Wood, nematodes, and the nematode-trapping fungus Arthrobotrys oligospora

Researchers have proposed that Arthrobotrys oligospora and related fungi trap soil nematodes to obtain nitrogen and thereby compete saprophytically for carbon and energy in nitrogen-poor environments, including litter and wood. The current study tested two hypotheses concerning this model. The first was that wood decomposition would be enhanced if both large numbers of nematodes (a potential nitrogen supply) and A. oligospora (a cellulolytic organism that can use that N supply) were present. The second was that A. oligospora trapping activity would increase if large numbers of nematodes were added to soil containing abundant carbon (a wood dowel or chip). Although the first hypothesis was supported by an in vitro experiment on agar (A. oligospora degraded much more wood when nematodes were present), neither hypothesis was supported by an experiment in vials containing field soil. In soil, wood decomposition was unaffected by the addition of A. oligospora or large numbers of nematodes. Whereas A. oligospora trapped virtually all nematodes added to agar cultures, it trapped few or no nematodes added to soil. Given that the fungal isolate was obtained from the same soil and that the fungus increased to large numbers (>1×10³ propagules g⁻¹ soil), the failure of A. oligospora to trap nematodes in soil is difficult to explain. Soil nitrate levels, however, were high (71 mg kg⁻¹), and it is possible that with lower nitrate levels, trapping in soil might be stimulated by wood and nematodes.     

Impact of entomopathogenic nematodes on adults of Phyllotreta spp. (Coleoptera: Chrysomelidae) under laboratory conditions

Abstract

In 2005, preparations of four species of the entomopathogenic nematodes Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora, and H. megidis, were tested under laboratory conditions for their ability to kill adult flea beetles, Phyllotreta spp. (Coleoptera: Chrysomelidae). The nematode preparations were tested at doses of 200, 1000, and 2000 IJs per adult and at temperatures 15°C, 20°C, and 25°C. The numbers of beetles killed were recorded two, four, six, and eight days after treatment. The nematodes were more effective at 20°C and 25°C than at 15°C. At 20°C, the nematodes had killed between 44% (H. megidis at the lowest dose tested) and 77% (S. feltiae at the lowest dose tested) of the beetles eight days after treatment. At the two highest doses tested, the S. feltiae, S. carpocapsae, and H. bacteriophora preparations each killed at least 74% of the beetles at 25°C. Steinernema feltiae was the most effective nematode (LC₅₀=483–1467 IJs/adult) and, as an alternative to chemical insecticides, appears to have the highest potential for controlling overwintered flea beetles (May) under field conditions. The nematodes S. feltiae, S. carpocapsae and H. bacteriophora would all be suitable for controlling adult flea beetles during warm summer months, when flea beetles occur in high numbers in Slovenia.     

Agricultural insect pest compromises survival of two endemic Braya (Brassicaceae)

Agro-ecosystems support a vast array of non-native insects, but the potential of these insects to invade and degrade natural ecosystems is largely unknown. Plutella xylostella L. (diamondback moth) is a global agricultural pest that is not native to North America. It feeds on members of the Brassicaceae family, including the endangered Braya longii (Fernald) (Long’s braya) and threatened B. fernaldii (Abbe) (Fernald’s braya) which are endemic to the limestone barrens of Newfoundland, Canada. The immigration of P. xylostella from overwintering sites in the United States to this rare natural ecosystem was monitored with pheromone traps between 2003 and 2005. After their mass immigration in early summer, females lay eggs on an average of 30% of the B. longii and 16% of the B. fernaldii population. Larval feeding reduces the mean seed output of infested plants by 60%, from 10.8 to 4.3 seeds/fruit, and damages 26% of their leaves. There are residual and long-term effects of this herbivory, as many dead braya had higher numbers of eggs, and subsequent leaf and fruit damage one to three years before they died. High summer air temperatures and low precipitation allowed this pest to become multivoltine, resulting in additive damage to braya individuals. Presently, insufficient attention is directed to the impacts of agricultural pests on native ecosystems and rare host plants; hence, there is a need for both the conservation and agricultural communities to cooperate in mitigating the impacts of these pests on native biodiversity.     

The nematode-trapping fungus Arthrobotrys oligospora in soilof the Bodega marine reserve: distribution and dependenceon nematode-parasitized moth larvae

Of the 13 nematode-trapping fungi previously detected at the Bodega Marine Reserve (BMR, Sonoma County, CA, USA), Arthrobotrys oligospora is by far the most abundant. Why A. oligospora is so abundant is unclear, but the answer may involve bush lupines (Lupinus arboreus), ghost moth larvae (Hepialus californicus), and insect-parasitic nematodes (Heterorhabditis marelatus). Previous research documented a dramatic increase of A. oligospora in BMR soil with the addition of an H. marelatus-parasitized moth larva. The current study tested two predictions based on the hypothesis that the H. marelatus-parasitized ghost moth larva is a unique and important resource for A. oligospora at BMR. First, because ghost moth larvae are concentrated in soil under bush lupines, we predicted that A. oligospora numbers would be greater under lupines than away from lupines. Second, we predicted that A. oligospora would be enhanced more by moth larvae containing living H. marelatus than by moth larvae containing dead H. marelatus or no H. marelatus or by nematodes alone. The first prediction was supported by data from a field study (A. oligospora population density was greater beneath lupines than in grasses 2 m away), but the difference was small. The second prediction was not supported by data from a laboratory experiment (dead moth larvae caused dramatic increases in A. oligospora numbers whether or not the dead moth larvae contained living nematodes). While H. marelatus are clearly unnecessary for the large increase in A. oligospora numbers, the importance of nematodes in general remains unclear because addition of dead moth larvae always resulted in large increases in bacterivorous nematodes and because addition of nematodes alone enhanced A. oligospora in one trial but not in two others.     

紫外辐射对9个昆虫病原线虫品系存活率和致病力的影响

为探明紫外辐射对昆虫病原线虫（entomopathogenic nematodes,EPN）的作用,以甘肃省分离得到的昆虫病原线虫为研究材料,用室内模拟生测法,测定了经20 W、40 W紫外线辐射后,斯氏属（Steinernema feltiae,S.kraussei）和异小杆属（Heterorhabditis brevicaudis,H.megidis）4种昆虫病原线虫9个品系的存活率,并以大蜡螟（Galleria mellonella）为供试昆虫,采用改良的ONE ON ONE法,测定了其对大蜡螟的致病力。结果表明：经UV-B辐射后供试的9个昆虫病原线虫品系存活率未发生显著变化（P>0.05）。20 W紫外辐射30 min后,S.feltiae 0639C和0663TD 2品系对大蜡螟的致病力提高,其中S.felitiae 0639C致病力显著升高（P<0.05）,侵染32 h后,寄主昆虫死亡率由13.3%升高到73.3%;S.felitiae 0661YM品系的致病力显著降低（P<0.05）,侵染32 h后,寄主昆虫死亡率率由66.7%降低到0;其余各品系致病力均无显著变化。不同辐射时长和辐射强度对S.felitiae 0639C和0663TD引起寄主昆虫死亡的时间有影响,较对照组而言,经辐射预处理后,2品系线虫引起寄主昆虫死亡的时间均提前,其中S.felitiae 0639C随着辐射强度的增大引起寄主昆虫开始死亡时间提前,而S.felitiae 0663TD随着辐射强度增加引起寄主昆虫开始死亡时间延后。不同紫外辐射时长对S.felitiae 0663TD和S.felitiae 0639C致病力无显著影响,30 min、60 min、90 min、120 min辐射处理之间差异不显著（P>0.05）。研究表明,9个供试昆虫病原线虫品系均对UV-B辐射具有一定的耐受能力;20 W UV-B辐射30 min,能刺激S.felitiae 0663TD和S.felitiae 0639C侵染和致死寄主昆虫;一定的辐射时长内（120 min）,辐射时长对S.felitiae 0663TD和S.felitiae 0639C的致病力无影响。     

Colonisation of seminal roots of wheat and barley by egg-parasitic nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and development of root-rot

This study provides evidence that egg-parasitic nematophagous fungi, Pochonia chlamydosporia, Pochonia rubescens and Lecanicillium lecanii, can also reduce root colonisation and root damage by a fungal pathogen. Interactions of nematophagous fungi with the take-all fungus, Gaeumannomyces graminis var. tritici (Ggt), and their influence on severity of the root disease it causes were studied in laboratory and pot experiments. In Petri dish experiments the three nematophagous fungi reduced colonisation of barley roots by Ggt and also reduced necrotic symptoms. On the contrary, root colonisation by nematophagous fungi was unaffected by Ggt. In growth tube experiments, the three nematophagous fungi again reduced Ggt root colonisation and increased effective root length of barley seedlings. This was true for both simultaneous and sequential inoculation of nematophagous fungi versus Ggt. In the pot experiments the inoculum of the tested fungi in soil was applied in the same pot, as a mixture or in layers, or in coupled pots used for wheat grown with a split-root system. The nematophagous fungi P. chlamydosporia (isolate 4624) and L. lecanii (isolate 4629), mixed with Ggt or in split root systems with the pathogen, promoted growth of wheat (i.e. increased shoot weight), although no disease reduction was found. In split root systems, lower levels of peroxidase activity were found in seedlings inoculated with Ggt in combination with the nematophagous isolates 4624 and 4629 than when the take-all fungus was applied alone.Our results show that nematophagous fungi reduce root colonisation by Ggt, root damage and stress induced senescence in Ggt-inoculated plants.     

Pre-symbiotic and symbiotic interactions between Glomus intraradices and two Paenibacillus species isolated from AM propagules. In vitro and in vivo assays with soybean (AG043RG) as plant host

Two indole-producing Paenibacillus species, known to be associated with propagules of arbuscular mycorrhizal (AM) fungi, were examined for their mycorrhization helper bacteria activity at pre-symbiotic and symbiotic stages of the AM association. The effects were tested under in vitro and in vivo conditions using an axenically propagated strain of the AM fungus Glomus intraradices and Glycine max (soybean) as the plant host. The rates of spore germination and re-growth of intraradical mycelium were not affected by inoculation with Paenibacillus strains in spite of the variation of indole production measured in the bacterial supernatants. However, a significant promotion in pre-symbiotic mycelium development occurred after inoculation of both bacteria under in vitro conditions. The Paenibacillus rhizosphaerae strain TGX5E significantly increased the extraradical mycelium network, the rates of sporulation, and root colonization in the in vitro symbiotic association. These results were also observed in the rhizosphere of soybean plants grown under greenhouse conditions, when P. rhizosphaerae was co-inoculated with G. intraradices. However, soybean dry biomass production was not associated with the increased development and infectivity values of G. intraradices. Paenibacillus favisporus strain TG1R2 caused suppression of the parameters evaluated for G. intraradices during in vitro symbiotic stages, but not under in vivo conditions. The extraradical mycelium network produced and the colonization of soybean roots by G. intraradices were promoted compared to the control treatments. In addition, dual inoculation had a promoting effect on soybean biomass production. In summary, species of Paenibacillus associated with AM fungus structures in the soil, may have a promoting effect on short term pre-symbiotic mycelium development, and little impact on AM propagule germination. These findings could explain the associations found between some bacterial strains and AM fungus propagules.     

Molecular detection of nematode predation and scavenging in oribatid mites: Laboratory and field experiments

Recent stable isotope analyses indicate that a number of putative detritivorous soil microarthropods is not typical detritivores but rather live as predators or scavengers. Using molecular gut content analyses the present study investigates if nematodes indeed form part of the diet of oribatid mites. First, in a no-choice laboratory feeding experiment two nematode species (Phasmarhabditis hermaphrodita and Steinernema feltiae) were offered to eight species of oribatid mites and one gamasid mite. Second, after feeding for 4 and 48 h on each nematode species the detection time of prey DNA in the oribatid mite species Steganacarus magnus was investigated. Third, in a field experiment nematode prey (P. hermaphrodita and S. feltiae) in the diet of microarthropods was investigated distinguishing between scavenging and predation. In the no-choice laboratory experiment not only the gamasid mite but also several of the studied oribatid mite species consumed nematodes. After feeding on nematodes for 4 h prey DNA was detectable in S. magnus for only 4 h, but after feeding for 48 h prey DNA was detectable for 128 h, indicating that the duration of feeding on prey is an important determinant for prey DNA detection. The field experiment confirmed that oribatid mite species including Liacarus subterraneus, Platynothrus peltifer and S. magnus intensively prey on nematodes. Interestingly, DNA of dead P. hermaphrodita was detectable to a similar degree as that of living individuals indicating that scavenging is of significant importance in decomposer food webs. Results of our study indicate that predation and scavenging on nematodes by “detritivorous” microarthropods in soil food webs need to be reconsidered.     

Hydrolytic enzyme activities in maize (Zea mays) and sorghum (Sorghum bicolor) roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices

Two strains of Gluconacetobacter diazotrophicus (Pal 5, UAP5541) and the arbuscular mycorrhizal fungus Glomus intraradices increased both the shoot and root dry weight of sorghum 45 days after inoculation, whereas they had no effect on the shoot and root dry weight of maize. Co-inoculation (Gluconacetobacter diazotrophicus plus Glomus mosseae) did not increase the shoot and root dry weight of either plant. There was a synergistic effect of Gluconacetobacter diazotrophicus on root colonization of maize by Glomus intraradices, whereas an antagonistic interaction was observed in the sorghum root where the number of Gluconacetobacter diazotrophicus and the colonization by Glomus intraradices were reduced. Plant roots inoculated with Gluconacetobacter diazotrophicus and Glomus intraradices, either separately or together, significantly increased root endoglucanase, endopolymethylgalacturonase and endoxyloglucanase activities. The increase varied according to the plant. For example, in comparison with non-inoculated plants, there were higher endoglucanase (+328%), endopolymethylgalacturonase (+180%) and endoxyloglucanase (+125%) activities in 45-day old co-inoculated maize, but not in 45-day old sorghum. The possibility is discussed that hydrolytic enzyme activities were increased as a result of inoculation with Gluconacetobacter diazotrophicus, considering this to be one of the mechanisms by which these bacteria may increase root colonization by AM fungi.     

Soil feedback effects to the foredune grass Ammophila arenaria by endoparasitic root-feeding nematodes and whole soil communities

In coastal foredunes, the grass Ammophila arenaria develops a soil community that contributes to die-back and replacement by later successional plant species. Root-feeding nematodes and pathogenic soil microorganisms are involved in this negative feedback. Regular burial by wind-blown beach sand results in vigorous growth of A. arenaria, probably because of enabling a temporary escape from negative soil feedback. Here, we examine the role of root-feeding nematodes as compared to the whole soil community in causing negative feedback to A. arenaria. We performed a 3-year sand burial experiment in the field and every year we determined the feedback of different soil communities to plant growth in growth chamber bioassays.In the field, we established A. arenaria in tubes with beach sand, added three endoparasitic root-feeding nematode species (Meloidogyne maritima, Heterodera arenaria and Pratylenchus penetrans) or root zone soil to the plants, and created series of ceased and continued sand burial. During three subsequent years, plant biomass was measured and numbers of nematodes were counted. Every year, bioassays were performed with the field soils and biomass of seed-grown A. arenaria plants was measured to determine the strength of feedback of the established soil communities to the plant.In the field, addition of root zone soil had a negative effect on biomass of buried plants. In the bioassays, addition of root zone soil also reduced the biomass of newly planted seedlings, however, only in the case when the field plants had not been buried with beach sand. Addition of the three endoparasitic root-feeding nematodes did not influence plant biomass in the field and in the bioassays. Our results strongly suggest that the negative feedback to A. arenaria is not due to the combination of the three endoparasitic nematodes, but to other components in the soil community, or their interactions with the nematodes.     

Growth promoting effects of corn (Zea mays) bacterial isolates under greenhouse and field conditions

Fertilizer costs are a major component of corn production. The use of biofertilizers may be one way of reducing production costs. In this study we present isolation and identification of three plant growth promoting bacteria that were identified as Enterobacter cloacae (CR1), Pseudomonas putida (CR7) and Stenotrophomonas maltophilia (CR3). All bacterial strains produced IAA in the presence of 100 mg l⁻¹ of tryptophan and antifungal metabolites to several soilborne pathogens. S. maltophilia and E. cloacae had broad spectrum activity against most Fusarium species. The only strain that was positive for nitrogen fixation was E. cloacae and it, and P. putida, were also positive for phosphate solubilization. These bacteria and the corn isolate Sphingobacterium canadense CR11, and known plant growth promoting bacterium Burkholderia phytofirmans E24 were used to inoculate corn seed to examine growth promotion of two lines of corn, varieties 39D82 and 39M27 under greenhouse conditions. When grown in sterilized sand varieties 39M27 and 39D82 showed significant increases in total dry weights of root and shoot of 10-20% and 13-28% and 17-32% and 21-31% respectively. Plants of the two varieties grown in soil collected from a corn field had respective increases in dry weights of root and shoot of 10-30% and 12-35% and 11-19% and 10-18%. In sand, a bacterial mixture was highly effective whereas in soil individual bacteria namely P. putida CR7 and E. cloacae CR1 gave the best results with 39M27 and 39D82 respectively. These isolates and another corn isolate, Azospirillum zeae N7, were tested in a sandy soil with a 55 and 110 kg ha⁻¹ of nitrogen fertility at the Delhi research Station of Agriculture and Agri-Food Canada over two years. Although out of seven bacterial treatments, no treatment provided a statistically significant yield increase over control plots but S. canadense CR11 and A. zeae N7 provided statistically significant yield increase as compared to other bacteria. The 110 kg rate of nitrogen provided significant yield increase compared to the 55 kg rate in both years.     

The effect of feeding behavior on Hg accumulation in the ecophysiologically different earthworms Lumbricus terrestris and Octolaseon cyaneum: A microcosm experiment

 A soil microcosm experiment was performed to assess the uptake of Hg from various Hg-spiked food sources (soil, leaf litter and root litter of Trifolium alexandrinum) by two earthworm species, Lumbricus terrestris (anecic) and Octolaseon cyaneum (endogeic). Treatments were applied in which one of the three food sources was Hg spiked and the other two were not. Additional treatments in which all or none of the food sources were Hg spiked were used as controls. Uptake of Hg from soil into tissues of both earthworm species was significantly higher than uptake of Hg from leaf litter or root litter, indicating that soil may be the most important pool for the uptake of Hg into earthworms. In addition, the anecic L. terrestris significantly accumulated Hg from all Hg-spiked food sources (leaf litter, root litter and soil), whereas the endogeic O. cyaneum took up Hg mainly from soil particles. Interestingly, there was no further increase in Hg in L. terrestris when all food sources were Hg spiked compared to the single Hg-spiked sources. This may be attributed to the relatively high Hg content in the soil, which may have influenced the feeding behavior of the earthworms, although their biomass did not significantly decline. We suggest that, in addition to the physiological differences, feeding behavior may also play a role in the contrasting uptake of Hg by the two earthworm species.     

First indications for the involvement of strigolactones on nodule formation in alfalfa (Medicago sativa)

Strigolactones have recently been suggested to be phytohormones that are present in all plants. Strigolactones are released by roots into the rhizosphere, stimulating the seed germination of parasitic plants such as Striga spp. and Orobanche spp. and play a crucial role in the interaction between plants and symbiotic arbuscular mycorrhizal fungi.By applying different concentrations of the synthetic strigolactone analogue GR24 to alfalfa (Medicago sativa) inoculated with Sinorhizobium meliloti we could show that in alfalfa nodulation is positively affected by the presence of the strigolactone analogue GR24. Moreover, we could show that this increased nodulation cannot be linked with a stimulatory effect of GR24 on the growth or the expression of nod genes of S. meliloti.Putative mechanisms operating in the plant in response to the addition of GR24 and leading to increased nodule formation by rhizobia are discussed.     

Soil texture and irrigation influence the transport and the development of Pasteuria penetrans, a bacterial parasite of root-knot nematodes

The transport of the spores of Pasteuria penetrans was studied in three contrasted textured soils (a sandy, a sandy-clay and a clay soils), cultivated with tomato, inoculated with juveniles of Meloidogyne javanica and watered with 25 or 150 mm day⁻¹. One month after inoculation of the nematodes, 53% of the spores inoculated were leached by water flow in the sandy soil but only 14% in the sandy-clay soil and 0.1% in the clay soil. No nematodes survived in the clay soil, while the population was multiplied both in the sandy and in the sandy-clay soils. But juveniles of M. javanica were more infected by P. penetrans in the sandy-clay soil than in the sandy soil. Comparing different combinations of bare soils containing 1.1-57% of clay showed that the best spore percolation and retention balance occurred in soils amended with 10-30% clay. However, the spore recoveries decreased when the soil was enriched with more than 30% clay. The role of clay particles on the extractability of spores and on their availability to attach to the nematode cuticle in the soil is discussed.     

Inferring species distributions in the absence of occurrence records: An example considering wolverine (Gulo gulo) and Canada lynx (Lynx canadensis) in New Mexico

Information about geographic distributions is required for species conservation and management. Ultimately, this information is derived from records of occurrence. However, the reliability and availability of occurrence records are variable. A conceptual framework for evaluating the reliability of occurrence records is provided. Only records associated with physical evidence, especially a museum voucher specimen, are considered verified. However, errors in species identification or location are possible even for verified records. In addition, biases exist in occurrence records because they generally are collected haphazardly. Other sources of bias include sampling error associated with small areas or range limits and aspects of the species’ biology that make it unlikely to be documented. A practical method is provided for interpreting a species’ distribution in a particular area given a paucity of reliable occurrence records. Factors that must be considered for including such areas of interest within the range of a species include: (1) plausible reason for the paucity of records; (2) continuous suitable habitat between the area of interest and localities of reliable occurrence; and (3) absence of biogeographic breaks in the distribution of other organisms with similar evolutionary histories. The possible distribution of wolverine (Gulo gulo) and Canada lynx (Lynx canadensis) in New Mexico provides a case study of this approach. It is concluded that the mountains of north-central New Mexico should be considered within the natural range of wolverine and Canada lynx.     

Conserving marginal populations of the food plant (Impatiens noli-tangere) of an endangered moth (Eustroma reticulatum) in a changing climate

Impatiens noli-tangere is scarce in the UK and probably only native to the Lake District and Wales. It is the sole food plant for the endangered moth Eustroma reticulatum. Significant annual fluctuations in the size of I. noli-tangere populations endanger the continued presence of E. reticulatum in the UK. In this study, variation in population size was monitored across native populations of I. noli-tangere in the English Lake District and Wales. In 1998, there was a crash in the population size of all metapopulations in the Lake District but not of those found in Wales. A molecular survey of the genetic affinities of samples in 1999 from both regions and a reference population from Switzerland was performed using AFLP and ISSR analyses. The consensus UPGMA dendrogram and a PCO scatter plot revealed clear differentiation between the populations of I. noli-tangere in Wales and those in the Lake District. Most of the genetic variation in the UK (H_T=0.064) was partitioned between (G_ST=0.455) rather than within (H_S=0.034) regions, inferring little gene flow occurs between regions. There was similar bias towards differentiation between metapopulations in Wales, again consistent with low levels of interpopulation gene flow. This contrasts with far lower levels of differentiation in the Lake District which suggests modest rates of gene flow may occur between populations. It is concluded that in the event of local extinction of sites or populations, reintroductions should be restricted to samples collected from the same region. We then surveyed climatic variables to identify those most likely to cause local extinctions. Climatic correlates of population size were sought from two Lake District metapopulations situated close to a meteorological station. A combination of three climatic variables common to both sites explained 81-84% of the variation in plant number between 1990 and 2001. Projected trends for these climatic variables were used in a Monte Carlo simulation which suggested an increased risk of I. noli-tangere population crashes by 2050 at Coniston Water, but not at Derwentwater. Implications of these findings for practical conservation strategies are explored.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

Reproductive biology and conservation implications of three endangered snapdragon species (Antirrhinum, Plantaginaceae)

About 32% of Antirrhinum species are considered to be endangered; however, no field studies have focused on their reproductive biology. In this work, several aspects of the reproductive biology (flowering phenology, floral biology, breeding system) and potential limits on seed quantity and quality (pollen limitation, inbreeding depression) were studied in natural populations of three endangered species of the genus (Antirrhinum charidemi, Antirrhinum subbaeticum, Antirrhinum valentinum). Results disclose that all three species need insect visitors for seed production since fruit set after autonomous self-pollination was lower than under hand cross-pollination. A. charidemi and A. valentinum were mainly self-incompatible, whereas A. subbaeticum was self-compatible but herkogamous. Supplementary pollination in open-pollinated flowers only increased fruit set and seed set relative to controls in a given population of A. valentinum. Preliminary data on inbreeding depression at early life-cycle stages of the self-compatible A. subbaeticum revealed that the cumulative level was low. Despite the three species being closely related and sharing many ecological characteristics, they show different mating systems, and different factors limit seed quantity and quality. Thus, caution should be taken when making a common conservation plan for a group of closely related taxa.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.