Performance of Trichoderma Harzianum Rifai as a Biological Control Agent for Basal Stem Rot of Oil Palm (Elaeis Guineensis Jacq.) Caused by Ganoderma Boninense Pat

Basal stem rot (BSR) is a major threat to the oil palm industry. The disease is caused by Ganoderma boninense, which rots the internal tissues at the trunk base resulting in stem fracture and death of palm. The present study investigated the efficacy of two isolates of Trichoderma harzianum (BIO T32 and BIO T66) as potential biological control agents against BSR based on in vitro and in vivo trials. The study revealed that treatment applied as a soil drench using conidial suspension (mean of 1.61 x lo8 sporeslml) of BIO T32 in addition to a Trichoderma-incorporated palm press fibre (ppf) surface mulch, performed better with a significant difference compared to the use of BIO T66. The disease severity index (DSI) of the former was 28.35 compared to 76.67 of the latter. BIO T32 was also a competent biological control agent in the delayed treatment given to pre-infected seedlings at 6 weeks before treatment with BIO T32, giving a DSI of 45, which was statistically significant compared to the infected and untreated control seedlings with a DSI of 86.67. In testing the synergistic effect by combining the 2 isolates, a poorer performance was observed based on the DSI and plant biomass compared to single application of BIO T32. Isolate BIO T66 which showed good antagonistic properties in the in vitro assessment was not found to display similar results in the in vivo trials. A series of treatments were evaluated for their potential as a BIO T32 carrier. Out of the 3 studies, only ppf and compost exhibited promising results in their capacity as surface 4 mulches, where treatments with either one gave a DSI of 30. Both are food base carriers for they increased the growth of oil palm seedlings significantly, with compost displaying better results. Treatment with compost in terms of vegetative growth gave the highest plant biomass, leaf area measurement, nitrogen, phosphorus and potassium (NPK) content in the seedlings compared to the other 2 treatments of ppf and the untreated control seedlings. 1 In total, the experiment revealed that the application of BIO T32 as a single inoculum was the best treatment, giving a DSI of 28.35. Trials using a single application of BIO T66 and BIO T66 mixed with BIO T32 performed poorly, giving a DSI of 76.67 each and were not significantly different from the infected non-treated control plants. An appropriate interval of conidial suspension's application played a pertinent role in the inhibition of disease as demonstrated in the delayed treatment. The application of compost was found to be an interesting alternative to ppf as surface mulch, which functions also as a Trichoderma carrier. Finally, in terms of vegetative growth both ppf and compost as food base carriers significantly increased plant biomass, total leaf area measurement and N uptake compared to the untreated control

An in vitro study of the antifungal activity of Trichoderma virens 7b and a profile of its non-polar antifungal components released against Ganoderma boninense

Ganoderma boninense is the causal agent of a devastating disease affecting oil palm in Southeast Asian countries. Basal stem rot (BSR) disease slowly rots the base of palms, which radically reduces productive lifespan of this lucrative crop. Previous reports have indicated the successful use of Trichoderma as biological control agent (BCA) against G. boninense and isolate T. virens 7b was selected based on its initial screening. This study attempts to decipher the mechanisms responsible for the inhibition of G. boninense by identifying and characterizing the chemical compounds as well as the physical mechanisms by T. virens 7b. Hexane extract of the isolate gave 62.60% ± 6.41 inhibition against G. boninense and observation under scanning electron microscope (SEM) detected severe mycelial deformation of the pathogen at the region of inhibition. Similar mycelia deformation of G. boninense was observed with a fungicide treatment, Benlate® indicating comparable fungicidal effect by T. virens 7b. Fraction 4 and 5 of hexane active fractions through preparative thin layer chromatography (P-TLC) was identified giving the best inhibition of the pathogen. These fractions comprised of ketones, alcohols, aldehydes, lactones, sesquiterpenes, monoterpenes, sulphides, and free fatty acids profiled through gas chromatography mass spectrometry detector (GC/MSD). A novel antifungal compound discovery of phenylethyl alcohol (PEA) by T. virens 7b is reported through this study. T. virens 7b also proved to be an active siderophore producer through chrome azurol S (CAS) agar assay. The study demonstrated the possible mechanisms involved and responsible in the successful inhibition of G. boninense

Differential transcriptomic profiles effected by oil palm phenolics indicate novel health outcomes

Abstract Background Plant phenolics are important nutritional antioxidants which could aid in overcoming chronic diseases such as cardiovascular disease and cancer, two leading causes of death in the world. The oil palm (Elaeis guineensis) is a rich source of water-soluble phenolics which have high antioxidant activities. This study aimed to identify the in vivo effects and molecular mechanisms involved in the biological activities of oil palm phenolics (OPP) during healthy states via microarray gene expression profiling, using mice supplemented with a normal diet as biological models. Results Having confirmed via histology, haematology and clinical biochemistry analyses that OPP is not toxic to mice, we further explored the gene expression changes caused by OPP through statistical and functional analyses using Illumina microarrays. OPP showed numerous biological activities in three major organs of mice, the liver, spleen and heart. In livers of mice given OPP, four lipid catabolism genes were up-regulated while five cholesterol biosynthesis genes were down-regulated, suggesting that OPP may play a role in reducing cardiovascular disease. OPP also up-regulated eighteen blood coagulation genes in spleens of mice. OPP elicited gene expression changes similar to the effects of caloric restriction in the hearts of mice supplemented with OPP. Microarray gene expression fold changes for six target genes in the three major organs tested were validated with real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and the correlation of fold changes obtained with these two techniques was high (R2 = 0.9653). Conclusions OPP showed non-toxicity and various pleiotropic effects in mice. This study implies the potential application of OPP as a valuable source of wellness nutraceuticals, and further suggests the molecular mechanisms as to how dietary phenolics work in vivo.</p

Assessment of Ganoderma infection in oil palm (Elaeis guineensis jacq.) by pre-inoculation of arbuscular mycorrhiza fungi and endophytic bacteria.

Basal stem rot of oil palm (Elaeis guineensis Jacq.) caused by Ganoderma spp is of major economic concern and it is the predominant disease of oil palm in Southeast Asia. The disease is a “white rot” process involving growth of the fungus within the oil palm as the lignin and cellulose are biodegraded. There is yet an effective measure to control the disease. Endophytic microorganisms such as arbuscular mycorrhizal fungi (AMF) and endophytic bacteria (EB) have been previously identified as potential biocontrol agents especially for soil borne diseases. These endophytes are able to suppress diseases with various mechanisms such as antibiosis, lysis, and production of antifungal compounds and induced systemic resistance (ISR). The use of endophytes is also preferred as they reside within the plant system avoiding rhizosphere competence such as competition for space and nutrient between other soil microbes. Therefore this study was designed with the specific objectives to (i) investigate the antagonistic potential and compatibility between both endophytes, (ii) explore the potential disease suppression and vegetative growth enhancement in pre-inoculated oil palm seedlings challenged with G. boninense, (iii) determine the biochemical responses and gene expression profile in pre-inoculated seedlings challenged with G.boninense, and (iv) evaluate the effect of single and a consortium of endophytic microorganisms in field trial using the seedling baiting technique. AMF (Glomus intraradices UT 126 and G. clarum BR152B)and EB (Pseudomonas strain UPMP3 and Burkholderia strain UPMB3)represent the endophytic microbes tested in this study. Symbiotic interactions were observed between AMF species; Glomus intraradices UT 126 and G. clarum BR152B with Pseudomonas strain UPMP3 and Burkholderia strain UPMB3. Both EB strains significantly increased germination and hyphal length of AMF spores by 180 to 240%,respectively. This is a novel finding as the EB strains were never reported as potential mycorrhizal helper bacteria (MHB). Antagonistic effect of EB strains was demonstrated by the percentage inhibition of radial growth of G. boninense at >86% and >74% by UPMP3 and UPMB3, respectively. Scanning electron micrographs revealed severe morphological deformities such as shrivelling, flattening and shrinking of G. boninense hyphae in the presence of UPMP3 and UPMB3 strains. In addition,production of POX, PPO, chitinase and β-1, 3-glucanase during pre and post infection were enhanced in pre-inoculated seedlings and reassessed with gene expression analysis. It is also proposed that PPO acts as a potential biomarker for induced resistance against G. boninense. The single application of UT126 (T2) was found to be superior in increasing the vegetative parameters such as height, girth, leaf area, frond count and fresh weight of leaf and root with significant difference at P<0.05. However, disease suppression was highest in treatment that had the application of both AMF and UPMP3 with a disease reduction of 57.53% (P<0.05). The disease incidence in treatment with both AMF and UPMP3 (T4) was delayed by 8 weeks indicating effective suppression of BSR infection. Similar treatments were subjected to field trial using seedling bait technique. Disease suppression in T4 was significantly highest with reduced disease severity of 4.33% (P<0.05) when compared to other treatments (ranging from 7 to 14% o infection), 20 months after planting. This is the first report of field seedling baiting technique to be successfully implemented in testing microbial pre-inoculation for disease suppression. Pre-inoculation with AMF and UPMP3 was most effective in reducing disease severity in oil palm either in nursery or field conditions

Expression of genes encoding manganese peroxidase and laccase of Ganoderma boninense in response to nitrogen sources, hydrogen peroxide and phytohormones

Ganoderma produces lignolytic enzymes that can degrade the lignin component of plant cell walls, causing basal stem rot to oil palms. Nitrogen sources may affect plant tolerance to root pathogens while hydrogen peroxide (H2O2), salicylic acid (SA) and jasmonic acid (JA) play important roles in plant defense against pathogens. In this study, we examined the expression of genes encoding manganese peroxidase (MnP) and laccase (Lac) in Ganoderma boninense treated with different nitrogen sources (ammonium nitrate, ammonium sulphate, sodium nitrate and potassium nitrate), JA, SA and H2O2. Transcripts encoding MnP and Lac were cloned from G. boninense. Of the three GbMnP genes, GbMnP_U6011 was up-regulated by all nitrogen sources examined and H2O2 but was down-regulated by JA. The expression of GbMnP_U87 was only up-regulated by JA while GbMnP_35959 was up-regulated by ammonium nitrate but suppressed by sodium nitrate and down-regulated by H2O2. Among the three GbLac genes examined, GbLac_U90667 was up-regulated by ammonium nitrate, JA, SA and H2O2; GbLac_U36023 was up-regulated by JA and H2O2 while GbLac_U30636 was up-regulated by SA but suppressed by ammonium sulphate, sodium nitrate, JA and H2O2. Differential expression of these genes may be required by their different functional roles in G. boninense

Comparison of real-time PCR, conventional PCR and RT-lamp for the detection of Coconut Cadang-Cadang Viroid variant in oil palm

Real-time quantitative polymerase chain reaction (real-time PCR) was designed for the detection of oil palm Coconut Cadang-cadang Viroid (CCCVd) 246 nt variant. The primers and probe specifically designed for the real-time PCR were optimised with 300 nM of probe against a primer concentration of 400 nM for the detection of oil palm CCCVd variants. Oil palm CCCVd 246 nt variant was successfully detected using realtime PCR in leaf samples collected from 14 symptomatic oil palm from various regions. The sensitivity of real-time PCR was compared with reverse transcription loop-mediated isothermal amplification (RT-LAMP) and conventional PCR. The latter two techniques were reported earlier to be able to detect CCCVd variants in oil palm. Conventional PCR analysis using CCCVd full-length primers detected the presence of CCCVd 246 nt variant in 10 while RT-LAMP detected only seven positive samples from the total 14 field samples. The real-time PCR was highly sensitive and reliable compared to the two detection techniques. The primers designed for the real-time PCR were specific as only the oil palm CCCVd variant plasmid was detected with high fluorescence and lowest quantification cycle (Cq value) compared to the other tested viroids (ASSVd, CTiVd, ELVd, HLVd and PLMVd). The present study has proven the reliability of the technique, thus, highly recommending the next step of developing the high-throughput diagnostic procedure for the eventual use in epidemiological monitoring programmes

Consolidated evaluation of polysaccharide degradation after oil palm wood pretreatment with indigenous white-rot hymenomycetes

The oil palm industry is the backbone of the Malaysian economy. However, this industry is threatened by basal stem rot (BSR) caused by the fungus Ganoderma boninense, which has caused severe losses over the past several decades. The study reported here is part of an ongoing project to reduce Ganoderma inoculum and manage oil palm waste on plantations, which could be a green technology approach to reduce BSR infection pressure. A preliminary search for potential white-rot hymenomycetes to accelerate the degradation of oil palm stumps has been initiated. As a continuity, the present study was designed to comprehensively investigate the changes in wood crystallinity at different stages of oil palm wood degradation after pretreatment with three white-rot hymenomycete species. The findings were used to limit potential white-rot candidate(s) for their secretion of cellulolytic, hemicellulolytic and amylolytic enzymes, selectively or simultaneously, using principal component analysis. The overall per cent crystallinity of healthy wood decayed by Lentinus tigrinus increased as degradation proceeded. Conversely, crystallinity generally increased at 75 d in the diseased wood pretreated with Trametes lactinea, owing to the preferential degradation of noncrystalline lignins and hemicelluloses by this strain. Anatomical characterization revealed penetration of fungal mycelia into the degraded wood vessels, phloem and parenchymal tissues with microscopic cavities. In advanced stages of pretreatment, the loosening of the parenchymal tissue, including the wood rays, was more rapid in the diseased wood than in the healthy wood. This study provides new insights into the biochemical and anatomical changes initiated by white-rot hymenomycetes during the degradation of oil palm wood blocks. The present findings could be efficiently utilized for sustainable biological stump pretreatment, particularly with respect to the polysaccharide components of lignocellulosic materials