Each experiment was replicated 3 times with 20 Pitavastatin solubility dmso pots in each replication. Quantification of endophytic population
of Ruboxistaurin chemical structure Lu10-1 Seedlings of mulberry raised as above were incubated in a growth chamber at 26°C, 90% RH, and 12 h of light. When the seedlings were about 10 cm tall, they were treated with Lum10-1 by drenching the soil with a 108 CFU mL-1 suspension and maintained by watering suitably in a growth chamber as described above. The control seedlings were treated with sterile LB medium. Root, stem, and leaf samples were obtained at different times after the treatment and were surfaced-disinfected as described before . The samples were triturated with a sterile mortar and pestle in potassium phosphate buffer (PB). Serial dilutions of the triturate were made in PB and the cultures grown on nutrient agar containing 100 μg mL-1 of rifampicin and streptomycin. The plates were incubated at 28°C for 48-72 h and colony counts were recorded. For each sampling date, the average of 3 plates of a given dilution was taken for calculating the number of viable cells in 1 mL suspension. For each kind of tissue, there were three replicates with five samples in each replicate. The data were analyzed as described above. Infection sites of Lu 10-1 in mulberry seedlings Mulberry seeds were surface-disinfected and germinated as described above. When no contamination was found on the plates,
it was confirmed that the seed surface was sterile. When the roots were selleck compound about 1 cm long, they were inoculated with Lu10-1 by dipping them in a cell suspension (106 CFU mL-1) for 1 h and then washed with sterile distilled water. Roots of the control seedlings were dipped in sterile distilled water. The treated seedlings were transplanted into 2.5 cm diameter Exoribonuclease tubes filled
with semisolid LB medium and incubated in a plant growth chamber at 25°C under a light regimen comprising 14 h of light alternating with 10 h of darkness. Root samples were obtained at 24 h and 48 h after inoculation. The root samples were fixed in 2.5% glutaraldehyde (v/v) in 0.05 M PB for 2 h, washed in the same buffer, and then fixed in 1% (w/v) osmium tetroxide for 1.5 h. Dehydration was effected with a graded series of ethanol (50%-100%, v/v), and the samples were dried with a critical-point dryer, mounted on stubs, and shadowed with gold (22 nm) for viewing under a SEM (JEM-S570) operating at 20 kV. All images were computer-processed. Construction of GFP-labelled Lu10-1 and microscopic observations on colonization in mulberry plant The plasmid, pGFP4412, containing one copy of constitutively expressed gfp and neomycin- and ampicillin-resistance genes in tandem, was donated by the College of Agronomy and Biotechnology, China Agricultural University, Beijing, China. This plasmid expresses the gfp genes constitutively from the rpsD promoter of Bacillus subtilis. The plasmid was introduced into Lu10-1 by electroporation as described in an earlier paper .