Effects of carbohydrate substrate on the vegetative mycelial growth of an ectomycorrhizal mushroom, <Emphasis Type="Italic">Tricholoma matsutake</Emphasis>, isolated from <Emphasis Type="Italic">Quercus</Emphasis>

We studied the characteristics of the utilization of carbohydrate substrates and the production of those hydrolyzing enzymes of the Tricholoma matsutake J-1 strain isolated from hardwood (Quercus sp.). In the culture medium, 5% glucose inhibited mycelial growth. The growth inhibition rate was remarkable in the Z-1 strain from softwood (Pinus densiflora) compared with that of the J-1 strain from hardwood. α-Amylase production varied with starches from different origins in contrast to mycelial growth. The range of the effect of 0.5%–15% soluble starch on vegetative mycelial growth was also investigated. The optimal concentration for mycelial growth was 15% for the J-1 strain but 10% for the Z-1 strain. Mycelial growth of the J-1 strain was strongly inhibited in PMML medium containing Sunpeal-CP prepared from sulfite pulp softwood waste, but that of the Z-1 strain was not inhibited by Sunpeal-CP. Moreover, mycelial growth of the J-1 strain from Quercus sp. dramatically decreased with the addition of CNF-HWSF (hot water-soluble fractions from corn fiber) to the PMML and PDL medium. However, inhibition by CNF-HWSF was not shown in the Z-1 strain from P. densiflora.     

Microbial diversity associated with <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> fruiting bodies

Endophytes play an important role in the growth and development of the host. However, the study of endophytes is mostly focused on plants and animals, and reports on microorganisms associated with fungus are relatively rare. We studied the microorganisms associated with Tricholoma matsutake fruiting bodies picked from three main T. matsutake-producing areas in Sichuan, China, by both culture-dependent and culture- independent methods. Altogether 13 fungus, 15 yeast and 14 bacterial strains were isolated from the T. matsutake fruiting bodies. The most abundant cultivable fungus, yeast and bacteria isolates were assigned as Fusarium solanis, Cryptococcus sp. and Pseudomonas sp., respectively. Terminal-restriction fragment length polymorphism analysis (T-RFLP) showed that the bacteria in T. matsutake were abundant and diverse. Betaand gamma-proteobacteria, Acidobacteria, Bacteroidetes and Sphingobacterium were found in samples from all collecting sites. Among these bacteria, we may find some strains that can promote the growth of T. matsutake.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

The mycorrhizal fungus<Emphasis Type="Italic"> Tricholoma matsutake</Emphasis> stimulates<Emphasis Type="Italic"> Pinus densiflora</Emphasis> seedling growth in vitro

While it has been suggested that Matsutake mycorrhizae might not be functional and that Matsutake may behave as a saprobic fungus in soil or even have some pathogenic activity on seedlings, we investigated the consequences of Matsutake inoculation on Pinus densiflora growth. Seventy-five days after inoculation, hyphae were anchored on short roots and well-developed Hartig net palmettis were observed. Compared to both control treatments—seedlings treated with distilled water and seedlings treated with autoclaved mycelium—inoculation significantly stimulated seedling total dry weight by 70.9% and 98.0%, respectively. These findings attest that some type of symbiotic relationship must be functional and favour host growth, ruling out claims of pathogenicity under the sterile conditions used here.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Multiple paternity in <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis> confirmed by microsatellite analysis

The aim of this study was to determine if individual ticks among the progeny of a single female Rhipicephalus (Boophilus) microplus tick removed from cattle under natural conditions are the result of mating with one or several males. To this end, simulations were run using an existing dataset of genotypes from 8 microsatellite loci to predict the number of samples required and the best locus. Subsequently, 14–22 progeny from each of 15 engorged female ticks removed from three cows, and the engorged females themselves, were genotyped for the BmM1 locus and the minimum number of potential male parents was determined for each progeny group. Of the 15 progeny groups, 10 must have been sired by more than one male, as indicated by the presence of five unique alleles among the progeny or three unique alleles that could not have been contributed by the female. This finding demonstrates multiple paternity in R. microplus.     

Root-associated bacteria influencing mycelial growth of <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> (pine mushroom)

Tricholoma matsutake is an ectomycorrhizal fungus usually associated with Pinus densiflora in South Korea. Fruiting bodies (mushrooms) of T. matsutake are economically important due to their attractive aroma; yet, T. matsutake is uncultivatable and its habitat is rapidly being eradicated due to global climate change. Root-associated bacteria can influence the growth of ectomycorrhizal fungi that co-exist in the host rhizosphere and distinctive bacterial communities are associated with T. matsutake. In this study, we investigated how these bacterial communities affect T. matsutake growth by isolating bacteria from the roots of P. densiflora colonized by ectomycorrhizae of T. matsutake and co-culturing rootassociated bacteria with T. matsutake isolates. Thirteen species of bacteria (27 isolates) were found in pine roots, all belonging to the orders Bacillales or Burkholderiales. Two species in the genus Paenibacillus promoted the growth of T. matsutake in glucose poor conditions, likely using soluble metabolites. In contrast, other bacteria suppressed the growth of T. matsutake using both soluble and volatile metabolites. Antifungal activity was more frequent in glucose poor conditions. In general, pine rhizospheres harbored many bacteria that had a negative impact on T. matsutake growth and the few Paenibacillus species that promoted T. matsutake growth. Paenibacillus species, therefore, may represent a promising resource toward successful cultivation of T. matsutake.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Transcriptional analysis of <Emphasis Type="Italic">Pinus sylvestris</Emphasis> roots challenged with the ectomycorrhizal fungus <Emphasis Type="Italic">Laccaria bicolor</Emphasis>

Background  

Symbiotic ectomycorrhizal associations of fungi with forest trees play important and economically significant roles in the nutrition, growth and health of boreal forest trees, as well as in nutrient cycling. The ecology and physiology of ectomycorrhizal associations with Pinus sp are very well documented but very little is known about the molecular mechanisms behind these mutualistic interactions with gymnosperms as compared to angiosperms.     

Isolation and characterization of microsatellite DNA markers for the flagfish <Emphasis Type="Italic">Jordanella </Emphasis><Emphasis Type="Italic">floridae</Emphasis>

The flagfish (Jordanella floridae) is commonly used in studies of wetlands ecology. Here we describe the isolation of ten microsatellite loci, six of which were polymorphic. The observed number of alleles ranged from 4 to 24 and observed heterozygosities ranged from 0.59 to 0.81 for the polymorphic loci. The isolation of these markers will enable estimations of genetic diversity in natural populations.     

DNA-based geographic typing of the gourmet mushroom <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> traded in China

Tricholoma matsutake is among the most valuable mushrooms in the world, and natural populations of this species from different geographic regions are priced very differently. To examine the geographic origins of ‘matsutake’ mushrooms traded in China, we analyzed the mushrooms from two major production and trading regions in China, the Southwest and the Northeast, using a recently published retroelement-based DNA marker. Our analyses showed that 67% of commercial matsutake claimed to be from the northeast were in fact genetically identical to those from the southwest but were different from authentic northeast samples. The finding suggests that caution should be applied to the authentication of commercial matsutake from northeast China.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Camellia nitidissima</Emphasis>

Camellia nitidissima Chi (Theaceae), a rare and famous plant with golden-yellow flowers, narrowly distributes in Southwest China and North Vienam. It is now endangered and its distribution is ongoing shrinked due to deforestation and collection of seeds for horticulture. From repetitive DNA enriched libraries, we reported here the development and characterization of ten microsatellite markers for this species. The number of alleles ranged from 5 to 12. Observed and expected heterozygosities ranged from 0.4583 to 0.9474, and 0.6976 to 0.9033, respectively. These ten polymorphic microsatellite loci is the first set of microsatellite markers for C. nitidissima, and they will be used for future study of conservation genetics of this species.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

A qPCR assay that specifically quantifies <Emphasis Type="Italic">Tricholoma matsutake</Emphasis> biomass in natural soil

Tricholoma matsutake is an ectomycorrhizal basidiomycete that produces prized, yet uncultivable, “matsutake” mushrooms along densely developed mycelia, called “shiro,” in the rhizosphere of coniferous forests. Pinus densiflora is a major host of this fungus in Japan. Measuring T. matsutake biomass in soil allows us to determine the kinetics of fungal growth before and after fruiting, which is useful for analyzing the conditions of the shiro and its surrounding mycorrhizosphere, predicting fruiting timing, and managing forests to obtain better crop yields. Here, we document a novel method to quantify T. matsutake mycelia in soil by quantifying a single-copy DNA element that is uniquely conserved within T. matsutake but is absent from other fungal species, including close relatives and a wide range of ectomycorrhizal associates of P. densiflora. The targeted DNA region was amplified quantitatively in cultured mycelia that were mixed with other fungal species and soil, as well as in an in vitro co-culture system with P. densiflora seedlings. Using this method, we quantified T. matsutake mycelia not only from shiro in natural environments but also from the surrounding soil in which T. matsutake mycelia could not be observed by visual examination or distinguished by other means. It was demonstrated that the core of the shiro and its underlying area in the B horizon are predominantly composed of fungal mycelia. The fungal mass in the A or A₀ horizon was much lower, although many white mycelia were observed at the A horizon. Additionally, the rhizospheric fungal biomass peaked during the fruiting season.     

Development and characterization of microsatellite markers for <Emphasis Type="Italic">Prunus verecunda</Emphasis> and <Emphasis Type="Italic">Prunus grayana</Emphasis> (Rosaceae)

Simple sequence repeat (SSR) markers were developed for Prunus verecunda and Prunus grayana to help understand the seed dispersal pattern of each species. We isolated and characterized nine microsatellite loci (four from P. verecunda and five from P. grayana). In P. verecunda, the number of alleles detected and the expected heterozygosity of five loci ranged from 11 to 24 and 0.59 to 0.92, respectively. In P. grayana, the number of alleles detected and the expected heterozygosity of five loci ranged from 4 to 14 and 0.62 to 0.86, respectively. These results show that the markers described here will be useful in studying the seed dispersal pattern of Prunus species.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.