In Vitro Assessment of Organic and Residual Fractions of Nematicidal Culture Filtrates from Thirteen Tropical Trichoderma Strains and Metabolic Profiles of Most-Active

The nematicidal properties of Trichoderma species have potential for developing safer biocontrol agents. In the present study, 13 native Trichoderma strains from T. citrinoviride, T. ghanense (2 strains), T. harzianum (4), T. koningiopsis, T. simmonsii, and T. virens (4) with nematicidal activity were selected and cultured in potato dextrose broth to obtain a culture filtrate (CF) for each. Each CF was partitioned with ethyl acetate to obtain organic (EA) and residual filtrate (RF) fractions, which were then tested on second-stage juveniles (J2s) of the nematodes Meloidogyne javanica and M. incognita in a microdilution assay. The most lethal strains were T. harzianum Th43-14, T. koningiopsis Th41-11, T. ghanense Th02-04, and T. virens Th32-09, which caused 51–100% mortality (%M) of J2s of both nematodes, mainly due to their RF fractions. Liquid chromatography–diode array detector-electrospray-high resolution mass spectrometry analysis of the most-active fractions revealed sesquiterpene and polyketide-like metabolites produced by the four active strains. These native Trichoderma strains have a high potential to develop safer natural products for the biocontrol of Meloidogyne species.

Transcriptomic Analysis of the White-rot Basidiomycete Lentinus Squarrosulus to Provide Insights Into Its Lignocellulose Biodegradation Ability

BackgroundBasidiomycetes are of special interest in biotechnological research for their versatile potential in the degradation of lignocellulosic biomass, chiefly attributed to ligninolytic enzymes along with exo, endo β-glucanases, xylanases, esterases, pectinases, mannanases, cellobiohydrolases, polysaccharide monooxygenases. Relatively little is known about the metabolic process and the subsequent polysaccharide degradation. Transcriptomic analysis of lignicolous fungi grown on different substrates, although attempted by researchers, has focused on a fairly small group of species reporting the expression of fungal genes in response to lignocellulosic biomass as a substrate. This study accordingly reports analysis of transcriptome of a white-rot Basidiomycete L.squarrosulus grown in simple potato dextrose broth supplemented with aromatic compound, reactive black dye to gain an insight into the degradation ability of the fungus. RNA was sequenced using Illumina NextSeq 500 to obtain 6,679,162 high-quality paired-end reads that were assembled de novo using CLC assembly cells to generate 25,244 contigs. Putative functions were assigned for the 10,494 transcripts based on sequence similarities through BLAST2GO 5.2 and Function annotator.ResultsFunctional assignments revealed enhanced oxidoreductase activity through the expression of diverse biomass-degrading enzymes and their corresponding coregulators. CAZyme analysis through dbCAN and CUPP revealed the presence of 6 families of polysaccharide lyases, 51 families of glycoside hydrolases, 23 families of glycoside transferases, 7 families of carbohydrate esterases and 10 families of auxiliary activities. Genes encoding ligninolytic enzymes and auxiliary activities among the transcript sequences were identified through gene prediction by AUGUSTUS and FGENESH. Biochemical analysis of several biomass-degrading enzymes substantiated the functional predictions.ConclusionIn essence, L. squarrosulus grown in a simple medium devoid of lignocellulosic substrate demonstrated the presence of a repertoire of lignocellulose-degrading enzymes, simplying that a source of lignocellulose is not required for the expression of these biomass-degrading enzymes. This study on the transcriptome analysis of L. squarrosulus revealed significant facts on this front and will definitely enhance the knowledge about the biodegradative ability of this fungus, potentially paving the way for efficient biotechnological applications utilizing its potency in biomass degradation and its future functional exploitation in biomass conversion applications.

Orange-Brown Pigment Production from an Endophytic Fungus Aspergillus Sp. N11 and its Potential Pharmaceutical Applications

Endophytic fungi are the main source of natural compounds including pigments having various industrial applications. Present study describes the production of extracellular orange-brown pigment from an endophytic fungal isolate Aspergillus sp. N11from Teucrium stocksianum. The optimum conditions for pigment production from this isolate was investigated and results showed that highest yield was observed in Potato dextrose broth, at pH 5 and 30 ℃ under shaking condition at 150 rpm for 7-10 days. The pigment was extracted in ethyl acetate and purified using column chromatography. Three different pigments were purified (yellow, light brown and orange-brown) and characterized based on Thin layer chromatography and Fourier transform infrared spectroscopy. The antimicrobial activity of purified fragments showed maximum zone of inhibition of 40 mm against S. aureus while for P. aeruginosa maximum zone of 50 mm and maximum antifungal activity of 20 mm against C. albicans. The antioxidant potential of purified pigment obtained from Aspergillus sp. N11 indicates that maximum scavenging activity of 67%. The results showed that purified pigments are astaxanthins belonging to oxygen containing carotenoids. The purified astaxanthins showed antibacterial, antifungal and antioxidant activities indicating its potential to be utilized in pharmaceutical and food industries.

First report of Macrophomina phaseolina causing charcoal root rot of Hebe (Veronica cupressoides, V. ochracea, and V. pinguifolia) in Oregon, U.S.A.

Hebes (Veronica spp. in the section Hebe) are ornamental perennials and shrubs grown for their flowers and symmetric, evergreen leaves. They are uncommon in U.S. horticulture and are only produced by a few nurseries regionally (Oregon and Washington). In June, July, and August (2016 to 2021), stems on 1 to 5-year-old Veronica cupressoides, V. ochracea, and V. pinguifolia in five landscape plantings around Benton County, OR (17 plants total, locations 2 to 37 km apart) began to wilt, turn brown, and die. At least nine of the plants originated from a single nursery. Initially, just one or two stems/plant were affected, but eventually the entire plant died. Stem tissues were discolored brown to black internally and the roots were dry and necrotic. Leaves turned brown and brittle, but remained attached. Stems from each plant were disinfested in 0.5% NaOCl (1 min), rinsed in 70% ethanol, and dried (2 min). Pieces (5 mm2) were then plated onto 1/2 strength potato dextrose agar amended with streptomycin (50 mg/liter) and incubated in the dark at 20°C. Three to five days later, greyish-white cultures producing black microsclerotia (75 × 110 µm, n = 50) grew out of all samples. No spores were produced. All isolates were identified as Macrophomina phaseolina by morphology and by ≥99% homology (566-570/571 nt) to the internal transcribed spacer sequence (primers ITS1 and ITS4) from the type specimen (GenBank KF766195) (Hyde et al. 2014). Three representative sequences were deposited in GenBank (MZ726450 to MZ726452). Inoculum was prepared from these isolates by growing cultures in 250 ml of potato dextrose broth on a shaker (125 rpm at 25°C). After 2 weeks, the broth was decanted and the fungal biomass was air dried for 3 days at 25°C before grinding into a powder with a mortar and pestle. Three plants each of 6-month-old V. ochracea 'James Stirling', V. cupressoides 'McKean', and V. pinguifolia 'Sutherlandii' were inoculated with each isolate by rinsing the soil off of the roots with tap water, trimming off 0.5 cm of the roots, and then soaking the rootball in a slurry of 1 g dried inoculum in 500 ml of 0.2% water agar (WA) for 10 minutes (Reyes Gaige et al. 2010). Three plants of each species that were soaked in plain 0.2% WA served as negative controls. Afterwards, plants were potted into soilless media (Metro-Mix 840, Sun Gro Horticulture, Agawam, MA) in 3.5 inch square pots and arranged in a completely randomized design in a greenhouse set at 28/24°C day/night. The experiment was conducted three times. One to three months later, inoculated plants began to turn yellow, wilt, and die whereas all control plants remained healthy. The same pathogen was reisolated from 90% of the inoculated plants, but never from negative controls. M. phaseolina was reported on strawberry in southern Oregon in 2014 (Pscheidt and Ocamb 2021), but has not been reported from locations further north in the state where soil temperatures are cooler. It is unusual that M. phaseolina was isolated from an uncommon host at five different locations in an area of the state where the pathogen was not known to occur. Based on this, and on the number of infected plants originating from a single source, it seems likely that M. phaseolina was accidentally spread on contaminated plants produced by the nursery industry, where the warmer temperatures in production greenhouses would provide a more conducive environment for the pathogen's growth and spread. Growers should keep watch for symptoms of this pathogen in their nurseries.

POTENTIAL OF ENDOPHYTIC FUNGI DERIVING FROM ASIATIC PENNYWORTH TO PRODUCE ANTIOXIDANTS

<p>Asiatic pennyworth is a medicinal plant that contains triterpenoids, saponin, flavonoids, and tannins which possess antioxidants. Endophytic fungi from the plant could produce a similar compound; therefore, antioxidants could be made in the laboratory if the fungi are isolated. This study aimed to evaluate the potential of endophytic fungi isolated from Asiatic pennyworth to produce antioxidants. The study used 34 endophytic fungal isolates from Asiatic pennyworth accessions of Malaysia (17 isolates) and Bengkulu, Indonesia (17 isolates) collected by the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development. The fungi were propagated in a potato dextrose broth medium, then mycelia mats and filtrates were separated and then freeze-dried. The antioxidant activities were first tested with 1,1-diphenyl-2- picrylhydrazyl (DPPH) using thin layer chromatography (TLC), then UV-Vis spectrophotometry λ517 nm with five variations of concentration. Results showed all 34 fungal isolates have antioxidant activities based on a yellowish-white color change after applying 0.002% DPPH solution of the TLC method and IC50 value of the UV-Vis spectrophotometer. The highest antioxidant activity was shown by <em>Aspergillus austroafricanus </em>MB 1 (IC50 = 12.08 ppm) from Bengkulu accession and <em>A. oryzae </em>MM 13 (IC50 = 10.29 ppm) from Malaysia accession. <em>A. austroafricanus </em>MB 1 produced more antioxidant compounds (seven) than <em>A. oryzae </em>MM 13 (six). The antioxidant compounds produced by both endophytic fungi included in the group of flavonoids, fatty acids, and carboxylic acids. The research implies that <em>A. austroafricanus </em>MB 1 and <em>A. oryzae </em>MM 13 could be further developed as sources of antioxidants.</p>

Biochemical Profile by GC–MS of Fungal Biomass Produced from the Ascospores of Tirmania nivea as a Natural Renewable Resource

The edible fruiting bodies of desert truffles are seasonally collected and consumed in many regions of the world. Although they are very expensive, they are bought and sold as a result of considerable scientific reports confirming their health and nutritional benefits. This study aimed to conduct laboratory production of the fungal biomass of Tirmania nivea as a natural renewable resource of many active biological compounds using an artificial growth medium. The T. nivea collected from Hafar Al-Batin, which is north of Saudi Arabia, and their ascospores were harvested and used to produce fungal biomass in potato dextrose broth. The cultivation was conducted using a shaking incubator at 25 °C for two weeks at 200 rpm. The crud extracts of the fungal biomass and mycelium-free broth were prepared using ethyl acetate, methanol and hexane. Preliminary gas chromatography–mass spectrometry (GC–MS) analysis and their biological activity as antimicrobial agents were investigated. The results showed that the crude extracts have biological activity against mold, yeast and bacteria. The preliminary GC–MS analysis reported that the fungal biomass and extracellular metabolites in the growth medium are industrial renewable resources of several biological compounds that could be used as antifungal, antibacterial, antiviral, anticancer, antioxidant, anti-trypanosomal and anti-inflammatory agents.

Diagnosis and Inhibition of the Virulence Factor Phaseolenone of the Pathogenic Fungus Macrophomina phaseolina Using Some Chemical and Biological Methods

Al-Jbory, A.A.A. and A.A Hasan. 2021. Diagnosis and Inhibition of the Virulence Factor Phaseolenone of the Pathogenic Fungus Macrophomina phaseolina Using Some Chemical and Biological Methods. Arab Journal of Plant Protection, 39(4): 252-256. https://doi.org/10.22268/AJPP-039.4.252256 Results obtained in this study confirmed the efficacy of some chemical and biological factors in reducing the concentration of Phaseolenone toxin produced by the fungal pathogen Macrophomina phaseolina grown on potato dextrose broth medium. The toxin concentrations were measured in different treatments using high performance liquid chromatography (HPLC). The citric acid treatment at 5% concentration surpassed the other treatments (lactic acid, acetic acid, SDS salts, EDTA, bacterial filtrates of Pseudomonas fluorescens and Bacillus subtilis). Citric acid treatment reduced by 5% the toxin concentration to 1.74 microgram/ml compared to 6.94 microgram/ml for the control treatment. This reduction in Phaseolenone concentration was also noticed through the reduction of the area under the curve which amounted to 1.684% for the citric acid treatment compared with 6.814% for the control treatment. Keywords: Phaseolenone, Macrophomina Phaseolina, charcoal rot, mycotoxin.

Nematicidal Activity of Cyclopiazonic Acid Derived From Penicillium commune Against Root-Knot Nematodes and Optimization of the Culture Fermentation Process

Among 200 fungal strains isolated from the soil, only one culture filtrate of Aspergillus flavus JCK-4087 showed strong nematicidal activity against Meloidogyne incognita. The nematicidal metabolite isolated from the culture filtrate of JCK-4087 was identified as cyclopiazonic acid (CPA). Because JCK-4087 also produced aflatoxins, six strains of Penicillium commune, which have been reported to be CPA producers, were obtained from the bank and then tested for their CPA productivity. CPA was isolated from the culture filtrate of P. commune KACC 45973. CPA killed the second-stage juveniles of M. incognita, M. hapla, and M. arearia with EC50–3 days 4.50, 18.82, and 60.51 μg mL–1, respectively. CPA also significantly inhibited egg hatch of M. incognita and M. hapla after a total of 28 days of treatment with the concentrations &gt; 25 μg mL–1. The enhancement of CPA production by P. commune KACC 45973 was explored using an optimized medium based on Plackett–Burman design (PBD) and central composite design (CCD). The highest CPA production (381.48 μg mL–1) was obtained from the optimized medium, exhibiting an increase of 7.88 times when compared with that from potato dextrose broth culture. Application of the wettable power-type formulation of the ethyl acetate extract of the culture filtrate of KACC 45973 reduced gall formation and nematode populations in tomato roots and soils under greenhouse conditions. These results suggest that CPA produced by P. commune KACC 45973 can be used as either a biochemical nematicide or a lead molecule for developing chemical nematicides to control root-knot nematodes.

Growth and Characteristics of Two Different Epichloë sinensis Strains Under Different Cultures

In the present study, two Epichloë sinensis endophyte strains isolated from different Festuca sinensis ecotypes were inoculated on potato dextrose agar (PDA) and potato dextrose broth (PDB) media with or without (control) exogenous additives. After 4weeks of growth, the growth (colony diameter, hyphal diameter, and mycelial biomass) and other characteristics (pH and antioxidant capacity of culture filtrate, mycelial ion contents, and hormone contents) were measured. The results showed that the culture conditions had significant effects (p&lt;0.05) on the hyphal diameter, mycelial biomass, and hormone content of the two strains. The mycelial biomass of the two strains in PDB was significantly higher (p&lt;0.05) than that on PDA. Except for strain 1 with indole-3-acetic acid (IAA) treatment and strain 84F with control and VB1 treatments, the hyphal diameter of the two strains in PDB under the other treatments was significantly higher (p&lt;0.05) than that on PDA. In most cases, the IAA, cytokinins (CTK), abscisic acid (ABA), and gibberlic acid (GA) contents in the mycelia on PDA of the two strains were significantly higher (p&lt;0.05) than those in PDB. The two E. sinensis strains exhibited significantly different performances (p&lt;0.05) under the five treatments. The indices, including colony diameter, mycelial biomass, scavenging ability of superoxide anion radicals and hydroxyl radicals, pH of culture filtrate, ion contents, hyphal diameter, and IAA, CTK, GA, and ABA contents were significantly different (p&lt;0.05) between the two strains, although the performance was inconsistent. Exogenous additives had significant effects (p&lt;0.05) on the performance of the two E. sinensis strains. Indole-3-acetic acid and VB1 treatments significantly promoted (p&lt;0.05) the growth of the two strains on both PDA and PDB. Indole-3-acetic acid treatment also significantly increased the hyphal diameters of the two strains in PDB (p&lt;0.05). Indole-3-acetic acid and VB1 treatments significantly reduced (p&lt;0.05) the antioxidant ability of these two strains in PDB. NaCl and ZnCl2 treatments had significant inhibitory effects (p&lt;0.05) on fungal growth and promotion effects on the antioxidant ability of the two strains. The treatments also had significant effects (p&lt;0.05) on hyphal diameters and ion and hormone contents, although the effects varied with different indices.

Anti-Fungal Effects 0f Ambon Banana Skin Extract (Musa paradisiaca Linn. Var. Sapientum) on Candida albicans ATCC® 10231 ™ (In Vitro)

Introduction: Disease is a global problem for health. One of the most common infectious diseases is a disease caused by fungi (mycosis), a species of Candida (candidiasis) caused by Candida albicans. These bananas are generally often consumed daily by Indonesians. Ambon banana skin extract (Musa Paradisiaca Linn. Var.Sapientum) is a nutritious medicinal plant, because Ambon banana skin waste has active antifungal compounds, namely tannins, flavonoids, quinones, phenols, and steroids. Which can damage fungal cell wall membrane proteins, damage the DNA chain causing brittle cell walls resulting in fungal cell death. Objectives: Research to determine the zone of inhibition, MIC and MBC of Ambon banana skin extract at concentrations of 100%, 90%, 80%, 70%, 60%, and 50% against Candida albicans ATCC® 10231™. Method: Laboratory experimental research with a post test only control group research design, the sample used was 18 samples, tested with the Anova test. In determining the inhibition zone using the well diffusion method, the measurement of the inhibition zone uses the calipers, MIC and MBC using the dilution method. Ambon banana skin was extracted by maceration method using 70% ethanol solvent, carried out 6 treatments with various concentrations. Each treatment was repeated 3 times. Results: The inhibition zone research at concentrations of 100%, 90%, 80%, 70%, 60%, and 50% with an average of 13.2mm 11.6mm 11.3mm, 10.5mm, 9.9mm, 8.3mm. The MIC of each concentration was clear and equalized according to the standard of 0.5 McFarland (1.5 x 108 CFU / ml) with potato dextrose broth medium, and the MBC was emphasized with potato dextrose agar (PDA) medium. Conclusion Inhibition zone concentrations of 100% -70% indicate strong criteria, 60-50% including weak criteria. MIC obtained at a concentration of 50%, MBC at a concentration of 60%. In this study, using a concentration of 70% indicates an average diameter of above 10mm, meaning that it is in accordance with the David and Stout method which states that the criteria are strong at an average of 10-20 mm. a concentration of 70% is used because it is effective in killing Candida albicans ATCC® 10231™, 100% -80% has a high toxicity which can cause toxic effects on all organisms such as the body, fungi, plants, etc.