Process development of oxalic acid production in submerged culture of Aspergillus niger F22 and its biocontrol efficacy against the root-knot nematode Meloidogyne incognita.

Oxalic acid has potent nematicidal activity against the root-knot nematode Meloidogyne incognita. In this study, fermentation parameters for oxalic acid production in submerged culture of Aspergillus niger F22 at 23, 25, and 30 °C were optimized in 5-L jar fermenters. The viscosity of the culture broth increased with increasing temperature. There was a negative correlation between oxalic acid production and the apparent viscosity; high volumetric productivity of oxalic acid was obtained at low apparent viscosity (less than 1000 cP), with a productivity of more than 100 mg/L h. When the apparent viscosity was over 2500 cP, the volumetric productivity decreased below 50 mg/L h. In addition, the volumetric mass transfer coefficient, K L a, positively correlated with volumetric productivity. When the K L a value increased from 0.0 to 0.017 /s, the volumetric productivity proportionally increased up to 176 mg/L h. When the temperature decreased, K L a increased due to the decrease in viscosity, leading to increased volumetric productivity. The highest productivity of 7453.3 mg/L was obtained at the lowest temperature, i.e., 23 °C. The nematicidal activity of culture filtrate was proportional to the content of oxalic acid. Based on a constant impeller tip speed, oxalic acid production was successfully scaled up to a 500-L pilot vessel, producing a final concentration comparable to that in the 5-L jar.

Development of a Biofungicide Using a Mycoparasitic Fungus Simplicillium lamellicola BCP and Its Control Efficacy against Gray Mold Diseases of Tomato and Ginseng.

To develop a commercial product using the mycoparasitic fungus Simplicillium lamellicola BCP, the scaleup of conidia production from a 5-l jar to a 5,000-l pilot bioreactor, optimization of the freeze-drying of the fermentation broth, and preparation of a wettable powder-type formulation were performed. Then, its disease control efficacy was evaluated against gray mold diseases of tomato and ginseng plants in field conditions. The final conidial yields of S. lamellicola BCP were 3.3 × 109 conidia/ml for a 5-l jar, 3.5 × 109 conidia/ml for a 500-l pilot vessel, and 3.1 × 109 conidia/ml for a 5,000-l pilot bioreactor. The conidial yield in the 5,000-l pilot bioreactor was comparable to that in the 5-l jar and 500-l pilot vessel. On the other hand, the highest conidial viability of 86% was obtained by the freeze-drying method using an additive combination of lactose, trehalose, soybean meal, and glycerin. Using the freeze-dried sample, a wettable powder-type formulation (active ingredient 10%; BCP-WP10) was prepared. A conidial viability of more than 50% was maintained in BCP-WP10 until 22 weeks for storage at 40°C. BCP-WP10 effectively suppressed the development of gray mold disease on tomato with control efficacies of 64.7% and 82.6% at 500- and 250-fold dilutions, respectively. It also reduced the incidence of gray mold on ginseng by 65.6% and 81.3% at 500- and 250-fold dilutions, respectively. The results indicated that the new microbial fungicide BCP-WP10 can be used widely to control gray mold diseases of various crops including tomato and ginseng.

Diffusible and Volatile Antifungal Compounds Produced by an Antagonistic Bacillus velezensis G341 against Various Phytopathogenic Fungi.

The aim of this study was to identify volatile and agar-diffusible antifungal metabolites produced by Bacillus sp. G341 with strong antifungal activity against various phytopathogenic fungi. Strain G341 isolated from four-year-old roots of Korean ginseng with rot symptoms was identified as Bacillus velezensis based on 16S rDNA and gyrA sequences. Strain G341 inhibited mycelial growth of all phytopathogenic fungi tested. In vivo experiment results revealed that n-butanol extract of fermentation broth effectively controlled the development of rice sheath blight, tomato gray mold, tomato late blight, wheat leaf rust, barley powdery mildew, and red pepper anthracnose. Two antifungal compounds were isolated from strain G341 and identified as bacillomycin L and fengycin A by MS/MS analysis. Moreover, volatile compounds emitted from strain G341 were found to be able to inhibit mycelial growth of various phytopathogenic fungi. Based on volatile compound profiles of strain G341 obtained through headspace collection and analysis on GC-MS, dimethylsulfoxide, 1-butanol, and 3-hydroxy-2-butanone (acetoin) were identified. Taken together, these results suggest that B. valezensis G341 can be used as a biocontrol agent for various plant diseases caused by phytopathogenic fungi.

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.

Antimicrobial activities of novel mannosyl lipids isolated from the biocontrol fungus Simplicillium lamellicola BCP against phytopathogenic bacteria.

The antagonistic fungus Simplicillium lamellicola BCP has been developed as a microbial biopesticide that effectively controls the development of various plant diseases caused by both pathogenic bacteria and pathogenic fungi. Antibacterial bioassay-directed fractionation was used to isolate mannosyl lipids from S. lamellicola BCP, and the structures of these compounds were elucidated using spectral analysis and chemical degradation. Three novel mannosyl lipids were characterized and identified as halymecins F and G and (3R,5R)-3-O-ß-D-mannosyl-3,5-dihydrodecanoic acid. Massoia lactone and (3R, 5R)-3-hydroxydecan-5-olide were also isolated from S. lamellicola BCP. The three novel compounds inhibited the growth of the majority of phytopathogenic bacteria that were tested, and halymecin F displayed the strongest antibacterial activity. Agrobacterium tumefaciens was the most sensitive to the three novel compounds, with IC50 values ranging from 1.58 to 24.8 µg/mL. The ethyl acetate extract of the fermentation broth from the antagonistic fungus effectively reduced the bacterial wilt caused by Ralstonia solanacearum on tomato seedlings. These results indicate that S. lamellicola BCP suppresses the development of plant bacterial diseases through the production of antibacterial metabolites.