Aggressiveness of Small-Spored Alternaria spp. and Their Sensitivity to Succinate Dehydrogenase Inhibitor Fungicides

Brown leaf spot of potato is caused by a number of small-spored Alternaria spp. Alternaria alternata sensu stricto, Alternaria arborescens, and Alternaria tenuissima have been reported with increasing frequency in commercial potato fields. Potato cultivars with resistance to small-spored Alternaria spp. have yet to be developed; therefore, the application of foliar fungicides is a primary management strategy. Greenhouse inoculation assays demonstrated that isolates of these three small-spored Alternaria spp. were pathogenic to potato. Significant differences in aggressiveness were observed across isolates; however, there was no trend in aggressiveness based on species. Significant fungicide by isolate interactions in in vitro fungicide sensitivity and significant differences between baseline and non-baseline isolates were observed in all three small-spored Alternaria spp. The ranges of in vitro sensitivity of A. alternata baseline isolates to boscalid (EC50 <0.010 to 0.89 µg/ml), fluopyram (<0.010 to 1.14 µg/ml) and solatenol (<0.010 to 1.14 µg/ml) were relatively wide when compared to adepidyn (<0.010 to 0.023 µg/ml). The baseline sensitivity of A. arborescens and A. tenuissima isolates to all four fungicides were less than 0.065 µg/ml. Between 10 and 21% of non-baseline A. alternata isolates fell outside the baseline range established for the four SDHI fungicides evaluated. In A. arborescens, 10 to 80% of non-baseline isolates had higher sensitivities than the baseline. A. tenuissima isolates fell outside the baseline for boscalid (55%), fluopyram (14%), and solatenol (14%) but none fell outside the baseline range for adepidyn. Evaluations of in vivo fungicide efficacy demonstrated that most isolates were equally controlled by the four SDHI fungicides. However, reduced boscalid efficacy was observed for four isolates (two each of A. arborescens and A. tenuissima) and reduced fluopyram control was observed in one A. alternata isolate. Results of these studies demonstrate that isolates of all three species could be contributing to the brown leaf spot pathogen complex and that monitoring both species diversity and fungicide sensitivity could be advantageous for the management of brown leaf spot in potatoes with SDHI fungicides.

Sensitivity and Resistance Risk Assessment of Puccinia striiformis f. sp. tritici to Triadimefon in China

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat that seriously threatens production safety in wheat-producing areas worldwide. In China, the disease has been largely controlled with fungicide triadimefon. Although high levels of fungicide resistance in other fungal pathogens have been reported, failure to control Pst with any fungicides has seldomly been reported and fungicide sensitivity of Pst has not been evaluated in China. The distribution of triadimefon-resistant Pst isolates was investigated in the present study. The baseline sensitivity of 446 Pst isolates across the country to triadimefon was determined, and the concentration for 50% of maximal effect (EC50) showed a unimodal distribution curve, with a mean value of 0.19 μg mL-1. The results indicated a wide range of sensitivity to triadimefon, with more insensitive isolates collected from Pst winter-increasing areas and northwest over-summering areas, whereas more sensitive isolates were collected from southwest over-summering areas and epidemic areas of Xinjiang and Tibet. The majority of the tested Pst isolates were sensitive to triadimefon; only 6.79% had developed varying degrees of resistance. Characterization of parasitic fitness revealed that the triadimefon-resistant isolates exhibited strong adaptive traits in urediniospore germination rate, latent period, sporulation intensity, and lesion expansion rate. Positive cross-resistance was observed between triadimefon and tebuconazole or hexaconazole, but not between pyraclostrobin or flubeneteram. The point mutation Y134F in the 14α-demethylase enzyme (CYP51) was detected in triadimefon-resistant isolates. A molecular method (Kompetitive Allele Specific PCR) was established for the rapid detection of Y134F mutants in the Pst population. Two genotypes with one point mutation Y134F conferred resistance to triadimefon in Pst. The risk of resistance to triadimefon in Pst may be low to moderate. This study provided important data for establishment of high throughput molecular detection methods, fungicide resistance risk management, and the development of new target fungicides.

Molecular characterization and overexpression of the difenoconazole resistance gene CYP51 in Lasiodiplodia theobromae field isolates

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicides are widely used for disease control in mango orchards. The baseline sensitivity to difenoconazole of 138 L. theobromae isolates collected from mango in the field in 2019 was established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible mechanism for L. theobromae resistance to difenoconazole was preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/mL. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole but not with non-DMI fungicides carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in the CYP51 protein, but they were unlikely to be related to the resistance phenotype. There was no alteration in the promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates compared to the susceptible isolates. These results are vital to develop effective mango disease management strategies to avoid the development of further resistance.

Feasibility and Effectiveness Assessment of SARS-CoV-2 Antigenic Tests in Mass Screening of a Pediatric Population and Correlation with the Kinetics of Viral Loads

The gold standard for diagnosis of SARS-CoV-2 infection has been nucleic acid amplification tests (NAAT). However, rapid antigen detection kits (Ag-RDTs), may offer advantages over NAAT in mass screening, generating results in minutes, both as laboratory-based test or point-of-care (POC) use for clinicians, at a lower cost. We assessed two different POC Ag-RDTs in mass screening versus NAAT for SARS-CoV-2 in a cohort of pediatric patients admitted to the Pediatric Emergency Unit of IRCCS—Polyclinic of Sant’Orsola, Bologna (from November 2020 to April 2021). All patients were screened with nasopharyngeal swabs for the detection of SARS-CoV-2-RNA and for antigen tests. Results were obtained from 1146 patients. The COVID-19 Ag FIA kit showed a baseline sensitivity of 53.8% (CI 35.4–71.4%), baseline specificity 99.7% (CI 98.4–100%) and overall accuracy of 80% (95% CI 0.68–0.91); the AFIAS COVID-19 Ag kit, baseline sensitivity of 86.4% (CI 75.0–93.9%), baseline specificity 98.3% (CI 97.1–99.1%) and overall accuracy of 95.3% (95% CI 0.92–0.99). In both tests, some samples showed very low viral load and negative Ag-RDT. This disagreement may reflect the positive inability of Ag-RDTs of detecting antigen in late phase of infection. Among all cases with positive molecular test and negative antigen test, none showed viral loads > 106 copies/mL. Finally, we found one false Ag-RDTs negative result (low cycle thresholds; 9 × 105 copies/mL). Our results suggest that both Ag-RDTs showed good performances in detection of high viral load samples, making it a feasible and effective tool for mass screening in actively infected children.

Baseline Sensitivity and Control Efficacy of Various Group of Fungicides against Sclerotinia sclerotiorum in Oilseed Rape Cultivation

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is a devastating disease of oilseed rape that may cause significant yield losses if not controlled by cultural management strategies and fungicide applications. Studies were conducted to evaluate the efficacy of different group of fungicides as well as a biopesticide, including azoxystrobin, boscalid, fludioxonil, prothioconazole, tebuconazole, azoxystrobin/tebuconazole, boscalid/pyraclostrobin, prothioconazole/fluopyram and Bacillus amyloliquefaciens, on baseline sensitivity of S. sclerotiorum isolates under in-vitro conditions as well as control of SSR in the field. Artificial inoculation and mist irrigation prompt to reproducible SSR infection in oilseed rape cultivation. All compounds significantly reduced 36.7% to 86.9% SSR severity and increased 55.2% to 98.7% yield, 1.5% to 7.0% thousand grain-weight, 1.5% to 5.9% oil and 0.1% to 1.3% protein content. Fludioxonil, boscalid/pyraclostrobin and fluopyram/prothioconazole achieved strongest fungicidal activity against SSR. The biopesticide provided 36% of disease control. Under in vitro conditions, B. amyloliquefaciens not only strongly inhibited mycelial growth but also the formation of sclerotia in all concentrations. Boscalid and fludioxonil exhibited the highest level of fungicidal activity against S. sclerotiorum, with mean EC50 values of 1.23 and 1.60 μg a.s. mL−1. The highest variability of EC50 values between isolates was observed towards prothioconazole and azoxystrobin.

Development of an Agar Bioassay Sensitivity Test in Alopecurus myosuroides for the Pre-Emergence Herbicides Cinmethylin and Flufenacet

Rapid and reliable tests for pre-emergence herbicide susceptibility in weeds are important to test a wider range of accessions on their baseline sensitivity, as well as to provide information on putative resistance. This study focused on the development of an agar quick test to determine sensitivity differences in Alopecurus myosuroides Huds. to pre-emergence herbicides containing flufenacet and cinmethylin. The new agar quick test and a standardized whole plant pot bioassay were conducted twice in 2019. For both test systems, seeds of 18 populations of A. myosuroides originated from Southwest Germany and Great Britain were used and treated with discriminating rates of herbicides in dose-response studies. After 28 days, the above-ground dry matter of the plants was determined and the resistance factors were calculated. The agar test was able to provide information on the resistance status of the tested biotype within 12 days. All populations did not show reduced sensitivity to cinmethylin. Within three populations, differences in sensitivity levels were observed between the two test systems. As cinmethylin is not yet marketed in Europe, these resistance factors can also be considered as a baseline sensitivity for A. myosuroides. For flufenacet, the resistance factors differed significantly from the whole plant pot bioassay and the agar test for the biotypes A (0.35, 13.1), C (0.56, 13.2), D (1.87, 12.4), E (15.5, 3.5) and H (2.95, 14). It was possible for the most part for the cinmethylin tested populations to confirm the results of the standardized whole plant pot bioassay in the agar bioassay sensitivity tests, and hence create a promising, faster test system.

Molecular Characterization and Overexpression of CYP51 gene of Difenoconazole Resistance in Lasiodiplodia Theobromae Field Isolates

Stem-end rot (SER) caused by Lasiodiplodia theobromae is an important disease of mango in China. Demethylation inhibitor (DMI) fungicide are widely used for diseases control in mango orchards. The baseline sensitivity to difenoconazole of 138 isolates collected in the field in 2019 from mango were established by the mycelial growth rate method. The cross-resistance to six site-specific fungicides with different modes of action were investigated using 20 isolates randomly selected. The possible reasons for L. theobromae resistance to difenoconazole were preliminarily determined through gene sequence alignment and quantitative real-time PCR analysis. The results showed that the EC50 values of 138 L. theobromae isolates to difenoconazole ranged from 0.01 to 13.72 µg/ml. The frequency of difenoconazole sensitivity formed a normal distribution curve when the outliers were excluded. Difenoconazole showed positive cross-resistance only with the DMI tebuconazole, but not with non-DMI carbendazim, pyraclostrobin, fludioxonil, bromothalonil, or iprodione. Some multifungicide-resistant isolates of L. theobromae were found. Two amino acid substitutions (E209k and G207A) were found in CYP51 protein, but they were not likely related to the resistance phenotype. There was no alteration in promoter region of the CYP51 gene. However, difenoconazole significantly increased the expression of the CYP51 gene in the resistant isolates when compared to the susceptible isolates. This study is important references to explore resistance mechanism. These results are vital to make effective mango diseases management strategies in order to avoid the development of further resistance.