Dynamics of solute/matric stress interactions with climate change abiotic factors on growth, gene expression and ochratoxin A production by Penicillium verrucosum on a wheat-based matrix
Introduction
Penicillium verrucosum is a predominantly soil-based xerotolerant species that also survives saprophytically on crop residue. It colonises temperate cereal grains during harvesting and delayed drying or poor post-harvest management which can lead to ochratoxin A (OTA) contamination of pockets of under-dried or moist grain (Lund and Frisvad, 2003; Lindblad et al., 2004; Magan and Aldred, 2007). Indeed, because OTA is considered to be a nephrotoxin and potentially carcinogenic for humans (International Agency for Cancer Research, 1993), there are legislative limits in cereals destined for food processing or for animal feed (European Union, 2006).
Previous ecological studies have shown that water availability, temperature and inter-granular atmosphere and their interactions have an impact on growth and OTA production in vitro and in situ in stored wheat grain and identified the optimum and boundary conditions for growth and OTA production (Cairns et al., 2005). It has also been shown that populations of P. verrucosum predominantly reside in soil and on crop residue which form the focal points for the development of the inoculum for contaminating cereals during harvesting and drying (Elmholt, 2003; Elmholt and Hostbjerg, 1999). Thus, an understanding of the relative tolerance of P. verrucosum to both soil water stress, mainly determined by the soil matric potential, and solute stress in crop residue is important. Abdelmohsen et al. (2020) recently showed that optimum growth and OTA production were at -7.0 MPa (=0.95 water activity, aw) and -1.4 MPa (=0.99 aw) respectively, regardless of whether solute or matric stress were imposed on P. verrucosum. However, this species was more sensitive to ionic solute stress (NaCl) with no growth at -19.6 MPa (=0.86 aw) while growth still occurred in the presence of the non-ionic solute (glycerol) and matric stress treatments.
Previous studies with non-xerophilic toxigenic fungi such as Fusarium graminearum, and xerophilic/xerotolerant species such as Aspergillus ochraceus (= A. westerdijkiae) and A. flavus have examined the relative tolerance to matric vs solute stress (Ramos et al., 1999; Ramirez et al., 2004; Giorni et al., 2008). These showed that for the non-xerophilic species both macroconidial germination and growth were more sensitive to matric than solute stress. In contrast, the xerotolerant/xerophilic species were more resilient and able to tolerate both matric and solute stress (Magan, 1988; Magan et al., 1995; Ramos et al., 1999; Ramirez et al., 2004). Subsequently Jurado et al. (2008) showed that the non-xerophilic mycotoxigenic species F. verticillioides, a pathogen of maize, grew relatively similarly under both ionic and non-ionic solute stress, but was also more sensitive to matric stress. The relative expression of the FUM1 gene involved in fumonisins biosynthesis reflected these differences.
OTA is a polyketide mycotoxin, with the biosynthetic pathway predominantly elucidated in P. nordicum (Wang et al., 2016). In this species, the gene cluster for OTA includes those encoding for a polyketide synthase (PKS) (otapksPN) and non-ribosomal peptide synthetase (NRPS) (otanrpsPN). Geisen et al. (2004) correlated the relative expression of the otapksPN from P. nordicum with OTA production. There is a good homology between the OTA biosynthetic pathways in both P. nordicum and P. verrucosum, with some differences related to the function of the PKS gene (otapks) (Geisen et al., 2006; Wang et al., 2016). Abdelmohsen et al. (2020) were able to show that P. verrucosum was able to express the otapksPV over a wide range of ionic/non-ionic solute stress conditions (-1.4 to -14.0 MPa; = 0.99–0.90 aw). Interestingly, the otanrpsPV gene was significantly up-regulated under matric stress, especially with relatively freely available water (-1.4 MPa = 0.99 aw). These studies focused on solute/matric stress and did not examine the effects of interactions with temperature or other abiotic factors.
There is now interest in the resilience of mycotoxigenic fungi to climate-related abiotic factors and whether this will stimulate or inhibit mycotoxin production. Such interacting factors have been shown to result in stimulation of biosynthetic genes involved in mycotoxin production and phenotypic toxin production including aflatoxins by A. flavus, OTA by A. westerdijkiae and T-2/HT-2 toxin by F. langsethiae (Akbar et al., 2016; Medina et al., 2017; Verheecke-Vaessen et al., 2019; Cervini et al., 2020). However, no studies have previously examined solute vs matric stress when combined with changes in temperature and exposure to existing or elevated CO2 may have on growth, biosynthetic genes involved in toxin production and the amounts of toxin production. This may be important in understanding the potential changes in the life cycle and ecological characteristics of this species especially in soil and on crop debris which will influence the inoculum potential for contamination of cereals with OTA, especially in the harvesting, drying and post-harvest phases.
Thus, the objectives of this work were to examine the effect of solute or matric stress (-2.8 or -7.0 MPA (=0.98 and 0.95 aw), temperature (25 or 30 °C) and exposure to CO2 (400 vs 1000 ppm) on: (a) growth, (b) relative expression of two key biosynthetic genes (otapksPV, otanrpsPV) involved in OTA biosynthesis and (c) OTA production by P. verrucosum on a milled wheat matrix.
Section snippets
Fungal strain
A strain of P. verrucosum (OTA11) was used in these studies. This was isolated from wheat grain and is a known producer of OTA (Cairns et al., 2005; Abdelmohsen et al., 2020). We are grateful to Dr. Monica Olsen (National Food Authority, Sweden) for the supply of the strain.
Inoculum preparation and inoculation
The fungal strain was sub-cultured on malt extract agar (30.0 g L−1 malt extract, 5.0 g L−1 peptone and 15.0 g L−1 agar) at 25 °C in the dark for up to 10 days. The spores were gently dislodged from the colony surface by
Effect of climate change-related interacting factors on relative growth rates at 25 °C and 30 °C on wheat-based matrices
Fig. 1a and b compares the effect of matric and solute stress, temperature (25 and 30 °C) and CO2 exposure (400 or 1000 ppm) on the relative growth of the P. verrucosum strain. Growth was significantly affected when exposed to 30 °C and -2.8 MPa (= 0.98 aw) and 1000 ppm CO2 where no growth occurred in the solute stress treatment. However, at -7.0 MPa (0.95 aw) and 1000 ppm CO2 there was an increased growth rate when compared to existing conditions. With matric stress there was no effect on
Discussion
This study has examined the effect of different types of water stress and their interaction with other climate change-related scenarios on the molecular ecology of P. verrucosum. To our knowledge, no previous studies have addressed this in the context of resilience of such mycotoxigenic fungi in relation to interacting abiotic stresses relevant to activity in soil and on crop residue. This OTA producing strain was able to grow at both the tested solute and matric imposed stress conditions
Acknowledgements
S.A. is grateful to the British Council and the Newton Musharraf Programme for financial support.
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2022, Current Opinion in Food ScienceCitation Excerpt :They found that the increase of more than 2.5-fold CO2 concentration even at a lower-temperature cycle (15–28 °C) resulted in an increase in colony growth and OTA production. Moreover, a similar study has been conducted on P. verrucosum by analyzing the effect of temperature (25 vs. 30 °C), CO2 (400 vs. 1000 ppm) and matric/solute stress (−2.8 vs. −7.0 MPa) on growth, OTA biosynthesis, and cluster regulation [72]. Overall, the growth rate under solute stress was slower in elevated CO2 than under matric stress when compared with existing conditions, and under elevated CO2 levels in matric stress treatments, otaA (otapksPV) gene expression was increased.
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2021, Fungal Biology ReviewsCitation Excerpt :Under acidic conditions (<pH 5) and low concentration of sodium chloride (NaCl), the expression of OTA gene (OTApks) was suppressed, leading to low production of OTA. However, pH of 6–8, as well as increasing concentration of NaCl and temperature of 25 °C, were found to be the optima conditions for the transcriptional activation of OTA biosynthetic genes and OTA production in P. nordicum and P. verrucosum (Abdelmohsen et al., 2021; Geisen, 2004; Schmidt-Heydt and Geisen, 2007). Regarding the effect of projected climate change scenarios on the transcriptional profile of OTA biosynthetic genes and OTA production, Cervini et al. (2021) examined the effect of current and predicted temperature (15–28 °C vs 18–34 °C) and CO2 concentrations (400 vs 1000 ppm) on the expression of OTA structural (OTApks, OTAnrps, OTAhal & OTAp450) and regulatory (OTAbZIP laeA & veA) genes in three A. carbonarius strains - ITEM 7444, 18515 and 5010.
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Present address: School of Life and Medical Sciences, University of Hertfordshire, Hatfield, Herts, AL10 9AB, UK.