PhD student at Monash University, aiming to improve the well-being of plants (palm oil)
Beneficial use of endophytes, plant-associated microbes, for plants' well-being
It is almost a sure thing that many of us love chocolates, ice-creams and biscuits. The luxurious taste and crunchiness bring comfort when we are down (especially with research). One thing these comforting foods have in common is palm oil as their main ingredient. Harvested from the oil palm trees, they can be used to manufacture a variety of products ranging from food, beverage to even cosmetics. Despite their promising uses, the trees face two major problems: (i) fungal attack and (ii) stressful growth condition in metal-contaminated sites. The presence of metal contaminant in the environment is undeniably harmful and will eventually be integrated into organisms.
Understanding the importance of this economically important plant, it is therefore essential to address problems faced by these plants in Asia. With these two problems, they give rise to my research. My project focuses on the well-being of the oil palms by not only attempting to fight against the pathogenic fungus but also intending to encourage the trees’ growth in metals. Luckily, a group of microorganisms capable of such purposes exists and this group is known as endophyte. These guardian-like microorganisms originally lived within host plant tissues and can be isolated for further understanding of their potentials. Studies have shown that they are capable of numerous capabilities, in terms of biocontrol as well as promoting plant growth.
The first step was to identify endophytes which are metal-tolerant, usually living within phytoremediator plants. Their capabilities were further studied through screening for the production of secondary metabolites as well as the interaction with the pathogen. Besides these mechanisms in inhibiting the growth of the pathogen, we also simultaneously study their capabilities in encouraging plant growth via nutrient mobilization. This highly potential group of microbes, if successful, will help us in addressing the problems faced by the poor oil palms in Asia.
Abstract: This study characterized the antagonistic activities of five fungal endophytes (Aspergillus calidoustous BTF07, Penicillium citrinum BTF08, Trichoderma asperellum T2, Diaporthe phaseolorum WAA02, Diaporthe phaseolorum MIF01) and evaluated their endophyte-host compatibility with the host plant (oil palm). The antifungal activities of the endophytes towards Ganoderma boninense (GB) were first established using the dual culture test, revealing antagonistic nature of endophytes via production of non-volatiles, volatiles and competitive exclusion. Endophyte-host compatibility was then assessed using a simple but rapid endophyte-calli dual-culture assay, and results validated using endophyte-ramet test. Results revealed that endophytes elicited different responses in oil palm calli. BTF08 had growth promoting effects towards the host tissues with the highest calli weight (1013 mg) obtained, while BTF07 appeared to inhibit calli (1006 mg) leading to browning and necrosis. This endophyte-calli test also revealed the influence of calli on endophyte growth. Isolates BTF08 and GB benefited from host association, with increased radial growth (2.36 cm and 2.31 cm, respectively) compared to growth in the absence of calli (2.10cm and 2.15 cm, respectively). Endophytes and GB were also isolated from host tissues, suggesting compatibility and ability to colonize host tissues (root, stem, leaf). This suggested the reliability of the endophyte-calli test as a rapid assay to provide an insight on the endophyte-host compatibility.
Pub.: 03 Dec '16, Pinned: 30 Jul '17
Abstract: The phenylpropanoid and flavonoid pathways are well known for their function towards plant development and defense. Oil palm seedlings from a single cross challenged with Ganoderma boninense displayed different severity of infection after six and fourteen months post inoculation (mpi). Transcripts from phenylpropanoid and flavonoid pathways, and transcription factors related to secondary cell wall formation were compared based on external and internal symptom classification. This long term infection study revealed that the oil palm roots responded to G. boninense and regulated both phenylpropanoid and flavonoid pathways during infection. The expression of PAL2 and PAL3 were significantly higher at 6 mpi in symptomatic compared to asymptomatic and control seedlings. However, the expression of all PAL genes were down-regulated at 14 mpi for both symptomatic and asymptomatic seedlings. In the monolignol and lignin pathways, down-regulation of COMT in symptomatic and CCR in asymptomatic seedlings was observed at 14 mpi but not at 6 mpi suggesting a possible change in lignin composition likely related to observed resistance. The expression of transcription factors for secondary cell wall and monolignol (MYB58, MYB63 and SND1) biosynthesis showed down-regulation in symptomatic seedlings, also suggesting a link between cell wall biosynthesis and disease resistance. Genes that regulate the flavonoid pathway were mostly down-regulated in both symptomatic seedlings at both 6 mpi and 14 mpi indicating a possibility of pathway suppression after challenge with G. boninense. Prolonged G. boninense exposure suggested that oil palm seedlings responded differently in terms of phenylpropanoid and flavonoid pathway expression in conferring defense and disease resistance.
Pub.: 15 Jul '16, Pinned: 17 Aug '17
Abstract: This study aimed to establish the colonization behavior and proliferation potential of three endophytes and one pathogen Ganoderma boninense (Gb) introduced into oil palm ramets (host model). The endophytes selected were Diaporthe phaseolorum (WAA02), Trichoderma asperellum (T2), and Penicillium citrinum (BTF08). Ramets were first inoculated with 100 mL of fungal cells (106 cfu mL-1) via soil drenching. For the next 7 days, ramets were sampled and subjected to three different assays to detect and identify fungal colonization, and establish their proliferation potential in planta. Plate assay revealed the presence of endophytes in root, stem and leaf tissues within 7 days after inoculation. Polymerase Chain Reaction (PCR) detected and identified the isolates from the plant tissues. The ergosterol assay (via High Performance Liquid Chromatography, HPLC) confirmed the presence of endophytes and Gb in planta. The increase in ergosterol levels throughout 49 days were however insignificant, suggesting that proliferation may be absent or may occur very slowly in planta. This study strongly suggests that the selected endophytes could colonize the host upon inoculation, but proliferation occurs at a slower rate, which may subsequently influence the biocontrol expression of endophytes against the pathogen.
Pub.: 03 Nov '16, Pinned: 17 Aug '17
Abstract: Basal stem rot disease caused by Ganoderma spp. is still considered a large threat to oil palm production in many countries, especially in Malaysia, which contributes to approximately 45% of the world's palm oil exports. In the last 10 years, studies on the oil palm-Ganoderma interaction have mainly focused on the response of the plant towards the fungus. In this study, a comparative proteomic analysis was conducted to investigate the change in protein expression of two Ganoderma spp., namely Ganoderma boninense, which is known as the most aggressive species and Ganoderma tornatum, previously reported as non-pathogenic. Our results showed that both species colonize and penetrate the oil palm root after only 72 h of inoculation. In addition, proteins were expressed differentially in both species that have either direct or indirect links to virulence and pathogenicity. Other proteins related to fungal growth, metabolism and stress from both species were also discussed in this study.
Pub.: 12 Feb '17, Pinned: 17 Aug '17
Abstract: Wood rot fungi form one of the main classes of phytopathogenic fungus. The group includes many species, but has remained poorly studied. Many species belonging to the Ganoderma genus are well known for causing decay in a wide range of tree species around the world. Ganoderma boninense, causal agent of oil palm basal stem rot, is responsible for considerable yield losses in Southeast Asian oil palm plantations. In a large-scale sampling operation, 357 sporophores were collected from oil palm plantations spread over peninsular Malaysia and Sumatra and genotyped using 11 SSR markers. The genotyping of these samples made it possible to investigate the population structure and demographic history of G. boninense across the oldest known area of interaction between oil palm and G. boninense. Results show that G. boninense possesses a high degree of genetic diversity and no detectable genetic structure at the scale of Sumatra and peninsular Malaysia. The fact that few duplicate genotypes were found in several studies including this one supports the hypothesis of spore dispersal in the spread of G. boninense. Meanwhile, spatial autocorrelation analysis shows that G. boninense is able to disperse across both short and long distances. These results bring new insight into mechanisms by which G. boninense spreads in oil palm plantations. Finally, the use of approximate Bayesian computation (ABC) modelling indicates that G. boninense has undergone a demographic expansion in the past, probably before the oil palm was introduced into Southeast Asia.
Pub.: 01 Feb '17, Pinned: 17 Aug '17
Abstract: Species of Trichoderma are widely recognized for their biocontrol abilities, but seldom studied collectively, for their plant growth promotion, abiotic stress tolerance and bioremediation properties. Our study is a concentrated effort to establish the potential of native isolate Trichoderma harzianum KSNM (T103) to tolerate biotic (root pathogens) and abiotic stresses [high salt (100-1000 mM); heavy metal (chromium, nickel and zinc: 1-10 mM); pesticides: malathion (100-600 ppm), carbofuran (100-600 ppb)], along with its ability to support plant growth. In vitro growth promotion assays with T103 treated Vigna radiata, Vigna mungo and Hordeum vulgare confirmed 'non-species specific' growth promotion effects of T103. At lower metal concentration, T103 treatment was found to completely negate the impact of metal stress [60 % increase in radicle length (RL) with no significant decrease in %germination (%G)]. Even at 10 mM metal, T103 inoculation gave 80 % increase in %G and >50 % increase in RL. In vitro experiments confirmed high metal reduction capacity (47 %-Cr, 35 %-Ni and 42 %-Zn) of T103 at concentrations as high as 4 mM. At maximum residual concentrations of malathion (440 ppm) and carbofuran (100 ppb) reported in agricultural soils, T103 maintained 80 and 100 % survivability, respectively. T103 treatment has improved %G and RL in all three hosts challenged with pesticide. Isolate T103 was found to effectively suppress growth of three major root pathogens: Macrophomina phaseolina (65.83 %) followed by Sclerotium rolfsii (19.33 %) and Fusarium oxysporum (19.18 %). In the light of these observations, native T. harzianum (T103) seems to be a competent biocontrol agent for tropical agricultural soils contaminated with residual pesticides and heavy metals.
Pub.: 25 Jun '16, Pinned: 30 Jul '17
Abstract: Oil palm is subjected to various diseases including Basal Stem Rot (BSR) which is mainly caused by the basidiomycetes fungi, Ganoderma. However, studies on the fungal infection mechanism especially at molecular level and the biological processes involved are still very limited. Fungal cyclophilin (CYP) has been reported to be involved in fungal pathogenicity but involvement of CYP in the pathogenicity of G. boninense has not been revealed yet. The main objective of this study is to isolate cDNAs encoding CYP and to profile the expression level of these transcripts during infection stages of G. boninense. In this study, five full-length cDNAs encoding CYP were successfully isolated from G. boninense. They were classified as different family members of CYP because significant differences could be observed on their coding, 5′ or 3′ un-translated regions (UTRs). The expression of these five CYP transcripts in different type of G. boninense tissues and the infecting mycelia tissues was studied using real time quantitative PCR (qPCR). Based on the expression results and analysis, the potential functions of these CYP transcripts were predicted to be involved in the development of fruiting body (GbCYP201) and fungal stress response or pathogenicity (GbCYP203 and GbCYP205).
Pub.: 24 May '16, Pinned: 17 Aug '17
Abstract: Ganoderma is a genus of medicinal mushrooms. This review deals with secondary metabolites isolated from Ganoderma and their biological significance. Phytochemical studies over the last 40years led to the isolation of 431 secondary metabolites from various Ganoderma species. The major secondary compounds isolated are (a) C30 lanostanes (ganoderic acids), (b) C30 lanostanes (aldehydes, alcohols, esters, glycosides, lactones, ketones), (c) C27 lanostanes (lucidenic acids), (d) C27 lanostanes (alcohols, lactones, esters), (e) C24, C25 lanostanes (f) C30 pentacyclic triterpenes, (g) meroterpenoids, (h) farnesyl hydroquinones (meroterpenoids), (i) C15 sesquiterpenoids, (j) steroids, (k) alkaloids, (l) prenyl hydroquinone (m) benzofurans, (n) benzopyran-4-one derivatives and (o) benzenoid derivatives. Ganoderma lucidum is the species extensively studied for its secondary metabolites and biological activities. Ganoderma applanatum, Ganoderma colossum, Ganoderma sinense, Ganoderma cochlear, Ganoderma tsugae, Ganoderma amboinense, Ganoderma orbiforme, Ganoderma resinaceum, Ganoderma hainanense, Ganoderma concinna, Ganoderma pfeifferi, Ganoderma neo-japonicum, Ganoderma tropicum, Ganoderma australe, Ganoderma carnosum, Ganoderma fornicatum, Ganoderma lipsiense (synonym G. applanatum), Ganoderma mastoporum, Ganoderma theaecolum, Ganoderma boninense, Ganoderma capense and Ganoderma annulare are the other Ganoderma species subjected to phytochemical studies. Further phytochemical studies on Ganoderma could lead to the discovery of hitherto unknown biologically active secondary metabolites.
Pub.: 16 May '15, Pinned: 17 Aug '17
Abstract: Ganoderma boninense is a white rot basidiomycete that causes basal stem rot disease of oil palm (Elaeis guineensis). The aims of this study were to identify endophytic basidiomycetes occurring naturally within oil palm and to assess their potential as biocontrol agents against G. boninense strain PER71 in vitro. In total, 376 isolates were recovered from samples collected from the root, stem and leaves of oil palm using Ganoderma‐selective medium. Ten of these isolates (2.7% of the total 376 isolates) were identified as basidiomycetes on the basis of clamp connections and the production of poroid basidiomes after incubation in glass jars containing PDA medium for 7–12 days. The isolates were identified using ITS rDNA sequencing as Neonothopanus nambi (five isolates), Schizophyllum commune (four isolates) and Ganoderma orbiforme (one isolate). The N. nambi isolates showed the greatest antagonistic activity against G. boninense, based on 73–85% inhibition of the radial growth measurements of G. boninense in dual culture and 76–100% inhibition of G. boninense growth in a culture filtrate assay. Possible modes of action for the antagonism shown by N. nambi against G. boninense in vitro include competition for substrate availability, space and the production of non‐volatile metabolites or antibiotics that inhibited the growth of G. boninense. Further in vivo investigations are required to determine the ability of N. nambi isolates to colonize oil palm seedlings and to protect oil palm from infection when challenged with G. boninense.
Pub.: 24 Jun '16, Pinned: 17 Aug '17
Abstract: Authors: Yit Kheng Goh ; Nurul Fadhilah Marzuki ; Teik Khiang Goh ; Suet Yee Tan ; You Keng Goh ; Kah Joo Goh Article URL: http://www.tandfonline.com/doi/full/10.1080/09583157.2016.1202192?ai=z4&mi=3fqos0&af=R Citation: Biocontrol Science and Technology Publication Date: 2016-08-12T09:44:04Z Journal: Biocontrol Science and Technology
Pub.: 12 Aug '16, Pinned: 17 Aug '17
Abstract: Understanding the mechanism of interaction between the oil palm and its key pathogen, Ganoderma spp. is crucial as the disease caused by this fungal pathogen leads to a major loss of revenue in leading palm oil producing countries in Southeast Asia. Here in this study, we assess the morphological and biochemical changes in Ganoderma disease infected oil palm seedling roots in both resistant and susceptible progenies. Rubber woodblocks fully colonized by G. boninense were applied as a source of inoculum to artificially infect the roots of resistant and susceptible oil palm progenies. Gas chromatography-mass spectrometry was used to measure an array of plant metabolites in 100 resistant and susceptible oil palm seedling roots treated with pathogenic Ganoderma boninense fungus. Statistical effects, univariate and multivariate analyses were used to identify key-Ganoderma disease associated metabolic agitations in both resistant and susceptible oil palm root tissues. Ganoderma disease related defense shifts were characterized based on (i) increased antifungal activity in crude extracts, (ii) increased lipid levels, beta- and gamma-sitosterol particularly in the resistant progeny, (iii) detection of heterocyclic aromatic organic compounds, benzo [h] quinoline, pyridine, pyrimidine (iv) elevation in antioxidants, alpha- and beta-tocopherol (iv) degraded cortical cell wall layers, possibly resulting from fungal hydrolytic enzyme activity needed for initial penetration. The present study suggested that plant metabolites mainly lipids and heterocyclic aromatic organic metabolites could be potentially involved in early oil palm defense mechanism against G. boninense infection, which may also highlight biomarkers for disease detection, treatment, development of resistant variety and monitoring.
Pub.: 04 Oct '16, Pinned: 17 Aug '17
Abstract: Verticillium wilt is a soil-borne disease, and severely limits the development of cotton production. To investigate the role of endophytic fungi on Verticillium wilt, CEF-818 (Penicillium simplicissimum), CEF-714 (Leptosphaeria sp.), CEF-642 (Talaromyces flavus.) and CEF-193 (Acremonium sp.) isolated from cotton roots were used to assess their effects against cotton wilt disease caused by a defoliating V. dahliae strain Vd080. In the greenhouse, all treatments significantly reduced disease incidence and disease index, with the control efficacy ranging from 26% (CEF-642) to 67% (CEF-818) at 25 days (d) after inoculation. In the disease nursery, compared to controls (with disease incidence of 33.8% and disease index of 31), CEF-818, CEF-193, CEF-714 and CEF-642 provided a protection effect of 69.5%, 69.2%, 54.6% and 45.7%, respectively. Especially, CEF-818 and CEF-714 still provided well protection against Verticillium wilt with 46.9% and 56.6% or 14.3% and 33.7% at the first peak of the disease in heavily infected field, respectively (in early July). These results indicated that these endophytes not only delayed but also reduced wilt symptoms on cotton. In the harvest, the available cotton bolls of plant treated with CEF-818 and CEF-714 increased to 13.1, and 12.2, respectively. And the seed cotton yield significantly increased after seed bacterization with CEF-818 (3442.04 kg/ha) compared to untreated control (3207.51 kg/ha) by 7.3%. Furtherly, CEF-818 and CET-714 treatment increased transcript levels for PAL, PPO, POD, which leads to the increase of cotton defense reactions. Our results indicate that seed treatment of cotton plants with CEF-818 and CET-714 can help in the biocontrol of V. dahliae and improve seed cotton yield in cotton fields. This study provided a better understanding of cotton-endophyte interactions which will aid in developing effective biocontrol agents for Verticillium wilt of cotton in futhre.
Pub.: 21 Jan '17, Pinned: 30 Jul '17
Abstract: Multi-parental populations are promising tools for identifying quantitative disease resistance loci. Stem rot caused by Ganoderma boninense is a major threat to palm oil production, with yield losses of up to 80% prompting premature replantation of palms. There is evidence of genetic resistance sources, but the genetic architecture of Ganoderma resistance has not yet been investigated. This study aimed to identify Ganoderma resistance loci using an oil palm multi-parental population derived from nine major founders of ongoing breeding programs. A total of 1200 palm trees of the multi-parental population was planted in plots naturally infected by Ganoderma, and their health status was assessed biannually over 25 yr. The data were treated as survival data, and modeled using the Cox regression model, including a spatial effect to take the spatial component in the spread of Ganoderma into account. Based on the genotypes of 757 palm trees out of the 1200 planted, and on pedigree information, resistance loci were identified using a random effect with identity-by-descent kinship matrices as covariance matrices in the Cox model. Four Ganoderma resistance loci were identified, two controlling the occurrence of the first Ganoderma symptoms, and two the death of palm trees, while favorable haplotypes were identified among a major gene pool for ongoing breeding programs. This study implemented an efficient and flexible QTL mapping approach, and generated unique valuable information for the selection of oil palm varieties resistant to Ganoderma disease.
Pub.: 09 Jun '17, Pinned: 17 Aug '17
Abstract: This work, for the first time, investigated the diversity of endophytic fungi harbored in the xylem and phloem of the root of Sophora tonkinensis Gapnep from three geographic localities with emphasis on the influence of the tissue type and geographic locality on endophytic fungal communities and their potential as biocontrol agents against phytopathogens of Panax notoginseng. A total of 655 fungal strains representing 47 taxa were isolated. Forty-two taxa (89.4%) were identified but not five taxa (10.6%) according to morphology and molecular phylogenetics. Out of identifiable taxa, the majority of endophyte taxa were Ascomycota (76.6%), followed by Basidiomycota (8.5%) and Zygomycota (4.3%). The alpha-diversity indices indicated that the species diversity of endophytic fungal community harbored in the root of S. tonkinensis was very high. The colonization and species diversity of endophytic fungal communities were significantly influenced by the geographic locality but not tissue type. The geographic locality and tissue type had great effects on the species composition of endophytic fungal communities. Forty-seven respective strains were challenged by three fungal phytopathogens of P. notoginseng and six strains exhibited significant inhibitory activity. It was noteworthy that endophytic Rhexocercosporidium sp. and F. solani strongly inhibited pathogenic F. solani and other fungal phytopathogens of P. notoginseng.
Pub.: 17 Mar '17, Pinned: 30 Jul '17
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