The fungal elicitor AsES requires a functional ethylene pathway to activate the innate immunity in strawberry.

Acremonium strictum Elicitor Subtilisin (AsES) is a fungal elicitor that activates innate immunity, conferring disease resistance in strawberry (Fragaria × ananassa Duch.), Arabidopsis and other plant species. The aim of the present work was to evaluate the involvement of the ethylene (ET) signalling pathway in AsES-mediated immune response in strawberry. Ethylene production and expression of the genes responsible for ET synthesis, perception and response were measured after AsES treatment. ROS (H2 O2 ) accumulation and immunity induced by AsES were studied after ET perception was blocked by 1-methylcyclopropene (1-MCP). Biochemical and molecular results showed that AsES induced a marked increase in local and systemic biosynthesis of ET, both in a biphasic manner. Blocking of ET perception by 1-MCP prior to AsES induction reduced production of ROS (H2 O2 ) and prevented AsES from eliciting defence against fungal pathogens having different lifestyles, such as Botrytis cinerea (necrotrophic) and Colletotrichum acutatum (hemibiotrophic). These findings contribute to elucidate the mode of action of the novel elicitor subtilase, AsES, specifically regarding the role of ET signalling in the activation of plant innate immunity, in addition to the multitude of processes regulated by ET in plants.

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth-promoting bacterium Azospirillum brasilense.

Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth-promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum-strawberry plant interaction. Ethylene production was evaluated by gas chromatography in plants inoculated or not with A. brasilense REC3. Also, plants were treated with AgNO3 , an inhibitor of ET biosynthesis; with 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth-promoting effect of A. brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT-PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes. Results showed that ET acts as a rapid and transient signal in the first 12 h post-treatment. A. brasilense REC3-inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up-regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A. brasilense REC3 induces production of this auxin, modulating ET signalling. Ethylene production and up-regulation of genes associated with ET signalling in strawberry plants inoculated with A. brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

The novel elicitor AsES triggers a defense response against Botrytis cinerea in Arabidopsis thaliana.

AsES (Acremonium strictum Elicitor and Subtilisin) is a novel extracellular elicitor protein produced by the avirulent isolate SS71 of the opportunist strawberry fungal pathogen A. strictum. Here we describe the activity of AsES in the plant-pathogen system Arabidopsis thaliana-Botrytis cinerea. We show that AsES renders A. thaliana plants resistant to the necrotrophic pathogen B. cinerea, both locally and systemically and the defense response observed is dose-dependent. Systemic, but not local resistance is dependent on the length of exposure to AsES. The germination of the spores in vitro was not inhibited by AsES, implying that protection to B. cinerea is due to the induction of the plant defenses. These results were further supported by the findings that AsES differentially affects mutants impaired in the response to salicylic acid, jasmonic acid and ethylene, suggesting that AsES triggers the defense response through these three signaling pathways.

Physiological, structural and molecular traits activated in strawberry plants after inoculation with the plant growth-promoting bacterium Azospirillum brasilense REC3.

The plant growth-promoting strain REC3 of Azospirillum brasilense, isolated from strawberry roots, prompts growth promotion and systemic protection against anthracnose disease in this crop. Hence, we hypothesised that A. brasilense REC3 can induce different physiological, structural and molecular responses in strawberry plants. Therefore, the aim of this work was to study these traits activated in Azospirillum-colonised strawberry plants, which have not been assessed until now. Healthy, in vitro micropropagated plants were root-inoculated with REC3 under hydroponic conditions; root and leaf tissues were sampled at different times, and oxidative burst, phenolic compound content, malondialdehyde (MDA) concentration, callose deposition, cell wall fortification and gene expression were evaluated. Azospirillum inoculation enhanced levels of soluble phenolic compounds after 12 h post-inoculation (hpi), while amounts of cell wall bound phenolics were similar in inoculated and control plants. Other early responses activated by REC3 (at 24 hpi) were a decline of lipid peroxidation and up-regulation of strawberry genes involved in defence (FaPR1), bacterial recognition (FaFLS2) and H2O2 depuration (FaCAT and FaAPXc). The last may explain the apparent absence of oxidative burst in leaves after bacterial inoculation. Also, REC3 inoculation induced delayed structural responses such as callose deposition and cell wall fortification (at 72 hpi). Results showed that A. brasilense REC3 is capable of exerting beneficial effects on strawberry plants, reinforcing their physiological and cellular characteristics, which in turns contribute to improve plant performance.

Elemental composition of strawberry plants inoculated with the plant growth-promoting bacterium Azospirillum brasilense REC3, assessed with scanning electron microscopy and energy dispersive X-ray analysis.

The elemental composition of strawberry plants (Fragaria ananassa cv. Macarena) inoculated with the plant growth-promoting bacterium Azospirillum brasilense REC3, and non-inoculated controls, was studied using scanning electron microscopy (SEM) and energy dispersive X-ray (EDS) analysis. This allowed simultaneous semi-quantification of different elements in a small, solid sample. Plants were inoculated and grown hydroponically in 50% or 100% Hoagland solution, corresponding to limited or optimum nutrient medium, respectively. Bacteria-inoculated plants increased the growth index 45% and 80% compared to controls when grown in 100% and 50% Hoagland solution, respectively. Thus, inoculation with A. brasilense REC3 in a nutrient-limited medium had the strongest effect in terms of increasing both shoot and root biomass and growth index, as already described for Azospirillum inoculated into nutrient-poor soils. SEM-EDS spectra and maps showed the elemental composition and relative distribution of nutrients in strawberry tissues. Leaves contained C, O, N, Na, P, K, Ca and Cu, while roots also had Si and Cl. The organic fraction (C, O and N) accounted for over 96.3% of the total chemical composition; of the mineral fraction, Na had higher accumulation in both leaves and roots. Azospirillum-inoculated and control plants had similar elemental quantities; however, in bacteria-inoculated roots, P was significantly increased (34.33%), which constitutes a major benefit for plant nutrition, while Cu content decreased (35.16%).

First Report of Anthracnose on Fruits of Duchesnea indica Caused by Colletotrichum acutatum in Northwestern Argentina.

Duchesnea indica (Andrews) Focke, a cosmopolitan wild species related to the cultivated strawberry that is widely distributed in northwestern Argentina, grows in close proximity to strawberry crops and has proven to be almost immune to Colletotrichum spp. isolated from diseased strawberry plants (1), hence it has never been considered a phytopathological risk. During a field survey of "La Heladera" (27°01'45″S, 65°39'20″W), Tafí del Valle (Tucumán, Argentina) from November 2009 to November 2010, a genotype of D. indica showing fruits with dark brown, necrotic, irregular, circular lesions of 5 to 20 mm in diameter were collected. Setose acervuli were observed on the center of the fruit lesions. Pathogens were obtained from 10 diseased fruit collected at random, and four fungal isolates were isolated per fruit on potato dextrose agar (PDA). To reduce the number of samples for evaluation, two isolates per fruit that were exhibiting stable but distinctive morphological features were chosen to continue the studies. Isolates were characterized by morphological, molecular, and phytopathological criteria. After 10 days of incubation on PDA medium at 28°C with continuous white light, colonies exhibited a gray, aerial mycelium, whereas the reverse of the colony is a pale maroon with a radial, pale salmon color. Masses of salmon-colored conidia formed in the center of the colonies. Conidia were hyaline, one celled, fusiform, tapered to a point at both ends, and measured 14.8 to 17.3 × 4.5 to 7.4 µm (n = 100). Setae were scarce and sclerotia were absent. All morphological characteristics that were observed indicated that the isolates were C. acutatum (3). To fulfill Koch's postulates and verify the pathogenicity on commercial varieties of strawberry, six healthy plants of D. indica and Fragaria × ananassa cv. Camarosa with mature fruits were used to test each isolate. Four plants were spray inoculated with conidial suspensions of the virulent isolates (1.5 × 106 conidia/ml) and two with sterile distilled water as controls. Both treatments were maintained under white light (2,000 lux, 12 h per day) at 28°C and 70% relative humidity. Nine days after the inoculation, dark brown lesions and salmon-colored masses of conidia were observed only in inoculated fruits of both genotypes. The fungus isolated from diseased fruits and the conidia that were produced were identical to the isolates used to inoculate the plants. To confirm pathogen identity, PCR amplification with the species-specific pair of primer CaInt2/ITS4 (4) were carried out using fungal total DNA from the original isolates and isolates obtained from inoculated fruits. An amplification product of approximately 490 bp, which is specific for C. acutatum, was observed in all DNA samples (4). Although C. acutatum has already been reported in Fragaria × ananassa in Argentina (2), to our knowledge, this is the first report of C. acutaum causing anthracnose in D. indica species. This result is relevant since this species grows close to strawberry fields and can be an alternative host and potential vector of the anthracnose disease agent. References: (1) M. E. Arias. Frutillas Silvestres y Especies Relacionadas con la Cultivada. EDUNT, Argentina, 2007. (2) C. J. Ramallo et al. Plant Dis. 84:706. 2000. (3) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990. (4) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996.

Genetic diversity of Pto-like serine/threonine kinase disease resistance genes in cultivated and wild strawberries.

Degenerate oligonucleotide primers, designed based on conserved regions of several serine-threonine kinases (STK) previously cloned in tomato and Arabidopsis, were used to isolate STK candidates in wild and cultivated strawberries. Seven distinct classes of STKs were identified from three related wild species, i.e., Fragaria vesca, Fragaria chiloensis, and Potentilla tucumanensis, and seven different Fragaria x ananassa cultivars. Alignment of the deduced amino acid sequences and the Pto R protein from tomato revealed the presence of characteristic subdomains and conservation of the plant STK consensus and other residues that are crucial for Pto function. Based on identity scores and clustering in phylogenetic trees, five groups were recognized as Pto-like kinases. Strawberry Pto-like clones presented sequences that were clearly identified as the activation segments contained in the Pto, and some of them showed residues previously identified as being required for binding to AvrPto. Some of the non-Pto-like kinases presented a high degree of identity and grouped together with B-lectin receptor kinases that are also involved in disease resistance. Statistical studies carried out to evaluate departure from the neutral theory and nonsynonymous/synonymous substitutions suggest that the evolution of STK-encoding sequences in strawberries is subjected mainly to a purifying selection process. These results represent the first report of Pto-like STKs in strawberry.

Isolation and diversity analysis of resistance gene analogues (RGAs) from cultivated and wild strawberries.

Degenerate oligonucleotide primers, designed based on conserved regions of Nucleotide Binding Site (NBS) domains from previously cloned plant resistance genes, were used to isolate Resistance Gene Analogues (RGAs) from wild and cultivated strawberries. Seven distinct families of RGAs of the NBS-LRR type were identified from two related wild species, Fragaria vesca and F. chiloensis, and six different Fragaria x ananassa cultivars. With one exception (GAV-3), the deduced amino acid sequences of strawberry RGAs showed strong similarity to TIR (Toll Interleukin I Receptor)-type R genes from Arabidopsis, tobacco and flax, suggesting the existence of common ancestors. GAV-3 seemed to be more closely related to the non-TIR type. Further studies showed that the recombination level and the ratio of non-synonymous to synonymous substitutions within families were low. These data suggest that NBS-encoding sequences of RGAs in strawberry are subject to a gradual accumulation of mutations leading to purifying selection, rather than to a diversifying process. The present paper is the first report on RGAs in strawberry.

Modeling of the controlled expression of a harmful protein by a three-plasmid harboring system.

A genetically structured mathematical model was developed and used to evaluate the influence of molecular parameters involved in the expression of a harmful recombinant protein (SPA::EcoRI). The system consists of the controlled expression of the endonuclease EcoRI cloned in the plasmid pMTC48. The control is exerted by the lambda CI repressor expressed from the plasmid pRK248cIts. The deleterious effect of the activity of the enzyme EcoRI on the host DNA is prevented by the action of the EcoRI methylase that is expressed constitutively from a third plasmid, pEcoR4. The model includes molecular mechanisms involved in the regulation of the expression of these genes and is used to determine cultural conditions that maximize the production of the recombinant protein.

In-well cell lysis technique reveals two new megaplasmids of 103.0 and 212.6 MDa in the multiple plasmid-containing strain V517 of Escherichia coli.

AIMS: Identification of two new plasmids in the multiple plasmid-containing strain V517 of Escherichia coli. METHOD AND RESULTS: By using an in-well mild cell lysis technique suitable for megaplasmids observation, two plasmids of 103.0 and 212.6 MDa were detected in the multiplasmid-containing E. coli V517. CONCLUSIONS: The two new megaplasmids that were completely overlooked when standard disruptive procedures were used, can now be added to the list of eight plasmids with molecular size from 1.36 to 35.84 MDa reported earlier. SIGNIFICANCE AND IMPACT OF THE STUDY: This finding allows to use the strain V517 not only as a size reference of small and moderately large plasmids but as a size reference of megaplasmids as well.

Effect of fragarin on the cytoplasmic membrane of the phytopathogen Clavibacter michiganensis.

Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganensis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dissipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability.

Azospirillum amazonense: su presencia en el área cañera de la provincia de Tucumßn / Azospirillum amazonense: its presence in the sugar cane area of the province of Tucumßn

We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumßn. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.

Azospirillum amazonense: su presencia en el área cañera de la provincia de Tucumßn / Azospirillum amazonense: its presence in the sugar cane area of the province of Tucumßn

We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumßn. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.(AU)

Plasmid effects on Escherichia coli metabolism.

The idea that plasmids replicate within hosts at the expense of cell metabolic energy and preformed cellular blocks depicts plasmids as a kind of molecular parasites that, even when they may eventually provide plasmid-carrying strains with growth advantages over plasmid-free strains, doom hosts to bear an unavoidable metabolic burden. Due to the consistency with experimental data, this idea was rapidly adopted and used as a basis of different hypotheses to explain plasmid-host interactions. In this article we critically discuss current ideas about plasmid effects on host metabolism, and present evidence suggesting that the complex interaction between plasmids and hosts is related to the alteration of the cellular regulatory status.

ADP modulates the dynamic behavior of the glycolytic pathway of Escherichia coli.

A mathematical model that includes biochemical interactions among the PTS system, phosphofructokinase (PFK), and pyruvate kinase (PK) is used to evaluate the dynamic behavior of the glycolytic pathway of Escherichia coli under steady-state conditions. The influence of ADP, phosphoenolpyruvate (PEP), and fructose-6-phosphate (F6P) on the dynamic regulation of this pathway is also analyzed. The model shows that the dynamic behavior of the system is affected significantly depending on whether ADP, PEP, or F6P is considered constant a steady state. Sustained oscillations are observed only when dADP/dt not equal 0 and completely suppressed if dADP/dt = 0 at any steady-state value. However, when PEP or F6P is constant, the system evolves toward the formation of stable limit cycles with periods ranging from 0.2 min to hours.

[Azospirillum amazonense: its presence in the sugar cane area of the province of Tucumán]. / Azospirillum amazonense: su presencia en el área cañera de la provincia de Tucumán.

We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumán. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.

First Report of Colletotrichum gloeosporioides on Strawberry in Northwestern Argentina.

Colletotrichum gloeosporioides was isolated from symptomatic strawberry (Fragaria × ananassa Duch. 'Chandler') growing in Lules (Tucumán, Argentina). Isolates were characterized based on several criteria. Potato dextrose agar (PDA) was used to evaluate cultural and morphological characteristics of the isolates. After 10 days on PDA at 28°C under continuous white light, colonies showed abundant aerial, cottony white to pale beige growth, with orange asexual fruiting bodies in older colonies. Isolates displayed cylindrical conidia, rounded at both ends, averaging 10.4 × 3.9 µm (length by width). A sexual phase (perithecia) was observed in all isolates in 2-month-old cultures on PDA at 28°C under continuous white light. Pathogenicity tests were conducted with healthy plants of cvs. Pájaro and Chandler. Spray inoculation with conidial suspensions (106 conidia per ml) resulted in disease symptoms (petiole and crown lesions with wilting of crown-infected plants) 7 days after inoculation. Infection progressed at a higher rate in Pájaro than in Chandler. Reisolations from infected strawberry lesions yielded isolates with characteristics identical to the isolate used to inoculate the host. Based on morphological and cultural characteristics, isolates were identified as C. gloeosporioides Penz. & Sacc. (teleomorph Glomerella cingulata Spauld & H. Schenk) (1). This is the first report of C. gloeosporioides causing strawberry anthracnose in northwestern Argentina. Reference: (1) P. S. Gunnell and W. D. Gubler. Mycol. 84:157, 1992.

Azospirillum amazonense: su presencia en el área cañera de la provincia de Tucumán. / [Azospirillum amazonense: its presence in the sugar cane area of the province of Tucumán]

We confirm the presence of Azospirillum amazonense in the sugar cane area of the Province of Tucumán. Several strains were isolated from sugar-cane roots in different places. Phenotypic and genotypic characteristics coincided with those described for this species. This new one is being added to the other two species, A. brasilense and A. lipoferum, already reported.

Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA.

Plasmid-free and plasmid-harbouring E. coli JM109 strains were investigated in shaken flasks, stirred tanks in batch and continuous operation. The shaken flask cultivations were performed in M9 minimal medium and in media with various protein supplements. The host hardly grows on M9 minimal medium as opposed to the plasmid-harbouring cells, which grow well on this medium. All of the investigated cells propagate well on protein-containing media. The influence of the combinations of repressor plasmid pRK248cI, the protection plasmid EcoR4 and the production plasmid pMTC48 were determined on the initial specific growth rate of the E. coli JM109 without gene expression, on the yield coefficient of cell growth, acetate concentration and acetate yield coefficient in the yeast extract-containing (HM) medium. The influence of various media on the induction of the gene expression were evaluated. In cultivation media with protein supplement, the growth rate and yield coefficient increased. The variation of the volumetric and specific beta-lactamase activities with the cultivation time were determined in a stirred tank reactor in HM medium. With increasing dilution rate the process performance decreased. Simple relationships exist between the substrate uptake rate and the specific growth rate of the continuous cultivated cells in M9 and HM media. The influence of the dilution rate on the cell mass concentration, colony forming units, acetate formation, yield coefficients of growth and acetate formation, substrate uptake rate, CO2 production rate, ammonium formation rate and beta-lactamase activity in M9 and HM media were determined as well. Carbon balances of the batch and continuous cultivations indicated high carbon recoveries. On account of the higher growth rate of plasmid-harbouring cells than than of the plasmid-free cells, the behaviour of the investigated plasmid-free and plasmid-harbouring E. coli JM109 cells deviates from the published properties of other plasmid-free and plasmid-harbouring E. coli cells.

Gene expression enhancement due to plasmid maintenance.

Analysis of the chromosomic beta-galactosidase activity in strains of Escherichia coli with and without plasmids indicated that plasmid maintenance enhances gene expression. Cyclic AMP (cAMP) determinations confirmed that the gene enhancement observed in strains carrying plasmids was due to a small increase in the intracellular concentration of cAMP. Also, cells carrying plasmids displayed higher specific glucose uptake rates than did cells without plasmids. The increases in the expression of beta-galactosidase and the glucose uptake rate suggest a cAMP-mediated release of the glucose effect due to plasmid maintenance. Our results suggested that this effect is independent of the host and type and number of plasmids.