PROIMI Biotecnologia

Beyond their biological roles, metals have a strong impact on the environment. It has been reported that metals are also inhibitory of Quorum Sensing (QS) mechanisms, ones of the best characterized signaling systems in bacteria and fungi. We analyzed the effect of CuSO4, CdCl2, and K2Cr2O7, on QS systems sharing or differing in the bacterial host or the QS signal. The results in this study show that CuSO4 can not only be inhibitory, but also stimulatory of QS activity: at 0.2 mM increased six fold the activity in Chromobacterium subtsugae CV026. This behavior is related to the concentration of the metal and the particular QS system: E. coli MT102 (pJBA132) was no affected, but CuSO4 decreased the QS activity of Pseudomonas putida F117 (pKR-C12) to half its control values. K2Cr2O7 increased four and three folds the QS activities of E. coli MT102 (pJBA132) and P. putida F117 (pAS-C8), respectively, but without effect when combined with CuSO4 or CdCl2. CdCl2 only showed a positive effect in CV026 when combined with CuSO4. Results suggest that factors related with the culture conditions impact on the influence of the metals, and reinforce the importance of the environment in the modulation of QS activity.

The corn leafhopper Dalbulus maidis is a specialist herbivore that attacks maize in the tropical and subtropical regions of the Americas. It is the vector of three relevant plant pathogens that are responsible for severe yield losses. Modern agriculture is dependent on the addition of fertilizers, especially nitrogen, which may influence the nutritional quality and/or the defense ability of the plants possibly with a subsequent increment of herbivorous insect populations. Through a field experiment, using a randomized design with four treatments with incremental levels of fertilization, we evaluated the effects of nitrogen in corn on the population level of the vector D. maidis, on the incidence of the diseases transmitted by it, and on the level of parasitism of the vectors’ eggs. The amount of nitrogen fertilizer used significantly influenced the density of the corn leafhopper and, as a consequence, the parasitism by egg parasitoids, but not the incidence of the diseases transmitted by it. Two weeks after fertilization, the vector density was significantly higher in the highly fertilized treatment. The disease incidence was not directly linked with the level of fertilization. However, the symptoms of the diseases were much less evident in plants that received higher fertilization. Levels of parasitism by egg parasitoids increased accordingly to the level of D. maidis populations. The management of nitrogen fertilization in corn crops can mitigate the negative effects of the corn stunting disease without affecting the levels of natural control performed by egg parasitoids.

Quorum Sensing (QS) signaling, which allows the coordination of the microbial physiology, can be influenced by environmental factors. However, the impact of copper-based fungicides on the QS of rhizosphere microorganisms is currently unknown. The simultaneous presence of other microorganisms may also alter the QS signaling. In this work we report that the fungicide CuSO4 modifies the expression from the promoter of ppuI, ppoR and rsaL, components of Pseudomonas capeferrum WCS358 QS system. ppuI and ppoR promoters showed a biphasic behavior reducing their activities as CuSO4 concentrations increased up to 1 mmol l−1, but with increased values at higher metal concentrations. rsaL promoter attained a minimal activity with 1 mmol l−1, though higher concentrations did not modify its expression. The impact of CuSO4 on biofilm formation by WCS358 was dependent on the bacterial QS activity. Dual biofilms with Papiliotrema laurentii YL2 yeast were also affected by the bacterial QS and the metal. CuSO4 was detrimental for the bacterial colonization, but YL2 exerted a protective effect in dual colonies, an effect that depended on the QS activity. These results suggest the importance of the WCS358 QS systems in the rhizosphere, where copper-based fungicides and concomitant microorganisms conform a complex system.

In the environment, communication among microorganisms is a key aspect of their lives. Through communication, single cells of a microbial population can coordinate their gene expression and, in consequence, their physiology. Quorum sensing systems (QS) are one of these mechanisms that allow this communication through the production and detection of signal molecules whose concentration is related to the density of the signaling cells. In several Gram-negative bacteria, these QS signals belongs to the N-acyl homoserine lactone (AHL) family, which can be detected in the laboratory by means of simple bioassays. In this report, we present for undergraduate students a detailed protocol that allows us to evidence QS activity in environmental samples. First, with a selective culture medium, Gram-negative bacteria are isolated from samples provided by the students. Then, isolates are screened with two biosensor strains for the detection of AHL production. Positive results are visualized by the induction of violacein production in the biosensors. These experiments evidence the presence of communicating microorganisms in the analyzed samples. This simple protocol allows the students to analyze a particular microbial activity in samples provided by them, which enhance their learning experiences.

Agrobacterium tumefaciens is considered a prominent phytopathogen, though most isolates are nonpathogenic. Agrobacteria can inhabit plant tissues interacting with other microorganisms. Yeasts are likewise part of these communities. We analyzed the quorum sensing (QS) systems of A. tumefaciens strain 6N2, and its relevance for the interaction with the yeast Meyerozyma guilliermondii, both sugarcane endophytes. We show that strain 6N2 is nonpathogenic, produces OHC8-HSL, OHC10-HSL, OC12-HSL and OHC12-HSL as QS signals, and possesses a complex QS architecture, with one truncated, two complete systems, and three additional QS-signal receptors. A proteomic approach showed differences in QS-regulated proteins between pure (64 proteins) and dual (33 proteins) cultures. Seven proteins were consistently regulated by quorum sensing in pure and dual cultures. M. guilliermondii proteins influenced by QS activity were also evaluated. Several up- and down- regulated proteins differed depending on the bacterial QS. These results show the QS regulation in the bacteria-yeast interactions.

Two new species of Protonephrocerus Collin, P. flavipilus Skevington, Marques & Rafael sp. nov. and P. misionensis Skevington sp. nov. are described. The only other currently recognized species in the genus, P. chiloensis Collin, is redescribed and genetic data ascribed to this species are transferred to P. flavipilus sp. nov. All previously published genetic data refer to P. flavipilus sp. nov. The first records of Protonephrocerus in Argentina are documented.

Predator/Parasitoid functional response is one of the main tools used to study predation behaviour, and in assessing the potential of biological control candidates. It is generally accepted that predator learning in prey searching and manipulation can produce the appearance of type III functional response. Holling proposed that in the presence of alternative prey, at some point the predator would shift the preferred prey, leading to the appearance of a sigmoid function that characterized that functional response. This is supported by the analogy between enzyme kinetics and functional response that Holling used as the basis for developing this theory. However, after several decades, sigmoidal functional responses appear in the absence of alternative prey in most of the biological taxa studied. Here, we propose modelling the effect of learning on the functional response by using the explicit incorporation of learning curves in the parameters of the Holling functional response, the attack rate (a), and the manipulation time (h). We then study how the variation in the parameters of the learning curves causes variations in the shape of the functional response curve. We found that the functional response product of learning can be either type I, II or III, depending on what parameters act on the organism, and how much it can learn throughout the length of the study. Therefore the presence of other types of curves should not be automatically associated with the absence of learning. These results are important from an ecological point of view because when type III functional response is associated with learning, it is generally accepted that it can operate as a stabilizing factor in population dynamics. Our results, to the contrary, suggest that depending on how it acts, it may even be destabilizing by generating the appearance of functional responses close to type I.

The reproductive traits of the mymarid wasp Cosmocomoidea annulicornis (Ogloblin) (Hymenoptera: Mymaridae) attacking eggs of the sharpshooter Tapajosa rubromarginata (Signoret) (Hemiptera: Cicadellidae) were evaluated under laboratory conditions. Bioassays were carried out to estimate the realized fecundity and egg load of females. The ovigeny index was calculated and different biological traits, such as body size, oöcyte length, gaster length and wing length, were analysed to assess significant associations between these traits and the species fitness. In addition, the effect of host availability and feeding on longevity and potential fecundity throughout life and the effect of female age on egg maturation dynamics were assessed. The results showed that C. annulicornis is a strongly synovigenic species. A positive correlation was found between fecundity and longevity of the females and between body size and oöcyte length. Contrary to expected, body size was not related to fecundity and longevity. Females lived significantly longer in the presence of hosts and honey than when they were host-deprived and honey-fed or both host and honey deprived. Host availability had a significant effect on the amount of eggs laid by C. annulicornis females. Female age was negatively associated with oöcyte length. Furthermore, females were able to mature additional eggs as they aged, nevertheless, when host-deprived, senescent females presented significantly less mature eggs than younger ones, suggesting a possible egg oosorption. These results might contribute to a better understanding of the reproductive potential of this species as a biocontrol agent.

A reduction in the use of agrochemicals requires the development of either alternatives or complementary control methods in order to limit their dangerousness. An alternative is the biological control of fungi by bacteria. The fungal cell wall is a unique structure of the fungi, composed of glucan, chitin, and glycoproteins. Therefore, bacteria producing mycolytic enzymes, like chitinases, are of great interest to degrade fungal cell-wall components. The objectives of this work were to isolate chitinolytic bacteria from the guano of insectivorous bats (Tadarida brasiliensis) and to verify the presence of antifungal activities against phytopathogenic fungi. From the guano samples, 28 bacterial isolates were obtained, 70% of which presented chitinolytic activity. Four isolates were selected since they showed the highest values of chitinase activity, and they were characterized as belonging to Bacillus genus, by analyzing the 16S ribosomal RNA gene sequence. Cell-free supernatants of bacterial cultures were used in inhibition tests on 16 fungi: Alternaria and Colletotrichum acutatum were the most affected. Chitinase and antifungal activities were observed in the cell-free supernatant regardless of the culture medium used. Both activities were stable to heat and proteinase K treatments. Finally, when the culture medium was supplemented with 1 ml of cell-free supernatants (0.33%) and incubated for 120 h, the inhibition of hyphae formation and germination spores of reporter fungus were observed under light microscopy. These results suggest the feasibility of using cell-free supernatants as eco-friendly fungicides. The use of them may contribute to reducing the dose of toxic chemicals.

Quorum sensing modulates bacterial collective behaviors including biofilm formation, motility and virulence in the important human pathogen Acinetobacter baumannii. Disruption of quorum sensing has emerged as a promising strategy with important therapeutic potential. In this work, we show that light modulates the production of acyl-homoserine lactones (AHLs), which were produced in higher levels in the dark than under blue light at environmental temperatures, a response that depends on the AHL synthase, AbaI, and on the photoreceptor BlsA. BlsA interacts with the transcriptional regulator AbaR in the dark at environmental temperatures, inducing abaI expression. Under blue light, BlsA does not interact with AbaR, but induces expression of the lactonase aidA and quorum quenching, consistently with lack of motility at this condition. At temperatures found in warm-blooded hosts, the production of AHLs, quorum quenching as well as abaI and aidA expression were also modulated by light, though in this case higher levels of AHLs were detected under blue light than in the dark, in a BlsA-independent manner. Finally, AbaI reduces A. baumannii's ability to kill C. albicans only in the dark both at environmental as well as at temperatures found in warm-blooded hosts. The overall data indicate that light directly modulates quorum network in A. baumannii.

Mating has profound physiological and behavioral consequences for female insects. During copulation, female insects typically receive not only sperm, but a complex ejaculate containing hundreds of proteins and other molecules from male reproductive tissues, primarily the reproductive accessory glands. The post-mating phenotypes affected by male accessory gland (MAG) proteins include egg development, attraction to oviposition hosts, mating, attractiveness, sperm storage, feeding, and lifespan. In the Mexican fruit fly, Anastrepha ludens, mating increases egg production and the latency to remating. However, previous studies have not found a clear relationship between injection of MAG products and oviposition or remating inhibition in this species. We used RNA-seq to study gene expression in mated, unmated, and MAG-injected females to understand the potential mating- and MAG-regulated genes and pathways in A. ludens. Both mating and MAG-injection regulated transcripts and pathways related to egg development. Other transcripts regulated by mating included those with orthologs predicted to be involved in immune response, musculature, and chemosensory perception, whereas those regulated by MAG-injection were predicted to be involved in translational control, sugar regulation, diet detoxification, and lifespan determination. These results suggest new phenotypes that may be influenced by seminal fluid molecules in A. ludens. Understanding these influences is critical for developing novel tools to manage A. ludens. This article is protected by copyright. All rights reserved.

The corn leafhopper Dalbulus maidis (DeLong) (Hemiptera: Cicadellidae), transmits three important plant pathogens that adversely affect corn crop and ranges from the USA to Argentina. The vector has a rich natural enemy complex that generates high levels of parasitism, but its populations are persistent and prevalent. We characterized the oviposition sites of D. maidis on young corn plants in order to verify the hypothesis that the vector has an oviposition strategy for mitigating parasitism. Oviposition locations on plants were assessed in the laboratory and eggs within corn plants were exposed to natural parasitism in a cornfield. Eggs were located mostly laid in the unfolded leaves and were attacked by five parasitoid species. Parasitism was significantly affected by the class of leaf and the position of the egg in the leaf. Anagrus virlai Triapitsyn was the most abundant parasitoid species, which emerged significantly higher in the basal blade than other species. Our results suggest that leafhoppers minimize egg parasitism by laying their eggs within concealed locations on the plant.

In the rhizosphere, the role of yeasts in microbial interactions and signaling is an open question. To study the influence of fungicides on yeast inactivation of N-acyl homoserine lactone quorum sensing signals, we evaluated the resistance of Papiliotrema laurentii strains from tomato rhizosphere to CuSO4 and Cu2(OH)3Cl fungicides, and the copper effect on yeast quorum quenching. Copper resistance profiles and colony morphologies allowed the distinction of two groups of P. laurentii strains: mucoid, green and resistant, and brown-orange and more sensitive. Most of the strains inactivated C6-HSL and C10-HSL QS signals. Inactivation and copper divided the strains in three with weak activity independently of the metal, and 11 with activities affected by copper. The lack of alkalinization allows the hypothesis of an enzymatic inactivation of the signals. These results suggest that yeasts contribute to communications in the rhizosphere, and that copper fungicides can modify their interactions with other rhizosphere microorganisms.

The parasitic ability of Cosmocomoidea annulicornis (Hymenoptera: Mymaridae) was tested on eggs of the sharpshooter Tapajosa rubromarginata (Hemiptera: Cicadellidae), vector of the bacterium Xylella fastidiosa that causes citrus variegated chlorosis (CVC), among other plant diseases. In the present study, we evaluated biological traits of C. annulicornis such as parasitism and emergence rates, developmental time, longevity and sex ratio in laboratory conditions on leaves of Citrus aurantium, C. limon, Zea mays and Sorghum halepense. In addition, the functional response of C. annulicornis on host egg masses laid on C. aurantium leaves was studied and new distribution sites of the parasitoid in Argentina are reported. The overall parasitism of C. annulicornis was 49.20 ± 2.90%. Of those parasitized eggs, 83.30 ± 6.51% adults emerged. Parasitism of C. annulicornis was significantly lower in host eggs laid on S. halepense and developmental time was longer in C. limon compared to the other host plant species tested. In all host plant species tested, females developed faster than males. Individuals lived on average 5.94 ± 0.23 days and statistically significant differences were found between the longevity of females and males. Sex ratio of parasitoids was balanced in all host plant species tested, excepted for maize plants, in which sex ratio was female biased. Cosmocomoidea annulicornis females exhibited a type III functional response, indicating a greater searching efficiency at high host densities compared to low densities. Previous reports of field parasitism in combination with our results of C. annulicornis performance on eggs of T. rubromarginata are indicators of a potential biological control agent against the vector of X. fastidiosa, though further studies are needed regarding its biology.

The sharpshooter Tapajosa rubromarginata (Signoret) (Hemiptera: Cicadellidae, Proconiini), a vector of the bacterium Xylella fastidiosa Wells et al. (Xanthomonadaceae) that causes Citrus Variegated Chlorosis, has more than 30 reported host‐plant species. The fitness of a phytophagous insect is determined by the host plant suitability, plant resistance, and the natural enemies. The aim of this study was to: (1) identify plant species utilized as oviposition substrate by T. rubromarginata in the field; (2) establish the relationship between plants and clutch size; (3) establish the relationship among host plants, clutch size, and level of parasitism; and (4) establish variations in parasitoid composition and abundance in the various host plants. Egg masses of the sharpshooter were surveyed on plants reported as hosts, or those that were abundant in the study site. The number of eggs of the sharpshooter and emerged parasitoids were recorded for all the collected masses. We found egg masses of T. rubromarginata on 12 out of 21 plant species sampled. The size of the egg masses was greatly influenced by the type of leaf venation and to a lesser extent by the plant species. Parasitism rates were influenced by both leaf venation and host plant. Trichogrammatidae species were mostly associated with egg masses in plants with parallel‐veined leaves, whereas Mymaridae attacked masses laid in reticular‐veined leaves. The choice between a good host plant, but heavily attacked by parasitoids, and the host plants that are less suitable for nymphs but less frequently attacked by natural enemies, was a trade‐off for T. rubromarginata females to increase their fitness. We conclude that the host plant utilization by T. rubromarginata females in the field could be influenced by leaf structure and the strategy to avoid parasitism by selecting plants that were less attractive for parasitoids.

Following damage by herbivores, many plants release volatiles that dissuade future conspecifics from feeding. In many crop plants however, induced volatiles mediating this kind of interactions among plants, herbivores and also their natural enemies have been altered through the process of domestication. The selection of crops for increased yield may have gone at a cost of defense, possibly including defense-related volatiles. Dalbulus maidis (Hemiptera: Cicadellidae), a specialist leafhopper that only feeds on Zea spp., is a vector of Corn Stunt Spiroplasma, a serious maize disease. Here, we compared the volatiles released following D. maidis attack by a maize landrace and two maize hybrids of temperate and tropical background. Also, we performed behavioral assays with the leafhopper contrasting healthy non-attacked maize seedlings versus attacked seedlings. The maize landrace produced more than 6-fold larger quantities of induced volatiles compared to the maize hybrids after herbivory. Corn leafhopper females were able to detect and significantly preferred the odors of healthy seedlings over the attacked ones only in the landrace. They did not discriminate between the attacked and non-attacked hybrids. Additionally, we found that the attraction of the parasitoid wasp Anagrus virlai (Hymenoptera: Mymaridae) to its host was diminished in the tested hybrids. The parasitoid was able to detect the odors of the attacked landrace, however it was unable to discriminate between healthy and attacked maize hybrid plants. These results suggest that those more domesticated germplasms may have lost the ability not only to release volatiles that avoid colonization of future herbivores, but also to attract their natural enemies in a tritrophic system.

Among the species belonging to 11 genera of Neotropical spittlebugs attacking graminaceous plants, Notozulia entreriana (Berg) (Hemiptera: Cercopidae) is one of the most important pests of grasses in several countries of South America. In this study we evaluate the influence of three economically relevant species of host plants (Poaceae) on life‐cycle traits of N. entreriana . We tested for differences in fitness responses (survival and fecundity) of insects on forage species which are frequent hosts for wild populations of spittlebugs. Comparison of life cycles on three host plants showed that only 2.5% of nymphs completed their development on Zea mays L., 22.5% on Chloris gayana Kunth, and 95% on Brachiaria decumbens Stapf. All the above suggests that C. gayana and B. decumbens are able to sustain complete development, behaving like natural hosts; the latter species is superior for artificial colonies because a shorter developmental period, higher survivorship in nymphs, and longer lifespan of adults were observed. Zea mays did not allow proper development, causing high mortality instead. We also developed a new approach for small‐scale breeding of N. entreriana , which will enable subsequent biological and behavioral studies about this important pest species.

The search for new biomaterials with superior mechanical properties is the focus in the area of materials science. A promising pathway is drawing inspiration from nature to design and develop materials with enhanced properties. In this work, a novel strategy to produce functionalized supramolecular bionanomaterials from the microbial biofilm is reported. Tuneable biofilms with specific characteristics were obtained by controlling the culture condition of the microorganism. When the exopolysaccharide (EPS) production was desired the tryptone was the best nutritional component for the EPS production into the biofilm. However, for the expression of a high amount of amyloid protein the combination of peptone and glucose was the best nutritional choice. Each biofilm obtained showed its owner rheology properties. These properties were altered by the addition of extracellular DNA, which increased the viscosity of the biofilm and induced a viscoelastic hydrogel behavior. Besides, as a proof of concept of bionanomaterial, a novel supramolecular polymeric hybrid EPS-Amyloid protein (EPAP) was obtained from the biofilm and it was tested as a new natural functionalized support for enzyme immobilization. The results suggest that this technology could be used as a new concept to obtain biomaterials from biofilms by controlling the nutritional conditions of a microorganism. Understanding environmental factors affecting biofilm formation will help the development of methods for controlling biofilm production and therefore obtaining new biomaterials.

Lippia integrifolia, commonly known as “incayuyo”, is a traditional aromatic and medicinal plant that grows wild in northwestern Argentina. Aqueous extracts of this species exert beneficial effects against affections of the gastrointestinal tract, mainly gastric inflammations. Across the wide distribution of L. integrifolia, there is a high variability in the essential oil composition and four chemotypes have been characterized based on the dominant terpenoids in their essential oil composition: trans-nerolidol, lippiafolienone, spathulenol/byciclogermacrene and trans-davanone. In the present work, the morphology, anatomy, chemical constituents and biochemical properties of the aqueous extract of different chemotypes of L. integrifolia were evaluated comparatively. The chemotype whose essential oil is dominated by trans-nerolidol showed significantly higher density of trichomes on the leaves and higher yield in aqueous extractions. Only quantitative variations of dominant metabolites in the aqueous extracts were found among the different chemotypes; those compounds were characterized as 6-methoxyluteolin-O-hexoside, 6-methoxyscutellarein-hexoside, B ring-dimethoxylated flavone-hexoside I, II and lippidulcine-A. In spite of the higher phenolic and flavonoid content, trans-nerolidol chemotype sample presented similar antioxidant properties compared with the other chemotypes. Additionally, the histochemistry of leaves of the trans-nerolidol chemotype was analyzed. The trans-nerolidol chemotype, which grows naturally in the province of La Rioja (Puerto Alegre), presents the best qualities for the medicinal use of the species, due to its higher yield in aqueous extractions and higher phenolic and flavonoid content.

Dalbulus maidis is the most important leafhopper pest of maize in the Americas. Anagrus virlai is an egg parasitoid commonly associated with the corn leafhopper. We evaluated whether the performance of A. virlai is dependent on different diets provided during 24 hr or throughout adult female lifetime. Additionally, functional response of A. virlai on D. maidis eggs using maize leaves containing honeydew plus honey was described. A. virlai is a mostly pro-ovigenic autogenic species whose females are able to parasitize eggs immediately after emergence. We found that wasps oviposit the majority of their eggs in the first day of their adult life. Realized lifetime fecundity and lifetime potential fecundity did not differ significantly among treatments, but longevity and egg production increased when honey was added to diet. Data were consistent with an intermediate functional response between type II and III, but closer to type II, indicating a high parasitism rate at low host densities and a decrease in the oviposition rate at high host densities, due to a possible egg limitation. Our results suggest that carbohydrate food sources (honeydew and honey) might not be the factor limiting reproductive success during the first 24 hr. Food supply, however, might influence egg maturation and survivorship of wasps, thus potentially enhancing biological pest control when hosts are scarce in the course of the first few days of adulthood.