Effect of fishmeal replacement by poultry by‐product meal on growth performance,proximate composition,digestive enzyme activity,haematological parameters and gene expression of gilthead seabream (Sparus aurata)

Two feeding trials examined the replacement of fishmeal (FM) with poultry by‐product meal (PBM) in the diet of juvenile Sparus aurata. In Feeding trial I (100 days), three diets were formulated, where FM protein was replaced by 50% (PBM50) and 100% (PBM100) PBM, while in Feeding trial II (110 days), four diets were formulated using the same FM control diet, but FM was replaced at lower levels: 25% (PBM25), and 25% (PBM25 + ) and 50% (PBM50 + ) with the supplementation of lysine and methionine amino acids. PBM protein can successfully replace 50% of FM protein in the diet of S. aurata without adverse effects on survival, feed intake, growth performance and feed utilization, given that the diet is balanced with lysine and methionine. The proximate composition of body and muscle was unaffected by the diet, but the total FM replacement resulted in reduced lipid and energy contents in fish. A fifty per cent FM replacement by PBM did not affect haematological parameters indicating a good fish health. Similarities in trypsin and chymotrypsin activities with FM‐fed fish suggest a high digestibility of PBM. High dietary levels of PBM reduced the liver gene expression of GH/IGF axis and of cathepsin D suppressing fish growth and modulating the protein turnover.     

Characterization of Laboratory Mutants of Botrytis cinerea Resistant to QoI Fungicides

Mutants of Botrytis cinerea with moderate and high resistance to pyraclostrobin, a Qo inhibitor of mitochondrial electron transport at the cytochrome bc ₁ complex, were isolated at a high mutation frequency, after nitrosoguanidine mutagenesis and selection on medium containing pyraclostrobin and salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strain by pyraclostrobin and SHAM, but not in the mutant isolates. Cross-resistance studies with other Qo and Qi inhibitors (QoIs and QiIs) of cytochrome bc ₁ complex of mitochondrial respiration showed that the mutation(s) for resistance to pyraclostrobin also reduced the sensitivity of mutant strains to other QoIs as azoxystrobin, fluoxastrobin, trifloxystrobin and picoxystrobin, but not to famoxadone and to the QiIs cyazofamid and antimycin-A. An increased sensitivity of pyraclostrobin-resistant strains to the carboxamide boscalid, an inhibitor of complex II, and to the anilinopyrimidine cyprodinil, a methionine biosynthesis inhibitor, was observed. Moreover, no effect of pyraclostrobin resistance mutation(s) on fungitoxicity of the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the benzimidazole benomyl, and to the phenylpyridinamine fluazinam, which affect other cellular pathways, was observed. Study of fitness parameters in the wild-type and pyraclostrobin-resistant mutants of B. cinerea showed that most mutants had a significant reduction in the sporulation, conidial germination and sclerotia production. Experiments on the stability of the pyraclostrobin-resistant phenotype showed a reduction of resistance, mainly in moderate resistant strains, when the mutants were grown on inhibitor-free medium. However, a rapid recovery of the resistance level was observed after the mutants were returned to a selective medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of high resistant mutants was higher than the moderate ones. Pathogenicity tests on cucumber seedlings showed that all mutant strains tested exhibited an infection ability similar with the wild-type parent strain. Preventive applications of the commercial product of F-500 25EC (pyraclostrobin) were effective against lesion development on cotyledons by the wild-type, but ineffective, even at high concentrations, against disease caused by the pyraclostrobin-resistant isolates. Boscalid (F-510 50WG) was found equally effective against the disease caused by the wild-type or pyraclostrobin-resistant mutants. This is the first report indicating the appearance of B. cinerea strains resistant to QoI fungicides by the biochemical mechanism of site modification and the risk for field resistance.     

Studies on the Inherent Resistance Risk to Fenhexamid in Botrytis cinerea

After chemical mutagenesis with N-methyl-N-nitrosoguanidine (MNNG) two phenotypes that were highly or moderately resistant to fenhexamid, were isolated from a wild-type strain of Botrytis cinerea, at a mutation frequency of 0.9 × 10^–5. Resistance factors, based on EC⁵⁰ values, were 460–570 and 10–15, respectively. The mutation(s) for resistance to fenhexamid did not affect the sensitivity of mutant strains to the benzimidazole benomyl, the phenylpyridinamine fluazinam, the anilinopyrimidine cyprodinil, the guanidine iminoctadine or to the sterol-biosynthesis-inhibiting fungicides fenarimol, fenpropimorph and tridemorph. On the contrary, an increased sensitivity (EC⁵⁰ ratios of 0.2–0.6) of fenhexamid-resistant strains to the phenylpyrrole fludioxonil and the dicarboximide iprodione was observed. Study of fitness parameters of fenhexamid-resistant isolates of both phenotypic classes showed that these mutation(s) had no effect on mycelial growth and sensitivity to high osmolarity, but they did affect one or more of some other characteristics, such as sporulation, conidial germination and sclerotia production. In tests on cucumber seedlings under greenhouse conditions, all highly fenhexamid-resistant isolates tested presented decreased infection ability compared with the wild-type. Preventive applications of a commercial formulation of fenhexamid, Teldor 50 WP, were effective against lesion development on cotyledons by the wild-type, but ineffective, even in high concentrations, against disease caused by the fenhexamid-resistant isolates. The risk of resistance problems arising during commercial use of fenhexamid is discussed.     

Fitness and cross-resistance of Alternaria alternata field isolates with specific or multiple resistance to single site inhibitors and mancozeb

European Journal of Plant Pathology - 42 field isolates of Alternaria alternata collected from infected tomato fruit of processing plants located in the region of Pelloponisos, South Greece, were...     

Molecular diagnostic for detecting the cytochrome b G143S - QoI resistance mutation in Cercospora beticola

The mutation G143S has been associated with high-level strobilurin resistance in laboratory mutant strains of Cercospora beticola, one of the most destructive pathogens in sugar beet plants. By using allele specific primers (PASA-PCR) and agarose gel visualization, a molecular diagnostic was developed for the detection of the G143S resistance mutation. This assay is simple and applicable in low tech laboratory settings, with high reliability when a relatively large proportion of mutated mitochondrial alleles are present in the resistant strains. To achieve detection of resistant alleles at low frequencies, a more sensitive Real Time PCR based assay capable of discriminating resistant (S143) genotypes in frequencies as low as 1:10,000 resistant:sensitive alleles was developed. Both diagnostics were successfully validated in laboratory strains. Subsequently, a large number of C. beticola isolates from QoI-treated sugar beet experimental fields in Greece were screened for resistance to Qo fungicides using these diagnostics and classic bioassays. No proportion of the 143S resistant allele was detected in all field isolates tested, which was in agreement with the phenotypes revealed by the biotests confirming that the efficacy of QoIs against C. beticola has been sustained in Greece 7 years after their introduction.     

Biological and molecular characterization of field isolates of <Emphasis Type="Italic">Alternaria alternata</Emphasis> with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC₅₀ values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Evaluation of compost amendments for suppressiveness against Verticillium wilt of eggplant and study of mode of action using a novel Arabidopsis pathosystem

The induced resistance potential of eleven compost samples that originated from four different countries (Greece, France, Netherlands and Israel) and were manufactured from various raw materials, was evaluated in an Arabidopsis thaliana–Verticillium dahliae pathosystem under greenhouse conditions using a novel Plexiglas chamber. Five out of eleven composts tested showed significant disease suppressiveness compared to the control treatment; three composts exhibited disease severity equal to the control, while in the other three composts, disease severity was higher than the control treatment. Two of the tested composts that showed strong or medium suppressiveness were further evaluated under field conditions against Verticillium wilt of eggplant. Neither of them significantly reduced disease severity or resulted in higher fruit yield in a semi-commercial field test although they could induce a systemic resistance response in the greenhouse. However, as a consequence of a growth-promoting effect, one of the compost samples tested in the field resulted in a significant yield increase compared with the other.     

The development of size variation in Dover sole,Solea solea and turbot,Scophthalmus maximus: genetic variability between different geographical and among year class farmed strains

Growth and variation in size of turbot and sole larvae originating from different genetic sources were compared at different stocking densities, including genetically based information that might lead to a better knowledge of heterogeneity in cultivated populations. There was no effect of density on growth rate in either species. However, between the two sole batches there was an earlier metamorphosis of faster growing fish from Norway. In sole groups after metamorphosis, variation was stable and therefore no development of size variation was observed. There was no density effect on size variation within turbot batches before, or after metamorphosis. Spreading rate and growth rate were positively correlated in sole larvae indicating that growth of the smaller fish was not equal to the larger ones, but size variation followed a different pattern in turbot. No evidence for either overall significant heterozygosity excess, or deficiency was evidenced in the farmed strains of either species . However, significant differences between farmed populations in terms of genetic variability (θ) were reported, which was rather attributed to within differentiation than between strains. One could safely argue that different strains of Dover sole originated from different genetic sources (factorial analysis). Moreover, when the analysis was extended to individual loci to assess whether specific loci had any consistently larger, or smaller effect on heterozygosity, locus specific significant trends were discerned in turbot. A positive Heterozygosity Fitness (growth rate) Correlation (HFC) suggests that heterozygotes reported enhanced growth rates compared with homozygous ones in flatfish larvae, thus special precautions should be taken into account whenever different genetic structure patterns emerge, because of various flatfish broodstock geographical origin and year class samples.