Fungal endophyte-infected grasses: Alkaloid accumulation and aphid response

The occurrence of the alkaloidsN-formyl andN-acetyl loline, peramine, lolitrem B, and ergovaline and the response of aphids to plants containing these compounds were determined in species and cultivars ofFestuca,Lolium, and other grass genera infected with fungal endophytes (Acremonium spp., andEpichloe typhina). Twenty-nine of 34 host-fungus associations produced one or more of the alkaloids, most frequently peramine or ergovaline. Three alkaloids (lolines, peramine, and ergovaline) were found in tall fescue and in perennial ryegrass infected withA. coenophialum, while peramine, lolitrem B, and ergovaline were present in perennial ryegrass and in tall fescue infected withA. lolii and inF. longifolia infected withE. typhina. WhileA. coenophialum andA. lolii produced similar patterns of alkaloids regardless of the species or cultivar of grass they infected, isolates ofE. typhina produced either no alkaloids or only one or two different alkaloids in the grasses tested. Aphid bioassays indicated thatRhopalosiphum padi andSchizaphis graminum did not survive on grasses containing loline alkaloids and thatS. graminum did not survive on peramine-containing grasses. Ergovaline-containing grasses did not affect either aphid.     

Effect of fungal metabolite peramine and analogs on feeding and development of argentine stem weevil (Listronotus bonariensis)

Peramine, a pyrrolopyrazine alkaloid produced by the fungal endophyte of perennial ryegrassAcremonium lolii, deterred the feeding of both adults and larvae of the graminacious herbivore, the Argentine stem weevil (Listronotus bonariensis), at 0.1 g/g and 10 g/g, respectively. In a no-choice test fewer stem weevil larvae fed and developed on diet containing as little as 2 g/g peramine. The proportion of larvae which did not develop beyond the first instar was higher on diet containing peramine and appeared to be due to a higher proportion of larvae which did not feed. For larvae which fed on the peramine-containing diet, feeding scores and times to pupation were not significantly different from those of controls. A number of simple peramine analogues showed feeding-deterrent activity against adult weevils, indicating the importance of the pyrrolopyrazine ring system of peramine in determining feeding-deterrent activity.     

Does decreased mowing frequency enhance alkaloid production in endophytic tall fescue and perennial ryegrass?

Tall fescue, Festuca arundinacea, and perennial ryegrass, Lolium perenne, are widely infected with fungal endophytes (Neotyphodium spp.). The symbiosis between plant and fungus leads to synthesis of alkaloids that have been shown to be either toxic or act as feeding deterrents against insect pests. As cultural practices have the potential to regulate production of plant secondary metabolites, we evaluated the influence of mowing frequency on the levels of major alkaloids in tall fescue and perennial ryegrass in the greenhouse. Tall fescue and perennial ryegrass maintained in 15-cm-diam. pots were cut to 5-cm height weekly or biweekly. Samples were taken monthly and the alkaloids extracted and analyzed by reverse-phase LC-MS. In tall fescue, ergovaline, ergonovine, and ergocristine were identified, whereas only ergocristine was identified in perennial ryegrass samples. In tall fescue, we observed a trend showing higher levels in samples cut biweekly than in those cut weekly. A similar pattern was seen in some putative alkaloids that were not identified. In perennial ryegrass, ergocristine and two putative alkaloids followed a pattern similar to that of alkaloids in tall fescue. A survey of a few samples of perennial ryegrass using extractions specific to peramine and lolitrem B yielded evidence suggesting their presence as well as several other identified alkaloids. These data support the hypothesis that decreased mowing frequency enhances alkaloid production/accumulation in tall fescue and perennial ryegrass.     

Pine Weevil (Hylobius abietis) Antifeedants from Lodgepole Pine (Pinus contorta)

Pine weevils (Hylobius abietis) fed less on bark of lodgepole pine (Pinus contorta) than on bark of Scots pine (P. sylvestris). Two pine weevil antifeedants, ethyl trans-cinnamate and ethyl 2,3-dibromo-3-phenyl-propanoate, were isolated from bark of lodgepole pine. These two compounds significantly reduced pine weevil feeding in a laboratory bioassay. In field assays, the second compound significantly decreased pine weevil damage on planted seedlings. Ethyl 2,3-dibromo-3-phenylpropanoate has not previously been reported as a natural product.     

Ergovaline and peramine production in endophyte-infected tall fescue: Independent regulation and effects of plant and endophyte genotype

Peramine and ergovaline have ecological and economical significance because they possess insect and/or mammalian toxicity properties. The relationship between these endophytically derived alkaloids in tall fescue (Festuca arundinacea Schreb.) is unknown. Seasonal and plant tissue effects on the concentration of peramine and ergovaline was investigated in field and greenhouse experiments. The relationship between the alkaloids and the regulatory effects of endophyte and plant on their content were investigated among progeny of reciprocal crosses between high- and low-ergovaline and peramine plant-endophyte combinations. Variation in peramine concentration ranged from 750 to 1742 µg/kg in greenhouse-grown plants, and there was no seasonal trend in peramine content. There was a correlation (r=0.69) between peramine and ergovaline content among leaf tissues of field-grown plants, but there was no correlation between the alkaloids in the culm (r=0.20) or panicle (r=0.17) tissues. Mean leaf ergovaline concentration of progeny derived from the low-ergovaline parent (163 µg/kg) was less than the midparent value (228 µg/kg), but mean of progeny from the high-ergovaline parent was not different from the mid-parent value. Ranges within each progeny set were approximately double their mean. Mean leaf peramine concentrations of the progeny sets were similar to the mid-parent value (3354 µg/kg) but ranges were from 1716 to 8753 µg/kg. There was no correlation between leaf peramine and ergovaline (r=0.01). These data suggest that endophytically produced alkaloids are independently regulated and are controlled by both plant and endophyte genotype.     

Peramine alkaloid variation in Neotyphodium-infected Arizona fescue: effects of endophyte and host genotype and environment

We determined concentrations of peramine, the only alkaloid produced by Neotyphodium-infected (E+) Arizona fescue plants (of the four major types typically assayed in infected grasses), in a long-term field experiment. Four plant genotypes with (E+) and without (experimentally removed, E–) their respective haplotypes (two haplotypes in two plant genotypes) of Neotyphodium were grown in the field under manipulated soil moisture and nutrients. Peramine production required the presence of the endophyte; plants without their endophytes did not contain peramine. Whereas the endophyte was necessary for peramine production, levels of peramine did not vary by Neotyphodium haplotype within plant genotypes. Furthermore, peramine levels did not differ among soil moisture and nutrient treatments, and growth and reproductive measures of the host grass explained little of variation in peramine levels. Instead, peramine levels differed significantly between plant genotypes harboring the same endophyte haplotype, suggesting that plant genotype, rather than endophyte haplotype or environment, largely determines levels of peramine in Arizona fescue. The results suggest that whereas the endophyte is required for peramine production, the plant genotypic background in which the endophyte is embedded, rather than endophyte haplotype or environmental factors, mostly influences peramine levels within this population of Arizona fescue.     

(Z)-oxacyclotridec-10-en-2-one does not appear to be responsible for resistance to adult alfalfa weevil feeding exhibited byMedicago rugosa

Steam distillate prepared from leaves ofMedicago rugosa Desr. could inhibit feeding by adult alfalfa weevils (Hypera postica (Gyll.) on membrane filters, whereas an equivalent amount of steam distillate prepared usingM. sativa L. had no effect on weevil feeding. Earlier work established that a 12-carbon lactone, (Z)-oxacyclotridec-10-en-2-one, was responsible for the feeding deterrent properties of the steam distillate fromM. rugosa. The concentration of this volatile varied with plant age and leaf position. The maximum concentration in leaves ofM. rugosa (area basis) was estimated, on the basis of tests using an inert substrate, to be too low to cause a detectable inhibition of feeding. No correlation between lactone content and weevil feeding was detected when leaves from similar positions on plants of different ages were used in a multiple choice bioassay. Although small amounts (< 12.5g) of (Z)-oxacyclotridec-10-en-2-one can inhibit adult alfalfa weevil feeding on 13-mm-diameter membrane filters, because leaf concentrations (area basis) of this compound are lower than required to cause a detectable inhibition of feeding on the inert substrate, and because concentrations do not correlate with weevil feeding, it is unlikely that this lactone is responsible for resistance to adult alfalfa weevil feeding exhibited byM. rugosa.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA and does not imply approval to the exclusion of other products or vendors that may also be suitable.Technical paper No. 9416 of the Agricultural Experiment Station, Oregon State University, Corvallis, Oregon 97331.     

(R *,S *)-5-hydroxy-4-methyl-3-heptanone male-produced aggregation pheromone ofSitophilus oryzae (L.) andS. zeamais motsch

A male-produced aggregation pheromone, common to the rice weevil,Sitophilus oryzae, and the maize weevil,S. zeamais, was isolated and identified from hexane extracts of highly absorbent paper disks exposed individually to young virgin male weevils. A combination of preparative column and gas-liquid chromatography of disk extracts yielded purified natural pheromone. When analyzed by nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry, the structure of the natural pheromone, sitophilure, proved to be (R ^*,S ^*)-5-hydroxy-4-methyl-3-heptanone, of unknown enantiomeric composition. Synthetic racemic pheromone was highly attractive to males and females of both rice and maize weevils. In addition, both sexes of the granary weevil,S. granarius (L.) were attracted to the racemic preparation.     

cis-Vaccenyl acetate as an aggregation pheromone inDrosophila melanogaster

Pentane extracts of matureDrosophila melanogaster males substantially increased the attractiveness of food odors to both males and females in a wind-tunnel olfactometer. Extracts of females caused no such increase. An active component of the extract was isolated and identified as (Z)-11-octadecenyl acetate (cis-vaccenyl acetate, cVA), and synthetic cVA was active in bioassay. Hydrolysis of the ester linkage or movement of the double bond to the 9 position destroyed the activity. Mature virgin males released cVA into their feeding vials, and amounts of synthetic CVA equal to that released per male caused significant bioassay responses. Females, which were known to receive cVA from males during copulation, were found to emit relatively large amounts of the ester into their feeding vials within 6 hr after mating. cVA had been demonstrated previously to be a close-range pheromone inD. melanogaster, discouraging males from courting other males or recently mated females; it now appears to have a longer-range function as well.     

Allelopathic potential and chemical constituents of volatiles from Ageratum conyzoides under stress

The allelopathic potential of Ageratum conyzoides was investigated under different environmental stress conditions, including nutrient deficiency, physical damage, 2,4-D treatment, competition with Bidens pilosa, infection with Erysiphe cichoracearum, and feeding by Aphiids gossypii. The inhibitory effects of A. conyzoides volatiles on peanut (Arachis hypogaea), redroot amaranth (Amaranthus retroflexus), cucumber (Cucumis sativus), and ryegrass (Lolium multiforum) increased when plants were grown under nutrient-deficient conditions or in competition with B. pilosa; however, there was no difference with physical damage or 2,4-D treatment. Phytoinhibitory effects decreased under fungal infection and aphid feeding. Volatiles from A. conyzoides plants infected with E. cichoracearum or exposed to A. gossypii feeding inhibited or killed fungi and insects. Precocenes and their derivatives, monoterpenes, and sesquiterpenes were the major volatile components of A. conyzoides.     

Pheromone chirality of african palm weevil,Rhynchophorus phoenicis (F.) and palmetto weevil,Rhynchophorus cruentatus (F.) (Coleoptera: Curculionidae)

There are four stereoisomers of both 3-methyl-octan-4-ol, the aggregation pheromone of the African palm weevil,Rhynchophorus phoenicis (F.) and 5-methyl-octan-4-ol, the aggregation pheromone of the palmetto weevil,Rhynchophorus cruentatus (F.). Synthetic stereoisomers of 3-methyl-octan-4-ol and 5-methyl-octan-4-ol were baseline-separated on a Cyclodex-B fused silica column. Use of this column in gas chromatographic-electroantennographic detection (GC-EAD) and GC-mass spectrometric (GC-MS) analyses revealed that only one stereoisomer, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol, is produced by maleR. phoenicis and maleR. cruentatus, respectively, and elicits good antennal responses by conspecific male and female weevils. In field trapping experiments, withR. phoenicis in Côte d'Ivoire andR. cruentatus in Florida, (3S,4S)-3-methyl-octan-4-ol and (4S,5S)-5-methyl-octan-4-ol strongly enhanced attraction of fresh palm tissue, whereas other stereoisomers were behaviorally benign. Stereoisomeric 3-methyl-octan-4-ol and 5-methyl-octan-4-ol may be utilized to monitor and/or manage populations of these two palm weevils.     

Different Levels of Protective Alkaloids in Grasses with Stroma-forming and Seed-transmitted Epichloë/Neotyphodium Endophytes

The three alkaloid groups—lolines, ergopeptides, and peramine— are typically associated with endophyte infection of grasses, with the main function to protect hosts against herbivores. We determined levels of N-formylloline, N-acetylloline, ergovaline, and peramine in 18 European grasses naturally infected with seed-transmitted Neotyphodium endophytes or sexual Epichloë species. Peramine was the most common alkaloid, whereas lolines and ergovaline were only detected in Festuca hosts infected with E. festucae, N. coenophialum, or N. uncinatum. Only ten of the grass species analyzed contained detectable amounts of one or more of these alkaloids. There was a clear tendency for plants associated with stroma-forming Epichloë species to be free of alkaloids, and those that did produce alkaloids contained only small levels of peramine. In contrast, plants infected with seed-transmitted Neotyphodium endophytes often contained extremely high levels of lolines. Lolines enhance host survival through increased protection from herbivores and, thus, may be particularly favored in asexual endophytes that depend on host seed-production for their dispersal.     

Identification of a novel moth sex pheromone inEriocrania cicatricella (Zett.) (Lepidoptera: Eriocraniidae) and its phylogenetic implications

Extracts from different body parts of adult femaleEriocrania cicatricella (Zett.) were tested for electrophysiological activity on conspecific male antennae. Extracts from the Vth abdominal segment, containing a pair of exocrine glands, elicited the largest electroantennographic response when compared to extracts of other body parts. Female extracts were analyzed by gas chromatography with simultaneous flame ionization and electroantennographic detection (EAD). The EAD active peaks were identified as (Z)-4-hepten-2-one, (2R)-heptane-2-ol, and (2R)-(Z)-4-hepten-2-ol by coinjection on a gas chromatography and by comparison of mass spectra with those of synthetic standards. In field tests, a blend of these three pheromone components was highly attractive to conspecific males, and a subtractive assay confirmed that the unsaturated alcohol is the major pheromone component, whereas no definite behavioral activity could be assigned to the ketone or the saturated alcohol. A bait containing the two alcohols withS-configuration was attractive to maleE. sparrmannella (Bosc), whereas no males ofE. cicatricella were found in these traps. The sex pheromone compounds inE. cicatricella are chemically similar to pheromones reported in Trichoptera and they are produced in homologous glands.     

Response ofNp mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts

TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned byNp, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10^–4 weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callus-inducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constitutent(s) was stable and nonpolar. Thus, theNp allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil.     

Influence of white pine watering regimes on feeding preferences of spring and fall adults of the white pine weevilPissodes strobi (Peck)

Spring and fall adults of the white pine weevil,Pissodes strobi (Peck), were exposed in no-choice and two-choice tests to bark from water-stressed and non-water-stressed white pine (Pinus strobus L.), which had also been exposed or not exposed to weevil attack. This experiment demonstrated that the weevils could discriminate between bark from water-stressed white pine and preferred bark from the nonstressed plants. The weevils also preferred bark from nonstressed plants that were previously exposed to weevil damage. Spring and fall adults displayed the same feeding preferences. No sex differences were found in feeding preferences. Less nitrogen, phosphorus, and potassium were found in bark from the nonstressed plants, and the potassium level was higher in damaged plants. We expect that the biological performance of the weevil should be favored by vigorously growing plants rather than by stressed plants.     

Natural and synthetic oviposition stimulants forCatolaccus grandis (Burks) females

Oviposition behavior was elicited fromCatolaccus grandis (Burks) (Hymenoptera: Pteromalidae) females, an ectoparasitoid of the boll weevil,Anthonomus grandis Boheman (Coleoptera: Curculionidae), by smears of freshly cut cotton bolls or smears of extracts prepared with boll weevil damaged or undamaged cotton boll tissues. Oviposition behavior was also elicited fromC. grandis females by smears made withn-pentane,n-hexane,n-heptane, and isooctane. This is the first report of oviposition behavior elicited for any parasitoid by these short-chain saturated hydrocarbons (alkanes), introducing a new concept on the chemical mediation of parasitoid behavior during host selection. Oviposition behavior was also elicited fromC. grandis females by volatiles emanating from an artificial diet devoid of insect components that was specifically developed for the in vitro rearing of ectoparasitoids. The possible use of a synergistic combination ofn-hexane and diet to optimize the mechanized production of noncontaminated eggs is also discussed.Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.     

Insect feeding and oviposition deterrents from western red cedar foliage

The feeding deterrent activity of fractions from the foliage of western red cedar,Thujaplicata Donn, was studied in laboratory bioassays using the white pine weevil,Pissodes strobi Peck, as a test insect. The most active fraction was the volatile mixture that comprises the leaf oil of this tree species. Further fractionation of the leaf oil indicated feeding deterrent activity in the monoterpene hydrocarbon, thujone, and terpene alcohol fractions. When tested alone, both (–)-3-isothujone and (+)-3-thujone, which made up 75–88% and 5–10% of the leaf oil, respectively, deterred feeding by the weevils. Western red cedar leaf oil also showed antifeedant activity with the alder flea beetle,Altica ambiens (Le Conte), and served as an oviposition deterrent for the onion root maggot,Hylemya antiqua Meigen. The leaf oil, however, had no inhibitory effect on the feeding of the leaf roller,Epinotia solandriana L., and the red-backed sawfly,Eriocampa ovata L.Research supported by the Natural Science and Engineering Research Council, Canada, Co-op Grant No. A0243 and Operating Grant Nos. A3881 and A3706, and by the British Columbia Ministry of Labour Youth Employment Program.     

Identification of sex pheromone produced by female sweetpotato weevil,Cylas formicarius elegantulus (Summers)

A sex pheromone of the sweetpotato weevil,Cylas formicarius elegantulus (Summers), was obtained from collections of volatiles from virgin females, and pheromone was isolated by means of liquid and gas chromatography. The purification procedure was monitored by quantitative laboratory and field bioassays and the compound was identified as (Z)-3-dodecen-1-ol (E)-2-butenoate by means of spectroscopic and microchemical methods. Synthesis, followed by laboratory and field bioassays, showed that the biological activity of the synthetic material was qualitatively and quantitatively indistinguishable from that of the purified natural product.Mention of a commercial or proprietary product does not constitute an endorsement by the USDA.     

Aggregation pheromone for the pepper weevil,Anthonomus eugenii cano (Coleoptera: Curculionidae): Identification and field activity

This study describes the identification of an aggregation pheromone for the pepper weevil,Anthonomus eugenii and field trials of a synthetic pheromone blend. Volatile collections and gas chromatography revealed the presence of six male-specific compounds. These compounds were identified using chromatographic and spectral techniques as: (Z)-2-(3,3-dimethylcyclohexylidene)ethanol, (E)-2-(3,3-dimethylcyclohexylidene)ethanol, (Z)-(3,3-dimethylcyclohexylidene)acetaldehyde, (E)-(3,3-dimethylcyclohexylidene)acetaldehyde, (E)-3,7-dimethyl-2,6-octadienoic acid (geranic acid), and (E)-3,7-dimethyl-2,6-octadien-1-ol (geraniol). The emission rates of these compounds from feeding males were determined to be about: 7.2, 4.8, 0.45, 0.30, 2.0, and 0.30µg/male/day, respectively. Sticky traps baited with a synthetic blend of these compounds captured more pepper weevils (both sexes) than did unbaited control traps or pheromone-baited boll weevil traps. Commercial and laboratory formulations of the synthetic pheromone were both attractive. However, the commercial formulation did not release geranic acid properly, and geranic acid is necessary for full activity. The pheromones of the pepper weevil and the boll weevil are compared. Improvements for increasing trap efficiency and possible uses for the pepper weevil pheromone are discussed. A convenient method for purifying geranic acid is also described.The mention of firm names or trade products does not imply endorsement or recommendation by the U.S. Department of Agriculture over other firms or similar products not mentioned. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status or handicap.     

Effect of steam distillate extracts of selected resistant cultivated and wild rices on behavior of leaffolderCnaphalocrocis medinalis (Guenée) (Lepidoptera: Pyralidae)

Rice plant volatiles extracted as steam distillates significantly affected the behavior of the rice leaffolder,Cnaphalocrocis medinalis (Guenée). Leaffolder moths laid significantly fewer eggs on TN1 rice plants treated with extracts of resistant wild species of rice,Oryza officinalis andOryza punctata, than on TN1 plants treated with extracts of resistant cultivated rices. Extracts ofO. officinalis andO. punctata adversely affected egg hatchability.O. officinalis andO. punctata extracts were more toxic to first-instar larvae than extracts of other resistant varieties. Leaf area consumed by the larvae was reduced on TN1 plants treated withO. officinalis andO. punctata extracts compared with that on plants treated with extracts of resistant cultivated rice varieties.