Resistance of Corynespora cassiicola from soybean to QoI and MBC fungicides in Brazil

Fungicide sprays on soybean in Brazil have contributed to the selection of less sensitive isolates of Corynespora cassiicola. We collected 59 isolates of C. cassiicola from three Brazilian states and two isolates from Paraguay. We investigated their EC₅₀ to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC), any cross-resistance to compounds within QoI and MBC groups, and characterized the polymorphisms in their cytb and β-tubulin genes. Local associations of polymorphisms identified in each gene were statistically correlated with assays results. In total, 79% and 74% of the isolates were classified as resistant to QoI and MBC fungicides, respectively. There was positive cross-resistance to active ingredients within QoI and MBC groups. For QoI, all isolates presented heteroplasmy in G143A of cytb gene; the mutations F129L and G137R were not found. For MBC, 63% of isolates possessed E198A and 21% possessed F200Y mutations, associated with reduced control by MBC fungicides. Heteroplasmy was identified in two and one isolates from Brazil with E198A and F200Y mutations, respectively. The resistance factor for isolates with E198A (10.9) was statistically similar to the isolate with F200Y (8.8) mutation. Genic association analysis of the in vitro assays using discriminatory doses proved them to be accurate. Reduced sensitivity of C. cassiicola to QoI and MBC was also identified in isolates from Paraguay and resistance to QoI and MBC was widely present in C. cassiicola isolates from the main soybean-producing states in Brazil. Thus, integrated management measures should be adopted to manage soybean target spot in these countries.     

Occurrence of <Emphasis Type="Italic">Bursaphelenchus mucronatus</Emphasis> (Nematoda; Aphelenchoididae) in France and association with <Emphasis Type="Italic">Monochamus galloprovincialis</Emphasis> (Coleoptera: Cerambycidae)

As a consequence of the recent introduction of the pine wood nematode Bursaphelenchus xylophilus in Portugal, nematodes of the genus Bursaphelenchus were looked for in various French pine forests, in trees attacked by Monochamus galloprovincialis, the vector insect of B. xylophilus, and in the insects themselves. Trap trees were felled in 12 localities distributed all over the country. Nematodes were extracted from transversal stem discs; insects emerging from the trap trees were studied. B. hellenicus, B. leoni, B. mucronatus and B. sexdentati were isolated, but not B. xylophilus. The presence of B. mucronatus and the absence of B. xylophilus were confirmed by molecular markers. B. mucronatus was isolated from several regions with an average prevalence of 19%. The infestation of M. galloprovincialis by B. mucronatus reached 26.7%. The wide distribution of B. mucronatus in France could have an effect on the extension of B. xylophilus in a case of an introduction.     

Inheritance of long juvenile period under short day conditions for the BR80-6778 soybean (Glycine max (L.) Merrill) line

The long juvenile period characteristic (LJP), which delays flowering under short day conditions, has been identified in soybean cultivars (Glycine max L. Merrill). This characteristic may be especially important as it increases the range of adaptation of soybean in low latitudes and gives greater flexibility for sowing periods within the same latitude. The inheritance of the long juvenile period was studied in the BR80-6778 soybean line to provide knowledge to support the development of cultivars adapted to short day conditions. Cultivars with classic flowering, Paraná, Bossier, Bragg and Davis, which flower early under short day conditions, were also used as parents in single crosses. They were crossed among themselves and with the genotypes with LJP, BR80-6778 and MG/BR 22 (Garimpo). The study was carried out under short day conditions (early sowings) in greenhouses and in the field at Embrapa National Soybean Research Center, Londrina, PR. Flowering was assessed daily. The results indicated that the BR89-6778 line shares a pair of alleles with the Paraná cultivar, and when associated with the cc allele retards flowering under short day conditions. The following genotype constitutions were attributed to the cultivars: Paraná (aaBBCC), Bossier (AabbCC), and BR80-6778 (aaBBcc). The combination of the genes aabb and aabbcc has a pronounced effect on the manifestation of the trait. This revised version was published online in July 2006 with corrections to the Cover Date.     

Single-chain variable fragments antibody specific to <Emphasis Type="Italic">Corynespora cassiicola</Emphasis> toxin,cassiicolin, reduces necrotic lesion formation in <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

Corynespora leaf disease poses a serious threat to rubber cultivation because infected leaves develop necrotic lesions and abscise, leaving the tree unproductive. The destructiveness of Corynespora cassiicola has been largely attributed to cassiicolin, a protein toxin secreted by the fungus. Recombinant antibody technology offers hope to curtail the disease whereby single-chain variable fragments (scFv) specific to cassiicolin could bind and deactivate the toxin in genetically modified rubber trees that harbour the antibody gene. A scFv phage library was constructed from heavy and light variable chains of IgG from cassiicolin immunized Balb/C mice spleen. Biopanning of the phage library yielded a scFv clone with high specificity to cassiicolin. The nucleotide sequence and deduced amino acid sequence information of the scFv were obtained. Hemagglutinin (HA)-tagged scFv expressed in Escherichia coli is discerned as a band at ca. 30 kDa on SDS-PAGE, and the corresponding band was detected by anti-HA IgG on a Western immunoblot. Deactivation of cassiicolin by the affinity-purified scFv was demonstrated in a detached-leaf bio-assay on selected susceptible Hevea clones (PB 260, RRIM 2020, RRIM 901 and RRIM 929). The assay was also performed on clones that are relatively more resistant to the fungus (RRIM 600 and GT-1), and the results were as expected. Thus, we have successfully demonstrated that the cassicolin-specific scFv can effectively reduce cassicolin toxicity.     

Effect of fast protein liquid chromatography fractionated salivary gland extracts from different ixodid tick species on interleukin-8 binding to its cell receptors

Interleukin-8 plays a critical role in inflammatory processes. Hence generation of molecules with anti-IL-8 activity is likely to be important for successful feeding and for survival of the ticks. Anti-IL-8 activity was studied in saliva of three ixodid tick species--Dermacentor reticulatus (Fabricius, 1794), Rhipicephalus appendiculatus Neumann, 1901, and Amblyomma variegatum (Fabricius, 1794). The greatest activity was shown in saliva prepared from D. reticulatus. The activity was attributed to tick salivary gland molecules that bind to IL-8, preventing binding of the chemokine to its specific receptor, rather than to occupation of the IL-8 cell receptor by the tick molecules. The distribution of anti-IL-8 activity in fast protein liquid chromatography (FPLC) fractions of salivary gland extracts (SGE) derived from adult female D. reticulatus, R. appendiculatus and A. variegatum was compared directly by both ELISA and receptor-binding inhibition assays. The correspondence in results with fractions of SGE from ELISA is consistent with detection of tick molecules that inhibit IL-8 binding to its receptor. As IL-8 is an important chemoattractant and activator of neutrophils, the presence of an anti-IL-8 activity in tick saliva indicates that neutrophils play an important role in the host response to parasitism by ticks.     

Irrigation practices may affect denitrification more than nitrogen mineralization in warm climatic conditions

Predicting the impact of irrigation practices on soil N mineralization and N balance is an important issue to optimize N fertilization and reduce the N losses towards the environment. The effect of summer irrigation on N dynamics was investigated in two arable fields in Southern France. Net N mineralization was assessed by combining frequent measurements of water and mineral N contents in soil and the use of a calculation model (LIXIM). It was first calculated assuming that denitrification was negligible. This hypothesis led to inconsistent results, apparent net N mineralized being smaller under irrigated than non-irrigated conditions and net mineralization kinetics being erratic. The occurrence of denitrification was confirmed by the use of ¹⁵NO₃ tracing in an experiment carried out in summer, including three irrigated treatments. The average ¹⁵N recovery varied from 45% to 85% and was smallest in the most frequently irrigated treatment. Over the 8-week experiment, the N losses varied from 30 to 38 kg ha⁻¹ in the irrigated treatments. They were satisfactorily simulated by a simple denitrification model (NEMIS). Combining the LIXIM model and the simulated or calculated denitrification allowed to predict satisfactorily the evolution of soil mineral N accounting for the effects of temperature and moisture. The net N mineralized for 8 weeks varied from 34 kg N ha⁻¹ in the un-irrigated to 46 kg N ha⁻¹ in the irrigated treatments. The drying–rewetting cycles did not induce a flush of N mineralization. Our results suggest that denitrification has to be accounted for in irrigated systems, particularly in warm conditions and when the topsoil contains high nitrate contents.     

The complex etiology of epidemic diseases in calves on large-capacity farms and their clinical and epizootic characteristics

It is not easy to exactly diagnose the etiology of the mass infections of new-born calves on large farms where considerable losses are suffered. On the basis of the complex epizootological, clinical and laboratory examination in four large calf-rearing facilities, rotaviruses, coronaviruses, the infectious bovine rhinotracheitis (IBR) virus and the bovine viral diarrhoea (BVD) virus, and in some cases also the enteropathogenic E. coli, were found to be etiologically involved in the mass rise of diarrhoea, complicated by respiratory symptoms already during the first days after birth. The clinical picture of the disease, therapeutically difficult and reminding of "pneumoenteritis", has often been observed in stocks where, in addition to rotaviruses and coronaviruses in the faeces, the IBR or BVD viruses (sometimes both at the same time) were detected and identified in the respiratory and enteral tract. The serological examination of a higher number of animals in the stocks of calves under study confirmed the considerable rate of spreading of all the four viruses in the cattle population and, at the same time, demonstrated the very unfavourable immunological profile of the herds. The high percentage of animals low in antibody titres and the serologically negative animals constitute the infection-sensitive part of population in the affected herds. With the high culling rate and with the open herd turnover it is impossible to reach the required immunity through natural disinfection. Loss-free rearing of healthy calves will be achieved on the basis of a well-oriented vaccination programme with a good combination of inoculants.     

Measuring agreement and discord among hemagglutination inhibition assays against different ophidian paramyxovirus strains in the Eastern massasauga (Sistrurus catenatus catenatus)

At present, the hemagglutination inhibition (HI) assay is the sole commercially available serologic method available to detect exposure to ophidian paramyxovirus (OPMV) in snakes. During 2006, 26 eastern massasaugas (Sistrurus catenatus catenatus) were collected, and blood was sampled to determine their OPMV status. Samples from each snake were divided into 3 aliquots and tested by using commercially available HI assays against the 4 OPMV isolates used in the 3 laboratories that offer the service. All snakes were tested for antibodies by using HI assays against the green tree python (GTP), San Lucan rattlesnake (SLR), and Aruba Island rattlesnake (AIR) isolates. Twenty-five snakes were tested for antibodies against the western diamondback rattlesnake (WDR) isolate. All samples tested against the GTP and SLR were positive (26/26), whereas 56% (14/25) of the WDR assays were positive, and none (0/26) of the AIR assays yielded a positive result. There was 100% agreement between the GTP and SLR assays, and complete disagreement between the SLR and AIR, as well as the GTP and AIR assays. Kappa statistics for the GTP-WDR, SLR-WDR, GTP-AIR, SLR-AIR, and WDR-AIR indicated that the assays had less than chance agreement. The results demonstrate that current HI assays are not reliable as a sole diagnostic assay in the eastern massasauga. Furthermore, HI assays need to be evaluated by using other parameters to determine OPMV exposure in eastern massasaugas.