Arbuscular mycorrhizal fungi in roots of non-photosynthetic plants, <Emphasis Type="Italic">Sciaphila japonica</Emphasis> and <Emphasis Type="Italic">Sciaphila tosaensis</Emphasis> (Triuridaceae)

The mycorrhizal fungi in the roots of achlorophyllous Sciaphila japonica and S. tosaensis (Triuridaceae) were identified by molecular methods. The habitats of S. japonica were in a tree plantation of Japanese cypress, Chamaecyparis obtusa, and bamboo forests, and those of S. tosaensis were in a camellia forest and a bamboo forest. In the root cortical cells of both plants, aseptate hyphal coils were observed, which suggested the Paris-type arbuscular mycorrhiza (AM). A phylogenetic analysis based on a partial sequence of an AM fungal nuclear small subunit ribosomal RNA gene showed that the fungal DNA sequences of S. japonica were separated into three closely related clades. Those of S. tosaensis were separated into two clades, which were also closely related to each other. The AM fungi of S. japonica and S. tosaensis were completely separated in the phylogenetic tree even among those found in the same habitat, which suggests the high specificities in the plant-fungal partnerships. All the detected AM fungi in these plants belonged to Glomus-group A. Even though the habitats are in quite common environments, both plant species are known as endangered species in Japan. Such a definite specificity in AM symbioses seems to restrict the distribution of the myco-heterotrophic plants.     

Morphology and colonization preference of arbuscular mycorrhizal fungi in <Emphasis Type="Italic">Clethra barbinervis</Emphasis>, <Emphasis Type="Italic">Cucumis sativus</Emphasis>, and <Emphasis Type="Italic">Lycopersicon esculentum</Emphasis>

Clethra barbinervis (Ericales), Cucumis sativus, and Lycopersicon esculentum were grown in soils collected from six different vegetation sites (cedar, cypress, larch, red pine, bamboo grass, and Italian ryegrass), and morphology and colonization preference of arbuscular mycorrhizal (AM) fungi were investigated by microscopic observation and PCR detection. C. barbinervis consistently formed Paris-type AM throughout the sites. C. sativus formed both Arum- and Paris-type AM with high occurrence of Arum-type AM. L. esculentum also formed both Arum- and Paris-type AM but with high occurrence of Paris-type AM. AM diversity within the same plant species was different among the sites. Detected AM diversity from AM spores in different site soils did not consistently reflect AM fungal diversity seen in test plants. Detected families were different, depending on test plants grown even in the same soil. AM fungi belonging to Glomaceae were consistently detected from roots of all test plants throughout the sites. Almost all the families were detected from roots of C. barbinervis and L. esculentum. On the other hand, only two or three families of AM fungi (Archaeosporaceae and/or Paraglomaceae and Glomaceae) but not two other families (Acaulosporaceae and Gigasporaceae) were detected from roots of C. sativus, indicating strong colonization preference of AM fungi to C. sativus among test plants. This study demonstrated that host plant species strongly influenced the colonization preference of AM fungi in the roots.     

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.     

<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.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

<Emphasis Type="Italic">Amanita concentrica</Emphasis>: a new species in <Emphasis Type="Italic">Amanita</Emphasis> section <Emphasis Type="Italic">Amanita</Emphasis> from Japan

Amanita concentrica sp. nov. is described from an evergreen broad-leaved forest of Chiba Prefecture with Castanopsis cuspidata var. sieboldii and Quercus glauca. The fungus is a medium to large, white to yellowish-white mushroom characterized by many pyramidal warts on pileus, striate margin of pileus, distinct ascending concen-tric rings at upper part of stipe bulb, and inamyloid basidiospores. It is considered to belong to the section Amanita. Received: March 30, 2001 / Accepted: October 1, 2001     

<Emphasis Type="Italic">Rf5</Emphasis> is able to partially restore fertility to Honglian-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Three-line japonica hybrids have been developed mainly on Chinsurah Boro II (BT)-type cytoplasmic male sterile (CMS) lines of Oryza sativa L., but the unstable sterility of some BT-type CMS lines, and the threat of genetic vulnerability when using a single cytoplasm source, have inhibited their use in rice cultivation. Previously, the sterility of Honglian (HL)-type japonica CMS lines derived from common red-awned wild rice (Oryza rufipogon) has been proven to be more stable than that of BT-type japonica CMS lines. Here, we genetically characterized HL-type japonica CMS lines and the restorer-of-fertility (Rf) gene for breeding HL-type japonica hybrids. HL-type japonica CMS lines displayed stained abortive pollen grains, unlike HL-type indica CMS lines. The BT-type japonica restorer lines, which contain Rf, had different capabilities to restore HL-LiuqianxinA (HL-LqxA), an HL-type japonica CMS line, and the restorers for the HL-type japonica CMS lines could be selected from the preexisting BT-type japonica restorers in rice production. A genetic analysis showed that the restoration of normal fertility to HL-LqxA was controlled by a major gene and was affected by minor effector genes and/or modifiers. The major Rf in SiR2982, a BT-type japonica restorer, was mapped to a ~100-kb physical region on chromosome 10, and was demonstrated to be Rf5 (Rf1a) by sequencing. Furthermore, Rf5 partially restored fertility and had a dosage effect on HL-type japonica CMS lines. These results will be helpful for the development of HL-type japonica hybrids.     

Fungal associates of <Emphasis Type="Italic">Pyrola rotundifolia</Emphasis>, a mixotrophic Ericaceae,from two Estonian boreal forests

Pyrola rotundifolia (Ericaceae, Pyroleae tribe) is an understorey subshrub that was recently demonstrated to receive considerable amount of carbon from its fungal mycorrhizal associates. So far, little is known of the identity of these fungi and the mycorrhizal anatomy in the Pyroleae. Using 140 mycorrhizal root fragments collected from two Estonian boreal forests already studied in the context of mixotrophic Ericaceae in sequence analysis of the ribosomal DNA internal transcribed spacer region, we recovered 71 sequences that corresponded to 45 putative species in 19 fungal genera. The identified fungi were mainly ectomycorrhizal basidiomycetes, including Tomentella, Cortinarius, Russula, Hebeloma, as well as some ectomycorrhizal and/or endophytic ascomycetes. The P. rotundifolia fungal communities of the two forests did not differ significantly in terms of species richness, diversity and nutritional mode. The relatively high diversity retrieved suggests that P. rotundifolia does not have a strict preference for any fungal taxa. Anatomical analyses showed typical arbutoid mycorrhizae, with variable mantle structures, uniseriate Hartig nets and intracellular hyphal coils in the large epidermal cells. Whenever compared, fungal ultrastructure was congruent with the molecular identification. Similarly to other mixotrophic and autotrophic pyroloids in the same forests, P. rotundifolia shares its mycorrhizal fungal associates with surrounding trees that are likely a carbon source for pyroloids.     

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.     

<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.     

<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.     

New combinations and typifications in <Emphasis Type="Italic">Bistorta</Emphasis>, <Emphasis Type="Italic">Persicaria</Emphasis>, <Emphasis Type="Italic">Polygonum,</Emphasis> and <Emphasis Type="Italic">Rumex</Emphasis> (Polygonaceae)

New combinations are proposed in anticipation of the Polygonaceae treatment in the forthcoming volume of Intermountain Flora: Polygonum kelloggii var. esotericum, P. kelloggii var. watsonii , Rumex densiflorus var. pycnanthus , R. salicifolius var. utahensis, and R. occidentalis var. tomentellus. Typifications are proposed to facilitate ongoing studies in Polygonaceae and to maintain current usage.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Rhizobium</Emphasis> strains in the biological control of the phytopathogenic fungi <Emphasis Type="Italic">Sclerotium</Emphasis> (<Emphasis Type="Italic">Athelia</Emphasis>) <Emphasis Type="Italic">rolfsii</Emphasis> on the common bean

Aims

To identify Rhizobium strains’ ability to biocontrol Sclerotium rolfsii, a fungus that causes serious damage to the common bean and other important crops, 78 previously isolated rhizobia from common bean were assessed.

Methods

Dual cultures, volatiles, indole-acetic acid (IAA), siderophore production and 16S rRNA sequencing were employed to select strains for pot and field experiments.

Results

Thirty-three antagonistic strains were detected in dual cultures, 16 of which were able to inhibit ≥84% fungus mycelial growth. Antagonistic strains produced up to 36.5 μg mL^?1 of IAA, and a direct correlation was verified between IAA production and mycelium inhibition. SEMIA 460 inhibited 45% of mycelial growth through volatile compounds. 16S rRNA sequences confirmed strains as Rhizobium species. In pot condition, common bean plants grown on S. rolfsii-infested soil and inoculated with SEMIA 4032, 4077, 4088, 4080, 4085, or 439 presented less or no disease symptoms. The most efficient strains under field conditions, SEMIA 439 and 4088, decreased disease incidence by 18.3 and 14.5% of the S. rolfsii-infested control.

Conclusions

Rhizobium strains could be strong antagonists towards S. rolfsii growth. SEMIA 4032, 4077, 4088, 4080, 4085, and 439 are effective in the biological control of the collar rot of the common bean.

     

Characterization and expression of <Emphasis Type="Italic">DjPreb</Emphasis> gene in the planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In this study, we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5’-UTR and a 60 bp 3’-UTR. Phylogenetic analysis shows that DjPreb is PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient express with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.     

Prevalence,intensity and associated factor analysis of <Emphasis Type="Italic">Tropilaelaps</Emphasis><Emphasis Type="Italic">mercedesae</Emphasis> infesting <Emphasis Type="Italic">Apis</Emphasis><Emphasis Type="Italic">mellifera</Emphasis> in China

Tropilaelaps mercedesae is a serious ectoparasite of Apis mellifera in China. The aim of this study was to investigate the infestation rates and intensity of T. mercedesae in A. mellifera in China, and to explore the relative importance of climate, district, management practices and beekeeper characteristics that are assumed to be associated with the intensity of T. mercedesae. Of the 410 participating apiaries, 379 apiaries were included in analyses of seasonal infestation rates and 352 apiaries were included in multivariable regression analysis. The highest infestation rate (86.3%) of T. mercedesae was encountered in autumn, followed by summer (66.5%), spring (17.2%) and winter (14.8%). In autumn, 28.9% (93) of the infested apiaries were in the north (including the northeast and northwest of China), 71.1% (229) were in the central and south (including east, southeast and southwest China), and 306 apiaries (82.9%) were co-infested by both T. mercedesae and Varroa. Multivariable regression analysis showed that geographical location, season, royal jelly collection and Varroa infestation were the factors that influence the intensity of T. mercedesae. The influence of beekeeper’s education, time of beekeeping, operation size, and hive migration on the intensity of T. mercedesa was not statistically significant. This study provided information about the establishment of the linkage of the environment and the parasite and could lead to better timing and methods of control.     

Arbuscular mycorrhizal fungi colonize decomposing leaves of<Emphasis Type="Italic"> Myrica parvifolia</Emphasis>,<Emphasis Type="Italic"> M. pubescens</Emphasis> and<Emphasis Type="Italic"> Paepalanthus</Emphasis> sp.

Hyphae and vesicles of arbuscular mycorrhizal fungi (AMF) were found within the decomposing leaves of Myrica parvifolia, M. pubescens and Paepalanthus sp. at three montane sites in Colombia. Hyphae, vesicles, and arbuscule-like structures were also found within scale-like leaves of the rhizomes of Paepalanthus sp. The litter found in the vicinity of the roots was divided into three decomposition layers. The highest AMF colonization occurred in the most decomposed leaves, which were in close association with roots. In contrast, there were no differences in AMF colonization of roots present in the different decomposition layers. Colonization of decomposing leaves by AMF did not differ between the two closely related species M. parvifolia and M. pubescens, nor between two sites (Guatavita and Zipacón, Colombia) differing in soil fertility. Occurrence of vesicles in decomposing leaves was correlated with abundant AMF extraradical hyphae among the leaves. We propose that AMF enter decomposing leaves mechanically through vascular tissue. As a consequence, AMF are well positioned to obtain and efficiently recycle mineral nutrients released by decomposer microorganisms before their loss by leaching or immobilization in soil.     

Feeding behaviour,efficiency and food preference in yabbies <Emphasis Type="Italic">Cherax</Emphasis><Emphasis Type="Italic"> destructor</Emphasis>

The influence of body size on the consumption of live zooplankton (Daphnia spp.) by freshwater crayfish was examined using yabbies (Cherax destructor) ranging from 5 to 45 g. Food preference between live zooplankton and inert pellets was also assessed under experimental conditions. In experimental tanks, yabbies of four size classes (<15, 15–24.9, 25–34.9 and 35–45 g) were presented with live Daphnia. All yabbies were held in separate tanks with five animals per size class. In yabbies less than 15 g, the feeding mode on zooplankton involved rapid searching and probing with the first two pairs of walking legs. Once a prey was located, the chelae on the end of these walking legs would grasp the zooplankton and then rapidly move it towards the mouthparts. Yabbies larger than 25 g tended to use their walking legs to push the Daphnia nearer to their third maxilliped which would then force or scoop the zooplankton towards the mouthparts. A short-term feeding trial showed that there was no significant difference between size classes in regards to zooplankton consumption (P > 0.05). Capture efficiency of live Daphnia by yabbies less than 15 g was significantly lower (76%, P = 0.008) than the three larger size classes (93.6%). Yabbies less than 15 g consumed a significantly (P < 0.001) higher percentage (5.2%) of their body weight than the other size classes (1.1%, 0.8%, and 0.6%, respectively). In the presence of both live zooplankton and a pellet diet, yabbies spent significantly (P = 0.005) more time feeding on zooplankton (85%) than on inert pellets (15%). This was the first study to quantify zooplankton consumption by yabbies and the results provide insights into understanding the trophic role of freshwater crayfish in structuring zooplankton communities and the husbandry management of crayfish farming. Handling editor: S. I. Dodson