Culture optimization of Streptomyces sp. KRA16-334 for increased yield of new herbicide 334-W4.

This study aimed to isolate actinomycetes that exhibit strong herbicidal activity, identify compounds active against weeds, and researching methods to improve the production of these compounds through culture optimization to establish a foundation for the development of environmentally friendly bioherbicides. 334-W4, one of the herbicidal active substances isolated from the culture broth of Streptomyces sp. KRA16-334, exhibited herbicidal activity against various weeds. The molecular formula of 334-W4 was determined to be C16H26N2O6, based on ESI-MS (m/z) and 1H and 13C NMR spectral data. It had molecular weight 365.1689 [M+Na] and 343.1869 [M+H], indicating the presence of the epoxy-ß-aminoketone moiety based on HMBC correlations. Additionally, selective culture was possible depending on the addition of trifluoroacetic acid (TFA) during culture with GSS medium. Experiments confirmed that exposure of the KRA16-334 strain to UV irradiation (254 nm, height 17 cm) for 45 seconds improved the yield of the active substance (334-W4) by over 200%. As a result of examining yields of active materials of four mutants selected through optimization of culture conditions such as temperature, agitation, and initial pH, the yield of one mutant 0723-8 was 264.7 ± 12.82 mg/L, which was 2.8-fold higher than that of wild-type KRA16-334 at 92.8 ± 5.48 mg/L.

Herbicidal Characteristics and Structural Identification of the Potential Active Compounds from Streptomyces sp. KRA17-580.

Weeds are notorious plant species exhibiting a harmful impact on crops. Biological weed control is an efficient and environmentally friendly technique, usually constitutes naturally derived compounds, including bioherbicidal metabolites produced by Streptomyces sp. The isolation and structural identification of phytotoxic compounds from Streptomyces have recently been proposed as an effective way to the discovery of novel bioherbicides. In the screening of bioherbicidal agents, isolated Streptomyces strain KRA17-580 demonstrated significant phytotoxic activity against Digitaria ciliaris. Phylogenetic analysis of the 16S rRNA sequence indicated that isolated KRA17-580 is similar to Streptomyces olivochromogenes. The bacterial culture conditions were optimized for temperature, agitation, and initial pH. Streptomyces strain KRA17-580 showed intense phytotoxic activity and high cell mass at an initial pH of 5.5-7.0, more than 150 rpm, and 25-30 °C. The herbicidal compounds isolated from the culture filtrate of strain KRA17-580 were purified by solvent partition, C18, Sephadex LH20 column chromatography, and high-performance liquid chromatography. By 1D-NMR, 2D-NMR, and electrospray ionization mass spectrometry analysis, the 580-H1 and 580-H2 compounds were identified as a cinnoline-4-carboxamide (MW, 173.0490; C9H7N3O2) and cinnoline-4-carboxylic acid (MW, 174.0503; C9H6N2O2), respectively. Only these two herbicidal compounds showed strong phytotoxic activity against D. ciliaris in foliar applications. However, compound 580-H2 was more phytotoxic than 580-H1 and the toxicity was dose-dependent. The herbicidal metabolite KRA17-580 produced by Streptomyces sp. is a new bioherbicidal candidate that may provide a new lead molecule for more efficient phytotoxic compounds.

In Vivo Disease Control Efficacy of Isoquinoline Alkaloids Isolated from Corydalis ternata against Wheat Leaf Rust and Pepper Anthracnose.

Phytochemicals have been considered as alternatives for synthetic fungicides because of their biodegradability and low toxicity. In this study, we found that the methanolic extract of Corydalis ternata suppressed the development of plant diseases caused by Puccinia triticina and Colletotrichum coccodes. As the antifungal substance, three isoquinoline alkaloids (dehydrocorydaline, stylopine, and corydaline) were isolated from C. ternata. These active compounds also exhibited in vivo antifungal activity against P. triticina and C. coccodes. Taken together, our results suggest that C. ternata and its active compounds can be used to control plant diseases.

In vivo assessment of plant extracts for control of plant diseases: A sesquiterpene ketolactone isolated from Curcuma zedoaria suppresses wheat leaf rust.

As an alternative to synthetic pesticides, natural materials such as plant extracts and microbes have been considered to control plant diseases. In this study, methanol extracts of 120 plants were explored for in vivo antifungal activity against Rhizoctonia solani, Botrytis cinerea, Phytophthora infestans, Puccinia triticina, and Blumeria graminis f. sp. hordei. Of the 120 plant extracts, eight plant extracts exhibited a disease control efficacy of more than 90% against at least one of five plant diseases. In particular, a methanol extract of Curcuma zedoaria rhizomes exhibited strong activity against wheat leaf rust caused by P. triticina. When the C. zedoaria methanol extracts were partitioned with various solvents, the layers of n-hexane, methylene chloride, and ethyl acetate showed disease control values of 100, 80, and 43%, respectively, against wheat leaf rust. From the C. zedoaria rhizome extracts, an antifungal substance was isolated and identified as a sesquiterpene ketolactone based on the mass and nuclear magnetic resonance spectral data. The active compound controlled the development of rice sheath blight, wheat leaf rust, and tomato late blight. Considering the in vivo antifungal activities of the sesquiterpene ketolactone and the C. zedoaria extracts, these results suggest that C. zedoaria can be used as a potent fungicide in organic agriculture.

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.

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.

Antibacterial activity of tannins isolated from Sapium baccatum extract and use for control of tomato bacterial wilt.

In the search for new antibacterial agents from natural sources, we revealed that a crude methanol extract of Sapium baccatum was highly active against Ralstonia solanacearum, a causal agent of a serious disease called bacterial wilt of tomato. The bioassay-guided fractionation of this extract resulted in the isolation of seven known active compounds, including gallic acid, methyl gallate, corilagin, tercatain, chebulagic acid, chebulinic acid, and quercetin 3-O-α-L-arabinopyranoside. Their chemical structures were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. An in vitro antibacterial bioassay using a broth microdilution method revealed that, except for quercetin 3-O-α-L-arabinopyranoside (MIC = 250 µg/mL), the isolated compounds exhibited strong antibacterial activity against R. solanacearum (MIC = 26-52 µg/mL). Among the seven compounds, methyl gallate exhibited the strongest broad-spectrum activity against most of the plant pathogenic bacteria tested (MIC = 26-250 µg/mL). In the in vivo experiments, the crude extract of S. baccatum at 2000 and 1000 µg/mL reduced the development of tomato bacterial wilt by 83 and 63%, respectively, under greenhouse conditions after 14 days of infection. The results suggested that the extracts of S. baccatum or isolated tannins could be used as natural bactericides for the control of bacterial wilt of tomato.

Pathotype Classification of Plasmodiophora brassicae Isolates Using Clubroot-Resistant Cultivars of Chinese Cabbage.

Clubroot disease caused by Plasmodiophora brassicae is one of the most serious diseases in Brassica crops worldwide. In this study, the pathotypes of 12 Korean P. brassicae field isolates were determined using various Chinese cabbage including 22 commercial cultivars from Korea, China, and Japan, and 15 inbred lines. All P. brassicae isolates exhibited the typical clubroot disease on non-clubroot resistant cultivar, indicating that the isolates were highly pathogenic. According to the reactions on the Williams' hosts, the 12 field isolates were initially classified into five races. However, when these isolates were inoculated onto clubroot-resistant (CR) cultivars of Chinese cabbage, several isolates led to different disease responses even though the isolates have been assigned to the same race by the Williams' host responses. Based on the pathogenicity results, the 12 field isolates were reclassified into four different groups: pathotype 1 (GN1, GN2, GS, JS, and HS), 2 (DJ and KS), 3 (HN1, PC, and YC), and 4 (HN2 and SS). In addition, the CR cultivars from Korea, China, and Japan exhibited distinguishable disease responses to the P. brassicae isolates, suggesting that the 22 cultivars used in this study, including the non-CR cultivars, are classified into four different host groups based on their disease resistance. Combining these findings, the four differential hosts of Chinese cabbage and four pathotype groups of P. brassicae might provide an efficient screening system for resistant cultivars and a new foundation of breeding strategies for CR Chinese cabbage.

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 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.

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.

Acid hydrolysis of Curcuma longa residue for ethanol and lactic acid fermentation.

This research examines the acid hydrolysis of Curcuma longa waste, to obtain the hydrolysate containing lactic acid and ethanol fermentative sugars. A central composite design for describing regression equations of variables was used. The selected optimum condition was 4.91% sulphuric acid, 122.68°C and 50 min using the desirability function under the following conditions: the maximum reducing sugar (RS) yield is within the limited range of the 5-hydroxymethylfurfural (HMF) and furfural concentrations. Under the condition, the obtained solution contained 144 g RS/L, 0.79 g furfural/L and 2.59 g HMF/L and was directly fermented without a detoxification step. The maximum product concentration, average productivity, RS conversion and product yield were 115.36 g/L, 2.88 g/L/h, 89.43% and 64% for L-lactic acid; 113.92 g/L, 2.59 g/L/h, 88.31% and 63.29% for D-lactic acid; and 55.03 g/L, 1.38 g/L/h, 42.66 and 30.57%, respectively, for ethanol using a 7-L jar fermenter.

Production of L- and D-lactic acid from waste Curcuma longa biomass through simultaneous saccharification and cofermentation.

Simultaneous saccharification and cofermentation (SSCF) of Curcuma longa waste biomass obtained after turmeric extraction to L- and D-lactic acid by Lactobacillus coryniformis and Lactobacillus paracasei, respectively, was investigated. This is a rich, starchy, agro-industrial waste with potential for use in industrial applications. After optimizing the fermentation of the biomass by adjusting nitrogen sources, enzyme compositions, nitrogen concentrations, and raw material concentrations, the SSCF process was conducted in a 7-l jar fermentor at 140 g dried material/L. The maximum lactic acid concentration, average productivity, reducing sugar conversion and lactic acid yield were 97.13 g/L, 2.7 g/L/h, 95.99% and 69.38 g/100 g dried material for L-lactic acid production, respectively and 91.61 g/L, 2.08 g/L/h, 90.53% and 65.43 g/100 g dried material for D-lactic acid production, respectively. The simple and efficient process described in this study could be utilized by C. longa residue-based lactic acid industries without requiring the alteration of plant equipment.

Effect of Gallotannins Derived from Sedum takesimense on Tomato Bacterial Wilt.

In the process of searching antibacterial agents from plants, we discovered that the methanol extract of Sedum takesimense showed potent antibacterial activity against Ralstonia solanacearum in vitro and in vivo. Eight antibacterial gallotannins were isolated from the aerial parts of S. takesimense and identified as gallic acid, methyl gallate, 4,6-di-O-galloylarbutin, 2,6-di-O-galloylarbutin, 2,4,6-tri-O-galloyl-glucose, 1,3,4,6-tetra-O-galloyl-ß-glucose, 1,2,4,6-tetra-O-galloyl-ß-glucose, and 1,2,3,6-tetra-O-galloyl-ß-glucose based on electrospray ionization mass spectrometry and proton nuclear magnetic resonance spectroscopy. These gallotannins displayed broad-spectrum activity against various plant-pathogenic bacteria, and the strongest in vitro antibacterial activities of these gallotannins were against R. solanacearum minimum inhibitory concentration = 0.02 to 0.10 g/liter). Among these gallotannins, methyl gallate and 1,2,3,6-tetra-O-galloyl-ß-glucose showed the strongest activities. In addition, synergistic or partial synergistic effects were observed in most combinations between major antibacterial compounds. The wettable powder formulation of the S. takesimense crude extract effectively reduced the development of tomato bacterial wilt caused by R. solanacearum under greenhouse conditions for 14 days after infection. This is the first report on the isolation of antibacterial compounds from S. takesimense. These results suggest that the extract from S. takesimense or the isolated gallotannins could be used as natural bactericides for the control of tomato bacterial wilt.

D-lactic acid production from dry biomass of Hydrodictyon reticulatum by simultaneous saccharification and co-fermentation using Lactobacillus coryniformis subsp. torquens.

D-lactic acid production from dry biomass of the microalga, Hydrodictyon reticulatum, was carried out in a 5-l jar fermentor (initial pH 6, 34 °C using CaCO(3) as a neutralizing agent) through simultaneous saccharification and co-fermentation using the Lactobacillus coryniformis subsp. torquens. After 36 h, 36.6 g lactic acid/l was produced from 80 g H. reticulatum/l in the medium containing 3 g yeast extract/l and 3 g peptone/l in the absence of mineral salts. The maximum productivity, average productivity and yield were 2.38 g/l h, 1.02 g/l h and 45.8 %, respectively. The optical purity of D-Lactic acid ranged from 95.8-99.6 %. H. reticulatum is thus a promising biomass material for the production of D-Lactic acid.

Production of l-lactic acid from a green microalga, Hydrodictyon reticulum, by Lactobacillus paracasei LA104 isolated from the traditional Korean food, makgeolli.

The freshwater microalga, Hydrodictyon reticulum, that contained 47.5% reducing sugars including 35% glucose was used as substrate for the production of l-lactic acid (LA) by LA-producing bacteria. Lactobacillus paracasei LA104 was selected for fermentation in a 5-l fermentor since it was able to grow at pH 3, 60g LA/l, 200g glucose/l, 125g NaCl/l, and 45°C and produced over 97.3% optically pure l-lactic acid with glucose as a substrate. Simultaneous saccharification and cofermentation from H. reticulum to l-LA using LA104 was investigated in a jar fermentor. The yield reached 46g/100g H. reticulum dry material, with a final concentration of 37.11g/l and a productivity of 1.03g/l/h. This is the first report of the production of l-LA from a microalga, and H. reticulum could be a potential feedstock for large-scale production of l-LA by LA104.

Potent in vivo antifungal activity against powdery mildews of pregnane glycosides from the roots of Cynanchum wilfordii.

Two new pregnane glycosides, kidjoranine 3-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1 → 4)-ß-D-cymaropyranoside (5) and caudatin 3-O-ß-D-glucopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 4)-α-L-cymaropyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-α-L-diginopyranosyl-(1 → 4)-ß-D-cymaropyranoside (6), were isolated from the roots of Cynanchum wilfordii along with four known compounds (1-4). The antifungal activities of the six compounds against barley powdery mildew caused by Blumeria graminis f. sp. hordei were compared to the antifungal activity of polyoxin B. The caudatin glycosides (1, 4, and 6) showed stronger antifungal activities than polyoxin B, whereas kidjoranine glycosides (2, 3, and 5) had weaker activities than polyoxin B. A wettable powder-type formulation (C. wilfordii-WP20) of the ethyl acetate extract from C. wilfordii roots prohibited the development of barley powdery mildew much more effectively than the commercial fungicide polyoxin B-WP10. In addition, C. wilfordii-WP20 effectively controlled strawberry powdery mildew caused by Sphaerotheca humuli under greenhouse conditions. Thus, the crude extract containing the pregnane glycosides can be used as a botanical fungicide for the environmentally benign control of powdery mildews.

Nematicidal and antifungal activities of annonaceous acetogenins from Annona squamosa against various plant pathogens.

The methanol extract of Annona squamosa seeds was highly active against two phytoparasitic nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. It efficiently suppressed plant diseases, caused by Phytophthora infestans and Puccinia recondita. Ten annonaceous acetogenins (AAs) were isolated, and their chemical structures were identified by mass and nuclear magnetic resonance spectral data. Out of 10 substances, eight displayed strong in vitro nematicidal activity against B. xylophilus with LD(50) values ranging 0.006 to 0.048 µg/mL. Squamocin-G showed potent nematicidal activity against M. incognita. Squamocin, squamocin-G, and squamostatin-A also displayed potent in vitro and in vivo antifungal activities against P. infestans causing tomato late blight. In addition, squamostatin-A effectively controlled the development of wheat leaf rust caused by P. recondita. Our findings suggested that A. squamosa seeds and its bioactive AAs can be an alternative resource of a promising botanical nematicide and fungicide to control various plant diseases.