Entomologia Hellenica

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ENTOMOLOGIA HELLENICA

Vol. 14, 2002

Entomopathogens of Anαcridium αegyptium L. in Crete

Roditakis Ν. Ε. NAGREF-Plant Protection Institute Heraclion Crete, 710 03 Katsabas, Heraclion -'Department of Biology University of Crete Kollaros D. Department of Biology University of Crete Legakis Α. Department of Biology University of Crete https://doi.org/10.12681/eh.14038

To cite this article:

Roditakis, Ν., Kollaros, D., & Legakis, Α. (2002). Entomopathogens of Anαcridium αegyptium L. in Crete. ENTOMOLOGIA HELLENICA, 14, 5-10. doi:https://doi.org/10.12681/eh.14038

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Entomopathogens of Anacridium aegyptium L. in Crete1

N. E. RODITAKIS2, D. KOLLAROS3 and A. LEGAKIS3

2NAGREF-Plant Protection Institute Heraclion Crete, 710 03 Katsabas, Heraclion -'Department of Biology University of Crete

ABSTRACT The entomopathogenic fungus Beauveria tassiana (Bals.) Vuil. was recorded for the first time on Anacridium aegyptium L. in Crete. The insects were fed on pieces of leaf subjected to a serial dilution of spores over three to four orders of magnitute. Comparative studies on the virulence of ß. bassiana (I 91612 local isolate) and Metarhizium anisopliae var. acridum (IMI 330189 standard isolate of IIBC) showed that M. anisopliae var. acridum was more virulent than B. bassiana at a conidial concentration lower or equal to 106 per ml while they were similarly virulent on first stage nymphs at 107 conidia per ml.

Introduction 40 ind./ha) in winter that reaches much higher lev els during some summers. The reasons of these A three year study ( 1990-1992) was started on lo sporadic outbreaks remain unknown. By contrast custs in Crete aiming to study Cretan acridofauna, it is always harmful on field vegetables in Chan including species composition and their seasonal dras, Sitia (East Crete) so the local Agricultural abundance on main crops, harmful species and Advisory Services suggests extensive control native biological agents. Grapes are the second measures based on insecticides. crop in order of importance of Cretan agriculture. Crop losses dued to locust Anacridium aegyptium It is known that microbial agents such as the had been noticed on certain locations in Crete in entomopathogenic fungi cause epizootics affect the past (Roditakis 1990). ing population size in the field. The main factors favoring epizootics are temperature and humidity Anacridium aegyptium L. is the most abundant conditions (>92-93%) (Walstad et al. 1970, (55%) species of the acridofauna of Cretan vine Burges and Hussey 1971, Ferron 1978) (wet and yards and it is also an insect of secondary impor rainy weather, irrigation etc) and microenviro- tance for grape vines in Crete (Roditakis 1990, ment (Moore 1973, Krammer 1980, Doberski Kollaros 1993). This species is very common in 1981). The pathogens are generally highly het all Greece as it is also in other countries of tropi erogenous species but there exist host-adapted cal and subtropical regions (Willemse 1985). The genotypes (Riba et Miergejewska 1986, Hajek population density is very low (5-10 adults/ha) in and Leger 1994) and geographically distinct pop central Crete not allowing this species to be gen ulations which show high degree of virulence erally harmful on grapevines. There are only two (Fargues et al 1992). One of them, Metarhizium restricted grapevine areas in Crete, a nothern one anisopliae var. acridum has recently been used in Kalessa and a southern one in Pompia, where extensively against very hazardous locusts such as we have noticed a higher population density (20- the desert locust Schistocerca gregaria (Prior 1990). The use of pathogens for pest control could re move some problems associated with insecticides Rcccivcd for publication January 1999 such as hazards to enviromental pollution and tox-

http://epublishing.ekt.gr | e-Publisher: EKT | Downloaded at 20/02/2020 21:50:57 | f. ENTOMOLOGIA HELLENICA Vol. 14 (2001-2002) icity to beneficial fauna. The current research followed by untreated ones during the next period. aims at discovering local pathogens of this locust The treated individuals were kept on small sepa in order to use them in IPM programmes either on rate transparent plastic boxes (10 cm diam, 5.5 cm vines or on vegetables. height). Four groups of 12 adults each (48 in total/treat Materials and Methods ment) were used came from a colony reared in the The adults of A. aegyptium were collected with laboratory during summer. In order to achieve the sweep nets in the field and reared in the laborato appropriate number of adults for laboratory tests, ry in small wooden cages (30 cm x 35 cm x 35 we reared the locusts for 3-4 generations under cm) in a rearing room under natural illumination laboratory conditions and natural illumination and 24±1°C. Fresh vine leaves and oat flakes were from May to September. These adults of A. ae offered as food. The cages were kept clean from gyptium were subjected to reproductive diapause faeces and remainders of food daily. The leaves in automn. Adults of A. aegyptium entered to re provided were rinsed with chlorinated tap water. productive diapause consumed fewer food (1/5- The females deposited their eggs on small trans 1/9 of food consumed by non diapause adults) parent plastic boxes filled with sterilized lightly (personal observations) and we took it into ac moistured sea sand. When one or two egg pods count during laboratory tests. The chi square was were laid per plastic box, the box was removed used for statitistcal analysis of percentage mortal and placed in a separate wooden cage. ity by Statgraphic statistical programme. Every insect suspected of being ill, was re Four groups of 16 first stage nymphs each (64 moved and placed in separate transparent plastic in total per treatment) were also used. The patho box ( 12 cm diameter and 8 cm height). After its genicity of local pathogens isolated was compared death, it was sectioned, spread, pinned and air with an IMI 330189 Metarhizium anisopliae var. dried. Some of the insects were kept on lightly acridum isolate provided by IIBC. The previous moistured cotton, until fungal hyphae erupted publications on the biological control of locusts from the cadaver. Following this, a one-conidium and grasshoppers using IMI 330189 referred as culture was initiated on potato dextrose agar M. flavoviride Games & Rozsypal (Drivers et al. (PDA). The pathogens isolated were sent to the 2000). The first stage nymphs used were very vo International Institute of Biological Control - In racious and ate the food offered. They consumed sect Pathology Laboratory (IIBC) for identifica much food (fresh vine leaves) (0.18 gr/24h) (Kol- tion. laros 1993) than the non diapause adults con sumed 0.5-0.9 gr/24h (fresh vine leaves) (person The pathogens isolated were tested both on al observations). Lt was calculated by a comput adults and on first stage nymphs in the laboratory. 50 er programme provided by the IOBC laboratory. We used conidia concentrations 2.5, 5, 10 and 20 x 106 per ml on adults and 2 x I03, 2 x 104, 2 χ Dead insects were kept on separate Petri dish 105, 2 x IO6 and 2 χ ΙΟ7 per ml conidia on first es with a lightly moistened filter paper on the bot stage nymphs. tom and closed firmly all around by parafilm. The conidia were harvested from Petri dishes There they remained until fungal hyphae were using sterile distilled water agitated by a bent projecting from the cadaver. This was interpreted glass rod. The conidia suspension was cleared of as confirmation of the pathogen-related death. hyphal debris by filtering through coarse-mashed muslin. After that, the coagulated conidia were TABLE 1. Mortality of adults of Anacridium aegyptium fed separated by a sonicator. Four Petri dishes (9 cm on pieces of lettuce leaves treated with different conidia diam) were enough in order to obtain 100 ml coni concentrations of fungi Beauveria bassiana (total number dia suspension of 20 x 106 conidia/ml concentra of adults per treatment 48). tion. Treatments Dead insects Corrected The tests for pathogenicity of isolated fungi conidia/ml on 10th day mortality '/< were carried out by administering small pieces 2.5 χ \{ψ 48 100 (5cm x 5cm) of lettuce leaves immersed in a cer 5.0 x IO6 40 80 tain conidium concentation for 10 seconds. One 10.0 x 106 36 70 or two small drops of Tween 80 was added as 20.0 x 10*' 36 70 emulsifier in each conidia concentration before Control 8 0 immersion of leaf. The treated pieces of lettuce * = Abbott's formula leaves were offered as a food the first two days χ2=13.2

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TABLE 2. Mortality (%) of first stage nymphs of Anacridium aegyptium fed on pieces of lettuce leaves treated with different conidia concentrations of Beauveria bassiana and Metarhizium anisopliae var. acridum (total insects per treatment 64).

I. Beauveria bassiana Days post inuculation" 20 days j uays Conidia/ No of dead Mortality No of dead Corrected No of dead Corrected ml insects (%)' insects mortality (%)" insects mortality (%)* Control 0 0.0 4 0.0 4 0.0 2x 10' 0 0.0 4 0.0 5 1.9 2x 104 0 0.0 12 13.6 12 13.6 2 x 10* 4 6.2 16 20.2 20 26.8 2x 10" 4 6.2 12 13.6 14 17.9 2x 10' 4 6.2 56 86.0 60 93.3 II. Metarhizium anisopliae var. acridum

Control 0 0.0 0 0.0 0 0.0 2x IO' 0 0.0 0 0.0 12 18.7 2x 104 8 12.5 24 37.5 36 56.2 2 χ \

Koch's postulates were satisfied by the reculture both fungi but it was longer at lower concentra

of fungus from the infected insects. tions (Table 3). The LT50 of M. flavoviride was generally shorter than that of B. bassiana at the Results lower concentration of 2 x 106 and 2 x 105 per ml. Five dead adults from 48 specimens collected was We observed also Aspergillus flavus Link in a firstly became reddish and when put on lightly moistened filter paper on a glass Petri dish, a rich few dead speciments in laboratory colonies where white mycelium covered all the body in 5-8 days. adults were crowded. Firstly, the mycelium covered head and antennae and later the thorax, abdomen and legs. The fun Discussion gus isolated was Beauveria bassiana (Bals.) Vuill. The entomopathogenic fungus ß. bassiana, This fungus proved virulent on adults (Table 1) known as muscardine disease is an ento- and first stage nymphs (Table 2) as well. Accord mopathogen in many locust species (Dresner ing to the data presented in Table 1, conidia con 6 1949, Moore and Erlandson 1988). This fungus centrations from 2.5 to 20 x 10 per ml caused was also isolated from grape berry moth larvae in high mortality ranging from 70-100%. The mor Crete in 1985 (IMI 294185). A. aegyptium inhab tality of first stage nymphs was 86 and 93.3% at 2 7 its several parts of Greece and this the first record x 10 conidia concentration after 10 and 20 days of B. bassiana on this locust in Greece. Even if after treatment respectively (Table 2). this locust is of secondary importance in agricul

The LT50 (Lethal time for 50% individuals) ture there are some other locusts very harmful on was 7 days at 2 x 107 conidia concentration for cereals and vegetables on which it could be a use- full biological control agent. There are three re gions in Crete where A. aegyptium is harmful, at TABLE 3. The lethal time for 50% (LT50) offirst stag e nymphs of Anacridium aegyptium at several conidia Kalessa (North Crete) Pompia (South Crete) concentrations of entomopathogenic fungi Beauveria where it attacks grape vines and at Chandras (East bassiana and Metarhizium anisopliae var. acridum. Crete) where it attacks vegetables. LT in days 50 In Chandras, Sitia (East Crete) A. aegyptium is Conidia Beauveria Metarhizium anisopliae very harmful and the local agricultural authorities concentrations bassiana var. acridum take extensive control measures based on insecti 2x 104 >20 >20 5 cides every year. Concerning vines this locust 2 χ 10 >20 16 2x 106 >20 8 should be under consideration in IPM programs 2 χ 10' 7 7 on grape vines in South Crete.

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Entomopathogenic fungi usually penetrate the e.g. microbial antagonists, host suitability, cuticle of insects and then cause mycosis which pathogen virulence, inoculum thresholds, sun spreads through haemolymph to all parts of the light, pesticides, temperature and humidity. Many body. Oral ingestion of fungal conidia could infect of the above constraints could be overcome with the insect either from gut wall cither from buccal formulation and application strategy (Zimmer cavity. There are very few reports that propose in mann 1994). Recently an oil formulation of fection through gut wall after ingestion of fungal Metarhizium anisopliae var. acridum was excel spores (reviewed by Dillon and Charnley 1991). lent against Schistocerca gregaria under low hu Dillon and Charnley (1995) noted that the germi midity condition (Batcman et al. 1993). The addi nation of conidia of Metarhizium was inhibited by tion of UV protectants can also increase signifi bacteria in the gut of desert locust due to antimi cantly their persistence in the field (Inglis et al. crobial phenols associated with Pantoea agglom- 1995). erans. Gut infection seems to occur often in Β. bassiana can be easily mass produced and aquatic insects (Sweeney 1979, Knight 1980). B. formulated in high conidia concentrations (Hall bassiana can infect Aedes aegyptii Rock larvae and Papierok 1982, Auld 1991). Its mass produc through the gut (Miranpuri and Khachatourians tion, formulation and application methods can al 1991). However there are also reports in which so be impoved considerably (Goctel and Roberts conidia germination in buccal cavity. Siebeneich- 1991, Bateman 1991). The virulence of B. er et al. (1992) showed that Solenopsis invida Bu bassiana compared to that of M. flavoviride was ren workers possessed germination β. bassiana lower at lower concentrations (<107) while it was conidia in their buccal cavities. Shabel (1976) virulent as well at 2 χ 106 conidia concentration demonstrated istologically that Metarhizium for both fungi. Considering that indigenous anisopliae conidia invade through the buccal cav pathogens have been suggested for use because of ity of the weevil Hylobius pales Herbst. We iso the risk of unknown effects from non indigenous lated B. bassiana from the hypopharynx and crop pathogens on the local fauna (Goettel and Roberts of A. aegyptium showed symptoms of illness. Un 1991, Prior 1990, 1991) the use of local strain der low humidity conditions, we observed a rich ß mycelium with conidiophorcs only inside the bassiana as a biological control agent in an IPM head capsule causing a whitish discoloration of programmes on vine pests should be considered. the head of dead insects. Dead insects kept under high humidity conditions on Petri dishes showed Acnowledgements visible mycelium growth firstly on antennae and head and later on rest parts of the its body. The ob I would like to thank Dr. Chris Prior of the Inter servations displayed above show that buccal cav national Institute of Biological Control Ascot ity of this locust could be initially the place of London U.K. for the determination of species of conidia germination, following with penetration entomopathogenic fungi and for providing the οι Metarhizium anisopliae var. acridum for of neigbour parts of its body. strain comparative studies.

A. flavus has often been isolated from insects References and mites (Moore and Erladson 1988, Pelagati el al. 1988, Wicklow and Dowed 1989). This fungus Auld. B.A. 1991. Mass production, formulation and appli cation of fungi as biocontrol agents. In C.J. Lomer and was isolated only from insects reared in our labo C. Prior (eds.) Biological control of Locusts and ratory. It was often isolated from our rearing units Grasshoppers, CAB International. U.K., pp. 219-229. where adults were crowded. A. flavus infections Bateman, R.P. 1991. Controlled droplet application of my- are fairly common in crowded insects due to copepticides to locusts. In C.J. Lomer and C. Prior stress and it might be the cause of its occurrence (eds.) Biological control of Locusts and Grasshoppers, in our laboratory colonies. It is known that the in- CAB International, U.K., pp. 249-254. Bateman. R. P., M. Carey, D. Moore and C Prior. 1993. The secticidal activity of A. flavus is mainly based on enhanced infectivity of Metarhizium anisopliae var. the aflatoxin complex. The aflatoxin complex is acridum in oil formulations to desert locusts at low hu toxic and carcinogenetic to vertebrates so it is un midities. Ann. Appi. Biol.:122: 145-152. likely to be used in microbial control. Following Burges, H.D. and N. W. Hussey. 1971. Microbial control of that, the present study was focused on the local insects and mites. Academic press London. Dillon. R. I., and A. K. Charnley. 1991. The fate of fungal strain of B. bassiana. spores in the insect gut. In G.T. Cole and H.C. Hoch (eds.). The fungal spore and disease initiation in plants The ability of entomopathogenic fungi to con and animals. Plenum New York, pp. 129-156. trol pests is affected by abiotic and biotte factors Dillon, R. I., and A. K. Charnley. 1995. Chemical barriers

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to gut infection in the desert locust: In vivo production Prior. C. 1991. Discovery and characterization of fungal of antimicrobial phenols assosiated with bacterium pathogens for locust and grasshopper control.In C.J. Pantoea agglomérons. J. Invertebr. Pathol. 66: 72-75. Lomer and C. Prior (eds.), Biological control of Lo Driver, F., R.J. Milner, and J. W. H. Trueman. 2000. A tax- custs and Grasshoppers, CAB International, U.K., pp. onomic revision of Metarhizium based on a phyloge- 159-180. netic analysis of rDNA sequence data. Mycological Riba, G. and E. Miezejewska. 1986. Alpha-esterases of Research 104: 134-150. twenty polish strains of Beauveria bassiana. Bull. Pol Doberski. J. W. 1981. Comparative laboratory studies on ish. Acad. Sci. Biol. Sci. 34:41-45 (from Tigano et al. three fungal pathogens of the elm bark beetle Scolytus 1990). scolytus: effect of temperature and humidity on infec Roditakis, Ν. E. 1990. Vine pests of secondary importance tion by Beauveria bassiana, Metarhizium anisopliae the last six years in Crete. Proc. meeting of EC experts and Paecilomyces farinosus. J. Invertebr. Pathol. 37: group / Thessaloniki. Greece / 6-8 Oct. 1987, Ed. A. 195-200. Balkema, Rotterdam, pp. 151 -156. Dresner, E. 1949. Culture and use of enlomogenus fungi for Schabel. H. G. 1976. Oral infection of hylobius pales by the control of insect pests. Contributions, Boyce Metarhizium anisopliae. J. Invertebr. Pathol. 27: 377- Thomson Institute for Plant Research 15: 319-335. 383. Farques. J.. N.K. Maniana, J.C. Dclmas and N. Smils. 1992. Siebencicher, S. R.. S. B. Vinson, and C. M. Kencrlcy. Influence de la température sur le croissance in vitro d' 1992. Infection of the red imported fire ant by Beauve hyphomycètes entomopathogènes. Agronomie 12: ria bassiana through various routes of exposure. J. In 557-564. vertebr. Pathol. 59: 280-285. Ferron, P. 1978. Biological control of insect pests by ento- Sweeney, A. W. 1979. Infection of mosquito larvae by mogenous fungi. Ann. Rev. Entomol. 23: 409-442. Culicinomyces sp. through anal papillae. J. Invertebr. Goettcl, M. S. and D. W. Roberts. 1991. Mass production, Pathol. 33:249-251. formulation and field application of entomopathogen Walstad, J. D. R.F. Anderson, and W.J. Stambaugh. 1970. ic fungi. In C.J. Lomer and C. Prior (eds.) Biological Effects of enviromcntal conditions on two species of control of Locusts and Grasshoppers, CAB Interna muscardine fungi (Beauveria bassiana and Metarhiz tional, U.K., pp. 230-238. ium anisopliae). J. Invertebr. Pathol. 16: 221-226. Hajek A. E. and R. J. St. Léger. 1994. Interaction between Wicklow, D.T. and Dowd P.F. 1989. Entomotoxigenic po fungal pathogens and insect hosts. Ann. Rev. of Ento- tential of wild and domesticated yellow - green as mol.:39: 293-322. pergini: Toxicity to corn earworm and fall armyworm Hall, R.A. and B. Papierok. 1982. Fungi as biological con larvae. Mycologia 81 (4): 561-566. trol agents of arthropods of agricultural and medical Willemse, F. 1985. Fauna Graeciac: A key to the Orthoptera importance:Parasitology 84: 205-240. species of Greece, Vol I. Ed. Hellenic Zoological Soci Inglis, G.D., M.S. Goetel and D.L Jonhson. 1995. Influence ety. of ultraviolet protectans on the persistence of the ento Zimmermann. G, 1994. Strategies for the utilization of en mopathogenic fungus Beauveria bassiana Biol. Cont. tomopathogenic fungi. Proc. 5th Int. Colloq. Invertebr. 5:581-590. Pathol, pp. 67-73. Knight, A. L. 1980. Host range and temperature require ments of Culicinomyces clavisporus. J. Invertebr. Pathol. 36: 423-425. Kollaros, D. 1993. Biology and ecology of superfamily Acridoidea (Orthoptera) on Giouchtas mountain (Crete. Greece). Thesis, Univ. of Crete. Krammcr, J.P. 1980. The house-fly mycosis caused by En- tomophtora muscae: influence of relative humidity on infectivity and germination. J. N.Y. Entomol. Soc. 88: 236-240. Miranpuri. G. S. and G. G. Khachatourians. 1991. Infection sites of the entomopathogenic fungus Beauveria bassiana in the larvae of the mosquito Aedes aegypti. Entomol. Exp. Appi. 59: 19-27. Moore, G. E. 1973. Pathogenicity of three entomogenus fungi to the Southern Pine Beetle at various tempera tures and humidities. Environ. Entomol. 2: 54-57. Moore, K.C. and M. A. Erlandson. 1988. Isolation of As pergillus parasiticus Speare and Beauveria bassiana (Bals.). Vuillemin from Mclanoplinc Grasshoppers (Orthoptera: Acrididae) and demonstration of their pathogenicity in Melanoplus sanguinipes (Fabricius). Can.Ent. 120:989-991. Pelagatti, Ο,. G. Frate and G. Caretta. 1988. Fungi isolati da insetti e acari. Redia, Vol. LXXI, 1: 255-266. Prior, C. 1990. The biological basis for regulating the re lease of micro-organisms, with particular reference to KEYWORDS: Anacridium aegyptium, Beauveria the use of fungi for pest control. Aspects of Applied bassiana, Metarhizium anisopliae var. acridum, Biology 24: 231-238. virulence, grape vines.

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Εντομοπαθογόνα του Anacridium aegyptium L. στην Κρήτη

Ν. E. ΡΟΔΙΤΑΚΗΣ1, Δ. ΚΟΛΛΑΡΟΣ2 και Α. ΛΕΓΑΚΗΣ3 ' Εθνικό Ίδρυμα Αγροτικής Έρευνας, Ινστιτούτο Προστασίας Φυτών Ηρακλείου Κρήτης, 710 03 Καταάμπα, Ηράκλειο 2 Τμήμα Βιολογίας, Πανεπιστήμιο Κρήτης

ΠΕΡΙΛΗΨΗ Στα πλαίσια ενός τριετούς ερευνητικού προγράμματος για τις ακρίδες στην Κρήτη ερευνήθηκε η ύπαρξη ιθαγενο5ν εντομοπαθογόνιον μικροοργανισμοί για την βιολογική καταπολέμηση τους. Απομονιόθηκε για πρώτη φορά ο μύκητας Beauveria bassiana (Bals) Vuill., αξιολογήθηκε η αποτελεσματικότητα του στα ακμαία και νύμφες πρώτης ηλικίας της ακρίδας Anacridium aegyp tium L., είδος βλαβερό σε αμπέλια και λαχανικά, και συγκρίθηκε με το στέλεχος του εντομοπαθογόνου μύκητα Metarhizium anisopliae var. acridum (IMI 330189). Τα πειράματα έδειξαν ότι το ιθαγενές στέλεχος του μύκητα Β. bassiana που απομονώθηκε είχε εξαιρετική αποτελεσματικότητα σε ακμαία και νύμφες πρώτης ηλικίας της ακρίδας Α. aegyptium, δεν υστερεί σε αποτελεσματικότητα από το μύκητα M.flavoviride κα ι ενδείκνυται για το βιολογικό έλεγχο και προγράμματα ολοκληρωμένης διαχείρισης.

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