Daniel Carrillo

An inordinate fondness for Fusarium: Phylogenetic diversity of fusaria cultivated by ambrosia beetles in the genus Euwallacea on avocado and other plant hosts

Ambrosia beetle fungiculture represents one of the most ecologically and evolutionarily successful symbioses, as evidenced by the 11 independent origins and 3500 species of ambrosia beetles. Here we document the evolution of a clade within Fusarium associated with ambrosia beetles in the genus Euwallacea (Coleoptera: Scolytinae). Ambrosia Fusarium Clade (AFC) symbionts are unusual in that some are plant pathogens that cause significant damage in naïve natural and cultivated ecosystems, and currently threaten avocado production in the United States, Israel and Australia. Most AFC fusaria produce unusual clavate macroconidia that serve as a putative food source for their insect mutualists. AFC symbionts were abundant in the heads of four Euwallacea spp., which suggests that they are transported within and from the natal gallery in mandibular mycangia. In a four-locus phylogenetic analysis, the AFC was resolved in a strongly supported monophyletic group within the previously described Clade 3 of the Fusarium solani species complex (FSSC). Divergence-time estimates place the origin of the AFC in the early Miocene ∼21.2 Mya, which coincides with the hypothesized adaptive radiation of the Xyleborini. Two strongly supported clades within the AFC (Clades A and B) were identified that include nine species lineages associated with ambrosia beetles, eight with Euwallacea spp. and one reportedly with Xyleborus ferrugineus, and two lineages with no known beetle association. More derived lineages within the AFC showed fixation of the clavate (club-shaped) macroconidial trait, while basal lineages showed a mix of clavate and more typical fusiform macroconidia. AFC lineages consisted mostly of genetically identical individuals associated with specific insect hosts in defined geographic locations, with at least three interspecific hybridization events inferred based on discordant placement in individual gene genealogies and detection of recombinant loci. Overall, these data are consistent with a strong evolutionary trend toward obligate symbiosis coupled with secondary contact and interspecific hybridization.

Discordant phylogenies suggest repeated host shifts in the Fusarium-Euwallacea ambrosia beetle mutualism.

The mutualism between xyleborine beetles in the genus Euwallacea (Coleoptera: Curculionidae: Scolytinae) and members of the Ambrosia Fusarium Clade (AFC) represents one of 11 known evolutionary origins of fungiculture by ambrosia beetles. Female Euwallacea beetles transport fusarial symbionts in paired mandibular mycangia from their natal gallery to woody hosts where they are cultivated in galleries as a source of food. Native to Asia, several exotic Euwallacea species were introduced into the United States and Israel within the past two decades and they now threaten urban landscapes, forests and avocado production. To assess species limits and to date the evolutionary diversification of the mutualists, we reconstructed the evolutionary histories of key representatives of the Fusarium and Euwallacea clades using maximum parsimony and maximum likelihood methods. Twelve species-level lineages, termed AF 1-12, were identified within the monophyletic AFC and seven among the Fusarium-farming Euwallacea. Bayesian diversification-time estimates placed the origin of the Euwallacea-Fusarium mutualism near the Oligocene-Miocene boundary ∼19-24 Mya. Most Euwallacea spp. appear to be associated with one species of Fusarium, but two species farmed two closely related fusaria. Euwallacea sp. #2 in Miami-Dade County, Florida cultivated Fusarium spp. AF-6 and AF-8 on avocado, and Euwallacea sp. #4 farmed Fusarium ambrosium AF-1 and Fusarium sp. AF-11 on Chinese tea in Sri Lanka. Cophylogenetic analyses indicated that the Euwallacea and Fusarium phylogenies were largely incongruent, apparently due to the beetles switching fusarial symbionts (i.e., host shifts) at least five times during the evolution of this mutualism. Three cospeciation events between Euwallacea and their AFC symbionts were detected, but randomization tests failed to reject the null hypothesis that the putative parallel cladogenesis is a stochastic pattern. Lastly, two collections of Euwallacea sp. #2 from Miami-Dade County, Florida shared an identical cytochrome oxidase subunit 1 (CO1) allele with Euwallacea validus, suggesting introgressive hybridization between these species and/or pseudogenous nature of this marker. Results of the present study highlight the importance of understanding the potential for and frequency of host-switching between Euwallacea and members of the AFC, and that these shifts may bring together more aggressive and virulent combinations of these invasive mutualists.

Ambrosia Beetles (Coleoptera: Curculionidae: Scolytinae) that Breed in Avocado Wood in Florida

ABSTRACT Laurel wilt is a destructive disease caused by the fungus Raffaelea lauricola, which is transmitted by the invasive redbay ambrosia beetle, Xyleborus glabratus. Here we document ambrosia beetles that emerged from wilted avocado trees throughout Florida. In addition, the ambrosia beetle fauna associated with wilted swampbay trees in Miami-Dade was studied. Fourteen species of scolytine beetles were found associated with avocado wood from different parts of Florida. Multiple species of ambrosia beetles were found breeding in avocado and swampbay wood infected by R. lauricola with or without the presence of its primary vector, X. glabratus. Work is under way to determine whether other ambrosia beetle species can carry R. lauricola and transmit this pathogen to healthy avocado and swampbay trees.

Host plant range of Raoiella indica (Acari: Tenuipalpidae) in areas of invasion of the New World

Raoiella indica has spread rapidly through the Neotropical region where the mite damages economically and ecologically important plants. Three studies were conducted to determine the host plant range of R. indica, using the presence of colonies containing all life stages as an indicator of reproductive suitability. Periodic surveys at the Fairchild Tropical Botanic Garden (Miami Dade County, FL, USA) and the Royal Botanical Gardens (Port of Spain, Trinidad and Tobago) identified 27 new reproductive host plants. The reproductive suitability of two dicotyledonous species and three native Florida palm species was examined. An updated list of reproductive host plants of R. indica is presented. All reported reproductive hosts (91 plant species) of R. indica are monocots from the orders Arecales (Arecaceae), Zingiberales (Heliconiaceae, Musaceae, Strelitziaceae, Zingiberaceae) and Pandanales (Pandanaceae). Most are palms of the family Arecaceae that originated in areas of the Eastern Hemisphere; about one fourth of the reported hosts are native to the New World and could be considered new host associations of R. indica. Six years after the initial detection in the Caribbean, R. indica has expanded its host plant range. Here we report 27 new reproductive host of R. indica that represent 30% of increase on previous host plant records. As this mite continues spreading in the Neotropical region a great diversity of plants is potentially affected.

A review of the natural enemies of the red palm mite, Raoiella indica (Acari: Tenuipalpidae)

A review of all the available information about the natural enemies reported in association with the red palm mite, Raoiella indica is presented. Twenty-eight species of predatory arthropods, including mites and insects, have been reported in association with R. indica in Asia, Africa and the Neotropics. In addition, pathogenic fungi associated with R. indica in the Caribbean have been reported. The available literature indicates that each site has a different natural enemy complex with only one predator species, Amblyseius largoensis (Acari: Phytoseiidae), present in all the geographical areas. The phytoseiids, Amblyseius caudatus Berlese, Amblyseius channabasavanni Gupta and A. largoensis, were regarded as important natural enemies of R. indica, and their predatory efficiency was studied in some detail. Among the predatory insects the coccinellids Stethorus keralicus Kapur and Telsimia ephippiger Chapin were reported as major predators of R. indica. The known distribution, abundance and relative importance of each species reported in association with R. indica are discussed.

First Report of Raoiella Indica (Acari: Tenuipalpidae) in Colombia

Raoiella indica Hirst (Acari: Tenuipalpidae), the red palm mite, is a phytophagous mite that recently invaded the Western Hemisphere. This mite was first detected in Martinique (Flechtmann & Etienne 2004) and it rapidly spread to multiple islands of the Caribbean [St. Lucia and Dominica (Kane et al. 2005), Guadeloupe and Saint Martin (Etienne & Flechtmann 2006), Puerto Rico and Culebra Island (Rodrigues et al. 2007), and Cuba (de la Torre et al. 2010) among other islands]. In 2007, the mite was found in West Palm Beach, Florida (FDACS 2007), and in the state of Sucre, Venezuela (Vasquez et al. 2008), and more recently, reported in the northern state of Roraima in Brazil (Marsaro Jr. et al. 2009), and Isla Mujeres and Cancun, Mexico (NAPPO 2009). In January 2010, high populations of R. indica were found attacking coconut (Cocos nucifera L.), banana (Musa acuminata Colla) and heliconia (Heliconia sp.) plants in the Tayrona National Park located in the Colombian Caribbean littoral, near the city of Santa Marta, Magdalena. The presence of multigenerational colonies and exuvia was confirmed in 18 coconut palms, 4 heliconias and multiple banana plants located near the coast in the northern part of the park (11°18’44”N 73°56’04”W). In further surveys R. indica infestations were detected in commercial coconut and banana groves in June 2010 at Los Naranjos, Magdalena (11°17’49”N -73°53’49”W), approximately 6 km East of the Tayrona Park along the coast. In this locality the predatory mite Amblyseius largoensis Muma (Acari: Phytoseiidae) was found showing a conspicuous red coloration of the alimentary tract indicating recent feeding on R. indica. Previous studies indicated that populations of A. largoensis increased in numbers after the arrival of R. indica to Florida and some areas in the Neotropics (Pena et al. 2009; Carrillo et al. 2010). Raoiella indica and A. largoensis specimens were collected (70% ethanol) and subsequently slide mounted, identified, and deposited in the collections of the Laboratory of Plant Quarantine reference collection, Embrapa Genetic Resources and Biotechnology, Brasilia, Brazil, and the Laboratory of Acarology from the Instituto Agroforestal Mediterraneo, Universidad Politecnica of Valencia, Spain. The experienced negative effects of R. indica on coconut production in the Caribbean, where yield reduction has been estimated in over 50% at some locations (CARDI 2010), indicate the importance of adopting regulatory and other control measures in areas of recent invasion. The establishment of chemical practices needed to allow movement of host plant material, and continuous surveying (pre and post-invasion) using sentinel sites, have been adopted to prevent R. indica’s rapid dissemination in Florida (Roda et al. 2008). An integrated approach combining all available control tactics should be adopted and natural enemies identified for managing this species (Pena et al. 2009; Carrillo et al. 2010). In addition, studies are needed to determine the potential host plant range of R. indica in Colombia and the rest of the Neotropical region. Strict sanitary measures and other management tactics should be implemented to minimize the damage caused by R. indica in Colombia and other countries in South and Central America.

Recovery plan for laurel wilt on redbay and other forest species caused by Raffaelea lauricola and disseminated by Xyleborus glabratus

This recovery plan is one of several disease-specific documents produced as part of the National Plant Disease Recovery System (NPDRS) called for in Homeland Security Presidential Directive Number 9 (HSPD-9). The purpose of the NPDRS is to insure that the tools, infrastructure, communication networks, and capacity required to mitigate the impact of high-consequence plant disease outbreaks are such that a reasonable level of crop production is maintained. Each disease-specific plan is intended to provide a brief primer on the disease, assess the status of critical recovery components, and identify disease management research, extension, and education needs. These documents are not intended to be stand-alone documents that address all of the many and varied aspects of plant disease outbreak and all of the decisions that must be made and actions taken to achieve effective response and recovery. They are, however, documents that will help USDA guide further efforts directed toward plant disease recovery.

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

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 Hamiltons 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.

Presence and Prevalence of Raffaelea lauricola, Cause of Laurel Wilt, in Different Species of Ambrosia Beetle in Florida, USA

Abstract We summarize the information available on ambrosia beetle species that have been associated in Florida with Raffaelea lauricola T.C. Harr., Fraedrich & Aghayeva, the primary symbiont of Xyleborus glabratus Eichhoff and cause of laurel wilt. In total, 14 species in Ambrosiodmus, Euwallacea, Premnobius, Xyleborus, Xyleborinus, and Xylosandrus were either reared from laurel wilt-affected host trees or trapped in laurel wilt-affected stands of the same, and assayed for R. lauricola. In six collections from native species in the southeastern United States [Persea borbonia (L.), Persea palustris (Raf.) Sarg., and Persea humilis Nash] and four from avocado (Persea americana Mill.), extracted mycangia or heads (taxa with mandibular mycangia) or intact bodies (taxa with mycangia in other locations) were surface-disinfested before assays on a semi-selective medium for the isolation of Raffaelea (CSMA+). Raffaelea lauricola was identified based on its characteristic phenotype on CSMA+, and the identity of a random subset of isolates was confirmed with taxon-specific microsatellite markers. The pathogen was recovered from 34% (246 of 726) of the individuals that were associated with the native Persea spp., but only 6% (58 of 931) of those that were associated with avocado. Over all studies, R. lauricola was recovered from 10 of the ambrosia beetle species, but it was most prevalent in Xyleborus congeners. This is the first record of R. lauricola in Ambrosiodmus lecontei Hopkins, Xyleborinus andrewesi (Blandford), and Xyleborus bispinatus Eichhoff. The potential effects of R. lauricolas promiscuity are discussed.

Potential of Contact Insecticides to Control Xyleborus glabratus (Coleoptera: Curculionidae), a Vector of Laurel Wilt Disease in Avocados

ABSTRACT Xyleborus glabratus Eichhoff (Coleoptera: Curculionidae: Scolytinae) is an invasive ambrosia beetle that vectors laurel wilt, a new disease that threatens avocado and other species in the Lauraceae Family. The lethal concentrations (LC50 & 90) of nine commercial insecticides to X. glabratus were determined by using a bolt-dip bioassay. Different formulations of bifenthrin, permethrin, fenpropathrin, z-cypermethrin + bifenthrin, l-cyhalothrin + thiamethoxam, malathion, chlorpyrifos, carbaryl, and methomyl were tested. Four concentrations of each insecticide were tested (0.5, 0.1, 0.03, and 0.01 of the label rate) and with water as a control. Beetles were exposed to treated bolts and mortality registered 48 h later. After 2 wk, bolts were destructively sampled to determine the number of beetles that constructed galleries and were alive inside the wood. Probit analysis was used to determine the LC50 & 90. Six pesticides were applied directly to the trunk and limbs of avocado trees in a commercial grove. Limbs of treated trees were cut weekly after the application and exposed to X. glabratus to determine the number of beetles boring into the logs. The toxicity of pesticides to X. glabratus was greatly reduced 2 wk after application. Among the tested pesticides, malathion and z-cypermethrin + bifenthrin provided the best suppression of X. glabratus. Among the insecticides registered for use in avocado, fenpropathrin and malathion were the most effective in protecting trees from attack by X. glabratus. Other pesticides that are currently not registered for use in avocados could be useful for managing this ambrosia beetle.