Isolation of Rhizospheric Pantoea sp. and Pseudomonas sp. Bacteria and Evaluation of Their Bio-control Ability against Pectobacterium carotovorum subsp. carotovorum Causes of Tubers and Vegetables Rot

Pectobacterium carotovorum carotovorum (Pcc) is one of the main causes of soft rot disease in many economically important vegetables and tubers such as carrot, cabbage, potato, onion, cucumber, eggplant, garlic, bell pepper, radish, sweet potato, squash and tomato. The use of chemical bactericides is not suitable to control soft rot bacteria due to their toxic side effects and the emergence of resistance in bacterial populations. The use of bio-control bacteria to inhibit bacterial pathogens such as Pcc is a practical and useful method to overcome the issue. In this study, in order to control this disease with the help of bacterial biological control agents, some bacteria were isolated from rhizosphere of vegetables and potato. The aim of this study was to find bacteria with different bio-control mechanisms which are able to inhibit different isolates of Pcc. On the other hand, these bacteria (with different bio-control mechanisms) were combined to investigate the possibility of increasing their bio-control ability in a mixed manner.

12 rhizospheric bacteria were isolated from vegetables and potato. Antibacterial properties were evaluated using chloroform vapor method. Also, 12 bacteria were isolated from rhizosphere of vegetables and potato by minimal medium to evaluate the quorum quenching (QQ) ability. Chromobacterium violaceum CV026 biosensor was used to assess QQ activity in bacteria. There were two types of bio-control tests: the first case was the application of bacteria individually and the second case was the mix of bacteria with different bio-control mechanisms, antibacterial property and QQ activity, and its application against two strains of Pcc including PccK and PccM. At first, before doing the second bio-control test, interaction test between bio-control bacteria was performed by culturing two bacteria perpendicular to each other on solid medium. Bio-control test of Pcc on plant tissues (potato tubers, carrots and bell peppers) was performed by calculating the weight of rotting tissue in comparison with the control (containing Pcc alone). Classical and molecular bacteriological tests were performed to detect these bacteria.

Among rhizospheric bacteria with antibacterial properties, A10 isolate had a large inhibitory halo, 16.67 mm, against one strain of Pcc (PccK) and it had the ability to bio-control of PccK on vegetables and potato tubers. Among bacteria, only SS5 isolate showed QQ ability in both biosensor and bio-control tests. A10 and SS5 were detected as Pantoea and Pseudomonas sp. respectively. Pseudomonas SS5 had moderate bio-control activity against two Pcc strains with QQ mechanism as inhibition percentage on potato tubers rot for PccM and PccK were 53.92 % and 68 %, respectively. Pantoea A10 with antibacterial activity which had low or high bio-control properties against Pcc strains as inhibition percentage on potato tubers rot for PccM and PccK were 5.81 % and 91.33 %, respectively. The bio-control effect of A10 and SS5 bacteria against Pcc strains on carrots and bell peppers was better than potatoes. The interaction of two bacteria on solid medium, SS5 and A10, was not visible as a transparent area at their intersection, so they did not show an inhibitory effect against each other. Therefore, it is possible to use combination of two bacteria, A10 and SS5, for bio-control tests. The results showed that the bio-control ability of these two bacteria in the mixed state was much higher than the single application of each bacterium. In mix of two bio-control bacteria, disease was further reduced at least for PccM, in other words, the bio-control property was increased. In the case of the combination of two bacteria against PccM, compared to SS5 and A10 application alone, it produced 2.21 and 4.53 times less rot tissue, respectively.

There are two main bio-control mechanisms in bio-control bacteria for Pcc rot reduction including bacteria with antibacterial properties and bacteria with QQ abilities. In this study, for the first time, it was found that two bacteria with different bio-control mechanisms, including Pantoea and Psudomonas sp., are able to control Pcc separately and in combination. For the first time, the improvement of the bio-control properties of these two bacteria in combination with each other was identified. It was also found that the effect of these different bio-control bacteria on the control of each Pcc isolate and even each host of vegetables or potato tuber could be different, so that one Pcc isolate was sometimes completely inhibited and the other Pcc isolate was not affected by the bio-control bacteria. Because QQ mechanism disrupts the basic common physiological pathways in all Pcc isolates. So, this mechanism in bio-control bacteria apparently has a more general effect than the antibacterial effect.