Two new records of entolomatoid fungi associated with rosaceous plants from Japan

Two entolomatoid species associated with rosaceous plants, Entoloma saepium and E. clypeatum f. hybridum, are reported as new records from Japan, and their morphological characters are described and illustrated.     

Inoculation of willows (Salix spp.) with ectomycorrhizal fungi on mined boreal peatland

Inoculation with ectomycorrhizal fungi was explored as a means to improve productivity of experimental short-rotation plantations of the willowsSalix viminalis andSalix dasyclados for biomass production on surface-mined peatlands in northern Finland. Both willow species formed ectomycorrhizas withAmanita spp.,Cortinarius purpurascens, Entoloma nidorosum, otherEntoloma spp.,Hebeloma crustuliniforme, H. pusillum, Laccaria bicolor, andPaxillus involutus in greenhouse experiments.Field trials on a mined peatland site revealed (after one growing season) statistically significant growth stimulation after inoculation due to mycorrhiza formation in both willow species: plants inoculated withEntoloma were sometimes twice as large as control plants. However, such effects were observed only in plots receiving normal phosphate fertilization as opposed to low phosphate application, and were not consistent from season to season. With the inoculum of other species (Cortinarius, Hebeloma andPaxillus) some evidence of growth enhancement was found in the field, but these results were sometimes attributable to non-symbiotic effects of inoculation.     

Preferential Colonization of Solanum tuberosum L. Roots by the Fungus Glomus intraradices in Arable Soil of a Potato Farming Area

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities of Solanum tuberosum L. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging to Glomus groups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding to Glomus intraradices, was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species, G. intraradices.     

Multiple origins of sequestrate basidiomes within Entoloma inferred from molecular phylogenetic analyses

The genus Entoloma comprises diverse trophic modes and basidiome morphologies. Although Entoloma includes some sequestrate species, their origins are not clearly understood in relation to phylogenetic position and trophic status. In this study, we collected 34 sequestrate Entoloma specimens in Japan over a 9-y period. Their identities and phylogenetic positions were determined by molecular analyses using three nuclear loci [internal transcribed spacer (ITS) and large subunit (LSU) regions of rDNA and RNA polymerase II second LSU (rpb2) gene]. Based on species delimitation of 97 % sequence matches in the ITS region, which is a suitable region for species-level identification of higher fungi, we identified four sequestrate Entoloma species. Molecular phylogenetic analyses that included all related sequences in the International Nucleotide Sequence Database revealed that the four sequestrate Entoloma species belonged to two major phylogroups. One of the phylogroups was Inocephalus–Cyanula, which is composed only of saprotrophic species. Three of the Japanese sequestrate species, as well as three previously known sequestrate species from other regions, fell into at least two independent clades in this phylogroup, indicating multiple origins of sequestrate forms within this saprotrophic lineage. Another phylogroup, Rhodopolioid, was also shown to include a sequestrate species for the first time. Because the Rhodopolioid phylogroup is composed exclusively of mycorrhizal species (ectomycorrhizal and tuberculate mycorrhizal species), the sequestrate form may also have evolved from a mycorrhizal ancestor. Our results suggest that sequestrate basidiomes have evolved multiple times, irrespective of their trophic status in Entoloma. Finally, based on molecular and morphological characteristics, here we describe two new sequestrate Entoloma species, i.e., Entoloma prismaticum Sasaki, Kinoshita et Nara, sp. nov. and Entoloma hypogaeum Sasaki, Kinoshita et Nara, sp. nov.     

Detection and biocontrol potential of <Emphasis Type="Italic">Verticillium leptobactrum</Emphasis> parasitizing <Emphasis Type="Italic">Meloidogyne</Emphasis> spp.

Three isolates of Verticillium leptobactrum proceeding from egg masses of root-knot nematodes (RKN) Meloidogyne spp. and soil samples collected in Tunisia were evaluated against second-stage juveniles (J2) and eggs of M. incognita, to determine the fungus biocontrol potential. In vitro tests showed that V. leptobactrum is an efficient nematode parasite. The fungus also colonized egg masses and parasitized hatching J2. In a greenhouse assay with tomato plants parasitized by M. incognita and M. javanica, V. leptobactrum was compared with isolates of Pochonia chlamydosporia and Monacrosporium sp., introducing the propagules into nematode-free or naturally infested soils. The V. leptobactrum isolates were active in RKN biocontrol, improving plants growth with a significant increase of tomato roots length, lower J2 numbers in soil or egg masses, as well as higher egg mortalities. In a second assay with M. javanica, treatments with three V. leptobactrum isolates reduced egg masses on roots as well as the density of J2 and the number of galls. To evaluate the fungus capability to colonize egg masses a nested Real-time polymerase chain reaction (PCR) assay, based on a molecular beacon probe was used to assess its presence. The probe was designed on a V. leptobactrum ITS region, previously sequenced. This method allowed detection of V. leptobactrum from egg masses, allowing quantitative DNA and fungal biomass estimations.     

Identification of host plants and description of sclerotia of the truffle <Emphasis Type="Italic">Mattirolomyces terfezioides</Emphasis>

The truffle, Mattirolomyces terfezioides, is a hypogeous ascomycete with uncertain host relationships. The fungus has been regularly collected on sandy soils in the Carpathian Basin. During the study of the natural host plants of the fungus, strange, amorphous, belowground hyphal aggregates incorporating soil and sand particles have been found attached to the surface of the roots. The fruitbodies of M. terfezioides develop from these hyphal aggregates. This structure, similar to that formed by morels, could be interpreted as a sclerotium. Sclerotia were found both on roots of woody and herbaceous plants. To detect the roots colonized by M. terfezioides, a species-specific polymerase chain reaction was developed. Seven natural hosts of the fungus were identified by this method. No specificity regarding taxa or life form of the plants was found. The colonization of the roots by the septate hyphae of M. terfezioides was weak, particularly compared to the colonization by arbuscular–mycorrhizal fungi. This suggests that this fungus is not the dominant fungal partner of these plants. Therefore, using M. terfezioides as the only inoculum may be inappropriate in truffle cultivation experiments. Nevertheless, further in vitro experiments are needed to develop reliable knowledge on the still ambiguous symbiotic strategy of this fungus.     

Cooccurring plants forming distinct arbuscular mycorrhizal morphologies harbor similar AM fungal species

Arbuscular mycorrhizal (AM) fungal spores were isolated from field transplants and rhizosphere soil of Hedera rhombea (Miq) Bean and Rubus parvifolius L., which form Paris-type and Arum-type AM, respectively. DNA from the spore isolates was used to generate molecular markers based on partial large subunit (LSU) ribosomal RNA (rDNA) sequences to determine AM fungi colonizing field-collected roots of the two plant species. Species that were isolated as spores and identified morphologically and molecularly were Gigaspora rosea and Scutellospora erythropa from H. rhombea, Acaulospora longula and Glomus etunicatum from R. parvifolius, and Glomus claroideum from both plants. The composition of the AM fungal communities with respect to plant trap cultures was highly divergent between plant species. Analysis of partial LSU rDNA sequences amplified from field-collected roots of the two plant species with PCR primers designed for the AM fungi indicated that both plants were colonized by G. claroideum, G. etunicatum, A. longula, and S. erythropa. G. rosea was not detected in the field-collected roots of either plant species. Four other AM fungal genotypes, which were not isolated as spores in trap cultures from the two plant species, were also found in the roots of both plant species; two were closely related to Glomus intraradices and Glomus clarum. One genotype, which was most closely related to Glomus microaggregatum, was confined to R. parvifolius, whereas an uncultured Glomeromycotan fungus occurred only in roots of H. rhombea. S. erythropa was the most dominant fungus found in the roots of H. rhombea. The detection of the same AM fungal species in field-collected roots of H. rhombea and R. parvifolius, which form Paris- and Arum-type AM, respectively, shows that AM morphology in these plants is strongly influenced by the host plant genotypes as appears to be the case in many plant species in natural ecosystems, although there are preferential associations between the hosts and colonizing AM fungi in this study.     

Chilli rhizosphere fungus Aspergillus spp. PPA1 promotes vegetative growth of cucumber (Cucumis sativus) plants upon root colonisation

A rhizosphere fungus was isolated from roots of chilli plants and identified as Aspergillus spp. PPA1. The fungus was tested for its ability to promote the growth of cucumber plants in a pot experiment. Cucumber seeds were sown in sterilised field soil amended with wheat grain inoculum (WGI) of PPA 1 at the rate of 0.5, 1 and 1.5% w/w, and plants were grown for 21 days in a net house. The treatment with PPA1 significantly increased shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, plant length, leaf area and leaf chlorophyll content of cucumber plants compared to non-treated control. The growth promotion rate increased with the increasing concentration of inoculum of PPA1 applied to the soil. The fungus was re-isolated from the roots of cucumber plants at higher frequencies. These results suggest that Aspergillus spp. PPA1 is a root colonising plant-growth promoting fungus for cucumber.     

Phylogenetic analysis of Glomeromycota by partial LSU rDNA sequences

We analyzed the large subunit ribosomal RNA (rRNA) gene [LSU ribosomal DNA (rDNA)] as a phylogenetic marker for arbuscular mycorrhizal (AM) fungal taxonomy. Partial LSU rDNA sequences were obtained from ten AM fungal isolates, comprising seven species, with two new primers designed for Glomeromycota LSU rDNA. The sequences, together with 58 sequences available from the databases, represented 31 AM fungal species. Neighbor joining and parsimony analyses were performed with the aim of evaluating the potential of the LSU rDNA for phylogenetic resolution. The resulting trees indicated that Archaeosporaceae are a basal group in Glomeromycota, Acaulosporaceae and Gigasporaceae belong to the same clade, while Glomeraceae are polyphyletic. The results support data obtained with the small subunit (SSU) rRNA gene, demonstrating that the LSU rRNA gene is a useful molecular marker for clarifying taxonomic and phylogenetic relationships in Glomeromycota.     

Molecular characterization of strawberry pathogen Gnomonia fragariae and its genetic relatedness to other Gnomonia species and members of Diaporthales

Gnomonia fragariae is a poorly studied ascomycete belonging to Diaporthales. Originally G. fragariae was considered a saprophyte occurring on dead tissues of strawberry plants. Recently this fungus was found in Latvia and Sweden, and it was proven to be the cause of severe root rot and petiole blight of strawberry. Thirteen isolates of this pathogen and several other Gnomonia species occurring on rosaceous hosts were characterized by molecular analysis using nucleotide sequences of partial LSU rRNA gene and the total ITS region. The homologous regions from relevant diaporthalean taxa available in the GenBank were also included and compared with the taxa sequenced in this study. Phylogenetic analyses revealed that G. fragariae, G. rubi, and Gnomonia sp. (CBS 850.79) were genetically different from G. gnomon, the type species of the genus, and other members of Gnomoniaceae. The analyses showed that G. fragariae and Hapalocystis were genetically very closely related, forming a phylogenetic clade, which is possibly presenting a new family in the Diaporthales. Morphological comparisons of the Gnomonia species on the basis of commonly used criteria for the taxonomy of Diaporthales, so far did not reveal any evident features for the polyphyletic status of Gnomonia.     

Hedgerow hawthorn (Crataegus spp.) and blackthorn (Prunus spinosa) as hosts of fruit tree viruses in Britain

Apple chlorotic leafspot virus (CLSV) was detected in 27 of 109 hawthorn and three of 67 blackthorn plants sampled in various parts of Britain. The CLSV isolates possessed similar properties to those isolated from other rosaceous species but differed in the severity of symptoms they induced in woody indicators. No seed or aphid transmission of CLSV was detected. Prunus necrotic ringspot (PNRV) and prune dwarf (PDV) viruses were detected in four and three respectively of 67 blackthorn plants. The PNRV and PDV isolates were serologically closely related to isolates from cherry. Arabis mosaic virus was detected in one blackthorn plant, but plum pox virus was not found in any of the tested plants.     

Diversity of arbuscular mycorrhizal fungi colonising roots of the grass species<Emphasis Type="Italic"> Agrostis capillaris</Emphasis> and<Emphasis Type="Italic"> Lolium perenne</Emphasis> in a field experiment

Analysis of arbuscular mycorrhizal (AM) fungal diversity through morphological characters of spores and intraradicular hyphae has suggested previously that preferential associations occur between plants and AM fungi. A field experiment was established to investigate whether AM fungal diversity is affected by different host plants in upland grasslands. Indigenous vegetation from plots in an unimproved pasture was replaced with monocultures of either Agrostis capillaris or Lolium perenne. Modification of the diversity of AM fungi in these plots was evaluated by analysis of partial sequences in the large subunit (LSU) ribosomal RNA (rDNA) genes. General primers for AM fungi were designed for the PCR amplification of partial sequences using DNA extracted from root tissues of A. capillaris and L. perenne. PCR products were used to construct LSU rDNA libraries. Sequencing of randomly selected clones indicated that plant roots were colonised by AM fungi belonging to the genera Glomus, Acaulospora and Scutellospora. There was a difference in the diversity of AM fungi colonising roots of A. capillaris and L. perenne that was confirmed by PCR using primers specific for each sequence group. These molecular data suggest the existence of a selection pressure of plants on AM fungal communities.     

Chlamydospore formation of <Emphasis Type="Italic">Entoloma clypeatum</Emphasis> f. <Emphasis Type="Italic">hybridum </Emphasis>on mycorrhizas and rhizomorphs associated with <Emphasis Type="Italic">Rosa multiflora</Emphasis>

 Chlamydospores of Entoloma clypeatum f. hybridum were described on the mycorrhizas and rhizomorphs associated with Rosa multiflora. Their developmental pattern seems to be the Nyctalis type. This is the first report on chlamydospore formation on the mycorrhizae in entolomatoid fungi. Received: January 17, 2002 / Accepted: November 5, 2002 Acknowledgments K.H. is grateful to Emeritus Professor N. Sagara in Kyoto University, in whose laboratory part of this study was undertaken. Thanks are due to Mr. D. Sakuma for allowing the specimens to be kept in Osaka Museum of Natural History. Correspondence to:H. Kobayashi     

Genetic diversity and plant-growth related features of <Emphasis Type="Italic">Burkholderia</Emphasis> spp. from sugarcane roots

Brazil is the largest sugarcane producer in the world, mainly due to the development of different management strategies. Recently, microbial-plant related studies revealed that bacterial isolates belonging to the genus Burkholderia are mainly associated with this plant and are responsible for a range of physiological activity. In this study, we properly evaluate the physiological activity and genetic diversity of endophytic and rhizospheric Burkholderia spp. isolates from sugarcane roots grown in the field in Brazil. In total, 39 isolates previously identified as Burkholderia spp. were firstly evaluated for the capability to fix nitrogen, produce siderophores, solubilise inorganic phosphates, produce indole-acetic acid and inhibit sugarcane phytopathogens in vitro. These results revealed that all isolates present at least two positive evaluated activities. Furthermore, a phylogenetic study was carried out using 16S rRNA and gyrB genes revealing that most of the isolates were affiliated with the Burkholderia cepacia complex. Hence, a clear separation given by endophytic or rhizospheric niche occupation was not observed. These results presented an overview about Burkholderia spp. isolates from sugarcane roots and supply information about the physiological activity and genetic diversity of this genus, given direction for further studies related to achieve more sustainable cultivation of sugarcane.     

Strategies for Amplification by Polymerase Chain Reaction of the Complete Sequence of the Gene Encoding Nuclear Large Subunit Ribosomal RNA in Corals

The nearly complete nuclear large subunit ribosomal RNA (LSU rRNA) gene in corals was amplified by primers designed from polymerase chain reaction (PCR) strategies. The motif of the putative 3′-terminus of the LSU rRNA gene was sequenced and identified from intergenic spacer (IGS) clones obtained by PCR using universal primers designed for corals. The 3′-end primer was constructed in tandem with the universal 5′-end primer for the LSU rRNA gene. PCR fragments of 3500 bp were amplified for octocorals and non-Acropora scleractinian corals. More than 80% of the Acropora LSU rRNA gene (3000 bp) was successfully amplified by modification of the 5′-end of the IGS primer. Analysis of the 5′-end of LSU rDNA sequences, including the D1 and D2 divergent domains, indicates that the evolutionary rate of the LSU rDNA differs among these taxonomic groups of corals. The genus Acropora showed the highest divergence pattern in the LSU rRNA gene, and the presence of a long branch of the Acropora clade from the other scleractinian corals in the phylogenetic tree indicates that the evolutionary rate of Acropora LSU rDNA might have accelerated after divergence from the common ancestor of scleractinian corals. Received February 17, 2000; accepted June 12, 2000.     

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants

Abstract The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed. Received: 16 March 1999; Accepted: 27 August 1999; Online Publication: 23 February 2000     

Diazotrophic bacteria associated with banana (Musa spp.).

Nitrogen-fixing bacteria were isolated from surface sterilized banana (Musa spp.) plants and constituted a minor proportion of banana endophytic bacteria. Some isolates were characterized by alloenzyme profiles, biochemical tests, 16S rRNA and rpoB partial gene sequences, plasmid profiles and plant colonization. A large group of enterobacterial isolates that could not be clearly affiliated, most of them ascribed to group I (with characteristics of Enterobacter cloacae) were the diazotrophs most frequently found in banana. Different Klebsiella spp. and Rhizobiumsp. were identified as well. Klebsiella spp. were isolated from inside the roots and stems of plants grown in the two geographical regions sampled and from tissue culture-derived plantlets. Rhizobium sp. isolates were obtained only from Colima where bananas are grown extensively. Group I isolates and Rhizobium sp. could be re-isolated from surface-sterilized banana derived from tissue culture at five months after inoculation and significant increases in stem and leave fresh weight were obtained with some of the isolates.     

Growth promotion effect of Fusarium spp. PPF1 from bermudagrass (Cynodon dactylon) rhizosphere on Indian spinach (Basella alba) seedlings are linked to root colonisation

A rhizosphere fungus was isolated from roots of bermudagrass (Cynodon dactylon) and identified as Fusarium spp. PPF1. A pot experiment was conducted to test its ability to promote the vegetative growth of Indian spinach seedlings (Basella alba). Indian spinach seeds were sown in sterilised field soil amended with wheat grain inoculum of PPF1 at the rate of 0.5 and 1.0% w/w, and plants were grown for 21?days in a net house. Significantly, higher germination percentage and vigour index were observed due to application of PPF1 in the potting soil. Treatment with PPF1 also significantly increased shoot length, shoot fresh weight, shoot dry weight, root length, root fresh weight, root dry weight, leaf area and leaf chlorophyll content of cucumber plants compared to non-treated control. The growth promotion rate increased with the increasing concentration of inoculum of PPF1 applied to the soil. The fungus was re-isolated from the roots of cucumber plants at higher frequencies, while a positive co-relation was found between the root colonisation ability and the plant growth enhancement by the isolate. These results suggest that growth promotion effect of Fusarium spp. PPF1 on Indian spinach (B. alba) are linked to root colonisation ability of the fungus.     

Molecular phylogeny of an unidentified <Emphasis Type="Italic">Haliphthoros</Emphasis>-like marine oomycete and <Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> inferred from nuclear-encoded small- and large-subunit rRNA genes and mitochondrial-encoded cox2 gene

The SSU rRNA, LSU rRNA, and cox2 genes of an unidentified Haliphthoros-like marine oomycete (NJM0034) and Haliphthoros milfordensis (NJM0131) were sequenced, and their phylogenetic relationships are analyzed and discussed. All phylogenetic trees showed that NJM0034 and NJM0131 were branched before separation of the two main saprolegnian and peronosporalean clades. These data suggest that the clear phylogenetic separation of those marine oomycete endoparasites from the two main oomycete clades. Excepting the LSU rRNA gene tree, NJM0034 and Haliphthoros spp. did not form a monophyletic group. On the other hand, H. milfordensis NJM0131 clustered with H. philippinensis SANK 15178, not with H. milfordensis NJM9434 in the cox2 amino acid sequence (COII) tree. This result strongly suggests that a taxonomic reinvestigation of the genus Haliphthoros should be considered.     

Soil and root populations of fluorescent Pseudomonas spp. associated with seedlings and field-grown plants are affected by sorghum genotype

Sorghum [Sorghum bicolor (L.) Moench] is valued for bioenergy, feed and food. Potential of sorghum genotypes to support differing populations of root- and soil-associated fluorescent Pseudomonas spp. or Fusarium spp., in two soils, was assessed. Culturable pseudomonads were enumerated from roots and soil of sorghum (Redlan and RTx433) and wheat (Lewjain) seedlings repeatedly grown in cycled soils in the growth chamber. Pseudomonads and Fusarium spp. were assessed from roots and soil of field-grown sorghum along with biological control traits hydrogen cyanide (HCN) and 2,4-diacetylphlorogluconol (phl) production. After four 4-week cycles, soil associated with Redlan seedlings had greater numbers of fluorescent pseudomonads than Lewjain. In dryland field conditions, RTx433 roots had greater numbers of pseudomonads than Redlan before anthesis but similar numbers after. There were no differences in numbers of pseudomonads from dryland soil or roots or soil of irrigated plants. Percentages of HCN-producing root isolates and phl soil isolates declined on irrigated Redlan plants, but percentages of HCN-producers increased in dryland conditions. Redlan roots had greater percentages of Fusarium isolates in the Gibberella fujikuroi complex. Results indicated that sorghum genotype affected root-associated populations of fluorescent Pseudomonas spp. and Fusarium spp. across soil environments.