Artificial Infection Techniques for the Study of Plant Pathogen Interaction

Artificial inoculation techniques have been established to advance the understanding of plant-pathogen interactions, such as Fusarium oxysporum in watermelon (Zhou and Everts, 2004), Phytophthora lateralis in Chamaecyparis

lawsoniana trees (Oh and Hansen, 2007) and G. boninense in coconut (Karthikeyan et al., 2007). These studies have been carried out in the field or shade house on

intact plant seedlings (in planta), where external abiotic and biotic stresses could affect the plant-pathogen interactions and disease developments.

Limitations in these in planta infection systems could be improved by conducting an artificial infection in sterile environment (in vitro). In vitro system refers to studies in experimental biology that are carried out in an enclosed environment by using an organism that has been previously isolated from its natural environment, to allow a more simple yet comprehensive analysis (Alberts

et al., 2002). This system provides a controlled environment (effects of

environmental factors exerted on the assay can be neglected), where one-to-one interaction between plant and compatible pathogens can be evaluated. To date, however, there is still lack of an infection assay of oil palm undertaken under in

vitro conditions.

Previous plant and fungal pathogen studies in model plants such as

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assays to infect host plants with compatible fungal pathogens. In Arabidopsis and tobacco plants, spore suspensions of Botrytis cinerea have been inoculated onto the leaves by injection using sterilised needle (Govrin and Levine, 2000; Stukkens

et al., 2005). In contrast, conidial suspension of rice blast fungus Magnaporthe grisea were inoculated onto the leaves of rice cultivars by spraying with an artist’s

air brush (Foster et al., 2003). After that, the plants were incubated in growth chamber or tissue culture room with controlled light intensity, humidity and temperature. However, spores or conidial suspensions may not be suitable as the source of inoculum for G. boninense due to the nature of this fungal pathogen. Under normal circumstances, G. boninense produces spores after the formation of fruiting bodies on host plants, causing difficulty of isolating its spores on nutrient medium. Besides, genetic variation in G. boninense varies greatly when spore cultures are used to artificially infect oil palm, as the heterothallic and tetrapollar mating system and the presence of multiple alleles at mating loci of G. boninense spores favours out-breeding of this fungus (Pilotti, 2005). Hence, mycelium infection remains as a better option for artificial infection of oil palm by G.

boninense.

In vitro infection assays using fungal mycelium culture were reported by

Baumgartner et al. (2010), which focused on Armillaria root disease on Vitis

vinefera. In their study, mycelium plugs of Armillaria mellea were co-cultivated

with grape rootstocks on MS agar-based medium in magenta boxes (Figure 2.4). Then, the plants were incubated in growth chamber with controlled light intensity, day light and temperature for up to 4 months before the nutrients in the agar medium became deficient. Their study successfully developed an in vitro infection assay using fungal mycelium of A. mellea. This was a platform for the development of an artificial infection assay in oil palm by G. boninense with controlled environment.

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Figure 2.4 (A) Inoculation of Armillaria mellea (arrows) in culture medium with grape rootstocks. (B) Non-inoculated culture (Baumgartner et al., 2010).

2.2.5.1 Rubber-wood block inoculation system in oil palm for BSR disease

Artificial infection of G. boninense has been carried out using rubber wood blocks (RWB) on healthy or injured roots (Lim et al., 1992; Sariah et al., 1994; Rees et al., 2007; Mohd Zainudin and Abdullah, 2008; Mohd As’wad et al., 2011; Alizadeh et al., 2011, Kok et al., 2013; Yeoh et al., 2013) and germinated seeds (Idris et al., 2006; Breton et al., 2006) of oil palm. This method successfully proved the pathogenicity of G. boninense to fulfil Koch’s postulates. RWB inoculations involve cultivation of G. boninense fungal mycelium on sterilised wood blocks that have been soaked in growth medium for a few weeks or months. After that, resulting RWB inoculum is either tied to the root collars (Rees et al., 2007) or placed in contact with the primary roots of potted oil palm in polythene bags (Sariah

et al., 1994; Mohd As’wad et al., 2011; Kok et al., 2013). Idris et al. (2006) applied

similar infection techniques using germinated seeds of oil palm by placing the RWB inoculum at 2.5 cm away from the seeds before planting them in polythene bags and growing in field conditions (Figure 2.5).

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Figure 2.5 RWB techniques for the inoculation of G. boninense on oil palm germinated seeds. (A) RWB inoculum was prepared by inoculating RWB with G.

boninense. (B) RWB inoculum was transferred into polythene bag containing

unsterilised soil. (C) Oil palm germinated seeds were planted in polythene bag containing RWB inoculum (Idris et al., 2006).

Although RWB inoculation technique has been widely accepted and is frequently used, it is considered time-consuming as it requires an initial step of inoculating sterilised RWB with desired fungal inoculum that can take one month. The first symptom development is usually manifested after several months post- inoculation. For instance, disease incidence is normally detected in infected oil palms approximately 6 to 9 months post-inoculation (Sariah et al., 1994; Idris et

al., 2006), with the shortest period of 2 months reported by Kok et al. (2013). This

is considered undesirable as a number of inocula may dry out and die before the infection takes place (Baumgartner et al., 2010).

In addition, RWB technique can be inaccurate and labour intensive because it is conducted in the field or shade house (Sariah et al., 1994; Idris et al., 2006; Rees et al., 2009; Kok et al., 2013), where external environmental factors and stresses are exerted on oil palms. Furthermore, saprophytes could be present in the RWB even after several rounds of sterilisation, resulting in high levels of contamination when the RWB is used as the fungal inoculum (Chong et al., 2012). Disease symptoms manifested on treated oil palm due to G. boninense

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pathogenesis are difficult to differentiate from those symptoms caused by other stress factors or contaminations.

Recently, the concentration of ergosterol, which is the fungal cell membrane principal sterol unit, has been correlated with the increase in G. boninense biomass in infected oil palm (Mohd As’wad et al., 2011; Chong et al., 2012). This compound provides quantitative measurement of infection by G. boninense, which is independent from disease symptoms on oil palm. However, measurement of ergosterol can be non-specific as this compound is also detected in unrelated fungi, algae and protozoa (Chong et al., 2012). Since the RWB infection system and the response variables gathered from this system may not be suitable and are insufficient to answer research questions about the infection biology and pathogenesis of G. boninense, or to understand the underlying defence mechanism in oil palm, there is a need to improve on the RWB infection system for oil palm BSR.