Detection and Investigation of Soil Biological Activity against Meloidogyne incognita

Abstract

Greenhouse experiments with two susceptible hosts of Meloidogyne incognita, a dwarf tomato and wheat, led to the identification of a soil in which the root-knot nematode population was reduced 5- to 16-fold compared to identical but pasteurized soil two months after infestation with 280 M. incognita J2/100 cm(3) soil. This suppressive soil was subjected to various temperature, fumigation and dilution treatments, planted with tomato, and infested with 1,000 eggs of M. incognita/100 cm(3) soil. Eight weeks after nematode infestation, distinct differences in nematode population densities were observed among the soil treatments, suggesting the suppressiveness had a biological nature. A fungal rRNA gene analysis (OFRG) performed on M. incognita egg masses collected at the end of the greenhouse experiments identified 11 fungal phylotypes, several of which exhibited associations with one or more of the nematode population density measurements (egg masses, eggs or J2). The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. The negative correlation between the densities of the P. chlamydosporia genes and the nematodes was corroborated by an analysis using a P. chlamydosporia-selective qPCR assay.

Fig. 1

Phylogenetic tree of the major fungal phylotypes in the M. incognita egg masses collected at the end of the greenhouse experiments. The unrooted parsimony tree was made using at least one representative small-subunit rRNA gene sequence from each of the major phylotypes (identified by the OFRG analysis) and their closest relatives determined by an analysis using BLAST (1). Sequences from this study are named by their clone number followed by their phylotype designation (Poch = Pochonia chlamydosporia, Fus = Fusarium oxysporum, Plect = Plectosphaerella cucumerina, Micro = Microdochium nivale, Sacch = Saccharomyces sp., Tetra = Tetracladium sp., Geomy = Geomyces pannorum, Mon. e = Monacrosporium ellipsosporum, Mon. g = Monacrosporium geophytum, Cerat = Ceratobasidium sp., Auric = Auricularia sp.). Reference sequences are designated by their accession number and taxon. The scale bar length is 0.1, and it represents the number of nucleotide changes per position.

Fig. 2

Relative frequencies of the fungal phylotypes from the M. incognita egg masses collected at the end of the greenhouse experiments. Relative frequencies are the number of clones per phylotype divided by the number of total clones per soil treatment. (2a) Trial one; (2b) Trial two. Soil treatments: the first character designates the experiment type [A = Soil transfer trials (ratios of non-treated suppressive soil to fumigated non-suppressive soil), B = Temperature trials (soils were exposed to different temperature treatments), C = Soil fumigation trials (soils were exposed to various dosages of methyl iodide)], the second character designates the treatment type within each experiment (Experiment A: 1 = 100, 2 = 50, 3 = 10, 4 = 1, 5 = 0% suppressive soil; Experiment B: 1 = room temperature, 2 = 30°C, 3 = 40°C, 4 = 50°C, 5 = 60°C; Experiment C: 1 = 0 μl, 2 = 12 μl, 3 = 24 μl, 4 = 48 μl, 5 = 96 μl), and the third character designates the trial number.

Fig. 3

Scatter plots of the population densities of three nematode measurements vs. P. chlamydosporia rRNA genes. Nematode values are the number of egg masses or eggs per plant and the number of J2 per 50-cm³ soil. Pochonia chlamydosporia rRNA gene values are copy numbers per egg mass. The trend lines in the plots are from linear regression analyses.