Pure isolates of all R. solanaceraum colonies were analyzed using
the DAS-ELISA technique, as described above. When the result was
positive, intracellular spaces of tobacco plant leaves were infiltrated
with aqueous suspensions of bacteria (108 cfu ml1) to test their
pathogenicity after the thermal treatments.
To determine the persistence of the pathogen in soil in association
with the host tissue (artificially infected tomato plants), three
pieces of infected stem were placed into sterilized tubes containing
6 g of the disinfected growing medium. Twelve tubes with soil were
prepared, sealed with parafilm to avoid water loss, and placed in
two incubators at 25 C and 45 C, respectively. On a weekly basis
over a 6-week period, the plant debriswas removed from two tubes
of soil incubated at each temperature. The plant pieces from each
tube were washed with sterilized water, and were homogenized
with sterilized water in a mortar. To determine the presence of
pathogenic bacteria, part of this bacterial solution was directly
infiltrated into intracellular spaces of tobacco leaves (about 200 ml
in each leaf) to measure phytopathogenicity. The other part of the
suspension was stored at 6 C.
For bacterial suspensions showing negative results in tobacco,
the remaining suspension was serially diluted and plated as
described above to ensure that no R. solanacearum colonies were
obtained.
To determine the persistence of R. solanacearum in association
with the host tissue, one piece of infected stem was put into each
sterilized Eppendorf tube to be processed as described above.
A total of 12 pieces of infected stems were used in the experiment.
Pure isolates of all R. solanaceraum colonies were analyzed using
the DAS-ELISA technique, as described above. When the result was
positive, intracellular spaces of tobacco plant leaves were infiltrated
with aqueous suspensions of bacteria (108 cfu ml1) to test their
pathogenicity after the thermal treatments.
To determine the persistence of the pathogen in soil in association
with the host tissue (artificially infected tomato plants), three
pieces of infected stem were placed into sterilized tubes containing
6 g of the disinfected growing medium. Twelve tubes with soil were
prepared, sealed with parafilm to avoid water loss, and placed in
two incubators at 25 C and 45 C, respectively. On a weekly basis
over a 6-week period, the plant debriswas removed from two tubes
of soil incubated at each temperature. The plant pieces from each
tube were washed with sterilized water, and were homogenized
with sterilized water in a mortar. To determine the presence of
pathogenic bacteria, part of this bacterial solution was directly
infiltrated into intracellular spaces of tobacco leaves (about 200 ml
in each leaf) to measure phytopathogenicity. The other part of the
suspension was stored at 6 C.
For bacterial suspensions showing negative results in tobacco,
the remaining suspension was serially diluted and plated as
described above to ensure that no R. solanacearum colonies were
obtained.
To determine the persistence of R. solanacearum in association
with the host tissue, one piece of infected stem was put into each
sterilized Eppendorf tube to be processed as described above.
A total of 12 pieces of infected stems were used in the experiment.
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