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

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chenguang Wang ◽  
Luxi Xu ◽  
Xiaoyu Liang ◽  
Jing Wang ◽  
Xinwei Xian ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Management Strategies ◽  
Cross Resistance ◽  
Pcr Analysis ◽  
Baseline Sensitivity ◽  
Lasiodiplodia Theobromae ◽  
Normal Distribution Curve ◽  
Dmi Fungicides ◽  
Rate Method ◽  
Important Disease

AbstractStem-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.

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

2021 ◽  
Author(s):  
Chenguang Wang ◽  
Luxi Xu ◽  
Xiaoyu Liang ◽  
Jing Wang ◽  
Xinwei Xian ◽  
...  
Keyword(s):  
Gene Sequence ◽  
Management Strategies ◽  
Resistance Mechanism ◽  
Cross Resistance ◽  
Pcr Analysis ◽  
Baseline Sensitivity ◽  
Lasiodiplodia Theobromae ◽  
Normal Distribution Curve ◽  
Rate Method ◽  
Important Disease

Abstract 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.

Sensitivity of C. fructicola and C. siamense of peach in China to multiple classes of fungicides and characterization of pyraclostrobin-resistant isolates

Plant Disease ◽  
2021 ◽  
Author(s):  
Hafiz Muhammad Usman ◽  
Qin Tan ◽  
Mohammad Mazharul Karim ◽  
Muhammad Adnan ◽  
Weixiao Yin ◽  
...  
Keyword(s):  
High Resistance ◽  
Mycelial Growth ◽  
Management Strategies ◽  
Cross Resistance ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Dmi Fungicides ◽  
Colletotrichum Gloeosporioides Species Complex ◽  
Cyt B Gene

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including C. fructicola and C. siamense, is a devastating disease of peach. The chemical control has been widely used for years and management failures have increased towards commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance and 26 % were low resistant, all of the C. siamense isolates were low resistant. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin, even they are members of the same quinone outside inhibitor (QoI) fungicide group, and between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and low-resistant isolates of C. fructicola. Molecular characterization of Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the current resistance status of C. fructicola and C. siamense to different fungicides and their future perspectives. Demethylation inhibitor (DMI) fungicides are the best option among different chemicals to control peach anthracnose in China.

scholarly journals Point Mutations in the β-Tubulin of Phytophthora sojae Confer Resistance to Ethaboxam

2019 ◽  
Vol 109 (12) ◽  
pp. 2096-2106 ◽  
Author(s):  
Qin Peng ◽  
Zhiwen Wang ◽  
Yuan Fang ◽  
Weizhen Wang ◽  
Xingkai Cheng ◽  
...  
Keyword(s):  
Fungicide Resistance ◽  
Germination Rate ◽  
Management Strategies ◽  
Resistance Management ◽  
Point Mutations ◽  
Phytophthora Sojae ◽  
Cross Resistance ◽  
Baseline Sensitivity ◽  
Resistant Mutants ◽  
Β Tubulin

Ethaboxam is a β-tubulin inhibitor registered for the control of oomycete pathogens. The current study was established to determine the ethaboxam sensitivity of the plant pathogen Phytophthora sojae and investigate the potential for the emergence of fungicide resistance. The effective concentration for 50% inhibition (EC50) of 112 Phytophthora sojae isolates exhibited a unimodal distribution with a mean EC50 for ethaboxam of 0.033 µg/ml. Establishing this baseline sensitivity provided critical data for monitoring changes in ethaboxam-sensitivity in field populations. The potential for fungicide resistance was investigated using adaptation on ethaboxam-amended V8 agar, which resulted in the isolation of 20 resistant mutants. An assessment of the biological characteristics of the mutants including mycelial growth, sporulation, germination rate and pathogenicity indicated that the resistance risk in Phytophthora sojae was low to medium with no cross-resistance between ethaboxam and cymoxanil, metalaxyl, flumorph, and oxathiapiprolin being detected. However, positive cross-resistance was found between ethaboxam and zoxamide for Q8L and I258V but negative cross-resistance for C165Y. Further investigation revealed that the ethaboxam-resistant mutants had point mutations at amino acids Q8L, C165Y, or I258V of their β-tubulin protein sequences. CRISPR/Cas9-mediated transformation experiments confirmed that the Q8L, C165Y, or I258V mutations could confer ethaboxam resistance in Phytophthora sojae and that the C165Y mutation induces high levels of resistance. Taken together, the results of the study provide essential data for monitoring the emergence of resistance and resistance management strategies for ethaboxam, as well as for improving the design of novel β-tubulin inhibitors for future development.

scholarly journals Baseline Sensitivity and Cross-Resistance to Succinate-Dehydrogenase-Inhibiting and Demethylation-Inhibiting Fungicides in Didymella bryoniae

Plant Disease ◽  
2012 ◽  
Vol 96 (7) ◽  
pp. 979-984 ◽  
Author(s):  
A. Thomas ◽  
D. B. Langston ◽  
K. L. Stevenson
Keyword(s):  
Succinate Dehydrogenase ◽  
Fungicide Resistance ◽  
Mycelial Growth ◽  
Effective Concentration ◽  
Cross Resistance ◽  
Didymella Bryoniae ◽  
Baseline Sensitivity ◽  
History Of ◽  
Dmi Fungicides ◽  
Positive Correlations

Didymella bryoniae, which causes gummy stem blight (GSB) of watermelon, has a history of developing resistance to fungicides, most recently the succinate-dehydrogenase-inhibiting (SDHI) fungicide boscalid. To facilitate fungicide resistance monitoring, baseline sensitivity distributions were established for demethylation-inhibiting (DMI) fungicides tebuconazole and difenoconazole and the SDHI fungicide penthiopyrad, and reestablished for the SDHI fungicide boscalid. In all, 71 isolates with no known prior exposure to SDHIs or DMIs were used to determine the effective concentration at which mycelial growth was inhibited by 50% (EC50). EC50 values for boscalid, penthiopyrad, tebuconazole, and difenoconazole were 0.018 to 0.064, 0.015 to 0.057, 0.062 to 0.385, and 0.018 to 0.048 μg/ml, with median values of 0.032, 0.026, 0.118, and 0.031 μg/ml, respectively. Significant positive correlations between the sensitivity to penthiopyrad and boscalid (P < 0.0001, r = 0.75) and between tebuconazole and difenoconazole (P < 0.0001, r = 0.59) indicate a potential for cross-resistance between chemically related fungicides. In 2009, 103 isolates from fungicidetreated watermelon fields were tested for sensitivity to boscalid and penthiopyrad using a discriminatory concentration of 3.0 μg/ml. Of the isolates tested, 82 were insensitive and 14 were sensitive to both fungicides. Because of the significant potential for cross-resistance between closely related fungicides, growers will be advised not to use both SDHIs or both DMIs successively in the same fungicide spray program.

scholarly journals Baseline Sensitivity of Monilinia fructicola from China to the DMI Fungicide SYP-Z048 and Analysis of DMI-Resistant Mutants

Plant Disease ◽  
2012 ◽  
Vol 96 (3) ◽  
pp. 416-422 ◽  
Author(s):  
F. P. Chen ◽  
J. R. Fan ◽  
T. Zhou ◽  
X. L. Liu ◽  
J. L. Liu ◽  
...  
Keyword(s):  
Brown Rot ◽  
Cross Resistance ◽  
Monilinia Fructicola ◽  
Lesion Area ◽  
Site Mutation ◽  
Baseline Sensitivity ◽  
Dmi Fungicides ◽  
Resistant Mutants ◽  
Beijing China

Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. In this study, the sensitivity of 100 Monilinia fructicola isolates from four unsprayed orchards and two packinghouses in Beijing, China, to the new DMI fungicide SYP-Z048 was evaluated and ranged from 0.003 to 0.039 and 0.016 to 0.047 μg/ml, respectively. Laboratory mutants resistant to SYP-Z048 were generated using UV irradiation but no mutants occurred spontaneously. Resistance was stable after 10 weekly consecutive transfers on fungicide-free medium. Three parameters, including growth rate, sporulation in vitro, and lesion area, were significantly different when sensitive isolates and resistant mutants were analyzed as groups. Mutants grew more slowly and developed significantly smaller lesions on detached fruit, and their sporulation ability in vitro was reduced. Cross resistance was found between SYP-Z048 and propiconazole (ρ = 0.82, P < 0.0001) but not between SYPZ048 and tridemorph, carbendazim, procymidone, azoxystrobin, or pyrimethanil. SYP-Z048 resistance in mutants exhibiting 50% mycelial growth inhibition values greater than 0.3 μg/ml was correlated with the presence of a mutation in the CYP51 gene that encodes the target protein for DMI fungicides. The mutation caused an amino acid change from tyrosine to phenylalanine at position 136 (Y136F). To our knowledge, this is the first baseline sensitivity of M. fructicola collected from China to a DMI fungicide. The inability of M. fructicola to generate spontaneous DMI-resistant mutants coupled with reduced fitness of Y136F mutants can explain why this target site mutation has not yet emerged as a DMI fungicide resistance determinant in M. fructicola field populations worldwide.

scholarly journals Acquisition of Resistance to Sterol Demethylation Inhibitors by Populations of Venturia inaequalis

1997 ◽  
Vol 87 (12) ◽  
pp. 1272-1278 ◽  
Author(s):  
Stefan Kunz ◽  
Holger Deising ◽  
Kurt Mendgen
Keyword(s):  
Venturia Inaequalis ◽  
Cross Resistance ◽  
Apple Orchards ◽  
Inhibitory Effects ◽  
Baseline Sensitivity ◽  
Resistance Factors ◽  
Fruit Growing ◽  
Dmi Fungicides ◽  
Sterol Demethylation Inhibitors

Acquisition of resistance to sterol demethylation inhibitors (DMIs) by populations of Venturia inaequalis was investigated using a microscopical method developed by C. Siebels and K. Mendgen. Microscopical analysis of conidiophore formation enabled the earlier detection of resistance and a clearer distinction between DMI-resistant and DMI-sensitive populations than other in vivo methods commonly used to analyze inhibitory effects of fungicides. In addition, because observations were made on the level of individuals, quantitative measures of the composition of conidial populations were obtained. The development of DMI sensitivity was followed over a period of 3 years in control apple orchards that had never been treated with fungicides and in orchards with DMI history. The 50% effective dose values determined by microscopical evaluation of conidio-phore development for untreated populations revealed the baseline sensitivities of 0.3, 0.96, 0.09, 1.22, and 1.92 mg/liter for flusilazole, fenarimol, difenoconazole, tebuconazole, and pyrifenox, respectively. As compared with the baseline sensitivity, all populations with DMI history showed significant resistance to flusilazole. A strong nonlinear correlation (R = 0.96) was found between the resistance factors and the sum of all DMI treatments of the 3 years before taking the sample. According to this correlation, resistance can be expected in all apple orchards of the fruit-growing area along Lake Constance, Germany, in which more than two DMI treatments per season have been applied. Due to cross-resistance, the recently introduced DMI fungicides difenoconazole, tebuconazole, and pyrifenox did not allow the control of V. inaequalis populations resistant to flusilazole.

scholarly journals Fusarium basal rot: profile of an increasingly important disease in Allium spp.

2021 ◽  
Author(s):  
Dung Le ◽  
Kris Audenaert ◽  
Geert Haesaert
Keyword(s):  
Disease Management ◽  
Cultural Practices ◽  
Current Knowledge ◽  
Management Strategies ◽  
Future Directions ◽  
Basal Rot ◽  
Major Constraint ◽  
Fusarium Basal Rot ◽  
Chemical Pesticides ◽  
Important Disease

AbstractFusarium basal rot (FBR) is a soil-borne disease that affects Allium species worldwide. Although FBR has long been recognized as a major constraint to the production of economically important Allium species, information that could support disease management remains scattered. In this review, the current knowledge on the causal agents, symptomology and epidemiology, impact, and management strategies of FBR is synthesized. We highlight that FPR is associated with different complexes of several Fusarium species, of which Fusarium oxysporum and F. proliferatum are the most prevalent. These pathogenic complexes vary in composition and virulence, depending on sites and hosts, which can be challenging for disease management. Research to improve disease management using chemical pesticides, resistance cultivars, biocontrol agents, and cultural practices has achieved both promising results and limitations. Finally, research needs and future directions are proposed for the development of effective FBR management strategies.

scholarly journals SNP and SCAR Markers for Specific Discrimination of Antler-Shaped Ganoderma lucidum

2019 ◽  
Vol 7 (1) ◽  
pp. 12 ◽  
Author(s):  
O-Chul Kwon ◽  
Chang-Soo Lee ◽  
Young-Jin Park
Keyword(s):  
Ganoderma Lucidum ◽  
Gene Sequence ◽  
Rapd Analysis ◽  
Random Amplified Polymorphic Dna ◽  
Morphological Characteristics ◽  
Pcr Analysis ◽  
Scar Markers ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Specific Fragment

In this study we identified single nucleotide polymorphism (SNP) and sequence characteristic amplification region (SCAR) markers for specific identification of antler-shaped Ganoderma lucidum strains. When the partial mitochondrial SSU rDNA gene sequence of various antler- and kidney-shaped G. lucidum strains were analyzed and aligned, an SNP was found only in the antler-shaped G. lucidum strain at position 456 bp. In addition, this SNP of antler-shaped strains was digested by HinfI restriction enzyme. We further analyzed the polymorphism of various G. lucidum strains by random amplified polymorphic DNA (RAPD) analysis. In RAPD analysis, we isolated and sequenced a fragment, specific for antler-shaped G. lucidum strains. Based on this specific fragment sequence, two sets of specific primer pairs for antler-shaped G. lucidum strains were designed. PCR analysis revealed that two specific bands were observed only from antler-shaped strains. These two molecular markers will be helpful for identification of morphological characteristics of G. lucidum.

scholarly journals Sensitivity of Mycosphaerella fijiensis from Banana to Trifloxystrobin

Plant Disease ◽  
2001 ◽  
Vol 85 (12) ◽  
pp. 1264-1270 ◽  
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.

scholarly journals First Report of Botrytis cinerea Causing Gray Mold Disease on Peach from Pakistan

2018 ◽  
Vol 7 (3) ◽  
pp. 131-131
Author(s):  
Raees Ahmed ◽  
Amjad S. Gondal ◽  
Muhammad Tariq Khan ◽  
Shazia Shahzaman ◽  
Sajjad Hyder
Keyword(s):  
Botrytis Cinerea ◽  
Mycelial Growth ◽  
Agar Medium ◽  
Management Strategies ◽  
Gray Mold ◽  
First Report ◽  
Average Production ◽  
Conducive Environment ◽  
Hyphal Tip ◽  
Important Disease

Gray mold caused by Botrytis cinerea is an important disease that attacks fruits, leaves and twigs of peach. Peach is grown on an area of 18,008 ha with an average production of 72,085 tons per year in Pakistan (FAO, 2017). During May 2017, brown spots on 33% of the peach fruits examined were observed in Swat district of KPK province of Pakistan. Infected fruits were incubated at 25±2 °C in a humid chamber resulted in greyish mycelial growth with light brown lesions. Hyphal growths on infected fruits were cultured on PDA media and purified by hyphal tip method. Morphologically whitish grey growth was observed on PDA and later on dark sclerotia were observed after 6-7 days of incubation. Hyphae were found septate with branched hyaline conidiophores having a bunch of ovoid conidia at their tips. Further confirmations were done by amplifying internal transcribed spacer regions (Andrew et al., 2009) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) region of the isolates (Li et al., 2012). Amplicons sequenced from Macrogen Korea were blasted and submitted in NCBI showed that ITS sequences (Accessions MH049690 and MH049691) were 99% identical with already reported (MG878388 and MG654661) sequences and the G3PDH gene sequences (Accessions MH560352 and MH560353) were 99 % identical with already reported (Accessions MG204876) sequences of B. cinerea. Pathogenicity was confirmed on healthy peach fruits disinfected with 50% ethanol, inoculated by placing a plug of about 1cm2 taken from the edge of actively growing B. cinerea isolate (BTS-16). Fruits were incubated at 25±2 °C in a humid chamber (Abata et al., 2016). A set of healthy fruits mock-inoculated with a plug of agar medium were used as control. Three days after inoculation, inoculated fruits showed sunken lesions with cottony greyish mycelial growth on their surface. Fungus isolated from these infections was re-confirmed as B. cinerea. Conducive environment for the disease progression in nearby areas can result into a huge loss in peach produce so there is a need to devise management strategies to cope with the pathogen. This is the first report of gray mold disease of peach caused by B. cinerea from Pakistan. 

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