Phomapyrones from blackleg causing phytopathogenic fungi: isolation, structure determination, biosyntheses and biological activity

The isolation and structure determination of phomapyrones D-G, three 2-pyrones and a coumarin, from a group of isolates of the fungal pathogen Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm, is reported. As well, phomenin B, infectopyrone, and polanrazines B and C were also obtained for the first time from these isolates. In addition, based on results of incorporations of 13C-labeled acetate and malonate, and deuterated methionine, a polyketide pathway is proposed for the biosyntheses of phomapyrones.     

Chemical defenses of crucifers: elicitation and metabolism of phytoalexins and indole-3-acetonitrile in brown mustard and turnip

The metabolism of the cruciferous phytoalexins brassinin and cyclobrassinin, and the related compounds indole-3-carboxaldehyde, glucobrassicin, and indole-3-acetaldoxime was investigated in various plant tissues of Brassica juncea and B. rapa. Metabolic studies with brassinin showed that stems of B. juncea metabolized radiolabeled brassinin to indole-3-acetic acid, via indole-3-carboxaldehyde, a detoxification pathway similar to that followed by the "blackleg" fungus (Phoma lingam/Leptosphaeria maculans). In addition, it was established that tetradeuterated brassinin was incorporated into the phytoalexin brassilexin in B. juncea and B. rapa. On the other hand, the tetradeuterated indole glucosinolate glucobrassicin was not incorporated into brassinin, although the chemical structures of brassinins and indole glucosinolates suggest an interconnected biogenesis. Importantly, tetradeuterated indole-3-acetaldoxime was an efficient precursor of phytoalexins brassinin, brassilexin, and spirobrassinin. Elicitation experiments in tissues of Brassica juncea and B. rapa showed that indole-3-acetonitrile was an inducible metabolite produced in leaves and stems of B. juncea but not in B. rapa. Indole-3-acetonitrile displayed antifungal activity similar to that of brassilexin, was metabolized by the blackleg fungus at slower rates than brassinin, cyclobrassinin, or brassilexin, and appeared to be involved in defense responses of B. juncea.     

Phytoalexins and polar metabolites from the oilseeds canola and rapeseed: differential metabolic responses to the biotroph Albugo candida and to abiotic stress

The metabolites produced in leaves of the oilseeds canola and rapeseed (Brassica rapa L.) inoculated with either different races of the biotroph Albugo candida or sprayed with CuCl(2) were determined. This investigation established consistent phytoalexin (spirobrassinin, cyclobrassinin, and rutalexin) and phytoanticipin (indolyl-3-acetonitrile, arvelexin, caulilexin C, and 4-methoxyglucobrassicin) production in canola and rapeseed in response to both biotic and abiotic elicitation. In addition, a wide number of polar metabolites were isolated from infected leaves, including six new phenylpropanoids and two new flavonoids. The extractable chemical components of zoosporangia of A. candida and the anti-oomycete activity of phytoalexins were determined as well. Overall, the results suggest that during the initial stage of the interaction, leaves of B. rapa have a similar response to virulent and avirulent races of A. candida, with respect to the accumulation of chemical defenses. After this stage, despite the higher phytoalexin concentration, the "compatible" races could overcome the plant defense system for further infection, but growth of the "incompatible" races was inhibited. Since results of bioassays showed that cyclobrassinin and brassilexin were more inhibitory to A. candida than rutalexin, the apparent redirection of the phytoalexin pathway towards rutalexin, avoiding cyclobrassinin and brassilexin accumulation might be caused by the pathogen. Alternatively, A. candida might be able to detoxify both cyclobrassinin and brassilexin, similar to necrotrophic plant pathogens. Overall, the correlation between phytoalexin production in infected or stressed leaves and the outcome of the plant-pathogen interaction suggested that A. candida was able to elude the plant defense mechanisms by, for example, redirecting the phytoalexin biosynthetic pathway.     

Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation

Arabidopsis thaliana is frequently used as a model for the study of oilseed biology and metabolism. However, the very small seeds of Arabidopsis can complicate analysis of their oil content and influence the application of results to larger-seeded plants. Here, we describe how seed anatomy, light, and plant-to-plant variation influence the content and measurement of oil in Arabidopsis seeds. The anatomy of Arabidopsis and Brassica napus seeds were compared and the distribution of mass, oil and the fatty acid composition of different seed parts were determined. In Brassica, 90% of the seed oil resides in the cotyledons that contribute 74% of seed mass. By contrast, the values for Arabidopsis are 60% and 45%, respectively, with a higher fraction of the oil deposited in the radicle, hypocotyl, endosperm and seed coat. Growth of Arabidopsis plants with 600 micromol m(-2) s(-1) light resulted in a two-fold higher seed yield, a 40% increase in mass per seed and a 60% increase in oil per seed compared to growth at 100 micromol m(-2) s(-1). Factors that influence the analysis of oil content were evaluated. Intact-seed transmethylation followed by gas chromatography (GC) analysis provided reproducible analysis of Arabidopsis seed oil. However, plant-to-plant variation in oil content is large and we analyzed how this influences the ability to detect statistically valid changes in oil between different genotypes. These observations establish a reference data set on the fatty acid composition and distribution of mass and oil between tissues of Arabidopsis seeds that should help to predict the applicability of results obtained with Arabidopsis to other oilseeds.     

The phytoalexins from cauliflower, caulilexins A, B and C: isolation, structure determination, syntheses and antifungal activity

Our continuous search for phytoalexins from crucifers led us to examine phytoalexin production in florets of cauliflower (Brassica oleracea var. botrytis) under abiotic (UV light) elicitation. Four known (isalexin, S-(-)-spirobrassinin, 1-methoxybrassitin, brassicanal C) and three new (caulilexins A-C) phytoalexins were isolated. The syntheses and antifungal activity of caulilexins A-C against the economically important pathogenic fungi Leptosphaeria maculans, Rhizoctonia solani and Sclerotinia sclerotiorum, and the first synthesis of brassicanal C are reported.     

Management of Rotylenchulus reniformis in Pineapple,Ananas comosus,by Intercycle Cover Crops

The effects of intercycle cover crops on Rotylenchulus reniformis population densities in pineapple were evaluated in one greenhouse and two field experiments. In the greenhouse, Crotalaria juncea, Brassica napus, and Tagetes erecta were planted for 3 months and then incorporated. These treatments were compared to weedy fallow with or without 1,3-dichloropropene (1,3-D) in three soils (Makawao fallow, Wahiawa fallow, and Wahiawa pineapple) naturally infested with R. reniformis. All cover crop incorporation suppressed R. reniformis numbers in cowpea more than did the weedy treatment in the Makawao (P < 0.05) but not in the Wahiawa soils. Crotalaria juncea treatment increased bacterivorous nematodes and nematode-trapping fungal population densities more than the other treatments in Makawao fallow and Wahiawa pineapple-planted soils. The field trials included the same plants as well as Sinapis alba. Treatments with Crotalaria juncea and 1,3-D maintained lower R. reniformis population densities on pineapple longer than other cover crops or weedy fallow treatments. Crotalaria juncea could have suppressed R. reniformis because it is a poor host and because it enhances nematode-trapping fungi when incorporated into soil. Treatment with 1,3-D reduced microbial activities but produced the greatest pineapple yield.     

The morphology of the larval stages of three species (Arrenurus mediorotundatus Thor, 1898; Arrenurus conicus Piers 1894; and Arrenurus cylindratus Piers, 1896) of the Megaluracarus subgenus (Acari: Hydrachnidia: Arrenuridae)

The morphology of the larval stages of three species (Arrenurus mediorotundatus, Arrenurus conicus and Arrenurus cylindratus) belonging to the Megaluracarus subgenus is described. Particular attention is paid to the common characters of the three species in which they differ from the other species belonging to the other subgenera. These are: the shape of the excretory pore plate, the shape of the dorsal plate, the length of coxal plate II medial margin and the C1-CpI, C4-CpIII distances. Other characters differ between the three species also. These are: the shape of the dorsal plate, the shape of coxal plate III, the shape of excretory pore plate and the presence or absence of secondary setae of the IFe3 setae.     

The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.     

Identified biosynthetic pathway of aspergiolide A and a novel strategy to increase its production in a marine-derived fungus Aspergillus glaucus by feeding of biosynthetic precursors and inhibitors simultaneously

Aspergiolide A is a novel anti-tumor anthraquinone derivant produced by marine-derived fungus Aspergillus glaucus. To identify its biosynthetic pathway and further improve the production, the effects of biosynthetic pathway specific inhibitors and precursors were investigated. Cerulenin and iodoacetamide, the specific inhibitors of polyketide pathway, could completely inhibit the aspergiolide A accumulation. Putative precursors of polyketide pathway could increase aspergiolide A production greatly, such as 6 mM acetate increased production by 135%. Simvastatin and citrate, the inhibitors of mevalonate pathway, stimulated the production by 63% and 179%, respectively. Considering that acetyl-CoA is the common starter unit in both polyketide and mevalonate pathway, a novel strategy was designed to stimulate the aspergiolide A accumulation. Combinations of 12 mM acetate with 0.3 mM simvastatin could increase the production by 151%, while the supplementation with 12 mM acetate and 12 mM citrate brought a 262% increase of aspergiolide A production. The strategy might be very useful to enhance the production of other secondary metabolites derived from polyketide pathway.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

Asexual propagation in the coral reef macroalga Halimeda (Chlorophyta, Bryopsidales): production, dispersal and attachment of small fragments

Siphonous, green macroalgae of the genus Halimeda are ubiquitous and ecologically important in tropical and subtropical marine environments. It has been hypothesized that the abundance of Halimeda on coral reefs is in part due to the ability of this genus to propagate asexually via vegetative fragmentation. However, vegetative fragmentation has only been documented for H. discoidea in a laboratory setting. To test the hypothesis that vegetative fragmentation contributes to field populations of Halimeda, we examined three aspects of fragmentation by H. tuna (Ellis and Solander) Lamouroux, H. opuntia (Linneaus) Lamouroux and H. goreaui Taylor on Conch Reef in the Florida Keys: (1) short-term (8 days) and long-term (14 weeks) fragment survival and rhizoid production in the laboratory and field (7 and 21 m), (2) size of the fragment pool and (3) influences of herbivory and water motion on production and dispersal of fragments. Although morphologically similar to H. discoidea, only a small percentage of H. tuna fragments survived. Fragments of H. opuntia and H. goreaui were more robust, and survival and rhizoid production were positively correlated with size in short-term trials. In 14-week field trials, one-third or fewer fragments of any species survived at 7 m, potentially because fragments were covered by large amounts of sediment. Survivors included some buried, seemingly dead individuals that turned green when exposed to light, highlighting the remarkable ability of this genus to survive disturbances. There was much less sediment accumulation at 21 m, where more fragments survived. Most (93%) eight-segment fragments of H. opuntia produced attachment rhizoids by the end of the 14-week trial. Overall, a range of 4.7-9.4 fragments of Halimeda m⁻² day⁻¹ were found on Conch Reef; most fragments were generated by H. goreaui. Fish bite marks were evident on 75-85% of the individuals of H. tuna and the number of bites per thallus ranged from 1 to 23. Herbivorous reef fish commonly fed on all three species of Halimeda. Some fish consumed the biomass, while others rejected most bites. For example, 83% of bites were rejected by the blue-striped grunt. Dispersal distances for rejected bites ranged from 0 to 31 m. Water motion was also responsible for fragment dispersal; experimentally produced fragments moved up to 48 cm day⁻¹. Results presented here suggest that asexual propagation of fragments of Halimeda is an important component of the life-history of this genus and vegetative fragmentation contributes to the abundance of this genus on coral reefs.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

Egg production of the copepod Acartia tonsa: The influence of hypoxia and food concentration

Low dissolved oxygen conditions, or hypoxia, occur in estuaries and impact more than just the obvious commercially important species. Copepods are an important link in the food web, and the influence of hypoxia upon them is relatively unstudied. Using the copepod Acartia tonsa, a study of the impact of hypoxia on egg production was conducted. A. tonsa had reduced egg production at low dissolved oxygen concentrations (DO), with the lowest egg production occurring at 0.53-ml/l O₂ concentration. Another experiment was conducted to determine if, by increasing food concentration, the affect of hypoxia could be mitigated. The results indicate that increased food did not offset the impact that hypoxia had on egg production of A. tonsa. These results suggest that as A. tonsa experiences hypoxia in the wild, population numbers will decrease. Thus, if hypoxic conditions increase in scope and duration, declines in copepod abundance may lead to a decline in the abundance of species that depend on them as food. These species may be of commercial importance (e.g., fish, crabs, and oysters).     

Cry1B and Cry3A are active against Hypothenemus hampei Ferrari (Coleoptera: Scolytidae)

Cry1B and Cry3 proteins from Bacillus thuringiensis are toxic to beetles such as the colorado potato beetle and the cottonwood leaf beetle. We report the development of a suitable rearing, bioassay method and the toxicity of these Cry proteins to coffee berry borer first instar larvae.     

The lethal and sub-lethal consequences of entomopathogenic nematode infestation and exposure for adult pine weevils, Hylobius abietis (Coleoptera: Curculionidae)

Entomopathogenic nematodes (EPN) frequently kill their host within 1-2 days, and interest in EPN focuses mainly on their lethality. However, insects may take longer to die, or may fail to die despite being infected, but little is known about the effects of EPN infection on insects, other than death. Here we investigate both lethal and sub-lethal effects of infection by two EPN species, Steinernema carpocapsae and Heterorhabditis downesi, on adults of the large pine weevil, Hylobius abietis. Following 12 h nematode-weevil contact in peat, S. carpocapsae killed a significantly higher proportion of weevils (87-93%) than H. downesi (43-57%) at all concentrations tested. Less than 10% of weevils were dead within 2 days, and weevils continued to die for up to 10 days after exposure (LT₅₀ of 3 days or more). In a separate experiment, live weevils dissected 6 days after a 24 h exposure to nematodes on filter paper harbored encapsulated and dead nematodes, showing that weevils could defend themselves against infection. Some live weevils also harbored live nematodes 6 days after they had been removed from the nematode infested medium. Feeding by weevils was not affected by infection with, or exposure to, either species of EPN. We discuss these results in relation to the use of EPN in biological control against H. abietis.     

Trichomonas vaginalis: The adhesins AP51 and AP65 bind heme and hemoglobin

Trichomonas vaginalis is the cause of human trichomoniasis, the most common non-viral sexually transmitted disease worldwide. Although acquisition of iron by binding to host hemoglobin through distinct receptor(s) has been described, no specific heme- or hemoglobin-binding site has been reported in this parasite.To determine the presence of hemoglobin-binding protein(s), membrane proteins were subjected to hemoglobin-affinity chromatography. Eluted proteins were analysed by SDS-PAGE. Two protein bands of 48 and 63 kDa were detected. Competition assay with an excess amount of hemoglobin or hemin in hemoglobin-affinity chromatography could block the 63- and 48-kDa bands, respectively.Further analysis by mass spectrometry indicated that the 48- and 63-kDa proteins had identity with two T. vaginalis adhesins: AP51 and AP65, respectively. This study confirms the existence of multifunctional proteins in T. vaginalis, and suggested that AP51 and AP65, besides serving as adhesion molecules, could also act as heme- and hemoglobin-binding proteins.