Mycelial Growth Assay Research Articles

Morphology, Pathogenicity and Management of Coniella Fruit Rot (Coniella granati) on Pomegranate

One of the objectives of the study was to identify the fungus involved in fruit rot on pomegranates in the Mediterranean Region of Turkey. The fungus designated as Coniella granati (Saccardo) Petrak and Sydow based on morphological characteristics. The fungus colonized the fruit after 5 to 8 days, followed by the appearance of fruit rot symptoms leading to the formation of abundant pycnidia covering the peel. Secondly, the efficacy of fungicides against C. granati was evaluated by mycelial growth and conidial germination assays. Tebuconazole, boscalid+pyraclostrobin and iprodione at 1.0, 25, and 50 μgml-1 concentrations, respectively, completely inhibited mycelial growth. In the azoxystrobin and dodine, relatively higher concentrations required to inhibit mycelial growth. Tebuconazole exhibited the greatest inhibition (82.2%) of mycelium growth. The EC50 values in mycelial growth of C. granati ranged from 0.13 to 151.9. The highest EC50 values occurred for tebuconazole (0.13μgml-1). Tebuconazole, boscalid+pyraclostrobin and iprodione at 200, 10 and 5 μgml-1 concentrations, respectively, were the highly effective in inhibiting conidial germination. Azoxystrobin exhibited a low effect (61%) on conidial germination. The EC50 values on conidial germination of C. granati ranged from 0.2 to 28.7. Tebuconazole had the lowest EC50 value, while boscalid+pyraclostrobin exhibited the highest EC50 value.

Biological and molecular characterization of field isolates of Alternaria alternata with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC50 values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.

Volatile organic compounds produced by Lysinibacillus sp. FJAT-4748 possess antifungal activity against Colletotrichum acutatum

ABSTRACTFJAT-4748 is a bacterial strain isolated from forest soil samples taken from Dongba Valley, Lijiang, Kunming, Yunnan Province, PR China. This strain was identified as Lysinibacillus sp. based on a 16S rRNA gene sequence analysis. FJAT-4748 has been shown to possess antifungal activity against different fungi, including Colletotrichum acutatum, Aspergillus niger, Fusarium solani, Fusarium moniliforme and Fusarium oxysporum. The results of the present study indicate that this antifungal activity results from volatile organic compounds (VOCs) produced by this strain. The observed inhibition rates of VOCs from FJAT-4748 against these fungi were 100%, 100%, 37.20%, 18.94% and 7.64%, respectively. GC-MS analysis identified 24 VOCs from FJAT-4748, which included different categories of compounds, such as aldehydes, ketones, alcohols, aromatic hydrocarbons and alkanes. Of these 24 VOCs, the most abundant compound was 2-ethyl-1-hexanol, which constituted 36.24% of the total VOCs based on the relative peak area. In the in vitro C. acutatum mycelial growth assay, 2-ethyl-1-hexanol exhibited the strongest activity, with an inhibitory rate of 100% using 10 µL/plate of this VOC. The activity of benzaldehyde was lower. 2-decanone showed the weakest activity among the compounds tested. The inhibitory activity of an artificial mixture of three VOCs against the C. acutatum increased with the amount of artificial mixture used. The inhibition rate reached 100% using 30 µL/plate of this artificial mixture in the plate test. Taken together, these results show that the antifungal VOCs produced by Lysinibacillus sp. FJAT-4748 are potentially useful as agents for controlling anthracnose caused by Colletotrichum acutatum.

Sensitivity of Colletotrichum acutatum to six fungicides and reduction in incidence and severity of chili anthracnose using pyraclostrobin

Anthracnose, caused by Colletotrichum acutatum, is one of the most destructive diseases of chili worldwide. Fungicides including picoxystrobin, azoxystrobin, difenoconazole, thiophanate-methyl and mancozeb have been used to manage anthracnose diseases for many years. Pyraclostrobin was introduced in China in 2016 to control chili anthracnose but has not seen widespread use. In this study, 40 isolates of C. acutatum were examined for their corresponding sensitivities to these fungicides using mycelial growth and spore germination assays. In greenhouse and field trials, the duration of protection of chili fruit from infection provided by pyraclostrobin against C. acutatum was determined. In vitro experiments showed that pyraclostrobin and azoxystrobin were inhibitory to both mycelial growth and spore germination, while mancozeb was only effective in inhibiting spore germination, and difenoconazole and thiophanate-methyl were effective in inhibiting mycelial growth. The results obtained suggest that these fungicides inhibited C. acutatum. It was observed that germ tube elongation and sporulation were reduced by pyraclostrobin. Pyraclostrobin provided protective activity for up to 96 h before inoculation, as well as curative activity up to 72 h after inoculation. In field trials, pyraclostrobin was effective in reducing the incidence and severity of anthracnose, and enhanced the marketable yield of chili. These results demonstrate that pyraclostrobin is a useful fungicide for the management of chili anthracnose.

Sensitivity of Exserohilum turcicum to demethylation inhibitor fungicides

Exserohilum turcicum, the causal agent of northern leaf blight (NLB) of corn (Zea mays), can cause significant yield reductions in Illinois. Metconazole, propiconazole, and prothioconazole are demethylation inhibitor (DMI) fungicides often applied as solo active ingredients or mixed with other fungicide groups to control NLB. Monitoring for shifts in DMI fungicide sensitivity in E. turcicum populations is important for making management decisions and maintaining fungicide efficacy. Sensitivity to metconazole, propiconazole, and prothioconazole were determined for E. turcicum isolates collected prior to DMI use on corn (baseline group collected prior to 1992) and E. turcicum isolates collected between 2009 and 2014. An in vitro mycelial growth assay was used to determine the effective fungicide concentration at which 50% of the fungal growth was inhibited (EC50) for each isolate-fungicide combination. Baseline EC50 value least-square means (lsmeans) for metconazole, propiconazole, and prothioconazole were 0.032, 0.060, and 0.254 μg/mL, respectively. When lsmeans of EC50 values for 2009, 2010, 2011, 2012, 2013, and 2014 E. turcicum isolates were compared to the lsmean of the baseline E. turcicum EC50 values, no significant (P ≤ 0.05) shift towards reduced sensitivity was observed for metconazole, propiconazole, or prothioconazole. Three E. turcicum isolates had EC50 values significantly higher (P ≤ 0.05) than the least sensitive baseline isolate for metconazole, and one isolate had an EC50 value significantly higher (P ≤ 0.05) than the least sensitive baseline isolate for propiconazole. These isolates will require further evaluation to determine if they demonstrate reduced field sensitivity. Isolate prothioconazole EC50 values were positively correlated (P ≤ 0.05) with those of metconazole and propiconazole (r = 0.1690 and r = 0.3269, respectively); however, no correlation was found between metconazole and propiconazole EC50 values. Positive correlations between EC50 values of prothioconazole and the other fungicides suggest the potential for cross-resistance between these DMI fungicides. To date, no loss of NLB control has been observed with the use of metconazole, propiconazole, and prothioconazole in the field.

CHALLENGES IN ASSESSING EFFICACY OF POLYOXIN-D ZINC SALT AGAINST COLLETOTRICHUM SPECIES

Polyoxin-D zinc salt was recently made available for disease control of small fruit pathogens in the United States, but very little is known about its efficacy against diseases other than gray mold. In this study, the inhibitory activity of polyoxin-D zinc salt against Colletotrichum species from peach, the causal agent of peach fruit anthracnose, was assessed based on mycelial growth and germ tube elongation assays on four growth media, and in detached fruit assays. EC50 values between isolates of different Colletotrichum species varied within and between media for both mycelial growth and germ tube elongation tests. Occasionally, variability in sensitivity to polyoxin-D zinc salt was also found between isolates within the same species on the same media. Polyoxin-D zinc salt applied on detached fruit at recommended label rates inhibited mycelial growth of C. nymphaeae (100%), followed by C. fructicola (88.50%), C. siamense (78.97%), C. fioriniae-subgroup 2 (75.47%), C. fioriniaesubgroup 1 (73.55%), and C. truncatum (58.90%). In our assays, polyoxin-D zinc salt was equal or superior in efficacy compared to penthiopyrad. The results indicate that the EC50 values determined for Colletotrichum species is strongly dependent on the medium used and that none of the media accurately reflected observations on detached fruit assays.

Sensitivity of Fusarium asiaticum to a novel succinate dehydrogenase inhibitor fungicide pydiflumetofen

Fusarium head blight (FHB) or scab mainly caused by Fusarium asiaticum is a devastating fungal disease in wheat. In China, carbendazim (MBC) and its co-formulated mixture with other fungicides are the main fungicides for controlling the disease. But resistant populations have developed severely in the field. The fungicides for controlling the disease are limited. So screening novel fungicides for controlling FHB is urgent. The fungicide pydiflumetofen (development code number: SYN545974),3-(Difluoromethyl)N-methyl-N-[1-methyl-2-(2,4,6-trichlorophenyl)ethyl]-1H-pyrazole-4-carboxami.d.e, is a novel succinate dehydrogenase inhibitor (SDHI) that was developed by Syngenta. In this study, the baseline sensitivities for different isolates of F. asiaticum from various regions of China to pydiflumetofen were established. The 50% effective concentration (EC50) value of pydiflumetofen on suppressing mycelial growth against 116 isolates of F. asiaticum was 0.019–0.2084 μg/ml and their average value was 0.0745 ± 0.0367 μg/ml. The EC50 value of pydiflumetofen on suppressing conidium germination was 0.0583–0.4237 μg/ml and their average value was 0.1813 ± 0.0861 μg/ml. The results indicated that the fungicide had strong inhibition effect on mycelial growth and conidium germination against F. asiaticum in vitro. The EC50 values distribution of 116 isolates measured by mycelial growth assay or by conidium germination assay was a unimodal curve and could be used for detecting any sensitivity changes of F. asiaticum populations to pydiflumetofen in the future. The results of cross-resistance analysis indicated that there was no positive or negative cross-resistance between pydiflumetofen and carbendazim/phenamacril. In field trials, though FHB was very severe, pydiflumetofen at 125, 150 and 200 g ai ha−1 all provided more than 80% control efficacy which was significantly higher than that of phenamacril at 375 g ai ha−1 or MBC at 600 g ai ha−1 in 2015 and 2016. In conclusion, pydiflumetofen had very high activity against F. asiaticum in vitro and excellent efficacy for controlling FHB could also manage carbendazim-resistant F. asiaticum populations in the field.

Baseline Sensitivity of Fusarium virguliforme to Fluopyram Fungicide.

Fluopyram, a succinate dehydrogenase inhibitor (SDHI) fungicide, was recently registered for use as a soybean seed treatment for management of sudden death syndrome (SDS) caused by Fusarium virguliforme. Although registered and now used commercially, in vitro baseline fungicide sensitivity of F. virguliforme to fluopyram has not yet been established. In this study, the baseline sensitivity of F. virguliforme to fluopyram was determined using in vitro growth of mycelium and germination of conidia assays with two collections of F. virguliforme isolates. A total of 130 and 75 F. virguliforme isolates were tested using the mycelial growth and conidia germination assays, respectively, including a core set of isolates that were tested with both assays. In the mycelial growth inhibition assay, 113 out of 130 isolates (86.9%) were inhibited 50% by effective concentrations (EC50) less than 5 µg/ml with a mean EC50 of 3.35 µg/ml. For the conidia germination assay, 73 out of 75 isolates (97%) were determined to have an estimated EC50 of less than 5 µg/ml with a mean EC50 value of 2.28 µg/ml. In a subset of 20 common isolates that were phenotyped with both assays, conidia germination of F. virguliforme was determined to be more sensitive to fluopyram (mean EC50 = 2.28 µg/ml) than mycelial growth (mean EC50 = 3.35 µg/ml). Hormetic effects were observed in the mycelial growth inhibition assay as 22% of the isolates demonstrated more growth on medium amended with the lowest fluopyram concentration (1 µg/ml), as compared with the nonfluopyram amended control. It was not possible to determine EC50 values for nine out of 185 isolates (4.8%), as those isolates were not inhibited by 50% even at the highest fluopyram concentrations of 100 µg/ml for mycelial growth and 20 µg/ml for conidia germination inhibition assays. On the whole, the F. virguliforme population appears to be sensitive to fluopyram, and this study enables future monitoring of fungicide sensitivity.

Sensitivity of Rhizoctonia solani AG-2-2 from Sugar Beet to Fungicides.

Rhizoctonia damping-off and crown and root rot caused by Rhizoctonia solani are major diseases of sugar beet (Beta vulgaris L.) worldwide, and growers in the United States rely on fungicides for disease management. Sensitivity of R. solani to fungicides was evaluated in vitro using a mycelial radial growth assay and by evaluating disease severity on R. solani AG 2-2 inoculated plants treated with fungicides in the greenhouse. The mean concentration that caused 50% mycelial growth inhibition (EC50) values for baseline isolates (collected before the fungicides were registered for sugar beet) were 49.7, 97.1, 0.3, 0.2, and 0.9 μg ml-1 and for nonbaseline isolates (collected after registration and use of fungicides) were 296.1, 341.7, 0.9, 0.2, and 0.6 μg ml-1 for azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole, respectively. The mean EC50 values of azoxystrobin, trifloxystrobin, and pyraclostrobin significantly increased in the nonbaseline isolates compared with baseline isolates, with a resistant factor of 6.0, 3.5, and 3.0, respectively. Frequency of isolates with EC50 values >10 μg ml-1 for azoxystrobin and trifloxystrobin increased from 25% in baseline isolates to 80% in nonbaseline isolates. Although sensitivity of nonbaseline isolates of R. solani to quinone outside inhibitors decreased, these fungicides at labeled rates were still effective at controlling the pathogen under greenhouse conditions.

Spatial and Temporal Sensitivity of Alternaria Species Associated With Potato Foliar Diseases to Demethylation Inhibiting and Anilino-Pyrimidine Fungicides.

Early blight and brown spot, caused by Alternaria solani and Alternaria alternata, respectively, are important foliar diseases of potato, affecting both tuber yield and quality. Most of the commercial cultivars lack resistance; therefore, the application of foliar fungicides remains a primary disease management strategy. Baseline sensitivities of A. solani to difenoconazole and metconazole (demethylation inhibitors) using mycelial growth assay exhibited similar intrinsic activity against the pathogen with mean EC50 (the effective concentration at which the fungal growth is inhibited by 50%) values of 0.09 μg/ml. However, the sensitivity of individual baseline A. solani isolates to each fungicide varied substantially, resulting in very low and nonsignificant correlation coefficients among fungicides. Mean EC50 values for baseline A. alternata isolates in response to difenoconazole and metconazole were 0.14 and 0.26 μg/ml, respectively. The sensitivity of the majority of A. solani and A. alternata isolates collected from 2010 to 2014 from various potato production states was consistent with baseline isolates, therefore, these potato pathogens remain sensitive to the two demethylation inhibitor chemistries used to manage it. Baseline sensitivity assays of pyrimethanil (anilino-pyrimidine) also indicated great intrinsic activity against both foliar pathogens with mean EC50 values of 0.44 and 0.35 μg/ml for A. solani and A. alternata, respectively. Although A. alternata remains largely sensitive to pyrimethanil, 6 out of 245 A. solani isolates collected from 2010 to 2014 exhibited reduced-sensitivity to the fungicide in in vitro assays. Reduced-sensitive isolates were not controlled at most pyrimethanil doses except at 100 μg/ml in greenhouse in vivo efficacy tests. These chemistries remain valuable options for fungicide rotation programs in areas of high disease pressure.

Sensitivity and cross-resistance patterns to demethylation-inhibiting fungicides in California populations of Alternaria alternata pathogenic on pistachio

Alternaria late blight (ALB) caused by Alternaria spp. is an annual production concern for pistachio growers in California. In this study, isolates of Alternaria alternata collected from California pistachio orchards between 1998 and 2003 (population A, n = 46) and in 2010 (population B, n = 38) prior to the registration and use of sterol demethylation-inhibiting (DMI) fungicides for ALB control, were tested for their sensitivity to difenoconazole, propiconazole, and tebuconazole using an in vitro mycelial growth assay. The 50% effective dose (EC50) values for isolates in population A to difenoconazole, propiconazole, and tebuconazole, respectively, ranged from 0.02 to 0.82 μg/mL (mean = 0.17), 0.25–5.73 μg/mL (mean = 1.29), and 0.02–2.76 μg/mL (mean = 0.96), and from 0.02 to 0.93 μg/mL (mean = 0.21), 0.13–5.36 μg/mL (mean = 1.25), and 0.17–5.47 μg/mL (mean = 1.03) for those in population B. Among 65 isolates collected in 2012 from orchards with very limited exposure to difenoconazole, propiconazole or metconazole (population C, n = 65), the EC50 values for tebuconazole ranged from 0.02 to 2.99 μg/mL (mean = 0.51), while EC50 values for difenoconazole of 55 of these isolates varied from 0.01 to 0.44 μg/mL (mean = 0.06). The DMI-exposed population C was found to be on average two to four times less sensitive compared with the populations A and B. Pearson correlation analyses of EC50 values for the three fungicides showed significant positive correlations between the sensitivities of tebuconazole and propiconazole (P < 0.01, r = 0.32), tebuconazole and difenoconazole (P < 0.01, r = 0.26), and difenoconazole and propiconazole (P < 0.01, r = 0.40). Results indicate a clear difference in fungicide intrinsic activity, with difenoconazole being more active than the two other DMIs. Although wide variations in sensitivities for tebuconazole and propiconazole were observed in the non- and DMI-exposed Alternaria populations, efficacy of DMI products has been excellent against ALB. The data collected here will be crucial for the rational use of DMIs in fungicide spray programs for ALB management and serve as a reference to detect any shifts in A. alternata sensitivity to these fungicides in subsequent years as they become more frequently used in California pistachios.

Imidazolium salts with antifungal potential for the control of head blight of wheat caused by Fusarium graminearum.

Evaluate the invitro effect of imidazolium salts (IMS) on the conidia germination and mycelial growth of Fusarium graminearum and their invivo efficacy for suppressing the symptoms of the disease and infection of kernels in wheat plants. The minimum inhibitory concentrations (MIC) of three IMS (C16 MImCl, C16 MImMeS and C16 MImNTf2 ) were determined for four F.graminearum isolates using serial broth dilution method. The MICs found for all IMS were either 3·12 or 6·25μgml(-1) across the isolates, with the former as the most frequent. In the mycelial growth assay on potato dextrose agar media, only the C16 MImCl among the IMS reduced 50% of mycelial growth of one isolate at an estimated concentration of 0·32mgml(-1) . The time-kill curves showed a strong fungicidal effect starting 1h after incubation at a concentration of 12·5μgml(-1) , representing a fourfold increase in the most frequent MIC. The C16 MImCl sprayed onto the spikes of potted wheat plants during the flowering stage reduced disease intensity at levels comparable to the commercial fungicide when applied preventatively (1h prior to fungal inoculation), rather than curatively, and at the higher dosage (2mgml(-1) ) rather than lower dosage (0·5mgml(-1) ). C16 MImCl proved to be a potent inhibitor of F.graminearum growth and provided good levels of control of the disease at levels comparable to a commercial fungicide, in wheat plants treated prior to fungal infection during flowering stages. This study suggests the potential of using IMS as alternative to the hazardous standard fungicides in the management of Fusarium head blight of wheat.

Baseline sensitivity of natural population and resistance risk of Peronophythora litchii to four novel QoI fungicides

Sixty-eight field isolates of Peronophythora litchii, the causal agent of litchi downy blight, were examined for sensitivity to four novel QoI fungicides (SYP-1620, SYP-2815, ZJ0712 and enestroburin) in vitro with a mycelial growth assay. Ranges and means of EC50 values (in μg/mL) were 0.047–0.288 and 0.138 for SYP-2815, 0.056–0.992 and 0.148 for ZJ0712, 0.430–7.529 and 2.720 for SYP-1620, and 0.180–1.772 and 0.810 for enestroburin; the distributions were approximately unimodal and skewed. Eight mutants resistant to the three novel QoI fungicides and azoxystrobin (as a known QoI fungicide) were obtained by exposing field isolates to fungicide-amended agar and to ultraviolet irradiation, while mutants resistant to enestroburin were not obtained. In laboratory assays, all eight mutants had a high and stable resistance, and most were as fit or more fit than sensitive isolates. There was cross-resistance among the five QoI fungicides. The resistance risk of P. litchii to the four novel QoI fungicides was inconsistent, and the probability that P. litchii will develop resistance to SYP-2815, ZJ0712, and SYP-1620 may be high if these fungicides are frequently applied in the field.

Chemical characterization and antifungal activities of four Thymus species essential oils against postharvest fungal pathogens of citrus

The aim of this study was to find an alternative to synthetic fungicides currently used in the control of postharvest fungal diseases of citrus. Antifungal activities of essential oils from four Thymus species were investigated against Penicillium digitatum, Penicillium italicum and Geotrichum citri-aurantii. The essential oils, obtained by hydrodistillation from aerial parts of four Thymus species were submitted to gas chromatography hyphenated with mass spectrometry analysis. The main constituents were carvacrol (76.94%) for Thymus leptobotrys, borneol (27.71%) and thymol (18.47%) for Thymus satureioides subsp. pseudomastichina, camphor (46.17%) and α-terpineol (7.69%) for Thymus broussonnetii subsp. hannonis and carvacrol (32.24%), γ-terpinene (19.60%) and p-cymene (13.52%) for Thymus riatarum. In the in vitro mycelial growth assay, complete inhibition of the three pathogens was obtained by T. leptobotrys and T. riatarum essential oils, at 1000μlL−1. The effect of essential oils on spore germination varied significantly between tested Thymus species. T. leptobotrys essential oil displayed the highest bioactivity, inhibiting completely the spore germination of G. citri-aurantii at 250μlL−1 and of P. digitatum and P. italicum at 500μlL−1. T. riatarum essential oil was able to inhibit completely the spore germination of G. citri-aurantii (2000μlL−1) and both Penicillium species used as target organisms (1000μlL−1). The essential oil of T. leptobotrys had the lowest minimum inhibitory concentration (MIC) for the three pathogens (MIC value<500μlL−1). These findings suggest that essential oils obtained from T. leptobotrys, T. riatarum and T. satureioides subsp. pseudomastichina may be useful and effective agents for control of citrus fungal pathogens.

Determination of Fungicide Resistance in Botrytis cinerea from Strawberry in the Central Coast Region of California

Grey mold, caused by Botryis cinerea, is one of the most important diseases of strawberry in California. Management of grey mold typically relies on repeated fungicide applications. The occurrence of fungicide resistance in B. cinerea was examined in the Central Coast strawberry production region of California. In mid-May 2013, 59 samples consisting of a single diseased fruit or plant part with gray mold symptoms were collected from six different strawberry fields. Single hyphal tip cultures were then used for mycelial growth assays to compare sensitivities to four different fungicides—boscalid, fenhexamid, iprodione, and pyraclostrobin. Each isolate was tested against discriminatory doses of each of the fungicides. In addition, representative highly sensitive and highly resistant isolates were tested against a range of fungicide concentrations to determine EC50 values. Although all of the 59 isolates were sensitive to iprodione, 37%, 31%, and 29% of the isolates were resistant to pyraclostrobin, fenhexamid, and boscalid, respectively. In some instances the isolates were dual and triple-resistant to these fungicides. EC50 values were often higher than 100 mg/liter, which was the highest concentration used. Therefore, appropriate fungicide resistance management measures should be employed in strawberry growing areas of the Central Coast region of California. Accepted for publication 19 February 2016. Published 2 March 2016.

Occurrence of Fludioxonil Resistance in Penicillium digitatum from Citrus in California

Penicillium digitatum is the causal agent of green mold, one of the most important postharvest diseases of citrus (Citrus spp.). Fludioxonil can be used alone or in combination with azoxystrobin for the control of green mold and other postharvest diseases on citrus. Baseline sensitivity to fludioxonil in P. digitatum populations from California citrus packinghouses has been previously established (Kanetis et al. 2008). To monitor resistance to fludioxonil in P. digitatum, 20 Penicillium spp. isolates were obtained from decayed oranges by isolation from decayed tissue and 22 isolates were recovered from potato dextrose agar plates amended with 0.5 mg/liter fludioxonil (Kanetis et al. 2006) and exposed to packinghouse air for 5 min each sampling time during March to July 2013 in two California citrus packinghouses. All isolates were single-spore cultured and identified as P. digitatum based on morphological characters (Pitt 1979). The sequence analysis of the internal transcribed spacer (ITS) region, using the primers ITS1/ITS4, was conducted to confirm the species-level identification. A MegaBLAST search showed that the sequences of all isolates had 99% homology (E-value = 0.0) with that of P. digitatum deposited at GenBank (Accession No. AF033471.1). In a mycelial growth assay, 15 of the 42 P. digitatum isolates were able to grow at 0.5 μg/ml, a previously established discriminatory concentration (Kanetic et al. 2006). EC50 values (the effective concentration that inhibits fungal growth by 50% relative to the control) of fludioxonil for the resistant isolates were >100 mg/liter following a method described by Li and Xiao (2008). To assess whether fludioxonil at the label rate was able to control fludioxonilresistant isolates, ‘Eureka’ lemons were wounded with a stainless steel rod (1 × 2 mm) and inoculated with 10 μl of conidial suspensions (6 × 10 conidia/ml) of a representative sensitive or a resistant isolate. After 4 h, inoculated fruit were dipped for 30 s in either a formulated product of fludioxonil, Graduate (50% active ingredient; Syngenta Crop Protection, Research Triangle Park, Raleigh, NC) at 1,198 mg/liter, or water as a control, and then stored at 20°C in air for 7 days. There were four 20-fruit replicates for each treatment and the experiment was performed twice. All inoculated water-dipped fruit were decayed, while fludioxonil completely controlled green mold on fruit inoculated with the fludioxonil-sensitive isolate. However, 100% fruit inoculated with the fludioxonil-resistant isolate and treated with fludioxonil were decayed. Fludioxonil-insensitive isolates have been reported to occur in natural populations of P. digitatum before its commercial use, but at very low frequency (1.4 to 2.5 × 10) (Kanetis et al. 2006). This is the first report of fludioxonil resistance in P. digitatum collected from commercial citrus packinghouses after the introduction of the fungicide on the market. These fludioxonil-resistant isolates were obtained from packinghouses where the fungicide had been used during citrus packing for 2 consecutive years, approximately for a 3-month period in each year, indicating that fludioxonil-resistant individuals emerged quickly in P. digitatum populations. Our results show that fludioxonil failed to control green mold incited by the fludioxonil-resistant isolate, suggesting that appropriate fungicide resistance management practices need to be implemented to ensure adequate control.

Detection of isolates of Alternaria alternata with multiple-resistance to fludioxonil, cyprodinil, boscalid and pyraclostrobin in California pistachio orchards

In this study, 229 isolates of Alternaria alternata collected from commercial pistachio orchards with (n = 198) and without (n = 31) a prior history of Switch (cyprodinil + fludioxonil) applications were examined for their sensitivity to fludioxonil and cyprodinil using in vitro mycelial growth assays. The 50% effective dose (EC50) values for the baseline isolates for cyprodinil and fludioxonil ranged from 0.001 to 1.184 μg/mL (average = 0.214) and from 0.010 to 4.875 μg/mL (average = 0.770), respectively. Out of 72 isolates assayed from the exposed population, 93% had EC50 values for fludioxonil ranging from 0.001 to 5.370 μg/mL (average = 0.472), but five isolates were highly resistant to this fungicide (EC50 > 100 μg/mL). About 98.55% of these isolates were sensitive to cyprodinil (0009 < EC50 < 0.415 μg/mL; average = 0.098). Only one isolate was resistant to cyprodinil (EC50 > 10 μg/mL). No cross-resistance relationship between cyprodinil and fludioxonil was observed (P = 0.2925, r = −0.1074). To determine the occurrence of multiple resistances, the radial growths of 126 isolates, collected from orchards with a history of Switch, Pristine, and strobilurin exposures, were measured on agar media amended with a single discriminatory dose of 10 μg fungicide/mL. High proportions of these isolates were resistant to boscalid (86.3%) and pyraclostrobin (92.9%). Most isolates were still sensitive to fludioxonil (77.8%) and cyprodinil (89.7%), but some were found to be independently or simultaneously resistant to all fungicides tested. Under conditions of excessive exposure to Switch resistant phenotypes may develop in A. alternata populations and ultimately affect fungicide performance. Therefore, monitoring studies must be continued and strict adherence to anti-resistance strategies followed and implemented in at risk orchards.

Sensitivity of Ascochyta Species Infecting Pea, Lentil, and Chickpea to Boscalid, Fluxapyroxad, and Prothioconazole.

Management of Ascochyta blight in pea, lentil, and chickpea relies on repeated fungicide applications, which has led to development of fungicide resistance and disease control failures in some systems. In vitro assays were conducted to determine baseline fungicide sensitivity in Mycosphaerella pinodes (Ascochyta pinodes), A. lentis, and A. rabiei populations to the demethylation-inhibiting fungicide prothioconazole and the succinate dehydrogenase-inhibiting fungicides boscalid and fluxapyroxad by determining the effective concentration at which 50% of germination or fungal growth was inhibited (EC50). Mean boscalid EC50 values from conidial germination assays were 0.669, 0.639, and 0.171 μg/ml and from mycelial growth assays were 0.258, 0.791, and 0.443 μg/ml for M. pinodes, A. lentis, and A. rabiei, respectively. Mean fluxapyroxad EC50 values were 0.050, 0.763, and 0.057 μg/ml for M. pinodes, A. lentis, and A. rabiei, respectively. Mean baseline EC50 values for prothioconazole with mycelial growth were 0.541, 0.604, and 0.283 μg/ml for M. pinodes, A. lentis, and A. rabiei, respectively. A single discriminatory fungicide concentration of 1 μg/ml was selected for all species. Established sensitivity profiles and discriminatory concentrations will be used to monitor sensitivity shifts in populations of Ascochyta spp. and to make effective disease management recommendations.

Aspergilli Response to Benzalkonium Chloride and Novel-Synthesized Fullerenol/Benzalkonium Chloride Nanocomposite.

A comprehensive comparative analysis of antifungal potential of benzalkonium chloride and newly synthesized fullerenol/benzalkonium chloride nanocomposite was conducted to assess the possible impact of carbon-based nanocarrier on antimicrobial properties of the commonly used biocide. Physical characterization of synthesized nanocomposite showed zeta potential of +37.4 mV and inhomogeneous particles size distribution, with nanocomposite particles' dimensions within 30–143 nm and maximum number of particles at 44 nm. The effect of pure and fullerenol nanocarrier-bound biocide was evaluated in eight Aspergillus species. In mycelial growth assay, nanocomposite was more potent, as fungicidal effect of 1.04/0.6 μg mL−1 was obtained in all but one of the isolates (A. niger), while proportional concentration of pure biocide (0.6 μg mL−1) completely inhibited mycelial growth of only three Aspergillus species. However, conidia appear to be less susceptible to nanocomposite treatment, as lower fungistatic (MIC) and fungicidal (MFC) concentrations were obtained with biocide alone (MIC in range from 0.03 to 0.15 μg mL−1 and MFC from 0.075 to 0.45 μg mL−1). To a different degree, both substances stimulated aflatoxin B1 production and inhibited ochratoxin A synthesis. Very low mycelium biomass yield, in range from 1.0 to 3.0 mg dry weight, was documented in both biocide and nanocomposite enriched medium.

Multiple resistance to QoIs and other classes of fungicides in Botrytis cinerea populations from strawberry in Zhejiang Province, China

Gray mold caused by Botrytis cinerea is a major fungal disease of strawberry in Zhejiang Province, which is considered as one of the most important strawberry-producing areas in China. Recently, effective management of this disease has become an urgent need for the farmers as a result of the failure of many fungicides in controlling this pathogen. A total of 486 B. cinerea isolates were collected from strawberry in nine locations of Zhejiang Province and screened for resistance to azoxystrobin, carbendazim, iprodione, pyrimethanil and fludioxonil. Among the 486 isolates, 353 (72.6 %) showed resistance to single or multiple fungicides, 254 (52.3 %) were simultaneously resistant to azoxystrobin, carbendazim, iprodione and pyrimethanil, but no fludioxonil-resistant (FluR) isolates were found, indicating that fludioxonil has great potential for control of gray mold on strawberry. In mycelial growth assays, we determined the EC50 values for 47 azoxystrobin-sensitive (AzoS), 41 azoxystrobin-resistant (AzoR), 48 pyrimethanil-sensitive (PyrS), 44 pyrimethanil-resistant (PyrR) and 100 fludioxonil-sensitive (FluS) isolates. The resistance factors (RF) of AzoR and PyrR isolates ranged from 21.7 to 4,359.0 and from 53.0 to 320.1, respectively. The EC50 values for FluS isolates ranged from 0.003 to 0.04 (mean = 0.01) μg/ml. All 322 AzoR isolates harboured the G143A point mutation. B. cinerea populations were divided into two types according to the structure of the cyt b gene, with (12.2 %) or without (87.8 %) the third intron Bcbi-143/144. The high frequency of isolates without the Bcbi-143/144 intron indicates that B. cinerea populations from strawberry have a high inherent risk for the development of resistance to QoIs.