Mycorrhizal fungi arbuscular in forage grasses cultivated in Cerrado soil

The Cerrado is one of the most important regions for agricultural development in the world and is the main productive breadbasket of the Americas. One of the main agricultural activities in the region is high-tech livestock. Cerrado soils are predominantly low in fertility, and arbuscular mycorrhizal fungi play a fundamental role in plant nutrition in this biome. Understanding the behavior of mycorrhizal fungi in the soil under pasture is essential for the development of more efficient and sustainable management practices. Thus, this work aims to verify the activity of arbuscular mycorrhizal fungi in different species of forage grasses cultivated in cerrado soil. To measure mycorrhizal activity, soil spore density factors and mycorrhizal colonization rates in roots of 14 forage grass genotypes were investigated. No significant differences were identified in spore density values between the investigated genotypes. Panicum maximum cv and Mombasa showed the lowest values of mycorrhizal colonization, and the highest values were found in the roots of Brachiaria decumbens. Among the identified genera associated with the rhizosphere of the genotypes studied, Gigaspora, Scutelospora and Sclerocysts are less frequent, which indicates that the association with these fungal genera is less recurrent than with the others.

www.nature.com/scientificreports/ on symbiotic systems may also reduce the use of herbicides, which have been used extensively for no-till systems in the tropical climates of Brazil 16 . Generally, Cerrados are environments that naturally offer adverse abiotic conditions for plant growth and development. With low phosphorus levels and irregular rainfall, vegetation depends directly on the performance of mycorrhizal fungi to resist surviving such conditions, which is attributed to the association between fungi and plants as an important factor in building resilience to stressful situations 1,[17][18][19] .
The association of mycorrhizal fungi with vegetation started its evolution in tropical regions, and there are even species that are found only in these regions 20 . Today, however, the presence of these fungi is reported in different regions of the planet, regardless of climate 19,[21][22][23][24][25] .
The average density of mycorrhizal fungal species in the soils of the Cerrado varies from 25 to 50 spores per 50 cm 3 of soil on average. In the neighboring region of the Caatinga, there is a variation in the number of propagules of these fungi, probably due to differences in the plant community, and in relation to chemical composition and land use, with ranges containing high phosphorus 6,26 .
For agricultural production, combined with the recovery of degraded areas, understanding the behavior of forage grass species with soil biology is essential for the development of more efficient practices for the management of natural resources. Cerrados are environments that offer adverse abiotic conditions for plant growth and development, with low levels of phosphorus and a limited water regime, and the development of their vegetation depends directly on the action of soil microorganisms. Under these conditions, mycorrhizal fungi stand out as organisms that promote plant growth and contribute to plant resilience to stressful situations 1,[17][18][19]27 . Therefore, this work aims to verify the mycorrhizal population dynamics in forage grass species in Cerrado soils.

Materials and methods
The experiment was conducted at the Agrostological Field of the Ricardo Fontoura Experimental Station of the Cerrado, which is part of the Evangelical College of Goianésia, in the state of Goiás, Brazil. The climate is classified as a tropical season (AW) characterized by two well-defined seasons: dry and rainy 14 . The density of spores and the mycorrhizal colonization rate of 14 varieties of forage grasses were evaluated ( Table 1).
Samples of rhizospherical soil containing the treatment roots described in Table 1 were collected. Each sample taken to the laboratory was composed of 3 simple samples randomly collected from each plot. The design had a completely updated design with 6 replicates. Sampling was carried out at the end of the dry season in September 2020.
The analyses were carried out in the laboratory of agricultural microbiology of the Evangelical College of Goianésia. The spores of arbuscular mycorrhizal fungi (AMF) were extracted from 50 cm 3 of rhizospherical soil by wet sieving 15 followed by centrifugation in water and a 50% sucrose solution. The spores were separated according to their phenotypic characteristics, such as color, size and shape, composing the different morphotypes under stereoscopic binocular magnifying glass 28 .
To determine the percentage of colonization, the roots were clarified and ordered with 0.05% Trypan Blue in lactoglycerol 16 and the colonization was evaluated under under a stereoscopic microscope, following the quadrant intersection technique 17 .
To identify the genera of AMF from morphological characteristics, the spores were separated according to their morphotypes and mounted on blades with pure polyvinyl-lactoglycerol (PVLG) and PVLG mixed with Melzer (1:1 v/v). To support the identification work, original articles from the descriptions of species were provided on the website of the "International Culture Collection of Arbuscular and Vesicular-Arbuscular Mycorrhizal Fungi" 18,29 . www.nature.com/scientificreports/ The data were submitted to variance analysis by the Assistat 30 analyses of canonical correspondence were performed by the Past 20,31 software. Spore density variables, and the rate of mycorrhizal colonization was determined by a 5% Tukey test. The presence of identified genera was used as the parameter for multivariate analysis.
The conduct of the experiment followed the International guidelines of the IUCN Policy Statement on Research Involving Species at Risk of Extinction. The plant material studied, as a perennial species, is available for study and review at the agrostological field of the Evangelical College of Goianésia.

Results and discussion
For the determination of ecological interactions between AMF and forage grasses, the values of density of spores in the soil, mycorrhizal colonization rate in the root, and the presence of genera of AMF associated with the rhizosphere are used as parameters. No significant difference was verified between the analyzed varieties when investigating the density of spores in rhizospherical soil of forage grass varieties in Cerrado soils (Fig. 1a). P values for spore density were 0.2868 and for colonization were 0.1662.
The absence of a significant difference in spore density values is because the varieties were installed in the same area and where they are probably being colonized by the same fungal species, since they present low specificity. It is expected that there is no difference in the sporulation of fungi from the same area, since spore production  www.nature.com/scientificreports/ is a response of the fungus and not of the host plant. The production of spores is a reflection of the fungus to environmental changes that, under stressful conditions, begin to produce spores as a resistance structure 21,22,32 .
The samplings were carried out at the end of the dry season. In the cerrado, the climate is classified as tropical seasonal (AW) characterized by two well-defined seasons: a dry period and a rainy period 33 . These climatic conditions are considered stressful for most soil organisms due to the absence of rainfall for more than 4 consecutive months, which explains the high values of spore density values in the soil 12,23 .
Forage plants do not present specificity for the colonization of mycorrhizal fungi and can be colonized by more than one species of fungus 24 . However, some plant species have higher mycorrhizal colonization rates than others. Different species may present different values of colonization in the same environment, which is a reflection of the evolutionary adaptability of this symbiotic association [34][35][36][37] .
Mycorrhizal colonization values indicate the intensity to which fungi have to associate with vegetation to assist with functions such as water and nutrient absorption (MOREIRA; SIQUEIRA, 2006). Because it is the same soil, the variation in mycorrhizal colonization values is explained by the physiological differences of plants and not fungi. This behavior can be observed when comparing forage plants, such as Megathyrsus maximus and Brachiaria brizanta, which presented similar colonization rates, regardless of cultivar [38][39][40] . www.nature.com/scientificreports/ Table 2 shows the genera identified in the soil of the grasses investigated. The genera Acaulospora and Glomus were identified in all plants investigated, while the genus Slerocystis was identified to be associated only with B. brizantha piatã.
Canonical correspondence analysis aims to identify the proximity of the presence of mycorrhizal fungi identified with the grass species investigated (Fig. 2).
The genera of mycorrhizal fungi identified were commonly found in the rhizosphere of all grasses investigated, except for the genera Gigaspora, Scutelospora and Sclerocysts, which indicates that the association with these genera of fungi is less recurrent than with the other genera. The genera Glomus and Acaulospora are commonly found in Cerrado soils 6 . When investigating the biodiversity of AMF in Cerrado soils, the same genera were found to be associated with bamboo 41 , sugarcane 42,43 , sorghum and corn 44 .
The grasses present substantial mycorrhizal colonization volume for the root system of the grasses, which favors fungal colonization. The adaptability of this plant family to the natural conditions of the Cerrado also favors the exposure of the plant to the action of the fungus when subjected to situations of environmental stress, especially water.

Conclusion
The spore density values do not vary among the species of fodder studied. This parameter is independent of the plant, as it is a physiological response of the fungus. On the other hand, Urochloa decumbens presented higher values of mycorrhizal colonization. The genera of mycorrhizal fungi identified are commonly found in the rhizosphere of all grasses investigated, except for the genera Gigaspora, Scutelospora and Sclerocysts, and these genera of fungi are less recurrent than the other genera. www.nature.com/scientificreports/