Study the Stability of SSR Repeats of pEA29 Plasmid of the Casual Agent of Fire Blight

Introduction Fire blight disease causes by Erwinia amylovora infects a wide variety of rosaceous plants. It was first recorded from pear trees in Karaj in year 1990. After that it was observed in many pear and apple orchards of the country. E. amylovora isolates differed slightly in virulence, symptoms and host range which ca be related to different plasmid content. The presence of an universal plasmid, pEA29, has been observed in majority of E. amylovora strains. Short-sequence DNA repeat with eight nt were repeated 3 to 15 times in the PstI fragment of the pEA29 plasmid. Here, the stability of SSR units and efficiency of this method to categorize strains was checked. For this reseaon two methods including amplification and cloning of whole and part of PstI fragment was done and the sequences were compared to eachother. In addition stability was evaluated based on three treatments including long time propagation, keeping strains in cold situation and re-isolation of infected tissues. Materials and Methods In this study 20 strains were purchased from the Iranian Plant Protection Research Institute. Their typical phenotypic tests were examined for all strains. All of then were checked with lateral flow immune chromatography in different serial dilutions. The pathogenicity were aassayed using complete fruit. Direct PCR with A/B primers and nested PCR with AJ75/AJ76 were applied for studied strains. Ten representative strains were selected snf part of PstI fragment amplified using RS1/RS2 primers. The PCR products were purified by QIAquick PCR purification kit (Qiagen, USA), cloned in pGEM-T and were sequenced (Macrogen Inc., Korea). Five of 10 were chosen for stability tests including long time propagation and sub culturing each two week for three months, keeping strains in refrigerator for three months and re-isolation of infected tissues. Results and Discussion In phenotypic tests all studies strains were facultative anaerobic growth, oxidase negative, catalase positive and non fluorescent on King's B. The biochemical tests for reduction of nitrate, tween 20 and growth at 36 °C were recorded negative. All E. amylovora strains induced the hypersensitive reaction (HR) on tobacco and pelargonium leaves. All phenotypic tests were agreed with standard references. There is no variation in lab expermints. Pathogenicity assay were checked using immature pear fruit in two separate treatments. Inoculation caused water soaking, tissue necrosis and sometimes necrosis in pear samples. No symptoms were observed in the negative controls. There is no variation in this assay. It seems that in most cases, the pEA29 plasmid can modify synthesis of amylovoran, levansucrase and finally affect pathogenicity in the host plant with respect to the environment. All strains were check by agri strips (Bioreba, Switzeland, Reinach) in lateral flow immune chromatography to confirm presence of E. amylovora. All strains of E. amylovora amplified a fragment of the expected size using primers A and B. The accuracy of the direct PCr was evaluated by nested PCR using primers AJ75/AJ76. The PCR products were visualized after electrophoresis on 1.2% agarose gels. A 3kb DNA ladder (Fermentas, Lithuania) was used as a molecular size marker in all experiments. In order to chracterize SSR units in thi study, part of PstI fragment was amplified by RS1/Rs2 primers. The relevant variability found in the length of the this fragment was explained by a variation in the number of copies of a SSRs of 8 bp, GAATTACA. It is recorded 4 and 8 times in apple and rose plants respectively. Recording the SSRs of 4, 5 and 7 in other hosts including pear, hawthorn and quince may be indicated that under natural conditions, a mixture of E. amylovora strains with different SSR numbers can be caused fire blight. Frequent transffering of bacterial isolats to new nutrient agar medium did not change the SSR units ater three months. Among three testd tretments, keeping in cold weather for at leat three months caused variation in repetitive units in IrGh59 isolates. According to SSR stability some researchers believed that mutation, antibiotic treatment, maintaing in specific suitations may changed the numbers of this units. But our results showed that SSR numbers of several strains remained constant under laboratory conditions. Conclusion Sequencing the whole PstI fragment may provide better information about SSR units and the flanking regions. The numbers of SSR are stable under experimental conditions and evaluation of E. amylovora isolaes with this method can apply for strain grouping

Problems Encountered with Nested PCR to Diagnosis of Phytoplasmas

Introduction: Phytoplasmas are phytopathogenic bacteria without cell walls that can be found in plant phloem, have been found associated with numerous diseases of plants worldwide. A sensitive and precise diagnostic test for detection of phytoplasma-infected plant is critical to avoid using infected planting material and dispersal of these agents. The detection of phytoplasma from plant tissues by PCR, requires using DNA extraction methods that extract high level phytoplasmas DNA with less plant inhibitors. Usually Phytoplasma diagnostics have been based on the 16S rRNA gene and the 16S–23S rRNA spacer region because universal primers that design for replication these regions could detect different groups of phytoplasmas but diagnostics based on these primers can be problematic, with occasional false positives, through amplification of some bacterial genomes that might be present in a plant sample. These primers also have sequence homology to chloroplasts and plastids and increase the risk of false positives so it is important to guard against false negatives and positive during such detection techniques. Materials and Methods: Health and infected Lime samples with Candidatus phytoplasma aurantifolia were used respectively as negative and positive samples. DNA was extracted by the CTAB methods, SDS method, Fermentas DNA extraction kit, column based Method (Genet Bio genomic DNA isolation kit) and compared to remove inhibitors and reduced false negative reactions in nested PCR detection method. DNA samples were tested for phytoplasma infection by direct PCR using the universal phytoplasma primer pair P1/P7 and nested PCR using primer pairs P1/P7-R16F2n/R16R2 and P1/P7-fU5/rU3. The PCR products were sequenced and subsequent analysis using GenBank database information at the national center for biotechnology was employed. Results and Discussion: Comparison of different DNA extraction methods indicated using suitable method can significantly reduce false negative reaction, but even in successful column-based DNA extraction method, false negative reactions were reported that were due to low phytoplasma concentration and irregular distribution within host tissues or could be caused by inhibitor presence in DNA samples. Based on results of sequencing, false positives were obtained sporadically, using primer pairs combination P1/P7- R16F2n/R16R2 that may be arising from cross over contamination or sequence homology with plant genome, so some primers can react probably with sequences of plant genome and false positives could be observed. Since false positives are also a major problem in PCR protocols, especially in nested PCR so single tube nested PCR (STNP) was optimized to avoid false positive reaction but regardless advantages of this method such as facility, cost and time effective and ability to detect low concentration of pathogen, false positive reactions were observed in a few samples. The advantage of STNP is that tubes do not have to be opened, so the risk of contamination minimized. Conclusion: PCR-based techniques for phytoplasma detection, appears to be the method of choice because of their high sensitivity and specificity. Using suitable method for extraction of DNA from infected plant tissues are getting more critical for precise detection through increasing DNA quality and quantity. In the other hand confirmation of phytoplasma presence must be accomplished at least by RFLP analyses or different primer pair combination to avoid false positive detection

Identification of Plant-parasitic Nematodes in Cotton Fields in Southern Khorasan Province

Introduction: Gossypium species are distributed in the arid to semiarid regions of the tropical and subtropical, generally shrubs or shrub-like plants. The leaves are broad and lobed with three to five lobes. The seeds are contained in a capsule called a "boll", each seed surrounded by fibers. Commercial species of cotton plant are Gossypium hirsutum (>90% of world production), G. barbadense (3-4%), G. arboreum and G. herbaceum (together, 2%). Cotton is grown as a cash crop; it is often grown in a monoculture system that favors the development of a nematode community dominated by one or a few parasitic species. The different genera of plant parasitic nematodes such as Haplolaimus, Helicotylenchus, Blanolaimus, Pratylenchus, Paratrichodorus, Meloidogyne, Merlinius, Xiphinema, Tylenchorynchus, Rotylenchulus, Scutellonema, Heterodera, Ditylenchus, Longidorus, Aphelenchoides, Aphelenchus were recorded from cotton fields in the world. The objective of this study was to investigate the plant parasitic nematodes associated with cotton fields in Southern Khorasan province of Iran. Materials and Methods: In order to identify the plant parasitic nematodes in cotton fields in Southern Khorasan province, 56 soil samples were collected from different areas during 2013 and 2014. Soil samples were washed and nematodes were extracted by combined sieving and centrifugal-flotation method of Jenkins (1964) and Whitehead tray method (1965). Nematodes were fixed and transferred to glycerin by using the method of De Grisse (1969). The permanent slides were prepared and the nematodes were studied by light microscope. Results and Discussion: In this study, 15 species from 10 genera related to order Tylenchomorpha, were identified as follows: Aphelenchus avenae, Basiria graminophila, Boleodorus clavicaudatus, B. pakistanensis, B. thylactus, Ditylenchus hexaglyphus, D. tenuidens, D. valveus, Geocenamus rugosus, Filenchus vulgaris, Pratylenchus neglectus, P. thornei, Scutylenchus quadrifer, Merlinius brevidens, Zygotylenchus guevarai. One genus of cotton-parasitic nematodes known to cause yield loss were found in this survey (Pratylenchus spp). Other parasitic nematodes not known to cause yield loss also were found. Three species, including Boleodorus pakistanensis, Ditylenchus hexaglyphus, D. valveus are reported as new records for Iran. Boleodorus pakistanensis is characterized by four incisures, head low, unstriated and conoid shape, stylet 8.5-10µm with cone short and slender knobs, non-muscular median bulb, Basal bulb pyriform, posterior vulva (v=68.1-72.4 ; v/=77.5-88.4). Tail elongate-conoid, central arcuate, ending in a finely rounded terminus. B. pakistanensis resembles, B. flexuosus B. teres, B. thylactus and B. cylindricus. B. thylactus but can be differentiated by the more posterior position of the opening of the dorsal gland (2 vs 3-5); B. flexuosus has a more anterior vulva (V=54-63 vs 68.1-72.4); B. teres has a more anterior vulva (V=54-63 vs 68.1-72.4), tail shape and stylet length (10-12 vs 8.5-10); B. cylindricus has a more anterior position of the opening of the dorsal gland (7 vs 3-5) and more tail (88-101 vs 72-88.5). Ditylenchus hexaglyphus is characterized by six lines in lateral fields, low head, stylet 8-9µm with cone shorter than the shaft and small elongated knobs, non-muscular median bulb, Posterior bulb offset, posterior vulva (v=82.3 -83.5; v/=87.8-89.6). Tail ventrally curved, terminus rounded. Having non-muscular median bulb, six incisures, short stylet and posterior vulva. D. hexaglyphus resembles D .medians, D. taylori, D. affinis, and D. tuberosus. However, none of these species have such small stylet knobs, and all but D. affinis have longer PUS than that observed in D. hexaglyphus. D. taylori more anterior vulva (V=75-77 vs 82.3-83.5) and tail appears to be thinner (c/=6-7 vs 2.9-3.8); D. affinis has a more anterior vulva (V=76-80 vs 82.3-83.5). Ditylenchus valveus is characterized by six incisures, annulated head, stylet 8-9µm with short cone and small rounded knobs, muscular median bulb with small. Posterior bulb offset, posterior vulva (v=74.3 -81.5; v/=84.9-89.2). Tail terminus rounded. Because of six incisures, short stylet, vulva position, oesophagus structure and tail shape, D. valveus resembles D. acutatus D. myceliophagus and D. medicaginis. D. myceliophagus differs from D.valveus by tail shape and generally overlapping oesophagus; D. medicaginis differs from D. valveus mainly by tail shape and also slightly more posterior vulva position (78-83 vs 74.3-81.5); D. acutatus differs from D. valveus mainly by tail shape. Conclusion: In this study, 15 species from 10 genera of plant parasitic nematodes were identified. Among these genera and species, three species including Boleodorus pakistanensis, Ditylenchus hexaglyphus, D. valveus are reported as new records for Iran

Evaluation of Colorimetric LAMP Assay for Visual Detection of Ralstoniasolanacearum in Potato Shipments at Quarantine Stops in Ira

Introduction: Race 3/ biovar 2 of this pathogen causes bacterial blight of solanaceous plants especially potato in both tropical and temperate regions and results in great economic losses worldwide. Infection is prevented via quarantine or incineration of infected plant materials. However, the use of healthy seed tubers is the most effective way to avoid dissemination of this harmful plant pathogenic bacterium to pathogen-free areas. Amplification of functional genes such as endoglucanase and hrpB and fliChas been used as an alternative to study R. solanacearumspecies complex. In order to facilitate detection of R. solanacearumin imported seed tubers and identify high-risk fields and stores where inoculums population is low, loop-mediated isothermal amplification (LAMP) reaction as a potentially fast and cost-effective method was used. The attention of the present study wason evaluation of latent infection in potato tubers with R. solanacearum bacterium targeting the fliC gene by colorimetric LAMP assay. The LAMP protocol was compared with the conventional PCR which routinely used at most quarantine stops. Materials and Methods: In this study, bacterial strains were isolated on tetrazolium chloride (TZC) agar medium. Pathogenicity assay was carried out on tomato and potato seedlings under greenhouse conditions. Total DNA of bacterial strains was prepared using Chen and Kao (1993) protocol. In some cases, the boiled filtrated potato extract was used directly in molecular experiments. Identification of R. solanacearum strains at species and phylotype levels and biovar determination were done based on literature. The PCR products were analyzed on 1.2 % agarose gels in TBE buffer and visualized with UV light. To detect R. solanacearumin symptomatic and symptomless tissues, conventional PCR and LAMP assay according to fliC gene were performed and compared with each other. In order to check amplified LAMP products in visual assessment, the existence of magnesium pyrophosphate precipitate in tested tubes was analyzed. Furthermore, change in colourdue to the reaction was evaluated bynaked eye and UV treatmentafter adding the calcein. Finally, the LAMP products were examined by electrophoresis through 2% agarose gel after staining with green viewer. To determine limit of the LAMP assay, seven dilution series (2×107 to 2×10 CFU/ml) were prepared and 2 μl of each dilution was used for LAMP. Results and Discussion: Bacterial colonies showed mucous and opaque appearance with red centre and whitish periphery on TZC agar medium were selected for further study.In plant bioassay two weeks after bacterial inoculation, different levels of wilting were observed on tomato and potato seedlings.The expected 281 and 372 bp PCR-amplified fragments was observed in all strains supporting species and phylotype identification, respectively. Moreover, utilization of carbon sources indicated that the strains were related to biovar 2. Furthermore, all strains from potato were screened using Ral-fliC and Rsol-fliC primers. A 400 bp PCR product specific to R. solanacearum was obtained from all strains. Sequencing three purified PCR products confirmed the right amplification of fliC gene specific to R. solanacearum. The amplified products were detected by visual observation which the white turbidity of the reaction mixture by magnesium pyrophosphate was seen after 55 min. An alternative indicator to visually check the positive reactionwas calceinwhich was based onobservation ofyellow (green) in colour at the absence (presence) of UV light in infected samples and clear colour in negative control. Detection limits in pure cultures and infected potato extract were also determined. In conventional fliC-PCR, the detection limit rangedapproximately from 10 3 to 10 4cfu ml−1in both infected potato extract and pure cultures. Moreover, the lowest amount of consistently tested positive through LAMP assay was 10 4cfu ml−1 for both cases. Although the sensitivity of the fliC LAMP assay wasequal or lower than that of the conventional PCR, the accuracy of fliC LAMP seems to be sufficient toreliably confirmthe presence of R. solanacearum in potato samples. In addition, LAMP protocol assay is time-consuming procedure, does not require expensive equipments, provides visually detection of positive reactions and can apply to survey possible infection in host plants. Conclusion: Consequently, LAMP assay with ashort nucleic acid extraction step like as boiling treatment and efficient visualization processes such as calcein provide suitable preliminary data for screening of pathogen–free tubers prior to storage and during transportation

Diseases Caused by Pectobacterium and Dickeya Species Around the World

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