A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.     

Users’ Reactions to the Primer on Tree Registration Policies: Lessons for Designing Extension Materials and Improving Information Flow

In the Philippines, only a few farmers and plantation owners register their tree farms with the Department of Environment and Natural Resources (DENR), the office in charge of tree registration. One of the major reasons is their lack of knowledge of government policy on tree registration. To help improve the flow of information and understanding of tree registration policies among smallholder tree farmers and other stakeholders, a Primer on Tree Registration, Harvesting, Transport and Marketing Policies on Private Land was developed. Produced through the participation of stakeholders, including tree farmers, the DENR, and local government officers, police, and timber dealers, the Primer is highly visual and has been released in English as well as Waray–Waray and Cebuano dialects. This paper reports findings of focus group discussions conducted to gauge user reactions to the Primer. The Primer was considered as a valuable source of information about tree registration policies. Aside from being comprehensible, FGD participants found it attractive, perceived it to be intended for them and free from content that confused them. However, farmers suggested that illustrations need to be improved by making the persons portrayed more presentable and reflective of their actual tree farm operations. Participants said that aside from the Primer, the flow of information on tree registration policies could be improved through the use of multi-media including posters, radio broadcasts, and presentation in seminars for farmers.     

Glyphosate-resistant weeds of South American cropping systems: an overview

Herbicide resistance is an evolutionary event resulting from intense herbicide selection over genetically diverse weed populations. In South America, orchard, cereal and legume cropping systems show a strong dependence on glyphosate to control weeds. The goal of this report is to review the current knowledge on cases of evolved glyphosate-resistant weeds in South American agriculture. The first reports of glyphosate resistance include populations of highly diverse taxa (Lolium multiflorum Lam., Conyza bonariensis L., C. canadensis L.). In all instances, resistance evolution followed intense glyphosate use in fruit fields of Chile and Brazil. In fruit orchards from Colombia, Parthenium hysterophorus L. has shown the ability to withstand high glyphosate rates. The recent appearance of glyphosate-resistant Sorghum halepense L. and Euphorbia heterophylla L. in glyphosate-resistant soybean fields of Argentina and Brazil, respectively, is of major concern. The evolution of glyphosate resistance has clearly taken place in those agroecosystems where glyphosate exerts a strong and continuous selection pressure on weeds. The massive adoption of no-till practices together with the utilization of glyphosate-resistant soybean crops are factors encouraging increase in glyphosate use. This phenomenon has been more evident in Argentina and Brazil. The exclusive reliance on glyphosate as the main tool for weed management results in agroecosystems biologically more prone to glyphosate resistance evolution.     

Development of real-time PCR systems based on SYBR? Green I and TaqMan? technologies for specific quantitative detection of Phoma tracheiphila in infected Citrus

Real-time PCR assays based on SYBR? Green I and TaqMan? technologies were developed for in planta detection and quantification of Phoma tracheiphila, the mitosporic fungus causing ‘mal secco’ disease on citrus. Primers and a hybridization probe were designed on the basis of the internal transcribed spacer (ITS) region of the nuclear rRNA genes. The real-time PCR assays were compared with a classic isolation method in two separate experiments carried out on 6 and 24 month-old sour orange seedlings, artificially inoculated with a conidial suspension of the pathogen. Both technologies made it possible to follow the progression of infection by P. tracheiphila, enabling detection and quantification of the target fungus prior to the development of symptoms. The detection limit was 10 copies of the cloned target sequence and 15 pg of genomic DNA extracted from fungal spores. The values of the cycle threshold (Ct) were linearly correlated with the concentration of the target DNA, indicating that the method is suitable as a qualitative and quantitative assay. The presence of non-target fungal DNA had no effect on the specificity of the assay, but resulted in a 10-fold reduction of sensitivity. Total inhibition of the reaction occurred when conidia of the target pathogen were mixed with an organic soil substrate before extracting DNA using the standard protocol, while an alternative purification kit resulted in a significant decrease in sensitivity. Compared to classic methods, real-time PCR proved faster and easier to perform and showed a higher sensitivity. These results suggest that real-time PCR, based on both chemistries, has a great potential for early diagnosis of ‘mal secco’ disease and for quantitative estimation of fungal growth within host tissue.     

Branch cankers on citrus trees in Spain caused by Phytophthora citrophthora

Considerable losses of citrus trees have been observed in the major citrus-growing areas of Spain. Samples were collected from 132 orchards, and isolations and pathogencity tests were conducted to determine the aetiology of a serious canker disease. Affected trees showed cankers on the scion that frequently began on the branches. Three Phytophthora species were identified based on their morphological, cultural, physiological and molecular profiles. Phytophthora citrophthora was the main species associated with this new syndrome in 114 orchards. Phytophthora nicotianae (syn. P. parasitica ) was isolated from nine orchards as the sole Phytophthora species and in coinfection with P. citrophthora from another nine orchards. Phytophthora citricola was isolated only from one orchard. In stem-inoculation studies conducted under greenhouse conditions, clementine mandarin cv. Hernandina and sweet orange cv. Navel Late were more susceptible to P. citrophthora than sour orange and Carrizo citrange rootstocks. Clementine cv. Hernandina was also highly susceptible in field inoculation experiments. In agreement with field surveys, clementine mandarin cultivars were the most affected, their rootstocks remaining healthy. Phytophthora citrophthora was found to be the predominant species in orchard soils; however, P. nicotianae was also isolated. This information changes the scenario of diseases caused by Phytophthora spp. in Spain and consequently, the present knowledge of epidemiology and the effectiveness of the current control measures should be reassessed.     

Auswirkungen des Herbizids Basta® auf die Weinrebe und den Falschen Mehltau

A field experiment was conducted in order to analyse the effects of the herbicide Basta^® with glufosinate-ammonium as the active ingredient (that inhibits specifically the enzyme glutamine-synthetase) on the grapevine plant and the infestation with Plasmopara viticola, the downy mildew pathogen. The herbicide was applied at sublethal concentrations and the toxic effects on leaves and berries based on different quality parameters were estimated. Low concentrations of glufosinate-ammonium (<?0,625?mM) did not affect the chlorophyll and amino acid concentrations in leaves. This was also the case for the content of salicylic acid and phenolic compounds. Furthermore, must parameters such as sugars (glucose and fructose), acids (tartaric acid, malic acid, total acids and volatile acids), and alcohol (ethanol and glycerine) also remained unchanged compared to the fungicide-treated or untreated control, whereas the concentrations of nitrogenous components metabolically available to yeasts increased with increasing concentrations of glufosinate-ammonium. The amount of berries harvested at the end of the experiment depended on the glufosinate-ammonium concentration applied. The harvest was highest when glufosinate-ammonium was applied as a 0.1?mM solution and differed not significantly from the fungicide-treated control, but decreased with increasing herbicide doses. However, the infestation of leaves with P. viticola was significantly reduced by the application of the herbicide, even though not each infection was eradicated due to the non-systemic mode of action of the herbicide. The particular influence of the herbicide onto this host-parasite interaction still remains unclear. Since applications of glufosinate-ammonium at low concentrations seem not to affect grapevine plants negatively, but were able to reduce the infestation with downy mildew, the specific mode of action may reveal new alternatives for the control of fungal pathogens.     

Effects of Quercetin on Performance and Lipid Metabolism in Laying Hens

Quercetin has a variety of biological activities. However, its application as a feed additive in poultry production is limited. In this study, the effects of quercetin on performance and lipid metabolism in Hessian laying hens were investigated and the possibility of quercetin to decrease cholesterol in eggs and Low Density Lipoprotein cholesterol(LDL-cholesterol) in laying hens were explored. A total of 240 laying hens at 29-week-old were randomly assigned to four treatments, with six replicates of 10 birds per replicate. The treatments were fed with basal diet supplemented with 0, 0.2, 0.4 and 0.6 g · kg~(-1) quercetin for 8 weeks. Layer performance, lipid concentration in livers, yolks and serum were measured at the end of the experiment. The results showed that laying rate had a quadratic correlation with the level of quercetin(p=0.056) and was maximized by the supplement level of 0.2 g · kg~(-1) diet. When the level of dietary quercetin increased, feed-egg ratio decreased. Crude fat and the total cholesterol of livers decreased by supplementation of quercetin. The total cholesterols of yolks also decreased and had a quadratic correlation with the levels of quercetin(p=0.019). Insulin increased by supplement of quercetin. In conclusion, quercetin improved the performance and reduced egg yolk cholesterol by affecting serum hormone levels.     

Infection process of <Emphasis Type="Italic">Pseudocercospora musae</Emphasis> on banana leaf

Yellow Sigatoka that is caused by Pseudocercospora musae is an important banana disease. The aim of this study was to elucidate the infection process of P. musae in banana leaves by scanning electron microscopy. Leaf samples were inoculated on the abaxial surface with P. musae and then analysed at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post inoculation (hpi) and at 36 and 50 days post inoculation (dpi). The conidia were found to be germinated between 24 and 36 hpi and penetrated through the stomata between 96 and 120 hpi, or more generally from 144 hpi. P. musae colonized the spongy parenchyma at 36 dpi and the palisade parenchyma at 50 dpi. Sporulation occurred at 50 dpi on the adaxial surface of leaves through the emergence of conidia on conidiophores through the stomata. Considering the importance of yellow Sigatoka in banana production, our results provide a better understanding of the life cycle of the fungus for treatment processes.     

Geographical distribution and first molecular detection of an <Emphasis Type="Italic">Emaravirus,High Plains wheat mosaic virus</Emphasis>, in Argentina

High Plains wheat mosaic virus (HPWMoV) has recently been assigned to genus Emaravirus and is the causal agent of High Plains disease. In this work the geographical distribution and first molecular detection of HPWMoV in Argentina are reported. The virus was detected in six provinces and nine hosts, including wheat, corn, oat and barley, as well as weeds, which play an important role in the epidemiology of this disease. Molecular and phylogenetic analyses of a portion of RNA3 nucleoprotein gene sequence showed that five HPWMoV isolates from different wheat growing regions of Argentina were identical, and suggest a single introduction of HPWMoV to this country, possibly through corn seeds. To the best of our knowledge, this is the first molecular detection of HPWMoV in South America. These results highlight the importance of certification of viruses-free cereal seeds and strict controls for material transfer between different countries to prevent the entry not only of new pathogens but also of new variants or strain at sites where the pest has already been detected.