Effects of a megafire on the arbuscular mycorrhizal fungal community and parameters in the Brazilian Cerrado ecosystem

Aim of study: To evaluate the effects of a mega-fire on the arbuscular mycorrhizal fungi (AMF) community and parameters in soils under Cerrado vegetation. Study area: Chapada dos Veadeiros National Park, Goiás, Brazil. This site suffered the biggest fire in its history on October 10, 2017, with an affected area of 66,000 ha. Materials and methods: We analyzed AMF spore density, roots’ mycorrhizal colonization rate, easily extractable glomalin (EEG), as well as the AMF genera present. These parameters were evaluated in burned and unburned areas of five common phytophysiognomies of the region. Main results: Fire presence immediately affected the mycorrhizal community parameters in Cerrado soils, which tended to increase afterwards. The presence of AMF genera did not differ between burned and unburned areas, with Acaulospora, Claroideglomus, Diversispora, Glomus, Funneliformis, Sclerocystis, and Gigaspora being present. The recovery of AMF community conditions in the Cerrado after fire events could also be observed in the mycorrhizal parameters evaluated, as the values of spore density, roots’ mycorrhizal colonization rate, and EEG were similar in the burned and unburned areas. Research highlights: AMF diversity, and especially their community parameters, show great recovery after fire events, since they are crucial in processes like nutrient cycling and soil aggregation.


Introduction
The Cerrado is the second largest Brazilian biome, extending over an area of 2,045,064 km² and covering eight States in Central Brazil in addition to the Federal District (Hunke et al., 2015). This biome is crossed by three of the largest hydrographic basins in South America (Amazonia, São Francisco, and Paraná), with constant and high rainfall levels, and very high levels of animal and plant endemism and biodiversity (Klink et al., 2020), only behind the Amazon in the country. The Cerrado is currently considered as a transitional biome and the last agricultural frontier in the Americas (Hunke et al., 2015;de Moura & Cabral, 2019), with direct geographical contact with other important South American biomes such as the Amazon, Caatinga, Atlantic Forest, Pantanal, and Chacos Bolivianos (de Moura & Cabral, 2019).
Besides strong anthropogenic effects, the environmental history of the Cerrado is determined by a set of natural elements related to its geology and ecology, and their effects on the formation and constitution of the Neotropical savannas that compose this biome (Ratter et al., 2006). Among these elements, natural wildfires stand out as a main factor configuring the Cerrado landscape (de Moura & Cabral, 2019). Nonetheless, intentional fire is the main tool used to expand new agricultural areas in the Cerrado, and as a result, big and irreversible damages have already occurred to this biome, as the fragmentation of previously integrated phytophysiognomies, native biodiversity loss, soil nutrient loss, soil degradation and compaction, soil erosion, pollution of water sources, changes in the natural fire regime, and imbalances in the carbon cycle, all of which contribute to regional climate change (Klink et al., 2020).
The biggest fire event in the history of the Cerrado, suspected as arson, occurred in the Chapada dos Veadeiros National Park in October 10, 2017, destroying about 66,000 ha (de Santana Leite et al., 2017). The Chapada dos Veadeiros National Park was created in 1961 and comprises an area of 240,614 ha, and in 2001 was declared a Natural World Heritage Site by UNESCO (de Santana Leite et al., 2017). The Cerrado ecosystem has several phytophysiognomies, including Cerradão (dense forest formation with several tree species), Cerrado sensu stricto (low and twisted trees, shrubs, subshrubs, and herbs), Veredas (hydromorphic soils surrounded by cleaned fields, represented by rows or groups of Mauritia flexuosa), Campo Limpo (herbaceous vegetation with few shrubs and no trees), and Campo Sujo (herbaceous-shrub vegetation with shrubs and subshrubs spaced among themselves) (Walter, 2006). Factors as the expansion of the agricultural frontier (especially soybean crops) in the region adjacent to the Chapada dos Veadeiros National Park, and the presence of exotic grasses introduced in the Cerrado since the XVIII century, favor over-combustion, altering the natural fire regime (Simon et al., 2009;Simon & Pennington, 2012), which has harmful consequences to this biome. These intentional fires (in most cases illegally), lead to new areas destined for agricultural production. The harmful consequences of these fires are more accentuated during the dry season (from April to September), where low humidity amplifies the intensity and severity of these events (Simon & Pennington, 2012).
Soil microorganisms are essential for the conservation of biodiversity and for the recovery of ecosystem services, after extreme events as wildfires (Mataix-Solera et al., 2009). Arbuscular mycorrhizal fungi (AMF), are generalists and obligate plant symbionts (Davison et al., 2015), and are extremely sensitive to environmental changes and as such, they are excellent bioindicators of environmental quality and recovery (de Souza et al., 2016). Several studies have shown the resilience of the AMF community and mainly its community parameters after fire events (Martínez et al., 2005(Martínez et al., , 2018de Oliveira et al., 2006;Paulino et al., 2009;Rivas et al., 2016). For AMF it is a common response to decrease in biodiversity but sustain or even increase their aggregate parameters such as roots` mycorrhizal colonization, glomalin production, soil aggregation, among others, immediately after disturbances (Paulino et al., 2009;Rivas et al., 2016). For example, some studies have shown a very interesting pattern: a decrease in AMF species richness (as determined by morphological analyses) but no effect on AMF spore density post-fire (Longo et al., 2014).
According to studies already carried out which show the crucial role of AMF in the recovery of the Cerrado ecosystem after disturbance (De Souza et al., 2016), we expected that in this ecosystem the action of fire would promote an increase of roots colonization by AMF, which would cause a population increase of some species, and in return a decrease in biodiversity -not immediately but after a while. Given their environmental similarity, we expect similar results for all phytophysiognomies. This study aimed to evaluate the effects of a megafire on the AMF community in Cerrado soils, by measuring parameters such as spore density, easily extractable glomalin (EEG), and the rate of successful root colonization, comparing burned and unburned areas in five commonly found phytophysiognomies in the region.

Study sites and sampling scheme
The study was carried out in the Chapada dos Veadeiros National Park, Goiás, Brazil (Fig. 1). Five phytophysiognomies were defined according to Walter's characterization (2006): Cerradão, Cerrado sensu stricto, Campo Limpo, Campo Sujo, and Veredas. Ten sample plots (20 m × 20 m; 7 in and 3 outside the Park) were selected, and for each phytophysiognomy, one burned and one unburned plot (Fig. 1). Sampling was carried out in four periods: first, on November 10, 2017, right after the containment of the flames; second, on March 11, 2018; the third sampling on June 23, 2018; and the fourth and final sampling was carried out on November 2, 2018. For each plot and sampling period, and after removing organic debris (litter, 3 Fire effects on Cerrado mycorrhizal fungi and parameters wood), a total of six soil samples (rhizospheric soil and roots of the A horizon; 0-10 cm soil depth) were analyzed (which in turn, consisted of 6 randomly collected subsamples). Thus, a total of 240 soil samples were analyzed in this study (10 plots × 4 sampling periods × 6 samples per plot). The samples were collected in the same sampling area in all periods.

Microbiological analyses
Microbiological analyses were performed at the Agricultural Microbiology Laboratory at the Faculdade Evangélica de Goianésia, Goiás, Brazil. Spores of AMF were extracted from a volume of 50 cm 3 of rhizospheric soil using the wet-sieving technique (Gerdemann & Nicolson, 1963), including centrifugation at 2000 rpm for 2 minutes in water and a 50% sucrose solution. The spores were separated under a microscope (400-fold magnification), and were taxonomically analyzed according to their phenotypic characteristics as color, size, and shape (Oehl et al., 2011), composing the different AMF genera. In order to identify the genera of AMF from the morphological characteristics, the spores were separated according to their morphotypes (Oehl et al., 2011), and mounted on slides with pure polyvinyl lactoglycerol (PVLG) and PVLG mixed with the Melzer solution (1:1 v/v).
To determine the percentage of root colonization by AMF, the roots were clarified and stained with 0.05% Trypan-Blue in lactoglycerol (Phillips & Hayman, 1970), and the root colonization was evaluated under a stereoscopic microscope, following the technique of quadrants' intersection (Giovannetti & Mosse, 1980). The extraction of EEG was done by adding 1 g of dried soil in 8 mL of 20 mM sodium citrate (pH 7.0), followed by autoclave for 30 min at 121 ºC; then, centrifugation was carried out for 20 min at 5000 rpm (Wright & Upadhyaya, 1996;Nichols & Wright, 2006). For EEG extraction, we followed the Bradford (1976) method modified by Wright & Upadhyaya (1996), where bovine serum albumin was used as the standard protein, using a spectrophotometer at a 595 nm reading. Caution should be taken when interpreting the EGG results, as currently there are no specific analytical tools to measure glomalin (sensu Rillig, 2004) without significant interference from other substances (Holátko et al., 2021). The evidence provided so far for glomalin detection, even when measuring antibodies (Gadkar & Rillig, 2006) in soil as a direct metabolic product of AMF is still just correlative (Bedini et al., 2007;Bolliger et al., 2008;Wilson et al., 2009).

Statistical analyses
Each phytophysiognomy was evaluated separately, resulting in a completely randomized design with the plots arranged in a 2 × 4 factorial scheme with six replicates. The first factor consisted on burning (or not), and the second factor corresponds to the four sampling periods. Before the analyses, the homogeneity of variances and the normality of the residuals were checked using the Bartlett test and graphical checks, respectively. Analyses of variance (ANOVAs with two factors), T-tests (at 5% means discrimination), and graphs of the obtained data were made using the GraphPad Prism 8 software (SWIFT, 2020). We also performed stepwise regressions (in both directions) of the redundancy analysis (RDA) for each measured mycorrhizal parameter (root colonization, spore density, and glomalin), assessing the effect of each tested factor (phytophysiognomy, fire effect, sampling time) on them. This was done using the ordistep function of the vegan package (Oksanen et al., 2020) in RStudio 2021.09.0+351 (R Core Team, 2020).

Results
Fire had varied effects in the AMF parameters (spore density, root colonization, and EEG) evaluated in the five phytophysiognomies assessed on this study ( Fig. 2; the specific values are given in Table S1 [suppl]); such effects were not very strong regarding AMF genera (Table  1). AMF spore density in soil was at its lowest and similar in the first sampling in the burned and unburned areas in all phytophysiognomies (58.667-173 spores/50 cm 3 of soil). In the second sampling, five months after the fire event, the spore density values were higher in the burned than in the unburned areas in the Campo Limpo (356.833 vs. 170.667 spores/50 cm 3 of soil, respectively) ( Fig. 2a) and Cerrado sensu stricto (224.667 vs. 90.333 spores/50 cm 3 of soil, respectively) ( Fig. 2j) phytophysiognomies, not differing in the other phytophysiognomies. In the third sampling, the burned areas had higher spore density than the unburned areas in all phytophysiognomies; and in the last sampling, 13 months after the fire event, spore density was not significantly different in burned and unburned areas.
Roots' mycorrhizal colonization rate was lower in the burned than in the unburned areas in the first three samplings only in the Campo Limpo phytophysiognomy (burned: 0.226, 0.403, and 0.462%; unburned: 0.544, 0.882, and 0.806%), being only relatively similar in the last sampling (burn: 0.64%; unburned: 0.681%) (Fig. 2b). The other four phytophysiognomies did not show significant differences regarding roots' mycorrhizal colonization rate between the burned and unburned areas, but values ten-ded to increase towards the third sampling period, eight months after the megafire (Fig. 2). EEG soil content was overall not significantly different between burned and unburned plots, in all the investigated phytophysiognomies, and was somewhat similar across the sampling periods, with slightly higher values in the third sampling period (5.718-7.085 mg/g of soil).
Stepwise regressions of RDAs (in both directions) of the mycorrhizal parameters of this study, show that the three factors tested (phytophysiognomy, fire effect, and sampling period) significantly affected AMF spore density and root colonization (Table 2). Specifically, the effect of phytophysiognomy in spore density was quite large, while root colonization was mostly affected by the sampling period, factor that also highly affected EGG content -but this parameter was not affected by fire, and only slightly affected by phytophysiognomy ( Table 2).
The AMF genera Acaulospora, Claroideglomus, Diversispora, Glomus, Funneliformis, Sclerocystis, and Gigaspora were identified in the rhizospheric soil of the investigated phytophysiognomies (Table 1). Overall, the presence of AMF genera did not differ between burned and unburned areas ( Table 1). The genera Acaulospora, Claroideglomus, Diversispora, Funneliformis, and Gigaspora were identified in all phytophysiognomies, and the genus Glomus was found in all phytophysiognomies, regardless of fire action. The genera Sclerocystis was not found in the rhizospheric soil of the Campo Limpo phytophysiognomy. In the Campo Limpo phytophysiognomy, with the exception of the genus Diversispora, which in the first sampling was only identified in unburned areas, the other genera were found both in burned and unburned plots (Table 1).

Discussion
Arbuscular mycorrhizal fungi (AMF), as well as any soil microorganism, need specific environmental conditions for their proliferation and colonization (de Moura & Cabral, 2019). Soil health and biodiversity recovery after fire will depend on many factors, among them fire intensity, the amount of subsequent rain, preventive actions after the fire, among others. Cerrado soils have mostly low natural fertility, condition that encourage the spread of AMF and their colonization in crop vegetables (de . Climatic conditions, soil movement, the use of chemical fertilizers, wildfires, among other factors, directly influence the amount of AMF spores in soil . All of these factors can affect microbial abundance, richness, and ecosystem functions (van der Heyde et al., 2017).
The AMF community and parameters investigated in our study mostly recovered after fire at the end of our sampling, a relatively short period (one year). Furthermore,

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Fire effects on Cerrado mycorrhizal fungi and parameters by the second or third sampling periods, the three AMF parameters investigated (spore density, roots colonization, and EEG content) seemed to have fully recovered. Longo et al. (2014) found that after 8 months, fire decreased the richness of AMF spores, but -and as in our study-spore density was not affected. Similarly, Dove & Hart (2017) found in a meta-analysis that mycorrhizal colonization of roots was not affected by fire, also a similar finding to our results, at least after certain time. Several other studies in South America (Martínez et al., 2005(Martínez et al., , 2018Paulino et al., 2009;Rivas et al., 2016) have found a similar pattern: a decrease in AMF biodiversity but no effect (or even an increase) in different community parameters related to their ecosystem functions. In our case, further morphological and molecular characterizations are required to identify spores at a species level, to truly evaluate changes

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Fire effects on Cerrado mycorrhizal fungi and parameters in AMF biodiversity. This seems to be common feature of AMF in most types of disturbances, which is not necessarily the case for other fungal guilds or even mycorrhizal types (Marín et al., 2017;Steidinger et al., 2020;Marín & Kohout, 2021;Rodriguez-Ramos et al., 2021). AMF niches are relatively broad, driven mainly by temperature and soil pH, and correlated by phylogeny (Davison et al., 2021), which together with their broad distribution and low worldwide endemism (Davison et al., 2015), might explain their high recovery capacity. These broad niches and biogeographic distributions could in part explain our results: related taxa, which arrives relatively quickly after the fire, given their ubiquity, ends up having similar performances (e.g. in EGG or roots` colonization) after disturbances. The AMF spore density results obtained here are similar to those obtained by Cordeiro et al. (2005), who found similar patterns when evaluating areas of the native Cerrado biome, more specifically the Cerrado sensu stricto phytophysiognomy, where they found a spore density of 368 spores per 50 cm³ of soil. The symbiotic association between plants and AMF is especially stimulated when the plant is in an stressful environment (de Souza et al., 2016). Fungi help the plant in its nutrition and water absorption -besides pathogen and drought resistance (among other disturbances), allowing it to survive several and severe environmental changes (van der Heyde et al., 2017). The highest values of spore density were found in areas under fire in all phytophysiognomies, in the second and third sampling periods, demonstrating the importance of the AMF response to a megafire event. In the fourth sampling period, when the Cerrado areas had partially recovered from fire, the spore density values of the burned and unburned areas were similar.
Fire promotes significant changes in vegetation and plays key roles in plant physiology and community structure (Veenendaal et al., 2018). The post-fire plant regeneration process can take decades. Even if some plants are fire-adapted, variation in fire severity and intensity can lead to irreversible damage to some plants (Klink et al., 2020). Low intensity fires mainly affect undergrowth, which consists of the understory, grasses, and lower vegetation (van der Werf et al., 2017;Klink et al., 2020). In such case, trees are mostly not affected due to the rapid passage of fire. However, in atypical environmental con-ditions, where flames can reach up to 2.5 m in height, tree crowns can be burned (Kauffman et al., 1994;Miranda et al., 2009). In the Cerrado ecosystem, plant recovery after the incidence of fire occurs gradually, and some species may start flowering a few days after the fire event, while other species take weeks or months. In our study, the first species to be recomposed are the constituents of the herbaceous layer, mainly grasses and other graminoids. Klink et al. (2020) determined four patterns of post-fire re-sprouting in the Cerrado ecosystem: 1) at the beginning of the rainy season; 2) in the middle of the rainy season; 3) in the second half of the rainy season; and 4) at the end of the dry season. At least 70% of plant biomass is recovered by the mid-next rainy season (Klink et al., 2020).
The roots' mycorrhizal colonization rate in four out of five of the investigated phytophysiognomies presented a similar temporal trend to that of spore density: in the first sampling, the values were lower in the burned than in the unburned plots. Nutrient bioavailability is an important factor that directly influences the rate of roots' arbuscular mycorrhizal colonization in plants ( van der Heyde et al., 2017). An stressful, nutrient-depleted condition might provide an environment conducive to the establishment of this symbiosis, because the plant needs the fungus to acquire nutrients otherwise unavailable, or else, colonization might be impaired in soils where the plant has at its disposal a greater nutritional support (Ezawa & Saito, 2018). This could explain why in our study, burned areas had lower colonization rates than unburned areas. Ash supply temporarily increases nutrient availability, impairing root colonization (Longo et al., 2014). Similar trends regarding mycorrhizal colonization and soil nutrients were found by Martínez et al. (2005), de Oliveira et al. (2006), and Rivas et al. (2016).
The amount of roots analyzed was the same for all treatments, not influencing the results. The passage of fire was fast and, in a way, does not affect the integrity of the roots in the short term. In our study, the first samples were collected soon after the fire was contained. Thus, root sampling was possible even in phytophysiognomies that had their vegetation consumed by fire. The phytophysiognomies Campo Limpo, Campo Sujo, and Cerrado sensu stricto are mostly constituted by plants that have a rapid recovery, which does not affect the amount of roots of subsequent collections (Klink et al., 2020). In the case Stepwise models (in both directions) of redundancy analyses, RDA (Akaike criterium (AIC) and F-values are shown, with p-values as asterisks) testing the effects of the different factors of this study on the mycorrhizal parameters measured. p<0.01: **; p<0.05: * of the Cerradão phytophysiognomy, the woody layer is not consumed by fire, not affecting plant survival (Walter, 2006). In the case of the Veredas phytophysiognomy, its soil is hydromorphic (Walter, 2006), which allows the rapid recovery of plants that constitute this phytophysiognomy. Other plants, such as Mauritia flexuosa, as well as Cerradão trees, are not consumed by fire due to their woody layer. Very few studies have examined AMF biodiversity and community parameters immediately after the fire -usually some weeks, months or years have passed in most studies. Bellgard et al. (1994) -one of those few studies-found reduced AMF colonization immediately after fire compared to pre-fire conditions, but no difference between burn and unburned sites -a similar result to ours. Similarly, after one year, other studied parameters like AMF spore numbers and diversity recovered, which could be related to the sprouting organs of the vegetation present (Bellgard et al., 1994). Very similar results were obtained by Aguilar-Fernández et al. (2009) in a tropical dry forest, with very few changes in the AMF community after forest conversion by slash-and-burn followed by cultivation. Studies where the first sampling was conducted some few months after the fire (Rashid et al., 1997;Longo et al., 2014;Xiang et al., 2015) seem to agree with the above-described pattern, overall showing low AMF resistance but high resilience to fire (Xiang et al., 2015). Longo et al. (2014) showed that such pattern can be explained by fire having direct effects on the AMF community, not necessarily mediated by the effects of fire in soil physical and chemical properties.
In our study we found that spore density and AMF colonization of roots immediately decreased after the fire, but the effect was not so drastic in EGG soil content. Thus, a reduced amount of AMF spores, colonizing less roots, possibly are over-producing EGG in order to immediately stabilize the post-fire soil conditions. Afterwards, EGG production probably stabilizes. Though, fire did not significantly increased EEG soil content in almost all the phytophysiognomies investigated. Although, and as previously mentioned, EGG measurements usually have interference from other (non-AMF) substances (Holátko et al., 2021), so these results should be taken as a proxy. Moya et al. (2019) found higher levels of glomalin in soil after fire, both in treatments with high and low severity fire -an aspect we should test in further studies, as the fire in our system had a very high severity. Similar results regarding glomalin content after fires in Chile have been reported by Rivas et al. (2016), but in a very different ecosystem such as the temperate rainforests. The mycorrhizal activity in these forests intensified due to the demand for vegetation in the stressed areas, which were reflected in a greater protein production. The EEG values reported here were similar to those found by Santos (2016) in Cerrado soils, which ranged from 2.1 to 4.4 mg g-1 of soil. Fokom et al. (2012), when evaluating agricultural areas of intercropping with peanut, corn, banana, and cassava crops, found values of 6.51 mg g -1 of soil, also similar to our results; in forested areas, though, they found higher values reaching up to 10.56 mg g -1 of soil.
Glomalin soil content varies according to the species of mycorrhizal fungi found colonizing roots. Wright & Upadhyaya (1999) found a difference in glomalin production according to the studied AMF species: Gigaspora rosea and Gigaspora gigantea had higher glomalin productivity than Glomus intaradices and Glomus etunicatum (Wright & Upadhyaya, 1996), when evaluated under cultivation in a culture medium. As in their study, AMF genera as Glomus and Gigaspora were ubiquitous in our study. Similar to our study, where sampling time highly affected EGG, Rivas et al. (2016), when carrying out soil sampling one day and four years after fires on soils in the Andes, found that the difference in glomalin-related protein content was significant, but limited to the first 10 cm of the soil horizon. In contrast to our results, Sharifi et al. (2018), when carrying out work in the Zagros forest in western Iran, found that glomalin soil content was greater in burned areas than in native unburned forests; they found that the greater the fire intensity the greater the glomalin soil content, and that these values tended to increase in follow-up samplings. Sharifi et al. (2018) strongly recommend the use of glomalin (or proxies) as an indicator of soil degradation by fire, albeit the current difficulties to correctly measure this protein in soil (Holátko et al., 2021).
In summary, the presence of fire influenced the AMF community and its parameters in Cerrado soils, especially during the first sampling period, right after the containment of the flames. The Cerrado's responses to fire can also be seen in the community parameters of AMF, in which the values of spore density, roots' colonization rate, and EGG soil content tended to match up in the burned and unburned areas. Similarly, with few exceptions, the genera of these fungi were similar in burned and unburned areas.