John J. Beck

The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus

Aspergillus flavus is a ubiquitous saprophyte that is able to produce the most potent natural carcinogenic compound known as aflatoxin B1 (AFB1). This toxin frequently contaminates crops including corn, cotton, peanuts, and tree nuts causing substantial economic loss worldwide. Consequently, more than 100 countries have strict regulations limiting AFB1 in foodstuffs and feedstuffs. Plants and microbes are able to produce volatile compounds that act as a defense mechanism against other organisms. Pichia anomala strain WRL-076 is a biocontrol yeast currently being tested to reduce AF contamination of tree nuts in California. We used the SPME-GC/MS analysis and identified the major volatile compound produced by this strain to be 2-phenylethanol (2-PE). It inhibited spore germination and AF production of A. flavus. Inhibition of AF formation by 2-PE was correlated with significant down regulation of clustering AF biosynthesis genes as evidenced by several to greater than 10,000-fold decrease in gene expression. In a time-course analysis we found that 2-PE also altered the expression patterns of chromatin modifying genes, MYST1, MYST2, MYST3, gcn5, hdaA and rpdA. The biocontrol capacity of P. anomala can be attributed to the production of 2-PE, which affects spore germination, growth, toxin production, and gene expression in A. flavus.

Hull Split and Damaged Almond Volatiles Attract Male and Female Navel Orangeworm Moths

A blend of volatiles derived from the emissions of almonds at hull split and mechanically damaged almonds was compared to almond meal, the current monitoring standard for the insect pest navel orangeworm (NOW). Field trapping studies were performed to determine the blends ability to attract adult NOW. The blend comprised racemic 1-octen-3-ol, ethyl benzoate, methyl salicylate, acetophenone, and racemic (E)-conophthorin. Ethyl acetate was used as a solvent with a blend component concentration of 100 mg/mL. The blend attracted both sexes of NOW when tested in five 2-week intervals spanning the first three flights of NOW in commercial almond orchards in the southern Central Valley of California. The blend demonstrated consistently higher capture rates for female NOW throughout the evaluation period, but unlike almond meal it significantly attracted males. Reported is a survey of the major and minor volatiles emitted from almonds at hull split, the key period of vulnerability to NOW infestation. Also reported is the attractancy of a formulated test blend based on the host plant volatile emissions, electroantennographic screening experiments, and field trapping studies. The results of this test blend highlight progress toward a host-plant-based attractant for NOW, a major insect pest of California tree nuts that presently lacks an adequate monitoring tool.

In situ volatile collection, analysis, and comparison of three Centaurea species and their relationship to biocontrol with herbivorous insects.

Centaurea solstitialis, commonly known as yellow starthistle, is an invasive plant listed as a noxious weed in the western areas of North America and is the target of classical biological control, which involves release of herbivores known to be specific to this plant. These insects often choose their host plant on the basis of the volatile organic compounds (VOCs) emitted. Accordingly, volatile analysis of host plants can provide insight into VOCs that may attract and/or repel the insect. To this end, solid-phase microextraction (SPME) and a customized collection bag were utilized to perform in situ volatile collection on intact and mechanically damaged leaves of Centaurea solstitialis, Centaurea cyanus, and Centaurea cineraria. Volatile identification was performed by GC-MS, and the VOC differences were determined. The plants C. solstitialis and C. cyanus have been reported to attract the weevil, Ceratapion basicorne, a candidate for biological control, whereas C. cineraria does not attract the weevil. Major VOCs unique to C. cineraria include the sesquiterpenes cyclosativene, alpha-ylangene, and trans-alpha-bergamotene. The compound trans-beta-farnesene was unique to C. solstitialis and C. cyanus.

In situ seasonal study of the volatile production of almonds (Prunus dulcis) var. 'Nonpareil' and relationship to navel orangeworm.

Nonpareil almonds, Prunus dulcis , account for the largest percentage of almond varieties grown in the Central Valley of California. Several studies have investigated the various nonvolatile and volatile components of various plant parts; however, the volatile organic compound (VOC) emission of almonds from a single cultivar has not been studied over the course of a growing season. This aspect is particularly relevant to research concerning the navel orangeworm (NOW), a major insect pest of almonds and other tree nuts. Despite the continued presence of NOW, the identification of particular VOCs and their relationship to NOW have not been addressed. The VOC emission of Nonpareil almonds was collected in situ over the course of a growing season by solid-phase microextraction (SPME). The VOCs (Z)-hex-3-enyl acetate, (Z)-hex-3-enyl butyrate, undecan-2-ol, beta-bourbonene, and tetradecane were present for the majority of the days investigated. Several VOCs exhibited positive electroantennographic signals from male and/or female NOW moths.

Generation of the Volatile Spiroketals Conophthorin and Chalcogran by Fungal Spores on Polyunsaturated Fatty Acids Common to Almonds and Pistachios

The spiroketal (E)-conophthorin has recently been reported as a semiochemical of the navel orangeworm moth, a major insect pest of California pistachios and almonds. Conophthorin and the isomeric spiroketal chalcogran are most commonly known as semiochemicals of several scolytid beetles. Conophthorin is both an insect- and plant-produced semiochemical widely recognized as a nonhost plant volatile from the bark of several angiosperm species. Chalcogran is the principal aggregation pheromone component of the six-spined spruce bark beetle. Recent research has shown conophthorin is produced by almonds undergoing hull-split, and both spiroketals are produced by mechanically damaged almonds. To better understand the origin of these spiroketals, the volatile emissions of orchard fungal spores on fatty acids common to both pistachios and almonds were evaluated. The volatile emission for the first 13 days of spores placed on a fatty acid was monitored. The spores investigated were Aspergillus flavus (atoxigenic), A. flavus (toxigenic), Aspergillus niger, Aspergillus parasiticus, Penicillium glabrum, and Rhizopus stolonifer. The fatty acids used as growth media were palmitic, oleic, linoleic, and linolenic. Spores on linoleic acid produced both spiroketals, those on linolenic acid produced only chalcogran, and those on palmitic and oleic acid did not produce either spiroketal. This is the first report of the spiroketals conophthorin and chalcogran from a fungal source.

Volatile Analysis of Ground Almonds Contaminated with Naturally Occurring Fungi

Aflatoxigenic aspergilli inflict major economic damage to the tree nut industry of California, with the highest negative impact to almonds. Aspergilli and fungi in general are known to emit volatiles in varying quantity and composition dependent upon their growth media. The goal of the study was to determine the volatile emission of whole and blanched almonds that had been picked out and labeled as inedible by processors. The aflatoxin content and number of colony forming units of each sample were also determined. A total of 23 compounds were consistently detected and identified. Several volatiles from the blanched almonds demonstrated significant increases when compared to the emissions of whole almonds. Several of these volatiles are considered fatty acid decomposition products and included hexanal, heptanal, octanal, nonanal, 3-octen-2-one, tetramethylpyrazine, and decanal. The almond samples investigated were characteristic of a typical postharvest environment and illustrative of potential contamination within a stockpile or transport container. Volatiles indicative of fatty acid decomposition were predominant in the samples that underwent some form of blanching. The emission amounts of hexanal, heptanal, octanal, and hexanoic acid increased 3-fold in samples contaminated with aflatoxin; however, due to variability between samples they could not be considered as indicator volatiles for aflatoxin content. The emission profile of volatiles from almond kernels contaminated with naturally occurring aspergilli and associated fungi is heretofore unreported.

Characterization of Microencapsulated Pear Ester, (2E,4Z)-Ethyl-2,4-decadienoate, a Kairomonal Spray Adjuvant against Neonate Codling Moth Larvae

Codling moth (CM), Cydia pomonella (Lepidoptera: Tortricidae), is the key pest of apples, pears, and walnuts worldwide. The pear-derived kairomone, ethyl (2E,4Z)-2,4-decadienoate, the pear ester (PE), evokes attraction and arrestment of CM larvae. Microencapsulated PE formulation (PE-MEC) enhances the control efficacy of insecticides when used as a spray adjuvant. Characterization of the microencapsulated kairomone, including microcapsule size, concentrations, emission rates, and larval response, was performed. Microcapsule diameter ranged from 2 to 14 mum, with 68% of capsules being 2-3 mum, and the concentration of microcapsules averaged 25.9 x 10(4) capsules per mL of field spray solution. Headspace collections showed emission of PE was related to PE-MEC concentration and was best described as first-order power decay. Neonate larvae responded to PE-MEC applications aged through 14 days. These results demonstrated that application of PE-MEC concurrent with insecticides may increase neonate foliar wandering, thereby disrupting host location and enhancing mortality by prolonging its exposure to insecticide.

Survey of ex situ fruit and leaf volatiles from several Pistacia cultivars grown in California.

BACKGROUND California is the second largest cultivator of pistachios, producing over 375 million pounds and a revenue of

Effect of mechanical damage on emission of volatile organic compounds from plant leaves and implications for evaluation of host plant specificity of prospective biological control agents of weeds

787 million in 2009. Despite the agricultural and economic importance of pistachios, little is known regarding their actual volatile emissions, which are of interest owing to their potential roles as semiochemicals to insect pests. RESULTS The ex situ volatile analysis of leaves from Pistacia atlantica, P. chinensis, P. lentiscus, P. palaestina, P. terebinthus, P. vera and P. weimannifolia demonstrated emission differences between species as well as between female and male leaves. Leaves from the female P. vera cultivars Bronte, Damghan, II, III, Kerman and Ohadi as well as fruits of P. atlantica, P. chinensis, P. lentiscus, P. palaestina, P. terebinthus and P. vera (cultivars II, III, Kaleh, Kerman, Momtaz and Ohadi) showed differences in the composition and relative quantity of major volatiles. The compounds in highest relative quantities from the various analyses were sabinene, Δ(3)-carene, β-myrcene, α-phellandrene, limonene, (Z)-ocimene, (E)-β-ocimene and α-terpinolene. CONCLUSION This is the first ex situ survey of fruit and leaf volatile emissions from California-grown Pistacia species and a number of corresponding cultivars. The study provides an overview of the major and minor volatile emissions and also offers evidence of chemotypes based on monoterpenes. The results highlight the dissimilarity of major components detected between ex situ volatile collection and essential oil analysis.

An overview of plant volatile metabolomics, sample treatment and reporting considerations with emphasis on mechanical damage and biological control of weeds.

Abstract Assessment of host plant specificity is a critical step in the evaluation of classical biological control agents of weeds which is necessary for avoiding possible damage to non-target plants. Volatile organic compounds (VOCs) emitted by plants likely play an important role in determining which plants attract and are accepted by a prospective arthropod agent. However, current methods to evaluate host plant specificity usually rely on empirical choice and no-choice behavioural experiments, with little knowledge about what chemical or physical attributes are stimulating the insect. We conducted experiments to measure the quantitative and qualitative effects on emission of VOCs caused by simple mechanical damage to leaves of plants known to differ in suitability and attractiveness to a prospective agent. More VOCs were detected from damaged than from undamaged leaves for all three species tested. Discriminant analysis was able to correctly distinguish the taxonomic identity of all plants based on their VOC profiles; however, the VOCs that discriminated species among undamaged leaves were completely different from those that discriminated among damaged leaves. Thus, damaged and undamaged plants present different VOC profiles to insects, which should be considered when conducting host plant specificity experiments. An unacceptable non-target plant, Centaurea cineraria, emitted all except one of the VOCs that were emitted by its preferred host plant, Centaurea solstitialis, indicating the importance of compounds that are repellant in host plant specificity. Centaurea cyanus emitted fewer VOCs than C. solstitialis, which suggests that it lacked some VOCs important for host plant recognition.