Mycelial Growth Inhibition Assay Research Articles

Multistate Sensitivity Monitoring of Fusarium virguliforme to the SDHI Fungicides Fluopyram and Pydiflumetofen in the United States.

Sudden death syndrome (SDS), caused by Fusarium virguliforme, is an important yield-limiting disease of soybean (Glycine max). From 1996 to 2022, cumulative yield losses attributed to SDS in North America totaled over 25 million metric tons, which was valued at over US $7.8 billion. Seed treatments are widely used to manage SDS by reducing early season soybean root infection by F. virguliforme. Fluopyram (succinate dehydrogenase inhibitor [SDHI] - FRAC 7), a fungicide seed treatment for SDS management, has been registered for use on soybean in the United States since 2014. A baseline sensitivity study conducted in 2014 evaluated 130 F. virguliforme isolates collected from five states to fluopyram in a mycelial growth inhibition assay and reported a mean EC50 of 3.35 mg/liter. This baseline study provided the foundation for the objectives of this research: to detect any statistically significant change in fluopyram sensitivity over time and geographical regions within the United States and to investigate sensitivity to the fungicide pydiflumetofen. We repeated fluopyram sensitivity testing on a panel of 80 historical F. virguliforme isolates collected from 2006 to 2013 (76 of which were used in the baseline study) and conducted testing on 123 contemporary isolates collected from 2016 to 2022 from 11 states. This study estimated a mean absolute EC50 of 3.95 mg/liter in isolates collected from 2006 to 2013 and a mean absolute EC50 of 4.19 mg/liter in those collected in 2016 to 2022. There was no significant change in fluopyram sensitivity (P = 0.1) identified between the historical and contemporary isolates. A subset of 23 isolates, tested against pydiflumetofen under the same conditions, estimated an absolute mean EC50 of 0.11 mg/liter. Moderate correlation was detected between fluopyram and pydiflumetofen sensitivity estimates (R = 0.53; P < 0.001). These findings enable future fluopyram and pydiflumetofen resistance monitoring and inform current soybean SDS management strategies in a regional and national context.

Antifungal and Antiaflatoxigenic Activities of Massoia Essential Oil and C10 Massoia Lactone against Aflatoxin-Producing Aspergillus flavus.

Fungal infection and mycotoxin contamination are major hazards to the safe storage and distribution of foods and feeds consumed by humans and livestock. This study investigated the antifungal and antiaflatoxigenic activities of massoia essential oil (MEO) and its major constituent, C10 massoia lactone (C10), against aflatoxin B (AFB)-producing Aspergillus flavus ATCC 22546. Their antifungal activities were evaluated using a disc diffusion assay, agar dilution method, and a mycelial growth inhibition assay with the AFB analysis using liquid chromatography triple quadrupole mass spectrometry. MEO and C10 exhibited similar antifungal and antiaflatoxigenic activities against A. flavus. C10 was a primary constituent in MEO and represented up to 45.1% of total peak areas analyzed by gas chromatography-mass spectrometry, indicating that C10 is a major compound contributing to the antifungal and antiaflatoxigenic activities of MEO. Interestingly, these two materials increased AFB production in A. flavus by upregulating the expression of most genes related to AFB biosynthesis by 3- to 60-fold. Overall, MEO and C10 could be suitable candidates as natural preservatives to control fungal infection and mycotoxin contamination in foods and feeds as Generally Recognized As Safe (GRAS) in the Flavor and Extract Manufacturers Association of the United States (FEMA), and MEO is a more suitable substance than C10 because of its wider range of uses and higher allowed concentration than C10.

Natural product osthole can significantly disrupt cell wall integrity and dynamic balance of Fusarium oxysporum

Dendrobium officinale Kimura et Migo is a traditional Chinese herbal medicinal plant. However, the frequent occurrence of soft rot disease (SRD) is one of the most harmful diseases in D. officinale production in recent years, which can seriously affect its yield and quality. In this study, the major pathogenic fungus (SR-1) was isolated from D. officinale with typical symptoms of SRD, and was identified as Fusarium oxysporum through morphological and molecular identification. The biological activities of five natural products were determined against F. oxysporum using a mycelial growth inhibition assay. The results showed that osthole had the highest antifungal activity against F. oxysporum, with an EC50 value of 6.40 mg/L. Scanning electron microscopy (SEM) showed that osthole caused F. oxysporum mycelia to shrink and deform. Transmission electron microscopy (TEM) showed that the organelles were blurred and the cell wall was thickened in the presence of osthole. The sensitivity of F. oxysporum to calcofluor white (CFW) staining was significantly enhanced by osthole. Relative conductivity measurements and propidium iodide (PI) observation revealed that osthole had no significant effect on the cell membrane. Further experiments showed that the activity of chitinase and β-1,3-glucanase were decreased, and expression levels of chitinase and β-1,3-glucanase related genes were significantly down-regulated after treatment with osthole. In conclusion, osthole disrupted the cell wall integrity and dynamic balance of F. oxysporum, thereby inhibiting normal mycelial growth.

Evidence of Phosphite Tolerance in Phytophthora cinnamomi from New Zealand Avocado Orchards.

There is a limited number of chemical control agents for managing Phytophthora root and collar rot diseases of avocado internationally; of these, phosphite is one of the most effective. To determine whether prolonged phosphite use in New Zealand avocado orchards has led to decreased sensitivity of Phytophthora cinnamomi to phosphite, 57 isolates were collected from phosphite-treated and -untreated avocado orchards and screened for tolerance using a mycelial growth inhibition assay. The inhibitory effect of phosphite on mycelial growth was tested in vitro using six concentrations of phosphite. Based on changes in mycelial growth using optical density measurements to calculate the effective concentration to reduce growth by 50% (EC50) estimates, three phosphite-susceptible (EC50 range = 18.71 to 29.26 µg/ml) and three tolerant (EC50 range = 81.85 to 123.89 µg/ml) isolates were selected. The effects of phosphite on the colonization of lupin (Lupinus angustifolius) seedling roots and sporangia and zoospore production of three susceptible and three tolerant isolates were determined. The three tolerant isolates colonized lupin roots more extensively than the three susceptible isolates in the presence of phosphite at 5 and 10 g/liter. The tolerant isolates were able to asymptomatically colonize further above the lesion margin in the lupin treated with phosphite at 5 g/liter relative to the phosphite-susceptible isolates but no isolates were completely resistant to phosphite. The tolerant isolates produced more sporangia and, consequently, zoospores in the presence of phosphite than the susceptible isolates. The detection of phosphite tolerance by P. cinnamomi in planta and in vivo is concerning for the future efficacy of phosphite to manage Phytophthora diseases.

First Report of Resistance to Pyraclostrobin in Pseudocercospora spp. from Blackberry (Rubus fruticosus) in Georgia, United States

Pseudocercospora leaf spot is frequently observed in blackberry production in the southeastern United States. To manage the impacts of leaf spot diseases, blackberry growers in the southeastern United States typically rely on the application of single-site fungicides, including the quinone outside inhibitors (QoIs) (FRAC 11) pyraclostrobin and azoxystrobin. In recent years, despite the regular application of chemical fungicides to manage leaf spots, blackberry growers and extension personnel in Georgia have noticed relatively poor control of these diseases. To investigate potential control failures due to fungicide resistance development, Pseudocercospora were isolated from symptomatic leaves collected from blackberry ( Rubus fruticosus) in five commercial fields in southeastern Georgia. Using a mycelial growth inhibition assay, isolates were screened for resistance to pyraclostrobin. Among the 13 isolates tested, 12 were determined to be resistant to the fungicide. Subsequent sequencing of the cytochrome b gene indicated the presence of the G143A mutation in all fungicide-resistant isolates. The identification of fungicide resistance in 12 of 13 isolates and 4 of 5 examined commercial blackberry sites in Georgia suggests that resistance to this fungicide may be prevalent among Pseudocercospora on blackberry in Georgia and that future fungicide resistance monitoring efforts in Georgia blackberry plantings may be warranted. This represents the first report of QoI fungicide resistance in Pseudocercospora spp. from blackberry.

First Report of In Vitro Insensitivity to Fluazinam of Clarireedia jacksonii Causing Dollar Spot of Turfgrass on New England Golf Courses

Dollar spot, caused by the newly renamed ascomycete fungus Clarireedia spp., contributes to the aesthetic and functional damage and ultimate death of turfgrass. Fluazinam, a protective fungicide belonging to the 2,6-dinitroaniline chemical group, has proven to be one of the most efficacious fungicide classes of managing a variety of turfgrass diseases, including dollar spot, due to its low risk for fungicide resistance as a broad-spectrum fungicide. Dollar spot isolates collected from six New England golf courses with suspected resistance to multiple fungicide classes had shown reduced sensitivity to fluazinam using a mycelial growth inhibition assay. This is the first report of reduced sensitivity to fluazinam in dollar spot field isolates in the United States. Briefly, in vitro sensitivity assays of 22 isolates collected from 12 golf courses and UMass turf research facilities showed that reduced fluazinam sensitivity of isolates was detected on six golf courses since 2017, demonstrating that fungi with higher exposure to fluazinam tend to develop reduced sensitivity over time. To date, there is no reported field resistance of fluazinam in C. jacksonii. However, the findings highlight the need for ongoing monitoring of in vitro and field sensitivity to fluazinam in C. jacksonii, as well as emphasizing judicious use of fluazinam in managing dollar spot disease to delay the development of fungicide resistance.

Functional enhancement of hydroxypropyl cellulose‐based bionanocomposite films incorporating chitosan nanoparticles

SummaryThe aims of the present study were to investigate the effect of the incorporation of chitosan nanoparticles (ChiNPs) on the physicochemical properties and antifungal activity of hydroxypropyl cellulose (HPC) bionanocomposite films. The films of 4% HPC and ChiNP at various dosages (0%, 0.1%, 0.5%, 1%, 1.5%, 3% w/w of HPC) were prepared. Atomic force microscopy (AFM) verified the dispersion of spherical‐shaped ChiNP in a nanoscale size range (14.75–56.74 nm). Incorporation of nanofiller affected the functional properties by increasing the film–water contact angle to &gt;53°, improving the optical properties, and reducing water vapour permeability by 5.73–12.54%. These results were influenced by surface roughness and microstructure characteristics of a bionanocomposite film, which were confirmed using AFM and scanning electron microscopy (SEM). Tensile assays of the films showed slight increases in tensile strength from 8.87 to 11.08 MPa and Young's modulus from 0.10 to 0.15 GPa with increasing proportions of ChiNP (1.5%). An effective concentration of ChiNP of 1.5% (w/w of HPC) was able to improve the performance of HPC and offered antifungal activity against Botrytis cinerea, as confirmed through mycelial growth inhibition assays and by membrane integrity changes. The HPC/ChiNP bionanocomposite films may have potential applications for active packaging, particularly for fresh fruit commodities.

Antifungal Activity against Botryosphaeriaceae Fungi of the Hydro-Methanolic Extract of Silybum marianum Capitula Conjugated with Stevioside.

Silybum marianum (L.) Gaertn, viz. milk thistle, has been the focus of research efforts in the past few years, albeit almost exclusively restricted to the medicinal properties of its fruits (achenes). Given that other milk thistle plant organs and tissues have been scarcely investigated for the presence of bioactive compounds, in this study, we present a phytochemical analysis of the extracts of S. marianum capitula during the flowering phenological stage (stage 67). Gas chromatography–mass spectroscopy results evidenced the presence of high contents of coniferyl alcohol (47.4%), and secondarily of ferulic acid ester, opening a new valorization strategy of this plant based on the former high-added-value component. Moreover, the application of the hydro-methanolic extracts as an antifungal agent has been also explored. Specifically, their activity against three fungal species responsible for the so-called Botryosphaeria dieback of grapevine (Neofusicoccum parvum, Dothiorella viticola and Diplodia seriata) has been assayed both in vitro and in vivo. From the mycelial growth inhibition assays, the best results (EC90 values of 303, 366, and 355 μg·mL−1 for N. parvum, D. viticola, and D. seriata, respectively) were not obtained for the hydroalcoholic extract alone, but after its conjugation with stevioside, which resulted in a strong synergistic behavior. Greenhouse experiments confirmed the efficacy of the conjugated complexes, pointing to the potential of the combination of milk thistle extracts with stevioside as a promising plant protection product in organic Viticulture.

Synergistic Action of Commercially Available Fungicides for Protecting Wheat from Common Root Rot Caused by Bipolaris sorokiniana in China.

Wheat (Triticum aestivum) common root rot (CRR) caused by predominant fungal pathogen Bipolaris sorokiniana occurs in all wheat-growing regions worldwide and is difficult to control. In this study, the efficacy of eight fungicides against Bipolaris sorokiniana was examined in in vitro assays, and we determined that the combined application of two fungicides significantly inhibits the growth of fungal mycelium. Half of the maximal effective concentration of a mixture containing fludioxonil and difenoconazole in the ratio 1:4 was 0.0372 mg/liter, and the cotoxicity coefficient was 160.14. Under an environmentally controlled pot assay, seed treatment with the mixture of fludioxonil and difenoconazole in the 1:4 ratio demonstrated the best control efficiency at seedling and adult stages, respectively. The best synergistic mixture on seed treatment was assessed in a 2-year field experiment at Hebei, China. The best control efficacy achieved at the seedling and adult stages was 82.65% and 68.48%, respectively. Overall, the in vitro mycelial growth inhibition assay and controlled-environment and field studies indicated that the synergistic action of a mixture of fludioxonil and difenoconazole provides effective control against wheat CRR. These findings highlight the potential application of the fungicide combination for controlling CRR and reducing the selection pressure on fungal pathogens by lessening the use of various fungicides in the field.

Sensitivity of Colletotrichum Isolates Collected from Strawberries in Georgia to Pyraclostrobin, a Quinone Outside Inhibitor (QoI) Fungicide

Anthracnose fruit rot disease, caused by Colletotrichum spp., is the most significant disease problem of commercial strawberry (Fragaria × ananassa) production in the southeastern United States. The hot, humid weather and continuous rainfall in Georgia make Colletotrichum-induced fruit rot a widespread problem in strawberry production. In order to control this disease, growers mainly rely on preventive fungicide applications from flower bud emergence to harvest. The most commonly used single-site fungicides are quinone outside inhibitors (QoIs); the QoI active ingredients azoxystrobin and pyraclostrobin are utilized to manage anthracnose fruit rot. In 2019, we collected 108 strawberry fruits with visible rot symptoms from seven different strawberry farms in Georgia. These farms had received multiple applications of QoI fungicides during the 2019 growing season, as well as in previous seasons. Sensitivities to pyraclostrobin were assessed on 1% malt extract agar using a mycelial growth inhibition assay. Our results demonstrated that a majority of Colletotrichum isolates collected in 2019 were not inhibited by pyraclostrobin, suggesting a growing resistance issue with the QoI fungicides. A PCR-restriction fragment length polymorphism assay showed the presence of the G143A mutation in all QoI “resistant” C. acutatum isolates and none for isolates labeled “reduced sensitivity” or “sensitive”. These results further prove that C. acutatum isolates with the G143A mutation are highly resistant to the QoI fungicide. These findings suggest that there is a high risk of resistance development associated with using pyraclostrobin (likely all QoIs) for controlling anthracnose fruit rot of strawberry in Georgia.

Evaluating the Sensitivities and Efficacies of Fungicides with Different Modes of Action Against Phomopsis asparagi.

Asparagus stem blight caused by Phomopsis asparagi is a major hindrance to asparagus production worldwide. Currently, fungicides are used to manage the disease in commercial production, but resistance to common fungicides has emerged in the wild population. In the present study, 132 isolates of P. asparagi collected from different provinces in China were tested for sensitivities to pyraclostrobin, tebuconazole, and fluazinam. We also determined the efficacies of six fungicides against P. asparagi. The frequency distributions of EC50 values of the isolates tested were unimodal, but the curves for pyraclostrobin and tebuconazole had long right-hand tails. The mean EC50 values for pyraclostrobin, tebuconazole, and fluazinam were 0.0426 ± 0.0029, 0.6041 ± 0.0416, and 0.0314 ± 0.0013 μg/ml, respectively. In addition, the EC50 values for pyraclostrobin were very similar with or without salicylhydroxamic acid (SHAM), 20 μg/ml, indicating that SHAM is not needed to determine the sensitivity of P. asparagi to pyraclostrobin when using the mycelial growth inhibition assay. In greenhouse assays, Merivon (42.4% fluxapyroxad plus pyraclostrobin SC), Frown-cide (500 g/liter fluazinam SC), Cabrio (250 g/liter pyraclostrobin EC), and Nativo (75% trifloxystrobin plus tebuconazole WG) showed excellent preventive efficacy against P. asparagi. And these fungicides were more effective before inoculation than when they were applied after inoculation (P < 0.05). Therefore, these fungicides should be applied prior to infection to control stem blight. In field trials, Frown-cide, Merivon, Nativo, and Cabrio also performed good control effects, ranging from 75.2 to 86.0% in 2017 and 75.4 to 87.1% in 2018. We demonstrated that Frown-cide, Merivon, Nativo, and Cabrio had considerable potential to manage asparagus stem blight. In addition, rotations of these fungicides are essential for precluding or delaying the development of resistance and for controlling the disease.

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.

Impact of epoxiconazole on Fusarium head blight control, grain yield and deoxynivalenol accumulation in wheat

Fusarium head blight (FHB) is a destructive disease of small grain cereals with Fusarium graminearum as one of the most important causal agents. FHB not only can reduce yield and quality of grains, but also lead to accumulation of mycotoxins in grain, thereby threatening human and animal health. In this study, we observed that epoxiconazole exhibits strong inhibitory effects on both carbendazim-resistant and phenamacril-resistant isolates using mycelial growth inhibition assays. The artificially inoculated field trials further showed that epoxiconazole increased the control efficacy of FHB by being able to control carbendazim-resistant and phenamacril-resistant isolates. Epoxiconazole triggered DON production and Tri5 expression in vitro. However, in addition to increased FHB control efficacy and grain yield, decreased DON levels were measured in field trials after epoxiconazole applications. FHB control, grain yields and DON levels were significantly correlated with each other, suggesting that the visual disease rating can be used as an indicator of grain yields and mycotoxin contamination. Meanwhile, the frequency of carbendazim-resistant alleles in F. graminearum populations was dramatically reduced after epoxiconazole applications. In addition, epoxiconazole seed treatments had no effect on seed germination but phytotoxicity was apparent through growth inhibition of wheat seedlings. Overall, these findings of this study provide useful information for wheat protection programs against toxigenic fungi responsible for FHB and the consequent mycotoxin accumulation in grains.

Sensitivity of Phacidiopycnis spp. Isolates from Pome Fruit to Six Pre- and Postharvest Fungicides.

Phacidiopycnis washingtonensis and P. pyri cause speck rot and Phacidiopycnis rot on apple and pear, respectively. Infection occurs in the orchard and remains latent, and symptoms appear after months of storage. Decay management relies on orchard sanitation and pre- and postharvest fungicides. In a 2017 survey, speck rot accounted for 6.4% of apple decay in central Washington, whereas Phacidiopycnis rot accounted for 3.9 and 6.7% of total pear decay in Washington and Oregon, respectively. Sensitivities of baseline populations of 110 P. washingtonensis and 76 P. pyri isolates collected between 2003 and 2005 to preharvest fungicides pyraclostrobin (PYRA) and boscalid (BOSC) and to postharvest fungicides thiabendazole (TBZ), fludioxonil (FDL), pyrimethanil (PYRI), and difenoconazole (DFC) were evaluated using a mycelial growth inhibition assay. Mean effective concentrations necessary to inhibit 50% growth (EC50) of P. washingtonensis were 0.1, 0.3, 0.8, 1.8, 2.1, and 4.8 µg/ml for FDL, PYRI, TBZ, DFC, PYRA, and BOSC, respectively. Respective mean EC50 values for P. pyri were 0.2, 0.6, 1.6, 1.1, 0.4, and 1.8 µg/ml. The sensitivity of exposed P. washingtonensis and P. pyri populations collected in 2017 revealed potential shifts toward BOSC and PYRA resistance. The efficacy of the six fungicides to control isolates of each pathogen with different in vitro sensitivity levels was evaluated on apple and pear fruit. FDL, DFC, and PYRI controlled both Phacidiopycnis spp. regardless of their EC50 values after 5 months of storage at 0°C in a regular atmosphere. The consistent occurrence of Phacidiopycnis spp. will require continuous monitoring and development of disease management strategies based on fungicide phenotypes and efficacy of existing fungicides assessed herein.

Antifungal potential and biochemical effects of monoterpenes and phenylpropenes on plant

To develop new natural fungicides, six monoterpenes and two phenylpropenes were tested for their antifungal activity against eight plant pathogenic fungi. The results of the mycelial growth inhibition assay showed that trans-cinnamaldehyde was the most potent compound against the eight tested fungi with EC&lt;sub&gt;50&lt;/sub&gt; values ranging between 0.75 and 3.19 mg/l. This compound caused the higher mycelial growth inhibition than carbendazim. Furthermore, (–)-menthone exhibited strong antifungal activity against Alternaria solani (EC&lt;sub&gt;50&lt;/sub&gt; = 9.31 mg/l), Penicillium digitatum (EC&lt;sub&gt;50&lt;/sub&gt; = 16.14 mg/l), and Rhizoctonia solani (EC&lt;sub&gt;50&lt;/sub&gt; = 24.69 mg/l). Likewise, eugenol showed potent antifungal activity against P. digitatum, R. solani, Fusarium solani, and A. solani, whereas EC&lt;sub&gt;50&lt;/sub&gt; values were less than 30.0 mg/l. In a separate experiment, trans-cinnamaldehyde, p-cymene, eugenol, and (–)-menthone were evaluated for their inhibitory effects on pectin methyl esterase and cellulase. The tested compounds exhibited the pronounced inhibition of enzyme activities with trans-cinnamaldehyde being the most potent inhibitor for both enzymes.

Assessing the Risk for Resistance and Elucidating the Genetics of Colletotrichum truncatum That Is Only Sensitive to Some DMI Fungicides.

The genus Colletotrichum contains a wide variety of important plant pathogens, and Colletotrichum truncatum is one of the most prevalent species of Colletotrichum on chili in China. Demethylation-inhibitor fungicides (DMIs) are currently registered chemical agents for the management of the anthracnose disease caused by Colletotrichum spp. To assess the risk for DMI resistance development, 112 C. truncatum isolates were collected from infected pepper in 13 regions of China. The sensitivity of C. truncatum isolates to five DMI fungicides was determined based on mycelial growth inhibition assay. C. truncatum was sensitive to prochloraz, epoxiconazole, and difenoconazole, but not to tebuconazole or myclobutanil. Baseline sensitivity using the 112 C. truncatum isolates was established for the first three effective DMIs. Prochloraz, epoxiconazole, and difenoconazole EC50 values were 0.053 ± 0.023, 1.956 ± 0.815, and 1.027 ± 0.644 μg/ml, respectively. Eleven stable DMI-resistant mutants all exhibited lower fitness levels than their wild-type parents, suggesting a low risk of DMI resistance in C. truncatum. By inducing gene expression, CtCYP51 expression increased slightly in the resistant mutants as compared to wild-types when exposed to DMI fungicides and thus contributed at least partially to resistance. Molecular docking with CYP51 structure models was used to explain differential sensitivity of the DMI fungicides in C. truncatum. Our results suggest that the M376L/H373N mutations in CYP51 changed the conformation of DMIs in the binding pocket. These changes prevented the formation of the Fe – N coordinate bond between the heme iron active site and tebuconazole or myclobutanil, and apparently contributed to tebuconazole and myclobutanil insensitivity of C. truncatum.

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.

Heavy metal tolerant Pseudomonas protegens isolates from agricultural well water in northeastern Algeria with plant growth promoting, insecticidal and antifungal activities

The application of plant growth promoting bacteria (PGPB) with biocontrol activities as inoculants of crop plants against phytopathogenic fungi and insect pests provide a biological alternative to the use of agrochemicals. Two Pseudomonas protegens strains were isolated from agricultural well water in a heavy metal contaminated area near Bejaia, northeastern Algeria. The isolates S4LiBe and S5LiBe had 16S rRNA gene sequence similarities of 99.4%–99.7% with P. protegens CHA0T and other P. protegens strains. The phenotypic profiles tested with BIOLOG-GN2-microplates showed differences in 12 of 95 carbon sources tested, as compared to the type strain P. protegens CHA0T. The isolates S4LiBe and S5LiBe showed plant growth promoting potential which is commonly associated with the production of the phytohormone indole acetic acid and siderophores and the solubilization of insoluble phosphate. In addition, they produce chitinase and other polymer degrading enzymes. As the strains S4LiBe and S5LiBe were isolated from heavy metal polluted well water, they are resistant against several heavy metals (2.0 mM K2Cr2O7 and 3.0 mM CoSO4, HgSO4, CdSO4 8H2O and PbCl2), while the reference strain P. protegens CHA0T was very sensitive to Hg2+ and Cd2+ and had lower tolerance towards Co2+ and Pb2+. The isolates S4LiBe and S5LiBe were active in mycelial growth inhibition assays against Botrytis cinerea, Verticillium dahliae, Fusarium graminearum, Aspergillus niger and Aspergillus flavus (growth inhibition between 88% and 48%). Furthermore, S4LiBe and S5LiBe showed effective insecticidal activities, when tested in the Galleria injection assay and they were tested positive for the insect toxin gene fitD alike the reference strain CHA0T. Finally, inoculation of barley seeds with S5LiBe in non-polluted agricultural soil significantly stimulated the germination rate and growth of seedlings, with increased shoot length (11.96 cm ± 0.59), shoot and root fresh weight (0.10 g ± 0.009, 0.04 g ± 0.006), shoot and root dry weight (0.075 g ± 0.003, 0.03 g ± 0.007) as compared to non-inoculated plants (10.23 cm ± 0.84, 0.06 g ± 0.007, 0.025 g ± 0.006, 0.047 g ± 0.006, and 0.016 g ± 0.004, respectively). In heavy metal contaminated soil, inoculation with strain S5LiBe resulted in similar increase of germination rate and growth parameters of barley like in the non-polluted soil, while P. protegens CHA0T inoculated plants were not stimulated. Thus, the heavy metal tolerant isolates S4LiBe and S5LiBe have a potential as beneficial bacteria for agricultural application even in heavy metal polluted soils, e.g. for the stimulation of biomass crops. The demonstration of successful isolation from agricultural well water may open more ready access for a wide variety of this kind of beneficial bacteria for agricultural application.

Natural products as sources of new fungicides (II): antiphytopathogenic activity of 2,4-dihydroxyphenyl ethanone derivatives

A series of 17 simple 1-(2,4-dihydroxyphenyl) ethanones were synthesised, and their structures characterised by 1H, 13C NMR and ESI-MS. Their in vitro antifungal activities were evaluated against five phytopathogenic fungi including Glomerella cingulate, Botrytis cirerea, Fusarium graminearum, Curvularia lunata and Fusarium oxysporum f. sp. vasinfectum by the mycelial growth inhibition assay. Compounds 2g and 2h exhibited broad-spectrum inhibitory activity against the mycelial growth of the tested pathogens with IC50 values in the range of 16–36 μg/mL, and in particular being more active to G. cingulate, with IC50 values of 16.50 and 19.25 μg/mL, respectively, than the other pathogens. Preliminary SAR indicated that an α,β-unsaturated ketone unit of the alkyl chain of the compounds is the structure requirement for fungicidal action. The results suggested that 2g and 2h may be promising leads in the development of new antifungal agents.

Growth stimulation of barley and biocontrol effect on plant pathogenic fungi by a Cellulosimicrobium sp. strain isolated from salt-affected rhizosphere soil in northwestern Algeria

The plant growth promoting effect of bacterial isolates from salt-affected agricultural rhizospheric soil from Bejaia, Algeria, on barley seedlings as well as biological control abilities of these isolates against phytopathogenic fungi were determined. Four isolates stimulated significantly germination and growth of barley seedlings in an axenic test system and in soil pots. Isolate S16 (Cellulosimicrobium sp.) stimulated the growth of barley seedlings by 185% (stem height of 13.0 ± 0.11 cm) over non-inoculated control seedlings (7.0 ± 0.12 cm). Cellulosimicrobium sp. S16 was found also superior in mycelial growth inhibition assays against the plant pathogenic fungi Botrytis cinerea, Fusarium oxysporum and Verticillium dahliae. Furthermore, several plant growth promoting traits (production of indole acetic acid, inorganic phosphate solubilization, siderophore production) and production of enzymes beneficial for soil fertility (protease, chitinase, amylase and urease) were identified. However, no evidence for nitrogen fixation was found by testing acetylene reduction and the presence of nif-genes. Based on comparative sequence analysis of almost full length 16S-rRNA coding gene fragments, Cellulosimicrobium sp. S16 exhibits the highest similarity of 99.7% to Cellulosimicrobium cellulans (accession number AY665978). Cellulosimicrobium sp. S16 could be a successful candidate for the application as a plant growth promoting inoculant.