Detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop using post-enrichment DAS-ELISA

The detection of Ralstonia solanacearum (biovar 2A) in stems of symptomless plants before harvest of the potato crop, instead of tubers, would not only save highly valued planting material but would be less time-consuming and would also enhance farmers' market decisions. Although pathogen detection in stems has been proven efficient for ring rot, this has never been investigated for bacterial wilt (BW). Therefore the possibility of detecting BW latent infection in stem pieces about three weeks before harvest was assessed in 57 fields of the Andean highlands of Peru. Two sensitive, specific and user-friendly serological methods were used to detect the pathogen in latently infected tubers and stems: double-antibody sandwich (DAS)-ELISA and indirect ELISA on nitrocellulose membrane (NCM) after enrichment of the plant extracts in a semi-specific broth. Optimum sample sizes of stems and tubers were evaluated for 37 potato crops showing between 0 and 0·1% BW incidence using a binomial distribution model to calculate the detection probabilities. Although results of detection using the two serological techniques had 100% concordance, detection probabilities were higher using DAS-ELISA, whatever the plant part tested. BW detection probabilities were higher for tubers than for stems; a 99% detection probability was obtained by analysing 400 stems sections or 250 tubers using DAS-ELISA. Detection of BW infection in symptomless plants 20 days before harvest using post-enrichment DAS-ELISA is a reliable and user-friendly technique that can easily be used by national plant protection services and seed programmes in developing countries.     

Assessment of latent infection frequency in progeny tubers of advanced potatoclones resistant to bacterial wilt: A new selection criterion

Summary This paper reports results of a 3-year evaluation of CIP advanced potato clones in a bacterial wilt-infested field (race 3) in Peru. Clones resistant or moderately resistant to wilt were selected and all tubers harvested from each clone were tested for latent infection byRalstonia solanacearum using a sensitive serological technique developed at CIP. A sampling strategy to estimate accurately the frequency of infected tubers in the clones has been evaluated. This method will allow consideration of tuber latent infection as a new selection criterion in breeding for resistance to bacterial wilt. Thirteen clones were found resistant to wilt in all three evaluations (i.e.≤6% wilt), from which five had no wilt in all trials. However, all clones harboured latent infection in tubers averaging 30%. Analysing 30 tubers/clone provides an accurate estimation of the proportion of infected tubers with a high precision level.     

Loading of VO2+ and Cu2+ to partially oxidized charcoal fines rejected from Brazilian metallurgical industry

Purpose

Charcoal is utilized in Brazil for the metallurgical industry. Small size pieces, called charcoal fines, are rejected and sometimes are used to produce energy by burning. This charcoal can be used as soil conditioner to improve retention of metal ions in soil. However, changing the charcoal's surface chemistry via oxidation may increase retention of metal ions.

Materials and methods

Two kinds of oxidants were employed, nitric acid and selenium dioxide/hydrogen peroxide. Vanadyl ion (VO²⁺) and copper ion (Cu²⁺) were utilized as probe to study the metal ion complexation by the partially oxidized charcoal obtained. FTIR and EPR spectroscopy were used to characterize the materials.

Results and discussion

Oxidation with nitric acid increased nitrogen content, while oxidation with SeO₂/H₂O₂ elevated carbon content of treated charcoals. Organic free radicals (OFRs) with the unpaired electron in p orbitals of aromatic structures were confirmed by the EPR g-factors that ranged from 2.0038 to 2.0031. The oxidation with selenium dioxide/hydrogen peroxide formed charcoal with largest quantity of OFR. Loading charcoal with VO²⁺ and Cu²⁺ resulted in formation of complexes were oxygen acted as coordination atom. The formed complexes were of axial symmetry. The configurations around the Cu²⁺ ions were: CUNCu²⁺ complex (more stable square planar symmetry) and CFNCu²⁺ complex (less stable distorted tetrahedral configuration). Both complexes had oxygen as coordinating atoms. CFNVO²⁺ and CFSeVO²⁺ complexes presented complexation sites of axial symmetry, C_4v, with oxygen as coordinating atoms.

Conclusions

The oxidative treatment of charcoal with SeO₂/H₂O₂ is more appropriate to produce soil organic conditioner for complexation of metal ions.     

Optimisation of sample size for the detection of latent infection byRalstonia solanacearum in potato seed tubers in the highlands of Peru

Summary A sampling strategy was evaluated in the Andean highlands of Peru to optimise the detection ofRalstonia solanacearum in seed tubers harvested from symptomless crops. A sensitive and specific serological method developed at CIP was used to detect the pathogen in latently infected tubers. Optimum sample size was evaluated for symptomless crops after analysing various numbers of composite samples and using a binomial distribution model to calculate the detection probabilities.R. solanacearum was detected in all lots from fields with visible symptoms, so validating the detection technique. About half of the seed lots from apparently healthy fields at altitudes of up to 3,100 m were found positive for the pathogen.R. solanacearum was detected with 99% probability in samples of 350 tubers from seed lots from symptomless crops. This number of seed tubers could feasibly be processed in a seed-health test without incurring too high a cost for labour and materials.     

Evaluation of Nile tilapia in monoculture and polyculture with giant freshwater prawn in biofloc technology system and in recirculation aquaculture system

Biofloc technology system (BFT), recirculation aquaculture system (RAS) and polyculture promote efficient use of water, area and nutrient recycling, which are essential practices for sustainable aquaculture development. The aim of this study was to evaluate the growth, feed efficiency, biofloc composition and water quality of Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in monoculture and polyculture with giant freshwater prawn Macrobrachium rosenbergii (De Man, 1906) in BFT and RAS, over a period of 30 days. Fish (n = 128; 7.29 ± 0.67 g) were distributed randomly in 16 experimental tanks (8 fish/tank). Prawn (n = 96; 0.50 ± 0.09 g) were allocated in 8 experimental tanks (12 prawn/tank) in a polyculture. The experimental design was completely randomized with four treatments with four replicates each, in a factorial design 2 × 2 (BFT and RAS vs. monoculture and polyculture). The experimental diet (28% of digestible protein; 3100 kcal kg−1 of digestible energy) was used both to fish and prawn in BFT and RAS. There was significant effect (p < 0.01) of the system and the culture for weight gain, apparent feed conversion and protein efficiency ratio. The average weight gain and apparent feed conversion of tilapia in monoculture (30.04 g and 1.39) and in polyculture (36.44 g and 1.27) were superior (p < 0.01) in BFT than in monoculture (23.64 g and 1.74) and in polyculture (24.14 g and 1.61) in RAS. Weight gain and survival of giant freshwater prawn was superior (p < 0.01) in BFT (0.43 g and 87%) compared to RAS (0.26 g and 79%). The data showed that BFT provides better growth performance responses in monoculture for Nile tilapia and in polyculture with giant freshwater prawn compared to RAS.     

Highly sensitive detection of Ralstonia solanacearum in latently infected potato tubers by post-enrichment enzyme-linked immunosorbent assay on nitrocellulose membrane

A post-enrichment enzyme-linked immunosorbent assay (ELISA) on nitrocellulose membrane (NCM-ELISA) is described for the detection of Ralstonia solanacearum in latently infected potato tubers. The polyclonal antiserum specificity was significantly improved by adsorption with cross-reacting bacteria. The detection efficiency after enrichment was compared with those of nucleic acid spot hybridization (NASH), double-antibody-sandwich immunoassay (DAS-ELISA) and plating on modified Kelman's medium. After 48 h of incubation of the tuber extracts with modified SMSA broth at 30°C, sensitivities of post-enrichment NCM-ELISA, DAS-ELISA and NASH were similar. As few as 10cells mL⁻¹ were detected in either inoculated or naturally infected tuber extracts. Of 255 field samples, no cross-reactivity of NCM-ELISA was observed. Post-enrichment NCM-ELISA thus provides a reliable and sensitive low-cost method that is rapid and easy to use, making it suitable for assessing susceptibility of breeding lines to bacterial wilt, ecological studies and seed quality control in developing countries.     

Effects of Ambient Nitrite on Amazon River Prawn,Macrobrachium amazonicum,larvae

The effects of nitrite concentration on larval development of Amazon river prawn, Macrobrachium amazonicum, were studied in laboratory. In Experiment 1, larvae were reared in 600‐mL glass beakers filled with 300‐mL water with nitrite concentration of 0, 0.2, 0.4, 0.8 and 1.6 mg/L NO₂‐N. In Experiment 2, total ammonia nitrogen (TAN, NH₃‐N + NH₄‐N) excretion were analyzed in zoea (Z) I, III, VII and IX exposed to 0, 0.4, 0.8 and 1.6 mg/L NO₂‐N. In both experiments each treatment was conducted in five replicates. The experiments were carried out in test solutions at 10 salinity, constant temperature 30 C and 12:12 h daylight : darkness regime. Survival, productivity, weight gain and larval stage index decreased linearly with increasing ambient nitrite concentration. However, there was no significant difference among larvae reared at concentration ranging from 0 to 0.8 mg/L NO₂‐N by ANOVA in all variables. Individual ammonia‐N and mass‐specific ammonia‐N excretion increased in ZI and ZIX, was almost constant in ZIII and decreased in ZVII from 0 to 1.6 mg/L NO₂‐N. The relationship between individual TAN and body mass suggested that 1.6 mg/L NO₂‐N stress the larvae. Despite of the effects of nitrite on larvae follow a dose‐dependent response and shows large variability among individuals, levels below 0.8 mg/L may be used as a general reference in commercial hatcheries, which should be applied carefully.     

Subunit influenza vaccine candidate based on CD154 fused to HAH5 increases the antibody titers and cellular immune response in chickens

World Health Organization has a great concern about the spreading of avian influenza virus H5N1. To counteract its massive spread, poultry vaccination is highly recommended together with biosecurity measures. In our study, a recombinant vaccine candidate based on the fusion of extracellular segments of hemagglutinin (HA) H5 of avian influenza virus and chicken CD154 (HACD) is tested with the aim of enhancing humoral and cellular immune responses in chickens. Protein expression was carried out by transducing several mammalian cell lines with recombinant adenoviral vectors. HACD purification was assessed by three distinct purification protocols: immunoaffinity chromatography by elution at acidic pH or with a chaotropic agent and size exclusion chromatography. Humoral and cellular immune responses were measured using the hemagglutination inhibition assay and the semiquantitative real time PCR, respectively. The results showed that humoral response against HACD was significantly higher than the obtained with HA alone after booster (P<0.01, P<0.05). From HACD molecules purified by distinct protocols, only the obtained by size exclusion chromatography generated hemagglutinationin-inhibition activity. IFN-γ levels indicated that cellular immune response was significantly higher with HACD, in its pure or impure form, compared to its counterpart HA (P<0.01). These data demonstrate that HACD is able to significantly enhance humoral and cellular immune responses against HA antigen, which make this fusion protein a promising subunit vaccine candidate against H5N1 virus outbreaks.     

An improved enrichment broth for the sensitive detection of Ralstonia solanacearum (biovars 1 and 2A) in soil using DAS–ELISA

A reliable, sensitive, low-cost and easy-to-use technique is described for the detection of Ralstonia solanacearum (the causal organism of bacterial wilt, BW) in soil. A total of 273 potato isolates belonging to five different biovars (Bv), originating from 33 countries worldwide, were tested and successfully detected by antibodies produced at the International Potato Center (CIP). Isolates of R. solanacearum belonging to Bv1 and Bv2A were successfully detected by double antibody sandwich–enzyme-linked immunosorbent assay (DAS–ELISA) at low population levels after incubation of soil suspensions for 48 h at 30°C in a new semiselective broth containing a potato tuber infusion. Detection thresholds of 20 and 200 CFU g⁻¹ inoculated soil were obtained for Bv1 and Bv2A, respectively. Sensitivity of detection of Bv2A was similar or even higher in five different inoculated soil types. No cross-reactions were obtained in DAS–ELISA after enrichment of soil suspensions (i) prepared from 23 different soils sampled in BW-free areas in six departments of Peru; and (ii) inoculated with 10 identified bacteria and 136 unknown isolates of soil microbiota isolated from eight different locations. Only the blood disease bacterium gave a low-level reaction after enrichment. In naturally infested soils, average sensitivities of 97·6 (SE 14·8) and 100·9 (SE 22·6) CFU g⁻¹ were obtained for biovars 1 and 2A, respectively. By making serial dilutions of the soil suspension before enrichment, densities of R. solanacearum could be determined in a semiquantitative way. Results also showed that composite samples of five soils could be analysed to assess field soil populations without reducing detection sensitivity.