JWSS - Journal of Water and Soil Science

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In modern programmes of animal breeding, the polymorphisms of the milk proteins can be used as marker systems. Beta-Lactoglobulin is the major milk whey protein in the ruminants. The BLG coding gene located on ovine chromosome 3. This protein, synthesis in the mammary glands during pregnancy and the lactation stages. Studies have indicated that this protein is polymorphic in the many breeds of sheep. This is the result of a single base pair substitution in the Beta-Lactoglobulin gene that also rises to the RsaI restriction fragment length polymorphism (RFLP). The aim of this work was to analyze the genotype distribution of Beta-Lactoglobulin in sheep. Blood samples were supplied from 142 sheep of the 5 breeds (Ghezel, Afshari, Moghani, Makoii and Arkharmerino). Genomic DNA was extracted from the 200ul blood sample according to Boom et al. (1989) method modified by Shaikhayev (1995). The Gel monitoring and the spectrophotometeric methods were used for determination of the DNA quality and quantity. the Primers BLG5 and BLG3 amplified a 452 bp fragment from the exon II of the ovine Beta-Lactoglobulin gene. the Products of the amplification were recognized by the electrophoresis on the 1% agarose gel stained with ethidium bromide. The RsaI enzyme was used for restriction of the PCR products. The digested products were separated by the electrophoresis on the 8% nondenaturant polyacrylamide gel and visualized after staining with the ethidium bromide on UV transillumination. The popGen32 software (ver.1.31) was used to estimate the allele and the genotype frequencies, the Hardy-Weinberg equilibrium and dendrogram of the genetic distance. The frequency of A-allele in Ghezel, Afshari, Moghani, Makoii and Arkharmerino breeds was 56%, 34%, 36%, 53% and 48% respectively. The populations were in Hardy-Weinberg equilibrium except to Afshari breed. The lowest genetic distance was observed between Moghani and Afshari breeds and the highest genetic distance between Ghezel and Afshari breeds. The results of this study indicated that PCR-RFLP is an appropriate tool for evaluating genetic variability in sheep.

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To carry out this study, total DNA was extracted from eggs and from second stage juveniles of several populations of Meloidogyne javanica and Meloidogyne incognita, using phenol / chloroform method. Following extraction, DNA was electrophoresed on 1% agarose gel to determine its quality and quantity. A specific primer pair (C₂F₃ / 1108 23 and 20 nucleotides, respectively) was used to discriminate M. javanica from M. incognita populations using polymerase chain reaction (PCR). Primer annealing sites were located in the 3′ portion of mitochondrial gene encoding cytochrome oxidase subunit II (COII) and in the 16S rRNA gene. Following PCR amplification, electrophoresis of amplified DNA showed 1.7 kb fragment in populations of both species. Digestion of 1.7 kb amplified product with HinfI restriction endonuclease resulted in the generation of two DNA fragments of 0.7 and 1.0 kb in M. javanica and three DNA fragments of 0.3, 0.4 and 1.0 kb in M. incognita. There were no differences in the digestion patterns among various populations of each species examined.

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Cucumber cultivars, grown in greenhouse in the Jiroft region, were surveyed for the relative incidence of Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus-2 (WMV-2), Cucumber mosaic virus (CMV) and Tomato spotted wilt virus (TSWV) from 2001 to 2004. Samples from 1294 plants representing different cultivars were analysed by Enzyme-linked immunosorbent assay (ELISA) and Dot immunobinding assay (DIBA). The data showed that green-house cucumbers are infected by CMV, ZYMV, TSWV and WMV-2. However, ZYMV was the most prevalent virus. Mixed infection including double and triple infection was identified in some samples. Transmission of aphid-borne viruses (CMV, ZYMV and WMV-2) by Aphis gossypii, A. fabae, A. craccivora and Myzus persicae revealed that ZYMV is most efficiently vectored by these aphids and, A. craccivora transmitted these viruses with more than 60% efficiency. In addition, WMV-2 was not transmitted by A. gossypii. In RT-PCR, ZYMV infection was confirmed by amplifying a PCR product of the predicated size 458 bp, using total RNA extracted from infected plants. All ZYMV infected samples reacted with monoclonal antibodies (705-1, 705-2 and 705-4) in TAS-ELISA test. These results showed that ZYMV isolate collected from Jiroft belongs to group A, cluster 1 or 2. In electron microscopy study, normal length of ZYMV flexuous particles in partial purified preparation was calculated as 790 nm. The molecular weight of coat protein of ZYMV was estimated at 36 KDa., using SDS-PAGE and western blotting. This is the first report of these viruses in greenhouse grown cucumber in the Jiroft region.

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Fire blight caused by Erwinia amylovora, is a serious disease of pome fruits in many areas in the world which causes evaluative necrosis. Indeed, E. amylovora can invade the whole tree solely by internal progression through the host tissues. In this research, symptoms of necrotic shoots and exudates production on infected pear trees in different areas of Guilan province (Astaneh Ashrafieh, Lahijan and Kiashahr) were surveyed. Samples were taken from infected tissues of diseased trees. For isolation of bacterial causal agent, the infected tissues were crushed in pepton water, then 100µl of the extracts were cultured on Sucrose Nutrient Agar (SNA) and Luria Berthani (LB) containing Cyclohexamid antibiotic (50 µg / ml). The isolated bacteria were rod-shaped , gram negative and facultatively an-aerobic. The bacteria produced Levan on media including sucrose , but could not produce fluorescent pigments on King’s B medium. All strains made hypersensitive reaction on tobacco leaves. All isolates were oxidase , nitrate, urease and indole negative and were not able to rot potato tuber slices, produce H₂S and grow in 36 °C. The isolates could use citrate, acetoin, sorbitol and trehalose and their gelatin test was positive. Based on morphological, biochemical, physiological characteristics and production of a 937 bp with specific primer Ea1 and Ea2 in PCR method, the strains were identified as E. amylovora. This is the first report of the existence of this bacterium on pear fruit trees in Guilan province.

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Studying genetic diversity is important because a decrease in genetic variability might result in a reduction of the plasticity of the crops to respond to changes in climate, pathogen populations, or agricultural practices. In this study, 72 Sardari wheat (Triticum aestivum L.) ecotypes were analyzed by AFLP markers and 17 phenotypic characters. Three pairs of EcoRI/MseI primer combinations produced 1582 polymorphic bands (with mean percentage of polymorphic 73.92%). Cluster analysis using Jaccard coefficient and the entire AFLP data divided all ecotypes into eight major groups. Mean, coefficient of variation, phenotypic, genotypic and environment variance were calculated in each quantitative character. Cluster analysis using Euclidian distance through the quantitative characters divided all ecotypes into six major groups. Comparison of genetic distances obtained from AFLP and agronomic data showed low correlation between the two diversity measurements (0.02). The results showed a high degree of genetic diversity between the Sardari ecotypes, suggesting that Sardari is not a single cultivar, but it is the mass of ecotypes and could be introduced in the gene bank.

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To characterize the geographical distribution of medicago-nodulating rhizobia in western regions of Iran, 950 Sinorhizobium isolates were trapped from a combination of two local alfalfa populations (Hamedani, Nikshahri) together with a foreign cultivar ( Kodi) and soil samples from eight sites across Kurdestan, Kermanshah, Eastern Azarbayjan and Lorestan provinces. Also, a total of 45 isolates were obtained from nodules of naturally grown Melilotus officinalis (14 isolates) and Trigonella foenum-graecum (31 isolates) plants in Isfahan. On the basis of PCR partial amplification of the plasmid born nod box gene and chromosomal mucR gene of the isolates,16S ribosomal DNA PCR-restriction fragment length polymorphism analysis, and the nucleotide sequence, three isolates from alfalfa, seven isolates from M. officinalis and 13 isolates from T. foenum-graecum were proved to be Sinorhizobium medicae. The remaining isolates (943 from alfalfa, seven from M. officinalis and 18 from T. foenum-graecum) were identified as S. melilloti. Both species, S. meliloti and S. medicae, were recovered from nodules of all the hosts although S. meloti was clearly more dominant in nodulating different populations of alfalfa. Taken together, these results indicated that the abundance of S. meliloti is independent of the site of isolation and have a wide geographical distribution. In this study, the banding pattern resulting from PCR amplification of 16S rRNA gene, followed by digestion with Rsa I, clearly differentiated S. meliloti and S. medica strains, showing that PCR-RFLP is an appropriate method to discriminate medicago-nodulating rhizobian with relative rapidity.