Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT-PCR for its detection in single and bulked leaf samples

The potential for large-scale use of a sensitive real time reverse transcription polymerase chain reaction (RT-PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS-ELISA. Using total RNA extracted with RNeasy (R) or leaf soak methods, real time RT-PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS-ELISA had previously detected TSWV, real time RT-PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT-PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to I infected in 800 samples with pepper but never detecting more than I infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT-PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Temporally dependent C, N, and P dynamics associated with the decay of Rhizophora mangle L. leaf litter in oligotrophic mangrove wetlands of the Southern Everglades

We performed two litter decomposition experiments using nearly-senesced red mangrove (Rhizophora mangle L.) leaves collected from an Everglades dwarf mangrove wetland to understand the short-term (3 weeks) and long-term (1 year) changes in mass, as well as C-, N-, and P-content of decomposing leaf litter. We expected that leaves decomposing in this oligotrophic environment would be short-term sources of C, N, and P, but potential long-term sinks for N and P. In May 1998, we conducted a 3-week leaching experiment, incubating fresh, individual leaves in seawater for up to 21 days. From May 1997 to May 1998, leaf litter in mesh bags decomposed on the forest floor at two dwarf mangrove sites. Leaching accounted for about 33% loss of dry mass from R. mangle leaves after 3 weeks. Leaching losses were rapid, peaking by day 2, and large, with leachate concentrations of total organic carbon (TOC) and total phosphorus (TP) increasing by more than an order of magnitude after 3 weeks. Mean leaf C:N increased from 105 to 115 and N:P increased from a mean of 74 to 95 after 21 days, reflecting the relatively large leaching losses of N and P. Loss of mass in the litterbags leveled off after 4 months, with roughly 60%dry mass remaining (DMR) after nearly 1 year of decomposition. The mass of carbon in each litterbag declined significantly after 361 days, but the mass of nitrogen and phosphorus doubled, indicating long-term accumulation of these constituents into the detritus. Subsequently, the leaf C:N ratio dropped significantly from 90 to 34 after 361 days. Following an initial 44-day increase, leaf N:P decreased from 222 to 144, reflecting high accumulation of P relative to N. A review of several estuarine macrophyte decomposition studies reveals a trend in nitrogen accumulation through time regardless of site, but suggests no clear pattern for C and P. We believe that the increase in litter P observed in this study was indicative of the P-limited status of the greater Everglades ecosystem and that decomposing mangrove litter may represent a substantial phosphorus pool in the system.

Spatio-temporal patterns and nutrient status of macroalgae in a heavily managed region of Biscayne Bay, Florida, USA

The coastal bays of South Florida are located downstream of the Florida Everglades, where a comprehensive restoration plan will strongly impact the hydrology of the region. Submerged aquatic vegetation communities are common components of benthic habitats of Biscayne Bay, and will be directly affected by changes in water quality. This study explores community structure, spatio-temporal dynamics, and tissue nutrient content of macroalgae to detect and describe relationships with water quality. The macroalgal community responded to strong variability in salinity; three distinctive macroalgal assemblages were correlated with salinity as follows: (1) low-salinity, dominated by Chara hornemannii and a mix of filamentous algae; (2) brackish, dominated by Penicillus capitatus, Batophora oerstedii, and Acetabularia schenckii; and (3) marine, dominated by Halimeda incrassata and Anadyomene stellata. Tissue-nutrient content was variable in space and time but tissues at all sites had high nitrogen and N:P values, demonstrating high nitrogen availability and phosphorus limitation in this region. This study clearly shows that distinct macroalgal assemblages are related to specific water quality conditions, and that macroalgal assemblages can be used as community-level indicators within an adaptive management framework to evaluate performance and restoration impacts in Biscayne Bay and other regions where both freshwater and nutrient inputs are modified by water management decisions.

Leaf Gas Exchange and Nutrient Use Efficiency Help Explain the Distribution of Two Neotropical Mangroves under Contrasting Flooding and Salinity

Rhizophora mangle and Laguncularia racemosa cooccur along many intertidal floodplains in the Neotropics. Their patterns of dominance shift along various gradients, coincident with salinity, soil fertility, and tidal flooding. We used leaf gas exchange metrics to investigate the strategies of these two species in mixed culture to simulate competition under different salinity concentrations and hydroperiods. Semidiurnal tidal and permanent flooding hydroperiods at two constant salinity regimes (10 g L−1 and 40 g L−1) were simulated over 10 months. Assimilation ( ), stomatal conductance ( ), intercellular CO2 concentration ( ), instantaneous photosynthetic water use efficiency (PWUE), and photosynthetic nitrogen use efficiency (PNUE) were determined at the leaf level for both species over two time periods. Rhizophora mangle had significantly higher PWUE than did L. racemosa seedlings at low salinities; however, L. racemosa had higher PNUE and and, accordingly, had greater intercellular CO2 (calculated) during measurements. Both species maintained similar capacities for A at 10 and 40 g L−1 salinity and during both permanent and tidal hydroperiod treatments. Hydroperiod alone had no detectable effect on leaf gas exchange. However, PWUE increased and PNUE decreased for both species at 40 g L−1 salinity compared to 10 g L−1. At 40 g L−1 salinity, PNUE was higher for L. racemosa than R. mangle with tidal flooding. These treatments indicated that salinity influences gas exchange efficiency, might affect how gases are apportioned intercellularly, and accentuates different strategies for distributing leaf nitrogen to photosynthesis for these two species while growing competitively.

Foliar phosphorus application enhances nutrient balance and growth of phosphorus deficient sugarcane

Although it is well known that nutrient imbalance in shoot tissues may impair plant performance, the interactive effect between foliar phosphorus (P) application and varying P availability in the rooting medium on the nutritional status of sugarcane has not been well studied. To fill this research gap, four sugarcane varieties (IAC91-1099, IACSP94-2101, IACSP94-2094 and IACSP95-5000) were evaluated using a combination of two concentrations of P in nutrient solution (P-deficient, PD = 0.02 mmol L^(−1) and P-sufficient, PS = 0.5 mmol L^(−1)) and foliar P application (none and 0.16 mol L^(−1)). The spray was applied until drip point three times during the experiment with 15 days intervals, after which the plants were harvested to quantify growth and shoot concentration of nitrogen (N), P, magnesium (Mg), sulphur (S) and manganese (Mn). The responses of sugarcane plants to foliar P spray at different levels of P supply in the rooting medium was not genotype-dependent. It was demonstrated for the averaged values across varieties, that foliar P application enhanced sugarcane performance under low P, as revealed by improvements of leaf area and dry matter production of shoot and root of PD plants. Under P limitation we also observed diminished shoot concentration of N, P, Mg, S and increased concentration of Mn. However, foliar P spray increased the concentrations of N, P, S and reduced shoot Mn. Furthermore, shoot P:N, P:Mg, P:S, P:Mn and Mg:Mn concentration ratios exhibited a positive relationship with shoot dry matter production. In conclusion, low P supply in the rooting medium impairs nutrient balance in shoot tissues of sugarcane at early growth; however, this effect was ameliorated by foliar P application which merits further study under field conditions.

Effect Of Aloe Vera Application On The Content And Molecular Arrangement Of Glycosaminoglycans During Calcaneal Tendon Healing.

Although several treatments for tendon lesions have been proposed, successful tendon repair remains a great challenge for orthopedics, especially considering the high incidence of re-rupture of injured tendons. Our aim was to evaluate the pharmacological potential of Aloe vera on the content and arrangement of glycosaminoglycans (GAGs) during tendon healing, which was based on the effectiveness of A. vera on collagen organization previously observed by our group. In rats, a partial calcaneal tendon transection was performed with subsequent topical A. vera application at the injury site. The tendons were treated with A. vera ointment for 7 days and excised on the 7(th) , 14(th) , or 21(st) day post-surgery. Control rats received ointment without A. vera. A higher content of GAGs and a lower amount of dermatan sulfate were detected in the A. vera-treated group on the 14(th) day compared with the control. Also at 14 days post-surgery, a lower dichroic ratio in toluidine blue stained sections was observed in A. vera-treated tendons compared with the control. No differences were observed in the chondroitin-6-sulfate and TGF-β1 levels between the groups, and higher amount of non-collagenous proteins was detected in the A. vera-treated group on the 21(st) day, compared with the control group. No differences were observed in the number of fibroblasts, inflammatory cells and blood vessels between the groups. The application of A. vera during tendon healing modified the arrangement of GAGs and increased the content of GAGs and non-collagenous proteins.

Anthocyanin synthesis, growth and nutrient uptake in suspension cultures of strawberry cells

Strawberry (Fragaria ananassa cv. Shikinari) cell suspension cultures carried out in shake flasks for 18 d were closely examined for cell growth, anthocyanin synthesis and the development of pigmented cells in relation to the uptake of carbohydrate, extracellular PO4, NO3, NH4, and calcium. Cell viability, extracellular anthocyanin content, pH and electrical conductivity of the broth were also monitored. The specific growth rate of strawberry cells at exponential phase was 0.27 and 0.28 d(-1) based on fresh and dry weight, respectively. Anthocyanin synthesis was observed to increase continuously to a maximum value of 0.86 mg/g fresh cell weight (FCW) at day 6, and was partially growth-associated. Anthocyanin synthesis was linearly related to the increase in pigmented cell ratio, which increased with time and reached a maximum value of ca. 70% at day 6 due to reduction in cell viability and depletion of substrate. Total carbohydrate uptake was closely associated with increase in cell growth, and glucose was utilized in preference to fructose. Nitrate and ammonia were consumed until 9 d of culture, but phosphate was completely absorbed within 4 d. Calcium was assimilated throughout the growth cycle. After 9 d, cell lysis was observed which resulted in the leakage of intracellular substances and a concomitant pH rise. Anthocyanin was never detected in the broth although the broth became darkly pigmented during the lysis period. This suggests that anthocyanin was synthesized only by viable pigmented cells, and degraded rapidly upon cell death and lysis. Based on the results of kinetic analysis, a model was developed by incorporating governing equations for the ratio of pigmented cells into a Bailey and Nicholson's model. This was verified by comparison with the experimental data. The results suggest Bat the model satisfactorily describes the strawberry cell culture process, and may thus be used for process optimization.

Dormancy release in Lolium rigidum seeds is a function of thermal after-ripening time and seed water content

Dormancy release in seeds of Lolium rigidum Gaud. (annual ryegrass) was investigated in relation to temperature and seed water content. Freshly matured seeds were collected from cropping fields at Wongan Hills and Merredin, Western Australia. Seeds from Wongan Hills were equilibrated to water contents between 6 and 18% dry weight and after-ripened at constant temperatures between 9 and 50degreesC for up to 23 weeks. Wongan Hills and Merredin seeds at water contents between 7 and 17% were also after-ripened in full sun or shade conditions. Dormancy was tested at regular intervals during after-ripening by germinating seeds on agar at 12-h alternating 15degreesC (dark) and 25degreesC (light) periods. Rate of dormancy release for Wongan Hills seeds was a positive linear function of after-ripening temperature above a base temperature (T-b) of 5.4degreesC. A thermal after-ripening time model for dormancy loss accounting for seed moisture in the range 6-18% was developed using germination data for Wongan Hills seeds after-ripened at constant temperatures. The model accurately predicted dormancy release for Wongan Hills seeds after-ripened under naturally fluctuating temperatures. Seeds from Merredin responded similarly but had lower dormancy at collection and a faster rate of dormancy release in seeds below 9% water content.

Dry matter production and boron concentrations of vegetative and reproductive tissues of canola and sunflower plants grown in nutrient solution

Canola (Brassica napus L.) and sunflower (Helianthus annuus L.), two important oilseed crops, are sensitive to low boron (B) supply. Symptoms of B deficiency are often more severe during the reproductive stage, but it is not known if this is due to a decreased external B supply with time or an increased sensitivity to low B during this stage. Canola and sunflower were grown for 75 days after transplanting (DAT) in two solution culture experiments using Amberlite (IRA-743) B-specific resin to maintain constant B concentration in solution over the range 0.6 - 53 muM. Initially, the vegetative growth of both crops was good in all treatments. With the onset of the reproductive stage, however, severe B deficiency symptoms developed and growth of canola and sunflower was reduced with less than or equal to 0.9 and less than or equal to 0.7 muM B, respectively. At these concentrations, reproductive parts failed to develop. The critical B concentration (i.e. 90% of maximum shoot dry matter yield) in the youngest opened leaf was 18 mg kg(-1) in canola and 25 mg kg(-1) in sunflower at 75 DAT. The results of this study indicate that the reproductive stage of these two oilseed crops is more sensitive than the vegetative stage to low B supply.

Growth and nutrient uptake of coffee seedlings cultivated in nutrient solution with and without silicon addition

In recent years, the application of silicon (Si) in crops, including coffee, has become a common practice. The objective of this study was to assess the silicon uptake by coffee seedlings and its effects on plant growth, water and macro and micronutrient uptake. The research was conducted using nutrient solution in a greenhouse at the Departamento de Fitotecnia da Universidade Federal de Viçosa, in a completely randomized design with two treatments (with and without silicon) and three replications. Each plot consisted of three plants grown in a 800 mL vessel containing the treatment solutions. At every three days, water consumption, the concentration of OH - and the depletion of Si and K were assessed in the nutrient solutions. After 33 days, the plants were assessed with regard to their fresh and dry weight of leaves, roots and stem, shoot height and total length of the plant (shoot and root). Number of leaves and internodes, and the content and accumulation of silicon, macro, and micronutrients were also determined. The consumption of water, the amount of potassium uptake and, biomass accumulation were greater in plants grown in solution without silicon addition. However, the concentration of OH- in the solution and the amount of silicon uptake were greater in plants grown in solution with added silicon. Silicon accumulation was greater in leaves than in stem and roots. Silicon decreased coffee plant accumulation of phosphorus, potassium, calcium, zinc, copper and iron.

Leaf area and epicuticular wax content of Sida spp

Morpho-physiological characteristics and chemical composition are directly related to superior competitive ability of crops. This study intended to make a comparative analysis of dry matter production, leaf area and amount of epicuticular wax of three species of Sida spp: S. urens L., S. rhombifolia L. and S. spinosa L. Plants were collected at three growth stages: V1: stage described as up to 10 fully expanded leaves; V2: between 11 leaves and flowering; and R: after flowering. At stages V2 and R, the highest number of leaves was recorded for S. rhombifolia, followed by S. spinosa at V2 and S. urens at R. These results were relatively proportional to leaf area for all species. S. spinosa at the vegetative stage produced the highest values of specific leaf area (SLA), with no significant differences between species at the stage R. The amount of wax per unit of leaf area between species at the same developmental stage was significantly different only at the reproductive stage, where S. spinosa produced 23.18 and 6.23 fold more wax than S. urens and S. rhombifolia respectively. Between the growth stages of each species, there was decrease in the amount of wax with plant age and increase in leaf area (AFE), number of leaves and dry matter. The leaves of the Sida species exhibit different characteristics and this information can be used to optimize the use of herbicides in the control of these weeds.

Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Organic material decomposition and nutrient dynamics in a mulch system enriched with leguminous trees in the Amazon

The new techniques proposed for agriculture in the Amazon region include rotational fallow systems enriched with leguminous trees and the replacement of biomass burning by mulching. Decomposition and nutrient release from mulch were studied using fine-mesh litterbags with five different leguminous species and the natural fallow vegetation as control. Samples from each treatment were analyzed for total C, N, P, K, Ca, Mg, lignin, cellulose content and soluble polyphenol at different sampling times over the course of one year. The decomposition rate constant varied with species and time. Weight loss from the decomposed litter bag material after 96 days was 30.1 % for Acacia angustissima, 32.7 % for Sclerolobium paniculatum, 33.9 % for Iinga edulis and the Fallow vegetation, 45.2 % for Acacia mangium and 63.6 % for Clitoria racemosa. Immobilization of N and P was observed in all studied treatments. Nitrogen mineralization was negatively correlated with phenol, C-to-N ratio, lignin + phenol/N ratio, and phenol/phosphorus ratios and with N content in the litterbag material. After 362 days of field incubation, an average (of all treatments), 3.3 % K, 32.2 % Ca and 22.4 % Mg remained in the mulch. Results confirm that low quality and high amount of organic C as mulch application are limiting for the quantity of energy available for microorganisms and increase the nutrient immobilization for biomass decomposition, which results in competition for nutrients with the crop plants.

Water-soluble nutrients in aerial plant parts of peanut and white oat as affected by lime and gypsum application

Lime and gypsum influence nutrient availability and uptake, as well as the content of organic acids in the aerial plant parts. These changes, quantified by plant analysis of soluble nutrients, may potentiate the effect of soil amendment, ensuring the sustainability of the no-tillage system. In this sense the effect of lime and gypsum surface application on the content of water-soluble nutrients in peanut and oat residues was evaluated. The experiment was conducted on an Oxisol in Botucatu (SP) in the growing seasons 2004/2005 and 2005/2006. It was arranged in a randomized block design in split plots with four replications, where lime rates represented the plots and presence or absence of gypsum application the subplots. Peanut was grown in summer and white oat in the winter in the entire experimental area. Gypsum applied to peanut increased soluble Ca only in the first season, due to the short period between product application and determination of soluble nutrient contents in the plant extract. Liming of peanut and oat increased soluble Ca, Mg, K contents, did not alter Cu content and reduced Zn, Mn and Fe contents in both years of cultivation. Gypsum on the other hand reduced the electrical conductivity of peanut (2004/2005 and 2005/2006) and white oat (2004/2005).

Effects of vapour pressure deficit and soil water content on leaf water potential between selected provenances of Eucalyptus microtheca in an irrigated plantation, eastern Kenya

Summary