Molecular analysis of the interaction between white rot pathogen (Rigidoporus microporus) and rubber tree (Hevea brasiliensis)

The white rot pathogen, Rigidoporus microporus (Basidiomycota) syn. Rigidoporus lignosus is the most destructive root pathogen in rubber plantations located in South East Asia and Africa. Most management practices for the disease have been carried out without adequate knowledge of the population genetics of the pathogen. Our primary objective in the first part of this work was to characterize isolates from rubber trees (Hevea brasiliensis) in Nigeria. The lack of global studies comparing isolates from Asia and Africa where the majority of rubber plantations are located necessitated extending our molecular phylogeny studies to include isolates from other regions of the world. We compared the Nigerian isolates with isolates from South East Asia and Central/South America. Forty isolates were analyzed by sequencing four gene regions: the nuclear ribosomal DNA ITS and LSU, β-tubulin and translation elongation factor 1-α (tef1) genes. The isolates form three distinctive clades corresponding to Africa, South East Asia and South/Central America. There was high intra-species variation within each clade, but no geographic pattern was detected among the Nigerian isolates and also among the West and Central African isolates. Our multi-gene molecular phylogenetic analysis suggests the presence of two distinctive species associated with the white rot disease. Phylogenetic analysis also placed the Rigidoporus genus within the Oxyporus clade of the Hymaenochataeles. This study supports the need for a thorough revision of the Rigidoporus genera within the Polyporales/Hymaenochataeles. To gain insights on the saprotrophic abilities of Rigidoporus microporus isolates associated with the white rot disease of rubber trees, we performed wood decay tests of selected isolates on rubber wood blocks. We included a non-pathogenic endophytic South American isolate for comparative analysis. The structural alterations caused due to fungal growth in the rubber wood blocks was also studied. The pathogenic isolates associated with rubber trees significantly decayed the rubber wood blocks suggesting a capacity to switch to saprotrophic growth when the tree is killed. The non-pathogenic isolate had very low saprotrophic ability. Structural alterations of wood cell walls caused in the wood blocks were typical for white rot basidiomycetes. The fact that Rigidoporus microporus can switch to saprotrophic growth on dead cells of the tree led us to investigate the transcriptomic profiles of the fungus during growth on dead rubber wood. We performed RNA-Seq de novo transcriptomic assembly which produced 25, 880 annotated unigenes. The transcriptome expressed over 400 genes encoding lignocellulose enzymes. A number of glycoside hydrolases (GHs) were highly induced in rubber wood including all nine members of GH43 genes in the transcriptome. Twenty-four manganese peroxidase encoding genes were among a large number of oxidoreductases that were significantly up-regulated in rubber wood. Several genes involved in fatty acid and rubber latex degradation were expressed in the transcriptome. Several ABC transporters and hydrophobin genes were also expressed in the transcriptome. Finally, pathway enrichment analysis of genes related to energy metabolism revealed some genes including alcohol dehydrogenase with potential biotechnological applications. This is the first study on the transcriptomic analysis of R. microporus on rubber wood. The data generated should serve as a useful resource for future studies. Understanding how rubber tree is able to defend itself against invading Rigidoporus microporus was an additional research goal. The expression of defence-related genes; pathogenesis related proteins (PR1, PR3, PR5, PR8 and PR9), cell wall modification genes (PAL and expansin), signal transduction genes (ACC oxidase, AOC, MAPK) and a Myb transcription factor were studied in two clones (RRIM612 and PR107) of H. brasiliensis with different levels of susceptibility. Clone RRIM612 had a higher lesion size than clone PR107 after 5 weeks post inoculation with R. microporus. A class I chitinase (PR3) was up-regulated in RRIM612 in response to the pathogen while a class IV peroxidase (PR9) was highly up-regulated in PR107. Signal transduction genes involved in ethylene and jasmonic acid signaling as well as Phenylalanine ammonia-lyase (PAL) were up-regulated almost equally in both clones. The predicted expansin like protein was up-regulated 40 fold in RRIM612 in response to the pathogen. The results obtained demonstrate the variability in defence responses in the two clones studied and provides the first set of defence genes expression profiles of the host-pathogen interaction of the white rot disease of rubber trees. This study sheds light on the phylogeny of pathogenic Rigidoporus microporus isolates associated with the white rot disease as well as evidence showing its ability to switch to saprotrophic growth. The transcriptome of the fungus during saprotrophic growth was also de novo assembled revealing a vast number of lignocellulose degrading enzymes. Finally, this work provides an overview of potential candidate defence genes in H. brasiliensis in response to infection by R. microporus.Rigidoporus microporus on Kaakkois-Aasiassa ja Afrikassa merkittävin kumipuun taudinaiheuttaja ja lahottaja. Luonnonkumin suuresta taloudellisesta merkityksestä huolimatta taudinaiheuttajan alkuperä ja biologia tunnetaan huonosti. Työssä tutkittiin sienen populaatiogenetiikkaa Aasiassa ja Afrikassa, jossa valtaosa kumista tuotetaan. Tutkimuksessa sekvensoitiin yhteensä neljästäkymmenestä sienikannasta neljä geeniä: ribosomia koodaavan DNA:n ITS ja LSU-alueet, β-tubuliini- ja tef1- geenit (translation elongation factor 1-α). Sienikannat jakaantuivat sekvenssien perusteella kolmeen selkeään sukupuun haaraan (Afrikka, Kaakkois-Aasia ja Etelä/Keski-Amerikka). Näiden ryhmien sisällä havaittiin vaihtelua, muttei tarkempaa maantieteellistä ryhmittymistä. Tulokset viittaavat siihen, että tautia aiheuttaisi kaksi erillistä sienilajia. Lisäksi Rigidoporus -suku sijoittui tämän tutkimuksen perusteella Hymaenochataeles-lahkon Oxyporus-sukuja sisältävään ryhmään, joten Rigidoporus-sukujen taksonomiaa tulisi vielä tarkistaa. Tutkimuksessa testattiin eri sienikantojen kykyä lahottaa kumipuuta. Vertailussa oli mukana Etelä-Amerikkalainen sienikanta, joka ei ole taudinaiheuttaja, vaan elää oireettomassa puussa endofyyttinä. Taudinaiheuttamiskykyiset sienikannat lahottivat puuta tehokkaasti, mikä viittaa sienen kykyyn vaihtaa ravinnonhankkimisstrategiaa kun puu kuolee. Puuaineen rakenteelliset muutokset olivat valkolaholle tyypillisiä. Koska havaittiin, että Rigidoporus microporus pystyy elämään saprotrofina, seuraavaksi tutkimuksessa analysoitiin sienen geenien transkriptiota. Sienen havaittiin ilmentävän yli 400 puun ligniinin ja selluloosan hajottamiseen liittyvää entsyymiä. Lisäksi kumipuun hajottamisessa indusoitui glykosidihydrolaaseja, suuri määrä oksidoreduktaaseja, kuten 24 mangaaniperoksidaasia sekä useita rasvahappojen ja kumilateksin hajottamiseen liittyviä geenejä. Energiametaboliareittien tarkempi tutkimus paljasti alkoholidehydrogenaaseja, joilla voisi olla bioteknisiä käyttösovelluksia. Kumipuun puolustusreaktioita tutkittiin tartuttamalla taudinaiheuttaja kahteen vastustuskyvyltään erilaiseen kumipuuklooniin (RRIM612 ja PR107). Vertailu tehtiin analysoimalla ns. PR-proteiineja (pathogenesis related; PR1, PR3, PR5, PR8 and PR9), soluseinään vaikuttavia entsymeitä (PAL ja expansin), signaalin transduktiogeenejä (ACC oxidase, AOC, MAPK) ja Myb transkriptiotekijää. Sieni levisi nopeammin kloonin RRIM612 puuaineessa, ja kloonin kitinaasin (PR3) tuotanto lisääntyi, kun taas peroksidaasin (PR9) tuotanto lisääntyi kloonissa PR107. Molemmissa puuklooneissa havaittiin viestinvientiin liittyvien yhdisteiden, kuten etyleenin ja jasmonihapon metabolian kuten myös fenyylialaniini-ammonia-lyaasin (PAL) ekspression voimistumista. Ekspansiinin kaltaisen proteiinin tuotanto 40-kertaistui kloonissa RRIM612 . Tulokset kertovat puolustusreaktioiden suuresta vaihtelusta ja ne ovat myös ensimmäiset molekyylitason tiedot kumipuun puolustautumisesta taudinaiheuttajaa vastaan

FOLK USES OF MUSHROOMS BY THE AKOKO-EDO PEOPLE OF NIGERIA

A study carried out on the folk uses and ethnomycology of wild edible and medicinal mushrooms by five selected communities: Igarra, Sasaru, Egbigere, Ikpeshi and Aghor in Akoko-Edo Local Government Area of Edo State, Nigeria showed that about 86% of respondents consumed mushrooms. About 12.5% of the respondents consumed mushrooms because of its taste, 54.2% used them as substitutes for meat or fish, 16.7% use certain mushrooms e.g. sclerotium of Pleurotus tuberregium and Schizophyllum commune while 9.2% sold them for extra incomes. Auricularia judae, Cortinarius melliolens, Macrolepiota procera, Lactarius edulis, Lycoperdon sp., Pleurotus tuberregium, P. squarrosulus, Termitomyces robustus and Tricholoma lobayense were identified as edible species, while Daldinia concentrica, Ganoderma lucidum, G. applanatum (Pers.) Pat. and Nothopanus sp. were used in traditional medicine practices. The source of utilitarian mushrooms remained forests, agroforests and farms in many communities studied, and their collection and/or sales reserved for women and childre

De novo transcriptomic assembly and profiling of Rigidoporus microporus during saprotrophic growth on rubber wood

Background: The basidiomycete Rigidoporus microporus is a fungus that causes the white rot disease of the tropical rubber tree, Hevea brasiliensis, the major source of commercial natural rubber. Besides its lifestyle as a pathogen, the fungus is known to switch to saprotrophic growth on wood with the ability to degrade both lignin and cellulose. There is almost no genomic or transcriptomic information on the saprotrophic abilities of this fungus. In this study, we present the fungal transcriptomic profiles during saprotrophic growth on rubber wood. Results: A total of 266.6 million RNA-Seq reads were generated from six libraries of the fungus growing either on rubber wood or without wood. De novo assembly produced 34, 518 unigenes with an average length of 217(bp. Annotation of unigenes using public databases; GenBank, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups (COG) and Gene Ontology (GO) produced 25, 880 annotated unigenes. Transcriptomic profiling analysis revealed that the fungus expressed over 300 genes encoding lignocellulolytic enzymes. Among these, 175 genes were up-regulated in rubber wood. These include three members of the glycoside hydrolase family 43, as well as various glycosyl transferases, carbohydrate esterases and polysaccharide lyases. A large number of oxidoreductases which includes nine manganese peroxidases were also significantly up-regulated in rubber wood. Several genes involved in fatty acid metabolism and degradation as well as natural rubber degradation were expressed in the transcriptome. Four genes (acyl-CoA synthetase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA acetyltransferase) potentially involved in rubber latex degradation pathway were also induced. A number of ATP binding cassette (ABC) transporters and hydrophobin genes were significantly expressed in the transcriptome during saprotrophic growth. Some genes related to energy metabolism were also induced. Conclusions: The analysed data gives an insight into the activation of lignocellulose breakdown machinery of R. microporus. This study also revealed genes with relevance in antibiotic metabolism (e.g. cephalosporin esterase) as well as those with potential applications in fatty acid degradation. This is the first study on the transcriptomic analysis of R. microporus on rubber wood and should serve as a pioneering resource for future studies of the fungus at the genomic or transcriptomic level.Peer reviewe

Genome sequencing of Rigidoporus microporus provides insights on genes important for wood decay, latex tolerance and interspecific fungal interactions

Correction: Scientific reports, vol. 10:1, art. 18199Fungal plant pathogens remain a serious threat to the sustainable agriculture and forestry, despite the extensive efforts undertaken to control their spread. White root rot disease is threatening rubber tree (Hevea brasiliensis) plantations throughout South and Southeast Asia and Western Africa, causing tree mortality and severe yield losses. Here, we report the complete genome sequence of the basidiomycete fungus Rigidoporus microporus, a causative agent of the disease. Our phylogenetic analysis confirmed the position of R. microporus among the members of Hymenochaetales, an understudied group of basidiomycetes. Our analysis further identified pathogen’s genes with a predicted role in the decay of plant cell wall polymers, in the utilization of latex components and in interspecific interactions between the pathogen and other fungi. We also detected putative horizontal gene transfer events in the genome of R. microporus. The reported first genome sequence of a tropical rubber tree pathogen R. microporus should contribute to the better understanding of how the fungus is able to facilitate wood decay and nutrient cycling as well as tolerate latex and utilize resinous extractives.Peer reviewe

Cadophora margaritata sp. nov. and other fungi associated with the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.Peer reviewe

Nigerian Mushrooms: Underutilized Non-Wood Forest Resources

Mushroom resources have been exploited in most developed economies because of their huge agro-industrial, medicinal and commercial benefits. Nigerians utilized mushroom-forming fungi only for food and folk medicine for many decades. Auricularia auricular Judae (Bull.) Quél, Lentinus squarrosulus Mont., Pleurotus tuberregium (Fr.) Singer and Volvariella volvacea (Bull.) Singer was some of the common edible mushrooms that were successfully cultivated in Nigeria on small-scale basis. The mushroom resources in Nigeria are grossly under-studied and their attractive potentials under-exploited for addressing economic and industrial development. Resourceful biotechnological approach in the application of mushrooms in agriculture, medicine, industry and environment is inchoate and uncommon in the country