2.4.2. Greenhouse experiments
Non-disinfected tomato seeds were germinated as indicated for
colonization experiments. Seedlings without visible contamination
were transferred to speedling trays (100 cells per tray of 25 ml each)
previously filled with pH 6.0 sterile vermiculite and placed in a
controlled growth chamber under the same conditions indicated
above. Sterile modified Hoagland’s solution with KNO3 as nitrogen
source (Hoagland and Arnon, 1950) was weekly added to each
cell throughout the experiment period in the growth chamber. Four
week-old seedlings were inoculated with 5 ml of a G. diazotrophicus
PAL 5 suspension (approximately 1.109 CFU ml−1) directly in each
cell and incubated overnight. Control plants received the same volume
of sterile water. Plants were then transplanted to the soil into
the greenhouse. Two independent experiments, from December
to April (4 months) in two consecutive seasons (2008–2009 and
2009–2010), were carried out in greenhouse with natural daylight.
Experiments comprised two treatments: non-inoculated control
and inoculated with G. diazotrophicus PAL 5. The experimental
design was a randomized complete block design with four replicates
per treatment and two plots per block with 20 plants each.
The plots were divided in 2 rows separated by a 1 m wide gap and
plants were placed at every 50 cm. Plants were not fertilized. All
treatments received water daily by overhead irrigation.
The nutrient composition of soil was: organic matter 3.45%;
organic carbon (Walkley and Black, 1974) 2.10%; Total nitrogen
(Bremner, 1960) 0.24; available Phosphorous (Bray and Kurtz,
1945) 40 ppm; nitrates 130 ppm; potassium 124 ppm; sodium
190 ppm; pH 6.2; conductivity 2.78 mho cm−1.
Crop yield was determined by measuring total tomato production
throughout the season. Fruit number and weight were
examined once a week since two months after inoculation and during
the following two months of the experiments. Tomato yield
production was expressed as means of the total measurements
per replicate during two months of evaluation. Statistical analysis
was performed using one way analysis of variance (ANOVA)
followed by Duncan’s test. P-values
≤0.05 were considered as
significant.