Metabolic Profile of Scytalidium parasiticum-Ganoderma boninense Co-Cultures Revealed the Alkaloids, Flavonoids and Fatty Acids that Contribute to Anti-Ganoderma Activity.

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.

Determining Soil Microbial Communities and Their Influence on Ganoderma Disease Incidences in Oil Palm (Elaeis guineensis) via High-Throughput Sequencing.

Basal stem rot (BSR), caused by Ganoderma boninense, is the most devastating oil palm disease in South East Asia, costing US$500 million annually. Various soil physicochemical parameters have been associated with an increase in BSR incidences. However, very little attention has been directed to understanding the relationship between soil microbiome and BSR incidence in oil palm fields. The prokaryotic and eukaryotic microbial diversities of two coastal soils, Blenheim soil (Typic Quartzipsamment-calcareous shell deposits, light texture) with low disease incidence (1.9%) and Bernam soil (Typic Endoaquept-non-acid sulfate) with high disease incidence (33.1%), were determined using the 16S (V3-V4 region) and 18S (V9 region) rRNA amplicon sequencing. Soil physicochemical properties (pH, electrical conductivity, soil organic matter, nitrogen, phosphorus, cation exchange capacity, exchangeable cations, micronutrients, and soil physical parameters) were also analyzed for the two coastal soils. Results revealed that Blenheim soil comprises higher prokaryotic and eukaryotic diversities, accompanied by higher pH and calcium content. Blenheim soil was observed to have a higher relative abundance of bacterial taxa associated with disease suppression such as Calditrichaeota, Zixibacteria, GAL15, Omnitrophicaeota, Rokubacteria, AKYG587 (Planctomycetes), JdFR-76 (Calditrichaeota), and Rubrobacter (Actinobacteria). In contrast, Bernam soil had a higher proportion of other bacterial taxa, Chloroflexi and Acidothermus (Actinobacteria). Cercomonas (Cercozoa) and Calcarisporiella (Ascomycota) were eukaryotes that are abundant in Blenheim soil, while Uronema (Ciliophora) and mammals were present in higher abundance in Bernam soil. Some of the bacterial taxa have been reported previously in disease-suppressive and -conducive soils as potential disease-suppressive or disease-inducible bacteria. Furthermore, Cercomonas was reported previously as potential bacterivorous flagellates involved in the selection of highly toxic biocontrol bacteria, which might contribute to disease suppression indirectly. The results from this study may provide valuable information related to soil microbial community structures and their association with soil characteristics and soil susceptibility to Ganoderma.

Biocontrol and Plant-Growth-Promoting Traits of Talaromyces apiculatus and Clonostachys rosea Consortium against Ganoderma Basal Stem Rot Disease of Oil Palm.

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-as well as Cr+Ta consortium-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 4.9-60% BSR disease reduction compared to the untreated control. Co-inoculation of Cr and Ta resulted in increased BSR control efficiencies by 18-26% (compared with Cr only) and 48-55% (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.

Experimental mixture design as a tool to optimize the growth of various Ganoderma species cultivated on media with different sugars.

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.

Scytalidium parasiticum sp. nov., a New Species Parasitizing on Ganoderma boninense Isolated from Oil Palm in Peninsular Malaysia.

A mycoparasite, Scytalidium parasiticum sp. nov., isolated from the basidiomata of Ganoderma boninense causing basal stem rot of oil palm in Johor, Malaysia, is described and illustrated. It is distinct from other Scytalidium species in having smaller asci and ascospores (teleomorphic stage), longer arthroconidia (anamorphic stage), hyaline to yellowish chlamydospores, and producing a fluorescent pigment. The phylogenetic position of S. parasiticum was determined by sequence analyses of the internal transcribed spacers and the small-subunit ribosomal RNA gene regions. A key to identify Scytalidium species with teleomorphic stage is provided.

Wetting properties of fungi mycelium alter soil infiltration and soil water repellency in a γ-sterilized wettable and repellent soil.

Soil water repellency (SWR) has a drastic impact on soil quality resulting in reduced infiltration, increased runoff, increased leaching, reduced plant growth, and increased soil erosion. One of the causes of SWR is hydrophobic fungal structures and exudates that change the soil-water relationship. The objective of this study was to determine whether SWR and infiltration could be manipulated through inoculation with fungi. The effect of fungi on SWR was investigated through inoculation of three fungal strains (hydrophilic -Fusarium proliferatum, chrono-amphiphilic -Trichoderma harzianum, and hydrophobic -Alternaria sp.) on a water repellent soil (WR-soil) and a wettable soil (W-soil). The change in SWR and infiltration was assessed by the water repellency index and cumulative infiltration respectively. F. proliferatum decreased the SWR on WR-soil and slightly increased SWR in W-soil, while Alternaria sp. increased SWR in both the W-soil and the WR-soil. Conversely T. harzianum increased the SWR in the W-soil and decreased the SWR in the WR-soil. All strains showed a decrease in infiltration in W-soil, while only the F. proliferatum and T. harzianum strain showed improvement in infiltration in the WR-soil. The ability of fungi to alter the SWR and enmesh soil particles results in changes to the infiltration dynamics in soil.

Natural fruit extracts as non-toxic fluorescent dyes for staining fungal chlamydospores.

Currently, most synthetic dyes utilized for fungal fluorescent staining are toxic, carcinogenic, or harmful to animals, humans, and the environment. This study proposes non-toxic extracts of fruits from the genera Rhamnus, Ribes, Sambucus, Viburnum, Sorbus and Beta as simple, safe, and ecological alternatives to chemical fluorescent dye for efficient staining of Fusarium chlamydospore cells using, as test strains, five different pathogenic Fusarium species.

qPCR quantification of Sphaerodes mycoparasitica biotrophic mycoparasite interaction with Fusarium graminearum: in vitro and in planta assays.

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.

Heat- and cold-shock responses in Fusarium graminearum 3 acetyl- and 15 acetyl-deoxynivalenol chemotypes.

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.

Rapid detection of ciprofloxacin effects on Fusarium graminearum and F. avenaceum cells in modulating environmental pH using a reactive, non-toxic food-dye indicator.

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 ≥40µg/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 mycoparasitica biotrophic mycoparasite of 3-acetyldeoxynivalenol- and 15-acetyldeoxynivalenol-producing toxigenic Fusarium graminearum chemotypes.

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 quadrangularis biotrophic mycoparasitism on Fusarium avenaceum.

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.

Sphaerodes mycoparasitica sp. nov., a new biotrophic mycoparasite on Fusarium avenaceum, F. graminearum and F. oxysporum.

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.

A novel method for identifying hydrophobicity on fungal surfaces.

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.