Classification Of Ganoderma Species Causing Bsr

Published Date: 02 Nov 2017

CHAPTER 2

Basal stem rot (BSR) is a devastating disease of oil palm in Malaysia and Indonesia, which caused by fungi of Ganoderma species (Idris et al., 2000). Four species of Ganoderma, namely G. zonatum, G. encidum, G. colossus and G. applanatum have been identified as the causal agent of BSR (Idris et al., 2000). Tuner (1981) has reported that fifteen species of Ganoderma are more likely the pathogens associated with BSR disease from different part of the world. Therefore, BSR is unlikely to be caused by only single type of Ganoderma species in any specific area. Recently, G. boninense, G. zonatum, G. miniatocinctum and G. tornatum have been identified to cause diseases of oil palm in Malaysia (Idris et al., 2000). G. boninense, G. zonatum, G. miniatocinctum are classified as the pathogenic species, however, the latter three species were less aggressive than G. boninense. The G. tornatum are found only on dead palms as saprophyte , thus, it is classified as the non-pathogenic type (Idris et al., 2000).

2.1.2 Symptoms of BSR disease

The earliest external symptoms of basal stem rot of oil palms normally occur in the foliage, generally after at least half of the cross-sectional area of the stem base has been destroyed. The supply of water and nutrient supply to the aerial parts of the plants will be restricted when the plants were decayed. The external symptoms of young palms infected with basal stem rot are yellowing or mottling of the lower fronds, followed by necrosis. The young unfolded leaves may become chlorotic and reduced in length, sometimes necrotic tips will also be observed. As the disease progresses, palms may have a pale appearance, spear leaves remaining unopened and growth of the palms was retarded. Mature palms infected with BSR will also have a similar symptom with multiple unopened spear leaves and a commonly pale leaf canopy. When the palms are affected with BSR, necrosis will begin at the oldest fronds and extending to younger regions of the crown. After the appearance of unexpanded spear leave, the palms normally will die within 6 to 12 months (Plantwise Knowledge Bank n.d.).

Furthermore, BSR disease also causes the rotting of internal tissues at the stem base or root bole. The fracturing of the stem or root bole of the infected palm will also be observed and the palm will collapse eventually. In addition, fungus also may be observed as a whitish skin-like layer on the inner surface of the exodermis of the older roots. Normally, the position of the infected area within the stem will be reflected by the position of basidiomata on the stem. (Plantwise Knowledge Bank n.d.). However, the basidiomata of Ganoderma will only appear at a very late stage of infection when more than half of the roots have decayed. Thus, this leaves no chance for the curing process to be carried out on the infected palms (Alizadeh et al., 2011).

2.2 Antagonistic bacteria against G. boninense as the biological control agents for basal stem rot disease

To control BSR, chemical, cultural and mechanical controls were conducted but satisfactory outcome has not been achieved. Therefore, recent control measures to overcome this Ganoderma problem are focused on seeking effective biological control agents. In a research done by Suryanto et al. (2012), chitinolytic bacterial isolates such as Enterobacter sp. KR05, Enterobacter cloacae LK08, Bacillus sp. BK13, Enterobacter sp. BK15, and Bacillus sp. BK17 can be utilized to control basal stem rot disease caused by Ganoderma boninense in oil palm seedling. Antagonistic assay of chitinolytic bacterial isolates to G. boninense was conducted in minimum salt medium (MSMC) agar with 2% colloidal chitin as sole carbon source. In the antagonistic assay, fungal cultures were grown at the center of MSMC agar. After that, two pieces of paper discs soaked with bacterial suspension were placed in the opposite direction about 3.5 cm from the fungal culture. The plates were incubated around 28 to 30°C. Clear zone was observed around the bacterial colonies on MSMC medium. Inhibition zone was measured from 5 to 10 days of incubation as the radius of the normal fungal growth subtracted with the radius of the inhibited fungal growth. The ability of the chitinolytic isolates in reducing the basal stem rot disease incidence was also being tested in the way of pouring the 3 to 4 months old of oil palm seedlings with chitinolytic bacterial isolates a day prior infestation of G. boninense spores. In this approach, the result showed that all chitinolytic isolates were able to inhibit the growth of G. boninense in vitro. Thus, Suryanto et al. (2012) concluded that chitinolytic isolates were able to reduce the disease incidence on the oil palm seedling to some extent and these isolates might infest into the oil palm seedling root as endophytes.

Bivi et al. (2010) had done a research on screening the potential endophytic bacteria to be used as a biological control agent for Ganoderma boninense, the major causal pathogen of Basal Stem Rot (BSR) disease in oil palm. Dual culture assay was carried out to screen whether the isolated bacteria possess antagonistic properties towards G. boninense in vitro. For dual culture assay, a five mm diameter agar disc was taken from the five day-old PDAculture of G. boninense and plugged centrally in nutrient agar plate. Then, colonies of endophytic bacteria were streaked three cm away from G. boninense plug. The entire dual culture assay plates were incubated at 28±2ºC. After 7 days of incubation, the percentage inhibition of radial growth (PIRG) of each assay plate was assessed by using the formula of , in which R1 = radial growth of G. boninense in the negative control plate and R2 = radial growth of G. boninense in the direction toward the bacteria. The most promising bacteria with PIRG value of more than 50% were kept for further study. In their research, twenty endophytic bacteria was isolated from symptomless oil palm roots but only seven isolates showed inhibitory effect by suppressing the mycelial growth of G. boninense. Four bacterial endophytes isolated (EB2, EB4, EB5 & EB6) may act as a potential biocontrol agents against G. boninense since they showed a PIRG value of 52.78, 83.33, 67.59 and 93.52%, respectively on NA medium. EB4 and EB6 showed a higher inhibitory effect toward G. boninense if compared with other bacteria isolates. By using the Biolog® System, EB6 was identified as Pseudomonas aeruginosa but EB4 could not be unidentified due to the limitation of the method used. EB4 and EB6 were both Gram negative, rod shaped bacteria.

Study of Zaiton et al. (2007) stated that Burkholderia cepacia (B3) and Pseudomonas aeruginosa (P3) isolated from symptomless oil palm root tissues possess antagonistic effect toward the spread of G. boninense. Burkholderia cepacia and Pseudomonas aeruginosa able to keep the G. boninense population below threshold for the initiation of BSR by restricting its entry and movement in the palm. In their study, the oil palm seedlings were inoculated with the respective endophyte treatments by drenching the soil with 150 mL of the inoculum suspensions. Seedlings treated with sterile distilled water (SDW) acted as the negative control. In the duration of 8 months after inoculation, BSR incidence was reduced by 76% in seedlings pre-inoculated with P. aeruginosa (P3). B. cepacia (B3) reduced incidence by 42% and the mixture of P. aeruginosa and B. cepacia by 54%. Zaiton et al. (2007) also concluded that endophytes may act as biocontrol agents against G. boninense since they are protected from the adverse conditions outside the soil and they are able to invade and proliferate in the plants to encounter the pathogens for BSR.

Dwi et al. (2011) had conducted a study on the isolation and characterization of chitinolytic bacteria and their potential to inhibit plant pathogenic fungi. Soil bacteria were isolated from Karo, Langkat, and Bangka, Sumatra. Fungus Ganoderma boninense, Fusarium oxysporum and Penicillium citrinum was used for the stock cultures of growth inhibition assay. All bacterial isolates inhibited the growth of G. boninense, F. oxysporum and P. citrinum at a different extent on the MSMC agar. MSMC agar is a modified salt medium which contains 2% chitin colloidal. In their research, bacteria LK08 showed the highest inhibition rate followed by BK07 and BK09. Antagonistic activity of the bacteria was measured as radius of uninhibited mycelia substracted by radius of inhibited mycelia by bacterial chitinolytic activity. However, P. citrinum was inhibited more by BK07 and BK09. Morphological and physiological traits of the bacterial isolates were also being studied in their research. LK08 and BK07 are Gram negative, rod shaped, catalase positive and motile bacteria. In addition, BK08 is a Gram negative, cocci, catalase negative and motile bacteria.

Chang et al. (2010) stated that by boiling and crushing the shellfish processing waste, shrimp and crab shell powder can be produced. Crab shell powder can be used as a substrate for the isolation of Bacillus subtilis NPU 001 (antifungal chitinase producing microorganism) from soil sample. By culturing the Bacillus subtilis in a medium containing 2% (w/v) shrimp and crab shell powder as the major carbon source, Bacillus subtilis was able to produce chitinase which had a molecular weight of 31 kDa and a pI of 5.4. In their research, colloidal chitin was used as a substrate for the chintinase assay. The purified chitinase worked best at pH 6.0, and the chitinase activity was stable from pH3.0 to pH7.0 for 30 minutes. The optimum temperature for chitinase activity was 50oC, followed by activity lost at 80oC over 10 minutes and completely denatured at 90oC over 10 minutes. The purified chitinase was able to inhibit the hyphal extension of the fungus Fusarium oxysporum. Compared with other known bacterial chitinases, NPU 001 chitinase possessed antifungal activity against plant-pathogenic fungi and chitotriose was produced as the major enzymatic hydrolysate from colloidal chitin.

In the study of Kamil et al. (2007), four hundred bacterial isolates were isolated from rhizosphere of deferent plants collected from three different localities in Egypt. From the 400 isolates tested, twenty of them possessed the chitinase activity. Then, isolates Bacillus licheniformis (MS1), Stenotrophomonas maltophilia(MS2), Bacillus licheniformis (MS3) and Bacillus Thuringiensis(MS4) showed the highest chitinase activity compared to the other selected isolates. Bacillus licheniformis (MS1) was rod shaped, motile, appeared as flat and brown in colour on the agar plate. Stenotrophomonas maltophilia (MS2) was rod shaped, motile, appeared as flat and yellow in colour on the agar plate. Bacillus licheniformis (MS3) was rod shaped, motile, appeared as flat and white in colour on the agar plate. Bacillus Thuringiensis(MS4) was rod shaped, motile, appeared as flat and white in colour on the agar plate. Four of the isolates (MS1, MS2, MS3 and MS4) showed positive result for the catalase, anaerobic growth, hydrolysis of casein, lipase test and able to grow at 30°C and 40°C. The isolates showed negative result for the formation of indole, ornithine , decarboxylase, gas from nitrate, H2S production test and unable to growth at 5°C, 10°C, 65°C and at NaCl, 12%. From the in vitro antifungal assay conducted, MS1, MS2, MS3 and MS4 were able to suppress the mycelial growth of phytopathogenic fungi like Rhizoctonia solani, Macrophomina phasiolina, Fusarium culmorum, Pythium sp, Alternaria alternata and Sclerotium rolfsii. MS1 and MS3 isolates possessed the highest antifungal activity, where the largest inhibition zones were recorded. Hence, Bacillus licheniformis may be a potential biological control agent against some plant diseases.

2.2.1 Mechanisms and metabolites involved in anti- fungal activities

The general mechanism of antagonistic bacteria as biological control agent against the pathogenic fungi can be divided into direct and indirect effects. Direct effects include competition for space and nutrients, production of antibiotic and lytic enzymes, inactivation of the pathogen’s enzymes and parasitism. Indirect effects include all those features that induce host plant biochemical and morphological changes such as tolerance to stress through enhanced root and plant development, solubilization or sequestration of inorganic nutrients, and induced resistance (Gohel et al., 2006).

Competition for substrates and site exclusion is a common anti- fungal mechanism possessed by bacteria where the antagonist (bacteria) and the pathogen are closely related. Endophytic bacteria, Burkholderia cepacia as an internal colonizers may compete in the vascular system of oil palm depriving Ganoderma boninense for nutrients and space (Azadeh et al., 2010). For example, efficient root colonizer Pseudomonas aeruginosa PNA1, isolated from rhizosphere of chicken pea plants, has been shown to be effective in combating a number of phytopathogenic fungi (Gohel et al., 2006).

Furthermore, antibiosis is also one of the important mechanisms in controlling the fungal infection (Gohel et al., 2006). Fluorescent Pseudomonas spp. able to produce the antibiotic of phenazine derivatives that contributes to disease suppression. Apart from that, anti- fungal metabolite such as iturin A and rhizocticin A are produced by Bacillus subtilis to encounter against the Candida spp. and Aspergillus spp. (Jonathan 1999).

Bacteria may also involve in the anti-fungal activities by inducing the systemic response of the plant toward the plant pathogenic fungi. Induced systemic response (ISR) is defined as the process of active resistance dependent on the host plant’s physical and chemical barriers, activated by biotic and abiotic agents (Gohel et al., 2006). Study of Kloepper et al. (1992) states that systemic resistance in plants toward the plant pathogens is induced by bacteria such as Serratia spp., Burkholderia spp., Bacillus spp., Pseudomonas spp. and Actinomycetes from the genera Streptomycetes, Streptosporangium and Nacardiopsis.

In addition, parasitism and production of extracellular enzymes is also one of the important mechanisms for control of plant diseases. The ability of bacteria Actinomycetes spp. to parasitize and degrade spores of fungal plant pathogens is well known. Furthermore, mycolytic enzyme such as chitinase is produced by Bacillus subtilis and Streptomyces bacteria to lyse fungal cell walls (Suryanto et al., 2011).