Fig 1.
Phylogenetic analysis of Tp WPM 39143.
The ITS nucleotide sequence of Tp WPM 39143 was aligned with similar sequences from 27 taxa of Trichoderma/Hypocrea species available in the GenBank. The neighbor-joining method was used to construct the phylogenetic tree. The bootstrap scores are based on 1,000 reiterations. Fusarium oxysporum CBS 129.24 and Neotyphodium uncinatum NBRC 32642 were used as outgroup.
Fig 2.
Growth comparisons of Tp WPM 39143 at different temperatures.
Tp WPM 39143 along with Th and Ta strains were point inoculated on the center of PDA (A) and YPD (B) agar plates and incubated at different temperatures (6°C-22°C) for 9 days and colony diameter was measured. The Tp WPM 39143 growth was significantly rapid (asterisk denotes p<0.001) as compared to Ta or Th at lower temperatures of 6°C and 10°C. Further incubation (20 days) revealed good growth of Th and restricted growth of Ta at 10°C, but no growth of Th or Ta at 6°C (C).
Fig 3.
Dual culture challenge on PDA agar medium.
Approximately 15 μl spore suspension of Pd (107/ml) was inoculated near the edge of the PDA plate. Following incubation at 15°C for 10 days, 15 μl of Tp WPM 39143 or Th conidial suspension (105 cells/ml) was inoculated on the opposite edge of the culture plate and the interaction between these two fungi was assessed at 14 and 28 days post-incubation. Pd colonies were white and restricted in the presence of Tp WPM 39143 or Th as compared to fluffy and pigmented when grown alone.
Fig 4.
Pd CFU recovery from dual culture challenge in soil (culture-dependent method).
Autoclaved soil sample was first inoculated with 100 μl of Pd conidia (104/g) and following incubation for 7 days at 15°C, one soil sample each containing Pd, was inoculated with 100 μl of Tp WPM 39143 or Th conidia (104/g) or 100 μl of water alone. Dual culture challenge samples and control samples were incubated at 15°C for another 35 days, and three aliquots of 100 mg soil from each treatment group were processed for the recovery of Pd in culture. Approximately, 4-logs reduction of Pd CFU by Tp WPM 39143 (p<0.0001) compared to 1.7-logs reduction of Pd CFU by Th (p<0.05) was observed.
Fig 5.
Pd genome copies recovery from dual culture challenge in soil (culture-independent method).
A, Standard curves of Pd intergenic spacer region (IGS) and alpha L-Rhamnosidase (ALR) gene by real-time PCR assays. The purified genomic DNA from Pd was used for the generation of standard curve against IGS and ALR targets, which was used to extrapolate Pd genome copies in the soil samples with or without biocontrol agents. All data points represent the mean Ct value of amplification reactions done in triplicates with error bars denoting standard deviation. The assay was linear over 6 orders of magnitude for IGS gene (y = –3.5886x+42.131, R2 = 0.9982) and over 4 orders of magnitude for ALR gene (y = –3.398x+45.716, R2 = 0.9994). B, Pd genome copies recovery by real-time PCR assays. Autoclaved soil sample was first inoculated with 100 μl of Pd conidia (104/g) and following incubation for 7 days at 15°C, one each soil sample containing Pd was inoculated with 100 μl of Tp WPM 39143 or Th conidia (104/g) or 100 μl of water alone. The dual challenge samples were incubated at 15°C for additional 35 days and three aliquots of 100 mg each soil sample from each treatment group were processed for Pd gDNA extraction followed by IGS and ALR real-time PCR assays. The mean Ct counts equivalent to DNA was extrapolated from the standard curve, and Pd genome copies were calculated based on the formula described in Materials and Methods. Substantial reduction of Pd genome copies observed in samples challenged with Tp WPM 39143 (p<0.0001) as compared to samples challenged with Th (p<0.05).
Fig 6.
Inhibition of Pd by Tp WPM 39143 and Th extracts.
YPD agar plates were streaked with 108 spore suspensions of Pd or Pp. Sterile filter discs (6 mm) were placed on the surface of the inoculated plate and saturated with 15 μl of Tp WPM 39143 or Th crude extract. Plates were incubated at 15°C for 10 days. Both Tp WPM 39143 and Th extract showed inhibitory activity against Pd (A) but not against Pp (B). The fermentation medium without metabolites, which went through similar extraction process as medium containing metabolites served as a negative control (NCo).
Fig 7.
Stability assessment of Pd inhibitory activity in Tp WPM 39143 crude extract.
YPD agar plates were streaked with 100 μl of Pd conidial suspension (108) and sterile filter discs (6 mm) were placed on agar plates and saturated with 15 μl of treated or untreated Tp WPM 39143 extract. Plates were incubated at 15°C for 10 days and were photographed for inhibition zone around discs. Abbreviations: PCo, positive control (untreated Tp WPM 39143 extract), NCo, negative control (fermentation medium alone); 1, Tp WPM 39143 extract exposed to regular light for 20 h; 2, Tp WPM 39143 extract exposed to proteinase K at 37°C for 20 h; 3, Tp WPM 39143 extract exposed to proteinase K at 45°C for 20 h; 4, Tp WPM 39143 extract exposed to 45°C for 20 h.
Fig 8.
Inhibitory activity of HPLC fractions obtained from Tp WPM 39143 extract.
A) Tp WPM 39143 crude extract was fractionated by HPLC (details in methods). Total of 15 sub-fractions were collected at the intervals of 5 min. B) Disc diffusion assay to measure inhibition of Pd growth by various HPLC sub-fractions. F1, 0–5 min sub-fraction; F2, 5–10 min sub-fraction; F3, 10–15 min sub-fraction; …………. F14, 65–70 min sub-fraction; F15, 70–75 min, 5% CH3OH in H2O elution time. Abbreviation: PCo, positive control (untreated Tp WPM 39143 crude extract).