Deciphering the mechanisms involved in reduced sensitivity to azoles and fengycin lipopeptide in Venturia inaequalis.

Apple scab, caused by the hemibiotrophic fungus Venturia inaequalis, is currently the most common and damaging disease in apple orchards. Two strains of V. inaequalis (S755 and Rs552) with different sensitivities to azole fungicides and the bacterial metabolite fengycin were compared to determine the mechanisms responsible for these differences. Antifungal activity tests showed that Rs552 had reduced sensitivity to tebuconazole and tetraconazole, as well as to fengycin alone or in a binary mixture with other lipopeptides (iturin A, pumilacidin, lichenysin). S755 was highly sensitive to fengycin, whose activity was close to that of tebuconazole. Unlike fengycin, lipopeptides from the iturin family (mycosubtilin, iturin A) had similar activity on both strains, while those from the surfactin family (lichenysin, pumilacidin) were not active, except in binary mixtures with fengycin. The activity of lipopeptides varies according to their family and structure. Analyses to determine the difference in sensitivity to azoles (which target the CYP51 enzyme involved in the ergosterol biosynthesis pathway) showed that the reduced sensitivity in Rs552 is linked to (i) a constitutive increased expression of the Cyp51A gene caused by insertions in the upstream region and (ii) greater efflux by membrane pumps with the involvement of ABC transporters. Microscopic observations revealed that fengycin, known to interact with plasma membranes, induced morphological and cytological changes in cells from both strains. Sterol and phospholipid analyses showed a higher level of ergosta-7,22-dien-3-ol and a lower level of PI(C16:0/C18:1) in Rs552 compared with S755. These differences could therefore influence the composition of the plasma membrane and explain the differential sensitivity of the strains to fengycin. However, the similar antifungal activities of mycosubtilin and iturin A in the two strains indirectly indicate that sterols are probably not involved in the fengycin resistance mechanism. This leads to the conclusion that different mechanisms are responsible for the difference in susceptibility to azoles or fengycin in the strains studied.

Anti-oomycete activities from essential oils and their major compounds on Phytophthora infestans.

Botanicals are various plant-based products like plant extracts or essential oils. Anti-fungal activities of selected essential oils were tested on the pathogen causing potato and tomato late blight (Phytophthora infestans). Tests to evaluate anti-oomycete activities of commercial essential oils and their major compounds were carried out in vitro in microplate in liquid media. Anti-oomycete activities on Phytophthora infestans strain were obtained from essential oils/major compounds: Eucalyptus citriodora/citronellal; Syzygium aromaticum (clove)/eugenol; Mentha spicata/D-Carvone, L-Carvone; Origanum compactum/carvacrol; Satureja montana (savory)/carvacrol; Melaleuca alternifolia (tea tree)/terpinen-4-ol, and Thymus vulgaris/thymol. As an active substance of mineral origin, copper sulfate was chosen as a control. All selected essential oils showed an anti-oomycete activity calculated with IC50 indicator. The essential oils of clove, savory, and thyme showed the best anti-oomycete activities similar to copper sulfate, while oregano, eucalyptus, mint, and tea tree essential oils exhibited significantly weaker activities than copper sulfate. Clove essential oil showed the best activity (IC50 = 28 mg/L), while tea tree essential oil showed the worst activity (IC50 = 476 mg/L). For major compounds, three results were obtained: they were statistically more active than their essential oils (carvacrol for oregano, D- and L-Carvone for mint) or as active as their essential oils sources (thymol for thyme, carvacrol for savory, terpinen-4-ol for tea tree) or less active than their original essential oils (eugenol for clove, citronellal for eucalyptus). Microscopical observations carried out with the seven essential oils showed that they were all responsible for a modification of the morphology of the mycelium. The results demonstrated that various essential oils show different anti-oomycete activities, sometimes related to a major compound and sometimes unrelated, indicating that other compounds must play a role in total anti-oomycete activity.

Antifungal Activity of Hop Leaf Extracts and Xanthohumol on Two Strains of Venturia inaequalis with Different Sensitivities to Triazoles.

Hop cones are well-known for their antimicrobial properties, attributed to their specialized metabolites. Thus, this study aimed to determine the in vitro antifungal activity of different hop parts, including by-products such as leaves and stems, and some metabolites against Venturia inaequalis, the causal agent of apple scab. For each plant part, two types of extracts, a crude hydro-ethanolic extract and a dichloromethane sub-extract, were tested on spore germination of two strains with different sensitivities to triazole fungicides. Both extracts of cones, leaves and stems were able to inhibit the two strains, whereas rhizomes did not show activity. The apolar sub-extract of leaves appeared as the most active modality tested with half maximal inhibitory concentrations (IC50) of 5 and 10.5 mg·L-1 on the sensitive strain and the strain with reduced sensitivity, respectively. Differences in activity level between strains were noticed for all active modalities tested. Sub-extracts of leaves were then separated into seven fractions by preparative HPLC and tested on V. inaequalis. One fraction, containing xanthohumol, was especially active on both strains. This prenylated chalcone was then purified by preparative HPLC and showed significant activity against both strains, with IC50 of 1.6 and 5.1 mg·L-1. Therefore, xanthohumol seems to be a promising compound to control V. inaequalis.

Assessment of Lipopeptide Mixtures Produced by Bacillus subtilis as Biocontrol Products against Apple Scab (Venturia inaequalis).

Apple scab is an important disease conventionally controlled by chemical fungicides, which should be replaced by more environmentally friendly alternatives. One of these alternatives could be the use of lipopeptides produced by Bacillus subtilis. The objective of this work is to study the action of the three families of lipopeptides and different mixtures of them in vitro and in vivo against Venturia inaequalis. Firstly, the antifungal activity of mycosubtilin/surfactin and fengycin/surfactin mixtures was determined in vitro by measuring the median inhibitory concentration. Then, the best lipopeptide mixture ratio was produced using Design of Experiment (DoE) to optimize the composition of the culture medium. Finally, the lipopeptides mixtures efficiency against V. inaequalis was assessed in orchards as well as the evaluation of the persistence of lipopeptides on apple. In vitro tests show that the use of fengycin or mycosubtilin alone is as effective as a mixture, with the 50-50% fengycin/surfactin mixture being the most effective. Optimization of culture medium for the production of fengycin/surfactin mixture shows that the best composition is glycerol coupled with glutamic acid. Finally, lipopeptides showed in vivo antifungal efficiency against V. inaequalis regardless of the mixture used with a 70% reduction in the incidence of scab for both mixtures (fengycin/surfactin or mycosubtilin/surfactin). The reproducibility of the results over the two trial campaigns was significantly better with the mycosubtilin/surfactin mixture. The use of B. subtilis lipopeptides to control this disease is very promising.

Antifungal Activities of Bacillus subtilis Lipopeptides to Two Venturia inaequalis Strains Possessing Different Tebuconazole Sensitivity.

Within the framework of biocontrol development, three natural substances produced by Bacillus subtilis, called lipopeptides, have been studied: fengycin (F), surfactin (S), and mycosubtilin (M). Their antifungal properties were tested in vitro, in liquid medium, on two strains of Venturia inaequalis, ascomycete fungi causing apple scab. These two strains were, respectively sensitive and less sensitive to tebuconazole, an active substance of the triazole family. These three molecules were tested on their own, in binary (FS, FM, SM) and ternary mixtures (FSM). The antifungal activities of lipopeptides were estimated by calculating an IC50, compared to tebuconazole chemical substance. In tests involving the sensitive strain, all lipopeptide modalities exhibited antifungal activity. However, modalities involving fengycin and its mixtures exhibited the best antifungal activities; the activity of fengycin alone being very similar to that of tebuconazole. Interestingly, regarding the strain with reduced sensitivity to tebuconazole, surfactin and fengycin alone were not efficient while mycosubtilin and the different mixtures showed interesting antifungal activities. Specifically, the antifungal activity of FS and FSM mixture were equivalent to that of tebuconazole. For both fungal strains, microscopic observations revealed important morphological modifications in the presence of fengycin and in a less important proportion in the presence of surfactin but not in the presence of mycosubtilin. Overall, this study highlights the diversity in mode of action of lipopeptides on apple scab strains.

Antifungal activity of essential oils on two Venturia inaequalis strains with different sensitivities to tebuconazole.

The antifungal activity of seven essential oils (eucalyptus, clove, mint, oregano, savory, tea tree, and thyme) was studied on Venturia inaequalis, the fungus responsible for apple scab. The composition of the essential oils was checked by gas chromatography-mass spectrometry. Each essential oil had its main compound. Liquid tests were performed to calculate the IC50 of essential oils as well as their majority compounds. The tests were made on two strains with different sensitivities to tebuconazole: S755, the sensitive strain, and rs552, the strain with reduced sensitivity. Copper sulfate was selected as the reference mineral fungicidal substance. IC50 with confidence intervals were calculated after three independent experiments. The results showed that all essential oils and all major compounds had in vitro antifungal activities. Moreover, it was highlighted that the effectiveness of four essential oils (clove, eucalyptus, mint, and savory) was higher than copper sulfate on both strains. For each strain, the best activity was obtained using clove and eucalyptus essential oils. For clove, the IC50 obtained on the sensitive strain (5.2 mg/L [4.0-6.7 mg/L]) was statistically lower than the IC50 of reduced sensitivity strain (14 mg/L [11.1-17.5 mg/L]). In contrast, for eucalyptus essential oil, the IC50 were not different with respectively 9.4-13.0 and 12.2-17.9 mg/L for S755 and rs552 strains. For mint, origano, savory, tea tree, and thyme, IC50 were always the best on rs552 strain. The majority compounds were not necessarily more efficient than their corresponding oils; only eugenol (for clove) and carvacrol (for oregano and savory) seemed to be more effective on S755 strain. On the other hand, rs552 strain seemed to be more sensitive to essential oils than S755 strain. In overall, it was shown that essential oils have different antifungal activities but do not have the same antifungal activities depending on the fungus strain used.

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides.

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α-demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI-resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real-time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC50 results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post-inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch.

Genetic diversity and population structure in French populations of Mycosphaerella graminicola.

Mycosphaerella graminicola populations were examined in France with microsatellite markers and PCR-SSCP analysis of partial actin and ß-tubulin encoding sequences. A total of 363 isolates was sampled in 2005 from 17 provinces, and genotypes from corresponding strains were characterized. Unique haplotypes comprised 84% of the population, and gene diversity was high nationwide (0.70) and locally. A moderate genetic differentiation (G(ST) = 0.18) was found and indicated that in France the M. graminicola population was more structured than in other previously studied European countries. Bayesian structure analysis identified three genetic clusters distributed among the 17 provinces. Our results highlighted the potential for the adaptation of the fungus to local conditions, leading to genetic clusters among the French population of the fungus as well as genotype flow between regional clusters.

Mating type idiomorphs from a French population of the wheat pathogen Mycosphaerella graminicola: widespread equal distribution and low but distinct levels of molecular polymorphism.

Septoria tritici blotch caused by the heterothallic ascomycete Mycosphaerella graminicola is currently the most frequent and the most economically damaging disease on wheat worldwide. Five hundred and ten strains of this fungus were sampled from 16 geographical locations representing the major wheat producing areas in France. Multiplex PCR amplification, PCR-RFLP-SSCP screening and sequencing of parts of mating type encoding sequences were performed in order to assess the distribution and molecular polymorphism of the mating type idiomorphs. The two idiomorphs were scored at similar frequencies within all sampled locations. Both mating types were also identified at the leaf spatial scale, on 42% of leaves from which two or three strains were isolated. No correlation was found between distribution of mating types and either host cultivars from which the sampling was carried out or in vitro colony phenotypes observed during the culture of strains on potato dextrose agar (PDA) medium. PCR-RFLP-SSCP assay highlighted only one MAT1-1 strain exhibiting a profile distinct from all other MAT1-1 strains, whereas ten MAT1-2 strains (among which two and four with same profiles, respectively) showed profiles differing from the other MAT1-2 strains. Sequencing revealed that all polymorphisms corresponded to single nucleotide variations and all strains displaying the same single strand conformation polymorphism (SSCP) profiles showed identical nucleotide sequences, thereby confirming the high sensitivity of SSCP. Only two out of the disclosed nucleotide variations were nonsynonymous. This study strongly suggests a large potential for sexual reproduction in the French population of M. graminicola and reports a high conservation of mating type sequences in the fungus at both nucleotide and population levels, with a great difference in molecular variability between the two idiomorphs.