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Basal stem rot disease of oil palm caused by Ganoderma boninense is one of the most devastating diseases in oil palm plantation resulting in low yield, loss of palm stands and shorter replanting cycle. To-date, there is no effective treatment for Ganoderma infected palms. Control measures, either chemical or cultural approaches, show varying degrees of effectiveness. The application of biological control agents which is environmental-friendly could be an attractive solution to overcome the problem. Earlier, we had isolated a mycoparasite, Scytalidium parasiticum, from the basidiomata of Ganoderma boninense. In vitro assay and nursery experiment showed that this fungus could suppress Ganoderma infection and reduce disease severity. However, metabolites which might contribute to the antagonistic or mycoparasitic effect remain unknown. In the current study, optimization of fungal sample processing, extraction, and analytical procedures were conducted to obtain metabolites from the maize substrate colonized by mycoparasitic ascomycetous Scytalidium parasiticum. This technique capable of producing sexual spores in sac-like organs. Untargeted metabolomics profiling was carried out by using Liquid Chromatography Time of Flight Mass Spectrometry (LC-ToF-MS). We found that S. parasiticum in both liquid- and solid-state cultivation gave higher metabolite when extracted with 60% methanol with 1% formic acid in combination with homogenisation methods such as ultrasonication and grinding. The findings from this study are useful for optimisation of metabolite extraction from other fungi-Ganoderma-plant interactions.

Soil pollution is rampant in recent years, attributed mainly to various anthropogenic activities. The pollutants, organic or inorganic in nature, reside in the soils and accumulate to levels exceeding permissible safe levels, posing hazards to living organisms. As a result, soils can harbour various pollutants such as polycyclic aromatic hydrocarbons (PAHs), persistent organic pollutants (chlorinated and brominated aromatic substances, organochlorine-based pesticides, and dioxins), heavy metals, and metalloids. And often, soils that are heavily contaminated are agricultural soils due to the extensive use of fertilizer and pesticides resulting in residual accumulation of heavy metals, PAHs, and dichlorodiphenyltrichloroethane. Conventional approaches to soil remediation include physicochemical means that is costly, laborious, and non-sustainable. In response, biological means is sought as an alternative due to its environmental-friendly, better sustainability, and cost-effective traits. The biological remediation utilizes plants (phytoremediation) or the typical soil microflora (microbial remediation). In this chapter, the alternative of exploring microbial biocontrol agents for soil remediation is proposed. Microbial biocontrol agents are often introduced into the soil to suppress disease development or to enhance growth of plants. They are introduced into various agricultural soils which may harbour pollutants. Biocontrol agents have metal-chelating and nutrient-solubilizing properties. This is further escalated by the discovery of metal-tolerant species as biocontrol agents. As such, this approach promotes the use of biocontrol agents as bioremediation agents as well, to manage disease and bioremediate soils at the same time. This chapter will describe microbial biocontrol agents and their potential, mechanisms, and applications for the remediation of polluted soils.

In solving the issue of basal stem rot diseases caused by Ganoderma, an investigation of Scytalidium parasiticum as a biological control agent that suppresses Ganoderma infection has gained our interest, as it is more environmentally friendly. Recently, the fungal co-cultivation has emerged as a promising method to discover novel antimicrobial metabolites. In this study, an established technique of co-culturing Scytalidium parasiticum and Ganoderma boninense was applied to produce and induce metabolites that have antifungal activity against G. boninense. The crude extract from the co-culture media was applied to a High Performance Liquid Chromatography (HPLC) preparative column to isolate the bioactive compounds, which were tested against G. boninense. The fractions that showed inhibition against G. boninense were sent for a Liquid Chromatography-Time of Flight-Mass Spectrometry (LC-TOF-MS) analysis to further identify the compounds that were responsible for the microbicidal activity. Interestingly, we found that eudistomin I, naringenin 7-O-beta-D-glucoside and penipanoid A, which were present in different abundances in all the active fractions, except in the control, could be the antimicrobial metabolites. In addition, the abundance of fatty acids, such as oleic acid and stearamide in the active fraction, also enhanced the antimicrobial activity. This comprehensive metabolomics study could be used as the basis for isolating biocontrol compounds to be applied in oil palm fields to combat a Ganoderma infection.

Basal stem rot (BSR) disease caused by Ganoderma boninense basidiomycetous fungus is the most economically important disease in oil palms in South East Asia. Unfortunately, there is no single most effective control measure available. Tremendous efforts have been directed in incorporation of environmentally friendly biocontrol approaches in minimizing BSR disease. This study investigated the performance of two potential biocontrol agents (BCAs), AAT0115 and AAB0114 strains recovered from oil palm on suppression of BSR in planta, and also assessed their plant-growth-promoting (PGP) performance. ITS rRNA-sequence phylogeny discriminated the two ascomycetous Talaromyces apiculatus (Ta) AT0115 and Clonostachys rosea (Cr) AAB0114 biocontrol species with PGP characteristics. In vitro studies have demonstrated both Ta and Cr are capable of reducing linear mycelial growth of G. boninense. Inoculation of individual Cr and Ta&mdash, as well as Cr+Ta consortium&mdash, induced a significant increment in leaf area and bole girth of oil-palm seedlings five months post-inoculation (MPI) under nursery conditions. At five months post-inoculation, shoot and root biomass, and nutrient contents (nitrogen, phosphorus, potassium, calcium, magnesium and boron) were significantly higher in Ta-inoculated seedlings compared to control treated with non-Ta-inoculated maize. Chlorophyll and carotenoids contents in rapidly growing oil-palm seedlings challenged with Cr, Ta or a combination of both were not negatively affected. Cr, Ta and Cr+Ta consortium treated seedlings had 4.9&ndash, 60% BSR disease reduction compared to the untreated control. Co-inoculation of Cr and Ta resulted in increased BSR control efficiencies by 18&ndash, 26% (compared with Cr only) and 48&ndash, 55% (compared with Ta only). Collectively, Cr and Ta, either individually or in consortium showed potential as BSR biocontrol agents while also possess PGP traits in oil palm.

Blenheim soil (Typic Quartzipsamment) and Bernam soil (Typic Endoaquept) with low and high Ganoderma basal stem rot (BSR) incidence, respectively, were studied to determine the role of naturally-occurring microbiota in suppressing basal stem rot (BSR) disease in oil palm. These coastal soils were also compared against a typical soil for nursery use, the inland Bungor soil (Typic Kandiudults). For each soil type, half of the soils were sterilized by autoclaving, and the other half were non-sterilized. These soils were then used for the microbiome analysis, evaluation of disease incidences, and effect on plant growth. Soil microbial communities were profiled using 16S and 18S ribosomal RNA gene amplicon sequencing on the Illumina MiSeq. Polybag trials were conducted over a period of 20 weeks. Results showed that by eliminating or inactivating the indigenous microbial communities from BSR-suppressive Blenheim soil through sterilization, this increased BSR incidences (from 50 to 92%) and severity (from 28 to 78%), and affected plant growth (reduction of 35% leaf area, 11% height, and 19% bole’s girth). Soil microbiome analysis revealed that Acidobacteriota, Nitrospirota, Latesbaciterota, Nanoarchaeota, Entotheonellaeta, Zixibacteria, Candidatus Yanofskybacteria (Patescibacteria), MB-A2-108 (Actinobacteriota), Cnidaria and Acaulospora (Mucoromycota) were less prevalent in sterilized Blenheim soil compared to non-sterilized Blenheim soil. Relative abundance of the genus Ganoderma was also lower in BSR-suppressive Blenheim soil compared to sterilized Blenheim soil. In contrast, disease development and plant growth remained unaffected by sterilization of BSR-conducive Bernam and Bungor soils, suggesting that naturally-occurring microbiota in these two soils played minimal roles in contributing to conducive conditions for disease development. Relative abundance of Ganoderma was higher in BSR-conducive Bernam and Bungor soils as well, compared to BSR-suppressive Blenheim. Results from this study provides the first insights into the soil microbiota in BSR suppressive soils and their role in BSR disease progression and growth of oil palms.

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. ...

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.

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 ...

The influence of different medium components (glucose, sucrose, and fructose) on the growth of different Ganoderma isolates and species was investigated using mixture design. Ten sugar combinations based on three simple sugars were generated with two different concentrations, namely 3.3% and 16.7%, which represented low and high sugar levels, respectively. The media were adjusted to either pH 5 or 8. Ganoderma isolates (two G. boninense from oil palm, one Ganoderma species from coconut palm, G. lingzhi, and G. australe from tower tree) grew faster at pH 8. Ganoderma lingzhi proliferated at the slowest rate compared to all other tested Ganoderma species in all the media studied. However, G. boninense isolates grew the fastest. Different Ganoderma species were found to have different sugar preferences. This study illustrated that the mixture design can be used to determine the optimal combinations of sugar or other nutrient/chemical components of media for fungal growth.

Sphaerodes mycoparasitica, a biotrophic mycoparasite of Fusarium species, improved wheat seed germination and seedling growth in vitro compared to Trichoderma harzianum, a necrotrophic mycoparasite. However, under phytotron conditions, both S. mycoparasitica and T. harzianum had positive impact on wheat seedlings growth in the presence of F. graminearum. Once exposed to the mycoparasites, the DNA quantity of F. graminearum in wheat root decreased. Observed shifts in DNA quantity using qPCR, a set of newly designed Sphaerodes-specific SmyITS primers, as well as Trichoderma-TGP4 and Fusarium-Fg16 N primers, demonstrated the mycoparasite's biocontrol effectiveness in planta. In the presence of F. graminearum, the concentration of S. mycoparasitica DNA remained stable in the root, whereas the amount of T. harzianum DNA decreased. The toxicity assays indicated that S. mycoparasitica's mycelia withstand higher concentrations of deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone mycotoxins than T. harzianum mycelia. This study compares the ability of two fungi to improve the wheat growth, decrease the root colonization of Fusarium, and withstand mycotoxins.

Fusarium graminearum Schwabe is the primary cause of Fusarium head blight (FHB) in North America. Chemically distinct F. graminearum sub-populations can be identified based on the type or composition of deoxynivalenol (DON) mycotoxin derivatives, including 3-acetyl (3-ADON) and 15-acetyl (15-ADON). The evaluation of randomly selected 3-ADON and 15-ADON isolates, collected from spring wheat throughout Canada, was performed using thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), ice-nucleation activity (INA), and heat and cold tolerance tests conducted within a temperature range of -70°C to 65°C. The results indicated that the 3-ADON sub-population, which is responsible for the highest disease severity and has rapidly displaced the 15-ADON sub-population, produces more DON and zearalenone (ZEA) than the 15-ADON sub-population when exposed to heat and cold. Following exposures (1 and 2 h) to extremely high or low temperatures, 3-ADON isolates exhibited faster mycelial growth than 15-ADON isolates. In addition, the warmest temperature at which INA activity occurred was in 3-ADON (-3.6°C) vs. 15-ADON (-5.1°C). Taken together, these features suggest that the newly emerging 3-ADON sub-population is more resilient than the resident 15-ADON sub-population. Overall, the differences between the two sub-populations could provide new insights into FHB epidemiology and if validated under field conditions, may provide important information for predicting future FHB epidemics.

Sucrose-water medium induced a high rate of macroconidia formation in Fusarium graminearum 3- and 15-acetyldeoxynivalenol (ADON) chemotypes. The F. graminearum isolates tested produced uniform macroconidia in large quantities and within a shorter incubation period in sucrose-water medium compared to other assessed media. At the same time, high rates of mycotoxin accumulation (trichothecenes: DON, 3-ADON, 15-ADON, and zearalenone: ZEA) in macroconidia were detected using high performance liquid chromatography. The proposed “saccharose-water” method represents a valuable alternative for conidia production in mycotoxigenic F. graminearum isolates, having advantages over the potentially mutagenic UV light, and potato-dextrose agar, which can degenerate macroconidia, and over expensive chemical compounds or natural substances for which the preparation protocols and handling procedures are relatively complex or time-consuming.

Fusarium spp. are economically important crop pathogens and causal agents of Fusarium head blight (FHB) of cereals worldwide. Of the FHB pathogens, Fusarium graminearum 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) are the most aggressive mycotoxigenic chemotypes, threatening food and feed quality as well as animal and human health. The objective of the study was to evaluate host specificity and fungal-fungal interactions of Sphaerodes mycoparasitica- a recently described mycoparasite - with F. graminearum 3- and 15-ADON strains by employing in vitro, microscopic and PCR techniques. Results obtained in this study show that the germination of mycoparasite ascospore in the presence of F. graminearum 3- and 15-ADON filtrates was greatly improved compared with Fusarium proliferatum and Fusarium sporotrichioides filtrates, suggesting a compatible interaction. Using quantitative real-time PCR with Fusarium-specific (Fg16N) and trichothecene Tri5 (Tox5-1/2)-specific primer sets, S. mycoparasitica was found to reduce the amount of F. graminearum 3-ADON and 15-ADON DNAs under separate coinoculation assays. Sphaerodes mycoparasitica was not only able to germinate in the presence of F. graminearum filtrates, but also established biotrophic mycoparasitic relations with two F. graminearum chemotypes and suppressed Fusarium growth.

Sphaerodes mycoparasitica is an ascomycetous mycoparasitic taxon commonly found growing and sporulating on Fusarium pathogens in Canada. Ascospores of S. mycoparasitica failed to germinate under different standard laboratory conditions (sterile distilled water, water agar, and commercially available media), even after heat- and cold-shock treatments. In contrast, spore germination was obtained on commercial potato dextrose agar medium amended with Fusarium filtrates. Significant improvement in the percentage of spore germination was obtained for the spores suspended in Fusarium filtrates. Fusarium avenaceum and Fusarium oxysporum filtrates induced the highest germination, whereas Fusarium sporotrichioides and Fusarium proliferatum triggered lower germination frequency. Filtrates of beneficial fungal inoculants, including Trichoderma harzianum (RootShield®), Penicillium bilaii (JumpStart®), and Chaetomium globosum , had no impact on germination. Ascospore suspensions showed a double-polar germination pattern in F. avenaceum filtrate and single-polar germination when challenged with F. oxysporum filtrate. Sphaerodes mycoparasitica grown on F. oxysporum maintained the same spore germination patterns when transferred on F. avenaceum for three subsequent offspring generations, indicating a stable genome-regulated expression. Preliminary phylogenetic analyses based on large subunit rDNA showed the evolution of a group of mycoparasitic Sphaerodes species from nonmycoparasitic ancestors, but no clear evidence of coevolution or cospeciation between known Sphaerodes and their Fusarium hosts.

Most natural and synthetic dyes currently used for microbial fluorescent staining are toxic or carcinogenic and are harmful to animals, humans and the environment. A food dye for microbial staining, brilliant blue FCF, was used as an alternative to lactofuchsin and lactophenol blue. Brilliant blue FCF shows pronounced microbial cell fluorescence staining of an array of pathogenic/toxigenic (Fusarium granunearum 3- and 15-acetyldeoxynivalenol chemotypes, and Escherichia coli O157:H7) and beneficial fungi and bacteria (Trichoderma harzianum and Bacillus subtilis). Brilliant blue FCF has no toxic effects on the microbes tested and is inexpensive.

The objective of this study was to investigate the relationships between the biotrophic mycoparasite Sphaerodes mycoparasitica and pathogenic Fusarium strains. To study the interactions between S. mycoparasitica and four different phytopathogenic Fusarium strains, macroscopic observations were performed using dual-culture assays and microscopic examinations in combination with light and fluorescent microscopy. Both macroscopic and microscopic techniques were also vital in determining the host specificity of S. mycoparasitica with F. avenaceum, F. oxysporum, F. proliferatum, and F. sporotrichioides. Our results suggest that S. mycoparasitica established haustorial contact with F. avenaceum and F. oxysporum. Data obtained from the dual-culture assay and parasitism interactions revealed that this newly described contact biotrophic mycoparasitic fungus was capable of reducing F. avenaceum and F. oxysporum linear growth and size of hyphal cells through infection and penetration.

Fungal surface hydrophobicity has many ecological functions and water contact angles measurement is a direct and simple approach for its characterization. The objective of this study was to evaluate if in-vitro growth conditions coupled with versatile image analysis allows for more accurate fungal contact angle measurements. Fungal cultures were grown on agar slide media and contact angles were measured utilizing a modified microscope and digital camera setup. Advanced imaging software was adopted for contact angle determination. Contact angles were observed in hydrophobic, hydrophilic and a newly created chronoamphiphilic class containing fungi taxa with changing surface hydrophobicity. Previous methods are unable to detect slight changes in hydrophobicity, which provide vital information of hydrophobicity expression patterns. Our method allows for easy and efficient characterization of hydrophobicity, minimizing disturbance to cultures and quantifying subtle variation in hydrophobicity.

A new species, Sphaerodes mycoparasitica (Ascomycetes, Melanosporales), was isolated from isolates of Fusarium avenaceum and Fusarium graminearum originating from wheat fields in Saskatchewan, and from Fusarium oxysporum originating from asparagus fields in Quebec, Canada. The species is characterized by a unique combination of ascospore size, shape (fusiform and triangular) and wall ornamentation (reticulate and smooth). Also, conidia are produced from simple phialides on the surface of ascoma peridial wall, on ascoma surrounding hyphae, and on irregularly branched conidiophores arising from hyphae. The closest relation of S. mycoparasitica is Sphaerodes quadrangularis, which has no detected anamorphic stage. The description of S. mycoparasitica, its phylogenetic position-based on DNA sequences of large subunit ribosomal RNA gene (LSU)-as well as a key for all known Sphaerodes species are provided.

Chlamydospores are vital asexual resting cells, which allow most of the Fusarium pathogenic strains to retain their longevity, thus ensuring survival of viable reproductive cells. This study suggested that both abiotic – extreme temperature and growth media, and biotic – antagonistic Bacillus amyloliquefaciens SMCD 518 and mycoparasititic Acremonium strictum SMCD 504 are natural stressors able to shift chlamydospores formation in Fusarium graminearum and F. sporotrichioides under in vitro conditions. In F. sporotrichioides, Minimal Conversion Media (MCM) with mannitol supplement induced high chlamydospore size, and chain abundance at optimal 21°C and extreme 37°C temperatures, respectively. F. graminearum showed low chlamydospore formation on MCM–mannitol, even when exposed to 37°C under prolonged 5 days incubation. Generally, F. sporotrichioides has higher chlamydospore abundance, longer chlamydospore chain, and production rapidity compared to F. graminearum in both abiotic and biotic treatments...

The objective of the study was to assess the effect of ciprofloxacin antibiotic on the physiological or phenotypic characteristics of food-borne toxigenic Fusarium graminearum and F. avenaceum molds under in vitro conditions. In the presence of ciprofloxacin, Fusarium mycelia growth and morphology were altered based on the antibiotic concentration range used. Results showed that ciprofloxacin in concentrations ≥40 μg/mL induced chlamydospore formation in Fusaria and as such, this antibiotic should be considered as an important abiotic stress factor and growth inhibitor. A novel method was investigated to correlate chlamydospore formation with the colour changes observed in FD&C Green Number 3, a common water soluble food dye. The antibiotic-treated F. graminearum and F. avenaceum isolates produced chamydospores, which in turn altered environmental pH with concomitant changes in the colour and intensity of the dye. The colour changes observed as a function of environmental pH were supported by instrumental methods (pH meter and spectroscopy), and a commercial pH indicator (thymol blue) results. In conclusion, we propose that FD&C Green Number 3 can be used as an accurate indicator for the rapid assessment of Fusarium molds when grown on ciprofloxacin antibiotic-containing substrate. Special emphasis should be given to an indirect risk assessment of antibiotic effects on toxic molds.

Sphaerodes quadrangularis Garcia, Stchigel & Guarro was shown to be a facultative contact biotrophic mycoparasite of Fusarium avenaceum (Fr.) Sacc. (teleomorph: Gibberella avenacea Cooke) that established an intimate relationship with its host by producing hook-shaped and clamp-like attachment structures that appeared to derive nutrients and other essential factors from living host cells. The contact cells of the mycoparasite surrounded and envelop host hyphae and, although they do not appear to penetrate the host, the parasitized hyphae were significantly smaller than unparasitized controls. Sphaerodes quadrangularis did not form specialized attachment structures when cultured together with strains representing F. oxysporum and F. graminearum, indicating some degree of host specificity in the parasite.

Aim: To determine whether assessing the penetration of solutions with different concentrations of ethanol (alcohol percentage test: APT) on fungal surfaces is effective in characterization of hydrophobicity on fungal surfaces. Methods and Results: APT and contact angle (CA) measurements were conducted on nine hydrophobic and two hydrophilic fungal strains from the phyla of Ascomycota, Basidiomycota and Zygomycota. There was a strong positive correlation (R2 = 0·95) between the APT and CA measurements from eight of the nine hydrophobic stains (four pathogenic and mycotoxigenic Fusarium taxa, one melanosporaceous biotrophic taxon, Alternaria sp, Penicillium aurantiogriseum and Cladosporium cladosporioides). Hydrophilic control strains, Mortierella hyalina and Laccaria laccata, had CAs