Phenylalanine ammonia lyase (PAL) activity
In general, PAL activity levels were recorded in all categories of seedlings of with or without treatment). In both treatment samples, Phenylalanine ammonia lyase activity was gradually increased after 3 h of incubation and its peak was obtained after 6 h in the elicitor treated samples and resistant seedlings. In contrast, PAL activity peaked at 9 h in susceptible seedlings. Inoculated seedlings had considerably higher PAL activity than uninoculated seedlings in all treatments and at all time periods assessed (Fig. 1).
Temporal pattern of PAL activity in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB-Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen-inoculated seedlings, PAL activity peaked at 6 hpi in resistant and elicitor treated seedlings, while in susceptible control seedlings PAL activity peaked at 9 hpi. The maximum PAL activity was noted in GB-treated seedlings, which were even significantly higher than resistant seedlings. Among the elicitor treatments, at 6 hpi, GB-treated seedlings recorded 42.66 units PAL activity, followed by CWG, LPS, and DCA treatments, which recorded 39.6, 38.56, and 29.15 units PAL activity, respectively, whereas in the control seedlings it showed 6.92 units PAL activity. Phenylalanine ammonia lyase activity in GB, CWS, LPS, and DCA treated seedlings was 6.16-, 5.72-, 5.57-, and 4.21-folds higher at 6 hpi than that of the control, respectively. Resistant seedlings showed 34.26 units PAL activity and it was 4.95-folds higher than the control. At 6 hpi GB-treated seedlings showed 1.07-, 1.11-, and 1.46-fold higher PAL activity than CWG, LPS, and DCA treatments, respectively.
In seedlings without pathogen inoculation, the pattern of PAL activity was similar to that of pathogen inoculated seedling but the level of activity was significantly lesser. Among the elicitor treatments at 6 h, GB-treated seedlings recorded maximum PAL activity by showing 18.75 activity followed by CWG, LPS, and DCA treatments which showed 17.28, 16.92, and 16.61 units PAL activity, respectively compared to control seedlings which recorded 2.96 PAL activity. The GB-treated seedlings showed 6.33-folds higher activity than the control at 6 h.
At 6 h PAL activity in challenge inoculation in treated seedlings of GB, CWG, LPS, and DCA, results showed 2.27-, 2.29-, 2.27-, and 1.75-folds higher than that of the un-inoculated samples, respectively.
Peroxidase (POX) activity
The constitutive POX activity was recorded in all categories of seedlings in both treatments (with or without pathogen inoculation). In both pathogen inoculated and uninoculated samples, POX enzyme activity gradually increased from 3 h onwards and peaked at 9 h in samples of elicitor treated and resistant seedlings, whereas in susceptible seedlings POX activity peaked at 12 h. The POX enzymatic activity was significantly higher in inoculated seedlings compared to the uninoculated seedlings in all treatments and at all tested time intervals (Fig. 2).
Temporal pattern of POX activity in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R-resistant, S-susceptible, DCA-3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen-inoculated seedlings the POX activity was peaked at 9 hpi in both resistant and elicitor treated seedlings whereas in susceptible control seedlings, its activity peaked at 12 hpi. The maximum POX enzymatic activity was observed in resistant seedlings, which recorded 70.28, units and it was 7.47-folds higher than the susceptible control seedlings. Among the elicitor treated samples, at 9 hpi, GB and LPS treated seedlings recorded 67.86 and 67.73 units POX activity, respectively which were not statistically significant from each other, followed by CWG and DCA treatments which recorded 65.9 and 65.33 POX activity, respectively, whereas in control seedlings it showed 9.4 units POX activities. At 9 hpi, POX activity in GB, LPS, CWS, and DCA treated seedlings was 7.22-, 7.20-, 7.01-, and 6.95-folds higher than control seedlings, respectively.
In seedlings without pathogen inoculation, the pattern of POX activity was similar to pathogen inoculated seedling, but the activity level was significantly lesser. Among the elicitor treatments at 9 h, GB-treated seedlings recorded maximum POX activity by showing 31.32 units activity followed by, CWG, DCA, LPS, and treatments which showed 28.65, 25.58, and 24.54 POX activity, respectively, compared to the control seedlings with 5.71 units POX activity. GB treatment showed 5.48-folds higher activity than the susceptible control at 9 h.
At 9 h POX activity in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 2.16-, 2.75-, 2.3-, and 2.55-folds higher than that of the uninoculated samples, respectively.
Polyphenoloxidase (PPO) activity
In general, the constitutive PPO activity was recorded in all categories of seedlings with or without pathogen inoculation. In both pathogen inoculated and uninoculated samples PPO activity gradually increased from 3 h onwards and peaked at 24 h in elicitor treated, resistant, and control seedlings. Polyphenoloxidase activity was significantly higher in inoculated seedlings compared to the uninoculated seedlings in all treatments and at all tested time intervals (Fig. 3).
Temporal pattern of PPO activity in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen inoculated seedlings, PPO activity peaked at 24 hpi, and maximum PPO activity was observed in GB-treated seedlings, which was even significantly higher than resistant seedlings. Among the elicitor treatments, at 24 hpi, GB-treated seedlings recorded 47.02 units PPO activity followed by LPS, CWG, and DCA treatments which recorded 42.66, 38.65, and 33.32 units PPO activity, respectively, whereas the control seedlings showed 13.09 PPO activity. Polyphenoloxidaseactivity at 24 hpi h in GB, LPS, CWS, and DCA-treated seedlings was 3.59-, 3.25-, 2.95-, and 2.54-folds higher than that of the control seedlings, respectively. Resistant seedlings showed 41.95 units PPO activity which was 3.2-folds higher than the control seedlings. At 24 hpi GB-treated seedlings showed 1.1-, 1.21-, and 1.41-folds higher PPO activity than LPS, CWG, and DCA treatments, respectively.
In seedlings without pathogen inoculation, the pattern of PPO activity was similar to that of pathogen inoculated seedling but the level of activity was significantly lesser. Among the elicitor treatments at 24 h, GB-treated seedlings recorded maximum PPO activity by showing 20.18 units activity followed by LPS, CWG, and DCA treatments, which showed 16.58, 13.37, and 11.94 units PPO activity, respectively compared to the control seedlings which showed 7.87 PPO activity. GB treatment showed 2.56-folds higher activity than the susceptible control at 24 h.
At 24 h PPO activity in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 2.33-, 2.57-, 2.89-, and 2.79-folds higher than that of the uninoculated samples, respectively.
β-1,3-glucanase activity
In general, constitutive levels of β-1,3-glucanase activity were recorded in all categories of seedlings with or without pathogen inoculation, however, the activities were significantly higher in resistant seedlings compared to the other categories of seedlings. In both pathogen inoculated and uninoculated samples β-1,3-glucanase activity gradually increased from 3 h onwards and peaked at 24 h in elicitor treated, resistant, and control seedlings. The β-1,3-glucanase activity was significantly higher in inoculated seedlings compared to the uninoculated seedlings in all treatments and at all tested time intervals (Fig. 4).
Temporal pattern of β-1,3-glucanase activity in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen inoculated seedlings, β-1,3-glucanase activity peaked at 24 hpi, and maximum activity was observed in GB-treated seedlings which was even significantly higher than resistant seedlings. Among the elicitor treatments, at 24 hpi, GB-treated seedlings recorded 46.86 units β-1,3-glucanase activity, followed by LPS, CWG, and DCA treatments which recorded 40.36, 37.26, and 31.36 units β-1,3-glucanase activity, respectively, whereas control seedlings showed 22.2 units -1,3-glucanase activity. At 24 hpi β-1,3-glucanase activity in GB, LPS, CWS, and DCA treated seedlings was 2.11-, 1.81-, 1.67-, and 1.41-folds higher than that of the control, respectively. Resistant seedlings showed 42.24 β-1,3-glucanase activity which was 1.9-folds higher than the control seedlings. At 24 hpi GB-treated seedlings showed 1.16, 1.25, and 1.49 folds higher β-1,3-glucanase activity than LPS, CWG, and DCA treatments, respectively.
In seedlings without pathogen inoculation, the pattern of β-1,3-glucanase activity was similar to that of pathogen inoculated seedling, but the activity level was significantly lesser. Among the elicitor treatments at 24 h, GB-treated seedlings recorded maximum β-1,3-glucanase activity by showing 18.16 units activity followed by CWG, LPS, and DCA treatments which showed 15.65, 14.18, and 13.15 units β-1,3-glucanase activity, respectively compared to the control seedlings which showed 8.45 β-1,3-glucanase activity. GB treatment showed 2.16-folds higher activity than the susceptible control at 24 h.
At 24 h β-1,3-glucanase activity in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 2.58-, 2.84-, 2.38-, and 2.40-folds higher than that of the uninoculated samples, respectively.
Lipoxygenase activity
In general, the constitutive level of LOX activity was recorded in all categories of seedlings with or without pathogen inoculation. In both pathogen inoculated and uninoculated samples LOX activity gradually increased from 3 h onwards and peaked at 24 h in elicitor treated, resistant, and control seedlings. LOX activity was significantly higher in inoculated seedlings compared to the uninoculated seedlings in all treatments and at all tested time intervals (Fig. 5).
Temporal pattern of LOX activity in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen inoculated seedlings LOX activity peaked at 24 hpi and maximum LOX activity was observed in DCA treated seedlings, which was even significantly higher than resistant seedlings. Among the elicitor treatments, at 24 hpi, DCA-treated seedlings recorded 42.88 units LOX activity, followed by CWG, LPS, and GB treatments, which recorded 29.63, 27, and 23.19 LOX activity, respectively, whereas the control seedlings showed 17.42 units LOX activity. LOX activity at 24 hpi in DCA, CWG, LPS, and GB-treated seedlings was 2.4-, 1.7-, 1.55-, and 1.33-folds higher than that of the control seedlings, respectively. Resistant seedlings showed 34.18 LOX activity which was 1.98-folds higher than the control seedlings. At 24 hpi DCA treated seedlings showed 1.45-, 1.59-, and 1.85-fold higher LOX activity than CWG, LPS, and GB treatments, respectively.
In seedlings without pathogen inoculation, the pattern of LOX activity was similar to that of pathogen inoculated seedling but the level of activity was significantly lesser. Among the elicitor treatments at 24 h, DCA-treated seedlings recorded maximum LOX activity by showing 16.83 activity followed by CWG, LPS, and GB treatments which showed 10.8, 9.47, and 8.2 units LOX activity, respectively compared to the control seedlings which showed 5.4 LOX activity. DCA treatment showed 3.12-folds higher activity than the susceptible control at 24 h.
At 24 h LOX activity in pathogen inoculated DCA, CWG, LPS, and GB-treated seedlings was 2.55-, 2.74-, 2.85-, and 2.82-folds higher than that of the uninoculated samples, respectively.
Hydroxyproline-rich glycoproteins (HRGPs) activity
In general, the constitutive level of HRGP activity was recorded in all categories of seedlings with or without pathogen inoculation. In both pathogen inoculated and uninoculated samples, HRGP activity gradually increased from 3 h onwards and peaked at 9 h in elicitor treated, and resistant seedlings, whereas in control seedlings HRGP activity peaked at 24 h. HRGP activity was significantly higher in inoculated seedlings compared to the uninoculated seedlings in all treatments and at all tested time intervals (Fig. 6).
Temporal pattern of accumulation of HRGPs in 2-day old P. glaucum seedlings with (I-inoculated) or without (U-uninoculated) Sclerospora graminicola inoculation. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Bars indicate standard errors; means with different superscripts are significantly different, as shown by Tukey’s HSD test (p ≤ 0.05).
In pathogen inoculated seedlings HRGP activity peaked at 9 hpi and maximum HRGP activity was observed in GB-treated seedlings, which showed 0.971 HRGP activity which was on par with resistant seedlings which showed 0.9766 units HRGP activity. Among the elicitor treatments, at 9 hpi, GB-treated seedlings recorded 0.971 HRGP activity, followed by LPS, CWG, and DCA treatments which recorded 0.866, 0.746, and 0.733 units HRGP activity, respectively, whereas the control seedlings showed 0.356 units HRGP activity. HRGP activity at 9 hpi in GB, LPS, CWS, and DCA treated seedlings was 2.72-, 2.43-, 2.09-, and 2.06-folds higher than that of the control seedlings, respectively. Resistant seedlings showed 2.74-fold higher HRGP activity than the control seedlings. At 9 hpi GB-treated seedlings showed 1.12-, 1.3-, and 1.32-fold higher HRGP activity than LPS, CWG, and DCA treatments, respectively.
In seedlings without pathogen inoculation, the pattern of HRGP activity was similar to that of pathogen inoculated seedling but the level of activity was significantly lesser. Among the elicitor treatments at 9 h, GB-treated seedlings recorded maximum HRGP activity by showing 0.398 units activity followed by LPS, DCA, and CWG treatments which showed 0.393, 0.368, and 0.357 HRGP activity, respectively compared to the control seedlings which showed 0.235 units HRGP activity. GB treatment showed 1.69-folds higher activity than the susceptible control at 9 h. At 9 h HRGP activity in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 2.44-, 2.20-, 2.01-, and 1.99-folds higher than that of the uninoculated samples, respectively.
Quantitative real time PCR analysis (qPCR) for defense enzymes, hydroxyproline-rich glycoproteins, and pathogenesis-related proteins
PAL gene expression
Phenylalanine ammonia lyase transcripts was detected in all categories of seedlings with or without pathogen inoculation and the expression level was higher in resistant and elicitor treated seedlings compared to the susceptible controls. In all sets of seedlings PAL gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 7). Among the pathogen inoculated seedlings the expression of PAL gene was highest at 6 hpi in GB treated seedlings which was even higher than the resistant seedlings. Among the elicitor treated seedlings, at 6 hpi highest PAL gene expression was observed in GB treated seedlings followed by CWG, LPS and DCA treatments, which were 5.34-, 5.19-, 4.78- and 3.96-folds higher than that of the susceptible control seedlings, respectively. In seedlings without pathogen inoculation, pattern of PAL expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lower. At 6 h, PAL gene expression in pathogen inoculated GB, CWG, LPS and DCA treated seedlings was 2.11-, 2.45-, 2.41-, and 2.02-folds higher than that of the uninoculated samples, respectively.
qRT-PCR determined relative expression of PAL genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates. The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
POX gene expression
Peroxidase transcripts was detected in all categories of seedlings with or without pathogen inoculation and the expression level was higher elicitor treated seedlings compared to the resistant check and susceptible controls. In all sets of seedlings POX gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 8). Among the pathogen inoculated seedlings the expression of POX gene was highest at 9 hpi in elicitor treated seedlings which was even higher than the resistant seedlings. Among the elicitor treated seedlings, at 9 hpi highest POX gene expression was observed in GB treated seedlings followed by LPS, CWG and DCA treatments, which were 7.12-, 6.77-, 6.88- and 6.14-folds higher than that of the susceptible control seedlings, respectively.
qRT-PCR determined relative expression of POX genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates. The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
In seedlings without pathogen inoculation, pattern of POX expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lower. At 9 h, POX gene expression in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 1.73-, 1.82-, 1.93-, and 2.75-folds higher than that of the uninoculated samples, respectively.
PPO gene expression
Polyphenoloxidase transcripts was detected in all categories of seedlings with or without pathogen inoculation and the expression level was higher in resistant seedlings compared to the elicitor treated and susceptible controls. In all sets of seedlings PPO gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 9). Among the pathogen inoculated seedlings, at 24 hpi, resistant seedlings recorded highest PPO gene expression which was 6.08-folds higher than that of the control. And at 24 hpi, among the elicitor treated seedlings, highest PPO gene expression was observed in GB treated seedlings followed by LPS, CWG, and DCA treatments, which were 5.36-, 5.24-, 4.79- and 4.04-folds higher than that of the susceptible control seedlings, respectively.
qRT-PCR determined relative expression of PPO genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates. The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
In seedlings without pathogen inoculation, pattern of PPO expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lesser. At 24 h, PPO gene expression in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 1.58-, 1.62-, 2.38-, and 2.29-folds higher than that of the uninoculated samples, respectively.
β-1,3-glucanase gene expression
β-1,3-glucanase transcripts was detected in all categories of seedlings with or without pathogen inoculation. In all sets of seedlings β-1,3-glucanase gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 10). Among the pathogen inoculated seedlings the expression of β-1,3-glucanase gene was highest at 24 hpi in all test samples. Maximum β-1,3-glucanase gene expression was observed in GB treated seedlings followed by LPS, CWG and DCA treatments. β-1,3-glucanase gene expression in GB, LPS, CWG and DCA treated seedlings was 2.85-, 2.27-, 2.17- and 2.09-folds higher than that of the control seedlings, respectively.
qRT-PCR determined relative expression of β-1,3-glucanase genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
In seedlings without pathogen inoculation, pattern of β-1,3-glucanase expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lesser. At 24 h, β-1,3-glucanase gene expression in pathogen inoculated GB, LPS, CWG and DCA treated seedlings was 1.76, 1.67, 1.66 and 2.10-folds higher than that of the uninoculated samples, respectively.
LOX gene expression
Lipoxygenase transcripts was detected in all categories of seedlings with or without pathogen inoculation and the expression level was higher in resistant and elicitor seedlings compared to the susceptible controls at all time intervals. In all sets of seedlings LOX gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 11). Among the elicitor treatments, maximum LOX gene expression was observed in DCA treated seedlings followed by CWG, LPS and GB treatments. LOX gene expression in DCA, CWG, LPS and GB treated seedlings was 2.22-, 1.58-, 1.52- and 1.27-folds higher than that of the control seedlings, respectively.
qRT-PCR determined relative expression of LOX genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates. The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
In seedlings without pathogen inoculation, pattern of LOX expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lesser. At 24 h, LOX gene expression in pathogen inoculated DCA, CWG, LPS and GB treated seedlings was 2.28-, 2.33-, 2.45- and 2.29-folds higher than that of the uninoculated samples, respectively.
HRGP gene expression
Initially HRGP transcripts was detected in all categories of seedlings with or without pathogen inoculation and the expression level was significantly higher in resistant seedlings compared to the elicitor treated and susceptible controls. In all sets of seedlings HRGP gene expression was higher in inoculated samples compared to the uninoculated samples at all time points (Fig. 12). Among the pathogen inoculated, elicitor treated seedlings; at 9 hpi highest HRGP gene expression was observed in GB treated seedlings followed by LPS, CWG and DCA treatments, which were 3.24-, 3.06-, 2.82- and 2.71-folds higher than that of the susceptible control seedlings, respectively. HRGP gene expression in GB treated seedlings was 1.06-, 1.15-, and 1.19-folds higher than that of LPS, CWG, and DCA treated seedlings.
qRT-PCR determined relative expression of HRGPs genes in 2-day-old P. glaucum seedlings with (I) or without (U) Sclerospora graminicola inoculation harvested 0, 3, 6, 9, 12, 24, 48, and 72 h. R—resistant, S—susceptible, DCA—3,5-Dichloroanthranilic acid treated, CWG—Cell Wall Glucans isolated from the endophyte Trichoderma hamatum UOM 13, LPS—Lipopolysaccharides isolated from bacteria Pseudomonas fluorescens UOM 14, GB—Glycinebetaine an amino acid derivative. Expression levels were measured by qPCR and normalized to the constitutive PP2A gene. Values are means of experiments carried out in four replicates. The bars indicate ± SE and the data were analyzed by one-way ANOVA followed by Tukey’s test and p ≤ 0.05.
In seedlings without pathogen inoculation, pattern of HRGP expression was similar to that of pathogen inoculated seedling but the level of expression was significantly lesser, except for the LPS treatment. At 9 h, HRGP gene expression in pathogen inoculated GB, LPS, CWG, and DCA treated seedlings was 2.55-, 1.63-, 2.35-, and 2.26-folds higher than that of the uninoculated samples, respectively.
