CAPÍTULO 2: DESARROLLO TEMATICO
III. RESULTADOS Y ANALISIS DE LOS HALLAZGOS
3.1. Resultado del estudio
2.5.1. Plant cultivars
Cultivars were chosen by the experimental stations for vegetables to be disease susceptible and depended on availability.
All plant assays with X. campestris pv. campestris were performed in cauliflower plants. Cultivars Clarina (Syngenta), Clapton (Syngenta) and Clarify (Syngenta) were chosen based on their resistance to clubroot (Plasmodiophora brassicae) as this pathogen caused major problems at Inagro in 2014 making disease assessment impossible. Furthermore, cultivars Balboa (Bejo), Clarina (Syngenta) and Giewont (Seminis) had demonstrated to be disease susceptible (Table 2.1).
The leek assays with P. syringae pv. porri were performed using the disease susceptible cultivars Harston (Nunhems), Kenton (Nunhems), Krypton (Nunhems) and Striker (Bejo) (Table 2.2).
Cultivar Seed company
2012 PCG 2013 Inagro 2015 PCG 2015 Inagro LMG 568 LMG 28776 LMG 28775 LMG 28773 Balboa Bejo -- - - Fortaleza Seminis - +/- -- Freebel Seminis - -- Giewont Seminis - - +/- --
Dexter Rijk Zwaan - -
Bonique Enza +/- +/-
Clarina Syngenta +/- +/- - +/-
Gohan Syngenta +/- -
Moby Dick Clause +/- +/-
Naruto Clause +/- +/- Seoul Hazera +/- +/- - Octopus Clause +/- + +/- Anique Enza +/- + Caddilac Syngenta + +/- Raoul Hazera + -- + David Syngenta - + +/-
Table 2.1 Cauliflower cultivars and their susceptibility to the bacterial pathogen X. campestris pv. campestris. -- very susceptible (red), - susceptible (orange), +/- moderately susceptible (yellow), + little susceptible (light green), ++ not susceptible (dark green). The scores were assigned by the experimental stations for vegetables based on field trials using different bacterial strains.
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Cultivar
Seed company
2012 2012 2012 2013 2013 2014 2015 2015 PSKW PCG PCG PSKW Inagro Inagro Inagro Inagro
Late autumn Late autumn Winter Late autumn Late autumn Early autumn Early autumn Late autumn + winter Belton Nunhems - +/- - +/- + Duraton Nunhems - +/- Harston Nunhems + -- + - - Jumper Bejo - - Kenton Nunhems +/- - Krypton Nunhems - +/- +/- - Lancaster Uniseeds - -- +/- Levis Syngenta -- +/- -- Mercurian Syngenta - +/- Poulton Nunhems +/- - - - + Striker Bejo - - Celcius Seminis +/-- + +/- Delmas Syngenta +/- +/- + - + Hawking Seminis +/- +/- + Lucretius Seminis - + - Oberon Syngenta +/- + - Walton Nunhems - +/- + + Aylton Nunhems +/- + + + +/- Curling Bejo + + +/- + Pluston Nunhems + +/- +/- + + + + Vitaton Nunhems + +/- + +/- -
2.5.2. Phage assays on cauliflower transplants and leek leaves
The ability of the X. campestris pv. campestris phages to prevent vascular infection was tested on cauliflower transplants with 3 to 4 mature leaves, cultivar Clarina. Transplants were planted in soil and prepared by growth under dry conditions to stimulate adsorption. Per phage, a droplet with a concentration of 107 CFU/ml of the host bacteria suspended in phage
buffer was placed onto the leaf axil of a cotyledon. A needle was used to stab through the droplet into the plant stem in order to stimulate vascular infection. Immediately after, a 50 µl droplet with a concentration of 109 pfu/ml phage was placed onto the leaf axil of a mature
leaf situated higher on the stem and again vascular entrance was stimulated by stabbing through the droplet with a sterile needle. Ten plants were inoculated per phage and the
Table 2.2 Leek cultivars and their susceptibility to the bacterial pathogen P. syringae pv. porri strain CFBP 1687. -- very susceptible (red), - susceptible (orange), +/- moderately susceptible (yellow), + little susceptible (light green), ++ not susceptible (dark green). The scores were assigned by the experimental stations for vegetables based on field trials.
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experiment was independently repeated on cauliflower plants cultivar Giewont using the same setup. Inoculations with only bacteria, only phage or buffer served as positive and negative controls. Plants were kept in the greenhouse at 25°C. Symptom development was monitored and disease severity was recorded after 8 and 13 days by counting the number of affected leaves.
In planta activity of the P. syringae pv. porri phages was tested by infiltration of phage and
bacteria suspensions into leek leaves. Leek plants of cultivar ‘Krypton’ were grown in separate pots until they had three fully developed leaves. Next, 0.1 ml bacterial suspension with a concentration of 107 CFU/ml was injected with a syringe into the leave. Subsequently, 3 cm
above the first injection spot, 0.1 ml of the phage suspension with a concentration of 109
pfu/ml was injected. Per phage, eight leaves were inoculated distributed over three plants. Plants were covered for 48 h with a plastic bag to maintain humid conditions and were kept at a temperature of 25 °C. Lesion lengths were measured after 10 days. Injections with only bacteria, only phage or buffer served as positive and negative controls.
This experiment was repeated but with a bacterial concentration of 106 CFU/ml and strains
CFBP 1687 and LMG 28496 as host. This time the plant cultivar ‘Striker’ was used and 10 leaves were injected per phage. The rest of the parameters remained the same.
2.5.3. Pot trials
Three types of pot trials were performed in order to obtain information about the mode of action of the phages in disease control. In a first trial, phages were poured into pots followed by bacteria after two hours to check the ability of the phages to prevent root infection. In a second trial, phages and bacteria were sprayed onto the leaves to investigate phage ability to control leaf infection and in a third trial, phages were added to pots followed by spraying leaves with bacteria in order to look for systemic movement of phages in plants leading to the prevention of leaf infection.
In cauliflower, the pot trials were performed with transplants of cultivar Giewont, containing three to four mature leaves, using phage SoPhi2 and strains LMG 28773 as bacterial host. Per trial, 20 transplants were planted in separate pots using substrate for professional use
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(Peltracom). Experiment setup consisted of four objects (water control, only phage, only bacteria, phage + bacteria) with each 5 plants in the three trials. A volume of 50 ml was used in the first trial with a phage concentration of 2*107 pfu/ml and a bacteria concentration of
5*106 CFU/ml. In the second trial, sprayed volumes were 10 ml with a phage concentration
of 108 pfu/ml + 0.01% Tween®20 and a bacteria concentration of 5*107 CFU/ml + 0.01%
Tween®20. In the third trial, 50 ml phage (2*107 pfu/ml) was poured into the pots and 10 ml
bacteria (5*107 CFU/ml + 0.01% Tween®20) was sprayed on the leaves. In the first and third
trial, plant roots were wounded after planting by stabbing three times with a knife into the plant pot from above to stimulate root entrance of bacteria and phage. The number of affected plants and the percentage affected leaf surface were recorded 14 and 19 days post inoculation. After treatment, all plants were covered with plastic foil for 48 hours to maintain humid conditions. Plants were kept at 25°C day and night with extra light 12 hours per day (Philips TL-D36W/840, product code 8711500285614, 56.1 µmol*s-1*m-2).
The second cauliflower pot trial was repeated with 100 transplants of cultivar Giewont divided over four objects. 5 ml of phage SoPhi2 (108 pfu/ml + 0.01% Tween®20) was sprayed onto the
leaves followed by 5 ml of LMG 28773 (6*107 CFU/ml + 0.01% Tween®20) after two hours.
Symptoms were recorded 8, 12, 16, 20 and 23 days post inoculation.
In leek, the pot trials were performed with plants of cultivar Striker using phage KIL3 and strain CFBP 1687 as bacterial host. The same experiment set up was used as in the cauliflower trial. In the first trial 250 ml phage solution in tap water with a concentration of 107 pfu/ml
was added to the pots. After two hours, 200 ml bacterial solution in tap water with a concentration of 2*107 CFU/ml was added. Volumes were chosen as the maximum amount
of water that the pots could retain. Controls were treated with a same volume of tap water. In the second trial, 10 ml phage solution (4*107 pfu/ml + 0.01% Tween®20) was sprayed onto
the plant leaves while the soil was covered with plastic. After two hours, 10 ml 2*107 CFU/ml
+ 0.01% Tween®20 bacteria solution was sprayed onto the leaves in the same way. In the third trial, 250 ml phage solution (107 pfu/ml) was poured into the pots followed by spraying the
leaves with 10 ml 2*107 CFU/ml + 0.01% Tween®20. After treatment, all plants were covered
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night with extra light 12 hours per day (Philips TL-D36W/840, product code 8711500285614, 56.1 µmol*s-1*m-2). Symptom development was monitored for two months.
2.5.4. Field trials
The field trials were performed by the experimental stations for vegetables PCG (Kruishoutem; N 50.94337°, E 3.52710°), PSKW (Sint-Katelijne Waver; N 51.078120°, E 4.528180°) and Inagro (Beitem; N 50.901508°, E 3.124464°). PCG (province of East Flanders) and Inagro (province of West Flanders) are located on a sandy-loam soil type and have a maritime climate with mild winters and cool summers. PSKW is situated in the center of Flanders in the province of Antwerp and has slightly warmer summers and cooler winters and a sandy soil type (Figure 2.1). The amount of rainfall is slightly higher at PCG and Inagro compared to PSKW.
In a first trial, cauliflower transplants were treated with phages before they were planted in an infested field. The field was infested by spraying a solution of X. campestris pv. campestris strain LMG 28773 (Inagro and PSKW) or strain LMG 28775 (PCG) with a concentration of 106
CFU/ml on a field at a rate of 1 000 l/ha at 1.5 bar with 0.01% Tween® 20 added as a surfactant. Before planting, plants were sprayed with a solution containing a mix of the seven different phages, each present at a concentration of 108 pfu/ml. This resulted in a
Figure 2.1: Location of the experimental stations for vegetables in Flanders. The map roughly presents soil types in Flanders. (1) Inagro, (2) PCG and (3) PSKW.
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concentration of 8x107 phages per plant in total. This trial was performed simultaneously at
the three different locations in Flanders: PCG, PSKW and Inagro.
The same experiments were performed with leek plants. First, the field was infested by spraying a solution of CFBP 1687 with a concentration of 106 CFU/ml on a field at a rate of 1
000 l/ha at 1.5 bar with 0.01% Tween® 20 added as a surfactant. The next day, before planting plants were submerged in a solution containing a mix of the six different phages, each present at a concentration of 107 pfu/ml. This resulted in a concentration of 6*107 phages per plant
in total. Four blocks of the phage-treated and non-treated objects were randomly distributed over the field, with each block containing 300 plants. At multiple time points after planting, the number of affected plants as well as the percentage of leaf surface affected was measured for 20 to 50 plants per block. This trial was performed simultaneously at the three different locations in Flanders mentioned above.
In a second trial, cauliflower transplants were planted in a non-infested field. Two months after planting, bacterial infection was performed with strain LMG 28773 (Inagro and PSKW) or strain LMG 28775 (PCG). A suspension with a concentration of 106 CFU/ml was spread on
the plants at a rate of 1 000 l/ha at 1,5 bar with 0.01% Tween® 20 added as a surfactant. The next day, a solution with the eight different phages was sprayed onto the plants, ensuring that each plant was covered with 108 pfu of each phage after spraying. Plants were covered
for 48 h and the disease incidence as well as disease severity (% of leaf surface affected) was measured for 20 to 50 plants per block, at multiple time points. This trial was performed in parallel on the three different locations in Flanders mentioned above.
This second trial was also performed on leek infected with strain CFBP 1687. A suspension with a concentration of 106 CFU/ml was spread on the plants at a rate of 1 000 l/ha at 1,5 bar
with 0.01% Tween® 20 added as a surfactant. The next day, a solution with the six different phages each present in a concentration of 109 pfu/ml and 0.01% Tween 20 was sprayed onto
the plants, ensuring that each plant was covered with 108 pfu of each phage after spraying.
Plants were covered for 48 h and the disease incidence as well as disease severity (% of leaf surface affected) was measured for 20 to 50 plants per block, at multiple time points. This trial was also performed in parallel at the three different locations in Flanders mentioned above.
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