Determination of in vitro biocontrol efficiencies of endophytic and epiphytic bacterial agents against botryosphaeria dieback disease agents on nut trees

As a result of the awareness of people about healthy nutrition, the interest in nut fruits is increasing day by day. Pistachio (Pistacia vera L.), walnut (Juglans regia L.), hazelnut (Corylus avellana L.) and almond (Prunus dulcis L.) are the leading fruits that are cultivated with economic importance in our country. Very recently, fungal disease agents, belonging to Botryosphaeria, Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum and Phomopsis, have been reported to cause symptoms of disease in the form of dieback, dryness of stems, branches and leaves in walnut, almond, pistachio and hazelnut trees in different region of our country. Since, there are very limited information available on the disease factors on walnut, almond, pistachio and hazelnut trees, studies on disease control is not currently available. Therefore, this study aimed for (i) isolation and (ii) identification of endophytic and epiphytic bacterial isolates from different healthy nut trees such as walnut, almond, pistachio and hazelnut and (iii) characterize in vitro biocontrol activities and (iv) possible antagonistic and plant growth promoting (PGP) mechanisms that play a role against Botryosphaeria dothidea, Neofusicoccum parvum and Neoscytalidium dimidiatum disease agents. A total of 74 bacterial isolates were obtained from the rhizosphere and endosphere of healthy trees, 48 of these isolates were identified by MALDI-TOF MS analysis and selected for antagonistic potential against fungal isolates In dual culture assay, Pseudomonas chlororaphis NKA10ep and Bacillus mojavensis NKA7en isolates were found as the most effective antagonistic bacterial isolates inhibiting mycelial growth of Neoscytalidium dimidiatum, Botryosphaeria dothidea and Neofusiccocum parvum by 80-84%. Bacterial isolates significantly varied in the production of siderophore, protease, hydrogen ciyanide [HCN], ammonia, indole acetic acid [IAA], phosphate solubilization. Among the tested 43 bacterial isolates, 43 were positive for siderophore production, 35 for ammonium production, 28 for protease and 4 for HCN production. The production of siderophore, protease and ammonium by these isolates may be the contributing factor(s) for their antagonistic properties against the suppression of the mycelial growth. All selected bacterial isolates produced IAA in a relatively varying amounts but 17 isolates solubilized the phosphate. Among the isolates, Pseudomonas aeruginosa NKC2ep and Acinetobacter dijkshoorniae NKA8en produced a relatively large amount of extracellular IAA and solubilised phosphate. In conclusion, the strong antagonistic (siderophore, protease, ammonium production) and PGP traits (IAA and phosphate solubilization potentials) displayed by effective isolates belonging to Bacillus spp and Pseudomonas spp. may be considered a good alternative as a biocontrol agent and have the potential to be developed as biological control agent for suppressing dieback plant pathogens tested in this study. This is the first study to investigate the biocontrol effect and mode of action of some of the antagonist bacterial species on dieback fungal disease agents of nuts tested in the present study.