In planta assays of biological control against Gnomoniopsis smithogilvyi, a fungal agent of chestnut brown rot and chestnut canker Jonathan Bourquin1, Matteo Conti1, Julien Crovadore1, Bastien Cochard1, Romain Chablais1, Mauro Jermini2, Francesco Bonavia3, & François Lefort1. 1Group Plants and pathogens , Institute Land Nature Environment, hepia, HES-SO//Genève, University of applied sciences and arts Western Switzerland, 150 route de Presinge, 1254 Jussy, Suisse. E-mail: francois.lefort@hesge.ch; 2Agroscope, Cadenazzo Research Centre, A Ramél 18, 6593 Cadenazzo, Switzerland; 3Vivaio forestale cantonale, Dipartimento del territorio, Divisione dell’ambiente, Sezione forestale Repubblica e Cantone Ticino, 6835 Morbio Superiore, Switzerland Experiment 1 - Selection of efficient antagonistic organisms against Gnomoniopsis smithogilvyi. Five fungal strains and 3 bacterial strains highly inhibitory of G. smithogilvyi growth 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. Challenge test between T. harzianum F1 et G. smithogilvyi. In vitro challenge tests have been carried out on Potato glucose agar (PGA) medium between Gnomoniopsis smithogilvyi and 9 strains of antagonistic fungi of 7 species : Trichoderma asperellum, T. atroviride, T. aureoviride, T. hamatum, T. harzianum F1, T. harzianum B05, Aureobasidium pullulans, A. pullulans éch.5 and Beauveria bassiana. Similar tests were performed on Luria Bertani Potato glucose agar (LBPGA) with 7 strains of antagonistic bacteria belonging to the following species: Pseudomonas putida, P. viridiflava, P. graminis, P. xanthomarina, Bradyrhizobium elkanii, Bacillus amyloliquefaciens Ba2 and B. amyloliquefaciens Ba4. Two different strains of G. smithogilvyi, isolated in Geneva and Ticino, were used for these experiments. Gnomoniopsis smithogilvyi is a fungus recently identified in Europe and Switzerland as the main agent of chestnut fruit rot and canker. As an endophytic fungus, it may evolve as a pathogen under conditions not yet described. It appeared to be associated with early and high mortality occurring in young chestnut orchards. In order to develop an effective biological control method against this pathogen, promising antagonists have been selected and are now tested in vivo. Experiment 3 – Evaluation of the biostimulant and protecting effect of Trichoderma hamatum UASWS1405 and Pseudomonas putida UASWS0946 on infected trees. Treatments and modalities : Three treatment modalities are tested: T. hamatum, P. putida, T. hamatum & P. putida. A negative control (water) has also been implemented. The antagonists are inoculated by watering. The treatment is performed three times. Twice in summer and once in autumn. Material : 200 chestnut trees of 2 years growing in nursery. Measured parameters : trunk circumference (mm) at pot height and main stem length (cm). Material and methods Experiment 4 – Grafting treatment (A) (B) (C) (D) Figure 7. (A) Scion sampling in nursery and forest. Then, removing of the leaves before soaking, (B) inoculation of the antagonists for 18 h at ambient temperature, (C) 5 to 8 buds are removed for grafting, (D) chip budding Scions were sampled from mother trees in the nursery or in the forest. The same modalities as experiment 3 were tested. Treatments : the scions on which the grafting buds are sampled were soaked in suspensions of antagonist microorganisms. Material : 635 chestnut trees of 1 years old Measured parameters: cork canker size (cm2), visual symptoms, presence / absence of fruiting bodies of G. smithogilvyi and C. parasitica. As G. smithogilvyi infection appears to develop from grafting, an application method was tested to prevent infection at this time Experiment 5 - Metagenomics Goal : Investigate changes in the endophyte community in G. smithogilvyi pre-infected scions treated with antagonists. The same protocol as experiment 1 is used (Figure 2) for the first part, but only with Trichoderma hamatum and Pseudomonas putida. A modality where scions are infected first with G. smithogilvyi is also added. Metagenomics : in order to study the endophytic microbial community after treatment, scions are then cut into strips. The total DNA is extracted and sequenced in an Illumina MiniSeq sequencer, and the results analyzed to calculate the fungal and bacterial endophytes relative abundance in scions. relative abundance of the endophytes (A) (B) (C) Figure 8 : (A) scions are cut in 4 parts uniformly taken over the entire length of the scion, (B) extraction of total DNA, (C) bioinformatics analysis and relative abundances calculation of the different endophytes 48h G. smithogilvyi Bacterial or fungi, water 3 weeks Figure 2. (A) Chestnut scions were cut into smaller pieces (20cm long, 1cm ⍉ ), (B) for each bacterial or fungal strain tested, 8 scions were inoculated by soaking for 48 hours in a aqueous suspensions of fungi or bacteria, (C) 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, (D) a suspension of G. smithogilvyi has then been applied on all scions of all modalities and half of the control scions and then returned in the climatic chamber. 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. (A) (B) (C) (D) Figures 3 & 4. Scions soaked in T. hamatum UASWS1405 (left) et P. putida UASWS0946 (right). These scions showed no symptoms of G. smithogilvyi or C. parasitica. Experiment 2 - Microbiological tests on chestnut scions To test the efficiency of the antagonists identified in the previous experiment, a microbiological test has been performed on living material. Following the first screening experiments, two promising biological controls, Pseudomonas putida UASWS0946 and Trichoderma hamatum UASWS1405 have been identified. Therefore, the two large scales in planta experiments on chestnuts trees should confirm that these antagonists of G. smithogilvyi could be used in a long term treatment methodology. The metagenomic analysis would provide with more information about the impact of the antagonists on the endophytes community and their ability to colonize chestnut trees as endophytes. Conclusion The bacterial strain Pseudomonas putida UASWS0946 and the fungal strain Trichoderma hamatum UASWS1405 totally inhibited the growth of Gnomoniopsis smithogilvyi and Cryphonectria parasitica. The absence of these fungi was confirmed by specific molecular diagnostics tests. Intermediate results Annual Biocontrol Industry Meeting ABIM 2017 23-25 October 2017, Basel, Switzerland