Characteristics and Prognostic Factors Associated With the Progression of Myopic Traction Maculopathy in Mexican Patients.

Background In this study, the characteristics and prognostic factors associated with the progression of myopic traction maculopathy (MTM) were evaluated in a Mexican population. Methods This is a retrospective observational study that analyzed patients with MTM who underwent optical coherence tomography (OCT). Clinical-ocular information, the MTM classification, and initial and final visual acuity (VA) were recorded. Results In total, 101 eyes of 84 patients (mean age 63.5 ± 10.7 years) were included (88.1% female and 11.9% male). The mean spherical equivalent was -16.8 ± 6.4 D, axial length was 29.6 ± 2.1 mm, and mean initial VA was 0.8 ± 0.5 logMAR. The mean follow-up time was 25.7 ± 27.6 months. The change in final VA from diagnosis to the last follow-up was +0.1 (0.2) (p = 0.001). Overall, 24.8% of patients progressed, 72.3% did not progress, and 3% showed regression. The patient-year progression rate was 0.20 ± 0.44. Factors associated with progression were initial logMAR VA (p= 0.012) and staphyloma (p= 0.001). Conclusions One in four patients with MTM progressed, and the patient-year progression rate was 0.5. The factors associated with disease progression were initial VA and the presence of staphyloma. The characteristics of Mexican patients with MTM are similar to those described in other populations.

Virulence-Related Assays for Investigation of the Acidovorax citrulli-Cucurbitaceae Pathosystem.

Acidovorax citrulli is one of the most important pathogens of cucurbit crops, mainly melon and watermelon. Although A. citrulli is able to infect all aerial parts of the plant, fruits are highly sensitive to the bacterium. Therefore, the disease is known as bacterial fruit blotch (BFB). The unavailability of effective tools for managing BFB, including the lack of resistant varieties, exacerbates the threat this disease poses to the cucurbit industry. However, despite the economic importance of BFB, still little is known about basic aspects of A. citrulli-plant interactions. Here, we present diverse techniques that have recently been developed for investigation of basic aspects of BFB, including identification of virulence determinants of the pathogen.

Isolation, Quantification, and Visualization of Extracellular Membrane Vesicles in Rhizobia Under Free-Living Conditions.

Rhizobia are a group of soil proteobacteria that are able to establish a symbiotic interaction with legumes. These bacteria are capable to fix atmospheric nitrogen into ammonia within specific plant root organs called nodules. The rhizobia-legume interaction is established by a complex molecular dialogue that starts with flavonoids exudated by the plant roots. In response, signaling molecules known as Nod factors (NFs) are secreted by the bacteria. These factors are sensed by specific plant receptors that trigger a downstream signaling cascade leading to rhizobium-specific intracellular colonization of the root hair via the formation of infection threads and the eventual development of nodules on roots. In these organs, rhizobia can fix nitrogen from the atmosphere for the plant in exchange for photosynthates and the appropriate environment for nitrogen fixation. Recently, it has been demonstrated that extracellular membrane vesicles (EMVs) produced by some rhizobia carry NFs. EMVs are proteolipidic structures that are secreted to the milieu from the bacterial membranes and are involved in several important biological processes, including intercellular communication. Thus far, little is known about rhizobia vesicles, and further studies are needed to understand their functions, including their role as transporting vessels of signaling molecules during the process of symbiosis. Here, we present a detailed protocol to isolate high-purity EMVs from free-living cultured rhizobia, test their integrity, and quantify their abundance.

Isolation of Rhizobial Extracellular Membrane Vesicles from Bacteroids.

Extracellular-membrane vesicles (EMVs) are spherical buds of the extracellular membrane, commonly produced by Gram-negative bacteria, known to mediate intricate inter-kingdom communication. In this context, comprehensive research dissecting the role of EMVs in one of the most complex nature-occurring molecular dialogues, rhizobium-legume symbiosis, has been so far neglected. During the different stages of the symbiotic process, rhizobia and their host plants establish a very specific and controlled intercellular trafficking of signal molecules. Thus, as conveyors of a broad range of molecules into the target cell, EMVs are gaining weight in the field. Here, we describe a detailed protocol to isolate EMVs from bacteroids of legume nodules, opening a new door for discovering new authors of the symbiotic process.

Natural variation in a short region of the Acidovorax citrulli type III-secreted effector AopW1 is associated with differences in cytotoxicity and host adaptation.

Bacterial fruit blotch, caused by Acidovorax citrulli, is a serious disease of melon and watermelon. The strains of the pathogen belong to two major genetic groups: group I strains are strongly associated with melon, while group II strains are more aggressive on watermelon. A. citrulli secretes many protein effectors to the host cell via the type III secretion system. Here we characterized AopW1, an effector that shares similarity to the actin cytoskeleton-disrupting effector HopW1 of Pseudomonas syringae and with effectors from other plant-pathogenic bacterial species. AopW1 has a highly variable region (HVR) within amino acid positions 147 to 192, showing 14 amino acid differences between group I and II variants. We show that group I AopW1 is more toxic to yeast and Nicotiana benthamiana cells than group II AopW1, having stronger actin filament disruption activity, and increased ability to induce cell death and reduce callose deposition. We further demonstrated the importance of some amino acid positions within the HVR for AopW1 cytotoxicity. Cellular analyses revealed that AopW1 also localizes to the endoplasmic reticulum, chloroplasts, and plant endosomes. We also show that overexpression of the endosome-associated protein EHD1 attenuates AopW1-induced cell death and increases defense responses. Finally, we show that sequence variation in AopW1 plays a significant role in the adaptation of group I and II strains to their preferred hosts, melon and watermelon, respectively. This study provides new insights into the HopW1 family of bacterial effectors and provides first evidence on the involvement of EHD1 in response to biotic stress.

Preterm preeclampsia screening using biomarkers: combining phenotypic classifiers into robust prediction models.

BACKGROUND: Preeclampsia screening is a critical component of antenatal care worldwide. Currently, the most developed screening test for preeclampsia at 11 to 13 weeks' gestation integrates maternal demographic characteristics and medical history with 3 biomarkers-serum placental growth factor, mean arterial pressure, and uterine artery pulsatility index-to identify approximately 75% of women who develop preterm preeclampsia with delivery before 37 weeks of gestation. It is generally accepted that further improvements to preeclampsia screening require the use of additional biomarkers. We recently reported that the levels of specific metabolites and metabolite ratios are associated with preterm preeclampsia. Notably, for several of these markers, preterm preeclampsia prediction varied according to maternal body mass index class. These findings motivated us to study whether patient classification allowed for combining metabolites with the current biomarkers more effectively to improve prediction of preterm preeclampsia. OBJECTIVE: This study aimed to investigate whether metabolite biomarkers can improve biomarker-based preterm preeclampsia prediction in 3 screening resource scenarios according to the availability of: (1) placental growth factor, (2) placental growth factor+mean arterial pressure, and (3) placental growth factor+mean arterial pressure+uterine artery pulsatility index. STUDY DESIGN: This was an observational case-control study, drawn from a large prospective screening study at 11 to 13 weeks' gestation on the prediction of pregnancy complications, conducted at King's College Hospital, London, United Kingdom. Maternal blood samples were also collected for subsequent research studies. We used liquid chromatography-mass spectrometry to quantify levels of 50 metabolites previously associated with pregnancy complications in plasma samples from singleton pregnancies. Biomarker data, normalized using multiples of medians, on 1635 control and 106 preterm preeclampsia pregnancies were available for model development. Modeling was performed using a methodology that generated a prediction model for preterm preeclampsia in 4 consecutive steps: (1) z-normalization of predictors, (2) combinatorial modeling of so-called (weak) classifiers in the unstratified patient set and in discrete patient strata based on body mass index and/or race, (3) selection of classifiers, and (4) aggregation of the selected classifiers (ie, bagging) into the final prediction model. The prediction performance of models was evaluated using the area under the receiver operating characteristic curve, and detection rate at 10% false-positive rate. RESULTS: First, the predictor development methodology itself was evaluated. The patient set was split into a training set (2/3) and a test set (1/3) for predictor model development and internal validation. A prediction model was developed for each of the 3 different predictor panels, that is, placental growth factor+metabolites, placental growth factor+mean arterial pressure+metabolites, and placental growth factor+mean arterial pressure+uterine artery pulsatility index+metabolites. For all 3 models, the area under the receiver operating characteristic curve in the test set did not differ significantly from that of the training set. Next, a prediction model was developed using the complete data set for the 3 predictor panels. Among the 50 metabolites available for modeling, 26 were selected across the 3 prediction models; 21 contributed to at least 2 out of the 3 prediction models developed. Each time, area under the receiver operating characteristic curve and detection rate were significantly higher with the new prediction model than with the reference model. Markedly, the estimated detection rate with the placental growth factor+mean arterial pressure+metabolites prediction model in all patients was 0.58 (95% confidence interval, 0.49-0.70), a 15% increase (P<.001) over the detection rate of 0.43 (95% confidence interval, 0.33-0.55) estimated for the reference placental growth factor+mean arterial pressure. The same prediction model significantly improved detection in Black (14%) and White (19%) patients, and in the normal-weight group (18.5≤body mass index<25) and the obese group (body mass index≥30), with respectively 19% and 20% more cases detected, but not in the overweight group, when compared with the reference model. Similar improvement patterns in detection rates were found in the other 2 scenarios, but with smaller improvement amplitudes. CONCLUSION: Metabolite biomarkers can be combined with the established biomarkers of placental growth factor, mean arterial pressure, and uterine artery pulsatility index to improve the biomarker component of early-pregnancy preterm preeclampsia prediction tests. Classification of the pregnant women according to the maternal characteristics of body mass index and/or race proved instrumental in achieving improved prediction. This suggests that maternal phenotyping can have a role in improving the prediction of obstetrical syndromes such as preeclampsia.

Membrane vesicle engineering with "à la carte" bacterial-immunogenic molecules for organism-free plant vaccination.

The United Nations heralds a world population exponential increase exceeding 9.7 billion by 2050. This poses the challenge of covering the nutritional needs of an overpopulated world by the hand of preserving the environment. Extensive agriculture practices harnessed the employment of fertilizers and pesticides to boost crop productivity and prevent economic and harvest yield losses attributed to plagues and diseases. Unfortunately, the concomitant hazardous effects stemmed from such agriculture techniques are cumbersome, that is, biodiversity loss, soils and waters contaminations, and human and animal poisoning. Hence, the so-called 'green agriculture' research revolves around designing novel biopesticides and plant growth-promoting bio-agents to the end of curbing the detrimental effects. In this field, microbe-plant interactions studies offer multiple possibilities for reshaping the plant holobiont physiology to its benefit. Along these lines, bacterial extracellular membrane vesicles emerge as an appealing molecular tool to capitalize on. These nanoparticles convey a manifold of molecules that mediate intricate bacteria-plant interactions including plant immunomodulation. Herein, we bring into the spotlight bacterial extracellular membrane vesicle engineering to encase immunomodulatory effectors into their cargo for their application as biocontrol agents. The overarching goal is achieving plant priming by deploying its innate immune responses thereby preventing upcoming infections.

Non-Ionic Osmotic Stress Induces the Biosynthesis of Nodulation Factors and Affects Other Symbiotic Traits in Sinorhizobium fredii HH103.

(1) Background: Some rhizobia, such as Rhizobium tropici CIAT 899, activate nodulation genes when grown under osmotic stress. This work aims to determine whether this phenomenon also takes place in Sinorhizobium fredii HH103. (2) Methods: HH103 was grown with and without 400 mM mannitol. ß-galactosidase assays, nodulation factor extraction, purification and identification by mass spectrometry, transcriptomics by RNA sequencing, motility assays, analysis of acyl-homoserine lactones, and indole acetic acid quantification were performed. (3) Results: Non-ionic osmotic stress induced the production of nodulation factors. Forty-two different factors were detected, compared to 14 found in the absence of mannitol. Transcriptomics indicated that hundreds of genes were either activated or repressed upon non-ionic osmotic stress. The presence of 400 mM mannitol induced the production of indole acetic acid and acyl homoserine lactones, abolished swimming, and promoted surface motility. (4) Conclusions: In this work, we show that non-ionic stress in S. fredii HH103, caused by growth in the presence of 400 mM mannitol, provokes notable changes not only in gene expression but also in various bacterial traits, including the production of nodulation factors and other symbiotic signals.

A complex regulatory network governs the expression of symbiotic genes in Sinorhizobium fredii HH103.

Introduction: The establishment of the rhizobium-legume nitrogen-fixing symbiosis relies on the interchange of molecular signals between the two symbionts. We have previously studied by RNA-seq the effect of the symbiotic regulators NodD1, SyrM, and TtsI on the expression of the symbiotic genes (the nod regulon) of Sinorhizobium fredii HH103 upon treatment with the isoflavone genistein. In this work we have further investigated this regulatory network by incorporating new RNA-seq data of HH103 mutants in two other regulatory genes, nodD2 and nolR. Both genes code for global regulators with a predominant repressor effect on the nod regulon, although NodD2 acts as an activator of a small number of HH103 symbiotic genes. Methods: By combining RNA-seq data, qPCR experiments, and b-galactosidase assays of HH103 mutants harbouring a lacZ gene inserted into a regulatory gene, we have analysed the regulatory relations between the nodD1, nodD2, nolR, syrM, and ttsI genes, confirming previous data and discovering previously unknown relations. Results and discussion: Previously we showed that HH103 mutants in the nodD2, nolR, syrM, or ttsI genes gain effective nodulation with Lotus japonicus, a model legume, although with different symbiotic performances. Here we show that the combinations of mutations in these genes led, in most cases, to a decrease in symbiotic effectiveness, although all of them retained the ability to induce the formation of nitrogen-fixing nodules. In fact, the nodD2, nolR, and syrM single and double mutants share a set of Nod factors, either overproduced by them or not generated by the wild-type strain, that might be responsible for gaining effective nodulation with L. japonicus.

Water Vapor Adsorption Provides Daily, Sustainable Water to Soils of the Hyperarid Atacama Desert.

Water is necessary for all life on Earth. Water is so critical that organisms have developed strategies to survive in hyperarid environments. These regions with extremely low water availability are also unique analogs in which to study the physico-chemical conditions of extraterrestrial environments such as Mars. We have identified a daily, sustainable cycle of water vapor adsorption (WVA) and desorption that measurably affects soil water content (SWC) in the hyperarid region of the Atacama Desert in southern Perú. We pair field-based soil temperature and relative humidity soil profiles with laboratory simulations to provide evidence for a daily WVA cycle. Using our WVA model, we estimate that one adsorptive period-one night-increases SWC by 0.2-0.3 mg/g of soil (∼30 µm rainfall). We can plausibly rule out other water inputs during our field campaign that could account for this water input, and we provide evidence that this WVA cycle is driven by solar heating and maintained by atmospheric water vapor. The WVA may also serve to retain water from infrequent rain events in these soils. If the water provided by WVA in these soils is bioavailable, it could have significant implications for the microorganisms that are endemic to hyperarid environments.

Preliminary Evaluation of a Blast Sprayer Controlled by Pulse-Width-Modulated Nozzles.

Precision spraying relies on the response of the spraying equipment to the features of the targeted canopy. PWM technology manages the flow rate using a set of electronically actuated solenoid valves to regulate flow rate at the nozzle level. Previous studies have found that PWM systems may deliver incorrect flow rates. The objective of the present study was to characterize the performance of a commercial blast sprayer modified with pulse-width-modulated nozzles under laboratory conditions, as a preliminary step before its further field validation. Four different duty cycles (25 percent, 50 percent, 75 percent and 100 percent) and four different pressures (400 kPa, 500 kPa, 600 kPa and 700 kPa) were combined to experimentally measure the flow rate of each nozzle. Results showed that the PWM nozzles mounted in the commercial blast sprayer, under static conditions, were capable of modulating flow rate according to the duty cycle. However, the reduction of flow rates for the tested duty cycles according to pressure was lower than the percentage expected. A good linear relation was found between the pressure registered by the control system feedback sensor and the pressure measured by a reference conventional manometer located after the pump. High-speed video recordings confirmed the accurate opening and closing of the nozzles according to the duty cycle; however, substantial pressure variations were found at nozzle level. Further research to establish the general suitability of PWM systems for regulating nozzle flow rates in blast sprayers without modifying the system pressure still remains to be addressed.

The Rhizobium tropici CIAT 899 NodD2 protein promotes symbiosis and extends rhizobial nodulation range by constitutive nodulation factor synthesis.

In the symbiotic associations between rhizobia and legumes, the NodD regulators orchestrate the transcription of the specific nodulation genes. This set of genes is involved in the synthesis of nodulation factors, which are responsible for initiating the nodulation process. Rhizobium tropici CIAT 899 is the most successful symbiont of Phaseolus vulgaris and can nodulate a variety of legumes. Among the five NodD regulators present in this rhizobium, only NodD1 and NodD2 seem to have a role in the symbiotic process. However, the individual role of each NodD in the absence of the other proteins has remained elusive. In this work, we show that the CIAT 899 NodD2 does not require activation by inducers to promote the synthesis of nodulation factors. A CIAT 899 strain overexpressing nodD2, but lacking all additional nodD genes, can nodulate three different legumes as efficiently as the wild type. Interestingly, CIAT 899 NodD2-mediated gain of nodulation can be extended to another rhizobial species, since its overproduction in Sinorhizobium fredii HH103 not only increases the number of nitrogen-fixing nodules in two host legumes but also results in nodule development in incompatible legumes. These findings potentially open exciting opportunities to develop rhizobial inoculants and increase legume crop production.

Fiabilidad y validez de constructo del Inventario de Autoestima de Coopersmith en estudiantes de medicina / Reliability and Construct Validity of the Coopersmith Self-esteem Inventory in Medical Students

RESUMEN Introducción: los problemas de autoestima constituyen uno de los principales motivos de consulta en las unidades de orientación estudiantil, en especial en las ciencias médicas; sin embargo, en el contexto cubano no existen instrumentos validados para la correcta evaluación. Los problemas de autoestima afectan seriamente el rendimiento académico y estimulan la deserción escolar, al no encontrar instrumentos validados en el contexto cubano para su correcta evaluación. Objetivo: determinar la fiabilidad y la validez de constructo del Inventario de Autoestima de Coopersmith en estudiantes de medicina de la Universidad de Ciencias Médicas de Pinar del Río, durante el año 2019. Métodos: estudio observacional, descriptivo y transversal en estudiantes de medicina de la Universidad de Ciencias Médicas de Pinar del Río durante el año 2019. La muestra estuvo constituida por 400 estudiantes cubanos de los seis años de la carrera. Se aplicó el Inventario de Autoestima de Coopersmith. Resultados: se obtuvieron valores muy elevados de Alfa de Crombach (0,91), lo cual es indicativo de una alta fiabilidad del instrumento. En el análisis factorial realizado, se obtuvieron cinco dimensiones o factores, en correspondencia con los resultados obtenidos por otros estudios, también se alcanzaron correlaciones adecuadas entre los ítems pertenecientes a cada factor. Conclusiones: el Inventario de Autoestima de Coopersmith mostró cualidades métricas adecuadas al ser validado en el contexto cubano, probó ser fiable y con una estructura multifactorial en correspondencia con resultados de estudios anteriores.

ABSTRACT Introduction: self-esteem problems constitute one of the main reasons for consultation in the units for students' counseling, especially in medical sciences; however, in the Cuban context there are no validated instruments for the correct evaluation. Self-esteem problems are a frequent reason for consultation in Student Counseling Unit in universities, especially in the medical sciences, seriously affecting academic performance and stimulating school desertion; however, in the Cuban context there are no validated instruments for their correct evaluation. Objective: to determine the reliability and construct validity of the Coopersmith Self-esteem Inventory in medical students from Pinar del Rio University of Medical Sciences during 2019. Methods: observational, descriptive and cross-sectional study in medical students from Pinar del Rio University of Medical Sciences during 2019. The sample was comprised of 400 Cuban students from the six academic years of the medicine major. The Coopersmith Self-esteem Inventory was applied. Results: very high values of Cronbach's Alpha (0,91) were obtained, that is an indication of a high reliability of the instrument. In the factor analysis carried out, five dimensions or factors were taken, in correspondence with the results reached by other studies; finding adequate correlations among the items belonging to each factor. Conclusions: Coopersmith Self-esteem Inventory showed adequate metric qualities when validated in the Cuban context, proving to be reliable and with a multifactorial structure in correspondence with the results of previous studies.

Knock-down of phosphoenolpyruvate carboxylase 3 negatively impacts growth, productivity, and responses to salt stress in sorghum (Sorghum bicolor L.).

Phosphoenolpyruvate carboxylase (PEPC) is a carboxylating enzyme with important roles in plant metabolism. Most studies in C4 plants have focused on photosynthetic PEPC, but less is known about non-photosynthetic PEPC isozymes, especially with respect to their physiological functions. In this work, we analyzed the precise roles of the sorghum (Sorghum bicolor) PPC3 isozyme by the use of knock-down lines with the SbPPC3 gene silenced (Ppc3 lines). Ppc3 plants showed reduced stomatal conductance and plant size, a delay in flowering time, and reduced seed production. In addition, silenced plants accumulated stress indicators such as Asn, citrate, malate, and sucrose in roots and showed higher citrate synthase activity, even in control conditions. Salinity further affected stomatal conductance and yield and had a deeper impact on central metabolism in silenced plants compared to wild type, more notably in roots, with Ppc3 plants showing higher nitrate reductase and NADH-glutamate synthase activity in roots and the accumulation of molecules with a higher N/C ratio. Taken together, our results show that although SbPPC3 is predominantly a root protein, its absence causes deep changes in plant physiology and metabolism in roots and leaves, negatively affecting maximal stomatal opening, growth, productivity, and stress responses in sorghum plants. The consequences of SbPPC3 silencing suggest that this protein, and maybe orthologs in other plants, could be an important target to improve plant growth, productivity, and resistance to salt stress and other stresses where non-photosynthetic PEPCs may be implicated.

Findings of uncertain significance by optical coherence tomography (OCT) as prognostic factors in neovascular age-related macular degeneration (nAMD) treated with ranibizumab.

BACKGROUND: Biomarkers hold great promise for personalized medicine as information gained from diagnostic or progression markers can be used to tailor treatment to the individual for highly effective intervention in the disease process. METHODS: The aim of this retrospective study was to evaluate the association between visual outcome and the presence of findings of uncertain significance by optical coherence tomography (OCT) pre and post loading dose in patients with neovascular age-related macular degeneration (nAMD) treated with ranibizumab. RESULTS: Univariate analysis revealed a higher letter gain in those with presence of onion sign (+ 5.6 ETDRS letters, p = 0.04) absence of prechoroidal cleft (+ 3.7 ETDRS letters, p = 0.04), intraretinal pseudocysts (+4.8 ETDRS letters, p = 0.002), subretinal pseudocysts (+ 4.6 ETDRS letters, p = 0.005) and choroidal caverns (+ 4.4 ETDRS, letters p = 0.0065). CONCLUSIONS: The presence of prechoroidal cleft, intraretinal and subretinal pseudocysts and choroidal caverns were associated with lower visual gains. Moreover, we found that the onion sign is related as a biomarker of good prognostics. Trial registration Registration number: 2021R13B2. Date of registration: 01/05/2020.

Effect of storage temperature and time on the behavior of Salmonella, Listeria monocytogenes, and background microbiota on whole fresh avocados (Persea americana var Hass).

Avocados are popular fruits; however, contamination of whole fresh avocados and avocado products with foodborne pathogens has raised concern about their safety. Recalls and import alerts of avocado products due to contamination with Listeria monocytogenes cause important economic losses. The behavior of Salmonella, L. monocytogenes, and background microbiota on whole fresh avocados at 5 and 25 °C as affected by temperature and time of storage was investigated. Whole fresh avocados were inoculated by immersion in suspensions containing six rifampicin-resistant strains of Salmonella or L. monocytogenes, and stored at 5 °C for 48 d, or at 25 °C for 11 d. At selected sampling times, avocados were removed from storage and pathogens enumerated. The log counts of both pathogens at each temperature were fitted to the Weibull distribution nonlinear model to estimate kinetic parameters including the time for the first 1-log reduction (δ), the shape of the curve (ρ), and the time for two (2-D) and three (3-D) log reductions. Salmonella and L. monocytogenes initial populations (approx. 7 log CFU/avocado) decreased during storage at 5 and 25 °C; L. monocytogenes mean counts were higher than those observed for Salmonella (P < 0.05). L. monocytogenes showed a lower rate of decline at 5 °C when compared to Salmonella. In general, the ability of both pathogens to survive on the surface of avocados stored at room temperature was similar. Salmonella and L. monocytogenes counts decline over time on the epicarp of whole avocados; however, if the initial number of cells is large enough, the pathogens could be present for large periods of time. Simultaneously, psychrotrophic microorganisms (PM), aerobic plate count (APC), coliforms (C) and yeasts/molds (Y/M) were enumerated from non-inoculated avocados stored at 5 and 25 °C. Initial mean counts for PM, APC, C and Y/M ranged from 6.1 to 6.6 log CFU/avocado and showed no change (P > 0.05) during storage at both temperatures. Good agricultural and handling practices from farm to fork are crucial to prevent or minimize contamination of whole avocados; otherwise, if large numbers of pathogens contaminate the fruit, they could survive and be transferred to the pulp, or to other ready to eat foods, representing a risk for consumers.

The nodD1 Gene of Sinorhizobium fredii HH103 Restores Nodulation Capacity on Bean in a Rhizobium tropici CIAT 899 nodD1/nodD2 Mutant, but the Secondary Symbiotic Regulators nolR, nodD2 or syrM Prevent HH103 to Nodulate with This Legume.

Rhizobial NodD proteins and appropriate flavonoids induce rhizobial nodulation gene expression. In this study, we show that the nodD1 gene of Sinorhizobium fredii HH103, but not the nodD2 gene, can restore the nodulation capacity of a double nodD1/nodD2 mutant of Rhizobium tropici CIAT 899 in bean plants (Phaseolus vulgaris). S. fredii HH103 only induces pseudonodules in beans. We have also studied whether the mutation of different symbiotic regulatory genes may affect the symbiotic interaction of HH103 with beans: ttsI (the positive regulator of the symbiotic type 3 protein secretion system), and nodD2, nolR and syrM (all of them controlling the level of Nod factor production). Inactivation of either nodD2, nolR or syrM, but not that of ttsI, affected positively the symbiotic behavior of HH103 with beans, leading to the formation of colonized nodules. Acetylene reduction assays showed certain levels of nitrogenase activity that were higher in the case of the nodD2 and nolR mutants. Similar results have been previously obtained by our group with the model legume Lotus japonicus. Hence, the results obtained in the present work confirm that repression of Nod factor production, provided by either NodD2, NolR or SyrM, prevents HH103 to effectively nodulate several putative host plants.

Secretion systems play a critical role in resistance to predation by Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, a Gram-negative predatory bacterium belonging to the Bdellovibrio and like organisms (BALOs), predates on Gram-negative bacteria. BALO strains differ in prey range but so far, the genetic basis of resistance against BALO predation is hardly understood. We developed a loss-of-function approach to screen for sensitive mutants in a library of strain M6, a predation-resistant strain of the plant pathogen Acidovorax citrulli. The screen is based on tracking the growth of a B. bacteriovorus strain expressing the fluorescent reporter Tdtomato in mutant pools to reveal predation-sensitive variants. Two independent loci were identified in mutant strains exhibiting significant levels of susceptibility to the predator. Genes in the two loci were analysed using both protein sequence homology and protein structure modeling. Both were secretion-related proteins and thus associated to the bacterial cell wall. Successful complementation of gspK, a gene encoding for a minor pseudopilin protein confirmed the involvement of the type II secretion system in A. citrulli M6 resistance. This proof of concept study shows that our approach can identify key elements of the BALO-prey interaction, and it validates the hypothesis that mutational changes in a single gene can drastically impact prey resistance to BALO predation.

One door closes, another opens: when nodulation impairment with natural hosts extends rhizobial host-range.

The rhizobium-legume symbiosis is the best-understood plant-microbe association. The high degree of specificity observed in this relationship is supported by a complex exchange of signals between the two components of the symbiosis. Findings reported in last years indicate that multiple molecular mechanisms, such as the production of a particular set of nodulation factors at a very specific concentration or a suitable arsenal of effectors secreted through the type III secretion system, have been adjusted during evolution to ensure and optimize the recognition of specific rhizobial strains by its legume host. Qualitative or quantitative changes in the production of these symbiotic molecular determinants are detrimental for nodulation with its natural host but, in some cases, can also result beneficial for the rhizobium since it extends the nodulation host-range to other legumes. Potential repercussion of the extension in the nodulation host-range of rhizobia is discussed.

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis.

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.