Production,characterization and interaction of single-spore isolates ofPlasmodiophora brassicae

Out of 164 plants of clubroot-susceptible Chinese cabbage inoculated with single resting spores ofPlasmodiophora brassicae, two plants developed clubroot symptoms. The two single-spore isolates (SSIs) extracted from these plants gave an identical reaction pattern on the European Clubroot Differential set (ECD) and seven doubled-haploid lines (DH-lines). Their reaction pattern differed from that of the original field isolate on four hosts: ECD hosts 06 and 07 were susceptible to the field isolate but resistant to both SSIs, while for DH-lines Bi and Pt the reverse was true. DH-line Pt was significantly less diseased by mixed inocula consisting of the field isolate and SSI-1 than by SSI-1 alone. It was concluded that the SSI-1 pathotype was a minor component of the field isolate, although it was isolated twice. The results also suggest that the alleviating effect of the field isolate in mixed inoculations with SSI-1 on DH-line Pt was due to induced resistance, rather than to competitive interactions.Abbreviations cv cultivar - DH-line doubled haploid line - ECD European Clubroot Differential set - SSI single-spore isolate     

Characterization and PCR-based detection of benzimidazole-resistant isolates of Monilinia laxa in California

Monilinia laxa is a pathogen of brown rot of stone fruit and almond in California, causing blossom blights and fruit rots. In this study, low-level resistance to the benzimidazole fungicides benomyl and thiophanate-methyl was detected in field isolates of M laxa collected from stone fruits and almonds in California. Low-resistant (LR) isolates grew in potato dextrose agar (PDA) plates amended with benomyl and thiophanate-methyl at 1 and 5 microg ml(-1), respectively, but not in plates amended with benomyl at 5 microg ml(-1) or thiophanate-methyl at 50 microg ml(-1). The benzimidazole LR isolates were characterized by temperature sensitivity and the DNA sequence of the beta-tubulin gene. The LR isolates showed high-temperature sensitivity, being sensitive to 1 microg ml(-1) of benomyl at 28 degrees C but resistant at 8-24 degrees C. Analysis of the DNA sequence of the beta-tubulin gene showed that the LR isolates had a point mutation at the amino-acid position 240, causing substitution of leucine by phenylalanine. Based on the point mutation, a pair of allele-specific PCR primers was developed for rapid detection of LR isolates of M laxa. In addition, a pair of PCR primers specific to M laxa was developed on the basis of the differences in the DNA sequence of the intron 6 of beta-tubulin gene from M laxa, M fructicola and other fungal species. The primer pair amplified the expected 376-bp DNA fragment from all M laxa isolates tested, but not from 14 other fungal species isolated from stone fruit and almond crops. The restriction endonuclease BsmA I recognized the sequence GTCTCC in the PCR products from sensitive (S) isolates only, but not the GTTTCC sequence in the PCR products from LR isolates. The endonuclease digested the 376-bp PCR products from S isolates to produce two bands (111 and 265 bp) on agarose gels. Thus, both allele-specific PCR and the PCR-restriction fragment length polymorphism (PCR-RFLP) methods could be useful for rapidly detecting benzimidazole-resistant isolates of M laxa from stone fruit and almond crops in California.     

Molecular identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. using a polymerase chain reaction–restriction fragment length polymorphism technique

Polymerase chain reaction (PCR) and polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) techniques were applied for establishing the reliable practice in identification of Echinochloa oryzicola Vasing. and E. crus-galli (L.) Beauv. (barnyardgrass). Total DNA was extracted from 18 accessions and 86 individuals of E. oryzicola , 33 accessions and 140 individuals of E. crus-galli var. crus-galli , 23 individuals of E. crus-galli var. praticola , and six individuals of E. crus-galli var. formosensis that were collected from Japan. A partial region of intergenetic spacer between trn T and trn L, and an intron of trn L were amplified separately using a trn-a and trn-b1 primer set, and a trn-c and trn-d primer set, respectively. All individuals of E. oryzicola showed the same fragment amplified by the trn-a and trn-b1 primer set. The fragment was 481 bp in length, and was undigested by Eco R I, whereas all individuals of E. crus-galli , including three botanical varieties, showed the same fragment with a 449-bp length. The fragment was digested by Eco R I into two fragments (178 and 271 bp). The fragment amplified by the trn-c and trn-d primer set in all individuals of E. oryzicola was digested by Alu I into two fragments (174 and 452 bp), but undigested by Dra I. In contrast, the fragment amplified by the trn-c and trn-d primer set in all individuals of E. crus-galli was digested by Dra I into two fragments (134 and 487 bp), but undigested by Alu I. There was no intraspecific variation in these regions; thus, these two species are easily identifiable by using our method.     

Rapid diagnosis of sulfonylurea‐resistant Schoenoplectus juncoides [Roxb.] Palla using polymerase chain reaction–restriction fragment length polymorphism and isogene‐specific direct sequencing

Rapid diagnostic methods to detect known mutations in acetolactate synthase (ALS) genes that confer sulfonylurea (SU) resistance to Schoenoplectus juncoides were developed in this study. By using 11 SU‐resistant accessions (nine accessions with a Pro₁₉₇ substitution in ALS1 or ALS2, one accession with an Asp₃₇₆Glu substitution in ALS2 and one accession with a Trp₅₇₄Leu substitution in ALS2), polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis for DNA fragments that were amplified simultaneously from genomic ALS1 and ALS2 and PCR–RFLP analysis for DNA fragments that were amplified from either of the genomic ALS1 or ALS2 were carried out. In each of the two PCR–RFLP analyses, a common PCR product was digested separately with the restriction enzymes, BspLI, MboI and MunI, in order to detect Pro₁₉₇ substitutions, an Asp₃₇₆Glu substitution and a Trp₅₇₄Leu substitution, respectively. In each of the lanes where the detection of SU‐resistant substitutions was aimed, a specific band to suggest the existence of the said substitutions was observed in theoretically assumable ways. Separately, a direct sequencing method also was established, which was able to selectively sequence ALS1 or ALS2 from common templates containing both ALS1 and ALS2 by the isogene‐selective primers that were designed to anneal either of the ALS genes. It is expected that these methods could be used for the genetic analysis of SU‐resistant S. juncoides by providing rapid and accurate diagnosis.     

Sexual mating preferences in vitro and in planta among Japanese isolates of Phytophthora infestans

The sexual preferences of Japanese isolates of Phytophthora infestans were determined by mating on agar, in broth, or in plants. The influence of their sexual preference was confirmed in the host tissues. Three wild-type isolates and a -glucuronidase (GUS) transformant were co-cultured to identify the origin of antheridia and oogonia. Japanese A1 isolate had a unique sexual preference compared with foreign isolates. It produced self-fertile oospores with about 40% of total gametangia but tended to form antheridia on V-8 agar medium. In addition, oospores were formed in plants, but their sexual preference could not be reflected in vitro.     

Differentiation of Xanthomonas campestris pv. Phaseoli from Xanthomonas campestris pv. Phaseoli var. Fuscans by PFGE and RFLP

Xanthomonas campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans, the causal agents of the common and fuscous bacterial blight of beans, appear to be phenotypically identical except that the latter can produce a melanin-like pigment in culture. Ten isolates of X. campestris pv. phaseoli and 12 isolates of X. campestris pv. phaseoli var. fuscans were examined using pulsed-field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP). The average genome sizes for X. campestris pv. phaseoli and X. campestris pv. phaseoli var. fuscans were 3850.6±48.9 and 3584.3±68.1kb respectively. The genetic relatedness of the isolates was determined from macrorestriction patterns generated using XbaI. Cluster analysis indicated that the non-fuscous and fuscous strains are distinct. RFLP results, based on the highly conserved hrp genes and a pectate lyase gene from Xanthomonas, also indicated that the two bacteria are genetically different. The results obtained in this study suggest that this pathovar can be segregated into two subgroups under a recently proposed reclassification of the Xanthomonas genus.     

Cultural characteristics, pathogenicity and genetic diversity of Fusarium oxysporum isolates from tobacco fields in Spain

Several formae speciales of Fusarium oxysporum are capable to produce disease in tobacco plants. Different authors have classified those isolates as a forma specialis or a race within on the basis of the severity of disease and host specificity. Fusarium wilt of tobacco plant in Extremadura (central Spain) tobacco fields have been recorded in the last years and F. oxysporum was isolated from symptomatic plants. The aim of our study was to characterize these F. oxysporum populations. For this purpose, the in vitro spore production and growth and the virulence (severity of disease) have been tested. Although all isolates behaved as pathogen, the virulence of isolates was different. The differences in growth could not be correlated with other characteristics but the two isolates with scarce spore production have also behaved as the weakest pathogen. We have analyzed intergenic spacer (IGS) region polymorphism of ribosomal DNA and random amplified polymorphic DNA (RAPD) markers to assess the genetic diversity within F. oxysporum isolates. These molecular analyses showed two major groups with different physiological capabilities that could reflect two different lineages. One group was characterized by medium–high sporulation, high virulence and the same IGS-RFLP pattern. The other group was more heterogeneous featuring low–medium sporulation and variable virulence and growth. This first experimental approach to pathogen population could be a good starting point for further studies including non-pathogenic isolates and a larger number of pathogen that could clarify if there are two or more genetic lineages.     

Pathogenic and molecular variability among isolates of Pyrenophora tritici-repentis, causal agent of tan spot of wheat in Argentina

Tan spot, caused by Pyrenophora tritici-repentis, is a common disease of wheat (Triticum aestivum) responsible for economic losses in some wheat growing areas worldwide. In this study the pathogenic and genetic diversity of 51 P. tritici-repentis isolates collected from different ecological regions of Argentina were analyzed. Virulence tests were conducted on 10 selected wheat cultivars: Buck Halcón, Chris, Gabo, Glenlea, Klein Dragón, Klein Sendero, Max, ND 495, ProInta Guazú and ProInta Imperial. Data revealed significant differences between all main factors evaluated and the interactions for 19 of the isolates analyzed. Based on the reaction type of each isolate/cultivar combination, 48 different pathogenic patterns were detected. The molecular analysis using Inter-Simple Sequence Repeats (ISSR) revealed the existence of 36 different haplotypes among 37 isolates of P. tritici-repentis originally selected for this study. These results indicate that P. tritici-repentis on wheat in Argentina is a heterogeneous fungus, implying that screening wheat germoplasm for resistance for tan spot disease requires a wide range of pathogen isolates.     

Histological study of giant cells formed by the root-knot nematodeMeloidogyne artiellia as compared withM. hapla andM. javanica in cabbage, turnip and barley

Feeding sites induced by the root-knot nematodeMeloidogyne artiellia in turnip (Brassica rapa), cabbage (Brassica oleracea) and barley (Hordeum vulgare) were examined by light and electron transmission microscopy, and compared with those formed byM. javanica andM. hapla. The feeding cells ofM. artiellia in turnip and cabbage showed hypertrophy, hyperplasia and vacuolization, and became multinucleate giant cells. In contrast to the typical simple-shaped giant cells ofM. javanica andM. hapla, those ofM. artiellia had an amoeboid structure containing ‘protuberances’, which are distinct from previously reported ‘projects’ of giant cells induced byM. incognita in other plants. Protuberances expanding between vascular cells were observed in young and developed giant cells. Since no cell-wall fragments were found at the bases of the protuberances, either by light or electron microscopy, it is strongly indicated that these structures were caused by local expansion of the giant cells rather than by fusion with adjacent cells. The giant cells ofM. artiellia in barley had regular shapes of giant cells without protuberances, and resembled those induced byM. javanica orM. hapla in turnip and cabbage. http://www.phytoparasitica.org posting Sept. 17, 2006.     

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.     

Genetic variation among <Emphasis Type="Italic">Fusarium</Emphasis> isolates from onion,and resistance to <Emphasis Type="Italic">Fusarium</Emphasis> basal rot in related <Emphasis Type="Italic">Allium</Emphasis> species

The aim of this research was to study levels of resistance to Fusarium basal rot in onion cultivars and related Allium species, by using genetically different Fusarium isolates. In order to select genetically different isolates for disease testing, a collection of 61 Fusarium isolates, 43 of them from onion (Allium cepa), was analysed using amplified fragment length polymorphism (AFLP) markers. Onion isolates were collected in The Netherlands (15 isolates) and Uruguay (9 isolates), and received from other countries and fungal collections (19 isolates). From these isolates, 29 were identified as F. oxysporum, 10 as F. proliferatum, whereas the remaining four isolates belonged to F. avenaceum and F. culmorum. The taxonomic status of the species was confirmed by morphological examination, by DNA sequencing of the elongation factor 1-α gene, and by the use of species-specific primers for Fusarium oxysporum, F. proliferatum, and F. culmorum. Within F. oxysporum, isolates clustered in two clades suggesting different origins of F. oxysporum forms pathogenic to onion. These clades were present in each sampled region. Onion and six related Allium species were screened for resistance to Fusarium basal rot using one F. oxysporum isolate from each clade, and one F. proliferatum isolate. High levels of resistance to each isolate were found in Allium fistulosum and A. schoenoprasum accessions, whereas A. pskemense, A. roylei and A. galanthum showed intermediate levels of resistance. Among five A. cepa cultivars, ‘Rossa Savonese’ was also intermediately resistant. Regarding the current feasibility for introgression, A. fistulosum, A. roylei and A. galanthum were identified as potential sources for the transfer of resistance to Fusarium into onion.     

陕西省小麦白粉菌群体毒性结构及遗传多样性

为明确近年来陕西省小麦白粉菌Blumeria graminis f. sp. tritici群体毒性及遗传变异情况,利用32份已知抗白粉病基因小麦品种(系)对2013年和2014年陕西省小麦白粉菌群体毒性结构进行测定,并应用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)标记对2014年陕西省西安市、咸阳市、渭南市、宝鸡市及陕西省毗邻的甘肃省天水市5个小麦白粉菌地理群体共118株白粉菌菌株进行遗传多样性分析。毒性监测结果显示,供试小麦白粉菌群体对Pm1c、Pm2、Pm3d、Pm4a、Pm4b、Pm5b、Pm13、Pm21、Pm24、Pm30、Pm2+6、Pm2+Mld、Pm2+6+?、Pm4b+5b、Pm4+?、Pm5+6的平均毒性频率在0～17.23%之间,表明这些基因抗性保持较好;对Pm1a、Pm3b、Pm3c、Pm3e、Pm3f、Pm7、Pm8、Pm19、Pm1+2+9的平均毒性频率介于69.17%～99.60%之间,表明这些基因抗性已丧失。用筛选出的6对AFLP标记共扩增出831个多态性位点,多态性位点百分比为94.86%;小麦白粉菌群体遗传多样性指数和香农信息指数分别为0.1151和0.2036,遗传变异主要来源于群体内变异。群体间基因流为4.7939,表明5个小麦白粉菌群体间基因交流广泛。群体间遗传距离和地理距离两者显著正相关。     

Pseudomonas for biological control of dutch elm disease. I. Labeling,detection and identification of pseudomonas isolates injected into elms; comparison of various methods

To understand the mechanisms involved in biological control of Dutch elm disease byPseudomonas, data were needed on the distribution of the introduced bacteria within elm and on the development of the bacterial population over a period of time.As traditional biochemical identification techniques are not suitable for distinguishment between individualPseudomonas isolates, three alternative approaches were compared.

Development of ZWIPERO, A Model Forecasting Sporulation and Infection Periods of Onion Downy Mildew based on Meteorological Data

The weather-based forecasting model ZWIPERO was developed by the German Weather Service and determines the risk of sporulation and infection of Peronospora destructor quantitatively based on actual as well as predicted weather data (temperature, relative humidity, leaf wetness, precipitation). The model allows precise planning of disease monitoring and infection-related application of fungicides. ZWIPERO is a more complex mathematical model than the previously published models for downy mildew. In order to operate ZWIPERO independently of the actual field location and season, the time of sunrise and sunset of the location are exactly determined by a subroutine. Another subroutine provides simulated microclimatic input variables based on local production data as well as actual and hourly predicted (up to 4 days) standard weather data. Starting at the time of 'sunrise + 7 h', ZWIPERO calculates the number of sporangia produced, the time of onset of sporangia release, as well as the number of infections possible and the number of sporangia which may survive the day for each 24-h time step. Field evaluations of sporulation periods of downy mildew showed that the simulated micrometeorological input variables are reliable. As the actual plant development, the susceptibility and the disease incidence in the field are not taken into account, ZWIPERO has to be considered primarily as a decision support system for extension services and growers.     

Sequence comparison and transmission of <Emphasis Type="Italic">Blackcurrant reversion virus</Emphasis> isolates in black,red and white currants with black currant reversion disease and full blossom disease symptoms

We collected samples from black, red and white currants showing symptoms of blackcurrant reversion disease (BRD) and full blossom disease (FBD), cultivated in the Czech Republic. Blackcurrant reversion virus (BRV) was detected in all symptomatic plants. After amplification, a substantial part of the 3′ non-translated region (3′-NTR) of RNA2 of 15 new isolates of BRV was sequenced and compared with sequences available in the literature and GenBank. We did not find significant sequence diversity among isolates associated with either FBD or BRD. BRV was graft-transmitted from FBD infected red currant to black currant where symptoms of BRD were observed. Further sequence analysis of BRV isolates resulted in a phylogenetic tree with four branches, each consisting of six to nine isolates. No correlation with geographic origin was visible on the tree as isolates from various countries occurred in all four branches. We also found no correlation between the host and the topology of the tree: most of black currant isolates occurred in branches 3 and 4, but also occurred in branches 1 and 2. Only one white currant and one red currant isolate occurred in branches 3 and 4, respectively. The sequence identity of the Czech isolates in this region ranged from 91.9 to 99.8%. The 17 plant species growing within and in the close vicinity of the BRD-infested plantation were tested negative for BRV by RT-PCR as natural hosts of BRV. BRV was successfully transmitted by mechanical inoculation from black currant to Nicotiana occidentalis and N. tabacum cv. Xanthi, the latter being a new host for BRV. The infection was confirmed by PCR and sequencing.     

Pathogenicity,colony morphology and diversity of isolates of <Emphasis Type="Italic">Guignardia citricarpa</Emphasis> and <Emphasis Type="Italic">G. mangiferae</Emphasis> isolated from <Emphasis Type="Italic">Citrus</Emphasis> spp.

In the present study, the pathogenicity of 36 isolates of Guignardia species isolated from asymptomatic ‘Tahiti’ acid lime fruit peels and leaves, ‘Pêra-Rio’ sweet orange leaves and fruit peel lesions, and a banana leaf were characterized. For pathogenicity testing, discs of citrus leaves colonized by Phyllosticta citricarpa under controlled laboratory conditions were kept in contact with the peels of fruit that were in susceptible states. In addition, pathogenicity was related to morphological characteristics of colonies on oatmeal (OA) and potato dextrose agar (PDA). This allowed the morphological differentiation between G. citricarpa and G. mangiferae. Polymerase chain reactions (PCRs) were also used to identify non-pathogenic isolates based on primers specific to G. citricarpa. A total of 14 pathogenic isolates were detected during pathogenicity tests. Five of these were obtained from leaf and fruit tissues of the ‘Tahiti’, which until this time had been considered resistant to the pathogen. Given that the G. citricarpa obtained from this host was pathogenic, it would be more appropriate to use the term insensitive rather than resistant to categorize G. citricarpa. A non-pathogenic isolate was obtained from lesions characteristic of citrus black spot (CBS), indicating that isolation of Guignardia spp. under these conditions does not necessarily imply isolation of pathogenic strains. This also applied to Guignardia spp. isolates from asymptomatic citrus tissues. Using fluorescent amplified fragment length polymorphism (fAFLP) markers, typically pathogenic isolates were shown to be more closely related to one another than to the non-pathogenic forms, indicating that the non-pathogenic isolates display higher levels of genetic diversity.     

Relationship between aggressiveness of <Emphasis Type="Italic">Stagonospora </Emphasis>sp. isolates on field and hedge bindweeds,and <Emphasis Type="Italic">in vitro</Emphasis> production of fungal metabolites cercosporin,elsinochrome A and leptosphaerodione

Stagonospora convolvuli LA39, an effective biocontrol agent of Convolvulus arvensis (field bindweed) and Calystegia sepium (hedge bindweed) produces phytotoxic metabolites leptosphaerodione and elsinochrome A. Stagonospora isolate 214Caa produces the toxin cercosporin. If toxic metabolite production is not linked to the pathogenic ability of the fungus on bindweeds, selection of aggressive strains with limited or no production of the metabolites would reduce any perceived risk of using strains of the fungus as a mycoherbicide. Therefore, 30 isolates of Stagonospora sp. including LA39 and 214Caa were characterised for aggressiveness on both bindweeds, and production of the three metabolites. Nine isolates were more aggressive than LA39 on both bindweeds. Classification of isolates based on metabolite type agreed largely with previous similar characterisation based on polymerase chain reaction-restriction fragment length polymorphism of internal transcribed spacer of ribosomal DNA. Cercosporin producers produced neither leptosphaerodione nor elsinochrome A and together with isolates that produce none of the three metabolites, were less pathogenic on bindweeds. Conversely, there was a positive correlation between elsinochrome A and leptosphaerodione production, and each was positively correlated with aggressiveness of isolates on both bindweeds. Generally, any isolate where elsinochrome A was not detected was not aggressive on any of the two bindweeds. This probably implies that selecting elsinochrome A-negative, but aggressive Stagonospora strain(s) may be difficult. However, aggressive isolates may not produce elsinochrome A in planta at levels that could constitute any risk in the environment. In a preliminary attempt to determine the levels of elsinochrome A and leptosphaerodione produced in diseased bindweeds, none of the toxins was detected in Stagonospora infected bindweed leaves. Detailed investigation focusing on the detection and quantification of in planta production of elsinochrome A by Stagonospora isolates, and determination of the fate of elsinochrome A in the environment, and its relationship with leptosphaerodione may be essential. Similarly, development of molecular tools to monitor the mycoherbicide following field application is vital.     

Preliminary studies of<Emphasis Type="Italic">in vitro</Emphasis> stimulation of sexual mating among isolates of<Emphasis Type="Italic">Mycosphaerella fijiensis</Emphasis>, causal agent of black sigatoka disease in bananas and plantains

Single-ascospore-derived isolates ofMycosphaerella fijiensis Morelet from false horn ‘Agbagba’ plantain leaves obtained from five different villages in southern Nigeria were stimulated to mate under artificial conditions. Pairs of isolates were incubated under blacklight on potato dextrose agar (PDA) with surface-sterilized plantain leaves or on PDA with autoclaved plantain leaves. Some isolates were observed to be sexually compatible by their ability to produce spherical to bulb-shaped fruiting body structures (FBS) and ascospores on pairing. FBS were observed to measure between 39–65 μm (smallest diameter) and 39–104 μm (largest diameter; mean 55.3×71.1 μm) in diameter, whereas ascospore lengths measured between 13.0 and 14.9 μm. Length of incubation time required for FBS production was dependent on the pair of isolates involved, the average being 40.1 days. With some pairs, ascospores were observed after 35 days of incubation. http://www.phytoparasitica.org posting Dec. 16, 2002.