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Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay

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ABSTRACT

This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL−1) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via high-performance liquid chromatography, HPLC) confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

No MeSH data available.


Ergosterol concentration (μg mL−1) and estimated fungal mycelium weight (g) in ramets that were inoculated with [a] G, [b] W, [c] T and [d] B detected at every time interval during 49 days incubation period (G = Gb-inoculated ramets, W = WAA02-inoculated ramets, T = T2-inoculated ramets, B = BTF08-inoculated ramets). Bars represent means ± SE (standard error) of triplicate treatments. Means with different letters are significantly different at P < 0.05, n = 3 using Tukey’s HSD test.
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f0015: Ergosterol concentration (μg mL−1) and estimated fungal mycelium weight (g) in ramets that were inoculated with [a] G, [b] W, [c] T and [d] B detected at every time interval during 49 days incubation period (G = Gb-inoculated ramets, W = WAA02-inoculated ramets, T = T2-inoculated ramets, B = BTF08-inoculated ramets). Bars represent means ± SE (standard error) of triplicate treatments. Means with different letters are significantly different at P < 0.05, n = 3 using Tukey’s HSD test.

Mentions: Ergosterol was detected in endophyte- and pathogen-inoculated ramets but the concentrations did not increase significantly throughout the 49 days of incubation (Fig. 3). Ergosterol was detected in G-ramets on all days except days 28, 35 and 49 (Fig. 3a) whereas ergosterol was detected in W-ramets during every sampling intermittent (Fig. 3b). For T-ramets, ergosterol was detected on all days except days 28 and 35 (Fig. 3c) while for B-ramets, ergosterol detection was positive on days 7, 14, 21 and 42 (Fig. 3d). Nevertheless, these ergosterol levels showed no significant increase over time, suggesting that the endophytic isolates WAA02, T2, BTF08 and pathogenic isolate Gb were present in the seedlings but were not be able to proliferate inside the ramets (internal tissues). This was further concurred by the insignificant P-value of 0.150, 0.079, 0.545, and 0.734 obtained, respectively, using Tukey’s HSD test. Further data on N (sample size used to generate data at each time point), mean values, standard error and significance (P values) is presented in Supplementary Data. Ergosterol was absent in non-inoculated ramets (treatment C) throughout the 49 days (data not shown).


Understanding colonization and proliferation potential of endophytes and pathogen in planta via plating, polymerase chain reaction, and ergosterol assay
Ergosterol concentration (μg mL−1) and estimated fungal mycelium weight (g) in ramets that were inoculated with [a] G, [b] W, [c] T and [d] B detected at every time interval during 49 days incubation period (G = Gb-inoculated ramets, W = WAA02-inoculated ramets, T = T2-inoculated ramets, B = BTF08-inoculated ramets). Bars represent means ± SE (standard error) of triplicate treatments. Means with different letters are significantly different at P < 0.05, n = 3 using Tukey’s HSD test.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5121152&req=5

f0015: Ergosterol concentration (μg mL−1) and estimated fungal mycelium weight (g) in ramets that were inoculated with [a] G, [b] W, [c] T and [d] B detected at every time interval during 49 days incubation period (G = Gb-inoculated ramets, W = WAA02-inoculated ramets, T = T2-inoculated ramets, B = BTF08-inoculated ramets). Bars represent means ± SE (standard error) of triplicate treatments. Means with different letters are significantly different at P < 0.05, n = 3 using Tukey’s HSD test.
Mentions: Ergosterol was detected in endophyte- and pathogen-inoculated ramets but the concentrations did not increase significantly throughout the 49 days of incubation (Fig. 3). Ergosterol was detected in G-ramets on all days except days 28, 35 and 49 (Fig. 3a) whereas ergosterol was detected in W-ramets during every sampling intermittent (Fig. 3b). For T-ramets, ergosterol was detected on all days except days 28 and 35 (Fig. 3c) while for B-ramets, ergosterol detection was positive on days 7, 14, 21 and 42 (Fig. 3d). Nevertheless, these ergosterol levels showed no significant increase over time, suggesting that the endophytic isolates WAA02, T2, BTF08 and pathogenic isolate Gb were present in the seedlings but were not be able to proliferate inside the ramets (internal tissues). This was further concurred by the insignificant P-value of 0.150, 0.079, 0.545, and 0.734 obtained, respectively, using Tukey’s HSD test. Further data on N (sample size used to generate data at each time point), mean values, standard error and significance (P values) is presented in Supplementary Data. Ergosterol was absent in non-inoculated ramets (treatment C) throughout the 49 days (data not shown).

View Article: PubMed Central - PubMed

ABSTRACT

This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100&nbsp;mL of fungal cells (106&nbsp;cfu&nbsp;mL&minus;1) via soil drenching. For the next 7&nbsp;days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7&nbsp;days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via high-performance liquid chromatography, HPLC) confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49&nbsp;days was however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.

No MeSH data available.