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Turfgrass, the most widely grown ornamental crop, is severely affected by fungal pathogens including Sclerotinia homoeocarpa, Rhizoctonia solani, and Magnaporthe poae. At present, turfgrass fungal disease management predominantly relies on synthetic fungicide treatments. However, the extensive application of fungicides to the soil increases residual detection frequency, raising concerns for the environment and human health. The bacterial volatile compound, 2,3-butanediol (BDO), was found to induce plant resistance. In this study, we evaluated the disease control efficacy of a combination of stereoisomers of 2,3-BDO and commercial fungicides against turfgrass fungal diseases in both growth room and fields. In the growth room experiment, the combination of 0.9% 2R,3R-BDO (levo) soluble liquid (SL) formulation and 9% 2R,3S-BDO (meso) SL with half concentration of fungicides significantly increased the disease control efficacy against dollar spot and summer patch disease when compared to the half concentration of fungicide alone. In field experiments, the disease control efficiency of levo 0.9% and meso 9% SL, in combination with a fungicide, was confirmed against dollar spot and large patch disease. Additionally, the induction of defense-related genes involved in the salicylic acid and jasmonic acid/ethylene signaling pathways and reactive oxygen species detoxification-related genes under Clarireedia sp. infection was confirmed with levo 0.9% and meso 9% SL treatment in creeping bentgrass. Our findings suggest that 2,3-BDO isomer formulations can be combined with chemical fungicides as a new integrated tool to control Clarireedia sp. infection in turfgrass, thereby reducing the use of chemical fungicides.

Since the protective activity of the Bordeaux mixture against plant disease caused by oomycetes was discovered, copper compounds have been used for more than a century as an effective plant protection strategy. However, the application of excessive copper can cause adverse effects through long-term heavy metal accumulation in soils. Therefore, it is necessary to develop new strategies to reduce or replace copper in pesticides based on organic and low-input farming systems. Organic acids are eco-friendly. In this study, we tested the antifungal and anti-oomycete activity of maleic acid (MA) and copper sulfate (CS) against 13 plant pathogens. Treatment with a mixture of MA and CS showed strong anti-oomycetes activity against Phytophthora xcambivora, P. capsici, and P. cinnamomi. Moreover, the concentration of CS in the activated mixture of MA and CS was lower than that in the activated CS only, and the mixture showed synergy or partial synergy effects on the anti-oomycete activity. Application of a wettable powder formulation of MA and CS mixture (MCS 30WP; 26.67% MA and 3.33% CS) had excellent protective activity in pot experiments with control values of 73% Phytophthora blight on red pepper, 91% damping-off on cucumber, and 84% Pythium blight on creeping bentgrass, which are similar to those of the CS wettable powder formulation (6.67% CS) containing two times the CS content of MCS 30WP. These observations suggest that the synergistic effect of the MA and CS combination is a sustainable alternative for effective management of destructive oomycete diseases.

Shot-hole disease (SH) is one of the most common and important diseases affecting flowering cherry (FC; Prunus × yedoensis Matsumura; Somei-yoshino) trees in South Korea every year, resulting in premature defoliation and reduced flowering in the following year. However, pathogens associated with the disease remain unknown, which has rendered disease management challenging. Here, the pathogens associated with SH, their biochemical characteristics, and their host range were elucidated. Detached-leaf and in planta assays revealed that two biofilm-forming bacteria—namely, Burkholderia contaminans and Pseudomonas syringae pv. syringae—caused SH of FC trees. These pathogens were recorded for the first time as the causes of SH of FC trees in South Korea. Additionally, the two pathogens induced similar disease symptoms in several stone fruit belonging to the genus Prunus, including peach (Prunus persica), plum (P. salicina), and apricot (P. mume), with peach being the most susceptible. These results indicate that B. contaminans and P. syringae pv. syringae caused SH on FC trees and presented a broad spectrum of hosts. Furthermore, Xanthomonas arboricola pv. pruni, the causative agent of leaf spot on stone fruit, incited brown spots and shot holes on FC leaves. Therefore, FC trees are susceptible to infections by various pathogenic bacteria, including B. contaminans, P. syringae pv. syringae, and X. arboricola pv. pruni. These findings will be of great importance as a reference for effective management of SH in the face of possible cross-infection between Prunus spp. in the future.

Grammicin (Gra) is derived from the endophytic fungus Xylaria grammica EL000614 and shows nematicidal activity against the devastating root-knot nematode Meloidogyne incognita in-vitro, in planta, and in-field experiments. However, the mechanism of the nematicidal action of Gra remains unclear. In this study, Gra exposure to the model genetic organism Caenorhabditis elegans affected its L1, L2/3, L4, and young adult stages. In addition, Gra treatment increased the intracellular reactive oxygen species (ROS) levels of C. elegans and M. incognita. Molecular docking interaction analysis indicated that Gra could bind and interact with GCS-1, GST-4, and DAF-16a in order of low binding energy, followed by SOD-3, SKN-1, and DAF-16b. This implies that the anthelmintic action of Gra is related to the oxidative stress response. To validate this mechanism, we examined the expression of the genes involved in the oxidative stress responses following treatment with Gra using transgenic C. elegans strains such as the TJ356 strain zIs356 [daf-16p::daf-16a/b::GFP + rol-6 (su1006)], LD1 ldIs7 [skn-1p::skn-1b/c::GFP + rol-6 (su1006)], LD1171 ldIs3 [gcs-1p::GFP + rol-6 (su1006)], CL2166 dvIs19 [(pAF15) gst-4p::GFP::NLS], and CF1553 strain muIs84 [(pAD76) sod-3p::GFP + rol-6 (su1006)]. Gra treatment caused nuclear translocation of DAF-16/FoxO and enhanced gst-4::GFP expression, but it had no change in sod-3::GFP expression. These results indicate that Gra induces oxidative stress response via phase II detoxification without reduced cellular redox machinery. Gra treatment also inhibited the nuclear localization of SKN-1::GFP in the intestine, which may lead to a condition in which oxidative stress tolerance is insufficient to protect C. elegans by the inactivation of SKN-1, thus inducing nematode lethality. Furthermore, Gra caused the mortality of two mutant strains of C. elegans, CB113 and DA1316, which are resistant to aldicarb and ivermectin, respectively. This indicates that the mode of action of Gra is different from the traditional nematicides currently in use, suggesting that it could help develop novel approaches to control plant-parasitic nematodes.

Though information exists regarding the pathogenesis of the shot-hole disease (SH) in flowering cherry (FC), there has been a lack of research focusing on SH management. Therefore, here, we investigated the inhibitory activities of antagonistic bacteria against SH pathogens both in vitro and in vivo as well as their biochemical characteristics and bioactive compounds. Two biosurfactant-producing bacterial antagonists, identified as Bacillus velezensis strains JCK-1618 and JCK-1696, exhibited the best effects against the growth of both bacterial and fungal SH pathogens in vitro through their cell-free culture filtrates (CFCFs). These two strains also strongly inhibited the growth of the pathogens via the action of their antimicrobial diffusible compounds and antimicrobial volatile organic compounds (VOCs). Crude enzymes, solvent extracts, and biosurfactants of the two strains exhibited antimicrobial activities. Liquid chromatography/electrospray ionization time-of-flight mass spectrometric analysis of the partially purified active fractions revealed that the two antagonists produced three cyclic lipopeptides, including iturin A, fengycin A, and surfactin, and a polyketide, oxydifficidin. In a detached leaf assay, pre-treatment and co-treatment of FC leaves with the CFCFs led to a large reduction in the severity of the leaf spots caused by Epicoccum tobaicum and Bukholderia contaminans, respectively. In addition, the two antagonists produced indole-3-acetic acid, siderophore, and a series of hydrolytic enzymes, along with the formation of a substantial biofilm. To our knowledge, this is the first report of the antimicrobial activities of the diffusible compounds and VOCs of B. velezensis against the SH pathogens and their efficiency in the biocontrol of SH.

In July 2018, a serious rust symptom was found throughout the fringe trees planted in Gangjin-gun, Korea. Yellow and brown spots were observed on the adaxial (topside) surface of the collected fringe tree leaves, and yellow color aecia were observed on the abaxial (underside) surface leaves. The size of aeciospore and urediniospores of JCK-KCFR1 strain were measured to 41.2 μm (Φ) and 28.84 μm (Φ) with a light microscope. Phylogenetic analysis of the small subunit rRNA, internal transcribed spacer, and large subunit rRNA region indicated that JCK-KCFR1 strain is novel species of the genus Puccinia and closely related to Puccinia kusanoi, which has been reported a rust pathogen on bamboo. In May 2019, rust symptoms were also discovered on the bamboo leaves planted around the fringe tree on Muwisa-ro, and their telia and teliospores were observed on the abaxial leaf surfaces of the bamboo with 100% sequence homology with the rust of the fringe tree. This is the first report that Puccinia sp. JCK-KCFR1 is a new species that requires both primary (fringe tree) and alternative (bamboo) host plants to complete its life cycle in Korea.

Multi-drug resistance in nematodes is a serious problem as lately several resistant phenotypes have emerged following the intermittent usage of synthetic nematicides. Contemporary research continues to focus on developing and/or repurposing small molecule inhibitors that are eco-friendly. Here, we describe the repurposing of the indole derivative, 5-iodoindole, as a nematicide for the root-knot nematode, Meloidogyne incognita. 5-Iodoindole effectively killed juveniles and freshly hatched juveniles by inducing multiple vacuole formation. Notably, at higher dosage (50 μg/mL), 5-iodoindole induced rapid juvenile death within 6 h. Microscopic analysis confirmed that the rapid death was due to the generation of reactive oxygen species (ROS). Computational docking attributed this ROS production to the antagonistic effect of 5-iodoindole on glutathione S-transferase (GST), which is known to play a critical role in the suppression of ROS in nematode models. Furthermore, 5-iodoindole also effectively reduced the gall formations and eggs masses of M. incognita on Solanum lycopersicum roots in pot experiments, and importantly it did not harm the physiological properties of the plant. Overall, the study provides valuable insights on the use of 5-iodoindole as an alternate measure to control root-knot nematodes. Overall, our findings suggest the efficacy of 5-iodoindole should be studied under field conditions.

Bacillus methylotrophicus DR-08 exhibited strong antibacterial activity against Ralstonia solanacearum, a causal agent of tomato bacterial wilt. This study aimed to identify the antibacterial metabolites and evaluate the efficacy of the strain as a biocontrol agent for tomato bacterial wilt. A butanol extract of the DR-08 broth culture completely inhibited the growth of 14 phytopathogenic bacteria with minimum inhibitory concentration (MIC) values of 1.95–500 μg/mL. R. solanacearum was highly sensitive to the DR-08 extract, with an MIC value of 12.62 μg/mL. Two antibacterial metabolites were isolated and identified as difficidin and oxydifficidin derivatives through bioassay-guided fractionation and instrumental analyses. Both metabolite derivatives inhibited the growth of most of the phytopathogenic bacteria tested and the oxydifficidin derivatives generally presented a stronger antibacterial activity than the difficidin derivatives. A 30% suspension concentrate of DR-08, at a 500-fold dilution, effectively suppressed the development of tomato bacterial wilt in pot and field experiments. It also effectively reduced the development of bacterial leaf spot symptoms on peach and red pepper. The results of this study suggests that B. methylotrophicus DR-08 can be utilized as a biocontrol agent for various bacterial plant diseases including tomato bacterial wilt.

Root-knot nematodes (RKNs) are among the most destructive plant-parasites worldwide, and RKN control has been attempted mainly using chemical nematicides. However, these chemical nematicides have negative effects on humans and the environment, thus necessitating the search for eco-friendly alternative RKN control methods. Here, we screened nematicidal lactic acid bacteria (LAB) isolated from kimchi and evaluated their efficacy as biocontrol agents against RKNs. Of 237 bacterial strains, Lactobacillus brevis WiKim0069 showed the strongest nematicidal activity against the second-stage juveniles (J2) of Meloidogyne incognita, M. arenaria, and M. hapla and inhibited the egg hatch of M. incognita. The culture filtrate of WiKim0069 had a pH of 4.2 and contained acetic acid (11,190 μg/ml), lactic acid (7,790 μg/ml), malic acid (470 μg/ml), and succinic acid (660 μg/ml). An artificial mixture of the four organic acids produced by WiKim0069 also induced 98% M. incognita J2 mortality at a concentration of 1.25%, indicating that its nematicidal activity was derived mainly from the four organic acids. Application of WiKim0069 culture filtrate suppressed the formation of galls and egg masses on tomato roots by M. incognita in a dose-dependent manner in a pot experiment. The fermentation broth of WiKim0069 also reduced gall formation on melon under field conditions, with a higher efficacy (62.8%) than that of fosthiazate (32.8%). This study is the first report to identify the effectiveness of kimchi LAB against RKNs and to demonstrate that the organic acids produced by LAB can be used for the RKN management.

The widespread use of synthetic nematicides has caused significant problems to the environment as well as human health. To address this issue, eco-friendly control measures, such as microbial nematicides, are being developed. During the screening of Myrothecium strains with nematicidal activity against the root-knot nematode (RKN) Meloidogyne incognita, we found that the acetone extract of Myrothecium sp. KACC 40321 was highly effective against hatched juveniles of M. incognita at 7 days after exposure. The fungus was identified as Meloidogyne verrucaria. Two macrocyclic trichothecenes verrucarin A and roridin A were isolated and identified as major active metabolites by bioassay-guided fractionation and instrumental analysis. When the second-stage juveniles were treated with the chemicals, no juvenile mortality was observed. However, they effectively killed juveniles from treated eggs. The hatched juvenile mortality was used to evaluate the in vitro nematicidal activity of the compounds against M. incognita. The median effective concentrations were 1.88 μg/mL for verrucarin A and 1.50 μg/mL for roridin A. Among various liquid media, commercial malt extract broth (cMEB) was found to be the best for the production of verrucarin A and roridin A, followed by potato dextrose broth. The cMEB culture filtrate effectively reduced the formation of galls and egg masses on tomato roots in a pot experiment. In addition, the culture filtrate reduced the formation of galls on the roots of melon plants and the number of RKNs in the soils under field conditions. These results suggest that M. verrucaria KACC 40321 can be used as a biocontrol agent against RKNs in various crops. To the best of our knowledge, this is the first study to report the effectiveness of verrucarin A and rorridin A against hatched juveniles of M. incognita.

Streptomyces species have been used as biocontrol agents for the management of fungal postharvest diseases. Reveromycin E (RE) and its new derivative, reveromycin E 1-methyl ester (REME), were isolated from Streptomyces sp. JCK-6141. Both compounds displayed broad-spectrum and extensive antifungal activities at acidic condition, but their activities were dramatically reduced at neutral or base conditions. These compounds effectively suppressed the cherry tomato gray mold and mandarin blue mold caused by Botrytis cinerea and Penicillium italicum, respectively, at 10 and 50 mg L−1. In in vitro and in vivo assays, RE showed stronger antifungal activity than REME. Reveromycin was known as an isoleucine tRNA synthetase (IleRS) inhibitor. However, there is unconsolidated result in confirming binding site of reveromycin in previous studies. A computational study revealed that RE had a lower binding energy than REME and both compounds tend to bind catalytic domain rather than editing domains of IleRS. Our results indicated that Streptomyces sp. JCK-6141 producing reveromycins can be widely used as a new microbial fungicide for the control of postharvest diseases on fruit.

The antimicrobial efficacies of extracts and constituents fractionated from Rheum tanguticum rhizomes were evaluated by using in vitro and in vivo bioassay against seven phytopathogenic fungi and ten pathogenic bacteria, respectively. Dichloromethane and ethyl acetate soluble extracts derived from R. tanguticum rhizomes effectively controlled plant diseases caused by five fungi Magnaporthe oryzae, Blumeria graminis f. sp. hordei, Colletotrichum coccodes, Phytophthora infestans, and Puccinia recondita in in vivo whole plant bioassay. In addition, these extracts showed strong inhibitory activity against the bacterial growth of Acidovorax avenae subsp. cattlyae (Aac), Clavibacter michiganensis subsp. michiganensis, Xanthomonas arboricola pv. pruni (Xap), and Pseudomonas syringae pv. actinidiae (Psa), with MIC values ranging from 125 to 250 μg/mL. The active extracts of R. tanguticum rhizomes were fractionated by using various and repeated chromatographic techniques to yield seven constituents that included three anthraquinones and four stilbenes. Notably, at concentrations ranging from 75 to 300 μg/mL, physcion (DH03) and chrysophanol (DH04) were significantly effective in suppressing the development of barley powdery mildew (BPM) caused by B. graminis f. sp. hordei, with control values from 80 to 96.7%. Moreover, stilbenes rhapontigenin (DH02) and desoxyrhapontigenin (DH05) had a broad spectrum and potent activity against phytopathogenic bacteria; DH05 effectively inhibited the bacterial growth of Aac, Burkholderia glumae and Psa at MICs ranging from 38 to 150 μg/mL. In this paper, we report their antibacterial activity for the first time. The botanical materials containing anthraquinones and stilbenes were active against both phytopathogenic fungi and bacteria. These results suggested that R. tanguticum rhizomes could be used as a new source of antimicrobial substances for developing a botanical fungicide and bactericide to control plant diseases.

BACKGROUND The endolichenic fungus Xylaria grammica KCTC 13121BP showed strong nematicidal activity against Meloidogyne incognita. This study aims 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 an EC50/72 h value of 15.9 μg/mL and EC50/14d value 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.

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 cine rea, Colletotrichum coccodes, Endothia parasitica, Fu sarium 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 antifun gal metabolite from the butanol extract was identified as iturin A by thin layer chromatography-bioautogra -phy In addition, volatile organic compounds produced by DA12 were able to inhibit mycelial growth of vari ous 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 amyloliquefa ciens 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.

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.

Flowering cherry (FC, Prunus x yedoensis Matsumura; Somei-yoshino cherry) is an ornamental tree, planted across South Korea and producing stunning flowers in spring. The seasonal blooms are annually celebrated during cherry blossom festivals in many locations across the country. The leaf spot disease is among the most common and important diseases affecting FC trees every year, resulting in premature defoliation and reduced flowering of cherry blossoms in the following year. In May 2018, brown spots (2 to 5 mm), circular to irregular and with dark borders were observed on FC leaves in Hadong, Gyeongsangnamdo, South Korea (35°07'48.9"N, 127°46'53.8"E), with a disease incidence of 55%. Single lesions often coalesced and were sometimes perforated, leaving shot holes. Sampled leaves were surface sterilized with 1% NaOCl for 1 min and 70% ethanol for 30 s, and then rinsed twice with sterile distilled water. About 2-mm-long infected leaf pieces from the margins of lesions were put onto water agar (WA, 1.5% agar) plates and incubated at 25oC for 72 h. Mycelia grown from symptomatic tissue were transferred to PDA plates, and five similar fungal isolates were obtained from hyphal tips. They produced a strong reddish-orange diffusible pigment on PDA after 5 d and exudates after 8 d. Conidia were globular to pear-shaped, dark, verrucose, multicellular, and 14.8 to 23.5 μm in diameter (av. = 18.7 μm, n = 30) for isolate JCK-CSHF10. These morphological characteristics were consistent with the Epicoccum genus. Three loci, ITS, tub2, and rpb2, from three isolates JCK-CSHF8, JCK-CSHF9, and JCK-CSHF10 were amplified using the primer pairs ITS1F/LR5 (Gardes and Bruns 1993; Vilgalys and Hester 1990), Btub2Fd/Btub4Rd (Woudenberg et al. 2009), and RPB2-5F2/RPB2-7cR (Liu et al. 1999; Sung et al. 2007), respectively. The ITS, tub2, and rpb2 sequences of the three isolates were deposited in Genbank (MW368668-MW368670, MW392083-MW392085, and MW392086-MW392088, respectively), showing 99.6 to 100% identity to E. layuense (E33), a later synonym for E. tobaicum (Hou et al. 2020). The phylogenetic tree using concatenated sequences of the three loci placed the three isolates in a cluster of E. tobaicum (CBS 232.59, CGMCC 3.18362, and CBS 384.36; Hou et al. 2020). Taken together, the three isolates were identified as E. tobaicum. The pathogenicity of JCK-CSHF10 was tested on 15 healthy leaves on three FC trees (cv. Somei-yoshino, 1.2 m in height) kept in a greenhouse. Five-mm-diameter plugs from 7-d-old fungal cultures grown on PDA or mycelia-free PDA plugs as controls were placed on the abaxial side of a leaf at three points, previously wounded by a sterile needle (Zlatkovic et al. 2016). Inoculation sites were covered with moist cotton plugs. Trees were then covered with a clear plastic bag and maintained in high humidity at 25oC in darkness for 24 h, followed by a 12-h photoperiod. Brown spots appeared on inoculated leaves after 7 d, identical to those observed in the field, while control leaves remained symptomless. This experiment was repeated three times. A fungus with the same morphology as JCK-CSHF10 was recovered from lesions, thus confirming Koch's postulates. E. layuense (syn. E. tobaicum) has been reported as a leaf spot-causing agent on Perilla sp. (Chen et al. 2017) and Camellia sinensis (Chen et al. 2020). To date, there is no report on the occurrence of E. tobaicum from leaf spots on FC. To our knowledge, this is the first report of E. tobaicum causing leaf spot on FC in South Korea.

Fusarium head bight (FHB) is a devastating disease on major cereal crops worldwide which causes primarily by Fusarium graminearum. Synthetic fungicides are generally used in conventional agriculture to control FHB. Their prolonged usage has led to environmental issues and human health problems. This has prompted interest in developing environmentally friendly biofungicides, including botanical fungicides. In this study, a total 100 plant extracts were tested for antifungal activity against F. graminearum. The crude extract of Pterocarpus santalinus heartwood showed the strongest antifungal activity and contained two antifungal metabolites which were identified as α-cedrol and widdrol by GC-MS analysis. α-Cedrol and widdrol isolated from P. santalinus heartwood extract had 31.25 mg/l and 125 mg/l of minimal inhibitory concentration against the spore germination of F. graminearum, and also showed broad spectrum antifungal activities against various plant pathogens. In addition, the wettable powder type formulation of heartwood extract of P. santalinus decreased FHB incidence in dose-dependent manner and suppressed the development of FHB with control values of 87.2% at 250-fold dilution, similar to that of chemical fungicide (92.6% at 2,000-fold dilution). This study suggests that the heartwood extract of P. santalinus could be used as an effective biofungicide for the control of FHB.

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 β-sitosterol (1), campesterol (2), stigmasterol (3), cauloside A (4) and a novel dipsacus saponin, named colchiside (3-O-β-d-xylopyranosyl-23-O-β-d-glucopyranosyl-28-O-β-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.

Ralstonia solanacearum species complex (RSSC) causes bacterial wilt, and it is one of the most important soil-borne plant pathogenic bacteria. RSSC has a large host range of more than 50 botanical families, which represent more than 200 plant species, including tomato. It is difficult to control bacterial wilt due to following reasons: the bacterial wilt pathogen can grow inside the plant tissue, and it can also survive in soil for a long period; moreover, it has a wide host range and biological diversity. In most previous studies, scientists have focused on developing biological control agents, such as antagonistic microorganisms and botanical materials. However, biocontrol attempts are not successful. Plant-derived metabolites and extracts have been promising candidates to environmentally friendly control bacterial wilt diseases. Therefore, we review the plant extracts, essential oils, and secondary metabolites that show potent in vivo antibacterial activities (in potted plants or in field) against tomato bacterial wilt, which is caused by RSSC.

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.

Paenibacillus elgii JCK1400 shows strong antifungal activity against various plant pathogenic fungi in vitro, but little is known about its mode of action. Four antifungal lipopeptides were isolated from P. elgii JCK1400 using bioassay-directed fractionation. Their chemical structures were determined to be pelgipeptins (PGPs) using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. Among the four lipopeptides, PGP-C showed the strongest mycelial growth inhibitory activity against several plant pathogenic fungi—with minimum inhibitory concentration (MIC) values ranging from 4 to 32 μg mL−1—followed by PGP-D, -A, and -B. In pot experiments, PGP-C also effectively suppressed the development of important fungal diseases in crops. In particular, PGP-C was effective in controlling tomato grey mold and wheat leaf rust, with control values of 91% and 73%, respectively, at a concentration of 125 μg mL−1. The fermentation broth of the antagonistic bacterium reduced the development of creeping bentgrass dollar spot and Kentucky bluegrass brown patch in a dose-dependent manner. However, our study on the effect of PGP-C on the fungal cell membrane—using microscopic observation with propidium iodide (PI) fluorescence—indicated that PGP-C does not target the fungal cell walls, but instead targets the cell membranes. This is the first study to report the in vitro and in vivo antifungal activity of PGP-C against various plant pathogenic fungi. Our results suggest that P. elgii JCK1400, which produces PGPs, could serve as a potential biocontrol agent for plant diseases caused by various fungi.

Five isolates of Curvularia inaequalis were obtained from blighted leaves of zoysia grass. The optimal growth temperature of the pathogen was 30°C and all isolates caused foliar blighting symptoms on zoysia grass, bent grass, and bermuda grass. Phytotoxic substances were associated with pathogenicity. Two phytotoxins were isolated from liquid cultures of C. inaequalis by ethyl acetate extraction and repeated silica gel column chromatography. On the basis of mass and nuclear magnetic resonance spectral analyses, the compounds were identified as pyrenocines A and B, phytotoxins produced by Pyrenochaeta terrestris. Pyrenocine A caused leaf necrosis in a leaf-wounding bioassay and inhibited growth of all plants tested in a whole plant test. Large crab grass and fall panicum were most susceptible to pyrenocine A. Pyrenocines A and B also caused significant electrolyte leakage from leaf tissues of bermuda grass. Pyrenocine B exhibited much weaker phytotoxic activity than pyrenocine A in all bioassays performed. Both compounds caused leaf tip dieback symptoms in turf grass plants similar to symptoms observed in the field. Thus, pyrenocines A and B are thought to be involved in the development of Curvularia blight disease of turfgrasses caused by C. inaequalis.

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-β-glucose, 1,2,4,6-tetra-O-galloyl-β-glucose, and 1,2,3,6-tetra-O-galloyl-β-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-β-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.

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.

Molecular mechanisms underlying the responses to environmental factors, such as nitrogen, carbon and pH, involve components that regulate the production of secondary metabolites, including mycotoxins. In this study, we identified and characterized a gene in the FGSG_02083 locus, designated as FgArt1, which was predicted to encode a Zn(II)2 Cys6 zinc finger transcription factor. An FgArt1 deletion mutant of Fusarium graminearum exhibited impaired starch hydrolysis as a result of significantly reduced α-amylase gene expression. The deletion strain was unable to produce trichothecenes and exhibited low Tri5 and Tri6 expression levels, whereas the complemented strain showed a similar ability to produce trichothecenes as the wild-type strain. In addition, FgArt1 deletion resulted in impairment of germination in starch liquid medium and reduced pathogenicity on flowering wheat heads. To investigate the roles of the FgArt1 homologue in F. verticillioides, we deleted the FVEG_02083 gene, and the resulting strain showed defects in starch hydrolysis, similar to the FgArt1 deletion strain, and produced no detectable level of fumonisin B1 . Fum1 and Fum12 expression levels were undetectable in the deletion strain. However, when the FvArt1-deleted F. verticillioides strain was complemented with FgArt1, the resulting strain was unable to recover the production of fumonisin B1 , although FgArt1 expression and starch hydrolysis were induced. Thus, our results suggest that there are different regulatory pathways governed by each ART1 transcription factor in trichothecene and fumonisin biosynthesis. Taken together, we suggest that ART1 plays an important role in both trichothecene and fumonisin biosynthesis by the regulation of genes involved in starch hydrolysis.

This study was conducted to establish an efficient screening system for resistant tomato to bacterial wilt caused by Ralstonia solanacearum. Under several conditions such as inoculation methods, growth stages of tomato seedlings, inoculum concentrations, and incubating temperatures after inoculation, development of bacterial wilt on nine resistant or susceptible cultivars of tomato was investigated. To inoculate by drenching the non-cut roots with the bacterial suspension was better to distinguish resistance and susceptibility of tomato cultivars than by drenching the cut roots using scalpel. And ‘Hawaii7996’ a resistant tomato to R. solanacearum showed high resistance at all the tested conditions including growth stages (3-, 6-, 8-, 10-leaf stages), inoculum concentrations (OD600=0.1-0.4) and incubation temperatures (25, 30, 35°C). On the other hands, susceptible cultivars represented disease index of 3.7 and 3.9 at 6- and 8-leaf stages, respectively. At 3- and 10-leaf stages, the cultivars demonstrated lower disease severity of 2.1 and 0.5, respectively, than at 6- and 8-leaf stages. When the inoculated seedlings were incubated in growth chambers of 25, 30 and 35°C, disease severity of susceptible cultivars was significantly greater at 30 and 35°C than at 25°C. In addition, the level of resistance of the tomato cultivars was not significantly affected by inoculum concentrations of OD600=0.1–0.4. On the basis of the results, we suggest an efficient screening method to measure resistance level of tomato cultivars to bacterial wilt. The eight-leaf stage seedlings transplanted 7 days before inoculation, are inoculated with R. solanacearum by drenching the non-cut roots with a bacterial suspensions (OD600=0.4) to give inoculum volume of 50 ml/soil l. The inoculated plants are incubated in a growth room at 30°C for 12-13 days with 12-hour light a day.

Colletotrichum species are major fungal pathogens that cause devastating anthracnose diseases in many economically important crops. In this study, we observed the hydrolyzing activity of a fungus-inducible pepper carboxylesterase (PepEST) on cell walls of C. gloeosporioides, causing growth retardation of the fungus by blocking appressorium formation. To determine the cellular basis for the growth inhibition, we observed the localization of PepEST on the fungus and found the attachment of the protein on surfaces of conidia and germination tubes. Moreover, we examined the decomposition of cell-wall materials from the fungal surface after reaction with PepEST, which led to the identification of 1,2-dithiane-4,5-diol (DTD) by gas chromatography mass spectrometry analysis. Exogenous DTD treatment did not elicit expression of defense-related genes in the host plant but did trigger the necrosis of C. gloeosporioides. Furthermore, the DTD compound displayed protective effects on pepper fruits and plants against C. gloeosporioides and C. coccodes, respectively. In addition, DTD was also effective in preventing other diseases, such as rice blast, tomato late blight, and wheat leaf rust. Therefore, our results provide evidence that PepEST is involved in hydrolysis of the outmost layer of the fungal cell walls and that DTD has antifungal activity, suggesting an alternative strategy to control agronomically important phytopathogens.

This study was conducted to establish a simple mass-screening method for resistant melon to Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (FOM). Root-dipping inoculation method has been used to investigate resistance of melon plants to Fusarium wilt. However, the inoculation method requires a lot of labor and time because of complicate procedure. To develop a simple screening method on melon Fusarium wilt, occurrence of Fusarium wilt on susceptible and resistant cultivars of melon according to inoculation method including root-dipping, soil-drenching, tip, and scalpel methods was investigated. Scalpel and tip methods showed more clear resistant and susceptible responses in the melon cultivars than root-dipping inoculation method, but tip method represented slightly variable disease severity. In contrast, in the case of soil-drenching inoculation method, disease severity of the susceptible cultivars was very low. Thus we selected scalpel method as inoculation method of a simple screening method for melon Fusarium wilt. By using the scalpel inoculation method, resistance degrees of the cultivars according to incubation temperature after inoculation (25 and 30°C) and inoculum concentration (1×106 and 1×107 conidia/ml) were measured. The resistance or susceptibility of the cultivars was hardly affected by all the tested conditions. To look into the effectiveness of scalpel inoculation methods, resistance of 22 commercial melon cultivars to FOM was compare with root-dipping inoculation method. When the melon cultivars were inoculated by scalpel method, resistance responses of all the tested cultivars were clearly distinguished as by root-dipping method. Taken together, we suggest that an efficient simple mass-screening method for resistant melon plant to Fusarium wilt is to sow the seeds of melon in a pot (70 ml of soil) and to grow the seedlings in a greenhouse (25±5°C) for 7 days, to cut the root of seedlings with a scalpel and then pour a 10 ml-aliquot of the spore suspension of 1×106 conidia/ml on soil. The infected plants were cultivated in a growth room at 25 to 30°C for about 3 weeks with 12-hr light a day.

Control of nematode has become difficult owing to the restricted use of effective soil fumigant, methyl bromide, and other non-fumigant nematicides. Therefore, it is urgently necessary to develop microbial nematicide to replace chemical nematicides. In this study, the 50% aqueous methanol extraction solution of fermentation broths of 2,700 actinomycete strains were tested for their nematicidal activity against second stage of juveniles (J2s) of Meloidogyne incognita. As the results, only the 50% aqueous methanol extraction solution of AN110065, at 20% equivalent to 10% fermentation broth, showed strong nematicidal activity with 78.9% of mortality 24 h after treatment and 94.1% of mortality at 72 h. The 16S rRNA gene sequencing showed that the strain sequence was 99.78% identical to Streptomyces netropsis. The extract of S. netropsis AN110065 fermentation broth was successively partitioned with ethyl acetate and butanol and then the ethyl acetate, butanol and water layers were investigated for their nematicidal activity against the M. incognita. At 1,000 mg/ml, ethyl acetate layer showed the strongest activity of 83.5% of juvenile mortality 72 h after treatment. The pot experiment using the fermentation broth of AN110065 on tomato plant against M. incognita displayed that it evidently suppressed gall formation at a 10-fold diluent treatment. The tomato plants treated with the fermentation broth of S. netropsis AN110065 did not show any phytotoxicity. The results suggest that S. netropsis AN110065 has a potential to serve as microbial nematicide in organic agriculture.

Clubroot and Fusarium wilt of cole crops (Brassica oleracea L.) are destructive diseases which for many years has brought a decline in quality and large losses in yields all over the world. The breeding of resistant cultivars is an effective approach to reduce the use of chemical fungicides and minimize crop losses. This study was conducted to evaluate the resistance of 60 cabbage (B. oleracea var. capitata) and 6 broccoli (B. oleracea var. italica) lines provided by The RDA-Genebank Information Center to clubroot and Fusarium wilt. To investigate resistance to clubroot, seedlings of the genetic resources were inoculated with Plasmodiophora brassicae by drenching the roots with a mixed spore suspension (1 : 1) of two isolates. Of the tested genetic resources, four cabbage lines were moderately resistant and ‘K166220’ represented the highest resistance to P. brassicae. The others were susceptible to clubroot. On the other hand, to select resistant plants to Fusarium wilt, the genetic resources were inoculated with Fusarium oxysporum f. sp. conglutinans by dipping the roots in spore suspension of the fungus. Among them, 17 cabbage and 5 broccoli lines were resistant, 16 cabbage lines were moderately resistant, and the others were susceptible to Fusarium wilt. Especially, three cabbage (‘IT227115’, ‘K161791’, ‘K173350’) and two broccoli (‘IT227100’, ‘IT227099’) lines were highly resistant to the fungus. We suggest that the resistant genetic resources can be used as a basic material for resistant B. oleracea breeding system against clubroot and Fusarium wilt.

The study was performed to establish an efficient screening method for resistant cucumber to Fusarium oxysporum f. sp. cucumerinum. The isolate KR5 was identified as F. oxysporum f. sp. cucumerinum based on molecular analyses of ITS and TEF genes and host-specificity test on cucurbits including melon, oriental melon, cucumber, and watermelon. Then four cucumber and two rootstock cultivars showing different resistance degrees to the Fusarium wilt pathogen KR5 were selected. And development of Fusarium wilt of the six cultivars according to several conditions, including incubation temperature after inoculation, inoculum concentration, root wounding, and growth stages of seedlings, was investigated. Disease severity of Fusarium wilt on the resistant cultivars was changed with incubation temperatures after inoculation. The resistant cultivars showed the higher resistance when inoculated plants were kept at 25 or 30oC than at 20oC. Among four different growth stages of the seedlings, seven-day-old seedling represented the most difference of resistance and susceptibility to Fusarium wilt. From above results, we suggest that an efficient screening method for resistant cucumber to F. oxysporum f. sp. cucumerinum is to dip the non-cut roots of seven-day-old seedlings in spore suspension of 1.0 × 106-1.0 × 107 conidia/ml and to transplant the seedling into a non-infected soil, and then to incubate the inoculated plants in a growth room at 25oC for 3 weeks to develop Fusarium wilt.

Fusarium graminearum is an important fungal plant pathogen that causes serious losses in cereal crop yields and mycotoxicoses in humans and livestock. In this study, we characterized an insertion mutant, Z39R9282, with pleiotropic defects in sexual development and virulence. We determined that the insertion occurred in a gene encoding an ortholog of yeast elongator complex protein 3 (ELP3). Deletion of elp3 led to significant defects in sexual and asexual development in F. graminearum. In the elp3 deletion mutant, the number of perithecia formed was reduced and maturation of perithecia was delayed. This mutant also produced morphologically abnormal ascospores and conidia. Histone acetylation in the elp3 deletion mutant was reduced compared with the wild type, which likely caused the developmental defects. Trichothecenes were not produced at detectable levels, and expression of trichothecene biosynthesis genes were significantly reduced in the elp3 deletion mutant. Infection of wheat heads revealed that the elp3 deletion mutant was unable to spread from inoculated florets to neighboring spikelets. Furthermore, the elp3 deletion mutant was more sensitive to oxidative stress than the wild type, and the expression of putative catalase genes was reduced. We demonstrate that elp3 functions in sexual and asexual development, virulence, and the oxidative stress response of F. graminearum by regulating the expression of genes involved in these various developmental processes.

Damping-off, caused by Rhizoctonia solani and Pythium ultimum, is a very important plant disease and affects seeds and seedlings of many plant species worldwide. To investigate control efficacy of Serenade formulation (1.34%, SC) against Rhizoctonia and Pythium damping-off diseases, seeds of chili pepper and cucumber were sown in soils inoculated with R. solani and P. ultimum and Serenade formulation was applied by soil-drenching with 9-, 19-, and 39-fold dilutions. Control values of Serenade formulation on Rhizoctonia damping-off of chili pepper was 58% and 29% for 9- and 19-fold dilutions, respectively. In the case of cucumber Rhizoctonia damping-off, Serenade treatments showed similar control efficacy with damping-off of chili pepper. On the other hand, control efficacy of Serenade formulation on Pythium damping-off of cucumber was less than control effects on Rhizoctonia damping-off. Only Pythium damping-off of chili pepper treated with 9-fold dilution Serenade was statistically different with untreated control. This result suggest that Serenade formulation could be effectively used for controlling Rhizoctonia and Pythium damping-off diseases.

Root-knot nematodes represent a significant problem in cucumber, causing reduction in yield and quality. To develop screening methods for resistance of cucumber to root-knot nematode Meloidogyne incognita, development of root-knot nematode of four cucumber cultivars (‘Dragonsamchuk’, ‘Asiastrike’, ‘Nebakja’ and ‘Hanelbakdadaki’) according to several conditions such as inoculum concentration, plant growth stage and transplanting period was investigated by the number of galls and egg masses produced in each seedling 45 days after inoculation. There was no difference in galls and egg masses according to the tested condition except for inoculum concentration. Reproduction of the nematode on all the tested cultivars according to inoculum concentration increased in a dose-dependent manner. On the basis of the result, the optimum conditions for root-knot development on the cultivars is to transplant period of 1 week, inoculum concentration of 5,000 eggs/plant and plant growth stage of 3-week-old in a greenhouse (25 ± 5°C). In addition, under optimum conditions, resistance of 45 commercial cucumber cultivars was evaluated. One rootstock cultivar, Union was moderately resistant to the root-knot nematode. However, no significant difference was in the resistance of the others cultivar. According to the result, we suggest an efficient screening method for new resistant cucumber to the root-knot nematode, M. incognita.

Black rot caused by Xanthomonas campestris pv. campestris (Xcc) is one of the most serious diseases ofcrucifers world-wide. To establish the efficient screening method for resistant cabbage to Xcc, differentinoculation methods, inoculation positions, growth stages of seedlings, and incubation temperatures afterinoculation were investigated with the seven cabbage cultivars showing different resistance degrees to thepathogen. Clipping with mouse-tooth forceps was better inoculation method than piercing with 18 pins orcutting with scissors to distinguish the level of resistance and susceptibility. In inoculation using mouth-toothforceps, clipping the edges of the leaves near veins is more effective than injuring the veins of the leavesdirectly. In addition, the inoculated plants kept at 22oC showed more clear resistant and susceptible responsesthan those kept at 26 or 30oC. On the basis of the results, we suggest that an efficient screening method forresistance of cabbage cultivars to black rot is to clip the edges of the leaves near veins of the four-week-oldseedlings with mouth-tooth forceps dipped in a suspension of Xcc at a concentration of 7 × 107 cfu/ml andincubate the inoculated plants in a growth room at 22oC with 12-hr light a day.

Diverse bacteria are known to colonize plants. However, only a small fraction of that diversity has been evaluated for their biopesticide potential. To date, the criteria for sampling and selection in such bioprospecting endeavors have not been systematically evaluated in terms of the relative amount of diversity they provide for analysis. The present study aimed to enhance the success of bio-prospecting efforts by increasing the diversity while removing the genotypic redundancy often present in large collections of bacteria. We developed a multivariate sampling and marker-based selection strategy that significantly increase the diversity of bacteria recovered from plants. In doing so, we quantified the effects of varying sampling intensity, media composition, incubation conditions, plant species, and soil source on the diversity of recovered isolates. Subsequent sequencing and high-throughput phenotypic analyses of a small fraction of the collected isolates revealed that this approach led to the recovery of over a dozen rare and, to date, poorly characterized genera of plant-associated bacteria with significant biopesticide activities. Overall, the sampling and selection approach described led to an approximately 5-fold improvement in efficiency and the recovery of several novel strains of bacteria with significant biopesticide potential.

Plants are attacked by various phytopathogenic fungi. For many years, synthetic fungicides have been used to control plant diseases. Although synthetic fungicides are highly effective, their repeated use has led to problems such as environmental pollution, development of resistance, and residual toxicity. This has prompted intensive research on the development of biopesticides, including botanical fungicides. To date, relatively few botanical fungicides have been registered and commercialized. However, many scientists have reported isolation and characterization of a variety of antifungal plant derivatives. Here, we present a survey of a wide range of reported plant-derived antifungal metabolites.

Rusts are plant diseases caused by pathogenic fungi of the order Pucciniales. Rusts can affect a variety of plants. Development of new effective and environmentally benign agents against rusts is of great interest. In this study, we investigated the disease control efficacy of the emulsion concentrate (EC10) and wettable powder (WP20) type formulations of the extract of Magnolia officinalis (Magjia90; containing honokiol and magnolol at 90%) against rust diseases of perilla and zoysiagrass in fields. The treatment of EC10 and WP20 of Magjia90 showed control values of 47.9% to 69.6% and Magjia90-WP20 reduced more effectively the development of rust symptoms on perilla plants than Magjia90-EC10. Magjia90-WP20 also highly suppressed zoysiagrass rust with control values of 65.7% to 80.5%. On the other hand, no harmful effect of Magjia90- EC10 and Magjia90-WP20 was observed on the perilla and zoysiagrass plants tested. The results strongly indicate that the extract of M. officinalis (Magjia90) can be used as a natural fungicide for the control of rust diseases.

Peroxisomes are organelles that are involved in a number of important cellular metabolic processes, including the β-oxidation of fatty acids, biosynthesis of secondary metabolites, and detoxification of reactive oxygen species (ROS). In this study, the role of peroxisomes was examined in Fusarium graminearum by targeted deletion of three genes (PEX5, PEX6, and PEX7) encoding peroxin (PEX) proteins required for peroxisomal protein import. PEX5 and PEX7 deletion mutants were unable to localize the fluorescently tagged peroxisomal targeting signal type 1 (PTS1)- and PTS2-containing proteins to peroxisomes, respectively, whereas the PEX6 mutant failed to localize both fluorescent proteins. Deletion of PEX5 and PEX6 resulted in retarded growth on long-chain fatty acids and butyrate, while the PEX7 deletion mutants utilized fatty acids other than butyrate. Virulence on wheat heads was greatly reduced in the PEX5 and PEX6 deletion mutants, and they were defective in spreading from inoculated florets to the adjacent spikelets through rachis. Deletion of PEX5 and PEX6 dropped survivability of aged cells in planta and in vitro due to the accumulation of ROS followed by necrotic cell death. These results demonstrate that PTS1-dependent peroxisomal protein import mediated by PEX5 and PEX6 are critical to virulence and survival of F. graminearum.

Biotransformation is an eco-friendly and efficient method for enhancing the bioavailability of biopesticide. Toincrease the antifungal activity of the crude extract of Cynanchum wilfordii roots against barely powderymildew, we performed biotransformation of wilfoside C1G using β-glucosidase (cellobiase from Aspergillusniger). The mixture (G sample) of partially purified wilfoside C1G and cynauricuoside A (K1G) was treatedwith β-glucosidase to remove a glucopyranosyl moiety. The enzyme completely converted C1G to C1N andK1G to K1N. Optimal conditions for enzymatic biotransformation of G sample were determined to be 10%ethanol, 1,555 μU β-glucosidase/ml, pH 5, and 45oC. In in vivo experiment, the G sample transformed by β-glucosidase showed stronger antifungal activity against barley powdery mildew than the non-treated Gsample. These results suggest that β-glucosidase biotransformation can be applied to increase the antifungalactivity of the crude extract of C. wilfordii roots against powdery mildews.

The problems caused by synthetic pesticides have led the need for effective biodegradable pesticides withgreater selectivity. Botanical pesticides are generally recognized as safe in agriculture systems. Thus, theyhave been regarded as attractive alternatives to synthetic chemical pesticides for the pest management. Bothlower efficacy and higher costs of production make botanicals more expensive to use than conventionalpesticides. Moreover, only a small portion of plant-derived metabolites among a number of bioactivemetabolites are in use because commercialization of botanicals is inhibited by several problems such astoxicity, or high production cost. However, with the growing acceptance of botanical pesticides as an efficientcrop protection alternative resulting in increasing demand, plant-based pesticides will play a significant rolein achieving sustainable agriculture in future.

Plants are the promising reservoirs for natural products with their diverse secondary metabolites. Many invasive plants have been introduced in Korea, which adversely affect on the native ecosystem but holds difficulty removing them due to their proliferation. In this study, we evaluated disease control efficacy of methanol extracts from four invasive plant species against 7 representative crop pathogens. Methanol extract of Phytolacca americana effectively suppressed rice blast, tomato gray mold, and tomato late blight in a dose dependent manner. The methanol extract of Amorpha fruticosa also exhibited potent antifungal activity against pepper anthracnose in a concentration dependent way. These data suggest that the extracts of P. americana and A. fruticosa can be developed as plant disease protection agents against rice blast, tomato gray mold, tomato late blight, and pepper anthracnose. Furthermore, more extensive research will be required to identify and isolate active compounds from problematic invasive plant species to develop valuable agrochemicals.

In 2006, pinewood nematode, Bursaphelenchus xylophilus, was isolated from about 50 years old trees of Pinus densiflora and P. koraiensis showing leaf-wilt and -drying symptoms in Gwangju, Gyeonggi-do and Chuncheon, Gangwon-do. Isolation of pinewood nematodes from sapwood of infected pine trees showed no difference in population density between tree species and among the sampling heights on the main stem. Migration of pinewood nematodes in the host tree were investigated by inoculation of red pine (P. densiflora, 3 years old) seedlings with B. xylophilus. The nematodes seemed to move in red pine seedlings prior to multiplication and it might have taken about 20 days to start multiplication and expression of symptoms including wilt and dieback. In initial time after inoculation, nematodes started migration through the cortical resin canal from inoculated site and further showed upward and downward movements. More nematodes were observed in cortical resin canal during early period of inoculation and later in resin canal of xylem and tracheid also while, the pith still remained free from nematode. The density of B. xylophilus was higher in seedlings of low-vigor with poor root growth than in seedlings of normal root growth. Seedlings showing high density of B. xylophilus exhibited stem discoloration and secondary infection by fungus at the inoculation site.

Clubroot caused by Plasmodiophora brassicae Woron. is one of the most important diseases in Brassica cropsworldwide. To establish more simple and reliable screening method for resistant cabbage and broccoli to P.brassicae, the development of clubroot on the plants according to inoculum concentration and incubationperiod after inoculating with the pathogen was investigated using P. brassicae GN1 isolate (race 9). Tofacilitate and acquire precise result of resistance screening of cabbage and broccoli to clubroot, 14-day- ldseedlings were inoculated by drenching roots with the spore suspension of P. brassicae to give inoculumdensity of 2.5× 109 spores/pot. To develop the disease, the inoculated seedlings were incubated in a growthchamber at 20oC for 3 days, and then cultivated in a greenhouse (20±5oC) for five weeks. Under the optimumconditions, 16 cabbage and 17 broccoli cultivars were tested for resistance to four field isolates (GN1, GN2,GS and YC) of P. brassicae collected from four regions in Korea. Among them, some cabbage and broccolicultivars showed different resistance response to three isolates (GN1, GN2 and GS) determined as race 9 byusing the differential varieties of Williams. On the other hand, all the tested cultivars were highly susceptibleto YC isolate (race 2). The results suggest that this method is efficient screening method of cabbage andbroccoli for resistance to P. brassicae.

A bacterial strain antagonistic to some fungal phytopathogens was isolated from the stem of a Persimmon tree in Yeongam, Korea. This bacterium was identified as Bacillus subtilis by 16S rRNA gene sequencing and designated as B. subtilis GDYA-1. In in vivo experiment, the fermentation broth exhibited antifungal activities against Magnaporthe oryzae on rice plants, Phytophthora infestans on tomato plants, and Puccinia recondita on wheat plants. We isolated one antifungal compound and its chemical structure was determined by mass and H-NMR spectral data. The antifungal substance was identified as benzoic acid. It inhibited mycelial growth of M. oryzae, Rhizoctonia solani, Sclerotinia sclerotiorum, and P. capsici with minimum inhibition concentration (MIC) values, ranging from 62.5 to 125 µg/ml. Moreover, the substance effectively suppressed Phytophthora blight of red pepper caused by P. capsici in a pot experiment. To the author’s knowledge, this is the first report on the antifungal activity of benzoic acid against phytopathogenic fungi. Benzoic acid and B. subtilis GDYA-1 may contribute to environmental-friendly protect crops from phytopathogenic fungi.

Blast is considered the most important fungal disease of rice due to its wide distribution and destructiveness under favorable conditions. Development of new effective and environmentally benign agents against the causal pathogen, Magnaporthe oryzae , is of great interest. In the course of a search for natural antifungal compounds in medicinal plants, we found that the methanol extract of Angelica gigas roots showed a potent control efficacy against rice blast caused by M. oryzae . We isolated antifungal coumarins from the extract, and they were identified as decursin and decursinol angelate. Antifungal activities of these compounds, along with kasugamycin, were tested on M. oryzae in vivo and in vitro . In an in vivo assay, the three compounds effectively suppressed the development of rice blast at concentrations more than 100 μg/mL. Coumarins showed relatively weak inhibitory effect on fungal mycelial growth when compared to kasugamycin. However, they strongly inhibited M. oryzae spore germination, which was not observed in kasugamycin treatments. This is the first report demonstrating that decursinol angelate can provide control against rice blast and that the two coumarins inhibit M. oryzae spore germination. In addition, the wettable powder formulation of the crude extract of A. gigas prohibited the development of blast symptoms on rice plants more effectively than liquid concentrate formulation of kasugamin, a commercial fungicide. Based on our study, we propose that coumarin compounds as well as the A. gigas root crude extract can be used as natural, benign fungicides for controlling rice blast.

Department of Applied Plant Science, Kangnung National University, Gangneung 210-702, Korea(Received on June 9, 2011; Revised on June 30, 2011; Accepted on June 30, 2011)Chitosan is a linear polysaccharide composed of randomly distributed s-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine. There have been many reports on the induced systemic resistance and in vivoantifungal activities of higher molecular weight chitosans with molecular weights over 3,000 amu (atomaticmass unit), but there are few papers on in vivo antifungal activities of low molecular weight chitosans(oligochitosans) with molecular weights less than 3,000 amu. In our study, an oligochitosan sample (320 −3,000amu) showed a potent 1-day protective activity with control values more than 94% at concentrations of 500and 1,000 µg/ml especially against tomato late blight caused by Phytophthora infestans under growth chamberconditions. It also displayed a moderate 1-day protective activity with control values of 67−89% atconcentrations of 500 and 1,000 µg/ml against wheat leaf rust and red pepper anthracnose. On the otherhand, it showed a 16-hr curative activity against red pepper anthracnose, but not against tomato late blightand wheat leaf rust. In field experiments, oligochitosan effectively suppressed the development of late blighton potato and tomato plants with control values of 72% and 48%, respectively. The results strongly indicatethat oligochitosan can be used as an eco-friendly organic material for the control of late blight on tomato andpotato plants.Keywords : Control, Oligochitosan, Organic material, Phytophthora infestans , Tomato late blight