Inoculation with the endophytic bacterium Herbaspirillum seropedicae promotes growth, nutrient uptake and photosynthetic efficiency in rice.

MAIN CONCLUSION: Higher vacuolar proton pump activity may increase plant energy and nutrient use efficiency and provide the nexus between plant inoculation with Herbaspirillum seropedicae and growth promotion. Global change and growing human population are exhausting arable land and resources, including water and fertilizers. We present inoculation with the endophytic plant-growth promoting bacterium (PGPB) Herbaspirillum seropedicae as a strategy for promoting growth, nutrient uptake and photosynthetic efficiency in rice (Oryza sativa L.). Because plant nutrient acquisition is coordinated with photosynthesis and the plant carbon status, we hypothesize that inoculation with H. seropedicae will stimulate proton (H+) pumps, increasing plant growth nutrient uptake and photosynthetic efficiency at low nutrient levels. Plants were inoculated and grown in pots with sterile soil for 90 days. Herbaspirillum seropedicae endophytic colonization was successful and, as hypothesized, inoculation (1) stimulated root vacuolar H+ pumps (vacuolar H+-ATPase and vacuolar H+-PPase), and (2) increased plant growth, nutrient contents and photosynthetic efficiency. The results showed that inoculation with the endophytic bacterium H. seropedicae can promote plant growth, nutrient uptake and photosynthetic efficiency, which will likely result in a more efficient use of resources (nutrients and water) and higher production of nutrient-rich food at reduced economic and environmental costs.

Coffea canephora Peptides in Combinatorial Treatment with Fluconazole: Antimicrobial Activity against Phytopathogenic Fungus.

The objective of the present study was to evaluate the antimicrobial activity of the Cc-LTP2 and Cc-GRP peptides isolated from Coffea canephora seeds and their possible synergistic activity with the azole drug fluconazole and characterize their mechanisms of action on cells of pathogenic fungi. Cc-LTP2 and Cc-GRP alone or in combination with 20 µg/mL of fluconazole were evaluated for their antimicrobial activity on the fungus Fusarium solani, and the effects of these peptides on the permeability of membranes and the induction of oxidative stress were determined. Our results show that these peptides at a concentration of 400 µg/mL combined with 20 µg/mL of fluconazole were able to inhibit the growth of the tested fungi, promote changes in their growth pattern, permeabilize the membrane, and induce reactive oxygen species (ROS). Some of these results were also observed with the peptides alone or with fluconazole alone, suggesting that the peptides act synergistically, promoting the potentiation of antimicrobial action. In this study, it was shown that Cc-LTP2 and Cc-GRP in combination with fluconazole were able to inhibit the growth of the fungus F. solani, to promote permeabilization of its membrane, and to induce the production of ROS, suggesting a combinatorial activity between the peptides and fluconazole.

Embryogenic Competence Acquisition in Sugar Cane Callus Is Associated with Differential H+-Pump Abundance and Activity.

Somatic embryogenesis is an important biological process in several plant species, including sugar cane. Proteomics approaches have shown that H+ pumps are differentially regulated during somatic embryogenesis; however, the relationship between H+ flux and embryogenic competence is still unclear. This work aimed to elucidate the association between extracellular H+ flux and somatic embryo maturation in sugar cane. We performed a microsomal proteomics analysis and analyzed changes in extracellular H+-flux and H+-pump (P-H+-ATPase, V-H+-ATPase, and H+-PPase) activity in embryogenic and non-embryogenic callus. A total of 657 proteins were identified, 16 of which were H+ pumps. We observed that P-H+-ATPase and H+-PPase were more abundant in embryogenic callus. Compared to non-embryogenic callus, embryogenic callus showed higher H+ influx, especially on maturation day 14, as well as higher H+-pump activity (mainly, P-H+-ATPase and H+-PPase activity). H+-PPase appears to be the major H+ pump in embryogenic callus during somatic embryo formation, functioning in both vacuole acidification and PPi homeostasis. These results provide evidence for an association between higher H+-pump protein abundance and, consequently, higher H+ flux and embryogenic competence acquisition in the callus of sugar cane, allowing for the optimization of the somatic embryo conversion process by modulating the activities of these H+ pumps.

Programmed cell death in yeast by thionin-like peptide from Capsicum annuum fruits involving activation of caspases and extracellular H+ flux.

CaThi is a thionin-like peptide isolated from fruits of Capsicum annuum, which has strong antimicrobial activity against bacteria, yeasts and filamentous fungi, and induced reactive oxygen species (ROS) in fungi. ROS are molecules that appear in the early stages of programmed cell death or apoptosis in fungi. Due to this fact, in this work we analyzed some events that may be related to process of apoptosis on yeast induced by CaThi. To investigate this possibility, we evaluated phosphatidylserine (PS) externalization, presence of active caspases and the ability of CaThi to bind to DNA in Candida tropicalis cells. Additionally, we investigated mitochondrial membrane potential, cell surface pH, and extracellular H+ fluxes in C. tropicalis cells after treatment with CaThi. Our results showed that CaThi induced PS externalization in the outer leaflet of the cell membrane, activation of caspases, and it had the ability for DNA binding and to dissipate mitochondrial membrane potential. In addition, the cell surface pH increased significantly when the C. tropicalis cells were exposed to CaThi which corroborates with ~96% inhibition on extracellular H+ efflux. Taking together, these data suggest that this peptide is capable of promoting an imbalance in pH homeostasis during yeast cell death playing a modulatory role in the H+ transport systems. In conclusion, our results strongly indicated that CaThi triggers apoptosis in C. tropicalis cells, involving a pH signaling mechanism.

Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition.

Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA) induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

Crop management as a driving force of plant growth promoting rhizobacteria physiology.

Crop management systems influence plant productivity and nutrient use efficiency, as well as plant growth-promoting rhizobacteria (PGPR), which are known to influence the growth of plants via phytohormone production, phosphate solubilization, nitrogen (N) fixation and antimicrobial activity. The objective of this study was to compare the influence of two crop management system on microbial PGPR features. PGPR isolated from the rhizospheres of Carica papaya L. grown under two distinct management systems (conventional and organic) were identified and characterized. The 12 strains most efficient in solubilizing inorganic phosphate belonged to the genera Burkholderia, Klebsiella, and Leclercia. N fixation was observed in the strains B. vietnamiensis from the conventional farming system and B. vietnamiensis, B. cepacia and Leclercia sp. from the organic farming system. The B. vietnamiensis, B. cepacia, Klebsiella sp. and Klebsiella sp. isolates showed antifungal activity, while Leclercia sp. did not. The strains B. vietnamiensis and Enterobcter sp. (isolated from the conventional farming system) and Klebsiella sp. (isolated from the organic farming system) were efficient at solubilizing phosphate, producing phytohormones and siderophores, and inhibiting the mycelial growth of various phytopathogenic fungi (Botrytis cinerea, Pestalotia sp., Alternaria sp., Phoma sp., Fusarium culmorum, Geotrichum candidum). Physiological differences between the isolates from the two crop management regimes were distinguishable after 10 years of distinct management.

Biochemical and ecophysiological responses to manganese stress by ectomycorrhizal fungus Pisolithus tinctorius and in association with Eucalyptus grandis.

At relatively low concentrations, the element manganese (Mn) is essential for plant metabolism, especially for photosynthesis and as an enzyme antioxidant cofactor. However, industrial and agricultural activities have greatly increased Mn concentrations, and thereby contamination, in soils. We tested whether and how growth of Pisolithus tinctorius is influenced by Mn and glucose and compare the activities of oxidative stress enzymes as biochemical markers of Mn stress. We also compared nutrient accumulation, ecophysiology, and biochemical responses in Eucalyptus grandis which had been colonized by the ectomycorrhizal Pisolithus tinctorius with those which had not, when both were exposed to increasing Mn concentrations. In vitro experiments comprised six concentrations of Mn in three concentrations of glucose. In vivo experiments used plants colonized by Pisolithus tinctorius, or not colonized, grown with three concentrations of Mn (0, 200, and 1000 µM). We found that fungal growth and glucose concentration were correlated, but these were not influenced by Mn levels in the medium. The anti-oxidative enzymes catalase and glutathione S-transferase were both activated when the fungus was exposed to Mn. Also, mycorrhizal plants grew more and faster than non-mycorrhizal plants, whatever Mn exposure. Photosynthesis rate, intrinsic water use efficiency, and carboxylation efficiency were all inversely correlated with Mn concentration. Thus, we originally show that the ectomycorrhizal fungus provides protection for its host plants against varying and potentially toxic concentrations of Mn.

Mechanistic basis for morphological damage induced by essential oil from Brazilian pepper tree, Schinus terebinthifolia, on larvae of Stegomyia aegypti, the dengue vector.

BACKGROUND: Dengue has become the subject of public health programs worldwide. The lack of a vaccine and the high environmental risk of synthetic insecticides, arouse the interest in natural products against this vector. This study aimed to determine the chemical composition of the essential oil of ripe fruits and seeds of Schinus terebinthifolia Raddi; to evaluate the essential oil effect on mortality of Stegomyia aegypti (Linnaeus, 1792) larvae; and to characterize the structural damage suffered by larvae and their association with different contents of essential oil. METHODS: Ripe fruits and seeds were crunched and their essential oil was extracted through hydrodistillation, purified, and its phytochemical analysis was carried out through High Resolution Gas Chromatography, coupled with Mass Spectrometry. This essential oil was diluted in a 10-point gradient of 86.22 - 862.20 ppm, at regular intervals of 86.22 ppm. Each point received 50 larvae and the assessments of surviving were made at 24, 48 and 72 hours after inoculation. Structural damage was assessed through measurements of thickness with exoskeleton, evaluating the integrity of the head, thorax, abdominal segments, and air siphon, using ImageJ software. Statistical data analysis was carried out through Logistic Regression and Discriminant Analysis. RESULTS: 56 substances were identified, corresponding to 81.67% of the essential oil composition. Larvae were dose-dependent susceptible to the essential oil; the concentration produced a significant effect on larval mortality. Among the major deformations found in the larvae, it was detected inhibition of chitin synthesis by the activity of the oil, thus reducing the deposition of cuticle layers. CONCLUSION: The essential oil caused death in exposed larvae after 72 hours, in a dose-dependent manner. It also changed the structure of exposed larvae, indicating a direct effect on larval exoskeleton. The results open up possibilities for the use of natural products as an alternative to control dipterans.

The essential oil of Brazilian pepper, Schinus terebinthifolia Raddi in larval control of Stegomyia aegypti (Linnaeus, 1762).

BACKGROUND: The ability of mosquitoes of the genus Aedes and its allies, such as Stegomyia, to transmit diseases such as dengue and yellow fever, makes them important in public health. This study aims to evaluate the use of the essential oil of Brazilian pepper in biological control of by assessing and quantifying the larvicidal effect against S. aegypti, the only available access to dengue control, and test its risk of genotoxicity with Salmonella typhimurium as an indicator of safety for its environmental use. RESULTS: The density of the oil was 0.8622 g mL-1. Gas chromatography coupled with mass spectrometry revealed six major constituents: δ-3-carene (55.43%), α-pinene (16.25%), sylvestrene (10.67%), germacrene D (2.17), ß-myrcene (1.99%), and isoterpinolene (1.4%). The minimum inhibitory dose to larvae development was 862.20 µg mL-1. The median lethal dose (LD50) of the essential oil for larvae was between the concentrations of 172.44-344.88 µg mL-1. There was no mutagenic risk for the essential oil, since there were no biochemical or morphological changes in S. typhimurium after exposure to the essential oil. CONCLUSIONS: The minimum inhibitory essential oil concentration and the median lethal dose pointed to the value of the use of water dispersions of Brazilian pepper essential oil as an environmental safe natural larvicidal for S. aegypti.

Plant-microbe symbioses: new insights into common roots.

Arbuscular mycorrhiza (AM), a type of plant-fungal endosymbiosis, and nodulation, a bacterial-plant endosymbiosis, are the most ubiquitous symbioses on earth. Recent findings have established part of a shared genetic basis underlying these interactions. Here, we approach root endosymbioses through the lens of the homology and modularity concepts aiming at further clarifying the proximate and ultimate causes for the establishment of these biological systems. We review the genetics that underlie interspecific signaling and its concomitant shift in genetic programs for either partner. Also, through the comparative analysis of genetic modules shared by AM and nodulation symbioses, we identify fundamental nodes in these networks, suggesting the elemental steps that may have permitted symbiotic adaptation. Here, we show that this approach, allied to recent technical advances in the study of genetic systems architecture, can provide clear testable hypotheses for the advancement of our understanding on the evolution and development of symbiotic systems.

A pH signaling mechanism involved in the spatial distribution of calcium and anion fluxes in ectomycorrhizal roots.

Mycorrhization is a typical example of a host-pathogen symbiotic interaction where the pathogen cell biology and the host immune response coevolved several functional links. Here, the role played by ion fluxes across the root concerning nutrient uptake, osmoregulation, growth and signaling events is addressed. An ion-selective vibrating probe system was used to determine the net fluxes of protons (H(+)), calcium (Ca(2+)) and anions (A(-)) along nonmycorrhizal and ectomycorrhizal (ECM) roots of Eucalyptus globulus colonized by Pisolithus sp. These data show that, from five root zones analyzed, the main effect of fungal colonization was localized to the elongation zone. Here, strong changes in ion dynamics and rhizosphere acidification capacity were observed. Additionally, ion fluxes exhibited periodic fluctuations. To verify whether these fluctuations corresponded to sustained oscillations, continuous wavelet time spectrum analysis was applied and it was determined that H(+) and A(-) fluxes from ECM roots had longer periods than nonmycorrhizal roots. By contrast, Ca(2+) oscillations were completely abolished following fungal interaction. These results are interpreted in the light of a working model in which nutrient uptake and stimulation of growth are mediated by ECM fungi and may be pH-dependent. Furthermore, the variations detected in ECM roots for H(+) and A(-) fluxes suggest a main contribution from the plant, while the results obtained for Ca(2+) point to a significant involvement of the fungus.

Arbuscular mycorrhizal fungi induce differential activation of the plasma membrane and vacuolar H+ pumps in maize roots.

Roots undergo multiple changes as a consequence of arbuscular mycorrhizal (AM) interactions. One of the major alterations expected is the induction of membrane transport systems, including proton pumps. In this work, we investigated the changes in the activities of vacuolar and plasma membrane (PM) H(+) pumps from maize roots (Zea mays L.) in response to colonization by two species of AM fungi, Gigaspora margarita and Glomus clarum. Both the vacuolar and PM H(+)-ATPase activities were inhibited, while a concomitant strong stimulation of the vacuolar H(+)-PPase was found in the early stages of root colonization by G. clarum (30 days after inoculation), localized in the younger root regions. In contrast, roots colonized by G. margarita exhibited only stimulation of these enzymatic activities, suggesting a species-specific phenomenon. However, when the root surface H(+) effluxes were recorded using a noninvasive vibrating probe technique, a striking activation of the PM H(+)-ATPases was revealed specifically in the elongation zone of roots colonized with G. clarum. The data provide evidences for a coordinated regulation of the H(+) pumps, which depicts a mechanism underlying an activation of the root H(+)-PPase activity as an adaptative response to the energetic changes faced by the host root during the early stages of the AM interaction.

Proton (H+) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi.

Ion dynamics are important for cell nutrition and growth in fungi and plants. Here, the focus is on the relationship between the hyphal H(+) fluxes and the control of presymbiotic growth and host recognition by arbuscular mycorrhizal (AM) fungi. Fluxes of H(+) around azygopores and along lateral hyphae of Gigaspora margarita during presymbiotic growth, and their regulation by phosphate (P) and sucrose (Suc), were analyzed with an H(+)-specific vibrating probe. Changes in hyphal H(+) fluxes were followed after induction by root exudates (RE) or by the presence Trifolium repens roots. Differential sensitivity to P-type ATPase inhibitors (orthovanadate or erythrosin B) suggests an asymmetric distribution or activation of H(+)-pump isoforms along the hyphae of the AM fungi. Concentration of P and Suc affected the hyphal H(+) fluxes and growth rate. However, further increases in H+ efflux and growth rate were observed when the fungus was growing close to clover roots or pretreated with RE. The H(+) flux data correlate with those from polarized hyphal growth analyses, suggesting that spatial and temporal alterations of the hyphal H(+)fluxes are regulated by nutrient availability and might underlie a pH signaling elicitation by host RE during the early events of the AM symbiosis.

pH signature for the responses of arbuscular mycorrhizal fungi to external stimuli.

Environmental and developmental signals can elicit differential activation of membrane proton (H(+)) fluxes as one of the primary responses of plant and fungal cells. In recent work,1 we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H(+) flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H(+)-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H(+) effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H(+) fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.