Application of the Scholander pressure bomb to studies on endophytic bacteria of plants

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Abstract
The Scholander pressure bomb system, which expresses vascular plant sap, was compared with the trituration method, in which roots are surface disinfested and triturated, for recovery of endophytic bacteria. The two methods were compared for recovery of indigenous and introduced endophytes from roots of several plant genera. The pressure bomb method was acceptable for routine recovery of endophytes from cotton (Gossypium hirsutum), soybean (Glycine max), and bean (Phaseolus vulgaris), but owing to tissue collapse under pressure, the method did not work reliably for cucumber (Cucumis sativa) or tomato (Lycopersicon esculentum) seedlings. High bacterial densities on the root surface, experimentally obtained by dipping cotton roots into a suspension of Enterobacter asburiae JM22 immediately prior to processing, did not affect the population densities of recovered indigenous endophytic bacteria by the pressure bomb technique but resulted in increased bacterial densities for the trituration method. Internal populations of JM22 following application as a seed treatment were statistically equivalent with the trituration and pressure bomb techniques. Analysis of taxonomic diversity of a group of indigenous endophytes recovered with the trituration and pressure bomb techniques indicated some differences between the two groups. The total number of bacterial genera and species recovered was greater using the pressure bomb method. Gram-positive species, such as Bacillus spp., were more frequently isolated with the trituration method than with the pressure bomb method. Agrobacterium radiobacter and less common species were more often isolated using the pressure bomb technique. Pseudomonas spp. and Phyllobacterium spp. were recovered with equal frequencies using both techniques. These results suggest that the two techniques sample two different internal habitats available for colonization by endophytic bacteria, i.e., the trituration method recovering mainly endophytes residing in the root cortex and the pressure bomb method detecting vascular colonists. A combination of both methods is recommended for understanding the full pattern of internal plant colonization by endophytic bacteria.Key words: endophytic bacteria, Scholander pressure bomb, isolation method, cotton.