Incidence of Myclobutanil- and Kresoxim-Methyl-Insensitive Isolates of Venturia inaequalis in Quebec Orchards

Sensitivity of baseline and exposed populations of Venturia inaequalis to myclobutanil and to kresoxim-methyl were evaluated in vitro. For myclobutanil, the population was constructed with 238 monoconidial isolates of V. inaequalis collected from 48 orchards. For kresoxim-methyl, the population was constructed with 251 monoconidial isolates collected from 49 orchards. Baseline populations were constructed with 34 and 29 monoconidial isolates collected from apple trees that had never been treated for myclobutanil and kresoxim-methyl, respectively. Sensitivity to fungicides was evaluated based on 50% effective dose (ED50) values. The V. inaequalis population that was not exposed to myclobutanil had a baseline sensitivity (mean ED50) of 0.064 μg/ml and showed a lognormal distribution. The V. inaequalis population constructed with isolates from commercial orchards had a mean ED50 of 2.600 μg/ml, which was significantly higher than the baseline sensitivity. The distribution of ED50 values did not follow a lognormal distribution. In response to declining levels of scab control with myclobutanil and other sterol demethylation inhibitor fungicides (DMIs), three orchards were more deeply investigated. The mean ED50 values were 1.618 (n = 23), 3.079 (n = 29), and 1.500 μg/ml (n = 20) in orchards one, two, and three, respectively. Resistant isolates, according to criteria set by other studies, accounted for 39, 76, and 85% of the isolates tested. The V. inaequalis population that had never been exposed to kresoxim-methyl had a baseline sensitivity (mean ED50) of 0.092 μg/ml and showed a lognormal distribution. The V. inaequalis population constructed with isolates from commercial orchards had a mean ED50 of 6.093 μg/ml, which was significantly higher than the baseline sensitivity. The distribution of ED50 values followed a lognormal distribution. However, when a subsample of isolates was retested for their sensitivity to kresoxim-methyl with the addition of salicylhydroxamic acid (an inhibitor of alternative oxidase) at 100 μg/ml to the growth medium, more than 98% inhibition was observed for all isolates. The results from in vitro tests showed a high level of resistance to myclobutanil and a low level of resistance to kresoxim-methyl, suggesting that the use of myclobutanil and DMIs should be discontinued or significantly reduced before practical resistance is reached.

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Baseline Sensitivities of Venturia inaequalis Populations to the Strobilurin Fungicide Kresoxim-methyl

Plant Disease ◽

10.1094/pdis.1999.83.3.274 ◽

1999 ◽

Vol 83 (3) ◽

pp. 274-278 ◽

Cited By ~ 58

Author(s):

Gilberto Olaya ◽

Wolfram Köller

Keyword(s):

Venturia Inaequalis ◽

Resistance Development ◽

Additional Tool ◽

Apple Leaves ◽

Scab Control ◽

In Vivo Tests ◽

Strobilurin Fungicide ◽

The Mean

The efficacies of the new strobilurin fungicide kresoxim-methyl for the protection of apple leaves from infection by baseline populations of Venturia inaequalis were uniform across five major apple growing regions in North America. The mean ED50 value determined for 25 populations was 0.35 μg ml-1, with values ranging from 0.11 μg ml-1 to 0.75 μg ml-1. The mean level of scab control achieved at the kresoxim-methyl dose of 4 μg ml-1 was 93%. For one of the five orchards sampled in each region, kresoxim-methyl sensitivities of germinating conidia were determined. Sensitivities of 250 isolates were broadly distributed, with ED50 values ranging from 0.003 μg ml-1 to 0.14 μg ml-1 and a mean of 0.02 μg ml-1. This broad range of in vitro sensitivities was not reflected for the in vivo efficacy of kresoxim-methyl in the protection of apple leaves from scab infections. The discrepancy between in vivo and in vitro sensitivities implies that in vivo tests are more useful for the monitoring of kresoxim-methyl sensitivities of orchard populations. Because it can be expected that only isolates resistant under both test conditions will be prone to future selection, such isolates will contribute to increased frequencies of the least sensitive isolates described in this baseline study. Testing of in vitro isolate sensitivities will, therefore, provide an additional tool in the monitoring of kresoxim-methyl resistance development.

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Sensitivity of Mycosphaerella fijiensis from Banana to Trifloxystrobin

Plant Disease ◽

10.1094/pdis.2001.85.12.1264 ◽

2001 ◽

Vol 85 (12) ◽

pp. 1264-1270 ◽

Cited By ~ 27

Author(s):

K. M. Chin ◽

M. Wirz ◽

D. Laird

Keyword(s):

Cross Resistance ◽

In Vitro Tests ◽

Mycosphaerella Fijiensis ◽

In Planta ◽

Baseline Sensitivity ◽

Single Spore ◽

Resistance Factors ◽

Development Site ◽

Disease Pressure

An ascospore germination method was developed and validated to assess the sensitivity of bulk samples of Mycosphaerella fijiensis to trifloxystrobin. Using this method, the sensitivity of 142 ascospore samples from banana plantations not treated with strobilurins was analyzed to establish a baseline of pathogen sensitivity. A bulk method was utilized for monitoring purposes because it avoids potential complications due to the isolation and propagation of single-spore isolates and enables the testing of larger samples. Following intensive use of strobilurins (6 to 11 applications per year) over 4 years, under conditions of high disease pressure and the absence of sanitary measures at a development site in Costa Rica, bulk samples with 50% effective concentration (EC50) resistance factors (RFs) in excess of 500 compared with the mean baseline sensitivity were detected. Single-ascospore isolates derived from spores germinating at the discriminatory dose of 3 μg/ml were also resistant, suggesting that the frequency of resistant individuals in bulk samples could be estimated from the relative numbers of ascospores growing at this dose. The resistance of selected isolates was confirmed in planta. In vitro tests with four resistant and two sensitive single-ascospore isolates collected from different locations and times indicated possible cross-resistance of trifloxystrobin to azoxystrobin, famoxadone, and fenamidone, but not to propiconazole.

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Resistance to Boscalid Fungicide in Alternaria alternata Isolates from Pistachio in California

Plant Disease ◽

10.1094/pdis-91-10-1345 ◽

2007 ◽

Vol 91 (10) ◽

pp. 1345-1350 ◽

Cited By ~ 105

Author(s):

Herve F. Avenot ◽

Themis J. Michailides

Keyword(s):

Spore Germination ◽

Alternaria Alternata ◽

Hyphal Growth ◽

Conidial Germination ◽

Prior History ◽

In Vitro Tests ◽

Fitness Parameters ◽

History Of ◽

The Mean

Boscalid is a new carboxamide fungicide recently introduced in a mixture with pyraclostrobin in the product Pristine for the control of Alternaria late blight of pistachio. In all, 108 isolates of Alternaria alternata were collected from pistachio orchards with (59 isolates) and without (49 isolates) prior exposure to boscalid. The sensitivity to boscalid was determined in conidial germination assays. The majority of isolates from two orchards without a prior history of boscalid usage had effective fungicide concentration to inhibit 50% of spore germination (EC50) values ranging from 0.089 to 3.435 μg/ml, and the mean EC50 was 1.515 μg/ml. Out of 59 isolates collected from an orchard with a history of boscalid usage, 52 isolates had EC50 values ranging from 0.055 to 4.222 μg/ml, and the mean EC50 was 1.214 μg/ml. However, in vitro tests for conidial germination and mycelial growth also revealed that seven A. alternata isolates, originating from the orchard exposed to boscalid were highly resistant (EC50 > 100 μg/ml) to this fungicide. Furthermore, in vitro tests showed no significant differences between wild-type and boscalid-resistant mutants in some fitness parameters such as spore germination, hyphal growth, sporulation, or virulence on pistachio leaves. Experiments on the stability of the boscalid-resistant phenotype showed no reduction of the resistance after the mutants were grown on fungicide-free medium. Preventative applications of a commercial formulation of boscalid (Endura) at a concentration which is effective against naturally sensitive isolates failed to control disease caused by the boscalid-resistant isolates in laboratory tests. To our knowledge, this is first report of field isolates of fungi resistant to boscalid.

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Resistance to Myclobutanil in Populations of Venturia inaequalis in Winchester, Virginia

Plant Health Progress ◽

10.1094/php-2007-1113-01-rs ◽

2007 ◽

Vol 8 (1) ◽

pp. 3 ◽

Cited By ~ 3

Author(s):

Sasha C. Marine ◽

David G. Schmale ◽

Keith S. Yoder

Keyword(s):

Fungicide Resistance ◽

Venturia Inaequalis ◽

Apple Scab ◽

Apple Orchards ◽

The Mean ◽

Moderately Resistant

Sterol-inhibiting (SI) fungicides are widely used to manage apple scab, caused by Venturia inaequalis. However, recent observations indicate that populations of V. inaequalis in orchards in Virginia have developed resistance to myclobutanil and other SI fungicides. Little is known about the frequency and distribution of fungicide resistance in apple scab populations in Virginia. Isolates of V. inaequalis were collected from three different apple orchards in Winchester, VA in 2006. Orchards were treated with myclobutanil on 12 April, 19 April, 1 May, 30 May, and 7 July. The sensitivity of 87 single-spored isolates of V. inaequalis to myclobutanil was determined by monitoring their growth on agar dishes amended with 0, 0.1, 0.5, or 1.0 μg/ml myclobutanil. A relative continuum of fungicide resistance was observed: 16 isolates were resistant, 40 isolates were moderately resistant, and 31 isolates were sensitive to myclobutanil. After 28 days, the mean growth of isolates collected from trees treated with myclobutanil was significantly greater than that of isolates collected from non-treated trees at all concentrations of myclobutanil tested in vitro. High levels of fungicide resistance found in populations of V. inaequalis suggest that replacement programs may need to be developed to manage apple scab in Virginia. Accepted for publication 4 September 2007. Published 13 November 2007.

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Biphasic Calcium Phosphate/Poly-(DL-Lactide-Co-Glycolide) Biocomposite as Filler and Blocks for Reparation of Bone Tissue

Materials Science Forum ◽

10.4028/www.scientific.net/msf.494.519 ◽

2005 ◽

Vol 494 ◽

pp. 519-524 ◽

Cited By ~ 14

Author(s):

N. Ignjatović ◽

P. Ninkov ◽

Z. Ajduković ◽

V. Konstantinović ◽

Dragan P. Uskokovic

Keyword(s):

Bone Tissue ◽

Vascular Tissue ◽

Average Diameter ◽

In Vitro Tests ◽

Scanning Calorimetry ◽

Tissue Reconstruction ◽

High Level ◽

Osseous Regeneration

Composite biomaterials, like calciumphosphate/bioresorbable polymer, offer excellent potential for reconstruction and reparation of bone tissue defects induced by different sources. In this paper synthesis of calciumphosphate/poly-DL-lactide-co-glycolide (BCP/DLPLG) composite biomaterial formed as filler and blocks was studied. BCP/DLPLG composite biomaterial was produced in the form of spherical granules of BCP covered by a DLPLG layer, average diameter of 150-250 µm. By cold and hot pressing of granules at up to 10000 kg/cm2, blocks with fine distribution of phases and porosity up to 3% were obtained. Characterization was performed by wide-angle X-ray structural analysis (WAXS), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), infrared spectroscopy (IR), and mechanical properties by defining the compressive strength. In vitro citotoxicity research was carried out on cellular cultures of fibroblasts of human (MRC5) and mouse (L929). In vivo research was performed in two steps. Reparatory ability of BCP/DLPLG in mice was examined in the first step, and then bone tissue reconstruction possibilities on 10 patients in the next step. In vitro tests showed very good fibroblast adhesion and non-citotoxicity of the composite. A material is considered non-cytotoxic if the cell survival is above 50 %, and in our case it was 90%. In vivo research on mice indicated high level of reparatory ability of this composite with formation of new bone and vascular tissue six weeks after reparation. Application of this composite for healing infrabone defects of patients showed a high level of osseous regeneration.

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Measurement of Activated Factor IX in Factor IX Concentrates: Correlation with In Vivo Thrombogenicity

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653839 ◽

1995 ◽

Vol 73 (04) ◽

pp. 675-679 ◽

Cited By ~ 35

Author(s):

Elaine Gray ◽

Jill Tubbs ◽

S Thomas ◽

A Oates ◽

M Boisclair ◽

...

Keyword(s):

Significant Positive Correlation ◽

Factor Ix ◽

In Vitro Tests ◽

Prothrombin Complex Concentrates ◽

Thrombotic Risk ◽

Chromogenic Assay ◽

The Mean ◽

Thrombogenic Potential

SummaryCurrent in vitro tests for thrombogenicity of FIX concentrates used for prothrombin complex concentrates (PCCs), are of little value when applied to high purity FIX (HP FIXs). In the present study, we have developed a chromogenic assay for activated FIX (FIXa) and evaluated its ability to predict in vivo thrombogenic potential of HP FIXs in a modified Wessler stasis model. Among the HP FIXs, only 1 out of 7 products had no detectable FIXa; this product also showed no in vivo thrombogenicity. In the other 6 products, FIXa content ranged from 0.15–1.2 U/1000 iu FIX, and all showed some evidence of in vivo thrombogenicity, with mean thrombus scores ranging from 0.25–4. There was a significant positive correlation (r = 0.55, p <0.02) between FIXa levels and in vivo thrombogenicity of HP FIXs. NAPTT data were not significantly correlated with the in vivo results and the TFCT also showed no direct correlation with the mean thrombus score. These results indicate that HP FIXs may still carry a small residual thrombotic risk and measurement of FIXa content of these products may be a better predictor of thrombogenicity than the current in vitro tests.

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Baseline Sensitivity of Guignardia citricarpa Isolates from Florida to Azoxystrobin and Pyraclostrobin

Plant Disease ◽

10.1094/pdis-03-13-0335-re ◽

2014 ◽

Vol 98 (6) ◽

pp. 780-789 ◽

Cited By ~ 15

Author(s):

Martha Hincapie ◽

Nan-Yi Wang ◽

Natalia A. Peres ◽

Megan M. Dewdney

Keyword(s):

Mycelial Growth ◽

Control Measure ◽

Black Spot ◽

Conidial Germination ◽

Baseline Sensitivity ◽

Qoi Fungicides ◽

Resistance Risk ◽

Guignardia Citricarpa ◽

The Mean

Citrus black spot (CBS), caused by Guignardia citricarpa, is an emerging disease in Florida. Fungicide applications are the main control measure worldwide. The in vitro activity and baseline sensitivity of G. citricarpa isolates to quinone outside inhibitor (QoI) fungicides (azoxystrobin and pyraclostrobin) were evaluated. The effective concentration needed to reduce mycelial growth or spore germination by 50% (EC50) was determined for 86 isolates obtained from Florida counties where CBS is found. The effect of salicylhydroxamic acid (SHAM) plus azoxystrobin and pyraclostrobin was also assessed for mycelial growth and conidial germination. The mean EC50 for mycelial growth for azoxystrobin was 0.027 μg/ml and that for pyraclostrobin was significantly lower at 0.007 μg/ml (P < 0.0001). Similarly, the mean EC50 for conidial germination for azoxystrobin was 0.016 μg/ml and that for pyraclostrobin was significantly lower at 0.008 μg/ml (P < 0.0001). There was no effect of SHAM on inhibition of mycelial growth or conidial germination by the QoI fungicides but SHAM slightly affected mycelium inhibition by pyraclostrobin. Cytochrome b was partially sequenced and three group 1 introns were found. One intron was immediately post G143, likely inhibiting resistance-conferring mutations at that site. It is surmised that the QoI resistance risk is low in the Florida G. citricarpa population.

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Influence of selected fungicides registered in the Czech Republic for winter oilseed rape on in vitro Sclerotinia sclerotiorum mycelial growth

Plant Protection Science ◽

10.17221/137/2016-pps ◽

2018 ◽

Vol 54 (No. 2) ◽

pp. 101-110 ◽

Cited By ~ 1

Author(s):

Poslušná Jana ◽

Plachká Eva ◽

Mazáková Jana

Keyword(s):

Czech Republic ◽

Oilseed Rape ◽

Sclerotinia Sclerotiorum ◽

Mycelial Growth ◽

Winter Oilseed Rape ◽

Baseline Sensitivity ◽

The Czech Republic ◽

Dose Rates ◽

The Mean

The baseline sensitivity of 55 isolates of Sclerotinia sclerotiorum, collected from oilseed rape in 6 regions of the Czech Republic, to selected fungicides was determined during the period 2013–2015. One single-component fungicide – Horizon (tebuconazole), and four multicomponent fungicides – Pictor (boscalid, dimoxystrobin), Efilor (boscalid, metconazole), Prosaro 250 EC (prothioconazole, tebuconazole), and Propulse (fluopyram, prothioconazole), were chosen as these are commonly used locally. The effect of each fungicide on the in vitro pathogen radial mycelial growth and EC50 values for the respective fungicides were determined. The following MIC values were estimated; for the fungicides Horizon 250 EW, Efilor, and Propulse the mean MIC values ranged between 0.125 and 0.250 µl/ml, for Prosaro 250 EC ranged between 0.0625 and 0.125 µl/ml, and for Pictor ranged from 0.00781 to 0.01562 µl/ml. No strains of S. sclerotiorum resistant to the tested fungicides were detected and the growth of all isolates was fully inhibited at concentrations corresponding to their registered dose rates. The highest fungicidal efficacy on the collected S. sclerotiorum isolates was recorded for Pictor, followed by Prosaro 250 with an EC50 value 0.05856 µl/ml and then the remaining fungicides Propulse, Efilor, and Horizon 250 EW (EC50 values 0.07277, 0.07221, and 0.08519 µl/ml, respectively).

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Overwintering of Conidia of Venturia inaequalis and the Contribution to Early Epidemics of Apple Scab

Plant Disease ◽

10.1094/pdis.2004.88.7.751 ◽

2004 ◽

Vol 88 (7) ◽

pp. 751-757 ◽

Cited By ~ 27

Author(s):

I. J. Holb ◽

B. Heijne ◽

M. J. Jeger

Keyword(s):

Venturia Inaequalis ◽

Apple Scab ◽

Early Spring ◽

Shoot Length ◽

Bud Break ◽

Phenological Stage ◽

Tight Cluster ◽

The Mean ◽

High Level ◽

Previous Autumn

Overwintering of conidia of Venturia inaequalis associated with shoots and buds was determined, and the contribution to early spring epidemics of apple scab was evaluated during three consecutive seasons (1999 to 2001) in the Netherlands. Examinations of shoot samples collected before bud break showed that the percentage of shoots with superficial black fungal mycelia or conidia was above 65%, and the mean number of conidia on a 1-cm piece of shoot length ranged from 581 to 1,033. However, germination tests showed that the viability of conidia on shoots was less than 1.5%. No macroscopic scab lesions were detected on the scales of dormant buds. However, microscopic examinations of individual bud tissues demonstrated that the number of conidia was >3,000 per 100 buds in each year. The mean viability of conidia associated with buds ranged from 0.7 to 1.9% and from 3.7 to 10.5% for the outer and inner bud tissues, respectively. Results of field assessments at tight-cluster phenological stage showed that the percentage of infection caused by the viable overwintered conidia ranged from 0.3 to 3.8% in the various treatments. Our results indicated that conidia were unlikely to overwinter on the surface of shoots or outer bud tissues, where they were exposed to fluctuating environmental conditions, and, consequently, were unlikely to play a role in initiating an early epidemic of apple scab in the spring. However, our results indicated a risk from overwintered conidia in the inner bud tissues arising from a high level of scab the previous autumn. Therefore, orchards with high levels of apple scab, where ascosporic inoculum is much reduced, e.g., by sanitation, should be protected in early spring by means of fungicide treatment at green tip.

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Occurrence of QoI Resistance and Detection of the G143A Mutation in Michigan Populations of Venturia inaequalis

Plant Disease ◽

10.1094/pdis-12-10-0898 ◽

2011 ◽

Vol 95 (8) ◽

pp. 927-934 ◽

Cited By ~ 30

Author(s):

Kimberley E. Lesniak ◽

Tyre J. Proffer ◽

Janna L. Beckerman ◽

George W. Sundin

Keyword(s):

Spore Germination ◽

Control Strategies ◽

Venturia Inaequalis ◽

Apple Scab ◽

Pathogenic Fungi ◽

Qoi Fungicides ◽

Widespread Occurrence ◽

Scab Control ◽

High Level ◽

Qoi Resistance

Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungicides such as the quinone-outside inhibitors (QoI) have been used in Michigan apple orchards for more than 11 years. In 2008, we sampled eight commercial orchards in the Fruit Ridge growing region of Michigan in which apple scab control failures were observed on ‘McIntosh’ apple following applications of kresoxim-methyl or trifloxystrobin. QoI resistance was assessed in 210 total isolates (a total of 17 orchards) using a spore germination assay and in 319 isolates using a polymerase chain reaction (PCR) assay to detect the G143A mutation located within the V. inaequalis cytochrome b gene (CYTB). The G143A mutation is known to confer high-level QoI resistance in plant-pathogenic fungi. QoI resistance was confirmed in 50 and 64% of the isolates tested with the spore germination and PCR assays, respectively, and there was a 97% concordance observed between the assays. In 2009, we sampled and examined an additional 1,201 V. inaequalis isolates from 64 orchards in Michigan and 86 isolates from four baseline sites in Ohio. All of these isolates were assayed for the G143A mutation and it was detected within 67 and 0% of the Michigan and Ohio isolates, respectively. Our results indicate the widespread occurrence of QoI resistance in Michigan commercial orchard populations of V. inaequalis. Loss of QoI fungicides further limits the arsenal of fungicides available to commercial apple growers for successful scab management.

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