BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Acromyrmex octospinosus (Hymenoptera: Formicidae) Management: Effects of TRAMILs Fungicidal Plant Extracts Author(s): Isabelle Boulogne, Harry Ozier-Lafontaine, Lionel Germosén-Robineau, Lucienne Desfontaines, and Gladys Loranger-Merciris Source: Journal of Economic Entomology, 105(4):1224-1233. 2012. Published By: Entomological Society of America DOI: http://dx.doi.org/10.1603/EC11313 URL: http://www.bioone.org/doi/full/10.1603/EC11313 BioOne (www.bioone.org ) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use . Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder.
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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers,academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research.
Acromyrmex octospinosus (Hymenoptera: Formicidae)Management: Effects of TRAMILs Fungicidal Plant ExtractsAuthor(s): Isabelle Boulogne, Harry Ozier-Lafontaine, Lionel Germosén-Robineau,Lucienne Desfontaines, and Gladys Loranger-MercirisSource: Journal of Economic Entomology, 105(4):1224-1233. 2012.Published By: Entomological Society of AmericaDOI: http://dx.doi.org/10.1603/EC11313URL: http://www.bioone.org/doi/full/10.1603/EC11313
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in thebiological, ecological, and environmental sciences. BioOne provides a sustainable onlineplatform for over 170 journals and books published by nonprofit societies, associations,museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicatesyour acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercialuse. Commercial inquiries or rights and permissions requests should be directed to theindividual publisher as copyright holder.
Acromyrmex octospinosus (Hymenoptera: Formicidae) Management:Effects of TRAMILs Fungicidal Plant Extracts
ISABELLE BOULOGNE,1–4 HARRY OZIER-LAFONTAINE,2 LIONEL GERMOSEN-ROBINEAU,3
LUCIENNE DESFONTAINES,2 AND GLADYS LORANGER-MERCIRIS1,2
J. Econ. Entomol. 105(4): 1224Ð1233 (2012); DOI: http://dx.doi.org/10.1603/EC11313
ABSTRACT Leaf-cutting ants, Acromyrmex octospinosus (Reich), are considering among the mostimportant pest species of the New World. Until now, the main insecticides used for controlling theseants were synthetic chemicals. Leaf-cutting ants live in obligate symbiosis with a basidiomycete fungus,Leucocoprinus gongylophorus (Heim) Moeller. The crucial role of this symbiotic partner in the nestof leaf-cutting ants has prompted us to focus on A. octospinosus management through the use offungicides in our study. Five parts of plants identiÞed for their antifungal potential through TRAMILethnopharmacological surveys were tested: 1) bulbs ofAllium cepa L.; 2) seed pods of Allium sativumL.; 3) green fruits of Lycopersicon esculentum L.; 4) leaves ofManihot esculenta Crantz; and 5) leavesof Senna alata (L.) Roxburgh. One plant extract with strong fungicidal activity (S. alata) against L.gongylophoruswas found. The other extracts had lesser fungistatic or fungicidal effects depending onthe concentrations used. The data presented in this study showed that TRAMILs fungicidal plantextracts have potential to control the symbiotic fungus of leaf cutting ants, in particular a foliage extractof S. alata.
KEYWORDS Integrated pest management (IPM), natural fungicides, Leucocoprinus gongylophorus(Heim) Moeller, MTT, leaf-cutting ants
Leaf-cutting ants (tribe Attini, genera Acromyrmexand Atta) are considered major herbivores and mainagricultural pests in the Neotropics (Fowler 1978,Hughes et al. 2002). Owing to the large quantity ofplant material they collect, these ants cause substantialdamage to agricultural and forestry production andgenerate economic losses estimated at several milliondollars per year (Bacci et al. 2009). Fresh vegetationgathered by the ants is used to cultivate a symbioticbasidiomycete fungus that is the ultimate food sourcefor the ants, and they are therefore known as fungus-growing ants (Mikheyev 2008). This fungus has neverbeen found living outside antsÕ nests, probably be-cause it depends on the ants that have developedseveral mechanisms to protect it (Pagnocca et al.2001). It is vertically transmitted, from a queen to heroffspring. Indeed each mated female (queen of a newcolony) leaves her parental nest with a piece of thefungus garden stored in the infrabuccal pocket (We-ber 1972).
The leaf-cutting ant, Acromyrmex octospinosus(Reich) cultivates the fungus Leucocoprinus gongylo-phorus (Heim) Moeller (Silva et al. 2006). In thissymbiosis, the fungus converts the plant polysaccha-rides into nutrients easily assimilated by the ants and,reciprocally, ants protect the symbiotic partner fromparasites and potential competitors like Escovopsis(Currie 2001, Miyashira et al. 2010). The fungus growsinside ant nests and is called a Ôfungus garden.Õ It hasa gray spongy morphology, is easily broken and canmeasure 100Ð6,000 cm3 according to the age of thecolony. There are three parts to a fungus garden: asuperÞcial and young part called the Ôgreen and blackzoneÕ; a middle one called the Ôwhite zone,Õ and a basalpart, which is the oldest, called the Ôyellow zoneÕ(Decharme 1978). In the white and yellow parts, thefungus produces staphylae, bundles of specializedspheroid hyphae known as gongylidia. This specialmycelia structure is rich in glycogen and provides foodfor ant larvae and the queen. The workers also ingestthe cytoplasm of gongylidia to supplement their diet ofplant sap. Sap and digested fungal material are trans-mitted throughout the colony by workers via trophal-laxic regurgitations (Erthal et al. 2007).
Synthetic pesticides like organochlorines (Echols1966), sulßuramid (Knight and Rust 1991), or Þpronil(Hooper-Bui and Rust 2000) are used worldwide forthe control of leaf-cutting ants, but they can also causeconsiderable environmental damage (Paoletti et al.2000, Chauzat et al. 2006) and their overuse leads to
1 Universite des Antilles et de la Guyane, UFR Sciences Exactes etNaturelles, Campus de Fouillole, 97157 Pointe-a-Pitre Cedex, Gua-deloupe.
2 INRA, UR1321, ASTRO Agrosystemes Tropicaux, 97170, Petit-Bourg (Guadeloupe), France.
3 TRAMIL (Program of Applied Research to Popular Medicine inthe Caribbean), Laboratoire de Biologie et de Physiologie Vegetales,Campus de Fouillole, Universite des Antilles et de la Guyane, 97157Pointe-a-Pitre Cedex, Guadeloupe.
0022-0493/12/1224Ð1233$04.00/0 � 2012 Entomological Society of America
insecticide resistance (Bourguet et al. 2000). This sit-uation has prompted an increasing interest in alter-native methods for control of leaf cutter ants.
TRAMIL (Program of Applied Research to PopularMedicine in the Caribbean), created in 1982, is anetwork for applied research on the traditional andmedicinal plant resources of the Caribbean basin.TRAMILs mission is to validate traditional plantsÕ uses.Plant species identiÞed in ethnobotanical surveyswere selected if they had a frequency of 20% or higherfor a particular use (Boulogne et al. 2011). The ob-jectives and experience of TRAMIL in traditionalCaribbean knowledge and sustainable utilization ofplants seems served as our starting point to Þnd alter-native control methods for A. octospinosus manage-ment.
A previous study dealt with an alternative methodfor the control of leaf-cutting ants, that is, the appli-cation of TRAMIL insecticidal plant extracts (Bou-logne et al. 2012). However, the crucial role of thesymbiotic fungalpartner in thenestof leaf-cuttingantsprovides the opportunity for fungicidal managementof the A. octospinosus in this current study. AlthoughL. gongylophorus is considered the most importantsymbiont of the leaf-cutting ant, there are few pub-lished reports on the management of this fungus withplant extracts (Pagnocca et al. 1990, 2006; Lapointe etal. 1996; Bigi et al. 2004). Until now, fungicidal plantsidentiÞed in the TRAMIL project had not evaluatedfor the management of the symbiotic fungus. Never-theless, these were the plants most cited in interviewsurveys for treatment of fungal ailments. Indeed ac-cording to several TRAMIL surveys, the parts of theplants used in this study are used as fungicides againsta large spectrum of dermatophytes (TRAMIL 2007,Boulogne et al. 2011).
The current study focuses on the management of L.gongylophorus because of its symbiosis with the leaf-cutting ant A. octospinosus. The aim of the study is toevaluate several plant extracts, cited by TRAMIL fortheir fungicidal properties, on in vitro culture of L.gongylophorus.We hypothesized that these plant ex-tracts will have various effects on the fungus, fromfungistatic to fungicidal effects. Previous studies offungicidal effects of plants extracts against L. gongy-lophorus used only solid medium bioassays. In ourstudy, we compared results from solid medium bio-assays with those from a liquid medium assay. More-over, we used others tools not previously used on this
symbiotic fungus: an assay with tetrazole bromideMTT and MESURIM software to measure fungalgrowth. Studies on the characterization of antifungalcompounds in plants have shown that secondary me-tabolites (particularly alkaloids, phenolic compounds,and terpenoids) are responsible for this activity(Grayer and Harborne 1994). Thus, in addition to theantifungal assays, a preliminary phytochemical anal-ysis for the presence of alkaloids, phenolic com-pounds, and terpenoids in the extracts was carried out.
Materials and Methods
FungalMaterial.ArtiÞcial fungal gardens were main-tained in the laboratory.Piecesof fungus were removedfrom the green and black zone of these artiÞcial gar-dens using sterile Þne forceps and inoculated ontosolid medium (20 g/L of malt extract, 5 g/L of pep-tone, 2 g/L of yeast extract, 20 g/L of agar, and 1 literof distillated water) (Miyashira et al. 2010) containingstreptomycin (200 �g/ml) (Decharme 1978) to pre-vent bacterial contamination. The symbiotic funguswas identiÞed on the basis of the very slow fungalgrowth of the white mycelia on the medium and mor-phological characteristics including the gongylidia ob-served by microscopy on a one-mo-old fungal culture(Pagnocca et al. 2001, Miyashira et al. 2010).PlantsMaterial. Five traditional plant extracts cited
in the participative surveys were tested. The wholepart of the plants used for the bioassays was freeze-dried, ground with a coffee mill, and sieved at 0.5 mm.Voucher specimens were deposited at the JBSD Her-barium (Botanical Garden of Santo Domingo in theDominican Republic, the ofÞcial Herbarium of theTRAMIL network). Vouchers numbers, plant partsused, the dried residue in grams per kilogram of freshplant after freeze-drying and the source of the plantsare shown in Table 1.Fungicidal Bioassays.Antifungal TestWith SolidMe-dium. Each experimental unit consisted of a petri dish(Ø 5 cm) with 10 ml of sterilized solid medium (asdescribed above but without streptomycin) contain-ing three concentrations of dried plant extracts (500,1,000 and 2,000, �g/ml). One disk of active growingmycelium was isolated from a 1-mo-old fungal culturewith a 1 mm cork borer and used to inoculate themedium (Seaman 1984). Plates were then maintainedin a growth chamber at 25�C for 30 d. Six replicationsof each plant extract and the control were made. The
Table 1. grVouchers numbers, parts of plants used, dried residue in grams per kilogram of fresh plant after freeze-drying and sourcesof plants used
Vouchers numbersPart of plant
usedDry residue after freeze-drying
(grams per kilogram of fresh plant)Sources of collected or
Guadeloupe FWI)S. alata Boulogne,Gd,10,UAG/INRA Leaves 295,04 Family garden (Petit-Bourg,
Guadeloupe FWI)
August 2012 BOULOGNE ET AL.: Acromyrmex octospinosus MANAGEMENT 1225
petri dishes were scanned with a resolution of 600 dpiand the images were analyzed with MESURIM soft-ware to determine the growing fungal surface insquare millimeters.Antifungal Test With Liquid Medium. A 1-mo-old
fungal culture was aseptically transferred and frag-mented in a ßask of sterile peptone (1 g/L). Thismixture was then diluted in a 0.9% aqueous NaClsolution to obtain a mycelial suspension for inocula-tion (Pagnocca et al. 2006). Each experimental unitconsisted of a 5 ml sterile culture tube with 3 ml ofsterilized liquid medium (as above but without agarand streptomycin) containing three concentrations ofdried plant extracts (500, 1,000, and 2,000 �g/ml). Onemilliliter of mycelial suspension was placed in eachculture tube. Tubes were then maintained in a growthchamber at 25�C for 30 d. Six replications of each plantextract and controls were made. Fungi were recov-ered in each culture tube with Þne forceps, dried at70�C for 7 d and weighed.MTT Test. The MTT test is a colorimetric assay
intended to measure the activity of enzymes that re-duce MTT (a yellow tetrazole bromide) into a darkblue formazan precipitate. This test has been previ-ously used to determine the cytotoxicity of potentialfungicidal plants (Hadacek and Greger 2000). Eachexperimental unit consisted of a 5 ml sterile tube withsterilized liquid medium containing three concentra-tions of dried plant extracts and 1 ml of mycelialsuspension as in the antifungal test with liquid me-dium. Six replications of each plant extract and con-trols were made and tubes were maintained in a
growth chamber at 25�C for 15 d. Then 1 ml of MTTwas placed in each tube and tubes maintained at 25�Cfor a further 15 d. If the color of the culture tubebecame blue, the extract was considered to be fungi-static. If the color of the culture tube remained yellow,the extract was considered to be fungicidal.Chemical Analysis. Each freeze-dried plant sample
was mixed with an Ultra-Turax at 100 mg/ml withdistilled water at room temperature. The extracts werethen Þltered through an absorbent cotton syringe Þl-ter (10 � 20 mm) with a speed vacuum and stored at4�C before analysis. The extracts were submitted tophytochemical tests for plant secondary metabolitesknown for their potential fungicidal activity: alkaloids,phenolic compounds, and terpenoids. The quantity ofeach compound was determined using a Varian Cary100 spectrophotemeter, according to the followingquantitative methods.
● Alkaloids were determined using MarquisÕs reagentusing a colorimetric method (Szabo et al. 2003). Theextracts (500 �l) were oxidized by 2.5 ml of theformaldehyde-sulfuric acid reagent. Absorbancewas measured at 600 nm. Alkaloõds were expressedas piperine equivalents (�g PI/gFw) by comparingthe absorbance values with a standard curve (0Ð1mg/ml of piperine).
● Total phenolic compounds were determined usingthe Folin-Ciocalteau colorimetric method (Single-ton et al. 1999, Heilerova et al. 2003). The extracts(500 �l) were oxidized by 2.5 ml of the Folin-Cio-calteau reagent at 0.2 N and the reaction was neu-
Fig. 1. Fungal surface area in square millimeters in the antifungal test with solid medium. Treatments without commonsuperscript letters differ signiÞcantly based on the KruskalÐWallis test (K � 88.188; DLL � 15; P� 0.0001) with DunnÕs multiplecomparison test (P � 0.05). Extract concentrations are 500 (light), 1,000 (medium), and 2,000 (dark) �g/ml.
1226 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 4
tralized with 2 ml of sodium carbonate at 75 g/L.Absorbance was measured at 760 nm. Total phenoliccompounds were expressed as gallic acid equiva-lents (�g GA/gFw) by comparing the absorbancevalues with a standard curve (0Ð50 �g/ml of gallicacid).
● Terpenoids were determined using the iron (III)chloride-o-phosphoric acid-sulfuric acid colorimet-ric method (Zak et al. 1957). The extracts (100 �l)were heated at 100�C, solubilized with 3 ml of aceticacid, then 2 ml of iron (III) chlorideÐsulfuric acidreagentwasadded.Absorbancewasmeasuredat450nm. Terpenoõds were expressed as ergosterol equiv-alents (�g EG/gFw) by comparing the absorbancevalues with a standard curve (0Ð1 mg/ml of ergos-terol).
All values were expressed as the mean (milligrams ofstandards per grams of freeze-dried fresh plant sam-ple) � SD for three replications.Statistical Analyses. The results of fungicidal tests
were analyzed on the basis of percentage of growthinhibition (PI):
PI �ST � SE
ST
� 100
ST: average surface area (in square millimeters) oraverage weight (in grams) of the fungus on controlmedium. SE: average surface area (in square millime-ters) or average weight (in grams) of the fungus onmedium containing extracts.
IC50 and IC99 values (concentrations that inhibit 50and 99% of mycelium growth) and their 95% CIs werecalculated with logistic regression by Probit analysis(Finney 1971, Hosmer and Lemeshow 2000). A non-parametric analysis was also carried out using theKruskalÐWallis test (Hollander and Wolfe 1999, Mc-Donald 2009) and multiple comparisons using theDunn method (Dunn 1964). All analyses were madeusing XLSTAT software.
Results
Antifungal Test With Solid Medium. Treatmentswith Allium sativum L. and Manihot esculenta Crantzextracts at 2,000 �g/ml had a fungicidal effect on L.gongylophorus. Senna alata (L.) Roxburgh extract hadthe strongest fungicidal effect at all concentrations(Fig. 1).AntifungalTestWithLiquidMedium.Results were
similar to those with the solid medium in that extractsof A. sativum and M. esculenta at 2,000 �g/ml had afungicidal effect. S. alatawas again the most bioactiveplant extract with a signiÞcant fungicidal effect at allconcentrations (Fig. 2).MTT Test. Only S. alata L. extract had fungicidal
activity at all the concentrations used. A. sativumand M. esculenta extracts had fungistatic activity at500 and 1,000 �g/ml and were fungicidal at 2,000�g/ml with 100% inhibition of fungal growth. All the
Fig. 2. Weight in grams of fungus in the antifungal test with liquid medium. Treatments without common superscriptletters differ signiÞcantly based on the KruskalÐWallis test (K � 86.747; DLL � 15; P � 0.0001) with DunnÕs multiplecomparison test (P � 0.05). Concentrations as in Fig. 1.
August 2012 BOULOGNE ET AL.: Acromyrmex octospinosus MANAGEMENT 1227
other extracts had fungistatic effects at the concen-trations used (Table 2).Inhibitory Concentrations (IC50 and IC99). S. alata
extract had the lowest IC50 and IC99 values (respec-tively, 251.51 and 350.78 �g/ml). A. sativum and M.esculenta extracts had an IC99 value lower than thehighest concentration used. A. cepa L. had an esti-mated IC99 greater than the highest concentrationused. L. esculentum L. extract also had estimated IC50
and IC99 values (respectively, 2262.29 and 3547.40�g/ml) greater than the highest concentration used inthe assays (Table 3).Chemical Analysis. S. alata and A. sativum extracts
had the greatest quantities of total alkaloids (Fig. 3part A). S. alata extract also had the most total terpe-noids (Fig. 3 part B). S. alata andM. esculenta extractshad the greatest quantities of total phenolic com-pounds (Fig. 3 part C).
Discussion
The S. alata foliar extract was fungicidal against L.gongylophorus (Figs. 1 and 2; Tables 2 and 3). Ourresults are consistent with previous studies reportingthat extracts obtained from this plant have fungicidalactivity against several fungi (Table 4). Our prelimi-nary chemical analysis revealed that S. alata foliagecontains alkaloids, phenolic compounds, and terpe-noids (Fig. 3). Previous studies have shown that thefungicidal activity of S. alata foliar extracts can beattributed to phenolic compounds such as anthraqui-nones (Somchit et al. 2003) like chrysophanol (Pa-lanichamy and Nagarajan 1990).
The foliar extract ofM. esculentawas fungistatic andfungicidal at concentrations over 1,500 �g/ml (Figs. 1and 2; Tables 2 and 3). The use of M. esculenta as afungicide has been recognized in some studies inwhich leaf extracts were active against some fungi
(Table 4). Our chemical analysis revealed that M.esculenta foliage contains alkaloids, phenolic com-pounds, and terpenoids (Fig. 3). This is consistentwith previous studies that revealed that fungicidalactivity of the foliage was caused by phenolic com-pounds such as coumarins (Buschmann et al. 2000)and ßavonoids (Kamil et al. 1974).
The extract of A. cepa bulbs had no fungicidal ac-tivity in our tests. Fungistatic activity exists at con-centrations over 5,500 �g/ml (Figs. 1 and 2; Tables 2and 3). Nevertheless, previous studies indicated thatthebulbdemonstrated fungicidal activityagainst somefungi (Table 4).
The extract of A. sativum pods was fungistatic andfungicidal at concentrations over 1,500 �g/ml (Figs. 1and 2; Tables 2 and 3). The use of A. sativum as afungicide has long been recognized and several stud-ies indicated that pod extracts were active againstnumerous fungi (Table 4). Our chemical analysis re-vealed that bulbs of A. cepa and pods of A. sativumcontain alkaloids, phenolic compounds, and terpe-noids (Fig. 3). However, many previous studies re-vealed that organosulfur compounds, such as allicinand ajoene, were responsible for antifungal activitiesin Allium species (Cavallito et al. 1944, Kim et al.2004). The low fungicidal effect of these Allium spe-cies, despite their well-known antifungal compounds,may be because of the degradation of allicin and ajo-ene during the freeze-drying and sterilization proce-dures; allicin and ajoene are the immediate thermo-sensitive products of the enzymic degradation of theprecursor alliin (present in intact garlic and onion)(Kimbaris et al. 1944).
The green fruit of L. esculentum had no fungicidalactivity in our tests, although fungistatic activityoccurs at concentrations over 3,548 �g/ml (Figs. 1and 2; Tables 2 and 3). Previous studies have indi-cated that the fruit demonstrated fungicidal activityagainst some fungi (Table 4). Our chemical analysisrevealed that L. esculentum fruit contains alkaloids,phenolic compounds, and terpenoids (Fig. 3). Manystudies suggest that the alkaloid tomatine is respon-sible for the antifungal activity (Wilson et al. 1961,Lydon and Duke 1989, Burden et al. 1990). Theabsence of a fungicidal effect of L. esculentum fruitextract, despite its well-known antifungal com-pound, is not surprising in that the glycoalkaloidcontent depends on the plant part. Mean concen-trations of tomatine (measured by high perfor-mance liquid chromatography [HPLC] with ultra
�, color of the culture tube became blue (the extract was fungi-static); Ð, color of the culture tube remained yellow (the extract wasfungicidal).
Table 3. Concentrations that inhibited 50 and 99% of the mycelium growth (IC50 and IC99) and their 95% CIs calculated with logisticregression by Probit analysis on the basis of percentage of growth inhibition (Wald �2, Likehood Ratio �2, and associated P values of theregression tests were also given)
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violet detection) in the stems, ßowers, or leaves are100 times greater than in green fruit (Barceloux2009).
Based on our results, we conclude that some ofTRAMILsfungicidalplantextractshavepotential tocon-trol the symbiotic fungus of leaf cutting ants, L. gongy-
Fig. 3. Quantitative chemical analysis of plant extracts with values expressed in milligrams of standards per grams offreeze-dried plant sample � SD for three replications. Alkaloids (part A), terpenoids (part B), and phenolic compounds (partC). Treatments without common superscript letters differ signiÞcantly based on the KruskalÐWallis Test (alkaloids: K �16.055; DLL � 4; P � 0.003; terpenoids: K � 13.659, DLL � 4; P � 0.008; phenolics: K � 17.437; DLL � 4; P � 0.002) withDunnÕs multiple comparison test (P � 0.05).
August 2012 BOULOGNE ET AL.: Acromyrmex octospinosus MANAGEMENT 1229
Table 4. Fungicidal activity of TRAMIL plant chosen on others fungi
Fungi References
Allium cepa Saccharomyces cerevisiae, Elnima et al. 1983Candida albicansAspergillus niger Guerin and Reveillere 1984Aspergillus fumigatus, Yin and Tsao 1999Aspergillus flavusNannizzia gypsea, Singh and Deshmukh 1984Nannizzia incurvata,Nannizzia fulvaHelminthosporium turcicum, Khan et al. 1998Ascochyta rabiei
Allium sativum Microsporum canis Prasad et al. 1982Microsporum gypseum Malaengpoothong 1980Aspergillus flavus, Yin and Chen 1998Aspergillus nigerCandida albicans Vaijayanthimala et al. 2000Candida stellatoidea, Moore and Atkins 1977Candida tropicalis,Candida krusei,Candida guilliermondii,Candida rugosa,Candida pseudotropicalis,Candida tenuis,Cryptococcus neoformans,Cryptococcus terreus,Cryptococcus uniguttulatus,Cryptococcus laurentii,Rhodotorula rubra,Torulopsis candida,Torulopsis inconspicua,Torulopsis glabrata,Trichosporon capitatum,Trichosporon pullulansRhyzopus arrhizus, Caporaso et al. 1983Mucor pusillus,Rhizopus rhizopodiformis,Aspergillus fumigatus,Cryptococcus albidus,Candida parapsilosis,Candida glabrataFusarium oxysporum Tariq and Magee 1990Saccharomyces cerevisiae Elnima et al. 1983Penicillium digitatum Guerin and Reveillere 1984Fusarium moniliforme Liu et al. 1985Trychophyton asteroides Ploddee and Palakornkol 1977Trychophyton violaceum, Malaengpoothong 1980Trychophyton rubrumTrychophyton mentagrophytes, Caceres et al. 1991Epidermophyton floccosumPhytophthora species, Soytong et al. 1985Rhizopus microsporus,Melanconium fuligineum,Myrothecium roridum,Thanatephorus cucumeris,Tricholoma crassum,Volvariella volvacea,Ceratocystis paradoxa,Colletotrichum denatium,Sclerotium rolfsii,Pythium aphanidermatum,Alternaria alternata,Fusarium solani,Geotrichum candidum,Ustilago maydisTrichoderma species, Imwidthaya et al. 1978Curvularia species,Basidiobolum meristosporusTrichoconiella padwickii Shetty et al. 1989Nannizzia fulva, Singh and Deshmukh 1984Nannizzia gypsea,Nannizzia incurvata
Continued on following page
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lophorus. The freeze-dried extract of S. alata foliage, inparticular, is promising for leaf-cutter ant managementbased on its activity to the antÕs symbiotic fungus.
Baits containing S. alata extract (tested in the cur-rent study against the mutualistic fungus) and baitscontaining Mammea americana extract (tested in ourprevious study) (Boulogne et al. 2012) should betested against whole ant colonies in artiÞcial nests andeventually in the Þeld to determine if these extractscould kill entire ant nests like they kill ants and sym-biotic fungus in vitro. The lyophilized leaves of S. alataand lyophilized aqueous extract ofM. americana seedswill be incorporated into an artiÞcial diet composed ofglucose, peptone, yeast extract, agar, and distilled wa-ter and will be fed to ant colonies.
Acknowledgments
The authors thank CEREGMIA and its director Fred Ce-limene for Þnancial support (to I.B.). We also thank CeciliaDelag, Herve Mauleon, Michele Salles, Fred Burner, AndeveMulciba, and Guy Gougougnan for their technical assistance.Special thanks to Pr. Murray Isman for his revision to improvethe English language.
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Received 19 September 2011; accepted 9 March 2012.
August 2012 BOULOGNE ET AL.: Acromyrmex octospinosus MANAGEMENT 1233
