Identification Of Pathotypes Research Articles

Synchytrium endobioticum

<i>Synchytrium endobioticum</i>

Study of European and Czech populations of potato cyst nematodes (Globodera rostochiensis and G. pallida) by RAPD method

Potato cyst nematodes (PCN) are the big problem in worldwide planting of potatoes and another Solanaceous plants. Identification of individual pathotypes according to international scheme is very demanding but a very important part of the phytosanitary process to control these pests. Molecular genetic identification of different plant and animal species or individuals is a very interesting way at the present time and let&amp;rsquo;s hope that it will be important in future. This report presents results of the RAPD study of nine different real PCN populations. There were five Globodera rostochiensis populations and four G. pallida populations. Pathotypes Ro2, Ro2/3, Ro4, Ro5, Pa2 and Pa3 were from European populations; population Ro1 and X were of Czech provenance. Genetics variable of these populations was described by a set of six decameric primers (OPA 07, OPG 03, OPG 05, OPG 08, OPG 10 and OPG 13). Genetic dissimilarity was by Gel Manager for Windows evaluated. Detectable differences behind all populations were found and the dendrogram was compiled. The unknown population X was sorted into group of Globodera pallida species subgroup of Pa2 consequently.

European virulence survey for leaf rust in wheat

With standardised near isogenic line (NIL) differentials co-operators were able to present the first comprehensive virulence survey of the European wheat leaf rust population (1996-1999). The work included pathotype identification of 2608 isolates and field tests of NILs. Lr9 and Lr19 were very effective all over Europe. Lr24, Lr25, and Lr28 were also effective, but in some countries and locations substantial virulence frequencies were observed. In addition, the genes Lr12, Lr13, Lr22a, Lr34, Lr35 and Lr37 were effective at the adult plant stage, but locally less so. In general, the indoor seedling tests and adult plant field tests showed good agreement. Virulence to Lr1, Lr2a, Lr24, Lr25, Lr28 and Lr29 tended to increase in the period, for the other Lr-genes the virulence frequency remained more or less stable. Among the 105 pathotypes identified none was clearly predominant in Europe.

Stem rusts on wheat, barley, and oat in Canada in 1996 and 1997

In 1996, stem rusts of wheat and barley [Puccinia graminis f. sp. tritici] and oat [P. graminis f. sp. avenae] were light in eastern Canada, and in western Canada they were confined mainly to the Red River Valley of Manitoba, where maximum severities of 40% occurred in lines of susceptible wheat in nurseries. In 1997 only traces of stem rust were found on any host at any location. There were no infections in commercial wheat fields in 1996 or 1997. Only trace levels of stem rust were found in cultivated barley both years. There was no stem rust in commercial oat fields in 1996 or 1997. Infections in wild oat did not exceed 15% in either year and occurred only in Manitoba. In 1997, most isolates collected from cultivated or wild barley, and about one-half of those collected from wheat, were rye stem rust [P. graminis f. sp. secalis]. Pathotype identification was made on only 14 collections obtained from wheat, and on 19 from wild or cultivated barley in Manitoba in 1997. During both years, 24 pathotypes of P. g. tritici were identified in collections from all sources. TPMKR was the most common in 1996 and 1997 on wheat and cultivated or wild barley in Manitoba and Ontario. From collections obtained from British Columbia in 1997, QFCSR and RCCJN were the most common. In 1996 QCCJD was common on cultivated and wild barley in Manitoba, but was not detected in 1997. A similar pathotype (QCCJN), however, was commonly found in 1997. All four isolates obtained from cultivated barley in British Columbia were QCCJN. All isolates of P. g. tritici collected from wheat in Canada and from cultivated and wild barley in Manitoba were virulent to the individual genes Sr5, Sr21, Sr9g, and Sr9d. All isolates were avirulent to genes Sr22, Sr24, Sr25, Sr26, Sr27, Sr29, Sr30, Sr3J, Sr32, Sr33, Sr35, Sr37, and Sr40; to a series of resistant lines (Mida-McMurachy-Exchange, Frontana-K58-Newthatch, CI8154/2Frocor, Alpha 16, BW197, RL6076, RL6088, RL6172); to the bread wheat cultivars Kenya Farmer, Agatha, Glenlea, Katepwa, Leader, Roblin, AC Taber, Grandin, and AC Domain; and to the durum cultivars Plenty, AC Melita, and Sceptre. From collections of P. g. avenae in Manitoba, NA29 was the most common in 1996 and 1997. Other pathotypes were NA18, NA27, NA28, NA30, and NA68. Only three collections were obtained from Ontario each year. In 1996 two were NA26 and one was NA32, and in 1997 all were NA25.

Fate of biological control introductions: monitoring an Australian fungal pathogen of grasshoppers in North America.

In North America there are two generally recognized pathotypes (pathotypes 1 and 2) of the fungus Entomophaga grylli which show host-preferential infection of grasshopper subfamilies. Pathotype 3, discovered in Australia, has a broader grasshopper host range and was considered to be a good biocontrol agent. Between 1989 and 1991 pathotype 3 was introduced at two field sites in North Dakota. Since resting spores are morphologically indistinguishable among pathotypes, we used pathotype-specific DNA probes to confirm pathotype identification in E. grylli-infected grasshoppers collected at the release sites in 1992, 1993, and 1994. In 1992, up to 23% of E. grylli-infected grasshoppers of the subfamilies Melanoplinae, Oedipodinae, and Gomphocerinae were infected by pathotype 3, with no infections > 1 km from the release sites. In 1993, pathotype 3 infections declined to 1.7%. In 1994 grasshopper populations were low and no pathotype 3 infections were found. The frequency of pathotype 3 infection has declined to levels where its long-term survival in North America is questionable. Analyses of biocontrol releases are critical to evaluating the environmental risks associated with these ecological manipulations, and molecular probes are powerful tools for monitoring biocontrol releases.

Pathotypes in the Entomophaga grylli species complex of grasshopper pathogens differentiated with random amplification of polymorphic DNA and cloned-DNA probes

The zygomycetous fungus Entomophaga grylli is a pathogen that shows host-specific variance to grasshopper subfamilies. Three pathotypes of the E. grylli species complex were differentiated by three molecular techniques. In the first method, the three pathotypes showed different fragment patterns generated by random amplification of polymorphic DNA (RAPD). There was little or no interisolate variability in RAPD fragment patterns within each pathotype. Passage of an isolate of pathotype 3, originally from an Australian grasshopper (Praxibulus sp.), through a North America grasshopper resulted in no differences in the resultant RAPD fragment patterns. In the second method, polymorphic RAPD fragments were used to probe the genomic DNA from the three pathotypes, and pathotype-specific fragments were found. In the third method, restriction fragments from genomic DNA of the three pathotypes were cloned and screened for pathotype specificity. A genomic probe specific for each pathotype was isolated. These probes did not hybridize to DNA from Entomophaga aulicae or from grasshoppers. To facilitate the use of RAPD analysis and other molecular tools to identify pathotypes, a method for extracting DNA from resting spores from infected grasshoppers was developed. The DNA from the fractured resting spores was of sufficient integrity to be blotted and probed with the pathotype-specific DNA probes, thus validating the use of these probes for pathotype identification in field-collected grasshoppers.

Characterization and Molecular Detection of the P4 Pathotype of Pea Seedborne Mosaic Potyvirus

The New York isolate of pea seedborne mosaic potyvirus (PSbMV) pathotype P4 was differentiated from the more prevalent PSbMV-P1 pathotype on selected pea cultivars, serologically and by sequence-specific reverse transcription-polymerase chain reaction (RT-PCR). PSbMV-P4 was seed-transmissible at much lower rates than P1 (0 to 0.7% versus 0 to 32%) in selected pea cultivars and was not detectable by enzyme-linked immunosorbent assay (ELISA) in reproductive tissues (pollen or embryo axes) of pea, whereas P1 was readily detectable in these tissues. P4 also was aphid-transmitted at lower rates (4 to 16%) than those of Pl (11 to 40%) and had distinct virus acquisition-time optima. RT-PCR detected approximately 10 pg of P4 RNA per 1.0 μg of total nucleic acids from P4-infected tissues and provided positive pathotype identification in single or mixed infections. RT-PCR and pathotype-selective ELISA were used to determine seed-transmission rates of the two pathotypes in seedling progeny of pea (Pisum sativum) cultivars inoculated with P1, P4, or P1 + P4. There were slight variations in the frequency of P1 seed transmission in the presence of P4; P4 seed-transmission rates did not increase in the presence of P1.

Pathotype identification of Leptosphaeria maculans with PCR and oligonucleotide primers from ribosomal internal transcribed spacer sequences

In order to develop a pathotype-specific assay, the internal transcribed spacer region 1 (ITS 1) of ribosomal RNA genes of highly and weakly virulent isolates of Leptosphaeria maculans were sequenced using primers from the flanking 17S and 5·8S ribosomal DNA (rDNA). The sequenced ITS 1 region had approximately 67% similarity among highly and weakly virulent isolates, and 37–46% similarity between L. maculans and other species of fungi. From the ITS 1 sequences, two pairs of oligonucleotide primers were synthesized which specifically amplified DNA from either the highly or weakly virulent pathotype of L. maculans using the polymerase chain reaction (PCR). Primer pair HV17S/5·8S was based on sequences from the highly virulent isolates, Leroy and LM26, and only amplified highly virulent isolates. Primer pair WV17S/5·8S was based on sequences from the weakly virulent isolate, Unity, and only amplified weakly virulent isolates. This PCR-based assay utilizing pathotype-specific primers was employed to detect an isolate of the highly virulent pathotype of L. maculans in infected plant tissue and shows promise as a diagnostic tool.

Rapid identification of genetic variation and pathotype of Leptosphaeria maculans by random amplified polymorphic DNA assay

Canadian isolates of Leptosphaeria maculans, the causal agent of blackleg of crucifers, were examined for genetic relatedness by the random amplified polymorphic DNA assay. DNA polymorphisms amplified with random decamer primers were used to distinguish three groups of isolates. Group 1 contained all isolates of the virulent pathotype, group 2 contained isolates of the avirulent pathotype from western Canada, and group 3 contained avirulent pathotype isolates from Ontario. These results agreed with other reports which showed many genetic differences between pathotypes and were consistent with the hypothesis that the virulent pathotype was recently introduced into Canada and has diverged relatively little. In contrast, the avirulent pathotype has probably been present in Canada for a longer time and has diverged with geographic isolation. In addition to establishing genetic relationships, DNA fingerprints generated by the random amplified polymorphic DNA assay have potential applications in pathotype identification and blackleg disease management.

DNA fingerprinting of Ascochyta rabiei with synthetic oligodeoxynucleotides

The ascomycete fungus Ascochyta rabiei, an important pathogen of the grain legume crop chickpea (Cicer arietinum L.) in the Mediterranean region, has not been adequately characterized in molecular terms. We therefore used DNA fingerprinting, with synthetic oligodeoxynucleotides complementary to simple repetitive sequences, to pathotype different isolates of the fungus. Six single-spored A. rabiei isolates were first categorized using a host differential set of nine chickpea genotypes. Seedlings were inoculated under controlled environmental conditions, and disease severity was recorded 9 days after inoculation. DNA was extracted from in vitro-grown mycelia of the six purified fungal isolates, restricted with EcoRI, HinfI, MboII and TaqI, and fingerprinted with radiolabeled (GATA)4, (GTG)5, (CA)8, and (TCC)5, respectively. High levels of polymorphism were detected with optimal enzyme/probe combinations that allow one to discriminate between the isolates. The potential of DNA fingerprinting with simple repetitive sequences can thus be expanded to the identification of fungal races and pathotypes. The characterization of the geographic distribution and genetic variability of pathotypes will facilitate the selection of suitable host cultivars to be grown in specific regions.

Detection and identification of pathotypes of verocytotoxigenicEscherichia coliisolated from weaned piglets using gene probes for sevenE. colitoxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.

Detection and identification of pathotypes of verocytotoxigenic Escherichia coli isolated from weaned piglets using gene probes for seven E. coli toxins

Seventy verocytotoxigenic (VTEC) and sixty-three non VTEC haemolytic Escherichia coli isolated from recently weaned piglets were examined by the colony hybridization assay using gene probes for three verocytotoxins: Edema disease principle (EDP) and Shiga-like toxins I and II (SLTI and SLTII). The results with the EDP and SLTII probes were identical. All VTEC hybridized with these two probes, while non VTEC did not. All 133 E. coli were negative for the SLTI probe. Hybridization of the plasmid content of 14 VTEC did not show any evidence for plasmid localization of the genes coding for the EDP. The 70 VTEC were also assayed with gene probes for heat-stable (STaP, STb) and heat-labile (LT, LTIIa) enterotoxins. Only the STb probe was hybridized by 36 of them. Most STb-positive isolates belonged to serotype O141: K85 biotypes 9 and 13 PC.

Morphological—taxonomical studies and pathotype classification in potato cyst nematodes1

Tests on differential clones for pathotype determination of potato cyst nematode populations were supplemented with morphological studies of male spicules. Such studies often speed up the process and are sometimes necessary for a proper pathotype identification.

Glucose phosphate isomerase polymorphism in field populations of the potato cyst nematodes, Globodera rostochiensis and G. pallida

SUMMARYThe isozymic variation of glucose phosphate isomerase (GPI) was compared in 20 field populations of potato cyst‐nematode (PCN) and related to variation in reference pathotypes of PCN. Populations pathotyped as G. rostochiensis Ro 1 by conventional differential cultivar tests were found to be different from populations of G. pallida. Variation within populations pathoptyped as G. pallida was seen and three populations were found to be similar to the reference G. pallida Pal pathotype. This seemed to be confirmed for two populations using cysts formed on a cultivar resistant to the Pa 1 pathotype when an isoenzyme pattern characteristic of G. palida Pa2 and Pa3 was found. The feasibility of the use of isozyme identification of PCN pathotypes is discussed in the light of these results.

Suggestions for Determination and Terminology of Pathotypes and Genes for Resistance in Cyst‐forming Nematodes, especially Heterodera avenae1

A short review of differentiation into pathotypes is given. Use of the word ≪ pathotype ≫ is recommended when a very clear difference is established between virulence of nematode populations. Our present knowledge makes it possible to differentiate between 10 pathotypes of Heterodera avenae. It is suggested that the pathotypes are given numbers, and corresponding terms should be used for genes for resistance in plants, e.g. a gene Hal on the barley chromosome gives resistance to nematode pathotypes 11,21,31,41 etc. Some proposals for improvement of pathotype identification are given.

Identification of Pathotypes of the Potato Cyst Nematode1

AbstractSince the discovery of pathotypes of the potato cyst nematode (Heterodera rostochiensis Wollenweber, 1923), surveys on the distribution of these pathotypes have been carried out in different European countries. Test varieties of potato were selected according to findings resulting in the present situation of varying distinctive host‐plants and of diverging programs for breeding potatoes resistant to the potato cyst nematode.With respect to rational selection and use of resistant potato varieties, there is a great need for standardization in pathotype identification (nomenclature), maintenance and supply of appropriate genotypes of Solanum species and methods for testing resistance in new potato varieties.Anticipating a cross‐experiment with pathotypes and test varieties from Great Britain, the Federal Republic of Germany and the Netherlands in 1974, a preliminary test with all recognized or presumed pathotypes from these countries, but with only part of the potential differential host‐plants was carried out by the Plant Protection Service at Wageningen. The results of this experiment are discussed.