African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5233

Full Length Research Paper

Fungal diversity and community structure in gut, mound and surrounding soil of fungus-cultivating termites

Huxley Mae Makonde
  • Huxley Mae Makonde
  • Department of Microorganisms, Leibniz-Institute DSMZ (German Collection of Microorganisms and Cell Cultures), Braunschweig, Germany.
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Romano Mwirichia
  • Romano Mwirichia
  • Department of Biological Sciences, Embu University, Embu, Kenya.
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Edith Mnyazi Muwawa
  • Edith Mnyazi Muwawa
  • Department of Biological Sciences, Pwani University, Kilifi, Kenya.
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Hans-Peter Klenk
  • Hans-Peter Klenk
  • School of Biology, Newcastle University, Newcastle upon Tyne, UK.
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Hamadi Iddi Boga
  • Hamadi Iddi Boga
  • School of Agriculture, Earth and Environmental Sciences, Taita Taveta University, Voi, Kenya.
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  •  Received: 13 February 2017
  •  Accepted: 09 March 2017
  •  Published: 28 March 2017

 ABSTRACT

The fungus-cultivating termites (Macrotermitinae) form part of diverse termite fauna in Africa, but information on their fungal symbionts is inadequate and poorly understood. In this study, the fungal communities and structure between termite gut, mound and surrounding soil were determined using the 454 pyrosequencing-based analysis of the internal transcribed spacer (ITS) gene sequences. Genomic DNA was extracted and purified from the guts of three termites (Odontotermes sp., Macrotermes michaelseni and Microtermes sp.), mound and surrounding soil samples for high-throughput sequencing. A total of 15,256 sequences were obtained and individual samples contained between 4 and 133 operational taxonomic units (OTUs). Termite gut had the least fungal diversity, dominated by members of the Basidiomycota (> 98%). More than 98% of the gut sequences were of the genus Termitomyces, while < 2% were related to the genera Chaetomium, Fusarium, Eupenicillium, Cladosporium, Curreya and Phaeosphaeria with between 95 and 98% pair-wise sequence identities.  Members of Ascomycota (> 94%) were the most abundant in the mound and soil, but significantly differed (P value of 0.04; R value = 0.909) between the mound and soil environments. The results confirm that the genus Termitomyces exist in a tight association with their hosts and that Termitomyces species are scarcely present in the mound and soil. In addition, by altering soil properties; the fungus-cultivating termites modify the fungal community composition and structure in the mound and surrounding soil environments.

 

Key words: 454-pyrosequencing, microtermitinae, mutualism, tropical mycology.


 INTRODUCTION

The diverse and numerous microorganisms in the soil perform  key    functions    within    the    environment   by participating in the cycling and flux of various nutrients, thereby influencing structure formation and sustenance of soil properties (Holt and Lepage, 2000; Harry et al., 2001). Termites, a group of social insects consisting of over 2 600 species worldwide (Ahmed et al., 2011), are part of soil organisms that influence soil properties (Holt and Lepage, 2000; Harry et al., 2001; Manuwa, 2009; Muwawa et al., 2014). They are known as “soil engineers” as they have a great influence on the soil characteristics (Holt and Lepage, 2000), hence controlling diversity and activity of other soil organisms (Jones et al., 1997; Lavelle et al., 1997). Their influence on the soil microbial component is as a result of their major construction activities of complex galleries and mounds that result into soil heterogeneity in the tropical regions (De Bruyn and Conacher, 1990; Holt and Lepage, 2000). The termite mound, thus, forms a specific habitat for soil microbiota since the physical and chemical properties are different from the surrounding soil (De Bruyn and Conacher, 1990; Holt and Lepage, 2000). The type of mound construction depends on the feeding habit of the termite species (Holt and Lepage, 2000). The fungus-growing termites build their mounds using soil and clay cemented by salivary secretions that make the mounds enriched with clay particles but impoverished in carbon (Harry et al., 2001). The nest-walls consist of organo-mineral aggregates, characterized by a low stability hence mineralize easily (Garnier-Sillam et al., 1988). They have a wider range of activity on the surrounding soil of 1 to 3 m in depth and within a range of a 2 to 8 m (Harry et al., 2001), which may influence the soil properties and fertility. The question is whether the fungus-feeding termites can be regarded as metabionts (Waid, 1999).
 
The Macrotermitinae comprises of the economically important termite species (Ahmed et al., 2011) that have been comprehensively studied (Mathew et al., 2012; Makonde et al., 2013; Otani et al., 2014, 2015; Muwawa et al., 2016). Previous studies have focused on the mutualistic symbiosis between Termitomyces sp. (Basidiomycota) and fungus-growing termites (Mohindra and Mukerji, 1982; Zoberi and Grace, 1990; Aanen et al., 2007, 2009; Osiemo et al., 2010; Nobre et al., 2010, 2011), parasitic fungi for termites (Traniello et al., 2002) and saprotrophic fungi such as Xylaria species that colonize termite nests (William, 1969; Moriya et al., 2005). Despite the termite activities influencing the microbial diversity and community structure, there is little information on comparative fungal community composition between termite gut, mound and corresponding soil environments. Therefore, in this study, we conducted a 454 pyrosequencing-based analysis of the ITS gene sequences to evaluate the gut fungal diversity associated with three fungus-cultivating termites. In addition, we evaluated on how, by altering soil properties; the fungus-cultivating termites modify the fungal community composition and structure in the mound and surrounding soil environments.


 MATERIALS AND METHODS

Study sites and sampling 
 
The samples were collected from Juja in Kiambu County, Kenya (latitude 1° 5' 54.68'' N, longitude 37° 1' 1.10'' W). The Odontotermes sp. (OTG1) [JQ247986] belonging to mound C, Macrotermes michaelseni (MTG4) [JQ247993] and Microtermes sp. (MIG7) [JQ247990] both colonizing mound D (~2 km far away from mound C) were sampled by excavating each mound to a depth of approximately 1.0 m and aseptically collecting the termites (n = 200 workers and 50 soldiers). Worker-caste termites were used in the experiments due to their foraging behavior. The identity of the termites was confirmed by sequencing the mitochondria cytochrome oxidase II gene in DNA extracted from the heads of soldiers (Austin et al., 2004) and comparing it to the sequences of previously identified specimens (Inward et al., 2007). In addition, soil samples (~40 g collected at ~5 cm depth) from termite mounds (OTN2 and MTN5) and surrounding soil samples (OTS3 and MTS6, collected at 3 m away from termite mounds C and D, respectively) were included in the analyses.
 
DNA extraction
 
DNA extraction was performed as described previously (Makonde et al., 2013). Briefly, the exterior surfaces of the termites were washed with 70% ethanol and then rinsed with sterile distilled water. The guts were aseptically removed with forceps. A total of 165 guts (~1 g) of the Odontotermes sp. (OTG1) and M. michaelseni (MTG4) and 198 guts (~1g) of Microtermes sp. (MIG7) were separately put into sterile micro tubes containing 0.5 ml of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). They were then homogenized using a sterile glass rod. The corresponding homogenates were then transferred into sterile tubes and used for total DNA extraction. The soil samples were homogenized separately and debris were removed. Subsequently, soil samples (~4 g) were used for total microbial DNA extraction. Total DNA extraction for all samples was performed using MoBio PowerMax Soil DNA isolation kit (MoBio Laboratories, Inc. CA, USA) according to the manufacturer’s protocol. DNA concentration was quantified using a NanoDrop Spectrophotometer (NanoDrop Technologies, USA) as recommended by the manufacturer.
 
Amplification of internal transcribed spacer (ITS) gene region and sequencing
 
The fungal DNA was PCR amplified using a set of the universal ITS gene primers (the ITS1 [5’-TCCGTAGGTGAACCTGCGG-3’] and ITS4 [5’-TCCTCCGCTTATTGATATGC-3’]) according to White et al.
 
 (1990). These fungal primer set were modified for 454 pyrosequencing by attaching an adaptor sequence, a key and a unique 12 Nucleotide MID for multiplexing purposes. Each PCR reaction (50 μL) contained forward and reverse primers (10 μM, each), dNTP’s (10 mM each), Phusion GC buffer (Finzymes), Phusion high fidelity polymerase (0.5 U μL-1) and 25 ng of template DNA. Amplifications occurred in an Eppendorf Mastercycler Thermal Cycler with the following program conditions: An initial heating at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s and extension at 72°C for 1 min, after which a final extension step at 72°C for 5 min was performed. The amplification was confirmed using gel electrophoresis of 2 µl of the PCR product on a 1% TAE agarose gel (40 mM Tris base, 20 mM glacial acetic acid, 1 mM EDTA, 1.5% (w/v) for 1 h at 100 V. Later three independent PCR products per sample were pooled in equal amounts, separated on a gel and extracted using the peqGOLD gel extraction kit (PeqLab Biotechnologie GmbH, Erlangen, Germany). Quantification of the PCR products was performed by using the Nanodrop (NanoDrop Technologies, USA) method and a Qubit fluorometer mbH, (Invitrogen GmbH Karlsruhe, Germany) as recommended by the manufacturer. Sequencing of the PCR amplicons was done at the Göttingen Genomics Laboratory using Roche GS-FLX 454 pyrosequencer (Roche, Mannheim, Germany) as recommended in the instructions of the manufacturer for amplicon sequencing.
 
Data analysis
 
Raw sequences were quality filtered according to Huse et al. (2007) using the QIIME release 1.9.0 software (Caporaso et al., 2010). Briefly, low quality sequences were removed from the analysis if they were less than 200 bp in length, contained ambiguous characters, did not contain the primer sequence or contained an uncorrectable barcode. The remaining sequences were assigned to samples based on the 12-nucleotide barcode. The denoised sequences were evaluated for potential chimeric sequences using UCHIME in the USEARCH package v.4.2.66 (Edgar, 2010). A sequence identity cutoff of 97% was used to pick OTUs from the quality filtered non-chimeric sequences. Representative OTUs were picked using the de novo OUT clustering (Rideout et al., 2014) with standard UCLUST method using the default settings as implemented in QIIME at 97% similarity level. OTU alignment was performed using the python implementation of the NAS algorithm, PyNAST (Caporaso et al., 2010). Taxonomy was assigned to representative sequences from each cluster using BLASTn against the SILVA SSU Reference 119 database at default e-value threshold of 0.001 in QIIME (Quast et al., 2013) at dissimilarity levels of 3, 5 and 10%. Rarefied datasets were generated with the multiple_rarefaction function in QIIME in order to remove sample heterogeneity before diversity assessment. Rarefaction curves and diversity indices were calculated and plotted for each sample using QIIME (Caporaso et al., 2010). To determine the amount of dissimilarity (distance) between any pair of bacterial communities, we used the UniFrac metric (Lozupone and Knight, 2005; Lozupone et al., 2007) that incorporates the degree of divergence in the phylogenetic tree of OTUs into Principal coordinates analysis (PCoA). A relatively small UniFrac distance implies that two communities are compositionally similar, harboring lineages sharing a common evolutionary history. In unweighted UniFrac, only the presence or absence of lineages is considered. We used the analysis of similarities (ANOSIM) (Clarke, 1993; Fierer et al., 2010) through 1000 to test for differences in community composition among the groups of samples. Additionally, the relative abundance of the genera was used in hierarchical clustering using the pearson correlation distance  metric  implemented in  MultiExperimentViewer version 4.9.0 (MeV 4.9.0). Fungal communities across the analyzed samples were compared based on the relative abundances of some selected fungal genera, using principal component analysis (PCA) as implemented in R (R Core Team, 2012). All pyrosequencing-derived ITS gene sequences datasets were deposited in the GenBank under accession number SRP019764.


 RESULTS

Distribution of Fungal phyla across the samples
 
The overall reads for the fungal samples were 18,294. After quality filtering and chimera check 15,256, the resulting sequences (≥ 200 bp) were clustered into 287 OTUs (Table 1) at 3% sequence divergence. Taxonomic assignment of the resulting sequences against the SILVA database showed ≥ 2 known phyla, but the major ones (> 90% of the analyzed sequences) were Ascomycota and Basidiomycota (Figure 1).
 
 
 
Fungal community composition across samples
 
The abundance of fungal composition at the phylum level differed across the samples (Figure 1; Table 1). Members of the phylum Basidiomycota were the most abundant (>98% of the analyzed sequences) in the gut samples [MIG7, MTG4 and OTG1] compared to those of mound [sample OTN2] and soil environments [samples OTS3 andMTS6], which were predominated by members of the phylum Ascomycota [>94% of the analyzed sequences] (Figure 1). There were no sequences for sample MTN5 due to some sequencing errors. At the class level, members affiliated with Agaricomycetes were the most abundant (>98%) in the gut samples [MIG7, MTG4 and OTG1], but least in the mound (OTN2) and soil (OTS3 and MTS6) samples (Table 2). Members of Sordariomycetes and Eurotiomycetes were the most abundant in the mound (89%) and soil (54-68%) samples, respectively. Other classes such as Dothideomycetes (4.5%), Eurotiomycetes (3.6%) and uncultured ascomycete (1.1%) were relatively abundant in the mound, while classes such as Sordariomycetes (≥12%), Dothideomycetes and Orbiliomycetes (>5%) were relatively abundant in the soil (Table 2).
 
 
At the order level, the relative abundances of the fungal communities in the samples were different. The order Agaricomycetidae was the most abundant group in the termite gut. Notably, the mound was dominated by the order Hypocreomycetidae while the soil was predominated by members of the order Eurotiomycetidae (Figure 2). Other orders such as Dothideomycetidae, Pleosporomycetidae, Chaetothyriomycetidae, Sordariomycetidae, Xylariomycetidae and Orbiliales were detected at varying relative abundances (1 to 22% of the analyzed sequences) in some samples (Figure 2). 
 
 
At the  genus level, the most abundant genus in the gut samples was Termitomyces (>98%), but was detected at low relative abundance (< 0.4%) in the mound and soil samples (Table 3). Notably, fungal species associated with Eupenicillium limosum, Monilinia fructicola and Fusarium oxysporum (with 96 to 98% sequence identities) were detected in the gut of Odontotermes sp. (sample OTG1). These fungi, however, constituted about 0.1% of the analyzed sequences. Likewise, in the gut of Microtermes sp. (sample MIG7), about 0.1% of the sequences were related to Trichocoma paradoxa and Cladosporium sp. CF-25 (with 96 to 98% sequence identities). The gut of M. michaelseni (sample MTG4), had about 1.3% of the analyzed sequences affiliated with Chaetomium globosum, Myrothecium sp. J3, Monodictys castaneae, Fusarium oxysporum, Penicillium purpurogenum, Cladosporium sp. CF-25, Phaeosphaeria avenaria and Curreya pityophila [with 96 to 99% sequence identities] (Table 3).
 
 
The genus Fusarium [17%] and particularly the genus Hypocrea [59%] were the most abundant genera in the mound (Table 3), but the soil samples  were   predominated   by   the   following genera; Aspergillus [45%], Eupenicillium [39%] and Xylaria [19%]. In the mound (OTN2), most of the fungal species were affiliated with H. koningii, Fusarium sp. CPCC 1400009 and C. globosum (with 97 to 99% sequence identities) while in the surrounding soil, the fungal species were between 97 and 98% affiliated with E. limosum, A. fumigatus, Xylaria hypoxylon and Hypocrea koningii (Table 3).
 
Fungal diversity and richness
 
Fungal diversity and richness for the analyzed sequences for each sample (Table 1) were evaluated by rarefaction (Figure not shown). At 3% sequence divergence, some rarefaction curves did not reached saturation, indicating that the surveying efforts did not fully cover the extent of taxonomic diversity at this genetic distance, but a substantial fraction of the fungal diversity within individual samples was evaluated. The diversity measures showed that MTS6 had the most genus-level taxa (133; Table 1) and MIG7 the least (4;  Table 1), that MTS6 was richest (Chao 1 index), while MIG7 was poorest. There was variation in community composition as indicated by the Simpson (1/D) and Shannon indices (Table 1).
 
Comparison of the individual samples using unweighted UniFrac PCoA (Figure 3) showed a distinct clustering by environment, but the p-value of 0.04 and R value of 0.909 indicated that at an alpha of 0.05; the grouping of samples is relative strong implying that there is dissimilarity between the groups. For instance, the gut samples (MIG7, OTG1 and MTG4) did not cluster together and with those of mound and soil (Figure 3 and 4), indicating dissimilarity in the fungal communities. Likewise, samples OTS3, MTS6 and OTN2 did not cluster together, indicating that each individual sample had almost different fungal communities. Notably, the mound sample (OTN2) did not cluster with its corresponding soil sample (OTS3), implying that the mound fungal community composition was different from that of its surrounding soil (Figures 3 and 4).
 
 
 
The PCA shows that the fungal communities within the termite gut are mainly impacted by the genus  Termitomyces  while  those  of  the mound are impacted by the genera Fusarium and Hypocrea (Figure 5). The surrounding soil is impacted by the genera Xylaria, Aspergillus and Eupenicillium. Their relative abundances varied across the samples (Table 3). On one hand, Termitomyces species were the most dominant fungal species in the gut of three fungus cultivating termites (M. michaelseni, Odontotermes and Microtermes species), while members of the genera Fusarium and Hypocrea were more dominant in the mound. On the other hand, members of the genera Xylaria, Aspergillus and Eupenicillium were more predominant in savannah soil (Table 3 and Figure 4).
 


 DISCUSSION

Defining the number of fungi on the planet has always been an area of debate (Hawksworth, 2001), but has recently gained prominence in scientific literature. This has provided the foundation for studies aimed at obtaining  a  better  understanding  of  fungal  biodiversity worldwide. Termites and their mounds harbor diverse organisms including fungi. There are two aspects of fungal diversity on termite mounds, namely, the presence of Termitomyces versus other fungi such as Xylaria/ or Pseudoxylaria species (Moriya et al., 2005; Okane and Nakagiri, 2007; Ju and Hsieh, 2007; Guedegbe et al., 2009; Visser et al., 2009; 2012) and the diversity within Termitomyces species.
 
Our study compared the fungal diversity and community structure in the termite gut, mound and surrounding soil. The results of our study revealed two major fungal phyla; Ascomycota and Basidiomycota whose members’ distribution differed significantly across the samples. The phylum Basidiomycota was the most abundant in the termite gut while the phylum Ascomycota dominated in the mound and surrounding soil. Furthermore, members of Ascomycota differed significantly between the mound and surrounding soil (Table 3 and Figures 2 and 4). The discrepancy of fungal composition between the mound and  soil  may  emanate  from  the  construction  activities of the termites. Such activities can chemically modify the organic matter in the mound (Holt and Lepage, 2000; Harry et al., 2001), hence creating ecological microniches suitable for more specialized fungi.
 
The most abundant fungal genus in the termite gut was Termitomyces, which was represented by over 98% of the analyzed sequences in each gut sample (Table 3). Notably, the gut Termitomyces symbiont differed in the host. For instance, an interesting scenario was noted in mound D, which was inhabited by two different termite species. Each termite species (M. michaelseni vs. Microtermes sp.) cultivated its own Termitomyces strain (Table 3). Since the Macrotermes and Microtermes termites colonized the lower and upper parts, respectively, the likelihood of horizontal transfer of the fungus should have been high as suggested previously (Makonde et al., 2013). But this was not the case; thus, the affected host-Termitomyces relationships are likely  to be too specialized to allow host switching. Literature indicates that some termite genera cultivate a restrictive set of fungal symbionts (Aanen et al., 2007; Osiemo et al., 2010). Nonetheless, it remains to be addressed how the termites exclusively select the right Termitomyces symbiont for their colony. The mound and soil samples (OTS3, MTS6 and OTN2) did not cluster together, indicating that each individual sample had almost different fungal communities.
 
Besides, the 454-pyrosequencing approach used in this study revealed other minor fungal species, which were previously often undetected by the traditional Sanger sequencing in the termite gut (Mathew et al., 2012; Makonde et al., 2013). This is because the use of the clone-based approach for microbial analyses in the previously mentioned studies could have been limited by PCR errors and  bias  in  selecting  representative  clones for sequencing. In this current study, fungal species affiliated with E. limosum, M. fructicola and F. oxysporum were detected in the gut of Odontotermes species (Table 3). This, however, constituted about 0.1% of the effective sequences just like in the gut of Microtermes species, where 0.1% of the sequences were associated with T. paradoxa and Cladosporium sp. CF-25 (with 96 to 98% sequence identities). In the gut of M. michaelseni, about 1.4% of the sequences were related to Chaetomium globosum, Myrothecium sp. J3, M. castaneae, F. oxysporum, P. purpurogenum, Cladosporium sp. CF-25, P. avenaria and C. pityophila (with 96 to 99% sequence identities). Elsewhere, Mathew et al. (2012) isolated yeasts closely related to Debaryomyces hansenii, Pichia guilliermondii and Candida inconspicua from the comb material and gut of Odontotermes formosanus using adapted cultivation techniques. However, these fungi/yeasts were detected insignificantly in quantitative terms, and it is unclear which role they play. In contrast to our  findings,   some   previous  studies  conducted  using clone-based approach (Mathew et al., 2012) could not identify fungal genera in some fungus-cultivating termites’ guts other than Termitomyces.
 
Though the results of this study do not support the physiological roles of the symbiotic fungi detected, several researchers have proposed roles associated with symbiotic fungi (Termitomyces species) in termites. For example, provision of glycosyl hydrolases (Martin and Martin, 1978), enrichment of nitrogen, which is advantageous as the dead plant material consumed by termites, is poor in nitrogen (Collins, 1983), and lignin degradation, which subsequently allow for cellulose digestion (Hyodo et al., 2000). Nonetheless, the significance of each role differs in value among termite species (Rouland-Lefévre, 2000; Hyodo et al., 2003). Hyodo et al. (2003) suggested that the important role of symbiotic fungi in Macrotermes species is to degrade lignin, hence allowing for efficient digestion of cellulose, whereas for Odontotermes species, Hypotermes makhamensis  and   Phidiana   militaris,  it  is  to  serve  a
 
nutritional role. However, it is still unclear whether the different roles of such fungi are directly dependent on termite taxonomy or variation in plant biomass used to make fungus comb (Hyodo et al., 2003).
 
The genus Fusarium and particularly the genus Hypocrea were the most abundant genera in the mound compared to the soil, which was dominated by the genus Aspergillus among others (Eupenicillium, Xylaria and Hypocrea). On one hand, the transformed soil properties in  the   mound  might  have  favored  the  proliferation  of particular fungi; especially those related to the genera Fusarium and Hypocrea. On the other hand, it might have limited the growth of other genera such as Aspergillus, Xylaria and Eupenicillium, which were mostly favored by conditions in the surrounding soil. Thus, the soil harbored a higher diversity of fungi most of which were different from those of the mound and gut. The differences in fungal communities between the mound and surrounding soil may partly be attributed by influence of the fungus- cultivating    termites    on    the    soil   properties,   which consequently modify the diversity and composition of fungal communities. Previously, Chen and Cairney (2002) demonstrated that perturbation of Australian forest soils affected the fungal composition while Landeweert et al. (2003) observed difference in basidiomycete community between the organic and mineral horizons. The activities of other biota can modify soil properties and might be the same factor affecting fungal diversity within the same region and vegetation type (McLean and Parkinson, 2000).
 
The current findings underline the difference on fungal community composition between the gut, mound and surrounding soil. The heterogeneity of the organic matter, occurrence of fungal inhibitors (Chen and Cairney 2002; Lamberty et al., 2001) and the creation of new substrates/or reduced access for fungi in such clay organic complexes could favor some specialized fungal species (Roose-Amsaleg et al., 2004). As a result, fungus-cultivating termites could be regarded, according to Waid (1999), as true metabionts since they create special micro-environments that support specific organisms such as fungi that may adapt, evolve and hence diversify. Such a scenario has been observed on soil-feeding termites; which by modifying the environment drastically   affected   the   soil    ascomycete   community
structure (Roose-Amsaleg et al., 2004).
 
Soil fungi mediate many biochemical interactions (Bridge and Spooner, 2001) including a variety of associations with plants as pathogens (e.g Fusarium spp.), while other genera such as Aspergillus, Penicillium and Xylaria could be saprophytes, necrophilia and even coprophile. Several species of fungi associated with Reticulitermes flavipes have been isolated (Zoberi and Grace, 1990), many of which were common saprophytic soil organisms (Barnett and Hunter, 1972). Mucor mucedo (L.) Fr. and Aspergillus niger Van Tieg. (Steinhaus, 1949) are known to be facultative insect pathogens while Mucor hiemalis Weh., was reported as a pathogen of bees. It is worthy of note that the multi-species fungal interactions such as competitive or parasitic interactions (Zoberi and Grace, 1990) among fungi promote termite survival as supported by a number of species associated with living termites. 


 CONCLUSION

The findings of this study have demonstrated that members of the genus Termitomyces exist in a tight association  with  their   hosts   (Rouland-Lefevre,  2000), hence Termitomyces species are scarcely present in the mound and soil. In addition, by altering the habitat, fungus-cultivating termites create microecological niches suitable for some specialized soil fungal species. The use of 454-pyrosequencing has demonstrated the existence of other minor fungal genera in the termite guts other than Termitomyces, which is the dominant fungus. This demonstrates that the mutualistic association of the Termitomyces with termites is important for their survival. Therefore, further studies should be focused on host-symbiont specificity and physiological roles of the host symbionts for further exploitation particularly in the field of biotechnology. 


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



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