Differentiation between human and ovine isolates of Bordetellaparapertussis using pulsed-field gel electrophoresis

Abstract The genetic relatedness of 18 human and 29 ovine isolates of Bordetella parapertussis was examined by macrorestriction digestion of DNA with the rarely cutting enzyme Xba I and resolution by pulsed-field gel electrophoresis. There was clear separation of human and ovine isolates and variation within host types. The human isolates were separated into three types as were the 24 Scottish ovine isolates. Species-specific bands were observed with the human isolates at 114, 134, 166, 213, 346 and 372 kb. No species-specific bands were found in the B. parapertussis ovine isolates. Isolates of B. parapertussis recovered from sheep in New Zealand gave a further two DNA banding patterns which were clearly different from the Scottish ovine and the human isolates. These results indicate that human and ovine isolates of B. parapertussis are genetically distinct and that variation exists within isolates from the same host species. Pulsed-field gel electrbphoresis therefore appears to be a powerful discriminatory tool for the classification of B. parapertussis .     

Characterisation of ovine Bordetella parapertussis isolates by analysis of specific endotoxin (lipopolysaccharide) epitopes, filamentous haemagglutinin production, cellular fatty acid composition and antibiotic sensitivity

Abstract Isolates of Bordetella parapertussis , recovered from sheep or man, were characterised by reaction with specific anti- Bordetella lipopolysaccharide monoclonal antibodies, production of filamentous haemagglutinin, fatty acid patterns, and antibiotic sensitivity. Generally, the isolates lay within one of four groups, with separation of the ovine isolates into two groups. Reactions with specific monoclonal antibodies against lipopolysaccharide separated the ovine isolates into these two groupings. Analysis of the cellular fatty acid compositions by cluster analysis differentiated between the human and the ovine strains and also showed variation within the ovine isolates. When the production of filamentous haemagglutinin was analysed in an ELIS A system, a similar pattern emerged. Varying concentrations of filamentous haemagglutinin (11–429 ng (mg total protein)⁻¹) were extracted from the human isolates and the one group of ovine isolates with no significant protein detected in the other ovine group. These studies demonstrate variation between and within B. parapertussis isolates recovered from two mammalian sources.     

Molecular and phenotypic characterization of Pseudomonas spp. isolated from milk

Putative Pseudomonas spp. isolated predominantly from raw and processed milk were characterized by automated ribotyping and by biochemical reactions. Isolates were biochemically profiled using the Biolog system and API 20 NE and by determining the production of proteases, lipases, and lecithinases for each isolate. Isolates grouped into five coherent clusters, predominated by the species P. putida (cluster A), P. fluorescens (cluster B), P. fragi (as identified by Biolog) or P. fluorescens (as identified by API 20 NE) (cluster C), P. fragi (as identified by Biolog) or P. putida (as identified by API 20 NE) (cluster D), and P. fluorescens (cluster E). Isolates within each cluster also displayed similar enzyme activities. Isolates in clusters A, C, and D were generally negative for all three enzyme activities; isolates in cluster B were predominantly positive for all three enzyme activities; and isolates in cluster E were negative for lecithinase but predominantly positive for protease and lipase activities. Thus, only isolates from clusters B and E produced enzyme activities associated with dairy product flavor defects. Thirty-eight ribogroups were differentiated among the 70 isolates. Ribotyping was highly discriminatory for dairy Pseudomonas isolates, with a Simpson's index of discrimination of 0.955. Isolates of the same ribotype were never classified into different clusters, and ribotypes within a given cluster generally showed similar ribotype patterns; thus, specific ribotype fragments may be useful markers for tracking the sources of pseudomonads in dairy production systems. Our results suggest that ribogroups are generally homogeneous with respect to nomenspecies and biovars, confirming the identification potential of ribotyping for Pseudomonas spp.     

Analyses of genetic and pathogenic variability among Botrytis cinerea isolates

Seventy nine isolates of Botrytis cinerea were collected from different host plants and different locations of India and Nepal. All the isolates were identified as B. cinerea based on morphological features and were confirmed using B. cinerea specific primers. Differentiation among the isolates was assessed using morphological, genetic and biochemical approaches. To analyze morphological variability, differences in conidial size, presence or absence of sclerotia and their arrangement were observed. Genetic variability was characterized using RAPD analysis, presence or absence of transposons and mating type genes. Cluster analysis based on RAPD markers was used for defining groups on the basis of geographical region and host. The biochemical approach included determining differences in concentration of oxalic acid and activity of lytic enzymes. All the isolates were categorized into different pathogenic groups on the basis their variable reaction towards chickpea plants. Isolates with higher concentration of oxalic acid and greater activity of lytic enzymes were generally more pathogenic. Pathogenicity was also correlated to transposons. Isolates containing transposa group showed some degree of correlation with pathogenic behavior. However, isolates could not be grouped on the basis of a single approach which provides evidence of their wide diversity and high evolution potential. Sensitivity of sampled isolates was also tested against five botryticides. Most of the isolates from same region were inhibited by a particular fungicide. This feature provided interesting cues and would assist in devising novel and more effective measures for managing the disease.     

Genotypic and Phenotypic Comparisons of Chromosomal Types within an Indigenous Soil Population of Rhizobium leguminosarum bv. trifolii

The relative genetic similarities of 200 isolates of Rhizobium leguminosarum bv. trifolii recovered from an Oregon soil were determined at 13 enzyme loci by multilocus enzyme electrophoresis (MLEE). These isolates represented 13 antigenically distinct serotypes recovered from nodules formed on various clover species. The MLEE-derived levels of relatedness among isolates of R. leguminosarum bv. trifolii were found to be in good agreement with the levels of relatedness established by using repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the PCR technique and with levels of relatedness from previously published DNA reassociation studies. BIOLOG substrate utilization patterns showed that isolates within an electrophoretic type (ET) were phenotypically more similar to each other than to isolates of other ETs. The soil isolates were represented by 53 ETs which could be clustered into seven groups (groups B, E, G, H1, H2, I, and J). Evidence for multilocus structure within the population was obtained, and group B was identified as the primary creator of the disequilibrium. Of 75 isolates belonging to the nodule-dominant serotype AS6 complex, 72 were found in group B. Isolates WS2-01 and WS2-02 representing nodule-dominant serotypes recovered from subclover grown at another Oregon site were also found in group B. Isolates representing the most numerous ETs in group B (ETs 2 and 3) were either suboptimally effective or completely ineffective at fixing nitrogen on six different clover species. Another four groups of isolates (groups A, C, D, and F) were identified when 32 strains of diverse origins were analyzed by MLEE and incorporated into the cluster analysis. Group A was most dissimilar in comparisons with other groups and contained strain USDA 2124 (T24), which produces trifolitoxin and has unique symbiotic characteristics.     

Biochemical characterization of some additional mycelial cultures of basidiomycetes

Twenty-six biochemical tests were used to study cultures of basidiomycetes isolated from roots of fruit-trees and other plants. The results enabled isolates to be placed into one of sixteen groups. Three of these groups were identified as Collybia drucei, Corticium utriculicum and Heteroporus biennis by matching their biochemical reactions with those of isolates from fruiting bodies of these fungi. This suggests that the other groups might also correspond to species. The three named fungi are indigenous, the first two not having been recorded elsewhere. Thus, root infections by these fungi may have originated from the indigenous fungal flora. Isolates of C. utriculicum and Stereum purpureum which were indistinguishable in culture were also separated using biochemical tests.     

How do Japanese isolates ofVerticillium dahliae correspond with standardized VCG testers?

We examined the vegetative compatibility of 56 Japanese isolates provisionally assigned to four subgroups ofV. dahliae to estimate the genetic relatedness with testers of the standardized VCGs. Subgroup J1 was assigned to VCG 2A/B as a new category of assignment. Subgroup J2, except isolate Vdt 110, was assigned to VCG 2A, and subgroup J3, except isolate Vdf 1, was assigned to VCG 2B. Isolates Vdf 1 and Vdt 110 were assigned to VCG 2A/B. Subgroup J4 was assigned to two subgroups, VCG 4B for Vde 1 and VCG 4A/B for FY 3 and HR 1. Four isolates were compatible with both VCG 2 and 4. Isolate U56 was compatible with VCG 2A/B and 4A. Isolates of VCG 2A, Vdt 9 and FF1, were compatible with either VCG 4A or 4A/B. One isolate of VCG 2B, Vdp-4, was compatible with VCG 4A. Three isolates of subgroup J2 showed weak reactions with the testers of VCG 4. These isolates may be “bridging strains”. Japanese isolates were composed of two VCGs, 2 and 4, “bridging strains” compatible with these VCGs, and some self-incopatible isolates. Testers of VCG 1 and VCG 3 did not show any reactions with the Japanese isolates.     

Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (> or =86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Fatty acid composition as a taxonomic characteristic for Microcystis and other coccoid cyanobacteria (blue-green alga) isolates

The taxonomic importance of fatty acid composition at genus and sub-genus level was evaluated by analysing the fatty acid composition of fourteen different Microcystis isolates and seven additional members of the order Chroococcales. Fatty acid composition proved to be consistent within isolates. Isolates were clustered into two major groups, namely A and B. Group B contained all the Microcystis isolates and was further divided into subgroups of varying similarity indicating the existence of different taxa. The Microcystis isolates were characterised by a high content of polyunsaturated fatty acids (27–44%) and a low content of palmitoleate. The test organisms were arranged in a scheme indicating their possible phylogenetic relationship based on fatty acid composition and other phenotypic characteristics. According to our data the toxic strains, represented by different isolates, of Microcystis appear as a distinct group. Furthermore two dubious species namely Microcystis incerta and a Synechocystis sp. could clearly be reasigned to different genera. The results demonstrated that fatty acid composition is an effective taxonomic tool in clarifying taxonomical problems of Microcystis isolates. Department of Microbiology, University of the Orange Free State     

Heterogeneity in expression of lipopolysaccharide by strains of Salmonella enterica serotype Typhimurium definitive phage type 104 and related phage types

AIMS: To investigate lipopolysaccharide (LPS) expression in Salmonella enterica serotype Typhimurium definitive phage type 104 (Salmonella Typhimurium DT104) and related phage types. METHODS AND RESULTS: Isolates were examined for the expression of LPS by SDS-PAGE and silver staining and subtyped by Pulsed Field Gel Electrophoresis (PFGE). The 100 isolates expressed one of two LPS profiles designated A (72%) and B (28%). LPS profiling was able to discriminate between isolates of identical PFGE type. Among 10 groups of outbreak isolates examined, each group was of a single LPS profile: A, 8/10 and B, 2/10. All 10 outbreaks were identical by PFGE analysis. CONCLUSIONS: Isolates of Salmonella Typhimurium DT104 and related phage types expressed one of two distinct LPS profiles. The two LPS profiles appear similar but shifted and in phase with one another, suggesting that the heterogeneity is due to changes in the LPS core region rather than among the repeating oligosaccharide units of the long-chain LPS. SIGNIFICANCE AND IMPACT OF THE SUTDY: LPS profiling provides a useful adjunct to PFGE and other molecular methods for the subtyping of this group of bacteria in epidemiological investigations.     

Phylogenetic Distribution of Phenotypic Traits in Bacillus thuringiensis Determined by Multilocus Sequence Analysis

Diverse isolates from a world-wide collection of Bacillus thuringiensis were classified based on phenotypic profiles resulting from six biochemical tests; production of amylase (T), lecithinase (L), urease (U), acid from sucrose (S) and salicin (A), and the hydrolysis of esculin (E). Eighty two isolates representing the 15 most common phenotypic profiles were subjected to phylogenetic analysis by multilocus sequence typing; these were found to be distributed among 19 sequence types, 8 of which were novel. Approximately 70% of the isolates belonged to sequence types corresponding to the classical B. thuringiensis varieties kurstaki (20 isolates), finitimus (15 isolates), morrisoni (11 isolates) and israelensis (11 isolates). Generally, there was little apparent correlation between phenotypic traits and phylogenetic position, and phenotypic variation was often substantial within a sequence type. Isolates of the sequence type corresponding to kurstaki displayed the greatest apparent phenotypic variation with 6 of the 15 phenotypic profiles represented. Despite the phenotypic variation often observed within a given sequence type, certain phenotypes appeared highly correlated with particular sequence types. Isolates with the phenotypic profiles TLUAE and LSAE were found to be exclusively associated with sequence types associated with varieties kurstaki and finitimus, respectively, and 7 of 8 TS isolates were found to be associated with the morrisoni sequence type. Our results suggest that the B. thuringiensis varieties israelensis and kurstaki represent the most abundant varieties of Bt in soil.     

Chararacteristics of Vagococcus salmoninarum isolated from diseased salmonid fish

Isolates of the salmonid pathogen Vagococcus salmoninarum were recovered from Atlantic salmon, rainbow trout and brown trout with peritonitis. The phenotypes of these isolates and the type strain of Vag. salmoninarum NCFB 2777 were determined by morphological, biochemical and physiological tests and whole cell protein profiles by SDS-PAGE. There was a high level of phenetic similarity between the salmonid isolates and the type strain. The species forms short Gram-positive rods, hydrolyses L-pyrrolidonyl-β-naphthylamide, is α-haemolytic on sheep's blood agar, grows at pH 9·6 and 10°C but not at 40°C or in 6·5% NaCl and is catalase-negative; a Lancefield group N antigen is not present. Vagococcus salmoninarum can be distinguished phenetically from similar fish pathogens including Carnobacterium piscicola, Enterococcus seriolicida and Lactococcus piscium.     

Phenotypic characteristics of Streptococcus iniae and Streptococcus parauberis isolated from olive flounder (Paralichthys olivaceus)

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis , respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges–Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.     

Variation within Pseudomonas syringae pv. philadelphi, the cause of a leaf spot of Philadelphus spp.

R oberts , S.J. 1985. Variation within Pseudomonas syringae pv. philadelphi , the cause of a leaf spot of Philadelphius spp. Journal of Applied Bacteriology 59 , 283–290
In pathogenicity tests on Philadelphus and other plant species, belonging to ten genera in seven families, isolates of Pseudomonas syringae from leaf spots on Philadelphus spp. in England did not produce symptoms on any plants other than Philadelphus . It is therefore proposed that these isolates should be designated a distinct pathovar of Ps. syringae with the name Pseudomonas syringae pv. philadelphi . Isolates of this new pathovar varied in their reactions to 6 of 57 biochemical tests. In phage typing tests isolates also varied in their sensitivity to five of seven bacteriophage strains. Four of the six biochemical tests (aesculin hydrolysis, utilization of DL-homoserine L-leucine and sorbitol) and all five of the phages (P11, Pls, P2, A15, and A26) were used to separate the isolates into seven groups. These groups had some relation to their geographical origin, species of Philadelphus from which they were originally isolated, and relative virulence on P. coronarius and P. x purpureo-maculatus . They may represent ecotypes of this new pathovar.     

Genomic heterogeneity of environmental and clinical aeromonads

Thirty-three isolates of Aeromonas from environmental sources and clinical samples were tested and the results, obtained using the pulsed field gel electrophoresis (PFGE) technique, were compared with those obtained by biochemical typing. On the basis of their biochemical characteristics 31 strains was assigned to one of the recognised groups or species within the Aeromonas genus and 2 strains to the species Vibrio fluvialis. These latter were nevertheless found to belong to the Aeromonas genus on the basis of the chromosomal DNA analysis. Among the clinical isolates the biochemical analysis showed greater uniformity. A low correlation between molecular and traditional typing methods was observed with a wider heterogeneity at the genomic level. The results showed the difficulty of discriminating Aeromonas isolates by conventional biochemical methods. The genomic analysis performed by PFGE can be a more effectual technique, which can be used for epidemiological and ecological studies of the microorganisms belonging to the Aeromonas group.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

A <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> isolation method utilizing a novel stain,low selection and high throughput produced atypical results

Background  

Bacillus thuringiensis is a bacterium known for producing protein crystals with insecticidal properties. These toxins are widely sought after for controlling agricultural pests due to both their specificity and their applicability in transgenic plants. There is great interest in isolating strains with improved or novel toxin characteristics, however isolating B. thuringiensis from the environment is time consuming and yields relatively few isolates of interest. New approaches to B. thuringiensis isolation have been, and continue to be sought. In this report, candidate B. thuringiensis isolates were recovered from environmental samples using a combination of a novel stain, high throughput and reduced selection. Isolates were further characterized by SDS-PAGE, light microscopy, PCR, probe hybridization, and with selected isolates, DNA sequencing, bioassay or Electron Microscopy.     

Multilocus phylogenetic analyses within Blumeria graminis, a powdery mildew fungus of cereals

Blumeria graminis, a powdery mildew fungus, is an important plant pathogen that causes serious damage to a variety of cereal crops. In spite of the importance of the pathogen, information on phylogenetic structure within B. graminis is scarce. In this study we conducted phylogenetic analyses of B. graminis based on the DNA sequences of four different DNA regions (ITS, 28S rDNA, chitin synthase 1, and beta-tubulin). The analyses revealed that the protein-coding regions have higher amounts of phylogenetic signals than rDNA regions and are useful for phylogenetic analyses of B. graminis. The present phylogenetic analyses revealed nine distinct groups in the B. graminis isolates used in this study, a result which was commonly supported by all trees constructed from the four DNA regions. Isolates from a single host genus belonged to a single group except for isolates from Lolium and Bromus, in which the isolates were split into two and three groups, respectively. Isolates from Agropyron, Secale and Triticum formed a distinct clade (Triticum clade) with identical or similar DNA sequences. The Hordeum clade was a sister of the Triticum clade, and Poa and Avena clades were distantly related to the Triticum and Hordeum clades. This phylogenetic relationship of B. graminis is well concordant with the level of reproductive isolation between formae speciales and also with phylogeny inferred from a cytological study. Shimodaira-Hasegawa and Templeton tests using sequences of four different DNA regions significantly rejected the tree topology of plants. Therefore, possibility of co-speciation between B. graminis and its host plants was obscure in this study.     

The association between frequency of thiabendazole treatment and the development of resistance in field isolates of Ostertagia spp. of sheep

Martin P. J., Anderson N., Lwin T., Nelson G. and Morgan T. E. 1984. The association between frequency of thiabendazole treatment and the development of resistance in field isolates of Ostertagia spp. of sheep. International Journal for Parasitology14: 177–181. Isolates of Ostertagia spp. were obtained from grazing sheep 3,4 and 5 years after nil, planned (five per year) and regular (3-weekly) treatments with thiabendazole (TBZ). Levels of resistance to TBZ were measured by an in vitro egg hatch assay and a controlled anthelmintic efficiency assay. Isolates from planned treatment groups showed an increase in the level of resistance; the lethal concentrations of TBZ to 50% of eggs (LC₅₀s) were 3, 3 and 6 times the LC₅₀s of isolates from nil treatment groups for years 3, 4 and 5 respectively. The LC₅₀s of isolates from regular treatment groups were 14 times higher than those from nil treatment groups in each year. To assess the potential for an increase in level of resistance, additional egg assays were done 14 days after treatment with 44 mg kg^t?1 of TBZ on sheep infected with the planned group isolates for each year. This treatment raised the LC₅₀S for years 3, 4 and 5 respectively by 3, 2 and 1–5 times the LC₅₀s of the same isolates which had not been exposed to additional TBZ treatment. The controlled anthelmintic efficiency assay using 44 mg kg^t?1 of TBZ produced a significant reduction in the number of adult and immature worms from the nil isolate but failed significantly to reduce the number of worms from the planned and regular isolates. A three component analysis resolved the nonlinear trends of the log dose-probit plots in egg hatch assays for isolates from planned treatment groups into subpopulations of susceptible, hybrid and resistant individuals each with different levels of resistance. The proportions of these subpopulations changed in accordance with the level of resistance observed in each year.