HEAVY METALS AND MICRONUTRIENTS IN THE SOIL AND GRAPEVINE UNDER DIFFERENT IRRIGATION STRATEGIES

Soils under natural conditions have heavy metals in variable concentrations and there may be an increase in these elements as a result of the agricultural practices adopted. Transport of heavy metals in soil mainly occurs in forms dissolved in the soil solution or associated with solid particles, water being their main means of transport. In this context, the aim of this study was to evaluate the heavy metal and micronutrient content in the soil and in the grapevine plant and fruit under different irrigation strategies. The experiment was carried out in Petrolina, PE, Brazil. The treatments consisted of three irrigation strategies: full irrigation (FI), regulated deficit irrigation (RDI), and deficit irrigation (DI). During the period of grape maturation, soil samples were collected at the depths of 0-10, 10-20, 20-40, 40-60, and 60-80 cm. In addition, leaves were collected at the time of ripening of the bunches, and berries were collected at harvest. Thus, the heavy metal and micronutrient contents were determined in the soil, leaves, and berries. The heavy metal and micronutrient contents in the soil showed a stochastic pattern in relation to the different irrigation strategies. The different irrigation strategies did not affect the heavy metal and micronutrient contents in the vine leaves, and they were below the contents considered toxic to the plant. In contrast, the greater availability of water in the FI treatment favored a greater Cu content in the grape, which may be a risk to vines, causing instability and turbidity. Thus, adoption of deficit irrigation is recommended so as to avoid compromising the stability of tropical wines of the Brazilian Northeast.

Early Changes in Soil Metabolic Diversity and Bacterial Community Structure in Sugarcane under Two Harvest Management Systems

Preharvest burning is widely used in Brazil for sugarcane cropping. However, due to environmental restrictions, harvest without burning is becoming the predominant option. Consequently, changes in the microbial community are expected from crop residue accumulation on the soil surface, as well as alterations in soil metabolic diversity as of the first harvest. Because biological properties respond quickly and can be used to monitor environmental changes, we evaluated soil metabolic diversity and bacterial community structure after the first harvest under sugarcane management without burning compared to management with preharvest burning. Soil samples were collected under three sugarcane varieties (SP813250, SP801842 and RB72454) and two harvest management systems (without and with preharvest burning). Microbial biomass C (MBC), carbon (C) substrate utilization profiles, bacterial community structure (based on profiles of 16S rRNA gene amplicons), and soil chemical properties were determined. MBC was not different among the treatments. C-substrate utilization and metabolic diversity were lower in soil without burning, except for the evenness index of C-substrate utilization. Soil samples under the variety SP801842 showed the greatest changes in substrate utilization and metabolic diversity, but showed no differences in bacterial community structure, regardless of the harvest management system. In conclusion, combined analysis of soil chemical and microbiological data can detect early changes in microbial metabolic capacity and diversity, with lower values in management without burning. However, after the first harvest, there were no changes in the soil bacterial community structure detected by PCR-DGGE under the sugarcane variety SP801842. Therefore, the metabolic profile is a more sensitive indicator of early changes in the soil microbial community caused by the harvest management system.

Organic and Nitrogen Fertilization of Soil under ‘Syrah’ Grapevine: Effects on Soil Chemical Properties and Nitrate Concentration

ABSTRACT Viticulture is an activity of great social and economic importance in the lower-middle region of the São Francisco River valley in northeastern Brazil. In this region, the fertility of soils under vineyards is generally poor. To assess the effects of organic and nitrogen fertilization on chemical properties and nitrate concentrations in an Argissolo Vermelho-Amarelo (Typic Plinthustalf), a field experiment was carried out in Petrolina, Pernambuco, on Syrah grapevines. Treatments consisted of two rates of organic fertilizer (0 and 30 m3 ha-1) and five N rates (0, 10, 20, 40, and 80 kg ha-1), in a randomized block design arranged in split plots, with five replications. The organic fertilizer levels represented the main plots and the N levels, the subplots. The source of N was urea and the source of organic fertilizer was goat manure. Irrigation was applied through a drip system and N by fertigation. At the end of the third growing season, soil chemical properties were determined and nitrate concentration in the soil solution (extracted by porous cups) was determined. Organic fertilization increased organic matter, pH, EC, P, K, Ca, Mg, Mn, sum of bases, base saturation, and CEC, but decreased exchangeable Cu concentration in the soil by complexation of Cu in the organic matter. Organic fertilization raised the nitrate concentration in the 0.20-0.40 m soil layer, making it leachable. Nitrate concentration in the soil increased as N rates increased, up to more than 300 mg kg-1 in soil and nearly 800 mg L-1 in the soil solution, becoming prone to leaching losses.

Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins

In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/L condensed tannins extracted from Acacia angustissima, banana (Musa sp.) skin, Desmodium ovalifolium, red grape (Vitis vinifera) skin and Inga edulis, or no tannins. Cells were rinsed with Tris buffer pH 7 containing either 8% PEG or 6% PVP prior to cell lysis. Total RNA samples rinsed with either PEG or PVP migrated through denaturing agarose gels. The 16S rRNA bands successfully hybridized with a R. albus species-specific oligonucleotide probe, regardless of tannin source. The effect of rinsing buffers on the density of 16S rRNA bands, as well as on the hybridization signals was compared. There were significant effects (P<0.01) when the controls were compared to either buffer treatments due to tannin type, buffer used and the interaction of tannin type and buffer. The significant interaction indicates the influence of tannin type on the parameters evaluated.

Bacterial diversity of soil under eucalyptus assessed by 16S rDNA sequencing analysis

Studies on the impact of Eucalyptus spp. on Brazilian soils have focused on soil chemical properties and isolating interesting microbial organisms. Few studies have focused on microbial diversity and ecology in Brazil due to limited coverage of traditional cultivation and isolation methods. Molecular microbial ecology methods based on PCR amplified 16S rDNA have enriched the knowledge of soils microbial biodiversity. The objective of this work was to compare and estimate the bacterial diversity of sympatric communities within soils from two areas, a native forest (NFA) and an eucalyptus arboretum (EAA). PCR primers, whose target soil metagenomic 16S rDNA were used to amplify soil DNA, were cloned using pGEM-T and sequenced to determine bacterial diversity. From the NFA soil 134 clones were analyzed, while 116 clones were analyzed from the EAA soil samples. The sequences were compared with those online at the GenBank. Phylogenetic analyses revealed differences between the soil types and high diversity in both communities. Soil from the Eucalyptus spp. arboretum was found to have a greater bacterial diversity than the soil investigated from the native forest area.

Rhizosphere bacterial communities of potato cultivars evaluated through PCR-DGGE profiles

The objective of this work was to determine the shifts on the PCR-DGGE profiles of bacterial communities associated to the rhizosphere of potato cultivars, in order to generate baseline information for further studies of environmental risk assessment of genetically modified potato plants. A greenhouse experiment was carried out with five potato cultivars (Achat, Bintje, Agata, Monalisa and Asterix), cultivated in pots containing soil from an integrated system for agroecological production. The experiment was conducted in a split plot randomized block design with five cultivars, three sampling periods and five replicates. Rhizosphere samples were collected in three sampling dates during plant development. DNA of rhizosphere microorganisms was extracted, amplified by PCR using bacterial universal primers, and analyzed through DGGE. Shifts on the rhizosphere bacterial communities associated to rhizosphere of different cultivars were related to both cultivar and plant age. Differences among rhizosphere bacterial communities were clearest at the earliest plant age, tending to decrease in later stages. This variation was detected among bacterial communities of the five tested cultivars. The characterization of soil microbial communities can be part of plant breeding programs to be used on studies of environmental risk assessment of genetically modified potatoes.

Time-related action of Lactobacillus plantarum in the bacterial microbiota of shrimp digestive tract and its action as immunostimulant

The objective of this work was to assess the time-related action of probiotic Lactobacillus plantarum in the bacterial microbiota of the digestive tract of Litopenaeus vannamei, and the relation of total haemocyte count and serum phenol oxidase activity of shrimp challenged with Vibrio harveyi. Shrimps were fed with a probiotic-supplemented diet, for eight days, then shifted to a commercial diet. Shrimps fed only with the commercial diet served as control. Evaluations were made on the 8th day of experiment and repeated two, four, six and eight days later. Total lactic bacteria in the digestive tract was higher until the 4th day of evaluation in the probiotic-supplemented group. Vibrio spp. counts were higher in the control at days zero and two. Until the 4th day of evaluation, the total haemocyte counts in shrimps after challenge with V. harveyi were higher in probiotic-supplemented group than in control group. Significant difference was not observed in phenol oxidase activity. On the 6th day after shifting from supplemented to control diet, all parameters were equal in both groups, suggesting that the time-related action of L. plantarum in shrimp is short.

Production of polyclonal antisera using recombinant coat proteins of Grapevine leafroll-associated virus 2 and Grapevine virus B

The objective of this work was to produce and characterize specific antisera against Brazilian isolates of Grapevine leafroll-associated virus 2 (GLRaV-2) and Grapevine virus B (GVB), developed from expressed coat proteins (CPs) in Escherichia coli, and to test their possible use for the detection of these two viruses in diseased grapevines. The coat protein (CP) genes were RT-PCR-amplified, cloned and sequenced. The CP genes were subsequently subcloned, and the recombinant plasmids were used to transform E. coli cells and express the coat proteins. The recombinant coat proteins were purified, and their identities were confirmed by SDS-PAGE and Western blot and used for rabbit immunizations. Antisera raised against these proteins were able to recognize the corresponding recombinant proteins in Western blots and to detect GLRaV-2 and GVB in infected grapevine tissues, by indirect ELISA, discriminating healthy and infected grapevines with absorbances (A405) of 0.08/1.15 and 0.12/1.30, respectively. Expressing CP genes can yield high amount of viral protein with high antigenicity, and GLRaV-2 and GVB antisera obtained in this study can allow reliable virus disease diagnosis.

Molecular characterization of grapevine from Santa Catarina, Brazil, using microsatellite markers

The objective of this work was to characterize the grape germplasm in Santa Catarina, Brazil, using microsatellite DNA markers (simple sequence repeats - SSR). The DNA samples were collected from leaves and shoots of accessions of public and private collections from the counties Urussanga, Nova Trento, Rodeio, São Joaquim, Campos Novos, Videira, and Água Doce. Ten SSR loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79, VVMD25, VVMD28, VVMD31, and VVMD32) were analysed by capillary electrophoresis. Molecular profiling was conducted for 190 grapevines (European, American, and hybrids), and 67 genotypes were obtained. The data were compared with each other and with those from the literature and from online databases, in order to identify varieties and discover cases of synonymy and homonymy. Forty molecular profiles corresponded to known varieties, while 27 genotypes were described for the first time. The existence of typical germplasm composed mainly of American and hybrid varieties is an important finding for local viticulture. Applications of the results rely on quality control and certification at the nursery level. Increasing precision in the characterization of grapevine genotypes may help breeding programs.

Bacterial spot and early blight biocontrol by epiphytic bacteria in tomato plants

The objective of this work was to evaluate in vitro and in vivo biocontrol of bacterial spot (Xanthomonas vesicatoria) and early blight (Alternaria solani) by the epiphytic bacteria Paenibacillus macerans and Bacillus pumilus. Tomato plants were previously sprayed with epiphytic bacteria, benzalkonium chloride and PBS buffer and, after four days, they were inoculated with A. solani and X. vesicatoria. To determine the phytopathogenic bacteria population, leaflet samples were collected from each treatment every 24 hours, for seven days, and plated on semi-selective medium. The effect of epiphytic bacteria over phytopathogens was performed by the antibiosis test and antagonistic activity measured by inhibition zone diameter. The epiphytic and benzalkonium chloride drastically reduced the severity of early blight and bacterial spot in comparison to the control (PBS). In detached leaflets, the epiphytic bacteria reduced in 70% the number of phytopathogenic bacteria cells in the phylloplane. The antibiosis test showed that the epiphytic bacteria efficiently inhibit the phytopathogens growth. In all the bioassays, the epiphytic bacteria protect tomato plants against the phytopathogens

Obtaining interspecific hybrids, and molecular analysis by microsatellite markers in grapevine

The objective of this work was to assess the potential of interspecific hybridization of Vitis labruscana and Muscadinia rotundifolia by using artificial cross-pollinations. Microsatellite markers were used to confirm interspecific hybridizations and the identity of the parental genotypes. In crosses in which M. rotundifolia was used as the female parent, no true hybrids were obtained. In the reciprocal crosses, 114 seedlings were identified as true V. labruscana x M. rotundifolia hybrids. Self pollination occurred in direct and in reciprocal crosses. The crossings between 'Bordo' x 'Carlos', 'Magnolia', 'Regale' and' Roanoke', and between' Isabel' x 'Bountiful', 'Carlos', 'Magnolia', 'Regale' and 'Roanoke' were confirmed. The 15 markers evaluated showed that two M. rotundifolia parental genotypes had the same fingerprint profile, indicating a like lyplanting error. The success of hybridization depends mainly on the species and on the cultivar used as the female parent. Microsatellite markers are efficient to confirm the paternity of interspecific F1 hybrids and to determine the correct identity of M. rotundifolia cultivars.

Shoot growth of Merlot and Cabernet Sauvignon grapevine varieties

The objective of this work was to evaluate shoot growth of the grapevine varieties Merlot and Cabernet Sauvignon, during 2006/2007, and Cabernet Sauvignon, during 2008/2009, in São Joaquim, SC, Brazil. The experiment was carried out in a commercial vineyard trained on a vertical trellis system. The shoots of the central part of the plants were selected, and the lengths from the base to the apex of 20 shoots per cultivar were evaluated. In 2006/2007, monitoring began at pruning, on 9/15/2006, and ended on 2/6/2007, totalizing 144 days of evaluation. During the 2008/2009 cycle, phenology and shoot growth for 'Cabernet Sauvignon' were assessed from grape development (1/13/2009) (pea-sized grapes) until shoot vegetative growth had ceased. Budburst occurred in the second half of September, and shoot-growth cessation occurred during ripening. Higher growth rates (about 4 cm per day) were observed in pre- and post-flowering, followed by reduction due to the competition for photosynthates for the formation of flowers and bunches. Temperature and photoperiod induce grapevine shoots to cease growth in the highland regions of Santa Catarina State, Brazil.

Short-term storage in vitro and large-scale propagation of grapevine genotypes

The objective of this work was to evaluate the large-scale propagation of grapevine genotypes after short-term storage in vitro. Microshoots from ten grapevine genotypes were used. The following storage temperatures were evaluated: 10, 20, and 25°C. After short-term storage, the shoots were propagated in up to five successive subcultures, to assess the large-scale propagation of the germplasm maintained under conditions of minimal growth. The propagated shoots were rooted in different concentrations of indolbutiric acid (IBA) and acclimatized in greenhouse. The best temperature for short-term storage in vitro and survival of the genotypes was 20°C. In the propagation phase, the highest number of shoots per explant was found in the subcultures 4 and 5, with averages of 4.9 and 4.8 shoots per explant, respectively. In the rooting phase, the best results for number of roots were obtained using a culture medium supplemented with 0.4 µmol L-1 of IBA, with an average of three roots per shoot. During the acclimation phase, a survival rate higher than 95% was achieved after 30 days in the greenhouse. Grapevine genotypes maintained for six months in vitro, at 20ºC, can be micropropagated in large scale.

Antibacterial activity of essential oils on Xanthomonas vesicatoria and control of bacterial spot in tomato

The objective of this work was to evaluate the effects of plant essential oils (EOs) on the growth of Xanthomonas vesicatoria, on bacterial morphology and ultrastructure, and on the severity of tomato bacterial spot. EOs from citronella, clove, cinnamon, lemongrass, eucalyptus, thyme, and tea tree were evaluated in vitro at concentrations of 0.1, 1.0, 10, and 100% in 1.0% powdered milk. The effect of EOs, at 0.1%, on the severity of tomato bacterial spot was evaluated in tomato seedlings under greenhouse conditions. The effects of citronella, lemongrass, clove, and tea tree EOs, at 0.1%, on X. vesicatoria cells were evaluated by transmission electron microscopy. All EOs showed direct toxic effect on the bacteria at a 10%-concentration in vitro. Under greenhouse conditions, the EOs of clove, citronella, tea tree, and lemongrass reduced disease severity. EOs of clove and tea tree, and streptomycin sulfate promoted loss of electron-dense material and alterations in the cytoplasm, whereas EO of tea tree promoted cytoplasm vacuolation, and those of citronella, lemongrass, clove, and tea tree caused damage to the bacterial cell wall. The EOs at a concentration of 0.1% reduce the severity of the disease.

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.