette de Savoie and cheese agar ingredients.

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## Funding

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OS benefited from a Ph. D grant from the Région Bretagne and the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation. This work was co-funded by the French Dairy Interbranch Organization (CNIEL) (DIFUS Project, 2019_00627).

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## Authors' contributions

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Océane Savary: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft. Monika Coton: Funding acquisition, Conceptualization, Supervision, Writing - Review & Editing. Jean-Luc Jany: Funding acquisition, Conceptualization, Supervision, Writing - Review & Editing. Louis Coroller: Methodology, Supervision, Writing - Review & Editing. Emmanuel Coton: Funding acquisition, Conceptualization, Supervision, Writing - Review & Editing.

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<table><caption>**Table 1: Nectriaceae spp. strains used for the inter- and intra-specific studies.**</caption><thead><tr><th>Species</th><th>Strain code</th><th>Substrate/Origin</th></tr></thead><tbody><tr><td rowspan="3">Bisifusarium allantoides</td><td>UBOCC-A-120035</td><td>Cheese (Manchego, Spain)</td></tr><tr><td>UBOCC-A-120036<sup>HT</sup></td><td>Soft cheese type (France)</td></tr><tr><td>UBOCC-A-120037</td><td>Soft cheese type (France)</td></tr><tr><td>Bisifusarium biseptatum</td><td>CBS 110311<sup>T</sup></td><td>Soil (South-Africa)</td></tr><tr><td>Bisifusarium delphinoides</td><td>CBS 120718<sup>HT</sup></td><td>Stem lesions on succulent plant (Hoodia gordonii) (South-Africa)</td></tr><tr><td>Bisifusarium dimerum</td><td>IHEM-10066</td><td>Human aortic valve (Unknown)</td></tr><tr><td rowspan="5">Bisifusarium domesticum</td><td>IHEM-14513</td><td>Human blood (Belgium)</td></tr><tr><td>IHEM-2314</td><td>Dwelling environment, bathroom (Belgium)</td></tr><tr><td>MNHN RF-05625<sup>ET</sup></td><td>Blood from patient with acute myeloid leukemia (Netherlands)</td></tr><tr><td>UBOCC-A-110149</td><td>Soft drink (France)</td></tr><tr><td>MNHN RF-05627<sup>NT</sup></td><td>Food (Belgium)</td></tr><tr><td rowspan="5">Bisifusarium lunatum</td><td>UBOCC-A-120001</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120002</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120003</td><td>Cheese (Raclette d'alpage, Switzerland)</td></tr><tr><td>UBOCC-A-120004</td><td>Commercial adjunct culture (France)</td></tr><tr><td>CBS 632.76<sup>NT</sup></td><td>Dark spots on cactus epidermis (Gymnocalcium damsii) (Germany)</td></tr><tr><td>Bisifusarium nectrioides</td><td>CBS 176.31<sup>LT</sup></td><td>Banana plantation humus (Honduras)</td></tr><tr><td>Bisifusarium penicilloides</td><td>UBOCC-A-120021<sup>HT</sup></td><td>Cheese (Mont d'Or, France)</td></tr><tr><td rowspan="2">Bisifusarium penzigii</td><td>UBOCC-A-120034</td><td>Cheese (Mont d'Or, France)</td></tr><tr><td>VTT-D-041022</td><td>Surface of sandstone building, Arboat Abbey (Scotland)</td></tr><tr><td>Longinectria lagenoides</td><td>CBS 317.34<sup>HT</sup></td><td>European beech decayed wood (Fagus sylvatica) (England)</td></tr><tr><td></td><td>ESE 00140</td><td>Cheese (Tilsit, France)</td></tr><tr><td rowspan="4">Longinectria verticilliforme</td><td>UBOCC-A-120038</td><td>Cheese (Raclette d'alpage, Switzerland)</td></tr><tr><td>UBOCC-A-120039<sup>HT</sup></td><td>Cheese (Vacherin Fribourgeois, Switzerland)</td></tr><tr><td>UBOCC-A-120041</td><td>Raw cow's milk cheese (Switzerland)</td></tr><tr><td>UBOCC-A-120043<sup>HT</sup></td><td>Cheese (Alpeggio, Italy)</td></tr></tbody></table>

877 Ttype, HTholotype, ETEpitype, NTneotype, LTlectotype strains.

878 CBS: CBS-KNAW culture collection, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherland;

879 IHEM: Institute of Hygiene and Epidemiology Mycology Laboratory, Belgium; MNHN: Muséum National

880 d'Histoire Naturelle, France; UBOCC: Université de Bretagne Occidentale Culture Collection, France.
<table><caption>Table 2: Estimated cardinal values for temperature, pH and *a*<sub>w</sub> of the eleven studied *Nectriaceae* spp..</caption><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin ±SD</th><th>Xopt ±SD</th><th>Xmax ±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="10">Temperature (°C)</td><td><i>B. allantoides</i><sup>C</sup><br>UBOCC-A-120036<sup>NT</sup></td><td>-1.8</td><td>1.1</td><td>22.3</td><td>0.4</td><td>36.0</td><td>1.1</td><td>8.68 x10<sup>4</sup></td><td>2.24 x10<sup>3</sup></td><td>14392</td><td>15</td><td>5–30</td></tr><tr><td><i>B. domesticum</i><sup>C</sup><br>MNHN-RF-05627<sup>NT</sup></td><td>0.7</td><td>0.2</td><td>22.4</td><td>0.2</td><td>30.6</td><td>0.1</td><td>1.00 x10<sup>5</sup></td><td>1.30 x10<sup>3</sup></td><td>9822</td><td>11</td><td>5–30</td></tr><tr><td><i>B. penicilloides</i><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>-7.9</td><td>2.9</td><td>20.1</td><td>0.6</td><td>29.8</td><td>1.9</td><td>7.83 x10<sup>4</sup></td><td>2.83 x10<sup>3</sup></td><td>11832</td><td>15</td><td>5–25</td></tr><tr><td><i>L. lagenoides</i><sup>C</sup><br>UBOCC-A-120039<sup>NT</sup></td><td>-0.6</td><td>0.4</td><td>21.8</td><td>0.2</td><td>30.6</td><td>0.1</td><td>8.20 x10<sup>4</sup></td><td>1.17 x10<sup>3</sup></td><td>5195</td><td>9</td><td>5–30</td></tr><tr><td><i>L. verticilliforme</i><sup>C</sup><br>UBOCC-A-120043<sup>NT</sup></td><td>-0.2</td><td>1.1</td><td>19.1</td><td>0.5</td><td>31.4</td><td>0.3</td><td>6.46 x10<sup>4</sup></td><td>1.94 x10<sup>3</sup></td><td>18087</td><td>18</td><td>5–30</td></tr><tr><td><i>B. biseptatum</i><sup>NC</sup><br>CBS 110311<sup>T</sup></td><td>-7.6</td><td>1.0</td><td>32.1</td><td>0.3</td><td>38.2</td><td>0.0</td><td>1.41 x10<sup>5</sup></td><td>3.32 x10<sup>3</sup></td><td>40092</td><td>22</td><td>5–38</td></tr><tr><td><i>B. delphinoides</i><sup>NC</sup><br>CBS 120718<sup>HT</sup></td><td>4.9</td><td>ND</td><td>26.1</td><td>0.3</td><td>47.3</td><td>-0.9</td><td>1.11 x10<sup>5</sup></td><td>1.79 x10<sup>3</sup></td><td>5745</td><td>10</td><td>10–38</td></tr><tr><td><i>B. dimerum</i><sup>NC</sup><br>MNHN-RF-05625<sup>ET</sup></td><td>0.4</td><td>1.1</td><td>29.4</td><td>0.4</td><td>41.2</td><td>0.2</td><td>1.23 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>55271</td><td>24</td><td>10–40</td></tr><tr><td><i>B. lunatum</i><sup>NC</sup><br>CBS 632.76<sup>NT</sup></td><td>-6.7</td><td>1</td><td>36.2</td><td>0.4</td><td>38.0</td><td>0.0</td><td>2.00 x10<sup>5</sup></td><td>5.36 x10<sup>3</sup></td><td>42355</td><td>24</td><td>5–38</td></tr><tr><td><i>B. nectrioides</i><sup>NC</sup><br>CBS 176.31<sup>LT</sup></td><td>1.6</td><td>1.1</td><td>27.3</td><td>0.5</td><td>38.2</td><td>0.2</td><td>1.20 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>35883</td><td>22</td><td>10–37</td></tr><tr><td><i>B. penzigii</i><sup>NC</sup><br>CBS 317.34<sup>HT</sup></td><td>1.5</td><td>1.2</td><td>26.2</td><td>0.9</td><td>40.9</td><td>0.6</td><td>1.19 x10<sup>5</sup></td><td>6.63 x10<sup>3</sup></td><td>300725</td><td>54</td><td>5–39</td></tr></tbody></table>
<table><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin ±SD</th><th>Xopt ±SD</th><th>Xmax ±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="10">pH</td><td><i>B. allantoides</i><sup>C</sup><br>UBOCC-A-120036<sup>NT</sup></td><td>3.7</td><td>0.3</td><td>7.5</td><td>1.2</td><td>ND</td><td>-</td><td>1.04 x10<sup>5</sup></td><td>4.34 x10<sup>3</sup></td><td>10595</td><td>19</td><td>4-12</td></tr><tr><td><i>B. domesticum</i><sup>C</sup><br>MNHN-RF-05627<sup>NT</sup></td><td>3.7</td><td>0.1</td><td>6.7</td><td>0.4</td><td>ND</td><td>-</td><td>1.11 x10<sup>5</sup></td><td>4.34 x10<sup>3</sup></td><td>40426</td><td>29</td><td>4-12</td></tr><tr><td><i>B. penicilloides</i><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>3.9</td><td>0.2</td><td>5.1</td><td>2.0</td><td>ND</td><td>-</td><td>7.73 x10<sup>4</sup></td><td>6.94 x10<sup>3</sup></td><td>17628</td><td>25</td><td>4-12</td></tr><tr><td><i>L. lagenoides</i><sup>C</sup><br>UBOCC-A-120039<sup>NT</sup></td><td>3.4</td><td>0.2</td><td>6.4</td><td>0.4</td><td>ND</td><td>-</td><td>9.28 x10<sup>4</sup></td><td>3.14 x10<sup>3</sup></td><td>6418</td><td>15</td><td>4-12</td></tr><tr><td><i>L. verticilliforme</i><sup>C</sup><br>UBOCC-A-120043<sup>NT</sup></td><td>ND</td><td>-</td><td>11.0</td><td>2.8</td><td>12.1</td><td>0.9</td><td>7.64 x10<sup>4</sup></td><td>1.96 x10<sup>4</sup></td><td>12201</td><td>21</td><td>4-12</td></tr><tr><td><i>B. biseptatum</i><sup>NC</sup><br>CBS 110311<sup>T</sup></td><td>2.8</td><td>0.2</td><td>4.9</td><td>0.5</td><td>ND</td><td>-</td><td>1.21 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>21564</td><td>20</td><td>3-12</td></tr><tr><td><i>B. delphinoides</i><sup>NC</sup><br>CBS 120718<sup>HT</sup></td><td>3.0</td><td>0.0</td><td>6.2</td><td>0.3</td><td>ND</td><td>-</td><td>1.19 x10<sup>5</sup></td><td>2.30 x10<sup>3</sup></td><td>13540</td><td>16</td><td>3-12</td></tr><tr><td><i>B. dimerum</i><sup>NC</sup><br>MNHN-RF-05625<sup>ET</sup></td><td>2.8</td><td>0.3</td><td>4.9</td><td>1.0</td><td>ND</td><td>-</td><td>1.05 x10<sup>5</sup></td><td>2.63 x10<sup>3</sup></td><td>17544</td><td>18</td><td>3-12</td></tr><tr><td><i>B. lunatum</i><sup>NC</sup><br>CBS 632.76<sup>NT</sup></td><td>2.9</td><td>0.1</td><td>4.8</td><td>0.3</td><td>ND</td><td>-</td><td>1.26 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>17921</td><td>18</td><td>3-12</td></tr><tr><td><i>B. nectrioides</i><sup>NC</sup><br>CBS 176.31<sup>LT</sup></td><td>2.9</td><td>0.0</td><td>5.8</td><td>0.2</td><td>ND</td><td>-</td><td>1.17 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>26464</td><td>23</td><td>3-12</td></tr><tr><td><i>B. penzigii</i><sup>NC</sup><br>CBS 317.34<sup>HT</sup></td><td>2.9</td><td>0.1</td><td>4.8</td><td>0.5</td><td>ND</td><td>-</td><td>1.24 x10<sup>5</sup></td><td>4.59 x10<sup>3</sup></td><td>39464</td><td>27</td><td>3-12</td></tr></tbody></table>
<table><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin ±SD</th><th>Xopt ±SD</th><th>Xmax ±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="15">a<sub>w</sub> (NaCl)</td><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120036<sup>NT</sup></td><td>0.87</td><td>0.01</td><td>-</td><td>-</td><td>7.20 x10<sup>4</sup></td><td>6.62 x10<sup>3</sup></td><td>20284</td><td>29</td><td>0.91–0.98</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>MNHN-RF-05627<sup>NT</sup></td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>7.54 x10<sup>4</sup></td><td>1.53 x10<sup>3</sup></td><td>156</td><td>4</td><td>0.97–0.98</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>0.90</td><td>0.01</td><td>-</td><td>-</td><td>7.06 x10<sup>4</sup></td><td>7.96 x10<sup>3</sup></td><td>14487</td><td>30</td><td>0.93–0.98</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120039<sup>NT</sup></td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>5.91 x10<sup>4</sup></td><td>7.28 x10<sup>3</sup></td><td>1556</td><td>14</td><td>0.97–0.98</td></tr><tr><td><b>L. verticilliforme</b><sup>C</sup><br>UBOCC-A-120043<sup>NT</sup></td><td>0.97</td><td>0.00</td><td>-</td><td>-</td><td>5.89 x10<sup>4</sup></td><td>3.49 x10<sup>3</sup></td><td>184</td><td>6</td><td>0.97–0.98</td></tr><tr><td><b>B. biseptatum</b><sup>NC</sup><br>CBS 110311<sup>T</sup></td><td>0.97</td><td>-</td><td>-</td><td>-</td><td>(1.53 x10<sup>5</sup>) ND</td><td></td><td>9683</td><td>40</td><td>0.98</td></tr><tr><td><b>B. delphinoides</b><sup>NC</sup><br>CBS 120718<sup>HT</sup></td><td>0.88</td><td>0.01</td><td>-</td><td>-</td><td>9.23 x10<sup>4</sup></td><td>5.62 x10<sup>3</sup></td><td>18989</td><td>25</td><td>0.93–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>MNHN-RF-05625<sup>ET</sup></td><td>0.89</td><td>0.00</td><td>-</td><td>-</td><td>8.56 x10<sup>4</sup></td><td>4.14 x10<sup>3</sup></td><td>11140</td><td>19</td><td>0.93–0.98</td></tr><tr><td><b>B. lunatum</b><sup>NC</sup><br>CBS 632.76<sup>NT</sup></td><td>0.97</td><td>-</td><td>-</td><td>-</td><td>(1.14 x10<sup>5</sup>) ND</td><td></td><td>15676</td><td>51</td><td>0.98</td></tr><tr><td><b>B. nectrioides</b><sup>NC</sup><br>CBS 176.31<sup>LT</sup></td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>1.03 x10<sup>5</sup></td><td>6.00 x10<sup>3</sup></td><td>1804</td><td>12</td><td>0.97–0.98</td></tr><tr><td><b>B. penzigii</b><sup>NC</sup><br>CBS 317.34<sup>HT</sup></td><td>0.89</td><td>0.01</td><td>-</td><td>-</td><td>7.99 x10<sup>4</sup></td><td>5.64 x10<sup>3</sup></td><td>22086</td><td>27</td><td>0.93–0.98</td></tr></tbody></table>

886 Minimal (X<sub>min</sub>), optimal (X<sub>opt</sub>) and maximal (X<sub>max</sub>) values and optimal growth rates (μ<sub>opt</sub>, expressed in Relative Nephelometric Unit/hour) for fungal growth are

887 represented for each tested abiotic factor. Standard deviation (SD) was calculated from confidence interval values. ND corresponds to values that could not be
888 estimated by the model. SSE (Sum of the Squared Error) and RMSE (Root Mean Square Error) were estimated during secondary modeling. The measurable

889 growth limits are the minimum and maximum values for which growth was detected in the tested conditions. C: cheese-associated, NC: "non-cheese".
890 Table 3: Estimated cardinal values for temperature, pH and *a*<sub>w</sub> of *B. allantoides*, *B. domesticum*, *B. penicilloides*, *L. lagenoides*, *L. verticilliforme* and *B. dimerum* ("non-cheese") isolates.

891 *Lagenoides*, *L. verticilliforme* and *B. dimerum* ("non-cheese") isolates.
<table><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin</th><th>±SD</th><th>Xopt</th><th>±SD</th><th>Xmax</th><th>±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="20">Temperature (°C)</td><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120035</td><td>-0.9</td><td>1.0</td><td>23.1</td><td>0.4</td><td>39.4</td><td>1.6</td><td>8.99 x10<sup>4</sup></td><td>1.96 x10<sup>3</sup></td><td>10118</td><td>13</td><td>5-30</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120036<sup>NT</sup></td><td>-1.8</td><td>1.1</td><td>22.3</td><td>0.4</td><td>36.0</td><td>1.1</td><td>8.68 x10<sup>4</sup></td><td>2.24 x10<sup>3</sup></td><td>14392</td><td>15</td><td>5-30</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120037</td><td>-3.2</td><td>1.4</td><td>22.7</td><td>0.5</td><td>34.0</td><td>0.9</td><td>9.51 x10<sup>4</sup></td><td>3.04 x10<sup>3</sup></td><td>21372</td><td>19</td><td>5-30</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>MNHN-RF-05627<sup>NT</sup></td><td>0.7</td><td>0.2</td><td>22.4</td><td>0.2</td><td>30.6</td><td>0.1</td><td>1.00 x10<sup>5</sup></td><td>1.30 x10<sup>3</sup></td><td>9822</td><td>11</td><td>5-30</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120002</td><td>-1.9</td><td>1.2</td><td>20.1</td><td>0.5</td><td>31.0</td><td>0.2</td><td>6.58 x10<sup>4</sup></td><td>2.02 x10<sup>3</sup></td><td>12822</td><td>17</td><td>5-30</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120001</td><td>3.0</td><td>0.4</td><td>20.1</td><td>0.4</td><td>31.4</td><td>0.3</td><td>5.36 x10<sup>4</sup></td><td>1.84 x10<sup>3</sup></td><td>11355</td><td>16</td><td>5-30</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120003</td><td>-4.8</td><td>1.7</td><td>20.7</td><td>0.6</td><td>32.6</td><td>0.5</td><td>6.22 x10<sup>4</sup></td><td>1.71 x10<sup>3</sup></td><td>9197</td><td>14</td><td>5-30</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120004</td><td>8.3</td><td>ND</td><td>21.0</td><td>0.5</td><td>33.7</td><td>2.3</td><td>6.27 x10<sup>4</sup></td><td>2.47 x10<sup>3</sup></td><td>5051</td><td>13</td><td>10-25</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>-7.9</td><td>2.9</td><td>20.1</td><td>0.6</td><td>29.8</td><td>1.9</td><td>7.83 x10<sup>4</sup></td><td>2.83 x10<sup>3</sup></td><td>11832</td><td>15</td><td>5-25</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120034</td><td>1.2</td><td>ND</td><td>21.5</td><td>1.8</td><td>41.8</td><td>6.5</td><td>7.27 x10<sup>4</sup></td><td>2.47 x10<sup>3</sup></td><td>18737</td><td>19</td><td>5-25</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>VTT-D-041022</td><td>-12.1</td><td>3.5</td><td>22.5</td><td>0.7</td><td>27.6</td><td>3.0</td><td>1.05 x10<sup>5</sup></td><td>7.47 x10<sup>3</sup></td><td>17608</td><td>19</td><td>5-25</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>ESE 00140</td><td>-0.3</td><td>0.5</td><td>21.6</td><td>0.3</td><td>31.1</td><td>0.1</td><td>8.70 x10<sup>4</sup></td><td>1.89 x10<sup>3</sup></td><td>12530</td><td>14</td><td>5-30</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120038</td><td>-2.1</td><td>2.4</td><td>22.9</td><td>0.6</td><td>27.3</td><td>3.4</td><td>6.85 x10<sup>4</sup></td><td>8.04 x10<sup>3</sup></td><td>20553</td><td>22</td><td>5-25</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120039<sup>NT</sup></td><td>-0.6</td><td>0.4</td><td>21.8</td><td>0.2</td><td>30.6</td><td>0.1</td><td>8.20 x10<sup>4</sup></td><td>1.17 x10<sup>3</sup></td><td>5195</td><td>9</td><td>5-30</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120041</td><td>-0.3</td><td>0.4</td><td>21.3</td><td>0.3</td><td>30.7</td><td>0.1</td><td>8.15 x10<sup>4</sup></td><td>1.40 x10<sup>3</sup></td><td>7412</td><td>11</td><td>5-30</td></tr></tbody></table>
<table><caption>893 Table 3 Continued</caption><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin</th><th>±SD</th><th>Xopt</th><th>±SD</th><th>Xmax</th><th>±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="5">Temperature (°C)</td><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-2314</td><td>1.0</td><td>2.1</td><td>29.1</td><td>0.9</td><td>41.7</td><td>0.5</td><td>1.22 x10<sup>5</sup></td><td>5.36 x10<sup>3</sup></td><td>60025</td><td>33</td><td>10–40</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-10066</td><td>-4.8</td><td>1.8</td><td>30.4</td><td>0.5</td><td>40.9</td><td>0.2</td><td>1.09 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>19025</td><td>19</td><td>10–40</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-14513</td><td>-8.5</td><td>2.1</td><td>31.8</td><td>0.5</td><td>40.6</td><td>0.1</td><td>9.19 x10<sup>4</sup></td><td>2.47 x10<sup>3</sup></td><td>15914</td><td>17</td><td>10–40</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>MNHN-RF-05625<sup>ET</sup></td><td>0.4</td><td>1.1</td><td>29.4</td><td>0.4</td><td>41.2</td><td>0.2</td><td>1.23 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>55271</td><td>24</td><td>10–40</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>UBOCC-A-110149</td><td>-9.7</td><td>4.5</td><td>30.0</td><td>0.9</td><td>40.9</td><td>0.3</td><td>9.64 x10<sup>4</sup></td><td>4.41 x10<sup>3</sup></td><td>50517</td><td>31</td><td>10–40</td></tr><tr><td rowspan="15">pH</td><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120035</td><td>3.5</td><td>0.2</td><td>6.6</td><td>0.4</td><td>ND</td><td>-</td><td>9.78 x10<sup>4</sup></td><td>2.73 x10<sup>3</sup></td><td>4994</td><td>13</td><td>4–12</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120036<sup>NT</sup></td><td>3.7</td><td>0.3</td><td>7.5</td><td>1.2</td><td>ND</td><td>-</td><td>1.04 x10<sup>5</sup></td><td>4.34 x10<sup>3</sup></td><td>10595</td><td>19</td><td>4–12</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br>UBOCC-A-120037</td><td>3.6</td><td>0.3</td><td>6.8</td><td>0.6</td><td>ND</td><td>-</td><td>1.08 x10<sup>5</sup></td><td>3.83 x10<sup>3</sup></td><td>7867</td><td>17</td><td>4–12</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>MNHN-RF-05627<sup>NT</sup></td><td>3.7</td><td>0.1</td><td>6.7</td><td>0.4</td><td>ND</td><td>-</td><td>1.11 x10<sup>5</sup></td><td>4.34 x10<sup>3</sup></td><td>40426</td><td>29</td><td>4–12</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120002</td><td>3.5</td><td>0.3</td><td>6.7</td><td>0.5</td><td>ND</td><td>-</td><td>7.61 x10<sup>4</sup></td><td>3.47 x10<sup>3</sup></td><td>10424</td><td>19</td><td>4–12</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120001</td><td>3.6</td><td>0.1</td><td>8.0</td><td>0.2</td><td>13.0</td><td>0.2</td><td>8.06 x10<sup>4</sup></td><td>1.81 x10<sup>3</sup></td><td>2964</td><td>11</td><td>4–12</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120003</td><td>2.9</td><td>0.2</td><td>8.0</td><td>0.2</td><td>13.7</td><td>0.3</td><td>8.55 x10<sup>4</sup></td><td>1.79 x10<sup>3</sup></td><td>2902</td><td>10</td><td>4–12</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120004</td><td>3.4</td><td>0.2</td><td>7.3</td><td>0.3</td><td>13.5</td><td>0.3</td><td>6.69 x10<sup>4</sup></td><td>2.17 x10<sup>3</sup></td><td>5166</td><td>14</td><td>4–12</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>3.9</td><td>0.2</td><td>5.1</td><td>2.0</td><td>ND</td><td>-</td><td>7.73 x10<sup>4</sup></td><td>6.94 x10<sup>3</sup></td><td>17628</td><td>25</td><td>4–12</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120034</td><td>3.0</td><td>0.9</td><td>7.3</td><td>0.9</td><td>ND</td><td>-</td><td>6.82 x10<sup>4</sup></td><td>4.21 x10<sup>3</sup></td><td>18293</td><td>26</td><td>4–12</td></tr></tbody></table>
<table><caption>Table 3 Continued</caption><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin</th><th>±SD</th><th>Xopt</th><th>±SD</th><th>Xmax</th><th>±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="15">pH</td><td><b>B. penicilloides</b><sup>C</sup><br/>VTT-D-041022</td><td>4.0</td><td>0.2</td><td>ND</td><td>-</td><td>ND</td><td>-</td><td>1.05 x10<sup>5</sup></td><td>ND</td><td>8828</td><td>18</td><td>4–12</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br/>ESE 00140</td><td>3.4</td><td>0.2</td><td>6.5</td><td>0.3</td><td>ND</td><td>-</td><td>9.28 x10<sup>4</sup></td><td>2.68 x10<sup>3</sup></td><td>4755</td><td>13</td><td>4–12</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br/>UBOCC-A-120038</td><td>3.9</td><td>0.1</td><td>6.3</td><td>0.3</td><td>ND</td><td>-</td><td>7.88 x10<sup>4</sup></td><td>1.35 x10<sup>3</sup></td><td>1253</td><td>7</td><td>4–12</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br/>UBOCC-A-120039<sup>NT</sup></td><td>3.4</td><td>0.2</td><td>6.4</td><td>0.4</td><td>ND</td><td>-</td><td>9.28 x10<sup>4</sup></td><td>3.14 x10<sup>3</sup></td><td>6418</td><td>15</td><td>4–12</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br/>UBOCC-A-120041</td><td>3.7</td><td>0.2</td><td>5.9</td><td>0.4</td><td>ND</td><td>-</td><td>9.27 x10<sup>4</sup></td><td>2.45 x10<sup>3</sup></td><td>3547</td><td>11</td><td>4–12</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br/>IHEM-2314</td><td>1.9</td><td>0.3</td><td>6.8</td><td>0.3</td><td>ND</td><td>-</td><td>1.13 x10<sup>5</sup></td><td>3.06 x10<sup>3</sup></td><td>7297</td><td>15</td><td>3–12</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br/>IHEM-10066</td><td>2.5</td><td>0.1</td><td>5.9</td><td>0.2</td><td>ND</td><td>-</td><td>9.89 x10<sup>4</sup></td><td>1.63 x10<sup>3</sup></td><td>2587</td><td>9</td><td>3–12</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br/>IHEM-14513</td><td>1.5</td><td>0.4</td><td>6.6</td><td>0.3</td><td>ND</td><td>-</td><td>8.71 x10<sup>4</sup></td><td>1.76 x10<sup>3</sup></td><td>3259</td><td>10</td><td>3–12</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br/>MNHN-RF-05625<sup>ET</sup></td><td>2.8</td><td>0.3</td><td>4.9</td><td>1.0</td><td>ND</td><td>-</td><td>1.05 x10<sup>5</sup></td><td>2.63 x10<sup>3</sup></td><td>17544</td><td>18</td><td>3–12</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br/>UBOCC-A-110149</td><td>2.7</td><td>0.1</td><td>6.0</td><td>0.4</td><td>ND</td><td>-</td><td>8.31 x10<sup>4</sup></td><td>2.27 x10<sup>3</sup></td><td>4260</td><td>12</td><td>3–12</td></tr><tr><td rowspan="6">aw (NaCl)</td><td><b>B. allantoides</b><sup>C</sup><br/>UBOCC-A-120035</td><td>0.87</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5.73 x10<sup>4</sup></td><td>6.05 x10<sup>3</sup></td><td>21272</td><td>30</td><td>0.91–0.98</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br/>UBOCC-A-120036<sup>NT</sup></td><td>0.87</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.20 x10<sup>4</sup></td><td>6.62 x10<sup>3</sup></td><td>20284</td><td>29</td><td>0.91–0.98</td></tr><tr><td><b>B. allantoides</b><sup>C</sup><br/>UBOCC-A-120037</td><td>0.86</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.38 x10<sup>4</sup></td><td>5.80 x10<sup>3</sup></td><td>15176</td><td>25</td><td>0.91–0.98</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br/>MNHN-RF-05627<sup>NT</sup></td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.54 x10<sup>4</sup></td><td>1.53 x10<sup>3</sup></td><td>156</td><td>4</td><td>0.97–0.98</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br/>UBOCC-A-120002</td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.53 x10<sup>4</sup></td><td>8.11 x10<sup>3</sup></td><td>1703</td><td>15</td><td>0.97–0.98</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br/>UBOCC-A-120001</td><td>0.93</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5.13 x10<sup>4</sup></td><td>4.84 x10<sup>3</sup></td><td>3579</td><td>17</td><td>0.95–0.98</td></tr></tbody></table>
<table><caption>896 Table 3 Continued</caption><thead><tr><th>Abiotic factor</th><th>Species</th><th>Xmin</th><th>±SD</th><th>Xopt</th><th>±SD</th><th>Xmax</th><th>±SD</th><th>μ<sub>opt</sub></th><th>±SD</th><th>SSE</th><th>RMSE</th><th>Measurable growth limits</th></tr></thead><tbody><tr><td rowspan="15">aw (NaCl)</td><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120003</td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.58 x10<sup>4</sup></td><td>3.20 x10<sup>3</sup></td><td>273</td><td>6</td><td>0.97–0.98</td></tr><tr><td><b>B. domesticum</b><sup>C</sup><br>UBOCC-A-120004</td><td>0.97</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>(5.14 x10<sup>4</sup>)</td><td>-</td><td>39</td><td>3</td><td>0.98</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120021<sup>NT</sup></td><td>0.90</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.06 x10<sup>4</sup></td><td>7.96 x10<sup>3</sup></td><td>14487</td><td>30</td><td>0.93–0.98</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>UBOCC-A-120034</td><td>0.90</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.52 x10<sup>4</sup></td><td>6.33 x10<sup>3</sup></td><td>12088</td><td>25</td><td>0.93–0.98</td></tr><tr><td><b>B. penicilloides</b><sup>C</sup><br>VTT-D-041022</td><td>0.91</td><td>0.01</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.43 x10<sup>4</sup></td><td>7.21 x10<sup>3</sup></td><td>14787</td><td>28</td><td>0.93–0.98</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>ESE 00140</td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>8.05 x10<sup>4</sup></td><td>7.01 x10<sup>3</sup></td><td>1063</td><td>12</td><td>0.97–0.98</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120038</td><td>0.96</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>(3.28 x10<sup>4</sup>)</td><td>-</td><td>867</td><td>15</td><td>0.98</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120039<sup>NT</sup></td><td>0.96</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>5.91 x10<sup>4</sup></td><td>7.28 x10<sup>3</sup></td><td>1556</td><td>14</td><td>0.97–0.98</td></tr><tr><td><b>L. lagenoides</b><sup>C</sup><br>UBOCC-A-120041</td><td>0.97</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.70 x10<sup>4</sup></td><td>1.13 x10<sup>4</sup></td><td>2934</td><td>19</td><td>0.97–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-2314</td><td>0.88</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>8.72 x10<sup>4</sup></td><td>5.55 x10<sup>3</sup></td><td>11813</td><td>22</td><td>0.91–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-10066</td><td>0.88</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>7.50 x10<sup>4</sup></td><td>3.68 x10<sup>3</sup></td><td>6022</td><td>16</td><td>0.91–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>IHEM-14513</td><td>0.86</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.67 x10<sup>4</sup></td><td>2.17 x10<sup>3</sup></td><td>2341</td><td>10</td><td>0.91–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>MNHN-RF-05625<sup>ET</sup></td><td>0.89</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>8.56 x10<sup>4</sup></td><td>4.14 x10<sup>3</sup></td><td>11140</td><td>19</td><td>0.93–0.98</td></tr><tr><td><b>B. dimerum</b><sup>NC</sup><br>UBOCC-A-110149</td><td>0.87</td><td>0.00</td><td>-</td><td>-</td><td>-</td><td>-</td><td>6.91 x10<sup>4</sup></td><td>3.58 x10<sup>3</sup></td><td>6168</td><td>16</td><td>0.91–0.98</td></tr></tbody></table>

897 Minimal ($X_{min}$), optimal ($X_{opt}$) and maximal ($X_{max}$) values and optimal growth rates ($\mu_{opt}$, expressed in Relative Nephelometric Unit/hour) for fungal growth are

898 represented for each tested abiotic factor. Standard deviation (SD) was calculated from confidence interval values. ND corresponds to values that could not be
899 estimated by the model. SSE (Sum of the Squared Error) and RMSE (Root Mean Square Error) were estimated during secondary modeling. The measurable

900 growth limits are the minimum and maximum values for which growth was detected in the tested conditions. C: cheese-associated, NC: "non-cheese".
901

<caption>Table 4: Optimal growth rate ($\mu_{opt}$) estimated on PDA, Cheese agar and Raclette</caption>

902

de Savoie for the studied *Nectriaceae* isolates.

<table><thead><tr><th>Species</th><th>Isolates</th><th>Media</th><th>μ<sub>opt</sub></th><th>±SD</th><th>RMSE</th><th>%</th></tr></thead><tbody><tr><td rowspan="3">B. allantoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120035</td><td>PDA</td><td>4.5</td><td>0.2</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.8</td><td>0.2</td><td>0.1</td><td>-15.6</td></tr><tr><td>Raclette</td><td>2.6</td><td>0.1</td><td>0.1</td><td>-42.2</td></tr><tr><td rowspan="3">B. allantoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120036<sup>T</sup></td><td>PDA</td><td>4.9</td><td>0.2</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>4.0</td><td>0.1</td><td>0.1</td><td>-18.4</td></tr><tr><td>Raclette</td><td>2.8</td><td>0.2</td><td>0.1</td><td>-42.9</td></tr><tr><td rowspan="3">B. allantoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120037</td><td>PDA</td><td>4.8</td><td>0.3</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td>3.9</td><td>0.1</td><td>0.1</td><td>-18.8</td></tr><tr><td>Raclette</td><td>2.6</td><td>0.1</td><td>0.1</td><td>-45.8</td></tr><tr><td rowspan="3">B. domesticum<sup>C</sup></td><td rowspan="3">MNHN-RF-05627<sup>NT</sup></td><td>PDA</td><td>3.1</td><td>0.1</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.4</td><td>0.1</td><td>0.1</td><td>+9.7</td></tr><tr><td>Raclette</td><td>2.5</td><td>0.3</td><td>0.2</td><td>-19.4</td></tr><tr><td rowspan="3">B. domesticum<sup>C</sup></td><td rowspan="3">UBOCC-A-120001</td><td>PDA</td><td>2.1</td><td>0.2</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>2.7</td><td>0.2</td><td>0.1</td><td>+28.6</td></tr><tr><td>Raclette</td><td>2.0</td><td>0.2</td><td>0.2</td><td>-4.8</td></tr><tr><td rowspan="3">B. domesticum<sup>C</sup></td><td rowspan="3">UBOCC-A-120002</td><td>PDA</td><td>2.6</td><td>0.3</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td>2.7</td><td>0.3</td><td>0.2</td><td>+3.8</td></tr><tr><td>Raclette</td><td>2.1</td><td>0.2</td><td>0.2</td><td>-19.2</td></tr><tr><td rowspan="3">B. domesticum<sup>C</sup></td><td rowspan="3">UBOCC-A-120003</td><td>PDA</td><td>2.0</td><td>0.0</td><td>0.0</td><td></td></tr><tr><td>Cheese agar</td><td>2.5</td><td>0.2</td><td>0.1</td><td>+25.0</td></tr><tr><td>Raclette</td><td>1.8</td><td>0.1</td><td>0.1</td><td>-10.0</td></tr><tr><td rowspan="3">B. domesticum<sup>C</sup></td><td rowspan="3">UBOCC-A-120004</td><td>PDA</td><td>2.5</td><td>0.1</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>2.6</td><td>0.1</td><td>0.1</td><td>+4.0</td></tr><tr><td>Raclette</td><td>1.5</td><td>0.1</td><td>0.1</td><td>-40.0</td></tr><tr><td rowspan="3">B. penicilloides<sup>C</sup></td><td rowspan="3">UBOCC-A-120021<sup>T</sup></td><td>PDA</td><td>3.5</td><td>0.1</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.5</td><td>0.1</td><td>0.1</td><td>0.0</td></tr><tr><td>Raclette</td><td>2.4</td><td>0.1</td><td>0.1</td><td>-31.4</td></tr><tr><td rowspan="3">B. penicilloides<sup>C</sup></td><td rowspan="3">UBOCC-A-120034</td><td>PDA</td><td>2.7</td><td>0.2</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>2.2</td><td>0.2</td><td>0.2</td><td>-18.5</td></tr><tr><td>Raclette</td><td>1.2</td><td>0.1</td><td>0.1</td><td>-55.6</td></tr><tr><td rowspan="3">B. penicilloides<sup>C</sup></td><td rowspan="3">VTT-D-041022</td><td>PDA</td><td>3.1</td><td>0.1</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.6</td><td>0.1</td><td>0.0</td><td>+16.1</td></tr><tr><td>Raclette</td><td>2.3</td><td>0.2</td><td>0.1</td><td>-25.8</td></tr><tr><td rowspan="3">L. lagenoides<sup>C</sup></td><td rowspan="3">ESE 00140</td><td>PDA</td><td>3.8</td><td>0.3</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.1</td><td>0.2</td><td>0.1</td><td>-18.4</td></tr><tr><td>Raclette</td><td>2.1</td><td>0.2</td><td>0.2</td><td>-44.7</td></tr><tr><td rowspan="3">L. lagenoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120038</td><td>PDA</td><td>3.5</td><td>0.1</td><td>0.0</td><td></td></tr><tr><td>Cheese agar</td><td>3.2</td><td>0.1</td><td>0.1</td><td>-8.6</td></tr><tr><td>Raclette</td><td>2.7</td><td>0.1</td><td>0.1</td><td>-22.9</td></tr><tr><td rowspan="3">L. lagenoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120039<sup>T</sup></td><td>PDA</td><td>3.8</td><td>0.1</td><td>0.1</td><td></td></tr><tr><td>Cheese agar</td><td>3.2</td><td>0.1</td><td>0.1</td><td>-15.8</td></tr><tr><td>Raclette</td><td>2.3</td><td>0.2</td><td>0.1</td><td>-39.5</td></tr><tr><td rowspan="3">L. lagenoides<sup>C</sup></td><td rowspan="3">UBOCC-A-120041</td><td>PDA</td><td>3.7</td><td>0.3</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td>3.2</td><td>0.3</td><td>0.1</td><td>-13.5</td></tr><tr><td>Raclette</td><td>2.4</td><td>0.2</td><td>0.1</td><td>-35.1</td></tr><tr><td rowspan="3">L. verticilliforme<sup>C</sup></td><td rowspan="3">UBOCC-A-120043<sup>T</sup></td><td>PDA</td><td>3.4</td><td>0.3</td><td>0.3</td><td></td></tr><tr><td>Cheese agar</td><td>3.1</td><td>0.2</td><td>0.2</td><td>-8.8</td></tr><tr><td>Raclette</td><td>2.2</td><td>0.3</td><td>0.2</td><td>-35.3</td></tr></tbody></table>

903

50
904 **Table 4 Continued**

<table><thead><tr><th>Species</th><th>Isolates</th><th>Media</th><th>μ<sub>opt</sub></th><th>±SD</th><th>RMSE</th><th>%</th></tr></thead><tbody><tr><td rowspan="3">B. biseptatum<sup>NC</sup></td><td rowspan="3">CBS 110311<sup>T</sup></td><td>PDA</td><td><strong>12.8</strong></td><td>0.3</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td><strong>10.7</strong></td><td>0.1</td><td>0.0</td><td>-16.4</td></tr><tr><td>Raclette</td><td><strong>6.1</strong></td><td>0.1</td><td>0.0</td><td>-52.3</td></tr><tr><td rowspan="3">B. delphinoides<sup>NC</sup></td><td rowspan="3">CBS 120718<sup>HT</sup></td><td>PDA</td><td><strong>8.6</strong></td><td>0.5</td><td>0.3</td><td></td></tr><tr><td>Cheese agar</td><td><strong>6.7</strong></td><td>0.5</td><td>0.3</td><td>-22.1</td></tr><tr><td>Raclette</td><td><strong>4.4</strong></td><td>0.5</td><td>0.3</td><td>-48.8</td></tr><tr><td rowspan="3">B. dimerum<sup>NC</sup></td><td rowspan="3">MNHN-RF-05625<sup>ET</sup></td><td>PDA</td><td><strong>9.2</strong></td><td>0.5</td><td>0.3</td><td></td></tr><tr><td>Cheese agar</td><td><strong>7.4</strong></td><td>0.2</td><td>0.1</td><td>-19.6</td></tr><tr><td>Raclette</td><td><strong>5.6</strong></td><td>0.3</td><td>0.1</td><td>-39.1</td></tr><tr><td rowspan="3">B. lunatum<sup>NC</sup></td><td rowspan="3">CBS 632.76<sup>NT</sup></td><td>PDA</td><td><strong>19.7</strong></td><td>0.8</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td><strong>18.7</strong></td><td>0.2</td><td>0.0</td><td>-5.1</td></tr><tr><td>Raclette</td><td><strong>9.6</strong></td><td>0.2</td><td>0.0</td><td>-51.3</td></tr><tr><td rowspan="3">B. nectrioides<sup>NC</sup></td><td rowspan="3">CBS 176.31<sup>LT</sup></td><td>PDA</td><td><strong>3.5</strong></td><td>0.2</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td><strong>4.6</strong></td><td>0.2</td><td>0.1</td><td>+31.4</td></tr><tr><td>Raclette</td><td><strong>2.7</strong></td><td>0.2</td><td>0.1</td><td>-22.9</td></tr><tr><td rowspan="3">B. penzigii<sup>NC</sup></td><td rowspan="3">CBS 317.34<sup>HT</sup></td><td>PDA</td><td><strong>10.1</strong></td><td>0.4</td><td>0.2</td><td></td></tr><tr><td>Cheese agar</td><td><strong>6.9</strong></td><td>0.3</td><td>0.2</td><td>-31.7</td></tr><tr><td>Raclette</td><td><strong>4.6</strong></td><td>0.2</td><td>0.1</td><td>-54.5</td></tr></tbody></table>

Optimal growth rates (μ<sub>opt</sub>) (expressed in mm/days) for fungal growth are represented for each tested medium. The standard deviation (SD) was calculated from confidence interval values. RMSE (Root Mean Square Error) were estimated during secondary modeling. The impact on the μ<sub>opt</sub> of cheese agar or Raclette vs PDA were calculated and highlighted in the column “μ<sub>opt</sub>” in purpe, red, orange, yellow for a reduction ≥40.0 %, between 40.0 % and 20.0 %, between 20.0 % and 10.0 %, between 10.0 % and 0.0 %, respectively and green for no impact or increase. C: cheese-associated, NC: non-cheese.
911 **Figure captions**

912

913 **Figure 1: Secondary models obtained for the cheese-associated *Nectriaceae* species type strains for temperature, pH and $a_w$ (using NaCl as depressor) on PDB medium.** Lines A to E respectively correspond to *Bisifusarium allantoides* 916 UBOCC-A-120036<sup>NT</sup>, *Bisifusarium domesticum* MNRN-RF-05627<sup>NT</sup>, *Bisifusarium* 917 *penicilloides* UBOCC-A-120021<sup>NT</sup>, *Longinectria lagenoides* UBOCC-A-120039<sup>NT</sup> and 918 *Longinectria verticilliforme* UBOCC-A-120043<sup>NT</sup>. Dots correspond to no observable 919 growth in the tested conditions. 920

920

921 **Figure 2: Secondary models obtained for the "non-cheese" *Nectriaceae* species type strains for temperature, pH and $a_w$ (using NaCl as depressor) on PDB medium.** Lines A to E respectively correspond to *B. biseptatum* CBS 110311<sup>T</sup>, *B. 923 delphinoides* CBS 120718<sup>HT</sup>, *B. dimerum* MNHN-RF-05625<sup>ET</sup>, *B. lunatum* CBS 924 632.76<sup>NT</sup>, *B. nectrioides* CBS 176.31<sup>LT</sup> and *B. penzigii* CBS 317.34<sup>HT</sup>. Dots correspond 925 to no observable growth in the tested conditions. 926

927

928 **Figure 3: Secondary models obtained for various isolates of the four *Nectriaceae* cheese-associated species and *Bisifusarium dimerum* ("non-cheese") for temperature, pH and $a_w$ (using NaCl as depressor) on PDB medium.** 929 Lines A to E respectively correspond to *B. allantoides* (curves A: UBOCC-A-120035, 930 B: UBOCC-A-120036<sup>NT</sup>, C: UBOCC-A-120037), *B. domesticum* (curves A: UBOCC- 931 A-120002, B: UBOCC-A-120001, C: UBOCC-A-120003, D: MNHN-RF-05627<sup>NT</sup>, E: 932 UBOCC-A-120004), *B. penicilloides* (curves A:VTT-D-041022, B: UBOCC-A-120034, 933 C: UBOCC-A-120021<sup>NT</sup>), *L. lagenoides* (curves A: UBOCC-A-120038, B: UBOCC-A- 934 935
936 120039NT, C: UBOCC-A-120041, D: ESE-00140) and *B. dimerum* (curves A: IHEM-2314, B: IHEM-10066, C: IHEM-14513, D: UBOCC-A-110149, E: MNHN-RF-05625ET). Dots correspond to no observable growth in the tested conditions.
937
938
939

940 **Figure 4: Principal component analysis on nine estimated parameters (i.e. T<sub>min</sub>,**
941 T<sub>max</sub>, T<sub>(μopt)</sub>, pH<sub>min</sub>, pH<sub>opt</sub>, pH<sub>(μopt)</sub>, a<sub>w min</sub>, a<sub>w (μopt)</sub>) for the 26 studied Nectriaceae
942 isolates. Individuals factor map (A) and Variables factor map (B) are represented, and
943 the colors correspond to the cheese-associated species (green) and "non-cheese"
944 species (blue). Samples are noted with the following abbreviations "Bbis_A", "Bdel_A",
945 "BL_A", "BN_A", "BP_A" corresponding to B. biseptatum CBS 110311, B. delphinoides
946 CBS 120718, B. lunatum CBS 632.76, B. nectrioides CBS 176.31 and B. penzigii CBS
947 317.34. "Bdim_A", "Bdim_B", "Bdim_C", "Bdim_D", "Bdim_E" corresponding to B.
948 dimerum IHEM-2314, IHEM-10066, IHEM-14513, MNHN-RF-05625, UBOCC-A-
949 110149 respectively, "Bdom_A", "Bdom_B", "Bdom_C", "Bdom_D", "Bdom_E"
950 corresponding to B. domesticum MNHN-RF-05627, UBOCC-A-120001, UBOCC-A-
951 120002, UBOCC-A-120003, UBOCC-A-120004 respectively. "BA_A", "BA_B", "BA_C"
952 corresponding to B. allantoides UBOCC-A-120035, UBOCC-A-120036, UBOCC-A-
953 120037, "Bpeni_A", "Bpeni_B", "Bpeni_C" corresponding to B. penicilloides UBOCC-
954 A-120021, UBOCC-A-120034, VTT-D-041022, "Llag_A", "Llag_B", "Llag_C", "Llag_D"
955 corresponding to L. lagenoides ESE00140, UBOCC-A-120038, UBOCC-A-120039
956 and "LV_A" corresponding to L. verticilliforme UBOCC-A-120043.
957

958 **Figure 5: Secondary models obtained for the studied cheese-associated**
959 **Nectriaceae isolates for temperature on PDA, cheese agar medium and Raclette**
960 **de Savoie.** Maximal growth rates estimated at 15 and 25 °C on PDA (blue), cheese
agar (green) and Raclette de Savoie (red) were fitted applying the estimated cardinal values on the nephelometric data (black). Models A to P respectively corresponds to *B. allantoides* UBOCC-A-120035, *B. allantoides* UBOCC-A-120036<sup>NT</sup>, *B. allantoides* UBOCC-A-120037, *B. domesticum* UBOCC-A-120001, *B. domesticum* UBOCC-A-120002, *B. domesticum* UBOCC-A-120003, *B. domesticum* UBOCC-A-120004, *B. domesticum* MNHN-RF-05627<sup>NT</sup>, *B. penicilloides* VTT-D-041022, *B. penicilloides* UBOCC-A-120034, *B. penicilloides* UBOCC-A-120021<sup>NT</sup>, *L. lagenoides* UBOCC-A-120038, *L. lagenoides* UBOCC-A-120039<sup>NT</sup>, *L. lagenoides* UBOCC-A-120041, *L. lagenoides* ESE-00140 and *L. verticilliforme* UBOCC-A-120043<sup>NT</sup>. Dots correspond to no observable growth in the tested conditions.

971

**Figure 6: Secondary models obtained for "non-cheese" *Bisifusarium* spp. type strains for temperature on PDA, cheese agar medium and Raclette de Savoie.**

Maximal growth rates estimated at 15.0 and 25.0 °C on PDA (blue), cheese agar (green) and Raclette de Savoie (red) were fitted applying the cardinal values estimated on the nephelometric data (black); Models A to F respectively corresponds to *B. biseptatum* CBS 110311<sup>T</sup>, *B. delphinoides* CBS 120718<sup>HT</sup>, *B. dimerum* MNHN-RF-05625<sup>ET</sup>, *B. lunatum* CBS 632.76<sup>NT</sup>, *B. penzigii* CBS 317.34<sup>HT</sup>, *B. nectrioides* CBS 176.31<sup>LT</sup>. The dots correspond to a growth not observed in the tested conditions.

972

973

974

975

976

977

978

979
<figure><img src="image_2.png" /><figcaption>Figure 1. Temperature</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Figure 2.
Temperature</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Temperature</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

pH

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

NaCl

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>
<figure><img src="image_19.png" /><figcaption>Figure 4.</figcaption></figure>

988

989

990

991

992
<figure><img src="image_20.png" /><figcaption>Figure 5.</figcaption></figure>
<figure><img src="image_21.png" /><figcaption>Figure 6.</figcaption></figure>
<table><caption>998 Table S1: *B. domesticum* isolates used on RAPD study.</caption><thead><tr><th>Species</th><th>Isolates code</th><th>Substrate/Host of origin</th></tr></thead><tbody><tr><td rowspan="20">B. domesticum</td><td>MNHN RF-03342</td><td>Cheese (Saint Nectaire, France)</td></tr><tr><td>MNHN RF-05627<sup>NT</sup></td><td>Food (Belgium)</td></tr><tr><td>MNHN-RF-05628</td><td>Food (Switzerland)</td></tr><tr><td>MUCL-38933</td><td>Cheese (Saint Nectaire, France)</td></tr><tr><td>MUCL-38934</td><td>Cheese (Saint Nectaire, France)</td></tr><tr><td>MUCL-39198</td><td>Unknown (France)</td></tr><tr><td>NNRL 37582</td><td>Unknown</td></tr><tr><td>UBOCC-A-109095</td><td>Cheese-maker environment (France)</td></tr><tr><td>UBOCC-A-117075</td><td>Cheese (France)</td></tr><tr><td>UBOCC-A-113010</td><td>Unknown (France)</td></tr><tr><td>UBOCC-A-120001</td><td>Commercial adjunct culture (Switzerland)</td></tr><tr><td>UBOCC-A-120002</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120003</td><td>Cheese (Raclette d'alpage, Switzerland)</td></tr><tr><td>UBOCC-A-120004</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120005</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120006</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120007</td><td>Commercial adjunct culture (France)</td></tr><tr><td>UBOCC-A-120008</td><td>Cheese (Saint Nectaire, France)</td></tr><tr><td>UBOCC-A-120009</td><td>Cheese (Raclette d'alpage, Switzerland)</td></tr><tr><td>UBOCC-A-120010</td><td>Cheese (Vacherin Fribourgeois, Switzerland)</td></tr><tr><td>UBOCC-A-120011</td><td>Cheese (Alpeggio, Italy)</td></tr><tr><td>UBOCC-A-120012</td><td>Cheese (Saint Nectaire, France)</td></tr><tr><td>UBOCC-A-120013</td><td>Cheese (Mont d'Or, France)</td></tr><tr><td>ESE-00042</td><td>Ewe cheese (France)</td></tr><tr><td>ESE-00064</td><td>Cow cheese (France)</td></tr></tbody></table>

999 NTneotype strain. MNHN: Muséum National d'Histoire Naturelle, France; MUCL: Mycothèque de l'Université Catholique de Louvain, Belgium; NRRL: Agricultural Research Service Culture Collection, USA; UBOCC: Université de Bretagne Occidentale Culture Collection, France; ESE: Laboratoire Ecologie Systématique Evolution, Université Paris Saclay, France.
1000
1001
1002
1003
<figure><img src="image_22.png" /></figure>

Figure S1. ari1 RAPD genetic profiles of the 25 *B. domesticum* isolates included in this study.

Lanes 1 to 25, *B. domesticum* isolates UBOCC-A-117075, UBOCC-A-109095, MNHN RF-03342, MNHN RF-05627, MNHN RF-05628, MUCL 38934, MUCL 39198, MUCL 38933, UBOCC-A-120005, UBOCC-A-120006, UBOCC-A-120007, NRRL 37582, UBOCC-A-120002, UBOCC-A-120008, UBOCC-A-120003, UBOCC-A-120009, UBOCC-A-120010, UBOCC-A-120011, UBOCC-A-120012, UBOCC-A-120013, ESE-00042, ESE-00064, UBOCC-A-120004, UBOCC-A-113010, UBOCC-A-120001. M: size ladder (100 bp ladder, Promega). Selected isolates for the intraspecific study are highlighted by an arrow.