Metarhizium species in soil from Brazilian biomes: a study of diversity, distribution, and association with natural and agricultural environments
Introduction
Metarhizium is a diverse genus of fungi belonging to the Clavicipitaceae family which contains both amorphic (asexual) and telemorphic (sexual) phases (Sung et al., 2007). It has a broad geographic distribution with important ecological functions in the environment, occurs naturally in the soil, causes diseases in different orders of arthropods, and colonizes plants as endophytes (Zimmermann, 2007; Vega et al., 2009; Lacey et al., 2015; Barelli et al., 2016). In Brazil, studies involving Metarhizium species composition have included control of insect vectors of human diseases such as Triatoma infestans (Luz et al., 2004; Rocha and Luz, 2011), Aedes aegypti (Luz et al., 2008; Santos et al., 2009; Leles et al., 2010), Anopheles gambiae s.s., and Anopheles arabiensis (Mnyone et al., 2009). However, its main use is to control cercopids in sugarcane and pasture areas, with 33 products currently registered in Brazil that contain Metarhizium anisopliae as an active ingredient to control Mahanarva fimbriolata, Notozulia entreriana, and Deois flavopicta (Hemiptera: Cercopidae) (AGROFIT, 2019).
The M. anisopliae sensu lato (s.l.), traditionally used in biological control, is a complex of at least nine species, according to multigenic analysis supported by morphology (Bischoff et al., 2009). Four of them (Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii, and Metarhizium brunneum) form the PARB clade that constitutes the inner core of the complex (Kepler et al., 2015). M. pingshaense is the most predominant species in Asia and Australia, M. anisopliae in South America and Africa; M. robertsii in North America; and M. brunneum in Europe (Rehner and Kepler, 2017). In Brazil, the occurrence of these four species and three other species, also from the Metarhizium anisopliae complex (Metarhizium lepidiotae, Metarhizium acridum, and Metarhizium anisopliae majus), have been reported before (Lopes et al., 2013a, 2014, 2013b; Rocha et al., 2013; Rezende et al., 2015; Iwanicki et al., 2019). In addition, two lineages are referred to as Metarhizium sp. indet. 1, sister lineage to M. anisopliae sensu stricto (s.s.) (Rocha et al., 2013; Lopes et al., 2014; Rezende et al., 2015) recently described as a new species, Metarhizium humberi sp. nov. (Journal of Invertebrate Pathology, 2019, doi: https://doi.org/10.1016/j.jip.2019.107216), and Metarhizium sp. indet. 2 (not taxonomically characterized), designated in M. majus and Metarhizium guizhouense, referred as MGT clade (Rezende et al., 2015). Moreover, Metarhizium pemphigi ( = Metarhizium flavoviride var. pemphigi) and Metarhizium blattodeae belonging to the M. flavoviride species complex (Rocha et al., 2013; Montalva et al., 2016); Metarhizium brasiliense, Metarhizium rileyi (Kepler et al., 2014), and the most recently identified, Metarhizium alvesii (Lopes et al., 2018) have also been reported in Brazilian regions.
Molecular techniques have been extensively applied in recent years to study entomopathogenic fungi, especially the Metarhizium genus. These studies have focused on: (1) genetic diversity and population structure of Metarhizium spp. on a small geographical scale (Bidochka et al., 2001, 2005; Enkerli et al., 2005); (2) isolates from locations geographically distant (Velásquez et al., 2007; Inglis et al., 2008; Enkerli et al., 2009); (3) isolates originated from a host species or from a group of similar species (Luan et al., 2013); and (4) isolates from the same type of agricultural environment (Steinwender et al., 2011, 2014; Hernández-Domínguez and Guzmán-Franco, 2017). Brazilian studies have been restricted to isolates deposited in collections of entomopathogens or from a certain type of habitat/host in a region or state (Sosa-Gómez et al., 2009; Lopes et al., 2013a, 2013b, 2014; Rocha et al., 2013; Rezende et al., 2015; Castro et al., 2016; Iwanicki et al., 2019). With the development of molecular tools scaled to the diversification of M. anisopliae complex, a refined view of Metarhizium diversity is emerging, particularly for species within the group widely used for biological control (Kepler et al., 2014). Our study is the first to characterize the diversity of indigenous Metarhizium from Brazilian soils representing a range biomes and land-use types.
Brazil has the highest tropical biodiversity in the world (Mittermeier et al., 2005). The Amazon biome contains the largest tropical forest, which is one of the richest biological diversity reserves, hosting an estimated one-quarter of all terrestrial species (Dirzo and Raven, 2003). The Cerrado is considered as a hotspot for global biodiversity, but a large part has recently been transformed into pastures and monocultures. The great richness of species in the Atlantic Forest, extending from the north to the south along the Atlantic coast, is threatened by the drastic reduction of its original area (only 8% remain). The Caatinga, predominantly located in the northeastern region of Brazil, contains typically dry and thorny vegetation due to strong climatic irregularity during the year. Finally, the Pampa biome, in southern Brazil, is associated with native fields, which are generally used for livestock rearing, and occupies only 2% of the national territory (IBGE, 2004; MMA, 2015). Maia et al. (2015) estimated there to be 5719 species of fungi distributed in 1246 genera, 102 orders and 13 phyla in Brazil. Atlantic Forest contains the largest number of records (3017 species), followed by the Amazon Rainforest (1050), Caatinga (999), Cerrado (638), and Pampa (84). These numbers are constantly changing due to the reduction of native areas converted for agriculture.
Despite the importance of Metarhizium as a biocontrol agent in Brazilian agriculture, the diversity and distribution of its species, which occur in either natural or agricultural environments associated with the different biomes, are still poorly known. Moreover, this diversity has not been exploited considering that only M. anisopliae subclade Mani 2 is used commercially in the country. In this study, we investigated the genetic diversity of Metarhizium species using isolates obtained from soil of: (1) three types of vegetation (native, annual and perennial crops); in (2) five Brazilian biome areas (Amazon, Cerrado, Atlantic Forest, Caatinga, and Pampa); during (3) 2 y (2012 and 2013) and two seasons (dry and humid) of collection; and using (4) two methods of fungal isolation (selective medium and insect bait). For this, we sequenced the nuclear intergenic region MzIGS3 developed for species level investigations within the M. anisopliae complex (Kepler and Rehner, 2013). The objective of this study was to characterize the diversity and distribution of haplotypes of Metarhizium species associated with native and agroecosystems within five representative areas of Brazilian biomes. Species assignment to the isolates and their phylogenetic relationship was determined using 5′-TEF sequences. This information will help elucidate the belowground composition of Metarhizium species and their diversity associated with different ecosystems in Brazil.
Section snippets
Soil sampling
Metarhizium sp. isolates were recovered from soil of five representative Brazilian biomes (Amazon, Caatinga, Cerrado, Atlantic Forest, and Pampa) (Table 1). Sampling was carried out in both humid and dry seasons, in 2012 and 2013. During the dry season, sampling was performed from June to October in the Amazon and Cerrado biomes and from March to April in the Caatinga, Atlantic Forest, and Pampa. In the humid season, sampling occurred in the Amazon and Cerrado between January and March, and in
Assignment of isolates into the recognized species limits of Metarhizium
The taxonomic assignment and phylogeny were first carried out with 53 Metarhizium isolates from this study and 55 other reference sequences based on the 5′-TEF marker (see section 2.4). The final sequence size obtained was 599 base pairs. A set of 36 haplotypes were identified at the species level and eight of them had not been described yet (Fig. 1). The Bayesian and Maximum likelihood phylogenetic analyses produced similar topologies. Metarhizium isolates, recovered from soils of Brazilian
Discussion
This is the first comprehensive study revealing the belowground genetic diversity and distribution of Metarhizium isolates from different species, including new lineages, associated with native vegetation and agricultural crops from five biomes, which represents approximately 93% of Brazilian land area and comprises different and distant ecosystems. The main findings in this study were: (1) M. robertsii was the most abundant species in the soils and the only species identified in the Pampa; (2)
Acknowledgements
The financial support and scholarship provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; Grant: 563233/2010-9), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; Grant: 2010/52342-4), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) are greatly appreciated. We thank Uemerson Silva da Cunha (FAEM/UFPel), Edmilson Santos Silva (UFAL), Fernando Hercos Valicente (EMBRAPA Milho e Sorgo), Erika P. J. Britto (UFMT), and their work
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