Identification by PCR analysis on plasmid pEA29 of isolates of Erwinia amylovora responsible of an outbreak in Central Europe

A collection of 127 strains of Erwinia amylovora, the causative agent of fire blight, was tested by PCR amplification of a fragment of the plasmid pEA29. A variability in the length of the DNA fragment obtained was observed after digestion by MspI and Sau3A restriction enzymes. Strains were distributed into three groups according to the length of the DNA product. Most of the strains analysed were placed into two groups. Thirteen strains were clustered into a third group which was linked with the geographical origin of strains: they were all isolates from recently reported outbreaks of fire blight in Austria and in southern Bavaria in Germany. The variation in the length of the amplified fragment is probably due to an insertion into this fragment.     

Development of symptoms caused by Erwinia carotovora ssp. atroseptica under field conditions and their effects on the yield of individual potato plants

Infection of seed tubers by pectinolytic Erwinia species can lead to the development of various symptoms during vegetative growth of potato crops, including non-emergence of plants, chlorosis, wilting, haulm desiccation and typical blackleg. The relationships between types of symptoms and yield are poorly documented, and are investigated by following the development of symptoms in potato plants grown under field conditions from seed tubers artificially inoculated with E. carotovora ssp. atroseptica ( Eca ) , and measuring the yield of each plant. Symptoms were classified into five main types (non-emergence, wilting/chlorosis, blackleg, haulm desiccation and plant death). Each plant was scored for types of symptom on four successive dates; plants without visible symptoms were scored as healthy. The method of inoculation and inoculum concentration proved major factors for the subsequent development of symptoms. Disease development was more severe after vacuum infiltration of bacteria into seed tubers than after shaking tubers in contaminated sand. Disease usually progressed from chlorosis and/or wilting to partial or total desiccation on a given plant. Yield losses varied according to symptom type, but the relationship between symptoms recorded and yield also depended on scoring dates. The data suggest that the beginning of tuber growth might be the most suitable stage for predicting yield losses from symptom observations. In both cultivars studied (Bintje, highly susceptible, and Désirée, moderately resistant), the yield of symptomless plants growing from inoculated seed tubers was significantly less than that of control plants, indicating that the presence of bacteria on the seed tuber was detrimental, even in the absence of visible symptoms. Differences in symptom expression in the field between cultivars matched the level of visible infection of tubers at harvest, as Bintje tubers showed a higher incidence of rot than Désirée tubers.     

Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’)

Nine bacteriophages infecting Dickeya spp. biovar 3 (‘Dickeya solani’) were isolated from soil samples collected in different regions in Poland. The phages have a typical morphology of the members of the order Caudovirales, family Myoviridae, with a head diameter of c. 90–100 nm and tail length of c. 120–140 nm. In host range experiments, phage ?D5 expressed the broadest host range, infecting members of all Dickeya spp., and phage ?D7 showed the narrowest host range, infecting isolates of Dickeya dadantii and ‘D. solani’ only. None of the phages was able to infect Pectobacterium spp. isolates. All phages were prone to inactivation by pH 2, temperature of 85°C and by UV illumination for 10 min (50 mJ cm^?2). Additionally, phages ?D1, ?D10 and ?D11 were inactivated by 5 m NaCl and phage ?D2 was inactivated by chloroform. Phages ?D1, ?D5, ?D7 and ?D10 were characterized for optimal multiplicity of infection and the rate of adsorption to the bacterial cells. The latent period was 30 min for ?D1, 40 min for ?D5, 20–30 min for ?D7 and 40 min for ?D10. The estimated burst size was c. 100 plaque‐forming units per infected cell. The bacteriophages were able to completely stop the growth of ‘D. solani’ in vitro and to protect potato tuber tissue from maceration caused by the bacteria. The potential use of bacteriophages for the biocontrol of biovar 3 Dickeya spp. in potato is discussed.     

Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.     

Characterization of bacterial isolates from rotting potato tuber tissue showing antagonism to Dickeya sp. biovar 3 in vitro and in planta

Possibilities for biocontrol of biovar 3 Dickeya sp. in potato were investigated, using bacteria from rotting potato tissue isolated by dilution plating on nonselective agar media. In a plate assay, 649 isolates were screened for antibiosis against Dickeya sp. IPO2222 and for the production of siderophores. Forty‐one isolates (6·4%) produced antibiotics and 112 isolates (17·3%) produced siderophores. A selection of 41 antibiotic‐producing isolates and 41 siderophore‐producing isolates were tested in a potato slice assay for control of the Dickeya sp. Isolates able to reduce rotting of potato tuber tissue by at least 50% of the control were selected. Isolates were characterized by 16S rDNA analysis as Bacillus, Pseudomonas, Rhodococcus, Serratia, Obesumbacterium and Lysinibacillus genera. Twenty‐three isolates belonging to different species and genera, 13 producing antibiotics and 10 producing siderophores, were further characterized by testing acyl‐homoserine lactone (AHL) production, quorum quenching, motility, biosurfactant production, growth at low (4·0) and high (10·0) pH, growth at 10°C under aerobic and anaerobic conditions and auxin production. In replicated greenhouse experiments, four selected antagonists based on the in vitro tests were tested in planta using wounded or intact minitubers of cv. Kondor subsequently inoculated by vacuum infiltration with an antagonist and a GFP (green fluorescent protein)‐tagged biovar 3 Dickeya sp. strain. A potato endophyte A30, characterized as S. plymuthica, protected potato plants by reducing blackleg development by 100% and colonization of stems by Dickeya sp. by 97%. The potential use of S. plymuthica A30 for the biocontrol of Dickeya sp. is discussed.     

In vitro somatic growth and reproduction of phenylamide-resistant and -sensitive isolates of Phytophthora erythroseptica from infected potato tubers in Idaho

Pink rot of potato, most commonly caused by Phytophthora erythroseptica , is a major field and post-harvest problem in southern Idaho, USA, particularly since 1998 when isolates resistant to the phenylamide fungicide metalaxyl-M (mefenoxam) were detected. Isolates of P. erythroseptica were collected from infected tubers in 2001 and 2002 from six Idaho counties and tested for resistance to metalaxyl-M on amended agar. Metalaxyl-M resistant (MR) and metalaxyl-M-sensitive (MS) isolates were identified in six counties; 160 isolates were highly resistant, seven moderately resistant and 57 sensitive to metalaxyl-M with mean EC₅₀ values of 182, 23 and 0·5 mg L⁻¹ ai metalaxyl-M, respectively. Mycelial growth rates and oospore production in agar were assessed for 20 MS and 20 MR isolates at 10, 15, 20, 25 and 30°C. Growth rates of MR isolates were between 2·5 and 3·1 times greater ( P  < 0·05) than those of MS isolates at 10, 15, 20 and 25°C, and oospore production was between 6·8 and 20·5 times greater ( P  < 0·0001) for MR than for MS isolates at the same temperatures. Colony growth in V8 broth at 18°C was greater for MR than MS isolates ( P  < 0·0032). However, zoospore production at 18°C was greater for MS than for MR isolates ( P  < 0·0109), and zoospore production m m ⁻¹ of colony circumference was also greater for MS than for MR isolates, 14 191 and 9959, respectively ( P  = 0·0109). Sexual reproduction of MR isolates in nature may be greater than MS isolates, but MS isolates may be more asexually fit based on the fitness parameters studied.     

Characterization of potato potyvirus Y (PVY) isolates from seed potato batches. Situation of the NTN, Wilga and Z isolates

A collection of 38 PVY isolates from seed potato batches, originating from several Western European countries, was characterized by using current biological, serological and molecular tools differentiating PVY strains and groups. The correlation between the three kinds of tests was good but not absolute. No single serological or PCR method was able to discriminate among the five isolate groups found. Twenty-nine isolates belonged to the PVY^N strain and six to the PVY^O strain. No PVY^C was found. Two other isolates reacted serologically like PVY^O, but were unable to elicit a hypersensitive response from the Ny_tbr gene and probably represent the PVY^Z group. At the molecular level, these two isolates showed a combination of both PVY^O and PVY^N and could be recombinants of these strains. Another isolate reacted serologically like PVY^O, but induced vein necrosis in tobacco, like PVY^N-Wilga. Some PVY^N isolates caused tuber ring necrosis in glasshouse conditions. These might belong to the PVY^NTN group. The PVY^NTN, PVY^N-Wilga and PVY^Z groups probably represent pathotypes within strains PVY^N and PVY^O, respectively. The present study also confirms previous reports showing a high genetic variation at the 5 end within the PVYN strain.     

Genetic diversity of Fusarium oxysporum and related species pathogenic on tomato in Algeria and other Mediterranean countries

In order to characterize the pathogen(s) responsible for the outbreak of fusarium diseases in Algeria, 48 Fusarium spp. isolates were collected from diseased tomato in Algeria and compared with 58 isolates of Fusarium oxysporum originating from seven other Mediterranean countries and 24 reference strains. Partial sequences of the translation elongation factor EF‐1α gene enabled identification of 27 isolates as F. oxysporum, 18 as F. commune and three as F. redolens among the Algerian isolates. Pathogenicity tests confirmed that all isolates were pathogenic on tomato, with disease incidence greater at 28°C than at 24°C. All isolates were characterized using intergenic spacer (IGS) DNA typing, vegetative compatibility group (VCG) and PCR detection of the SIX1 (secreted in xylem 1) gene specific to F. oxysporum f. sp. lycopersici (FOL). No DNA polymorphisms were detected in the isolates of F. redolens or F. commune. In contrast, the 27 Algerian isolates of F. oxysporum were shown to comprise nine IGS types and 13 VCGs, including several potentially new VCGs. As none of the isolates was scored as SIX1+, the 27 isolates could be assigned to F. oxysporum f. sp. radicis‐lycopersici (FORL). Isolates from Tunisia were also highly diverse but genetically distinct from the Algerian isolates. Several Tunisian isolates were identified as FOL by a PCR that detected the presence of SIX1. The results show that isolates from European countries were less diverse than those from Tunisia. Given the difference between Algerian populations and populations in other Mediterranean countries, newly emergent pathogenic forms could have evolved from local non‐pathogenic populations in Algeria.     

Biochemical and molecular characterization of Bacillus amyloliquefaciens, B. subtilis and B. pumilus isolates with distinct antagonistic potential against Xanthomonas campestris pv. campestris

Fifty-one Bacillus isolates were characterized by fatty acid methyl ester (FAME) analysis; universal primer polymerase chain reaction (UP-PCR) fingerprinting; production of secondary metabolites and antagonistic activity against Xanthomonas campestris pv. campestris (causal agent of black rot in cabbage) in vitro and in vivo . Based on FAME analysis and/or PCR fingerprinting, the isolates were clustered into three different groups, named as Bacillus amyloliquefaciens , B . subtilis and B . pumilus . Seed treatment with Bacillus spp. generally reduced germination of seeds and incidence of black rot, but no relationship was found between the results of in vitro and in vivo experiments. The B .  amyloliquefaciens group contained isolates that were generally the most effective at reducing attack of black rot in vivo . The metabolic profiles of these isolates suggested that they produced surfactin, iturin, bacillomycine and/or azalomycin F. Isolates belonging to the B . subtilis group were mostly able to synthesize surfactin and arthrobactin. Surfactin, amphomycin, arthrobactin and valinomycin were generally found in culture extracts of isolates belonging to the B . pumilus group. No effect on growth of the pathogen was detected when the activity of filtered culture extracts and selected metabolites produced by the three different Bacillus species was tested in vitro against X . c . pv. campestris . However, inhibition was seen when bacterial liquid cultures were used. When the ability to colonize cabbage endophytically was examined for seven selected isolates with different antagonistic potential against black rot, it was found that the ability was related to the species and not to the antagonistic activity of the isolates.     

Aggressive and mild Potato virus Y isolates trigger different specific responses in susceptible potato plants

Differences in the early responses of two potato cultivars, Igor and Nadine, to two isolates of Potato virus Y (PVY), the aggressive PVY^NTN and the mild PVY^N, were monitored. Microarray and quantitative real‐time PCR analyses were carried out to identify differentially expressed genes after inoculation with each virus isolate. Additionally, symptom severity and development was observed and the amount of virus isolate accumulated in systemically infected leaves was evaluated, where a significantly higher amount of PVY^NTN was detected. Microarray analysis revealed 572, 1288 and 1706 differentially expressed genes at 0·5, 12 and 48 h post‐inoculation, respectively in cv. Igor, with a similar pattern observed in cv. Nadine. Microarray and quantitative real‐time PCR results implied an earlier accumulation of sugars and lower photosynthesis in leaves inoculated with the aggressive isolate than in leaves inoculated with the mild isolate. The PVY^NTN isolate did not activate early differential expression of the Fe‐superoxide dismutase and pectin methylesterase inhibitor (PMEI) genes, indicating a delay in plant response relative to that following PVY^N inoculation. Differences in the expression of the β‐glucanase‐I gene were also observed in early plant responses to inoculation with each virus isolate.     

<Emphasis Type="Italic">Helicobasidium mompa</Emphasis> isolates from sweet potato in continuous monoculture fields

 Isolates of the violet root rot fungus Helicobasidium mompa were collected from herbaceous and tree plants. Their host preference was studied by inoculation experiments using carrots, sweet potatoes, and apple stocks. It was found that sweet potato isolates from Kyushu produced infection cushions on carrots and sweet potatoes but not on apple stocks. Other isolates did not show host preference. Sweet potato isolates were also characterized by ready hyphal mass (sclerotium) production. They were thought to have adapted to the habitat with high disturbance by annual tillage. Received: July 15, 2002 / Accepted: September 26, 2002     

Molecular characterization of Endothia gyrosa isolates from Eucalyptus in South Africa and Australia

Endothia gyrosa is a canker pathogen best known as the causal agent of pin oak blight in North America, and causes cankers on other woody hosts such as Castanea spp. and Liquidambar spp. In South Africa, Australia and Tasmania, a fungus identified as E. gyrosa has been recorded on Eucalyptus spp. Some morphological differences exist between the North American fungus and the isolates from Eucalyptus . Phylogenetic relationships between E. gyrosa from North America and E. gyrosa from South Africa and Australia, as well as that of the related fungi Cryphonectria parasitica and C. cubensis , were studied using PCR-based restriction fragment length polymorphism (RFLP) and sequences of the internal transcribed spacer (ITS) region of the rRNA operon. Endothia gyrosa isolates from South Africa produced the same RFLP banding patterns as those from Australia, which differed markedly from North American isolates of E. gyrosa . In a phylogram based on the DNA sequences, the Australian and South African isolates of E. gyrosa resided in a single, well resolved clade, distinct from North American isolates. Isolates of C. parasitica grouped in the same clade as the South African and Australian isolates of E. gyrosa , but C. cubensis was distantly related to them. The molecular data suggest that the E. gyrosa isolates from South Africa and Australia represent a distinct taxon, and probably belong to the genus Cryphonectria .     

Role of co‐infection by Pectobacterium and Verticillium dahliae in the development of early dying and aerial stem rot of Russet Burbank potato

Potato early dying (PED) is a disease complex primarily caused by the fungus Verticillium dahliae. Pectolytic bacteria in the genus Pectobacterium can also cause PED symptoms as well as aerial stem rot (ASR) of potato. Both pathogens can be present in potato production settings, but it is not entirely clear if additive or synergistic interactions occur during co‐infection of potato. The objective of this study was to determine if co‐infection by V. dahliae and Pectobacterium results in greater PED or ASR severity using a greenhouse assay and quantitative real‐time PCR to quantify pathogen levels in planta. PED symptoms caused by Pectobacterium carotovorum subsp. carotovorum isolate Ec101 or V. dahliae isolate 653 alone included wilt, chlorosis and senescence and were nearly indistinguishable. Pectobacterium wasabiae isolate PwO405 caused ASR symptoms including water‐soaked lesions and necrosis. Greater Pectobacterium levels were detected in plants inoculated with PwO405 compared to Ec101, suggesting that ASR can result in high Pectobacterium populations in potato stems. Significant additive or synergistic effects were not observed following co‐inoculation with these strains of V. dahliae and Pectobacterium. However, infection coefficients of V. dahliae and Ec101 were higher and premature senescence was greater in plants co‐inoculated with both pathogens compared to either pathogen alone in both trials, and V. dahliae levels were greater in basal stems of plants co‐inoculated with either Pectobacterium isolate. Overall, these results indicate that although co‐infection by Pectobacterium and V. dahliae does not always result in significant additive or synergistic interactions in potato, co‐infection can increase PED severity.     

Characterization ofRhizoctonia solani isolates from potatoes in turkey and screening potato cultivars for resistance to AG-3 isolates

A total of 304Rhizoctonia solani isolates and 60 binucleateRhizoctonia-like fungi were recovered from stems and tubers of infected potato plants over a 2-yr period in northeast Turkey.R. solani isolates were identified to 11 anastomosis groups (AGs): AG-1 (0.66%), AG-2-1 (5.6%), AG-2-2 (0.99%), AG-3 (83.9%), AG-5 (4.6%), AG-6 (0.66%), AG-8 (1.32%), AG-9 (0.33%), AG-10 (1.32%), AG-12 (0.33%), and AG-13 (0.33%). In the greenhouse tests, most of the AG-3 isolates were significantly more virulent than isolates belonging to other AGs on potato cv. Batum. Isolates of other anastomosis groups differed in their virulence. Results indicated that AG-3 is an important pathogen on potatoes grown in the study area. Five of 22 commercial and local potato cultivars evaluated for their reaction toR. solani AG-3 isolates (TP-2) under greenhouse conditions were highly resistant; the remaining cultivars exhibited different levels of susceptibility to the pathogen isolate. http://www.phytoparasitica.org posting July 14, 2005.     

Genetic, phenotypic and pathogenic diversity among xanthomonads isolated from pistachio (Pistacia vera) in Australia

Repetitive extragenic palindromic polymerase chain reaction (rep-PCR), sequencing of the 16S−23S rDNA internal transcribed spacer (ITS), biochemical and physiological tests, the Biolog microplate system, polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins, and pathogenicity tests were used to characterize variability among xanthomonads isolated from pistachio trees suffering from bacterial dieback in four regions of Australia. ITS sequencing and rep-PCR revealed two distinct genotypes among the strains. The ITS sequencing suggested that the pistachio strains were closely related to Xanthomonas translucens pathovars, in particular X. translucens pv . poae . Results of physiological and biochemical tests, as well as Biolog microplate analysis and protein profiling, confirmed the existence of two groups. Furthermore, pathogenicity and host-range studies indicated that the two groups were biologically different. There was an association between the two groups and the geographical origin of the strains.     

Chestnut blight fungus in Croatia: diversity of vegetative compatibility types,mating types and genetic variability of associated Cryphonectria hypovirus 1

In order to improve understanding of its diversity, 338 isolates of Cryphonectria parasitica, the causal agent of chestnut blight, were sampled from 10 chestnut populations throughout chestnut‐growing coastal and continental areas of Croatia. Eighteen vegetative compatibility (VC) types were identified. The VC type EU‐1 was the most widespread, comprising 42·9% of the isolates, followed by EU‐2 (21%) and EU‐12 (14·2%). In respect to the occurrence of the main VC types, the C. parasitica populations in Croatia combined features of both northwestern and southeastern European populations. Perithecia and mating‐type ratios of approximately 1 : 1 were found in all populations, suggesting that sexual reproduction of the fungus is common in Croatia. Natural hypovirulence was also evident in all populations, with incidence of hypovirus‐infected isolates ranging from 12·7% in Istria‐Buje to 66·6% in the continental part of the country. A total of 36 hypovirus‐infected isolates sampled throughout Croatia were analysed in ORF‐A and ORF‐B by RT‐PCR/RFLP analysis. All viral isolates belonged to the Italian subtype of Cryphonectria hypovirus 1 (CHV‐1) and were closely related to the isolates found in other European countries. The RFLP patterns found were also identical or similar to the patterns of three isolates collected in Croatia 22 years ago, suggesting a slow evolution of the hypovirus.     

French isolates of Phytophthora infestans from potato and tomato differ in phenotype and genotype

Prior to 1996, the A2 mating type of Phytophthora infestans was not detected on potato in France, but was found at one site on tomato in 1995. This finding lead to the question of the extent of differences and relationships existing between the populations of P. infestans present on each host. A collection of 76 isolates collected in France, mainly in 1996, from potato and tomato was characterised for mating type, allozyme genotype at the Gpi and Pep loci, and mitochondrial DNA haplotype; 74 of these isolates were also characterised for multilocus RFLP fingerprint, and 62 for virulence. All isolates except four showed allozyme genotypes (Gpi 90/100 or 100/100, Pep 83/100 or 100/100) and mtDNA haplotypes (Ia or IIa) characteristic of the populations introduced into Europe in the late 1970s. The four exceptions were isolates collected from tomato in Southern France in 1988-1991, which showed some characteristics of the former European populations (Gpi 86/100, Pep 92/100, mtDNA Ib). Both mating types were present among the collections from both hosts, but isolates with the A2 mating type were found on potato only in one garden crop, adjacent to tomato. Nine different RG57 fingerprints were observed, with a greater diversity among tomato isolates. Furthermore, tomato and potato collections differed markedly in the frequencies of genotypes present. Finally, tomato isolates generally had a lower virulence complexity than potato isolates. These data suggest that P. infestans populations on tomato and potato are largely separated, despite the occurrence of limited gene flow.     

Comparison of conventional and molecular methods for the detection of Rosellinia necatrix in avocado orchards in southern Spain

White root rot, caused by Rosellinia necatrix , is one of the most important diseases in avocado orchards and is particularly widespread on the Mediterranean seaboard of southern Spain. In this study, the presence of the pathogen in soil samples collected from the base of 47 plants showing different symptoms of canopy decline was assessed with a molecular detection method based on real-time Scorpion PCR. Results were compared with symptoms in the canopy and with the traditional method of isolation of R. necatrix from roots and/or bark. The fungus was isolated from 24 samples by the traditional method and from 37 soil samples by the molecular method (cycle threshold values 25·8 to 47·1), demonstrating the higher sensitivity and reliability of the molecular method. A single real-time PCR amplification was sufficient to detect R. necatrix in naturally infested soils. The avoidance of nested PCR has important practical implications because of the reduced costs and risk of cross contamination. Also, it enables faster sample analysis and is more appropriate for quantitative detection. A modified molecular method was also developed to detect R. necatrix in roots and in soils with very low populations of the pathogen.