Influence of Pythium oligandrum biocontrol on fungal and oomycete population dynamics in the rhizosphere

Abstract

Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.

FIG. 1.

Phylogenetic tree of the Pythium strains used in this study (in bold), based on ITS-1 and -2 and the 5.8S gene of nuclear rDNA. The sequences of representative strains (CBS numbers) and strains from the LUBEM laboratory (LMSA numbers) were compared against all available entries in GenBank (GI numbers), as described by Lévesque and De Cock (26). Recently reported P. oligandrum sequences are included. The strains used in this study are shown in bold with an asterisk. This is one of 18 equally parsimonious trees (length, 147; consistency index, 0.925; retention index, 0.926; rescaled consistency index, 0.856).

FIG. 2.

Electrophoretic patterns on polyacrylamide gels and corresponding immunoblots of concentrated culture filtrates of P. oligandrum strains. (a and b) Coomassie blue-stained 12.5% SDS-PAGE gels. The same culture filtrates were subjected to 12.5% SDS-PAGE, and the proteins were transferred to nitrocellulose membranes. (c and d) The membranes were probed with rabbit anti-oligandrin serum. Each lane was loaded with 25 μg of proteins. Lanes M, molecular mass standard (Bio-Rad); other lanes, culture filtrates of the Pythium strains listed in Table 3.

FIG. 3.

(a) Root colonization (%) by P. dissotocum (a1) and P. oligandrum (a2), assessed by plate counting. The values reported are means for eight samples collected each month. The signed rank test indicates preferential colonization of the control plants by P. dissotocum (P = 0.029) and a more extensive colonization of the inoculated plants by P. oligandrum (P = 0.0000123). (b) Root colonization by P. dissotocum (b1) and P. oligandrum (b2), assessed by real-time PCR. DNA quantities correspond to amounts of Pythium DNA (fg) per unit of tomato DNA (ng). Median values are represented on the graphs (medians are suitable markers for dissymmetrical distributions). P. oligandrum DNA quantities were higher on inoculated than on control plants (signed rank test; P = 0.0000123), and those of P. dissotocum on control and inoculated plants were not significantly different.

FIG. 4.

Pythium oligandrum ISSR electrophoretic patterns. Lane 1, BenchTop 100-bp ladder (Promega, France); lane 2, P. dissotocum LMSA 1.01.531; lane 3, P. oligandrum CBS 109981; lane 4, P. oligandrum CBS 530.74; lane 5, P. oligandrum LMSA 1.01.631; lanes 6 to 10, P. oligandrum strains collected from tomato roots at the end of the cropping season.

FIG. 5.

SSCP fingerprints of fungal microfloras from control and P. oligandrum-inoculated plants at the beginning (April) and end (September) of the cropping season in the two greenhouses.

FIG. 6.

Comparison of genetic structures of fungal communities of the rhizosphere associated with inoculated and control plants by PCA, using fungal 28S rRNA profiles obtained by SSCP. For each sample, the first number represents the date of sampling (1, start; 2, middle; 3, end of the cropping season), the second number refers to the compartment (1, greenhouse 1; 2, greenhouse 2), the letters T and P are for control plants and P. oligandrum-inoculated plants, respectively, and the last letter indicates the sample.