Growth modulation and metabolic responses of Ganoderma boninense to salicylic acid stress

Various phenolic compounds have been screened against Ganoderma boninense, the fungal pathogen causing basal stem rot in oil palms. In this study, we focused on the effects of salicylic acid (SA) on the growth of three G. boninense isolates with different levels of aggressiveness. In addition, study on untargeted metabolite profiling was conducted to investigate the metabolomic responses of G. boninense towards salicylic acid. The inhibitory effects of salicylic acid were both concentration- (P < 0.001) and isolate-dependent (P < 0.001). Also, growth-promoting effect was observed in one of the isolates at low concentrations of salicylic acid where it could have been utilized by G. boninense as a source of carbon and energy. Besides, adaptation towards salicylic acid treatment was evident in this study for all isolates, particularly at high concentrations. In other words, inhibitory effect of salicylic acid treatment on the fungal growth declined over time. In terms of metabolomics response to salicylic acid treatment, G. boninense produced several metabolites such as coumarin and azatyrosine, which suggests that salicylic acid modulates the developmental switch in G. boninense towards the defense mode for its survival. Furthermore, the liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) analysis showed that the growth of G. boninense on potato dextrose agar involved at least four metabolic pathways: amino acid metabolism, lipid pathway, tryptophan pathway and phenylalanine pathway. Overall, there were 17 metabolites that contributed to treatment separation, each with P<0.005. The release of several antimicrobial metabolites such as eudistomin I may enhance G. boninense’s competitiveness against other microorganisms during colonisation. Our findings demonstrated the metabolic versatility of G. boninense towards changes in carbon sources and stress factors. G. boninense was shown to be capable of responding to salicylic acid treatment by switching its developmental stage.

Identification of a Suitable Machine Learning Model for Detection of Asymptomatic Ganoderma boninense Infection in Oil Palm Seedlings Using Hyperspectral Data

In Malaysia, oil palm industry has made an enormous contribution to economic and social prosperity. However, it has been affected by basal stem rot (BSR) disease caused by Ganoderma boninense (G. boninense) fungus. The conventional practice to detect the disease is through manual inspection by a human expert every two weeks. This study aimed to identify the most suitable machine learning model to classify the inoculated (I) and uninoculated (U) oil palm seedlings with G. boninense before the symptoms’ appearance using hyperspectral imaging. A total of 1122 sample points were collected from frond 1 and frond 2 of 28 oil palm seedlings at the age of 10 months old, with 540 and 582 reflectance spectra extracted from U and I seedlings, respectively. The significant bands were identified based on the high separation between U and I seedlings, where the differences were observed significantly in the NIR spectrum. The reflectance values of each selected band were later used as input parameters of the 23 machine learning models developed using decision trees, discriminant analysis, logistic regression, naïve Bayes, support vector machine (SVM), k-nearest neighbor (kNN), and ensemble modelling with various types of kernels. The bands were optimized according to the classification accuracy achieved by the models. Based on the F-score and performance time, it was demonstrated that coarse Gaussian SVM with 9 bands performed better than the models with 35, 18, 14, and 11 bands. The coarse Gaussian SVM achieved an F-score of 95.21% with a performance time of 1.7124s when run on a personal computer with an Intel® Core™ i7-8750H processor and 32 GB RAM. This early detection could lead to better management in the oil palm industry.

Distinct Detection of Ganoderma Boninense On Metal Oxides-Gold Nanoparticle Composite Deposited Interdigitated Electrode DNA sensor

Oil palms suffer severe losses due to Ganoderma boninense infection that causes Basal Stem Rot (BSR). The available detection measuring the severity of BSR disease have not proved satisfactory output. Due to the influence of oil palm industry in country’s economy, effective and efficient means of diagnostic measure is mandatory. Among the available diagnostic tools, biosensors were redeemed to yield the most rapid and selective results. To overcome the current issues, herein Interdigitated Electrode (IDE) electrochemical DNA biosensor to detect Ganoderma boninense was successfully designed and fabricated by thermal deposition. Lift-off photolithography fabrication process was applied followed by the surface chemical functionalization via seed deposition. Zinc Oxide (ZnO) and Titanium Dioxide (TiO2) were overlaid and the functionalized metal oxides IDE surfaces were used to detect DNA sequence complementation from Ganoderma boninense. Furthermore, gold nanoparticles were doped to increase the surface to volume ratio and enhance biocompatibility. Characterizations were made by validating the sensor’s topology characteristics and electrical characteristics. From the results recorded, it has been justified that IDE with ZnO doped with gold nanoparticles surface serves as an excellent DNA sensor for the detection of Ganoderma boninense with a remarkable current of 290 nA and 176 nA for immobilization and hybridization respectively.

Antagonistic capacity of dark septate endophytes (DSE) against Ganoderma boninense from oil palm (Elaeis guinensis Jacq.)

Ganoderma boninense, a causal agent of basal stem rot, as one of the major problems in oil palm plantation in Indonesia. The potential of dark septate endophytes (DSE) as biocontrol agents for G. boninense is not widely studied. Therefore, this study aims to screen DSE strains to obtain high antagonistic strains. The antagonistic capacity of 10 DSE strains was determined based on the growth inhibition in the dual culture, and volatile compounds challenge against G. boninense, in vitro. Volatile compound profiling of selected DSE strains that showed the highest challenge capacity was done using Pyrolysis Gas Chromatography-Mass Spectrometry. Among 10 DSE strains tested, TKC 2.2a had the highest percentage of G. boninense inhibition in dual cultures and the volatile compound challenge. This strain inhibited better if grown in media seven days earlier than grown simultaneously with G. boninsense. The volatile compounds of 14 days old TKC 2.2a were of 2H-1-Benzopyran-7-ol, 3,4-dihydro-5-methoxy-6-methyl-2-phenyl; 4-Allyl-2,6-dimetho-xyphenol; and hexa-decanoic acid, which were reported as having either antimicrobial or antifungal activities. These results indicate that DSE TKC 2.2a can be a candidate for a biocontrol agent against G. boninense in oil palm, depending on further research on its ability to reduce basal stem rot symptoms.

Temporal Changes Analysis of Soil Properties Associated with Ganoderma boninense Pat. Infection in Oil Palm Seedlings in a Controlled Environment

Basal stem rot (BSR) disease of oil palm (Elaeis guineensis Jacq.) spreads through the contact of the plant roots with Ganoderma boninense (G. boninense) Pat. inoculum in the soil. The soil properties can be altered by growing seedlings with or without G. boninense inoculum. In the early stage of infection, the symptoms are difficult to detect. Therefore, an understanding of the environmental soil conditions of the plant is crucial in order to indicate the presence of the fungus. This paper presents an analysis of the temporal changes of the soil properties associated with the G. boninense infection in oil palm seedlings. A total of 40 seedlings aged five months were used in the study, comprising 20 inoculated (infected seedlings: IS) and 20 control (healthy seedlings: HS) seedlings. The seedlings were grown in a greenhouse for six months (24 weeks) under a controlled environmental temperature and humidity. The data of the soil moisture content (MC in %), electrical conductivity (EC in µS/cm), and temperature (T in °C) for each seedling were collected daily using three MEC10 soil sensors every hour and then transferred to the ThingSpeak cloud using a 3G Internet connection. Based on the results, the mean MC and EC showed a decreasing trend, while the mean T showed an increasing trend in both HS and IS during the six-month monitoring period. The overall mean in both the monthly and weekly analysis of MC, EC, and T was higher in HS than IS. However, in the monthly analysis, a Student’s t-test at a 5% significance level showed that only the soil MC and EC were significantly different between HS and IS, while in the weekly analysis, HS was significantly different from IS in all parameters. This study suggests that soil MC, EC, and T can be used as indicators of the G. boninense infection, especially for the weekly data.

Identification of Oil Palm’s Consistently Upregulated Genes during Early Infections of Ganoderma boninense via RNA-Seq Technology and Real-Time Quantitative PCR

Basal stem rot (BSR) disease caused by pathogenic fungus Ganoderma boninense is a significant concern in the oil palm industry. G. boninense infection in oil palm induces defense-related genes. To understand oil palm defense mechanisms in response to fungal invasion, we analyzed differentially expressed genes (DEGs) derived from RNA-sequencing (RNA-seq) transcriptomic libraries of oil palm roots infected with G. boninense. A total of 126 DEGs were detected from the transcriptomic libraries of G. boninense-infected root tissues at different infection stages. Functional annotation via pathway enrichment analyses revealed that the DEGs were involved in the defense response against the pathogen. The expression of the selected DEGs was further confirmed using real-time quantitative PCR (qPCR) on independent oil palm seedlings and mature palm samples. Seven putative defense-related DEGs consistently showed upregulation in seedlings and mature plants during G. boninense infection. These seven genes might potentially be developed as biomarkers for the early detection of BSR in oil palm.

Hexaconazole-Micelle Nanodelivery System Prepared Using Different Surfactants for Ganoderma Antifungal Application

Reports on fungicide-based agronanochemicals in combating disastrous basal stem rot disease in the oil palm industry are scant. Herein, we describe the potential of fungicide nanodelivery agents based on hexaconazole-micelle systems produced using three different surfactants; sodium dodecylbenze sulfonate (SDBS), sodium dodecyl sulfate (SDS) and Tween 80 (T80). The resulting nanodelivery systems were characterized and the results supported the encapsulation of the fungicide into the micelles of the surfactants. We have investigated in detail the size-dependent effects of the as-synthesized micelles towards the inhibition growth of Ganoderma Boninense fungi. All the nanodelivery systems indicate that their size decreased as the surfactant concentration was increased, and it directly affects the fungal inhibition. It was also found that Tween 80, a non-ionic surfactant gave the lowest effective concentration, the EC50 value of 2, on the pathogenic fungus Ganoderma boninense compared to the other anionic surfactants; SDBS and SDS. This study opens up a new generation of agronanofungicide of better efficacy for Ganoderma disease treatment.