Characterization of Alternaria porri causing onion purple blotch and its antifungal compound magnolol identified from Caryodaphnopsis baviensis.

Alternaria porri (Ellis) Clf. causes purple blotch disease on Allium plants which results in the reduction of crop yields and quality. In this study, to efficiently find natural antifungal compounds against A. porri, we optimized the culture condition for the spore production of A. porri and the disease development condition for an in vivo antifungal assay. From tested plant materials, the methanol extracts derived from ten plant species belonging to the families Cupressaceae, Fabaceae, Dipterocarpaceae, Apocynaceae, Lauraceae, and Melastomataceae were selected as potent antifungal agents against A. porri. In particular, the methanol extract of Caryodaphnopsis baviensis (Lec.) A.-Shaw completely inhibited the growth of A. porri at a concentration of 111 µg/ml. Based on chromatographic and spectroscopic analyses, a neolignan compound magnolol was identified as the antifungal compound of the C. baviensis methanol extract. Magnolol showed a significant inhibitory activity against the spore germination and mycelial growth of A. porri with IC50 values of 4.5 and 5.4 µg/ml, respectively. Furthermore, when magnolol was sprayed onto onion plants at a concentration of 500 µg/ml, it showed more than an 80% disease control efficacy for the purple blotch diseases. In terms of the antifungal mechanism of magnolol, we explored the in vitro inhibitory activity on individual oxidative phosphorylation complexes I-V, and the results showed that magnolol acts as multiple inhibitors of complexes I-V. Taken together, our results provide new insight into the potential of magnolol as an active ingredient with antifungal inhibitory action to control purple blotch on onions.

Clerodane Diterpenoids Identified from Polyalthia longifolia Showing Antifungal Activity against Plant Pathogens.

In the search for new natural resources showing plant disease control effects, we found that the methanol extract of Polyalthia longifolia suppressed fungal disease development in plants. To identify the bioactive substances, the methanol extract of P. longifolia was extracted by organic solvents, and consequently, four new 2-oxo-clerodane diterpenes (1-4), a new 4(3 → 2)-abeo-clerodane diterpene (5), together with ten known compounds (6-16) were isolated and identified from the extracts. Of the new compounds, compound 2 showed a broad spectrum of antifungal activity with moderated minimum inhibitory concentration (MIC) values in a range of 50-100 µg/mL against tested fungal pathogens. Considering with the known compounds, compound 6 showed the most potent antifungal activity with an MIC value in the range of 6.3-12.5 µg/mL. When compound 6 was evaluated for an in vivo antifungal activity against rice blast, tomato late blight, and pepper anthracnose, compound 6 reduced the plant disease by at least 60% compared to the untreated control at concentrations of 250 and 500 µg/mL. Together, our results suggested that the methanol extract of twigs and leaves of P. longifolia and its major compound 6 could be used as a source for the development of eco-friendly plant protection agents.

Discovery of New Triterpenoid Saponins Isolated from Maesa japonica with Antifungal Activity against Rice Blast Fungus Magnaporthe oryzae.

While searching for new antifungal compounds, we revealed that a methanol extract of plant species Maesa japonica has a potent antifungal activity in vivo against rice blast fungus Magnaporthe oryzae. To identify the antifungal substances, the methanol extract of M. japonica was extracted by organic solvents, and consequently, six active compounds were isolated from the n-butanol layer. The isolated compounds were five new acylated triterpenoid saponins including maejaposide I (1), maejaposides C-1, C-2, and C-3 (2-4), and maejaposide A-1 (5), along with a known one, maejaposide A (6). These chemical structures were determined by NMR and a comparison of their NMR and MS data with those reported in the literature. Based on the in vitro antifungal bioassay, the five compounds (2-6) exhibited strong antifungal activity against M. oryzae with MIC values ranging from 4 to 32 µg/mL, except for maejaposide I (1) (MIC > 250 µg/mL). When the compounds were evaluated at concentrations of 125, 250, and 500 µg/mL for an in vivo antifungal activity against rice blast, compounds 2-6 strongly reduced the development of blast by at least 85% to 98% compared to the untreated control. However, compound 1 did not show any in vivo antifungal activity up to a concentration of 500 µg/mL. Taken together, our results suggest that the methanol extract of M. japonica and the new acylated triterpenoid saponins can be used as a source for the development of natural fungicides.

Identification of novel compounds, oleanane- and ursane-type triterpene glycosides, from Trevesia palmata: their biocontrol activity against phytopathogenic fungi.

Plants contain a number of bioactive compounds that exhibit antimicrobial activity, which can be recognized as an important source of agrochemicals for plant disease control. As part of our search for new antimicrobial agents from natural sources, we found that a crude methanol extract of Trevesia palmata exhibited a promising antifungal activity against phytopathogenic fungi, such as Magnaporthe oryzae and Botrytis cinerea. Furthermore, based on activity-guided fractionation, we isolated five antifungal compounds from the methanol extract of T. palmata: two new triterpene glycosides (TPGs), TPG1 (hederagenin-3-O-ß-D-glucopyranosyl-(1 → 3)-α-L-rhamnopyranosyl-(1 → 2)-α-L-rhamnopyranosyl-(1 → 2)-α-L-arabinopyranoside) and TPG5 (3-O-α-L-rhamnopyranosyl asiatic acid), along with three known TPGs (TPG2 [macranthoside A], TPG3 [α-hederin], and TPG4 [ilekudinoside D]). The chemical structures of the TPGs were determined by spectroscopic analyses and by comparison with literature data. An in vitro antifungal bioassay revealed that except for TPG4 (ilekudinoside D; IC50 >256 µg/ml), the other TPGs exhibited strong antifungal activities against the rice blast pathogen M. oryzae with IC50 values ranging from 2-5 µg/ml. In particular, when the plants were treated with compound TPG1 (500 µg/ml), disease control values against rice blast, tomato grey mold, tomato late blight, and wheat leaf rust were 84, 82, 88, and 70%, respectively, compared to the non-treatment control. Considering the in vitro and in vivo antifungal activities of the TPGs and the T. palmata methanol extracts, our results suggest that T. palmata can be a useful source to develop new natural fungicides.

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.

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.

Efficacies of quorum sensing inhibitors, piericidin A and glucopiericidin A, produced by Streptomyces xanthocidicus KPP01532 for the control of potato soft rot caused by Erwinia carotovora subsp. atroseptica.

To discover potential inhibitors of the quorum sensing (QS) system, a library of microbial culture extracts was screened with Chromobacterium violaceumCV026 strain. The culture extract of Streptomyces xanthocidicus KPP01532 contained quorum-sensing inhibitors (QSIs) of the CV026 strain. The active constituents of the culture extract of strain KPP01532 were purified using a series of chromatographic procedures, and based on data from NMR and mass spectroscopy, piericidin A and glucopiericidin A were identified. Erwinia carotovora subsp. atroseptica (Eca) is a plant pathogen that causes blackleg and soft rot diseases on potato stems and tubers. The virulence factors of Eca are regulated by QS. The expression of virulence genes (pelC, pehA, celV and nip) under the control of QS was monitored using quantitative real-time PCR (qRT-PCR). The transcription levels of the four genes were significantly lower when Eca was exposed to piericidin A or glucopiericidin A. These two compounds displayed similar control efficacies against soft rot caused by Eca in potato slices as furanone C-30. Therefore, piericidin A and glucopiericidin A are potential QSIs that suppress the expression of the virulence genes of Eca, suggesting that they could have potential use as control agents of soft rot disease on potato tubers.

Diversities in virulence, antifungal activity, pigmentation and DNA fingerprint among strains of Burkholderia glumae.

Burkholderia glumae is the primary causal agent of bacterial panicle blight of rice. In this study, 11 naturally avirulent and nine virulent strains of B. glumae native to the southern United States were characterized in terms of virulence in rice and onion, toxofalvin production, antifungal activity, pigmentation and genomic structure. Virulence of B. glumae strains on rice panicles was highly correlated to virulence on onion bulb scales, suggesting that onion bulb can be a convenient alternative host system to efficiently determine the virulence of B. glumae strains. Production of toxoflavin, the phytotoxin that functions as a major virulence factor, was closely associated with the virulence phenotypes of B. glumae strains in rice. Some strains of B. glumae showed various levels of antifungal activity against Rhizoctonia solani, the causal agent of sheath blight, and pigmentation phenotypes on casamino acid-peptone-glucose (CPG) agar plates regardless of their virulence traits. Purple and yellow-green pigments were partially purified from a pigmenting strain of B. glumae, 411gr-6, and the purple pigment fraction showed a strong antifungal activity against Collectotrichum orbiculare. Genetic variations were detected among the B. glumae strains from DNA fingerprinting analyses by repetitive element sequence-based PCR (rep-PCR) for BOX-A1R-based repetitive extragenic palindromic (BOX) or enterobacterial repetitive intergenic consensus (ERIC) sequences of bacteria; and close genetic relatedness among virulent but pigment-deficient strains were revealed by clustering analyses of DNA fingerprints from BOX-and ERIC-PCR.