Inonotus rickii (Agaricomycetes, Hymenochaetaceae) in Brazilian Cerrado: Expanding Its Geographic Distribution and Host List

Inonotus rickii (Pat.) Reid (Agaricomycetes: Hymenochaetaceae) is a poroid fungus characterized by the expressive production of chlamydospores, in vivo and in vitro, especially during its anamorphic stage. The species plays important ecological roles, standing out as a phytopathogen, affecting several species of ornamental and wild trees, mainly in tropical and subtropical regions. The infected trees develop canker and white rot of the wood, showing symptoms of reduced vegetative vigor and decline of leaves and branches which causes death in some cases. The first record of I. rickii for the Cerrado biome (Brazilian Savanna) and the first record as causal agent of canker in Schinus molle L. in Brazil is reported here. In addition, we present a checklist of its worldwide geographical distribution and known hosts, from an extensive bibliographic search in Google Scholar, SciELO, Scopus, and Web of Science databases. The species is widespread in tropical and subtropical zones; common in the American continent, especially in Central and South America and the Mediterranean region, and rare in temperate zones. We found specimens growing in both living and dead hosts, totalizing 70 species of hosts, distributed in 43 genera and 22 families. Of these, Acer negundo L. (10.5%), Celtis australis L. (6.5%), and Platanus acerifolia (Aiton) Willd. (4.8%), and the Fabaceae (30%), Fagaceae (10%), and Sapindaceae (8.6%) families were the most frequent. We present morphological descriptions and illustrations, as well as the growth characteristics in culture medium. Our study expands the known geographical distribution of I. rickii, including the Cerrado biome, as well as its structural, physiological characteristics, and its hosts.


INTRODUCTION
Inonotus rickii (Pat.) Reid is a poroid fungus that belongs to the family Hymenochaetaceae, class Agaricomycetes. It was described in South America in 1896 as Ptychogaster cubensis Pat., from a review of specimens from Cuba by Patouillard (1896). However, in 1908, the author observed that the species should be the anamorphic stage of a poroid fungus collected by Ricki in Brazil, recognized as Xanthochrous rickii Pat. (Patouillard, 1908); later the nomenclature was changed to Polyporus rickii (Pat.) Sacc. and Trotter (Saccardo and Trotter, 1912). In 1957, the species was recognized by Reid as I. rickii (Reid, 1957), currently accepted nomenclature, and its anamorphic stage as P. cubensis.
The relationship between the two life stages of the species has been confirmed by experimental studies carried by Davidson et al. (1942) and Stalpers (2000) and by molecular analysis of specimens collected from different geographical origins (Gottlieb et al., 2002;De Simone et al., 2011). I. rickii has a wide distribution in tropical, subtropical, and Mediterranean zones (Mazza et al., 2008;Ramos et al., 2008), where it is frequently found in trees in urban environments, mainly in its anamorphic stage (Mazza et al., 2008), which is considered a potential phytopathogen.
As a pathogen of woody plants, I. rickii infects branches and stems, causing canker and wood decay (Mazza et al., 2008;Annesi et al., 2010;De Simone et al., 2011). Adhesion and colonization of the substrate occurs due to the degradation of wood components, especially cellulose, hemicellulose and lignin, due to taxon enzymatic activity (Robles et al., 2014). When parasitizing the host, I. rickii can reach the heartwood, sapwood, and cambium, as well as provoke deep lesions resulting from the death of the bark tissues, characterizing the canker (Ramos et al., 2008;Annesi et al., 2010). Infected trees may show reduced vegetative growth, crown and branches decline and sparse foliage, which leads, in some cases, to death (Mazza et al., 2008;Ramos et al., 2008;Annesi et al., 2010).
The knowledge of this species-host began with reports describing the presence of a powdery mass of abundant ferruginous chlamydospores, which characterizes the anamorphic stage of the taxon in different plant hosts, but without mentioning its pathogenicity (Davidson et al., 1942). However, in the beginning of 2000, the diseases symptoms and pathological aspects became more evident in Europe. In Italy, the species was recorded in public gardens and wooded boulevards on Acer negundo L. and Albizia julibrissin Durazz. (Mazza et al., 2008;Annesi et al., 2010), and in Portugal, on Celtis australis L. (Ramos et al., 2008) causing serious damage or death of tree. Other reports in Chile (Sepúlveda et al., 2016), China (Cui et al., 2014), and Egypt (Shehata and Abdel-Wahab, 2013) also presented a similar situation regarding Schinus molle L., Acacia richii, A. Gray, Citrus spp. and Vitis spp. respectively. Based on the morphology of the basidiome and species of the host plants, the fungus called by different popular names e.g., "mapúa cheap, " which means "mascarilla de la mapúa, " in the Wayuu indigenous community of Colombia (Villalobos et al., 2017), or "Florcita de espinillo, " "Florcita de molle, " "Florcita de palo, " "Hongo de espinillo, " "Hongo de molle, " and "Hongo de palo, " in La Paz, Córboda, Argentina (Flamini et al., 2015).
In Brazil, information on I. rickii is mystery or fragmented and restricted to the holotype with unknown localities (Patouillard, 1908) and to occurrence records in the Caatinga biomes, on Spondias sp. (Umbuzeiro) (Drechsler-Santos et al., 2013;Maia et al., 2015); Atlantic Forest and Pampa without host identification (Campos-Santana and Loguercio-Leite, 2010;Maia et al., 2015). Here we report for the first time I. rickii in the Cerrado biome, which constitutes the first record of this species causing canker disease in S. molle. In addition, we present detailed morphological descriptions of both anamorphic and teleomorphic stages supported with illustrations, and updated checklist of its worldwide geographical distribution and known hosts up till now.  (Figure 1).

Study Area
The Brazilian Cerrado is considered the richest savanna in the world and is the dominant biome in the Central Plateau of Brazil which covers part of the North, Northeast, South, and Southeast regions, representing approximately 25% of the country's territory. The Cerrado characterized by a rainy tropical climate, with high temperatures in summer and dry winter, this biome is known for its diverse landscapes represented by a vegetation mosaic, which includes forest, savanna, and grassland formations, where trees, shrubs, and undergrowth predominate, respectively (Klink and Machado, 2005;Ribeiro and Walter, 2008;WWF, 2015). Despite its great diversity, only 61% of the original Cerrado vegetation remains preserved (Sano et al., 2010). Sampling of our target species occurred in mesophilic forest (semideciduous dry forest) in which there is a predominance of tree species and canopy formation, occurring in interfluves and rich soil presented different levels of deciduous vegetation in the dry season (Ribeiro and Walter, 2008).

Morphological Characterization
Collected samples were dried at 40 • C in oven and deposited in the Herbarium of the Universidade Estadual de Goiás (HUEG) and the Universidade Estadual Paulista (SJRP). Micro and macrophenotypic identification of basidiomata were carried out according to the relevant identification keys. For macroscopic characterization, the shape, consistency, color, dimension, and number of pores per mm of basidiomata were considered. For the description of the microscopic criteria, cross sections through the basidiomata in distilled water and 3% KOH were microscopically examined. Hyphal structure, hymenial setae, setal hyphae, basidia, basidiospores, and chlamydospores and their ornaments were examined according to Ryvarden (2004Ryvarden ( , 2005 and Ramos et al. (2008). Melzer reagent was used to test the amyloid reaction of the microstructures (Kirk et al., 2008). All microscopic criteria were observed by Olympus CX31 optical microscope (1000×) magnification, and the measurements were performed using the Piximètre software version 5.10 R 1541 (Henriot and Cheype, 2017). Abbreviations used for measurements of basidiospores and chlamydospores are: Q = quotient between length and width, Qm = medium value of Q and, N = number of measured structures. All photographs and measurements were made using 3% KOH as mounting medium. The color indication for all evaluated characters was based on Kornerup and Wanscher color cards (Kornerup and Wansher, 1978).
Fresh collected samples were plated out on potato dextrose agar (PDA) supplemented with 0.025 g/ml −1 of chloramphenicol as bactericidal and incubated in a BOD incubator at 25 • C. Recovered colonies were characterized based on macro (shape, color, texture, and presence of exudates) and micromorphological criteria (hyphae, setal hyphae, and chlamydospores) according to relevant identification keys. The cultures were preserved according to Castellani (1963) and deposited in the collection of fungal cultures of the Laboratório de Micologia Básica, Aplicada e Divulgação Científica (FungiLab) of the Universidade Estadual de Goiás, Brazil.

Checklist of Geographical Distribution and Hosts
We collected the data from an extensive bibliographic search of Google Scholar 1 , SciELO 2 , Scopus 3 , and Web of Science 4 by using different keywords: "Inonotus rickii" OR "Polyporus rickii" OR "Ptychogaster cubensis" OR "Xanthochrous rickii." Our data 1 scholar.google.com.br 2 www.scielo.org 3 www.scopus.com 4 www.webofknowledge.com included all published articles, books, chapters, and abstracts available for access. For the worldwide geographical distribution of the species, we consider the specific location described by the authors and the geographic coordinates; when not available, they were obtained using the Latlong coordinate system (Latitude and Longitude Finder, 2020). The geographic distribution map was constructed using the Quantum GIS software (QGIS Development Team, 2020) and the climatic classification was determined between the North and South zones of both hemispheres as: tropical between 23 • 27 , subtropical 23 • 27 and 46 • 54 , temperate 46 • 54 and 66 • 33 , and polar 66 • 33 and 90 • , respectively (Peel et al., 2007;Eccles et al., 2019).
For the host checklist, we considered the absolute frequency the number of times the species was reported and relative frequency the quotient between the absolute frequency and the total of citations. The system of nomenclature, hierarchical classification and the name authority of the plant species followed The plant list (The Plant List, 2020).

Description
Teleomorphic stage-basidiomata annual, 7.0-11 × 6.0-7.8 cm in size, solitary or aggregated in the form of semicircular shelves, sessile, pileate, and strongly attached to the substrate. Pileus applanate to ungulate, margin obtuse to undulate, soft and spongy consistency when fresh and firm and crumbly when dry. Pileal surface covered by a dense powdery layer formed by reddish brown chlamydospore (8D7). Pore surface also covered by chlamydospore, circular to angular pores, 2-4 pores per mm, thin and lacerated. Thick context and positive KOH reaction. Anamorphic stage-semicircular or cushionshaped, soft and robust, velvety to the touch, reddish orange (8B8) when young and reddish brown (8E7) when mature, structured by a dense mass of chlamydospore attached to hyphae, forming a structure similar to capillitium, which facilitates dispersion by anemophilia.
In culture presents velutinous to cottony mycelium, occupying the entire length of the petri dish (90 × 15 mm) in 2 weeks of cultivation, yellowish white (2A2) when young, becoming grayish yellow (4B4) to dark yellow (4C8) when mature, production of exudates in the form of reddish orange droplets (8A8). Abundant production of chlamydospores of various shapes, occasionally attached to setal hyphae, also abundant and parallel in the hyphalic trama. The color and size characteristics of the cultures' microstructures are in accordance with those observed in the basidiomata.

Geographical Distribution in Brazil
There are records in the Atlantic Forest areas in Pernambuco, Rio Grande do Sul and Santa Catarina, Caatinga areas in Alagoas and Bahia (record not shown in Figure 1, as the specific location was not found), Pampa areas in Rio Grande do Sul, and Cerrado areas (present study) in Goiás and São Paulo (Figure 1; Campos-Santana and Loguercio-Leite, 2010; Maia et al., 2015).

Habitat and Substrate
We collect our specimens in the mesophilic forest in both stages of life, on dead wood and an unknown living host, and in urban areas in the anamorphic stage on living and dead S. molle trees. In the data set listed in the present review, we found 70 plant species distributed in 43 genera and 22 families ( Table 2). Of these hosts, A. negundo (representing 10.5% of the total species), C. australis 6.5% and Platanus acerifolia 4.8%. On the family level Fabaceae came first by accounted 30% out of all host families followed by Fagaceae (10%) and Sapindaceae (8.6%). In the checklist, the host species referred as sp. or spp., were accounted for as a single species.

Comments
The morphological characteristics observed in collected samples coincide with those described by many authors (Gottlieb et al., 2002;Melo et al., 2002;Ryvarden, 2005). Measurements of setal hyphae recorded by Ryvarden (2005) and Campos-Santana and Loguercio-Leite (2010) were up to 250 µm or more long. Although these measurements are similar to our samples, we recorded smaller hyphal setae ranged between 110 and 200 µm in the examined materials and pure cultures. I. rickii is similar to Inonotus patouillardii (Rick) Imazeki in the field but differentiating itself by the abundant presence of chlamydospores. Our specimens were collected during both rainy
In Europe, especially in Italy  and Portugal (Ramos et al., 2008), it has been reported that canker caused in plant tissues has reduced the number of ornamental tree species in urban environments. In addition, in the Wayuu indigenous community in Colombia, it has been reported that the fungus has had an impact on local native vegetation, also parasitizing species of daily use, such as Parkinsonia praecox and Haematoxylon brasiletto, used for resin and paint extraction, respectively (Villalobos et al., 2017).
The most frequent host species of I. rickii are widely known as ornamental trees. However, some of the reported hosts are used as a food source (Barolo et al., 2014;Viveros Garciìa et al., 2018) or for extracting compounds for different applications (Sharma et al., 2018;Milena et al., 2019). This warns of the need to know the impact of I. rickii on species of local, regional and worldwide economics.
Our specimens were detected growing at the base of the stem and on main branches of living and dead trees, usually in the anamorphic stage. Only specimens SJRP 28714 and HUEG 12067 were sampled in their teleomorphic stage. Although the species produces reproductive structures in both stages of the life cycle, the teleomorphic phase occurs occasionally, with the anamorphic stage usually frequent (Mazza et al., 2008;Ramos et al., 2008).
The propagation of the species occurs mainly by the production and release of chlamydospores, which are more abundant in the anamorphic phase. At this stage, a structure sensitive to touch appears, which releases a large number of chlamydospores. On this occasion, in urban environments, dispersal is facilitated by tactile gardening activities in the maintenance of squares and parks, by anemophilia, by the contact of insects and other animals, including the flux of human traffic . We observed that all specimens collected in an urban area (HUEG 11993, 12062, and 12063) were in the anamorphic stage and the dispersion of chlamydospores may have been facilitated by the activities mentioned before. Infection and development of the fungus occur when its spores (basidiospores or chlamydospores) are deposited on susceptible hosts, preferably due to the presence of dead wounds, scars, or fragments; these spores then germinate and promote the growth of the fungus within wood tissues (Phillips and Burdekin, 1989).
In the present study, we report for the first time occurrence of I. rickii on S. molle in Brazil. The fungus was found either as saprobic or as a pathogen, causing canker of the host plant. Belonging to the Anacardiaceae family, S. molle is an arboreal and perennial species, popularly known as pepper tree, pink pepper, American pepper, false pepper, aroeira salsa (Brazil) or aroeira mansa (Brazil). It is native to South America, but it was introduced and naturalized in several regions of the world, mainly as an ornamental in urban environments. Its great popularity as a cultivated plant is associated with its high tolerance to water and temperature availability, high growth rate and medicinal properties (Kramer, 1957;Goldstein and Coleman, 2004;Habte et al., 2020).
Anamorphic samples infected S. molle (HUEG 11993, 12062, and 12063) were developed over the trunk and main branches of the living and dead host. We observed that the individuals growing in the dead host (HUEG 12062 and 12063) occupied a large extension of the wood between the bark and bark, projecting over the crevices of the stem and reaching to the heart (Figure 5). On the other hand, samples that grew on a living host (HUEG 11993), initial canker development was observed, possibly due to the time of maturity of the fungus that was in the early stages of development (Figure 5). The presence of I. rickii in this plant has already been reported in Portugal (Melo et al., 2002), Chile, and Spain (Intini and Tello, 2003;Sepúlveda et al., 2016).
Parallel to studies of phytopathological interest, I. rickii has also been the subject of studies to prospect for secondary metabolites and lignocellulolytic enzymes involved in attacking the host and degrading the constituents of wood (Xavier-Santos et al., 2004;Tura et al., 2009;Chen et al., 2014), aiming, above all, at an option sustainable in the bioremediation of recalcitrant compounds. The species is also associated with cultural events in the Wayuu indigenous community, Colombia, in which women apply a layer of chlamydospores to their face, previously treated with sheep tallow to protect themselves from solar radiation (Villalobos et al., 2017). Moreover, taxonomic and phylogenetic aspects are still performed, specially to understand its anamorphic stage.
Although molecular studies carried on different samples confirmed the relationship between anamorphic and teleomorphic phases of I. rickii. A preliminary phylogenetic analysis of samples (anamorphic and teleomorphic) collected from different geographical origins showed came from Florida, United States, were separated from those came from Asia, South America and Europe (De Simone et al., 2011;Cui et al., 2014). An interesting aspect to consider that all species from the Florida were collected in the anamorphic stage and the teleomorphic stage has never been recorded in North America. These results suggest the existence of two possible distinct species and are reinforced by morphophysiological, ecological data and climatic conditions (De Simone et al., 2011;Cui et al., 2014). However, studies that analyze isolates from North America and other regions, as well as the holotype of both stages of life, are necessary to confirm the distinction.

CONCLUSION
Our study expands the knowledge of geographical distribution of I. rickii, including the Cerrado biome, as well as of its structural, physiological characteristics, and its hosts. Although common in tropical and subtropical regions, the specimens described here constitute the first documented records of the species for the Brazilian Cerrado and for the Midwest and Southeast regions, in addition to the first report on S. molle within Brazil. The checklist of hosts provided may assist in the development of practices for the control of the pathogen in urban areas with a high incidence of infections. Therefore, it should be mentioned here that, although the present study adds new data to information concerning I. rickii, this updated checklist must be considered as a provisional one always waiting for continuous supplementation.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

AUTHOR CONTRIBUTIONS
LL-S and SX-S designed this study, collected and analyzed the samples, and wrote the manuscript text. LL-S prepared the figures and tables. AA-A reviewed and edited the manuscript. All authors have read and approved to the published version of the manuscript.