Analysis of quantitative interactions between two species of arbuscular mycorrhizal fungi, Glomus mosseae and G. intraradices, by real-time PCR

Arbuscular mycorrhizal fungi (AMF) are obligate biotrophs, known to play an important role in ecological processes. Conventional light microscopy is the most common method used to detect their presence in planta, but this method fails to discern the presence of multiple AMF species and is not quantitative. These two factors are critically important in ecological studies, where the symbiotic contribution of each isolate needs to be defined. This paper describes the use of quantitative real-time PCR (qRT-PCR) as a detection system to address this issue. We used two Glomus spp., namely, G. intraradices and G. mosseae, to show that it is possible to study the interactions between these two isolates during the cocolonization of a single root system. Three different physiological studies were set up to assess how the interactions affected the occupancy of these fungi intraradically on a temporal basis. These treatments included saline and phosphorus stress, spatial distribution in the root zone, and preference for a particular host. qRT-PCR could prove a valuable tool in the area of AMF field ecology, where such data are critically important for defining the role of each species in the community structure.     

Effects of estrone and 17 β-estradiol on vegetative growth of Medicago sativa

Alfalfa ( Medicago sativa L.) plants were grown in the absence or presence of the steroidal estrogens, estrone and 17β-estradiol, under varying conditions. Plants were analysed for the following parameters: plant weight, estrogen content, phytoestrogen content, degree of nodulation and nitrogen fixation activity. It was found that under controlled greenhouse conditions: (1) Treatment with estrogens in the range of 0.005 to 0.5 μg 1⁻¹ increases both shoot and root dry weitht. In contrast, estrogen in concentrations of 50 to 500 μg 1⁻¹ decreases plant growth. (2) The effect of estrogen of growth is most marked in the absence of nitrogen. (3) Estrone is more effective in increasing growth than 17 β-estradiol. (4) In the plants where estrogen induced growth there was no significant increase in nitrogen fixation activity and nodule number. (5) Endogenous estrogen content of the plant did not increase at concentrations (0.005-0.5 μg 1⁻¹) which increased vegetative growth. (6) Endogenous estrogen content of the plant did increase at concentrations of estrogen (50-500 μg 1⁻¹ which inhibited vegetative growth and nodule weight. It can be concluded that estrogen in concentrations found in sewage water (0.3 μg estrogen 1⁻¹) can affect the vegetative growth of alfalfa plants.     

The Role of EDTA in Lead Transport and Accumulation by Indian Mustard

Indian mustard (Brassica juncea) plants exposed to Pb and EDTA in hydroponic solution were able to accumulate up to 55 mmol kg⁻¹ Pb in dry shoot tissue (1.1% [w/w]). This represents a 75-fold concentration of Pb in shoot tissue over that in solution. A threshold concentration of EDTA (0.25 mm) was found to be required to stimulate this dramatic accumulation of both Pb and EDTA in shoots. Below this threshold concentration, EDTA also accumulated in shoots but at a reduced rate. Direct measurement of a complex of Pb and EDTA (Pb-EDTA) in xylem exudate of Indian mustard confirmed that the majority of Pb in these plants is transported in coordination with EDTA. The accumulation of EDTA in shoot tissue was also observed to be directly correlated with the accumulation of Pb. Exposure of Indian mustard to high concentrations of Pb and EDTA caused reductions in both the transpiration rate and the shoot water content. The onset of these symptoms was correlated with the presence of free protonated EDTA (H-EDTA) in the hydroponic solution, suggesting that free H-EDTA is more phytotoxic than Pb-EDTA. These studies clearly demonstrate that coordination of Pb transport by EDTA enhances the mobility within the plants of this otherwise insoluble metal ion, allowing plants to accumulate high concentrations of Pb in shoots. The finding that both H-EDTA and Pb-EDTA are mobile within plants also has important implications for the use of metal chelates in plant nutritional research.The synthetic chelate EDTA forms a soluble complex with many metals, including Pb (Kroschwitz, 1995), and can solubilize Pb from soil particles (Means and Crerar, 1978). Recently, application of EDTA to Pb-contaminated soils has been shown to induce the uptake of Pb by plants (Jøgensen, 1993; Huang and Cunningham, 1996; Blaylock et al., 1997; Huang et al., 1997), causing Pb to accumulate to more than 1% (w/w) of shoot dry biomass (Huang and Cunningham, 1996; Blaylock et al., 1997; Huang et al., 1997). For the in situ remediation of Pb-contaminated soils it appears that this chelate-assisted phytoextraction strategy (Salt et al., 1998) may be more effective than a strategy based on the natural ability of certain wild plant species for metal hyperaccumulation (Chaney, 1983; Baker et al., 1988).For more than 40 years, synthetic chelates have been used to supply plants with micronutrients in both soil and hydroponics. Yet the mechanisms by which chelates enhance metal accumulation are still not well characterized (Wallace and Wallace, 1992), and what is known appears contradictory. For example, some evidence suggests that the Fe-chelate EDTA can be absorbed by plants and translocated to shoots (Weinstein et al., 1954; Hill-Cottingham and Llyod-Jones, 1961, 1965). However, Tiffin et al. (1960) concluded that Fe-chelates are excluded from root tissue, and this was supported by Chaney et al. (1972), who demonstrated that Fe is taken up by plants only after first being split from the Fe-chelate complex by the action of a specific plasma membrane-bound Fe-chelate reductase.To optimize the process of chelate-assisted phytoextraction, it is important to understand the biological mechanisms responsible for this process. Because of the stimulatory role of chelate application in the uptake of Pb and other metals by plants, we have investigated the role of EDTA in Pb accumulation in plants. In this study we have demonstrated that the previously described EDTA-enhanced Pb accumulation in Indian mustard (Brassica juncea) is based on the ability of EDTA to chelate and transport Pb from soil into shoot tissue.     

A Vesicular Arbuscular Mycorrhizal Fungus (Glomus intraradix) Induces a Defense Response in Alfalfa Roots

Flavonoid accumulation and activities of phenylalanine ammonia-lyase (PAL), chalcone isomerase (CHI), and chitinase were followed during early colonization of alfalfa roots (Medicago sativa L. cv Gilboa) by vesicular arbuscular (VA) fungi (Glomus intraradix). Formononetin was the only flavonoid detected that showed a consistent increase in the inoculated roots. This increase depended only on the presence of the fungus in the plant rhizosphere; no colonization of the root tissue was required. CHI and chitinase activities increased in inoculated roots prior to colonization, whereas the increase in PAL activity coincided with colonization. After reaching a maximum, activities of all enzymes declined to below those of uninoculated roots. PAL inactivation was not caused by a soluble inhibitor. Our results indicate that VA fungi initiate a host defense response in alfalfa roots, which is subsequently suppressed.     

Mycorrhizal association between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum alters plant physiology and fitness to arid conditions

The host plant Helianthemum sessiliflorum was inoculated with the mycorrhizal desert truffle Terfezia boudieri Chatin, and the subsequent effects of the ectomycorrhizal relationship on host physiology were determined. Diurnal measurements revealed that mycorrhizal (M) plants had higher rates of photosynthesis (35%), transpiration (18%), and night respiration (49%) than non-mycorrhizal (NM) plants. Consequently, M plants exhibited higher biomass accumulation, higher shoot-to-root ratios, and improved water use efficiency compared to NM plants. Total chlorophyll content was higher in M plants, and the ratio between chlorophyll a to chlorophyll b was altered in M plants. The increase in chlorophyll b content was significantly higher than the increase in chlorophyll a content (2.58- and 1.52-fold, respectively) compared to control. Calculation of the photosynthetic activation energy indicated lower energy requirements for CO₂ assimilation in M plants than in NM plants (48.62 and 61.56 kJ mol⁻¹, respectively). Continuous measurements of CO₂ exchange and transpiration in M plants versus NM plants provided a complete picture of the daily physiological differences brought on by the ectomycorrhizal relationships. The enhanced competence of M plants to withstand the harsh environmental conditions of the desert is discussed in view of the mycorrhizal-derived alterations in host physiology.     

An active factor from tomato root exudates plays an important role in efficient establishment of mycorrhizal symbiosis

Root exudates play an important role in the early signal exchange between host plants and arbuscular mycorrhizal fungi. M161, a pre-mycorrhizal infection (pmi) mutant of the tomoto (Solanum lycopersicum) cultivar Micro-Tom, fails to establish normal arbuscular mycorrhizal symbioses, and produces exudates that are unable to stimulate hyphal growth and branching of Glomus intraradices. Here, we report the identification of a purified active factor (AF) that is present in the root exudates of wild-type tomato, but absent in those of M161. A complementation assay using the dual root organ culture system showed that the AF could induce fungal growth and branching at the pre-infection stage and, subsequently, the formation of viable new spores in the M161 background. Since the AF-mediated stimulation of hyphal growth and branching requires the presence of the M161 root, our data suggest that the AF is essential but not sufficient for hyphal growth and branching. We propose that the AF, which remains to be chemically determined, represents a plant signal molecule that plays an important role in the efficient establishment of mycorrhizal symbioses.     

Suitability of mycorrhiza-defective mutant/wildtype plant pairs (Solanum lycopersicum L. cv Micro-Tom) to address questions in mycorrhizal soil ecology

The ectomycorrhizal (ECM) fungi associated with Pinus thunbergii seedlings grown on sand dune were identified by molecular method, and the diversity of bacteria associated with ECM and Extraradical mycelium were examined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified 16S rDNA. The mycorrhizal formation rate of 1-year old P. thunbergii seedlings was more than 95%. Cenococcum geophilum was the most dominant ECM fungus, followed by T01, RFLP-8, Russula spp., and Suillus sp. Bacterial community was most diverse with C. geophilum- and RFLP-8-mycorrhiza. Sequencing analysis showed that Burkholderia spp. and Bradyrhizobium spp. were on the surface of ECM short root of seven ECM. The fungi detected as extraradical mycelium using DGGE of 18S rDNA were Suillus bovinus and RFLP-8-mycorrhiza. Bacterial community on the extraradical mycelium was more diverse than those on ECM root tip. Burkholderia spp. and Bradyrhizobium spp. were found also on extraradical mycelium.     

Signal Transduction Pathways in Mycorrhizal Associations: Comparisons with theRhizobium–Legume Symbiosis

A number of genera of soil fungi interact with plant roots to establish symbiotic associations whereby phosphate acquired by the fungus is exchanged for fixed carbon from the plant. Recent progress in investigating these associations, designated as mycorrhizae (sing., mycorrhiza), has led to the identification of specific steps in the establishment of the symbiosis in which the fungus and the plant interact in response to various molecular signals. Some of these signals are conserved with those of theRhizobium–legume nitrogen-fixing symbiosis, suggesting that the two plant–microbe interactions share a common signal transduction pathway. Nevertheless, only legume hosts nodulate in response toRhizobium,whereas the vast majority of flowering plants establish mycorrhizal associations. The key questions for the future are: what are the signal molecules produced by mycorrhizal fungi and how are they perceived by the plant?