Diversity and distribution of species of Ganoderma in south western Cameroon

The genus Ganoderma is one of the most important group of Basidiomycetes due to their medicinal effects and also because they cause decay in a very wide range of tree species all over the world. Opportunistic sampling was used to collect 57 samples of Ganoderma from oil palm and other hosts for identification using comparative morphology and supported by molecular studies of the ITS and mtSSu gene regions. The objectives were to identify the species associated with different hosts, and to generate a checklist of species of Ganoderma in south western Cameroon. Morphological and molecular characterization of the 57 specimens showed that they belonged to 17 species of Ganoderma. Two species, Ganoderma tornatum and Ganoderma chalceum are known records for Cameroon. Four species, Ganoderma weberianum, Ganoderma cupreum, Ganoderma steyaertanum and Ganoderma zonatum are new records for Cameroon. The remaining 11 species Ganoderma ryvardense, Ganoderma lobenense, and Ganoderma species 1–9 with different affinities might be new to science. Six plant species were identified as hosts to different species of Ganoderma. They are Elaeis guineensis, Cassia sp., Acacia sp., Pinus sylvestris, Avocado sp. and unidentified hardwoods, with E. guineensis, hosting the highest number of species. With supplementary literature survey, a check-list of 23 species was established.

rubber (Hevea brasiliensis Muell.Arg.), temperate hardwoods, coconut (Cocos nucifera L.) and betel nut palms (Areca catechu L.) (Singh, 1991).Several species cause basal stem rot of oil palm (Elaeis guineensis) (Kinge et al., 2012), and other tropical forest trees.The fruit body of Ganoderma, for its perceived health benefits, has gained wide popular use as a dietary supplement in China, Japan, North America and other regions of the world, including Cameroon.Ganoderma species are also used in folk medicine to cure various diseases, and strains are commercially cultivated for the preparation of health tablets or drinks.As a kind of health food, it has also been used to prevent and treat immunological diseases, such as hypertension, tumorigenesis, etc. (Liu et al., 2002;Kinge et al., 2011).The many medicinal benefits of Ganoderma were reviewed by Jong and Birmingham (1992).On the other hand, some Ganoderma species play an important role in plant pathogens.Several species cause severe diseases in plantations or in forests (Steyaert, 1967;Bakshi et al., 1976).However, some of them have been shown to selectively delignify wood and are recognized as a potentially important source of lignindegrading enzymes (Otjen et al., 1987).
The genus Ganoderma was divided into two distinct groups.The laccate including the Ganoderma lucidum complex is characterised by the presence of a cutex layer on the outer surface of the fruiting body that renders it shiny.The non-laccate lacks the cutex layer and is referred to as Ganoderma applanatum complex.Over 290 taxonomic names have been published in the genus Ganoderma, indicating that the genus is morphologically complex (Ryvarden, 2000).This led Ryvarden (1991) to describe the state of Ganoderma taxonomy as being in crisis.Traditional identification of Ganoderma species has been based on morphological features, physiological and developmental characters and chemical components such as secondary metabolites (Takamatsu, 1998).Species concept in the genus Ganoderma is thus not universally accepted neither well established (Gottlieb et al., 2000).
It has been shown that morphology and culture characteristics of species from the same genus can be greatly affected by growth conditions (Moncalvo, 2000).This signifies that a large number of synonyms may exist due to the number of species that have been identified based on morphology (Moncalvo, 2000).The shape of basidiocarp (fruiting body) has been demonstrated to be greatly influenced by the environment (Chen, 1993), and the basidiospores by latitude and altitude (Steyaert, 1975).In some species, the context colour was darker in collections from southern latitudes than northern latitudes on the European continent (Steyaert, 1972).Age and environment have been shown to have a marked effect on the colour, size and brightness of the fruiting body, as well as length of stipe (Moncalvo, 2000).Identification of Ganoderma based on these characteristics have contributed greatly to the confusion in the naming of species within this genus, and have resulted in traditional taxonomic methods being inconclusive for establishing a stable classification system for Ganoderma species (Hong et al., 2002;Hseu et al., 1996).Traditional identification methods are being supplemented with new identification methods such as restriction fragment length polymorphism (RFLP) (Miller et al., 1999), sequence analysis (Hong et al., 2002;Moncalvo et al., 1995a, b;Smith and Sivasithamparam, 2000a) and isoenzyme electrophoresis (Gottlieb et al., 1998;Gottlieb and Wright, 1999;Smith and Sivasithamparam, 2000b).It is the phylogenetic analysis of amino acid or DNA sequences that is known to have the highest resolving power (Bruns et al., 1991).These modern techniques have helped to clarify the distribution of different species complexes in the genus Ganoderma, and have revealed some instances of misidentification (Gottlieb et al., 1998;Moncalvo et al., 1995a, b).
Despite advances in taxonomic techniques, the species diversity of Ganoderma and other polypores in Africa have received very little attention.In Cameroon, the following species have been reported: Ganoderma tornatum var.tornatum, Ganoderma hildebrandii, G. lucidum, Ganoderma cf.multiplicatum, Ganoderma resinaceum, Ganoderma carocalcareus and Ganoderma ryvardense (Turner, 1981;Nunez and Daniels, 1999;Douanla-Meli and Langer, 2009;Kinge, 2012).Moncalvo and Ryvarden (1997) listed 49 Ganoderma species from Africa.Apart from the work published by Douanla-Meli and Langer (2009), Kinge (2012) and Kinge et al. (2012) all other reports based their identification on morphology alone.It is therefore reasonable to suggest that a wealth of information is waiting to be discovered.As very little is known about the diversity of Ganoderma in Cameroon.In order to understand the diversity of Ganoderma species, the knowledge of their distribution and association with their substrates are essential.The substrates such as dead and decaying wood and its associated fungi and invertebrates are vital elements of the forest ecosystem and their decay processes represent a key path for nutrient and carbon recycling (Bobiec et al., 2005).Thus, the objectives of this research were as follows: to identify the different hosts of species of Ganoderma in the study area, to prepare a check-list of species of Ganoderma in the study area and to produce a species distribution map.

Study area and sampling
Collection trips were done between 2008-2011 from Lobe, Bai, Dibombari, Idenau, Bota, Mondoni, Mungo and Beneo estates as well as on forest areas in Buea, Idenau, Ekona and Bafia in the Mount Cameroon Region all in South Western Cameroon (Figure 1).An opportunistic sampling method was used in collecting the samples.Collection sites were geo-referenced by GPS points using the Garmin Etrex Venture GPS.

External and internal morphology
Prior to examination of the basidiomata, specimens were photographed from above as per Steyaert (1972).External and internal morphological characters and confirmation by molecular methods have been described by Kinge et al. (2012).
After identification, a species distribution map based on the presence or absence of species was produced using GIS software (ArC GIS 9.3).A thorough literature search was also made to supplement data from the present field survey and a checklist for species of Ganoderma in Cameroon was produced.

Species diversity
A total of 57 samples were examined morphologically representing 17 species.Of the 17 identifiable entities, 6 were identified with known species.The 11 others had affinities with existing species and appeared to be new to science.Two of these, G. ryvardense and G. lobenense have been described as a new species (Kinge, 2012;Kinge and Mih, 2014).

Disease symptoms of Ganoderma on different hosts
Symptoms of Ganoderma disease was found on different hosts in the study area.This was evident with the presence of basidiocarp at the base of the stem in some cases.In oil palm, external symptoms observed included a one sided yellowing, or mottling of the lower fronds, followed by necrosis.The newly unfolded leaves were shorter than normal and chlorotic, and additionally the tips were necrotic in some plants.Also, with the progression of the disease within the plant, an overall pale appearance, with retarded growth was noticed and the spear leaves remain unopened.
Dead desiccated fronds droop at the point of attachment to the trunk or fracture at some point along the rachis and hang down to form a skirt of dead leaves.There is also the creation of bole at the base of the trunk, after which the palm breaks and falls over.In Cassia sp., wilting of the leaves was observed followed by yellowing of the leaves, defoliation and finally dieback was observed in the crown.In Acacia sp., there was wilting of the leaves, death branches and finally death of the tree.Only basidiocarp formation was observed on Pinus sylvestris, Avocado sp. and several unidentified hardwood (Figure 2).Ganoderma sp. 8 Unidentified hardwood Ganoderma sp. 5, Ganoderma sp. 2, Ganoderma sp. 4, Ganoderma sp.6, Ganoderma sp. 7, Ganoderma sp. 8, Ganoderma sp. 9

Natural range and distribution of species of Ganoderma
The plant species that were host to various species of Ganoderma are shown on Table 1.Of the 17 species obtained from the study area, eight were restricted to oil palm and nine species to ornamentals and forest trees.The species were well distributed except for species like G. lobenense, G. weberianum and Ganoderma species 1-9 with different affinities which were restricted to specific locations (Figure 3).There were six species reported in literature of which just one species, Ganoderma tornatum, was found in the present study.

Checklist of species of Ganoderma in Cameroon
Apart from the 17 species of Ganoderma collected from the study area, a review of the literature identified 8 other species from Cameroon, giving a total of 25 species of Ganoderma in Cameroon (Table 2).

DISCUSSION
The present study shows that 17 species of Ganoderma can be discerned based on comparative morphology.Comparative morphology remains the cheapest and most available tool for identification in the developing economies.This is evident with many authors who have used comparative morphology to study the taxonomy of Ganoderma from Cameroon.For example, Turner (1981) reported the occurrence of G. tornatum var tornatum from Cameroon but without specifying the locality.Nunez and Daniels  Species are annotated by: + = New species, -= known species and # = new records, NS = not stated.
the taxonomic status of species of Ganoderma in Cameroon, it is confirmed that till date, 25 valid species, some with affinities have been reported from Cameroon of which 17 species are reported in the present study.
The work facilitates the understanding of species diversity of Ganoderma from Cameroon.Due to the high variability of morphological characters, the genus Ganoderma has been described as a fairly character poor genus by Moncalvo and Ryvarden (1997) and Douanla-Meli and Langer (2009).
As per the study, E. guineensis, Cassia sp., unidentified hardwood, Acacia sp. and P. sylvestris were found to be most susceptible hosts to species of Ganoderma.Oil palms of the CDC plantation were the most susceptible and showed high incidence of infection causing threat to the plantation.Seven species of Ganoderma (G. ryvardense, G. lobenense, G. tornatum, G. chalceum, G. steyaertanum, G. zonatum and Ganoderma sp. 3) were found associated with basal stem rot disease of oil palm.Six species out of the seven species were found to be host specific and found only in association with oil palm while G. ryvardense had dual host distribution because it was found to be pathogenic on oil palm and Cassia sp.Other species of Ganoderma encountered in the study area on landscape plants such as (Acacia sp., Cassia sp., P. sylvestris), unidentified hardwood, and forest trees are G. cupreum, G. weberianum and Ganoderma species with different affinities.
Species of Ganoderma have traditionally been reported to be a problem on oil palm and thus of economic importance to agriculture (Utomo and Niepold, 2000;Utomo et al., 2005).The present study showed that members of this genus can be of significant importance in horticulture, infecting landscape plants (Pinus sp., Acacia sp., Cassia sp.) and fruit trees (Avocado).They can also be of importance in forestry.Whereas some species were host-specific; others attached more than one host.This showed that the diseases caused by Ganoderma could rise to epidemic proportions as was observed in Mungo.Our study and observations strongly suggested that there is a dearth of information on Ganoderma species diversity and distribution in Cameroon, emphasizing the point made by Douanla-Meli and Langer (2009) about the poor state of knowledge on macrofungi in Cameroon.The checklist presented in this study contains only those Ganoderma taxa for which a reasonably confident identification has been obtained.G. tornatum and G. chalceum are known records for Cameroon.Ganoderma weberianum, Ganoderma cupreum, Ganoderma steyaertanum and Ganoderma zonatum are new records for Cameroon.Ganoderma ryvardense, Ganoderma lobenense, and Ganoderma species 1-9 with different affinities might be new to science and some are supported with molecular data.
The distribution of some species of Ganoderma at certain sites and hosts and not in others may be due to dispersal, if spores have just begun and they have not had enough time to expand their range into other habitats.It might also be because the species are habitat specific or because the viability of their spore is short.The species were well distributed except for species like viz.G. lobenense, G. weberianum and Ganoderma species with different affinities which are restricted to specific locations and hosts.The abundance of certain species such as G. ryvardense might be due to the fact that spores have acquired the capacity to remain dormant during unfavourable periods.Symptoms of basal stem rot disease on different hosts were evident on Cassia sp., Acacia sp., Avocado sp., E. guineensis, Pinus sylvestris and many unidentified hardwood.Different species of Ganoderma have been reported as the causal agents for basal stem rot disease of oil palm in Malaysia, Indonesia, Papua New Guinea and Cameroon.The most widely reported fungi associated with root-rot disease of tropical Acacia are species of Ganoderma (Glen et al., 2006).In Papua New Guinea an unnamed Ganoderma species was associated with root and butt rot in a plantation of Acacia mangium (Arentz and Simpson, 1988) and a sporocarp of G. weberianum (Bres.& Henn.ex Sacc.)Steyaert was also collected from a decayed stump in this plantation.This shows that species of Ganoderma have diverse hosts.

Figure 1 .
Figure 1.Sampling sites for diversity studies of species of Ganoderma in south western Cameroon.

Figure 3 .
Figure 3. Distribution map of species of Ganoderma in south western Cameroon.

(
1999) identified Ganoderma hildebrandii, G. lucidum and G. cf.multiplicatum from the Dja biosphere reserve and recently, Douanla-Meli (2007) described G. hildebrandii, G. lucidum and Ganoderma resinaceum and Ganoderma australe from the Mbalmayo forest reserve.This survey represents the first major investi-gation establishing a checklist of species of Ganoderma in Cameroon.A small number of collections have already been published, principally by different authors and are compared with the present study.We checked and studied a total of 57 collections representing 17 species of Ganoderma from the study area.Understanding

Table 2 .
Checklists of species of Ganoderma in Cameroon.