Guido Lingua

Effects of three AM fungi on growth, distribution of glandular hairs, and essential oil production in Ocimum basilicum L. var. Genovese

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 α-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.

Do interactions between plant roots and the rhizosphere affect parasitoid behaviour

Multitrophic interactions are powerful forces shaping the structure of living communities. Plants encounter a great diversity of organisms in their environment: some of these interactions are beneficial (e.g. symbiotic fungi and insect pollinators) while some are detrimental (e.g. herbivorous insects and pathogenic micro-organisms). Multitrophic interactions between below-ground and above-ground organisms are receiving increasing attention because they may influence plant defences against biotic and abiotic stresses (van Dam et al., 2003). Plant defences can be constitutive or induced (Agrawal et al., 1999; Walling, 2000), and may also be direct (e.g. toxic compounds like glucosinolates in Brassicaceae) or indirect. Indirect defences typically involve the production of volatile semiochemicals that are attractive towards natural enemies of herbivorous insects (Dicke, 1999). These semiochemicals have been referred to as synomones to stress the mutual benefit of the partners involved (Vet & Dicke, 1992). Some of these volatiles are released as a specific response to the attack of a specific herbivore (see Agrawal et al., 1999 and references therein), a feature termed induced indirect defence. Several studies show that the release of induced volatiles is not confined exclusively to the organ attacked but involves all the plant through the circulation of systemic elicitors (Mattiacci et al., 1995; Alborn et al., 1997; Guerrieri et al., 1999; Dicke & Dijkman, 2001). The growing evidence that any colonising organism alters the profile of plant volatiles suggests that this may have intriguing and often unpredictable consequences for the performance of higher trophic levels (Dicke et al., 2003). In this paper we report on the interactions between below-ground interactions and indirect defences. Arbuscular mycorrhizal symbioses are mutualistic interactions between plant roots and soil fungi, and have been reported for more than 80% of higher plants (Smith & Read, 1997). Colonisation by arbuscular mycorrhizal fungi induces resistance or tolerance to a variety of pathogens in tomato and in other plants (Cordier et al., 1996; Trotta et al., 1996; Lingua et al., 2002). These changes are mediated by a variety of mechanisms, including the up-regulation and down-regulation of specific genes (TahiriAlaoui & Antoniw, 1996) that result in localised and systemic responses by the plant. These responses include the synthesis of new isoforms of chitinases and glucanases and the thickening of the cell walls (Azcón-Aguilar et al., 2002; Pozo et al., 2002) that may affect herbivore colonisation. The effects of arbuscular mycorrhizal symbiosis on aboveground herbivores has been investigated with contrasting results (van Dam et al., 2003 and references therein). More recently, the effects of different species of arbuscular mycorrhizal fungi on parasitism rates have been reported (Gange et al., 2003) but this study did not demonstrate a direct link between arbuscular mycorrhizae and attraction of insect parasitoids. In this study we tested the hypothesis that an arbuscular mycorrhizal symbiosis makes tomato plants significantly more attractive towards aphid parasitoids. Fig. 1a shows the multitrophic system used for this study: at the base of the system is tomato (Lycopersicon esculentum Miller) whose roots were colonised by the arbuscular mycorrhizal fungus Glomus mosseae Nicol & Gerd (Gerdemann & Trappe) BEG 12. Although tomato plants are characterised by high levels of constitutive defences (e.g. glandular trichomes, a-tomatine, Kennedy, 2003), induced defence mechanisms are nevertheless important (reviewed in Agrawal et al., 1999 and references therein). The herbivore (the potato aphid, Macrosiphum euphorbiae Thomas) is a key pest of tomato all over the world, causing direct and indirect damage to plants, including Correspondence: Emilio Guerrieri, Istituto per la Protezione delle Piante, CNR, Sez. Portici, Via Università 133, 80055 Portici (NA), Italy. E-mail: guerrieri@ipp.cnr.it Ecological Entomology (2004) 29, 753–756

Synergistic interactions between the ACC deaminase-producing bacterium Pseudomonas putida UW4 and the AM fungus Gigaspora rosea positively affect cucumber plant growth

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.

Mycorrhiza-induced differential response to a yellows disease in tomato.

Abstract. 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.

Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on heavy metal-contaminated soil, and this is associated with upregulation of foliar metallothionein and polyamine biosynthetic gene expression

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.

Clonal differences in survival capacity, copper and zinc accumulation, and correlation with leaf polyamine levels in poplar: a large-scale field trial on heavily polluted soil.

Three ex situ collections of poplar clones from natural populations of Populus alba and P. nigra growing in northern Italy were assessed for their genetic dissimilarity (GD) by means of amplified fragment length polymorphism (AFLP). The high GD evidenced within populations was exploited for screening 168 clones in a field trial on heavy metal-polluted soil. After one growth season, clonal differences in plant survival and growth were observed. On the basis of performance, six clones were singled out, and used to evaluate copper and zinc accumulation in different organs. Clonal differences in metal concentrations were most evident for leaves and stems; one clone of P. alba (AL35) had a distinctly higher concentration of both metals in the roots. Leaf polyamine (putrescine, spermidine, spermine) profiles correlated with tissue metal concentrations, depending on the clone, plant organ and metal. In particular, the high metal-accumulating clone AL35 exhibited a dramatically higher concentration of free and conjugated putrescine. Overall, the results indicate that, given the high GD of Populus even within populations, it is possible to identify genotypes best suited for soil clean-up, and useful also for investigating physiological markers associated with high metal accumulation/tolerance.

Colonization pattern of primary tomato roots by Pseudomonas fluorescens A6RI characterized by dilution plating, flow cytometry, fluorescence, confocal and scanning electron microscopy

Early colonization of primary tomato roots, grown in vitro, by Pseudomonas fluorescens A6RI, introduced by seed bacterization, was monitored for 7 days in three different root zones (zone A, apex+elongation+young hairy zone; zone B, hairy zone; zone C, old hairy zone+collar). Bacterial quantification was assessed by enumeration of (i) colony forming units (cfu) after dilution plating and of (ii) total bacterial cells by flow cytometry. Bacterial distribution and organization in the root zones were analyzed by fluorescence, confocal and scanning electron microscopy. For all sampling dates and zones, the densities of total bacterial cells were significantly higher than those of the cfu. The kinetics of cfu densities varied according to the root zone. Their density decreased with time in zone A, while no variation with time was recorded in zones B and C. Densities of total bacterial cells did not show any significant temporal variation for any of the root zones. Microscopic analyses allowed the characterization of the distribution and organizational patterns of the bacterial cells according to time and space. In 3-day-old plants, bacteria were mostly present as single cells and were evenly distributed in the two root zones analyzed (A and B). In 5- and 7-day-old plants, distribution and organization differed according to the root zone. In zone A, only few single cells were observed, whereas zones B and C were mostly covered by cells localized between epidermal root cells and organized in pairs and strings, respectively.

Proteomic analysis of Pteris vittata fronds: two arbuscular mycorrhizal fungi differentially modulate protein expression under arsenic contamination.

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.

Preferential colonization of Solanum tuberosum L. roots by the fungus Glomus intraradices in arable soil of a potato farming area.

ABSTRACT 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.

Effects of heavy metals and arbuscular mycorrhiza on the leaf proteome of a selected poplar clone: a time course analysis.

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.