Optimization of Taxol Extraction Process Using Response Surface Methodology and Investigation of Temporal and Spatial Distribution of Taxol in Taxus mairei.

Taxus mairei is an important source for industrial extraction of taxol in China. However, the standard and steps of extraction are currently not uniform, which seriously affects the taxol yield. In the present study, the influence of four factors (methanol concentration, solid-liquid ratio, ultrasonic extraction temperature, and ultrasonic extraction time) on the taxol yield was successively explored in T. mairei. A response surface methodology (RSM) was used to optimize the extraction process based on the single-factor experiments above. The optimal conditions were as follows: methanol concentration was 90%, solid-liquid ratio was 1:15 (g/mL), ultrasonic extraction temperature was 40 °C and ultrasonic extraction time was 60 min. Moreover, the twigs and needles from T. mairei with different tree ages were treated by the optimum extraction process, which further revealed temporal and spatial distribution of taxol in the reproducible tissues. Interestingly, the taxol content was relatively higher in needles of T. 'Jinxishan' (a cultivar from T. mairei with yellow aril, FY), but was less in FY twigs. The accumulation of taxol in twigs and leaves of females (with red aril, FR) was significantly higher than that of males (M); however, the content showed a decreasing trend with the increasing tree ages. Therefore, it is suitable to increase the proportion of female trees especially the FY leaves as raw materials for the industrial production of taxol from T. mairei, and the tree ages should be better controlled at 3-7 years.

Fast and cost-effective preparation of plant cells for scanning electron microscopy (SEM) analysis.

The analysis of plant cell structure provides valuable information about its morphological, physiological, and biochemical characteristics. Nowadays, scanning electron microscope (SEM) is widely used to provide high-resolution images at the surface of biological samples. However, biological specimens require preparation, including dehydration and coating with conductive materials for imaging by SEM. There are several techniques for providing images with maximum maintenance of cell structure and minimum cellular damage, but each requires the use of expensive and hazardous materials, which can be damaging to the cell in many cases. Therefore, the provision of new and effective preparation methods based on maintaining cell structure for imaging can be very practical. In the present study, a fast and cost-effective protocol was first performed for chemical fixation and preparation of the plant cells for imaging by SEM. Taxus baccata and Zhumeria majdae cells were chemically fixed using glutaraldehyde and then successfully dried with different percentages of ethanol including 70, 80, 90, and 100%. In addition, SEM was performed for imaging the cell surface in different micro-scales. This protocol can be used by plant cell biologists and biotechnologists who are interested in studying structural and biochemical responses of treated or stressed plant cells by SEM.

Seed and pollen gene dispersal in Taxus baccata, a dioecious conifer in the face of strong population fragmentation.

Background and Aims: Dispersal is crucial due to its direct impact on dynamics of a species' distribution as well as having a role in shaping adaptive potential through gene flow. In plants forming scarce and small populations, knowledge about the dispersal process is required to assess the potential for colonizing new habitats and connectivity of present and future populations. This study aimed to assess dispersal potential in Taxus baccata, a dioecious gymnosperm tree with a wide but highly fragmented distribution. Methods: Seed and pollen dispersal kernels were estimated directly in the framework of the spatially explicit mating model, where genealogies of naturally established seedlings were reconstructed with the help of microsatellite markers. In this way, six differently shaped dispersal functions were compared. Key Results: Seed dispersal followed a leptokurtic distribution, with the Exponential-Power, the Power-law and Weibull being almost equally best-fitting models. The pollen dispersal kernel appeared to be more fat-tailed than the seed dispersal kernel, and the Lognormal and the Exponential-Power function showed the best fit. The rate of seed immigration from the background sources was not significantly different from the rate of pollen immigration (13.1 % vs. 19.7 %) and immigration rates were in agreement with or below maximum predictions based on the estimated dispersal kernels. Based on the multimodel approach, 95 % of seeds travel <109 m, while 95 % of pollen travels <704 m from the source. Conclusions: The results showed that, at a local spatial scale, yew seeds travel shorter distances than pollen, facilitating a rapid development of a kinship structure. At the landscape level, however, although yew exhibits some potential to colonize new habitats through seed dispersal, genetic connectivity between different yew remnants is strongly limited. Taking into account strong population fragmentation, the study suggests that gene dispersal may be a limiting factor of the adaptability of the species.

Dormancy release and germination of Taxus yunnanensis seeds during wet sand storage.

Dormancy is an innate constraint on germination that occurs across all life forms. In this study, we investigated the seed dormancy release and germination characters of Taxus yunnanensis by exploring the seed morphology, permeability, germination inhibitors, endogenous hormones, and embryo germination in vitro during wet sand storage. Our results showed that seeds and embryos grew to a critical size to germination and permeability increased with the extension of storage. Seed coat and kernel methanol extracts reduced Brassica campestris seed vigor index. The in vitro embryo germination rate increased by 12.20% after storage for 30-360 d, whereas seed germination occurred after 450 d. Gibberellic acid and zeatin riboside contents were relatively stable, whereas abscisic acid (ABA) content decreased; indole acetic acid (IAA) content and the IAA/ABA ratio showed increasing trends. These results indicate that ABA is the key inhibitor of germination in Taxus. The chemical(s) in seed coat and kernel cause the inhibition of seed germination. Taken together, Taxus seeds have morphophysiological dormancy, in which the embryos can continue to grow and hormone imbalance inhibits further development and germination. Further, seed dormancy is active even during the middle of storage and shows "double peaks" during the entire dormancy process.

Dispersal of remnant endangered trees in a fragmented and disturbed forest by frugivorous birds.

Most endangered plant species in a fragmented forest behave as a unique source population, with a high dependence on frugivorous birds for recruitment and persistence. In this study, we combined field data of dispersal behavior of birds and GIS information of patch attributes to estimate how frugivorous birds could affect the effective dispersal pattern of Chinese yew (Taxus chinensis) in a fragmented and disturbed forest. Nine bird species were observed to visit T. chinensis trees, with Urocissa erythrorhyncha, Zoothera dauma and Picus canus being the most common dispersers. After foraging, six disperser species exhibited different perching patterns. Three specialist species, P. canus, Turdus hortulorum, and Z. dauma stayed in the source patch, while three generalist species, U. erythrorhyncha, Hypsipetes mcclellandii, and H. castanonotus, could perch in bamboo patches and varied in movement ability due to body size. As a consequence of perching, dispersers significantly contributed to the seed bank, but indirectly affected seedling recruitment. Moreover, the recruitment of T. chinensis was also affected by patch attributes in a fragmented forest (distances to source patch, patch type, size). Our results highlighted the ability of unique source population regeneration of T. chinensis in a fragmented forest, with high dependence on both frugivorous birds and patch attributes, which should be considered in future planning for forest management and conservation.

Effects of light acclimation on shoot morphology, structure, and biomass allocation of two Taxus species in southwestern China.

Acclimation to changing light conditions plays a crucial role in determining the competitive capability of tree species. There is currently limited information about acclimation to natural light gradient and its effect on shoot structure and biomass in Taxus species. We examined the acclimation of the leaf and shoot axis morphology, structure and biomass allocation of Taxus yunnanensis and T. chinensis var. mairei under three different natural light environments, full daylight, 40-60% full daylight and <10% full daylight. The leaf biomass, nitrogen content per unit area, leaf carbon content per dry mass and leaf dry mass to fresh mass ratio increased with light in both species, demonstrating an enhanced investment of photosynthetic biomass and structural investment under high light. The number of leaves per unit shoot axis length and the leaf dry mass per unit shoot axis length increased with light in both species. However, the light increase did not result in the increase of the total shoot mass. T. yunnanensis produced larger leaves under low light and a higher shoot axis length per unit dry mass under high light, whereas the leaf size and biomass yield of T. chinensis var. mairei were not sensitive to light.

[Not Available]. / Characteristic and protection of rare and endangered Taxus chinensis var. maireiin the Taihang Mountains.

The endangered causes of Taxus chinensis var. maireiin the Taihang Mountains are analyzed in three sides in connection with the situation that is resources increasing attenuation.The first is biological factors such as pollination barriers, deeply dormancy seed, cannot vegetative propagation under natural conditions, poor adaptability of seedling to environment and slow growth. The second is environmental factors such as very limited distribution environment and position in community. The third is interference of persons and other animals.According to these factors, we provide three measures to protect Taxus chinensis var. maireiin three sides that protect existing resources, breed subsequent resources and find new pathway of producing taxol.

How does the functional diversity of frugivorous birds shape the spatial pattern of seed dispersal? A case study in a relict plant species.

Genetic markers used in combination with network analysis can characterize the fine spatial pattern of seed dispersal and assess the differential contribution of dispersers. As a case study, we focus on the seed dispersal service provided by a small guild of frugivorous birds to the common yew, Taxus baccata L., in southern Spain. We build the spatial networks of seed dispersal events between trees and seed-plots within the studied population-local network-and the spatial network that includes all dispersal events-regional network. Such networks are structured in well-defined modules, i.e. groups of tightly connected mother trees and seed-plots. Neither geographical distance, nor microhabitat type explained this modular structure, but when long-distance dispersal events are incorporated in the network it shows a relative increase in overall modularity. Independent field observations suggested the co-occurrence of two complementary groups, short- and long-distance dispersers, mostly contributing to the local and regional seed rain, respectively. The main long-distance disperser at our site, Turdus viscivorus, preferentially visits the most productive trees, thus shaping the seed rain at the landscape scale and affecting the local modular organization. We end by discussing how DNA barcoding could serve to better quantify the role of functional diversity.

Seasonal-based temporal changes fluctuate expression patterns of TXS, DBAT, BAPT and DBTNBT genes alongside production of associated taxanes in Taxus baccata.

KEY MESSAGE: Environmental cues have synergistic or antagonistic regulatory roles on transcription activity and taxanes accumulation in yew, though DBAT activity is less influenced, could be accordingly a rate-limiting enzyme. The current work was undertaken to elucidate the consequences of some environmental cues (i.e., day length, temperature, sunlight and relative humidity) on the expression patterns of TXS, DBAT, BAPT and DBTNBT genes contributed to the taxol biosynthetic pathway along with the accumulation of some taxanes in needles and stems of Taxus baccata over year 2013-2014. In both tissues, light intensity and temperature correlated with the production of 10-DAB III and total taxanes, and TXS activity, while a lack of significant association was deduced for day length and relative humidity. Furthermore, in both tissues, a weak correlation was observed between BAC III and light intensity, temperature, day length and relative humidity, and the corresponding gene, DBAT. Surprisingly, DBAT activity was not co-induced with TXS in both tissues, and remained expressed at basal levels over year, supporting that the conversion of 10-DAB III into BAC III could presumably be a rate limiting step in the taxol biosynthetic pathway. Similar to BAC III, no strong correlation was detected between production of taxol in both tissues and all the meteorological data, while the corresponding genes BAPT and DBTNBT, in some cases, exhibited significant correlated results. Notably, despite higher activities of BAPT and DBTNBT in both tissues over year, taxol production was still in small quantities, probably owing to the low amounts of its precursors rather than low volumes of BAPT and DBTNBT transcripts. The results, altogether, could provide us new insights towards the potential regulatory roles of environmental cues on the production of taxanes in yew trees.

Mechanisms and effective control of physiological browning phenomena in plant cell cultures.

Browning phenomena are ubiquitous in plant cell cultures that severely hamper scientific research and widespread application of plant cell cultures. Up to now, this problem still has not been well controlled due to the unclear browning mechanisms in plant cell cultures. In this paper, the mechanisms were investigated using two typical materials with severe browning phenomena, Taxus chinensis and Glycyrrhiza inflata cells. Our results illustrated that the browning is attributed to a physiological enzymatic reaction, and phenolic biosynthesis regulated by sugar plays a decisive role in the browning. Furthermore, to confirm the specific compounds which participate in the enzymatic browning reaction, transcriptional profile and metabolites of T. chinensis cells, and UV scanning and high-performance liquid chromatography-mass spectrometry (HPLC-MS) profile of the browning compounds extracted from the brown-turned medium were analyzed, flavonoids derived from phenylpropanoid pathway were found to be the main compounds, and myricetin and quercetin were deduced to be the main substrates of the browning reaction. Inhibition of flavonoid biosynthesis can prevent the browning occurrence, and the browning is effectively controlled via blocking flavonoid biosynthesis by gibberellic acid (GA3 ) as an inhibitor, which further confirms that flavonoids mainly contribute to the browning. On the basis above, a model elucidating enzymatic browning mechanisms in plant cell cultures was put forward, and effective control approaches were presented.

Differential contribution of frugivorous birds to dispersal patterns of the endangered Chinese yew (Taxus chinensis).

The contribution of forest generalists and specialists to the dispersal pattern of tree species is not well understood. Specialists are considered low-quality dispersers because their dispersal distance is often short. However, disregard for seed deposition site may result in underestimation of the dispersal quality of specialists. The present study estimated the contribution of generalist and specialist species to the dispersal patterns of the endangered Chinese yew (Taxus chinensis) in a subtropical patchy forest in Southeast China. A relatively diverse assemblage of frugivorous birds visited T. chinensis source trees, and specialist Hypsipetes leucocephalus and generalist Urocissa erythrorhyncha were by far the highest-quantity dispersers. Considering dispersal effectiveness, the quantity aspect of effectiveness differed between the specialist assemblage and generalist assemblage; the contribution of specialists to the quantity part of effectiveness was significantly higher than that of generalists despite the relatively low diversity of specialists. After foraging, both specialist H. leucocephalus and generalist U. erythrorhyncha significantly contributed to the number of seedlings, and their contributions to seedling recruitment did not differ with regard to quality. Our results highlight the ability of T. chinensis to recruit an effective disperser assemblage in patchy habitats, thus increasing its persistence in this disturbed habitat.

Loss of nuclear flavanols during drought periods in Taxus baccata.

Normally, needles of Taxus baccata during the growth period prominently stain blue for nuclear flavanols with the histochemical DMACA procedure. However, under excess heat and drought conditions, nuclear flavanols of current-year needles decline to zero. Nevertheless, greenish-yellow-coloured flavonols (quercetin derivatives) were still observed in nuclei. All of these yellow nuclei were in a silenced state and without mitosis. This link between drought and loss of nuclear flavanols was found in 3 years, 2003, 2007 and 2010. In 2007, exceptional drought occurred in early spring, interrupted by short rains. This, in turn, led to flushing of new sprouts, a characteristic feature in which nuclei were overloaded with flavanols. By the end of three drought periods, all nuclei developed blue-coloured nuclear flavanols. The flavanols seem to be associated with the histone proteins of chromatin. The oxidative degradation of catechin in Tris buffer (pH 8.0) containing MgCl2 was studied in the presence of the H4-core fragment TYTEHAKRKTVTAMD, modified according to the epigenetic histone code. The results show that catechin degradation can be significantly inhibited by the non-modified peptides and the methylated peptides (methylation at both lysine residues). The acetylated and formylated peptides do not show this behaviour. These observations indicate that flavanol association at chromosomes appears to be regulated by the epigenetic histone code.

Climate warming and the decline of Taxus airborne pollen in urban pollen rain (Emilia Romagna, northern Italy).

Woody plant performance in a changing global environment has always been at the centre of palaeoenvironmental and long-term climate reconstructions carried out by means of pollen analysis. In Mediterranean regions, Taxus constitutes the highest percentage in past pollen diagrams from cold or cool periods, and therefore it is generally considered a good index to infer climate features from past records. However, a comparison of these inferences with the true current trends in pollen production has not been attemped until now. This study reports the decline of airborne pollen of Taxus observed in Emilia Romagna, a region of northern Italy, during the period 1990-2007. Phenological observations on four male specimens and microscopic examination of fresh pollen were made in order to check Taxus flowering time and pollen morphology. Airborne pollen was monitored through continuous sampling with a Hirst volumetric sampler. In the 18-year long period of investigation, Taxus pollen production has decreased, while total woody pollen abundance in air has increased. The trend of the Taxus pollen season shows a delay at the beginning, a shortening of the pollen period, and an advance of the end of the pollen season. This was interpreted as a response to climate warming. In particular, Taxus follows the behaviour of winter-flowering plants, and therefore earlier pollination is favoured at low autumn temperatures, while late pollination occurs more often, most likely after warm autumn temperatures.

A population balance equation model of aggregation dynamics in Taxus suspension cell cultures.

The nature of plant cells to grow as multicellular aggregates in suspension culture has profound effects on bioprocess performance. Recent advances in the measurement of plant cell aggregate size allow for routine process monitoring of this property. We have exploited this capability to develop a conceptual model to describe changes in the aggregate size distribution that are observed over the course of a Taxus cell suspension batch culture. We utilized the population balance equation framework to describe plant cell aggregates as a particulate system, accounting for the relevant phenomenological processes underlying aggregation, such as growth and breakage. We compared model predictions to experimental data to select appropriate kernel functions, and found that larger aggregates had a higher breakage rate, biomass was partitioned asymmetrically following a breakage event, and aggregates grew exponentially. Our model was then validated against several datasets with different initial aggregate size distributions and was able to quantitatively predict changes in total biomass and mean aggregate size, as well as actual size distributions. We proposed a breakage mechanism where a fraction of biomass was lost upon each breakage event, and demonstrated that even though smaller aggregates have been shown to produce more paclitaxel, an optimum breakage rate was predicted for maximum paclitaxel accumulation. We believe this is the first model to use a segregated, corpuscular approach to describe changes in the size distribution of plant cell aggregates, and represents an important first step in the design of rational strategies to control aggregation and optimize process performance.

Asymmetric somatic hybridization between Bupleurum scorzonerifolium Willd. and Taxus chinensis var. mairei.

In an attempt to transfer the genetic components needed for taxol synthesis into a more tractable plant, protoplasts of Taxus chinensis var. mairei were UV irradiated for various times prior to their fusion with protoplasts of Bupleurum scorzonerifolium. Of the 60 presumptive hybrid calli obtained, selections of the 9 most rapidly growing were recognized as hybrid clones based on a combination of their callus morphology, esterase profile, chromosome number, and RAPD genotype. The RAPD data showed that 82.4-96.8% of the hybrid genome was inherited from the recipient (B. scorzonerifolium) and 4.6-13.9% from the donor (T. chinensis), with 1.6-6.9% of the RAPD fragments being not present in either parent. None of the hybrid clones expressed a detectable quantity of taxol, but four produced more of the triterpenoid oleanolic acid than did calli of the recipient. With the use of semi-quantitative reverse transcriptase PCR, it was possible to show variation in the expression of several triterpenoid biosynthetic pathway related genes between B. scorzonerifolium and the hybrids. Increasing the concentration of oleanolic acid requires that the expression levels of the genes encoding IPP isomerase, squalene synthase, squalene epoxidase and ß-amyrin synthase are increased, while at the same time, those of the genes encoding the branching enzymes cycloartenol synthase and lupeol synthase need to be reduced.

Increased inbreeding and strong kinship structure in Taxus baccata estimated from both AFLP and SSR data.

Habitat fragmentation can have severe genetic consequences for trees, such as increased inbreeding and decreased effective population size. In effect, local populations suffer from reduction of genetic variation, and thus loss of adaptive capacity, which consequently increases their risk of extinction. In Europe, Taxus baccata is among a number of tree species experiencing strong habitat fragmentation. However, there is little empirical data on the population genetic consequences of fragmentation for this species. This study aimed to characterize local genetic structure in two natural remnants of English yew in Poland based on both amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. We introduced a Bayesian approach that estimates the average inbreeding coefficient using AFLP (dominant) markers. Results showed that, in spite of high dispersal potential (bird-mediated seed dispersal and wind-mediated pollen dispersal), English yew populations show strong kinship structure, with a spatial extent of 50-100 m, depending on the population. The estimated inbreeding levels ranged from 0.016 to 0.063, depending on the population and marker used. Several patterns were evident: (1) AFLP markers showed stronger kinship structure than SSRs; (2) AFLP markers provided higher inbreeding estimates than SSRs; and (3) kinship structure and inbreeding were more pronounced in denser populations regardless of the marker used. Our results suggest that, because both kinship structure and (bi-parental) inbreeding exist in populations of English yew, gene dispersal can be fairly limited in this species. Furthermore, at a local scale, gene dispersal intensity can be more limited in a dense population.

Molecular phylogeny and paclitaxel screening of fungal endophytes from Taxus globosa.

We studied the endophytic mycoflora associated with Taxus globosa, the Mexican yew. The study localities; Las Avispas (LA), San Gaspar (SG), and La Mina (LM) were three segments of cloud forest within the range of Sierra Gorda Biosphere Reserve, México. Overall, 245 endophytes were isolated and 105 representative Ascomycota (morphotaxons) were chosen for phylogenetic and genotypic characterization. Maximum likelihood analyses of large subunit of ribosomal RNA (LSU) rDNA showed well-supported clades of Dothideomycetes, Eurotiomycetes, Leotiomycetes, Pezizomycetes, and Sordariomycetes. Analyses of ITS rDNA groups showed 57 genotypes (95% sequence similarity), in general consistent with the phylogenetically delimitated taxa based on LSU rDNA sequences. The endophyte diversity measured by Fisher's α, Shanonn, and Simpson indices was ca. three-fold and ca. two-fold greater in LM than in LA and SG respectively. A screening for paclitaxel using a competitive inhibition enzyme immunoassay showed 16 positive isolates producing between 65 and 250 ng l(-1). The isolates included Acremonium, Botryosphaeria, Fusarium, Gyromitra, Nigrospora, Penicillium, three novel Pleosporales, and Xylaria.

Metabolic profiling as a tool for understanding defense response of Taxus cuspidata cells to shear stress.

To obtain a better understanding of responsive mechanism of plant cells in response to hydrodynamic mechanical stress, a metabolic profiling approach was used to profile metabolite changes of Taxus cuspidata cells under laminar shear stress. A total of 65 intracellular metabolites were identified and quantified, using gas chromatography coupled to time-of-flight mass spectrometry. Potential biomarkers were found by the principal component analysis as well as partial least squares combined with variable influence in the projection. Trehalose, sorbitol, ascorbate, sucrose, and gluconic acid were mainly responsible for the discrimination between shear stress induced cells and control cells. Further analysis by mapping measured metabolite concentrations onto the metabolic network revealed that shear stress imposed restrictions on primary metabolic pathways by inhibiting tricarboxylic acid cycle, glycolysis, and N metabolism. To adapt to the shear condition, cells responded by starting defensive programs. These defensive programs included coinduction of glycolysis and sucrose metabolism, accumulation of compatible solutes, and antioxidative strategy. A strategy of defense mechanisms at the level of metabolites for T. cuspidata cells when challenged with the shear stress was proposed.

Analysis of phospholipids, sterols, and fatty acids in Taxus chinensis var. mairei cells in response to shear stress.

Plant cell culture has been developed as an alternative method for the production of an anticancer drug, paclitaxel. However, the sensitivity of plant cells to shear stress has been one of the main obstacles to the scale-up of the plant cell culture. To gain a better understanding of the mechanism of plant cells' response to hydrodynamic mechanical stress, lipid profiling of suspension-cultured Taxus chinensis (Chinese yew) var. mairei cells under shear stress was carried out using liquid chromatography-tandem MS and gas chromatography-time-of-flight MS. T. chinensis var. mairei cells cultured in a Couette-type shear reactor responded with an increase of cell membrane permeability compared with control cells, which indicated that the adaptation to shear stress altered membrane lipid composition. The main changes of lipid profiles in the shear-stress-induced cells were the following: (a) the total phospholipid content decreased, especially that of structural phospholipids such as phosphatidylglycerol, phosphatidylethanolamine and phosphatidylcholine; (b) cells showed increased stigmasterol at the expense of sitosterol and campesterol; (c) the stigmasterol/phospholipid ratio increased; (d) the mono-unsaturated-fatty-acid content increased; (e) the shear-stress-induced cells accumulated very-long-chain saturated fatty acids (C22:0, C23:0, C24:0 and C25:0). These findings suggested alterations in membrane composition and hinted at a possible molecular basis for the mechanotransduction processes induced by shear stress in T. chinensis var. mairei cells.

Clavatol and patulin formation as the antagonistic principle of Aspergillus clavatonanicus, an endophytic fungus of Taxus mairei.

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2',4'-dihydroxy-3',5'-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.