Biocontrol of angular leaf spot disease and colonization of cucumber (Cucumis sativus L.) by endophytic bacteria

Plant materials, bacterial isolates, and growth conditions

In vivo screening of endophytic bacteria against Pseudomonas syringae pv. lachrymans

In vivo tests were carried out on plantlets emerging from seeds. The 24 selected endophytic isolates were tested in vivo for their biocontrol against Psl on cucumber plants (25 °C day, 22 °C night, 16-h illumination, 45 days). EB isolates were inoculated twice: once before sowing, as seed coating, and again after transplanting, as soil drench.

Both EB and pathogen were grown on King’s B (KB) medium at 24 °C for 24–48 h (Schaad et al. 2001). The growing EB colonies were suspended in carboxymethyl cellulose (CMC, 1% v/v) for the first EB treatment, i.e., seed bacterization. The suspensions were adjusted to an OD₆₀₀ of 0.1 using a spectrophotometer (PG Instruments T60 UV/VIS). The seeds were rinsed three times with sterile distilled water after being surface sterilized with 1% sodium hypochlorite for 1 min and soaked for 30 min in EB suspensions amended with CMC. In the control treatment, the seeds were covered with CMC alone. The seeds were left on blotting papers in a sterile cabinet for 24 h before sowing. After bacterization, the seeds were sown individually in separate plastic pots filled with sterile peat and maintained under standard growth chamber conditions. The second EB treatment (soil drench) was carried out at the true leaf stage. The suspensions (OD₆₀₀, 0.1) for the second EB treatment were prepared by growing colonies on KB medium at 24 °C for 24–48 h. They were suspended in sterile distilled water. The plants were treated with the suspensions in the form of a soil drench (20 mL/plant) at the first true leaf stage 1–2 days before pathogen inoculation (Lwin and Ranamukhaarachchi 2006). Control plants only received distilled water.

The inoculum was prepared from Psl colonies grown on KB medium at 28 °C for 24 h. They were suspended in sterile distilled water and the concentration was adjusted to approx. 10⁷ CFU/mL (OD₆₀₀, 0.05) using a spectrophotometer (Klement et al. 1990). A single drop of Tween 20 was added to each liter of inoculum to ensure effective distribution of the pathogen on the leaves. Cucumber seedlings at the second true leaf stage were inoculated by spraying Psl suspensions on the underside of each true leaves 1–2 days after soil drenching. After inoculation, the seedlings were incubated at 24 °C for 2–3 days at 95–100% relative humidity (RH). The seedlings were then placed in a transparent cabinet to maintain high RH (95–100%) post-inoculation. The cucumber plants were kept in the growth chamber for 14 days after inoculation to monitor disease symptoms. Cucumber plants obtained from non-bacterized seeds and inoculated with the pathogen were used as positive controls. Cucumber plants that had received neither EB nor pathogen were evaluated as negative controls (Fig. 1d).

The experiments were arranged in a completely randomized block design consisting of 10 replicates (one plant per replicate). The experiments were repeated twice under similar conditions. Disease development was monitored for 14 days after Psl inoculation. Disease severity was assessed after 14 days using a visual rating scale from 0 to 4 in which 0 = no symptoms, 1 = necrotic lesions covering < 25% of the leaf surface, 2 = necrotic lesions covering 25–50% of the leaf surface, 3 = necrotic lesions covering 50–75% of the leaf surface, and 4 = necrotic lesions covering >75% of the leaf surface and plant death (Raupach and Kloepper 2000). The disease index (DI) was calculated by using the formula adapted from Liu et al. (1995b): DI = [Σ (Number of plants in the rating × Rating number)/(total number of plants × the highest rating)] × 100%.

Monitoring endophytic bacteria and pathogen colonization

Taking into account the results obtained from the in vitro and in vivo experiments and the availability of suitable plant material and growth chamber conditions, two EB isolates showing significant biological control against Psl in the in vitro and in vivo experiments were selected to monitor population density in the cucumber plant root and shoot over a 30-day period from sowing under the same conditions described above.

Treated plant samples [5 g each of roots and shoots (including stem segments, petioles, and leaves) of the whole plant] were surface sterilized with 70% ethanol for 5 min followed by 1% sodium hypochlorite for 1 min then washed three times in distilled water (1 min per rinse). The samples were then ground and processed as described above to determine the microbial populations in them. Root and shoot extracts (typically 100 μL for standard 90 mm Ø Petri dish) were serially diluted tenfold and spread on Petri dishes containing KB medium amended with rifampicin (200 ppm). The plates were incubated at 24 °C for 3 days, then the EB (Rif ⁺) colonies were calculated.

To determine pathogen colonization, the plant samples were taken at different time intervals after inoculation, as shown in Table 2. Plant shoots were gently rinsed in sterile distilled water. Shoot tissues (5 g/plant) were macerated with a manual stomacher in sterile polyethylene bags containing 100 mL phosphate-buffered saline solution. Macerates were then shaken for approximately 30 min at 24 °C on a rotary shaker (120 rpm). Extracts (typically 100 μL for standard 90 mm Ø Petri dish) were serially diluted tenfold and plated on modified KBZ medium (Anonymous 2012) known to be selective for Psl. Pathogen colonies were counted after 3 days incubation at 24 °C. The pathogenicity tests conducted on healthy cucumber plants containing bacterial colonies supported Koch’s postulates (Agrios 2005).

Endophyte identification by bacterial 16S rRNA gene sequencing

In preparation for DNA extraction, EB isolates CB361-80 and CC372-83 were grown on KB medium for 24–48 h at 24 °C. DNA was extracted from EB suspensions by the boiling lysis method. EB suspensions were prepared in sterile distilled water at an OD₆₀₀ of 0.1 then centrifuged at 15,000×g for 10 min. The pellets were resuspended in 40 μL ultrapure water, boiled at 100 °C for 10 min, cooled on ice, centrifuged at 15,000×g for 10 s, then stored at − 20 °C. The DNA extract was used as a template for PCR amplification (Omar et al. 2014). The universal primers 27F (5′ AGAGTTTGATCMTGGCTCAG 3′) and 1492R (5′ TACGGYTACCTTGTTACGACTT 3′) were used in the PCR to amplify approximately 1428 bp of 16S rDNA. The primers were designed by Lane and associates in 1991 for the 27th and 1492nd positions in the Escherichia coli 16S rRNA gene region and reviewed in Hodkinson and Lutzoni 2009. The final PCR reaction mixture contained 100 ng DNA extract, 10× Taq KCI reaction buffer (Fermentas), 1 mM of each primer, 1.5 mM MgCl₂ (Fermentas), 0.2 mM dNTP (Fermentas), and 1 U Taq DNA polymerase (recombinant 5 U/μL, Fermentas). The amplification proceeded as follows: for 35 cycles, there was an initial denaturation at 95 °C for 3 min, followed by denaturation at 94 °C for 1 min, then annealing at 50 °C for 1 min, then extension at 72 °C for 2 min, and a final extension at 72 °C for 5 min in a thermal cycler (Eppendorf® Mastercycler Personal). The PCR products were then subjected to electrophoresis on 1.5% agarose gel in 0.5× TAE buffer (50× Tris-acetate-EDTA, Fermentas) with RedSafeT nucleic acid staining solution (20.000×) (iNtRON Biotechnology). The gel was run at 80 V for 90 min. DNA bands were visualized under UV light using a GeneRuler™ 1 kb DNA Ladder (Fermentas).

The amplified products were purified with a QIAquick Gel Extraction Kit (QIAGEN). Sequencing was performed by a Macrogen online sequencing order system (Macrogen, Netherlands). Sequence editing and inspection were done with Sequencher V5.4.5. The DNA sequences of the PCR products were compared with GenBank sequences using BLASTn software (http://blast.ncbi.nlm.nih.gov/). The 16S rRNA sequences of the EB in this study were deposited with accession numbers in the GenBank database.

Statistical analysis

Endophytic bacterial population data were separately transformed to log root and shoot CFU/g before averaging. All data were analyzed with Microsoft Excel 2010 and SPSS (IBM SPSS Statistics, version 24.0). The data obtained were subjected to ANOVA (analysis of variance). The population data were transformed to log (CFU/g). Means were compared using Duncan’s t test with a level of significance P < 0.05, and the SD were calculated.

In vivo biocontrol effects of endophytic bacterial isolates against Pseudomonas syringae pv. lachrymans

Monitoring the colonization of endophytic bacteria and Pseudomonas syringae pv. lachrymans in cucumber plants

Rifampicin-resistant mutants (Rif ⁺) of EB isolates CB361-80 and CC372-83 were selected to monitor EB colonization rates inside the cucumber plants. The populations of these bacterial isolates on surface-sterilized seeds were 3 × 10⁶–7.1 × 10⁷ CFU/g 1 day after bacterization. Endophytic colonization continued in cucumber plant tissues at the first true leaf stage. EB populations there ranged from 1.4 × 10⁴ to 1.7 × 10⁶ CFU/g shoot. In the absence of Psl, EB populations in the shoots remained between 3.8 × 10⁴ and 8 × 10⁴ CFU/g. In plants exposed to Psl, EB population levels were from 1.9 × 10⁵ to 2.2 × 10⁵ CFU/g 45 days after seed bacterization.

The population level of EB isolates CB361-80 and CC372-83 in the root tissues were between 1.6 × 10⁵ and 6.3 × 10⁵ CFU/g at the first true leaf stage. In the absence of Psl, EB populations ranged from 2 × 10⁴ to 7.5 × 10⁴ CFU/g. EB populations in roots inoculated with Psl were between 5.8 × 10⁴ and 2.7 × 10⁴ CFU/g 45 days after seed bacterization.

EB isolates CB361-80 and CC372-83 successfully colonized cucumber shoot and root (Fig. 2a, b). The colonizations of EB isolates CB361-80 and CC372-83 were not significantly affected by Psl inoculation during the entire sampling period.

Psl population levels were also monitored in cucumber plants treated and not treated with EB isolates. Psl levels in cucumber plants receiving the pathogen alone (no EB) reached from 4.5 × 10⁴ to 9.5 × 10⁸ CFU/g 14 days after inoculation.

Psl colonization rates in cucumber plants treated with EB isolates CC372-83 and CB361-80 were between 3.3 × 10³ and 4.2 × 10⁴ CFU/g, respectively, 1 day after Psl inoculation. Psl levels were between 3 × 10⁷ and 2.6 × 10⁸ CFU/g, respectively, 14 days after Psl inoculation (Fig. 3).

Psl populations in cucumber plants treated with EB isolate CC372-83 were lower than those in cucumber plants treated with EB isolate CB361-80, according to the results of monitoring 14 days after Psl inoculation. These data were confirmed by statistical analysis.

Identification of promising endophytes

EB isolates CB361-80 and CC372-83 were the most promising bacteria for the biological control of Psl. They were identified using 27F/1492R universal 16S rRNA primers. The products of the 1428-bp sequence were obtained by PCR using DNA from EB isolates CB361-80 and CC372-83. Partial 16S rRNA gene sequencing and their analysis via BLASTn in NCBI database revealed that EB isolates (CB361-80) and (CC372-83) have sequences that are highly similar (99%) to those of Ochrobactrum pseudintermedium and Pantoea agglomerans, respectively. Therefore, these EB isolates were identified as Ochrobactrum sp. and Pantoea sp. due to the complex taxonomy of genus Ochrobactrum and Pantoea. The 16S rRNA sequences of the EB determined in this study have been deposited in the GenBank database under accession numbers KX589254-KX589255.

Endophytic bacteria have been identified in several different genera that include both non-pathogens and plant/human pathogens (Rosenblueth and Martínez-Romero 2006; Melnick et al. 2008; Assumpção et al. 2009). According to sequence analysis of the 16S rRNA of the EB isolates, endophytes of CB361-80 and CC372-83 isolated from cucumber plants were identified as Ochrobactrum sp. and Pantoea sp., respectively. The previous studies reported that 16S rRNA gene sequence-based bacterial identification can better identify poorly described, rarely isolated, or phenotypically aberrant strains. It was also considered as a precise method of bacterial identification because it can provide bacterium species-specific signature (Clarridge 2004; Preveena and Bhore 2013). However, because of the complex taxonomy of genus Ochrobactrum and Pantoea, it is necessary to perform other identification tests prior to confirming their identity, such as the use of the approach of Teyssier et al. (2007). According to this study, 16S rRNA gene sequence-based identification method of bacteria could be used for rapid identification, but it is necessary to perform other special identification tests prior to confirming bacterial identity. This method is not enough alone to approve to the identity of bacterial species in some genera. In the next phase of this study, the further diagnostic tests i.e. morphological, physiological, and biochemical tests for these bacterial isolates will be perform to confirm their identity.

Promising experimental data have shown that the application of antagonistic bacteria could serve as an alternative approach to the biological control of plant diseases (Poon et al. 1977; Chen et al. 1995; Sturz and Matheson 1996). It has been reported that Pseudomonas putida 89B-27 and Serratia marcescens 90-166 could reduce angular leaf spot disease severity and incidence in cucumber via rhizobacteria-mediated induced systemic resistance (ISR) (Liu et al. 1995a). The ability of EB to suppress disease in cucumber depends mainly on its antagonism against the pathogen (Psl) colonization in the internal tissues and on its production of various inhibitory substances and bacteriocins. It is possible that several mechanisms such as antibacterial compounds, siderophore production, nutrient competition, niche exclusion, and induction of systemic resistance (ISR) play a role in the biological control demonstrated by endophytes (Cook and Baker 1983). The results of our previous study indicated that while isolates CB361-80 and CC372-83 produced IAA and siderophores and solubilize phosphate, it did not produce effective antibacterial compounds. These properties have been directly or indirectly correlated with their biocontrol activity of the two endophytes (Ozaktan et al. 2015). Some of these mechanisms play a role in the biological control of Psl demonstrated by CB361-80 and CC372-83 isolates. Our results suggest that ISR or nutrient competition may be important factors of biocontrol of Psl by these endophyte isolates.

Results demonstrated that both EB strains colonized internal cucumber host tissues. This study also showed that CB361-80 and CC372-83 could form and sustain populations in the internal root and shoot tissues of both Psl-infected and uninfected cucumber plants. The EB population density in surface-sterilized cucumber seeds was in the range of 3 × 10⁶–7.1 × 10⁷ CFU/g, so EB successfully penetrated the cucumber seeds. EB populations in cucumber shoots and roots changed over time, but on average, they colonized the plant organs at the rate of 10⁴–10⁵ CFU/g under growth chamber conditions over 30 days from sowing. In this study, the large standard errors are indicative of variation among replications within some treatments of the colonization studies presented here in (Figs. 2a, b and 3). The lower populations were included as zero in the data set. Chen et al. 1995 suggested that endophytic populations, particularly at lower levels, were probably underestimated. Our results corroborate those of previous research on EB colonization rates on sugarcane (Dong et al. 1994), alfalfa (Gagné et al. 1987), and cotton (Misaghi and Donndelinger 1990; Mclnroy and Kloepper 1995). Previous studies suggested that EB population densities vary not only among plant species (Quadt-Hallmann and Kloepper 1996) but also in different plant organs (highest in roots and lowest in shoots (Mclnroy and Kloepper 1995). Nevertheless, our data on CB361-80 and CC372-83 population density did not significantly differ between cucumber root and shoot tissues. In a previous study, it was reported that Pantoea sp. and Ochrobactrum sp. effectively colonized the internal rice root tissues. Nevertheless, Pantoea sp. was more effective at colonizing rice tissues than Ochrobactrum sp. (Assumpção et al. 2009). These studies showed that the EB P. agglomerans LRC 8311 reduced bacterial wilt disease severity in bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens and very effectively colonized bean seedlings (Hsieh et al. 2005). Ochrobactrum sp. colonized internal soybean tissues and was antagonistic to three phytopathogenic fungi: Fusarium oxysporum (soybean, cotton, and bean), Fusarium semitectum, and Cercospora kikuchii (both soybean) according to Assumpção et al. (2009). The ability of bacterial endophytes to colonize internal host plant tissues may in part account for their effectiveness in controlling the systemic invasion of angular leaf spot. Competition between Psl and endophytic isolate CC372-83 tends to reduce the pathogen population in their common habitat. This fact could be strong evidence for the biocontrol ability of the endophyte CC372-83 against Psl.

Both EB isolates decreased disease severity caused by Psl on cucumber plants for the result of in vivo tests. Nevertheless, while CC372-83 had decreased Psl population density to a level tenfold less than that in plants treated with Psl alone, CB361-80 has not decreased Psl population density. In addition, the population densities of CB361-80 and CC372-83 inside the cucumber tissues have not decreased following Psl inoculation. (Pieterse et al. 1998) reported that disease severity suppression without a concomitant decrease in pathogen population density could be explained by the presence of rhizobacteria inducing systemic resistance (ISR) in the host plants. Bacterial endophytes and their roles in ISR have been reviewed by Kloepper and Ryu 2006. This study suggests the possibility that enhanced host defenses will be responsible for the reduction of symptom severity of Psl because of other mechanisms, such as antibacterial compounds, nutrition competition, and niche exclusion which are less likely according to in vitro test results.

EB inoculation should promote the establishment of the microorganism in the soil and on the plant and support host penetration and colonization. Seed inoculation is an efficient and convenient way of introducing EB to plants (Bejarano et al. 2014). Our results showed that EB applied to the pathosystem via seed bacterization and soil drenching effectively inhibited Psl disease symptom development in cucumber. When the endophyte population began to decrease inside the plants, the soil drenching treatment with endophytes is applied to the plants. Following that, the populations of endophytes in plant shoot and root maintained not to change too much until the end of the experiments. This situation of endophyte populations could be sourced from shortly monitoring time (only 14 days) after treatment.