The Efficacy of Three Species of Trichodew for the control of Basal Stem Rot in Oil Palm Seedlings

This study evaluated the potential of three Trichoderma species, namely T. harzianum t (isolate BIO T32), T. longibrachiatum (BIO T28) and T. virens (BIO T128) for the control of Ganoderma boninense (EGB Ol), the causal pathogen of basal stem rot (BSR) of oil palms in nursery trials. Besides their spore production and antagonistic properties, h s study also investigated the growth response of each of the species towards a wide range of temperature and pH conditions. All three species exhibited particular strengths in the growth parameters studied but BIO T32 exhibited consistent and relatively good antagonistic properties and was used as the main inoculant in 'nursery trials against G. boninense. The type and size of wood block were found to influence the success and consistency of the inocula in establishing disease during artificial infection of seedlings. Very low infectivity rates were acheved when inoculum blocks were half to a quarter of the standard 6 x 6 x 12 cm; this size was found to give consistent infection rates leading to approximately 85% mortality. In nursery trials, seedlings treated with a single inoculum of T. lzarzianum (TI) gave the lowest and most significant disease severity index (DSI) of 28.34. The conidial drench was stopped at week 14 and the first sign of disease was only observed on week 20. The uninfected and untreated control seedlings gave a DSI of 0 where as, the infected, untreated controls gave a DSI of 86.87. Soils under treatment using a single (TI), two mixed (T2) and three mixed (T3) inocula showed an increase in spore count based on colony forming units (cfu) starting from two weeks after application. When the soil drench was tenninated at week 14, the spore count was peak on the 18&, 1 4 a~nd 1 0 ~ week for TI, T2 and T3 treatments respectively. Spore counts of BIO T32 were not significantly different on the upper (5 cm) and deeper (15 cm) layer of the treated soils. This study found that when T. lzarzianum (BTO T32) was used as a single inoculum, it f gave the most significant and effective performance as a biological control agent. This was only followed by a mixture of T. hurzianum and T. longibrachiatum. Lastly, the use of a combination of three Trichoderma species were found to give the poorest disease control, giving a DSI that was not statistically different from the infected, untreated control experiment

Development of simple sequence repeat (SSR) markers for oil palm and their application in genetic mapping and fingerprinting of tissue culture clones

This study describes the application of a simple and effective method to isolate SSR markers from oil palm genomic sequences. A total of 12 informative SSR markers are described. The SSR markers were found suitable for genome analysis and DNA fingerprinting of oil palm tissue culture clones. Eleven of the 12 SSR markers exhibited expected Mendelian segregation ratios when tested on a mapping population, indicating their suitability for genetic mapping studies. The markers identified in the species Elaeis oleifera also showed applicability in a second species, that is Elaeis guineensis. Apart from genetic mapping, the SSR markers also showed promise as molecular probes for DNA fingerprinting of oil palm tissue culture clones. The SSR markers can be used for clonal identification, monitoring line uniformity between and within clones and detecting culture mix-up

Evidence-based gene models for structural and functional annotations of the oil palm genome

The advent of rapid and inexpensive DNA sequencing has led to an explosion of data waiting to be transformed into knowledge about genome organization and function. Gene prediction is customarily the starting point for genome analysis. This paper presents a bioinformatics study of the oil palm genome, including comparative genomics analysis, database and tools development, and mining of biological data for genes of interest. We have annotated 26,059 oil palm genes integrated from two independent gene-prediction pipelines, Fgenesh++ and Seqping. This integrated annotation constitutes a significant improvement in comparison to the preliminary annotation published in 2013. We conducted a comprehensive analysis of intronless, resistance and fatty acid biosynthesis genes, and demonstrated that the high quality of the current genome annotation. 3,658 intronless genes were identified in the oil palm genome, an important resource for evolutionary study. Further analysis of the oil palm genes revealed 210 candidate resistance genes involved in pathogen defense. Fatty acids have diverse applications ranging from food to industrial feedstocks, and we identified 42 key genes involved in fatty acid biosynthesis in oil palm. These results provide an important resource for studies of plant genomes and a theoretical foundation for marker-assisted breeding of oil palm and related crops

Genome sequencing and analysis of G. boninense, G. zonatum, G. miniatocinctum and G. tornatum towards understanding basal stem rot of oil palm

Oil palm (Elaeis guineensis) is renowned as the golden crop of Malaysia and Indonesia for its’ high yields per hectare of land compared to other oil crops globally. Malaysia contributes 25.93% of the world’s palm oil supply with export revenue of RM73.25 billion in the year 2020. As with any other crop, oil palm faces challenges, such as pests and diseases in sustaining the oil palm plantation industry. In Malaysia, four Ganoderma species are commonly found in the oil palm plantations, whereby G. boninense (isolate PER71), G. zonatum (isolate POR69) and G. miniatocinctum (isolate 337035) are associated with high incidences of basal stem rot (BSR) disease, with the former being the most pathogenic of all species. The fourth species, G. tornatum (isolate NPG1) is a saprophyte. Besides being causal agents of BSR, G. boninense and G. zonatum are also causal agents of upper stem rot (USR) disease in Malaysia. For decades, studies have been conducted to understand the mode of infection and epidemiology, genetic diversity, and discover ways to curb these diseases from spreading to adjacent trees or new plantings. Most studies focused primarily on the host and host-pathogen interactions but not on the pathogen itself. Understanding the pathogen is essential and that too at the molecular level. With continuous developments in DNA sequencing technologies, PacBio single molecule-real time (SMRT) offers long reads with high accuracy to decipher many organisms. Although there are many extraction protocols available for Ganoderma species, the high molecular weight of good quality nucleic acid is a prerequisite of SMRTbell library construction. Therefore, the first objective of this study was to extract high quality genomic DNA for the four Ganoderma species required for PacBio sequencing. This study described modified DNA extraction protocols for G. boninense, G. zonatum, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol was suitable for G. boninense and G. miniatocinctum, while the modified high salt low pH protocol was suitable for G. tornatum. These modified nucleic acid extraction protocols were able to produce high quality DNA and total RNA of ~140-160 µg/g and ~80 µg/g of mycelia, respectively. As for G. zonatum, the Boehm protocol was the most rapid and efficient protocol with a yield of 208.95±4.52 µg DNA per gram of tissue. In the second objective, using the PacBio (Sequel platform) sequenced long read data, two draft genomes G. boninense the most pathogenic species, and G. tornatum, a saprophyte were assembled. The diploid aware assemblers FALCON and FALCON-Unzip phased assembled the two Ganoderma genomes generating ~67 Mbp and ~76 Mbp size genomes with 297 and 246 contigs for the former and latter species, respectively. Next, gene prediction was performed using four gene prediction pipelines on these two genomes, and MAKER2 was selected as the best pipeline generating 19,851 and 23,989 gene models, respectively. In the third objective, molecular markers were identified to distinguish pathogens from non-pathogens in oil palm plantations. This was performed using the four assembled Ganoderma genomes (454 sequencing technology). Species-specific simple sequence repeats (SSRs) were mined from the four Ganoderma genomes based on BUSCO (Benchmarking Universal Single-Copy Orthologs) and ortholog analysis. One hundred SSRs were synthesized and screened against the four Ganoderma species. Based on the screening results, 13 SSR markers were evaluated on 20 Ganoderma isolates collected from four states in West Malaysia. Data from the absence and presence of amplicons from the 25 Ganoderma isolates generated a phylogenetic tree. However, these 13 SSR markers were not able to distinguish the pathogenic isolates/species from the non-pathogens. Therefore, published internal transcribed spacers (ITS) were also used for species identification. For isolates with low quality ITS sequences, new ITS primers were designed based on the assembled G. boninense genome (PacBio) from this study. Molecular markers, together with morpho-taxonomy were able to facilitate the identification of Ganoderma isolates from West Malaysia. In the fourth objective, G. boninense culture was grown in limited carbon (C) and nitrogen (N) medium. This starved environment with minimal simple sugar and sawdust was to mimic as closely as possible to the field conditions. In response to the starved condition, differentially expressed genes (DEGs) in numerous metabolic pathways were identified, with four significant pathways explored in this study. The results provided first insights into possible genes that were up- and down-regulated in the pathways, namely secondary metabolite biosynthesis, carbohydrate metabolism, glycan metabolism and mycotoxin biosynthesis. A cocktail of enzymes was released by G. boninense to be associated with the degradation of cellulose, hemicellulose, lignin and pectin. The DEGs involved in the mycotoxin biosynthesis pathway are of interest, as they have not been reported in Ganoderma species. In the fifth objective, using comparative genome analysis genes associated with pathogenicity between the two Ganoderma species were identified. A workflow to detect small secreted proteins (SSPs) was used on the Ganoderma gene models of G. boninense and G. tornatum. A total of 18 candidate SSPs were identified in G. boninense, predicted to be involved in numerous functions associated with host-pathogen interactions. This study reported on optimized nucleic acid extraction protocols for four Ganoderma species that were meant for PacBio sequencing. This study also reported on novel genes that were differentially expressed during C-N starvation in four metabolic pathways of G. boninense. The 18 candidate SSPs associated with pathogenicity in G. boninense have not been reported yet. Further investigation on these genes needs to be verified in a wet lab to confirm their presence in the pathogen

The efficacy of three species Trichoderma for the control of basal stem rot in oil palm seedlings

This study evaluated the potential of three Trichoderma species, namely T. harzianum (isolate BIO T32), T. longibrachiatum (BIO T28) and T. virens (BIO T128) for the control of Ganoderma boninense (EGB 01), the causal pathogen of basal stem rot (BSR) of oil palms in nursery trials. Besides their spore production and antagonistic properties, this study also investigated the growth response of each of the species towards a wide range of temperature and pH conditions. All three species exhibited particular strengths in the growth parameters studied but BIO T32 exhibited consistent and relatively good antagonistic properties and was used as the main inoculant in nursery trials against G. boninense. The type and size of wood block were found to influence the success and consistency of the inocula in establishing disease during artificial infection of seedlings. Very low infectivity rates were achieved when inoculum blocks were half to a quarter of the standard 6 x 6 x 12 cm; this size was found to give consistent infection rates leading to approximately 85% mortality. In nursery trials, seedlings treated with a single inoculum of T. harzianum (T1) gave the lowest and most significant disease severity index (DSI) of 28.34. The conidial drench was stopped at week 14 and the first sign of disease was only observed on week 20. The uninfected and untreated control seedlings gave a DSI of 0 where as, the infected, untreated controls gave a DSI of 86.87. Soils under treatment using a single (T1), two mixed (T2) and three mixed (T3) inocula showed an increase in spore count based on colony forming units (cfu) starting from two weeks after application. When the soil drench was terminated at week 14, the spore count was peak on the 18th, 14th and 10th week for T1, T2 and T3 treatments respectively. Spore counts of BIO T32 were not significantly different on the upper (5 cm) and deeper (15 cm) layer of the treated soils. This study found that when T. harzianum (BIO T32) was used as a single inoculum, it gave the most significant and effective performance as a biological control agent. This was only followed by a mixture of T. harzianum and T. longibrachiatum. Lastly, the use of a combination of three Trichoderma species were found to give the poorest disease control, giving a DSI that was not statistically different from the infected, untreated control experiment

Genome sequencing and analysis of G. boninense, G. zonatum, G. miniatocinctum and G. tornatum towards understanding basal stem rot of oil palm

Oil palm (Elaeis guineensis) is renowned as the golden crop of Malaysia and Indonesia for its’ high yields per hectare of land compared to other oil crops globally. Malaysia contributes 25.93% of the world’s palm oil supply with export revenue of RM73.25 billion in the year 2020. As with any other crop, oil palm faces challenges, such as pests and diseases in sustaining the oil palm plantation industry. In Malaysia, four Ganoderma species are commonly found in the oil palm plantations, whereby G. boninense (isolate PER71), G. zonatum (isolate POR69) and G. miniatocinctum (isolate 337035) are associated with high incidences of basal stem rot (BSR) disease, with the former being the most pathogenic of all species. The fourth species, G. tornatum (isolate NPG1) is a saprophyte. Besides being causal agents of BSR, G. boninense and G. zonatum are also causal agents of upper stem rot (USR) disease in Malaysia. For decades, studies have been conducted to understand the mode of infection and epidemiology, genetic diversity, and discover ways to curb these diseases from spreading to adjacent trees or new plantings. Most studies focused primarily on the host and host-pathogen interactions but not on the pathogen itself. Understanding the pathogen is essential and that too at the molecular level. With continuous developments in DNA sequencing technologies, PacBio single molecule-real time (SMRT) offers long reads with high accuracy to decipher many organisms. Although there are many extraction protocols available for Ganoderma species, the high molecular weight of good quality nucleic acid is a prerequisite of SMRTbell library construction. Therefore, the first objective of this study was to extract high quality genomic DNA for the four Ganoderma species required for PacBio sequencing. This study described modified DNA extraction protocols for G. boninense, G. zonatum, G. miniatocinctum and G. tornatum, and an RNA extraction protocol for G. boninense. The modified salting out DNA extraction protocol was suitable for G. boninense and G. miniatocinctum, while the modified high salt low pH protocol was suitable for G. tornatum. These modified nucleic acid extraction protocols were able to produce high quality DNA and total RNA of ~140-160 µg/g and ~80 µg/g of mycelia, respectively. As for G. zonatum, the Boehm protocol was the most rapid and efficient protocol with a yield of 208.95±4.52 µg DNA per gram of tissue. In the second objective, using the PacBio (Sequel platform) sequenced long read data, two draft genomes G. boninense the most pathogenic species, and G. tornatum, a saprophyte were assembled. The diploid aware assemblers FALCON and FALCON-Unzip phased assembled the two Ganoderma genomes generating ~67 Mbp and ~76 Mbp size genomes with 297 and 246 contigs for the former and latter species, respectively. Next, gene prediction was performed using four gene prediction pipelines on these two genomes, and MAKER2 was selected as the best pipeline generating 19,851 and 23,989 gene models, respectively. In the third objective, molecular markers were identified to distinguish pathogens from non-pathogens in oil palm plantations. This was performed using the four assembled Ganoderma genomes (454 sequencing technology). Species-specific simple sequence repeats (SSRs) were mined from the four Ganoderma genomes based on BUSCO (Benchmarking Universal Single-Copy Orthologs) and ortholog analysis. One hundred SSRs were synthesized and screened against the four Ganoderma species. Based on the screening results, 13 SSR markers were evaluated on 20 Ganoderma isolates collected from four states in West Malaysia. Data from the absence and presence of amplicons from the 25 Ganoderma isolates generated a phylogenetic tree. However, these 13 SSR markers were not able to distinguish the pathogenic isolates/species from the non-pathogens. Therefore, published internal transcribed spacers (ITS) were also used for species identification. For isolates with low quality ITS sequences, new ITS primers were designed based on the assembled G. boninense genome (PacBio) from this study. Molecular markers, together with morpho-taxonomy were able to facilitate the identification of Ganoderma isolates from West Malaysia. In the fourth objective, G. boninense culture was grown in limited carbon (C) and nitrogen (N) medium. This starved environment with minimal simple sugar and sawdust was to mimic as closely as possible to the field conditions. In response to the starved condition, differentially expressed genes (DEGs) in numerous metabolic pathways were identified, with four significant pathways explored in this study. The results provided first insights into possible genes that were up- and down-regulated in the pathways, namely secondary metabolite biosynthesis, carbohydrate metabolism, glycan metabolism and mycotoxin biosynthesis. A cocktail of enzymes was released by G. boninense to be associated with the degradation of cellulose, hemicellulose, lignin and pectin. The DEGs involved in the mycotoxin biosynthesis pathway are of interest, as they have not been reported in Ganoderma species. In the fifth objective, using comparative genome analysis genes associated with pathogenicity between the two Ganoderma species were identified. A workflow to detect small secreted proteins (SSPs) was used on the Ganoderma gene models of G. boninense and G. tornatum. A total of 18 candidate SSPs were identified in G. boninense, predicted to be involved in numerous functions associated with host-pathogen interactions. This study reported on optimized nucleic acid extraction protocols for four Ganoderma species that were meant for PacBio sequencing. This study also reported on novel genes that were differentially expressed during C-N starvation in four metabolic pathways of G. boninense. The 18 candidate SSPs associated with pathogenicity in G. boninense have not been reported yet. Further investigation on these genes needs to be verified in a wet lab to confirm their presence in the pathogen

General workflow of oil palm tissue culture. Explant (E0) is cultured to form callus (<i>C</i>) which is transferred to a new medium (C1) to form embryoids.

<p>Cultures not forming callus (<i>NC</i>) are transferred to a fresh medium (<b>E1</b>–<b>E3</b>) and undergo the same process again. Embryoids (<i>EC</i>) generated from <b>C1</b> proceed to polyembryoid culture (<b>PE1</b>–<b>PE15</b>) for the regeneration of plantlets. Callus cultures that fail to generate embryoids (<i>NEC</i>) are transferred to a fresh medium (<b>C2</b>–<b>C4</b>) and undergo the same process again.</p

Alignment of the ENL48 (left) and ML161 (right) maps using co-segregating markers.

<p>Markers showing distorted segregation are indicated by asterisk (*) representing significance at p<0.1; (**) p<0.05; (***) p<0.01; (****) p<0.05 and; (******) p<0.0005.</p

QTLs detected for PLnCR and PordER using Multi-trait QTL analysis, GenStat 14.

<p>Upper panel shows the QTL profiles at –log10 (P-value) which resulted from interval mapping scanning. The horizontal line shows the genome-wide significant threshold determined by Li and Ji (P = 3.5). Lower panel shows the QTL effects (green square) resulting from multi-trait interactions: QTL on LGD4b was affected by PLnCR (dark blue square) and PordER (light blue square) while; QTL on LGP16b only contains effect from PordER (brown square).</p

SSR markers mapped on both the ENL48 and ML161 parental maps and their accession numbers.

<p>Putative IDs were deduced for the SSR-containing sequences by comparing to the non-redundant protein database (Blastx for EST sequences) and nucleotide database of GenBank (tBlastx for genomic sequences). A threshold score of >80 was used to assign significant similarity.</p>a<p>Two SSR markers were mapped.</p>b<p>SSRs developed from oil palm sequences from NCBI GenBank.</p>c<p>Accession numbers of NCBI GenBank.</p>d<p>Probe Unique Identifiers (PUIDs) of NCBI Probe Database.</p