Foliar flavonoids of eastern North AmericanVitis (Vitaceae) north of Mexico

Twenty-one flavonoid glycosides were isolated from the leaves of 22 North AmericanVitis L. taxa, representing two subgenera and five series. Three chemical groups were evident: one producing flavonols, flavones, and C-glycosylflavones, a second producing flavonols and flavones, and a third producing only flavonols. These three chemical groups did not correspond to any of the subgeneric groupings based on morphology. However, flavonoid distributions within series in each subgenus correlate well with morphological data. Parallel flavonoid evolution within each series is thought to account for this lack of subgeneric and interserial flavonoid distinction. The flavonoid data indicate that seriesCordifoliae of subgenusVitis, particularlyV. vulpina L., is the most closely related group to subgenusMuscadinia (Planch.)Rehder, and represents an evolutionary link between the two subgenera.     

Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in <Emphasis Type="Italic">Vitis cinerea</Emphasis> for marker-assisted selection

Background

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes.

Results

Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F₁ V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM.

Conclusion

The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

     

Cloning and Partial Characterization of Endopolygalacturonase Genes from Botrytis cinerea

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.     

Flavonoids as taxonomic markers in old world Lupinus species

In a leaf survey of 54 specimens of 11 Old World Lupinus species three classes of flavonoids were detected: flavones (in 82%), flavonols (in 36%) and flavone C-glycosides (in 55%). The rough-seeded species were clearly distinguished from the smooth-seeded taxa by the presence of a novel 2′-hydroxyflavone, luteolin and flavone C-glycosides as major leaf constituents and by the absence of flavonols. Within the smooth-seeded species, there are three flavonoid patterns: (a) flavonols only, L. albus; (b) flavones and flavonols, L. luteus, L. hispanicus and L. angustifolius; and (c) flavones only, L. micranthus. L. angustifolius further differed in uniquely producing diosmetin as a major leaf constituent. These divisions coincide exactly with previous groupings based on alkaloidal and morphological data. Amongst the 12 samples of L. angustifolius three chemical races were distinguished and a number of diosmetin glucoside malate esters detected. The flower flavonoid aglycone patterns of the nine Old World species surveyed differed markedly from the corresponding leaf profiles by the presence of flavones: luteolin and apigenin in eight and chrysoeriol in seven species as major constituents, while flavone C-glycosides were found only in trace amount in three species. In a leaf flavonoid survey of 13 representative New World Lupinus taxa, glycoflavones were major leaf components, a variety of methylated flavones were identified and flavonols were absent. The presence of the novel 2′-hydroxyflavone in five New World species may indicate some evolutionary link with the rough seeded taxa of the Old World.     

Systematic studies on mexican coreopsis (compositae). Coreopsis mutica: Flavonoid chemistry,chromosome numbers,morphology, and hybridization

Coreopsis mutica is a highly variable species occurring in the highlands from Central Mexico southeastward barely into El Salvador and Honduras. It is not continuous over this range, however, but is found in three geographic population centers: one in Guatemala and Chiapas, a second in Oaxaca, and the third in Central Mexico. Populations in Guatemala and Chiapas are uniform in chromosome number (2n = 56), leaf flavonoid chemistry, and morphology. Var.microcephala is proposed to accommodate these assemblages. Plants comprising populations centered around Cd. Oaxaca have a chromosome number of 2n = ca. 112. This large complex consists of two distinct varieties and their putative hybrids. Those plants to the northwest of Cd. Oaxaca (var.subvillosa) are constant in leaf flavonoid chemistry (producing only flavones) and possess a combination of distinctive morphological traits. To the southeast of Cd. Oaxaca plants invariably contain flavonols and anthochlors in their leaves in addition to flavones. Moreover, these plants (the newly proposed var.carnosijolia) are readily separable from var.subvillosa by a number of morphological features. Evidence is presented that the two taxa hybridize in the vicinity of Cd. Oaxaca. On the southeastern edge of the var.subvittosavar. carnosifolia complex a population was encountered which has a chromosome number of 2n = 56 and a very distinctive morphology and flavonoid chemistry. These plants have been accorded taxonomic status as var.multiligulata. Two morphologically similar, yet distinguishable, varieties occur in Central Mexico. It has been determined that the two differ also in chromosome number and leaf flavonoid chemistry. One taxon (var.leptomera) has a chromosome complement of 2n = 56 and produces only flavones in its leaves, whereas var.mutica has a chromosome number of 2n = ca. 112 and produces flavones, flavonols, and anthochlors.     

Poskeanolide,a seco-germacranolide and other sesquiterpene lactones from Vernonia species

The aerial parts of Vernonia cinerea, collected in Costa Rica, gave four new hirsutinolides while a sample from Malawi afforded glaucolide E and two related lactones. From V. poskeana a seco-germacranolide biogenetically closely related to a new glaucolide was isolated. The investigation of two Critoniopsis species gave in addition to known lactones a further glaucolide and a vernopatensolide. A related species, V. flexipappa, only gave glaucolide B. The structures were elucidated by highfield NMR spectroscopy.     

Botrydial confers Botrytis cinerea the ability to antagonize soil and phyllospheric bacteria

The role of the sesquiterpene botrydial in the interaction of the phytopathogenic fungus Botrytis cinerea and plant-associated bacteria was analyzed. From a collection of soil and phyllospheric bacteria, nine strains sensitive to growth-inhibition by B. cinerea were identified. B. cinerea mutants unable to produce botrydial caused no bacterial inhibition, thus demonstrating the inhibitory role of botrydial. A taxonomic analysis showed that these bacteria corresponded to different Bacillus species (six strains), Pseudomonas yamanorum (two strains) and Erwinia aphidicola (one strain). Inoculation of WT and botrydial non-producing mutants of B. cinerea along with Bacillus amyloliquefaciens strain MEP₂18 in soil demonstrated that both microorganisms exert reciprocal inhibitory effects; the inhibition caused by B. cinerea being dependent on botrydial production. Moreover, botrydial production was modulated by the presence of B. amyloliquefaciens MEP₂18 in confrontation assays in vitro. Purified botrydial in turn, inhibited growth of Bacillus strains in vitro and cyclic lipopeptide (surfactin) production by B. amyloliquefaciens MEP₂18. As a whole, results demonstrate that botrydial confers B. cinerea the ability to inhibit potential biocontrol bacteria of the genus Bacillus. We propose that resistance to botrydial could be used as an additional criterion for the selection of biocontrol agents of plant diseases caused by B. cinerea.     

Oviposition preference and larval performance of Epiphyas postvittana (Lepidoptera: Tortricidae) on Botrytis cinerea (Helotiales: Sclerotiniaceae) infected berries of Vitis vinifera (Vitales: Vitaceae)

In this paper we tested the behavior of gravid Epiphyas postvittana in selecting the most‐appropriate site for oviposition thus benefitting offspring performance. Our hypothesis was built on Jaenike's preference–performance hypothesis (also referred to as the “mother‐knows‐the‐best” hypothesis). To test this, we used the interacting Epiphyas postvittana, its host Vitis vinifera, and the pathogenic microbe Botrytis cinerea system. Populations of E. postvittana and B. cinerea often exist concurrently on V. vinifera in Australasia and their interaction and mutual influence are currently being explored, although the suggestion presently is that the relationship between E. postvittana and B. cinerea is mutualistic. We tested the effect of volatiles from B. cinerea‐infected berries and uninfected (control) berries of V. vinifera on the oviposition behavior of E. postvittana. We also characterized the effects of B. cinerea infection on the berries of V. vinifera on the growth and development of E. postvittana. Contrary to the preference–performance hypothesis, oviposition choices made by gravid E. postvittana did not result in the best offspring survival, development, and performance. The preference for oviposition by E. postvittana was strongly influenced by the olfactory and tactile cues. She laid fewer eggs on B. cinerea‐infected berries compared to uninfected berries of V. vinifera. The larvae of E. postvittana showed no preference to uninfected berries of V. vinifera. The larvae fed on B. cinerea‐infected berries of V. vinifera showing greater survival rate, shorter time to pupation, greater pupal mass, and on becoming adults they laid more numbers of eggs than the larvae that were enabled to feed on uninfected berries. The larvae of E. postvittana transport the conidia of B. cinerea and transmit grey‐mould disease to uninfected berries of V. vinifera.     

Botrytis pseudocinerea Is a Significant Pathogen of Several Crop Plants but Susceptible to Displacement by Fungicide-Resistant B. cinerea Strains

Botrytis cinerea is one of the most important pathogens worldwide, causing gray mold on a large variety of crops. Botrytis pseudocinerea has been found previously to occur together with B. cinerea in low abundance in vineyards and strawberry fields. Here, we report B. pseudocinerea to be common and sometimes dominant over B. cinerea on several fruit and vegetable crops in Germany. On apples with calyx end rot and on oilseed rape, it was the major gray mold species. Abundance of B. pseudocinerea was often negatively correlated with fungicide treatments. On cultivated strawberries, it was frequently found in spring but was largely displaced by B. cinerea following fungicide applications. Whereas B. cinerea strains with multiple-fungicide resistance were common in these fields, B. pseudocinerea almost never developed resistance to any fungicide even though resistance mutations occurred at similar frequencies in both species under laboratory conditions. The absence of resistance to quinone outside inhibitors in B. pseudocinerea was correlated with an intron in cytB preventing the major G143A resistance mutation. Our work indicates that B. pseudocinerea has a wide host range similar to that of B. cinerea and that it can become an important gray mold pathogen on cultivated plants.     

Characterization of Serine Proteinase Expression in Agaricus bisporus and Coprinopsis cinerea by Using Green Fluorescent Protein and the A. bisporus SPR1 Promoter

The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

Morphological and biochemical characteristics of genetically transformed roots of Scutellaria andrachnoides

Genetically transformed roots (hairy roots) and callus tissue of skullcap (Scutellaria andrachnoides Vved.) were for the first time introduced in the in vitro culture. S. andrachnoides is the endemic plant of the Kyrgyzstan. These cultures were characterized by active and stable growth in the hormone-free liquid Gamborg nutrient medium. The growth rate of undifferentiated callus tissue was higher than that of hairy roots, which were the source of this callus. The composition of secondary metabolites in hairy roots, callus tissue, and also the roots of seedlings and adult S. andrachnoides plants was analyzed. It was found that S. andrachnoides hairy roots and callus culture retained the ability for the synthesis of flavones typical for the roots of intact plants. Substantial quantitative differences in secondary metabolites were observed between the roots of juvenile and adult plants. In the seedling roots, which like hairy roots have no secondary thickening, wogonoside, a wogonin glucuronide, predominated among flavones. In the roots of adult plants growing due to the secondary thickening, balcalin, a baicalein glucuronide, was a dominating flavon. It is proposed to use the large-scale in vitro cultivation of roots and especially the rapidly growing callus tissue of S. andrachnoides with a profitable content of only one group of flavones for the development of the biotechnological method for producing wogonin and creating on its basis a new drug — a valuable anticancer agent of plant origin with selective cytotoxic activity.     

Heat-induced oxidative injury contributes to inhibition of Botrytis cinerea spore germination and growth

The inhibitory effect of heat treatment (HT) on Botrytis cinerea, a major postharvest fungal pathogen, and the possible mode of action were investigated. Spore germination and germ tube elongation of B. cinerea were both increasingly and significantly inhibited by HT (43 °C) for 10, 20 or 30 min. HT-induced gene expression of NADPH oxidase A, resulted in the intracellular accumulation of reactive oxygen species. HT-treated B. cinerea spores exhibited higher levels of oxidative damage to proteins and lipids, compared to the non-HT control. These findings indicate that HT resulted in oxidative damage which then played an important role in the inhibitory effect on B. cinerea. In the current study, HT was effective in controlling gray mold, caused by B. cinerea, in pear fruits. Understanding the mode of action by which HT inhibits fungal pathogens will help in the application of HT for management of postharvest fungal diseases of fruits and vegetables.     

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose, and pectin, and its composition can change upon stress. BETA-XYLOSIDASE4 (BXL4) belongs to a seven-member gene family in Arabidopsis (Arabidopsis thaliana), one of which encodes a protein (BXL1) involved in cell wall remodeling. We assayed the influence of BXL4 on plant immunity and investigated the subcellular localization and enzymatic activity of BXL4, making use of mutant and overexpression lines. BXL4 localized to the apoplast and was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea in a jasmonoyl isoleucine-dependent manner. The bxl4 mutants showed a reduced resistance to B. cinerea, while resistance was increased in conditional overexpression lines. Ectopic expression of BXL4 in Arabidopsis seed coat epidermal cells rescued a bxl1 mutant phenotype, suggesting that, like BXL1, BXL4 has both xylosidase and arabinosidase activity. We conclude that BXL4 is a xylosidase/arabinosidase that is secreted to the apoplast and its expression is upregulated under pathogen attack, contributing to immunity against B. cinerea, possibly by removal of arabinose and xylose side-chains of polysaccharides in the primary cell wall.

A putative bifunctional xylosidase/arabinofuranisodase localizes to the apoplast and is important for immunity against the necrotrophic pathogen Botrytis cinerea.     

Macromolecular Toxins Secreted by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Induce Programmed Cell Death in Arabidopsis Leaves

Botrytis cinerea causes grey mold disease in crops and horticultural plants. It is suspected to kill plant cells via secreted toxins and to derive nutrients from dead or dying cells. However, whether macromolecular phytotoxins (MPs) secreted by B. cinerea induce necrosis or also trigger a programmed cell death (PCD) remains to be determined. We have previously partially characterized MPs secreted by B. cinerea. Here we isolated MPs from B. cinerea culture and applied them to leaf cells, assessing PCD over the following 120 h. Cell death was assessed by propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. Catalase (CAT), peroxidase (POD) activity and the cytochrome c/a ratio were assessed by spectrophotometer. POD isomers were measured using the benzidine acetate method. In Arabidopsis thaliana (L.) Heynh. exposed to B. cinerea MPs, we observed chromatin condensation and marginalization, nuclear substance leakage and accumulation of autofluorescent materials in the cell wall. Furthermore, B. cinerea MPs induced release of cytochrome c from the mitochondria into the cytosol. Moreover, CAT and POD activity was upregulated and the POD isoenzyme pattern was altered. In conclusion, A. thaliana exposed to B. cinerea MPs exhibits multiple hallmarks of PCD, suggesting that B. cinerea induces PCD in host cells through secreted macromolecules.     

Effects of fermentation treatments on Polygonatum odoratum flavones’ antioxidant activities

The main aim of this study is to analyze antioxidant properties of Polygonatum odoratum fermented with bacteria, fungi and yeast. Antioxidant activities (1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, hydroxyl radical scavenging, and anti-lipid peroxidation abilities) were assessed in samples of flavones isolated from fermented P. odoratum (Mill.) druce samples. Fermentations using Lactobacillus, yeast and Aspergillus were investigated. Results showed that the antioxidant ability of Polygonatum odoratum flavones was decreased by the fermentation of Lactobacillus and yeast. Aspergillus niger fermentation improved the antioxidant ability of P. odoratum flavones. In this study, effective antioxidant activity was achieved in flavones fermented with Aspergillus niger than yeast and Lactobacillus species.