A Rapid Method to Quantify Fungicide Sensitivity in the Brown Rot Pathogen Monilinia fructicola

Management of brown rot of stone fruit relies upon the application of effective fungicides that may be compromised by the development of fungicide resistance. We evaluated fungicide resistance in the brown rot pathogen, Monilinia fructicola, using Alamar blue (AB) dye, or resazurin, a chromogenic substrate that can be used as an indicator of respiration, in a 96-well microtiter format. We compared the AB method to traditional mycelial growth assays for resistance screening using 10 isolates of M. fructicola that represented a range of sensitivities to fenbuconazole. Using traditional mycelial growth assays, isolate sensitivity ranged from 17.7 to 115.3% growth on medium amended with fenbuconazole at 0.03 μg/ml relative to that on nonamended medium. Concordant results between both assays were obtained (R2 = 0.9943, P < 0.0001), but the AB method provided results within 24 h, as opposed to the 3- to 5-day period required for mycelial growth assays. We found that sensitive isolates reduced AB less than resistant isolates in the presence of fungicide. Spore density influenced the reduction of AB by M. fructicola; spectrophotometric discrimination of fungicide sensitivity was best achieved at a density of 105 spores/ml.

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Phylogenetic Analysis and Fungicide Baseline Sensitivities of Monilia mumecola in China

Plant Disease ◽

10.1094/pdis-11-18-1953-re ◽

2019 ◽

Vol 103 (9) ◽

pp. 2231-2236 ◽

Cited By ~ 1

Author(s):

L. F. Yin ◽

S. F. Du ◽

C. Chaisiri ◽

R. Cheewangkoon ◽

C. X. Luo

Keyword(s):

Genetic Diversity ◽

Phylogenetic Analysis ◽

Growth Inhibition ◽

Mycelial Growth ◽

Brown Rot ◽

Effective Concentration ◽

Fungicide Sensitivity ◽

Significant Difference

Monilia mumecola is one of the causal agents of peach brown rot in China. In this study, M. mumecola isolates from different locations and hosts were used to analyze the genetic diversity and to assay the sensitivity to four generally used fungicides: carbendazim, tebuconazole, azoxystrobin, and boscalid. Results showed that isolates from different locations tended to be separated. Interestingly, isolates from different hosts (e.g., peach and apricot) at the same locations generally clustered together, indicating that the M. mumecola isolates may infect different hosts in the same areas. The fungicide sensitivity assay of 93 M. mumecola isolates showed that the average effective concentration for 50% mycelial growth inhibition values for carbendazim, tebuconazole, azoxystrobin, and boscalid were 0.103, 0.034, 0.325, and 0.419 µg/ml, respectively. The sensitivity distributions of the tested isolates to the four fungicides showed continuous unimodal curves, indicating no qualitative shift of resistance. No significant difference of sensitivity to tested fungicides was observed among isolates from either different locations or different hosts.

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Validation of the Lipbalm Tube Assay for Evaluation of Fungicide Sensitivity in Field Isolates of Monilinia fructicola

Plant Health Progress ◽

10.1094/php-2009-1118-01-rs ◽

2009 ◽

Vol 10 (1) ◽

pp. 4 ◽

Cited By ~ 5

Author(s):

Achour Amiri ◽

Phillip M. Brannen ◽

Guido Schnabel

Keyword(s):

Storage Temperature ◽

Brown Rot ◽

Petri Dish ◽

Colony Growth ◽

Penicillium Expansum ◽

Colletotrichum Acutatum ◽

Monilinia Fructicola ◽

Fungicide Sensitivity ◽

Qoi Fungicides ◽

Field Isolates

Simple and rapid techniques for monitoring fungicide sensitivity of pathogen populations can be helpful to optimize spray programs. In this study, a lipbalm tube assay developed previously in our laboratory was investigated for shelf life duration, ability to differentiate M. fructicola from fungal contaminants, and ability to accurately determine the sensitivity offield isolates of Monilinia fructicola to sterol demethylation inhibitor (DMI), benzimidazole (BZI), and quinone outside inhibitor (QoI) fungicides. The sensitivity of isolates with different DMI, BZI, and QoI sensitivity phenotypes to the above-mentioned fungicide classes was not altered on agar disks sliced from 30-day-old, fungicide-amended potato dextrose agar (PDA) tubes stored at 4°C compared to disks from freshly prepared fungicide-amended PDA. At the storage temperature of 22°C, however, the sensitivity started to decrease after 10 days. Colony growth of M. fructicola on PDA disks was visually distinguishable from Alternaria alternata, Aspergillus niger, Cladosporium herbarum, Gilbertella persicaria, Penicillium expansum, and Rhizopus stolonifer but not Colletotrichum acutatum after 72 h of incubation at 22°C. The sensitivity of 40 M. fructicola field isolates to DMI, BZI, and QoI fungicides was determined by direct inoculation of lipbalm tube disks with conidia from field-derived peach fruit with toothpicks under semi-sterile conditions and by the traditional Petri dish assay. A strong correlation was observed between the two assays in determining fungicide sensitivity of M. fructicola field isolates. Because of its simplicity and reliability, the lipbalm tube assay will be a useful tool for trained crop consultants to determine orchard-specific resistance profiles outside a research laboratory for precision brown rot management. Accepted for publication 11 September 2009. Published 18 November 2009.

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Laboratory Evaluation of Three Rapid, Agar-Based Assays to Assess Fungicide Sensitivity in Monilinia fructicola

Plant Disease ◽

10.1094/pdis-92-3-0415 ◽

2008 ◽

Vol 92 (3) ◽

pp. 415-420 ◽

Cited By ~ 7

Author(s):

A. Amiri ◽

H. Scherm ◽

P. M. Brannen ◽

G. Schnabel

Keyword(s):

Growth Inhibition ◽

Mycelial Growth ◽

Fungal Growth ◽

Petri Dish ◽

Laboratory Evaluation ◽

Monilinia Fructicola ◽

Fungicide Sensitivity ◽

Thiophanate Methyl ◽

Average Percentage ◽

Low Levels

Three rapid, agar-based assays were compared with a traditional petri dish method for assessing the sensitivity of Monilinia fructicola to propiconazole (0.3 and 2.0 μg/ml), thiophanate-methyl (1.0 and 50 μg/ml), and azoxystrobin (1.0 and 35 μg/ml) in the laboratory. The three assays were based on mycelial growth inhibition on agar disks sliced from lipbalm tubes filled with fungicide-amended potato dextrose agar (PDA), on PDA-coated cotton swabs, or in PDA-filled microcentrifuge tubes. Mycelial growth inhibition of eight previously characterized isolates (two resistant to propiconazole, two highly resistant to thiophanate-methyl, two with low levels of resistance to thiophanate-methyl, and two sensitive to all three fungicides) was determined visually 24, 48, and 72 h after inoculation. The 48-h time point was the earliest suitable time to collect data for all methods because insufficient growth was recorded in the petri dish and tube assays after 24 h. With the exception of the swab assay, all methods classified the isolates previously determined to be fungicide sensitive correctly (i.e., no fungal growth was observed for these isolates). For propiconazole-resistant isolates, the lipbalm assay resulted in levels of growth inhibition very similar to the petri dish method, whereas the swab assay and the tube assay overestimated and underestimated, respectively, the level of resistance. Both the lipbalm and the swab assays classified isolates correctly as being thiophanate-methyl resistant, and both were able to discriminate the isolates previously classified as having low versus high levels of resistance when treated with this fungicide at 50 μg/ml, as was the petri dish method. None of the eight isolates which previously were determined to be azoxystrobin sensitive grew on azoxystrobin-amended media, regardless of the assay type. Overall, the average percentage of correct isolate classifications (relative to their previously determined resistance status) on propiconazole- and thiophanate-methyl-amended media after 48 h ranged from 87.5 to 100, 85.3 to 100, 63.2 to 94.5, and 50.5 to 81.0% for the petri dish, lipbalm, swab, and tube assays, respectively. The lipbalm assay provided the most accurate assessments (85.3 to 100%) after only 24 h of incubation, supporting its use as a rapid and simple tool to monitor resistance levels in M. fructicola field populations.

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Reduced Sensitivity in Monilinia fructicola Field Isolates from South Carolina and Georgia to Respiration Inhibitor Fungicides

Plant Disease ◽

10.1094/pdis-94-6-0737 ◽

2010 ◽

Vol 94 (6) ◽

pp. 737-743 ◽

Cited By ~ 44

Author(s):

A. Amiri ◽

P. M. Brannen ◽

G. Schnabel

Keyword(s):

South Carolina ◽

Mycelial Growth ◽

Disease Incidence ◽

Brown Rot ◽

Conidial Germination ◽

Cross Resistance ◽

Monilinia Fructicola ◽

Significant Difference

Quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitor (SdhI) fungicides are respiration inhibitors (RIs) used for preharvest control of brown rot of stone fruit. Both chemical classes are site-specific and, thus, prone to resistance development. Between 2006 and 2008, 157 isolates of Monilinia fructicola collected from multiple peach and nectarine orchards with or without RI spray history in South Carolina and Georgia were characterized based upon conidial germination and mycelial growth inhibition for their sensitivity to QoI fungicides azoxystrobin and pyraclostrobin, SdhI fungicide boscalid, and a mixture of pyraclostrobin + boscalid. There was no significant difference (P = 0.05) between EC50 values for inhibition of conidial germination versus mycelial growth. The mean EC50 values based upon mycelial growth tests for 25 isolates from an orchard without RI-spray history were 0.15, 0.06, 2.23, and 0.09 μg/ml for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. The respective mean EC50 values for 76 isolates from RI-sprayed orchards in South Carolina were 0.9, 0.1, 10.7, and 0.13 μg/ml and for 56 isolates from RI-sprayed orchards in Georgia were 1.2, 0.1, 8.91, and 0.17 μg/ml. Overall, mean EC50 values of populations from RI-sprayed orchards increased three-, two-, five-, and twofold between 2006 and 2008 for azoxystrobin, pyraclostrobin, boscalid, and pyraclostrobin + boscalid, respectively. A subset of 10 M. fructicola isolates representing low and high EC50 values for azoxystrobin, boscalid, and boscalid + pyraclostrobin was selected for a detached fruit assay to determine disease incidence and severity following protective treatments of formulated RI fungicides at label rates. Brown rot incidence was greater than 50% when fruit were inoculated with isolates having EC50 values of 2, 4, and 0.6 μg/ml for azoxystrobin, boscalid, and pyraclostrobin + boscalid, respectively. Pyraclostrobin failed to control any of the isolates tested in detached fruit assays. Based on minimum inhibitory concentration and brown rot incidence data, we recommend using 3 and 0.75 μg/ml as discriminatory doses to distinguish between sensitive isolates and those with reduced sensitivity to azoxystrobin and pyraclostrobin + boscalid, respectively. Results from our in vitro and in vivo assays indicate a shift toward reduced sensitivity in M. fructicola from the southeastern United States. No cross-resistance was observed between the QoI and the SdhI fungicides, which implies that rotation or tank mixtures of these two chemical classes can be used as a resistance management strategy.

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Genetic diversity assessments of brown rot pathogen Monilinia fructicola based on the six simple sequence repeat loci

Journal of Plant Diseases and Protection ◽

10.1007/s41348-021-00504-4 ◽

2021 ◽

Author(s):

Hakan Guven ◽

Sydney E. Everhart ◽

Rita Milvia De Miccolis Angelini ◽

Hilal Ozkilinc

Keyword(s):

Genetic Diversity ◽

Simple Sequence Repeat ◽

Brown Rot ◽

Monilinia Fructicola ◽

Sequence Repeat ◽

Simple Sequence

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Improving the Biocontrol Potential of Bacterial Antagonists with Salicylic Acid against Brown Rot Disease and Impact on Nectarine Fruits Quality

Agronomy ◽

10.3390/agronomy11020209 ◽

2021 ◽

Vol 11 (2) ◽

pp. 209

Author(s):

Nadia Lyousfi ◽

Rachid Lahlali ◽

Chaimaa Letrib ◽

Zineb Belabess ◽

Rachida Ouaabou ◽

...

Keyword(s):

Salicylic Acid ◽

Disease Severity ◽

Mycelial Growth ◽

Brown Rot ◽

Antagonistic Bacteria ◽

Biological Treatments ◽

Rot Disease ◽

The Impact

The main objective of this study was to evaluate the ability of both antagonistic bacteria Bacillus amyloliquefaciens (SF14) and Alcaligenes faecalis (ACBC1) used in combination with salicylic acid (SA) to effectively control brown rot disease caused by Monilinia fructigena. Four concentrations of salicylic acid (0.5%, 2%, 3.5%, and 5%) were tested under in vitro and in vivo conditions. Furthermore, the impact of biological treatments on nectarine fruit parameters’ quality, in particular, weight loss, titratable acidity, and soluble solids content, was evaluated. Regardless of the bacterium, the results indicated that all combined treatments displayed a strong inhibitory effect on the mycelial growth of M. fructigena and disease severity. Interestingly, all SA concentrations significantly improved the biocontrol activity of each antagonist. The mycelial growth inhibition rate ranged from 9.79% to 88.02% with the highest reduction rate recorded for bacterial antagonists in combination with SA at both concentrations of 0.5% and 3.5%. The in vivo results confirmed the in vitro results with a disease severity varying from 0.00% to 51.91%. A significant biocontrol improvement was obtained with both antagonistic bacteria when used in combination with SA at concentrations of 0.5% and 2%. The lowest disease severity observed with ACBC1 compared with SF14 is likely due to a rapid adaptation and increase of antagonistic bacteria population in wounded sites. The impact of all biological treatments revealed moderate significant changes in the fruit quality parameters with weight loss for several treatments. These results suggest that the improved disease control of both antagonistic bacteria was more likely directly linked to both the inhibitory effects of SA on pathogen growth and induced fruit resistance.

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Combination of Suspension Array and Mycelial Growth Assay for Detecting Multiple-Fungicide Resistance in Botrytis cinerea in Hebei Province in China

Plant Disease ◽

10.1094/pdis-07-18-1269-re ◽

2019 ◽

Vol 103 (6) ◽

pp. 1213-1219

Author(s):

Zehua Su ◽

Xin Zhang ◽

Jianjiang Zhao ◽

Wenqiao Wang ◽

Lei Shang ◽

...

Keyword(s):

Botrytis Cinerea ◽

High Throughput ◽

Fungicide Resistance ◽

Mycelial Growth ◽

Accurate Method ◽

Hebei Province ◽

Combination Method ◽

Major Type ◽

Suspension Array ◽

Growth Assay

To provide a high-throughput, efficient, and accurate method to monitor multiple-fungicide resistance of Botrytis cinerea in the field, we used the suspension array, sequencing, and mycelial growth assay in our research. Discriminating-dose bioassays for detecting carbendazim, diethofencarb, boscalid, and iprodione resistance (CarR, DieR, BosR, and IprR, respectively) were used to analyze 257 isolates collected from Hebei Province in China during 2016 and 2017. High resistance frequencies to carbendazim (100%), diethofencarb (92.08%), and iprodione (86.59%) were detected. BosR isolates accounted for 11.67% of the total. In addition, 103 isolates were randomly selected for phenotype and genotype detection. The high-throughput suspension array was utilized to detect eight genotypes simultaneously, including BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S, which were associated with resistance toward carbendazim or diethofencarb, boscalid, iprodione, and fenhexamid (FenR), respectively. Most of the benzimidazole-resistant isolates (81.55%) possessed the E198V mutation in the BenA gene. Ninety-three isolates with dual resistance to carbendazim and diethofencarb showed the E198V/K mutation. All BosR isolates carried the H272R mutation in the SdhB gene. The I365S and Q369P+N373S (66.99%) mutations in the BcOS1 gene were more frequently observed. No mutation was detected in the erg27 gene in Hebei isolates. There were 13 resistance profile phenotypes. Phenotypes with triple resistance were the most common (83.50%), and CarRDieRBosSIprRFenS was the major type. CarR isolates that carried E198V/K/A were all highly resistant (HR) and only one F200Y mutant was moderately resistant (MR) to carbendazim. Isolates that possessed E198V/K were MR or HR to diethofencarb. BosR isolates that possessed H272R mutation were lowly resistant (LR). IprR isolates were all LR or MR. The distribution of half maximal effective concentrations of CarR isolates with E198V/K mutations and IprR isolates with Q369P+N373S mutations significantly increased from 2016 to 2017. Combined with our observations, a combination method of the high-throughput suspension array and the mycelial growth assay was suggested to accurately monitor multiple resistance of B. cinerea in the field.

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Fungicide Sensitivity of Pythium spp. Associated with Cavity Spot of Carrot in California and Michigan

Plant Disease ◽

10.1094/pdis-07-11-0562 ◽

2012 ◽

Vol 96 (3) ◽

pp. 384-388 ◽

Cited By ~ 7

Author(s):

Xiao Hong Lu ◽

R. Michael Davis ◽

S. Livingston ◽

J. Nunez ◽

Jianjun J. Hao

Keyword(s):

United States ◽

Growth Inhibition ◽

Active Ingredient ◽

Mycelial Growth ◽

The United States ◽

Fungicide Sensitivity ◽

First Report ◽

Cavity Spot ◽

Pythium Violae

The identity of 172 isolates of Pythium spp. from cavity spot lesions on carrot produced in California and Michigan was determined, and their sensitivity to three fungicides was examined. Pythium violae accounted for 85% of California isolates, with P. irregulare, P. dissotocum (the first report as a carrot pathogen in the United States), P. ultimum, and P. sulcatum making the balance. P. sulcatum, P. sylvaticum, and P. intermedium were the most commonly recovered (85%) species in Michigan; others from Michigan included P. intermedium, P. irregulare, and an unclassified strain, M2-05. On fungicide-amended media, 93% of isolates were sensitive to mefenoxam (inhibition of mycelial growth was >60% at 10 μg active ingredient [a.i.]/ml); however, two of five isolates of P. irregulare from California were highly resistant (≤60% inhibition at 100 μg a.i./ml); about half of the isolates of P. intermedium and P. sylvaticum and a single isolate of P. violae were highly or intermediately resistant to mefenoxam (>60% inhibition at 100 μg a.i./ml, or ≤60% inhibition at 10 μg a.i./ml). P. dissotocum, P. irregulare, P. sulcatum, M2-05, and three of seven isolates of P. intermedium were insensitive to fluopicolide (effective concentrations for 50% growth inhibition [EC50] were >50 μg a.i./ml), while P. sylvaticum, P. ultimum, P. violae, and some isolates in P. intermedium were sensitive (EC50 < 1 μg a.i./ml). All isolates were sensitive to zoxamide (EC50 < 1 μg a.i./ml). Sensitivity baselines of P. violae to zoxamide and fluopicolide were established.

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Overexpression of a Redox-Regulated Cutinase Gene, MfCUT1, Increases Virulence of the Brown Rot Pathogen Monilinia fructicola on Prunus spp.

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-2-0176 ◽

2010 ◽

Vol 23 (2) ◽

pp. 176-186 ◽

Cited By ~ 28

Author(s):

Miin-Huey Lee ◽

Chiu-Min Chiu ◽

Tatiana Roubtsova ◽

Chien-Ming Chou ◽

Richard M. Bostock

Keyword(s):

Gene Expression ◽

Redox Regulation ◽

Brown Rot ◽

Gus Expression ◽

Apple Fruit ◽

Gus Activity ◽

Monilinia Fructicola ◽

Gus Reporter ◽

Flanking Regions ◽

Prunus Spp

A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H2O2 enhanced MfCUT1 gene expression. A β-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.

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