Amplified Fragment Length Polymorphism Groups Research Articles

Virulence and Molecular Diversity in the Kabatiella zeae Population Causing Maize Eyespot in China.

Maize eyespot, caused by Kabatiella zeae, has become a major yield-limiting factor in maize planting areas in northeast China. Limited information is available on pathotypes, virulence, and the genetic diversity of the K. zeae population. We analyzed virulence and genetic diversity of 103 K. zeae isolates collected from six provinces in China with differential hosts and the amplified fragment length polymorphism (AFLP) technique, respectively. To evaluate the virulence, 103 isolates were inoculated on nine differential hosts (maize inbred lines)-E28, Shen137, Qi319, B73, Danhuang34, Zi330, Mo17, Huangzaosi, and CN165-and grouped into 23 pathotypes and three virulence groups according to the coded triplet nomenclature system on differential hosts. AFLP analysis resolved the set of isolates into four genetic diversity clusters (DICE similarity values of 76%). Genetic variation of K. zeae among and between pathotypes revealed that the pathogen population had a high genotypic diversity. The correlation between pathotypes, virulence, and genetic diversity grouping was low. A correlation between AFLP groups and geographic locations was detected.

Variability among isolates of Fusarium udum and the effect on progression of wilt in pigeonpea

Vascular wilt (caused by Fusarium udum) is the most important disease of pigeonpea. Breeding strategies are needed to develop durable resistance against this pathogen. Knowledge of the genetic and pathogenic variability among isolates of F. udum is essential for effective deployment of resistance. Thirteen isolates of F. udum, collected from three regions of India were studied using cultural characteristics, molecular variability and pathogenicity. The isolates of F. udum showed variability in aerial and radial mycelium growth, colour of mycelia, substrate pigmentation, length and septation of macro- and microconidia. Amplified fragment length polymorphism (AFLP) analysis of the isolates determined seven phylogenetic groups. Pathogenesis and progress of wilt on susceptible pigeonpea cultivars varied and pathogenic isolates from the same phylogenetic group exhibited similarities in timing of infection and colonization. Non-pathogenic isolates formed separate clusters in the phylogenetic tree. The timing of fungal invasion, vascular clogging, drooping of shoots, and wilt establishment were demonstrated for the first time in pigeonpea. Vascular clogging occurred with susceptible cultivars at 78–84, 54–60, 48–60 and 36–48 h post inoculation (HPI) when inoculated with isolates of F. udum from AFLP groups 1, 3, 6 and 7, respectively. Subsequently, susceptible cultivars inoculated with isolates from the same groups exhibited wilt at 144–168, 120–144, 96–120 and 72–96 HPI, respectively. This study contributes to characterising vascular wilt of pigeonpea.

Subtyping and antimicrobial susceptibility of Clostridium difficile PCR ribotype 078/126 isolates of human and animal origin

The Clostridium difficile PCR ribotype complex 078/126 (RT078/126) is often involved in human disease and is also frequently isolated from diverse animal species. The high genetic relatedness between human and animal RT078/126 isolates found in different regions has encouraged discussion about the zoonotic potential of this lineage. We compared for the first time the genetic diversity and antimicrobial susceptibility profiles of human and animal C. difficile RT078/126 isolates from Spain. A collection of 96 isolates (50 of human and 46 of animal origin; 63 and 33 of ribotypes 078 and 126, respectively) was subtyped by an improved amplified fragment length polymorphism (AFLP) fingerprinting method and tested for in vitro antimicrobial susceptibility. A total of 67 genotypes were distinguished, three of which grouped together isolates of human and animal origin. Furthermore, two main groups of isolates that mostly correlated with PCR ribotypes could be distinguished in the AFLP dendrogram. Human origin was significantly associated with resistance to ertapenem, erythromycin and moxifloxacin; resistance to clindamycin and erythromycin was associated with RT126 and AFLP group 1. Twenty-nine isolates (30.2% of total) displayed heteroresistance to metronidazole. Substantial differences were observed in the susceptibility profiles of isolates belonging to a same genotype. Altogether, these results provide a valuable baseline for future studies on the epidemiology of C. difficile RT078/126.

Genotypic and Antimicrobial Susceptibility of Carbapenem-resistant Acinetobacter baumannii: Analysis of is Aba Elements and bla OXA-23-like Genes Including a New Variant.

Carbapenem-resistant Acinetobacter baumannii (CR-AB) causes serious nosocomial infections, especially in ICU wards of hospitals, worldwide. Expression of blaOXA genes is the chief mechanism of conferring carbapenem resistance among CR-AB. Although some blaOXA genes have been studied among CR-AB isolates from Iran, their blaOXA-23-like genes have not been investigated. We used a multiplex-PCR to detect Ambler class A, B, and D carbapenemases of 85 isolates, and determined that 34 harbored blaOXA-23-like genes. Amplified fragment length polymorphism (AFLP) genotyping, followed by DNA sequencing of blaOXA-23-like amplicons of CR-AB from each AFLP group was used to characterize their blaOXA-23-like genes. We also assessed the antimicrobial susceptibility pattern of CR-AB isolates, and tested whether they harbored insertion sequences ISAba1 and ISAba4. Sequence comparison with reference strain A. baumannii (NCTC12156) revealed five types of mutations in blaOXA-23-like genes; including one novel variant and four mutants that were already reported from China and the USA. All of the blaOXA-23-like genes mutations were associated with increased minimum inhibitory concentrations (MICs) against imipenem. ISAba1 and ISAba4 sequences were detected upstream of blaOXA-23 genes in 19 and 7% of isolates, respectively. The isolation of CR-AB with new blaOXA-23 mutations including some that have been reported from the USA and China highlights CR-AB pervasive distribution, which underscores the importance of concerted national and global efforts to control the spread of CR-AB isolates worldwide.

Phylogeography of the heathers Erica arborea and E. trimera in the afro-alpine ‘sky islands’ inferred from AFLPs and plastid DNA sequences

The ericaceous vegetation zone of the unique and highly fragmented afro-alpine environment in the eastern African high mountains is typically dominated by the heather Erica arborea, often in combination with its close relative E. trimera. Both species are shrubs or small trees with tiny seeds, potentially capable of dispersal by wind over long distances. While E. arborea is widely distributed in Africa, the Middle East and Europe, E. trimera is endemic to the afro-alpine region where it is restricted to higher altitudes than E. arborea. We used Amplified Fragment Length Polymorphisms (AFLPs) and variation in non-coding plastid DNA sequences to test whether these two morphologically and ecologically very similar species display similar phylogeographic patterns in the afro-alpine region. We predict that the more high-altitudinal E. trimera shows more distinct genetic structuring than E. arborea, because dispersal of the latter may have been facilitated by formation of interglacial forest bridges between mountains. Based on extensive field sampling in most of the high mountains of Ethiopia and East Africa, we show that the two species are clearly distinct at AFLP and plastid DNA loci. Both showed low levels of overall AFLP diversity, suggesting bottlenecking in small refugial populations during unfavourable climatic periods. However, their genetic structuring and inferred phylogeographic histories were conspicuously different. The more high-altitudinal E. trimera consisted of three to four distinct AFLP groups, which also had different plastid DNA haplotypes and different geographic distributions, suggesting long-term restriction to several refugia (at least one in Ethiopia and two in East Africa). In contrast, E. arborea showed little geographic structuring at AFLP loci and only a single, widespread plastid DNA haplotype, which may suggest recent colonization of the entire study area from a single source population, likely via a combination of gradual expansion via forest bridges and long-distance dispersals. The source population of E. arborea may be situated in (or north of) Ethiopia, which harbours most genetic diversity.

Analisis Keragaman Genetik Temulawak (Curcuma xanthorrhiza Roxb.) Menggunakan Penanda Amplified Fragment Length Polymorphism (AFLP)

Curcuma xanthorrhiza Roxb, which is well-known as Java turmeric, has been extensively used in pharmaceutical industries in Indonesia. In spite of this commercial value, the identity of this species is commonly mistaken from other similar orange rhizomes Curcuma. Correct identity of these species is vital in pharmaceutical industries. The objective of the study was to determine genetic diversity of 32 accession Curcuma xanthorrhiza Roxb. Genomic DNA was extracted from leaf using Sodium Dodesyl Sulphate (SDS) modification. Amplified fragment length polymorphism (AFLP) was carried out according to the protocol ofAFLPTM plant mapping kit and the final polymerase chain reaction (PCR) products were separated using The Agilent 2100 Bioanalyzer. The number of fragment produced by 12 pairs primer combination of AFLP ranged from 42 to 60 with an average of 52. Data obtained was analyzed by the NTSys program. From the AFLP amplification on 32 DNA samples, it was proven that the accession of Curcuma xanthorrhiza Roxb. had a high degree of diversity. Based on analysis of AFLP and unweighted pair group with arithme average (UPGMA) it was shown that the accession of Curcuma xanthorrhiza Roxb. could be grouped into two cluster at relative ecludian distance of 0.10 (10%). Cluster I for accession from Palembang, Pacitan and Ciamis 2. Cluster II for accession from Makale, Pontianak, Kulonprogo, Mataram, Boyolali, Salatiga, Sumberejo, Bali, P. Seram, Sentolo, Purworejo, Samas Bantul, Ciamis1, Blora, Semarang, Poso, Kalsesl, Tagari, Merapi Farm, Salakaria, NTB, Menoreh, Karang Anyar, Mangunan, Medan, Toraja, dan Solok.

Identification of bacteria in drinking water with Raman spectroscopy

Raman spectroscopy was used to discriminate between Legionella strains and between E. coli and coliform strains. The relationship between triplicate Raman spectra derived from Legionella bacteria was compared with that derived from a blind set of samples and amplified fragment length polymorphism (AFLP) data from the same strains. Triplicate Raman spectra of E. coli and coliform bacteria were compared with their 16S phylogeny. In all cases Raman spectra were reproducible and could be distinguished from spectra of other organisms down to the strain level. All samples in a blind fourth set were identified correctly. Raman spectra of organisms of the same coliform species clustered according to 16S rRNA gene phylogeny, except for Enterobacter spp. At higher taxonomic levels the relationship between species was less comparable. For Legionella strains the Raman spectra grouped according to AFLP groups, based on the dataset used in this study. Raman spectroscopy could correctly distinguish E. coli from other coliform bacteria and L. pneumophila from non-pneumophila strains. Incubation of Legionella strains in different types of drinking water at different temperatures over a period of one week introduced so little variation in the Raman spectra that only very closely related L. pneumophila strains could not be distinguished from each other. Temperature, ageing and water type did not influence the identification potency of Raman spectroscopy in all cases. Given the accuracy, speed and simplicity of the Raman spectroscopy technique this method seems a welcome addition to the current tools for identification of waterborne bacteria.

AFLP analysis shows high incongruence between genetic differentiation and morphology-based taxonomy in a widely distributed tortoise

Morphology has traditionally been used to diagnose the taxa of various taxonomic ranks. However, there is growing evidence that morphology is not always able to reveal cryptic taxa, and that pronounced morphological variation could reflect phenotypic plasticity rather than evolutionary divergence. Spur-thighed tortoises (the Testudo graeca complex), distributed in the western Palaearctic region, are characterized by high morphological variability and complicated taxonomy, which are under debate. Previous molecular studies using mainly mitochondrial DNA (mtDNA) sequences have revealed incongruence between genetic differentiation and morphology-based taxonomy, suggesting that morphological variability is the result of phenotypic plasticity and stabilizing selection, which masks the true genealogies. In the present study, we used a range-wide sampling and nuclear Amplified fragment length polymorphism (AFLP) markers to investigate genetic differentiation within the T. graeca complex. We found that spur-thighed tortoises are differentiated into four geographically well-defined AFLP groups: Balkans–Middle Eastern, western Mediterranean, Caucasian and central-eastern Iranian. Compared with the distribution of mtDNA lineages, the groups are largely concordant, although the AFLP markers are less sensitive and distinguish fewer groups than do mtDNA sequences. The AFLP groups show an allopatric or parapatric distribution. The AFLP differentiation conflicts with the previously proposed morphology-based taxonomy of the complex, suggesting that local adaptation to different environmental conditions may have led to the great extent of morphological variation within the same lineages. We propose a re-evaluation of the taxa that were confirmed genetically using a thorough morphological analysis corrected for phenotypic plasticity. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●●, ●●–●●.

Genome mapping in F1 population of crossbred Italia and Mercan grape varieties: establishment of AFLP and SSR linkage groups towards significant morphological characters and fungal diseases.

This research aims at the establishment of a genome map for F 1 population developed by crossbreeding of Italia and Mercan grape varieties. A co-dominant marker SSR (Simple Sequence Repeats) and a dominant marker AFLP (Amplified Fragment Length Polymorphism) were used in the study. Double-pseudotestcross mapping technique was used for genome mapping. The study analyzed 60 plants selected from the F 1 population as well as the parental grape varieties, which differed in regards to resistance to diseases and some morphological characteristics. Amplification products were evaluated as “available” and “not available” at the end of silver staining for SSR and radioactive marking for AFLP. Polymorphic loci were determined according to their segregation ratios ranging between 1: 1 and 3: 1 at the end of X 2 tests. 3.0 LOD value was used in Mapmaker/Exp 3.0 package program for mapping. Two separate genetic linkage maps (maternal and paternal) were observed which included 6 and 1 linkage groups, respectively. Linkage of the loci located on the linkage groups to the observed diseases and morphological characters were analyzed with regression and variance analyses. A total of 818 primer pairs were tested on the two parents (Italia and Mercan), 60 F 1 (Italia × Mercan population) and two reference grape cultivar (Cabarnet Sauvignon and Merlot), successfully amplifying 112 markers. When the resistance traits to fungal diseases were analyzed during the study, no markers related with resistance to Botrytis cinerea and downy mildew could be found. However, it was found that the markers (VrZAG29a, VrZAG29c) related with the control of resistance to powdery mildew were on a locus which was in linkage group. Keywords: Vitis vinifera L., grape, SSR, AFLP, mapping African Journal of Biotechnology Vol. 9(36), pp. 5879-5886, 6 September, 2010

The molecular grouping of Fusarium isolates on cassava in Nigeria

The Fusarium isolates molecularly grouped in this experiment were isolated from the roots of three cassava genotypes (TMS 30572, TMS 4(2)1425 and TME-1) harvested in Ibadan derived savanna, Sabongidda-Ora humid forest and the Onne humid forest regions of Nigeria in 2004 and 2005. Isolates were previously identified using conventional morphological characters at the Plant Pathology Laboratory of the International Institute of Tropical Agriculture, Ibadan. Molecular grouping was done using amplified fragment length polymorphism at the Fusarium Laboratory of the Kansas State University, Kansas, USA. The molecular groupings agreed with the conventional morphological identification. Ten distinct amplified fragment length polymorphism (AFLP) groups of Fusarium were distinguishable, each group probably a distinct species, and many of them might represent previously undescribed Fusarium species. The two largest of the AFLP groups correspond to F. oxysporum and F. solani species complex. F. solani is an important root rot pathogen of cassava Nigeria.

The extreme Beringian/Atlantic disjunction in Saxifraga rivularis (Saxifragaceae) has formed at least twice

AbstractAim The oceanic Saxifraga rivularis L. presents one of the most extreme disjunctions known in the arctic flora: it has a small amphi‐Beringian range and a larger amphi‐Atlantic one. It was recently suggested to have had a single allopolyploid origin in Beringia at least one glacial cycle ago, followed by gradual expansion in a more humid period and differentiation into two allopatric subspecies (the Atlantic ssp. rivularis and the Beringian ssp. arctolitoralis). Here we explore the history of its extreme disjunction.Location The amphi‐Beringian and northern amphi‐Atlantic regions.Methods We obtained amplified fragment length polymorphisms (AFLPs) and chloroplast DNA sequences from 36 populations (287 individuals) and 13 populations (15 individuals), respectively. The data were analysed using principal coordinates analyses, Bayesian clustering methods, and analyses of molecular variance.Results Two distinctly divergent AFLP groups were observed, corresponding to the two described subspecies, but, surprisingly, four of the West Atlantic populations belonged to the supposedly Beringian endemic ssp. arctolitoralis. This was confirmed by re‐examination of their morphological characteristics. The overall AFLP diversity in the species was low (26.4% polymorphic markers), and there was no variation in the five investigated chloroplast DNA (cpDNA) regions. There was little geographic structuring of the AFLP diversity within each subspecies, even across the extreme disjunction in ssp. arctolitoralis, across the Bering Sea, and across the Atlantic Ocean, except that most plants from the arctic Svalbard archipelago formed a separate genetic group with relatively high diversity.Main conclusions The extreme disjunction in S. rivularis has evidently formed at least twice. The first expansion from Beringia was followed by allopatric differentiation into one Beringian and one Atlantic subspecies, which are distinctly divergent at AFLP loci but still harbour identical cpDNA haplotypes, suggesting that the expansion was quite recent but before the last glaciation. The next expansion from Beringia probably occurred by means of several long‐distance dispersals in the current interglacial, resulting in the colonization of the western Atlantic region by ssp. arctolitoralis. The poor geographic structuring within each subspecies suggests frequent long‐distance dispersals from two main Weichselian refugia, one Beringian and one western‐central European, but it is possible that the genetic group in Svalbard originates from an additional refugium.

How did an annual plant react to Pleistocene glaciations? Postglacial history of Rhinanthus angustifolius in Europe

The impact of climate fluctuations during the Pleistocene on the geographic structure of genetic variation in plant populations is well documented, but there is a lack of studies of annual species at the European scale. The present study aimed to infer the history of the widespread European annual Rhinanthus angustifolius C. C. Gmelin (Orobanchaceae). We explored variation in chloroplast DNA (cpDNA) sequences and amplified fragment length polymorphism (AFLP) in twenty-nine populations covering the entire distribution area of the species. Five AFLP groups were identified, suggesting at least two glacial refugial areas: one area in southwestern Europe and one large eastern area in the Balkan/Caucasus. Recolonization of previously glaciated areas mainly took place from the east of Europe. Despite the difference in life-history traits, the patterns found for the annual R. angustifolius show similarities with those of perennial species in terms of genetic diversity and geographic organization of genetic variation. Although organelle markers have typically been preferred in phylogeographic studies, the cpDNA variation in R. angustifolius did not show any clear geographic structure. The absence of geographic structure in the cpDNA variation may reflect persistence of ancestral polymorphisms or hybridization and introgression with closely-related species.

Aggressiveness ofFusarium graminearum sensu strictoIsolates in Wheat Kernels in Argentina

This study was designed to investigate the degree of aggressiveness of Fusarium graminearum sensu stricto isolates and its relationship with trichothecene production. In order to characterize Fusarium strains aggressiveness, disease severity was visually assessed as the percentage of spikelets bleached per ear. The severity ranged from a minimum of 27.19% seven days after inoculation to a maximum of 84.73% at the end of the experiment. At maturity the ears were harvested and threshed for grain weight determination. All treatments showed significant differences in kernel weight with respect to the control plants, with a yield reduction of 35-85% in comparison with the yield of the control. Grains infected by F. graminearum may contain significant levels of mycotoxins like trichothecenes. No correlation was found between aggressiveness and the toxins in the kernels at maturity, taking into consideration not only the quantity but also the type of toxin (nivalenol, deoxynivalenol or their acetylderivatives). Kernel weight reduction was a better estimator of the presence of deoxynivalenol in the kernels than the area under the disease progress curve (AUDPC) calculated with severity ratings. The amplified fragment length polymorphism (AFLP) technique was used to establish genetic relationships between 18 Argentinean isolates and eight reference strains of the Fusarium graminearum complex. All the isolates studied grouped with the two F. graminearum s. str. reference isolates, with a similarity coefficient greater than 75%. The other reference strains of the F. graminearum complex were clearly separated, with similarities ranging between 55 and 73%. The AFLP groups had no relationship with toxin accumulation on kernels or with the geographical origin of the isolates. Great heterogeneity was found in the AUDPC, yield reduction and toxin accumulation values across the regions.

Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

VCG and AFLP analyses identify the same groups in the causal agents of mango malformation in Brazil

The causal agents of mango malformation disease in Brazil are a new Fusarium lineage in the Gibberella fujikuroi species complex and Fusarium sterilihyphosum; however information on the genetic and geographical diversity of these pathogens in Brazil is missing. Vegetative compatibility group (VCG) and amplified fragment length polymorphism (AFLP) analyses were used to measure the genetic diversity within these populations. Both techniques identified the same genetic groups. Six VCG and AFLP groups were identified amongst isolates of the new lineage from Brazil. FB-VCG 1/AFLP I was the most widespread group, found in seven of the 13 sites sampled. The second most frequent group was recovered from three sites. The remaining four groups were recovered from single-sites. We think that this lineage represents a genetically and geographically diverse indigenous population that reproduces clonally. In F. sterilihyphosum, group FS-VCG 1/AFLP VII was found at three sites in the southeast region of Brazil. FS-VCG 2/AFLP VIII contained isolates from South Africa but not from Brazil. Fusarium mangiferae isolates from India and South Africa formed one group, while isolates from Egypt and the USA formed a second group. F. sterilihyphosum at present is represented by a small population that might have been introduced only once into a restricted area. The clonal nature of the observed populations suggests that these fungi either occur naturally on indigenous hosts and have jumped to the introduced mango host (introduced in Brazil) or that they originated with mango and went through a severe population bottleneck when they were introduced to Brazil from India or Southeast Asia.

Nuclear vs. plastid data: complex Pleistocene history of a circumpolar key species

To fully understand the contemporary genetic structure of plants, both nuclear and plastid markers are needed. Three chloroplast DNA (cpDNA) lineages, which probably diverged before the major Pleistocene glaciations, have been identified in the circumpolar/circumboreal Vaccinium uliginosum. Here we investigate its nuclear DNA variation using nuclear ribosomal internal transcribed spacer (ITS) sequences, DNA ploidy level measurements and amplified fragment length polymorphisms (AFLPs). We also extend the cpDNA dataset. Two ITS lineages, corresponding to diploids and tetraploids, respectively, were identified. However, both main sequence types apparently occurred in most individual plants but showed ploidy-biased homogenization and possibly reflect paralogy predating the origin of V. uliginosum. The ploidy levels were largely consistent with the cpDNA lineages, suggesting that the initial cpDNA divergence followed early polyploidizations. Five main AFLP groups were identified, consistent with recent glacial refugia in Beringia, western Siberia, the southern European mountains and areas south/east of the Scandinavian and Laurentide ice sheets. Except from the southern European mountains, there has been extensive expansion from all refugia, resulting in several contact zones. Surprisingly, the presumably older ploidy and cpDNA patterns were partly inconsistent with the main AFLP groups and more consistent with AFLP subgroups. A likely major driver causing the inconsistencies is recent nuclear gene flow via unreduced pollen from diploids to tetraploids. This may prevent cytoplasmic introgression and result in overlayed patterns formed by processes dominating at different time scales. The data also suggest more recent polyploidizations, as well as several chloroplast capture events, further complicating this scenario. This study highlights the importance of combining different marker systems to unravel intraspecific histories.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Amplified fragment length polymorphism of Streptococcus suis strains correlates with their profile of virulence-associated genes and clinical background

Amplified fragment length polymorphism (AFLP) typing was applied to 116 Streptococcus suis isolates with different clinical backgrounds (invasive/pneumonia/carrier/human) and with known profiles of virulence-associated genes (cps1, -2, -7 and -9, as well as mrp, epf and sly). A dendrogram was generated that allowed identification of two clusters (A and C) with different subclusters (A1, A2, C1 and C2) and two heterogeneous groups of strains (B and D). For comparison, three strains from each AFLP subcluster and group were subjected to multilocus sequence typing (MLST) analysis. The closest relationship and lowest diversity were found for patterns clustering within AFLP subcluster A1, which corresponded with sequence type (ST) complex 1. Strains within subcluster A1 were mainly invasive cps1 and mrp+ epf+ (or epf*) sly+ cps2+ strains of porcine or human origin. A new finding of this study was the clustering of invasive mrp* cps9 isolates within subcluster A2. MLST analysis suggested that A2 correlates with a single ST complex (ST87). In contrast to A1 and A2, subclusters C1 and C2 contained mainly pneumonia isolates of genotype cps7 or cps2 and epf- sly-. In conclusion, this study demonstrates that AFLP allows identification of clusters of S. suis strains with clinical relevance.

Genetic variation and population structure of Fusarium oxysporum f.sp. vasinfectum in Australia

Genetic variation among 348 isolates of Fusarium oxysporum f.sp. vasinfectum (Fov) collected from diseased cotton plants in 31 fields in six cotton‐growing regions in New South Wales and Queensland in 2002 and 2004 was analysed using amplified fragment length polymorphisms (AFLPs). Twenty‐eight haplotypes were identified based on 146 polymorphic bands generated with four EcoRI and MseI and four HindIII and MseI primer combinations. The haplotypes separated into two distinct groups (37% similarity), with 21 in group I and seven in group II. The two unique vegetative compatibility groups of Fov known to occur in Australia (VCG 01111 and VCG 01112) were correlated to the two AFLP groups, with both VCG 01111 reference isolates being included in group I and both VCG 01112 reference isolates in group II. Group I was widespread, occurring in all regions sampled and all but one of the fields, while group II was limited to three fields in the Boggabilla region. Group I was further divided into two subgroups. The two haplotypes in subgroup I‐B (I‐20 and I‐21) may represent the emergence of a new form of Fov based on their marked genetic discrimination from the subgroup I‐A haplotypes. No spatial population differentiation was discernible at the national level, as only 3·9% of total genetic variation was attributed to differences among regions (P = 0·4868). When each region was analysed separately, clear differentiation was found in the Boggabilla region, with 86·3% of total genetic variation resulting from differences among fields (P < 0·0001).

Fusarium Species from the Cassava Root Rot Complex in West Africa

ABSTRACT Fusarium species are a significant component of the set of fungi associated with cassava root rot. Yield losses due to root rot average 0.5 to 1 ton/ha but losses >3 ton/ha, an equivalent of 15 to 20% yield, often occur. This paper reviews previous work on cassava root rot and summarizes a few recent studies on Fusarium species associated with the disease. Our studies in Cameroon showed that 30% of rotted tubers were infected by Fusarium spp. 12 months after planting and represented 25% of all the fungal isolates recovered. Other commonly recovered fungi were Botryodiplodia theobromae and Armillaria spp. Numerous and diverse species of Fusarium were associated with rotted cassava roots in Nigeria and Cameroon. At least 13 distinct amplified fragment length polymorphism (AFLP) groups of Fusarium were distinguishable, each group probably a distinct species, and many of them might represent previously undescribed Fusarium species. The two largest of the AFLP groups correspond to F. oxysporum and F. solani species complex. The distribution of Fusarium spp. varied among countries and among locations within a country, suggesting that germ plasm resistant at one location may not be resistant at another. Fusarium spp. also cause seedling blight of cassava and can be recovered from the stems of infected plants up to 1 m above the ground. Therefore, the pathogen can spread with stems cut as planting material. Fusarium spp. also can colonize Chromolaena odorata, the dominant weed in short fallows, which could further complicate management efforts by serving as an alternative host for strains that colonize cassava.