3.3. Determination of the persistence of the pathogen in association
with the growing medium and host tissue
After thermally treating the substrate (mix of peat moss and
sand) at 25 C, high population densities of the pathogen were detected in the YDA and TTC media (Table 2). Pure cultures from
recovered colonies were obtained and analyzed by DAS-ELISA with
positive results. The bacterial solutions from pure cultures with
a positive ELISA result were inoculated into tobacco leaves, and all
tests for pathogenicity were positive. This result showed that the
R. solanacearum cells introduced as free cells into the soil mixture
remained infective after six weeks treatment at 25 C. In contrast,
R. solanacearumwas not detected after any of the soil treatments at
45 C.
The growth of the different saprophyte bacteria recovered
during the thermal treatments was evaluated in both culture media
(Fig. 1). The number of saprophyte colonies was similar for both
thermal treatments and culture media. Neither the antibiosis
reactions were not observed in different plates nor were the
saprophyte bacteria identified by PCR and sequencing.
The effect of thermal treatment of different duration on the
population dynamics of R. solanacearum and counted saprophytes
at 25 C are shown in Fig. 2. Similar results were obtained for both
assayed nutrient media. The regression analysis for all the results
reveals a slight decrease of the pathogen and the saprophytic
populations.
After treatments at 25 C and 45 C, the bacterial suspensions
obtained directly from the recovered tomato plant residues (and
mixed with the assayed growing media) were analyzed by DASELISA.
Positive results were obtained from week one to week six
of thermal treatment. However, negative reactions were obtained
when these bacterial suspensions were infiltrated in tobacco leaves.
Similar reactions were obtained with the tomato tissues without
substrate (without soil).