Evaluating changes in growth and pigmentation of Cladosporium cladosporioides and Paecilomyces variotii in response to gamma and ultraviolet irradiation

Gamma source and dose modeling

The general literature contains conflicting results on whether the energies of photons interacting with fungi affects the radiotrophic response. As such, we sought to control critical variables while irradiating the fungi with ionizing radiation from a sealed Cs-137 source and a UV source. The Cs-137 source emitted a photon at 662 keV along with other lower energy photons near 30 keV (Table S1).

A review of previous studies was conducted to identify the gamma dose rate and total dose that should be targeted for exposure (Table S2). Those dose rates ranged from 600,000 rad delivered in 1.5 h to 0.08 rad delivered in 16 h. Even among studies examining the same fungi attributes, the total dose varied dramatically. For the present study, we used a Health Physics code to target a 50-rad dose over a one-week exposure. This dose was selected as it changes blood count observed in most humans²⁴. We hypothesized that this dose would induce physiological changes in the fungi without causing a high rate of lethality. A MicroShield (Grove Software, Inc.) model was created to identify the quantity of radioactive material and distance between source and sample necessary to achieve the dose of 50 rad in seven days. From a sensitivity analysis of the MicroShield model, it was determined that ~ 350 µCi of Cs-137 would create a dose rate of ~ 50 rad in seven days (Fig. 1; Table S3), if placed 1.8 cm from the surface of the fungi. It should be noted that Microshield values are often conservative and likely underestimate the actual dose on target. In addition, 50 rad falls in the middle of the large range for energies previously reported in the general literature (Table S2).

The dose from the Cs-137 source on the fungal mycelium is also dependent on the radial growth of the fungus from the center plug used to initiate growth. As the fungus grows away from the source, the leading edge will experience a lower total dose of radiation. Although a uniform dose would have been ideal, a source with activity sufficient to create a uniform radiation field would have initiated a variety of safety controls deemed impractical for this experiment. The background radiation dose at the testing site in Albuquerque is approximately 10 µrem h⁻¹; the dose at the outermost area of the Petri dish was measured at 65,553 µrad h⁻¹. As this dose was primarily from gamma emissions, rad and rem can be considered equivalent. To validate the simulation, a dose rate study was performed using thermoluminescent dosimeters (TLD) placed at varying distances from the center of the source. The TLD placed directly under the source measured ~ 100 rad over the seven-day exposure, which is double the prediction from the simulation (50 rad; Fig. 2A). However, at a radial distance of 3.5 cm, the measured and estimated total dose over seven days were much closer, 12.3 and 11.4 rad, respectively. A comparison of the measured and estimated dose on target demonstrated a non-linear correlation (Fig. 2B), in which the simulation better approximated the dose at larger radial distances from the source.

In order to normalize the energy deposited in the fungi from Cs-137 and the UV lamp sources, the units of MeV g⁻¹ s⁻¹ were selected for additional simulations. Monte Carlo N-Particle transport code (MCNP) simulations were used to determine this quantity for the Cs-137. The materials and geometry of the Petri dish and fungus used for these simulations are shown in Fig. 3. The Cs-137 was simulated as a point source located 1.5 cm from the top of the fungi. The Petri dish was set on a bakelite table. The setup was located in the center of a notional 5 m × 5 m × 5 m room with 30 cm thick concrete walls and filled with air. Leads bricks set on the table surrounded the petri dish and source. The International Commission on Radiological Protection (ICRP) material definitions did not contain data for fungal mycelia. Thus, we selected for skin as the closest approximation of the properties of the fungal mycelium²⁵. This simulation gave a result for the energy deposited per particle as 6.53 × 10^–4 MeV g⁻¹, which for a 350 μCi activity, the rate of energy deposition was determined to be 7907 MeV g⁻¹ s⁻¹.

UV source and irradiation

Our intent was to match the energy absorbed by the fungi to control for all variables except the photon energy difference between the Cs-137 source and UV lamp. The spectrum of energies emitted from the Cs-137 source varied significantly from those of the UV lamp, which in this case was a 30 W deuterium lamp that emitted from 185 to 400 nm (Fig. S1). This wide bandwidth represented photon energies ranging from 3.1 to 6.7 eV. The bandwidth of the UV exposure was limited to 300–350 nm using a 50-nm bandpass filter centered at 325 nm to ensure that incident photons would be in the UV energy range and not form ozone. Because we chose to match the overall energy deposited from the UV source to the gamma source it was necessary to attenuate the beam to the right power level. We assumed that all the UV energy would be absorbed near the surface rather than in the bulk since the fungi were melanized. This simplified the calculations and reduced risk, given the challenge of accurately estimating the absorbance of the fungi. The power deposited by the gamma source was calculated as the rate of energy deposition was determined to be 7907 MeV g⁻¹ s⁻¹ (1.3 nW g⁻¹ s⁻¹). Given the initial size of the plug was 1 cm in diameter, the desired lamp fluence needed to be ~ 2.8 nW cm⁻². Across the spectrum of interest, the lamp power was determined to be 3.202 × 10^–4 mW, thus requiring an attenuation of 8.7 × 10^–9 (OD 8.06), reducing the lamp power to ~ 3 pW cm⁻² and achieving a reasonably close power density to the target. Due to the sensitivity of UV detectors, the required power densities could not be measured directly. Alternatively, we measured the neutral density filters to verify the prescription was indeed correct.

Response of P. variotii to irradiation

Uniform plugs (~ 5-mm in diameter) of actively growing mycelia of P. variotii were cut using the end of a Pasteur pipette and transferred a Petri plate containing potato dextrose agar (PDA) one day prior to initiating exposure experiments. The diameter of the mycelium was measured from four images, separated by precisely six hours, over the course of seven days and used to measure the growth rate. Differences in the pigmentation of the fungi under the different conditions was quantified in Fiji²⁶ through analysis of grayscale images collected at day seven, following the method described by Brilhante et al.²⁷ A ratiometric value was derived from the grayscale values and the white background, which corrected for variations in lighting across or between images.

Significant differences in the pigmentation but not growth rates of P. variotii were associated with exposure to UV and gamma to irradiation, based on One-Way ANOVA analyses (Fig. 4A; Table S4). P. variotii is a ubiquitous filamentous fungus commonly inhabiting soil, decaying plants, and food products and was reported to be present on the surface of the walls of Unit-4 at ChNPP^22,28. P. variotii is also a common food contaminant and is resistant to high temperature and metals^29,30, despite being more sensitive to gamma irradiation than other fungi such as Aspergillus fumigatus³¹. In the present study, we hypothesized that positive radiation-induced effects in P. variotii would result in enhanced growth rates due to gamma irradiation. Across all conditions, the average growth rate of P. variotii was ~ 5.6 ± 0.9 mm d⁻¹ (mean ± standard deviation). While the growth rate of P. variotii exposed to gamma irradiation was greater compared with the control and UV-irradiated samples (Fig. 4A), the difference in the mean growth rates was not significant (P = 0.255) by ANOVA.

We also hypothesized that the pigmentation of P. variotii would increase with exposure to gamma and UV irradiation. While P. variotti does not produce melanin, it does produce a pigment, Ywa1, from a polyketide synthesis (PKS) gene cluster and has been shown to protect the fungus against UV-C irradiation²⁸. In some melanized fungi, Ywa1 serves as precursor and can be hydrolyzed to 1,3,6,8-tetrahydroxynaphthalen (T4HN). T4HN may then be converted to 1,8-dihydroxynaphthalene (1,8-DHN) melanin through the DHN pathway³². However, Lim et al.²⁸ concluded that P. variotii does not produce true melanin as the pigmentation was maintained when the DHN-melanin pathway was inhibited. Significant differences in the pigmentation of P. variotii were observed among the three different sample types (P < 0.001; Table S4). Here, the gamma irradiation produced the least amount of pigmentation, followed by the control, and the UV-irradiated produced the greatest level of pigmentation (Fig. 4A). The high level of pigmentation in the UV-irradiated samples is consistent the prior report of enhanced PKS production to protect against UV-C exposure²⁸. Further, the lower levels of pigmentation in gamma irradiated samples of Paecilomyces are consistent with its reported sensitivity to gamma irradiation³¹.

Because the fungus grew radially outward from a plug placed at the center of the Petri plate, the total dose on a given region of the fungus is dependent upon both the distance from the source and the duration that a specific region is exposed. For example, P. variotii grew at an average rate of 6.7 mm d⁻¹, almost completely covering the surface of a Petri plate. Using the measured growth rate value and the dose at given distances away from the source, based on TLD data, we estimated the total dose of the fungal mycelium as it grew from the central plug (Fig. 4B), which displayed an exponential decrease as a function of distance from the plug. Figure 4C provides a graphical representation of this effect, where each 1-cm ring of fungal mycelium experienced considerably less gamma dose than the prior ring. This relationship is critical to evaluate within the context of radiotropism/radiotolerance, as the total dose on the mycelium is anisotropic. As such, in our experiment, the leading growth edge of the fungus is continuously moving away from the irradiation source and experiencing smaller and smaller total doses of radiation.

Response of C. cladosporioides to irradiation

Similar to the results for P. variotii, significant differences in the pigmentation, but not the growth rate of C. cladosporioides were observed in samples exposed to gamma or UV irradiation (Fig. 5; Table S5). C. cladosporioides is a melanized filamentous fungus that is pervasive in outdoor and indoor air, and on decaying organic matter³³. Isolates of C. cladosporioides were collected from the walls of Unit-4 of the Chernobyl power plant in locations with both weak and severe levels of radioactive contamination²². Reports further suggest that C. cladosporioides exhibits radiotropism, i.e., preferential growth toward radioactive particles^3,5,34,35. This behavior has been linked to the high production of melanin pigments by Cladosporium species, which not only protect the fungus from ionizing radiation, but also may enable its exploitation as an energy source^13,17. A radiotrophic isolate of C. cladosporioides from ChNPP, IMV 00236, was also reported to have increased production of several secondary metabolites following irradiation with 200–300 nm light for 30 min, suggesting physiological changes potentially related to radiotropism³⁶. With respect to the latter, melanized cells of C. sphaerospermum displayed enhanced growth under limited nutrients and with radiation from a ¹⁸⁸Re/¹⁸⁸W generator¹⁷. In the present work, the average growth rate of C. cladosporioides across all treatments was 3.4 ± 0.3 mm d⁻¹, and no significant differences in the growth rate were observed due to irradiation (P = 0.615; Table S5).

Significant differences in the pigmentation of C. cladosporioides cultures were observed among the different treatments (P < 0.001; Table S5). The darkest pigmentation was observed for cultures exposed to UV irradiation, followed by the control, and then the gamma-irradiated sample (Fig. 5A). The observation for high pigmentation in UV-irradiated samples is consistent with the role of melanin in protecting fungi from UV³⁷. Cladosporium produces melanin by through the DHN pathway and has been implicated in radiotolerance/radiotropism^17,38,39. The lower level of pigmentation in gamma irradiated samples, however, contrasts prior reports that suggest melanin provides enhanced protection against gamma irradiation^13,37,40. Similarly, gamma irradiation has been reported to change the electronic properties of melanin⁴¹, allowing it to be used as an energy source in melanized fungi^13,17. In our work, C. cladosporioides produced significantly lower levels of pigments when exposed to gamma irradiation as compared to the control and UV-irradiated samples (P < 0.01; Table S5). This difference, at least in part, may be related to the fact that this environmental isolate had not been directly exposed to a gamma-rich environment prior to this experiment, compared to those isolated from ChNPP, which were exposed to radiation for years to decades. In addition, fungi in all of our experiments were grown on nutrient rich media (PDA). In prior work, Dadachova et al.¹⁷ reported enhanced growth C. sphaerospermum and Wangiella dermatitidis induced by ionizing radiation under conditions of limited nutrient availability. Thus, the nutrient-rich conditions in our experiments may have masked potential radiation-induced enhancement in growth.

As noted above, the radiation exposure profile may be estimated based on the growth rate of the fungus and the total dose on target, which is dependent on the distance from the source and duration on target. The average growth rate of C. cladosporioides exposed to gamma irradiation in our experiments was 3.3 mm d⁻¹ (Fig. 5A), which was approximately half the rate observed for P. variotii (6.7 mm d⁻¹; Fig. 4A). As such, the radiation exposure profile for C. cladosporioides (Fig. 5B,C) is also considerably different. Here the total dose on target ranged from 85.2 rad at the central mycelial plug to 6.1 rad as the growing edge of the mycelium (Fig. 5C), approximately 2 cm from the central plug. In contrast, the growing edge of P. variotii experienced a much lower total dose, < 1 rad at 4.5 cm from the central plug. This difference is important to consider when comparing the response of fungi to radiation as the growing edge of the mycelium is metabolically active while the older hyphae become vacuolated and lose their cytoplasm.

Source: Ecology - nature.com