RFLP Groups Research Articles

Diversity in clinical isolates of Ictalurid herpesvirus 1 (IcHV1) from U.S. farm-raised catfish and virulence assessment in channel and channel × blue catfish hybrids.

Ictalurid herpesvirus 1 (IcHV1) is the most significant viral agent in U.S. catfish aquaculture. Little is known regarding the genetic stability and antigenic variability of IcHV1. Herein, the genetic and antigenic diversity of IcHV1 field isolates was assessed by restriction fragment length polymorphism (RFLP) analysis and serum neutralization assays. RFLP analysis identified two distinct genotypes (IcHV1A and IcHV1B), both discrete from blue catfish alloherpesvirus (BCAHV). Neutralization assays with anti-IcHV1 monoclonal antibody Mab-95 indicate shared antigenic determinants for IcHV1A and IcHV1B that are absent from BCAHV, which Mab-95 did not neutralize. Virulence assessments with representative isolates demonstrate significant differences between isolates within RFLP groups and pooled RFLP group data suggest IcHV1B (pooled survival [mean ± SE]: 58.3% ± 2.6) may be more virulent than IcHV1A (survival: 68.6% ± 2.4). Rechallenges with representative IcHV1A and IcHV1B isolates indicate a cross-protective effect, with fish surviving initial exposure to IcHV1A or IcHV1B showing robust protection when subsequently re-exposed to IcHV1A or IcHV1B. This work demonstrated significant differences in virulence between case isolates, identifying two discrete IcHV1 lineages, distinct from BCAHV, with similar virulence in channel and channel × blue catfish hybrids and a cross-protective effect in catfish surviving exposure to either lineage.

Attenuation of a Field Strain of Infectious Laryngotracheitis Virus in Primary Chicken Culture Cells and Adaptation to Secondary Chicken Embryo Fibroblasts

The establishment of commercial infectious laryngotracheitis virus (ILTV) live-modified vaccines has relied on serial passaging in chicken embryo (CEO) and tissue culture (TCO) for attenuation. The objective of this study was to attenuate and adapt a virulent CEO-related ILTV field strain (6340) in immortalized cells (LMH), primary chicken embryo kidney cells (CEK), chicken embryo liver cells (CEL), and chicken embryo fibroblasts (CEF). CEFs were refractory to parent ILTV, LMH cells produced low virus yields (~2.5 log10 TCID50 per mL), while CEK and CEL cells produced higher viral titers (≥log10 6.0 TCID50 per mL). After 52 passages in CELs, the cytopathic effect (CPE) was observed not only in hepatocytes but also in CEL fibroblasts. Once CPE was evident in CEL fibroblasts, 20 further passages in CEFs with viral titers reaching yields of ~4.4–5.5 log10 TCID50 per mL were performed. The attenuation of CEF-adapted viruses was evaluated after intra-tracheal and conjunctival inoculation in 28-day-old broilers by assessing clinical signs at five days post-inoculation (DPI). Virus CEL cells passages 80, 90, and 100, and CEF passages 10 and 20 were significantly attenuated compared to the parental strain. This is the first report of the attenuation of a virulent field CEO-related ILTV strain (RFLP Group V) in CEF cells—a cell type from a different embryonic germ layer (mesoderm) than ILTV target cells—the respiratory epithelium (endoderm). This finding underscores the potential use of CEF adaptation for the development of a live-attenuated ILTV vaccine.

Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes

ObjectiveThe motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay. MethodsA Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar. ResultsThe highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d). ConclusionsC. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

MOLECULAR CHARACTERIZATION AND MATING STUDY OF Fusarium proliferatum FROM VARIOUS HOSTS IN MALAYSIA

Fusarium proliferatum is a well-known plant pathogenic fungus-infected many crops. The present study was carried out to molecularly identified and characterized morphologically identified F. proliferatum from various hosts and substrates. The species identity of the isolates was verified as F. proliferatum based on TEF-1α sequences and phylogenetic analysis indicated high intraspecific variations. RFLP-IGS analysis also indicated high intraspecific variations of which the isolates were clustered into three RFLP Groups (I, II, and III) comprising 67 IGS haplotypes. Seventy isolates were crossed-fertile and proven to be members of mating population D (MP-D) of Gibberella fujikuroi while four isolates were infertile. A high level of intraspecific variations is vital for F. proliferatum adaptation and survival in the host and environment. Correct species identification of F. proliferatum is important as the fungus is a well-known plant pathogen and mycotoxin producer. Correct species identity is also essential to strategize suitable disease control methods as well as to predict their host range and mycotoxin production.

Diversity and abundance of culturable nitrogen-fixing bacteria in the phyllosphere of maize.

The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N-fixing epiphyte bacteria on the phyllosphere of maize in arid and semi-arid regions of Iran. Leaf samples of the maize variety, 'single cross 704' (Zea mays L.) were collected from different locations in Iran. The community of culturable N-fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR-polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S-RFLP). Approximately, 31·82% of the 242 isolates were identified as N-fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N-fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N-fixing bacteria. BOXAIR-PCR showed that there was a low similarity level among the N-fixing bacteria isolates, and genetic differentiation of individual strains was relatively great. Our findings suggest that nitrogen-fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi-arid ecosystem. This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N-fertilizers for providing nitrogen to crops arid and semi-arid regions.

Molecular diversity of "Candidatus Phytoplasma mali" strains associated with apple proliferation disease in Bulgarian germplasm collection.

A quarantine organism, "Candidatus Phytoplasma mali," is the causal agent of apple proliferation, one of the most important apple diseases in Europe. The genetic diversity of this pathogen in Central and Southern Europe has already been reported; however, almost no data exists from Eastern Europe. In this study, "Ca. P. mali" strains, which were identified in 14 apple trees from the Bulgarian germplasm collection, were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of four genomic loci. In total, nine distinct genetic lineages were recognized based on the combination of the following detected RFLP profiles: two profiles for the 16S-23S rDNA region (16SrX-A2, -A3), four profiles for the secY gene (one previously known: secY(X)-A, and three new: secY-C, secY-D, secY-E), three profiles for the rpl22-rps3 genes (rpX-A, rpX-B, rpX-F), and one profile for the nitroreductase- and rhodanese-like gene (AT-1). Phylogenetic analysis of the Bulgarian and other European "Ca. P. mali" strains based on 16S-23S rRNA gene sequences confirmed RFLP grouping, regardless of the phytoplasma origin. In a phylogenetic tree based on the secY data, only German strains formed separate clade from the other strains. The tree based on rp genes did not correspond to RFLP profiles. Unexpectedly, when using nitroreductase and rhodanese-like gene sequences, the Bulgarian strains clustered separately from the other European strains. Apart from the identification of different "Ca. P. mali" strains, the paper also recommends the unification of the rpX-subgroup nomenclature to avoid future confusions. Both aims of this paper provide valuable tools to understand the epidemiology of this quarantine pathogen.

Natural fruits, flowers, honey, and honeybees harbor Helicobacterpylori-positive yeasts.

For controlling Helicobacterpylori infection in humans, its environmental reservoir should be determined. In this study, yeast isolates from an isolated village in Iran were studied for the intracellular occurrence of H.pylori. In this study, yeasts were isolated from 29 samples, including oral swabs from villagers (n=7), flowers and fruits (n=6), honey and honeybees (n=12) and miscellaneous samples (4). Yeasts were classified into 12 RFLP groups and identified by amplification of 26S rDNA and sequencing. DNA extracted from the yeast cells was examined for the presence of H.pylori using PCR. Of the 29 yeasts, 27 were members of different genera of Ascomycete. H.pylori was detected in 5 of 9 Candida (55.5%), 4 of 5 Komagataella (80%), 3 of 4 Pichia (100%), 2 of 2 Cytobasidia (100%), 2 of 2 Hansenia (100%), 1 of 1 Meyerozyma (100%) and 2 of 3 not sequenced (66.6%) yeasts. Distribution of 19 of 29 (65.5%) H.pylori-positive yeasts within 4 groups was as follows: 1 of 7(14.3%) in oral swabs, 5 of 6 (83.3%) in flowers and fruits, 10 of 12 (83.3%) in honey and the bee group and 3 of 4 (75%) in miscellaneous. Different genera of osmotolerant yeasts from flowers, fruits, honey, and honeybees contained H.pylori in their vacuole. High frequency of H.pylori-positive yeasts in these samples might be related to their high sugar content. Insects such as honeybees that facilitate transfer and easy access of these yeasts to nectars serve as the main reservoirs of these yeasts, playing an important role in their protection and dispersal. Accordingly, H.pylori inside these yeasts can be carried by honeybees to different sugar- and nutrient-rich environments. Sugar-rich environments and honeybees play an important role in distribution of H.pylori-positive yeasts in nature.

Epidemics of group 16SrI-A phytoplasmas in a garden of Vilnius region in Lithuania

Abstract Here we report on a plant disease caused by insect-transmitted unculturable plant pathogenic bacteria detected in a private garden in Vilnius region. Samples of symptomatic peas (Pisum sativum L.), nasturtium (Tropaeolum majus L.), strawberry (Fragaria × ananassa Duchesne) and zucchini (Cucurbita pepo var. giromontina) plant tissues were collected. Based on the molecular technique, the Internet tools and phylogenetic analysis, these pathogens were identified as phytoplasmas and classified in phytoplasma RFLP (restriction fragment length polymorphism) group 16SrI, subgroup A. Because this pathogen may be spread by insect-vector that comes from the wild nature, the phytoplasmas could cause a problem in agriculture of Lithuania.

Criteria for phytoplasma 16Sr group/subgroup delineation and the need of a platform for proper registration of new groups and subgroups

As phytoplasmas are discovered at an ever-increasing pace in emerging and re-emerging plant diseases worldwide, the scheme for classification of phytoplasmas into 16S rRNA gene RFLP (16Sr) groups and subgroups is experiencing an ongoing rapid expansion. Improper delineation or designation of new groups and subgroups can open potential conflicts in classifying newly identified phytoplasma strains. To maintain the integrity of the classification scheme, criteria for the delineation of new groups and subgroups must be followed, and proper registration should be required to track established groups and subgroups.

Coccidioides species determination: does sequence analysis agree with restriction fragment length polymorphism?

Fifteen Coccidioides isolates were previously examined for genetic diversity using restriction fragment length polymorphism (RFLP); two fragment patterns were observed. Two isolates demonstrated one banding pattern (designated RFLP group I), while the remaining 13 isolates demonstrated a second pattern (designated RFLP group II). Recently, molecular studies supported the division of the genera Coccidioides into two species: Coccidioides posadasii and Coccidioides immitis. It has been assumed that the species division corresponds to the RFLP grouping. We tested this hypothesis by amplifying the ribosomal DNA internal transcribed spacer region as well as the dioxygenase, serine proteinase, and urease genes from 13 isolates previously examined by RFLP and then sequencing the PCR products. The appropriate species for each isolate was assigned using phylogenetically informative sites. The RFLP grouping agreed with the Coccidioides species assignment for all but one isolate, which may represent a hybrid. In addition, polymorphic sites among the four genes examined were in agreement for species assignment such that analysis of a single gene may be sufficient for species assignment.

Community composition and cellulase activity of cellulolytic bacteria from forest soils planted with broad-leaved deciduous and evergreen trees

Cellulolytic bacteria in forest soil provide carbon sources to improve the soil fertility and sustain the nutrient balance of the forest ecological system through the decomposition of cellulosic remains. These bacteria can also be utilized for the biological conversion of biomass into renewable biofuels. In this study, the community compositions and activities of cellulolytic bacteria in the soils of forests planted with broad-leaved deciduous (Chang Qing Garden, CQG) and broad-leaved evergreen (Forest Park, FP) trees in Wuhan, China were resolved through restriction fragment length polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. All of the isolates exhibited 35 RFLP fingerprint patterns and were clustered into six groups at a similarity level of 50 %. The phylogeny analysis based on the 16S rRNA gene sequence revealed that these RFLP groups could be clustered into three phylogenetic groups and further divided into six subgroups at a higher resolution. Group I consists of isolates from Bacillus cereus, Bacillus subtilis complex (I-A) and from Paenibacillus amylolyticus-related complex (I-B) and exhibited the highest cellulase activity among all of the cellulolytic bacteria isolates. Cluster II consists of isolates belonging to Microbacterium testaceum (II-A), Chryseobacterium indoltheticum (II-B), and Flavobacterium pectinovorum and the related complex (II-C). Cluster III consists of isolates belonging to Pseudomonas putida-related species. The community shift with respect to the plant species and the soil properties was evidenced by the phylogenetic composition of the communities. Groups I-A and I-B, which account for 36.0 % of the cellulolytic communities in the CQG site, are the dominant groups (88.4 %) in the FP site. Alternatively, the ratio of the bacteria belonging to group III (P. putida-related isolates) shifted from 28.0 % in CQG to 4.0 % in FP. The soil nutrient analysis revealed that the CQG site planted with deciduous broad-leaved trees has a richer organic nutrient (total organic carbon and total nitrogen) than the FP site planted with evergreen broad-leaved trees. Against this background, the population density and the diversity of cellulolytic bacteria in the CQG site are clearly higher than those in the FP site, and the latter was dominated with high-cellulase-activity Bacillus- and Paenibacillus-related bacteria. The canonical correspondence analysis further indicated that the distribution of these groups is correlated with the FP site, whereas groups II and III are correlated with the organic nutrient-rich CQG site.

Biological and molecular variation of Iranian Cauliflower mosaic virus (CaMV) isolates

Seventeen provinces of Iran were surveyed during 2003-2012 to find Brassicaceae hosts of Cauliflower mosaic virus (CaMV). A total 397 samples were collected from plants with virus-like symptoms. Among those tested by ELISA, 255 samples (67.2%) were found to be infected with CaMV. Mechanical transmission tests showed that the Iranian isolates have similar biological properties on a number of Brassica and Raphanus plant species and cultivars tested. However, the isolates varied in the severity of symptoms they induced and in the capacity to infect B. oleracea var. capitata, on the basis of which they were grouped into two distinct biotypes L/MMo (latent/mild mottle) and severe (S) infection. The molecular diversity of natural population of CaMV were investigated based on the complete sequences of OFR 6 of 36 Iranian isolates collected from different geographically distant regions in Iran alongside the sequences of 14 previously reported isolates. Phylogenetic analyses indicated that the Iranian CaMV isolates belong to two groups (GI and GII). Most of the Iranian isolates fell into GI with other exotic isolates; however, the isolates from North-East Iran with Xinjiang from China fell into GII. The phylogenetic group GII (the North-East Iranian isolates) closely corresponded to the S biological group however other Iranian isolates corresponded to the L/MMo biological group. The within-population diversity was lower than the between population diversity suggesting the contribution of a founder effect on diversification of CaMV isolates. The Iranian isolates were differentiated from other exotic CaMV isolates and clustered into two RFLP groups using Hpy99I which closely corresponded to the biological and phylogenetic groups. This study showed the evolutionary process in CaMV isolates is shaped by a combination of host range differentiation and nucleotide substitution using the approach of population genetics.

Occurrence of Pectobacterium wasabiae in potato field samples

During the growing seasons of 1996 and 1997, samples of potato stems and tubers with symptoms of blackleg and soft rot were collected in different regions in Poland. After growing to pure cultures on crystal violet pectate (CVP) medium, isolates of bacteria were identified as Pectobacterium spp. on the basis of their ability to degrade pectate and with the use of biochemical tests. About 43 % strains isolated from 122 different plant samples were identified as Pectobacterium carotovorum subsp. carotovorum, whereas the rest of the pectinolytic bacteria was identified as Pectobacterium atrosepticum. A recent screening of these isolates with recA PCR-RFLP allowed identification of 18 different RFLP groups within the tested P. c. subsp. carotovorum strains. The third largest group of the tested P. c. subsp. carotovorum strains (14 %), which were assigned to the profile 3 recA PCR-RFLP, was re-identified as Pectobacterium wasabiae (formerly Erwinia carotovora subsp. wasabiae) on the basis of recA and 16S rRNA genes sequences. About 50 % of P. wasabiae isolated from potato, in contrast to horseradish isolates of P. wasabiae, have an ability to grow at 37°C and some of them grow on media containing 5 % of NaCl. In a pathogenicity test with 11 strains of P. wasabiae these strains showed a high capacity to rot potato tubers.

PCR–RFLP identifies differences in hrpZ sequences to distinguish two genetic groups of Pseudomonas syringae pv. syringae strains from barley and wheat with bacterial black nod

Differences in hrpZ sequences determined by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) were used to investigate the molecular epidemiology of Pseudomonas syringae pv. syringae (PSS) strains that were isolated from diseased barley and wheat plants in Okayama, Japan. PCR–RFLP using HhaI separated PSS strains into two groups (A and B). Although specific PCR–RFLP groups of PSS strains were not always isolated from specific cultivars or seeds produced in a specific area, many strains isolated from barley and wheat belonged to PCR–RFLP groups A and B, respectively.

‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described 'Candidatus Phytoplasma' species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described 'Candidatus Phytoplasma' species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1(R) as representative of a novel taxon, 'Candidatus Phytoplasma pruni'. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the 'Ca. Phytoplasma pruni' lineage. We propose that the term 'Ca. Phytoplasma pruni' be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and--within the tolerance of a single base difference in one unique sequence--peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of 'Candidatus Phytoplasma' taxa.

Genetic diversity of Iranian potato soft rot bacteria based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis

Bacterial soft rot diseases caused by Pectobacterium-Dickeya complex are the most important and yield losses diseases of potato crop worldwide. Loss due to these diseases in some years/fields under Iran condition is huge and destructive. To screen and characterize the causal agents, thirty bacterial soft rot isolates including 10 authentic pectolytic strains were investigated. Based on biochemical and physiological tests, the Iranian strains were identified as either Pectobacterium carotovorum subsp. carotovorum or P. carotovorum subsp. wasabiae. Sequence analysis of recA gene revealed that the strains closely related to Pectobacterium spp. To assess the genomic diversity, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed. PCR amplification of the recA gene followed by RFLP revealed 14 distinct RFLP groups. Here also, the same results were obtained and all 19 Iranian strains were assigned as either Pectobacterium carotovorum subsp. carotovorum or P. carotovorum subsp. wasabiae . Key words : Genetic diversity, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), recA gene, Pectobacterium spp.

Genetic diversity and spatial distribution of rhizobial bacteria nodulating on black locust (Robinia pseudoacacia) rhizospheres

Black locust (Robinia pseudoacacia L.) is an economically and ecologically important tree in Japan. The species is widely used for afforestation because of its symbiotic relationship with nitrogen-fixing rhizobia. In this study, genetic diversity of rhizobia isolated from black locust nodules and spatial distribution of their genotypes were examined. From a coastal forest, six black locust saplings including the whole root systems were collected and positions of nodules on the roots were recorded. Bacteria were isolated from each nodule and then genotyped by Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) and sequencing analysis of the 16S rRNA gene. Dendrogram analysis based on PCR-RFLP and sequencing analysis indicated that Mesorhizobium species dominantly colonized black locust roots in this forest, occupying at least 77% of living nodules. Positions and PCR-RFLP genotypes of the nodules revealed that rhizobia isolated from neighbouring nodules tended to have the same genotype in some cases. Statistical analysis has supported this fact. The clustering distribution of nodules in the same RFLP groups should be considered to contribute to the large genetic diversity of rhizobia.

Colonization of wheat seedlings ( Triticum aestivum L.) by strains of Pseudomonas spp. with respect to their nutrient utilization profiles

Nutrients that leach out from roots constitute a major source of food for root-colonizing bacteria. The role of specific nutrients in this interaction is unclear. The aim of the present study was to examine whether colonization ability could be attributed to specific nutrient utilization profiles. Twenty fluorescent pseudomonads were tested for colonization ability of 48-h-old wheat seedlings. Analyses of RFLPs of amplified 16S rRNA gene and of BIOLOG GN2 data demonstrated that colonization ability did not associate with any particular RFLP or metabolic group. The best colonizers PPS96, PSR2, PSR21, good colonizer PSR6 and four of ineffective colonizers were identified through 16S sequence analysis as Pseudomonas reactans. The best and good colonizers distinguished themselves from the less efficient colonizers by specifically utilizing: p-hydroxyphenylacetic acid, bromosuccinic acid, benzoic acid, methyl pyruvate, N-acetyl- d-glucosamine, d-trehalose and adonitol. However, there was no specific sole nutrient utilization profile that predicted excellent root colonization ability of wheat, since the best colonizers did not have identical profiles. This work indicates that strains of P. reactans are present in the rhizosphere of oil seed rape and wheat and that some of them are effective colonizers of wheat roots.

English

From six different districts of Punjab, Pakistan, 85 isolates of Citrus tristeza virus (CTV) were collected and characterized based on coat protein gene (CPG) analysis. All isolates were collected from field trees showing various CTV symptoms such as decline in most citrus varieties, inverse pitting on some sour orange rootstocks below bud union, mild-to-moderate stem-pitting on the trunk of some sweet orange. The CTV CP gene of all isolates was amplified by reverse transcriptase polymerase chain reaction (RT–PCR) using CP gene-specific primers yielding 672 bp. The maximum disease incidence was found in sweet orange followed by mandarin and grapefruit. These isolates were then subjected to CPG/Hinf I restriction fragment length polymorphism (RFLP) analysis. Mixed infection of CTV isolates was found very common in the field tress in Pakistan. The most dominant CPG/Hinf I RFLP groups III, I and VI are the basic causal epidemic in Pakistan. Moreover, based on symptoms in the field trees, CPG/Hinf I RFLP groups III, I and VI are considered to be the obvious causes of decline and stem-pitting in Pakistan. Key words: Citrus tristeza virus, CPG/Hinf I restriction fragment length polymorphism (RFLP) groups, decline, stem-pitting.

Isolation and characterization of eight bacteriophages infecting Erwinia amylovora and their potential as biological control agents in British Columbia, Canada

Nineteen active bacteriophages against Erwinia amylovora, the causal agent of fire blight, were collected from apple and pear orchards in the Okanagan and Fraser Valleys of British Columbia. Eight survived the isolation, purification and storage processes. Five bacteriophage isolates included in this study lysed more than 50% of the 20 E. amylovora strains tested from BC. Examination by transmission electron microscopy revealed that all eight phages belong to the order Caudovirales, the tailed phages, and included members of the families Myoviridae and Podoviridae. Bacteriophages were characterized by digestion of the phage DNA with four restriction endonucleases and two sets of PCR primers. Two novel groups, RFLP groups 7 and 8, were identified based on differences in restriction fragment patterns. Phages ΦEa1337-26 and ΦEa2345-6 reduced infection by 84% and 96%, respectively, when tested on detached pear blossoms using the epiphyte bacterium Pantoea agglomerans Eh21-5 as a carrier. In addition, bacteriophage ΦEa2345-6, applied in combination with Eh21-5, reduced infection of fire blight on apple flowers of potted apple trees by 56% and compared well with the antibiotic streptomycin.