Leaf senescence and the presence or absence of GBLS mycelium on stems were used to categorise oil palm plantlets into different disease severity classes, as these were the key visible symptoms that developed in plantlets within the 8 day experimental period. Figure 4.3 shows that disease scores of Ganoderma- infected, T3 plantlets increased linearly to as high as 70.37% at the end of the experimental period, and was significantly higher (P < 0.001, Appendix C1) than T1 (7.41%) and T2 plantlets (25.00%), indicating that T3 plantlets are severely infected by G. boninense via the in planta infection system.
Figure 4.3 Disease severity index (DSI) in oil palm plantlets within 8 days of incubation. Standard errors of mean (SEM) of replicate readings from three rounds of experiments was represented by the error bars.
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High DSI values in T3 plantlets were supported by a number of previous artificial infection studies of oil palms with G. boninense, where the rises in disease severity of oil palms were closely associated with the presence of G. boninense, GBLS and the duration of infection (Sapak et al., 2008; Mohd As’wad et al., 2011; Kok et al., 2013). However, the disease severity scored in infected and control oil palms in previous studies were significantly different after 10 to 14 weeks post- inoculation (Mohd Zainudin and Abdullah, 2008; Kok et al., 2013), with the shortest duration of 5 weeks post-inoculation reported by Naher et al. (2012a). As compared to their studies, DSI scores in T1 and T3 plantlets of the present study were significantly different (P < 0.05) from one another after 2 DPI, justifying the efficiency of the in planta infection assay.
From Figure 4.3, apparent disease scores were detected in T1 and T2 plantlets at low levels at the end of the experiment (7.41% and 25.00% respectively), as a result of leaf senescence due to mechanical injury during the transfer of plantlets and artificial wounding process (Philosoph-Hadas et al., 1994). Previous studies explained that the wounding of plants could stimulate the rate of ethylene production and plant respiration that lead to chlorophyll degradation and leaf senescence (Rushing, 1990; Philosoph-Hadas et al., 1994). Besides this, in the present study wounding processes in T2 plantlets were performed on the basal stem region, which impaired the internal xylem and disrupted the water translocation system, ultimately causing leaf senescence in T2 plantlets. Since early symptoms of Ganoderma infection in oil palm were similar to wilting effects, it was essential to wound the oil palm plantlets artificially in the experiment so that the symptoms that arose from the wounding effects and those of infection by G.
boninense could be differentiated.
However, there were no apparent differences (P > 0.05, Appendix C2-C5) in the height, weight, root length and stem diameters of oil palm plantlets under different treatments (T1, T2 and T3) over 8 days of incubation (Figure 4.4).
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Figure 4.4 (A) Height, (B) weight, (C) root length and (D) stem diameter of oil palm plantlets within 8 days of incubation. SEM of replicate readings from three rounds of experiments was represented by the error bars.
The height, weight and root lengths were weakly correlated (R < 0.25) with the disease severity index of oil palm plantlets, and there were no significant differences (P > 0.05) between their correlations (Table 4.2). The observation period in this experiment was probably too short for these growth characteristics to have significant changes, despite different treatments that were conducted on the oil palm plantlets. Similar results were reported in a previous study on healthy regeneration of oil palm plantlets that were maintained in MS culture medium after 4 to 6 weeks (Suranthran et al., 2011), where no significant changes were found in plant height, stem diameter and root lengths. In addition, a former in vivo infection study in oil palm by Ganoderma showed significant changes in the plant
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height, stem diameter and root mass of oil palm only after 8 months of infection (Sapak et al., 2008).
Table 4.2 Correlations between the physical growth parameters and disease severity index in oil palm plantlets.
Growth Parameters Disease Severity Index (DSI)
Pearson R R2 P value Summary
Height -0.1093 0.01194 0.4749 ns
Weight -0.1942 0.03770 0.2012 ns
Stem diameter 0.3175 0.1008 0.0336 *
Root length 0.2193 0.0481 0.1477 ns
Chlorophyll content -0.9299 0.8646 <0.0001 ***
Nevertheless, stem diameters of T2 (4.89 mm) and T3 (4.64 mm) plantlets were greater than T1 (4.36 mm) in overall observations (Figure 4.4D). The stem diameter of oil palm plantlets was positively correlated with disease severity (R = 0.3175, P < 0.05) (Table 4.2), where an increase of disease severity resulted in a 10.08% increase in oil palm stem diameter [r2: (0.3175)2 x 100]. This indicated that stem regions of T2 and T3 plantlets were swollen due to both mechanical injury and fungal infection. Swelling effects on plant stems due to wounding and infection were also reported in Pinus spp., where a massive destruction of the internal tissues including cambial zone, secondary phloem and xylem was observed (Liphschitz and Mendel, 1989; Nagy et al., 2000).
On the other hand, foliar conditions (the intensity of green colour and quantity of chlorophyll) are good indicators for plant health, as healthy plants normally have a higher quantity of chlorophyll content and greener leaves as compared to the diseased plants. Leaf chlorophyll content is one of the photosynthetic parameters that link directly to the senescence process, which can be used to verify chlorosis of leaves and yellowing development in oil palm plantlets. However, standard methods for the quantification of chlorophyll content in foliar tissues are time consuming and destructive to plant samples (Coste et al., 2010),
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making further analyses of plant specimens impossible. With advanced technologies, foliar greenness could be measured in a relatively simple, rapid, inexpensive and non-destructive manner using a chlorophyll meter such as SPAD- 502 by Konica Minolta, Osaka, Japan. Chlorophyll meter SPAD-502 is a lightweight handheld spectrophotometer and is sensitive to the chlorophyll pigments in leaves. The signal is accepted by a microprocessor and computed into a unit-less SPAD (Soil Plant Analysis Development) value (Wood et al., 1993a). According to Krugh
et al. (1994), chlorophyll meter SPAD-502 can be used to classify foliar “greenness”
of a plant. In their study on maize, mean SPAD values of 17.35 and 40.02 were obtained for yellow-green and green foliar respectively. Nevertheless, this meter was unable to pick up chlorophyll content in yellow foliar tissue, suggesting a possible “yellow” class of foliar tissues would be scoring SPAD values that are very near to 0. Higher SPAD value represents higher levels of chlorophyll content present in oil palm leaves and vice versa (Santos, 2001).
In Figure 4.5, it was observed that the SPAD value of T1 plantlets was higher (SPAD 30 and above) and remained constant throughout the experimental period. In contrast, the SPAD values of T2 and T3 plantlets were reduced almost linearly, with T3 plantlets having the lowest SPAD values consistently. This signified that wounding and infection reduced chlorophyll contents in leaf tissues of oil palm. SPAD values of T3 plantlets were significantly lower than of T1 (P < 0.001) and T2 plantlets (P < 0.01) on Day 8 (Appendix C6). Besides, there was a significant negative correlation between the chlorophyll content and disease severity (R = - 0.9299, P < 0.001) (Table 4.2). The value of r2 [(-0.9299)2 x 100] indicated that the increase of DSI led to a decrease in leaf chlorophyll content in oil palm plantlets by 86.37%.
Similar results were obtained in previous infection studies on Vitis vinefera, where infection by compatible fungal pathogens reduced chlorophyll content in leaves (Santos et al., 2005). Reduction in leaf chlorophyll contents are commonly
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noticed in wounded Arabidopsis (Riou et al., 2002) and infected plants (Seo et al., 2000; Milavec et al., 2001; Scarpari et al., 2005). This phenomenon is directly correlated with a decline in the level of chlorophyll a and b photosynthetic pigments (Scarpari et al., 2005; Santos et al., 2005) and an increase in carotenoid to chlorophyll ratio (Milavec et al., 2001). These chlorophyll pigments are degraded by H2O2 and phenolic compounds (Smart, 1994). In the current study, reduction of chlorophyll content was the first symptom that was observed in wounded and infected plantlets as compared to the other physical symptoms. This was because plant chloroplasts consist of the highest amount of proteins in leaf cells, which makes them the first organelle to undergo catabolism during leaf senescence upon mechanical stress and infection (Quirino et al., 2000).
Figure 4.5 Chlorophyll contents of oil palm plantlets within 8 days of incubation. SEM of replicate readings from three rounds of experiments was represented by the error bars.
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4.3.2 Effect of G. boninense Pathogenesis on the Degree of Colonisation at