Symbiotic fungi produce laccases potentially involved in phenol degradation in fungus combs of fungus-growing termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

培菌白蚁菌圃微生物降解木质纤维素的研究进展

白蚁及其共生微生物协同降解植物细胞壁的机理一直被世界各国科学家所关注。培菌白蚁作为高等白蚁,相比低等食木白蚁具有更多样化的食性,其利用外共生系统“菌圃”,对多种植物材料进行处理。本文综述了菌圃微生物降解木质纤维素的研究进展,以期为深入研究菌圃中木质纤维素降解过程及其机制,并挖掘利用菌圃降解木质纤维素的能力及仿生模拟菌圃开发新的生物质利用系统提供参考。培 菌白蚁在其巢内利用由植物材料修建的多孔海绵状结构——“菌圃”来培养共生真菌鸡枞菌Termitomyces spp.,形成了独特的木质纤维素食物降解和消化策略,使木质纤维素在培菌白蚁及其共生微生物协同作用下被逐步降解。幼年工蚁取食菌圃上的共生真菌菌丝组成的小白球和老年工蚁觅得食物并排出粪便堆积到菌圃上成为上层菌圃。这一过程中,被幼年工蚁取食的共生真菌释放木质素降解酶对包裹在植物多糖外部的木质素屏障进行解聚。菌圃微生物(包括共生真菌)对解聚的木质素基团进一步降解,将多糖长链或主链剪切成短链,使菌圃基质自下而上被逐步降解。最后下层的老熟菌圃被老年工蚁取食,其中肠的内源酶系及后肠微生物将这些短链进一步剪切和利用。因此,蚁巢菌圃及其微生物是培菌白蚁高效转化利用木质纤维素的基础。化学层面的研究表明,菌圃能够实现对植物次生物质解毒和植 物纤维化学结构解构。对共生真菌相关酶系的研究显示可能其在菌圃的植物纤维化学结构和植物次生物质的降解中发挥了作用,但不同属共生真菌间其效率和具体功能不尽相同。而菌圃中的细菌是否发挥了作用和哪些细菌类群发挥了作用等仍有待进一步的研究。相比于低等食木白蚁利用其后肠共生微生物降解木质纤维素,培菌白蚁利用菌圃降解木质纤维素具有非厌氧和能处理多种类型食物两大优势,仿生模拟菌圃降解木质纤维素的机制对林地表面枯枝落叶的资源化利用具有重要意义。     

Comparison of gut-associated and nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis)

Abstract The digestion of cellulose by fungus-growing termites involves a complex of different organisms, such as the termites themselves, fungi and bacteria. To further investigate the symbiotic relationships of fungus-growing termites, the microbial communities of the termite gut and fungus combs of Odontotermes yunnanensis were examined. The major fungus species was identified as Termitomyces sp. To compare the micro-organism diversity between the digestive tract of termites and fungus combs, four polymerase chain reaction clone libraries were created (two fungus-targeted internal transcribed spacer [ITS]– ribosomal DNA [rDNA] libraries and two bacteria-targeted 16S rDNA libraries), and one library of each type was produced for the host termite gut and the symbiotic fungus comb. Results of the fungal clone libraries revealed that only Termitomyces sp. was detected on the fungus comb; no non-Termitomyces fungi were detected. Meanwhile, the same fungus was also found in the termite gut. The bacterial clone libraries showed higher numbers and greater diversity of bacteria in the termite gut than in the fungus comb. Both bacterial clone libraries from the insect gut included Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, Nitrospira, Deferribacteres, and Fibrobacteres, whereas the bacterial clone libraries from the fungal comb only contained Firmicutes, Bacteroidetes, Proteobacteria, and Acidobacteris.     

Fungal partnerships stimulate growth of Termitomyces clypeatus stalk mycelium in vitro

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.     

培菌白蚁菌圃和粪便微生物多样性分析

【背景】培菌白蚁是属于白蚁科的一类与鸡枞菌属真菌共生的高等白蚁,其与体内肠道微生物和体外菌圃微生物形成三维共生体系。【目的】分析培菌白蚁菌圃和粪便的微生物多样性,并与肠道微生物进行比较。【方法】通过Illumina MiSeq高通量测序方法对培菌白蚁菌圃和粪便样品进行细菌16S rRNA基因和真菌ITS测序分析。【结果】高通量测序获得培菌白蚁菌圃和粪便样品细菌和真菌的有效序列和OTU数目。5个样品细菌OTU数目在90-199之间,而真菌OTU在10-58之间,细菌的种类多样性明显大于真菌。不论是细菌还是真菌,粪便样品的OTU数目多于菌圃样品。经物种分类分析,菌圃样品主要优势细菌是变形菌门(Proteobacteria),其相对含量超过82.4%;其次是拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes);粪便样品中优势细菌为拟杆菌门,其次是变形菌门,粪便优势菌属为别样杆菌属和营发酵单胞菌属,这与培菌白蚁肠道菌多样性组成一致。培菌白蚁菌圃和粪便样品共生真菌主要为担子菌门(Basidiomycota)和子囊菌门(Ascomycota)。菌圃优势真菌为鸡枞菌属(Termi...     

白蚁菌圃真菌多样性研究进展

白蚁菌圃存在于白蚁巢中,具有硬而脆的多孔结构,是特殊的真菌生存环境。当有白蚁在白蚁巢内活动时,蚁巢伞Termitomyces是菌圃上的优势菌;当白蚁巢被废弃,炭角菌Xylaria成为菌圃上的优势真菌。菌圃中还存在其他微生物如无性型真菌（anamorphic fungi）和酵母等。菌圃中的真菌很多具有潜在药用价值或其他经济价值。从蚁巢伞、炭角菌等主要真菌类群出发,结合分子生态学研究菌圃真菌多样性的方法,综述了白蚁菌圃真菌多样性的研究进展,揭示了目前的研究热点及存在的问题,并针对这些问题提出可能的发展方向。     

白蚁巢菌圃真菌多样性研究

培菌性白蚁能在存在于蚁巢或分散在其周围土壤中的菌圃上培养真菌。菌圃在无白蚁存在下培养会生长出炭角菌的子实体。对分别采集自我国西南四川、云南两省的4个土白蚁属菌圃采用原位培养法分离并纯化得到40株炭角菌,划分为13个形态型,ITS1-5.8S-ITS2序列分析确定为两种炭角菌。采用建立ITS基因文库的方法分析了白蚁菌圃真菌群落多样性,结果表明有白蚁存在的菌圃,蚁巢伞为单一优势菌;废弃的蚁巢中的菌圃,木霉、炭角菌等其他真菌成为优势菌。     

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.     

Phylogenetic relationships in Termitomyces (Family Agaricaceae) based on the nucleotide sequence of ITS: a first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi

Termitomyces constitutes a very poorly known genus of fungi whose essential characteristic is that all representatives of the genus are cultivated by termites (Macrotermitinae) in their nest and that all the fungi cultivated by termites belong to this genus. For the first time, the phylogenetic relationships of several African Termitomyces species was studied by the sequencing of their internal transcriber spacer region (ITS1--5.8S--ITS2). It appeared that this group is clearly monophyletic and belongs to the Tricholomataceae family. The total homology of the ITS zone of several Termitomyces symbionts of different termite genera indicated that the specific diversity of this group is in fact less important than previously supposed. Finally, the comparison between the Termitomyces phylogenetic tree and the taxonomic tree of Macrotermitinae showed that if for certain genera the hypothesis of termite/fungus coevolution is acceptable, it should not be applied for all symbiosis.     

培菌白蚁与鸡枞菌研究进展

培菌白蚁起源于非洲,蚁巢内具有复杂的社会分工.培菌白蚁依靠独特的蚁巢结构维持内部稳态和气体循环.菌圃是白蚁培育鸡枞菌的场所.鸡枞菌隶属于担子菌亚门,但其传播方式和生活史具有区别于其它担子菌的特点.鸡枞菌协助白蚁进行植物纤维的消化,白蚁则为鸡枞菌提供合适的生长环境,并控制鸡枞菌的遗传结构.培菌白蚁和鸡枞菌形成紧密的共生关...     

Yeasts and filamentous fungi carried by the gynes of leaf-cutting ants

Insect-associated microbes exhibit a wide range of interactions with their hosts. One example of such interactions is the insect-driven dispersal of microorganisms, which plays an essential role in the ecology of several microbes. To study dispersal of microorganisms by leaf-cutting ants (Formicidae: Attini), we applied culture-dependent methods to identify the filamentous fungi and yeasts found in two different body parts of leaf-cutting ant gynes: the exoskeleton and the infrabuccal pocket. The gynes use the latter structure to store a pellet of the ants’ symbiotic fungus during nest founding. Many filamentous fungi (n = 142) and yeasts (n = 19) were isolated from the gynes’ exoskeleton. In contrast, only seven filamentous fungi and three yeasts isolates were recovered from the infrabuccal pellets, suggesting an efficient mechanism utilized by the gynes to prevent contamination of the symbiotic fungus inoculum. The genus Cladosporium prevailed (78%) among filamentous fungi whereas Aureobasidium, Candida and Cryptococcus prevailed among yeasts associated with gynes. Interestingly, Escovopsis, a specialized fungal pathogen of the leaf-cutting ant-fungus symbiosis, was not isolated from the body parts or from infrabuccal pellets of any gynes sampled. Our results suggest that gynes of the leaf-cutter ants Atta laevigata and A. capiguara do not vertically transmit any particular species of yeasts or filamentous fungi during the foundation of a new nest. Instead, fungi found in association with gynes have a cosmopolitan distribution, suggesting they are probably acquired from the environment and passively dispersed during nest foundation. The possible role of these fungi for the attine ant–microbial symbiosis is discussed.     

Termite-regulated fungal monoculture in fungus combs of a macrotermitine termite Odontotermes formosanus

The mechanism of the exclusive growth of Termitomyces in fungus combs with fungi-growing termites, O. formosanus was examined using laboratory scale fungus combs. In the combs without the termites, vigorous growth of unidentified fungi was observed although no significant change was found in the case of the combs with termites. In addition, these results were reproducible even when incubated in a separated dish, suggesting that the physicochemical conditions were not the reason for the growth. With the molecular based analysis for the microbial communities in the combs, monoculture of the Termitomyces in the combs with termites was confirmed while the bacterial communities were independent either with or without termites. Possible mechanism of the exclusive growth of Termitomyces, such as the selective grazing of pathogenic fungi or contribution of antifungal activity giving actinomycetes were also discussed.     

Genetic variation of symbiotic fungi cultivated by the macrotermitine termite Odontotermes formosanus (Isoptera: Termitidae) in the Ryukyu Archipelago

Fungus-growing termites have a mutualistic relationship with their cultivated fungi. To improve understanding of genetic aspects of this relationship, we examined molecular markers in the fungus-growing termite Odontotermes formosanus and its fungi Termitomyces spp. from the Ryukyu Archipelago. Based on the polymorphic band patterns obtained from arbitrarily primed polymerase chain reaction methods, we constructed cladograms for related colonies of the termites and fungi. The resulting trees indicated that the termites display little genetic variation among the colonies, while the symbiotic fungi consist of two major genetic types. In addition, molecular phylogenetic trees of the symbiotic fungi based on internal transcribed spacer and 18S rDNA suggested that these two types of fungi are different species. We also demonstrated that the fungi comprising the fruiting bodies and fungus combs are identical, and that fungus combs are probably a monoculture within a single termite colony. Our results indicate that horizontal transmission of symbiotic fungi among termite colonies occurred during the evolutionary history of this symbiosis.     

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus-growing termite Macrotermes gilvus

The fungus-growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T-RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T-RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood-feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co-evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.     

云南杓兰菌根真菌组成及共生关系研究

杓兰属(Cypripedium)植物因具有较高的观赏和药用价值而长期被过度采集,已成为濒危植物。利用菌根技术进行杓兰属植物的保护和人工栽培,需要获得其可培养的菌根真菌。该研究采用分离培养法和共生回接方法,研究了云南杓兰菌根真菌菌群组成及其共生关系。结果表明:(1)从10株云南杓兰300块毛根组织中分离获得126株内生真菌,归属为3个菌属,分别是胶膜菌属(Tulasnella)73株、伏革菌属(Corticium)36株、角担菌属(Ceratobasidium)17株。其中,胶膜菌属为优势菌群,占总菌株数量的57.94%。(2)6株供试菌株中,4株菌株可显著缩短种子的萌发过程,6株菌株对幼苗的生长有显著的促进作用。(3)从中筛选获得一株CY-18高效促生真菌,对云南杓兰种子共生萌发和幼苗共生生长有极显著的促进作用。该研究结果为更好地利用菌根技术进行杓兰属植物资源的保护与可持续利用奠定了基础。     

Distribution patterns of four Termitomyces species cultivated by a fungus-growing termite,Odontotermes formosanus,in Taiwan

Fungus-growing termites inoculate the obligate symbiotic fungus Termitomyces into the fungus comb in their colonies. In this study, Taiwanese Termitomyces species were determined by diagnostic PCR using the metagenome of the body of Odontotermes formosanus (the only host termite living in Taiwan) as a template. Phylogenetic analyses revealed that four different Termitomyces species are cultivated by O. formosanus in Taiwan. Three have previously been registered in the DNA database, but one was first recorded in Taiwan. Only Termitomyces sp. Type C was distributed in all areas investigated in Taiwan, whereas the other three species were distributed regionally. Field observations indicated that the flush period and the number of fruit bodies in each colony varied between species. The distribution patterns of Termitomyces spp. in Taiwan may be related to the Taiwanese climate and/or the fruiting pattern.     

Distribution pattern of Termitomyces types symbiotic with the fungus‐growing termite Odontotermes formosanus on Okinawa Island

Fungus‐growing termites (subfamily Macrotermitinae) cultivate the symbiotic basidiomycete fungus Termitomyces in their fungus comb to digest cellulosic materials and to supply nitrogen‐rich fungal diet. In Japan, the fungus‐growing termite Odontotermes formosanus is found on the Yaeyama Islands and Okinawa Island, Okinawa Prefecture. Odontotermes formosanus is thought to have been recently and artificially introduced to Okinawa Island as its distribution is discontinuous and restricted to small areas. Previous DNA analyses revealed that two types of Termitomyces, namely Termitomyces sp. Type A and Termitomyces sp. Type B, whose fruiting bodies correspond to Termitomyces microcarpus‐like pseudorhiza‐lacking small mushroom and Termitomyces intermedius, respectively, are cultivated by O. formosanus on the Yaeyama Islands. However, information about the Termitomyces types cultivated by O. formosanus on Okinawa Island is limited. To define the fungal types cultivated by O. formosanus on Okinawa Island, I developed a diagnostic polymerase chain reaction method using primer sets specific to the nuclear ribosomal DNA sequences consisting of the internal transcribed spacers (ITS1 and ITS2) and 5.8S rDNA of Termitomyces not using fungal mycelium, but using the termite gut metagenome including fungal DNA as a template. The results indicated that the same two Termitomyces types from Iriomote Island are cultivated by O. formosanus on Okinawa Island. The distribution pattern of Termitomyces types on Okinawa Island showed that Termitomyces sp. Type A is limited to the mountainous side of Sueyoshi Park, despite Termitomyces sp. Type B being widely distributed in the area in which O. formosanus is found. This finding implies that O. formosanus on Okinawa Island was recently introduced from Iriomote Island to Sueyoshi Park.     

低等白蚁肠道共生微生物的多样性及其功能

低等白蚁肠道里存在着复杂的微生物区系,包括真核微生物鞭毛虫和原核生物,细菌及古细菌。低等白蚁的后肠以特别膨大的囊形胃及其氢氧浓度的明显梯度分布和丰富的微生物区系为特征,是白蚁进行木质纤维素消化的主要器官。后肠内的鞭毛虫能将纤维素水解并发酵为乙酸,二氧化碳和氢,为白蚁提供营养和能源。系统发育研究表明,低等白蚁肠道共生细菌的主要类群为白蚁菌群1、螺旋体、拟杆菌,低G C mol%含量的革兰氏阳性菌和紫细菌等。而古细菌主要为甲烷短杆菌属的产甲烷菌。共生原核生物与二氧化碳的还原和氮的循环等代谢有关。但肠道共生微生物的具体功能和作用机制还有待进一步的揭示。