Antigenic characteristics as taxonomic criterion of differentiation of Alternaria spp., pathogenic for carrot and parsley

Identification of Alternaria genus species is a very complicated process which demands broadly designed investigations and studying of great number of properties which together can be considered as satisfying taxonomic criteria. The main objective of these investigations was examining the possibilities of applying the antigenic characteristics of Alternaria spp. phytopathogenic fungi as a taxonomic criterion, as well as introducing the serological methods for their identification. Conducting the examination of Alternaria spp., pathogenic for Apiaceae plants in Serbia, several isolates were obtained and identified as Alternaria radicina, A. petroselini, A. dauci and A. alternata, based on the conventional mycological methods and host range, as well as on molecular detection and partial characterization. The investigation included 12 isolates from plant leaves, seeds and soil which were pathogenic mainly to carrot and parsley and were identified as A. radicina, A. petroselini, A. dauci and A. alternate. Investigated isolates were compared with each other, as well as with standard isolates for the mentioned species (a total of 5 isolates, originating from USA and EU). During the investigation of serological characteristics of Alternaria spp. firstly a polyclonal antiserum was prepared against one isolate from Serbia identified as A. dauci. This antiserum was specific to Alternaria genus while there was no reaction with antigens from other phytopathogenic fungi genera (Fusarium, Rhizoctonia and Agaricus). Antiserum titer, determined by slide agglutination test, was 1/32. Antigenic characteristics of Alternaria genus fungi were examined by Electro-Blot-Immunoassay serological method (EBIA, Western blot), i.e. their protein profiles were compared. Investigated Alternaria spp. isolates showed different protein band profiles in gel and on nitrocellulose paper, and the observed differences were in complete correlation with the results of the previous identification. All investigated isolates, both domestic and the standards, were similar to each other, and they could be correctly identified to the species level using EBIA. Besides grouping to the species level, antigenic characteristics indicated similarities and differences among the isolates within the same and different species, showing their complex relationships which properly reflect their diversity in nature. In all the previous investigations of Alternaria genus fungi up to now, there have been no data about their serological characteristics as possible taxonomic criteria. Introduction of this group of characteristics represents an important contribution both to the taxonomy and implementation of fast and accurate methods of phytopathogenic fungi identification.

Low cost production and purification of polyclonal antibodies to staphylococcal enterotoxin A

An immunization scheme for production of antiserum to staphylococcal enterotoxin A (SEA) is proposed. The reference method of Robbins and Bergdoll was modified to reduce the number of doses and the amount of toxin used per animal. The best immunization scheme used injections in days 0, 8, 24, 59, 62 and 67. The amount of toxin at each injection was 5, 6, 20, 50, 100 and 200<FONT FACE="Symbol">m</font>g, respectively. The total amount of toxin was 381<FONT FACE="Symbol">m</font>g, which corresponded to a reduction of 107<FONT FACE="Symbol">m</font>g in the amount of toxin for each animal when compared to the reference method. The average antiserum titer using the Optimum Sensitivity Plate - OSP was 1:60 and using ELISA the titer was 1:100.000. The lack of cross-reactivity with other staphylococcal enterotoxins indicated high specificity of the antibody to SEA. The proposed immunization scheme was adequate to produce specific SEA antisera, with high titers and the aditional advantage of reducing the amount of purified SEA required for immunization.

Banana Die-Back Virus - a New Virus Infecting Banana in Nigeria

Two viruses naturally infect Musa in Nigeria: banana streak badnavirus (BSV) and cucumber mosaic cucumovirus (CMV). During a recent field survey at Ibadan (Nigeria), some severely stunted banana plants (cv. Valery) were found that tested negative for CMV, banana bunchy-top virus, and BSV. The plants had symptoms of leaf crinkling, leaf necrosis, and cigar-leaf die-back. Subsequent suckers from the same mats were progressively more stunted. A 28- to 30-nm isometric virus was purified, and used for the production of antibodies, from the affected plants with (NH4)2SO4 to precipitate the virus. The antiserum (titer of 1:10,000) was used in enzyme-linked immunosorbent assay and immunosorbent electron microscopy to detect the virus. Mechanical inoculation with partially purified virus preparations resulted in stunting and development of pinpoint chlorotic lesions on Vigna unguiculata TVu-76 and symptomless systemic infection of Nicotiana occidentalis. The virus was not mechanically transmissible from N. occidentalis to banana. A serological relationship between this virus, banana die-back virus (BDBV), and tobacco ringspot, tomato ringspot, and cacao necrosis nepoviruses was found. The nematode species around the affected banana plants were isolated: Helicotylenchus multicinctus (Cobb) Golden was the dominant species, low numbers of H. dihystera (Cobb) Sher were present, but no virustransmitting nematodes were found in soil or banana roots. Further studies are needed to determine the mode of spread of BDBV, the implications for banana/plantain production in sub-Saharan Africa, and the safe international movement of germplasm.

Method for testing antiserum titer and avidity in nephelometric systems.

Antiserum performance in a nephelometric system can be characterized by parameters derived from measuring reaction rates. The characterization process is derived from a series of dose-response curves (elicited nephelometric response vs antigen concentration) generated from various dilutions of the antiserum being tested. Antiserum titer can then be calculated by plotting the antigen concentration found at one-half the maximum nephelometric response (Hmax) of each dose-response curve (C50) vs the corresponding antiserum dilution. Antiserum avidity can be calculated by plotting Hmax against its corresponding antiserum concentration. After general expressions are determined for C50 and Hmax vs antiserum concentration, a single dose-response curve suffices for characterizing antisera with respect to titer and avidity. Direct evidence is provided for the validity of C50 and Hmax as measures of titer and avidity by correlating these parameters with antiserum binding strength and with the number of antibodies eluted from immobilized antigen. This method can be applied to evaluate and compare different antiserum lots having the same specificity, to identify reagent inadequacies by comparing antisera of different specificity, and to predict the optimal antiserum dilution to use in performing an assay.