Incidence of fungal contamination in fresh ginseng samples and mycotoxigenic potential of representative fungal isolates.

BACKGROUND: Fresh ginseng is typically accompanied by soil after harvest, leading to contamination with harmful fungi during storage and distribution. In this study, we investigated the incidence of fungal contamination in fresh ginseng (5-6 years old) purchased from 22 different stores in Geumsan, Korea. RESULTS: The incidence of fungal contamination in the samples was 67.4-111.5%. Fusarium solani was the most abundant species in the head (38.5%) and fine root (19.3%) parts of the ginseng samples, whereas F. oxysporum was the most abundant in the main root (22.0%) part. We isolated Aspergillus, Fusarium and Penicillium spp. (total number of isolates: 395) from the ginseng samples, and 138 isolates were identified using phylogenetic analysis. Polymerase chain reaction-based screening of 65 mycotoxin-producing species revealed that two P. expansum isolates were positive for citrinin and/or patulin, and five F. oxysporum isolates were positive for fumonisin biosynthesis gene. One P. expansum isolate produced 738.0 mg kg-1 patulin, and the other produced 10.4 mg kg-1 citrinin and 12.0 mg kg-1 patulin on potato dextrose agar (PDA) medium. Among the 47 representative F. oxysporum isolates, 43 (91.5%) produced beauvericin (0.1-15.4 mg kg-1) and four of them (8.5%) produced enniatin B and enniatin B1 (0.1-1.8 mg kg-1) as well. However, none of these toxins was detected in fresh ginseng samples. CONCLUSION: Fusarium solani and F. oxysporum were the most abundant species in fresh ginseng samples. Most F. oxysporum (43) and P. expansum (2) strains isolated from fresh ginseng produced beauvericin and enniatins (B and B1), and patulin or citrinin, respectively, on PDA medium. This is the first report of the mycotoxigenic potential of P. expansum and F. oxysporum strains isolated from fresh ginseng. © 2024 Society of Chemical Industry.

Evaluation of Barley and Wheat Germplasm for Resistance to Head Blight and Mycotoxin Production by Fusarium asiaticum and F. graminearum.

Fusarium head blight (FHB) is one of the most serious diseases in barley and wheat, as it is usually accompanied by the production of harmful mycotoxins in the grains. To identify FHB-resistant breeding resources, we evaluated 60 elite germplasm accessions of barley (24) and wheat (36) for FHB and mycotoxin accumulation. Assessments were performed in a greenhouse and five heads per accession were inoculated with both Fusarium asiaticum (Fa73, nivalenol producer) and F. graminearum (Fg39, deoxynivalenol producer) strains. While the accessions varied in disease severity and mycotoxin production, four wheat and one barley showed <20% FHB severity repeatedly by both strains. Mycotoxin levels in these accessions ranged up to 3.9 mg/kg. FHB severity was generally higher in barley than in wheat, and Fa73 was more aggressive in both crops than Fg39. Fg39 itself, however, was more aggressive toward wheat and produced more mycotoxin in wheat than in barley. FHB severity by Fa73 and Fg39 were moderately correlated in both crops (r = 0.57/0.60 in barley and 0.42/0.58 in wheat). FHB severity and toxin production were also correlated in both crops, with a stronger correlation for Fa73 (r = 0.42/0.82 in barley, 0.70 in wheat) than for Fg39.

Characterization of Nivalenol-Producing Fusarium asiaticum That Causes Cereal Head Blight in Korea.

Fusarium asiaticum of the F. graminearum species complex causes head blight in small-grain cereals. The nivalenol (NIV) chemotypes of F. asiaticum is more common than the deoxynivalenol (DON) chemotypes of F. asiaticum or F. graminearum in Korea. To understand the prevalence of F. asiaticum-NIV in Korean cereals, we characterized the biological traits of 80 cereal isolates of F. asiaticum producing NIV or 3-acetyl-deoxynivalenol (3-ADON), and 54 F. graminearum with 3-ADON or 15-acetyl-deoxynivalenol (15-ADON). There was no significant difference in mycelial growth between the chemotypes, but F. asiaticum isolates grew approximately 30% faster than F. graminearum isolates on potato dextrose agar. Sexual and asexual reproduction capacities differed markedly between the two species. Both chemotypes of F. graminearum (3-ADON and 15-ADON) produced significantly higher numbers of perithecia and conidia than F. asiaticum-NIV. The highest level of mycotoxins (sum of trichothecenes and zearalenone) was produced by F. graminearum-3-ADON on rice medium, followed by F. graminearum-15-ADON, F. asiaticum-3-ADON, and F. asiaticum-NIV. Zearalenone levels were correlated with DON levels in some chemotypes, but not with NIV levels. Disease assessment on barley, maize, rice, and wheat revealed that both F. asiaticum and F. graminearum isolates were virulent toward all crops tested. However, there is a tendency that virulence levels of F. asiaticum-NIV isolates on rice were higher than those of F. graminearum isolates. Taken together, the phenotypic traits found among the Korean F. asiaticum-NIV isolates suggest an association with their host adaptation to certain environments in Korea.

Control of root-knot nematodes using Waltheria indica producing 4-quinolone alkaloids.

BACKGROUND: Waltheria indica exhibited strong nematocidal activity against Meloidogyne incognita, a causal agent of root-knot nematode disease. This study aimed to characterize the nematocidal metabolites and to evaluate the efficacy of the formulated extract of W. indica in the biological control of M. incognita under both pot and field conditions. RESULTS: Three 4-quinolone alkaloids, 5'-methoxywaltherione A, waltherione A and waltherione C, were isolated and characterized as nematocidal metabolites. 5'-Methoxywaltherione A and waltherione A caused high mortality in juveniles of Meloidogyne arenaria, Meloidogyne hapla, M. incognita and Bursaphelenchus xylophilus, whereas waltherione C exhibited significant nematocidal activity against only root-knot nematodes. In pot experiments, application of a wettable powder-type formulation of the ethyl acetate extract of W. indica (W. indica WP20) at 26.7, 53.4 and 106.8 mg a.i. kg-1 soil significantly reduced the formation of galls and egg masses on the roots of tomato plants in a dose-dependent manner. In addition, application of 20 mg a.i. per plant W. indica WP20 effectively reduced gall formation on the roots of melon plants and population density of nematode in soil compared with untreated control under field conditions. CONCLUSION: W. indica can be used as an effective botanical nematicide in the eco-friendly control of root-knot nematode disease. © 2019 Society of Chemical Industry.

Nematicidal activity of grammicin produced by Xylaria grammica KCTC 13121BP against Meloidogyne incognita.

BACKGROUND: The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aimed to identify the nematicidal metabolites and to evaluate the efficacy of the strain as a biocontrol agent under pot and field conditions. RESULTS: Bioassay-guided fractionation and instrumental analyses led to grammicin being identified as the nematicidal metabolite. Because patulin is a mycotoxic isomer of grammicin and is known to have strong antibacterial and cytotoxic activities, several biological activities of the two compounds were compared. Grammicin showed strong second-stage juvenile killing and egg-hatching inhibitory effects, with a 50% effective concentration at 72 h (EC50/72 h ) of 15.9 µg/mL and a 50% effective concentration at 14 days (EC50/14 days ) of 5.87 µg/mL, respectively, whereas patulin was virtually inactive in both respects. Patulin was strongly active toward various phytopathogenic bacteria in vitro, whereas grammicin was weakly so. Patulin at the concentration range of 0.1-10 µg/mL also showed dose-dependent cytotoxicity toward the human first-trimester trophoblast cell line SW.71, whereas grammicin was not toxic toward this cell line. In pot and field experiments, a wettable powder-type formulation and fermentation broth filtrate of X. grammica KCTC 13121BP effectively suppressed the development of root-knot nematode disease on tomato and melon plants. CONCLUSION: The results suggest that X. grammica and grammicin may have potential applications for control of root-knot nematode disease of various crops. © 2017 Society of Chemical Industry.

Characterization of Bacillus amyloliquefaciens DA12 Showing Potent Antifungal Activity against Mycotoxigenic Fusarium Species.

In an attempt to develop a biological control agent against mycotoxigenic Fusarium species, we isolated Bacillus amyloliquefaciens strain DA12 from soil and explored its antimicrobial activities. DA12 was active against the growth of mycotoxigenic F. asiaticum, F. graminearum, F. proliferatum, and F. verticillioides both in vitro and in planta (maize). Further screening using dual culture extended the activity range of strain DA12 against other fungal pathogens including Botrytis cinerea, Colletotrichum coccodes, Endothia parasitica, Fusarium oxysporum, Raffaelea quercus-mongolicae, and Rhizoctonia solani. The butanol extract of the culture filtrate of B. amyloliquefaciens DA12 highly inhibited the germination of F. graminearum macroconidia with inhibition rate 83% at a concentration of 31.3 µg/ml and 100% at a concentration of 250 µg/ml. The antifungal metabolite from the butanol extract was identified as iturin A by thin layer chromatography-bioautography. In addition, volatile organic compounds produced by DA12 were able to inhibit mycelial growth of various phytopathogenic fungi. The volatile compounds were identified as 2-heptanone, 5-methyl heptanone and 6-methyl heptanone by gas chromatography-mass spectrometry (GC-MS) analysis. These results indicate that the antagonistic activity of Bacillus amyloliquefaciens DA12 was attributable to iturin A and volatile heptanones, and the strain could be used as a biocontrol agent to reduce the development of Fusarium diseases and mycotoxin contamination of crops.

Antifungal activity of sterols and dipsacus saponins isolated from Dipsacus asper roots against phytopathogenic fungi.

The in vivo antifungal activity of crude extracts of Dipsacus asper roots was evaluated against the phytopathogenic fungi Botrytis cinerea, Colletotrichum coccodes, Blumeria graminis f. sp. hordei, Magnaporthe grisea, Phytophthora infestans, Puccinia recondita and Rhizoctonia solani using a whole-plant assay method. Ethyl acetate and acetone extracts, at 1000µg/mL, suppressed the development of tomato gray mold (TGM) and tomato late blight (TLB) by 90%. Through bioassay-guided isolation, five antifungal substances were isolated from the D. asper roots and identified as ß-sitosterol (1), campesterol (2), stigmasterol (3), cauloside A (4) and a novel dipsacus saponin, named colchiside (3-O-ß-d-xylopyranosyl-23-O-ß-d-glucopyranosyl-28-O-ß-d-(6-O-acetyl)-glucopyranosyl hederagenin) (5). Of those, cauloside A (4) displayed the greatest antifungal efficacy against rice blast, TGM and TLB. Colchiside (5) moderately suppressed the development of TLB, but exhibited little effect against the other diseases. The synergistic effects of the isolated compounds against TLB were also assessed. Synergistic and additive interactions were observed between several of the sterol compounds. This study indicated that the crude extracts of, and bioactive substances from, the roots of D. asper suppress TGM and TLB. In addition, cauloside A (4) and colchiside (5) could be used as antifungal lead compounds.

Biological Control of Meloidogyne incognita by Aspergillus niger F22 Producing Oxalic Acid.

Restricted usage of chemical nematicides has led to development of environmentally safe alternatives. A culture filtrate of Aspergillus niger F22 was highly active against Meloidogyne incognita with marked mortality of second-stage juveniles (J2s) and inhibition of egg hatching. The nematicidal component was identified as oxalic acid by organic acid analysis and gas chromatography-mass spectroscopy (GC-MS). Exposure to 2 mmol/L oxalic acid resulted in 100% juvenile mortality at 1 day after treatment and suppressed egg hatching by 95.6% at 7 days after treatment. Oxalic acid showed similar nematicidal activity against M. hapla, but was not highly toxic to Bursaphelenchus xylophilus. The fungus was incubated on solid medium and dried culture was used for preparation of a wettable powder-type (WP) formulation as an active ingredient. Two WP formulations, F22-WP10 (ai 10%) and oxalic acid-WP8 (ai 8%), were prepared using F22 solid culture and oxalic acid. In a field naturally infested with M. incognita, application of a mixture of F22-WP10 + oxalic acid-WP8 at 1,000- and 500-fold dilutions significantly reduced gall formation on the roots of watermelon plants by 58.8 and 70.7%, respectively, compared to the non-treated control. The disease control efficacy of the mixture of F22-WP10 + oxalic acid-WP8 was significantly higher than that of a chemical nematicide, Sunchungtan (ai 30% fosthiazate). These results suggest that A. niger F22 can be used as a microbial nematicide for the control of root-knot nematode disease.

Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita.

The fungal strain EML-DML3PNa1 isolated from leaf of white dogwood (Cornus alba L.) showed strong nematicidal activity with juvenile mortality of 87.6% at a concentration of 20% fermentation broth filtrate at 3 days after treatment. The active fungal strain was identified as Aspergillus oryzae, which belongs to section Flavi, based on the morphological characteristics and sequence analysis of the ITS rDNA, calmodulin (CaM), and ß-tubulin (BenA) genes. The strain reduced the pH value to 5.62 after 7 days of incubation. Organic acid analysis revealed the presence of citric acid (515.0 mg/kg), malic acid (506.6 mg/kg), and fumaric acid (21.7 mg/kg). The three organic acids showed moderate nematicidal activities, but the mixture of citric acid, malic acid, and fumaric acid did not exhibit the full nematicidal activity of the culture filtrate of EML- DML3PNa1. Bioassay-guided fractionation coupled with (1)H- and (13)C-NMR and EI-MS analyses led to identification of kojic acid as the major nematicidal metabolite. Kojic acid exhibited dose-dependent mortality and inhibited the hatchability of M. incognita, showing EC50 values of 195.2 µg/ml and 238.3 µg/ml, respectively, at 72 h postexposure. These results suggest that A. oryzae EML-DML3PNa1 and kojic acid have potential as a biological control agent against M. incognita.

Nematicidal activities of 4-quinolone alkaloids isolated from the aerial part of Triumfetta grandidens against Meloidogyne incognita.

The methanol extract of the aerial part of Triumfetta grandidens (Tiliaceae) was highly active against Meloidogyne incognita, with second-stage juveniles (J2s) mortality of 100% at 500 µg/mL at 48 h post-exposure. Two 4-quinolone alkaloids, waltherione E (1), a new alkaloid, and waltherione A (2), were isolated and identified as nematicidal compounds through bioassay-guided fractionation and instrumental analysis. The nematicidal activities of the isolated compounds against M. incognita were evaluated on the basis of mortality and effect on egg hatching. Compounds 1 and 2 exhibited high mortalities against J2s of M. incognita, with EC50 values of 0.09 and 0.27 µg/mL at 48 h, respectively. Compounds 1 and 2 also exhibited a considerable inhibitory effect on egg hatching, which inhibited 91.9 and 87.4% of egg hatching, respectively, after 7 days of exposure at a concentration of 1.25 µg/mL. The biological activities of the two 4-quinolone alkaloids were comparable to those of abamectin. In addition, pot experiments using the crude extract of the aerial part of T. grandidens showed that it completely suppressed the formation of gall on roots of plants at a concentration of 1000 µg/mL. These results suggest that T. grandidens and its bioactive 4-quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture.

Identification of orfamide A as an insecticidal metabolite produced by Pseudomonas protegens F6.

The use of biosurfactants for agricultural crop protection has been gaining interest because they are generally biodegradable and environmentally friendly. In the present study, we identified an insecticidal biosurfactant produced by Pseudomonas protegens F6 (F6) and examined its use for aphid control. The growth of F6 was accompanied by increased aphid mortality and decreased water surface tension. Bioassay-guided chromatography coupled with instrumental analyses, nuclear magnetic resonance (NMR), and time-of-flight mass spectrometer (TOF MS) identified orfamide A as a major metabolite that showed insecticidal activity against green peach aphid ( Myzus persicae ). Orfamide A revealed a dose-dependent mortality against aphids, producing a LC50 value at 34.5 µg/mL, and caused a considerable decrease in the surface tension value of water, giving about 35.7 mN/m at 10 µg/mL. Laboratory and greenhouse mortality bioassays suggested that orfamide A may be applicable to control aphids in organic agriculture. This is the first report of orfamide A as an insecticidal metabolite against Myzus persicae .