Hypothenemus hampei (Ferrari) from 2 different populations (Tapachula, ... from 90% of ï¬eld-collected female beetles from Tapachula, Chiapas, Mexico,.
ECOLOGY AND POPULATION BIOLOGY
Association Between Hypothenemus hampei (Coleoptera: Scolytidae) and Fusarium solani (Moniliales: Tuberculariaceae) M. GUADALUPE ROJAS, JUAN A. MORALES-RAMOS,
AND
THOMAS C. HARRINGTON1
Formosan Subterranean Termite Unit, USDAÐARS-SRRC, New Orleans, LA 70179
Ann. Entomol. Soc. Am. 92(1): 98Ð100 (1999)
ABSTRACT The fungus Fusarium solani (Martius) Saccardo was isolated from adult females of Hypothenemus hampei (Ferrari) from 2 different populations (Tapachula, Chiapas, Mexico; and Allada, Benin, Africa). F. solani was isolated from 60% of H. hampei females reared on an artiÞcial diet for 3 generations, from 90% of Þeld-collected female beetles from Tapachula, Chiapas, Mexico, and from 100% of Þeld-collected female beetles from Allada, Benin. The sequences of the internal transcribed spacer regions of the nuclear rDNA of the isolates from Mexico and Benin were similar to each other and to published sequences of F. solani (asexual states of Nectria hematococca). The possibility of a symbiotic association between F. solani and H. hampei is discussed. KEY WORDS Hypothenemus hampei, Fusarium solani, Nectria hematococca, ambrosia, coffee
THE COFFEE BERRY borer, Hypothenemus hampei (Ferrari), is the worldÕs most destructive insect pest of coffee (Coffea spp.). The origin of H. hampei is is believed to be Angola, from where it spread to the rest of Africa by the late 1920s. It was introduced to Java (1909, into Borneo in 1919, and into the Brazilian State of Sao Paulo in 1924 (Corbett 1933). The borer then spread from Brazil into all South American coffeeproducing countries, reaching Guatemala in 1971 and Mexico in 1978 (Baker 1984). Hypothenemus hampei can colonize and reproduce in 12 species of coffee. Although H. hampei has been observed attacking plants of several other genera, it has never been reported to reproduce in such plants (Johanneson and Mansingh 1984). Females colonize coffee berries by burrowing through the nipple (Corbett 1933). Mature red and yellow berries are preferred over green berries for colonization and breeding (Igboekwe 1984). Females of H. hampei oviposit 2 d after colonization (Baker et al. 1992). The developmental period lasts 30, 42, and 49 d at means of 26, 23.4, and 20.88C, respectively (Baker et al. 1992). The sex ratio (or proportion of females) is highly female-biased with a mean ratio of 13 females per male (Corbett 1933), although Baker et al. (1992) reported a mean of 10 females per male in colonies of up to 200 individuals. Males are unable to ßy and remain inside the berries for their entire life (Corbett 1933). However, females are not parthenogenetic and require fertilization to produce viable eggs (Corbett 1933, Barrera et al. 1995). The observed sex bias has been explained by the existence of pseudoarrhenotoky from indirect evidence based on inheri-
1 Department of Plant Pathology, Iowa State University, Ames, IA 50011.
tance of insecticide resistance (Borsa and Kjellberg 1996). An association between H. hampei and a fungal species has not been reported. Beaver (1986) reports the presence of mycangia on a congener, Hypothenemus curtipennis (Schedl), a rainforest scolytid of Western Samoa. Mycangia or mycetangia are specialized organs for the transport of symbiotic fungi (Batra 1963, Giese 1965). The presence of these structures suggests H. curtipennis may have a symbiotic association with some unknown fungi. Mycangia in H. hampei have not been reported. The objectives of our study were to determine whether an association between H. hampei and a fungus exists and to identify the associated fungal species. Materials and Methods Biological Materials. Three colonies from 2 populations of H. hampei were brought to the USDA-APHIS quarantine facility at the Biological Control Center in Mission, TX. Two colonies originated from Tapachula, Chiapas, Mexico. Colony 1 was obtained from Barrera after having been reared on the artiÞcial diet reported by Villarcorta and Barrera (1993, 1996) for 2Ð3 generations, before their introduction. The 2nd colony was isolated from infested coffee berries collected from coffee Þelds (from the ground and trees) near Tapachula, Chiapas. The 3rd colony was isolated from infested coffee berries collected from coffee plantations (from the ground and trees) near Allada, Benin, Africa. Fungal Isolation Method. Beetles collected from Þeld samples were cleaned with sorbic acid to reduce microbial contaminants before attempting to isolate any associated fungi. Insects were 1st rinsed with sterile water for 1 min, then placed between sterile paper
January 1999
ROJAS ET AL.: ASSOCIATION BETWEEN H. hampei AND F. solani
towels (50 insects at a time). These paper towels with the insects were soaked in 10 ml of 0.2% sterile sorbic acid solution for 5 min and then rinsed 3 times with sterile water for another 1 min. Rinsed beetles were dried by placing them on sterile paper inside a sterile glass petri dish for 24 h. Beetles from the artiÞcial medium were not cleaned because they were assumed to be free of contaminants. The artiÞcial medium included potent antifungal ingredients and remained free of fungal growth (Villacorta and Barrera 1993, 1996). Under aseptic conditions, 100 beetles from each colony were individually placed inside sterile glass petri dishes containing an artiÞcial diet devoid of microbial inhibitors (M. G. Rojas et al. to be submitted for a patent). The 300 petri dishes, each with a single beetle, were incubated for 1 wk at 27 6 28C, 78 6 15% RH, and total darkness, then examined for fungal growth, and, Þnally, the total number of dishes with fungal growth was recorded. Fungal Identification. Samples of fungi that grew from the beetle and on the coffee berry borer diet, including mycelia with conidiophores and conidia, were dried and sent for identiÞcation to the Department of Plant Pathology, Iowa State University, Ames. Both morphological and DNA characteristics were used for fungal identiÞcation. Template DNA of the fungal isolates was obtained by scraping mycelia lightly with a pipette tip, and DNA was ampliÞed as described by Harrington and WingÞeld (1995). The primers used for polymerase chain reaction (PCR) and sequencing were ITS-1 F (Gardes and Bruns 1993) and ITS-4 (White et al. 1990), which amplify the internal transcribed spacer regions (ITS) of the nuclear ribosomal DNA. PCR products were puriÞed using Microcon Microconcentrators (Amicon, Cleveland, OH) and sequenced with the ABI PRISM 377 genetic analyzer (Perkin-Elmer, Applied Biosystems, Foster City, CA) in the DNA Sequencing Facility at Iowa State University. Results Of the 100 beetles screened from each colony, fungal growth was found in 60, 90, and 100% of the petri dishes from colonies 1 (BarreraÕs colony), 2 (from infested berries collected near Tapachula), and 3 (infested berries collected near Allada), respectively. Mycelial of all the isolates was uniform, and morphology veriÞed that all 3 colonies derived from a single species. Three fungal isolates from each of the Mexican and Benin colonies were used in PCR. The ITS region, which included ITS-1, the 5.8s rDNA gene, and ITS-2, was 477 or 478 bp long in each of the 6 isolates. The ITS sequences of the 3 Mexican isolates were identical (AF059206). The 3 Benin isolates had sequences identical to each other (AF059207), but differed slightly from that of the Mexican isolates, which had an extra base and 2 apparent base substitutions in the ITS-2 region. The DNA sequences from the Mexican and Benin isolates were used in BLAST searches of the GeneBank database (National Center for Bio-
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technology Information, U.S. National Library of Medicine, Bethesda, MD). The closest DNA sequence found was that of N. hematococca mating population VI (L36620), which differed from the Benin isolates in only 2 single base substitutions in ITS-1. The next closest sequence to those of the beetle isolates was that of an isolate of N. hematococca mating population V (L36619). Fusarium solani (Martius) Saccardo is the asexual state name for these mating populations of N. hematococca, and the morphology of the coffee berry borer isolates Þt the morphology of F. solani.
Discussion The 1st report of an association between fungi and a Cryphalini beetle was reported for Hypothenemus curtipennis (Schedl) (Beaver 1986). These scolytids have a pair of structures in the sides of the pronotum that have been described as mycangia; however, the associated fungi were not identiÞed (Beaver 1986). The results of the current study places H. hampei as the 2nd Cryphalini scolytid to be associated with a fungus. The consistency of isolation of F. solani from beetle populations from 2 continents suggests a close association. The survival of F. solani through surface cleansing with sorbic acid suggests that the fungus might be internal to the beetle, perhaps in a mycangium, in the gut of the beetle, or protected by specialized external cuticular structures of the beetle; electron microscopy studies are currently being done to determine the presence of mycangia. F.solani is a complex of poorly understood species, some of which are asexual states of N. hematococca, and which have been further divided into different mating populations (van Etten and Kistler 1988). F. solani has been reported associated with other Scolytidae, including Corthylus columbianus Hopkins (Kabir and Giese 1966), Xyleborus ferrugineus (F.) (Baker and Norris 1968), Xylosandrus compactus (Eichhoff) (Ngoan et al. 1976), and Xyleborus volvulus (F.) (Morales-Ramos 1982). The association between F. solani and X. ferrugineus is symbiotic, the beetle depending nutritionally on the fungus for development and reproduction (Baker and Norris 1968), and the fungus is maintained and disseminated by the beetle (Batra 1963). Other Fusarium spp. also have been found associated with ambrosia beetles, such as Xyleborus destruens (Balduf) and Xylosandrus germanus (Blandford) (Norris 1979). Also important is F. solaniÕs pathogenicity to a wide range of plants and a crustacean (van Etten and Kistler 1988) infecting the roots of many crops, such as tomato (Garg and Gupta 1979), potato (Braslavska 1977), peanut (Lima and Aguillar 1978), eggplant (Kumar et al. 1983), soybean (OÕDonnell and Gray 1995), and pine seedlings (Eusebio and Quimio 1977). Baker (1972) reports F. solani as as one of the agents causing root rot in coffee plants in Kenya. However, there are no reports linking the presence of coffee berry borer with root infections of coffee plants. It is possible that F. solani pathologically colonizes coffee berries and renders them more suitable for beetle development.
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ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Acknowledgments
We thank R. James (Subtropical Agricultural Research Center, USDA-ARS, Weslaco, TX) and J. Reeve (SRS, USDAForest Service, Pineville, LA) for review of the manuscript and also special thanks to J. Reeve for introducing me to T. Harrington.
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Received for publication 9 July 1998; accepted 11 September 1998.
