N605ab,a specific molecular marker for <Emphasis Type="Italic">Phytophthora infestans</Emphasis>, distinguishes three genotypes in Japan

Random amplified polymorphic DNA (RAPD) analysis using the OPG-06 primer generated specific patterns for Japanese genotypes US-1, JP-1, and a new A1 (JP-2, JP-3, and JP-4) of Phytophthora infestans. N605, a specific RAPD fragment, was cloned and sequenced. PCR primers BD1/BD2 were constructed based on the N605 sequence and were used to clarify the genotypes. PCR products using the BD1/BD2 primers (N605ab marker) easily distinguished the new A1 from US-1 and JP-1. This technique provides a simple and effective method for rapid genotype discrimination that can be used in ecological experiments and forecasts for the occurrence of late blight.     

Comparison of Chinese and Japanese A1 isolates of <Emphasis Type="Italic">Phytophthora infestans</Emphasis>

The mating type, glucose-6-phosphate isomerase (Gpi) and peptidase (Pep) genotypes, RG57 fingerprint, and mitochondrial DNA (mtDNA) haplotype of Chinese isolates of Phytophthora infestans collected in Hebei and Gansu in 1996 were compared with those of Japanese isolates collected during 1997–2000. The Chinese isolates were divided into four genotypes, one of which was identical to the dominant Japanese genotype, A1-A (mating type A1; Gpi 100/100; Pep 100/100; RG57 100010001100110100011001110: 1–25, 14a, and 24a; and mtDNA haplotype IIa). Comparison of the genotypes with reported data revealed that some completely and partially identical genotypes occur in Russia and parts of Europe. The other two A1 genotypes and one A2 genotype were also detected in Gansu (Gpi 100/100, Pep 100/100, and mtDNA haplotype Ia), which were regarded as unique to this region.     

Use of microsatellite marker Pi26 for rapid discrimination of five Japanese genotypes of Phytophthora infestans

Microsatellite markers were tested to rapidly discriminate five Japanese genotypes (US-1, JP-1, JP-2, JP-3, and JP-4) of Phytophthora infestans. Collected from 1958 to 2007, 111 isolates of Japanese P. infestans were examined using a fluorescent-labeled primer and capillary electrophoresis. Microsatellite marker Pi26 generated specific products for each genotype without any differences in terms of isolation area or year for a particular genotype. The Pi26 marker is a powerful tool for obtaining information on the structure of Japanese populations of P. infestans.     

New multilocus genotypes of <Emphasis Type="Italic">Phytophthora infestans</Emphasis> in Japan

Twelve isolates of Japanese Phytophthora infestans, which differed from the major genotypes US-1, JP-1, JP-2, and JP-3, were analyzed for RG57 fingerprints, mtDNA haplotypes, two allozyme genotypes, and mating types. Genotypes JP-1.1, JP-2.1, JP-2.2, JP-3.1, and JP-4 were newly defined. JP-1.1 and JP-2.1 were isolated discontinuously from potato fields in several years, and JP-1.1 was found in Hokkaido and Kagoshima. These results show that some minor genotypes can overwinter and disperse from their original site.     

Characterization of some Asian isolates of Phytophthora infestans

A total of 401 isolates of Phytophthora infestans were collected from eight Asian regions (Korea, India, Taiwan, Indonesia, Thailand, Nepal, China and Japan) between 1992 and 2000 – 318 from potato and 83 from tomato. The isolates were analysed for mating type, metalaxyl resistance, RG57 fingerprinting, mitochondrial DNA (mtDNA) haplotype and the polymorphism of three allozyme loci, i.e. glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ) and malic enzyme ( Me ). The isolates were multilocus-genotyped based on RFLP (RG57) fingerprint, dilocus allozyme genotype, mtDNA haplotype and mating type. Twenty multilocus genotypes were identified among 125 isolates. Of these genotypes, 14 had not been previously reported. Some of the multilocus genotypes were common to isolates from several geographical regions, suggesting migration. The metalaxyl-resistant isolates belonged to the multilocus genotypes JP-1, JP-2, and JP-3. Multilocus genotypes coexisting in a single field were found in following regions: Thailand (1994), central China (1996), Nepal (1997) and Japan (1998 and 2000). The possible origins of certain genotypes are discussed, including the possibility of sexual recombination within the P. infestans populations in Nepal and perhaps Thailand.     

Regulation of insecticide resistance-related cytochrome P-450 expression in Drosophila melanogaster

Two protein bands, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, account for most of the cytochrome P-450 in Drosophila melanogaster. P-450-A is ubiquitous among strains tested; whereas P-450-B is unique to certain strains. Dimethylnitrosamine demethylase activity is associated with P-450-B. Biochemical and genetic analyses showed that genes located on chromosome II were required for P-450-B expression. These genes were mapped within an interval that includes a major insecticide-resistance locus. Regulatory loci on chromosome III were required for maximum expression of P-450-B. One of these regulatory loci was mapped at another major resistance locus on chromosome III. There was a good correlation between P-450-B expression and resistance to phenylurea among the different strains tested. These results indicate that Drosophila can be a useful model to study the molecular mechanisms of insecticide resistance.     

Random amplified polymorphic DNA markers reveal low genetic variation and a single dominant genotype in Eichhornia crassipes populations throughout China

Genetic variation within and between 34 populations of Eichhornia crassipes (water hyacinth) in China was surveyed using random amplified polymorphic DNA (RAPD) markers. A total of 1009 individuals were analysed, for which 12 RAPD primers amplified 69 reproducible bands, with 22 (32%) being polymorphic. The percentage of polymorphic loci (p) within a population ranged from 4.4% to 17.4%, and the mean Nei's gene diversity (H_e) was 0.046 ± 0.0145, indicating a low genetic diversity of E. crassipes in China. Each population contained at least four RAPD phenotypes (genotypes), and the same particular genotype was invariably dominant in all the populations sampled. The mean proportion of distinguishable genotypes was 0.29. Analysis of molecular variance revealed a large proportion of genetic variation (83.9%) residing within populations and a slightly larger proportion (88.1%) within localities, indicating a low genetic differentiation of E. crassipes populations, both locally and regionally. Human-mediated dispersal, vigorous clonal growth, and a generally low level of sexual reproduction were thought to be responsible for such a pattern of genetic structure.     

Pathogenicity and infectivity of Phytophthora ramorum vary depending on host species,infected plant part,inoculum potential,pathogen genotype,and temperature

A total of 25 ornamental plant species representing 10 families were inoculated using three genotypes, each representing one of the genetic lineages NA1, NA2, and EU1 of the pathogen Phytophthora ramorum. Leaves were inoculated using suspensions with two zoospore concentrations and exposure at three temperatures, while stems were inoculated using agar plugs colonized by mycelia. Susceptibility was determined by measuring either the success of pathogen reisolation or lesion length caused by the pathogen. Infectivity was determined by counting sporangia in washes of inoculated leaves or stems. Results from all three pathogen genotypes combined were used to rank each of the 25 plant species for susceptibility and infectivity, while pooled results per genotype from all 25 hosts combined were employed for a preliminary comparison of pathogenicity and infectivity among genotypes. Statistical analyses showed that leaf results were affected by the concentration of zoospores, temperature, plant host, pathogen genotype, and by the interaction between host and pathogen genotype. Stem results were mostly affected by host and by the interaction between host and pathogen genotype. Hosts ranked differently when looking at the various parameters, and differences in rankings were also significant when comparing stem and leaf results. Differences were identified among the 25 hosts and the three pathogen genotypes for all parameters: results can be used for decision-making regarding regulations or selection of plants to be grown where infestations by P. ramorum are an issue.     

Variation in genotype, pathotype and anastomosis groups of Colletotrichum lindemuthianum isolates from Mexico

Patterns of variation were determined in anastomosis, pathotype and genotype of Colletotrichum lindemuthianum samples collected from individual plants of common bean cultivars from four locations in the Mexican highlands. In Chihuahua, 22 polymorphic AFLP bands in isolates taken from a single plant identified five distinct genotypes. In Michoacán, nine genotypes were identified based on a total of only six polymorphic bands. Combined cluster analysis of all isolates from individual plants grouped them geographically. In Chihuahua, in isolates from 16 individual plants, only nine genotypes were identified and all samples were found to belong to the same anastomosis group. However, analysis of selected isolates revealed two new pathotypes not reported previously from Chihuahua (races 449 and 467). In contrast, in Michoacán all 13 isolates from individual plants were found to have distinct genotypes, and in Mexico State only two pairs of isolates among 20 samples had identical genotypes. Although no pathotype variation was determined, five anastomosis groups were identified in Michoacán and three in Mexico State. These results suggest that patterns of variation in genotype and anastomosis groups are complex in the different locations sampled, and that no strong relationship exists between genotype, pathotype or anastomosis group.     

The role of aggressiveness and competition in the selection of Phytophthora infestans populations

Selection within populations of Phytophthora infestans was investigated by comparing the aggressiveness of single‐lesion isolates on detached leaflets of four potato cultivars with differing levels of race‐nonspecific resistance to P. infestans. The isolates included 23 representative of Northern Ireland genotypes from the early 2000s, used to inoculate previously reported field trials on competitive selection (2003–2005), plus 12 isolates recovered from the 2003 trial. The cultivars were those planted in the previous trials: Atlantic (blight‐susceptible) and Santé, Milagro and Stirling (partially resistant). Very highly significant variation for latent period, infection frequency and lesion area was found between genotypes and cultivars; differences between genotypes were more marked on the more resistant cultivars, but no one genotype was the most aggressive across all. Detached leaflets were also inoculated with mixtures of isolates from each genotype group at three sporangial concentrations: differences in aggressiveness between genotypes were more apparent at lower concentrations and on the more resistant cultivars. Genotype groups that were the most aggressive on the more resistant cultivars tended to be those selected by the same cultivars in the field. A mixture of all isolates of all genotypes was used to inoculate detached leaflets of the same cultivars. With one exception, single spore isolates recovered from any one leaflet belonged to a single genotype, but different genotypes were recovered from different cultivars. Phytophthora infestans isolates from Northern Ireland showed significant variation for foliar aggressiveness, and pathogen genotypes exhibited differential aggressiveness to partially resistant cultivars and interacted competitively in genotype selection.     

Effect of competition between Vicia faba and Camelina sativa as a model weed in breeding for organic conditions

Weeds can have a detrimental effect on faba bean (Vicia faba) crops in organic farming. Breeding for competitive cultivars is one option for weed control in organic conditions. The aim of this study was to analyse the impact of heterozygosity and heterogeneity levels and plant height on the competitive ability of faba bean genotypes. A set of 24 genotypes, classified as genotype groups (eight inbred lines, eight polycross progenies, two inbred line bulks, two hybrid bulks and four controls), was tested under two treatments: with and without a model weed. Each group of genotypes was equally composed of tall and short genotypes. The competitive ability of the faba bean genotypes was tested with Camelina sativa (false flax) as the model weed in two German locations over the course of 2 years. Yield loss due to weed competition, as recorded per faba bean genotype, was mainly dependent on the heterozygosity level of the genotypes; hybrid bulks were the most competitive genotype group with an average loss of yield of only 6%, whereas the inbred lines were the least competitive genotype group and recorded an average yield loss of 35%. Within the genotype group, no correlations were found between either the yield performance of the genotypes or their plant height and their competitive responses. The competitive ability of faba bean against weeds was mainly determined by their level of heterozygosity; thus, highly heterozygous cultivars should be promoted for breeding.     

Host resistance mediated inter-genotype competition and temporal variation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">vasinfectum</Emphasis>

Temporal variation in Fusarium oxysporum f. sp. vasinfectum (Fov) populations was determined by comparing the genetic diversity of pathogen isolates recovered from three consecutive cotton crops (2002, 2004 and 2006) in the Boggabilla area of New South Wales, Australia. A total of 288 isolates were collected, among which 25 distinct AFLP genotypes were identified. These genotypes were classified into two main groups corresponding to known vegetative compatibility groups (VCG)—01111 and 01112. The Fov populations were dominated by four genotypes (I-A, I-B, II-A, II-B) that accounted for 87.5% of the isolates. Significant temporal variation was observed in both sampled fields with 6.8% and 10.7% of total genetic variation being attributed to differences among collections in different years. Genetic diversity based on Nei’s gene diversity and the Shannon-Wiener index increased over time. Significant changes in the frequency of the dominant Fov genotypes were observed in one field, where genotype I-A declined from 84.8% to 40.0% over the study period (2002–2006), while genotype I-B increased from 7.6% to 35.4%. Strong inter-genotype competition was detected in glasshouse bioassays with 93.4% of symptomatic plants sampled from dual inoculation trials being infected by single genotypes. Competition was differentially mediated by cotton cultivars as the competitive ability of pathogen genotype I-B was enhanced on the resistant cultivar Sicot 189 relative to the susceptible cultivar Siokra 1–4. This suggests that host-mediated inter-genotype competition may play an important role in temporal variation in Fov populations in the field.     

Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL180

In plant–pathogen interactions, strong structural and biochemical barriers may induce a cascade of reactions in planta, leading to host resistance. The kinetic speed and amplitudes of these defence mechanisms may discriminate resistance from susceptibility to necrotrophic fungi. The infection processes of two Ascochyta lentis isolates (FT13037 and F13082) on the recently identified ascochyta blight (AB)‐resistant Lens orientalis genotype ILWL180 and two cultivated genotypes, ILL7537 (resistant) and ILL6002 (susceptible), were assessed. Using histopathological methods, significant differences in early behaviour of the isolates and the subsequent differential defence responses of the hosts were revealed. Irrespective of virulence, both isolates had significantly lower germination, shorter germ tubes and delayed appressorium formation on the resistant genotypes (ILWL180 and ILL7537) compared to the susceptible genotype (ILL6002); furthermore, these were more pronounced on genotype ILWL180 than on genotype ILL7537. Subsequently, host perception of pathogen entry led to the faster accumulation and notably higher amounts of reactive oxygen species and phenolic compounds at the penetration sites of the resistance genotypes ILWL180 and ILL7537. In contrast, genotype ILL6002 responded slowly to the A. lentis infection and reaffirmed previous gross disease symptomology reports as highly susceptible. Interestingly, quantification of H₂O₂ was markedly higher in ILWL180 particularly at 12 h post‐inoculation compared to ILL7537, potentially indicative of its superior resistance capability. Faster recognition of A. lentis is likely to be a major contribution to the superior resistance observed in genotype ILWL180 to the highly aggressive isolates of A. lentis assessed.     

Genotypic and phenotypic characterization of the European A2 isolates of <Emphasis Type="Italic">Phytophthora ramorum</Emphasis>

Phytophthora ramorum, a recently described North American and European pathogen, has three clonal lineages. The NA1 and NA2 lineages are found in North American forests and nurseries, while the EU1 lineage appears mainly in European nurseries. P. ramorum is heterothallic, having two mating types A1 and A2. All NA1 and NA2 isolates are of A2 mating type. When first collected, all EU1 isolates were of A1 mating type, with the exception of one A2 isolate collected in Belgium in 2002. Screening 410 other Belgian isolates for mating type revealed two additional EU1-A2 isolates collected in 2002 and 2003. PCR-RFLP, AFLP and SSR markers were used to determine the nature of the mating type change. The three isolates show no indications of sexual recombination or mitotic crossing over, indicating that mutation or mitotic gene conversion is the most likely explanation for the mating type change. We compared the pathogenicity and sporulation characteristics of the EU1-A2 isolates to those of EU1-A1 and NA1-A2 isolates on four host plants. Despite small differences in pathogenicity on some hosts, the EU1-A2 isolates were similarly aggressive to each other and to the EU1-A1 isolates and more aggressive than the NA1-A2 isolates. Sporulation characteristics were also comparable among EU1-A2 isolates and between EU1-A1 and EU1-A2 isolates, except for EU1-A2 isolate BBA 26/02. The limited genotypic and phenotypic differences between EU1-A2 isolates probably evolved after the mating type change, which may have occurred several years before the isolates were detected. There are strong indications that the EU1-A2 population has been eradicated from Belgium.     

Genetic diversity of Puccinia striiformis from cereals in Alberta,Canada

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici has recently become a production problem on wheat in Alberta, Canada, and stripe rust of barley caused by P. striiformis f. sp. hordei occurs regularly. A total of 261 isolates of P. striiformis were collected from wheat, barley, Hordeum jubatum and triticale plants in Alberta, Canada from 2007 to 2012, and compared to isolates from other provinces and the USA. The genetic diversity of the pathogen was assessed using 11 simple sequence repeat (SSR) markers and by examining a length polymorphism in the ribosomal DNA (rDNA) intergenic spacer 1 (IGS1) region. A total of 28 SSR genotypes were detected within Alberta. The 13 genotypes common on wheat (P. striiformis f. sp. tritici) were distinct from the 15 genotypes common on barley (P. striiformis f. sp. hordei). Four SSR genotypes, two within each forma specialis, represented 85% of the isolates recovered. Genotypic diversity was low, population genetic analysis indicated a clonal structure, and the genotypes were widely dispersed. In both formae speciales, the dominant genotype varied between years. The second most common P. striiformis f. sp. hordei genotype was found to be more closely related to older P. striiformis f. sp. tritici genotypes from the USA than to other P. striiformis f. sp. hordei genotypes.     

Genetic resistance to bacterial blight disease in Persian walnut

Bacterial blight disease of Persian walnut (Juglans regia, L.), caused by Xanthomonas arboricola pv. juglandis (Xaj), leads to significant nut losses in northern, central and western areas of Iran. To identify the natural sources of resistance to disease in the endemic walnut genotypes of Iran, sixteen walnut genotypes, collected from different areas of Hamedan province, were inoculated with Xaj in a randomized complete block design with five replicates for each genotype. Two-year old genotypes were gently sprayed with a suspension of bacteria adjusted to approximately 2 × 10⁹ cfu ml⁻¹ of distilled water in May. Infected leaves were rated for disease 28 and 42 days after inoculation, using a 0 to 5 severity scale, based on the number, size and distribution of lesions on the leaves. Data analyses showed that there were variations among genotypes in response to pathogen. Upon inoculation by bacterial suspension genotype 94 showed the highest resistance to both disease incidence and its progress after 4–6 weeks of infection. Genotype 65 showed high susceptibility to disease and genotype 69 showed high susceptibilities both to disease incidence and its progress after 4–6 weeks of infection.     

Electrophoretic karyotyping and mapping of pathotype-specific DNA sequences in Japanese isolates of <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

The chromosome number and electrophoretic karyotype of Japanese isolates of Verticillium dahliae were investigated. In a genomic Southern blot analysis of seven isolates probed with a telomere consensus sequence (TTAGGG)₅, 12 or 14 bands were observed. Furthermore, pulsed-field gel electrophoresis (PFGE) of these isolates revealed five or six chromosomal bands. A band (approx. 3.5 Mbp) common to all isolates apparently contained more than two chromosomes. From these results, we concluded that each isolate’s chromosome number is six (an eggplant pathotype isolate) or seven (all isolates of tomato and sweet pepper pathotypes). Although the chromosome sizes differed among isolates, karyotypes were similar within tomato and sweet pepper pathotypes. A small chromosome (approx. 1.8 Mbp) was observed only in the sweet pepper pathotype. Subsequent PFGE-Southern hybridization analyses revealed that the three DNA fragments specific to tomato pathotype are located on the same chromosome. These results suggest that the tomato-pathotype-specific DNA sequences might coexist on one chromosome.     

The potential for selecting wheat varieties strongly competitive against weeds

The competitive abilities of a wide range of genotypes of wheat (Trilicvm aestivum L.) and durum wheat (Triticum durum Desf.) against Lolium rigidum Gaud, (annual ryegrass) were examined 1o determine the potential for breeders to select strongly competitive varieties, Considerable potential within the wheat genome to breed varieties with greater competitive ability was demonstrated. In 1993, 250 genotypes from around the world were screened and in 1994 a subset of 45 (mainly Australian) genotypes were further examined. A uniform density of L. rigidum reduced grain yield of wheat by up to about 80% in 1993 and to 50% in 1994, depending on wheat genotype. Reduction in grain yield was correlated with L. rigidum dry matter. Wheats varied in competitive ability with source, and durum wheats were less competitive than T. aestivum. The ‘old’ standard wheat varieties (released between 1880 and 1950) suppressed the weed more than all the current varieties, with the exception of eight F₁ hybrids. A doubling of the crop seeding rate of 10 of the genotypes in 1994 reduced the biomass of L. rigidum by an average of 25% compared with the standard seeding rate. Ranking of competitive ability of varieties at high density was consistent at both seeding rates. The strongly competitive genotypes had high early biomass accumulation, large numbers of tillers, and were tall with extensive leaf display. The potential for breeding enhanced competitive ability in wheat is discussed.