Taxonomical study of the genus Amanita from Western Burkina Faso

1 Department of Plant Biology and Physiology, Training and Research Unit in Life and Earth Sciences (UFR / SVT), University Joseph KI-ZERBO, BP. 7021, Ouagadougou, Burkina Faso. 2 Norbert ZONGO University, Higher Normal School, BP 376 Koudougou, Burkina Faso. 3 Institute for Agricultural Environment and Research (INERA/Kamboinsé), BP 8645, Ouagadougou, Burkina Faso. 4 New Dawn University, Département de Phytopathologie, Faculty of Agronomy, 06 BP: 9283 Ouagadougou, Burkina Faso.


INTRODUCTION
This article discusses the genus Amanita Pers. as part of the in-depth study on macromycetes by many mycologists in Burkina Faso. No scientific study has yet been carried out taxonomically by a mycologist on this genus in this country and many species will disappear without being known to the scientific world. There is therefore a need to make them known through inventories. The history of work on the diversity of macromycetes in Burkina Faso can be traced to the research carried out by Heim (1936) on "Overview of Madagascar mycological flora III: Three giant boletus from Africa and Madagascar". Currently, the main focus is to collect additional data on Burkina Faso fungi through Burkina Faso's national fungal biodiversity inventory, and to date, work carried out as part of a regional project coordinated by the University of Ouagadougou, has documented an extremely diverse macrofungal community . The genus Amanita is a group of fungi cosmopolitan in distribution and especially well represented in tropical Africa, but most previous taxonomic studies were largely based solely on morphology .
Studies on Amanita have focused much attention on toxin-producing species (Hawkeswood, 2006 1982; Cai et al., 2014;Zhang et al., 2010Zhang et al., , 2015. There are about 58 endemic taxa in sub-Saharan Africa (Tulloss and Possiel, 2017). Indeed, the genus Amanita has been the subject of advanced documentation through many studies carried out in Africa: Beeli (1931Beeli ( , 1935, Gilbert (1940Gilbert ( , 1941 and Bas (1969) in Congo, Bouriquet (1941Bouriquet ( , 1943 in Madagascar, Pegler and Piearce (1980) and Pegler and Shah-Smith (1997) in Zambia, Bas (1982) in Malawi, Reid and Eicker (1991) and Eicker et al. (1993) in South Africa, Härkönen et al. (1994) in Tanzania. Up to now, our survey on Amanitaceae in Burkina Faso shows that there are at least 20 species of Amanita in the country; among them, a number will certainly be new to science and Burkina Faso. To date, 1000 species have been described worldwide in the Amanitaceae family (Yang et al., 2018), including about 50 species in tropical Africa. The genus Amanita (Amanitaceae) is described as an ectomycorrhizal genus establishing a symbiotic association with partner plants (Onguene and Kuyper, 2012). Most species in the genus are considered to be ectomycorrhizal (EcM) and they are distributed in forests and heaths including Betulaceae, Dipterocarpaceae, Fabaceae, Myrtaceae, Pinaceae, and Salicaceae. They play a critical role in forest ecosystems worldwide (Zhang et al., 2004) and thus contribute to the improvement of water and mineral nutrition of partner plants (Onguene and Kuyper, 2012).
In temperate regions, the genus has a very bad reputation due to powerful and deadly toxins present in a number of species. Amanita Pers. is an important genus of mushrooms that includes several species that are widely recognized as the most toxic mushrooms in the world (Thongbai et al., 2016). However, few species are consumed. In tropical Africa, several species of edible amanites, including the very large Amanita loosii, appear to be widespread in open African forests (De Kesel et al., 2017).
Despite these contributions, the amanites of Burkina Faso are still very poorly regarded; they are non-existent compared to those of some other regions of the world. Further studies seem necessary to know and document the fungal diversity of Burkina Faso's natural ecosystems through inventories. Our work aims to do a taxonomic study of amanite species in Burkina Faso.

Study area
Located in the extreme southwest of Burkina Faso, the city of Niangoloko, bordering the nature reserve, is 18 km from the border with Côte d'Ivoire. Since 1936, the Niangoloko classified forest has been part of the State's classified forest estate. It is located in southwestern Burkina Faso ( Figure 1) and covers an area of 7295.83 ha between 4° 50' and 4° 58' W and between 10° 10' and 10° 17' N latitude (Ouoba et al., 2019). The natural reserve extends over an area of 7300 ha. The climate of this region, the rainiest of the country, is of Sudanian type (Guinko, 1984). The average annual rainfall is 1115 mm. The average temperature is 27.35°C with minimum of 18.3°C in December and maximum of 36.6°C in  (Ouoba, 2006). The characteristic vegetation is a mosaïc of gallery forests, shrubby savannas, wooded savannas and wooded savannas. All around the reserve, towns and villages of the Goin (farmers) and Peulh (herders) ethnic groups have settled. The main authorized activities within the forest are fruit picking, honey harvesting, medicinal plant harvesting and dead wood collection.

Collection, macroscopy and microscopy
The methodology used is similar to that of Tulloss (2008). The samples were collected in the Cascades region in July and August 2018, a timeframe during which rainy season is well established in the area. The samples were carefully collected from the ground with a pocket knife in order to obtain a complete carpophore, often necessary for the determination of a collection.
Photographs of the species were taken in situ using an OLYMPUS LENS camera (24 wide optical zoom ED 4.5-108, 0 mm 1: 3.0-6.9). Their substrate has been recorded. The environmental and macroscopic characterization was based on characteristics such as the color, size and shape of the cap, the outer surface of the cap, the texture of the cap surface, the blades and the presence or absence of latex, the geographical coordinates and that of the host substrate. Macroscopic descriptions were made subjectively and using a description sheet proposed by De Kesel et al. (2002). Anatomical studies were carried out on exsiccata (dried samples). Microscopic examination of the tissues was made with 5% KOH and stained with 1% ammoniacal Congo Red. Spores were observed in the Melzer's reagent to test the amyloidity. This amyloidity is best observed by soaking the tissues overnight in Melzer's reagent and observing in fresh reagent the next morning. Biometric variables such as spores and basidia sizes were measured by using a millimeter ocular lens. At least 50 spores and 20 basidia per sample were measured. Anatomical details were obtained using a NIKON Eclipse Ci optical microscope with a drawing tube, slides and cover plates.

Amanita citrina (Schaeffer) Persoon
Pileus 40-120 mm in diam., solitary, or rarely in pairs or gregarious; small size with a cap first hemispherical when young, then slightly spread out; it becomes convex when mature and slightly depressed at the center; dry but has a mucilaginous appearance when humid; the flesh is white, full, firm, slightly thick with a smell of fresh flour or tuber and a slightly sweet taste; with a surface almost entirely covered with small lying squamules, adhering from white to yellowish-white, sometimes disappearing when mature and leaving the cap smooth; these squames also form large or small patches. Lamellae, free, 6 to 8 mm high, white, fragile, uneven.Stipe 40-90 mm, cylindrical, central; 5-10 mm thick; bulbous volva in the shape of a circle or even circumcised, sometimes split; well-developed annulus in a superior position, white or yellowish, persistent, skirt shape; white flesh, full.

DISCUSSION
The biodiversity of mushrooms in Burkina Faso remains poorly known because less attention has been paid to them than to higher plants. The genus Amanita Pers. is an example. The three species recorded share the same ecological characteristics, host or substrate. Specifically, most wild mushrooms species belonging to the Russulaceae, Cantharelaceae, Polyporaceae, Boletaceae and Amanitaceae families share the same habitat and ecological characteristics (Chelela et al., 2015); however, A. citrina is found in slightly brighter areas while the other two are found in both shadows and light under broadleaved areas. This is due to the characteristics of the region (Cascades) and the nature of its habitat (Sudanian classified forests with groups of trees of Berlinia grandiflora, Isoberlinia doka). The high coverage rate of these different plant groups provides shade and humidity conducive to the growth of fungi.
Amanita pulverulenta shares a morphology almost similar to that of A. virido-odorata: macroscopically, the two taxa are characterized by the color and shape of the warts in the hat and the shape of the stipe; warts in the first species detach very easily leaving the hat smooth and covered with powdery spots while those in the second species are tough, small and fused to the hat. Both this study and other recent studies Dabiré, 2017) exemplify the high diversity of fungal species in Burkina Faso, suggesting that there are still more taxa to be discovered and recorded.
Up to now, most taxonomic research on fungi in Burkina Faso has focused on Russulaceae (Sanon et al., 2014;Sanon, 2015) scleroderms (Sanon et al., 2009; and in general (Guissou, 2005;Guissou et al., 2015Guissou et al., , 2008Bicaba, 2013) in the western, central plateau, central and central regions and the discoveries of amanite species in the cascade region suggest that further studies are needed in other regions of the country with different mycorrhizal hosts and ecology.
Our own experiences and field observations reveal that amanites have a different phenological appearance. Indeed, they appear well after the other groups of fungi, resulting in a late appearance. This observation was made by Guinberteau and Courtecuisse (1997) where they classified the genus Amanita as a late mycorrhizal genus by qualifying them as "early stage fungi". Madamo et al. (2017) add that this phenology would be greatly influenced by humidity. Tulloss et al. (2016) report that Amanita is a genus of fungi suspected of being ectomycorrhizal. These three species have been found in some parts of Africa. Amanita citrina, Amanita pulverulenta and A. virido-odorata are known in Benin Yorou et al., 2014;Aignon, 2016;Laourou, 2016), A. pulverulenta in Niger , Pulverulenta in Togo (Kamou et al., 2017;Kamou, 2012;De Kesel and Guelly, 2007); A. pulverulenta and A. virido-odorata in Burundi, Congo in Zambia (Bas, 1969;Buyck 1994). Ectomycorrhizal fungi are mainly forest fungi and their best substrates are the presence of ectotrophic species and their roots. These results are similar to those of Zhang et al. (2004) who indicated that the abundance and diversity of ectomycorrhizal fungi are influenced in forest ecosystems dominated by tropical and temperate plants of great economic importance such as Caesalpinioideae, Dipterocarpaceae, and Phyllanthaceae. Gévry (2010) argues that at the local level, the abundance of precipitation would be an excellent indicator of the diversity and structure of fungal communities. The preservation of the forest influences the subsistence of mushrooms. Fungal distributions are mainly determined by genetic, ecological and morphological constraints (Pringle et al., 2009). For example, ecological and phylogenetic studies have shown that some fungal species may be virtually free of dispersal barriers (Queloz et al., 2011), while others have strong biogeographic structures (Matheny et al., 2009) that may be related to host and habitat limitations.

Conclusion
This study the second of its kind gives an inventory, characterizes and identifies the wild plant genetic resources of fungi of the genus Amanita Pers. Mycological investigations have shown that there is a great diversity of Amanita species in Burkina Faso, which play a particular role in ecosystems. However, many of