Real-time RT-qPCR assay to detect sequences in the Piscine orthoreovirus-1 genome segment S1 associated with heart and skeletal muscle inflammation in Atlantic salmon

During the last decade, Piscine orthoreovirus was identified as the main causative agent of heart and skeletal muscle inflammation (HSMI) in Atlantic Salmon, Norway. A recent study showed that PRV-1 sequences from salmonid collected in North Atlantic Pacific Coast (NAPC) grouped separately from the Norwegian sequences found in Atlantic Salmon diagnosed with HSMI. Currently, the routine assay used to screen for PRV-1 in NAPC water and worldwide cannot differentiate between the two groups of PRV-1. Therefore, this study aimed at developing a real-time polymerase chain reaction (RT-qPCR) assay to target the PRV-1 genome segments specific for variants associated with HSMI. The assay was optimized and tested against 71 tissue samples collected from different regions including Norway, Chile and both coast of Canada and different hosts farmed Atlantic Salmon, wild Coho Salmon and escaped Atlantic Salmon collected in British Columbia, West Coast of Canada. This assay has the potential to be used for screening salmonids and non-salmonids that may carry PRV-1 potentially causing HSMI.     

Special issue “Deepen knowledge in plant pathology for innovative agroecology”

This study aimed to determine the genetic variability of isolates of rotting of pineapple fruitlet core in Brazil on the states of Paraiba, Pernambuco and Rio Grande do Norte, based on phylogenetic analysis of the RPB2 gene, morphocultural markers and aggressiveness of the isolates. The phylogenetic reconstruction of maximum parsimony and bayesian inference of the isolates were performed. Twenty-seven phylogenetic lineages were characterized with morphocultural markers on potato dextrose agar and synthetic nutrient-poor agar. The aggressiveness of these lineages were characterized in leaves and fruits of pineapple ‘Pérola’ cultivar. The Phylogenetic reconstruction showed close relationship between isolates of this study and phylogenetic lineages of F. guttiforme, F. ananatum and F. oxysporum by analysis of RPB2 gene. Phylogenetic lineages of this study shared significant morphocultural markers similar to those described for this species. Overall, the lineages related with Fusarium oxysporum Species Complex were more aggressive to the fruits of the Fusarium lineages related with Fusarium fujikuroi Species Complex. It is possible that F. ananatum and F. guttiforme or other lineages closely related to these species or F. oxysporum are present in the fields causing pineapple rotting fruitlet core in fruits of pineapple ‘Pérola’ in Brazil. The used markers determined high genetic variability in isolates analyzed in leaves and pineapple fruit ‘Pérola’ in the states of Paraíba, Pernambuco and Rio Grande do Norte of Brazil and the pathogenic lineages analyzed were better adapted to the fruits than to the detached leaves in pineapple ‘Pérola’ analyzed.     

Piscine reovirus in wild and farmed salmonids in British Columbia,Canada: 1974–2013

Piscine reovirus (PRV) was common among wild and farmed salmonids in British Columbia, western Canada, from 1987 to 2013. Salmonid tissues tested for PRV by real‐time rRT‐PCR included sections from archived paraffin blocks from 1974 to 2008 (n = 363) and fresh‐frozen hearts from 2013 (n = 916). The earliest PRV‐positive sample was from a wild‐source steelhead trout, Oncorhynchus mykiss (Walbaum), from 1977. By histopathology (n = 404), no fish had lesions diagnostic for heart and skeletal muscle inflammation (HSMI). In some groups, lymphohistiocytic endocarditis affected a greater proportion of fish with PRV than fish without PRV, but the range of Ct values among affected fish was within the range of Ct values among unaffected fish. Also, fish with the lowest PRV Ct values (18.4–21.7) lacked endocarditis or any other consistent lesion. From 1987 to 1994, the proportion of PRV positives was not significantly different between farmed Atlantic salmon, Salmo salar L. (44% of 48), and wild‐source salmonids (31% of 45). In 2013, the proportion of PRV positives was not significantly different between wild coho salmon, Oncorhynchus kisutch (Walbaum), sampled from British Columbia (5.0% of 60) or the reference region, Alaska, USA (10% of 58).     

Tunisian Population of the Wheat Pathogen Mycosphaerella graminicola is Still Fully Sensitive to Strobilurin Fungicides

Mycosphaerella graminieola （anamorph： Zymoseptoria tritici） is the causal agent of Septoria tritici blotch, the most frequently occurring disease on wheat crops worldwide. A set of 163 monoconidial isolates of this fungus were sampled in 2012 from five geographical locations of Tunisia （Bizerte, B6ja, Kef, Jendouba and Siliana） in order to examine the status of strobilurin resistance of M. graminicola in this country. The resistance was assessed by using PCR-based mismatch mutation assay that determined the cytochrome b substitution G143A responsible for strobilurin resistance. All isolates were found sensitive since they possessed the wild-type allele G143 conferring sensitivity. This study confirms previous reports on the fungus in Tunisia and reveals that the Tunisian population of M. graminicola remains fully sensitive to strobilurin fungicides. An appropriate management of strobilurin applications in Tunisia is thereby recommended to prevent local development and widespread of resistance, as in Europe, where pathogen populations are fully resistant to strobilurins today.     

Correlation of in planta endo‐beta‐1,4‐xylanase activity with the necrotrophic phase of the hemibiotrophic fungus Mycosphaerella graminicola

The association of the cell wall degrading enzyme endo‐beta‐1,4‐xylanase (EC 3.2.1.8) with pathogenicity of Mycosphaerella graminicola was examined in planta. The enzyme production of two M. graminicola isolates (T0372 and T0491), as well as their ability to infect seedlings of susceptible wheat cv. Scorpion, was first compared. No significant difference was found between the two isolates regarding spore germination rates, mycelial growth on the leaf surface or direct and stomatal penetrations. However, restricted hyphal growth was observed inside leaves inoculated with T0372, whereas successful mesophyll colonization with a strong intercellular fungal growth was found in leaves infected with T0491. Likewise, T0372 was unable to induce lesions bearing pycnidia and to produce endo‐beta‐1,4‐xylanase activity until 22 days post‐inoculation (d.p.i.). On the other hand, significant high increases of both diseased leaf area bearing pycnidia and endo‐beta‐1,4‐xylanase activity were observed between 16 and 22 d.p.i. for T0491 (r = 0·98). The investigation of 24 additional isolates, including the IPO323 and IPO94269 reference isolates, highlighted a strong correlation between endo‐beta‐1,4‐xylanase activity and disease development levels (r = 0·94). This study demonstrates that differences in pathogenicity in M. graminicola are not linked to events on the leaf surface or to frequency of leaf penetration, but to the ability of the fungus to colonize the mesophyll and to produce the cell wall degrading enzyme endo‐1,4‐beta‐xylanase during the necrotrophic phase.     

Azoxystrobin resistance of French Mycosphaerella graminicola strains assessed by four in vitro bioassays and by screening of G143A substitution

We tested here five distinct in vitro screening methods assessing Mycosphaerella graminicola resistance to strobilurin fungicides: (i) spotting on agar medium, (ii) spore germination on agar medium, (iii) growth in microplates without and (iv) with the addition of Alamar blue and (v) screening of the cytochrome b substitution conferring resistance G143A. The assays were performed by assessing resistance to azoxystrobin of 32 French M. graminicola strains and the two reference strains IPO323 and IPO94269 from the Netherlands. The microplate Alamar blue assay displayed high standard deviations from all growth averages, hence reporting the strong lack of reliability of this method. As expected, the two reference strains were found sensitive with all other methods. In agreement with the disruptive resistance of M. graminicola to single-site fungicides, the half maximal inhibitory concentration (IC₅₀) values of sensitive and resistant strains displayed a bimodal distribution pattern with each type of assay. All strains carrying G143 or A143 alleles were found sensitive or resistant respectively with each bioassay. Although the absolute IC₅₀ value for each strain varied among assays, the ranking of strains according to their IC₅₀ values was overall conserved with all assays. Finally, the frequency of strobilurin resistant haplotypes within France was investigated by screening G143A in 82 strains isolated in 2005 year from 12 localities. Results showed a marked gradual decrease in resistance distribution from 70% in northern to 30% and 0% in central and southern France, respectively. The accuracy of each method as well as the widespread and incidence of strobilurin resistance within the French population of M. graminicola have been discussed.     

Bodenentwicklung und Schwermetallgehalte einer Straßenkehrichtdeponie

Soil development and heavy metal contents of a street sweepings dump Soils from an unsealed street sweepings dump of the city of Kiel are inhomogeneous in their properties and in their distribution pattern. The deposited material is contaminated with copper, zinc, lead, and at lower concentrations with cadmium. The high pH-values lead to predominating immobility of heavy metals. However, heavy metals bonded in organic complexes are liable to be leached into the underground because of a high portion of large pores in these soils. Since there are only few other possible bonding mechanisms than organics, after the leaching of lime, pH decline, and mineralization of organic matter mobilization of heavy metals may be predicted. Thus, in the unsealed dump a danger of groundwater pollution with heavy metals remains. The site should be treated accordingly permanently after closing.