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Fungus-growing termites engage in an obligate mutualistic relationship with Termitomyces fungi, which they maintain in monocultures on specialised fungus comb structures, without apparent problems with infectious diseases. While other fungi have been reported in the symbiosis, detailed comb fungal community analyses have been lacking. Here we use culture-dependent and -independent methods to characterise fungus comb mycobiotas from three fungus-growing termite species (two genera). Internal Transcribed Spacer (ITS) gene analyses using 454 pyrosequencing and Illumina MiSeq showed that non-Termitomyces fungi were essentially absent in fungus combs, and that Termitomyces fungal crops are maintained in monocultures as heterokaryons with two or three abundant ITS variants in a single fungal strain. To explore whether the essential absence of other fungi within fungus combs is potentially due to the production of antifungal metabolites by Termitomyces or comb bacteria, we performed in vitro assays and found that both Termitomyces and chemical extracts of fungus comb material can inhibit potential fungal antagonists. Chemical analyses of fungus comb material point to a highly complex metabolome, including compounds with the potential to play roles in mediating these contaminant-free farming conditions in the termite symbiosis.

Fungus-growing termites (subfamily Macrotermitinae) mix plant forage with asexual spores of their plant-degrading fungal symbiont Termitomyces in their guts and deposit this blend in fungus comb structures, within which the plant matter is degraded. As Termitomyces grows, it produces nodules with asexual spores, which the termites feed on. Since all comb material passes through termite guts, it is inevitable that gut bacteria are also deposited in the comb, but it has remained unknown which bacteria are deposited and whether distinct comb bacterial communities are sustained. Using high-throughput sequencing of the 16S rRNA gene, we explored the bacterial community compositions of 33 fungus comb samples from four termite species (three genera) collected at four South African geographic locations in 2011 and 2013. We identified 33 bacterial phyla, with Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Candidate division TM7 jointly accounting for 92 % of the reads. Analyses of gut microbiotas from 25 of the 33 colonies showed that dominant fungus comb taxa originate from the termite gut. While gut communities were consistent between 2011 and 2013, comb community compositions shifted over time. These shifts did not appear to be due to changes in the taxa present, but rather due to differences in the relative abundances of primarily gut-derived bacteria within fungus combs. This indicates that fungus comb microbiotas are largely termite species-specific due to major contributions from gut deposits and also that environment affects which gut bacteria dominate comb communities at a given point in time.

Fungus-growing termites rely on symbiotic microorganisms to help break down plant material and to obtain nutrients. Their fungal cultivar, Termitomyces, is the main plant degrader and food source for the termites, while gut bacteria complement Termitomyces in the degradation of foodstuffs, fixation of nitrogen, and metabolism of amino acids and sugars. Due to the community complexity and because these typically anaerobic bacteria can rarely be cultured, little is known about the physiological capabilities of individual bacterial members of the gut communities and their associations with the termite host. The bacterium Trabulsiella odontotermitis is associated with fungus-growing termites, but this genus is generally understudied, with only two described species. Taking diverse approaches, we obtained a solid phylogenetic placement of T. odontotermitis among the Enterobacteriaceae, investigated the physiology and enzymatic profiles of T. odontotermitis isolates, determined the localization of the bacterium in the termite gut, compared draft genomes of two T. odontotermitis isolates to those of their close relatives, and examined the expression of genes relevant to host colonization and putative symbiont functions. Our findings support the hypothesis that T. odontotermitis is a facultative symbiont mainly located in the paunch compartment of the gut, with possible roles in carbohydrate metabolism and aflatoxin degradation, while displaying adaptations to association with the termite host, such as expressing genes for a type VI secretion system which has been demonstrated to assist bacterial competition, colonization, and survival within hosts., (Copyright © 2015, Sapountzis et al.)

The symbiotic relationship between termites and Termitomyces fungi, which allows the termite to digest cellulose-rich food sources, is poorly understood. In this study, in vitro mixed symbiotic relationships between Termitomyces clypeatus and fungi isolated from individual fungus-comb communities using a culture-dependent method were analyzed. Twenty-day-old stalk cultures of three T. clypeatus isolates were co-cultured with cellulase-producing fungi on potato dextrose agar. The high cellulase-producing fungal isolate no. 18, which showed 99 % ITS sequence identity to Sordariomycetes endophyte isolate 2171 (EU687039), increased growth of T. clypeatus 18/50 by 85.7 %. The high xylanase-producing isolate no. 13, which showed 88 % ITS sequence identity to Arthrinium sacchari isolate L06 (HQ115662), stimulated T. clypeatus 18/50 growth by 58.6 %. The high cellulase- and xylanase-producing isolate no. 50, which showed 90 % ITS sequence identity to the fungal endophyte isolate 2196 (EU687056), improved T. clypeatus 18/50 growth by 45.7 %. A Gigantropanus sp. promoted the growth of T. clypeatus 18/50 and 20/50 by 45.7 and 44.1 %, respectively, and that of T. clypeatus 19/50 by 10.6 %. These results indicated the most beneficial potential partnership of T. clypeatus might involve cellulase-producing fungi isolated from the same ecological niche. The Gigantropanus sp. is a potential partner of T. clypeatus but is likely to be less common than cellulase-producing fungi isolated from fungus combs owing to the lower host specificity of the Gigantropanus sp. This study provides an interesting method to culture Termitomyces using an in vitro mixed culture method for production of Termitomyces fruiting bodies in the future.

The microbial communities harbored in the gut and fungus comb of the fungus-growing termite Odontotermes formosanus were analyzed by both culture-dependent and culture-independent methods to better understand the community structure of their microflora. The microorganisms detected by denaturing gradient gel electrophoresis (DGGE), clonal selection, and culture-dependent methods were hypothesized to contribute to cellulose-hemicellulose hydrolysis, gut fermentation, nutrient production, the breakdown of the fungus comb and the initiation of the growth of the symbiotic fungus Termitomyces. The predominant bacterial cultivars isolated by the cultural approach belonged to the genus Bacillus (Phylum Firmicutes). Apart from their function in lignocellulosic degradation, the Bacillus isolates suppressed the growth of the microfungus Trichoderma harzianum (genus Hypocrea), which grew voraciously on the fungus comb in the absence of termites but grew in harmony with the symbiotic fungus Termitomyces. The in vitro studies suggested that the Bacillus sp. may function as mutualists in the termite-gut-fungus-comb microbial ecosystem., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Objective: Two models of domestication and cultivation of termite-mushroom were discussed: the cultivation of termitomyces model, which method of woodrotting fungi cultivation was emphasized and the original ecological model, which multiplication of symbiotic termites was focused. The problems and possible solutions during termite-mushroom cultivation were also discussed.

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.

It is unclear how mutualistic relationships can be stable when partners disperse freely and have the possibility of forming associations with many alternative genotypes. Theory predicts that high symbiont relatedness should resolve this problem, but the mechanisms to enforce this have rarely been studied. We show that African fungus-growing termites propagate single variants of their Termitomyces symbiont, despite initiating cultures from genetically variable spores from the habitat. High inoculation density in the substrate followed by fusion among clonally related mycelia enhances the efficiency of spore production in proportion to strain frequency. This positive reinforcement results in an exclusive lifetime association of each host colony with a single fungal symbiont and hinders the evolution of cheating. Our findings explain why vertical symbiont transmission in fungus-growing termites is rare and evolutionarily derived.

Fungus-growing termites live in obligate mutualistic symbiosis with species of the basidiomycete genus Termitomyces, which are cultivated on a substrate of dead plant material. When the termite colony dies, or when nest material is incubated without termites in the laboratory, fruiting bodies of the ascomycete genus Xylaria appear and rapidly cover the fungus garden. This raises the question whether certain Xylaria species are specialised in occupying termite nests or whether they are just occasional visitors. We tested Xylaria specificity at four levels: (1) fungus-growing termites, (2) termite genera, (3) termite species, and (4) colonies. In South Africa, 108 colonies of eight termite species from three termite genera were sampled for Xylaria. Xylaria was isolated from 69% of the sampled nests and from 57% of the incubated fungus comb samples, confirming high prevalence. Phylogenetic analysis of the ITS region revealed 16 operational taxonomic units of Xylaria, indicating high levels of Xylaria species richness. Not much of this variation was explained by termite genus, species, or colony; thus, at level 2-4 the specificity is low. Analysis of the large subunit rDNA region, showed that all termite-associated Xylaria belong to a single clade, together with only three of the 26 non-termite-associated strains. Termite-associated Xylaria thus show specificity for fungus-growing termites (level 1). We did not find evidence for geographic or temporal structuring in these Xylaria phylogenies. Based on our results, we conclude that termite-associated Xylaria are specific for fungus-growing termites, without having specificity for lower taxonomic levels.