Paul F. Rugman-Jones

Nuclear-Mitochondrial Barcoding Exposes the Global Pest Western Flower Thrips (Thysanoptera: Thripidae) as Two Sympatric Cryptic Species in Its Native California

ABSTRACT n Over the past three decades, Western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), has become a major worldwide pest of many agricultural and horticultural crops. In response, much time, money, and effort have been put into pure and applied research focusing on the biology and control of this pest, Western flower thrips is native to Western North America and widespread in California. High levels of variation in basic biology, pest status, and resistance to insecticides bring into question the specific status of Western flower thrips. We used nuclear-mitochondrial barcoding to compare DNA sequences of nuclear and mitochondrial genes between Western flower thrips populations across California, looking for association between these unlinked loci. Sequences of D2 domain of 28S and cytochrome c oxidase I gene revealed the existence of two distinct but sympatric genetic entities, and we describe a simple polymerase chain reactionbased method for diagnosing these entities. The complete association of these nuclear and mitochondrial loci in areas of sympatry is indicative of reproductive isolation and the existence of two cryptic species, both of which key out to Western flower thrips by using morphological characters. The finding that Western flower thrips is a complex of two species has important implications for past, current, and most importantly future research on these pests.

Host Range of Fusarium Dieback and Its Ambrosia Beetle (Coleoptera: Scolytinae) Vector in Southern California

The polyphagous shot hole borer (PSHB) is an invasive ambrosia beetle that forms a symbiosis with a new, as-yet-undescribed Fusarium sp., together causing Fusarium dieback on avocado and other host plants in California and Israel. In California, PSHB was first reported on black locust in 2003 but there were no records of fungal damage until 2012, when a Fusarium sp. was recovered from the tissues of several backyard avocado trees infested with PSHB in Los Angeles County. The aim of this study was to determine the plant host range of the beetle-fungus complex in two heavily infested botanical gardens in Los Angeles County. Of the 335 tree species observed, 207 (62%), representing 58 plant families, showed signs and symptoms consistent with attack by PSHB. The Fusarium sp. was recovered from 54% of the plant species attacked by PSHB, indicated by the presence of the Fusarium sp. at least at the site of the entry hole. Trees attacked by PSHB included 11 species of California natives, 13 agriculturally important species, and many common street trees. Survey results also revealed 19 tree species that function as reproductive hosts for PSHB. Additionally, approximately a quarter of all tree individuals planted along the streets of southern California belong to a species classified as a reproductive host. These data suggest the beetle-disease complex potentially may establish in a variety of plant communities locally and worldwide.

Molecular and morphological diagnoses of five species of Trichogramma: biological control agents of Chrysodeixis chalcites (Lepidoptera: Noctuidae) and Tuta absoluta (Lepidoptera: Gelechiidae) in the Canary Islands

Prospecting for potential natural enemies of the invasive lepidopteran tomato pest Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) and the banana pest Chrysodeixis chalcites (Esper) (Lepidoptera: Noctuidae) on the Canary Islands archipelago, where no Trichogramma species were previously recorded, has led to the discovery of five distinct species. T. achaeae Nagaraja & Nagarkatti, T. bourarachae Pintureau & Babault, T. euproctidis (Girault) and T. evanescens Westwood are relatively widespread species. The fifth is close to T. brassicae Bezdenko, but differs sufficiently in the sequence of the ITS2 region of ribosomal RNA to warrant further investigation as a species probably new to science. Each species is treated in detail in order to facilitate identification in future using molecular and/or morphological characters, or a combination of both. All species are newly recorded for the Canary Islands, and the distribution of each within the islands and elsewhere is provided. Known host records are given within the Canary Islands and elsewhere. The most common species found, T. achaeae, is already being used in biological control programmes against T. absoluta in mainland Spain and field trials are ongoing to evaluate its effectiveness as a biological control agent of C. chalcites in banana crops.

Can the destruction of California's oak woodlands be prevented? Potential for biological control of the goldspotted oak borer, Agrilus auroguttatus.

The goldspotted oak borer (GSOB), Agrilus auroguttatus Schaeffer (Coleoptera: Buprestidae), is an introduced and aggressive phloem/wood borer infesting native oaks in southern California. Elevated levels of oak mortality have occurred continually for the last nine years on three oak species in San Diego Co., California, USA. Biological control is being assessed as an option for long-term and widespread management of the invasive population of GSOB. Foreign exploration in the native ranges of GSOB and a related sibling species (Agrilus coxalis Waterhouse) was conducted to determine life history information, to assess the natural enemy complex, and to collect specimens for molecular analyses that could help to identify the area of origin of California’s introduced population. Two species of parasitoids, Calosota elongata Gibson (Eupelmidae) and Atanycolus simplex Cresson (Braconidae), were discovered with GSOB populations in Arizona and California. No insect natural enemies were found with populations of A. coxalis in southern Mexico. However, Quercus conzatti Trel. and Quercus peduncularis Nee in Oaxaca and Chiapas, respectively, were recorded as the first known hosts of A. coxalis. A comparative analysis of our understanding of the natural enemy complexes for other pestiferous Agrilus with that of GSOB suggests that more effort should be directed at uncovering potential egg parasitoids and microbial pathogens of GSOB. Analyses of mitochondrial and nuclear ribosomal DNA (rDNA) revealed that the California population of GSOB was more similar to the Arizona population. Specimens of A. coxalis from southern Mexico were confirmed as a separate species. Additional surveys and sampling are needed across the complete native range of the GSOB species complex to develop a comprehensive inventory of parasitoid species that could be considered for use in a classical biological control program in California and to delineate the area of origin of California’s population.

A molecular key to the common species of Trichogramma of the Mediterranean region.

A molecular key for the identification of common Trichogramma (Hymenoptera: Trichogrammatidae) species found in agricultural settings around the Mediterranean is developed based on the sequence of the internal transcribed spacer 2 of the ribosomal cistron. Using the size of the ITS2 PCR product and restriction fragment length polymorphisms of the amplicon, ten Trichogramma species (T. bourarachae Pintureau and Babault, T. brassicae Bezdenko, T. cacoeciae Marchal/T. embryophagum Hartig, T. cordubensis Vargas and Cabello, T. dendrolimi Matsumura, T. euproctidis Girault, T. evanescens Westwood, T. nerudai Pintureau and Gerding, T. oleae Voegelé and Pointel, and T. pintoi Voegelé) can be distinguished.

Taxon-specific multiplex-PCR for quick, easy, and accurate identification of encyrtid and aphelinid parasitoid species attacking soft scale insects in California citrus groves

Citricola scale, Coccus pseudomagnoliarum Kuwana (Hemiptera: Coccidae), is a serious pest of citrus in California’s San Joaquin Valley, but not in southern California where a complex of Metaphycus spp. Mercet (Hymenoptera: Encyrtidae) suppress it. This has created interest in using these (and other Metaphycus) species for biological control in the San Joaquin Valley. A critical step in assessing an organism’s potential for biological control is the ability to accurately identify it. For Metaphycus spp., this currently requires slide mounted adult specimens and expert taxonomic knowledge. We present a simple, quick and accurate method to identify any life stage of the ten major parasitoids of soft scales in California citrus, based on amplification of ribosomal DNA, using the polymerase chain reaction (PCR). Three multiplex-PCR protocols amplify products of taxon-specific sizes, allowing direct diagnosis of taxa accommodated by the PCR, and reducing identification time to a fraction of that of existing methods.

Population genetics of goldspotted oak borer, Agrilus auroguttatus Schaeffer (Coleoptera: Buprestidae): investigating the origin of an invasive pest of native oaks in California

The goldspotted oak borer, Agrilus auroguttatus Schaeffer, is an invasive woodborer in California USA that is native to oak woodlands across southern Arizona USA. Developing a classical biological control program for this pest in southern California is a high priority due to the continuing ecological and economic damage caused by this insect since its recent introduction into the area. In an attempt to determine the area of origin for this invasive beetle, analyses of the mitochondrial cytochrome oxidase and ribosomal nuclear D2 domain of the 28S gene regions were undertaken and provided insight into the phylogeographic relationship between and within populations of A. auroguttatus in Arizona and California. The area of origin for the invasive population of goldspotted oak borer in California was not determined conclusively, although our molecular data suggests the Dragoon Mountains in Cochise Co., Arizona as a possible source for the California population of A. auroguttatus. Results also confirmed that individuals collected from populations across southern Arizona and California are all A. auroguttatus, and are not part of a cryptic species complex comprised of the morphologically similar A. coxalis. Future surveys for natural enemies of A. auroguttatus will focus on the Dragoon Mountains as a potential source for co-evolved enemies for use in a classical biological control program against this invasive woodborer in southern California.

Molecular identification of the exotic slug Arion subfuscus sensu stricto (Gastropoda: Pulmonata) in California, with comments on the source location of introduced populations

Arion subfuscus sensu lato (s. l.) refers to a cryptic slug species complex that is native to Europe and which, from there, has been introduced into the northeastern U.S. The species complex was detected in California for the first time in 2005 and recently 12 specimens were collected during statewide surveys. The genital morphology of these specimens suggested that only A. subfuscus sensu stricto (s. s.) was present and partial sequences of mitochondrial 16S rDNA (443xa0bp) showed that they all belonged to a single haplotype of the mitochondrial lineage, S1 sensu Pinceel et al. (Genetica 125: 155–171, 2005). This result was corroborated by a parallel analysis of a 655xa0bp COI DNA barcode. The 16S rDNA S1 haplotype (S1-03) of the Californian specimens is hitherto known only from the northeastern U.S. Hence, it is likely that populations may have been introduced to California from the northeastern states, rather than directly from the native area of the S1 haplogroup in Europe.

First Report of Geosmithia pallida Causing Foamy Bark Canker, a New Disease on Coast Live Oak (Quercus agrifolia), in Association with Pseudopityophthorus pubipennis in California

Declining coast live oak (Quercus agrifolia) trees have been observed since 2012 throughout urban landscapes in Los Angeles, Orange, Riverside, Santa Barbara, Ventura, and Monterey counties in California. Symptoms causing branch dieback and tree death included a cinnamon-colored gum seeping through multiple 0.95-mm-diameter entry holes on the bole, followed by a prolific, cream-colored foamy liquid. Beneath the outer bark was phloem and xylem necrosis. Fifty 1- to 2.5-mm adult and larval beetles were collected. Adults fit the morphological description of Pseudopityophthorus pubipennis (western oak bark beetle) (R. Rabaglia, personal communication), and ~800 bp of the mitochondrial COI gene was amplified for three beetles using primer pairs and methods previously described (2,3). All three sequences were identical (GenBank Accession Nos. KJ831289 to 91) and a BLAST search confirmed the closest match (94%) as P. pubipennis. Necrotic wood tissues collected from two trees in each county were cultured on potato dextrose agar amended with 0.01% tetracycline (PDA-tet), and incubated at 25°C for 1 week. Ochre-colored cultures with plane or radially furrowed velutinous mycelium were consistently produced. Fifty conidia each measured from two isolates were 3.66 ± 0.04 μm × 1.77 ± 0.03 μm, and arranged in non-persistent conidial chains, at first roughly parallel, becoming tangled with age. These fungal colonies were observed within gallery walls. The rDNA internal transcribed spacer (ITS) was amplified using primer pairs and methods previously described (5). Three isolates were sequenced and matched 100% to known sequences of Geosmithia pallida in GenBank; sequences of two isolates (UCR2208 and UCR2210) were deposited in GenBank (KJ468687 and KJ468688). Pathogenicity tests were performed by inoculating twelve 27.0-cm detached coast live oak shoots for each isolate with a spore suspension of G. pallida (UCR2208 and UCR2210) and sterile distilled water for controls. A 2-mm-wide, 3-mm-deep hole was drilled into the center of each shoot, 20 μl of a 106 conidia/ml spore suspension was pipetted into the hole, and sealed with Vaseline and Parafilm. The experiment was repeated twice. After 4 weeks in a moist chamber at 25°C, lesions produced by G. pallida averaged 8.3 cm and was significantly longer (ANOVA; P < 0.0001) from the control (average 0.4 cm). G. pallida was re-isolated from all inoculated plants and identified by colony morphology. P. pubipennis is a native beetle, common as a secondary agent, and previously not associated with disease. However, cryptic species may be common among bark and ambrosia beetles (4). A larger sample (i.e., populations and loci) is needed to determine the precise taxonomic status of P. pubipennis. G. pallida was shown to inhibit root growth of Q. petraea by 25% in Europe (1), appears to have affinities with a range of subcorticolous insects, and is widely distributed (5), but there is no published record of the fungus occurring in the United States. This is the first report of G. pallida causing foamy bark canker in association with P. pubipennis on Q. agrifolia in California. Results suggest this new disease complex is causing decline of Q. agrifolia throughout the state. References: (1) D. Cizková et al. Folia Microbiol. 50:59, 2005. (2) A. I. Cognato and F. A. H. Sperling. Mol. Phylogenet. Evol. 14:445, 2000. (3) A. I. Cognato et al. Mol. Phylogenet. Evol. 36:494, 2006. (4) B. H. Jordal and M. Kambestad. Mol. Ecol. Res. 14:7, 2014. (5) M. Kolarík et al. Mycol. Res. 108:1053, 2004.

Synonomy of Five Scirtothrips Species (Thysanoptera: Thripidae) Described from Avocados (Persea americana) in Mexico

Abstract The holotypes of Scirtothrips aguacatae, S. kupandae, S. manihotifloris, S. tacambarensis, and S. uruapaniensis were examined and compared with specimens of Scirtothrips perseae from Mexico, Guatemala, and California. The chaetotaxy of the pronotum and head of each of these 5 holotypes was found to fall within the range observed on specimens identified as S. perseae based DNA analyses and subsequent slide mounting and morphological examination of the body surface of specimens from which analyzed DNA was extracted. As a result of morphological examination and complementary DNA analyses, these 5 species are considered synonyms of S. perseae. This synonomy should reduce potential quarantine disputes over avocado imports from Mexico that could arise from concerns raised over the large number of Scirtothrips species previously considered as pests of avocados in Mexico that might inadvertently accompany imports.