1. Evaluation of bacterial and fungal isolates efficient for controlling Gnomoniopsis smithogilvyi, the agent of chestnut fruit rot and chestnut canker
Matteo Conti1, Julien Crovadore1, Bastien Cochard1, Romain Chablais1, Mauro Jermini3 & François Lefort1.
1Groupe Plantes et pathogènes, Institut Terre Nature et Environnement, hepia, HES-SO//Genève 150 route de Presinge, 1254 Jussy, Suisse.
2Agroscope, Cadenazzo Research Centre, A Ramél 18, 6593 Cadenazzo, Switzerland
Results
Gnomoniopsis smithogilvyi is a fungal species recently identified in Europe and Switzerland as the main agent of chestnut fruit rot but also as a cork canker agent of chestnut trees. It is an endophytic fungus which can evolve as a pathogen in not yet described conditions. It also seems to be associated to an early and high mortality occurring in orchards of young chestnut trees. It is therefore important to find a biological control method against this pathogen.
Most organisms, which demonstrated an efficacy in in vitro challenge tests against Gnomoniopsis smithogilvyi, unfortunately did not maintain this efficacy in biological tests on scions. Almost all scions exhibited superficial fungal contaminations, mostly by Cryphonectria parasitica, which was present over 25% to 100% of the scions surfaces.
On the other hand, the bacterial strain Pseudomonas putida UASWS0946 and the fungal strain Trichoderma hamatum UASWS1405 totally inhibited the growth of Gnomoniopsis smithogilvyi and Cryphonectria parasitica. This observation was confirmed by specific molcecular diagnostics tests (Fig.6 and Fig.7)
The challenge test between G. smithogilvyi et C. parasitica did not show any predominance of any fungus: they grew at an equal speed until hyphal contact and growth ceased after contact.
Material and methods
Selection of efficient antagonistic organisms
In vitro challenge tests have been carried out in cultures on Potato glucose agar (PGA) medium between Gnomoniopsis smithogilvyi et 9 strains of antagonistic fungi of 7 species : Trichoderma asperellum, Trichoderma atroviride, Trichoderma aureoviride, Trichoderma hamatum, Trichoderma harzianum F1, Trichoderma harzianum B05, Aureobasidium pullulans, Aureobasidium pullulans éch.5 and Beauveria bassiana. Similar challenge tests were performed on Luria Bertani Potato glucose agar (LBPGA) with 7 strains of antagonistic bacteria belonging to the following species: Pseudomonas putida, Pseudomonas viridiflava, Pseudomonas graminis, Pseudomonas xanthomarina, Bradyrhizobium elkanii, Bacillus amyloliquefaciens Ba2 and B. amyloliquefaciens Ba4. Two different strains of G. smithogilvyi, isolated in Geneva (GE1) and Ticino (TI1), corresponding to 2 different genotypes were used for these experiments. A challenge test between G. smithogilvyi and C. parasitica was also performed.
These challenge tests allowed to select the most efficient antagonistic organisms in these given experimental conditions. Five fungal strains and 3 bacterial strains demonstrating a high inhibitory activity of the growth of G. smithogilvyi were retained for a microbiological control experiment on chestnut scions: T. harzianum B05, T. harzianum F1, T. hamatum UASWS1405 , T. aureoviride et T. asperellum for the fungi; P. putida UASWS0946, B. amyloliquefaciens UASWS Ba4 et B. amyloliquefaciens UASWS Ba2 for the bacteria.
Figure 1.
Contamination of a scion by Cryphonectria parasitica.
Figure 2.
Challenge test between T. harzianum F1 et G. smithogilvyi.
Figure 3.
Scions soaked in a T. harzianum F1 suspension and showing a high contamination.
Figures 4 et 5.
Scions soaked in T. hamatum UASWS1405 (left) et P. putida UASWS0946 (right). These scions showed no symptoms of G. smithogilvyi or C. parasitica.
Microbiological tests on chestnut scions
The chestnut scions were sampled in a forest in Canton Ticino and brought to Geneva under federal authorization (Notification A161711). For each bacterial or fungal tested strain, 8 scions were inoculated by soaking for 48 hours. The bacterial and fungal strains were grown between 2 and 5 days in 500 mL liquid cultures (Potato Glucose Broth for the fungi, Luria Bertani Broth for the bacteria). Bacterial and fungal cultures were concentrated by centrifugation and re-suspended in 250 mL sterile demineralized water. The aqueous suspensions of microorganisms were transferred to 2 L sterile Duran bottles and 8 scions (20 cm long; 1 cm diam.) were inserted in these bottles, with their ends soaking in the aqueous suspensions. After soaking for 48h at room temperature in closed bottles, each scion was transferred individually to an in vitro culture glass tube and placed in a climatic chamber (T°C: 26°C ; Photoperiod: 16 h day/ 8 h night; RH: 70%), for 3 weeks, in order to allow a uniform endophytic installation of the antagonists. A suspension of G. smithogilvyi has then been applied on all scions of all modalities and half of the control scions. The development of fructifications on the bark of the scions as well as the aspect of the scions were observed before and after inoculation for a total duration of 6 weeks.
Figure 6.
Scions soaked in P. putida UASWS0946 et T. hamatum UASWS1405 and inoculated with G. smithogilvyi.
Figure 7.
Scions soaked in P. putida UASWS0946 et T. hamatum UASWS1405 and inoculated with C. parasitica.
Conclusion
These experiments dit not give the results which could have been expected from the challenge tests at the exception of two biological control agents : Pseudomonas putida UASWS0946 et Trichoderma hamatum UASWS The scions had not been superficially disinfected in order to remain as close as possible to real conditions and a strong contamination mainly due to Cryphonectria parastica was revealed.
In the trials with Pseudomonas putida et Trichoderma hamatum, additionnally to the absence of visual symptoms, the absence of Gnomoniopsis smithogilvyi and Cryphonectria parasitica was confirmed by specific molecular diagnostics. These two strains of Pseudomonas putida et Trichoderma hamatum could remarkably control Gnomoniopsis smithogilvyi and Cryphonectria parasitica and be efficiently used in nurseries and orchards.
11th Annual Biocontrol Industry Meeting ABIM
Congress Center Basel, Basel, Switzerland
October 2016