Pre-harvest nitrogen and Azoxystrobin application enhances postharvest shelf-life in Butterhead lettuce

Two trials were carried out on Butterhead lettuce (March–May 2008 and April–June 2009) to investigate the effect of the application of nitrogen fertilizer (0, 50 and 100 kg ha⁻¹ of N) and of strobilurin (Azoxystrobin, methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy]phenyl}-3-methoxyacrylate) on (i) yield and morphological traits at harvest, (ii) physical (weight loss and dry matter), visual (chlorophyll content and main colour indices), physiological (relative water content, osmotic potential, and electrolytic leakage), and nutritional (ascorbic acid, nitrate, and polyphenol content) quality of raw material and their changes after storage of fresh-cut leaves. Cool storage lasted 7 and 12 days in the first and second experiment, respectively. In the first cycle, under early-spring conditions, lettuce yield was lower by 38% and, even if the product was lighter coloured [higher L* (+6%) and lower CHL (−21%)], it had lower dry matter content (−32%), higher electrolyte leakage (EL) (+14%) and WL_Trans (+8%) compared with the raw product from the second cycle. In both years, the increase of N supply and the application of Azoxystrobin improved yield (by 8.5% and 10%, respectively). The response in N fertilization was more evident under early-spring (2008) compared to late-spring (2009) conditions (12.3% vs. 4.8%), and when (2008) the highest N rate interacted with the application of Azoxystrobin (+12.9% compared with the other treatments). The nitrate content in leaves was always reduced by Azoxystrobin application (−43%) and increased with the N supply (+53%). In the second experiment, when storage was prolonged for 12 days, strobilurin improved postharvest shelf-life by reducing chlorophyll degradation (−27%), senescence (−19%, measured as EL), and browning (−53%, measured as h° index decrease). Azoxystrobin lowered also the total polyphenol content of raw material (−12.5%), which can be linked to less browning during storage. During postharvest storage, irrespective to the preharvest dose, N supply kept the visual quality and physiological senescence indices constant (L*, h° and EL). The suitability of the Butterhead lettuce to fresh-cut processing depends on climatic growing conditions. Preharvest Azoxystrobin supply improves the nutritional quality of the raw material, reducing leaf nitrate content, and the shelf-life in prolonged storage. The N rate of 100 kg ha⁻¹ of N is suitable under less favourable growing conditions, while the rate of 50 kg ha⁻¹ is better for more favourable climatic conditions, especially if a moderate contribution in available N from soil organic matter mineralization and no leaching from heavy rains is expected.     

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.     

Delaying senescence of wheat with fungicides has interacting effects with cultivar on grain sulphur concentration but not with sulphur yield or nitrogen:sulphur ratios

Winter wheat was grown in three field experiments, each repeated over two or three seasons, to investigate effects of extending flag leaf life by fungicide application on the concentration, kg ha⁻¹ and mg grain⁻¹ of nitrogen (N) and sulphur (S) as well as N:S ratio and sodium dodecyl sulphate (SDS) sedimentation volume. The experiments involved up to six cultivars and different application rates, timings and frequencies of azoxystrobin and epoxiconazole. For every day the duration to 37% green flag leaf area (m) was extended, N yield was increased by 2.58 kg ha⁻¹, N per grain by 0.00957 mg, S yield by 0.186 kg ha⁻¹ and S per grain by 0.000718 mg. The N:S ratio decreased by 0.0135 per day. There was no evidence that these responses varied with cultivar. In contrast, the relationship between flag leaf life and N or S concentration interacted with cultivar. The N and S concentrations of Shamrock, the cultivar that suffered most from brown rust (Puccinia recondita), increased with the extension of flag leaf life whereas the concentrations of N and S in Malacca, a cultivar more susceptible to Septoria tritici, decreased as flag leaf senescence was delayed. This was because the relationships between m and N and S yields were much better conserved over cultivars than those between m and thousand grain weight (TGW) and grain yield ha⁻¹.     

Effect of fungicides on the complex of <Emphasis Type="Italic">Fusarium</Emphasis> species and saprophytic fungi colonizing wheat kernels

Fusarium head blight of wheat, often associated with mycotoxin contamination of food and feed is caused by various Fusarium species. The efficacy of fungicide sprays for the control of the disease and mycotoxins varies from being highly effective to even increasing mycotoxin levels. The potential role of antagonistic fungi in this variability was investigated assessing sensitivity of Fusarium species and saprophytic fungi colonizing wheat kernels to fungicides. Saprophytes were tested for their antagonistic activity to the prevalent Fusarium species Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, and Fusarium poae. Fungal isolates from mature winter wheat kernels were Alternaria alternata, Arthrinium sp., Aspergillus niger, Epicoccum sp., Microdochium spp., Rhizopus oryzae and Trichoderma sp. In dual culture A. niger, R. oryzae and Trichoderma hamatum were more effective in reducing mycelial growth of Fusarium species than Microdochium majus; A. alternata and Epicoccum sp. were ineffective because of slow growth rates. Saprophytic fungi were sensitive to triazoles; however, prothioconazole and tebuconazole had stronger effects on mycelial growth of Fusarium species. ED₅₀ values also indicated significant differences in the sensitivity of Fusarium species to triazoles (range 0.1–1.7 mg l⁻¹). Azoxystrobin and fluoxastrobin were largely ineffective in inhibiting in vitro growth of Fusarium spp.; sensitivity of the other fungi was generally lower, except for M. majus which was highly sensitive. Due to differences in fungicide sensitivity among Fusarium spp. and ear-colonizing fungi antagonistic to Fusarium spp. fungicides are likely to modify the balance within the mycoflora of wheat ears which may also affect the mycotoxin contamination of grain.     

Resistance to azoxystrobin in Alternaria isolates from pistachio in California

Failure to control Alternaria late blight in a few California pistachio orchards was observed after only 3-4 years of consecutive applications of azoxystrobin-based fungicide programs. A total of 72 isolates of Alternaria alternata, Alternaria tenuissima, and Alternaria arborescens, the causal organisms of Alternaria late blight, were collected from pistachio orchards with (58 isolates) and without (14 isolates) a prior history of azoxystrobin applications. The sensitivity to azoxystrobin was determined in conidial germination assays. Isolates from orchards with a history of azoxystrobin applications had EC₅₀ values greater than 100 μg/ml, whereas isolates from orchards without a prior history of azoxystrobin usage had EC₅₀ values ranging from 0.008 to 0.045 μg/ml. Azoxystrobin resistance correlated with a single mutation in the cytochrome b (cyt b) gene causing a change of glycine to alanine at amino acid position 143. A pair of PCR primers AF and AR was developed that amplified a 226-bp DNA fragment of the cyt b gene containing the mutation site from all three Alternaria species but not from 30 other fungal species frequently found on pistachio. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction enzyme Fnu4HI allowed differentiation of the PCR fragment of wild type cyt b gene from that of mutated gene. This method will aid in a fast detection of azoxystrobin resistance in these three Alternaria species.     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.     

Effects of physical properties on the translaminar activity of fungicides

Translaminar redistribution is a key component of activity for many fungicides. The influence of physical properties (including water solubility, lipophilicity, melting point, and molar volume) on translaminar activity, however, is not well understood. Cucumber powdery mildew was used as a biological indicator to examine the influence of physical properties on translaminar activity of 61 fungicides in simple, uniform formulations, including three modes-of-action and a range of physical properties. Results were modeled using multiple regression and ordinal logistic fit. We confirmed that translaminar activity is a frequent attribute of fungicides and that lipophilicity and water solubility are important predictors of translaminar activity. The hypothesis that melting point drives translaminar movement and translaminar activity was not supported. Translaminar movement (driven only by physical properties) could be differentiated with the models from fungitoxicity-influenced translaminar control. Translaminar activity is a complex attribute and differences in inherent activity as well as physical properties and formulations must be considered when comparing compounds for relative translaminar activity.     

Comparative efficacy of strobilurin fungicides against downy mildew disease of pearl millet

Three commercial formulations of strobilurins, viz., azoxystrobin, kresoxim-methyl, and trifloxystrobin were evaluated for their efficacy against pearl millet downy mildew disease caused by Sclerospora graminicola. In vitro studies revealed inhibition of S. graminicola sporulation, zoospore release, and zoospore motility at 0.1-2 μg ml⁻¹ of all the three fungicides. The fungicides were evaluated for phytotoxic effects on seed quality parameters and for their effectiveness against downy mildew disease by treating pearl millet by: (1) seed dressing, (2) seed dressing followed by foliar spray, and (3) also by foliar spray alone. The highest non-phytotoxic concentrations of 5, 10, and 10 μg ml⁻¹ for azoxystrobin, trifloxystrobin, and kresoxim-methyl, respectively, were selected for further studies. Under greenhouse conditions, these fungicides showed varying degrees of protection against downy mildew disease. Among the three fungicides, azoxystrobin proved to be the best by offering disease protection of 66%. Further, seed treatment along with foliar application of these fungicides to diseased plants showed enhanced protection against the disease to 93, 82, and 62% in treatments of azoxystrobin, kresoxim-methyl and trifloxystrobin respectively. Foliar spray alone provided significant increase in disease protection levels of 91, 79, and 59% in treatments of azoxystrobin, kresoxim-methyl, and trifloxystrobin, respectively. Disease curative activity of azoxystrobin was higher compared to trifloxystrobin and kresoxim-methyl. Tested fungicides showed weaker translaminar activity, as the disease inhibition was marginal when applied on adaxial leaf surface. Partial systemic activity of azoxystrobin was evident by root uptake, while trifloxystrobin and kresoxim-methyl showed lack of systemic action in pearl millet. A trend in protection against downy mildew disease similar to greenhouse results was evident in the field trials. Grain yield was significantly increased in all strobilurin fungicide treatments over control and maximum increase in yield of 1673 kg ha⁻¹ was observed in combination treatments of seed treatment and foliar spray with azoxystrobin.     

Impacts of foliar fungicides on infection of soybean by Phomopsis spp. in Iowa, USA

Fungicides pyraclostrobin (strobilurin) and tebuconazole (triazole) were applied to soybean [Glycine max (L.) Merr.] at growth stages R3, R5 or R3 + R5, in 2008 and 2009 at two locations in Iowa. Incidence of infection of stems and seeds by Phomopsis spp. was evaluated, along with yield and seed quality. Stem infection by Phomopsis spp. was reduced in both years by pyraclostrobin applied at R3 + R5, and in 2008 by pyraclostrobin at R5, by approximately three-fold compared to the untreated control. In 2009, treatments including applications of tebuconazole at R3 and pyraclostrobin at R5 significantly reduced infection of seed by Phomopsis spp., by approximately two-fold compared to the untreated control. Only the application of pyraclostrobin at R3 + R5 reduced both stem and seed infection by Phomopsis spp. in 2009. None of the treatments had a significant effect on yield, or seed quality, evaluated by warm and cold germination tests. Seed infection by Phomopsis spp. was negatively correlated with seed quality. Fungicides applied at these growth stages can have an impact on infection by Phomopsis spp., but their effectiveness varies with environment and disease intensity.