Nested Allele-Specific PCR Primers Distinguish Genetic Groups of Uncinula necator

Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14α-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season.     

Genetic similarity of flag shoot and ascospore subpopulations of Erysiphe necator in Italy

The overwintering mode of the grape powdery mildew fungus, Erysiphe necator (syn. Uncinula necator), as mycelium in dormant buds (resulting in symptoms known as flag shoots) or as ascospores in cleistothecia, affects the temporal dynamics of epidemics early in the growing season. We tested whether distinct genetic groups (I and III) identified previously in E. necator correlate to overwintering modes in two vineyards in Tuscany, Italy, to determine whether diagnostic genetic markers could be used to predict overwintering. Samples from one vineyard were collected from flag shoots; the other vineyard, 60 km away, had no flag shoots, and mildew colonies were assumed to be derived from ascospores. Genetic markers putatively diagnostic for groups I and III showed that both types were common in the flag shoot subpopulation. Both genetic types were found in the ascospore population, although group III was dominant. We did not find strong genetic differentiation between the two subpopulations based on inter-simple sequence repeat markers. Although there was significant (P < 0.001) genetic differentiation between these subpopulations in 1997 and when 1997 and 1998 subpopulations were pooled (theta = 0.214 and 0.150, respectively), no differentiation was evident between vineyards in 1998 (theta = 0.138, P = 0.872). Moreover, we did not observe distinct lineages corresponding to overwintering modes, as observed in previous studies. We could not determine if differentiation resulted from biological differences or restricted gene flow between the two vineyards. Our samples were taken from both subpopulations early in the epidemic, while previous studies confounded overwintering mode and sampling time. These results do not support a strong correlation between overwintering and genetic groups, highlighting the need to base population biology studies on sound biological and epidemiological knowledge.     

Random Amplified Polymorphic DNA and Polymerase Chain Reaction Markers for the Differentiation and Detection of Stenocarpella maydis in Maize Seeds

The genetic relationship of 34 isolates of Stenocarpella maydis from different geographic regions in South Africa was analysed by random amplified polymorphic DNA (RAPD) and ribosomal DNA markers. Two genetic groups were differentiated by using three RAPD primers and correlated to the cultural morphology of the isolates. Of all the isolates tested, 79.4% were clustered into RAPD group I (RG I), which did not sporulate when cultured on potato dextrose agar (PDA) at 25°C for 10 days. The rest of the isolates designated as RG II sporulated on PDA medium and showed a higher genetic variation. Ribosomal DNA (rDNA) was amplified using polymerase chain reaction (PCR) with the universal primers, internal transcribed spacer (ITS) 1 and ITS 4. Restriction digestion of PCR products displayed three types (RF A, RF B and RF C) of profiles. RF A was in accordance with RG I. RF B was consistent with RG II except for one isolate, U5. However, U5 displayed a unique profile and had no restriction sites for Hpa II and Hae III. The results indicate that two distinct genetic groups exist among S. maydis isolates from maize in S. Africa. The ITS1 and ITS2 regions of rDNA were sequenced and primers were designed. The designed primer pair P1/P2 permitted a sensitive and specific detection of S. maydis .     

Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses

Current identification methods for the soft rot erwinias are both imprecise and time-consuming. We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification. Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques. The ITS was amplified from Erwinia and other genera using universal PCR primers. After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III). Group I comprised all Erwinia carotovora subsp. atroseptica and subsp. betavasculorum isolates. Group II comprised all E. carotovora subsp. carotovora, subsp. odorifera, and subsp. wasabiae and E. cacticida isolates, and group III comprised all E. chrysanthemi isolates. To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes. Digestion with CfoI identified E. carotovora subsp. atroseptica and subsp. betavasculorum (group I) and E. chrysanthemi (group III) isolates, while digestion with RsaI identified E. carotovora subsp. wasabiae, subsp. carotovora, and subsp. odorifera/carotovora and E. cacticida isolates (group II). In the latter case, it was necessary to distinguish E. carotovora subsp. odorifera and subsp. carotovora using the alpha-methyl glucoside test. Sixty suspected soft rot erwinia isolates from Australia were identified as E. carotovora subsp. atroseptica, E. chrysanthemi, E. carotovora subsp. carotovora, and non-soft rot species. Ten "atypical" E. carotovora subsp. atroseptica isolates were identified as E. carotovora subsp. atroseptica, subsp. carotovora, and subsp. betavasculorum and non-soft rot species, and two "atypical" E. carotovora subsp. carotovora isolates were identified as E. carotovora subsp. carotovora and subsp. atroseptica.     

Multiple Mutations and Overexpression in the CYP51A and B Genes Lead to Decreased Sensitivity of Venturia effusa to Tebuconazole

Multiple demethylation-inhibiting (DMI) fungicides are used to control pecan scab, caused by Venturia effusa. To compare the efficacy of various DMI fungicides on V. effusa, field trials were conducted at multiple locations applying fungicides to individual pecan terminals. In vitro assays were conducted to test the sensitivity of V. effusa isolates from multiple locations to various concentrations of tebuconazole. Both studies confirmed high levels of resistance to tebuconazole. To investigate the mechanism of resistance, two copies of the CYP51 gene, CYP51A and CYP51B, of resistant and sensitive isolates were sequenced and scanned for mutations. In the CYP51A gene, mutation at codon 444 (G444D), and in the CYP51B gene, mutations at codon 357 (G357H) and 177 (I77T/I77L) were found in resistant isolates. Expression analysis of CYP51A and CYP51B revealed enhanced expression in the resistant isolates compared to the sensitive isolates. There were 3.0- and 1.9-fold increases in gene expression in the resistant isolates compared to the sensitive isolates for the CYP51A and CYP51B genes, respectively. Therefore, two potential mechanisms—multiple point mutations and gene over expression in the CYP51 gene of V. effusa isolates—were revealed as likely reasons for the observed resistance in isolates of V. effusa to tebuconazole.     

Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides.

We examined the genetic and plasmid diversity within natural populations of Pseudomonas syringae isolated from three ornamental pear nurseries in eastern Oklahoma. The bactericide spray regimen differed at each nursery; copper and streptomycin, only copper, and no bactericides were applied at nurseries I, II, and III respectively. Resistance to copper (Cur) and resistance to streptomycin (Smr) were determined for 1,938 isolates of P. syringae; isolates from nurseries I and II were generally Cur Sms; whereas most isolates from nursery III were Cus Sms. The plasmid profiles of 362 isolates were determined, and six, one, seven, and four plasmid profiles were obtained for Cur, Smr, Cur Smr, and Cus Sms isolates, respectively. All Smr plasmids contained sequences homologous to the strA and strB Smr genes from broad-host-range plasmid RSF1010 and were associated with Smr transposon Tn5393. Plasmids were placed into two groups on the basis of hybridization to the oriV and par sequences from pOSU900, a cryptic plasmid in P. syringae pv. syringae. A total of 100 randomly chosen P. syringae isolates from nurseries I and III were analyzed for genetic diversity by using the arbitrarily primed PCR (AP-PCR) technique. An analysis of chromosomal genotypes by AP-PCR revealed a high degree of genetic diversity among the isolates, and the results of this analysis indicated that the isolates could be clustered into two distinct groups. The plasmid profiles were specific to isolates belonging to particular AP-PCR groups. Within each AP-PCR group, identical plasmid profiles were produced by isolates that had different chromosomal genotypes, implying that plasmid transfer has played an important role in the dissemination of Cur and Smr within the populations studied.     

Molecular characterization of Pisolithus spp. isolates by rDNA PCR-RFLP

 Variation within ribosomal DNA (rDNA) genes of 19 isolates of Pisolithus from different geographic origins and hosts was examined by polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis. The primers utilized amplify rDNA regions in a wide range of fungi. One amplified region includes the internal transcribed spacer (ITS), which has a low degree of conservation. The ITS amplification products (640–750 bp) were digested with a variety of restriction endonucleases. Cluster analysis based on the restriction fragments grouped the isolates into three distinct groups: group I contained isolates collected in the northern hemisphere, except Pt 1, group II contained those collected in Brazil and group III contained isolate Pt 1. Additional analysis of other rDNA regions, IGS, 17 S and 25 S rDNA, resulted in similar groups. The data suggest that the taxonomy and systematics of this ectomycorrhizal fungus should be revised. Accepted: 16 September 1998     

Distribution of papG alleles among uropathogenic Escherichia coli isolated from different species

The distribution of alleles I, II and III of the P adhesin gene papG among Escherichia coli isolated from urinary tract infections in humans, dogs and cats was studied by PCR. Allele I was present in 6% and 5% of the human and cat isolates. Allele II as such was present in 30% and 22%, or in association with allele III in 12% and 2% of the human and canine isolates, respectively. Allele III was present in 33% of the human strains and predominated largely over allele II in E. coli isolates from cystitis of animal origin (72% in dog and 95% in cat strains). The three different classes of the PapG adhesin have been suggested to play a role in host specificity, for example human versus canine specificity. Recent studies, however, showed papG III positive human and dog cystitis isolates to be largely indistinguishable. We found the Class II allele in animal isolates and detected for the first time in Europe the Class I allele in a different genetic background than the J96-like clonal group. Our findings show that uropathogenic E. coli isolates from different species can have the same papG alleles and thus may have zoonotic potential.     

Presence of a novel 16S-23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.     

Sequence variation in the CYP51 gene of Blumeria graminis associated with resistance to sterol demethylase inhibiting fungicides

Resistance to sterol 14alpha-demethylase inhibiting fungicides (DMIs) has been correlated with mutations in the CYP51 gene, which encodes the target enzyme eburicol 14alpha-demethylase. To test the hypothesis that variation in the CYP51 gene explains variation for DMI sensitivity in barley and wheat powdery mildew species, this gene was sequenced from isolates of Blumeria graminis f.sp. hordei (Bgh) and f.sp. tritici (Bgt), respectively, which differed in their responses to DMIs in agricultural populations in the UK. Two single-nucleotide mutations in the CYP51 gene, which resulted in the amino acid substitutions Y136F and K147Q, were detected. K147Q is a novel mutation present only in Bgh isolates expressing very high levels of resistance. Sequence analysis of the CYP51 gene from the progeny of a cross between DMI-sensitive and resistant Bgh isolates showed that both mutations segregate with resistance, which is consistent with CYP51 controlling a major portion of DMI resistance. However, genetic analysis of resistance to the DMI triadimenol indicates that mutation of the CYP51 gene is not the only mechanism of resistance operating in B. graminis.     

Determination of genetic diversity among Indian isolates of Rhizoctonia bataticola causing dry root rot of chickpea

Genetic diversity among 27 isolates (23 from chickpea and 4 from other host crops) of Rhizoctonia bataticola representing 11 different states of India was determined by random amplified polymorphic DNA (RAPD), internal transcribed spacer restriction fragment length polymorphism (ITS-RFLP) and ITS sequencing. The isolates showed variability in virulence test. Unweighted paired group method with arithmetic average cluster analysis was used to group the isolates into distinct clusters. The clusters generated by RAPD grouped all the isolates into six categories at 40% genetic similarity. High level of diversity was observed among the isolates of different as well as same state. Some of the RAPD (OPN 4, OPN 12, and OPN 20) markers clearly distinguished majority of the isolates into the area specific groups. The ITS I, 5.8rDNA and ITS II regions of 11 isolates representing different RAPD groups were amplified with primers ITS 1 and ITS 4 and digested with seven restriction enzymes. The restriction enzymes DraI, MboI, RsaI, and AluI were found to be suitable for differentiating the isolates into five categories by showing isolate specific ITS-RFLP patterns. The isolates were variable in their nucleotide sequences of the ITS regions. This is the first study on genetic diversity among chickpea isolates of R. bataticola.     

The Cytochrome P450 Lanosterol 14α-Demethylase Gene Is a Demethylation Inhibitor Fungicide Resistance Determinant in Monilinia fructicola Field Isolates from Georgia

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14α-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 μg of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position −117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.     

Genetic Diversity and Structure of the Invasive Toxic Cyanobacterium <Emphasis Type="Italic">Cylindrospermopsis raciborskii</Emphasis>

Strains of the invasive toxic cyanobacteria Cylindrospermopsis raciborskii were genetically evaluated with four genetic markers encompassing in total 2.9 kb (16S rRNA, ITS longer spacer, ITS shorter spacer and rpoC1) to assess the phylogenetic relationships, genetic variation and population differentiation of the species across all five continents. The phylogenetic analysis showed that the C. raciborskii strains grouped into three well-supported distinct clusters: (I) European (II) African/American, and (III) Asian/Australian. The European group presented a high genetic similarity with the Asian and the Australian isolates than with the African and American isolates. Several Portuguese isolates were analyzed (n = 7) and revealed a low genetic differentiation with little geographical structure. The genetic distance among groups and phylogenetic relationships obtained in this study suggest that the recent invasion of C. raciborskii in Portuguese and other European temperate environments could have had its origin in the Asian and/or Australian continents.     

Biofilm formation by strains of Burkholderia cepacia complex in dependence of their phenotypic and genotypic characteristics

Biofilm formation was studied in 54 strains of Burkholderia cepacia complex isolated in 7 Moscow hospitals. 80% of strains (biofilm groups I and II) had the capacity to biofilm formation and only 16.7% of strains (group III) were not capable to biofilm formation. Molecular genetic methods allowed to identify one of the epidemic markers (CBL, IS hybrid sequence, Burkholderia Cepacia Epidemic Strain Marker - BCESM) in 46.7, 23.3, and 33.3% of strains from group I, II, and III respectively. Gene cepR from the Quorum Sensing system was identified in three biofilm groups in nearly equal frequency (92.3, 96.2 and 100% for group I, II, and III respectively), whereas cepl gene was found more often in group I (76.9%) and II (65.4%). Strains from all three groups had protease and lipase activity and 13.3% of group I strains had chitinolytic activity. B. cepacia strains from group I produced hemolysin in 33.3% of cases, from group II--in 26.6%, and from group III--in 11.1% of cases. The majority of Moscow hospital strains of B. cepacia complex were identified as B. cenocepacia (genomovar III, group A). RAPD-PCR method enabled to differentiate isolated strains into several genotypic variants. 13.3% of strains from group I were susceptible to imipenem/ciprofloxacin, as well as 33.3% of isolates from group II and 44.4% of isolates from group III.     

大豆疫霉根腐病菌的rDNA ITS序列分析

采用真菌核糖体基因转录间隔区(ITS)通用引物,PCR扩增了大豆疫霉根腐病菌具有差异的17个菌株的ITSI与ITS2,经过与DL2000的标准分子量DNA进行比较,得到了大约800~1000bp左右的片段,并对PCR产物进行了序列测定。以USA为外类群利用最大简约法构建了大豆疫霉根腐病菌的系统发生树,并分析了菌株之间的遗传进化关系。结果表明:不同菌株ITS1和ITS2在碱基构成上有很大差异,17个菌株大致分为4个谱系中,且来自于同一地区的菌株大都分布在同一谱系中,显示出地理上的差异。     

Brief Report of a New Highly Divergent Variant of Grapevine leafroll‐associated virus 3 (GLRaV‐3)

The alignment of the complete genomes of genetic variants of Grapevine leafroll‐associated virus 3 (GLRaV‐3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA‐dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein (dCP) gene. Cloning and sequencing of the 549‐bp RT‐PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV‐3, which shared only 69.6–74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517‐bp amplicon of the HSP70 gene of GLRaV‐3 generated in RT‐nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3–78.7% nt similarity with other variants of GLRaV‐3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT‐nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards.     

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.     

Oogonial biometry and phylogenetic analyses of the Pythium vexans species group from woody agricultural hosts in South Africa reveal distinct groups within this taxon

Pythium vexans fits into the internal transcribed spacer (ITS) clade K sensu Lévesque & De Cock (2004). Within clade K, P. vexans forms a distinct clade containing two enigmatic species, Pythium indigoferae and Pythium cucurbitacearum of which no ex-type strains are available. In South Africa, as well as in other regions of the world, P. vexans isolates are known to be heterogeneous in their ITS sequences and may consist of more than one species. This study aimed to investigate the diversity of South African P. vexans isolates, mainly from grapevines, but also citrus and apple using (i) phylogenetic analyses of the ITS, cytochrome c oxidase (cox) I, cox II, and β-tubulin regions and (ii) seven biometric oogonial parameters. Each of the phylogenies clustered P. vexans isolates into a single well-supported clade, distinct from other clade K species. The β-tubulin region was phylogenetically uninformative regarding the P. vexans group. The ITS phylogeny and combined cox I and II phylogenies, although each revealing several P. vexans subclades, were incongruent. One of the most striking incongruences was the presence of one cox subclade that contained two distinct ITS subclades (Ib and IV). Three groups (A-C) were subjectively identified among South African P. vexans isolates using (i) phylogenetic clades (ITS and cox), (ii) univariate analysis of oogonial diameters, and (iii) multivariate analyses of biometric oogonial parameters. Group A is considered to be P. vexans s. str. since it contained the P. vexans CBS reference strain from Van der Plaats-Niterink (1981). This group had significantly smaller oogonial diameters than group B and C isolates. Group B contained the isolates from ITS subclades Ib and IV, which formed a single cox subclade. The ITS subclade IV isolates were all sexually sterile or produced mainly abortive oospores, as opposed to the sexually fertile subclade Ib isolates, and may thus represent a distinct assemblage within group B. Although ITS subclade Ib included the P. indigoferae ex-type sequence, this group was considered to be P. vexans since South African isolates in this clade produced globose sporangia. Group C contained four apple isolates that were related to, but distinct from P. cucurbitacearum. Although P. vexans groups A-C might be distinct species, they are not described here as such due to (i) these groups only representing some of the known diversity in P. vexans, (ii) conflicting gene tree phylogenies preventing phylogenetic species identification, and (iii) sexually sterile isolates preventing the broad application of biometrical data.     

A comparison of Olpidium isolates from a range of host plants using internal transcribed spacer sequence analysis and host range studies

Olpidium brassicae is a ubiquitous obligate root-infecting fungal pathogen. It is an important vector of a wide range of plant viruses. Olpidium isolates that infected brassica plants did not infect lettuce plants and vice-versa. Host range tests, PCR amplification and sequencing of the internal transcribed spacer (ITS) and 5.8S regions of 25 Olpidium isolates from brassica, carrot, cucumber and lettuce originating from four continents revealed differences between isolates. Based on their ability to infect lettuce and brassicas and the differences between their ITS1, 5.8S and ITS2 regions they could be separated into a number of distinct groups. Comparisons with other published sequences revealed two distinct genetic groups of brassica-infecting isolates, two distinct groups of lettuce-infecting isolates, one of which contained a carrot-infecting isolate and a distinct group comprising a cucumber-infecting isolate and a melon-infecting isolate. The possibility of the isolates belonging to three distinct species is discussed.     

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae)

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.