Tea Shot Hole Borer Research Articles

Evaluation of Spore Acquisition, Spore Production, and Host Survival Time for Tea Shot-Hole Borer, Euwallacea perbrevis, Adults after Exposure to Four Commercial Products Containing Beauveria bassiana.

Euwallacea perbrevis, the tea shot-hole borer (TSHB), is an invasive ambrosia beetle that vectors several fungal pathogens that cause Fusarium branch dieback in avocado trees in southern Florida. This study assessed the potential of four commercial products containing the entomopathogenic fungus Beauveria bassiana (Bb) for managing adult TSHB beetles. Formulated products containing Bb strains to which adult beetles were exposed were BioCeres WP, BotaniGard WP, BotaniGard ES, and Velifer ES. Controls consisted of water only and BotaniGard ES and Velifer ES supernatant with spores removed. Acquisition of spores by adult beetles dipped in product suspensions with 2.5 ± 0.1 × 106 spores/mL was assessed. Survival time of beetles after residual exposure to the Bb-based products in an in vivo avocado bark plug bioassay was determined. Production of Bb spores on beetles after being dipped in product suspensions and placed in a moistened bark-plug assay with water only was assessed. Significantly more spores were acquired by beetles exposed to Velifer ES and BotaniGard ES than beetles exposed to the other fungal products. Beetles exposed to Velifer ES and BotaniGard ES died faster (6-8 days) compared to beetles dipped in the other fungal products (10-11 days) and controls (12 days). Percentage of mycosis was highest with beetles exposed to Velifer ES (63%). Spore production on cadavers of beetles dipped in Velifer ES (20 ± 6.4 × 105 spores/cadaver) was the highest among all treatments, whereas it was the lowest on cadavers of beetles dipped in BotaniGard ES (1 ± 0.2 × 105 spores/cadaver). All Bb-based products, especially Velifer ES, demonstrated potential to manage TSHB populations under laboratory conditions. These Bb-based fungal products should be tested under field conditions to confirm these laboratory results.

Piperitone (p-Menth-1-En-3-One): A New Repellent for Tea Shot Hole Borer (Coleoptera: Curculionidae) in Florida Avocado Groves.

The tea shot hole borer, Euwallacea perbrevis, has been recently established in Florida, USA, where it vectors fungal pathogens that cause Fusarium dieback in avocado. Pest monitoring uses a two-component lure containing quercivorol and α-copaene. Incorporation of a repellent into IPM programs may reduce the incidence of dieback in avocado groves, particularly if combined with lures in a push-pull system. This study evaluated piperitone and α-farnesene as potential repellents for E. perbrevis, comparing their efficacy to that of verbenone. Replicate 12-week field tests were conducted in commercial avocado groves. Each test compared beetle captures in traps baited with two-component lures versus captures in traps containing lures plus repellent. To complement field trials, Super-Q collections followed by GC analyses were performed to quantify emissions from repellent dispensers field-aged for 12 weeks. Electroantennography (EAG) was also used to measure beetle olfactory response to each repellent. Results indicated that α-farnesene was ineffective; however, piperitone and verbenone were comparable in repellency, achieving 50-70% reduction in captures, with longevity of 10-12 weeks. EAG responses to piperitone and verbenone were equivalent, and significantly greater than response to α-farnesene. Since piperitone is less expensive than verbenone, this study identifies a potential new E. perbrevis repellent.

In-vitro evaluation of copper nanoparticles as a potential control agent against the fungal symbionts of the invasive ambrosia beetle Euwallacea fornicatus

The tea shot-hole borer (THSB), a member of the Euwallacea fornicatus (Eichhoff 1868) complex of species, harboring the plant pathogenic symbiotic fungi (AF-6, AF-8, and Fusarium sp.) (Curculionidae: Scolytinae) attacks avocado (Persea americana Miller) and other tree species in Florida. Currently, their management is limited to prevention and sanitation; therefore, the development of novel control alternatives is critical to mitigate pest outbreaks and avoid massive crop losses. Here, we report on the antifungal activity of copper nanoparticles (Cu-NPs) against the beetle/fungus complex and their effect on the beetle's reproduction. The results revealed that the synthesized Cu-NPs were pure metallic Cu with an average size of 240.88 ± 62.66 nm measured by electron microscopy. The antifungal test for symbionts showed that 0.75 mg/mL of Cu-NPs caused growth inhibition above 60%. However, strong alterations in the morphology of fungal cells were observed at Cu-NPs concentrations as low as 0.1 mg/mL. Additionally, treatments with 0.25, 0.5, and 1 mg/mL of Cu-NPs led to a reduction in offspring production in a concentration dependent manner. According to our findings, the application of Cu-NPs could be potentially considered as an alternative for the control of the beetle-fungi complex or even be integrated into novel disease management strategies.

One becomes two: second species of the Euwallacea fornicatus (Coleoptera: Curculionidae: Scolytinae) species complex is established on two Hawaiian Islands.

The cryptic species that make up the Euwallacea fornicatus species complex can be readily distinguished via their DNA sequences. Until recently, it was believed that the Hawaiian Islands had been invaded by only one of these cryptic species, E. perbrevis (tea shot hole borer; TSHB). However, following the 2016 deposition of a DNA sequence in the public repository GenBank, it became evident that another species, E. fornicatus (polyphagous shot hole borer; PSHB), had been detected in macadamia orchards on Hawaiʻi Island (the Big Island). We surveyed the two most-populous islands of Hawaiʻi, Big Island and Oʻahu, and herein confirm that populations of TSHB and PSHB are established on both. Beetles were collected using a variety of techniques in macadamia orchards and natural areas. Individual specimens were identified to species using a high-resolution melt assay, described herein and validated by subsequent sequencing of specimens. It remains unclear how long each species has been present in the state, and while neither is currently recognized as causing serious economic or ecological damage in Hawaiʻi, the similarity of the newly-confirmed PSHB population to other damaging invasive PSHB populations around the world is discussed. Although the invasive PSHB populations in Hawaiʻi and California likely have different geographic origins within the beetle’s native range, they share identical Fusarium and Graphium fungal symbionts, neither of which have been isolated from PSHB in that native range.

Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan

Carrillo, J.D., Rugman-Jones, PF., Husein, D., Stajich, J.E., Kasson, M.T., Carrillo, D., Stouthamer, R., and Eskalen, A. 2019. Members of the Euwallacea fornicatus species complex exhibit promiscuous mutualism with ambrosia fungi in Taiwan. A number of ambrosia beetles have come to prominence in recent years because of the damage they inflict on a variety of trees within invaded habitats across the globe. Ambrosia beetles rely on symbiotic microorganisms, mainly fungi, as a dedicated food source and carry those microorganisms around with them within specialized organs termed mycangia. Investigation of members of the Euwallacea fornicatus species complex and their fungal symbionts in Taiwan revealed promiscuous symbioses with ambrosial Fusaria clade (AFC) members, Graphium spp., and Paracremonium spp. based on co-phylogenetic analyses. For AFC members, a novel diagnostic PCR assay targeting mating type genes MAT1-1-1 and MAT1-2-1 was developed and validated by amplicon size and sequencing. Mating type screening of AFC members revealed the isolates screened are all heterothallic (self-sterile), with both MAT types represented and recovered from fungi vectored by E. fornicatus (tea shot hole borer), E. kuroshio (Kuroshio shot hole borer), and E. whitfordiodendrus (polyphagous shot hole borer) in Taiwan. Members of the Euwallacea fornicatus species complex and the variety of ambrosia fungi they utilize further confirms that their relationship with these fungi are more likely promiscuous in native areas, as opposed to strictly obligate to a specific combination of fungi as observed in invaded areas.

Naphthoquinone Metabolites Produced by Monacrosporium ambrosium, the Ectosymbiotic Fungus of Tea Shot-Hole Borer, Euwallacea fornicatus, in Stems of Tea, Camellia sinensis.

The tea shot-hole borer beetle (TSHB, Euwallacea fornicatus) causes serious damage in plantations of tea, Camellia sinensis var. assamica, in Sri Lanka and South India. TSHB is found in symbiotic association with the ambrosia fungus, Monacrosporium ambrosium (syn. Fusarium ambrosium), in galleries located within stems of tea bushes. M. ambrosium is known to be the sole food source of TSHB. Six naphthoquinones produced during spore germination in a laboratory culture broth of M. ambrosium were isolated and identified as dihydroanhydrojavanicin, anhydrojavanicin, javanicin, 5,8-dihydroxy-2-methyl-3-(2-oxopropyl)naphthalene-1,4-dione, anhydrofusarubin and solaniol. Chloroform extracts of tea stems with red-colored galleries occupied by TSHB contained UV active compounds similar to the above naphthoquinones. Laboratory assays demonstrated that the combined ethyl acetate extracts of the fungal culture broth and mycelium inhibited the growth of endophytic fungi Pestalotiopsis camelliae and Phoma multirostrata, which were also isolated from tea stems. Thus, pigmented naphthoquinones secreted by M. ambrosium during spore germination may prevent other fungi from invading TSHB galleries in tea stems. The antifungal nature of the naphthoquinone extract suggests that it protects the habitat of TSHB. We propose that the TSHB fungal ectosymbiont M. ambrosium provides not only the food and sterol skeleton necessary for the development of the beetle during its larval stages, but also serves as a producer of fungal inhibitors that help to preserve the purity of the fungal garden of TSHB.

Pheromones of three ambrosia beetles in the Euwallacea fornicatus species complex: ratios and preferences.

Three cryptic species in the Euwallacea fornicatus species complex were reared in laboratory colonies and investigated for the presence of pheromones. Collections of volatiles from combinations of diet, fungus, beetles, and galleries from polyphagous shot hole borer (Euwallacea sp. #1) revealed the presence of 2-heneicosanone and 2-tricosanone only in the presence of beetles, regardless of sex. Subsequent examination of volatiles from the other two species, tea shot hole borer (Euwallacea sp. #2) and Kuroshio shot hole borer (Euwallacea sp. #5), revealed these two ketones were present in all three species but in different ratios. In dual choice olfactometer behavioral bioassays, mature mated females were strongly attracted to a synthetic binary blend of ketones matching their own natural ratios. However, females in each species were repelled by ketone blends in ratios corresponding to the other two species. Males of each species responded similarly to females when presented with ratios matching their own or the other two species. The presence of these compounds in the three beetle species, in ratios unique to each species, and their strong species-specific attraction and repellency, suggests they are pheromones. The ecological function of these pheromones is discussed. In addition to the pheromones, the previously known attractant (1S,4R)-p-menth-2-en-1-ol (also known as quercivorol) was discovered in the presence of the fungal symbionts, but not in association with the beetles. Quercivorol was tested in a dual-choice olfactometer and was strongly attractive to all three species. This evidence suggests quercivorol functions as a kairomone for members of the E. fornicatus species complex, likely produced by the symbiotic fungi.

α-Copaene is an attractant, synergistic with quercivorol, for improved detection of Euwallacea nr. fornicatus (Coleoptera: Curculionidae: Scolytinae).

The tea shot-hole borer, Euwallacea fornicatus Eichhoff, is an ambrosia beetle endemic to Asia and a pest of commercial tea, Camellia sinensis (L.) Kuntze. Recently, a complex of species morphologically similar to E. fornicatus has been recognized, which includes new pests established in Israel and the USA, both in California and Florida. Collectively termed E. nr. fornicatus, these cryptic species carry symbiotic Fusarium spp. fungi, some of which cause dieback disease in susceptible hosts, which include avocado, Persea americana Miller. Due to the threat to this economically important crop, research was initiated to evaluate efficacy of kairomone-based lures for detection of the beetle in Florida (termed the Florida tea shot hole borer, FL-TSHB). A series of field tests were conducted in 2016 in commercial avocado groves known to have FL-TSHB at various population levels. All tests evaluated lures containing quercivorol (p-menth-2-en-1-ol) and α-copaene, presented separately and in combination; and one test evaluated effect of trap type on beetle captures. In addition, electroantennography (EAG) was used to quantify female olfactory responses to lure emissions. This study identified (-)-α-copaene as a new attractant for FL-TSHB, equivalent in efficacy to quercivorol (the standard lure for Euwallacea detection in the USA); however, the combination of lures captured significantly more FL-TSHB than either lure alone. This combination resulted in synergistic attraction at two field sites and additive attraction at a third site. Sticky panel traps captured more FL-TSHB than comparably-baited Lindgren funnel traps. Females engaged in host-seeking flight from 11:00 to 16:00 hr (EST), with peak numbers observed between 12:00 and 13:00 hr. EAG analyses confirmed olfactory chemoreception of both kairomones, with a higher response elicited with the combination of volatiles. Results indicate that detection of pest E. nr. fornicatus in Florida can be improved by using a two-component lure consisting of p-menth-2-en-1-ol and (-)-α-copaene.

Biology of two members of the Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae), recently invasive in the U.S.A., reared on an ambrosia beetle artificial diet

Abstract Recent molecular studies have found that the ambrosia beetle Euwallacea fornicatus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is a complex of cryptic species, each carrying a different species of symbiotic fungus, in the genus Fusarium, which they farm within galleries inside woody hosts. Several of these beetle species have become invasive pests around the world for attacking and infecting healthy trees with their phytopathogenic fungal symbionts. Diet and rearing protocols were developed for two members of the E. fornicatus species complex, polyphagous shot hole borer (PSHB) and tea shot hole borer (TSHB), using sawdust from host trees, allowing collection of data on beetle biology, phenology and sex ratios. Adults developed within 22 days at 24 °C. Single PSHB or TSHB foundresses averaged 32.4 and 24.7 adult female offspring, respectively, and up to 57 and 68 female adults within 6–7 weeks. A strong predictor of the number of offspring in a colony was the number of entry holes. Average sex ratios (% male) for PSHB and TSHB, respectively, were 7.4% and 7.2%. Being haplodiploid, virgin PSHB foundresses were able to produce and mate with male offspring, then subsequently produce female offspring, confirming that they have arrhenotokous reproduction. A cold tolerance study found significant mortality rates among PSHB colonies exposed to −5° or −1 °C but not colonies exposed to 0°, 1° or 5 °C. Given Hamilton's local mate competition (LMC) theory, a number of LMC predictions were violated. PSHB sex ratios were not affected by the number of foundresses; approximately 14% of broods did not contain males; males did not usually eclose before females but eclosed around the same time (22–23 days); and PSHB males were found walking outside of their natal galleries on the trunk of a heavily infested tree in the field. Alternatives to LMC are considered, such as early forms of sociality (maternal care, cooperative brood care), local resource enhancement and kin selection.

Tea Shot-Hole Borer Euwallacea fornicatus (Eichhoff, 1868) (Insecta: Coleoptera: Curculionidae: Scolytinae)

The tea shot-hole borer is an Asian ambrosia beetle introduced to Florida in the early 2000s. In Florida it does not have any known economic impact, but it is a serious pest of tea around the world and is one of the few ambrosia beetles that can infest healthy plants. The tea shot hole borer has a symbiotic relationships with the ambrosia fungus Fusarium ambrosium (Gadd & Loos), which is also a weak plant pathogen. This 4-page fact sheet includes a diagnosis section with guidance for avoiding misidentification. Written by You Li, Andrea Lucky, and Jiri Hulcr, and published by the UF Department of Entomology and Nematology, June 2015.

Fusarium euwallaceae sp. nov.—a symbiotic fungus of Euwallacea sp., an invasive ambrosia beetle in Israel and California

The invasive Asian ambrosia beetle Euwallacea sp. (Coleoptera, Scolytinae, Xyleborini) and a novel Fusarium sp. that it farms in its galleries as a source of nutrition causes serious damage to more than 20 species of live trees and pose a serious threat to avocado production (Persea americana) in Israel and California. Adult female beetles are equipped with mandibular mycangia in which its fungal symbiont is transported within and from the natal galleries. Damage caused to the xylem is associated with disease symptoms that include sugar or gum exudates, dieback, wilt and ultimately host tree mortality. In 2012 the beetle was recorded on more than 200 and 20 different urban landscape species in southern California and Israel respectively. Euwallacea sp. and its symbiont are closely related to the tea shot-hole borer (E. fornicatus) and its obligate symbiont, F. ambrosium occurring in Sri Lanka and India. To distinguish these beetles, hereafter the unnamed xyleborine in Israel and California will be referred to as Euwallacea sp. IS/CA. Both fusaria exhibit distinctive ecologies and produce clavate macroconidia, which we think might represent an adaption to the species-specific beetle partner. Both fusaria comprise a genealogically exclusive lineage within Clade 3 of the Fusarium solani species complex (FSSC) that can be differentiated with arbitrarily primed PCR. Currently these fusaria can be distinguished only phenotypically by the abundant production of blue to brownish macroconidia in the symbiont of Euwallacea sp. IS/CA and their rarity or absence in F. ambrosium. We speculate that obligate symbiosis of Euwallacea and Fusarium, might have driven ecological speciation in these mutualists. Thus, the purpose of this paper is to describe and illustrate the novel, economically destructive avocado pathogen as Fusarium euwallaceae sp. nov. S. Freeman et al.

New insect-disease complex strikes Southland trees

Science brief G. Arakelian RIVERSIDE, Calif. — UC Riverside scientists have identified a new insect-disease complex that is threat- ening avocado, coast live oak, box elder and other trees in Southern California. The complex includes a new shot hole borer and a symbiotic Fusarium spe- cies, which causes branch dieback. The same beetle and fungus have also been found in Israel, where the complex has caused severe damage to avocado trees since 2009. Entomologists led by Richard Stouthamer pro- posed the name polyphagous shot hole borer (PSHB) for the tiny insect, an ambrosia beetle. Smaller than a sesame seed (the female is 0.1 inch), the insect was earlier misidentified as tea shot hole borer. Subsequent DNA sequencing by UCR sci- entists distinguished the in- sect as a different, potentially new, species. After it bores into a tree, Polyphagous shot the insect deposits the fungus, hole borer which causes branch dieback and eventual death of the tree. The yet unnamed Fusarium sp. was identified by Akif Eskalen, UC Riverside Cooperative Extension plant pathologist. Eskalen has linked the insect-disease complex to the branch dieback and general decline of several avocado and landscape trees found in February and March this year in Los Angeles and Orange County residential neighborhoods. The insect and fungus have a symbiotic relationship, Eskalen says. “When the fe- male beetle burrows into the tree, it lays its eggs in galleries; the eggs develop into larvae, which feed on the fungus,” he explains. “At the same time, the fungus attacks the vascular tis- sue of the tree, disturbing water and nutrient flow, and causing branch dieback and — if not stopped — the tree’s death.” Symptoms in avocado include the appearance If infected branches are of white, powdery exudate in association cut off early enough, trees can with a single beetle exit hole on the bark of the trunk and main branches of the tree. This be saved. It is important that exudate could be dry, or it could appear as a growers and homeowners not wet discoloration. move infested avocado wood out of the infested area. If you suspect you have found the PSHB or seen symptoms of Fusarium dieback in your avocado grove or in other host plants, please contact your local farm advisor, county agricultural commissioner, or UC Riverside’s Eskalen at akif.eskalen@ucr.edu or (951) 827-3499. — Iqbal Pittalwalla and Janet White Correction To our readers: Managing editor Byron moves on After 13-plus years with California Agricul- ture journal, managing editor Janet Byron left in September to pursue a new career opportunity. Byron was first hired in June 1999 as a special editor to help develop the journal’s “Future in Focus: 2000–2025” editions, which were Janet Byron published in 2000. She became managing editor in February 2001. When Byron started, California Agriculture was mailing out paper reviews, scanning slides and posting abstracts on its website. “We have undergone a lot of changes to bring the journal into the digital age, but our commit- ment to publishing high-quality, peer-reviewed research in a beautiful, easily accessible format has never wavered,” she said. “Janet has been an unfailing source of creativity and inspiration in the challenging task of deliver- ing sound science to a lay audience,” said Janet White, executive editor. “She responds to chal- lenges with optimism and new ideas. She has been a key collaborator in the team effort to make the journal widely accessible on the Web — and will be greatly missed.” During her tenure, the journal won numerous awards from the Association of Communication Excellence in Agriculture, Natural Resources, and Life and Human Sciences (ACE) for editing, writ- ing, and technical and electronic publications. “I have enjoyed working with everyone in ANR, especially the hundreds of scientists who have shared their research results and patiently submit- ted to editing over the years,” Byron said. Byron’s new position is senior communications specialist for the Kaiser Permanente Division of Research in Oakland, where she will help dissemi- nate medical research results. She can be reached at janetbyron@att.net. In a letter from Dilipsinh M. Gaekwar published in the July-September 2012 issue, the year that Justin Smith Morrill began serving in Congress was incorrect. The correct year is 1855, not 1885. http://californiaagriculture.ucanr.edu • October–December 2012 121 Photos: Eskalen Lab, UC Riverside New insect-disease complex strikes Southland trees

First Report of a Fusarium sp. and Its Vector Tea Shot Hole Borer (Euwallacea fornicatus) Causing Fusarium Dieback on Avocado in California.

Per capita consumption of avocado in the United States has nearly doubled between 2000 and 2010. The California avocado industry supplies almost 40% of U.S. demand and the remaining 60% is supplied by imports from Latin America and New Zealand. The Tea Shot Hole Borer (TSHB) is an ambrosia beetle from Asia that forms a symbiosis with a new, yet undescribed Fusarium sp. and is a serious problem for the Israeli avocado industry (3). The beetle also causes severe damage on the branches of tea (Camelia sinensis) in Sri Lanka and India (1). In California, TSHB was first reported on black locust (Robinia pseudoacacia) in 2003, but there are no records of fungal damage (4). In 2012, nine backyard avocado trees (cvs. Hass, Bacon, Fuerte, and Nabal) exhibiting branch dieback were observed throughout the residential neighborhoods of South Gate, Downey, and Pico Rivera in Los Angeles County. Upon inspection, symptoms of white powdery exudate, either dry or surrounded by wet discoloration of the outer bark in association with a single beetle exit hole, were found on the trunk and main branches of the tree. Examination of the cortex and wood under the exit hole revealed brown discolored necrosis. The TSHB was also found within galleries that were 1 to 4 cm long going against the grain. Symptomatic cortex and sapwood tissues were plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet). The TSHB was dissected and plated onto PDA-tet after surface disinfestation following methods described by Kajimura and Hijii (2). After 5 days of incubation at room temperature, regular fungal colonies with aerial mycelia and reddish brown margins were produced. Single spore isolations were used to establish pure culture of the fungus. Fifty conidia were hyaline, clavate with a rounded apex, and initially aseptate (4.1 to 12.0 × 2.4 to 4.1 μm) becoming one- to three-septate (7.6 to 15.1 × 2.8 to 4.5 μm, 9.2 to 17.2 × 3.4 to 4.8 μm, and 13.5 to 17.6 × 4.3 to 4.7 μm, respectively). Identity of the fungal isolates was determined by amplification of the rDNA genes with primers ITS4/5 and EF1/2, respectively. Sequences were deposited into GenBank under Accession Nos. JQ723753, JQ723760, JQ723756, and JQ723763. A BLASTn search revealed 100% similarity to Fusarium sp. (Accession Nos. JQ038020 and JQ038013). Detached green shoots of healthy 1-year-old avocado were wounded to a depth of 1 to 2 mm and 5-mm mycelial plugs from 5-day-old cultures (UCR 1781 and UCR 1837) were placed mycelial side down onto the freshly wounded surfaces and then wrapped with Parafilm. Control shoots were inoculated with sterile agar plugs and five replicates per treatment were used. Shoots were incubated at 25 ± 1°C in moist chambers for 3 weeks. Lesions were observed on all inoculated shoots except for the control. Mean lesion lengths were 10.7 and 12.8 cm for UCR1781 and UCR1837, respectively, and were significantly different (P ≤ 0.05) from the control. Both isolates were reisolated from 100% of symptomatic tissues of inoculated shoots to complete Koch's postulates. This experiment was conducted twice and similar results were obtained. To our knowledge, this is the first report of Fusarium sp. and its vector E. fornicatus causing Fusarium dieback on Avocado in California.

Response of the shot-hole borer of tea, Xyleborus fornicatus (Coleoptera: Scolytidae) to conspecifics and plant semiochemicals

The responses of the tea shot-hole borer beetle, Xyleborus fornicatus, to conspecific and host plant odours were tested in an olfactometer. Males showed greater attraction to unmated females than to mated females and mated females showed a negative density dependent response with extremely significant repulsion shown when 10 mated females were present. Plants of the susceptible tea cultivar TRI2025 were significantly more attractive to females than those of the less susceptible cultivar TRI2023, although stem extracts of the susceptible cultivar were not found to be attractive to beetles. Beetles were found to be attracted to ethanol and to the known tea volatiles eugenol, hexanol, α- and β-pinene, geraniol, and methyl salicylate. The attraction of the tea volatiles was found to be enhanced when they were mixed with ethanol, indicating that ethanol has a broad synergistic effect. In a study on phagostimulant effects of tea extracts, sugars, and caffeine, the methanol extract of cultivar TRI2025 and the sugars glucose and sucrose were found to be attractive, and caffeine repulsive, to beetles. Pellets containing the extract were found to be preferred over those containing glucose. Beetles were attracted to pellets containing glucose/inositol mixture with glucose:inositol in the 3:1 ratio found in the susceptible cultivar TRI2025, being preferred to the mixture in 5:1 ratio found in stems of the less susceptible cultivar TRI2025. Both volatile and nonvolatile constituents of tea appear to play a role in attracting the female beetle to the plant.