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Sample collection and isolation

Knot samples were collected from infected olive trees in Marmara regions of Turkey between the years of 2010-2015: Balıkesir (Bandırma, Burhaniye, Edremit, Erdek), Çanakkale (Ayvacık, Geyikli, Ezine, Küçükkuyu, Gökçeada), Aegean (İzmir (Karaburun), Manisa (Akhisar) and Mediterranean (Hatay (Iskenderun). Symptoms observed were recorded.

Greenish-grey young knots from shoots, leaf or fruit stalks and fruit spots were surface disinfected, cut aseptically to small pieces (1-2 mm) and macerated in 1 mL sterile distilled water. An aliquot (50 µL) of suspension was streaked on King’s medium B (KB; King et al., 1954) and PVF-1 agar (Surico & Lavermicocca, 1989). Plates were incubated at 26°C for 3 to 5 days. Presumptive weak fluorescent, creamy colonies were selected and purified on KB medium. Isolates were stored in 20% glycerol at –80°C for further identification.

 

Identification of bacterial isolates

Biochemical tests. LOPAT (levan production, oxidase, potato soft rot, arginine dihydrolase and hypersensitivity reaction on ‘’White Burley’’ tobacco leaves) and some additional biochemical tests growth at 37°C; hydrolysis of gelatin and aesculin; acid from carbohydrates (melezitose, inositol, arabinose, dulcit, raffinose, sucrose, xylose, ethanol); growth at 2% NaCl were performed as recommended by Lelliott & Stead (1987).

PCR. Genomic DNAs were extracted from pure cultures of selected isolates. A cell suspension from each putative strain was prepared from 24 hr-old bacteria grown on KB medium. Bacterial cells were lysed by heating 15 min at 95°C, and subsequently cool on ice for 10 min. Then suspensions were centrifuged for 1 min at 10,000 rpm and supernatants were used as target DNA in the PCR. DNAs were stored at -20°C until usage. Two specific primers designed by Penyalver et al. (2000) — iaaF (5’-GGCACCAGCGGCAACATCAA-3’); iaaLR (5’-CGCCCTCGGAACTGCCATAC-3’) of the bacterial gene iaaL responsible for indole-3-acetyl-L-lysine synthetase ın IAA biosynthesis — were used for DNA amplification. PCR reaction mixture in a final volume of 20 µL included 10× Taq buffer (Thermo), 1.5 mM MgCl2, 5% formamide, 0.2 mM concentrations of each deoxynucleoside triphosphate (Thermo), 0.6 μM concentrations of each primer, and 1.5 U of Taq DNA polymerase (Thermo) per reaction. Then, 5 μL of the DNA extract from pure culture was added to each reaction. The PCR was performed in a thermal cycler (Eppendorf Master cycler Gradient) with the following program: 1 cycle of 94°C (5 min), followed by 35 cycles of 94°C (30 s), 62°C (30 s), and 72°C (30 s) and then 1 cycle of 72°C for 5 min. PCR-products were separated on electrophoresis gel (1.5% agarose). The gel was stained with 5 mg mL-1 ethidium bromide and DNA bands were visualized under an ultraviolet (UV) transilluminator. The expected fragment length of amplicons was 454 bp.

 

Pathogenicity tests

The isolates were inoculated on the 6-month-old olive plant (cv. ‘Manzanilla’) to verify their pathogenicity. The inoculum was prepared from 24-48 hr old bacterial growth on nutrient agar (NA) medium at 27 ± 2°C, suspended in sterile distilled water (SDW) and spectrophotometrically adjusted to 106 CFU mL-1. The young plants were stem-inoculated by putting 10 μL aliquots of bacterial suspension into wounds open in stem bark with a sterile scalpel. Three plants per isolate and 3 wounded sites per plant were inoculated. SDW was used for inoculation of control plants. Inoculated plants were kept at 60-80% relative humidity, 22-26°C, and 16 hr daylight until knot formation. Approximately 4 weeks post-inoculation the disease symptoms were recorded.

Immature healthy fruits ’Ayvalık’ were inoculated by injecting 5 μL aliquots of bacterial suspensions prepared as described above, in order to confirm the ability of the pathogen to cause fruit spots on olive fruits. Three isolates from spots on fruit ’Ayvalık’ (Ayvacık, Aegean region) were used. For each isolate 5 fruits were injected at 4 points per fruit. Control fruits were inoculated with SDW. Inoculated fruits were kept in a humid chamber at 27 ± 2°C. Ten days later the symptoms were recorded.

 

Virulence tests

Virulence tests were performed with selected pathogenic isolates in order to determine high virulent ones for further cultivar susceptibility experiments. The isolates representing different geographical areas with various environmental conditions or distinct locations in the same area and/or isolated from different plant parts were included in these tests. In general, for the same orchard only one isolate from the knots was chosen. A second isolate was included only if they were isolated from fruit spots which were not common for all orchards.

Comparison of the means of the average knot weights (g) of all tested isolates from diverse locations indicated significant differences in the ANOVA test (Tukey’s HSD test, p ≤ 0.05). Akhisar, Edremit, Burhaniye and Hatay isolates were in the same statistical group, but they differed from Ayvacık, Erdek and Karaburun isolates in the weight of produced knots (Fig. 2).

 

 

Figure 1.  Knot weights generated by selected Pseudomonas savastanoi pv. savastanoi isolates in virulence tests on cv. ‘Manzanilla’. Isolates: Akh1, Akh2, Akh3, Akh6, Akh7b from Akhisar (Manisa, the Aegean region); Ayv1, Ayv2, Ayv3, Ayv4a, Ayv5a, Ayv6 from Ayvacık (Çanakkale, Marmara region); Bur 2, Bur 2a, Bur 5 from Burhaniye (Balıkesir, the Marmara region); Edrmt 1, Edrmt 3, Edrmt 4, Edrmt 4a, Edrmt 6, Edrmt 6d from Edremit (Balıkesir, the Marmara region); Erd 1, Erd 2, Erd 3, Erd 5, Erd 7b, Erd 9, Erd 10, Erd 12 from Erdek (Balıkesir, the Marmara region); Hat1, Hat2; Hat3; Hat8 from Hatay (the Mediterranean region); Kar1, Kar3, Kar4, Kar7, Ka8, Kar10 from Karaburun (İzmir, the Aegean region). Isolates names marked only with number indicate they were isolated from knots, and those marked with number and letter were isolated from fruit spots. Psav: P. savastanoi pv nerii (Kavak & Ustun, 2009) positive control. Negative control: SDW (not included in the graph). Different letters above the histogram bars indicate differences between groups at p ≤ 0.05 (Tukey’s HSD test).

 

 

Figure 2.  Average knot weights (g) of Pseudomonas savastanoi pv. savastanoi isolates from different locations of Turkey. Average knot weight values (g) were calculated as a mean for all selected isolates from each location. Number of tested isolates for each location are: Akhisar (5), from Manisa, Aegean region; Burhaniye (3), Edremit (6) and Erdek (8) from Balıkesir, Marmara region; Ayvacık (6) from Çanakkale, Marmara region; Karaburun (6) from Izmir, Aegean region; Hatay (4) from Mediterranean region. Psav: P. savastanoi pv nerii, positive control. Negative control: water. Different letters above the histogram bars indicate differences between groups at p ≤ 0.05 (Tukey’s HSD test).

 

 

IAA biosynthesis of the isolates

IAA production by different isolates was determined using Salkowski’s reagent (Gordon & Weber, 1951; Mayer, 1958). The bacterial suspensions containing 108 CFU mL-1 from 24 hr old freshly grown cultures were prepared, and 0.1 mL from each suspension was transferred to 500 mL Erlenmeyer flasks including liquid 200 mL nutrient broth medi lm supplemented with 0.5 M L-tryptophan. Bacterial suspensions were shaken at 120 rpm at 28°C for 24 hr. The broth was then centrifuged for 5 min at 10,000 rpm and in the supernatant equal volume of Salkowski’s reagent (1.0 mL 0.5 M FeCl2 solution + 50 mL of 35% of perchloric acid) was added. The contents were mixed and allowed to stand at room temperature for 30 min to develop color. The OD was then recorded at 535 nm. Uninoculated broth served as control. Standard curve was prepared with 0-1000 (0, 10, 20, 30, 40, 50, 60, 70, 100, 200, 1000) μg mL-1 of IAA (Sigma Chemicals). The linear regression equation y=bx+a of the curve was used for quantification.

Absorbance values for 45 isolates ̶ 43 olive isolates; P. savastanoi pv. nerii (Kavak & Ustun, 2009) as positive control; and P. syringae pv lacrymans (Plac) as negative control, because it does not produce IAA ̶ were compared with the IAA standard curve and the IAA quantity of each strain was determined by using the formula y=bx+a of the linear regression equation.

 

 

Figure 3.  Absorbance values and IAA amount of Pseudomonas savastanoi pv. savastanoi isolates from the Aegean, Marmara and Mediterranean regions of Turkey. Isolates: Akh2, Akh3, Akh 4, Akh6, Akh7, Akh 12 from Akhisar (Manisa, Aegean region); Ayv1, Ayv2, Ayv3, Ayv4, Ayv5 from Ayvacık and GA1 and GA 2 from Gökçeada (Çanakkale, Marmara region); Bur 2, Bur 5 from Burhaniye (Balıkesir, Marmara region ); Edrmt 1, Edrmt 3, Edrmt 4, Edrmt 4a, Edrmt 6, Edrmt 6d from Edremit (Balıkesir, Marmara region); Erd 1, Erd 2, Erd 3, Erd 5, Erd 6, Erd 7, Erd 8, Erd 9, Erd 10 from Erdek (Balıkesir, Marmara region); Hat1, Hat2; Hat3; Hat 4, Hat 5, Hat 8 from Hatay (Mediterranean region); Kar1, Kar2, Kar4, Kar7, Ka8 from Karaburun (Izmir, Aegean region). Absorbance and IAA values of each isolates are means of three replications. Different letters above the histogram bars indicate differences between groups at p ≤ 0.05 (Tukey’s HSD test). Psav: positive control. Plac: negative control.

 

 

Figure 4.  Average IAA values of Pseudomonas savastanoi pv. savastanoi isolates from different locations of Turkey. IAA values were calculated as a mean for all isolates from each location. The numbers of isolates tested for each location are: Karaburun (5), Burhaniye (2), Edremit (7), Erdek (9), Hatay (6), Ayvacık (5), Gökçeada (2), Akhisar (5). Psav: positive control. Plac: negative control. Different letters above the histogram bars indicate differences between groups at p ≤ 0.05 (Tukey’s HSD test).

 

 

Cultivar susceptibility

The ten following native common grown olive genotypes were evaluated in two separate experiments: ‘Ayvalık’ (olive oil, Aegean Region ), ‘Domat’ (table consumption, Aegean Region), ‘Edincik’ (table consumption, Marmara Region), ‘Gemlik’ (olive oil and table consumption, Marmara and others regions), ‘Memecik’, ‘Memeli’ and ‘Uslu’ (olive oil and table consumption, Aegean Region), ‘Kilis’ and ‘Saurani’ (olive oil, Mediterranean Region), ‘Sari Ulak’ (table consumption, Mediterranean Region). The cultivar ‘Manzanilla’ (introduced) was used as control.

In the first experiment ‘Manzanilla’, ‘Ayvalık’, ‘Gemlik’, ‘Sarı Ulak’, ‘Memecik’, ‘Domat’ and ‘Memeli’ were inoculated separately with two isolates (Akh3 and Hat 3b) defined as the most virulent in virulence tests. In the second experiment ‘Edincik’, ‘Saurani’, ‘Kilis’, and ‘Uslu’ and the same isolates (Akh3 and Hat 8) were used. For inoculum preparation, bacterial isolates were grown on NA for 24 h and suspended in SDW. The suspensions were spectrophotometrically adjusted to 106 CFU mL-1. Stems and shoots of 2-yr-old plants of each genotype were wounded (10 wounds per plant) with a sterile scalpel and 0.1 mL of bacterial suspensions were put into wounds. For control plants, SDW was used instead of bacterial suspensions. Ten plants (2 plants × 5 replications) per genotype were inoculated by each isolate. Three months post-inoculation knots were cut and weighed separately and the average knot weight (g) per plant was estimated.

The genotypes were evaluated, as proposed by Penyalver et al. (2006), as highly susceptible (H; cultivars with significantly higher weight of knots per diseased plant than the average value of the trial), medium susceptible (M; cultivars with no significant differences with the average value), and low susceptible (L; cultivars with a significantly lower weight than the average value).

 

Statistical analysis

Analysis of variance (ANOVA) was performed by using XLSTAT software (by Addinsoft). Tukey’s HSD (Tukey's honestly significant difference test) tests (p ≤ 0.05) were used to define the differences between the isolates in virulence tests (knot weights) and their IAA biosynthesis. The test was used also to compare the genotypes for their susceptibility to two different isolates. The linear regression equation y=bx+a was used to quantify the IAA amount produced by each isolate.

Pearson correlation coefficient (Pearson, 1895) was calculated to determine the correlation between IAA biosynthesis and knot weight reproduced by the isolates by using the formula r = ∑((X-My)(Y-Mx))/√(SSx)(SSy) , (r= correlation coefficient; x = X values; y = Y values; Mx = mean of X values; My = mean of Y values; X-Mx & Y-My = deviation scores; (X-Mx)2 & (Y-My)2 = deviation squared; (X-M_x)(Y-M_y)= product of deviation scores). Scores near zero mean no (or weak) correlation; scores near 1 positive correlation; and -1 negative correlation.

In Exp. 2, a one-way ANOVA was performed to study the effect of each application method (seed coating and soil drenching) on the length of cotyledon for each fungus separately against Control. No statistical analysis was performed for comparing wheat seed germination since all of them germinated.

In Exp. 3, the number of plants colonized by EPF (B. bassiana or M. brunneum) depending on factor combinations [soil S/NS × seed D/ND] were analysed by Chi-square test (independent group), while the differences between sampling dates were performed through Cochran tests (dependent group). It should be noted that Control plants were not included in the analysis because fungal colonization was always zero, as expected. Finally, the leaf chlorophyll content (SPAD values) was analysed by a factorial ANOVA with fungus, sterilization, and disinfection as factors. The fungus × disinfection interaction was significant in the first factorial and let us split the data into plants whose seeds were previously disinfected and non-disinfected and perform a second factorial ANOVA with fungus and sterilization as factors. The comparison was made between Control against each fungus, separately.

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