Serological Methods for Detection of Polymyxa graminis, an Obligate Root Parasite and Vector of Plant Viruses

A purification procedure was developed to separate Polymyxa graminisresting spores from sorghum root materials. The spores were used as im-munogen to produce a polyclonal antiserum. In a direct antigen coating enzyme-linked immunosorbent assay (DAC ELISA), the antiserum could detect one sporosorus per well of the ELISA plate. In spiked root samples, the procedure detected one sporosorus per mg of dried sorghum roots. The majority of isolates of P. graminis from Europe, North America, and India reacted strongly with the antiserum. Interestingly, P. graminis isolates from the state of Rajasthan (northern India), from Pakistan, and an isolate from Senegal (West Africa) reacted weakly with the antiserum. The cross-reactivity of the serum with P. betae isolates from Belgium and Turkey was about 40% of that observed for the homologous isolate. There was no reaction with common fungi infecting roots or with the obligate parasite Olpidium brassicae. However, two isolates of Spongospora sub-terranea gave an absorbance similar to that observed with the homologous antigen. The DAC ELISA procedure was successfully used to detect various stages in the life cycle of P. graminis and to detect infection that occurred under natural and controlled environments. A simple procedure to conjugate antibodies to fluorescein 5-isothiocyanate (FITC) is described. Resting spores could be detected in root sections by using FITC-labeled antibodies. The potential for application of the two serological techniques for studying the epidemiology of peanut clump disease and for the characterization of Polymyxa isolates from various geographical origins is discussed.     

P0 protein of cotton leafroll dwarf virus-atypical isolate is a weak RNA silencing suppressor and the avirulence determinant that breaks the cotton Cbd gene-based resistance

Cotton blue disease (CBD) is the most important disease present in cotton crops in South America and cotton leafroll dwarf virus (CLRDV) is the causal agent. The disease has been controlled by sowing cotton varieties resistant to CLRDV. However, in the 2009/10 growing season, an outbreak due to an atypical CLRDV isolate (CLRDV-at) occurred in northwest Argentina. Although CLRDV and CLRDV-at genomes are very closely related, the symptoms they produce in cotton plants are quite different. P0 is the most divergent protein between the isolates and in CLRDV is a silencing suppressor protein. This work characterized the silencing suppressor activity of the P0 protein encoded by CLRDV-at (P0^CL-at) and evaluated its role in Cbd-resistance break in cotton plants. It was demonstrated that P0^CL-at, despite having a mutation in the consensus of the F-box-like motif, was able to suppress local RNA silencing, but displayed lower activity than P0^CL. P0^CL and P0^CL-at showed no differences in the interaction with Gossypium hirsutum SKP1 orthologue (GSK1) and Nicotiana benthamiana SKP1 and both P0 proteins triggered destabilization of ARGONAUTE1. However, when the ability to enhance PVX symptoms was evaluated, P0^CL-at was shown to be a weaker pathogenicity factor than P0^CL in N. benthamiana. Interestingly, trans-expressed P0^CL-at enabled CLRDV to systemically infect CBD-resistant plants, and a chimeric CLRDV-P0^CL-at infectious clone succeeded in establishing infection in CBD-resistant cotton varieties with symptoms resembling those produced by CLRDV-at. These results strongly suggest that P0^CL-at is the avirulence (Avr) determinant involved in breaking cotton Cbd gene-based resistance.     

Argentinian potato leafroll virus P0 protein: Novel activities for a previously known suppressor

The potato leafroll virus (PLRV) P0 protein (P0^PL) is a suppressor of RNA silencing. In this study, we showed that P0 protein from an Argentinian isolate of PLRV (P0^PL-Ar) has an additional activity not described for other PLRV or P0 proteins from poleroviruses. Besides reporting that P0^PL-Ar displays both local and systemic silencing suppressor activity, we demonstrated, for the first time, that P0^PL-Ar impedes accumulation of dsRNA-derived siRNAs. We also showed that P0^PL-Ar interacts with Solanum tuberosum SKP1 orthologue (StSKP1) and triggers destabilization of ARGONAUTE 1 (AGO1) and that these actions are mediated by the F-box-like domain. A mutant in the GW/WG motif within the P0^PL-Ar F-box-like motif lost the suppression activity, the interaction with StSKP1 and abolished AGO1 decay. Interestingly, a mutant in the L76/P77 residues within the P0^PL-Ar F-box-like motif, which lost the suppression activity and the interaction with StSKP1, retained the capacity to enable AGO1 decay. Thus, unlike other P0 proteins of previously characterized poleroviruses, P0^PL-Ar seems to have a dual activity, according to the findings of this study. This protein would act at both an upstream and a downstream step of the RNA silencing pathway: upstream of Dicer-like enzyme (DCL)-mediated primary siRNA production and downstream at the RNA-induced silencing complex (RISC) complex level. Our results contribute to the understanding of the different ways PLRV P0 proteins function as silencing suppressors.     

Production of polyclonal antibodies against ochratoxin A and its detection in chilies by ELISA

Polyclonal antibodies were produced for Ochratoxin A (OA) by injecting OA-bovine serum albumin (BSA) conjugate subcutaneously at multiple sites into a New Zealand White inbred rabbit. Antiserum could be used at a dilution exceeding 1:100 000 in an indirect competitive enzyme-linked immunosorbent assay (ELISA), and detected OA concentrations up to 0.1 ng/mL. The 50% inhibition binding (I(50)) of OA was 5 ng/mL. Antibodies did not react with ochratoxin B, coumarin, 4-hydroxycoumarin, L-phenylalanine, and aflatoxin B1. OA contamination in chilies (Capsicum annum L.) collected from commercial markets and cold storage units was determined. The mean recoveries from OA-free chilies spiked with 1 to100 microg of OA per kg of chili sample were 90-110% with a standard deviation of <10%. Of 100 chili samples tested, 26 were found to contain over 10 microg/kg of OA. In 12 samples the OA concentration varied from 10 to 30 microg/kg, in 10 samples from 30 to 50 microg/kg, in 3 samples from 50 to100 microg/kg, and in one sample it was 120 microg/kg. This is the first record in India of OA in chilies, a major component of cooked foods in this country, and it is noteworthy that OA contamination exceeded the permissible limit for human consumption of less than 20 microg/kg in over 26% of the market samples tested.     

Contribution of SO3 to the acid neutralizing capacity of Andosols exposed to strong volcanogenic acid and SO2 deposition

Soil response to acid and sulphur inputs is influenced largely by the soil's physico‐chemical properties. We studied the effects of such depositions in two types of Andosols exposed to volcanogenic emission (Masaya, Nicaragua), namely Eutric Andosols rich in allophanic constituents, and Vitric Andosols rich in volcanic glass. Small mineral reserves and large contents of secondary short‐range ordered minerals indicate a more advanced weathering of the Eutric than the Vitric Andosols. Strong correlations between soil specific surface and oxalate‐extractable Al, Si and Fe contents highlight the predominant contribution of short‐range ordered minerals to surface area. Both types of Andosols showed a decrease in pH upon acid input. Sulphur deposition increased the soil's S content to 5470 mg S kg^?1. However, the acid neutralizing capacity of the soil solid phase (ANC_s) was not significantly affected by the acid and S inputs. Non‐exchangeable (mineral reserve) and exchangeable cations and total contents of sulphur and phosphorus dictate most of the ANC_s variation. In the Vitric Andosols, mineral reserves contributed up to 97% to these four additive pools, whereas the exchangeable cations accounted for 1–4%. In the Eutric Andosols, the contribution of mineral reserves was less (71–92%), but the exchangeable cation content was greater (1–20%), whereas the contribution of sulphur and phosphorus was significant at 1–15% and 2–7%, respectively. The main process involved in H⁺ consumption is mineral weathering in Vitric Andosols and ion exchange in Eutric Andosols.     

Direct evidence of basic aluminium sulphate minerals in an S-impacted Andosol

The formation of basic aluminium sulphate (BAS) minerals [(K,Na)_nAl_x(OH)_y(SO₄)_z] has often been invoked to explain sulphate retention in soils. These minerals have not yet, however, been directly observed in the soil. We extracted the clay fractions of Andosols intercepting large inputs of volcanogenic sulphur dioxide and acids (HCl, HF), simply by dispersing clays with Na⁺‐resins in deionized water without any other chemical treatment. Clay fractions concentrate 39–63% of total sulphur content of soil. Transmission electron microscopy coupled with energy‐dispersive analysis revealed the presence of BAS particles, appearing as nodules and spheres. These particles have an equivalent diameter smaller than 0.2 µm. They have an Al:S ratio close to 2.2 and 3.8 and are possibly amorphous aluminite or basaluminite, respectively. They seem to have been formed in microenvironments enriched in sulphate, but also in fluoride anions. Their formation seems to have been enhanced by the combination of large inputs of acids and SO₂ and an effective Al supply from weathering of volcanic glass.     

Supplementation with groundnut haulms for sheep fattening in the West African Sahel

Groundnut haulms along with cowpea hay are major crop residues used for animal fattening in the West African Sahel. In traditional sheep fattening, feeds are always provided ad-hoc and in an unregulated fashion, which is rather wasteful. As a preliminary study to establish the optimal feeding levels of groundnut haulms for profitable sheep fattening, a feeding trial was conducted for 70 days with four levels of groundnut haulms (0, 150, 300 and 450 g/day) and a basal diet of bush hay. The effects of supplementation with groundnut haulms on feed intake, water consumption, live weight changes and economic return were determined. Twenty-four Peuhl Oudah rams with average initial weight of 28.6 kg (SD = 1.4) were randomly allocated to four treatments defined by the four levels of groundnut haulms in the diet. Faeces and urine were collected in weeks 5 and 9 of the trial. Digestible organic matter intake (g/(kg LW)^0.75) and nitrogen intake (g/day) increased linearly with the level of groundnut haulms offered. Sheep that were fed only bush hay lost 18.4 g/day, while those that were offered 150, 300 and 450 g of groundnut haulms gained 1.4, 19.3 and 40.2 g/day, respectively. The gross return ranged from 1883 to 4946 FCFA per ram. Net benefit, after removing the feed and veterinary costs from the gross return, ranged from 368 to 1400 FCFA per ram.     

Evaluation of sample pre-treatment and potential contamination of soluble carbon in soil extracts during lyophilisation

Lyophilisation of K₂SO₄ soil extracts has been proposed as a sample preparation technique before elemental analysis of carbon or nitrogen. However, previous measurements, based on wet oxidation or catalytic combustion, indicated that C measurements in lyophilised samples not always proved to be accurate. To determine whether the C analysis was affected by the lyophilisation process, an exploratory study was conducted to investigate the potential effects of the sample pre-treatment and of the lyophilisation process itself. This paper puts forward that the use of soil extracts, previously stored at −20 °C, may affect the recovery of salt in the samples and that contamination of the soluble carbon with exogenous C during lyophilisation is feasible. Therefore we recommend to use freshly prepared soil extracts for lyophilisation and always to include an internal standard among the unknown samples to account for a possible contamination.     

A comparison between visual estimates and image analysis measurements to determine septoria leaf blotch severity in winter wheat

Methods to estimate disease severity vary in accuracy, reliability, ease of use and cost. Severity of septoria leaf blotch (SLB, caused by Zymoseptoria tritici) was estimated by four raters and by image analysis (assumed actual values) on individual leaves of winter wheat in order to explore accuracy and reliability of estimates, and to ascertain whether there were any general characteristics of error. Specifically, the study determined: (i) the accuracy and reliability of visual assessments of SLB over the full range of severity from 0 to 100%; (ii) whether certain 10% ranges in actual disease severity between 0 and 100% were more prone to estimation error compared with others; and (iii) whether leaf position affected accuracy within those ranges. Lin's concordance correlation analysis of all severities (0–100%) demonstrated that all raters had estimates close to the actual values (agreement: ρ_c = 0·92–0·99). However, agreement between actual SLB severities and estimates by raters was less good when compared over short 10% subdivisions within the 0–100% range (ρ_c = ?0·12 to 0·99). Despite common rater imprecision at estimating low and high SLB severities, individual raters differed considerably in their accuracy over the short 10% subdivisions. There was no effect of leaf position on accuracy or precision of severity estimate on separate leaves (L1–L3). Pursuing efforts in understanding error in disease estimation should aid in improving the accuracy of assessments, making visual estimates of disease severity more useful for research and applied purposes.