Tuber aestivum association with non-host roots

Mycorrhizal fungi provide direct and functional interconnection of soil environment with their host plant roots. Colonization of non-host plants have occasionally been described, but its intensity and functional significance in complex plant communities remain generally unknown. Here, the abundance of ectomycorrhizal fungus Tuber aestivum was measured in the roots of host and non-host (non-ectomycorrhizal) plants in a naturally occurring T. aestivum colony using a quantitative PCR approach. The roots of non-host plant species found inside the brûlé area were extensively colonized by T. aestivum mycelium, although the levels were significantly lower than those found in host Carpinus betulus roots. However, fungal biomass concentration in the non-host roots was one to two orders of magnitude higher than that in the surrounding soil. This indicates existence of an important biotic interaction between T. aestivum mycelium and the non-host, mostly herbaceous plants. Roots, either host or non-host, thus probably constitute hot spots of T. aestivum activity in the soil ecosystem with as yet uncovered functional significance.     

Quantification of roots and seeds in soil with real-time PCR

Study of roots and associated organisms in soil particularly in mixed plant populations, such as pastures, is limited by difficulties in quantification of root growth and function. The research evaluated the potential of DNA quantification by real-time PCR to improve our capacity to study and understand roots in such contexts. Probes and primers were developed for two common pasture species, Trifolium subterraneum and Lolium perenne (and closely related Lolium spp.), and evaluated for specificity and sensitivity in TaqMan assays on DNA extracted from soil. Further experiments examined the ability to detect DNA in dead roots, the changes in root DNA levels of plants defoliated or treated with herbicide and the relationship between DNA and root dry weight for single and mixed plant species grown in pots. T. subterraneum DNA/PCR 200 fg/µl was detected at 17.5 cycles and L. perenne at 19.5 cycles. The assay for T. subterraneum was species specific but the L. perenne assay, as anticipated from the choice of probe, also detected some closely related species. The assays were sensitive and capable of detecting equivalent to <2 mg roots/kg of dry soil and able to quantify targets in mixed populations. DNA concentration varied with plant age and genotype and DNA in dead roots found to decay rapidly over a few days. DNA concentrations in roots were found to respond more rapidly to defoliation and herbicide treatments than root mass. This approach appears to offer a new way to study roots in soil and indicates that quantifying root DNA could provide insights into root function and responses not readily provided by other methods.     

Influence of inoculation with Glomus mosseae or Acaulospora morrowiae on arsenic uptake and translocation by maize

A split root device was designed to assess the possible role of AMF in translocation and detoxification of As by maize plants. Half of each maize root system grew in As-amended or unamended soil and the remainder was inoculated with either Glomus mosseae or Acaulospora morrowiae. External mycelium was collected from a third compartment. Neither shoot nor root As concentrations were affected by inoculation with either fungus. Soil As amendment produced higher As concentrations in roots in the second compartment and in the external mycelium. The As concentrations in the matrix solution of the second root compartment were lower in mycorrhizal treatments with no differences in soluble As in the hyphal compartments. Mycorrhiza exerted little effect on As translocation within plants but may have influenced root As efflux. Deposition of As in external mycelium indicates a possible role of mycorrhizal fungi in the detoxification of As in the host plants.     

The symbiotic recapture of nitrogen from dead mycorrhizal and non-mycorrhizal roots of tomato plants

Aims

The aim was to quantify the nitrogen (N) transferred via the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices from both a dead host and a dead non-host donor root to a receiver tomato plant. The effect of a physical disruption of the soil containing donor plant roots and fungal mycelium on the effectiveness of N transfer was also examined.

Methods

The root systems of the donor (wild type tomato plants or the mycorrhiza-defective rmc mutant tomato) and the receiver plants were separated by a 30 μm mesh, penetrable by hyphae but not by the roots. Both donor genotypes produced a similar quantity of biomass and had a similar nutrient status. Two weeks after the supply of ¹⁵?N to a split-root part of donor plants, the shoots were removed to kill the plants. The quantity of N transferred from the dead roots into the receiver plants was measured after a further 2 weeks.

Results

Up to 10.6 % of donor-root ¹⁵N was recovered in the receiver plants when inoculated with the arbuscular mycorrhizal fungus (AMF). The quantity of ¹⁵N derived from the mycorrhizal wild type roots clearly exceeded that from the only weakly surface-colonised rmc roots. Hyphal length in the donor rmc root compartments was only about half that in the wild type compartments. The disruption of the soil led to a significantly increased AMF-mediated transfer of N to the receiver plants.

Conclusions

The transfer of N from dead roots can be enhanced by AMF, especially when the donor roots have been formerly colonised by AMF. The transfer can be further increased with higher hyphae length densities, and the present data also suggest that a direct link between receiver mycelium and internal fungal structures in dead roots may in addition facilitate N transfer. The mechanical disruption of soil containing dead roots may increase the subsequent availability of nutrients, thus promoting mycorrhizal N uptake. When associated with a living plant, the external mycelium of G. intraradices is readily able to re-establish itself in the soil following disruption and functions as a transfer vessel.     

Studies on transmission of Indian peanut clump virus disease by Polymyxa graminis*

The plasmodiophoromycete fungus, Polymyxa graminis was observed in the roots of Sorghum bicolor, S. sudanense, Pennisetum glaucum, Triticum aestivum, Cyperus rotundus, Eleucine coracana, Zea mays, Tridax procumbens and Arachis hypogaea collected from Indian peanut clump virus (IPCV)-infested fields. Examination of roots of IPCV-infected S. bicolor, S. sudanense, P. glaucum and T. aestivum grown in previously air dried field soil also showed the presence of cystosori of P. graminis. IPCV-infested soil stored at room temperature for 3 years transmitted the virus to A. hypogaea, T. aestivum and S. bicolor. Roots extracted from IPCV-infected P. glaucum and S. bicolor containing cystosori, and dried root fragments incorporated into sterile soil, transmitted the virus to A. hypogaea and T. aestivum. The root extracts contained primary zoospores of the fungus, presumably arising from cystosori. Utilising root fragments of S. sudanense containing cystosori as inoculum P. graminis was shown to infect both monocotyledonous and dicotyledonous plants. Profuse cystosorus production in rootlets only occurred in monocotyledonous plants. In dicotyledonous plants, in general, only few rootlets showed cystosori. Indian isolates of P. graminis appear to differ from isolates from temperate soils in that they can infect dicotyledonous plants and have a much wider host range.     

Effects on plant growth and root-knot nematode infection of an endophytic GFP transformant of the nematophagous fungus Pochonia chlamydosporia

Pochonia chlamydosporia (Pc123) is a fungal parasite of nematode eggs which can colonize endophytically barley and tomato roots. In this paper we use culturing as well as quantitative PCR (qPCR) methods and a stable GFP transformant (Pc123gfp) to analyze the endophytic behavior of the fungus in tomato roots. We found no differences between virulence/root colonization of Pc123 and Pc123gfp on root-knot nematode Meloidogyne javanica eggs and tomato seedlings respectively. Confocal microscopy of Pc123gfp infecting M. javanica eggs revealed details of the process such as penetration hyphae in the egg shell or appressoria and associated post infection hyphae previously unseen. Pc123gfp colonization of tomato roots was low close to the root cap, but increased with the distance to form a patchy hyphal network. Pc123gfp colonized epidermal and cortex tomato root cells and induced plant defenses (papillae). qPCR unlike culturing revealed reduction in fungus root colonization (total and endophytic) with plant development. Pc123gfp was found by qPCR less rhizosphere competent than Pc123. Endophytic colonization by Pc123gfp promoted growth of both roots and shoots of tomato plants vs. uninoculated (control) plants. Tomato roots endophytically colonized by Pc123gfp and inoculated with M. javanica juveniles developed galls and egg masses which were colonized by the fungus. Our results suggest that endophytic colonization of tomato roots by P. chlamydosporia may be relevant for promoting plant growth and perhaps affect managing of root-knot nematode infestations.     

The survival on chrysanthemum roots of epiphytic mycelium of Mycosphaerella ligulicola

Mycosphaerella ligulicola has been shown to survive as epiphytic mycelium on the root surface of chrysanthemum cuttings: such survival could continue throughout the life of the glasshouse crop. Symptomless surface colonization of roots of cuttings could be induced in non-sterile soil from an inoculum of (a) mycelium and sclerotia or (b) conidia (Ascochyta state); the colonization could spread upwards over the root surface. After 12 weeks survival as an epiphyte on chrysanthemum roots the fungus was still pathogenic to unrooted cuttings. Although the root surfaces of twelve other plants could be colonized by M. ligulicola the fungus survived on these roots for not more than 8 weeks.     

Field persistence of the edible ectomycorrhizal fungus Lactarius deliciosus: effects of inoculation strain, initial colonization level, and site characteristics

Pinus pinea plants were inoculated with different strains of the edible ectomycorrhizal fungus Lactarius deliciosus. The inoculated plants were established in six experimental plantations in two sites located in the Mediterranean area to determine the effect of the initial colonization level and the inoculated strain on fungal persistence in the field. Ectomycorrhizal root colonization was determined at transplantation time and monitored at different times from uprooted plants. Extraradical soil mycelium biomass was determined from soil samples by TaqMan® real-time polymerase chain reaction (PCR). The results obtained indicate that the field site played a decisive role in the persistence of L. deliciosus after outplanting. The initial colonization level and the selection of the suitable strain were also significant factors but their effect on the persistence and spread of L. deliciosus was conditioned by the physical–chemical and biotic characteristics of the plantation soil and, possibly, by their influence in root growth. Molecular techniques based on real-time PCR allowed a precise quantification of extraradical mycelium of L. deliciosus in the field. The technique is promising for non-destructive assessment of fungal persistence since soil mycelium may be a good indicator of root colonization. However, the accuracy of the technique will ultimately depend on the development of appropriate soil sampling methods because of the high variability observed.     

In vitro mycorrhization of the rubber tree Hevea brasiliensis Müll Arg

In vitro cultivation systems of arbuscular mycorrhizal fungi are useful tools to study the interaction between plants and their fungal symbiont, and also to develop new biotechnologies. Plantlets of the latex-producing species Hevea brasiliensis clone PB 260 were grown in a dense extraradical mycelium network of the arbuscular mycorrhizal fungus Rhizophagus irregularis MUCL 41833 developed from a mycelium donor plant (Medicago truncatula A17). The factors indole-3-butyric acid (IBA), 2-morpholineoethanesulfonic acid monohydrate (MES) buffer, and carbon dioxide (CO₂) were tested on root development and colonization by the fungus. No colonization was observed in the presence of plantlets pre-treated with IBA. The highest levels of root colonization were obtained when plantlets were mycorrhized under a high CO₂ concentration (1,000 μmol?mol^?1) with MES (10 mM) added to the growth medium. Widespread root colonization (with presence of arbuscules, intraradical mycelium, and spores/vesicles) was predominantly observed in newly produced roots. Therefore, it appears essential to improve root initiation and growth for improving in vitro mycorrhization of H. brasiliensis. We demonstrated the potential of the “mycelium donor plant” in vitro culture system to produce colonized H. brasiliensis plantlets before their transfer to ex vitro conditions.     

Soil nutrient patchiness and plant genotypes interact on the production potential and decomposition of root and shoot litter: evidence from short-term laboratory experiments with Triticum aestivum

Aims

The purpose of this study was to test the hypotheses that soil nutrient patchiness can differentially benefit the decomposition of root and shoot litters and that this facilitation depends on plant genotypes.

Methods

We grew 15 cultivars (i.e. genotypes) of winter wheat (Triticum aestivum L.) under uniform and patchy soil nutrients, and contrasted their biomass and the subsequent mass, carbon (C) and nitrogen (N) dynamics of their root and shoot litters.

Results

Under equal amounts of nutrients, patchy distribution increased root biomass and had no effects on shoot biomass and C:N ratios of roots and shoots. Roots and shoots decomposed more rapidly in patchy nutrients than in uniform nutrients, and reductions in root and shoot C:N ratios with decomposition were greater in patchy nutrients than uniform nutrients. Soil nutrient patchiness facilitated shoot decomposition more than root decomposition. The changes in C:N ratios with decomposition were correlated with initial C:N ratios of litter, regardless of roots or shoots. Litter potential yield, quality and decomposition were also affected by T. aestivum cultivars and their interactions with nutrient patchiness.

Conclusions

Soil nutrient patchiness can enhance C and N cycling and this effect depends strongly on genotypes of T. aestivum. Soil nutrient heterogeneity in plant communities also can enhance diversity in litter decomposition and associated biochemical and biological dynamics in the soil.     

Association of the Plant-beneficial Fungus Verticillium lecanii with Soybean Roots and Rhizosphere

Colonization of soybean roots by the biocontrol fungus Verticillium lecanii was studied in vitro and in situ. For in vitro experiments, V. lecanii was applied to soybean root tip explant cultures. Four weeks after inoculation, the fungus grew externally on at least half of the roots (all treatments combined), colonizing 31% to 71% of root length (treatment means). However, when a potato dextrose agar plug was available as a nutrient source for the fungus, root tips inoculated soon after transfer were not colonized by V. lecanii unless Heterodera glycines was present. Scanning electron microscopy of colonized roots from in vitro cultures revealed a close fungus-root association, including fungal penetration of root cells in some specimens. In the greenhouse, soybeans in sandy soil and in loamy sand soil were treated with V. lecanii applied in alginate prills. The fungus was detected at greater depths from the sandy soil than from the loamy sand soil treatment, but fungus population numbers were small and variable in both soils. Root box studies coupled with image processing analysis of the spatial distribution of V. lecanii in sandy soil supported these findings. Verticillium lecanii was detected randomly in the rhizosphere and soil of root boxes, and was rarely extensively distributed. These in vitro and in situ experiments indicate that V. lecanii can grow in association with soybean roots but is a poor colonizer of soybean rhizosphere in the soil environment.     

Quantifying the effect of soil compaction on three varieties of wheat (Triticum aestivum L.) using X-ray Micro Computed Tomography (CT)

Aims

X-ray Micro Computed Tomography (CT) enables interactions between roots and soil to be visualised without disturbance. This study examined responses of root growth in three Triticum aestivum L. (wheat) cultivars to different levels of soil compaction (1.1 and 1.5?g?cm^?3).

Methods

Seedlings were scanned 2, 5 and 12?days after germination (DAG) and the images were analysed using novel root tracking software, RootViz3D?, to provide accurate visualisation of root architecture. RootViz3D? proved more successful in segmenting roots from the greyscale images than semi-automated segmentation, especially for finer roots, by combining measurements of pixel greyscale values with a probability approach to identify roots.

Results

Root density was greater in soil compacted at 1.5?g?cm^?3 than at 1.1?g?cm^?3 (P?=?0.04). This effect may have resulted from improved contact between roots and surrounding soil. Root diameter was greater in soil at a high bulk density (P?=?0.006) but overall root length was reduced (P?=?0.20). Soil porosity increased with time (P?<?0.001) in the uncompacted treatment.

Conclusions

RootViz3D? root tracking software in X-ray CT studies provided accurate, non-destructive and automated three dimensional quantification of root systems that has many applications for improving understanding on root-soil interactions.     

Suppression of the Biocontrol Agent Trichoderma harzianum by Mycelium of the Arbuscular Mycorrhizal Fungus Glomus intraradices in Root-Free Soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal ³³P transport and β-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal ³³P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

Changes in the fungus-specific,soluble-carbohydrate pool during rapid and synchronous ectomycorrhiza formation of Picea abies with Pisolithus tinctorius

A simple and convenient culture system has been developed for the analysis of ectomycorrhiza formation under controlled conditions. Rapid and synchronous mycorrhiza synthesis was observed when thin and even layers of Pisolithus tinctorius (Pers.) hyphae were brought at once into contact with the entire root system of 3-month-old Picea abies (L. Karst) plants. Suitable fungal layers were grown on cardboard with limiting glucose supply in the medium to maximize radial growth. The glucose was almost consumed by the time the fungus had spread over the whole cardboard and was ready for inoculation of the roots. At this stage, the fungus contained trehalose and arabitol as the main soluble carbohydrates. A few hours after the assembly of the culture system, contacts between roots and aerial hyphae were observed and a sheath was formed 3 days later, suggesting very rapid ectomycorrhiza formation under these conditions. The pool of soluble carbohydrates of the inoculum, i.e. the extramatrical mycelium, declined after inoculation of the roots and was almost zero after 2 weeks. The supply of carbon by the plant was then sufficient for the fungus to expand the soluble pool efficiently in both the mycorrhizas and the extramatrical mycelium. The kinetics of the carbohydrate pool and the observed differentiation of the short roots to mycorrhizas imply that in our culture system fully functional symbiosis was established no later than 14 days after the plants were inoculated with the fungus.     

Mycorrhizal Hyphal Turnover as a Dominant Process for Carbon Input into Soil Organic Matter

The atmospheric concentration of CO₂ is predicted to reach double current levels by 2075. Detritus from aboveground and belowground plant parts constitutes the primary source of C for soil organic matter (SOM), and accumulation of SOM in forests may provide a significant mechanism to mitigate increasing atmospheric CO₂ concentrations. In a poplar (three species) plantation exposed to ambient (380 ppm) and elevated (580 ppm) atmospheric CO₂ concentrations using a Free Air Carbon Dioxide Enrichment (FACE) system, the relative importance of leaf litter decomposition, fine root and fungal turnover for C incorporation into SOM was investigated. A technique using cores of soil in which a C₄ crop has been grown (δ¹³C −18.1‰) inserted into the plantation and detritus from C₃ trees (δ¹³C −27 to −30‰) was used to distinguish between old (native soil) and new (tree derived) soil C. In-growth cores using a fine mesh (39 μm) to prevent in-growth of roots, but allow in-growth of fungal hyphae were used to assess contribution of fine roots and the mycorrhizal external mycelium to soil C during a period of three growing seasons (1999–2001). Across all species and treatments, the mycorrhizal external mycelium was the dominant pathway (62%) through which carbon entered the SOM pool, exceeding the input via leaf litter and fine root turnover. The input via the mycorrhizal external mycelium was not influenced by elevated CO₂, but elevated atmospheric CO₂ enhanced soil C inputs via fine root turnover. The turnover of the mycorrhizal external mycelium may be a fundamental mechanism for the transfer of root-derived C to SOM.     

唐古特大黄药材提取物对小麦和垂穗披碱草种子萌发和幼苗生长的影响

测定了唐古特大黄（Rheum tanguticum Maxim. ex Balf.）药材水提液、蒽醌粗提液和多糖粗提液对小麦(Triticum aestivum Linn.)和垂穗披碱草（Elymus nutans Griseb.）种子萌发及幼苗生长的影响。结果表明：唐古特大黄药材水提液存在明显的化感作用,水提液在高浓度下对小麦种子萌发和芽长及垂穗披碱草种子萌发、根长和芽长均有明显的抑制作用,而在低浓度下对小麦的根长和芽长具有促进作用。蒽醌粗提液仅对垂穗披碱草幼苗生长有抑制作用,其作用随浓度的提高而增强。多糖处理液抑制了小麦、垂穗披碱草根的生长,同时高浓度的处理液延迟了垂穗披碱草种子发芽。     

Carbon Uptake and the Metabolism and Transport of Lipids in an Arbuscular Mycorrhiza

Both the plant and the fungus benefit nutritionally in the arbuscular mycorrhizal symbiosis: The host plant enjoys enhanced mineral uptake and the fungus receives fixed carbon. In this exchange the uptake, metabolism, and translocation of carbon by the fungal partner are poorly understood. We therefore analyzed the fate of isotopically labeled substrates in an arbuscular mycorrhiza (in vitro cultures of Ri T-DNA-transformed carrot [Daucus carota] roots colonized by Glomus intraradices) using nuclear magnetic resonance spectroscopy. Labeling patterns observed in lipids and carbohydrates after substrates were supplied to the mycorrhizal roots or the extraradical mycelium indicated that: (a) ¹³C-labeled glucose and fructose (but not mannitol or succinate) are effectively taken up by the fungus within the root and are metabolized to yield labeled carbohydrates and lipids; (b) the extraradical mycelium does not use exogenous sugars for catabolism, storage, or transfer to the host; (c) the fungus converts sugars taken up in the root compartment into lipids that are then translocated to the extraradical mycelium (there being little or no lipid synthesis in the external mycelium); and (d) hexose in fungal tissue undergoes substantially higher fluxes through an oxidative pentose phosphate pathway than does hexose in the host plant.     

Influence of colonisation by an arbuscular mycorrhizal fungus on the growth of seedlings of<Emphasis Type="Italic"> Banksia ericifolia</Emphasis> (Proteaceae)

Tap and primary lateral roots of seedlings of the putatively non-mycorrhizal Banksia ericifolia became marginally colonised when grown in an established mycelium of an arbuscular mycorrhizal (AM) fungus in the laboratory. A similar degree of colonisation was found in seedlings from an open woodland. All colonies lacked arbuscules. Two factors influencing colonisation and associated growth of host plants were examined experimentally: concentration of P in the soil and organic energy associated with the fungus. While some inoculated seedlings were slightly smaller when colonised by AM fungi, the results were inconsistent and never statistically significant. Seedlings take up insignificant quantities of soil P during early growth, even in the presence of abundant added P. Though colonisation was minor in all cases, an existing mycelium, whether or not connected to a companion plant, slightly increased the amount of root of B. ericifolia colonised by an AM fungus. All seedlings grew slowly. Shoots were significantly larger than roots, until the initiation of proteoid roots which commenced at about 40 days after germination, with both relatively high and low P supply.     

A Mechanistic Model of PCR for Accurate Quantification of Quantitative PCR Data

Background

Quantitative PCR (qPCR) is a workhorse laboratory technique for measuring the concentration of a target DNA sequence with high accuracy over a wide dynamic range. The gold standard method for estimating DNA concentrations via qPCR is quantification cycle () standard curve quantification, which requires the time- and labor-intensive construction of a standard curve. In theory, the shape of a qPCR data curve can be used to directly quantify DNA concentration by fitting a model to data; however, current empirical model-based quantification methods are not as reliable as standard curve quantification.

Principal Findings

We have developed a two-parameter mass action kinetic model of PCR (MAK2) that can be fitted to qPCR data in order to quantify target concentration from a single qPCR assay. To compare the accuracy of MAK2-fitting to other qPCR quantification methods, we have applied quantification methods to qPCR dilution series data generated in three independent laboratories using different target sequences. Quantification accuracy was assessed by analyzing the reliability of concentration predictions for targets at known concentrations. Our results indicate that quantification by MAK2-fitting is as reliable as standard curve quantification for a variety of DNA targets and a wide range of concentrations.

Significance

We anticipate that MAK2 quantification will have a profound effect on the way qPCR experiments are designed and analyzed. In particular, MAK2 enables accurate quantification of portable qPCR assays with limited sample throughput, where construction of a standard curve is impractical.