BLACKLEG in cattle has been recognized since 1938 in Iran that mostly affects cattle in enzootic farm. Main object of this study was to prepare and formulate a concentrated potent vaccine for immunization of cattle against BLACKLEG in Iran. Experimental concentrated BLACKLEG vaccine was prepared according to the method described by FAO. The medium (Modified medium for production of experimental C. chauvoei vaccine by fermenter) consisting of peptone, glucose, sodium chloride, cysteine hydrochloride and yeast extract was prepared by fermenter and inoculated by Clostridium chauvoei strain for preparation of BLACKLEG vaccine. Aluminum hydroxide gel adjuvant was added to the high yield vaccine. The vaccine was also concentrated by the method of precipitation. None of tested animals showed any local or general adverse reactions. All of vaccinated guinea pigs resisted the challenge with 4 MLD of virulent C. chauvoei.

BLACKLEG disease of rapeseed, which is caused by Leptosphaeria maculans (Phoma lingam), is considered as one of the destructive diseases of crucifera family and has resulted in total yield loss in some locations. It appears as an endemic disease in many areas of the world, especially in rapeseed fields. In this study, the response of six winter cultivars of rapeseed to the two isolates of the fungus (Aliabad and Bandar Torkman) was evaluated. Plants were inoculated in different growth stages: cotyledon, and stages, 2.3-2.4, 2.1, and 3.2, using spore suspension under 23±1°C on the basis of established procedures for a successful infection. Inoculated plants were kept under plastic covers for 48 hours in order to provide a 100% relative humidity. After inoculation period, disease severity was assessed in cotyledon stage by the method of William and Delwich, in stage 3.2 by McNabb and Rimmer, and in stage 5 on the basis of Newman and Bailey, and also Mc Nabb and Rimmer. It was revealed that responses of Okapi and SLM046 to disease were homogenous in all stages. Okapi was recognized more susceptible and SLM046 more resistant than other cultivars under study. Talaych, Regent × Cobra, Fomax and Ceres which showed different reaction during different stages of inoculation, indicated the heterogenicity of response to BLACKLEG disease in these cultivars.          

In all biological systems growth is defined as increase of chemical compounds. Bacteria can achieve to balanced growth if they are growing in a medium, which are completely adapted to it.Clostridium chauvoei, (Clostridium feseri) is an anaerobic, spore forming, motile, and polymorph bacteria, which its size varies from 0.5-1 to 3-8 micron and could be observed as individual bacterium, diplo, and rarely streptococcus. BLACKLEG is a fatal disease of young cattle. It produces an acute local infection, and the resulting blood poisoning leads to rapid death. Clostridium chauvoei and, less frequently, Clostridium septicum are the most commonly responsible organisms. Vaccination is the only effective means for controlling of BLACKLEG disease. Several kinds of vaccine are available commercially. It is 4 decades that BLACKLEG vaccine is produced in Razi institute and because of enhanced demand of country, decision was made to improve the production procedure of this vaccine using large-scale fermenter, so the aim of this study was adaptation of Clostridium chauvoei to growth and proliferation in fermenter for preparation of a potent vaccine. Accordingly attempts were made to prepare and formulate the ingredients in order to obtain high yield of Clostridium chauvoei in culture medium by fermenter. All experiments were done in two sets: A-growth in glass bottles using conventional culture medium and B-growth in fermenter using conventional culture medium similar to A and also enriched culture medium. Results showed high yield of Clostridium chauvoei suspension in fermenter after 10 hours, using enriched culture medium (more than 1, 480, 000, 000 organisms/ml), but no significant changes was obtained in glass bottles conditions comparing to the fermenter conditions. The safety and potency of the prepared vaccine was determined in sheep and guinea pigs according to British pharmacopoeia (veterinary) with satisfactory results. Since this research has been successfully done in Razi research institute, so the mono valent inactivated BLACKLEG vaccine, using the enriched culture medium is currently producing by fermenter and is used for immunization of cattles in Iran.

For detection and identification of the causal agents of potato BLACKLEG in Isfahan, the potato fields were surveyed during 2003-2004 growing seasons. Fifty four strains of Pectobacterium and Dickeya were isolated from the diseased stems and tubers. According to their biochemical and physiological test results, 43 isolates were identified as D. chrysanthemi. The remaining 11 isolates were placed in the second group which were identical with P. carotovorum subsp. carotovorum in biochemical characteristics. None of the isolates appeared to be P. atrosepticum. The results were confirmed by polymerase chain reaction technique using the recommended specific primer sets. Use of G1/L1 primer pair made it possible to detect Pa, Pcc and Dch in one PCR reaction. By using this primer set, the ITS regions of the bacteria were amplified. On the basis of the banding patterns obtained with G1/L1 the isolates were placed in three groups. The first group of ITS- PCR comprised only the standard isolate Eca SCRI1043. Isolates which were identified as Pcc by biochemical tests were placed in the second group and the third group included isolates that were identified as Dch on the basis of their biochemical and physiological characteristics. Results of the present study indicate that Pcc and Dch are the predominant causal agents of potato BLACKLEG and soft rot in Isfahan.

BLACKLEG caused by Leptosphaeria maculans is one of the important diseases widely affecting canola production worldwide. Effectiveness of three plant defence related genes transferred from pea (Pisum sativum) to canola was tested against BLACKLEG in two experiments. In the first experiment, three transgenes including PR10.1, Chitinase, and DRR206 were transferred from transgenic Wester variety into four commercial cultivars (Apollo, Sentry, OAC Triton, and MillenniUM 03) via backcrossing to examine the effects of different genetic backgrounds on disease response. In the second experiment, to study the effect of gene pyramiding on level of disease resistance, three transgenic Wester lines were crossed. Cotyledon inoculation was performed for indoor disease screening tests of BLACKLEG. DRR206 in Sentry and Chitinase×DRR206 appeared to be the best combinations conferring disease resistance. Overall, DRR206 had the highest impact on disease, probably due to delayed infection development. Promising lines of this research could be utilized in breeding programs and cultivar release projects after field trials.

Many isolates of Trichoderma spp. and Bacillus subtilis are natural biological agents of plant pathogenic fungi and bacteria. Trichoderma spp. and B. subtilis isolates were separated from the rhizosphere of infected and non-infected rapeseed plants. Phoma lingam, the causal agent of rapeseed black leg, was isolated from different tissues of infected plants, and infested soil. Dual cultures were used to examine the antagonistic effects of Trichoderma and B. subtilis isolates on P. lingam. In the greenhouse, the antagonistic effects of the isolates were tested through treating rapeseed seeds and rapeseed above-ground parts with the isolates. Totally, 20 isolates of P. lingam, 15 isolates of Trichoderma species and 17 isolates of B. subtilis were separated. Bacterial isolates BE3, B31, B66, B67, B68, B69, and B70, and Trichoderma isolates Tr.2901, Tr.2903, Tr.2904, Tr.2910, and Tr.2913 showed antagonistic effects on the pathogen. The greenhouse results revealed that in the seed treatment experiment, the bacterial isolates B70 and B67, and Trichoderma isolates Tr.2910 and Tr.2901 decreased the rapeseed BLACKLEG to 43.3, 80, 16.6 and 56.6%, respectively. Treating the above-ground parts with bacterial isolates did not reduce rapeseed BLACKLEG at all. Treating above-ground parts by Tr.2910 and Tr.2901 isolates of Trichoderma decreased the disease to 60 and 79.6%, respectively. Trichoderma isolates belonged to several species where the differentiation of the isolates from species was impossible based on the morphological characteristics. The two effective bacterial isolates were related to B. subtilis.

Numerous species of bacteria employ a mechanism of intercellular communication known as quorum sensing (QS). Amongst protobacteria, several functions including production of virulence determinants in Pectobacterium atrosepticum are regulated in this way. The signal molecules involved in quorum sensing inP. atroseptium belong to the group of N- acyl homoserine lactones (N-AHLs). In this research, ten strains of AHL degrading Rhizobacteria were isolated from potato rhizosphere. According to ribotyping analysis, these isolates fell into three groups which members were identified as genera Bacillus, Arthrobacter, and Pseudomonas. All these isolates were capable to degrade both synthetic and natural N-AHL produced by P. atrosepticum strain SM1. In biocontrol experiment, Bacillus sp. and Arthrobacter sp. were significantly reduced the pathogenicity of P. atrosepticum strain in potato tubers and totally suppressed tissue maceration and BLACKLEG symptoms on potato plants. Out of these results, AHL degrading bacteria might be highlighted and considered as useful agents in the biocontrol of bacterial plant diseases.

Canola stem canker or BLACKLEG, caused by Phoma lingam is a disease of world-wide importance on oilseed rape. Pathogenicity of ten isolates on Hayola 401 cultivar of canola under greenhouse conditions was studied and the most virulent isolate with 17% disease severity was selected for cultivars resistance studies. The relative resistance of ten cultivars namely Hayola 401, Hayola 308, Talaye, Zarfam, Sarigol, Fornax, Modena, Okapi, Option 500 and RGS 003 to P. lingam was studied in greenhouse conditions, using randomized complete block design with three replications. Disease severity was assessed ten days after inoculation with pathogen, by measuring the chlorotic and necrotic area in leaves by means of leaf area meter apparatus. Analysis of variance indicated significant differences among the cultivars in resistance to this fungus (P£0.01). According to mean disease severity in inoculation experiments the cultivars were placed into four groups. Sarigol and Hayola 401 with disease severity of 19.35 and 17.5 percent were the most susceptible cultivars. In contrast, Fornax and Talaye with disease severity of 5.86 and 5.82 percent were the least susceptible cultivars. Other cultivars were of intermediate susceptibility. This is the first study on evaluation of relative resistance of some commonly grown canola cultivars in Iran to a virulent Iranian isolate of P. lingam.

Cell-surface proteins of Clostridium chauvoei were purified using a simple method. Bacterial cultures were centrifuged and agitated vigorously in phosphate buffered saline with or without further glycine treatment and ammonium sulfate precipitation. Rabbits were immunized subcutaneously with a BLACKLEG disease vaccine twice with a two-week interval. Immunized sera were collected one week after the second injection. Enzyme-linked immunosorbent assay (ELISA) was performed using the proteins purified by the second method as the coating antigen. Bradford assay results showed a higher protein concentration in the second than the first method. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis showed multiple bands for the cell-surface proteins of C. chauvoei in the first method and a sharp band equivalent to flagellin protein in the second method. The ELISA results indicated that the purified proteins were capable of detecting antibodies against BLACKLEG disease vaccine. The purified protein would be an alternative antigen for indirect ELISA in order to monitor the immune response in vaccinated farm animals.