Soil fungal community in eucalyptus cultivation cycles

: The short production cycle, high biomass production, and adaptability to various edofoclimatic conditions have led the eucalyptus to a prominent position in the world forestry sector. However, little is known about fungal community structure in these crops. This work aimed to evaluate the structure of soil fungal communities in eucalyptus forests as a function of rhizosphere effect and crop cycles of this crop. We used the independent PCR-DGGE method to evaluate the structure of the soil fungal community in Eucalyptus urograndis plantations located in the Vale do Rio Doce region, Minas Gerais, Brazil. The rhizospheric and non-rhizospheric portions of the soil were sampled in an area of recent forest establishment and in an area under multiple cycles. The principal component analysis revealed that the rhizosphere microenvironment is the dominant component in structuring the fungal communities in the areas studied, and this effect is more pronounced in the area of recent establishment of eucalyptus culture. The same pattern was found for the richness and diversity values, with the greatest differences found in the recently established area. In both studied areas the dominance of the order Agaricales prevailed, evidencing the role of the fungal community in the cycling of nutrients in the soil.


Introduction
Eucalyptus is the most planted industrial wood species in the tropics, covering 20 million hectares, and is exploited mainly for the excellent properties of its pulp and for its short production cycle and adaptability to various soil and climate conditions (FAO, 2020).
In forest crops, trees contribute large amounts of carbon to the soil in the form of residues and root exudates, and root exudation is one of the main determinants of microbial activity in the rhizosphere (Andresen et al., 2020).
The rhizospheric environment provides the microbiota of this habitat with two faces, the first facing the root, full of readily available carbon compounds, and the other facing the soil, an often oligotrophic environment where survival requires great metabolic plasticity (Omotayo & Babalola, 2020). The rhizospheric habitat hosts a large network of microorganisms, mostly sustained and driven by the high input of organic carbon from root exudation (Lance et al., 2020).
It is known that the formation of specific communities associated with the rhizosphere of plant species is determined by complex gene interactions (Gong et al., 2019) and that the molecular approach has contributed to the better understanding of these interactions (Braga et al., 2016). Fingerprinting techniques such as Denaturing Gradient Gel Electrophoresis (DGGE) are able to provide short-term and cost-effective data on the genetic diversity of a specific environment as well as provide comparisons between different environments (Green et al., 2015).
Colonization and permanence in rhizospheric habitat seems to require more than simple craving for energy, being determined primarily by complex gene interactions between plants and rhizospheric microorganisms (Trivedi et al., 2020).
Most of the plants studied on the planet have successful interactions with a wide variety of microorganisms, many of them colonizing the rhizosphere. In this complex habitat, fungi play essential roles, either as mutualistic decomposers or pathogens (Mommer et al., 2018).
Fungi are heterotrophic organisms dependent on external sources of reduced carbon (Marupakula et al., 2020). Studies on the contribution of root exudates of Fagus sylvatica L. to the soil reveal the ability of photoassimilates to shape the fungal community of this habitat (Baumert et al., 2018).
Microbial diversity is seen as a critical factor for soil quality (Schloter et al., 2018). Despite the existence of several papers evidencing the induction of plant species on pathogenic and mycorrhizal fungal communities, few papers report the rhizosphere effect on non-symbiont fungal communities in the soil.
Considering the importance of plant-micro-organism interactions in the rhizosphere for soil carbon retention, nutrient cycling, and the sustainability of forest systems, understanding the factors that determine the structure of fungal communities in these ecosystems is critical. Thus, the present work aimed to evaluate the structure of soil fungal communities in eucalyptus forests as a function of crop cycles and rhizospheric effect in Eucalyptus urograndis located in the Vale do Rio Doce region, Minas Gerais, Brazil.

Area description
To conduct the experiments, areas cultivated with Eucalyptus urograndis were selected and located in the Vale do Rio Doce region, Minas Gerais, Brazil. Two plots were selected in areas with different crop cycles, the first fifth cycle (Area 1) and the second in the first cycle (Area 2), both containing 18-month old trees. In the experimental areas there is a predominance of intemperate soils, with low natural fertility and with an undulating relief, from wavy to strongly undulated, being the yellow Latosol the most representative soil class (Table 1). The average annual temperature is 22.2 °C and the average annual precipitation 1212 mm.

Soil sampling
The 1000 g soil samples from the 0-10 cm layer were collected from the rhizospheric and non-rhizospheric region of each of the areas. For the rhizospheric soil, the average collection radius was set so as to intercept the growth zone of fine roots near the soil surface. For this study, the rhizospheric portion was considered to be the entire volume of soil in contact with the root system of the eucalyptus plants. Nonriphospheric soil was collected in the center of the planting rows in a region where root growth was not observed.

Extraction of metagenomic DNA, PCR-DGGE and identification of UTOs of interest
For DNA extraction from soil fungal populations, the PowerSoil® DNA Isolation Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) was used with modifications. Twenty-five mg of each soil sample was transferred into PowerBead tubes provided by the kit followed by incubation in a water bath at 55 °C for 30 minutes. After cooling to room temperature, the sample was processed using the kit according to the manufacturer's recommendation. Table 1. Chemical and physical characteristics of the soils of areas and stands of Eucalyptus urograndis located in the Vale do Rio Doce region, Minas Gerais, Brazil, with different crop cycles, the first fifth cycle (Area 1) and the second in the first cycle (Area 2), both containing 18-month-old trees.

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The DNA obtained was used to amplify a fragment spanning the V1-V9 regions of the 18S rDNA (May et al., 2001;Oros-Sichler et al., 2006) and the products of the first PCR used as a template to amplify a fragment between the V7-V8 regions (Vainio & Hantula, 2000) ( Table 2).
The separation of the different amplicons was performed by applying 20 µL of the PCR product in Denaturing Gradient Gel Electrophoresis (DGGE) with 35% to 55% urea/formamide gradient and electrophoresis conducted at a temperature of 60 o C, constant voltage of 60 V for 20 hours in DCodeTM Universal Mutation Detection System equipment (BIO-RAD-, CA, USA), following the manufacturer's recommendations. The gel was stained with SYBR® Gold (Invitrogen, Carlsbad, CA, USA).

Identification of the UTOs
After visual analysis of the DGGE gel the bands corresponding to the most representative OTUs from the rhizospheric and non-rhizospheric soils were excised from the gel and eluted in 30 µL of sterile water. The elution product was used as a template for further amplification of the region of interest (Table 2). The amplicons obtained by PCR were sent for sequencing at Macrogen Inc., Korea.

Statistical analysis
Fungal community structure analysis was based on Dice's similarity coefficient and the UPGMA method (Unweighted Pair Group Method with Arithmetic) to build the clusters, using Bionumerics software, version 7.1 (Applied Maths, Kortrijk, Belgium). Richness (R) and Shannon diversity (H) indices were calculated using Paleontological Statistics (PAST) software from the presence and absence matrices generated from the analysis of the DGGE profiles. The data from the richness and diversity analyses were evaluated with the help of ASSISTAT Version 7.7.

Fungal community structure
Principal component analysis (PCA) showed the formation of distinct groups between the fungal communities of rhizospheric and non-rhizospheric soils, revealing that the input of carbon compounds into the rhizosphere is the dominant component in structuring fungal communities in eucalyptus forest soils (Figure 1).
A higher similarity was observed between the clusters formed by the rhizospheric and non-rhizospheric soil samples collected in the area under the first cycle (A2), about 60 % similarity, when compared to the rhizospheric and nonrhizospheric soil clusters collected in the area under multiple cycles (Area 1), about 43 % similarity ( Figure 2).
Significant differences in richness (R) and diversity (H) values were observed between the rhizospheric and nonrhizospheric soils in the recent crop establishment area (A2), with the rhizospheric soil showing higher R and H values. Similar R and H values were found between rhizospheric and
The migration pattern of DNA fragments in DGGE revealed distinct UTOs of fungi in the analyzed soils (Figure 3), of which only four are present in all sampled soils. Six UTOs were found to be unique to rhizospheric soil, while only three are present only in non-rhizospheric soil. The rhizospheric soil from the area under multiple crop cycles (A1) showed four distinct OTUs, the rhizospheric soil from area 2, one OTU, and the non-rhizospheric soils, from areas 1 and 2, only one distinct OTU (Figure 4).

Discussion
Principal component and cluster analysis obtained from the PCR-DGGE data reveals the differences between the fungal   communities in the rhizospheric soil and the non-rhizospheric soil in both areas studied. This observation confirms the hypothesis that the high input of carbon compounds from the aerial part into the rhizosphere is able to shape the structure of the fungal community in this habitat.
There is much evidence to support the hypothesis that the supply of carbon through root exudation is a major determinant of soil microbial community structure. In Cunninghamia lanceolata forest (He et al., 2016) in China, the disruption of carbon flux to the rhizospheric environment by tree girdling was found to be a major component determining soil microbial community structure. Also in China, tree ringing in Pinus elliottii forests has considerably altered the soil microbial community structure (Wan et al., 2019).
Diversity and community structure data obtained by PCR-DGGE have to be analyzed with caution, as heterogeneity 5/7  Table 4. Closest species obtained from comparison of sequences obtained from bands excised from the DGGE gel and sequences from the GenBank/EMBL/DDBJ database. within the analyzed regions or the occurrence of PCRpreferential may overestimate the diversity of a species or underestimate the diversity of infrequently present species (Iacumin et al., 2020). Popularly known as rare biosphere, these species are individually present in small numbers, but can account for 75 % of the biomass of some microbial communities .
The smaller distinction between the clusters formed by the rhizospheric communities in the area under multiple cropping cycles when compared to those formed in the first-cycle areas shows that the rhizospheric effect is more pronounced when forests are planted. It turns out that successive crops in monoculture are capable of shaping the structure of the entire soil fungal community, whether rhizospheric or non-rhizospheric.
Studies conducted in forest soils highlight that the introduction of new plant species is able to significantly alter the structure and activity of the soil microbial community (Arafat et al., 2019). There is evidence that anthropogenic restructuring of microbial communities is mediated by changes in the quality and quantity of carbon input to the soil (Domeignoz-Horta et al., 2020).
The similarity between the richness and diversity values of rhizospheric and non-rhizospheric soils in areas under multiple cycles of E. urograndis cultivation reaffirms the hypothesis that monoculture is able to shape and stabilize the soil microbial community.
When these parameters, richness and diversity, are analyzed in areas of recent crop establishment, a more pronounced rhizospheric effect is observed, which considerably increases the richness and diversity values. We infer this result to the priming effect leveraged by the addition of carbon to the rhizospheric environment in a region previously degraded by pasture. In E. uroplylla crops in southern China, forest monoculture was found to lead to increased fungal diversity in the soil, with the result being attributed to the possible favoring of these communities by stabilizing the chemical composition of the rhizosphere environment (Zhu et al., 2020).
It is proposed that the priming effect does not involve just one mechanism, but is the result of a complex interplay of sequential mechanisms directly linked to the structure of soil microbial communities (Li et al., 2018).

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Most of the identified OTUs belong to the order Agaricales, which highlights the role of the fungal community in organic matter mineralization and nutrient availability in eucalyptus forests. The order Agaricales is the most diverse of the phylum Basidiomycota, comprising about 9387 species in 347 genera. Representatives of this order are recognized as critical components in organic matter cycling and for their high potential for producing antimicrobial metabolites (Kyaschenko et al., 2017).
On forest sites the soil fungal community plays an essential role in the carbon, phosphorus and nitrogen cycles, acting, primarily through enzymatic mechanisms, in making these nutrients available and enabling the establishment of other microbial populations (Shi et al., 2019). In Eucalyptus grandis forests in Ethiopia it was found that fungal community structure is directly correlated to the presence of roots (Castaño et al., 2019).

Conclusions
There was the formation of distinct groups between the fungal communities in rhizospheric and non-rhizospheric soils, demonstrating that the rhizospheric effect contributes expressively to determining the structure of the communities in E. urograndis forest soils. Verificou-se ainda que, em menor escala, o cultivo sucessivo contribui para moldar a estrutura destas comunidades.
The lowest similarity between the fungal communities in the rhizospheric and non-rhizospheric soils was seen in the area under multiple cultivation when compared to the first cycle area, showing that the rhizospheric effect is more pronounced at forest planting. The same pattern was found for the richness and diversity values, with the greatest differences found in the recently established area.
Among identified fungal OTUs, mostly belonging to the order Agaricales, most were present in rhizospheric and nonrhizospheric soils, except for Serpula and Pleuroflammula OTUs, which were present only in rhizospheric soil.