Diversity of arbuscular mycorrhizal fungi of Panax quinquefolius cultivated in Shandong province / 中国中药杂志

In this study, the colonization, diversity and relative abundance of arbuscular mycorrhizal fungi(AMF) in the roots of Panax quinquefolius in different habitats of Shandong province were analyzed by staining-microscopy and high-throughput sequencing. The data were analyzed by bioinformatics tools and statistical software. The results showed that the roots of P. quinquefolius in different habitats were colonized by AMF with different rates and intensities. The AMF in roots of P. quinquefolius belong to three genera, three families, three orders, one class and one phylum. At the level of order, the AMF mainly included Paraglomerales(52.48%), Glomerales(25.60%) and Archaeosporales(3.08%). At the level of family, the AMF were dominated by Paraglomeraceae(52.48%), Glomeraceae(18.94%) and Claroideoglomeraceae(3.05%). At the level of genus, Paraglomus(51.46%), Glomus(20.01%) and Claroideoglomus(3.52%) accounted for a large proportion, of which Paraglomus and Glomus were dominant. Cluster analysis showed that the AMF in roots of P. quinquefolius with close geographical locations could be clustered together. In this study, the diversity and dominant germplasm resources of AMF in roots of P. quinquefolius cultivated in the main producing areas were identified, which provi-ded basic data for revealing the quality formation mechanism of P. quinquefolius medicinal materials from the perspective of environment.

Arbuscular mycorrhizal fungal communities in the rhizosphere of a continuous cropping soybean system at the seedling stage

Abstract Arbuscular mycorrhizae (AM) fungi play a crucial role in the growth of soybean; however, the planting system employed is thought to have an effect on AM fungal communities in the rhizosphere. This study was performed to explore the influence of continuous soybean cropping on the diversity of Arbuscular mycorrhizal (AM) fungi, and to identify the dominant AM fungus during the seedling stage. Three soybean cultivars were planted under two and three years continuous cropping, respectively. The diversity of AM fungi in the rhizosphere soil at the seedling stage was subsequently analyzed using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). The results showed that an increase in cropping years improved the colonization rate of AM in all three soybean cultivars. Moreover, the dominant species were found to be Funneliformis mosseae and Glomus species. The results of cluster analysis further confirmed that the number of years of continuous cropping significantly affected the composition of rhizospheric AM fungal communities in different soybean cultivars.

Tripartite symbiosis of Sophora tomentosa, rhizobia and arbuscular mycorhizal fungi

ABSTRACT Sophora tomentosa is a pantropical legume species with potential for recovery of areas degraded by salinization, and for stabilization of sand dunes. However, few studies on this species have been carried out, and none regarding its symbiotic relationship with beneficial soil microorganisms. Therefore, this study aimed to evaluate the diversity of nitrogen-fixing bacteria isolated from nodules of Sophora tomentosa, and to analyze the occurrence of colonization of arbuscular mycorrhizal fungi on the roots of this legume in seafront soil. Thus, seeds, root nodules, and soil from the rhizosphere of Sophora tomentosa were collected. From the soil samples, trap cultures with this species were established to extract spores and to evaluate arbuscular mycorhizal fungi colonization in legume roots, as well as to capture rhizobia. Rhizobia strains were isolated from nodules collected in the field or from the trap cultures. Representative isolates of the groups obtained in the similarity dendrogram, based on phenotypic characteristics, had their 16S rRNA genes sequenced. The legume species showed nodules with indeterminate growth, and reddish color, distributed throughout the root. Fifty-one strains of these nodules were isolated, of which 21 were classified in the genus Bacillus, Brevibacillus, Paenibacillus, Rhizobium and especially Sinorhizobium. Strains closely related to Sinorhizobium adhaerens were the predominant bacteria in nodules. The other genera found, with the exception of Rhizobium, are probably endophytic bacteria in the nodules. Arbuscular mycorrhizal fungi was observed colonizing the roots, but arbuscular mycorhizal fungi spores were not found in the trap cultures. Therefore Sophora tomentosa is associated with both arbuscular mycorhizal fungi and nodulating nitrogen-fixing bacteria.

Arbuscular mycorrhizal fungi in Mimosa tenuiflora (Willd. ) Poir from Brazilian semi-arid

Abstract Many plant species from Brazilian semi-arid present arbuscular mycorrhizal fungi (AMF) in their rhizosphere. These microorganisms play a key role in the establishment, growth, survival of plants and protection against drought, pathogenic fungi and nematodes. This study presents a quantitative analysis of the AMF species associated with Mimosa tenuiflora, an important native plant of the Caatinga flora. AMF diversity, spore abundance and root colonization were estimated in seven sampling locations in the Ceará and Paraíba States, during September of 2012. There were significant differences in soil properties, spore abundance, percentage of root colonization, and AMF diversity among sites. Altogether, 18 AMF species were identified, and spores of the genera Acaulospora, Claroideoglomus, Dentiscutata, Entrophospora, Funneliformis, Gigaspora, Glomus, Racocetra, Rhizoglomus and Scutellospora were observed. AMF species diversity and their spore abundance found in M. tenuiflora rhizosphere shown that this native plant species is an important host plant to AMF communities from Brazilian semi-arid region. We concluded that: (a) during the dry period and in semi-arid conditions, there is a high spore production in M. tenuiflora root zone; and (b) soil properties, as soil pH and available phosphorous, affect AMF species diversity, thus constituting key factors for the similarity/dissimilarity of AMF communities in the M. tenuiflora root zone among sites.

Mycorrhizal fungi isolated from native terrestrial orchids of pristine regions in Córdoba (Argentina) / Hongos micorrízicos aislados de orquídeas terrestres nativas de regiones pristinas en Córdoba (Argentina)

Orchidaceae is a highly dependent group on the Rhizoctonia complex that includes Ceratorhiza, Moniliopsis, Epulorhiza and Rhizoctonia, for seed germination and the development of new orchid plants. Thus, the isolation and identification of orchid mycorrhizal fungi are important to understand the orchid-fungus relationship, which can lead to the development of efficient conservation strategies by in vivo germination of seeds from endangered orchid plants. The aim of our work was to isolate and characterize the different mycorrhizal fungi found in roots of terrestrial orchids from Córdoba (Argentina), and, to learn about the natural habit and fungal associations in the Chaco Serrano woodland pristine region. In this study, bloomed orchid root and rhizosphere soil samples were obtained in two times from Valle de Punilla during spring of 2007; samples were kept in plastic bags until processed within 48 hours, and mycorrhizal condition confirmed assessing peloton presence. A total of 23 isolates of the orchideous mycorrhizal Rhizoctonia complex were obtained. The isolates were studied based on morphological characters and ITS-rDNA sequences. Morphological characteristics as color of colonies, texture, growth rate, hyphal diameter and length and presence of sclerotia were observed on culture media. To define the number of nuclei per cell, the isolates were grown in Petri dishes containing water-agar (WA) for three days at 25°C and stained with Safranine-O solution. The mycorrhizal fungi were grouped into binucleate (MSGib, 10 isolates) and multinucleate (MSGim, 13 isolates) based on morphological characteristics of the colonies. We obtained the ITS1-5.8s-ITS4 region that was amplified using primers ITS1 and ITS4. Based on DNA sequencing, isolates Q23 and Q29 were found to be related to species of Ceratobasidium. Isolates Q24 and Q4 were related to the binucleated anastomosis group AG-C of Rhizoctonia sp. The rest of the isolates grouped in the Ceratobasidium clade without grouping. From our knowledge this is the first report of the association of the AG-C testers with terrestrial orchids. A high specificity was observed in the symbiotic relationship. As the mycorrhizal fungal isolates were obtained from native orchids, they could be incorporated in conservation programes of endangered orchids in Argentina.

La Familia Orchidaceae se encuentra estrechamente relacionada con hongos micorrízicos que pertenecen al complejo Rhizoctonia, e incluyen los géneros Ceratorhiza, Moniliopsis, Epulorhiza y Rhizoctonia. Esta asociación es esencial para el desarrollo de nuevas plantas ya que favorecen el proceso de germinación de las semillas. Por lo tanto, el conocimiento de la naturaleza de esta interacción es importante para que los resultados de los programas de conservación de orquídeas sean efectivos. La fragmentación del bosque Chaqueño Serrano en el centro de Argentina, ha alcanzado un punto crítico en los últimos años, afectando el funcionamiento del ecosistema. El objetivo de este trabajo fue: a) aislar y caracterizar hongos micorrízicos presentes en orquídeas terrestres de la provincia de Córdoba (Argentina) y b) conocer el hábitat natural y las asociaciones fúngicas que se establecen en esta región prístina. A partir de las raíces de orquídeas terrestres, se obtuvieron 23 aislamientos de hongos micorrízicos que pertenecen al complejo Rhizoctonia. Estos aislamientos fueron caracterizados con base en caracteres morfológicos y moleculares. Las características morfológicas (color y textura de las colonias, cinética de crecimiento, diámetro y largo de la hifa y presencia de esclerocios) fueron observados en PDA y MEA a 25ºC. El número de núcleos por célula se observó en cultivos crecidos en AA (agar-agua) y teñidos con una solución de Safranine-O. La región ITS se amplificó usando los primers ITS1 e ITS4. Con base en las características morfológicas de la colonia, los aislamientos fueron agrupados en binucleados (MSGib) y multinucleados (MSGim). De acuerdo al cladograma obtenido con las secuencias de ADN, los aislamientos Q23 y Q29 están relacionados a especies de Ceratobasidium, aisladas de raíces de orquídeas. Los aislamientos Q24 y Q4 se asocian con el grupo de anastomosis de Rhizoctonia AG-C. Finalmente, se observó una alta variabilidad en el grado de especificidad existente en la simbiosis que se establece entre las raíces de estas orquídeas terrestres y los aislamientos obtenidos a partir de ellas. Este es el primer reporte de la asociación entre el grupo de anastomosis AG-C y orquídeas terrestres. Dado que estos aislamientos se obtuvieron de orquídeas terrestres nativas, podrían ser incorporados como nuevos patrones para micorrizas de orquídeas terrestres en Argentina. Este trabajo contribuye al conocimiento de la relación simbiótica que se establece entre orquídeas y hongos micorrízicos, así como también al desarrollo de estrategias de conservación de orquídeas terrestres nativas del bosque Chaco Serrano.

Response of aerobic rice to Piriformospora indica.

Rice cultivation under aerobic condition not only saves water but also opens up a splendid scope for effective application of beneficial root symbionts in rice crop unlike conventional puddled rice cultivation where water logged condition acts as constraint for easy proliferation of various beneficial soil microorganisms like arbuscular mycorrhizal (AM) fungi. Keeping these in view, an in silico investigation were carried out to explore the interaction of hydrogen phosphate with phosphate transporter protein (PTP) from P. indica. This was followed by greenhouse investigation to study the response of aerobic rice to Glomus fasciculatum, a conventional P biofertilizer and P. indica, an alternative to AM fungi. Computational studies using ClustalW tool revealed several conserved motifs between the phosphate transporters from Piriformospora indica and 8 other Glomus species. The 3D model of PTP from P.indica resembling “Mayan temple” was successfully docked onto hydrogen phosphate, indicating the affinity of this protein for inorganic phosphorus. Greenhouse studies revealed inoculation of aerobic rice either with P. indica, G. fasciculatum or both significantly enhanced the plant growth, biomass and yield with higher NPK, chlorophyll and sugar compared to uninoculated ones, P. indica inoculated plants being superior. A significantly enhanced activity of acid phosphatase and alkaline phosphatase were noticed in the rhizosphere soil of rice plants inoculated either with P. indica, G. fasciculatum or both, contributing to higher P uptake. Further, inoculation of aerobic rice plants with P. indica proved to be a better choice as a potential biofertilizer over mycorrhiza.

Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have beneficial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were collected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporocarps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effectiveness of the techniques used to amplify DNA polymorphic sequences, which enhances the ­characte­rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.

Evaluación de un sistema para la micorrización in vitro en plantas demora de castilla (Rubus glaucus, Benth) / Evaluation of an in vitro mycorrhization system of blackberry plants (Rubus glaucus, Benth) / Avaliação de um sistema para micorrização in vitro em plantas de amora-preta (Rubus glaucus, Benth)

Objetivo. Obtener un sistema de micorrización in vitro en sistemas de cultivo autotrófico para plantas de mora de castilla (Rubusglaucus, Benth). Materiales y métodos. Se utilizaron esporas y fragmentos de raíces con vesículas del Hongo Formador de MicorrizaArbuscular (HFMA) Glomus sp. (GEV02). Se estableció un sistema de cultivo autotrófico para plántulas de mora, comparando dosmétodos de inoculación directa con el HFMA. Se cuantificó el número de esporas producidas, la longitud del micelio extraradical; asícomo el porcentaje de colonización del HFMA. Adicionalmente se midió la longitud aérea y radical, el peso fresco y seco de la partefoliar y radical para determinar el desarrollo de las plantas. Resultados. El sistema de cultivo autotrófico fue exitoso para plantas demora de castilla (Rubus glaucus, Benth); observándose un óptimo crecimiento de la parte aérea y radical de la planta. Adicionalmenteen este estudio se pudo obtener un sistema que permitió el desarrollo de Glomus sp (GEV02) bajo condiciones in vitro, con formaciónde estructuras típicas de la simbiosis como una buena colonización intraradical, con producción de arbúsculos y vesículas, así comoel desarrollo de micelio extraradical con hifas ramificadas y la formación de nuevas esporas. Conclusión. Las plantas de moramicropropagadas se asociaron con éxito, por primera vez, con un hongo formador de micorriza arbuscular bajo condiciones in vitro,permitiendo el desarrollo del sistema simbiótico HFMA Glomus sp., asociado a las raíces de plántulas de mora castilla micropropagadas...

Objective. Obtain an in vitromycorrhization system in autotrophic culture systems of blackberry plants (Rubus glaucus, Benth). Materials and methods. We usedspores and root fragments with vesicles of Arbuscular Mycorrhizal Fungus (AMF) Glomus sp (GEV02). We established an autotrophicculture system of blackberry plantlets comparing two methods of direct inoculation of the AMF. We measured the number of sporesproduced, the length of the extraradical mycelium as well as the percentage of colonization of the AMF. Additionally, we measuredthe shoot and root length, and the fresh and dry weight of the leaf and root parts to determine the plant development. Results. Theautotrophic culture system was successful for blackberry plants (Rubus glaucus, Benth; an optimal shoot and root growth was observed.Additionally, we obtained a system that allowed the development of Glomus sp. in in vitro conditions, with the formation of structurestypical of the symbiosis as well as a good intraradical colonization, with the production of arbuscules and vesicles, development ofextraradical mycelium with branched hyphae, and formation of new spores. Conclusion. For the first time, micropropagated blackberryplants associated successfully with an AMF under in vitro conditions, enabling the development of the symbiotic system AMF Glomussp. associated to roots of micropropagated blackberry plantlets...

Objetivo. Obter umsistema de micorrização in vitro em sistemas de cultura autotróficos para plantas de amora-preta (Rubus glaucus, Benth). Materiais emétodos. Foram usados esporos e fragmentos de raízes com vesículas do Fungo Formador Micorrízico Arbuscular (FFMA) Glomus sp.(GEV02). Foi estabelecido um sistema de cultivo autotrófico para mudas de amora-preta, comparando dois métodos de inoculação diretacom o FFMA. Foi quantificado o número de esporos produzidos, o comprimento do micélio extra radicular; bem como a porcentagemde colonização do FFMA. Além disso, foi medido o comprimento e o peso fresco e seco da parte folhar e radicular para determinar odesenvolvimento das plantas. Resultados. O sistema de cultivo autotrófico foi bem-sucedido para as plantas de amora-preta (Rubusglaucus, Benth), onde foi observado um crescimento ótimo da parte aérea e da raidicular da planta. Além disso, neste estudo foi obtido umsistema que permitiu o desenvolvimento de Glomus sp (GEV02) sob condições in vitro, com formação de estruturas típicas da simbiosecomo uma boa colonização intra radicular, com produção de arbúsculos e vesículas, assim como o desenvolvimento de micélio extraradicular com hifas ramificada e a formação de novos esporos. Conclusão. As plantas de amora-preta micropropagadas associaram-secom sucesso, pela primeira vez, com um fungo formador micorrízico arbuscular em condições in vitro, permitindo o desenvolvimentodo sistema simbiótico FFMA Glomus sp., associado às raízes das plântulas de amora-preta micropropagadas...

Expression of phenazine biosynthetic genes during the arbuscular mycorrhizal symbiosis of Glomus intraradices

To explore the molecular mechanisms that prevail during the establishment of the arbuscular mycorrhiza symbiosis involving the genus Glomus, we transcriptionally analysed spores of Glomus intraradices BE3 during early hyphal growth. Among 458 transcripts initially identified as being expressed at presymbiotic stages, 20% of sequences had homology to previously characterized eukaryotic genes, 30% were homologous to fungal coding sequences, and 9% showed homology to previously characterized bacterial genes. Among them, GintPbr1a encodes a homolog to Phenazine Biosynthesis Regulator (Pbr) of Burkholderia cenocepacia, an pleiotropic regulatory protein that activates phenazine production through transcriptional activation of the protein D isochorismatase biosynthetic enzyme phzD (Ramos et al., 2010). Whereas GintPbr1a is expressed during the presymbiotic phase, the G. intraradices BE3 homolog of phzD (BGintphzD) is transcriptionally active at the time of the establishment of the arbuscular mycorrhizal symbiosis. DNA from isolated bacterial cultures found in spores of G. intraradices BE3 confirmed that both BGintPbr1a and BGintphzD are present in the genome of its potential endosymbionts. Taken together, our results indicate that spores of G. intraradices BE3 express bacterial phenazine biosynthetic genes at the onset of the fungal-plant symbiotic interaction.

Seasonality and host preference of arbuscular mycorrhizal fungi of five plant species in the inner Mongolia steppe, China

The seasonal change and host preference of arbuscular mycorrhizal (AM) colonization and community composition of five common plant species Agropyron cristatum, Anemarrhena asphodeloides, Cleistogenes squarrosa, Leymus chinensis, and Stipa grandis in the Inner Mongolia steppe were investigated. The AM root length colonization rates were different among the five plant species and were generally high in early (May and June) and late (September) growth seasons and low in August. A total of 18 AM fungal species representing five genera were isolated from rhizosphere soils of the five plant species, and most AM fungi had not host specificity, except that Acaulospora sp., Glomus constrictum, G. diaphanum and Glomus sp. showed a certain degree of host preference. Glomus albidum, G. etunicatum and G. geosporum were the dominant species and showed various sporulation patterns in the five plants during the growth seasons. The AM fungal spore densities and species richness increased from May to September and decreased in October and were different in the same month in the five plants. Multivariate analyses revealed that season and host significantly co-affected the AM fungal spore density, species richness, and Shannon-Wiener diversity index, and the season had higher influence than the host.

Occurrence and diversity of arbuscular mycorrhizal fungi in trap cultures from soils under different land use systems in the Amazon, Brazil / Ocorrência e diversidade de fungos micorrízicos arbuculares em culturas armadilhas de solos sob diferentes sistema de uso na Amazônia, Brasil

The aim of this work was to evaluate the occurrence of arbuscular mycorrhizal fungi (AMF) species diversity in soil samples from the Amazon region under distinct land use systems (Forest, Old Secondary Forest, Young Secondary Forest, Agroforestry systems, Crops and Pasture) using two distinct trap cultures. Traps established using Sorghum sudanense and Vigna unguiculata (at Universidade Regional de Blumenau - FURB) and Brachiaria decumbens and Neonotonia wightii (at Universidade Federal de Lavras - UFLA) were grown for 150 days in greenhouse conditions, when spore density and species identification were evaluated. A great variation on species richness was detected in several samples, regardless of the land use systems from where samples were obtained. A total number of 24 AMF species were recovered using both methods of trap cultures, with FURB';s traps yielding higher number of species. Acaulospora delicata, A. foveata, Entrophospora colombiana and two undescribed Glomus species were the most abundant and frequent species recovered from the traps. Number of species decreased in each genus according to this order: Acaulospora, Glomus, Entrophospora, Gigaspora, Archaeospora, Scutellospora and Paraglomus. Spore numbers were higher in Young Secondary Forest and Pastures. Our study demonstrated that AMF have a widespread occurrence in all land use systems in Amazon and they sporulate more abundantly in trap cultures from land uses under interference than in the pristine Forest ecosystem.

O objetivo deste trabalho foi avaliar a ocorrência de espécies de fungos micorrízicos arbusculares (FMAs) em amostras de solos da região Amazônica sob diferentes sistemas de uso da terra (Floresta, Capoeira Velha, Capoeira Nova, Sistema Agroflorestal, Culturas e Pastagens) usando dois métodos de culturas armadilhas. Culturas armadilhas estabelecidas com Sorghum sudanense e Vigna unguiculata (na Universidade Regional de Blumenau - FURB) e Brachiaria decumbens e Neonotonia wightii (na Universidade Federal de Lavras - UFLA) foram cultivadas por 150 dias em condições de casa-de-vegetação e avaliadas para o número de esporos e identificação das espécies. Uma grande variação na riqueza de espécies foi detectada em várias amostras, independente do sistema de uso da terra de onde as amostras foram obtidas. Um total de 24 espécies de FMAs foram recuperadas usando ambas metodologias de culturas armadilhas e as culturas estabelecidas na FURB produziram um número maior de espécies. Acaulospora delicata, A. foveata, Entrophospora colombiana e duas espécies não descritas de Glomus foram as espécies mais abundantes e freqüentes recuperadas das culturas armadilhas. O número de espécies diminui em cada gênero na seguinte ordem: Acaulospora, Glomus, Entrophospora, Gigaspora, Archaeospora, Scutellospora e Paraglomus. Número de esporos foi maior na Capoeira jovem e Pastagens. Nosso estudo demonstrou que os FMAs possuem uma ampla dispersão em todos os sistemas de uso na Amazônia e que eles esporulam mais abundantemente em culturas armadilhas estabelecidas de solos sob interferência antrópica do que em solos de Floresta nativa.

Communities of P-solubilizing bacteria, fungi and arbuscular mycorrhizal fungi in grass pasture and secondary forest of Paraty, RJ - Brazil

Avaliaram-se as comunidades de bactérias, fungos solubilizadores de fosfato e fungos micorrízicos arbusculares em dois diferentes ecossistemas. Foram feitas amostragens em duas áreas de Mata Atlântica, em Paraty - RJ, Brasil, uma de floresta secundária e outra de pastagem. Foram avaliados quatro meios de cultura: GL (glicose e extrato de levedura), GES (glicose, extrato de solo, KNO3, CaCl2, MgSO4, NaCl, FeEDTA solução de micronutrientes), GAGES (glicose, extrato de solo, arabinose, glicerol, CaCl2, MgSO4 e NaCl) e GELP (glicose, extrato de solo, extrato de levedura, peptona, CaCl2, MgSO4 e NaCl) para isolamento de microrganismos solubilizadores de fosfato. A identificação das bactérias solubilizadoras foi baseada na análise do 16 S rDNA, enquanto que os fungos solubilizadores e os micorrízicos arbusculares foram identificados pela sua morfologia. O maior número de bactérias solubilizadoras foi obtido usando os meios GL e GELP. O maior número de fungos solubilizadores foi obtido com os meios GAGES e GES. As bactérias solubilizadoras foram identificadas como Enterobacteriaceae e Bacillus sp., e os fungos como Aspergillus sp. Glomus macrocarpum e Glomus etunicatum foram as espécies de fungos micorrízicos dominantes nas áreas de floresta secundária e pastagem, respectivamente.

A new approach for enhanced multiplication of arbuscular mycorrhizal fungi and isolation of ITS regions from Glomus deserticola and Laccaria fraterna.

Rootlets induced from the petiole base of L. purpureus, using IAA and kinetin was used for enhanced multiplication of arbuscular mycorrhizal (AM) fungus, G. deserticula. Using conserved short arbitrary oligonucleotides, as specific primers, we amplified the ITS-region, a molecular marker for fungal identification, from the genomic DNA extracted from cultured spores of G. deserticola, and genomic DNA extracted from the mycelium of L. fraterna. The capacity of fungal colonization and subsequent spore formation of G. deserticola, compared with the natural root system was evaluated. This technology would provide a simple way to multiply AM fungi and to produce spores without microbial contamination useful for further molecular characterization.