Host plant defenses of black (Solanum nigrum L.) and red nightshade (Solanum villosum Mill.) against specialist Solanaceae herbivore Leptinotarsa decemlineata (Say)

Black nightshade (Solanum nigrum, S. nigrum L.) and red nightshade ( Solanum villosum, S. villosum Mill.) are medicinal plants from the Solanaceae family that synthesize glycoalkaloids and other secondary metabolites. To recognize the potential insecticide activity of these compounds, leaf extracts (containing glycoalkaloid and methanol fractions) were tested for enzyme inhibition, antifeedant activity and toxicity. For in‐vitro glutathione S‐transferase (GST) inhibition activity, we used insecticide‐resistant Colorado potato beetle, Leptinotarsa decemlineata ( L. decemlineata; Say) midgut and fat‐body homogenate. In‐vivo toxicity and the antifeedant activity were performed using larval bioassays. The methanol extracts had greater GST inhibitory activity compared to the glycoalkaloids, as well as greater 2nd instar larvae mortality and antifeedant activity. Furthermore, the green leaf volatile compound, cis‐hex‐3‐enyl acetate, at the concentration of 5 ppm, caused 50% mortality of 2nd instar larvae. Our findings suggest the potential usefulness of S. nigrum and S. villosum extracts to control L. decemlineata.     

Antifungal Activity of Volatile Compounds Produced by an Edible Mushroom Hypsizygus marmoreus against Phytopathogenic Fungi

Volatile compounds with antifungal activity produced by edible mushrooms have potential as biological control agents to combat fungal diseases and reduce fungicide use in agriculture. Here we investigated the antifungal activity of volatile compounds produced by the edible mushroom Hypsizygus marmoreus (TUFC 11906) against eight phytopathogenic fungi. The results showed that volatile compounds from the mycelia and culture filtrates (CFs) of H. marmoreus had antifungal activity against some phytopathogenic fungi. Among them, the mycelial growth and conidial germination of Alternaria brassicicola were significantly inhibited by 60 and 100%, respectively. Moreover, the volatile compounds from CFs inhibited the lesion formation of A. brassicicola on detached cabbage leaves by 94%. The volatile compounds had higher antifungal activity against A. brassicicola than other fungi. With the removal of the volatile compounds from conidia of A. brassicicola, the conidia began to germinate, which indicates fungistatic activity of the compounds. The volatile compounds were isolated from the CFs of H. marmoreus, and the major volatile compound with antifungal activity was estimated to be 2‐methylpropanoic acid 2,2‐dimethyl‐1‐(2‐hydroxy‐1‐methylethyl)propyl ester. As the volatile compound produced by H. marmoreus is a product of an edible mushroom and has fungistatic activity against some phytopathogenic fungi, especially A. brassicicola, it may be possible to use the compounds as a novel safe agent for protecting crops in the field and during storage.     

Biological containment of potato (Solanum tuberosum): outcrossing to the related wild species black nightshade (Solanum nigrum) and bittersweet (Solanum dulcamara)

The biological containment of the potato (Solanum tuberosum) was assessed by establishing the crossability of this tuberous crop with the related wild non-tuberous species in The Netherlands, black nightshade (S. nigrum) and bittersweet (S. dulcamara). To circumvent crossability barriers, genotypes with different ploidy number were employed and crosses were performed under different environmental conditions. S. dulcamara was shown to be incongruent with potato at all ploidy levels, while S. nigrum displayed unilateral incompatibility. If S. nigrum was emasculated and used as female, fertilization by potato pollen resulted in berry set and seed development. Emasculation of S. nigrum was essential in this cross, because analysis of the fertilization process demonstrated that this species is highly self-compatible and potato pollen was outcompeted by pollen of S. nigrum. The hybrid seeds derived from this cross did not mature and appeared not to be viable. By application of the technique of embryo rescue of immature embryos, hybrid plants could be obtained. However, these hybrid plants proved to be sterile. These data demonstrate that gene flow by pollen dispersal from potato to its most common wild relatives in Western Europe is highly unlikely. The potato is thus a naturally contained species in this part of the world.     

Dieter Seidel‐65 Jahre

Fruit extract of Solanum xanthocarpum was evaluated for its toxicity against Alternaria brassicae, the causal agent of Alternaria blight of Indian mustard [Brassica juncea (L.) Czern. &; Coss]. The mass obtained after vacuum drying of the crude methanolic extract was utilised for further sequential fractionation using n-hexane, ethyl acetate, n-butanol and methanol. Among the crude and different fractions tested, methanolic fraction was most effective with a minimum inhibitory concentration (MIC) of 62.5 μg/ml. The methanolic fraction was further fractionated using open column liquid chromatography into five subfractions (I–V) to identify the antifungal bioactive compounds. Among the five subfractions (SFs) tested SF IV was most effective at inhibiting A. brassicae conidial germination and thereby inhibited lesion development of Alternaria blight at a concentration of 15.625 μg/ml or higher. Furthermore, bioautography of SF IV with Alternaria alternata and diagnosis with Dragendorff reagent indicated that SF IV contains a mixture of bioactive alkaloids, namely a₁ (R_f = 0.12) and a₂ (R_f = 0.22). The potential of using S. xanthocarpum as a resource for the development of biofungicides is discussed.     

Antibacterial and antifungal activities of andrachne cordifolia muell

The crude methanolic extract of Andrachne cordifolia Muell. (Euphorbiaceae) and its various fractions in different solvent systems (chloroform, ethyl acetate and n- butanol) were screened for antibacterial and antifungal activities. Crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities against the tested pathogens. Highest antibacterial activity was displayed by both chloroform and ethyl acetate fractions (100%) followed by the crude extract (68%) against Salmonella typhi. Similarly, crude extract and its subsequent fractions showed mild to excellent activities in antifungal bioassay with maximum (76%) antifungal activity against Microsporum canis by the chloroform fraction followed by the crude extract (65%).     

Effect of extracts from in vitro-grown shoots of <Emphasis Type="Italic">Quillaja saponaria</Emphasis> Mol. on <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Pers.

The ethanolic and aqueous extracts from in vitro shoots of Quillaja saponaria Mol. (Quillay) were studied for their antifungal activity against the phytopathogenic fungus Botrytis cinerea Pers. These extracts reduced conidial germination and mycelial growth of B. cinerea, ethanolic extracts being more active than aqueous extracts. In addition, the damage areas produced by this fungus on tomato leaves and strawberry fruits pre-treated with quillay extracts were diminished. The fungitoxic effect of in vitro-grown quillay extract was similar to those obtained with commercial fungicides of both natural (BC-1000) and synthetic (iprodione–dicarboximide) origin. On the other hand, the antifungal action of quillay extracts obtained from adult trees naturally grown was only slightly superior to the fungitoxic activity of the extract from in vitro plants. HPLC analysis of the extract showed that it contained saponins and some phenolic compounds such as chlorogenic, caffeic, vanillic, and salicylic acids, and scopoletin, which have been identified as antifungal agents on phytopathogenic fungi. The results obtained in this work, suggests that extracts of in vitro-grown quillay have an important protective effect against B. cinerea and support the use of an in vitro culture system as a biotechnological alternative to obtain environmental safe antifungal quillay extracts to control B. cinerea, contributing to the preservation of this indigenous Chilean species.     

Enhancing the adhesion of Epicoccum nigrum conidia to peach surfaces and its relationship to the biocontrol of brown rot caused by Monilinia laxa

Aims: To find a formulation of Epicoccum nigrum conidia that enhances its adhesion to peach surfaces and improves its biocontrol efficacy against brown rot caused by Monilinia laxa. Methods and Results: The stickers, glycerol, sodium alginate and methylcellulose; the desiccants, silica powder and talc; and a commercial adhesive (NU FILM 17^®) were added at two different points during the production of an E. nigrum conidial formulation to improve conidial adhesion to peach surfaces. Conidial adhesion levels were determined from the number of E. nigrum conidia that adhered to glass slides or peach surfaces and conidial viability of adherent E. nigrum conidia was determined from the number of colony‐forming units of glass or peach‐adherent E. nigrum that grew on Petri dishes that contained potato dextrose agar. Compared to dried E. nigrum conidia without additives, the adhesion and viability of adherent E. nigrum conidia to peach surfaces were enhanced when either 1·25% sodium alginate or 2·5% methylcellulose was added to the conidial mass after fluid‐bed drying, and when 2·5% methylcellulose was added to the conidial mass after its production and before fluid‐bed drying. Epicoccum nigrum conidial formulations with 2·5% methylcellulose were more effective than dried E. nigrum conidia without additives in reducing the incidence of brown rot in peaches caused by M. laxa. Conclusions: When 2·5% methylcellulose is incorporated into an E. nigrum conidial formulation, the adhesion of E. nigrum conidia to peach surfaces improves and results in efficacious biocontrol of brown rot. Significance and Impact of the Study: A new improved formulation of a biocontrol agent has been developed to improve the control of M. laxa on peaches.     

Antibacterial and antifungal activities of teucrium royleanum (Labiatea)

The crude methanolic extract and subsequent fractions of Teucrium royleanum (Labiatea) were screened for antibacterial and antifungal activities. Against tested pathogens, crude extract and subsequent fractions demonstrated moderate to excellent antibacterial activities. Highest antibacterial activity was displayed by the ethyl acetate fraction against S. typhi (100%), against E.coli (76.7%) and against P. aerugenosa (70.8%) followed by the chloroform fraction against S. typhi (85.7%). Similarly, the crude extract and its subsequent fractions showed mild to excellent activities in the antifungal bioassay with maximum antifungal activity against M. canis (87%) by the chloroform fraction followed by the ethyl acetate (71%) and n-butanol (70%) fractions.     

Production of a Fungistatic Substance by <Emphasis Type="Italic">Pseudallescheria boydii</Emphasis> Isolated from Soil Amended with Vegetable Tissues and Its Significance

Four fungal isolates that were able to use vegetable tissues for multiplication in soil were isolated and identified as Pseudallescheria boydii based on morphological characteristics and ITS sequence similarity. When grown in broth prepared from the same vegetable tissues used in soil amendment, all these isolates of P. boydii produced a substance capable of reducing the disease incidence of black leaf spot of spoon cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The substance, which was fungistatic, was very stable under high temperature and high or low pH value. It was soluble in polar solvents and insoluble in non-polar solvents. Molecular weight estimation and ion exchange ability tests suggest that the fungistatic compound has a molecular weight between 500 and 1,000 and has no charge on its molecule. Results from this study suggest the possession of a strong competitive saprophytic ability by P. boydii, which in turn may explain the widespread occurrence of this human pathogen in soil. Production of a fungistatic substance when P. boydii was grown in broth prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soil.     

Antimicrobial activities of Gloriosa superba Linn (Colchicaceae) extracts

The methanol extract of the rhizomes of Gloriosa superba Linn (Colchicaceae) and its subsequent fractions in different solvent systems were screened for antibacterial and antifungal activities. Excellent antifungal sensitivity was expressed by the n-butanol fraction against Candida albicans and Candida glaberata (up to 90%) and against Trichophyton longifusus (78%) followed by the chloroform fraction against Microsporum canis (80%). In the antibacterial bioassay, the crude extract and subsequent fractions showed mild to moderate antibacterial activities. Chloroform fraction displayed highest antibacterial sensitivity against Staphylococcus aureous (88%) followed by the crude extract (59%). The total phenol content of the crude extract and fractions of the plant expressed no significant correlation with the antimicrobial activities.     

Leaf position-dependent effect of Alternaria brassicicola development on host cell death,photosynthesis and secondary metabolites in Brassica juncea

During the first 24 hours of infection, Alternaria brassicicola developmental parameters such as conidial germination, germ tubes and appressoria formation on each of the five mature Brassica juncea leaves, correlated with a leaf position showing stronger development of the pathogen on older leaves than on young ones. As a consequence of fungal development, the black spot disease was observed during 96 hours of infection on a macroscopic scale, as well as via confocal microscopy. Degradation of the chloroplast thylakoids and plastoglobule appearance during infection, followed by the decrease in chlorophyll a fluorescence parameters i.e. maximum quantum yield of PSII (F_v/F_m), non-photochemical quenching (NPQ) and chlorophyll a:b ratio, have been observed. Also, after an initial increase of carbohydrates (glucose, fructose and sucrose), content far below the respective control values was found. The content of secondary metabolites such as flavonoids and glucosinolates increased in a leaf position-dependent manner in infected leaves, with a lower level in older leaves than in younger ones. Although, the total phenolic compounds (TPCs) content did not differ significantly in infected leaves compared to control leaves, TPCs level in both control and infected leaves was leaf position-dependent. To the best of our knowledge, this is the first report on leaf position-dependent effect on the B. juncea biochemical response to A. brassicicola infection.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

Isolation of 12 polymorphic microsatellite loci in the phytopathogenic fungus Alternaria brassicicola

Twelve polymorphic microsatellite markers were isolated from the phytopathogenic fungus Alternaria brassicicola, the causal agent of black spot of crucifers. An enrichment protocol was used to isolate microsatellite loci, which were then analysed in a collection of 46 isolates sampled from seven different countries. The number of alleles detected in 12 loci ranged from two to 10 (mean 3.5). Investigation of cross‐species amplifications showed that the designed primers were specific to A. brassicicola.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Cardioactive properties of Solanaceae plant extracts and pure glycoalkaloids on Zophobas atratus

Glycoalkaloids, the biologically active secondary metabolites produced by Solanaceae plants, are natural defenses against animals, insects and fungi. In this paper, the effects of glycoalkaloids present in extracts of Solanaceae plants (potato, tomato and black nightshade) or pure commercial glycoalkaloids on the coleopteran Zophobas atratus F. were evaluated by in vitro and in vivo bioassays using heart experimental models. Each tested extract induced a dose‐dependent cardioinhibitory effect. The perfusion of Zophobas atratus semi‐isolated heart using the highest potato and tomato extract concentration (1 mmol/L) caused irreversible cardiac arrests, while extract from black nightshade produced fast but reversible arrests. Pure commercial glycoalkaloids caused similar but less evident effects compared with extracts. Our results showed that the bioactivity of tested compounds depended on their structure and suggested the existence of synergistic interactions when combinations of the main glycoalkaloids of potato and black nightshade were used for trials. Surprisingly, injection of tomato and potato extracts in 1‐day‐old pupae of Zophobas atratus induced reversible positive chronotropic effects and decreased the duration of the both phases (anterograde and retrograde) of the heart contractile activity. Furthermore, these extracts affected the amplitude of the heart contractions.     

Chitinolytic and antifungal activity of a <Emphasis Type="Italic">Bacillus pumilus</Emphasis> chitinase expressed in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.     

The impact of temperature regimes on development,dormancy breaking and germination of dwarf shrub seeds from arctic,alpine and boreal sites

It has been suggested that the infrequent sexual reproduction of arctic dwarf shrubs might be related to the harsh environmental conditions in which they live. If this is the case, then increases in temperature resulting from global climate change might drastically affect regeneration of arctic species. We examined whether recruitment of Empetrum nigrum ssp. hermaphroditum and Vaccinium uliginosum (hereafter E. nigrum and V. uliginosum) was affected by temperature during three reproductive stages: seed development, dormancy breakage and germination. Seeds were collected from an arctic, an alpine (only E. nigrum) and a boreal site with different climates; stored at different winter temperatures and incubated for germination at different temperatures. Seeds of V. uliginosum developed in the boreal region had a higher percentage germination than did seeds developed in the Arctic. In contrast, seeds of E. nigrum from the arctic site had a higher or similar percentage germination than did seeds from the alpine and boreal sites. Increased winter temperatures had no significant effect on resulting germination percentage of E. nigrum. However, V. uliginosum seeds from the arctic site suffered increased fungal attack (and thus decreased germination) when they were stratified under high winter temperatures. Seeds of both species increased germination with increased incubation temperatures. Our results suggest that both species would increase their germination in response to warmer summers. Longer summers might also favour the slow-germinating E. nigrum. However, increased winter temperatures might increase mortality due to fungal attack in V. uliginosum ecotypes that are not adapted to mild winters.     

Comparison of cytotoxic activities betweenin vitro and field grown plants ofTyphonium flagelliforme (Lodd.) blume

Anin vitro cytotoxicity screening of theTyphonium flagelliforme extracts indicated high cytotoxicity effect on human lung carcinoma NCl-H23 cells and human mammary gland carcinoma T-47D cells, but the extracts were not active on human liver carcinoma HepG2 cells. NCl-H23 cells were more susceptible toT. flagelliforme extracts than T-47D cells. EDP₅₀ values of the hexane fractions of the mature plant and thein vitro plantlet ofT. flagelliforme on NCl-H23 cells were less than 2 μg/mL Extract from the mature plant was relatively more cytotoxic than the one fromin vitro plantlet except for the hexane fraction. The chloroform and butanol fraction of the mature plant had higher cytotoxicity effect than the fraction fromin vitro plantlet on NCl-H23 cells. All the 3 fractions (hexane, chloroform, and butanol) of the mature plant exhibited higher cytotoxicity effects on human mammary gland carcinoma T-47D cells than the 3 fractions ofin vitro plantlet. However, the human liver carcinoma cells were resistant toT. flagelliforme extracts except for higher concentration of hexane fractions of both the mature and thein vitro plants and the chloroform fraction of the mature plant. Micropropagated plantlets ofT. flagelliforme could hence be used as herbal materials for the treatment of human lung and breast cancers.     

1‐Phenyl‐3‐pentanone,a Volatile Compound from the Edible Mushroom Mycoleptodonoides aitchisonii Active Against Some Phytopathogenic Fungi

Volatiles produced by mycelia of mushrooms with aromatic odour were investigated for their antifungal activity against plant‐pathogenic fungi. The results of the screening of 23 species of basidiomycetes revealed that volatile substances from mycelia of Mycoleptodonoides aitchisonii (TUFC10099), an edible mushroom, strongly inhibited the mycelial growth, spore germination and lesion formation on host leaves of some plant‐pathogenic fungi including Alternaria alternata, A. brassicicola, A. brassicae, Colletotrichum orbiculare and Corynespora cassiicola. The volatile compounds were isolated from the culture filtrate of M. aitchisonii, and 1‐phenyl‐3‐pentanone was identified as a major antifungal volatile. The compound had significantly inhibitory activity against plant‐pathogenic fungi at 35 ppm. This is the first report that the volatile compound produced by mycelia of M. aitchisonii has antifungal activity against plant‐pathogenic fungi.