Table 1.
Closest rDNA sequence matches (BLASTN) of the endophytic fungal isolates.
Fig 1.
Effect of endophytic fungi Trichoderma spp. ReTk1 and ReTv2 inoculation on growth promotion of rapeseed.
The experiment was carried out under greenhouse conditions. Field soil and planting mix were inoculated with ReTk1 and ReTv2 (1x107 conidia/gm soil) by soil drench method. (A) The phenotypes of rapeseed plants without treatment (a) or treatment with ReTk1 (b) and ReTv2 (c). (B) The growth promotion of rapeseed plants. The proliferation of the first true leaf diameter was measured 25 days after seeding and other growth parameters were quantified 42 days after seeding. The mean average of six replications and capped lines represent standard error. The symbol “*” on top of the bar indicates a significant difference compared to the control at P<0.05.
Fig 2.
Inhibition of endophytic fungi Trichoderma spp. ReTk1 and ReTv2 on resting spore germination of P. brassicae (Pb) in root exudate solution (RES).
RES amended with culture filtrates of ReTk1 & ReTv2 over six days incubation period. The germination of resting spores was counted by staining with orcein-acetic acid. Germinated spores were empty under light microscope, in contrast to red non-germinated spores (A). The germination also checked under CLSM (B). The black and yellow arrowheads in the figure indicate germinated and non-germinated resting spores, respectively. (C) The mean average of six replications and capped lines represent standard error. The symbol “*” on top of the bar indicates a significant difference compared to the control at P<0.05.
Fig 3.
Effect of Trichoderma spp. ReTk1 and ReTv2 on infection of rapeseed root hairs by P. brassicae at 7 and 14 days after seeding (DAS).
Roots of three treated plants were uprooted, washed and rinsed to remove most of the microbes, cut into 1 cm pieces, and fixed in 70% ethanol. Roots of control plants (pathogen-inoculated only) were processed similarly. Root pieces were stained overnight with aceto-carmine (1%) and examined using a light microscope for the presence of primary plasmodia or zoosporangia in root hairs (arrowhead). Root hairs of control plants showing damage due to infection by P. brassicae (A). Root haris of ReTk1 (B) and ReTv2-treated (C) plants showing less damage to the roots due to infection by P. brassicae. Bars indicate the average of six replications and the experiment repeated thrice (D). Capped lines represent standard error. The symbol “*” on top of the bar indicates a significant difference compared to the control at P<0.05.
Table 2.
Effect of Trichoderma spp. ReTk1 and ReTv2 on clubroot incidence and severity index of rapeseed in field soil and planting mix.
Table 3.
Effect of application timing of Trichoderma spp. ReTk1 and ReTv2 on clubroot severity index in field soil.
Fig 4.
Suppression of clubroot formation and promotion of plant growth of rapeseed.
The experiment was conducted with filed soil and planting mix under greenhouse conditions. The growth parameters and root index were quantified after 42 days of seeding. (A) Phenotypes of rapeseed plants without treatment (a) or treatment with ReTk1 (b) and ReTv2 (c). (B) Growth promotion of infected plants of rapeseed with or without Trichoderma-treatment and (C) root index. Root index means the fresh root weight of infected plants to the fresh root weight of uninfected plants (Ri/Rni). The smaller root index means less gall development and the larger root index means more gall development and shorter root length during infection. Bars represent the average six replications and capped lines standard errors. The symbol “*” on top of the bar indicates a significant difference compared to the control at P<0.05.
Fig 5.
Colonization of rapeseed roots by Trichoderma spp. ReTk1 and ReTv2.
Trichoderma transformant expressing rfp. (A & C) Root colonization by ReTktr-1(5). (B & D) Root colonization by ReTvtr-2(23).
Fig 6.
Transcript level analysis of nine selected plant-defense-related genes in rapeseed seedlings treated with a soil drench of Trichoderma spp. or water (control) was performed using quantitative reverse-transcriptase polymerase chain reaction (qPCR).
The primers used in this experiment for quantifying BnPR-1, BnPR-2 and BnPR-5 were based on Potlakayala et al. [45] and the rest on Zhao et al. [46]. Gene expression was normalized using the reference Actin gene [46] in qPCR. The genes included in this experiment are three pathogenesis-related (BnPR-1, BnPR-2 and BnPR-5), genes that control ethylene (BnSAM3 and BnACO), auxin (BnAA01), jasmonic acid (BnOPR2), or phenylpropanoid (4-cournarate CoA ligase (BnOPCL) and c-innamoyl CoA reductase (BnCCR) pathways were assessed using root and first true leaf samples taken at 14 days after seeding (DAS). The relative levels of the transcript were calculated by the comparative Ct method. Bars represent the means and capped lines standard error (three replications). A gene was considered up-regulated when its expression level was substantially higher in treated plants relative to that of the control (least significant difference, P < 0.05).