Young oil palms (AA Hybrida, Applied Agricultural Resources SB, Malaysia) were obtained as 3-month-old rooted ramets and were grown and maintained in a shade house. The bio-fertiliser (commercially known as living organic fertiliser (LOF)) was derived from an industrial composting process of empty fruit bunch (EFB), decanter cake, palm oil mill effluent (POME) and boiler ash while flaky palm kernel shell biochar was a by-product from the extraction of crude palm oil extraction. Both products were obtained from Greenplant Organics Sdn Bhd, Palong, Malaysia. All research activities in this study were carried out at the University of Nottingham Malaysia, Semenyih, Malaysia.

Trichoderma asperellum 4A (Genbank accession number: KM456217.1) was originally isolated from Balau Estate, Semenyih, Malaysia and was maintained fortnightly in potato dextrose agar (PDA) (Oxoid).

Trichoderma asperellum 4A was grown in PDA supplied with 250 µg/l of streptomycin (HiMedia) and 250 µg/l of chloramphenicol (Nacalai Tesque) for 14 days (Gil et al., 2009). Cultures were dislodged from standard 9 mm Petri dishes using autoclaved distilled water before being gently scraped with a glass microscope slide (Musa et al., 2018). The conidial cultures were diluted in sterile malt extract broth (MEB) (Oxoid) at 1:10 dilution and allowed to grow at room temperature, overnight in an orbital shaker at 100 rpm. The liquid cultures was adjusted to a concentration of 10⁶ spore/ml using MEB and were applied directly to oil palm pots. For T-mix, the same concentration of liquid cultures was mixed with PKS biochar at 100% w/v. T-mix was stored in high density polyethylene (HDPE) bags for up to six months at room temperature.

Ganoderma boninsense GBLS was obtained from Lian Seng Estate, Malaysia and characterised from a previous study (Goh et al., 2018). Ganoderma boninsense cultures was maintained in PDA. Two-week-old GBLS cultures grown in 9 mm Petri dish with PDA were inoculated on untreated 6cm × 6cm × 6cm rubber wood blocks (RWBs) (Rubber Research Institute Malaysia) by adding mycelia from one Petri dish per RWB. The RWBs were then placed in zip lock bags and stored away from sunlight to grow for six weeks (Figure 1) (Sariah et al., 1994).

Plant inoculation experiment was carried out with 180 young oil palms arranged in a complete block design. Each of the four blocks of treatments were given three levels of biological control with 15 palms for each level (Table 1). The fertiliser treatments were:

The three levels of biological control were:

Assay was conducted according to (Izzati and Abdullah, 2008) with slight modification. Three-month old oil palm plants were transplanted from their germination tray pots into polyethylene planting bags measuring 25cm × 25cm and allowed to acclimatise for one month before treatments were delivered.

The soil was prepared by mixing topsoil and river sand at a ratio of 1:1, and each planting bag contained approximately 4 kg of soil. LOF was applied according to manufacturer’s recommendation by mixing with soil in treatments T2, T4, T6, T8, T10 and T12. The plants were watered every day for 30 minutes using a garden hose with a spray nozzle. Upon acclimatisation, plants in diseased treatments (T3, T4, T7, T8, T11 and T12) were transplanted into new planting bags containing the same soil mix as before but with added RWBs inoculated with G. boninense. One week later, 100g of T-mix was applied on the surface of the soil for T9-T12 and for treatments with T. asperellum 4A only, T5-T8, 100 ml of liquid conidial suspension was delivered directly to the soil surface.

All plants received grower standard practice of inorganic fertiliser such as Bayfolan^® foliar fertiliser for three months and subsequent rock phosphate and NPK fertiliser as recommended by AARSB, Malaysia.

The plants were destructively sampled at the 2^nd, 4^th, and 6^th month post treatment. Root samples were obtained at every time point and root development parameters (total root surface area, total root length, average root diameter) were analysed using WinRhizo 2013e (Regent Instruments).

Rhizosphere DNA was collected from soil attached to roots at 6 months post inoculation. DNA wasextracted using DNA Powersoil Pro kit (Qiagen) according to manufacturer’s recommendation.The ITS1 region was amplified from gDNA using the primer pair BITS ACCTGCGGARGGATCA and B58S3 GAGATCCRTTGYTRAAAGTT (Klindworth et al., 2013; García-López et al., 2020). An additional 5 bases of inline barcode and partial Illumina adapter were incorporated at the 5’ end of the primers to enable inline barcoding (Glenn et al., 2019). Different samples were amplified using different combinations of the forward and reverse inline primers. PCR was performed using Rediant II PCR mastermix (Apical Scientific, Malaysia) using the PCR profile of: 95C for 2 minutes followed by 30 cycles of 95C for 15 s, 50C for 30s and 72C for 30s. The barcoded amplicons were subsequently visualised on gel and purified using 0.8 X vol. of SPRI bead. The purified amplicons were used as the template for 8 cycles of index PCR to incorporate the complete Illumina adapter and Illumina-compatible dual-index barcodes.

The constructed libraries were subsequently size-selected using 0.8 X vol of SPRI bead and pooled into a single tube. Quantification of the pooled libraries used Denovix high sensitivity assay. Sequencing of the pooled libraries was performed on a NovaSEQ6000 (Illumina, San Diego) using the 2x150bp paired-end sequencing configuration.

Demultiplexing and primer trimming of the raw paired-end reads used cutadapt v1.18 (Martin, 2011). The trimmed reads were subsequently merged usingfastp v0.21 (Chen et al., 2018). The processed readswere imported into QIIME2 v.2023.9 (Bolyen et al., 2019) and denoised into Amplicon Sequence Variant (ASV) with dada2 v.1.26.0 (Callahan et al., 2016). Taxonomic assignment of the ASV used q2-feature-classifier (Bokulich et al., 2018) that has been trained on the latest Unite V9 (McDonald et al., 2023).

ASVs (amplicon sequence variants) with taxonomic assignment to at least the phylum level were selected for subsequent analysis. Following data rarefaction to the sample with the lowest number of reads, alpha diversity analysis was conducted in QIIME2. Diversity indices including Chao1, ACE, Shannon, Simpson, and Faith_PD were calculated to assess within-sample diversity. For beta diversity analysis, distances based on Jaccard, Bray Curtis, weighted UniFrac, and unweighted UniFrac were computed using the rarefied dataset.

The results obtained were analysed using IBM SPSS Statistics version 28. For Trichoderma viability experiment, independent samples T-test was used to analyse the data to identify any significant difference between the two concentrations of Trichoderma. The root development parameter results were analysed using tests of between-subjects effects, and Tukey’s multiple comparisons test was carried to analyse the difference of means between treatments.

Overall, T5 and T9 displayed the highest results, with T5 having an almost 45% increase in total root length more than T1, and T9 having almost double the total root length of T1 after 6 months (Figure 2). This indicates the ability of Trichoderma spp. to induce root growth in young oil palms. In a related study, T. harzianum-induced total root length in tomato plants is regulated by the qid74 gene, which is shown to not only increase the length of lateral roots and secondary root hairs but increased the total absorptive surface of the roots and thus allowing for an increased uptake in nutrients (Samolski et al., 2012). T9 performed better than T5, suggesting that a mixture of Trichoderma spp. and biochar performs better than an application of pure Trichoderma spp. in boosting total root length (Figure 3). This is evidenced by findings by Hasan et al. in 2020 which showed that a mixture of T. asperellum and coconut fibres performed better than an application of pure Trichoderma alone.

In retrospective, healthy treatments that were supplied with LOF (T2, T6 and T10), recorded relatively low total root lengths. Addition of organic carbon may increase the overall required C/N ratio, affecting root growth (Nie et al., 2013). However, T10 had a significantly higher total root length than T6, which suggests that a combination of Trichoderma spp. with biochar was more effective at mitigating the inhibitory effects of LOF when compared to a liquid Trichoderma spp. application. This hypothesis can be further supported by comparing the total root length between treatments 8 and 12. The combination of Trichoderma spp. with biochar produced a higher total root length in diseased plants than a liquid Trichoderma spp. application. Treatments 3, 4, and 6 had no significant difference in root length, suggesting that a pure Trichoderma spp. application in tandem with LOF on healthy plants yielded the same results as diseased plants. The lower root lengths observed in treatments that received LOF suggest that in this study, LOF may have negatively affected root growth. There is evidence to show that organic fertilisers may contain trace levels of heavy metals (Wang et al., 2013). Heavy metals such as chromium have been shown to depress root elongation despite supplementation with nitrates (Panda and Patra, 2000). Although, in this study, no trace metal was detected in LOF, batch to batch variation could occur in the production facility.

The diseased treatments all recorded significantly lower total root length than the healthy plants that did not receive LOF application (Figure 4). Ganoderma spp. infection led to necrosis and decay of oil palm roots, which will in turn reduce total root length. The secretion of CWDEs not only reduces root surface area, but root length as well, as the action of enzymes such as cellulases, pectinases and polygalacturonases result in the destruction of root cell walls (Ghasemi et al., 2020; Zhang et al., 2022; Rees et al., 2009; Gawade et al., 2017). The degradation of root cell walls in turn causes the root cells to weaken and collapse, thus resulting in a reduced total root length. Ganoderma spp. is also shown to affect the hormonal balance in oil palms. Oil palms infected with Ganoderma spp. have been shown to display an upregulation of auxin regulators, which in turn limit auxin production and signalling (Bahari et al., 2018). As auxin plays a role in root growth and elongation, a disruption in auxin signalling results in reduced total root length. Another negative effect of Ganoderma spp. on oil palm roots is the induction of oxidative stresses. Bahari et al. observed the upregulation of reactive oxygen species (ROS) elicitors in oil palms infected with Ganoderma spp. A common plant defense mechanism is the promotion of ROS production through an oxidative burst. As early colonisation of Ganoderma spp. takes place in the roots, it is possible that the high levels of ROS produced during an oxidative burst may inhibit root growth due to DNA damage in the root elongation zone (Tsukagoshi, 2016).

Diseased plants treated with liquid Trichoderma spp. (T7) recorded a higher total root length than the diseased treatment that had both Trichoderma spp. and LOF (T8). This is similar to the difference observed between Treatments 11 and 12, in which the diseased treatment with T-mix and LOF performed better than the diseased treatment with only T-mix. This further strengthens the suggestion that T-mix was more effective at controlling the possible harmful effects of LOF, as improvements can be seen even in diseased young oil palms. However, an application of pure Trichoderma spp. was shown to be more effective than T-mix at increasing the total root length of diseased oil palms that did not receive LOF.

All the non-LOF treatments (Treatments 1, 3, 5, 7, 9 and 11) recorded a higher average root diameter than their counterparts that received LOF (Treatments, 2, 4, 6, 8, 10 and 12) (Figure 5). In following with the trend observed in the previous root growth parameters, it can be established that LOF is not beneficial to the root development of young oil palms, regardless of plant health. For young oil palm requiring high amounts of nitrogen for growth, reduced organic input is suggested. This is the opposite for mature palms within a plantation, where the organic input is necessary to maintain long term soil health (Sundram et al., 2019; Pauli et al., 2014);.

T5 and T9 had the highest average root diameter, which was consistent with the findings of the previous root development parameters. While the difference between Treatment 5 and the absolute control was significant, the difference was not as stark as that between Treatment 9 and the absolute control. Treatment 9 had a 31% increase in average root diameter when compared to Treatment 1, and a 25% increase when compared to Treatment 5. The application of Trichoderma spp.-biochar combination was more effective at increasing the average root diameter in healthy young oil palms than liquid Trichoderma spp. alone. To the author’s knowledge this is the first study to determine the effect of Trichoderma spp. application on the root health in young oil palm in combination with palm biowaste such as biochar. In a related study, Sani et al., in 2020 reported that a combination of Trichoderma spp. and biochar significantly boosted the health and root growth of tomato plants when compared to an application of Trichoderma spp. alone.

The oil palms seedlings that were infected with Ganoderma spp. all displayed significantly lower average root diameter when compared to healthy oil palms without LOF. Ganoderma spp. has been shown to affect the hormonal balance of oil palms. According to Bahari et al., a Ganoderma spp. attack can result in the downregulation of ethylene and gibberellin signalling pathways. As ethylene and gibberellin play an important role in root radial growth, a downregulation would result in a reduced root diameter (Tadeo et al., 1997). The accumulation of ROS in root cell walls due to oxidative bursts can also result in cell wall degradation, and thus a reduction in average root diameter. On a larger scale, the widespread damage caused by Ganoderma spp. on the collective root architecture of oil palms will reduce the average root diameter, leading to higher disease incidence and palm death in a plantation.

T5 and T9, which contain Trichoderma spp., consistently recorded the highest total root surface area, suggesting that Trichoderma spp. aids in increasing root surface area (Figure 6). Meng et al., 2019 reported the function of a T. guizhouense protein, TgSWO, that behaved similarly to plant expansion proteins and played a promoted the growth of cucumber roots. Trichoderma viride was shown to increase the total root surface area in liquorice plants (He et al., 2020). A study by Yedidia et al. highlighted the positive effect that T. harzianum had on the root surface area of cucumbers. The significant differences between the two treatments in the 2^nd and 4^th month of sampling also suggest that Trichoderma spp. may boost root surface area when used in tandem with a waste resource such as biochar, as evidenced by Vecstaudza et al. in 2018. However, the final month of sampling showed no significant differences in total root surface area between T5 and T9. Further studies of longer than six months may be required to determine the efficacy between applying liquid Trichoderma spp. and solid Trichoderma spp. mixed in biochar.

T2, T4 and T6, with LOF, displayed similar levels of root surface area growth as T3, the Ganoderma disease control. T2 and T6, despite not being infected with Ganoderma spp., showed consistently low total root surface area throughout the six months. Parallels between the low total root surface area can be seen across all three levels of biological control in the healthy treatments supplied with LOF (Treatments 2, 6 and 10) in comparison with the healthy treatments that were only supplied with chemical fertiliser, as evidenced in Treatments 6 and 10 in comparison to Treatments 5 and 9 respectively. The absence of any significant differences between Treatments 2, 3, 4 and 6 suggest that LOF did not support root surface area in young oil palms.

The treatments that were infected with Ganoderma spp. all displayed significantly lower total surface area than the healthy treatments without LOF. The difference between the total surface area of the diseased treatments and the healthy treatments were also shown to increase over the duration of six months. Ganoderma spp. primarily colonises oil palm through the roots, hence the extent of the damage to the roots is highly visible. Through the secretion of cell wall degrading enzymes (CWDEs), Ganoderma spp. can penetrate deeper and further damage root cell walls of oil palms (Rees et al., 2009). Alexander et al., in 2017 were able to show through scanning electron microscopy the effects of Ganoderma spp. on eight-month-old oil palm seedlings, in which Ganoderma spp. negatively modifies the root architecture of infected seedlings, resulting in a collapsed root cortex and disintegrated root epidermal and cortical cells. This results in a severely diminished total root surface area.

Diseased plants which received Trichoderma spp. and T-mix (T7 and T11), showed significantly higher total root surface area compared to untreated disease control (T3). The total root surface area recorded in the 6^th month was almost four times higher in Treatments 7 and 11 in comparison with Treatment 3. This large difference in results proves that Trichoderma spp. is highly capable at suppressing the effects of Ganoderma spp. Trichoderma spp. is shown to suppress the activity of Ganoderma spp. through the inducing of peroxidase and polypherol oxidase in oil palm seedlings, as well as boost plant growth (Musa et al., 2018). Muniroh et al., in 2019 showed that T. asperellum boosted plant growth through the production of indole acetic acid (IAA), a plant growth promoter, as well as phosphate solubilisation and siderophore production, both of which allowed for easier access to nutrients for plants. By comparing these findings with the total root surface area data observed by Treatments 7 and 11, it can be surmised that Trichoderma spp. and T-mix is successful at both controlling Ganoderma spp. and boosting the root development of young oil palms.

Diseased treatments that received LOF and a biological control (T8 and T12) had a higher total root surface area when compared to the healthy plants with a biological control that were supplied with LOF (T6 and T10). T8 and T12 also had a total root surface area that was about 4 times higher than T4, which was the diseased treatment that did not receive any biological control. This highlights the ability of Trichoderma spp. in improving the total root surface area of diseased young oil palms. However, the absence of significant differences between Treatments 7 and 8, and between Treatments 11 and 12 suggest that while the addition of Trichoderma spp. helps negate the inhibitory effects of LOF on the total root surface area, this does not improve the total root surface area of diseased young oil palms in comparison with just the addition of Trichoderma spp. alone.