Biocontrol agent (3 Pseudomonas fluorescens and 2 Serratia plymuthica) were tested in vitro as well as in vivo on pear fruits and potato tubers to evaluate their efficacy against the 2 pathogens (E. amylovora and P. carotovorum).
Sampling and isolation of bacteria
Bacteria isolates were recovered from loquat leaves and from rhizosphere or soil of potato plots in different locations (Oran, Mostaganem, and Relizane) in Algeria in 2017. Small tissue fragments of leaves were macerated in distilled water for 30 min according to the procedure of Sharifazizi et al. (2017). From soil samples, bacteria isolation was carried out according to the method described by Vidhyasekaran et al. (1997). Briefly, 1 g of soil was shaken with 9 ml sterile distilled water for 6–8 s, and serial decimal dilutions were made (up to 10−6 cfu/ml). The 10−x dilution was streaked onto King’s B medium agar plates (King et al. 1954), and the plates were incubated at 26–28°C for 48 h.
Biochemical characterization of bacterial isolates
After purification, biochemical characterization of bacterial isolates was made based on standard bacteriological methods. These included oxidase reaction, arginine dihydrolase, levan formation, and nitrate reduction, fluorescent production on King’s B medium, hypersensitive reaction (HR) induction on tobacco plant (Nicotiana tabacum), and tested on potato (LOPAT test). Moreover, biochemical characterization by miniaturized systems, such as API20E and API20NE (bioMérieux, Craponne, France), was also performed.
Molecular identification of bacterial isolates
Molecular characterization of the selected bacterial isolates was performed by sequencing the 16S RNA gene (Genetic PCR Solutions™, Elche, Spain). The completed 16S rRNA gene sequencing was used for Serratia strain identification by sequencing partial 794 bp for MB1 and 803 bp for MB2 (types of primers). The sequences were compared to a database of NCBI nucleotide sequences using the Blast program (http://www.ncbi.nlm.nih.gov/). The nucleotide sequences were aligned using the ClustalW program (Tamura et al. 2013) available in the BioEdit Sequence Alignment Editor 7.0.9.0 software (http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Alignments were manually adjusted where necessary. Neighbor joining phylogenetic analysis was performed on data sets by using the MEGA version 6.06 (Molecular Evolutionary Genetics Analysis) software (http://www.megasoftware.net) (Chenna et al. 2003).
For accurate identification of isolates of the P. fluorescens complex, a set of conventional PCR protocols according to Garrido-Sanz et al. (2017) was carried out. The primers were as follows:
DGPf_0 DGPf_0F: CATCGCAATCGCACRATGATY
DGPf_0R: GAAAGTCTTGACCAGCARVAG
DGPf_1 DGPf_1F: TGCAGGRAGACGGSAARG
DGPf_1R: CCRAGGAAGCCCAGGGAN
DGPf_2 DGPf_2F: GTRGTSTTCATCGGBGGHGG
DGPf_2R: TGGCARTACCAGACGTTRTCCG
DGPf_3 DGPf_3R: CCATGGCCGACCACCACGTCATCATCAARC
DGPf_3R: GCAGTTCCCAGTCGGTKATBCGYCGGTCG
DGPf_4 DGPf_4F: CGCTGATCCTCTCGTTGTCTGC
DGPf_4R: ACGCCCTTGTCCACATCG
DGPf_5 DGPf_5F: CGGCGTGGGTGTCGATCRR
DGPf_5R: GAGTTCGCAGAAAACCGTGACCG
DGPf_6 DGPf_6F: GCSTTGCGHTAYTTCCACGAGG
DGPf_6R: GCCAGGCTYTTCTGCACYTCC
DGPf_7 DGPf_7F: CYGARATCGAGGGGCTKTGGA
DGPf_7R: GCTGAARTCTGGVAGCAGGGC
DGPf_8 DGPf_8F: CCCACCGACAGCCAGCAACG
DGPf_8R: CGGTCTTGTCGCTGATGCCG
The total PCR reaction (25 μl) comprising 2 μl dNTPs (10 mM), 0.125 μl each primer (100 mM), 2.5 μl PCR buffer (10x), 0.2 Taq DNA polymerase (5U/μl), and 5 μl genomic DNA (30 ng/μl). The PCR program included an initial denaturation at 95 °C for 5 min, followed by 30 cycles of 30 s at 95 °C for denaturation, 30 s at 64 °C for annealing (or 30 s at 67 °C for DGPf5, DGPf6 et DGPf8 primers), and 1 min and 30 s at 72 °C for extension, with a final extension at 72 °C for 7 min. After amplification, the PCR amplicon of each bacterial isolate was analyzed on a 1.5% agarose gel and sequenced by ABI 3130 XL (Applied Biosystems, CA, USA).
Bacterial strains used and growth conditions
Various strains of different phytopathogenic bacterial species were employed: E. amylovora CFBP 1430, P. carotovorum Ecc 194.
Antagonism tests against Erwinia amylovora and Pectobacterium carotovorum
To determine if the bacterial isolates showed an antagonistic effect against E. amylovora and P. carotovorum, 2 methods were employed: (1) the antagonistic activity was determined using double-layer agar according to Iacobellis et al. (2005) procedure. Briefly, 1 ml of the pathogen suspension at concentration 1 × 108 cfu/ml (48 h old) was added to 9 ml of 0.8% (v/v) semi-liquid Luria-Bertani (LB) medium. Afterwards, the mixture was poured into Petri dishes containing 1.5% solid LB and left until solidification (5 min). A colony of the candidate isolate was picked up, and the culture was deposited on the plate and incubated at 25°C for 48 h. This test was repeated twice with 3 replicates, and the diameter of the inhibition zone was assessed as described by Montesinos et al. (1996). (2) The candidate isolates were cultivated in liquid King’s B medium under slight shaking. After 48 h, the grown culture was centrifuged at 13,000 rpm for 10 min, and then, the supernatant recovered, filtered, and kept at 4°C until use. The supernatant confrontation was studied using the protocol described by Valgas et al. (2007) and Sharifazizi et al. (2017). Briefly, a 1-m volume of the pathogen suspension was diluted with 9 ml of 0.8% (v/v) semi-liquid LB medium and then, poured into Petri dishes containing 1.5% LB solid. After solidification (5 min), wells were made in the LB medium containing the pathogen. Finally, 50-μl volume of the filtrate was deposited in these wells and incubated at 25°C for 48 h. The diameter of the inhibition halo was measured as described below. The experiment was repeated 3 times.
Bioassays on immature pear fruit
Bioassays were conducted under controlled laboratory conditions on detached immature pear fruits to evaluate the effects of candidate isolates against E. amylovora infection. Firstly, pear fruits were disinfected by immersion in sodium hypochlorite 1.5% for 30 min, after washing with sterile distilled water three times. Each fruit was wounded to 5 mm of every well. Suspension of candidate isolates was prepared to 1 × 108 cfu/ml by using strain about 48 h; the fruit was dipping in this suspension for a few seconds and then, placed in a plastic box in a controlled environment chamber at 25±1 °C and high relative humidity (43%). After 24 h of incubation, the fruit wounds were inoculated with 10 μl of a suspension of E. amylovora CFBP 1430 at 1 × 107 cfu/ml (Vanneste et al. 1992; Sharifazizi et al. 2017). Sterile distilled water was used as a negative control. The boxes were incubated at 26±0.2 °C. Positive results were recorded when bacterial exudates or necrosis was detected in the well. The results were determined between 3 and 9 days.
Bioassays on potato slice
Potatoes from Spunta cultivar were used according to the method of Benada et al. (2018), with some modifications; the potato tubers were first cleaned with water and surface disinfected by soaking them in 70% ethanol and briefly passing under the flame of a Bunsen burner. Sliced cuts were made, and then, wells in each slice were made (4 wells per slice of about 2-mm diameter and 5-mm depth). A 48-h-old bacterial suspension of the candidate isolates was prepared; the potato slices were emerged in this bacterial suspension with a concentration of 108 cfu/ml for a few minutes, then placed in petri dishes containing 1 ml sterile water (water required for a high relative humidity). After a 24-h incubation period, the wells were inoculated with 10 μl of bacterial suspension (108 cfu/ml) of P. carotovorum. The plates were then wrapped with parafilm to limit gas exchange and incubated in an oven at 28 °C for 48 h. After this period, the reading was made by observing the presence or absence of rot in the wells. The number of tubers infected with soft rot was recorded and expressed as a percentage according to the formula described by Abd-El-Khair and Haggag (2007).
$$ \mathrm{Infection}\ \left(\%\right)=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{infected}\ \mathrm{tubers}}{\mathrm{Total}\ \mathrm{no}.\mathrm{of}\ \mathrm{tubers}} \times 100 $$
Statistical analysis
The results are expressed in the form of means ± SD. The differences among the inhibition zones were the subject of an analysis of variance (ANOVA), followed by a comparison of means (Dunnett’s test or Tukey’s test) when the conditions of normality and homogeneity of the variances were respected (test Kolmogorov-Smirnov), or by the non-parametric test of multiple comparisons of Kruskal-Wallis, followed by pairwise comparison with Dunn’s test post hoc, if necessary. Moreover, a Chi-square test was used to compare the infection index. Statistical significance was considered at p < 0.05, using the Statistical Package for the Social Sciences software (SPSS) (version 20, IBM).