Generally, there were higher TPC in the leaves of both TKM6 and TN1 than in their stems (Tables 3 and 4). These same results were also observed in stemborer-infested resistant and susceptible maize varieties (Praveen et al., 2013) and in another study looking at the TPC in the leaves and stems of Monsonia burkeana (Mamphiswana et al., 2010). Surprisingly, TPC of the susceptible TN1 was generally higher than that of the resistant TKM6, both for the infested leaves and stems. It is hypothesized that the resistant genotypes have higher TPC than the susceptible. In contrast, the results obtained in the current study is different from the results of Praveen et al. (2013) on maize varieties and Punithavalli et al. (2013) on rice genotypes against leaf folder. With lower TPC than the susceptible, TKM6 showed promising significant phenolics-induced defense mechanism.
This mechanism may hint on the possible biochemical mechanism of resistance of TKM6 amidst biological enemy such as YSB larvae, which can be explored in succeeding studies. In contrast, TN1 had high TPC even before larvae introduction; thus, the slight increase in TPC may not be necessarily attributable to YSB infestation but to other biological stress, which the plant detected during the experiment.
LC Conditioning and Adjustments for Identification of Peaks and Compounds
Different mobile phase system and column temperatures were tested to determine the optimal chromatographic conditions to separate analytes. The peak of serotonin standard tends to elute very early while the peaks of trans-cinnamic acid tend to elute very late, and often does not appear using other elution conditions. Some peaks also tend to co-elute, which is probably due to the same retention properties, such as in the case of vanillic acid and syringic acid. Peak of trans-cinnamic acid was observed, and peaks of vanillic acid and syringic acid were slightly separated at 254 nm wavelength using the optimized mobile phase system used (Table 5).
Table 5. Optimized mobile phase system.
Minutes % Absorbance % Concentration
0 95 5
5 85 15
10 85 15
20 75 25
30 75 25
35 60 40
45 75 25
50 95 5
55 95 5
Identification of Peaks in Standards and Leaf Extracts
In RP-HPLC, polarity of phenolic compounds determines the elution order with the most polar one eluting first followed by the less polar ones (Zhang et al., 2013). Most polar compounds eluting first were serotonin (5.35 min), followed by tryptamine (9.53 min), 4 hydroxybenzoic acid (10.79 min), vanillic acid (12.25 min), syringic cid (12.74 min), p-coumaric acid (17.87 min), and lastly, trans-cinnamic acid (36.34 min) (Figure 3).
Vanillic acid (12.113 min) and p-coumaric acid (18.266 min) were detected at 254 nm wavelength in the diluted TKM6 extract (Figure 4). It is plausible that these phenolics found in TKM6 may be among the biochemical natures for its resistance against YSB.
Both vanillic and p-coumaric acid are well-studied phenolics in relation to plant resistance and insect herbivory. In a study conducted on castor plant by Usha Rani and Jyothsna (2014), vanillic acids delayed the egg laying of Achaea janata L. and Spodoptera litura F. On the other hand, a decrease in A. janata and S. litura larval body weights was evident in coumaric acid treatments confirming the antifeedant effects of the phenolics tested. In maize, the amount of free p-coumaric acid was correlated with the resistance level. Higher quantities of p-coumaric in the pith could contribute to general resistance to stemborer attack. Jointly with ferulic acid, p-coumaric acid could provide resistance mechanisms through cell wall fortification and lignification. The vanillic acid showed a decreased tendency after silking, when maize is most attractive for Sesamia nonagrioides, suggesting this acid could act as a chemoattractant for S. nonagrioides larvae or adults (Santiago et al., 2005).
The dietary exposure to vanillic acid led to enhanced activities of detoxifying enzymes, đ˝-glucosidase, carboxyl esterase, glutathione S-transferase, and glutathione reductase in the midgut tissues of all the larval instars of Spodoptera litura, indicating the toxic nature of these compounds in Capsicum annum.
RICE-BASED BIOSYSTEMS JOURNAL (2020) 7: 49-58 57
YSB varied in terms of released phenolic compound.
The resistant genotypes exhibited multiple defense mechanism in terms of higher expression of phenolic compound particularly when plants perceived threat following YSB infestation. Moreover, the optimized biochemical screening method from this study, in addition to existing breeding methods, can aid in the selection of materials to be used in the development of lines/variety that are resistant to stemborer in particular. However, correlation of results with the parallel studies on morphological (trichome density, orientation, and distribution) and physiological factors (lignin thickness and content) is necessary to explain the ways rice cope and to provide a complete picture plants protecting themselves from insect pest infestation such as stemborer.
Acknowledgment
This research is a component of the project
âMechanisms of rice insect pest and disease resistance in traditional rice varieties and development of genetic stocks with novel sources of resistant genesâ
funded by the Department of Agriculture - Bureau of Agricultural Research (DA-BAR).
The Authors are grateful to GSRillon, GDCSantiago, GFEstoy, BMTabudlong, PSTorrena, ABEstoy, NLManigbas, and NVDesamero for the comments, suggestions, and assistance in the experimental set up and design. The authors also grateful to JM Manalo IV for editing/proofreading the drafts of the paper.
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