plant surface microbiology.pdf

490
Donna M. Penrose and Bernard R. Glick
Table 1. ACC-utilizing bacterial strains isolated from Waterloo, Ontario, Canada and
California, USA
Genus and species Strain Soil location Plant source
Pseudomonas putida UW1 Waterloo, Ontario, Canada Bean
Enterobacter cloacae UW2 Waterloo, Ontario, Canada Clover
Pseudomonas putida UW3 Waterloo, Ontario, Canada Maize
Enterobacter cloacae UW4 Waterloo, Ontario, Canada Reeds
Pseudomonas fluorescens CAL1 San Benito, California, USA Oats
Enterobacter cloacae CAL2 King City, California, USA Tomato
Enterobacter cloacae CAL3 Fresco, California, USA Cotton
Our method of isolating bacteria entails screening soil bacteria for the ability
to use ACC as a sole nitrogen source, a trait that is a consequence of the
presence of the activity of the enzyme, ACC deaminase. One gram of soil is
added to 50 ml of sterile medium containing 10 g proteose peptone, 10 g
casein hydrolysate, 1.5 g anhydrous MgSO 4 , 1.5 g K 2 HPO 4 and 10 ml glycerol
(PAF medium) in a 250-ml flask. The flask and its contents are incubated in a
shaking water bath (200 rpm) at either 25 or 30 °C depending on the geographic
location of the soil samples, i.e., the samples collected in the cooler
Canadian climate of Waterloo, Ontario were grown at 25 °C and those from
the warmer weather of California, USA were grown at 30 °C.After 24-h, a 1-ml
aliquot is removed from the growing culture, transferred to 50 ml of sterile
PAF medium in a 250-ml flask and incubated at 200 rpm in a shaking water
bath for 24 h, at either 25 or 30 °C, the same temperature as the first incubation.
Following these two incubations, the population of pseudomonads is
enriched and the number of fungi in the culture is reduced.
A 1-ml aliquot is removed from the second culture and transferred to a
250-ml flask containing 50 ml of sterile minimal medium, DF salts (Dworkin
and Foster 1958; per litre): 4.0 g KH 2 PO 4 , 6.0 g Na 2 HPO 4 , 0.2 g MgSO 4 ·7H 2 O,
2.0 g glucose, 2.0 g gluconic acid and 2.0 g citric acid with trace elements: 1 mg
FeSO 4·7H 2O, 10 mg H 3BO 3, 11.19 mg MnSO 4·H 2O, 124.6 mg ZnSO 4·7H 2O,
78.22 mg CuSO 4·5H 2O, 10 mg MoO 3, pH 7.2 and 2.0 g (NH 4) 2SO 4 as a nitrogen
source. In our lab the DF minimal medium is prepared as follows: (1) the trace
elements (10 mg H 3BO 3, 11.19 mg MnSO 4◊H 2O, 124.6 mg ZnSO 4◊7H 2O,
78.22 mg CuSO 4◊5H 2O, and 10 mg MoO 3) are dissolved in 100 ml of sterile distilled
water and then stored in the refrigerator for up to several months; (2)
FeSO 4 ◊7H 2 O (1 mg) is dissolved in 10 ml of sterile distilled water and is stored
in the refrigerator for up to several months; (3) all of the other ingredients
including 4.0 g KH 2PO 4, 6.0 g Na 2HPO 4, 0.2 g MgSO 4·7H 2O, 2.0 g glucose, 2.0 g
gluconic acid, 2.0 g citric acid, 2.0 g (NH 4) 2SO 4 and 0.1 ml of each of the solutions
of trace elements and FeSO 4◊7H 2O are dissolved in 1 l of distilled water