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Antagonistic Efficacy of Trichoderma harzianum and Bacillus cereus against Ganoderma Disease of Oil Palm via Dip, Place and Drench (DPD) Artificial Inoculation Technique.

Byline: S.A. Nusaibah, Ghazala Saad and Tan Geok Hun

Abstract

Sustainability of the oil palm industry is crucial to ensure Malaysia's gross domestic product (GDP) by the agricultural sector. It is crucial to discover a sustainable and eco-friendly remedy for the most devastating Ganoderma disease of oil palm. The effects of pre-inoculation of oil palm seedlings with either Trichoderma harzianum and/or Bacillus cereus on their vegetative growth and the suppression of Ganoderma boninense were investigated. The dip, place and drench (DPD) artificial inoculation method was used to assure disease development. Disease severity was assessed based on the root symptoms (DS), disease incidence (DI) and disease reduction (DR). Application of a mixture of T. harzianum and B. cereus had the highest contribution to the vegetative growth of oil palm seedlings.

However, single application of B. cereus was found to be the most effective treatment in suppressing Ganoderma disease of oil palm with a disease reduction of 94.75% followed by single applications of T. harzianum (78.98%) and mixture of both T. harzianum and B. cereus (68.49%).

Keywords: Dip, place and drench (DPD); Bacillus cereus; Trichoderma harzianum; Biocontrol

Introduction

Oil palm (Elaeis guineensis Jacq.) is a tropical perennial crop, purposely for oil. The oil extracted from this crop is used world-wide for different industrial applications including cosmetics, oleo-chemicals, food and biofuel (Murphy, 2009; Paterson et al., 2009). This golden crop has been contributing generously to Malaysia's economy. However a major disease caused by Ganoderma boninense has been a treat to Malaysia's oil palm industry for many years and the estimated lost per annum was recorded as earnings of US$20 billion in terms of export earnings (MPOB, 2011) Therefore, there is an obvious need for understanding and developing an early control that will contribute to a sustainable environment along with catering this catastrophic disease (Paterson et al., 2009; Flood et al., 2010).

Government legislature for reducing the consumption of chemical fungicides had triggered an increasing public awareness on the hazards and the importance in attempting the search for alternatives of synthetic chemical fungicides. Hence, efforts to find an alternative way to control Ganoderma disease via biological control agents (BCAs) and utilization of resistant oil palms (Susanto et al., 2005) have been a better approach. In vitro studies have shown that Trichoderma spp., Aspergillus spp., and Penicillum spp. are antagonistic agents towards Ganoderma spp. (Bruce and Highley, 1991; Badalyan et al., 2004). These antagonists, particularly Trichoderma spp., are good BCAs of Ganoderma spp. (Bruce and Highley, 1991; Susanto et al., 2005). A part from fungus, endophytic bacteria was reported with the ability to enhance plant's immune system against pathogen attack.

This is done by winning competition, antibiosis, induced resistance and promoting plant growth. Endophytic bacteria such as Pseudomonas and Bacillus spp. were found to be potent BCAs against fungal pathogens on crops such as cotton, oilseed rape, tomato, cucumber and peas (Chen et al., 1995; Alstrom, 2001). In addition, some members of the genus Bacillus such as B. cereus are often considered as microbial factories of biologically active molecules that are potential inhibitors of fungal growth (Perez-Garcia et al., 2011).

Besides that, recent control measures to overcome Ganoderma infection in palms are now focused on the use of BCAs. Subsequently, research on the use of BCAs for Ganoderma disease control has gained momentum.

Consequently, in the present study, mixture of different BCAs was studied in an effort to add more potency to biological control solutions for Ganoderma disease.

In addition to that, an attempt to design a new artificial vivo trials has been taken in this study. To date the existing method using Rubber Wood Blocks (RWBs) (Khairudin, 1990; Nur Ain Izzati and Abdullah, 2008) is costly and it has been challenging to obtain the rubber wood supply due to the decreasing production. Apart from that, preparation of RWB inoculums are time consuming (approximately two months based on the RWB sizes) and the possibility of contamination with other fungi are high. Therefore, an alternative Ganoderma artificial inoculation method such as DPD would be desirable.

Hence, the objectives of this study were (i) to establish Ganoderma infection via a newly developed Ganoderma artificial inoculation method and (ii) to determine the efficiency of in vivo interaction of Trichoderma harzianum and/or B. cereus mixture for suppression of G. boninense infection in oil palm seedlings.

Materials and Methods

Maintenance of Microbes

All the microbes used in this study were isolated from oil palm roots in a preliminary study. These isolates were then sequenced, identified and sustained in the Department of Plant Protection, Universiti Putra Malaysia. Isolate UPM13 (G. boninense) and UPM29 (T. harzianum) cultures were maintained on Potato Dextrose Agar (PDA) (Difco(tm)) and incubated at room temperature (27 +- 1oC) for eight to ten days prior to usage. While pure freshly cultured bacterium UPM15 (B. cereus) cultures were maintained on Nutrient Agar (NA) (Difco(tm)) at 4+- 1oC for short term storage.

Plant Material

Oil palm seedlings of three months old, commercial GH500 variant (DuraxPisifera) placed in trays following normal nursery practices and certified as Ganoderma-free were purchased from Sime Darby Seeds and Agricultural Services Sdn Bhd., Banting, Selangor. The Seedlings were placed in a nursery, shaded with two layers of polynet 30/70 at Ladang 15 Nursery, Faculty of Agriculture, UPM, Serdang. Watering was done twice daily, before 11.00 am and after 4.00 pm.

The trays containing 3 months old seedlings were left in the nursery for two weeks to stabilize and adapt to the nursery environment before transferring them to polythene bags (polybags) 30 cm x 38 cm with a thickness of 500 gauge (0.125 mm) (Halimah et al., 2010) containing 3 kg of sterile soil mixture (3:2:1 v/v/v topsoil: peat: sand) prior to pre-inoculation with the BCAs and artificial inoculation with UPM13 (G. boninense). In vivo nursery trial was conducted with eight treatments (Table 1), replicated twice with six seedlings per replicate.

Table 1: Percentage of disease incidence (DI) in oil palm seedlings after inoculation with UPM13 (Ganoderma boninense)

Treatment###Disease Incidence (%)#

###2 MAI## 3 MAI 4 MAI 5 MAI 6 MAI

Ganoderma boninense (G)###0###16.6a 33.3b 66.7c 83.3d

Bacillus cereus+Trichoderma###0###0###16.6a 16.6a 33.3b

harzianum+Ganoderma

boninense (GTB)

Trichoderma###0###0###0###16.6a 33.3b

harzianum+Ganoderma

boninense (GT)

Bacillus cereus+Ganoderma###0###0###0###0###16.6a

boninense (GB)

Table S1: Treatments for experiment

Treatment###Description

T1 (TB)###plant + T. harzianum + B. cereus

T2 (T)###plant + T. harzianum

T3 (B)###plant + B. cereus

T4 (G)###plant + G. boninense

T5 (GTB)###plant + G. boninense + B. cereus + T. harzianum

T6 (GT)###plant + G. boninense + T. harzianum

T7 (GB)###plant + G. boninense + B. cereus

T8 (C)###+plant (Untreated negative control)

Inoculum of Biological Control Agents

Preparation and application of bacterium inoculum: UPM15 (B. cereus) inoculum suspension was prepared using 24 h old growing culture on NA. One mL of the 24 h old suspension was dislodged from the NA culture and pipetted into a test tube containing 9 mL of sterilized distilled water labeled as 10-1. A series of serial dilution up to 10-7 was carried out and then 0.1 mL bacterial suspension of each dilution factor were spread using sterile L-shaped glass rod on individual NA plates and incubated for 24 h at 27+-2degC. After 24 h, the presence of bacterial colonies on the NA plates were counted and expressed as colony forming unit (CFU mL-1). The suspensions were adjusted to a concentration of 1x10-8 CFU mL-1 for seedling pre-inoculation purpose.

Two weeks prior to artificial inoculation, the seedlings were pre-inoculated with UPM15 by drenching the soil with 150 mL of the suspension according to the designed treatments (Table S1). In addition to that, a booster application of the same concentration at pre-inoculation, UPM15 was applied again onto the seedling soil in the polybags after 25 days of artificial inoculation with UPM13.

Preparation and Application of Conidia Suspension

Conidia of UPM29 (T. harzanium) were harvested from seven day-old cultures on PDA. Ten milliliters of sterile distilled water was pipetted onto the PDA plate and the conidia were gently dislodged with a L-shaped glass rod.

Subsequently, the mixture was filtered through filter paper (Whatman(r) Grade 1, diameter: 9 cm, Pore Size: 11 um) to remove mycelial debris. Distilled water was added to make 1 L. Two weeks prior to artificial inoculation with G. boninense inoculum, seedlings were pre-inoculated with 250 mL of fresh conidia suspension of UPM29 by drenching it onto the soil surrounding the stem of the seedling of each treatment according to the designed treatments (Table S1). In addition to that, a booster application in the same amount applied at pre-inoculation of UPM29 was applied after 30 days of artificial inoculation of the oil palm seedlings with UPM13.

Ganoderma Artificial Inoculation with Dip, Place and Drench (DPD) Technique

Mycelia of G. boninense was grown in 250 mL of potato dextrose broth (PDB) for ten days without shaking, and subsequently blender using a kitchen electric grinder (MX-800S, Panasonic Malaysia). The recipe mentioned is for a suspension size for artificial inoculation of one seedling. Once the inoculum suspension was ready, it was transported to the nursery. The oil palm seedling in the polybag was then carefully uprooted by taking out half of the soil from the polybag. The uprooted roots were then immersed or dipped into the suspension of G. boninense fragments with approximately 250 mL of the inoculum per seedling. Subsequent to that, two plates of fully-grown G. boninense on PDA was placed on the remaining soil in the polybag.

The inoculum dipped oil palm seedling roots were then placed in the polybag on top of the G. boninense cultures and the remaining suspension for the dip step were drenched on the roots before covering with the same soil mixture taken out previously. The seedlings were watered twice daily throughout the experiment. The treatments designed for this study have been given in Table S1. Infection in the roots was confirmed by re-isolating G. boninense from the inoculated seedling roots after six months of inoculation on Ganoderma Selective Media (GSM) and via DNA detection using Ganoderma specific primer (Utomo and Niepold, 2000). This new DPD technique was validated thrice in a preliminary trial prior to the present study.

Effect of BCAs on Ganoderma Disease Affecting Oil Palm Seedlings

Assessment on the oil palm vegetative growth: Plant height, total root and top weight and bole girth were recorded onset, and 6 months after the treatment duration.

Ganoderma Disease Assessment at Nursery Trial

Disease development was monitored by measuring Disease Incidence (DI) percentage at monthly intervals. DI of seedlings was assessed as diseased visually (chlorosis and necrosis of leaves, with or without the production of fruiting body) (Idris et al., 2006). DI referred to the number of seedlings showing symptoms mentioned above in relation to the total number of seedlings assessed by the formula by Campbell and Madden (1990):

% Disease incidence (DI) = [Number of seedlings infected/Total number of seedlings assessed x 100]

A reduction in the disease incidence compared with the control would be a measure of the treatment effectiveness in suppressing the disease. Disease progress curve (AUDPC) was calculated using the formula by Campbell and Madden (1990):

(Equation)

Where by: n = the number of assessment time; y = Disease incidence (DI); t = Observation time (months).

The efficacy of treatments in Ganoderma disease reduction was calculated with the following formula:

Disease reduction (DR) (%) = (AUDPC in positive control - AUDPC in treatment) x 100%/AUDPC in positive control

In addition, the disease development in the seedlings was also rated as Disease Severity (DS). DS referred to the total area of plant tissues that exhibits disease symptoms. The percentage of DS of the oil palm root tissues were scored based on the disease rating scale by Breton et al. (2006) (Table 3) and the disease percentage was calculated according to the following formula:

DS (%) = S (Number of seedlings in the scale x Severity scale) x 100/Total number of seedlings assessed x Highest scale

Experimental Design and Statistical Analysis

The treatments designed for this study are shown in Table S1. The nursery trial experimental design used was Randomized Complete Block Design (RCBD) with eight treatments and six biological replicates. The percentage data were transformed by arcsine square root and analyzed by ANOVA with means compared by the Least Significant Difference (LSD) at P [?] 0.05. The vegetative growth data recorded were subjected to ANOVA and the significant data was determined using Tukeys's Studentized Range (HSD) Test at 5% probability level. All statistical analysis was done using SAS(r) software (v 8.1), Institute Inc. 1995.

Detection of G. boninense Genomic DNA on Treated Oil Palm Roots at 24 Weeks to Confirm Disease Establishment

DNA extraction, quantification, amplification and sequencing: Extraction method for total DNA was done according to the manual provided in Qiagen DNeasy Plant Mini Kit with a slight optimization according to Nusaibah et al. (2011). Seedlings were uprooted and the roots were harvested using a cutter and brought back to the lab that was located just 5 min' walk from the nursery.

Table 2: The effect of biological control agents (BCAs) on the development of Ganoderma disease in oil palm seedlings after artificially infected with Ganoderma boninense for six months

Treatment###AUDPC1###DR2 (%)

Ganoderma boninense (G)###158.25###-

Bacillus cereus + Trichoderma harzianum +###49.85###68.49

Ganoderma boninense (GTB)

Trichoderma harzianum+Ganoderma boninense (GT)###33.25###78.98

Bacillus cereus+Ganoderma boninense (GB)###8.3###94.75

Table 3: Scale used to score disease severity index based on rotten root tissues of oil palm seedlings by UPM13 (Ganoderma boninense) (Breton et al., 2006)

Scale###Symptoms

0###healthy no internal rot

1###20% rotting of tissue

2###20% to 50% rotting of tissues

3###>50% rotting of tissues

4###> 90% rotting of tissues

Subsequently the roots washed under running tap water until no soil was visible, dipped in 95% ethanol for 3 min and 5 min in sterile distilled water prior to the extraction process. Only rotten and necrotic secondary roots were subjected to the genomic DNA extraction. The extracted genomic DNA was checked for its concentration and purity using Nanodrop in MultiSkanGo instrument.

Polymerase chain reaction (PCR) amplification of G. boninense treated oil palm root genomic DNA was performed using the Ganoderma specific primers; Gan1: 5' - TTG ACT GGG TTG TAG CTG - 3' and Gan2: 5' - GCG TTA CAT CGC AAT ACA - 3' (Utomo and Niepold, 2000). Amplification was performed according to the protocols of Qiagen TopTaq Master Mix. Eppendorf Mastercycler(r) ep Gradient S Thermal Cycler (Hamburg, Germany) was used to run the polymerase chain reaction (PCR).The PCR started with denaturation for 2 min at 95degC. This was followed by 35 cycles of denaturation for 1 min at 94degC, annealing for 30 sec at 59.9degC and extension for 2 min at 72degC. The final step of extension was carried out for 10 minutes at 72degC, before it was maintained at 4degC.

The Amplified PCR Products were Run on 1.7% Agarose Gel, Stained with Ethidium Bromide (EtBr) and Visualized under a UV Transiluminator

Next, DNA sequencing was done to confirm reliability of amplified products. DNA sequencing for forward and reverse primer amplified products were done by purifying the amplified PCR products using QIAquick Gel Extraction Kit (QIAGEN, Germany). And then sequencing service was outsourced to a commercial service provider (First BASE Laboratories Sdn. Bhd. Malaysia). The sequence similarity was matched via BLASTN tool in National Center for Biotechnology Information (NCBI) against the non-redundant nucleotide database in the GenBank for sequence identification purpose.

Results

Effect of BCAs on Vegetative Oil Palm Growth

In the present study, application of BCAs without G. boninense inoculation as single or mixture significantly increased plant height compared to the positive control (Fig. 1). From the data obtained, mixture application of TB treatment contributed the most in the progression of oil palm seedlings plant height (50.4 cm) followed by the single application of UPM16 (T. harzianum) with 49.3 cm. The lowest plant height was recorded in treatment GTB with 38.6 cm, which is lower than the positive control (43.4 cm). Nevertheless, significant growth of plant height was observed in between onset and after 24 weeks regardless of treatment.

Based on the data obtained on root dry weight of the treated seedlings, it was shown clearly that B. cereus contributed enormously on the growth enhancement of oil palm seedling root mass regardless of challenged or not challenged with pathogenic G. boninense (Fig. 2). Comparison on the single application of both BCAs pointed out that B. cereus (13.3 g) was the BCA accountable in contributing to the root weight significantly unlike T. harzianum (3.65 g), where the contribution was about 72% higher. However, seedlings treated with the mixture of BCAs gave the highest significant dry root weight of 17.4 g compared to all other treatments. Surprisingly, the Ganoderma control treatment had a significant higher root dry weight (3.32 g) than the negative positive control (1.99 g).

Conversely, the data obtained on the aerial or top dry weight of the oil palm seedlings, single application of T. harzianum displayed a higher top dry weight of 14.1 g compared to B. cereus (8.27 g). On the other hand, the positive control with G. boninense exhibited the lowest top dry weight (4.66 g). However, again the highest top dry mass was yielded by mixture treatment of both BCAs (14.6 g) (Fig. 2).

Bole girth or circumference of treated oil palm seedlings only exhibited real significant differences in the treatments with unchallenged seedlings treated with the mixture of B. cereus and T. harzianum (4.53 cm) compared to the positive and negative controls (1.83 cm and 3.50 cm respectively) (Fig. 3). Unexpectedly, Ganoderma treated seedlings applied with single application of B. cereus (GB) demonstrated a bole size of 4.03 cm, which is 80.6% growth increase even though it is a diseased treatment compared to onset bole girth size. In general, the best vegetative growth of the bole by the TB treatment showed a growth increase of 82.6% within 24 weeks of inoculation period compared to onset bole girth size.

Effects of BCAs on Ganoderma Disease Suppression

The nursery trial was conducted to evaluate the in vivo efficacy of BCAs against Ganoderma disease of oil palm. After 6 months of treatment duration with G. boninense via dip, place and drench (DPD) artificial inoculation technique, seedlings treated with BCAs regardless of single or mixture demonstrated significantly lower percentage of DI (Table 1). The initial DI was observed in G. boninense challenged seedlings three months after inoculation (MAI) with 16.6%.

Nevertheless, a DI (16.6%) was detected in the mixture treatment with BCAs at four MAI. The least DI (16.6%) was exhibited in single treatment with B. cereus at six MAI compared with T. harzianum (33.3%) on the same MAI.

While the efficacy of the selected BCAs as a mixture or single application to reduce Ganoderma disease symptoms was expressed as the percentage of DR derived from the values of AUDPC (Table 2). The AUDPC values suggest the amount of disease developed in each treatment, where the treatment with the lowest AUDPC value indicates the effectiveness of the biocontrol in reducing the disease. The treated seedlings with B. cereus gave the highest DR of 94.8%, followed by T. harzianum (79.0%) and mixture of BCAs (68.5%).

Root disease severity index was scored after 6 months of inoculation with UPM13 (G. boninense) (Fig. 4) based on Table 3 disease scale by Breton et al. (2006). Ganoderma disease establishment was directly visualized based on the disease severity exhibited by the rotten and necrotic oil palm seedling root tissues. Visual colonization of G. boninense mycelium was also observed on both primary and secondary roots at 24 weeks. Based on the score carried out, treatment with B. cereus displayed the lowest DS (8.33%) followed by mixture of both BCAs (25.0%), T. harzianum (33.3%) and the highest DS observed in G. boninense challenged seedlings without any treatment (83.3%).

Detection of G. boninense on Inoculated Roots as Proof of Disease Establishment after Six Months of Inoculation Via Dip, Place and Drench Technique

In order to validate the disease establishment of the newly invented artificial inoculation method (DPD), presence of G. boninense in the challenged oil palm roots were detected via DNA and sequenced for identification after six months of post-inoculation period in a qualitative analysis. To date, detection based on DNA is the most reliable and accurate method. Based on the gel electrophoresis results on amplified PCR products obtained, high intensity of amplified PCR product band was present in the disease control seedlings (Fig. S1 and L1). However, in seedlings treated with BCAs (L2, L3 and L4), low intensity bands were detected, displaying the efficacy of BCAs in suppression or inhibition of G. boninense growth. Nonetheless, no bands or detection of G. boninense in L5-L9, indicates the reliability of this analysis.

In addition, sequenced PCR products also gave 100% similarity with the UPM 13 isolate that was used for Ganoderma disease establishment in the nursery trial conducted. While Fig. S2 (supplementary data) displays visual proof on the reliability of the new artificial inoculation method (DPD) in establishing Ganoderma disease in oil palm seedlings. Rotten and necrotic primary and secondary root tissues were demonstrated in seedlings inoculated with UPM13 (G. boninense) compared to un-inoculated seedling. To further strengthen the disease establishment results, harvested roots (for treatment G, GTB, GT and GB) were yielded on GSM for UPM13 isolation and identification, and all the results obtained were positive on the GSM with UPM 13.

Discussion

In order to assess control options, in in vivo trials, the reliability of an artificial inoculation method for disease establishment is crucial. The current established method for Ganoderma artificial inoculation requires rubber wood blocks (RWBs), which is costly and difficult to obtain nowadays. In addition to that, preparations of RWB inoculum are time consuming (approximately two months according to RWB sizes) and the possibility of contamination with other fungi is high. In addition to that, most importantly a fully colonized RWB in standard sizes used (6 cm x 6 cm x 6 cm and 6 cm x 6 cm x 12 cm) often exaggerates the disease severity on the young oil palm seedlings as old as 3 to 5 months that is often used in the nursery trials. This is way far from the actual disease establishment in the real-time environment where abundant inoculums present on-site. Therefore, an alternative potent Ganoderma artificial inoculation method such as DPD is essential.

The present study presents a new artificial Ganoderma disease inoculation method the Dip, Place and Drench (DPD). Based on the trials conducted, this method can be classified as effective on disease establishment, economical, non-laborious and not time consuming.

The current study evaluated two different BCAs as single and consortium application on oil palm seedlings. The first was the effect on the vegetative growth of oil palm seedlings, while the second was the ability of BCAs to suppress Ganoderma disease. In general, higher vegetative growth was recorded in all BCA treated seedlings when compared to the untreated seedlings. However, mixture of both T. harzianum and B. cereus proved to be significantly superior among all the treatments in enhancement of vegetative growth. This was followed by the single application of T. harzianum and B. cereus, which also significantly improved the vegetative growth of oil palm seedlings studied. Nevertheless, it was noticed that T. harzianum (T) application contributed to the most significant higher dry weight for top among all treatments. These show that, T. harzianum contributes enormously in growth of oil palm foliar and stem, which classifies it as an excellent plant growth promoter.

This was also in line with studies carried out by Harman et al. (2004) and Bal and Altintas (2006) where they observed increased growth response induced by Trichoderma species in several crops. Apart from that, several work that also yielded positive results on Trichoderma sp. as oil palm growth promoter and BCA were reported by Susanto et al. (2005), Nur Ain Izzati and Abdullah (2008) and Naher et al. (2012). Trichoderma spp. increases the uptake and concentration of soil nutrients.

Work by Dawwam et al. (2013) recommended B. cereus isolated from roots of potato plant as a biofertilizer component due to its excellent plant growth promoting characteristics. In addition, Zhao et al. (2011) also classified B. cereus isolated from Sophora alopecuroides root nodules as a potent plant growth promoter. Thus, these studies were in harmony with the present study.

On the other hand, both BCAs studied in the present work demonstrated its ability in suppressing Ganoderma disease of oil palm in vivo via DR and DS of roots analyses. Trichoderma spp. have been one of the most established BCA used worldwide due to its potent antagonistic effects against numerous plant fungal pathogens, including Fusarium spp., Sclerotinia spp. and Rhizoctonia spp. (Pandey et al., 2005; Bernal et al., 2009). To date, strains of Trichoderma including T. harzianum are currently being commercialized because they exert strong competitive effects for space and nutrients; more importantly, they produce toxins against phytopathogenic species, thus making them excellent biocontrol agents (Zimand et al., 1996; Susanto et al., 2005; Sharoni et al., 2006). Hence, this BCA would be the most preferred component in an amendment of a biological control compost or biofertilizer.

According to Pugliese et al. (2011) and Harman et al. (2004), Trichoderma as a component in compost could lead to a substrate with broader-range suppressive effects.

However, this present study demonstrated that consortium of T. harzianum and B. cereus was less effective in controlling Ganoderma disease of oil palm at nursery trial compared to individual application of the BCAs. This may be due to some incompatibility between them, as BCAs were typically selected based on their individual antagonistic behavior towards pathogens in vitro rather than for their combined potency (Leeman et al., 1996; Meyer and Roberts, 2002). Even though, this consortium did show some level of Ganoderma disease reduction by 68.49%.

Remarkably, it was also found single B. cereus treatment was able to display significantly higher percentage of Ganoderma disease suppression compared to T. harzianum with 94.75% DR rate. In harmony to the present study, Zaiton et al. (2008) and Ramli et al. (2016) also listed B. cereus as one of potential endophytic bacteria against G. boninense growth in vitro. However, Zaiton et al. (2008) and Ramli et al. (2016) did not test the ability of B. cereus in vivo or in Ganoderma disease suppression in oil palm nursery trials. This shows that, contrary results may be obtained in in vivo trials compared to in vitro experiments, as these are very different environments. Besides that, the reduction in DS suggested that T. harzanium and B. cereus played an independent role in the inhibition of Ganoderma disease symptoms.

In line to that, most bacteria used as BCAs are endophytes or common rhizosphere-colonizing bacteria such as Bacillus spp., Enterobacter spp., Pseudomonas spp., Serratia spp., and Burkholderia spp. (Van Loon and Bakker, 2004).

Conclusion

Promising results were obtained on the vegetative growth enhancement and suppression of Ganoderma disease by B. cereus and T. harzianum in the in vivo nursery trial on oil palm seedlings. The association and effect of these endophytic microbes on these abilities need to be assessed further based on the biochemical changes and gene expression at the molecular level.

Acknowledgements

The authors acknowledge the financial support from the Yayasan Pak Rashid UPM (Vote No. 6300817) through a short-term grant to conduct this current project. The authors also would like to thank all staffs of Department of Plant Protection, Faculty of Agriculture, Universiti Putra Malaysia whom had contributed guidance and hand in completing the current project.

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Author:Nusaibah, S.A.; Saad, Ghazala; Hun, Tan Geok
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9MALA
Date:Apr 30, 2017
Words:5941
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