Isoborneol as a natural sporulation quenching agent to control Aspergillus flavus

Abstract In an effort to seek natural antisporulating agents used in the control of Aspergillus flavus, 54 essential oil compounds were employed to evaluate their antisporulating activity against A. flavus at the concentration of 100 μg/mL. The results indicated that isoborneol could inhibit spore production at 100 μg/mL. The light microscopy and scanning electron microscopy (SEM) observations revealed that A. flavus did not produce any conidia, vesicles, phialides and conidiophores after treatment with isoborneol at 80 μg/mL, confirming the effectiveness of isoborneol. The in vivo bioassay results demonstrated that isoborneol could prevent the peanuts from A. flavus contamination by inhibiting the sporulation when treated with isoborneol at concentrations higher than 100 μg/mL. RT-qPCR results suggested that isoborneol exerts its antisporulating activity by suppressing the fluG expression. These results proved that isoborneol could be used as a natural and safe antisporulating agent for commercial applications to control spore infections of A. flavus.


Introduction
Aspergillus flavus, a ubiquitous and saprophytic fungus, is a common fungal pathogen of plants, animals and humans (Lan et al. 2018). The Aspergillus fungi have a high sporulation capacity, which leads to a ubiquitous presence, both indoors and outdoors, of high atmospheric spore concentrations (1 À 100 conidia/m 3 ) (Guerra et al. 2018). These spores are not only the inoculum of toxigenic A. flavus contamination in preharvest and postharvest seed crops and foodstuffs but also the causal agents of invasive and non-invasive aspergillosis in humans, animals, and insects (Yu et al. 2005). Besides, they also cause superficial infections and allergic reactions in humans (Weber et al. 2009). In light of these facts, inhibition of sporulation is a promising approach to reducing or ceasing the incidences of A. flavus contamination and spore-related diseases.
Due to the noticeable adverse impacts of synthetic fungicides (Brzezinska and Jankiewicz 2012;Mateo et al. 2017;Shakeel et al. 2018), recently essential oil compounds (EOCs) have gained tremendous popularity and scientific interest as alternatives to the chemical agents in the control of A. flavus, since they are effective, eco-friendly, renewable, cost-effective and easy to degrade (Seethapathy et al. 2016). Generally, these EOCs have been reportedly mainly concentrated on the antifungal activities against mycelial growth or the spore germination of A. flavus (Mahmoud 1994;Popovici et al. 2008;Singh et al. 2010;Tang et al. 2018;Lasram et al. 2019). Although some crude essential oil mixtures, such as Curcuma longa L., Zataria multiflora Boiss., cinnamon and lemongrass essential oils (Paranagama et al. 2003;Gandomi et al. 2009;Ferreira et al. 2013;Manso et al. 2013), have been reported to inhibit the sporulation of A. flavus, the explicit antisporulating EOCs have been less widely focused.
To seek the antisporulating EOCs, we herein evaluated the effects of 54 EOCs on the sporulation of A. flavus. In addition, in vivo antisporulating activity of the potent compound isoborneol and the sporulation-related genes were subsequently examined to help understand the antisporulating activity.

Results and discussion
According to the obtained results (Tables S1 and S2), most EOCs did not inhibit the spore production in their contact agar phase at the concentration of 100 lg/mL. Noticeably, the only antisporulating effect was observed for compound isoborneol at 100 lg/mL. From the ageing zone to the peripheral zone (illustrated in Figure S1), the isoborneol-treated PDA plates were covered by a fluffy white colony, while the control plates were covered by a greenish colony ( Figure S2a). Further light microscopy examinations of the colonies by scraping them into water confirmed the sporulation quenching activity of isoborneol at 100 lg/mL and excluded its potential for blocking the colony pigmentation. Besides, no significant colonial growth inhibition was found for isoborneol. Interestingly, borneol ( Figure S3), the enantiomer of isoborneol, did not inhibit the spore production ( Figure S2a), indicating that the inhibition effect of isoborneol is stereospecific. The result of the further sporulation quenching bioassay ( Figure S2b) identified that the minimum sporulation quenching concentration of isoborneol was 80 lg/mL. Meanwhile, supplementary light microscopy and SEM observation corroborated the effectiveness of isoborneol at 80 lg/mL ( Figure S4). Figure S5a and b, the in vivo bioassay revealed that isoborneol did not inhibit the mycelial growth in peanuts at the concentrations of 80 lg/mL, 100 lg/ mL and 150 lg/mL after inoculation for 15 d, with contamination rates higher than 90% ('T' sides in Petri dishes). Nevertheless, the isoborneol treatments at 100 lg/mL and 150 lg/mL significantly decreased the spore production with the sporulation rates of 5.6% and 2.2%, respectively. Correspondingly, it was found that the isoborneol treatments at 100 lg/mL and 150 lg/mL could completely prevent the peanuts from infection after inoculation for 15 d ('U' sides in Petri dishes).

As shown in
The results of RT-qPCR analysis ( Figure S5c) showed that genes fluG, brlA and abaA in A. flavus were significantly downregulated in comparison to the untreated group when the isoborneol was applied at the sporulation quenching concentration of 100 lg/mL. However, below the minimum sporulation quenching concentration (80 lg/mL), isoborneol treatment (50 lg/mL) increased the expression of these three genes compared with the control group. This increase might be ascribed to the fact that A. flavus were fruiting on the isoborneol-treated (50 lg/mL) plates after inoculation for 7 d while the fruiting process was almost finished in the control group.
Isoborneol has been well used as an inexpensive source of fragrance ingredients in many industries (Bl€ aske et al. 2003;Bhatia et al. 2008). Notably, it has been granted the status of generally recognized as safe (GRAS) and has been approved by the Food and Drug Administration (FDA), USA, for food use (Bl€ aske et al. 2003). Besides that, isoborneol has been found to possess diverse biological activity for manifold applications (Armaka et al. 1999;Tian et al. 2007;Su et al. 2013;Querido et al. 2022). Recently, it was reported that the isoborneol treatment at 100 lg/mL rendered a growth rate of 93.2% ± 17.4% for A. flavus and completely inhibited aflatoxin production in potato dextrose broth (Moon et al. 2018). Our in vitro bioassay results indicated that the colonial growth inhibition rate of isoborneol was 22.0% at 100 lg/mL on a PDA plate, which was in accord with the results reported above. However, to the best of our knowledge, the antisporulating activity of isoborneol against A. flavus has not been reported till now. As it is well known, chiralcenter enantiomers differ significantly in biological activity, pharmacodynamics, pharmacokinetics and toxicity (LaPlante et al. 2011). There is no exception for ECOs; for example, the two enantiomers of limonene behaved differently in antifungal activity and pharmacological properties (Lis-Balchin et al. 1996). In our case, borneol, the isomers of isoborneol, did not inhibit the spore production at 100 lg/mL, according with the known facts.
Generally, there is a putative central regulatory pathway to control conidiation-specific gene expression and asexual developmental processes ( Figure S1b) (Park and Yu 2012;Lee et al. 2016). In our case, the RT-qPCR results demonstrated that isoborneol could significantly suppress the fluG expression at the sporulation quenching concentration of 100 lg/mL, which confers complete repression of the expression of conidiation-specific genes brlA and abaA and conidiation. Collectively, these results suggested that isoborneol might exert the antisporulating action by inhibiting the fluG transcription or its upstream signaling pathway.

Experimental
Detailed experimental procedures are available in the supplementary materials.

Conclusions
Compound isoborneol was screened out and its minimum sporulation quenching concentration was 80 lg/mL on PDA plate. The in vivo bioassay results indicated that it could be used as a natural antisporulating agent to provide a promising and alternative approach to prevent A. flavus secondary infection in storage or to sanitize the indoor environment by reducing spores. RT-qPCR results suggested that isoborneol inhibits the spore production by suppression of the fluG expression.

Disclosure statement
No potential conflict of interest was reported by the authors.