Pathogenicity of Indigenous Strains of Three Entomopathogenic Fungi to the Sisal Weevil, Scyphophorus Acupunctatus (Gyllenhal) (Coleoptera: Curculionidae)

Hellenic Plant Protection Journal 8: 46-54, 2015 DOI 10.1515/hppj-2015-0007

Pathogenicity of indigenous strains of three entomopathogenic fungi to the sisal weevil, Scyphophorus acupunctatus (Gyllenhal) (Coleoptera: Curculionidae)

V.T. Gkounti, D. Markoyiannaki and D.Ch. Kontodimas*

Summary The pathogenicity of indigenous isolates of Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea was evaluated in the laboratory against larvae and adults of the sisal weevil, Scyphophorus acupunctatus. Inoculation was achieved via immersion of individuals into conidia suspensions of diff erent concentrations. All three fungal species proved high pathogenicity against larvae of the weevil, causing 100% mortality in most of the treatments. Beauveria bassiana caused the highest mortality of the adults (86.67±12%), followed by M. anisopliae (46.67±17.8%) and I. fumosorosea (40±17.5%). Mean survival time also diff ered signifi cantly among treatments and life stages of the weevil. In total, larvae survived signifi cantly fewer days than adults post infection. Results of the present study indicate the potential of indigenous strains of entomopathogenic fungi as biological control agents against the invasive weevil.

Additional keywords: agave, Beauveria bassiana, Curculionidae, Isaria fumosorosea, Metarhizium anisopliae

Introduction by plant’s fi bers to complete their development (Lock, 1965). In addition to the de- The sisal weevil, Scyphophorus acupuncta- structive activities of the larvae, the adults tus (Gyllenhal), is amongst the most severe are often natural vectors of plant pathogen- pests of both cultivated and ornamental ic bacteria, such as Erwinia carotovora (Dye) agave plants (Pott, 1975; Valenzuela-Zapata, (Aquino Bolanos et al., 2011). In Central and 1994; Camino Lavin et al., 2002). In its place South American agave cultivations chem- of origin, the Nearctic region, it has been ical insecticides are commonly applied to found to cause severe economic losses by control the sisal weevil, however there are strongly damaging Agave tequilana (We- many problems and controversies connect- ber) the tequila producing agave (Solis et ed to their use (Solis et al., 2001; Terán-Var- al., 2001). The sisal weevil was introduced gas et al, 2012). Firstly, as the weevil com- in Europe around 1980, when it was fi rst re- pletes its development within a protected corded on imported Yucca sp. in the Neth- habitat, inside the agave leaves, insecticides erlands (van Rossem et al., 1981). Since then have a low eff ectiveness (Figueroa-Castro et it has been found in many European coun- al., 2013). Secondly, their extended applica- tries (i.e. Italy, France, Greece) on ornamen- tion harbors the risk of resistance develop- tal plants (Colombo, 2000; EPPO 2008; Kon- ment in the pest species (Terán -Vargas et todimas and Kallinikou, 2010). al., 2012). Finally, there are certain restric- The females use the basal parts of the tions in the use of synthetic insecticides in agave leaves for feeding and as oviposition urban landscape areas, where the weevil is sites. Furthermore, once the neonate lar- mainly found, due to their eff ects on the en- vae hatch they start to bore galleries with- vironment and health risks. Therefore, the in the plant’s head and form a cocoon made development of biological and biotechnical control methods is of great environmental and economic importance. Department of Entomology and Agricultural Zoolo- Although a few arthropod natural ene- gy, Benaki Phytopathological Institute, 8 St. Delta Str., GR-145 61 Kifi ssia, Attica, Greece mies of the sisal weevil could potentially be * Corresponding author: [email protected] regarded as biological control agents in its

© Benaki Phytopathological Institute Pathogenicity of entomopathogenic fungi on sisal weevil 47 site of origin (Velazquez et al., 2008), these digenous isolates of fungal entomopatho- arthropods are not found in the invaded re- gens is advantageous, as they are already gions. Entomopathogenic fungi and nema- adapted in specifi c environmental condi- todes have been reported as potential bio- tions and could be part of a conservation bi- logical control agents of the weevil (Hueso ological control strategy. et al., 2005; Velazquez et al., 2008). Both the fungi and the nematodes are of great importance for the biological control of S. acu- Material and Methods punctatus, as they are able to overcome the barrier of the concealed environment in Entomopathogenic fungi which the weevil develops. While the nem- All entomopathogenic fungi strains used atodes are able to move within the agave in the laboratory assays were obtained from leaves and are easily able to reach the con- the entomopathogenic fungi collection of cealed pest (Dembilio et al., 2010a), the en- the Benaki Phytopathological Institute. The tomopathogenic fungi infect their hosts strain of B. bassiana IBB 010 was initially iso- by direct contact and subsequent penetra- lated from a Rhynchophorus ferrugineus (Ol- tion of their cuticle (Dembilio et al., 2010b). ivier) cadaver, found in Ellinikon region This makes insects susceptible both to di- (Athens, Greece). Both strains IBB020 of I. fu- rect contact with the pathogen, but also to mosorosea and TMB04 of M. anisopliae were passive, horizontal transmission, between isolated from soil samples with the Galleria healthy and infected individuals vector- bait method (Zimmerman, 1986) from the ing spores (Klein and Lacey, 1999; Quesada- Agios Stefanos and Marathon regions (At- Moraga et al., 2004). tica, Greece), respectively. The molecular It has been widely accepted that ento- identifi cation of the strains of I. fumosorosea mopathogenic fungi show high intra-spe- and M. anisopliae was previously conduct- cifi c diversity, which results in diff erent lev- ed by Beris et al. (2013). The fungal isolations els of pathogenicity among diff erent strains were cultured on Sabouraud dextrose agar of the same species (Ferron, 1978). Further- (SDA) at 25°C, under dark regulated condi- more, the virulence of a specifi c strain to tions. Spore (conidia) suspensions were pre- specifi c insect genera has been proved to pared by scraping the surface of 2 week old depend upon the host insect identity from cultures into an aqueous solution of 0.2% which it was isolated (Fargues and Robert, Tween 80, after which the suspensions were 1983; Maurer et al., 1997). This is attributed vortexed for 60 s and fi ltered twice using a to the strong selective pressure of the host sterile nylon membrane to remove myceli- insect environment on the pathogen (Roy um. Subsequently, the concentration of the et al., 2006) through which the origin of the spore suspensions was determined by the entomopathogens could aff ect their patho- use of a standard haemocytometer. The genicity against pests (Cherry et al., 2005). concentrations of spore suspensions used Despite the importance of alternative in bioassays were 2.12 x 107 and 2.12 x 106 methods for the control of the sisal weevil, spores/ml for B. bassiana, 1.27 x 107 and 1.27 research on the evaluation of potential bio- x 106 spores/ml for M. anisopliae and 7.45 x logical control agents of the pest is very lim- 106 spores/ml for I. fumosorosea. Spore con- ited (Molina-Ochoa et al., 2004; Valdes Estra- centrations are not equal due to the diff er- da et al., 2014). In the present study we aim ent developmental rate of each fungus (Kon- to evaluate the virulence of locally isolat- todimas and Gkotsi, 2009). Spore viability ed strains of Beauveria bassiana (Balsamo), measured over 96% for all selected strains. Metarhizium anisopliae (Metchnikoff ) So- rokin and Isaria fumosorosea (Wize) to adults Insects and larvae of the introduced weevil S. acu- Both adult and larvae of the sisal wee- punctatus. It is expected that the use of in- vil were collected from ornamental aga-

© Benaki Phytopathological Institute 48 Gkounti et al. ve plants, Agave americana L., in the ur- α=0.05. The analysis included life stage (lar- ban landscape area of Ardittos hill (Athens, val and adult), species of fungi (B. bassiana, Greece). The infestation of the agave plants I. fumosorosea and M. anisopliae), and con- was spotted in the summer of 2012 and no centration of spore suspensions (106 and action for controlling the pest was taken pri- 107 spores/ml) as variables. Additionally, or to the collection of the weevils. The adults the average survival times were calculated were collected by hand at site, while whole and compared by the Kaplan-Meier surviv- plants were cut from the basis and taken to al analysis (Kaplan and Meier, 1958). All the the laboratory for the isolation of 4th instar GLM procedures and survival analysis were larvae. The adults were kept in polyester carried out in R (v. 2.1; R Foundation for Sta- cages (30 x 30 x 30cm) and the larvae in plas- tistical Computing, Vienna, AT). tic boxes (20 x 20 x 20cm), with an opening covered by mesh for ventilation. All insects were kept at controlled laboratory condi- Results tions (25±1°C, 65±5% R.H. and 14:10 L:D) and fed on a natural diet (agave slices; twice per The examined strains of entomopathogenic week) until they were used in the bioassays. fungi caused diff erent levels of mortality to S. acupunctatus. The mean mortality of wee- Bioassays vils diff ered signifi cantly at diff erent life stag- The application of the spore suspen- es, and fungus species, but it did not diff er sions was achieved through direct cuticle between diff erent concentrations of spore contact. Groups of fi ve, 4th instar larvae and suspensions (X2=22.939, df=1, P<0.0001; adults were immersed in the prepared aque- X2=9.5758, df=2, P<0.0001 and X2=2.5984, ous suspensions for 30 sec after which each df=1, P=0.1070, respectively). No mortali- group of insects was transferred in 1L rearing ty was recorded for adult weevils or larvae boxes and provided with fresh natural diet. when treated with the aqueous solution of The treatment was repeated 15 times (rep- 0.2% Tween 80 (i.e. control treatment). Inter- licates) for each fungus strain and concen- actions among the variables did not aff ect tration per life stage. Furthermore, a control the mortality levels, thus, they were omitted treatment was included for both adults and from the initial model. larvae, where insects were immersed into All treatments with fungi resulted in al- an aqueous solution of 0.2% Tween 80. All most all concentrations at 100% mortality of bioassays were carried out in controlled en- larvae (Figure 1a). Mortality in adults depend- vironment conditions (25±1°C, 65±5% R.H. ed on fungus species but not on the concen- and 14:10 L:D). The mortality was recorded tration of the suspensions (X2=11.4223, df=2, daily for up to 11 and 21 days for larvae and P=0.0033 and X2=2.9638, df=1, P=0.0851, re- adults, respectively. When a weevil died, the spectively). The treatment with B. bassiana cadaver was transferred individually to a Pe- at the concentration of 2.12 x 107 spores/ml tri dish lined with a moistened sterile fi lter caused the highest mortality to adult wee- paper to ascertain the involvement of the vils (86.67±12%) compared to M. anisopliae entomopathogens to the death of weevils. and I. fumosorosea treatments (Figure 1b). The Petri dishes were sealed with parafi lm The lowest mortality levels to adults were and kept at room temperature, in darkness, caused by M. anisopliae, when applied at the to monitor for external signs of fungal infec- low spore concentration (26.67±15.8%). tion. The examined strains of B. bassiana, M. anisopliae and I. fumosorosea developed ex- Statistical analyses ternal fungal growth and sporulated on all General Linear Model (GLM) with a bino- treated cadavers of both adults (Figure 2) mial distribution was used to obtain Anal- and larvae, regardless the spore concentra- ysis of Deviance for a level of signifi cance tion.

Figure 1. Mean mortality ± SE of (A) larvae and (B) adults of Scyphophorus acupunctatus after immersion into spore suspensions of entomopathogenic fungi. *Diff erent letters within the same chart indicate signifi cant diff erences (LR test, α=0.05).

(a) (b) (c) Figure 2. Development of external fungal mycelium on cadavers of Scyphophorus acupunctatus adults: Beauveria bassiana (a), Metarhizium anisopliae (b) and Isaria fumosorosea (c).

The mean survival time of the treat- vil. Although all strains managed to fully ed larvae diff ered signifi cantly at diff erent complete their life cycle by sporulating on fungi species and diff erent concentrations, S. acupunctatus cadavers, B. bassiana proved namely the low concentration of B. bassi- to be the most pathogenetic to adult wee- ana (6.80±0.251 days) compared to the oth- vils. Several studies have pointed out the re- er treatments (4.73-5.67 days) (X2=8.3, df=4, lation between strains of entomopathogens P=0.0807; Table 1). isolated from specifi c hosts and the high vir- The mean survival time diff ered signifi - ulence towards the host genera (Fargues cantly for adult weevils that received diff er- and Robert, 1983; Maurer et al., 1997). There- ent treatments (X2=17.5, df=4, P=0.0016; Ta- fore, the high virulence of the strain IBB010 ble 1). Adult weevils treated with the high of B. bassiana might be related to its source concentration of B. bassiana exhibited the of isolation, an infected red palm weevil ca- shortest survival time (10.20±1.055 days) daver, as both the red palm weevil and the (Table 1). Overall, the survival time of larvae sisal weevil belong to the same tribe of was signifi cantly shorter than that of adults Rhynchophorini. (X2=105, df=1, P<0.001). Until now, B. bassiana is the only entomopathogenic fungus species mentioned as a natural enemy of the weevil in its place Discussion of origin (Aquino Bolanos et al., 2011). Our results showed that the concentration of 2.12 All species of fungi proved to be highly ef- x 107 spore suspension of B. bassiana caused fective on larvae of S. acupunctatus. Fur- a quicker death of adults compared to spore ther wider dose range tests would be neces- suspensions of M. anisopliae and I. fumoso- sary to show the dose eff ect, the diff erences rosea. Moreover, a 10-fold increase of the among strains and the potential of the test- diluted spore suspension of the B. bassiana ed strains in lower concentrations. Howev- strain from 2.12 x 106 to 2.12 x 107 decreased er, our results showed that the examined the mean survival time for adults by almost strains of entomopathogenic fungi are able 4 days . to infect larvae and adults of the sisal wee- The sisal weevil has identical life hab-

Table 1. Mean survival time ± SE (days after treatment) for larvae and adults of Scyphopho- rus acupunctatus treated with Beauveria bassiana, Metarhizium anisopliae and Isaria fumosorosea spore suspensions.

Mean survival time ± SE (days) Treatment Concentration (spores/ml) Larvae Adults 2.12 x 107 5.47±0.351a 10.20±1.055a B. bassiana 2.12 x 106 6.80±0.251b 14.74±0.999b 1.27 x 107 5.00±0.359a 15.65±1.031bc M. anisopliae 1.27 x 106 5.67±0.308ab 17.20±1.210 c I. fumosorosea 7.45 x 106 4.73±0.314a 17.50±1.025c * Mean survival times within columns followed by the same letter are not signifi cantly diff erent (LR test, α=0.05) * Mortality was recorded for up to 11 and 21 days for larvae and adults, respectively its with the red palm weevil and its devel- from the natural infection, such high mor- opment is completed within its host plant, tality levels in controlled conditions sup- adults are the only exposed stage to ento- port these strains as candidate biological mopathogenic fungal treatments (Gindin control agents. While the larvae of the wee- et al., 2006; Dembilio et al., 2010b). There- vil are the most destructive stage they are fore, the longer survival of an infected host also the most concealed life stage (Cami- increases the possibility of interacting with no Lavin et al., 2002; Lopez-Martinez et al., other individuals and probably of trans- 2011), challenging the application of the en- mitting the pathogen. In this sense, a low- tomopathogenic fungi. It is, however, pos- er spore concentration of B.bassiana might sible that entomopathogenic fungal endo- be more advantageous in a fi eld applica- phytes are eff ective against larval stages of tion, especially because our study showed the weevil. Among many others, the ento- that the mean mortality did not diff er sig- mopathogenic fungi B. bassiana, M. anisopli- nifi cantly between the two concentrations. ae and I. fumosorosea have been reported as However, other aspects of the application endophytes of several plants (Gomez-Vid- of these entomopathogenic fungi are not al et al., 2006; Quesada-Moraga et al., 2006; yet fully studied. For example, potential Vega, 2008) and attempts have been made horizontal transmission between adults or to introduce entomopathogens into host from adults to larvae and vertical transmis- plants for the control of pests (Gomez-Vid- sion from infected adults to their off spring al et al., 2006, Posada et al., 2007; Valdes Es- should be elucidated (Glare et al., 2002; Gin- trada et al., 2014). If the strains studied here din et al., 2006; Dembilio et al., 2010b). Fur- could be introduced into agave plants they thermore, M.anisopliae and B. bassiana have could possibly act as successful biocon- been found to reduce the fecundity of R. fer- trol agents due to their high pathogenicity rugineus (Gindin et al., 2006; Dembilio et al., against larvae. 2010b). If a similar eff ect can be proved on S. In conclusion, our results prove that the acupunctatus, then even strains that caused strain IBB 010 of B. bassiana is highly patho- lower levels of mortality might be a useful genic to both adults and larvae of S. acu- tool in the control of the weevil. punctatus and could be considered as a Another important fi nding of the pres- biological control agent of the weevil. Addi- ent study is the high mortality of larvae tionally, application of lower concentrations caused by all the strains tested. In most of IBB 010 was shown to prolong the adults’ of the cases, mortality in the larval stage survival time, without reducing the mor- reached 100%. Although our methodology tality levels. This should be taken into con- of immersing the larvae diff ers considerably sideration, as a positive eff ect for strategies

© Benaki Phytopathological Institute 52 Gkounti et al. based on attracting and infecting adults. idae) in Phoenix canariensis. Pest Management Strains IBB020 and TMB04 of I. fumosorosea Science, 66: 365-370. and M. anisopliae, respectively, caused low- Dembilio O., Quesada-Moraga E., Santiago-Alvaro C. and Jacas J.A. 2010b. Potential of an indige- er mortality levels to adults, but were equal- nous strain of the entomopathogenic fungus ly eff ective on larvae. Hence, they could be Beauveria bassiana as a biological control agent considered as potential control agents into against the red palm weevil, Rhynchophorus fer- an IPM plan against the pest. rugineus. Journal of Invertebrate Pathology, 104: 214-221. Despite the lack of fi eld data on the ef- EPPO. 2008: First record of Scyphophorus acupuncta- fectiveness of these strains, to the best of tus in France. EPPO Reporting Service 2008/220. our knowledge, this is the fi rst attempt of Fargues, J.F. and Robert, P.H. 1983. 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© Benaki Phytopathological Institute 54 Gkounti et al. Παθογένεια ιθαγενών στελεχών τριών εντομοπαθογόνων μυκήτων επί του εντομολογικού εχθρού της αγαύης Scyphophorus acupunctatus (Gyllenhal) (Coleoptera: Curculionidae)

Β.Τ. Γκουντή, Δ. Μαρκογιαννάκη και Δ.Χ. Κοντοδήμας

Περίληψη Η παθογένεια ιθαγενών απομονώσεων των εντομοπαθογόνων μυκήτων Beauveria bassiana, Metarhizium anisopliae και Isaria fumosorosea αξιολογήθηκε στο εργαστήριο εναντίον προ- νυμφών και ακμαίων του εντομολογικού εχθρού της αγαύης Scyphophorus acupunctatus. Ο πειραματι- σμός διεξήχθη με εμβάπτιση ατόμων του εντόμου σε διαφορετικές συγκεντρώσεις εναιωρημάτων κο- νιδίων των μυκήτων. Και τα τρία είδη μυκήτων έδειξαν υψηλή παθογένεια εναντίον των προνυμφών του σκαθαριού, προκαλώντας 100% θνησιμότητα στις περισσότερες επεμβάσεις. Ο μύκητας Beauveria bassiana προκάλεσε την υψηλότερη θνησιμότητα στα ακμαία (86.67±12%), ακολουθούμενος από τον Metarhizium anisopliae (46.67±17.8%) και τον Isaria fumosorosea (40±17.5%). Παρατηρήθηκαν στατιστι- κώς σημαντικές διαφορές στο μέσο όρο του χρόνου επιβίωσης μεταξύ των επεμβάσεων και των βιο- λογικών σταδίων του σκαθαριού. Σε όλες τις επεμβάσεις οι προνύμφες επιβίωσαν σημαντικά λιγότε- ρες ημέρες συγκριτικά με τα ακμαία. Τα αποτελέσματα της παρούσας μελέτης δείχνουν τη δυνατότητα χρήσης αυτών των ιθαγενών στελεχών ως παράγοντα βιολογικής αντιμετώπισης του S. acupunctatus.

Hellenic Plant Protection Journal 8: 46-54, 2015