Biological control agent Bacillus subtilis formulated as Theia is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (emulsifiable concentrate [EC] and wettable powder) and metconazole (EC and water-dispersible granule) at 0, 10, 50, 100, and 150 µg/ml of active ingredient. After cocultivation, the optical density (OD₆₀₀) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the wettable powder or water-dispersible granule formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia and each formulated DMI at 50 µg/ml of active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia with EC formulations showed weaker disease reduction due to antagonism. Only Theia plus non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared with the respective solo treatments and anthracnose disease incidence of cherry compared with the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia with a lower-than-label-rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects, especially when non-EC formulations were used.

Brown rot caused by Monilinia fructicola is one of the most important diseases affecting peach production in the southeastern United States. Management often involves the use of demethylation inhibitor (DMI) fungicides, but efficacy can be compromised because of overexpression of the MfCYP51 gene encoding the 14α-demethylase of the ergosterol biosynthesis pathway. This study aimed to investigate the influence of the biorational fungicide Howler EVO containing Pseudomonas chlororaphis ASF009 metabolites on the expression of MfCYP51 in M. fructicola and associated synergy with a DMI fungicide for control of DMI-resistant strains. Mycelia from two DMI-sensitive and three DMI-resistant M. fructicola isolates were exposed or not to propiconazole (0.3 μg/ml), Howler (88.1 μg/ml), or the combination propiconazole + Howler for 6 h prior to RNA extraction. Real-time PCR indicated that Howler reduced the constitutive expression of MfCYP51 in DMI-sensitive and two of three DMI-resistant isolates. Propiconazole-induced expression of the DMI target gene was significantly reduced by Howler and by the mixture of Howler plus propiconazole in all isolates. Detached fruit studies on apple revealed that the combination of Howler plus a reduced label rate of Mentor (50 μg/ml propiconazole) was synergistic against brown rot caused by a DMI-resistant isolate in high and low inoculum spore concentration experiments (synergy values of 40.1 and 4.9, respectively). We hypothesize that the synergistic effects against M. fructicola resistant to DMI fungicides based on MfCYP51 gene overexpression can be attributed to reduced 14α demethylase production due to transcription inhibition, which may necessitate fewer DMI fungicide molecules to arrest fungal growth. The use of Howler/DMI mixtures for brown rot control warrants further investigation because such mixtures could potentially allow for reduced DMI fungicide use rates in the field without compromising yield or increased resistance selection.

Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below-label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry ( Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Real-time PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.

Brown rot, caused by Monilinia fructicola, is one of the most economically important diseases of peach. Demethylation inhibitor (DMI) fungicides play an important part in managing brown rot in the Southeastern United States, but over the last 20 years, reduced efficacy to DMIs has been reported in field isolates overexpressing the DMI target enzyme encoding the MfCYP51 gene. Metabolites of the biocontrol agent (BCA) Pseudomonas chlororaphis strain AFS009 suppressed the MfCYP51 gene in sensitive and resistant M. fructicola isolates previously, but it is not known what molecule was responsible. The goals of this study were to determine the presence and role of pyrrolnitrin (PRN), a common metabolite of P. chlororaphis and chemical analog to fludioxonil with antifungal activity, in the suppression of the MfCYP51 gene and to investigate whether MfCYP51 expression can also be suppressed by Bacillus subtilis (Theia). High-performance liquid chromatography detected PRN at 1.75 μg/mg in P. chlororaphis metabolites formulated as Howler EVO (Howler). PRN at 0.1 μg/ml, fludioxonil at 0.1 μg/ml, and Howler applied at a dose that contained 0.1 μg/ml PRN significantly reduced the MfCYP51 gene expression at similar levels in DMI-resistant isolates. Furthermore, MfCYP51 expression in DMI-sensitive and three DMI-resistant isolates treated with Howler (88.1 μg/ml), Theia (209.5 μg/ml), propiconazole (0.3 μg/ml), or the mixture of either Howler or Theia + propiconazole revealed that Howler significantly reduced the MfCYP51 target gene expression in two of three sensitive and all three resistant M. fructicola isolates. On the other hand, Theia showed no suppressive effect and even increased the MfCYP51 gene expression level in two of three resistant isolates. In detached fruit assays on apple with a DMI-resistant isolate, only the mixture of Howler + 50 μg/ml propiconazole resulted in synergism. The results indicate that suppression of MfCYP51 target gene is BCA dependent and can be induced by PRN.

Strategic mixtures of biological control agents (BCA) with demethylation inhibitor (DMI) fungicides to control diseases of fruit crops can result in additive interaction, synergism, or antagonism. To evaluate the compatibility of eight commercial DMI fungicides with the BCA Bacillus subtilis AFS032321, marketed as Theia, bacteria were cultured in nutrient broth with 0, 10, 50, 100, and 150 μg/ml of each DMI fungicide at 30°C on a shaker rotating at 180 rpm, and the optical density was measured after 24 and 48 h. In addition, nutrient agar (NA) was enriched with the same concentrations, and an endospore suspension was streaked out to count colony-forming units (CFU/ml) and measure the colony diameter. Results showed vegetative cell growth was strongly inhibited at ≥50 μg/ml of difenoconazole and ≥100 μg/ml of tebuconazole. All other DMI fungicides had little impact on B. subtilis AFS032321 cell growth at any of the concentrations tested. Interestingly, mefentrifluconazole significantly promoted colony growth (diameter) at all concentrations on NA. Theia applied at the label rate combined with vegetative growth-promoting DMI fungicide mefentrifluconazole (formulated as Cevya) or vegetative growth-suppressing DMI fungicide difenoconazole (formulated as Inspire) at 150 μg/ml of active ingredient were investigated against gray mold of cherry. Disease incidence and severity 5 days after inoculation indicated the best control efficacy and synergism (+10.2) for the mixture Theia + Cevya. For the mixture Theia + Inspire, an antagonistic effect (−6.8) was calculated. Our results indicate that compatibility between biological and conventional fungicides must be considered if they are used in mixtures or together in integrated spray programs.

Venturia inaequalis, the cause of apple scab, readily develops resistance to fungicides with specific modes of action. Knowledge of the spatial and temporal pattern of resistance development is therefore relevant to fruit producers and their consultants. In the Lower Elbe region of Northern Germany, a two-year survey based on a conidial germination test was conducted, examining fungicide resistance in 35 orchards under Integrated Pest Management (IPM), 16 orchards of susceptible cultivars as well as a further 12 orchards of scab-resistant (Vf) cultivars under organic management, and 34 abandoned or unmanaged sites. No evidence of resistance to SDHI compounds (fluopyram, fluxapyroxad) was found after >5 yr of their regular use. Resistance to anilinopyrimidines (cyprodinil, pyrimethanil) had disappeared 15 yr after its widespread occurrence. Isolates from a few IPM orchards showed a reduced sensitivity to dodine. Double resistance to the MBC compound thiophanate-methyl and the QoI trifloxystrobin was rare in V. inaequalis strains that had achieved breakage of Vf-resistance, but very common (>50%) on scab-susceptible cultivars in IPM, organic and abandoned orchards in the ‘Altes Land’ core area of the Lower Elbe region, and in IPM orchards in the periphery. We conclude that resistance to QoI and MBC fungicides is persistent even decades after their last use, and that the core area harbours a uniform population adapted to intensive crop protection, whereas isolated orchards in the periphery are colonised by discrete populations of V. inaequalis.