Monilinia fructigena Suppressing and Plant Growth Promoting Endophytic Pseudomonas spp. Bacteria Isolated from Plum

Abstract

Brown rot caused by Monilinia spp. fungi causes substantial losses in stone and pome fruit production. Reports suggest that up to 90% of the harvest could be lost. This constitutes an important worldwide issue in the food chain that cannot be solved by the use of chemical fungicides alone. Biocontrol agents (BCAs) based on microorganisms are considered a potential alternative to chemical fungicides. We hypothesized that endophytic bacteria from Prunus domestica could exhibit antagonistic properties towards Monilinia fructigena, one of the main causative agents of brown rot. Among the bacteria isolated from vegetative buds, eight isolates showed antagonistic activity against M. fructigena, including three Pseudomonas spp. isolates that demonstrated 34% to 90% inhibition of the pathogen's growth when cultivated on two different media in vitro. As the stimulation of plant growth could contribute to the disease-suppressing activity of the potential BCAs, plant growth promoting traits (PGPTs) were assessed for bacterial isolates with M. fructigena-suppressing activity. While all isolates were capable of producing siderophores and indole-3-acetic acid (IAA), fixating nitrogen, mineralizing organic phosphate, and solubilizing inorganic phosphate and potassium, only the Pseudomonas spp. isolates showed 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Overall, our study paves the way for the development of an eco-friendly strategy for managing M. fructigena pathogens by using BCAs including Pseudomonas spp. bacteria, which could also serve as growth stimulators.

Keywords: Monilinia fructigena; bacterial control agents; plant growth promoting traits.

Figure 1

Inhibition of the growth of Monilinia fructigena in vitro on two different media (maltose-based and Potato Dextrose Agar (PDA) with a standard composition) as percentages ± SE. The red line denotes 30% radial growth inhibition, which is considered to be the minimum requirement for a biocontrol agent, as per Ulrich et al. [56]. The test was performed using the Kruskal–Wallis one-way analysis of variance on ranks, followed by pairwise comparisons with Dunn’s test. Significant divergences from the control are marked as * (p ≤ 0.05).

Figure 2

Dual culture antagonism assay of the Monilinia fructigena pathogen and the endophytic bacteria isolate Pseudomonas graminis SENP33v2 from Prunus domestica buds (a) on a maltose medium and (b) on a Potato Dextrose Agar, as compared with the control plates (shown on top).

Figure 3

Physiological test results of oxygen requirements (thioglycollate test), biofilm formation, catalase and oxidase production, and antibiotic susceptibility against ampicillin (AM), cefotaxime (CTX), chloramphenicol (C), kanamycin (K), streptomycin (STP), and ticarcillin (TIC), where the numbers beside the letters indicate the concentrations of antibiotics in micrograms. The dotted line by the biofilm formation test indicates the separation of only one isolate (SENP55) with moderate biofilm formation. The red and green rectangles in the diagram indicate the genus of the isolates. The separation into sub-morphotypes was caused by variations in the results of antibiotic susceptibility testing.

Figure 4

Plant growth promoting traits of the bacterial isolates. (A). Representative plant growth promoting trait tests on selective media: (a) siderophore production by SENP41 (a color change in the medium from blue to yellow), (b) phosphate mineralization by SENP33 (a clear zone around the colonies), (c) phosphate solubilization by SENP55 (clear zones around the colonies), (d) potassium solubilization by SENP53 (clear zones around the colonies), (e) ACC deaminase activity by SENP41 (growth on the medium with ACC as the only nitrogen source and no growth on the negative control plate without any nitrogen), and (f) nitrogen fixation by SENP81 (growth on the medium without any nitrogen source). (B). Identification of endophytic bacteria morphotypes based on IAA production. Starting from the middle of the circle, the isolates were separated by genus, species, and isolate according to the amount of IAA produced. The penultimate circle indicates the sub-morphotype of the species, and the color differs according to their IAA production (µg/mL). The outside circle indicates the morphotype of the isolates.