Fungicide Sensitivity Tests Research Articles

Cytochrome c Oxidase Influences Pyraclostrobin Sensitivity in Fusarium graminearum by Regulating FgAox Through Transcription Factors FgAod2 and FgAod5.

Cytochrome c oxidase (Cox) is a crucial terminal oxidase in the electron transport chain. In this study, we generated 14 Cox gene deletion or overexpression mutants in Fusarium graminearum. Fungicide sensitivity tests revealed that 11 Cox gene deletion mutants displayed resistance to pyraclostrobin, while 10 overexpression mutants showed hypersensitivity. RNA-Seq and RT-qPCR analyses demonstrated the upregulation of FgAox (alternative oxidase in F. graminearum), FgAod2, and FgAod5 (alternative oxidase deficiency in F. graminearum) in ΔFgCox4-2 and ΔFgCox17-75 mutants. In 11 Cox gene deletion mutants, FgAox expression was significantly upregulated, whereas in 10 Cox gene overexpression mutants, it was significantly downregulated. FgAox overexpression mutants exhibit resistance to pyraclostrobin, while FgAox deletion mutants show hypersensitivity to pyraclostrobin. FgAod2 and FgAod5 were identified as transcription factors for FgAox. Our findings reveal that FgCox influences pyraclostrobin sensitivity by regulating FgAox through FgAod2 and FgAod5. Understanding pyraclostrobin resistance mechanisms in F. graminearum could help develop better fungicide rotation and application strategies to manage resistance and guide the creation of new fungicides targeting different pathways.

Identification and Fungicide Sensitivity of Fusarium spp. Associated with Root Rot of Scutellaria baicalensis in Shanxi Province, China.

Fusarium root rot is usually classified as an extremely destructive soilborne disease. From 2020 to 2021, Fusarium root rot was observed in production areas and seriously affected the yield and quality of Scutellaria baicalensis in Shanxi Province, China. Based on morphological characteristics and combined analysis of the internal transcribed spacer region of ribosomal DNA and translation elongation factor 1-alpha sequences, 68 Fusarium isolates obtained in this work were identified as F. oxysporum (52.94%), F. acuminatum (20.59%), F. solani (16.17%), F. proliferatum (5.88%), F. incarnatum (2.94%), and F. brachygibbosum (1.47%). In the pathogenicity tests, all Fusarium isolates could infect S. baicalensis roots, presenting different pathogenic ability. Among these isolates, F. oxysporum was found to have the highest virulence on S. baicalensis roots, followed by F. acuminatum, F. solani, F.proliferatum, F. brachygibbosum, and F.incarnatum. According to fungicide sensitivity tests, Fusarium isolates were more sensitive to fludioxonil and difenoconazole, followed by carbendazim, thiophanate-methyl, and hymexazol. In brief, this is the first report of Fusarium species (F. oxysporum, F. acuminatum, F. solani, F. proliferatum, F. incarnatum, and F. brachygibbosum) as causal agents of root rot of S.baicalensis in Shanxi Province, China. The fungicide sensitivity results will be helpful for formulating management strategies of S. baicalensis root rot.

Identification and fungicide sensitivity of Xylogone ganodermophthora causing yellow rot in Ganoderma sichuanense in Jilin, China

Ganoderma sichuanense is a prominent medicinal fungus extensively cultivated across China. In 2022, symptoms of yellow rot were observed on the fruiting bodies of G. sichuanense in a mushroom farm located in Panshi City, Jilin Province, China. We isolated and purified a pathogenic fungus, identified as Xylogone ganodermophthora through morphological characteristics and multigene phylogenetic analysis. Pathogenicity of X. ganodermophthora was confirmed through inoculation of G. sichuanense fruiting bodies. This represents the first report of X. ganodermophthora causing yellow rot disease in G. sichuanense. Fungicide sensitivity tests were conducted using a mycelial growth assay, revealing high sensitivity of X. ganodermophthora to Carbendazim, Chlorothalonil, Fludioxonil, Prochloraz, and Prochlorza-Mn, with EC50 values ranging from 0.003 to 0.046 μg mL−1. These findings offer valuable insights into the effective management of yellow rot of G. sichuanense.

Colletotrichum Diversity within Different Species Complexes Associated with Fruit Anthracnose in South Korea and Their Fungicides In-Vitro Sensitivity

Anthracnose, caused by the <i>Colletotrichum</i> genus, comprises a significant number of plant pathogens and poses a major threat to fruit production worldwide, including South Korea. <i>Colletotrichum</i> species were identified associated with anthracnose in fruits such as apple, persimmon, plum, peach, jujube, walnut, and grape. A polyphasic approach, including morphology, multigene phylogenetics, and pathogenicity testing, was used. Additionally, the <i>in-vitro</i> sensitivity of identified <i>Colletotrichum</i> species to common fungicides was also evaluated. A total of nine <i>Colletotrichum</i> species within two complexes, namely gloeosporioides and acutatum, have been identified as the causal agents of anthracnose in common fruits in South Korea. In the gloeosporioides complex, we found <i>Colletotrichum aenigma, C. fructicola, C. gloeosporioides, C. horii, C. siamense</i>, and <i>C. viniferum</i>. Meanwhile, in the acutatum complex, <i>C. fioriniae, C. nymphaeae</i>, and <i>C. orientalis</i> were identified. Notably, <i>C. fructicola, C. siamense, C. fioriniae</i>, and <i>C. nymphaeae</i> were reported for the first time from apple, <i>C. siamense, C. fioriniae</i> and <i>C. nymphaeae</i> from plum, <i>C. siamense, C. fructicola</i>, and <i>C. fioriniae</i> from peach, <i>C. siamense</i> and <i>C. horii</i> from persimmon, <i>C. fioriniae</i> from Omija (<i>Schisandra</i>), <i>C. orientalis</i> from walnut, <i>C. nymphaeae</i> from jujube, and <i>C. aenigma, C. fructicola</i>, and <i>C. siamense</i> from grape. Fungicide sensitivity tests revealed significant variation in the EC<sub>50</sub> values among specific <i>Colletotrichum</i> species when exposed to different fungicides. Moreover, the same <i>Colletotrichum</i> species isolated from different host plants displayed varying sensitivity to the same fungicide.

The sphinganine C4-hydroxylase FgSur2 regulates sensitivity to azole antifungal agents and virulence of Fusarium graminearum

Lipid rafts consisting of ergosterol and sphingolipids in the lipid membrane of cells play important roles in various cellular processes. However, the functions of sphingolipids and their synthetic genes in phytopathogenic fungi have not been well understood yet. In this study, we conducted genome-wide searches and carried out systematic gene deletion analysis of the sphingolipid synthesis pathway in Fusarium graminearum, a causal agent of Fusarium head blight of wheat and other cereal crops worldwide. Mycelial growth assays showed that deletion of FgBAR1, FgLAC1, FgSUR2 or FgSCS7 resulted in markedly reduced hyphal growth. Fungicide sensitivity tests showed that the sphinganine C4-hydroxylase gene FgSUR2 deletion mutant (ΔFgSUR2) exhibited significantly increased susceptibility to azole fungicides. In addition, this mutant displayed a remarkable increase in cell membrane permeability. Importantly, ΔFgSUR2 was defective in deoxynivalenol (DON) toxisome formation, leading to dramatically decreased DON biosynthesis. Moreover, the deletion of FgSUR2 resulted in dramatically decreased virulence of the pathogen on host plants. Taken together, these results indicate that FgSUR2 plays an important role in regulating the susceptibility to azoles and virulence of F. graminearum.

Fungicide sensitivity levels in the Lithuanian Zymoseptoria tritici population in 2021.

Zymoseptoria tritici causes the disease known as septoria leaf blotch in winter wheat and is a major factor in yield loss worldwide. Farmers are inclined to use fungicides to protect their crops; however, the efficacy of these measures is rapidly decreasing due to the natural mechanisms of mutation emergence in pathogen populations. Increasing fungicide resistance is being recorded worldwide, therefore, screening of the current situation in Lithuania is essential to determine the subsequent steps of crop protection strategies. In this study, in vitro fungicide sensitivity tests, mutation detection, and field experiments were carried out. The mean EC50 values for prothioconazole-desthio and mefentrifluconazole were 0.14 and 0.28 mg/l, respectively. Increased frequency of the mutation S524T, linked to DMIs resistance, was observed. Results revealed that the dominant point mutation in the gene CYP51 was I381V, and the most frequent CYP51 haplotype was D13 (V136C, I381V, Y461H, S524T). The mutation G143A, linked to QoI resistance, was detected in ¾ of the population. Mutations conferring resistance to SDHIs were not detected in single pycnidium isolates. Two-year field experiments likewise showed no decline in field efficacy of SDHI fungicide in Lithuania. Moreover, the baseline sensitivity of the Lithuanian Z. tritici population to QiI fungicide fenpicoxamid was established. The findings of this study provide an update on the current status of fungicide resistance in the Lithuanian Z. tritici population.

Characterization, pathogenicity, and fungicide sensitivity of Alternaria isolates associated with preharvest fruit drop in California citrus

Alternaria rot has been recently described as an emerging fungal disease of citrus causing significant damage in California groves. A survey was conducted to determine latent infections on fruits, twigs, and leaves and investigate their seasonal patterns during 2019 and 2020. On fruits, latent infections were more associated with the stem end than with the stylar end, except during spring when a significantly high percentage of flowers (86%) had latent infections. Latent infections on twigs varied markedly between years (28% in 2019 and 9.5% in 2020), while Alternaria spp. were also recovered from citrus leaves. Alternaria isolates collected during the survey were identified based on multigene sequence analysis, confirming that Alternaria alternata and Alternaria arborescens are the two species associated with infections of citrus fruits. Of the 23 isolates, 19 were identified as A. alternata and demonstrated the dominance of this species over A. arborescens. Isolates representing populations of these two species were selected as representative isolates for physiological and morphological studies. A. alternata and A. arborescens showed similar conidial dimensions but differed in the number of conidia produced. Growth rates demonstrated that A. alternata grows faster than A. arborescens at all the temperatures evaluated, except at 25 and 35 °C. The growth patterns were similar for both species. The sporulation rate of the Alternaria isolates was influenced differently by temperature. This parameter also influenced conidial germination and appressorium formation, and no significant differences were observed between Alternaria species. Pathogenicity and aggressiveness tests on detached fruit demonstrated the ability of A. alternata and A. arborescens to cause internal lesions and produce fruit drop in the orchards with no quantitative differences between them (disease severity indexes of 58 and 68%, respectively). The fungicide sensitivity tests showed that DMI fungicides are the most effective fungicides in reducing mycelial growth. The SDHI fungicides had intermediate activity against the mycelial growth but also suppressed spore germination. The spore germination assay suggested that some of the isolates included in this study might have some level of resistance to QoI and SDHI fungicides. The findings of this study provide new information about the pathogens associated with the excessive fruit drop recently observed in some California citrus groves.

Comparing the Fungicide Sensitivity of Sclerotinia sclerotiorum Using Mycelial Growth and Ascospore Germination Assays.

The infection of the floral tissues of snap bean and other crops by Sclerotinia sclerotiorum, the causative agent of white mold, is by ascospores. Irrespective of the fungicide mode of action being evaluated, invitro fungicide sensitivity tests are conducted almost exclusively using mycelial growth assays. This is likely because of difficulties and time involved in sclerotial conditioning required to produce apothecia and ascospores. The objective of this research was to compare estimates of fungicide sensitivity between mycelial growth and ascospore germination assays for S. sclerotiorum. Sensitivity assays were conducted using serial doses of three fungicides commonly used to control white mold: boscalid, fluazinam, and thiophanate-methyl. A total of 27 isolates were evaluated in replicated trials conducted for each fungicide and assay type. The effective concentration to reduce mycelial growth or ascospore germination by 50% (EC50) was estimated for each isolate, fungicide, and assay type. The median EC50 values obtained from ascospore germination assays were 52.7, 10.0, and 2.7 times higher than those estimated from the mycelial growth for boscalid, fluazinam, and thiophanate-methyl, respectively. No significant correlation was found between EC50 values estimated by the two methods. These findings highlight differences that may be important in evaluating the sensitivity of S. sclerotiorum given the fungicide mode of action and how they will be used in the field.

Epidemiological Studies of Pan-Azole Resistant Aspergillus fumigatus Populations Sampled during Tulip Cultivation Show Clonal Expansion with Acquisition of Multi-Fungicide Resistance as Potential Driver.

Pan-azole resistant isolates are found in clinical and environmental Aspergillus fumigatus (Af) populations. Azole resistance can evolve in both settings, with Af directly targeted by antifungals in patients and, in the environment, Af unintendedly exposed to fungicides used for material preservation and plant disease control. Resistance to non-azole fungicides, including methyl benzimidazole carbamates (MBCs), quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs), has recently been reported. These fungicide groups are not used in medicine but can play an important role in the further spread of pan-azole resistant genotypes. We investigated the multi-fungicide resistance status and the genetic diversity of Af populations sampled from tulip field soils, tulip peel waste and flower compost heaps using fungicide sensitivity testing and a range of genotyping tools, including STRAf typing and sequencing of fungicide resistant alleles. Two major clones were present in the tulip bulb population. Comparisons with clinical isolates and literature data revealed that several common clonal lineages of TR34/L98H and TR46/Y121F/T289A strains that have expanded successfully in the environment have also acquired resistance to MBC, QoI and/or SDHI fungicides. Strains carrying multiple fungicide resistant alleles have a competitive advantage in environments where residues of multiple fungicides belonging to different modes of action are present.

Characterization of Fusariumsambucinum isolates associated with potato dry rot and evaluation of cultivar susceptibility and fungicides

Potato dry rot disease (Fusarium spp.) is one of the most important fungal storage rots on potato tubers after harvesting. The aims of this study were to identify the Fusarium species that cause dry rot in potato storages, evaluate the most virulent Fusarium species in vitro and in vivo for fungicide sensitivity and determine the susceptibility of the cultivars. In this context, as a result of surveys carried out in potato storages of Ödemiş and Torbalı counties in İzmir province in 2015-2016; Fusarium species obtained from diseased potato tubers were identified as Fusarium sambucinum, F. oxysporum, F. avenaceum and F. equiseti, using morphological and molecular methods. The most virulent species was determined according to the pathogenicity tests. To determine the susceptibility to disease, 13 potato cultivars, commonly stored in the region throughout the working period, were tested against the most virulent Fusarium species. The pathogenicity tests showed variation between the rank order of susceptibility of the cultivars to the most virulent pathogen. Fungicide sensitivity tests have been conducted with the most susceptible among these cultivars. There are not any plant protection products licensed against this disease in Turkey. On fungicide sensitivity tests, fludioxonil, flutolanil, thiophanate methyl, imazalil, fludioxonil + sedaxane, fluxapyroxad and tolclophos-methyl + thiram efficacy were investigated on the mycelial growth of F. sambucinum. In vitro assays; fludioxonil, fludioxonil + sedaxane and imazalil were the most effective fungicides preventing mycelial growth of the pathogen. In addition to chemicals that are effective in vitro tests, efficacy of Bacillus subtilis QST 713 strain 1.34%, Pseudomonas fluorescens strain Pf1 1.5% and 42% carboxylic acid were also evaluated. In vivo tests were conducted with Russet Burbank, Lady Olympia, Granola cultivars and fungicides were applied in two different methods including before and after inoculation. Results of this study indicated that the treatments with fludioxonil + sedaxane had the highest efficacy and treatments before inoculation was more effective.

Cultural and morphological characterisation of Colletotrichum fragrans of dracaena using various nutrient sources along with fungicide sensitivity tests

Dracaena, is an ornamental plant which helps in air purification. The foliage is damaged by innumerable diseases. Various carbon containing media viz. Potato dextrose agar, Peptone salt agar, Czepek’s Dox Agar, dextrose in Czepek’s Dox Agar medium was replaced by the same amount of sucrose and lactose and Oat meal agar was used to study the radial growth of Colletotrichum fragrans from Dracaena. Among these six carbon media, the most effective medium for its rapid growth was Peptone salt agar medium. Also, the morphological characters differed with change in media constituents, wherein Oat meal agar medium was found to be effective for the production of acervuli, conidia, setae of higher dimensions. Bioassay was conducted using four fungicides viz. Blitox, Mancozeb, Chlorothalonil and Difenoconazole with six different concentrations, Difenoconazole with EC50 value 299.2µ/ml gave best results. Also, in-vivo studies confirmed that Difenoconazole could inhibit the diseased lesion most effectively than others.

Fungicide Sensitivity Shifting of Zymoseptoria tritici in the Finnish-Baltic Region and a Novel Insertion in the MFS1 Promoter.

Septoria tritici blotch (STB) is caused by the ascomycete Zymoseptoria tritici and one of the predominating diseases in wheat (Triticum aestivum) in Europe. The control of STB is highly reliant on frequent fungicide applications. The primary objective of this study was to assess sensitivity levels of Z. tritici to different fungicide groups. The fungicides included in this study were epoxiconazole, prothioconazole-desthio, tebuconazole, and fluxapyroxad. A panel of 63 isolates from Estonia, Latvia, and Lithuania, and 10 isolates from Finland were tested. Fungicide sensitivity testing was carried out as a bioassay analyzing single pycnidium isolates on different fungicide concentrations. The average EC50 value in Baltic countries and Finland to epoxiconazole was high ranging from 1.04 to 2.19 ppm. For prothioconazole-desthio and tebuconazole, EC50 varied from 0.01 to 0.24 ppm, and 1.25 to 18.23 ppm, respectively. The average EC50 value for fluxapyroxad varied from 0.07 to 0.33 ppm. To explain the range of sensitivity, the samples were analyzed for CYP51 and Sdh mutations, as well as cytb G143A, CYP51 overexpression, and multidrug resistance (MDR). Frequencies of ZtCYP51 mutations D134G, V136A/C, A379G, I381V, and S524T in the Finnish-Baltic region were lower than in other European countries, but have increased compared to previous years. The frequency of cytb G143A conferring strobilurin resistance also augmented to 50–70% in the Z. tritici populations from Estonia, Finland, Latvia, and Lithuania. No Sdh mutations were found in this study, and neither strains of MDR phenotypes. However, we found a strain harboring a previously unknown transposon insertion in the promoter of the MFS1 gene, involved in drug efflux and multi-drug resistance. This new insert, however, does not confer an MDR phenotype to the strain.

Genetic Diversity and Azole Fungicide Sensitivity in Pseudocercospora musae Field Populations in Brazil.

Pseudocercospora musae, causal agent of Sigatoka leaf spot, or yellow Sigatoka disease, is considered a major pathogen of banana (Musa spp.). Widely disseminated in Brazil, this study explored the genetic diversity in field populations of the pathogen from production areas in the Distrito Federal and the States of Bahia, Minas Gerais, and Rio Grande do Norte. Resistance to demethylation inhibitor (DMI) fungicides was also examined. For 162 isolates from 10 banana growing regions, analysis of mating type idiomorph frequency was conducted, together with estimation of genetic diversity at 15 microsatellite loci. A total of 149 haplotypes were identified across the examined populations, with an average genetic diversity of 4.06. In general, populations displayed 1:1 proportions of idiomorphs MAT1-1 and MAT1-2, providing evidence for sexual recombination. Multilocus linkage disequilibrium also indicated asexual reproduction contributing to the genetic structure of certain populations. AMOVA revealed that 86.3% of the genetic differentiation of the pathogen occurred among isolates within populations. Discriminant Analysis of Principal Components (DAPC) identified six most probable genetic groups, with no population structure associated with geographic origin or collection site. Although genetic similarity was observed among certain populations from different states, data revealed increasing genetic differentiation with increasing geographic distance, as validated by Mantel’s test (r = 0.19, P < 0.001). On the basis of DMI fungicide sensitivity testing and CYP51 gene sequence polymorphism, isolates from the Distrito Federal separated into two main groups, one with generally higher EC50 values against eight DMI fungicides. A clear phenotype-to-genotype relationship was observed for isolates carrying the CYP51 alteration Y461N. Conventionally adopted fungicides for control of Sigatoka leaf spot are likely to be overcome by combined sexual and asexual reproduction mechanisms in P. musae driving genetic variability. Continued analysis of pathogen genetic diversity and monitoring of DMI sensitivity profiles of Brazilian field populations is essential for the development of integrated control strategies based on host resistance breeding and rational design of fungicide regimes.

Clonality, spatial structure, and pathogenic variation in Fusarium fujikuroifrom rain-fed rice in southern Laos.

Bakanae disease, caused by the fungal phytopathogen Fusarium fujikuroi, can be detected in most rice (Oryza sativa L.) growing areas worldwide. In this study, we investigated the population structure of this fungus in southern Lao PDR, a country located near the geographic origin of rice domestication. Microsatellites (SSRs) and mating type (MAT) analyses, pathogenicity and fungicide sensitivity tests were integrated in the study. The first key finding is that the population genetic structure of F. fujikuroi in Lao PDR is consistent with high clonal reproduction. Indeed, (i) “true” clones were identified; (ii) within populations, MAT types were frequently skewed from 1:1 ratio, (iii) linkage disequilibrium (among SSRs as also among SSRs and MAT) was present, and (iv) gene-flow between opposite MAT types within the same population is restricted. The presence of genetic divergence among areas and populations and the occurrence of positive spatial autocorrelation of genetic variation, indicate that migration is restricted, and that genetic drift plays an important role in the evolution of this fungus. Two main well-defined groups of isolates were detected (FST = 0.213) that display a non-random spatial distribution. They differ in the ability to induce seedlings death but not seedlings elongation (the typical Bakanae symptom) suggesting that the pathogen’s ability to induce the two symptoms is under different genetic control. Finally, we compared two agroecosystems with contrasting characteristics: low-input and traditional (Lao PDR) vs high-input and modern (Italy). We found differences in the level of population structuring and of spatial autocorrelation. This suggests that the evolutionary potential of the fungus not only depends on its intrinsic characteristics, but is strongly influenced by other external factors, most likely by the dynamics of infested seed exchange. Thus, quarantine and chemical treatments are a way to reduce population connectivity and hence the evolutionary potential of this pathogen.

Pathotype Grouping of Cercospora sojina Isolates on Soybean and Sensitivity to QoI Fungicides.

Frogeye leaf spot (FLS), caused by Cercospora sojina, is a common disease of soybean in the southern and northern United States and causes significant yield loss. The use of the current race scheme for classification for C. sojina does not take into account the range of disease severity reactions within each differential. The objective of this research was to better understand the diversity among C. sojina isolates through the development and use of pathogenicity groups. In this study, 83 isolates acquired from 2006 to 2009 were screened using 12 soybean (Glycine max) differentials. Disease severity on the 12 differentials ranged from 0 to 9, where 0 is immune and 9 is very susceptible. The average severity for each isolate across differentials ranged from 1 to 7. The 83 isolates were grouped into five pathogenicity groups (PG): PG1, PG2, PG3, PG4, and PG5, reflecting the severity grouping. Using the 12 differentials, PG1 isolates were differentiated by the lack of infection on Davis, Peking, Kent, Palmetto, Hood, CNS, Tracy, and Richland. PG2 had a range of infections on a scale of 1 to 2 on all differentials except on Davis; PG3 isolates had severity ranging from 3 to 4 except on Davis. PG4 isolates caused no infection on Davis, a maximum disease severity of 5 on Peking, while the rest of differentials had severities from 5 to 6. PG5 isolates caused no infection on Davis, severity of 7 on CNS, and severity of 8 on Kent, Hood, and Palmetto. The remaining seven differentials had severities of 9. Across the geographical locations, the predominant pathotypes were PG3 and PG4 and represented 84% of the tested isolates. Azoxystrobin fungicide sensitivity tests showed that 88% of the isolates were sensitive and dominated the population, while only 6% had a high level of fungicide resistance, suggesting that FLS resistance to the QoI fungicide group was not yet completely developed and had not spread to other areas at the time when these isolates were acquired. The overall virulence profile of the isolates indicated that there was variation in disease severity, suggesting that selection of resistance for each PG may produce lines with more precisely defined interactions to specific pathotypes of C. sojina. This may improve the screening and selection of useful resistance genes that could be pyramided for resistance to each pathogenicity group.

Occurrence of sdh Mutations in German Alternaria solani Isolates and Potential Impact on Boscalid Sensitivity In Vitro, in the Greenhouse, and in the Field.

The fungus Alternaria solani is the main pathogen causing early blight on potatoes (Solanum tuberosum L.). An increase in the development of resistance to the succinate dehydrogenase inhibitor (SDHI) boscalid, one of the main active ingredients for the control of early blight, has been reported. For this study, monitoring data from Germany were collected between 2013 and 2016 and an increase in the occurrence of A. solani succinate dehydrogenase (SDH) mutant isolates was observed. In addition to the known point mutations in sdh complex II, a new mutation in subunit C was found in German isolates (SdhC-H134Q). SDHI fungicide sensitivity testing was performed in the laboratory, greenhouse, and field. Reduced boscalid sensitivity was shown for mutant isolates (SdhB-H278Y and SdhC-H134R) both in vitro and in vivo. In addition, field trials with artificial inoculation were performed in 2016 and 2017. In both years, fungicide efficacy was significantly reduced after mutant inoculation compared with wild-type inoculation.

First Report of Resistance to Pyraclostrobin, Boscalid, and Thiophanate-methyl in Colletotrichum gloeosporioides from Blueberry in Georgia

Colletotrichum gloeosporioides causes anthracnose fruit rot and leaf spot on blueberries. For controlling anthracnose, blueberry growers mostly rely on pre- and postharvest fungicide applications in addition to orchard sanitation. Single-site fungicides including quinone outside inhibitors (QoIs), such as pyraclostrobin and azoxystrobin as well as fungicides containing the succinate dehydrogenase inhibitor (SDHI) boscalid are used frequently to control anthracnose rots and other diseases on blueberry; however, development of fungicide resistance is a real risk because a limited number of fungicides are now available for blueberry disease management. In 2019, three isolates of C. gloeosporioides were cultured from blueberry fruit collected from southern highbush blueberry cultivar ‘Farthing’ in two commercial blueberry fields in Pierce County, Georgia, where ripe rot had been a problem. Fungicide sensitivity tests were conducted using a mycelial growth inhibition assay as described previously. A total of nine fungicides were evaluated to determine the sensitivity of these C. gloeosporioides isolates. All three isolates were resistant to thiophanate-methyl, the QoI fungicide pyraclostrobin, and the SDHI fungicide boscalid. These findings suggest that continuous monitoring of fungicide resistance is necessary to avoid the unwarranted application of single-site fungicides.

Azole sensitivity in Leptosphaeria pathogens of oilseed rape: the role of lanosterol 14\u03b1-demethylase

Lanosterol 14-α demethylase is a key enzyme intermediating the biosynthesis of ergosterol in fungi, and the target of azole fungicides. Studies have suggested that Leptosphaeria maculans and L. biglobosa, the causal agents of phoma stem canker on oilseed rape, differ in their sensitivity to some azoles, which could be driving pathogen frequency change in crops. Here we used CYP51 protein modelling and heterologous expression to determine whether there are interspecific differences at the target-site level. Moreover, we provide an example of intrinsic sensitivity differences exhibited by both Leptosphaeria spp. in vitro and in planta. Comparison of homologous protein models identified highly conserved residues, particularly at the azole binding site, and heterologous expression of LmCYP51B and LbCYP51B, with fungicide sensitivity testing of the transformants, suggests that both proteins are similarly sensitive to azole fungicides flusilazole, prothioconazole-desthio and tebuconazole. Fungicide sensitivity testing on isolates shows that they sometimes have a minor difference in sensitivity in vitro and in planta. These results suggest that azole fungicides remain a useful component of integrated phoma stem canker control in the UK due to their effectiveness on both Leptosphaeria spp. Other factors, such as varietal resistance or climate, may be driving observed frequency changes between species.

Characterization of Pythium spp. associated with root rot of tobacco seedlings produced using the float tray system in Zimbabwe

AbstractThe study was undertaken to identify and characterize Pythium isolates associated with root rot disease of tobacco seedlings as a first step towards developing management strategies for the pathogen. A total of 85 Pythium isolates were collected from diseased tobacco seedlings during 2015–2016 tobacco growing season. The isolates were identified to species level using sequencing of the internal transcribed spacer region. Thereafter, a subset of the isolates was tested for sensitivity to the commonly used fungicides, metalaxyl, azoxystrobin and a combination of fenamidone/propamocarbby growing isolates on Potato Dextrose Agar plates amended with the fungicides. The sequence analysis of the ITS‐rDNA identified Pythium myriotylum as the dominant Pythium species associated with the root rot of tobacco seedlings in Zimbabwe. Pythium aphanidermatum and P. insidiosum were also identified albeit at lower frequencies. Phylogenetic analyses of the ITS region of the P. myriotylum isolates showed little sequence diversity giving rise to one distinct clade. The fungicide sensitivity tests showed that metalaxyl provided the best control of P. myriotylum in vitro, as compared to other fungicides. To the best of our knowledge, this is the first comprehensive study to determine and characterize Pythium species associated with root rot of tobacco in the float seedling production system in Zimbabwe.

Boscalid-resistance in Alternaria alternata and Alternaria solani populations: An emerging problem in Europe

The application of fungicides is the most common and most effective method to control Alternaria diseases in potatoes. Boscalid, a member of the succinate dehydrogenase inhibiting (SDHI) fungicide group, is frequently used in combination with Quinone outside inhibitors (QoIs) such as strobilurins in spraying programs. However, due to the frequent application of these fungicides, resistant populations can develop. Indeed, resistance towards SHDIs is an emerging problem in both A. alternata and A. solani populations. To gain deeper knowledge on the prevalence of SDHI mutants, 83 A. solani and 53 A. alternata isolates, collected during 2014 and 2015 in Belgium, situated in the center of Europe, were screened for the presence of amino acid substitutions in the different subunits of the succinate dehydrogenase gene (SdhB, SdhC and SdhD). The isolate screening revealed that mutations, causing a reduced sensitivity towards SDHIs, were widespread in the Belgian Alternaria population; 70% of the A. solani and 41% of the A. alternata isolates possessed one or more mutations. Mutations in the subunit SdhC were most frequently detected in the A. solani population, while mutations in the subunit SdhD were absent. In the A. alternata population, mutations were found in the three Sdh subunits. Mutations in the subunit SdhB were predominant, whereas mutations in the subunit SdhD were only marginally detected. Furthermore, it was seen that most isolates with a QoI mutation, providing resistance towards strobilurins, simultaneously carried substitutions in the Sdh gene. This means that 40% of the A. solani and 58% of the A. alternata population possesses a dual fungicide resistance. This is very important since SDHIs and QoIs are often formulated together in one fungicide. The fungicide sensitivity tests confirmed that the isolates with mutations were significantly less sensitive towards the SDHI boscalid compared to the isolates with neither a SDHI nor a QoI mutation. No significant differences in resistance level between isolates with mutations in the different subunits were detected. Furthermore, based on mycelium growth and spore germination capacity, there was no fitness cost associated with these mutations.