Varietal screening of Solanaceae accessions against R. solanacearum strains, agent of bacterial wilt in Côte d’Ivoire

Bacterial wilt caused by R. solanacearum is a constraint that limits the production of solanaceous in Côte d'Ivoire. The genetic control, which is more widely used, comes up against the genetic and phenotypic variability of the strains. The objective of this study was to screen commercial and traditional varieties of Solanaceae against Ivorian strains of different phylotypes to assess their spectrum of resistance to the disease. The level of virulence of the five strains of R. solanacearum was evaluated in semi-controlled conditions against eight varieties of Solanaceae (pepper, eggplant and tomato). Four groups of strains were identified according to the level of virulence. Group 4, composed of strains (RUN 1794 and RUN 1854) of phylotype III and I, respectively, and originating from the localities of Kondoukro and Yamoussoukro, was very virulent. The resistant varieties were for the F1 kalenda eggplant, for the yellow chilli from Burkina and for the F1 Lindo tomato. These varieties could be used in the fight against bacterial wilt in Côte d'Ivoire.

Host Range-Associated Clustering Based on Multilocus Variable-Number Tandem-Repeat Analysis, Phylotypes, and Virulence Genes of Atypical EnteropathogenicEscherichia coliStrains

ABSTRACTAtypical enteropathogenicEscherichia coli(aEPEC) strains (36 Japanese and 50 Bangladeshi) obtained from 649 poultry fecal samples were analyzed by molecular epidemiological methods. Clermont’s phylogenetic typing showed that group A was more prevalent (58%, 50/86) than B1 (31%, 27/86). Intimin type β1, which is prevalent among human diarrheal patients, was predominant in both phylogroups B1 (81%, 22/27) and A (70%, 35/50). However, about 95% of B1-β1 strains belonged to virulence group I, and 77% of them were Japanese strains, while 17% (6/35) of A-β1 strains did. Multilocus variable-number tandem-repeat analysis (MLVA) distributed the strains into 52 distinct profiles, with Simpson’s index of diversity (D) at 73%. When the data were combined with those of 142 previous strains from different sources, the minimum spanning tree formed five zones for porcine strains, poultry strains (excluding B1-β1), strains from healthy humans, bovine and human patient strains, and the B1-β1 poultry strains. Antimicrobial resistance to nalidixic acid was most common (74%) among the isolates. Sixty-eight percent of them demonstrated resistance to ≥3 antimicrobial agents, and most of them (91%) were from Bangladesh. The strains were assigned into two groups by hierarchical clustering. Correlation matrix analysis revealed that the virulence genes were negatively associated with antimicrobial resistance. The present study suggested that poultry, particularly Japanese poultry, could be another reservoir of aEPEC (phylogroup B1, virulence group I, and intimin type β1); however, poultry strains seem to be apart from patient strains that were closer to bovine strains. Bangladeshi aEPEC may be less virulent for humans but more resistant to antibiotics.IMPORTANCEAtypical enteropathogenicEscherichia coli(aEPEC) is a diarrheagenic type ofE. coli, as it possesses the intimin gene (eae) for attachment and effacement on epithelium. Since aEPEC is ubiquitous even in developed countries, we previously used molecular epidemiological methods to discriminate aEPEC as a human pathogen. The present study assessed poultry as another source of human diarrheagenic aEPEC. Poultry could be the source of aEPEC (phylogroup B1, virulence group I, and intimin type β1) found among patient strains in Japan. However, the minimum spanning tree (MST) suggested that the strains from Japanese poultry were far from Japanese patient strains compared with the distance between bovine and patient strains. Bangladeshi avian strains seemed to be less diarrheagenic but are hazardous as a source of drug resistance genes.

Divergence between the Highly Virulent Zoonotic Pathogen Helicobacter heilmannii and Its Closest Relative, the Low-Virulence “Helicobacter ailurogastricus” sp. nov.

Helicobacter heilmanniinaturally colonizes the stomachs of dogs and cats and has been associated with gastric disorders in humans. Nine felineHelicobacterstrains, classified asH. heilmanniibased onureABand 16S rRNA gene sequences, were divided into a highly virulent and a low-virulence group. The genomes of these strains were sequenced to investigate their phylogenetic relationships, to define their gene content and diversity, and to determine if the differences in pathogenicity were associated with the presence or absence of potential virulence genes. The capacities of these helicobacters to bind to the gastric mucosa were investigated as well. Our analyses revealed that the low-virulence strains do not belong to the speciesH. heilmanniibut to a novel, closely related species for which we propose the nameHelicobacter ailurogastricus. Several homologs ofH. pylorivirulence factors, such as IceA1, HrgA, and jhp0562-like glycosyltransferase, are present inH. heilmanniibut absent inH. ailurogastricus. Both species contain a VacA-like autotransporter, for which the passenger domain is remarkably larger inH. ailurogastricusthan inH. heilmannii. In addition,H. ailurogastricusshows clear differences in binding to the gastric mucosa compared toH. heilmannii. These findings highlight the low-virulence character of this novelHelicobacterspecies.

First Report of Root Rot Caused by Pythium spiculum Affecting Cork Oaks at Doñana Biological Reserve in Spain

Cork oaks (Quercus suber L.) are key tree species at Doñana Biological Reserve (DBR), Huelva, Spain. Sampling was conducted on a total of 13 trees exhibiting symptoms of decline (foliar wilting and defoliation, branch dieback, and root necrosis). In 2008. Phytophthora cinnamomi was isolated from feeder roots of one tree and Pythium spiculum from two additional oaks. In 2011, both pathogens were isolated from six oaks, only P. cinnamomi from three oaks, and only Py. spiculum from one oak. This expansion was associated with high winter rainfall levels since 2009 that led to long periods of soil flooding. While P. cinnamomi is well known to cause a root disease on Q. suber (2), P. spiculum is a newly described species isolated from Quercus, Vitis, Prunus, Castanea, and Celtis species, but its pathogenicity was demonstrated only on Q. ilex (syn. Q. rotundifolia) (1). Pathogenicity tests were conducted on 4-year-old Q. suber plants. Inocula consisted of two isolates of Py. spiculum from DBR (DO8 and DO36 from Q. suber). For comparison with these, three isolates previously tested on Q. ilex (1) were included: two isolates of Py. spiculum, PA54 (from Q. suber) and PE156 (from Q. ilex); and one isolate of P. cinnamomi, PE90 (from Q. ilex). All these isolates came from the Andalucía region, stored at the oomycete collection of the University of Córdoba, and showed a 99 to 100% homology with their expected ITS sequences in GenBank (DQ196131 for Py. spiculum and AY943301 for P. cinnamomi). Inoculum was prepared by shaking and mixing propagule-bearing mycelium produced in carrot broth petri dishes (20°C, 4 weeks) in sterile water, to produce a concentration of 3 × 104 oospores × ml−1 (Py. spiculum) or 3 × 104 chlamydospores × ml−1 (P. cinnamomi). One hundred milliliters of inoculum was applied to each root (1). There were 10 inoculated plants per isolate and 10 non-inoculated control plants. All plants were waterlogged 2 days per week to favor root infection and maintained in an acclimatised greenhouse (12–28°C). Three months later, the inoculated plants showed symptoms of root necrosis that resulted in foliar wilting followed occasionally by defoliation. Control plants did not develop foliar symptoms nor root necrosis. Root damage severity assessed on a 0 to 4 scale (3) exhibited significant differences (P < 0.05) in relation to the control plants for all the isolates tested, with isolate PE90 (P. cinnamomi) and isolates PA54, DO8, and DO36 (P. spiculum) all averaging a root necrosis value of 2.5. Isolate PE156 of P. spiculum produced values of root necrosis (1.6 in average) significantly lower (P < 0.05) than the rest. This isolate belongs to the low virulence group of P. spiculum described on Q. ilex (1). The inoculated oomycete was always reisolated from necrotic roots and never from roots of control plants. To the best of our knowledge, this is the first report of P. spiculum as the cause of root rot of Q. suber. References: (1) Romero et al. J. Phytopathol. 155:289, 2007. (2) Sánchez et al. For. Pathol. 32:5, 2002. (3) Sánchez et al. For. Pathol. 35:115, 2005.

Specific Properties of Enteropathogenic Escherichia coli Isolates from Diarrheal Patients and Comparison to Strains from Foods and Fecal Specimens from Cattle, Swine, and Healthy Carriers in Osaka City, Japan

ABSTRACTFor exhaustive detection of diarrheagenicEscherichia coli, we previously developed a colony-hybridization method using hydrophobic grid-membrane filters in combination with multiplex real-time PCR. To assess the role of domestic animals as the source of atypical enteropathogenicE. coli(aEPEC), a total of 679 samples (333 from foods, fecal samples from 227 domestic animals, and 119 from healthy people) were examined. Combining 48 strains previously isolated from patients and carriers, 159 aEPEC strains were classified by phylogroup, virulence profile, and intimin typing. Phylogroup B1 was significantly more prevalent among aEPEC from patients (50%) and bovine samples (79%) than from healthy carriers (16%) and swine strains (23%), respectively. Intimin type β1 was predominant in phylogroup B1; B1-β1 strains comprised 26% of bovine strains and 25% of patient strains. The virulence profile groups Ia and Ib were also observed more frequently among bovine strains than among porcine strains. Similarly, virulence group Ia was detected more frequently among patient strains than strains of healthy carriers. A total of 85 strains belonged to virulence group I, and 63 of these strains (74%) belonged to phylogroup B1. The present study suggests that the etiologically important aEPEC in diarrheal patients could be distinguished from aEPEC strains indigenous to humans based on type, such as B1, Ia, and β1/γ1, which are shared with bovine strains, while the aEPEC strains in healthy humans are different, and some of these were also present in porcine samples.

Biological characterisation of Globodera pallida from Idaho

The white potato cyst nematode, Globodera pallida, was recently found in the USA and was identified using molecular and morphometric assessment. This paper examined some biological characteristics of the Idaho population of G. pallida. The samples of the original cysts showed that they had low rates of juvenile hatch and, consequently, low rates of reproduction when compared with glasshouse-reared populations. Virulence tests using sources of partial resistance from Solanum vernei and S. tuberosum ssp. Andigena CPC 2802 compared the Idaho population to European and South American populations of G. pallida. The Idaho population showed a virulence pattern comparable to other European populations in the Pa2/3 virulence group. The overall level of reproduction of the Idaho population on the resistant cultivars was the lowest of all the populations tested, suggesting that it is not highly virulent. These results are compatible with molecular characterisation, which indicates that this population is similar to the European gene pool.

Differences in virulence of Diplocarpon earlianum isolates on selected strawberry cultivars

Eight isolates of Diplocarpon earlianum were evaluated for virulence, measured as the ability to produce percent leaf area with symptoms (LAS), on 18 cultivars of strawberry (Fragaria x ananassa). Significant differences were observed from isolate and cultivar effects, and isolate x cultivar interaction. Four of the eight isolates, on the average of 18 strawberry cultivars, had LAS ranging from 6.7-9.6%, and were classified as a group of low virulence. The remaining four isolates had LAS ranging from 25.9-45.8% and formed a high virulence group. Of the cultivars tested, 'Vibrant' was resistant and 'Micmac' was susceptible to all isolates in both virulence groups; 'Honeoye', 'Redcoat', 'Scotland', 'St. Clair' and 'Vantage' were resistant to the low virulence group, but had differential interactions to isolates in the high virulence group; the remaining 11 cultivars were susceptible to the high virulence group, but had differential interactions to isolates in the low virulence group. It is suggested that a number of different pathogen isolates are required to test strawberry genotypes for leaf scorch resistance. A leaf disk assay was developed in this study and can be used for laboratory evaluations of strawberry genotypes for susceptibility to D. earlianum.

Small subunit ribosomal DNA phylogeny of microsporidia that infect Daphnia (Crustacea: Cladocera)

Glugoides intestinalis, Microsporidium sp., Ordospora colligata, Gurleya vavrai, Larssonia obtusa and Flabelliforma magnivora are microsporidian parasites of planctonic freshwater crustaceans Daphnia spp. We performed a phylogenetic analysis of the small subunit ribosomal DNA which revealed their positions as polyphyletic. G. intestinalis, Microsporidium sp. and O. colligata, which are horizontally transmitted gut parasites with small spores and low virulence, group with different lineages. G. intestinalis is related to 2 microsporidia infecting lepidopterans and to Vittaforma corneae, which has been described as a human pathogen. It is thought that V. corneae may have an invertebrate as its natural host. Microsporidium sp. is a relative of the genera Enterocytozoon and Nucleospora, pathogens of man and fish respectively. O. colligata is the first species found to be closely related to the genus Encephalitozoon, which is comprised of 3 species that are parasites of homeothermic vertebrates. G. vavrai and L. obtusa are sister taxa that branch close to the Amblyosporidae, the only microsporidia with known intermediate hosts. This finding supports the presumption of secondary hosts for G. vavrai and L. obtusa, as it has not been possible to maintain these species in Daphnia in the laboratory. F. magnivora roots deep at the base of the phylum microsporidia with no close relative found so far.

Virulence of Xanthomonas oryzae pv. oryzae on Rice Lines Containing Single Resistance Genes and Gene Combinations

Fifty isolates of Xanthomonas oryzae pv. oryzae were collected from different rice-producing districts of Nepal and evaluated for their virulence on these 11 rice lines having from one to four resistance genes: IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB14 (Xa14), and IRBB21 (Xa21), two-gene combination AY4+5 (Xa4 and xa5), three-gene combinations NH21-37-1-1 (Xa4, xa5, and xa13) and NH24-10-1-3 (Xa4, xa5, and Xa21), and four-gene combination NH56-1-44-4 (Xa4, xa5, xa13, and Xa21). The ability of an isolate to cause lesions with different lengths across the lines was interpreted as virulence. Isolates that were consistently associated with high or low virulence were statistically differentiated. Most isolates produced large lesions on near-isogenic lines with single genes and small lesions on lines with different gene combinations. Based on infection responses on the two-, three-, and four-gene combinations, five virulence groups were identified. Isolates in virulence group I were widely distributed. The line × isolate interactions were generally not significant with gene combinations, indicating a low possibility of specificity. A line with a four-gene combination, NH56, showed wider spectrum and higher level of resistance to X. oryzae pv. oryzae than the other lines. The results of this study will facilitate the deployment of effective resistance to X. oryzae pv. oryzae in Nepal.

Evolutionary changes in myxoma virus in Britain: An examination of 222 in naturally occurring strains obtained from 80 counties during the period October–November 1962

Samples of lesions containing viable myxoma virus were collected from 222 infected wild rabbits captured in 80 counties of Britain during October–November 1962. They were dispatched by air to Australia and passaged once in domestic rabbits before being tested for their virulence by the intradermal inoculation of groups of six laboratory rabbits with small doses of virus.The results showed that a wide range of viruses of differing properties now coexist in Britain. Their virulence ranges from very high (99 % lethal) to low (about 50 %), but the majority of strains fall into the grade III virulence group, with estimated case mortality rates varying between 70 and 95%. The viruses also produce disease of varying symptomatology, skin lesions being very prominent, raised or flat. There was no obvious association between lesion type and virulence.Tests made on the wild rabbits from which the samples were obtained, and on Inoculated domestic rabbits, showed that virulence could not be predicted by gel-diffusion tests.