Defense related phytohormones regulation in arbuscular mycorrhizal symbioses depends on the partner genotypes.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Modulation of the hormonal and transcriptional profiles, including changes related to defense signalling, has been reported in many host plants during AM symbioses. These changes have been often related to the improved stress tolerance common in mycorrhizal plants. However, results on the alterations in phytohormones content and their role on the symbiosis are controversial. Here, an integrative analysis of the response of phylogenetically diverse plants (i.e., tomato, soybean, and maize) to two mycorrhizal fungi -Funneliformis mosseae and Rhizophagus irregularis- was performed. The analysis of the defense-related hormones salicylic acid, abscisic acid, and jasmonates, and the expression of marker genes of the pathways they regulate, revealed significant changes in the roots of mycorrhizal plants. These changes depended on both the plant and the AM fungus (AMF) involved. However, general trends can be identified: roots associated with the most effective colonizer R. irregularis showed fewer changes in these defense-related traits, while the colonization by F. mosseae led to significant modifications in all plants tested. The up-regulation of the jasmonate pathway by F. mosseae was found to be highly conserved among the different plant species, suggesting an important role of jasmonates during this AM interaction. Our study evidences a strong influence of the AMF genotype on the modulation of host defense signalling, and offers hints on the role of these changes in the symbiosis.

Multiple exocytotic markers accumulate at the sites of perifungal membrane biogenesis in arbuscular mycorrhizas.

Arbuscular mycorrhizas (AMs) are symbiotic interactions established within the roots of most plants by soil fungi belonging to the Glomeromycota. The extensive accommodation of the fungus in the root tissues largely takes place intracellularly, within a specialized interface compartment surrounded by the so-called perifungal membrane, an extension of the host plasmalemma. By combining live confocal imaging of green fluorescent protein (GFP)-tagged proteins and transmission electron microscopy (TEM), we have investigated the mechanisms leading to the biogenesis of this membrane. Our results show that pre-penetration responses and symbiotic interface construction are associated with extensive membrane dynamics. They involve the main components of the exocytotic machinery, with a major participation of the Golgi apparatus, as revealed by both TEM and in vivo GFP imaging. The labeling of known exocytosis markers, such as v-SNARE proteins of the VAMP72 family and the EXO84b subunit of the exocyst complex, allowed live imaging of the cell components involved in perifungal membrane construction, clarifying how this takes place ahead of the growing intracellular hypha. Lastly, our novel data are used to illustrate a model of membrane dynamics within the pre-penetration apparatus during AM fungal penetration.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Laparoscopic appendectomy for complicated acute appendicitis.

The role of laparoscopic appendectomy in complicated appendicitis is still not widely accepted. The authors report their retrospective study performed to evaluate the effectiveness of the laparoscopic approach in the management of complicated appendicitis. From January 2003 to October 2008, 552 patients underwent appendectomy in our surgical department. Among these, 358 were not complicated appendicitis while 194 were complicated. Of the 194 cases of complicated appendicitis, 121 patients underwent laparoscopic appendectomy while the remaining 73 cases were treated by conventional open surgery. The average length of hospital stay was 5.7 days, with a range from 4 to 13 days. Post-operative complications were observed in a total 11 patients (9.1%), including 3 cases of intra abdominal abscess (2.5%), 2 cases of umbilical wound infection (1.6%) and 6 cases of prolonged ileus (4.9%). Our experience suggests that the laparoscopic procedure is a valid, safe and feasible option to manage acute complicated appendicitis.

The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops.

Arbuscular mycorrhizal fungi (AMF) are a main component of soil microbiota in most agrosystems. As obligately mutualistic symbionts, they colonize the roots of the majority of plants, including crop plants. We used molecular techniques to investigate how different tillage systems (moldboard, shred-bedding, subsoil-bedding, and no tillage) can influence the AM fungal community colonizing maize, bean, and sorghum roots in an experimental site located in northern Tamaulipas, Mexico. Roots from 36 plants were analyzed using AM fungal-specific primers to partially amplify the small subunit (SSU) of the ribosomal DNA genes. More than 880 clones were screened for restriction fragment length polymorphism (RFLP) variation, and 173 of these were sequenced. Ten AM fungal types were identified and clustered into three AM fungal families: Gigasporaceae, Glomaceae, and Paraglomaceae. Glomus was the dominating taxon in all the samples. Four of the 10 identified types were distinct from any previously published sequences and could correspond to either known unsequenced species or unknown species. The fungal diversity was low in the four agriculture management systems, but the multidimensional scaling (MDS) analysis and log-linear-saturated model indicated that the composition of the AMF community was significantly affected by the tillage system. In conclusion, since some fungal types were treatment specific, agricultural practices could directly or indirectly influence AM biodiversity.

[Primary duodenal adenocarcinoma: report of three cases, prognostic factors and therapeutic approach]. / Adenocarcinoma primitivo del duodeno: tre casi clinici e considerazioni prognostico-terapeutiche.

Three cases of histologically proven primary non-ampullary adenocarcinoma of the duodenum, observed in our Department from 2001 to 2004, are described. The cases were treated by pancreaticoduodenectomy, duodenal resection and transduodenal excision, respectively. The rarity of this pathology is documented by few retrospective studies and justifies discussion about the main prognostic factors and the best therapeutic approach. We analyze diagnostic, therapeutic and prognostic factors after a revision of literature.

[Radiological study of anastomotic leakages following colorectal surgery]. / Il controllo radiologico delle deiscenze anastomotiche in chirurgia colo-rettale.

The anastomotic leakage is one of the most serious complications following colorectal surgery. The incidence rate is between 3% and 21% considering the different experiences, pathology and surgical techniques. Our aim is to verify the role of radiological study in 45 patients with clinical and subclinical colorectal anastomotic leakage total anastomoses = 252). In 31 patients at risk, the operation was concluded with a loop ileostomy. The radiological study gastrografin enema was performed in all patients (26 symptomatic and 19 asymptomatic patients with loop ileostomy). The dehiscence incidence resulted 5.5%: 14 of 252 patients. In our experience the radiological study of selected colorectal anastomoses allowed to show the site and the flow of the leakage and to plan the proper management. In asymptomatic patients the study allowed to programme a specific follow up in patients with higher risk of postinflammatory stenosis or perhaps neoplastic relapse.

[Surgical timing in bleeding liver adenoma: case report]. / Timing chirurgico nell'adenoma epatico sanguinante: case report.

The diagnosis of liver adenoma, which etiopathogenesis most often involves a prolonged assumption of estrogen (90% of adenomas occurs in women after more than 5 years of estrogen therapy), always imposes a surgical resection. The reason depend from neoplasia characteristics like the malignant evolution (4%) and the high risk of abdominal/intratumoral bleeding (30-50%), that increases during pregnancy and postpartum period. Regression of lesion after discontinuation of hormone therapy is rare and does not remove the degeneration and/or haemorrhagic risk. Liver resection should be performed with appropriate selective endovascular embolization, considering that an inept emergency surgery may impose a greater risk ot the liver, exposing the patient to major risk of morbidity and mortality. The correct timing from embolization to elective surgery is not yet standardized in the literature. The surgeon's personal experience and mainly a careful patient follow-up suggest the timing of surgery after embolization. The authors relate their own experience about the therapeutic strategy and surgical timing in a case of bleeding liver adenoma.

[Covered perforation of solitary cecal diverticulum: case report]. / Perforazione coperta di un diverticolo solitario del cieco: case report.

Acute right lower abdominal pain is often clinically difficult to diagnose. The diagnosis, especially in young patients, is frequently oriented to appendicular disease. Surgical exploration only confirms diagnosis or surprises the surgeon, revealing an unexpected right colon diverticulitis. This emergency condition challenges the surgeon with the dilemma about the best therapeutic choice: conservative or radical treatment? The elective localization of diverticulitis to the right colon is very rare (6.6-14%). The authors report a case of covered perforation of a solitary cecal diverticulum.

Intraradical colonization by arbuscular mycorrhizal fungi triggers induction of a lipochitooligosaccharide receptor.

Functional divergence of paralogs following gene duplication is one of the mechanisms leading to evolution of novel pathways and traits. Here we show that divergence of Lys11 and Nfr5 LysM receptor kinase paralogs of Lotus japonicus has affected their specificity for lipochitooligosaccharides (LCOs) decorations, while the innate capacity to recognize and induce a downstream signalling after perception of rhizobial LCOs (Nod factors) was maintained. Regardless of this conserved ability, Lys11 was found neither expressed, nor essential during nitrogen-fixing symbiosis, providing an explanation for the determinant role of Nfr5 gene during Lotus-rhizobia interaction. Lys11 was expressed in root cortex cells associated with intraradical colonizing arbuscular mycorrhizal fungi. Detailed analyses of lys11 single and nfr1nfr5lys11 triple mutants revealed a functional arbuscular mycorrhizal symbiosis, indicating that Lys11 alone, or its possible shared function with the Nod factor receptors is not essential for the presymbiotic phases of AM symbiosis. Hence, both subfunctionalization and specialization appear to have shaped the function of these paralogs where Lys11 acts as an AM-inducible gene, possibly to fine-tune later stages of this interaction.

Bacterial associations with mycorrhizal fungi: close and distant friends in the rhizosphere.

Bacteria and mycorrhizal fungi in the rhizosphere interact with each other at different levels of cellular integration, ranging from apparently simple association, through surface attachment, to intimate and obligatory symbiosis. This synergism may not only be important in promoting plant growth and health, but may also be significant to rhizosphere ecology.

Differential Localization of Carbohydrate Epitopes in Plant Cell Walls in the Presence and Absence of Arbuscular Mycorrhizal Fungi.

Two monoclonal antibodies (McAbs) generated against rhamnogalacturonan I and characterized as specific for a terminal [alpha]-(1->2)-linked fucosyl-containing epitope (CCRC-M1) and for an arabinosylated [beta]-(1,6)-galactan epitope (CCRC-M7) were used in immunogold experiments to determine the distribution of the epitopes in four plants. Allium porrum, Zea mays, Trifolium repens, and Nicotiana tabacum plants were chosen as representatives of monocots and dicots with different wall structures. Analyses were performed on root tissues in the presence and absence of arbuscular mycorrhizal fungi. A differential localization of the two cell wall epitopes was found between tissues and between species: for example, in leek, CCRC-M1 labeled epidermal and hypodermal cells, whereas CCRC-M7 labeled cortical cells only. Clover walls were labeled by both McAbs, whereas maize and tobacco were only labeled by CCRC-M7. In the presence of the arbuscular mycorrhizal fungi, labeling was additionally found in an apoplastic compartment typical of the symbiosis (the interface) occurring around the intracellular hyphae. Epitopes binding both McAbs were found in the interfacial material, and their distribution mirrored the pattern found in the host cell wall. These findings demonstrate that the composition of the interface zone in a fungus-plant symbiosis reflects the composition of the wall of the host cell.

Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots.

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.

The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells.

The role of the Lotus japonicus LjSym4 gene during the symbiotic interaction with Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi was analyzed with two mutant alleles conferring phenotypes of different strength. Ljsym4-1 and Ljsym4-2 mutants do not form nodules with M. loti. Normal root hair curling and infection threads are not observed, while a nodC-dependent deformation of root hair tips indicates that nodulation factors are still perceived by Ljsym4 mutants. Fungal infection attempts on the mutants generally abort within the epidermis, but Ljsym4-1 mutants allow rare, successful, infection events, leading to delayed arbuscule formation. On roots of mutants homozygous for the Ljsym4-2 allele, arbuscule formation was never observed upon inoculation with either of the two AM fungi, Glomus intraradices or Gigaspora margarita. The strategy of epidermal penetration by G. margarita was identical for Ljsym4-2 mutants and the parental line, with appressoria, hyphae growing between two epidermal cells, penetration of epidermal cells through their anticlinal wall. These observations define a novel, genetically controlled step in AM colonization. Although rhizobia penetrate the tip of root hairs and AM fungi access an entry site near the base of epidermal cells, the LjSym4 gene is necessary for the appropriate response of this cell type to both microsymbionts. We propose that LjSym4 is required for the initiation or coordinated expression of the host plant cell's accommodation program, allowing the passage of both microsymbionts through the epidermis layer.

A novel class of ectomycorrhiza-regulated cell wall polypeptides in Pisolithus tinctorius.

Development of the ectomycorrhizal symbiosis leads to the aggregation of fungal hyphae to form the mantle. To identify cell surface proteins involved in this developmental step, changes in the biosynthesis of fungal cell wall proteins were examined in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhizas by two-dimensional polyacrylamide gel electrophoresis. Enhanced synthesis of several immunologically related fungal 31- and 32-kDa polypeptides, so-called symbiosis-regulated acidic polypeptides (SRAPs), was observed. Peptide sequences of SRAP32d were obtained after trypsin digestion. These peptides were found in the predicted sequence of six closely related fungal cDNAs coding for ectomycorrhiza up-regulated transcripts. The PtSRAP32 cDNAs represented about 10% of the differentially expressed cDNAs in ectomycorrhiza and are predicted to encode alanine-rich proteins of 28.2 kDa. There are no sequence homologies between SRAPs and previously identified proteins, but they contain the Arg-Gly-Asp (RGD) motif found in cell-adhesion proteins. SRAPs were observed on the hyphal surface by immunoelectron microscopy. They were also found in the host cell wall when P. tinctorius attached to the root surface. RNA blot analysis showed that the steady-state level of PtSRAP32 transcripts exhibited a drastic up-regulation when fungal hyphae form the mantle. These results suggest that SRAPs may form part of a cell-cell adhesion system needed for aggregation of hyphae in ectomycorrhizas.

Impact on arbuscular mycorrhiza formation of pseudomonas strains used as inoculants for biocontrol of soil-borne fungal plant pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 &mgr;M DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.

Actin versus tubulin configuration in arbuscule-containing cells from mycorrhizal tobacco roots.

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells. Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus. These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant-fungus interactions during the mycorrhizal symbiosis.

Development and good breeding in legume models: poise and peas?: Molecular Genetics of Model Legumes.

John Innes Centre, Norwich, UK, June 2000 Genomics research involving legumes, an area that is attracting major funding, has two distinct branches - work involving model species, and work involving crops. This meeting aimed to stimulate communication between these two groups. The major research areas covered included leaf, flower and seed development, establishment of symbioses, pathogen interactions and applied aspects (from the conservation of legume ecotypes to products required by the market).

A combined morphological and molecular approach to characterize isolates of arbuscular mycorrhizal fungi in Gigaspora (Glomales).

• Morphological features of resting spores and information from nucleotide sequences of ribosomal RNA were used to characterize seven mycorrhizal fungal isolates in Gigaspora from different geographical areas. • Detailed observations were made under the light microscope on single spores mounted in Melzer's reagent and polyvinyl alcohol-lactic acid-glycerol medium to resolve size, colour and cell wall structures. Neighbour-joining analyses were carried out on a portion of the 18S gene and on the internal transcribed spacer (ITS) region amplified by PCR from multisporal DNA preparations. • Combined data allowed us to design oligonucleotides that unambiguously distinguished Gi. rosea from Gi. margarita and Gi. gigantea and also identified two isolates as Gi. rosea that had been previously diagnosed as Gi. margarita. ITS sequences revealed substantial genetic variability within clones of a single isolate of Gi. rosea as well as among geographically disjunct Gi. rosea isolates. • The results show how complementary morphological and molecular data can clarify relationships among species of low morphological divergence. Sequence information allowed the extent of genetic divergence within these species to be investigated and provided useful PCR primers for detection and identification.