PROIMI Biotecnologia

After a time away from the classrooms and laboratories due to the global pandemic, the return to teaching activities during the semester represented a challenge to both teachers and students. Our particular situation in a Microbial Physiology course was the necessity of imparting in shorter time, laboratory practices that usually take longer. This article describes a 2-week-long laboratory exercise that covers several concepts in an interrelated way: conjugation as a gene transfer mechanism, regulation of microbial physiology, production of secondary metabolites, degradation of macromolecules, and biofilm formation. Utilizing a Quorum Quenching (QQ) strategy, the Quorum Sensing (QS) system of Pseudomonas aeruginosa is first attenuated. Then, phenotypes regulated by QS are evidenced. QS is a regulatory mechanism of microbial physiology that relies on signal molecules. QS is related in P. aeruginosa to several virulence factors, some of which are exploited in the laboratory practices presented in this work. QQ is a phenomenon by which QS is interrupted or attenuated. We utilized a QQ approach based on the enzymatic degradation of the P. aeruginosa QS signals to evidence QS-regulated traits that are relevant to our Microbial Physiology course. Results obtained with the same test performed by a random group of students before and after the activities show the positive effectiveness of the approach presented in this work.

The "fall armyworm", Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a significant pest of many crops, particularly maize. To control it, genetically modified plants and synthetic pesticides are the main strategies employed, which has led to the emergence of resistant populations to both methods. The earwig Doru lineare (Eschscholtz) (Dermaptera: Forficulidae) has been observed in Argentina consuming both eggs and early instars S. frugiperda larvae. To contribute to the implementation of a rational pest management system, field cage tests were conducted to assess the efficiency of D. lineare as a predator against S. frugiperda and its potential protection of maize seedlings from the pest. The results of the tests showed that the survival rate of S. frugiperda larvae was significantly affected by the presence of earwigs. When two earwigs were released into a cage containing ten maize seedlings infested with twenty S. frugiperda larvae, after ten days, the dry weight of the plants was significantly higher and the level of damage was significantly lower compared to those without earwig release. These results confirm the protective effect of D. lineare against S. frugiperda, suggesting that earwigs should be considered as promising predators in conservation control strategies for maize crops.

The corn leafhopper Dalbulus maidis (Hemiptera: Cicadellidae) is a serious maize pest widely distributed on the American continent. This is the most common leafhopper feeding on corn crops in Argentina and Brazil, causing major economic losses due to its capability to transmit important pathogens, such as corn stunt spiroplasma, and considerably decreasing crop yield. One way to increase crop yield is to elevate fertilizing doses of nitrogen and phosphorus, although this could translate into an increase in pest populations, since changes in host plant quality makes them more attractive to herbivorous insects, positively influencing their population dynamics. This study aims to assess the oviposition preference and the olfactory response of D. maidis to corn plants subject to three levels of nitrogen fertilization (100 ppm, 200 ppm and 300 ppm). Olfactory response was evaluated in a static-air four-way olfactometer using maize plants with different nitrogen fertilization levels as odor sources. Additionally, plants with different nitrogen fertilization levels were offered to females in a multiple-choice assay to assess their oviposition preference. The olfactory response of D. maidis was influenced by nitrogen fertilization, as females oriented toward plants treated with high fertilization levels. Similarly, oviposition preference assays showed that D. maidis females preferred to lay its egg in plants with high fertilization levels. The results herein reported could contribute to the design of integrated pest management strategies that promote balance between crop fertilization to improve yield and insect vector management.

Vinasse is a by-product with a key role in circular economy. In this work, we analyze sugarcane vinasse as culture medium for obtaining single and mixed inoculants. Trichoderma harzianum MT2 was cultured in single and sequential co-culture with Pseudomonas capeferrum WCS358 or Rhizobium sp. N21.2. Fungal biomass in single culture was more than three folds higher in vinasse than in a standard medium, and was higher in co-culture with Rhizobium sp. N21.2 than with P. capeferrum WCS358. Bacterial growths in vinasse, in particular P. capeferrum WCS358, were improved in co-culture with T. harzianum MT2. Residual vinasses, obtained after microbial growth, presented almost neutral pH and lower conductivities and toxicity than raw vinasse. Fertigation with residual vinasses modifies characteristics of soil evidenced in the total N, cation exchange capacity, urease and acid phosphatase, and microbial metabolic diversity, in comparison to raw vinasse. In general, soil fertigation with residual vinasse from co-culture with P. capeferrum WCS358 is more similar to irrigation with water. Treatment evaluation indicates that vinasse is suitable for the production of mixed inoculants containing T. harzianum. The co-culture with P. capeferrum WCS358 improves the characteristics of the residual vinasse allowing a fertigation with less detrimental effect in soil in comparison to Rhizobium sp. N21.2. Obtaining valuable biomass of single or mixed inoculants in vinasse with lower ecological impact is relevant for the circular and green economy.

We evaluated the induction of plant volatile organic compounds (VOCs) in maize after the attack of two piercing-sucking maize hoppers: the specialist Dalbulus maidis and the oligophagous Peregrinus maidis. We analyzed VOCs in plants after feeding and oviposition by females of both species. We also contrasted VOCs induced by D. maidis males with those induced by females’ infestation that comprised oviposition, to evaluate which VOC could be responsible for attracting an egg parasitoid. We quantified the damage performed by both hoppers and tested the behavioral response of the egg parasitoid Anagrus virlai to VOCs emitted by infested and healthy plants. Plants infested by D. maidis females released significantly more VOCs than the other treatments: healthy plants, plants infested by P. maidis females and plants infested by D. maidis males. β-Caryophyllene, α-bergamotene, β-farnesene and cis-3-hexenyl acetate dominated the volatile blend (46%) in plants infested by D. maidis females, while in those plants infested by P. maidis these compounds represented only 17%. Females of both hoppers inflicted a comparable amount of damage, in terms of feeding injuries and number of eggs laid. β-Caryophyllene and cis-3-hexenyl acetate were not induced in plants infested by D. maidis males, that had no oviposited eggs. Finally, females of the egg parasitoid oriented toward VOCs from plants infested by females of the specialist D. maidis, rather than those infested by the oligophagous P. maidis or those infested by D. maidis males.

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.

Vinasse is a high pollutant liquid residue from bioethanol production. Due to its toxicity, most vinasse is used not disposed of in water bodies but employed for the fertigation of sugarcane crops, potentially leading to soil salinization or heavy metal deposition. The anaerobic digestion of vinasse for energy production is the main alternative to fertigation, but the process cannot eliminate colored compounds such as melanoidins, caramels, or phenolic compounds. The treatment of raw vinasse with white-rot fungi could remove colored and persistent toxic compounds, but is generally considered cost-ineffective. We report the treatment of vinasse by an autochthonous Trametes sp. strain immobilized in polyurethane foam and the concomitant production of high titers of laccase, a high value-added product that could improve the viability of the process. The reuse of the immobilized biomass and the discoloration of raw vinasse, the concentration of phenolic compounds, BOD and COD, and the phytotoxicity of the treated vinasse were measured to assess the viability of the process and the potential use of treated vinasse in fertigation or as a complementary treatment to anaerobic digestion. Under optimal conditions (vinasse 0.25X, 30 °C, 21 days incubation, 2% glucose added in the implantation stage), immobilized Trametes sp. causes a decrease of 75% in vinasse color and total phenolic compounds, reaching 1082 U L⁻¹ of laccase. The fungi could be used to treat 0.50X vinasse (BOD 44,400 mg O2 L⁻¹), causing a 26% decolorization and a 30% removal of phenolic compounds after 21 days of treatment with maximum laccase titers of 112 U L⁻¹, while reducing COD and BOD from 103,290 to 42,500 mg O2 L⁻¹ (59%) and from 44,440 to 21,230 mg O2 L⁻¹ (52%), respectively. The re-utilization of immobilized biomass to treat 0.50X vinasse proved to be successful, leading to the production of 361 U L⁻¹ of laccase with 77% decolorization, 61% degradation of phenolic compounds, and the reduction of COD and BOD by 75% and 80%, respectively. Trametes sp. also reduced vinasse phytotoxicity to Lactuca sativa seedlings. The obtained results show that the aerobic treatment of vinasse by immobilized Trametes sp. is an interesting technology that could be employed as a sole treatment for the bioremediation of vinasse, with the concomitant the production of laccase. Alternatively, the methodology could be used in combination with anaerobic digestion to achieve greater decolorization and reduction of phenolic compounds, melanoidins, and organic load. Graphical abstract

Parasitoids are known to exploit volatile cues emitted by plants after herbivore attack to locate their hosts. Feeding and oviposition of a polyphagous herbivore can induce the emission of odor blends that differ among distant plant species, and parasitoids have evolved an incredible ability to discriminate them and locate their hosts relying on olfactive cues. We evaluated the host searching behavior of the egg parasitoid Cosmocomoidea annulicornis (Ogloblin) (Hymenoptera: Mymaridae) in response to odors emitted by two taxonomically distant host plants, citrus and Johnson grass, after infestation by the sharpshooter Tapajosa rubromarginata (Signoret) (Hemiptera: Cicadellidae), vector of Citrus Variegated Chlorosis. Olfactory response of female parasitoids toward plants with no herbivore damage and plants with feeding damage, oviposition damage, and parasitized eggs was tested in a Y-tube olfactometer. In addition, volatiles released by the two host plant species constitutively and under herbivore attack were characterized. Females of C. annulicornis were able to detect and significantly preferred plants with host eggs, irrespectively of plant species. However, wasps were unable to discriminate between plants with healthy eggs and those with eggs previously parasitized by conspecifics. Analysis of plant volatiles induced after sharpshooter attack showed only two common volatiles between the two plant species, indole and β-caryophyllene. Our results suggest that this parasitoid wasp uses common chemical cues released by many different plants after herbivory at long range and, once on the plant, other more specific chemical cues could trigger the final decision to oviposit.

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.

Abstract Predator/parasitoid functional response is one of the main tools used to study predation behavior, 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 a 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 modeling 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.

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.

Bioprospecting sub-explored environments such as Antarctic locations leads to finding out diverse activities, reducing harmful chemical usage that affects both human health and the environment. In this study, ~ 7000 cold-adapted bacterial strains were isolated from samples around Melchior Antarctic Base at 5 °C and more than 13,000 at 15 °C. Out of them, 900 different colony morphotypes were evaluated for antimicrobial production, and 13 isolates demonstrated antibacterial and antifungal activities. One isolate, closely related to Burkholderia gladioli according to 16S rDNA (99.8%), gyrB (99.6%) and Cpn60 (99.4%) gene sequence analysis, showed a consistent, broad antimicrobial spectrum against both pathogenic and phytopathogenic bacteria. Its potent antifungal activity inhibits the growth of various plant pathogenic fungi, whereas it was mainly studied against Penicillium digitatum and Macrophomina phaseolina, the causal agents of blue mould in postharvest fruits and charcoal rot in soybean crops, respectively. The antibacterial compound exhibited low molecular weight (< 6000 Da), resistance to lytic enzymes and stability in a broad range of temperature and pHs. Observations of the B. gladioli MB39 antifungal effects over M. phaseolina mycelia by scanning electron microscopy showed alterations in hyphal structures, reduced hyphal extension, and severe cell morphology changes such as cytoplasmic leakage, flattened and empty mycelia. Here we report the isolation and identification of a cold-adapted B. gladioli strain. The results describe the effectiveness of the antarctic strain for bacterial and fungal phytopathogens biocontrol and its potential for crop protection plans

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.