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Artemisia annua L. is a medicinal plant appreciated for the production of artemisinin, a molecule used for malaria treatment. However, the natural concentration of artemisinin in planta is low. Plant nutrition, in particular phosphorus, and arbuscular mycorrhizal (AM) fungi can affect both plant biomass and secondary metabolite production. In this work, A. annua plants were ino- culated or not with the AM fungus Funneliformis mosseae BEG12 and cultivated for 2 months in controlled conditions at three different phosphatic (P) concentrations (32, 96, and 288 µM). Plant growth parameters, leaf photosynthetic pigment concentrations, artemisinin production, and mineral uptake were evaluated. The different P levels significantly affected the plant shoot growth, AM fungal colonization, and mineral acquisition. High P levels negatively influenced mycorrhizal colonization. The artemisinin concentration was inversely correlated to the P level in the substrate. The fungus mainly affected root growth and nutrient uptake and significantly lowered leaf artemisinin concentration. In conclusion, P nutrition can influence plant biomass production and the lowest phosphate level led to the highest artemisinin concentration, irrespective of the plant mineral uptake. Plant responses to AM fungi can be modulated by cost–benefit ratios of the mutualistic exchange between the partners and soil nutrient availability.

Bacterial endophytes can colonize plant tissues without harming the plant. Instead, they are often able to increase plant growth and tolerance to environmental stresses. In this work, new strains of bacterial endophytes were isolated from three economically important crop plants (sorghum, cucumber and tomato) grown in three different regions in soils with different management. All bacterial strains were identified by 16S rRNA sequencing and characterized for plant beneficial traits. Based on physiological activities, we selected eight strains that were further tested for their antibiotic resistance profile and for the ability to efficiently colonize the interior of sorghum plants. According to the results of the re-inoculation test, five strains were used to inoculate sorghum seeds. Then, plant growth promotion activity was assessed on sorghum plants exposed to salinity stress. Only two bacterial endophytes increased plant biomass, but three of them delayed or reduced plant salinity stress symptoms. These five strains were then characterized for the ability to produce the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which is involved in the increase of stress tolerance. Pseudomonas brassicacearum SVB6R1 was the only strain that was able to produce this enzyme, suggesting that ACC deaminase is not the only physiological trait involved in conferring plant tolerance to salt stress in these bacterial strains.

Salinity and drought are the most important abiotic stresses hampering crop growth and yield. It has been estimated that arid areas cover between 41% and 45% of the total Earth area worldwide. At the same time, the world’s population is going to soon reach 9 billion and the survival of this huge amount of people is dependent on agricultural products. Plants growing in saline/arid soil shows low germination rate, short roots, reduced shoot biomass, and serious impairment of photosynthetic efficiency, thus leading to a substantial loss of crop productivity, resulting in significant economic damage. However, plants should not be considered as single entities, but as a superorganism, or a holobiont, resulting from the intimate interactions occurring between the plant and the associated microbiota. Consequently, it is very complex to define how the plant responds to stress on the basis of the interaction with its associated plant growth-promoting bacteria (PGPB). This review provides an overview of the physiological mechanisms involved in plant survival in arid and saline soils and aims at describing the interactions occurring between plants and its bacteriome in such perturbed environments. The potential of PGPB in supporting plant survival and fitness in these environmental conditions has been discussed.

Previous works have suggested that arbuscular mycorrhizal (AM) fungi can enhance the quality of plant products and fruits. Therefore, the aim of the present study was to investigate the effects of AM fungi on Cucumis melo plant growth, yield, fitness and fruit quality. Inoculated and uninoculated plants of two melon cultivars (Baggio and Expò) were grown in field under common agronomic practices. In addition, a greenhouse test was run on cv. Expò in order to assess the effects of AM inoculation on shoot, leaf and flower development in semi-controlled conditions. In comparison to controls, AM plants flowered earlier, produced more flowers and their pulp contained more sugars and carotenoids. The symbiosis also improved seed size and weight, as well as their germination and root elongation in the first stages of growth in field. Concluding, AM inoculation significantly improved fruit quality in field, under commercial production conditions, also leading to the development of seeds with higher germination rate and increasing plant fitness.

Plant Growth Promoting Bacteria (PGPB) and Arbuscular Mycorrhizal Fungi (AMF) can positively affect plant nutrition and growth. Recent studies have also shown that rhizospheric microorganisms can result in improved fruit features. Aim of this work was to evaluate, in an industrial farming, the effects of three selected biostimulants (consisting of a mix of Plant Growth Promoting Bacteria and Arbuscular Mycorrhizal Fungi), employed in conditions of reduced fertilization on yield, fruit quality and nutritional value. Tomato plants were inoculated with AM fungi and Pseudomonas sp. 19Fv1T or P. fluorescens C7, transplanted and grown in open field under conditions of reduced fertilization. The impact of the microorganisms on the fruit yield and nutritional value was assessed by measuring the production, fruit size and concentration of soluble sugars, organic acids, carotenoids and ascorbate. The size and biomass of tomato fruits were affected by the inocula. Sugar concentration was increased by the selected microorganisms. All the mixtures induced an enhancement of malic acid, while double colonization with AMF and PGPB increased β-carotene concentration in fruits if compared to controls. The results of the present study show that inoculation with soil microorganisms can help to drastically reduce the use of chemical fertilization, maintaining and, in some cases, even improving the tomato fruit yield and quality. This can lead to economical, environmental and human health benefits in relation to the increased sustainability.

Wastewaters generated by the pyrolytic process require treatments to reduce the risks of contamination in rivers, lakes, and coastal waters. Utilizing constructed wetlands is one of the possible approaches according to a Circular Economy System. Plant Growth-Promoting Bacteria (PGPB) and Arbuscular Mycorrhizal Fungi (AMF) can improve plant growth and enhance the bioremediation of wastewater. Two experiments were set up: in the first, a pilot mesocosm was designed to evaluate the effects of a consortium of AM fungi and a PGPB strain on Phragmites australis. After 60 days, the highest plant growth was obtained after inoculation with the combination of microorganisms. In the second experiment, a constructed wetland was built to remediate wastewaters from gasification plant. The plants were efficient in scavenging biological oxygen demand (BOD5), chemical oxygen demand (COD), total fat and oils, hydrocarbons, phenols, aldehydes, surfactants, fluorides, sulfites, sulfates, nitrate, and phosphorus. These data suggest that inoculation of P. australis with AMF and PGPB strains significantly improve the depuration process of wastewaters from gasification plants via constructed wetlands.

Phaseolus vulgaris cv. Billo is an important crop, widely cultivated in Western Piedmont (Italy). It can form symbioses both with arbuscular mycorrhizal fungi (AMF) and bacteria belonging to the Rhizobium genus: AMF can improve plant nutrition and fruit/seed quality, whereas R. leguminosarum is a Gram-negative bacterium able to fix atmospheric nitrogen. In this study, 15 Rhizobium strains, isolated either from root nodules or soil, were characterized by molecular and biochemical point of view (P-solubilization, siderophore and IAA production, intrinsic antibiotic resistance) and one of the selected strains was used for field trial. The aims were: i) to select highly efficient symbiotic bacteria adapted to local conditions and ii) to assess bean seed yield and quality, in response to AMF/rhizobia inoculation at reduced fertilization level. At harvest, yield (pod and seed number and weight) and seed quality (starch, protein, fiber and mineral concentrations), root nodulation and mycorrhizal colonization were measured. Inoculation did not influence yield and fiber concentration. On the contrary, the total seed protein amount was higher in the seeds of rhizobium-inoculated plants in combination or without AMF than in uninoculated plants. The starch content in seeds was increased by rhizobia inoculation. Mg, K and Zn seed accumulation was positively affected by AMF, while the Mn concentration was higher in the presence of rhizobia. In conclusion, plant inoculation strongly affected seed characteristics, improving bean quality also in conditions of reduced fertilization.

International audience; Arbuscular mycorrhizal fungi (AMF) colonize the roots of most terrestrial plant species, improving plant growth, nutrient uptake and biotic/abiotic stress resistance and tolerance. Similarly, plant growth promoting bacteria (PGPB) enhance plant fitness and production. In this study, three different AMF (Funneliformis mosseae, Septoglomus viscosum, and Rhizophagus irregularis) were used in combination with three different strains of Pseudomonas sp. (19Fv1t, 5Vm1K and Pf4) to inoculate plantlets of Fragaria x ananassa var. Eliana F1. The effects of the different fungus/bacterium combinations were assessed on plant growth parameters, fruit production and quality, including health-promoting compounds. Inoculated and uninoculated plants were maintained in a greenhouse for 4 months and irrigated with a nutrient solution at two different phosphate levels. The number of flowers and fruits were recorded weekly. At harvest, fresh and dry weights of roots and shoots, mycorrhizal colonization and concentration of leaf photosynthetic pigments were measured in each plant. The following fruit parameters were recorded: pH, titratable acids, concentration of organic acids, soluble sugars, ascorbic acids, and anthocyanidins; volatile and elemental composition were also evaluated. Data were statistically analyzed by ANOVA and PCA/PCA-DA. Mycorrhizal colonization was higher in plants inoculated with R. irregularis, followed by F mosseae and S. viscosum. In general, AMF mostly affected the parameters associated with the vegetative portion of the plant, while PGPB were especially relevant for fruit yield and quality. The plant physiological status was differentially affected by inoculations, resulting in enhanced root and shoot biomass. Inoculation with Pf4 bacterial strain increased flower and fruit production per plant and malic acid content in fruits, while decreased the pH value, regardless of the used fungus. Inoculations affected fruit nutritional quality, increasing sugar and anthocyanin concentrations, and modulated pH, malic acid, volatile compounds and elements. In the present study, we show for the first time that strawberry fruit concentration of some elements and/or volatiles can be affected by the presence of specific beneficial soil microorganisms. In addition, our results indicated that it is possible to select the best plant-microorganism combination for field applications, and improving fruit production and quality, also in terms of health promoting properties.

Anthocyanins are a group of common phenolic compounds in plants. They are mainly detected in flowers and fruits, are believed to play different important roles such as in the attraction of animals and seed dispersal, and also in the increase of the antioxidant response in tissues directly or indirectly affected by biotic or abiotic stress factors. As a major group of secondary metabolites in plants commonly consumed as food, they are of importance in both the food industry and human nutrition. It is known that arbuscular mycorrhizal (AM) fungi can influence the plant secondary metabolic pathways such as the synthesis of essential oils in aromatic plants, of secondary metabolites in roots, and increase flavonoid concentration. Plant Growth-Promoting Bacteria (PGPB) are able to increase plant growth, improving plant nutrition and supporting plant development under natural or stressed conditions. Various studies confirmed that a number of bacterial species living on and inside the root system are beneficial for plant growth, yield and crop quality. In this work it is shown that inoculation with AM fungi and/or with selected and tested Pseudomonas strains, under conditions of reduced fertilization, increases anthocyanin concentration in the fruits of strawberry.

Phytoremediation is a cost-effective and environment friendly in situ technique for the reclamation of heavy metal-polluted soils. The efficacy of this technique, which relies on tolerant plant species, can be improved by the use of chelating agents. A pot experiment was carried out to evaluate the phytoextraction and phytostabilisation capacities of a white poplar (Populus alba L.) clone named AL35 previously selected for its marked tolerance to copper (Cu) and zinc (Zn). Cuttings were grown on agricultural soil highly contaminated with Cu and Zn, in the presence or not (controls) of a chelant mixture (EDTA/EDDS) known to enhance metal bioavailability and, hence, uptake by plant roots, or the not yet investigated synthetic, highly biodegradable polyaspartic acid (PASP). Both chelant treatments improved the phytostabilisation of Cu and Zn in AL35 plants, whilst the phytoextraction capacity was enhanced only in the case of Cu. Considering that the effectiveness of PASP as phytostabilizer was comparable or better than that of EDTA/EDDS, the low cost of its large-scale chemical synthesis and its biodegradability makes it a good candidate for chelant-enhanced metal phytoextraction from soil while avoiding the toxic side-effects previously described for both EDTA and EDDS.

The aim of this work was to assess the effects of plant-beneficial microorganisms (two Pseudomonas strains and a mixed mycorrhizal inoculum, alone or in combination) on the quality of tomato fruits of plants grown in the field and subjected to reduced fertilization. Pseudomonas strain 19Fv1T was newly characterized during this study. The size and quality of the fruits (concentration of sugars, organic acids and vitamin C) were assessed. The microorganisms positively affected the flower and fruit production and the concentrations of sugars and vitamins in the tomato fruits. In particular, the most important effect induced by arbuscular mycorrhizal (AM) fungi was an improvement of citric acid concentration, while bacteria positively modulated sugar production and the sweetness of the tomatoes. The novelty of the present work is the application of soil microorganisms in the field, in a real industrial tomato farm. This approach provided direct information about the application of inocula, allowed the reduction of chemical inputs and positively influenced tomato quality.

SPE IPM INRA UB CT1; International audience; Arbuscular mycorrhizal (AM) fungi are associated with about 80% of land plants. AM fungi provide inorganic nutrients to plants and in return up to 20% of the plant-fixed CO2 is transferred to the fungal symbionts. Since AM fungi are obligate biotrophs, unraveling how sugars are provided to the fungus partner is a key for understanding the functioning of the symbiosis. In this study, we identified two new monosaccharide transporters from Rhizophagus irregularis (RiMST5 and RiMST6) that we characterized as functional high affinity monosaccharide transporters. RiMST6 was characterized as a glucose specific, high affinity H(+) co-transporter. We provide experimental support for a primary role of both RiMST5 and RiMST6 in sugar uptake directly from the soil. The expression patterns of RiMSTs in response to partial light deprivation and to interaction with different host plants were investigated. Expression of genes coding for RiMSTs was transiently enhanced after 48h of shading and was unambiguously dependent on the host plant species. These results cast doubt on the 'fair trade' principle under carbon-limiting conditions. Therefore, in light of these findings, the possible mechanisms involved in the modulation between mutualism and parasitism in plant-AM fungus interactions are discussed. Copyright 2016 Elsevier Masson SAS. All rights reserved.

Soil microorganisms like arbuscular mycorrhizal fungi (AMF) and plant growth-promoting rhizobacteria (PGPR) are known to positively affect plant health, growth, and nutrition. Their use was often suggested in order to reduce the input of chemicals in agriculture. Recently, it has been observed that AMF and PGPR can also affect some quality features of various crops. In this chapter we review the literature concerning the effects of soil microbes, focusing on AMF and PGPR as the most common plant-associated microorganisms, on the quality of crops. Such effects were considered according to commercial and agronomic plant categories. Current limitations of the available information and some possible future developments have been indicated.

Plants growing under zinc excess store it in leaves, the organs in charge with carbon assimilation. Therefore, leaf modifications can help to understand the plant responses to environmental stresses. In this study, we tested the hypothesis that high zinc concentration can affect leaf anatomy and physiology of Populus alba cv. Villafranca, a tree species with interesting perspectives of application in phytoremediation. Plants were supplemented or not with 300 mg zinc per kg of soil and grown for six months. Green and macroscopically symptom-less leaves from poplars of each treatment were analyzed by light and electron microscopy (TEM, SEM-EDS) and by Raman spectroscopy. LHCII and D1–D2 protein expression were considered to evaluate PSII functioning. Zinc was histochemically detected in the leaves. Zinc strongly altered leaf morphology and ultrastructure, and negatively affected PSII activity decreasing D1 and D2, but not LHCII, protein expression. Zinc was mostly detected in the cell walls of the xylem and of the parenchyma cells surrounding the bundles of zinc treated plants, but not in the controls. A significant increase in the number of calcium oxalate crystals was observed in the leaves of zinc treated plants, but the latter metal was not incorporated in crystals, suggesting an increase of free calcium following zinc accumulation in the cell walls.

Arbuscular mycorrhizae (AM) are the most widespread mutualistic symbioses between the roots of most land plants and a phylum of soil fungi. AM are known to influence plant performance by improving mineral nutrition, protecting against pathogens and enhancing resistance or tolerance to biotic and abiotic stresses. The aim of this study was to investigate the frond proteome of the arsenic hyperaccumulator fern Pteris vittata in plants that had been inoculated with one of the two AM fungi (Glomus mosseae or Gigaspora margarita) with and without arsenic treatment. A protective role for AM fungi colonisation in the absence of arsenic was indicated by the down-regulation of oxidative damage-related proteins. Arsenic treatment of mycorrhizal ferns induced the differential expression of 130 leaf proteins with specific responses in G. mosseae- and Gi. margarita-colonised plants. Up-regulation of multiple forms of glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and enolase, primarily in G. mosseae-inoculated plants, suggests a central role for glycolytic enzymes in arsenic metabolism. Moreover, a putative arsenic transporter, PgPOR29, has been identified as an up-regulated protein by arsenic treatment.

† Background and Aims It is increasingly evident that plant tolerance to stress is improved by mycorrhiza. Thus, suitable plant –fungus combinations may also contribute to the success of phytoremediation of heavy metal (HM)-polluted soil. Metallothioneins (MTs) and polyamines (PAs) are implicated in the response to HM stress in several plant species, but whether the response is modulated by arbuscular mycorrhizal fungi (AMF) remains to be clarified. The aim of the present study was to check whether colonization by AMF could modify growth, metal uptake/translocation, and MT and PA gene expression levels in white poplar cuttings grown on HM-contaminated soil, and to compare this with plants grown on non-contaminated soil. † Methods In this greenhouse study, plants of a Populus alba clone were pre-inoculated, or not, with either Glomus mosseae or G. intraradices and then grown in pots containing either soil collected from a multimetal(Cu and Zn) polluted site or non-polluted soil. The expression of MT and PA biosynthetic genes was analysed in leaves using quantitative reverse transcription–PCR. Free and conjugated foliar PA concentrations were determined in parallel. † Results On polluted soil, AMF restored plant biomass despite higher Cu and Zn accumulation in plant organs, especially roots. Inoculation with the AMF caused an overall induction of PaMT1, PaMT2, PaMT3, PaSPDS1, PaSPDS2 and PaADC gene expression, together with increased free and conjugated PA levels, in plants grown on polluted soil, but not in those grown on non-polluted soil. † Conclusions Mycorrhizal plants of P. alba clone AL35 exhibit increased capacity for stabilization of soil HMs, together with improved growth. Their enhanced stress tolerance may derive from the transcriptional upregulation of several stress-related genes, and the protective role of PAs.

Although Rhizoctonia solani is a cosmopolitan soilborne pathogen, the genus includes isolates with different pathogenicity ranging from high virulence to avirulence. The biocontrol strain Pseudomonas fluorescens P190r and the arbuscular mycorrhizal (AM) fungus Glomus mosseae BEG12 were inoculated alone or in combination in tomato plants infested by the mildly virulent pathogen R. solani #235. Plant growth as well as root morphometric and topological parameters were evaluated. The infection of R. solani was significantly reduced by all the combinations of the beneficial microorganisms. Root systems of R. solani‐infected plants were weakly developed but highly branched with a herring‐bone pattern, while those inoculated with the AM fungus, alone or in combination with the bacterial strain, were longer and more developed, and displayed a dichotomous pattern. The interactions among these three microorganisms affected plant growth and root architecture of tomato plants.

Aims: After the determination of the toxic but nonlethal concentration of NaCl for cucumber, we examined the interaction between an ACC (1-aminocyclopropane-1-carboxylate) deaminase producing bacterial strain and an arbuscular mycorrhizal fungus (AMF) and their effects on cucumber growth under salinity. Methods and Results: In the first experiment, cucumber seedlings were exposed to 0·1, 50, 100 or 200 mmol l−1 NaCl, and plant biomass and leaf area were measured. While seeds exposed to 200 mmol l−1 NaCl did not germinate, plant growth and leaf size were reduced by 50 or 100 mmol l−1 salt. The latter salt cancentration caused plant death in 1 month. In the second experiment, seeds were inoculated with the ACC deaminase-producing strain Pseudomonas putida UW4 (AcdS+), its mutant unable to produce the enzyme (AcdS−), or the AMF Gigaspora rosea BEG9, individually or in combination and exposed to 75 mmol l−1 salt. Plant morphometric and root architectural parameters, mycorrhizal and bacterial colonization and the influence of each micro-organism on the photosynthetic efficiency were evaluated. The AcdS+ strain or the AMF, inoculated alone, increased plant growth, affected root architecture and improved photosynthetic activity. Mycorrhizal colonization was inhibited by each bacterial strain. Conclusions: Salinity negatively affects cucumber growth and health, but root colonization by ACC deaminase-producing bacteria or arbuscular mycorrhizal fungi can improve plant tolerance to such stressful condition. Significance and Impact of the Study: Arbuscular mycorrhizal fungus and bacterial ACC deaminase may ameliorate plant growth under stressful conditions. It was previously shown that, under optimal growth conditions, Ps. putida UW4 AcdS+ increases root colonization by Gi. rosea resulting in synergistic effects on cucumber growth. These results suggest that while in optimal conditions ACC deaminase is mainly involved in the bacteria/fungus interactions, while under stressful conditions this enzyme plays a role in plant/bacterium interactions. This finding is relevant from an ecological and an applicative point of view.

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities of Solanum tuberosum L. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging to Glomus groups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding to Glomus intraradices , was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species, G. intraradices .

Bacteria producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase modulate plant ethylene levels. Decreased ethylene levels increase plant tolerance to environmental stresses and promote legume nodulation. On the contrary, the role of ethylene in mycorrhizal symbiosis establishment is still controversial. In this work, the ACC deaminase-producing strain Pseudomonas putida UW4 AcdS+ and its mutant AcdS−, impaired in ACC deaminase synthesis, were inoculated alone or in combination with the AM fungus Gigaspora rosea on cucumber. Mycorrhizal and bacterial colonization as well as plant growth and morphometric parameters were measured. The influence of each microorganism on the photosynthetic efficiency was evaluated on the second and fourth leaf. The strain AcdS+, but not the AcdS− mutant, increased AM colonization and arbuscule abundance. The mycorrhizal fungus, but not the bacterial strains, promoted plant growth. However, the AcdS+ strain, inoculated with G. rosea, induced synergistic effects on plant biomass, total root length and total leaf projected area. Finally, the photosynthetic performance index was increased by the strain UW4 AcdS+ inoculated in combination with G. rosea BEG9. These results suggest a key role of this enzyme in the establishment and development of AM symbiosis.

It was previously shown that arbuscular mycorrhizal fungi (AMF) exert a significant improvement of growth in a tolerant white poplar (Populus alba L.) clone (AL35) grown on Cu- and Zn-polluted soil via foliar alterations in the levels of defence/stress-related transcripts and molecules. However, nothing is known about the epigenetic changes which occur during tolerance acquisition in response to heavy metals (HMs) in the same mycorrhizal vs. non-mycorrhizal poplar plants. In order to analyse the epigenome in leaves of AL35 plants inoculated or not with AMF and grown in a greenhouse on multimetal polluted or unpolluted soil, the Methylation Sensitive Amplification Polymorphism (MSAP) approach was adopted to detect cytosine DNA methylation. Modest changes in cytosine methylation patterns were detected at first sampling (4 months from planting), whereas extensive alterations (hypomethylation) occurred at second sampling (after 6 months) in mycorrhizal plants grown in the presence of HMs. The sequencing of MSAP fragments led to the identification of genes belonging to several Gene Ontology categories. Seven MSAP fragments, selected on the basis of DNA methylation status in treated vs control AL35 leaves at the end of the experiment, were analysed for their transcript levels by means of qRT-PCR. Gene expression varied in treated samples relative to controls in response to HMs and/or AMF inoculation; in particular, transcripts of genes involved in RNA processing, cell wall and amino acid metabolism were upregulated in the presence of AMF with or without HMs.

Aromatic hydrocarbons are pollutants which have mutagenic and carcinogenic properties as well as relatively high hydrosolubility. Their presence in soils makes techniques such as bioremediation an important topic for research. In this work, the effect of arbuscular mycorrhiza (AM) on the persistence of benzene, toluene, ethylbenzene and xylene (BTEX) in artificially contaminated substrates was evaluated. Leek plants were grown with three AM fungal species using a specially designed mesocosm system, in which internal air and substrate samples were analyzed by gas chromatography for BTEX content. Strong reductions in the BTEX concentration in substrates were generally observed in the presence of mycorrhizal plants. Residual BTEX content ranged between nearly total disappearance (

I norder to better understand which are the factors involved in the control of nuclear size, and more pre- cisely, to clarify why nuclear hypertrophy doesn't always follow genome increase, biological techniques combined with a strict mathematical approach have been applied to study the nuclei of root cortical cells of Lycopersicon escu- lentum Mill cv Early Mech. Tomato is a multiploid plant, with three different levels of DNA content, therefore it is especially suitable to study nucleus size according to genome size. In addition, as arbuscular mycorrhizal coloniza- tion strongly affects nucleus organization, part of the plants were inoculated with the arbuscular mycorrhizal fungus Glomus mosseae BEG 12. Quantitative and qualitative nuclear changes have been analyzed in mycorrhizal and non- mycorrhizal roots of tomato, by means of microscopy, immuno-labelling and flow cytometry. The results, supported by mathematical analysis, clearly show that increased ploidy is necessary, but not sufficient, to explain nuclear hyper- trophy, in which chromatin decondensation is also involved and related to cell metabolic activity.

The protective effects induced by arbuscular mycorrhizal (AM) fungi against a phytoplasma of the Stolbur group have been investigated in tomato by morphometry and flow cytometry. Symptoms induced by the phytoplasma were less severe when the plants also harboured AM fungi. Morphological parameters such as shoot and root fresh weight, shoot height, internode length, leaf number and adventitious root diameter were closer to those of healthy plants when arbuscular mycorrhiza were present. Reduced nuclear senescence was observed in AM plants infected with phytoplasmas; the percentages of nuclear populations with different ploidy levels were intermediate between AM and phytoplasma-infected plants. The mechanisms underlying these interactions are discussed and a direct action of the AM fungus is hypothesized.

High nitrogen concentration in wastewaters requires treatments to prevent the risks of eutrophication in rivers, lakes and coastal waters. The use of constructed wetlands is one of the possible approaches to lower nitrate concentration in wastewaters. Beyond supporting the growth of the bacteria operating denitrification, plants can directly take up nitrogen. Since plant roots interact with a number of soil microorganisms, in the present work we report the monitoring of nitrate concentration in macrocosms with four different levels of added nitrate (0, 30, 60 and 90 mg l(-1)), using Phragmites australis, inoculated with bacteria or arbuscular mycorrhizal fungi, to assess whether the use of such inocula could improve wastewater denitrification. Higher potassium nitrate concentration increased plant growth and inoculation with arbuscular mycorrhizal fungi or bacteria resulted in larger plants with more developed root systems. In the case of plants inoculated with arbuscular mycorrhizal fungi, a faster decrease of nitrate concentration was observed, while the N%/C% ratio of the plants of the different treatments remained similar. At 90 mg l(-1) of added nitrate, only mycorrhizal plants were able to decrease nitrate concentration to the limits prescribed by the Italian law. These data suggest that mycorrhizal and microbial inoculation can be an additional tool to improve the efficiency of denitrification in the treatment of wastewaters via constructed wetlands.

There is increasing interest in the quality of crops because of the implications concerning health, economic revenue, and food quality. Here we tested if inoculation with a mixture of arbuscular mycorrhizal fungi (AMF) and/or two strains of plant growth-promoting bacteria (PGPB), in conditions of reduced chemical inputs, affects the quality and yield of strawberry fruits. Fruit quality was measured by concentrations of soluble sugars, various organic acids, and two vitamins (ascorbic and folic acid). Co-inoculation with the AMF and each of the two PGPB resulted in increased flower and fruit production, larger fruit size, and higher concentrations of sugars and ascorbic and folic acid in comparison with fruits of uninoculated plants. These results provide further evidence that rhizospheric microorganisms affect fruit crop quality and show that they do so even under conditions of reduced chemical fertilization and can thus be exploited for sustainable agriculture.

Poplar is a suitable species for phytoremediation, able to tolerate high concentrations of heavy metals (HMs). Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with the roots of most land plants; they improve nutrient uptake and enhance phytoextraction of HMs while alleviating stress in the host plant. This review summarizes previous results from field and greenhouse studies conducted by us and dealing with this topic. In a field trial on a highly Zn- and Cu-contaminated site, differences in plant survival and growth were observed among 168 clones originating from natural populations of Populus alba L. and Populus nigra L. from northern Italy. After two and a half years from planting, the density, activity and metabolic versatility of the culturable fraction of the soil bacteria in the HM-polluted field was higher in the soil close to where larger poplar plants were growing, in spite of comparable HM concentrations recorded in these soils. One well-performing clone of P. alba (AL35), which accumulated a higher concentration of both metals and had high foliar polyamine (PA) levels, was used for further investigation. In a greenhouse study, AL35 cuttings pre-inoculated with AMF (Glomus mosseae or Glomus intraradices) and then transferred to pots containing soil, collected from the HM-polluted site, displayed growth comparable to that of controls grown on unpolluted soil, in spite of higher Cu and Zn accumulation. Such plants also showed an overall up-regulation of metallothionein (MT) and PA biosynthetic genes, together with increased PA levels. A genome-wide transcriptomic (cDNA-AFLP) analysis allowed the identification of a number of genes, mostly belonging to stress-related functional categories of defense and secondary metabolism, that were differentially regulated in mycorrhizal vs. non mycorrhizal plants. A proteomic analysis revealed that, depending on sampling time, changes in protein profiles were differentially affected by AMF and/or HMs. It is concluded that soil-borne microorganisms affect plant performance on HM-polluted soil. In particular, mycorrhizal plants exhibited increased capacity for phytostabilization of HMs, together with improved growth. Their greater stress tolerance may derive from the protective role of PAs, and from the strong modulation in the expression profiles of stress-related genes and proteins.

In this study we investigated if the symbiosis with the arbuscular mycorrhizal fungus Glomus mosseae, which contributes to alleviate heavy metal stress in plants, may affect the transcription of genes involved in the stress defence in the white poplar clone ‘AL35’ grown on a multimetal (Cu and Zn) contaminated soil. The results obtained showed that the symbiosis with G. mosseae reduced transcript abundance of genes involved in antioxidant defence in leaves and roots of ‘AL35’ plants grown on the heavy metal-polluted soil. Moreover, the interaction between this poplar clone and the arbuscular mycorrhizal fungus induced the gene coding for phytochelatin synthase in leaves, whereas the expression of genes involved in heavy metal homeostasis did not change in roots. The present results suggest that, in presence of high levels of heavy metals, inoculation with G. mosseae may confer to ‘AL35’ a more efficient control of the oxidant level. Moreover, in mycorrhizal plants heavy metal chelation pathways appear involved in the defence strategies in leaves, whereas in roots they do not seem to contribute to increase the plant tolerance of heavy metals.

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with the roots of most plant species. While receiving photosynthates, they improve the mineral nutrition of the plant and can also increase its tolerance towards some pollutants, like heavy metals. Although the fungal symbionts exclusively colonize the plant roots, some plant responses can be systemic. Therefore, in this work a clone of Populus alba L., previously selected for its tolerance to copper and zinc, was used to investigate the effects of the symbiosis with the AM fungus Glomus intraradices on the leaf protein expression. Poplar leaf samples were collected from plants maintained in a glasshouse on polluted (copper and zinc contaminated) or unpolluted soil, after four, six and sixteen months of growth. For each harvest, about 450 proteins were reproducibly separated on 2DE maps. At the first harvest the most relevant effect on protein modulation was exerted by the AM fungi, at the second one by the metals, and at the last one by both treatments. This work demonstrates how importantly the time of sampling affects the proteome responses in perennial plants. In addition, it underlines the ability of a proteomic approach, targeted on protein identification, to depict changes in a specific pattern of protein expression, while being still far from elucidating the biological function of each protein.

We performed a field trial to evaluate the response of different poplar clones to heavy metals. We found that poplar plants of the same clone, propagated by cuttings, had a marked variability of survival and growth in different zones of the field that were characterized by very similar physical–chemical prosperities. Since metal uptake and its accumulation by plants can be affected by soil microorganisms, we investigated soil microbial populations that were collected in proximity to the roots of large and small poplar plants. We used microbiological and molecular tools to ascertain whether bacterial strains or species were associated with large, or small poplars, and whether these were different from those present in the bulk (without plants) soil. We found that the culturable fraction of the bacteria differed in the three cases (bulk soil, small or large poplars). While some taxa were always present, two species ( Chryseobacterium soldanellicola and Variovorax paradoxus ) were only found in the soil where poplars (large or small) were growing, independently from the plant size. Bacterial strains of the genus Flavobacterium were prevalent in the soil with large poplar plants. The existence of different microbial populations in the bulk and in the poplar grown soils was confirmed by the DGGE profiles of the bacterial culturable fractions. Cluster analysis of the DGGE profiles highlighted the clear separation of the culturable fraction from the whole microbial community. The isolation and identification of poplar-associated bacterial strains from the culturable fraction of the microbial community provided the basis for further studies aimed at the combined use of plants and soil microorganisms in the remediation of heavy metal polluted soils.

Heavy metal pollution is a major worldwide environmental concern that has recently motivated researchers to develop a variety of novel approaches towards its cleanup. As an alternative to traditional physical and chemical methods of environmental cleanup, scientists have developed phytoremediation approaches that include the use of plants to remove or render harmless a range of compounds. Both plant growth promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) can be used to facilitate the process of phytoremediation and the growth of plants in metal-contaminated soils. This review focuses on the recent literature dealing with the effects of plant growth-promoting bacteria and AM fungi on the response of plants to heavy metal stress and points the way to strategies that may facilitate the practical realization of this technology.

Bacteria producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase modulate plant ethylene levels. Decreased ethylene levels increase plant tolerance to environmental stresses and promote legume nodulation. On the contrary, the role of ethylene in mycorrhizal symbiosis establishment is still controversial. In this work, the ACC deaminase-producing strain Pseudomonas putida UW4 AcdS+ and its mutant AcdS(-), impaired in ACC deaminase synthesis, were inoculated alone or in combination with the AM fungus Gigaspora rosea on cucumber. Mycorrhizal and bacterial colonization as well as plant growth and morphometric parameters were measured. The influence of each microorganism on the photosynthetic efficiency was evaluated on the second and fourth leaf. The strain AcdS+, but not the AcdS(-) mutant, increased AM colonization and arbuscule abundance. The mycorrhizal fungus, but not the bacterial strains, promoted plant growth. However, the AcdS+ strain, inoculated with G. rosea, induced synergistic effects on plant biomass, total root length and total leaf projected area. Finally, the photosynthetic performance index was increased by the strain UW4 AcdS+ inoculated in combination with G. rosea BEG9. These results suggest a key role of this enzyme in the establishment and development of AM symbiosis.

Because of their enormously large range of plant hosts and role in plant nutrition, arbuscular mycorrhizal (AM) fungi represent an extraordinarily fascinating field of study. Plant growth promotion effects by AM fungi were described as early as 1900 (Sthal 1900) and several data obtained in the second half of the last century support the idea that these microrganisms can act as biocontrol agents (BCA). The extent of root colonization is variable in different plants and under different environmental conditions (Giovannetti and Hepper 1985). Some effects of AM colonization on plants have been reported to be dependent on the degree of root colonization, while others have not. Root exudation and pH are modified by the presence of AM fungi (Bansal and Mukerji 1994; Bago et al. 1996), therefore AM fungi can affect the growth of rhizobacteria. Similarly, both root colonization by AM fungi and their effects on the plant can be affected by the presence of rhizobacteria, which can be plant growth-promoting, mycorrhiza helper or biocontrol agents. Although several genera of microorganisms have been reported to behave as biocontrol agents (BCA) or plant growth-promoting rhizobacteria (PGPR), over these years attention has been focused mainly on fluorescent pseudomonads, because they are common inhabitants of rhizosphere and phyllosphere environments, synthesize a wide range of metabolites and enzymes (O’Sullivan and O’Gara 1992), are easily isolated from natural environments, utilize a large variety of substrates (Latour and Lemanceau 1997), and are easy to culture and manipulate genetically (Walsh et al. 2001). Altogether, these characters make them more reliable to scientific experimentation (Whipps 1997; Haas and Defago 2005). Due to their impressive capacity in producing different types of antibiotics such as 2,4-diacetylphloroglucinol, phenazines, oomycin, pyoluteorin, pyrrolnitrin

Two registered clones of poplar were grown in soil contaminated or not with copper. Some of the plants were pre-inoculated with arbuscular mycorrhizal (AM) fungi. Copper effects on plant growth were evaluated, as well as the role of AM fungi in alleviating metal stress. Polyamine content, a class of molecules involved in stress re- sponses, metal content and P content were used to describe plant conditions. The two clones showed several differ- ences in relation to copper pollution and AM symbiosis, confirming the importance of accurate plant selection for phytoremediation purposes. The large accumulation of copper in the roots suggested that poplar is suitable for phy- tostabilization strategies when this kind of pollution is present.

The essential oils of basil are widely used in the cosmetic, pharmaceutical, food, and flavoring industries. Little is known about the potential of arbuscular mycorrhizal (AM) fungi to affect their production in this aromatic plant. The effects of colonization by three AM fungi, Glomus mosseae BEG 12, Gigaspora margarita BEG 34, and Gigaspora rosea BEG 9 on shoot and root biomass, abundance of glandular hairs, and essential oil yield of Ocimum basilicum L. var. Genovese were studied. Plant P content was analyzed in the various treatments and no differences were observed. The AM fungi induced various modifications in the considered parameters, but only Gi. rosea significantly affected all of them in comparison to control plants or the other fungal treatments. It significantly increased biomass, root branching and length, and the total amount of essential oil (especially alpha-terpineol). Increased oil yield was associated to a significantly larger number of peltate glandular trichomes (main sites of essential oil synthesis) in the basal and central leaf zones. Furthermore, Gi. margarita and Gi. rosea increased the percentage of eugenol and reduced linalool yield. Results showed that different fungi can induce different effects in the same plant and that the essential oil yield can be modulated according to the colonizing AM fungus.

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.

Arbuscular mycorrhizal (AM) colonization can strongly affect the plant cell nucleus, causing displacement from the periphery to the center of the cell, hypertrophy and polyploidization. The hypertrophy response has been shown in a variety of AM plants whilst polyploidization has been reported only in Lycopersicon esculentum, a multiploid species with a small genome. In order to determine whether polyploidization is a general plant response to AM colonization, analyses were performed on Allium porrum, a plant with a large genome, which is much less subject to polyploidization than L. esculentum. The ploidy status of leaves, complete root systems and four zones of the adventitious roots was investigated in relation to phosphorus content, AM colonization and root differentiation in A. porrum plants grown under two different regimes of phosphate nutrition in order to distinguish direct effects of the fungus from those of improved nutrition. Results showed the presence of two nuclear populations (2C and 4C) in all treatments and samples. Linear regression analyses suggested a general negative correlation between phosphorus content and the proportion of 2C nuclei. The percentage of 2C nuclei (and consequently that of 4C nuclei), was also influenced by AM colonization, differentiation and ageing of the root cells, which resulted in earlier occurrence, in time and space, of polyploid nuclei.

We have isolated a cDNA clone, Le-MI-13 (Lycopersicon esculentum mycorrhizal induced) by differential screening of a cDNA library prepared from mRNA extracted from tomato roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus mosseae. The Le-MI-13 clone encodes a polypeptide that shows a high degree of amino acid sequence similarity with members of the recently identified multigene family, the cullins. Northern blot analyses demonstrated that the Le-MI-13 transcript accumulated in tomato roots forming arbuscular mycorrhizal symbiosis. Only very little Le-MI-13 transcript was detected in control roots. Tomato roots infected by the pathogenic fungus Phytophthora nicotianae var. parasitica did not accumulate Le-MI-13 transcript, indicating that upregulation of the Le-MI-13 gene is specific to roots forming arbuscular mycorrhizal symbiosis. Indirect evidence suggesting that a Le-MI-13-mediated cell-cycle-like control might operate in AM-colonized cells came from flow cytometry and static micro fluorimetry analysis. There was a strong correlation between nuclear polyploidization and AM colonization.Key words: tomato, arbuscular mycorrhizae, Phytophthora, cullins, polyploidy.

The nuclei of plant cells show marked differences in chromatin organisation, related to their DNA content, which ranges from the type with large strands of condensed chromatin (reticulate or chromonematic nuclei) to one with mostly decondensed chromatin (chromocentric or diffuse nuclei). A loosening of the chromatin structure generally occurs in actively metabolising cells, such as differentiating and secretory cells, in relation to their high transcriptional activity. Endoreduplication may occur, especially in plants with a small genome, which increases the availability of nuclear templates, the synthesis of DNA, and probably regulates gene expression. Here we describe structural and quantitative changes of the chromatin and their relationship with transcription that occur in differentiated cells following an increase of their metabolism. The nuclei of root cortical cells of three plants with different 2C DNA content (Allium porrum, Pisum sativum and Lycopersicon esculentm) and their modifications induced by arbuscular mycorrhization, which strongly increase the metabolic activity of colonised cells, are taken as examples.

Nuclear changes in roots of tomato (Lycopersicon esculentum), a plant with a small genome, during the establishment of arbuscular mycorrhizal (AM) colonization were studied using light and electron microscopy, as well as flow and static cytometry. Nuclei of mycorrhizal root cortex cells were larger and had more decondensed chromatin than those of controls. Significant ploidy distribution differences were observed between nuclei of AM colonized and control roots, and a strong correlation between nuclear polyploidization and AM colonization was found. Polyploidization and decondensation are usually associated with high metabolic activity. The metabolic activity of mycorrhizal root cells, evaluated in this work as respiratory activity by using a cytochemical assay for succinate dehydrogenase combined with image analysis, increased in comparison to controls. The meaning of polyploidization is discussed in relation to the structural and metabolic modifications induced by mycorrhization.

We have investigated nuclear changes induced in tomato (Lycopersicon esculentum) root plants by two different pathogens, a phytoplasma of the stolbur group and the soil-borne fungus Phytophthora nicotianae var. parasitica, using light and electron microscopy as well as flow cytometry. Our results show that both pathogens strongly influenced nuclear structure, causing pyknosis and chromatolysis, and induced variations in proportion of nuclear populations with different DNA content, increasing the 2C and decreasing the 4C nuclear populations. These latter results suggest a block of the endoreduplication process in the presynthetic phase of the host cell cycle. Moreover, in pathogen-infected roots, nuclei of the same DNA content were less fluorescent, and therefore had a lower DNA content, compared to those of controls, as confirmed by morphological analyses. Our findings suggest that different stimuli may evoke similar nuclear changes, and confirm the usefulness of flow cytometry as a tool to recognize the stress conditions related to the presence of a pathogen.

Agrobacterium-mediated transformation of white poplar explants was performed using the pMAT-MT construct derived from the ipt-type MAT22 plasmid carrying the PsMTA1 cDNA encoding a pea metallothionein. In vitro shoot regeneration occurred on antibiotic-free medium and transformants were identified by visual selection. Beside the expected abnormal ipt-shooty phenotype, shoots with normal morphology were recovered. Interestingly, an additional phenotype characterised by a remarkable root development was also observed.