Stem rots of oil palm caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis>: Pathogen biology and epidemiology

Oil palm (Elaeis guineensis Jacq.) has been grown in Papua New Guinea since the early 1960s. The most important disease of oil palm in PNG is a stem rot of the palm base. This is the same disease that constitutes a major threat to sustainable oil palm production in SE Asia. Investigations into the causal pathogen have revealed that the stem rots in PNG are caused predominantly by the basidiomycete Ganoderma boninense, with a minor pathogen identified as G. tornatum G. tornatum was found to have a broad host range whereas G. boninense appears to be restricted to palms. The population structure of G. boninense was investigated using inter-fertility studies between isolates collected from basal stem rots on oil palm. Although the G. boninense field populations are predominantly comprised of distinct individuals, a number of isolates were found that share single mating alleles. This indicates that out-crossing had occurred over several generations in the resident or wild population of G. boninense prior to colonization of oil palm. No direct hereditary relationship between isolates on neighbouring diseased palms was found, although an indirect link between isolates causing upper stem rot and basal stem rot was detected.     

Mycoparasitic Scytalidium parasiticum as a potential biocontrol agent against Ganoderma boninense basal stem rot in oil palm*

The objective of this study was to assess the interactions between Scytalidium parasiticum (Sp) and Ganoderma boninense, the causal agent of basal stem rot (BSR) in oil palm (Elaeis guineensis). When compared with Scytalidium ganodermophthorum and Scytalidium sphaerosporum, Sp showed greater inhibition towards all Ganoderma isolates during dual-culture assays. At the interaction zone, coiling of host hyphae, formation of short lateral enlarged contact structures, and production of appressorium-like organs organs were observed in Sp on G. boninense. These were followed by the degradation, shrinkage, and deformation of G. boninense mycelia. Sp reduced mycelial survival and fruiting body regeneration of G. boninense. Sp's non-volatile metabolites suppressed the growth of G. boninense. Our results show that Sp could be a necrotrophic mycoparasite of G. boninense. Nursery experiments revealed that Sp was non-pathogenic to oil palm seedlings, and it could suppress Ganoderma infection and reduce disease severity. Sp increased the height of palms in the positive control with non-Ganoderma-inoculated rubber wood block and Sp inoculum compared to similar control without Sp. Leaf area was greater in the G. boninense G8 inoculated palms when Sp was present compared to without Sp. These results show that Sp might be a potential biocontrol candidate against BSR.     

An insight into spore dispersal of <Emphasis Type="Italic">Ganoderma boninense</Emphasis> on oil palm

The disease of oil palm caused by Ganoderma boninense, although universally referred to as Ganoderma basal stem rot, occurs in three very distinct phases, with basal stem rot only part of the disease cycle. G. boninense also causes a seedling disease and an upper stem rot. An understanding of spore dispersal provides an insight into where spores of G. boninense have a role in the infection process. This role will be discussed in relation to each of these three infection phases. This understanding is a critical component of developing a successful disease control strategy.     

Quantification and characterisation of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. from different ecosystems

Basal stem rot of oil palm caused by Ganoderma boninense is of major economic importance. Observations of the low incidence of disease due to Ganoderma species in natural stands, suggest that the disease is kept under control by some biological means. Trichoderma spp. are saprophytic fungi with high antagonistic activities against soil-borne pathogens. However, their abundance and distribution are soil and crop specific. Trichoderma species have been found to be concentrated in the A1 (0–30 cm) and Be soil horizons (30–60 cm), although the abundance of Trichoderma was not significantly different between the oil palm and non-oil palm ecosystems. Characterisation of Trichoderma isolates based on cultural, morphological and DNA polymorphism showed that T. harzianum, T. virens, T. koningii and T. longibrachiatum made up 72, 14, 10 and 4% of the total Trichoderma isolates isolated. As Trichoderma species are present in the oil palm ecosystem, but at lower numbers and in locations different from those desired, soil augmentation with antagonistic Trichoderma spp. can be developed as a strategy towards integrated management of basal stem rot of oil palm.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Current strategies and perspectives in detection and control of basal stem rot of oil palm

The rapid expansion of oil palm (OP) has led to its emergence as a commodity of strategic global importance. Palm oil is used extensively in food and as a precursor for biodiesel. The oil generates export earnings and bolsters the economy of many countries, particularly Indonesia and Malaysia. However, oil palms are prone to basal stem rot (BSR) caused by Ganoderma boninense which is the most threatening disease of OP. The current control measures for BSR management including cultural practices, mechanical and chemical treatment have not proved satisfactory. Alternative control measures to overcome the G. boninense problem are focused on the use of biological control agents and many potential bioagents were identified with little proven practical application. Planting OP varieties resistant to G. boninense could provide the ideal long-term solution to basal stem rot. The total resistance of palms to G. boninense has not yet been reported, and few examples of partial resistances have been observed. Importantly, basidiospores are now recognized as the method by which the disease is spread, and control methods require to be revaluated because of this phenomenon. Many methods developed to prevent the spread of the disease effectively are only tested at nursery levels and are only reported in national journals inhibiting the development of useful techniques globally. The initial procedures employed by the fungus to infect the OP require consideration in terms of the physiology of the growth of the fungus and its possible control. This review assesses critically the progress that has been made in BSR development and management in OP.     

A preliminary study on the effects of salicylic and jasmonic acids on Ganoderma boninense growth,mycelial hydrophobicity,and media pH under in vitro assays

Potato dextrose agar amended with salicylic acid (SA) suppressed growth of all Ganoderma boninense isolates. However, mycelial growth inhibitory effects varied with times, SA concentrations and G. boninense isolates. At 150 ppm of SA or higher, growth suppression diminished with time and mycelial growth recovery was observed. In contrast, jasmonic acid (JA) unexpectedly improved growth of G. boninense. At 21 day-after-inoculation, pH changes and hydrophobicity for the melanised mycelia were lower compared to non-melanised mycelia. The degree of inhibitory or promotional effects on the growth of G. boninense and the level of G. boninense mycelial hydrophobicity by SA and JA were isolate-dependent.     

Morphological and transcript changes in the biosynthesis of lignin in oil palm (Elaeis guineensis) during Ganoderma boninense infections in vitro

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4‐hydroxylase (EC 1.14.13.11), caffeic acid O‐methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ‐hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.     

<Emphasis Type="Italic">Streptomyces sanglieri</Emphasis> which colonised and enhanced the growth of <Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq. seedlings was antagonistic to <Emphasis Type="Italic">Ganoderma boninense</Emphasis> in in vitro studies

Actinomycete strain AUM 00500 was 99.5 % similar to Streptomyces sanglieri NBRC 100784^T and was evaluated for antagonistic activity towards Ganoderma boninense, the causative fungus of basal stem rot of oil palm. The strain showed strong antifungal activity towards G. boninense in in vitro and SEM analysis showed various modes of inhibition of the fungus. Ethyl acetate extracts of single culture and inhibition zone of cross-plug culture by HPLC indicated that strain AUM 00500 produced two different antibiotics of the glutarimide group namely cycloheximide and actiphenol. In greenhouse trials, oil palm seed treated with spores of S. sanglieri strain AUM 00500 at 10⁹ cfu/ml showed significant (P < 0.05) increase in oil palm seedlings growth when compared to the control. Streptomyces sanglieri strain AUM 00500 successfully colonised the epidermal surface of the roots of treated oil palm seedlings and it was recovered from root fragments plated on starch casein agar.     

Trichoderma aureoviride URM 5158 and Trichoderma hamatum URM 6656 are Biocontrol Agents that act against Cassava Root rot through different Mechanisms

Trichoderma has been used to manage a large number of pathogens, but there is a gap in the mechanisms used by these biocontrol agents regarding the physiological response of cassava plants (Manihot esculenta) when it is subjected to cassava root rot. The aims of this study were to investigate the antagonist activity of ten Trichoderma isolates against Fusarium solani on potato dextrose Agar (PDA), to quantify the chitinase production, to select and test in vivo the best isolate from each experiment and to assess the physiological response of cassava to the production of oxidative enzyme complex production (ascorbate peroxidase, catalase, peroxidase and polyphenol oxidase). All Trichoderma isolates have shown competitive capability against F. solani, and Trichoderma hamatum URM 6656 showed the highest inhibition of pathogen growth (88.91%). All isolates have shown chitinase activity, but Trichoderma aureoviride URM 5158 produced the highest amount of chitinase. T. hamatum URM 6656 and T. aureoviride URM 5158 were selected to be applied in vivo. The two Trichoderma strains reduced 64 and 60% of the disease severity in the shoot and 82 and 84% in the root. Cassava plants infected with Trichoderma have shown the highest peroxidase and ascorbate peroxidase production. Our results have indicated that T. aureoviride URM 5158 is an effective biocontrol agent against cassava root rot caused by F. solani, because it presented competitive antagonist capability in vitro, the highest chitinase production, and reduced the cassava root rot severity. The application of T. aureoviride has led to the maximum enzyme activity of reactive oxygen species group in cassava plants.     

Evaluation of Clove Essential Oil as a Mycobiocide Against Rhizopus stolonifer and Fusarium solani,Tuber Rot Causing Fungi in Yam (Dioscorea rotundata Poir.)

To exploit natural products for plant disease control, the essential oil of Syzygium aromaticum (L.) Merr. & Perr. (clove) was investigated for its antifungal activity against Rhizopus stolonifer and Fusarium solani, the postharvested yam tuber rot pathogens. The essential oil was obtained by hydrodistillation using a Clevenger‐type apparatus. The chemical composition of the oil was determined by gas chromatography and gas chromatography coupled with mass spectrometry. Antifungal activities of the oil were tested in vitro against mycelia growth and spores germination. In situ tests were conducted on healthy yam tubers, and necrosis symptoms were assessed. Results showed that eugenol (79.4%), eugenylacetate (9.2%) and isocaryophyllene (7.0%) were the major components. The oil exerted antifungal activities with total inhibition (TI) of the mycelia growth of R. stolonifer and F. solani was recorded at 200 and 300 ppm, respectively, while TI of spores germination was recorded at 31.25 and 250 ppm, respectively. For the standard fungicide (Ridomil^®), TI value of mycelia growth was 1600 ppm for the both pathogens, while TI of spores germination were 200 ppm and 1600 ppm, respectively, for Rhizopus and Fusarium. In situ tests showed complete inhibition of yam tuber rot when the essential oil was applied at 2000 ppm for preventive tests. This oil also reduced significantly (P ≤ 0.05) necrosis development on yam tuber for curative test at the same concentration. Total inhibition of the necrosis by Ridomil (3000 ppm) was observed only for Rhizopus on preventive test. There were positive correlations between the oil concentration and the reduction of necrosis cause by R. stolonifer and F. solani. These findings showed that clove essential oil may serve as environmental friendly bio‐fungicide for the management of postharvest yam tuber rot.     

Following basal stem rot in young oil palm plantings

The PCR primer GanET has previously been shown to be suitable for the specific amplification of DNA from Ganoderma boninense. A DNA extraction and PCR method has been developed that allows for the amplification of the G. boninense DNA from environmental samples of oil palm tissue. The GanET primer reaction was used in conjunction with a palm-sampling programme to investigate the possible infection of young palms through cut frond base surfaces. Ganoderma DNA was detected in frond base material at a greater frequency than would be expected by comparison with current infection levels. Comparisons are made between the height of the frond base infected, the number of frond bases infected, and subsequent development of basal stem rot. The preliminary results suggest that the development of basal stem rot may be more likely to occur when young lower frond bases are infected.     

Characterization of <Emphasis Type="Italic">Streptomyces</Emphasis> spp. isolated from the rhizosphere of oil palm and evaluation of their ability to suppress basal stem rot disease in oil palm seedlings when applied as powder formulations in a glasshouse trial

Ganoderma boninense, the main causal agent of oil palm (Elaeis guineensis) basal stem rot (BSR), severely reduces oil palm yields around the world. To reduce reliance on fungicide applications to control BSR, we are investigating the efficacy of alternative control methods, such as the application of biological control agents. In this study, we used four Streptomyces-like actinomycetes (isolates AGA43, AGA48, AGA347 and AGA506) that had been isolated from the oil palm rhizosphere and screened for antagonism towards G. boninense in a previous study. The aim of this study was to characterize these four isolates and then to assess their ability to suppress BSR in oil palm seedlings when applied individually to the soil in a vermiculite powder formulation. Analysis of partial 16S rRNA gene sequences (512 bp) revealed that the isolates exhibited a very high level of sequence similarity (>?98%) with GenBank reference sequences. Isolates AGA347 and AGA506 showed 99% similarity with Streptomyces hygroscopicus subsp. hygroscopicus and Streptomyces ahygroscopicus, respectively. Isolates AGA43 and AGA48 also belonged to the Streptomyces genus. The most effective formulation, AGA347, reduced BSR in seedlings by 73.1%. Formulations using the known antifungal producer Streptomyces noursei, AGA043, AGA048 or AGA506 reduced BSR by 47.4, 30.1, 54.8 and 44.1%, respectively. This glasshouse trial indicates that these Streptomyces spp. show promise as potential biological control agents against Ganoderma in oil palm. Further investigations are needed to determine the mechanism of antagonism and to increase the shelf life of Streptomyces formulations.     

Comparing interfertility data with random amplified microsatellites DNA (RAMS) studies in <Emphasis Type="Italic">Ganoderma</Emphasis> Karst. Taxonomy

The taxonomy of the causal pathogen of basal stem rot of oil palms, Ganoderma is somewhat problematic at present. In order to determine the genetic distance relationship between G. boninense isolates and non-boninense isolates, a random amplified microsatellites DNA (RAMS) technique was carried out. The result was then compared with interfertility data of G. boninense that had been determined in previous mating studies to confirm the species of G. boninense. Dendrogram from cluster analysis based on UPGMA of RAMS data showed that two major clusters, I and II which separated at a genetic distance of 0.7935 were generated. Cluster I consisted of all the biological species G. boninense isolates namely CNLB, GSDK 3, PER 71, WD 814, GBL 3, GBL 6, OC, GH 02, 170 SL and 348781 while all non-boninense isolates namely G. ASAM, WRR, TFRI 129, G. RES, GJ, and CNLM were grouped together in cluster II. Although the RAMS markers showed polymorphisms in all the isolates tested, the results obtained were in agreement with the interfertility data. Therefore, the RAMS data could support the interfertility data for the identification of Ganoderma isolates.     

Biological Control of Stem Canker and Black Scurf on Potato by Date Palm Compost and its Associated Fungi

The suppressive effects of two different types of date palm composts and some of their indigenous microorganisms were evaluated in vitro and on potato plants inoculated with Rhizoctonia solani. Fungi isolated from composts screened against R. solani by dual cultural assays on PDA showed a significant inhibition of pathogen mycelium growth as compared with untreated control. The type of hyphal interactions between R. solani and each tested antagonist was observed by light microscopy. Microscopic observations carried out at the confrontation zone of both agents showed different mechanisms of actions: mycelia lyses, mycoparasitism and/or formation of mycelia cords via anatomosis between mycelia filaments. Unsterilized and sterilized compost extracts were tested for efficacy against R. solani using agar‐well diffusion method or by pouring the extracts on PDA. Two sterilization methods were used: a filtration through a microfilter of 0.22 microns or autoclaving. Results showed that compost extract lost its activity after heating or filtration, confirming that chemical factors in compost had no direct inhibiting effect on the pathogen. The suppressiveness of composts was mainly due to their biotic component. Series of greenhouse trials showed that black scurf and stem canker incidence and severity were significantly reduced in peat–sand amended with compost compared with the untreated control. However, the potential suppressive effect of cattle manure and date palm compost (CMC) was higher than sheep manure and date palm compost (SMC). On potato seed tubers pre‐inoculated with the selected fungal isolates from compost, there was variability in the reduction of disease severity among treatments. Plant growth was unaffected by the application of fungal antagonists or by CMC amendment; however, an increase in the total yield was observed by the SMC potting mix compared with untreated control.     

Life expectancy of oil palm (Elaeis guineensis) infected by Ganoderma boninense in coastal soils,Malaysia: a case study

Ganoderma boninense basal stem rot poses a serious threat to the oil palm industry. The effects of external disease symptoms and coastal soils (Briah – Typic Endoaquepts, Jawa – Typic Sulfaquepts, and Selangor – Typic Humaquepts) on the life expectancy of the infected palms, from disease detection to death, were studied. Six-monthly censuses on disease classes for each palm were recorded between 2004 and 2012. Survival curves of disease symptoms and soil types were compared using Kaplan–Meier and log-rank methods, respectively. Ganoderma-infected palms in acid-sulphate (AS) and potential AS soils recorded lower life expectancy. Survival duration of infected palms with foliar symptoms was 12-months shorter. External factors, such as soil type may influence the survival of infected palms and soil types may pre-dispose oil palm to higher risk of Ganoderma infection. More effective Ganoderma management for palms planted on Coastal soils (with and without AS layer) have been proposed.     

Antagonistic Potential of Bacillus pumilus L1 Against Root‐Knot Nematode,Meloidogyne arenaria

This study was conducted to estimate the potential of Bacillus pumilus L1 against root‐knot nematode, Meloidogyne arenaria, in both in vitro and in vivo conditions. B. pumilus L1 was found to produce both protease and chitinase. When various concentrations (1–10%) of the bacterial culture (BC) or 0.02–0.11 mg/ml of the crude enzymes produced by B. pumilus L1 were used to treat M. arenaria eggs and second‐stage juveniles (J2), inhibition of hatching and J2 mortality were significantly increased under in vitro conditions. In addition, the hatching inhibition and J2 mortality rate were improved with increasing concentrations of BC and the crude enzymes. Similarly, these effects also increased over time after treatment with BC. Moreover, the crude enzymes caused partial degradation of the eggshell and juvenile body when treated at 0.11 mg/ml. The pot experiment also demonstrated that the application of BC to potted soil caused significant reduction in the number of galls and egg masses in the plant roots and of the J2 population as compared to the untreated control 6 weeks after M. arenaria infestation. In addition, the simultaneous application of BC upon nematode inoculation proved more effective than application 2 days postinoculation with nematode. B. pumilus L1 inoculation (BC, BCs and BC2) also promoted tomato plant growth as compared to the controls (TW, Ne, GM and NeT). Thus, our results demonstrated the ability of B. pumilus L1 as a potential biocontrol agent against root‐knot nematode, with additional activity as a plant growth promoter for tomato.     

Carvacrol and eugenol effectively inhibit Rhizopus stolonifer and control postharvest soft rot decay in peaches

Aims

This study aimed to investigate the antifungal mechanism of carvacrol and eugenol to inhibit Rhizopus stolonifer and the control of postharvest soft rot decay in peaches.

Methods and Results

To investigate the antifungal mechanism, the effects of carvacrol and eugenol on the mycelium growth, leakages of cytoplasmic contents, mycelium morphology, cell membrane and membrane composition of R. stolonifer were studied. Carvacrol and eugenol both exhibited dose‐dependent antifungal activity against R. stolonifer, carvacrol at a concentration of 2 μl per plant and eugenol at a concentration of 4 μl per plant inhibited fungal growth completely. The two essential oils (EOs) increased cell membrane penetrability and caused the leakage of cytoplasm, nucleic acid and protein content. The observation using scanning electron microscopy and fluorescent microscopy showed modification of the hyphal morphology and breakage of the cell plasma membrane. Decreased ergosterol contents confirmed that the two EOs could destroy the membrane of R. stolonifer. For the in vivo test, the inhibition of soft rot disease and the induction of defence‐related enzymes were investigated. Carvacrol and eugenol significantly reduced the incidence and severity of soft rot decay in inoculated peaches. The best treatments for controlling soft rot decay were obtained at 0·5 μl l^?1 for carvacrol and 1 μl l^?1 for eugenol. The activities of defence‐related enzymes in peaches were also enhanced by fumigation with two EOs.

Conclusion

This study showed that carvacrol and eugenol could effectively inhibit the growth of R. stolonifer in vitro and successfully control the incidence of soft rot decay in honey peaches.

Significance and Impact of the Study

The above findings may be the main antifungal mechanism of carvacrol and eugenol on R. stolonifer. Furthermore, carvacrol and eugenol are helpful for their commercial application on the preservation of fresh fruit.