Geographical Distribution and Host Range of Entomophthorales Infecting the Green Spruce Aphid Elatobium abietinum Walker in Iceland

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Journal of Invertebrate Pathology 78, 72–80 (2001)doi:10.1006/jipa.2001.5045, available online at http://www.idealibrary.com on

Geographical Distribution and Host Range of EntomophthoralesInfecting the Green Spruce Aphid Elatobium abietinum

Walker in IcelandCharlotte Nielsen,* Jørgen Eilenberg,* Susanne Harding,* Edda Oddsdottir,† and Gudmundur Halldorsson†*Department of Ecology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, 1871 Frederiksberg C., Denmark; and

†Iceland Forest Research Station, Mogilsa, IS-270 Mosfellsbær, Iceland

Received January 29, 2001; accepted July 12, 2001

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Entomophthora planchoniana and Neozygites frese-ii caused infection in populations of the green sprucephid, Elatobium abietinum, in Iceland. On this aphidpecies En. planchoniana was exclusively found in theestern part of Iceland, while N. fresenii was exclu-

ively found in the eastern part of Iceland. This dis-rete and nearly nonoverlapping geographical distri-ution correlates with the distribution of two differ-nt populations of El. abietinum found in Iceland. Onther aphid species N. fresenii, En. planchoniana,andora neoaphidis, and Conidiobolus obscurus wereocumented throughout the country. Transmissionxperiments showed that Pa. neoaphidis and En. plan-honiana could infect the eastern population of El.bietinum, although they have never been found onhis population in nature. This strongly indicates thathere is little or no interaction among El. abietinum,ther aphids, and their respective entomophthoraleanungi in the field. Furthermore, this study is the first toecord epizootics caused by N. fresenii and En. plan-honiana in the subpolar region. © 2001 Academic Press

Key Words: Entomophthorales; Neozygites fresenii;Entomophthora planchoniana; Erynia neoaphidis;Pandora neoaphidis; natural occurrence; host range;bioassay; Iceland; aphid; Elatobium abietinum.

INTRODUCTION

Sitka spruce, Picea sitchensis (Bong.) Carr., is anmportant conifer in reforestation programs in IcelandPetursson, 1999; Snorrason et al., 2000). The cultiva-ion of Sitka spruce has, however, been negatively af-ected by a series of major outbreaks of the greenpruce aphid Elatobium abietinum Walker. The feed-ng of El. abietinum on needles causes chlorosis andremature abscission and may, in some years, lead toxtensive defoliation and consequently a reduction inree growth (Bejer-Petersen, 1962; Straw et al., 1998).

720022-2011/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

robably on Christmas trees imported from Denmark.he first individuals of El. abietinum were thus ob-erved in Reykjavik in 1959 and have since spreadhroughout Iceland. In the first 3 decades after thentroduction, El. abietinum colonized spruce planta-ions in the southern and the eastern part of Icelandut from the 1990s the aphid was also found in plan-ations in the west and north (Carter and Halldorsson,998). Sigurdsson et al. (1999) found that El. abieti-um in Iceland consisted of two genetically distinctopulations, one in the eastern and the other in theestern part of the country. These populations origi-ate most probably from two distinct introductions.In oceanic regions of Europe, El. abietinum developsainly anholocyclicly. In Iceland males or eggs of El.

bietinum have never been found, which means that inceland the development is solely anholocyclic (Carternd Halldorsson, 1998). In the northwestern part ofurope outbreaks are usually seen in spring and earlyummer, except in Iceland where outbreaks alwaysccur in fall or early winter (Carter and Halldorsson,998). It has been suggested that this dissimilarity inphid population development can be partly explainedy a lack of important natural enemies in IcelandCrute and Day, 1990; Austarå et al., 1997).

Previous to the present study seven different speciesf entomopathogenic fungi all belonging to the orderntomophthorales (Zygomycotina) have been recordedttacking El. abietinum in the northwestern part ofurope (Austarå et al., 1997; Nielsen et al., 2000). So

ar only two species, Entomophthora planchonianaornu and Neozygites fresenii (Nowakowski) Re-audiere & Keller, have been reported from Iceland

Nielsen et al., 2000). However, only few observationsave been included in the earlier reported studies fromceland. Furthermore, samplings over several yearseem to be essential since studies in other ecosystemsave shown that the composition of the aphid patho-

an

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73ENTOMOPHTHORALES ON Elatobium abietinum

and locality (Byford and Reeve, 1969; Thoizon, 1970;Coremans-Pelseneer et al., 1983; Steenberg and Eilen-berg, 1995). Therefore, it is not confirmed whether thefindings of only two species of entomopathogenic fungiaffecting El. abietinum in Iceland reflect coincidence or

lack of several species of Entomophthorales acting asatural enemies.Laboratory experiments have shown that transmis-

ion of entomophthoralean fungi between differentphid host species is possible, even between aphid hostpecies from different genera (Milner et al., 1984; Feng

and Johnson, 1991; Steenberg and Eilenberg, 1995).Other aphid species in the surroundings of Sitkaspruce plantations may thus interact with El. abieti-num and serve as a reservoir for natural enemies dur-ing periods with low densities of El. abietinum, such asduring the summer. If these other aphids can posi-tively act as a reservoir of Entomophthorales, they mayprovide the possibility of developing a low-cost controlstrategy against the green spruce aphid. Aphids fromsuch host plants have, however, never been sampled ordiagnosed for entomopathogenic fungi in Iceland.

The objectives of this study were, therefore, to inves-tigate the natural occurrence of entomophthoraleanfungi infecting El. abietinum over several years and todetermine their geographic distribution in Iceland.Furthermore, the ecological host range of entomoph-thoralean fungi on aphids in Iceland was assessed for arange of different aphid species. Finally, the physiolog-ical host range was investigated in a series of experi-ments exploring the transmission of entomophtho-ralean fungi from various aphid hosts to Icelandic El.abietinum.

MATERIAL AND METHODS

Natural Occurrence of Entomophthoralean FungiInfecting El. abietinum

Sampling of El. abietinum for diagnosis of infectionby entomophthoralean fungi was conducted over a3-year period from 1997 to 1999. The aphids weresampled from 19 localities covering spruce plantationsin the western, southern, and eastern part of Iceland(localities 1–19 in Fig. 1).

Shoots of Sitka spruce were cut from trees andbrought to the laboratory where they were inspectedunder a microscope. Fresh sporulating cadavers werecarefully removed from the shoots and placed in humidchambers for 24 h over microscope slides to allowconidia to discharge. Living aphids were incubated incohorts of four to five in small plastic cups (30 ml)containing 3% water agar in the bottom to keep humid-ity high. Alternatively, the aphids were incubated inboxes with moist tissue paper in the bottom. In bothcases, incubation of the aphids took place at room

light/dark conditions). Shoots of Sitka spruce were pro-vided as food. The mortality was recorded daily for upto 1 week, and dead aphids were removed and placed inhumid chambers. Microscope slides with projectedconidia were mounted in lactic acid, and entomophtho-ralean fungi were identified under the microscope(4003) based on morphological characters (Keller,1987, 1991; Humber, 1989, 1997). Further, nonsporu-lating cadavers were squashed and microscopicallychecked for the presence of resting spores. In the fol-lowing text conidia originating from one infected indi-vidual will be considered one isolate. Morphologicalfeatures were carefully measured from a computerscreen for selected isolates, using the software programOlyLite, ver. 2.0 (Olympus A/S DK, Denmark).

Ecological Host Range

Sampling of aphid species other than El. abietinumwas conducted in the summer and fall of 1999. Aphidswere sampled from seven localities in the southernpart of Iceland (localities 6, 10, 15, 16, and 20–22 inFig. 1). The most intensively studied localities wereparks in Reykjavik (locality 6) and a national park atSkaftafell (locality 16). Both localities consisted mainlyof birch, willow, Sitka spruce, and several herbaceousplants. In addition, the parks in Reykjavik had manybeds of roses. Live aphids were sampled from trees,bushes, and herbaceous plants. Whole plants or shootsof plants were cut and incubated either in small plasticcups (30 ml) containing 3% water agar in the bottom orin plastic bags. The host plant was provided as foodduring incubation. Altogether more than 1500 aphidsfrom willow, 300 from birch, 2500 from roses, and 350from herbaceous plants were incubated during thisstudy. Mortality was recorded daily for up to 1 week.Dead aphids were removed and examined in the sameway as described for El. abietinum. From each host

FIG. 1. Sampling sites in Iceland. Elatobium abietinum weresampled from localities 1–19 and other aphids were sampled fromlocalities 6, 10, 15, 16, and 20–22. Numbered localities are listed inTable 1.

plant some specimens of healthy aphids were stored in

74 NIELSEN ET AL.

ethanol and later identified to the species level (Heie,1980, 1982, 1986, 1992, 1994).

Physiological Host Range

Two qualitative bioassays were performed to test thepathogenicity of Pandora neoaphidis (Remaudiere &Hennebert) Humber and En. planchoniana against theeastern population of El. abietinum. In the first bioas-say, El. abietinum sampled from Djupivogur (locality18 in Fig. 1) were exposed to En. planchoniana conidiaobtained from cadavers of El. abietinum sampled atThrastasskogur (locality 9). In the second bioassay, El.abietinum obtained from Eldhraun (locality 22) wereexposed to conidia of Pa. neoaphidis originating fromcadavers of Acyrthosiphon pelargonii Kalt. sampled onAlchemilla vulgaris L. at Geysir (locality 20). In bothbioassays, sporulating aphid cadavers were fixed bymeans of petroleum jelly to a lid over mixed stages ofnoninfected El. abietinum for 24 h. During the bioassaythe aphids were kept on needles of Sitka spruce in30-ml plastic cups containing 3% water agar. The cupswere incubated at room temperature available (ap-proximately 20°C) and varying light/dark regime. Themortality was recorded on a regular basis.

Time-mortality was recorded for apterous adults ofEl. abietinum exposed to conidia of a well-sporulatingin vitro culture of Pa. neoaphidis (ARSEF 5272 isolatedfrom a Danish Brevicoryne brassicae L.). El. abietinumwas obtained from a laboratory colony originally col-lected at Kirkjubæjarklaustur (locality 15). The fungalmaterial was obtained by producing a fungal mat asdescribed by Papierok and Hajek (1997). Ten healthyapterous adults were transferred to a twig of Sitkaspruce placed in 3% water agar in a 30-ml plastic cup.The sporulating fungal mat was fixed with petroleumjelly to a lid and placed over the aphids. After 1 h ofincubation in light at 18°C, the lid with the sporulatingmat was removed and replaced with PVC cling film toavoid condensation during the incubation. The aphidswere incubated at 18°C and a 16-h photoperiod. Con-trols consisted of aphids, which had not been exposedto the conidial shower, but were otherwise treated likethe rest of the aphids. The mortality was recordeddaily. The experiment was duplicated.

Statistical Methods

For time-mortality data both nonparametric andparametric tests were used to analyze the effect ofinfection with Pa. neoaphidis. In the nonparametrictest, average survival time (AST) was calculated byKaplan–Meier survival estimators in SAS, ver. 6.12(SAS Institute, Inc.). In the parametric test, medianlethal time (LT50) was estimated in Mathematica, ver.4.0 (Wolfram Research, Inc.), by the method describedby Throne et al. (1995). The proportion of insects killed

rected for control mortality using Abbott’s formula (Ab-bott, 1925). LT50 was then estimated by complemen-tary log–log, logit, or probit transformation of the pro-portion of aphids killed by the fungus and with timeused either directly or after log transformation. Byregression, the best-fit line was found, and a x2 good-ness-of-fit test was used to determine how well theregression line fit the observed data.

RESULTS

Natural Occurrence of Entomophthoralean FungiInfecting El. abietinum

Over the 3 years of sampling, a total of 12,753 aphidswere incubated, and fungi were identified from 3689cadavers (Table 1). N. fresenii and En. planchonianawere the only entomophthoralean species found on El.abietinum under natural conditions and both specieswere occasionally observed to cause epizootics in pop-ulations of El. abietinum. For En. planchoniana thehighest infection level was observed at Thrastaskogur(locality 9) in 1999 where a prevalence of 31% wasmeasured based on 84 incubated aphids. For N. freseniithe highest prevalence measured was 61% (of 191 in-cubated individuals) at Skaftafell (locality 16) in Octo-ber 1997. During the epizootics many fresh sporulatingcadavers were observed in the field. In the field the twofungi could be distinguished by macroscopic symptomson infected aphids. When N. fresenii conidia wereformed, El. abietinum was usually fixed to the needleby its proboscis and the abdomen was elevated fromthe needles. In contrast, the abdomen of El. abietinumsporulating with conidia of En. planchoniana was heldclose to the plant material by rhizoids. Cadavers sporu-lating with En. planchoniana were in most cases fixedto the stem, in contrast to cadavers sporulating with N.fresenii which were always fixed to the needles. Cadav-ers filled with resting spores of N. fresenii were swollenand black. The cadavers were fragile and when touchedthe black resting spores were released in a liquid mass.No cadavers filled with resting spores of En. planchoni-ana were found.

En. planchoniana was the only species found west ofKverkin and Gjogur (localities 11 and 12), and N. fre-senii was the only species found east of these twolocalities (Fig. 2A). At the two localities from the bor-derline between the areas of distribution, En. plan-choniana and N. fresenii were found sympatrically.

Ecological Host Range

In total, eight different aphid species were collectedand incubated during this study (Table 2). Except forBetulaphis quadrituberculata Kalt. entomophtho-ralean fungi were found on all sampled aphid species in

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75ENTOMOPHTHORALES ON Elatobium abietinum

and roses both Pa. neoaphidis and En. planchonianaere documented. For Cavariella aegopodii Scop. feed-

ng on willow the following four species were docu-ented: Pa. neoaphidis, En. planchoniana, Conidiobo-

us obscurus (Hall & Dunn) Remaudiere & Keller, and. fresenii. The geographical distribution of these four

pecies of Entomophthorales on aphid hosts other thanl. abietinum is illustrated in Fig. 2B. As illustrated,ll four species were found in Geysir (locality 20). En.lanchoniana was present on other aphid hosts in theastern part of Iceland where only N. fresenii wasound on El. abietinum (Skaftafell, locality 10). Sincen. planchoniana and N. fresenii were found on bothl. abietinum and Ca. aegopodii, a detailed morpholog-

cal comparison between isolates from the two aphidosts was made. The differences between isolates couldot be related to aphid host species for either En.lanchoniana or N. fresenii (Table 3).

hysiological Host Range

El. abietinum from the eastern part of Iceland could,

TABNatural Occurrence of Entomophthorales on

LocalityYear of

sampling

1 Barmahlıd 199719981999

2 Hvammur 19993 Stykkisholmur 1997

19981999

4 Ingunnarstadir 19995 Danielslundur 19996 Reykjavik 19997 Selskogur 19998 Hveragerdi 19999 Thrastasskogur 1999

10 Selfoss 199911 Breidas 199912 Kverkin 1999

13 Gjogur 199719981999

14 Tumastadir 199715 Kirkjubæjarklaustur 1997

199816 Skaftafell 199717 Hofn 199818 Djupivogur 1997

199819 Stodvarfjordur 1997

1998

In total

a Epizootics.

nder laboratory conditions, be infected with both En. d

lanchoniana and Pa. neoaphidis. Time-mortality datare shown graphically in Fig. 3. For El. abietinumnfected with ARSEF 5372 (Pa. neoaphidis), AST wasetermined as 4.7 days with a standard error of 0.15.or estimation of LT50 logit transformation of mortality

data and time used directly without transformationgave the most satisfactory results (P of a greater x2:0.54). LT50 was estimated at 3.9 days (3.3–4.4).

DISCUSSION

Only N. fresenii and En. planchoniana were docu-ented in populations of El. abietinum in Iceland. This

s in contrast to Denmark, where six species of ento-ophthoralean fungi have been recorded from this

ost aphid (Austarå et al., 1997; Nielsen et al., 2000).he only species documented in the western part ofceland was En. planchoniana, while N. fresenii washe only species found in the eastern part of Iceland.his pattern seemed to be general since intensive stud-

es were carried out over several years. The geographic

1latobium abietinum in Iceland (1997–1999)

of sampledaphids

No. of funguskilled aphids Fungal pathogens

176 1 En. planchoniana932 27 En. planchoniana203 5 En. planchoniana373 11 En. planchoniana11 2 En. planchoniana

649 8 En. planchoniana350 3 En. planchoniana400 3 En. planchoniana10 1 En. planchoniana

208 7 En. planchoniana400 26 En. planchoniana103 4 En. planchoniana84 26 En. planchonianaa

84 1 En. planchoniana65 1 En. planchoniana

291 1 En. planchoniana7 N. fresenii

16 1 N. fresenii94 4 N. fresenii

129 3 En. planchoniana24 3 N. fresenii

607 176 N. fresenii379 0

5307 3280 N. freseniia

158 56 N. freseniia

601 4 N. fresenii10 2 N. fresenii

1071 26 N. fresenii18 0

12753 130 En. planchoniana3559 N. fresenii

LEE

No.

istributions of the two entomophthoralean fungi were

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76 NIELSEN ET AL.

identical to the known distributions of the two popula-tions of El. abietinum earlier documented in Iceland(Sigurdsson et al., 1999). Sigurdsson et al. (1999) foundthat both populations of aphids were present in Gjogur(locality 13), while west of this locality only one popu-lation of aphids was present and east of this localityanother genetically distinct population was present.The border between the two aphid populations coin-cides with the border between the two species of ento-mophthoralean fungi infecting El. abietinum. Sigurds-son et al. (1999) proposed that the genetic separation inIceland was caused by an introduction from Denmarkof two genetically distinct populations at two differenttimes, the first in the late 1950s and the second in theearly 1990s. It is therefore possible that at each intro-duction El. abietinum carried only one of the six spe-cies of entomophthoralean fungi occurring in Den-mark. If this hypothesis is true, the dispersal of the

FIG. 2. A: Natural occurrence of Entomophthorales on Elato-bium abietinum. B: Natural occurrence of Entomophthorales onother aphid species than El. abietinum. F, Entomophthora plan-choniana; q, Neozygites fresenii; h, Pandora neoaphidis; , Conid-obolus obscurus.

fungi has apparently followed that of the host aphid

population. The spread of El. abietinum and, conse-quently, of its fungal diseases has probably been slowbecause most spruce plantations in Iceland are smalland isolated.

N. fresenii and En. planchoniana were here recordedor the first time causing epizootics in the subpolaregion (64–65°N). In particular, N. fresenii has beenegarded as a species specially adapted to hot, humid,nd even tropical conditions (Keller, 1997; Steinkraus,998). In the present study it was documented that inceland, the prevalence of N. fresenii in populations ofl. abietinum reached values above 60% at Skaftafell

n November when the mean temperature is below 3°CEinarsson, 1976). N. fresenii strains found in Iceland

ay be better adapted for cooler conditions thantrains occurring in tropical conditions. Conidial di-ensions of N. fresenii from Iceland matched those in

he description for this species (Keller, 1991, 1997).owever, the mean length of capilliphores from the

celandic material was measured to 50.9–67.7 mm,hereas Keller (1991, 1997) noted the maximum

ength of capilliphores always to be shorter than 60mm. Nevertheless, Barrachina (1999) found, in Spain,capilliphores of the same length as those measured inthis study. Morphological studies are, however, usuallynot sufficient to distinguish strains and isolates of en-tomophthoralean fungi. In order to determine whetherany geographical adaptation in the species N. freseniihas taken place, molecular studies of isolates are es-sential. It is, however, likely that some evolutionarychanges have occurred in N. fresenii in geographicaldistinct populations since it has been shown to happenfor Pa. neoaphidis, another pathogen of aphids (Rohelt al., 1997; Nielsen et al., 2001b).On the other aphid species occurring in the sur-

oundings of Sitka spruce, four species of entomoph-horalean fungi were documented. In Skaftafell (local-ty 16) Pa. neoaphidis was frequently found to infect aange of aphids species feeding on herbaceous plantsnd En. planchoniana was found on Ca. aegopodii. Theact that only N. fresenii was found on El. abietinum athis locality clearly indicates that there is little or nonteraction between El. abietinum and other aphid spe-ies in the area with respect to entomopathogenicungi. Nevertheless, under laboratory conditions it washown that Pa. neoaphidis from various aphid hostsere able to infect El. abietinum and that En. plan-

honiana was able to infect the eastern population ofl. abietinum. In earlier studies it has been shown thatntomopathogens could infect hosts in the laboratoryhat were never known to be infected in the field (Wild-ng, 1981; Hajek et al., 1996). Cameron and Milner1981) similarly showed that sympatric Australianopulations of Acyrthosiphon kondoi Shinji and

Acyrthosiphon pisum Harris were dominated by differ-ent entomophthoralean species but that under labora-

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77ENTOMOPHTHORALES ON Elatobium abietinum

Several factors may explain why El. abietinum issusceptible to Pa. neoaphidis and En. planchoniana inthe laboratory bioassays but was never observed to beinfected in the field. The most likely explanations couldbe due either to a separation in time and space or todifferences in the intrinsic host ranges of these fungalspecies. Concerning a separation in time, most aphidspecies in Iceland except El. abietinum peak duringsummer (Lambers, 1955) and during this study most ofthe aphid species were sampled in the summer period.However, in Reykjavik in October 1999 aphids infectedwith Pa. neoaphidis were found on roses and Shep-herd’s purse in the same parks as El. abietinum wassampled. It is therefore unlikely that the separation intime is the only explanation for the lack of interactionbetween El. abietinum and other aphid species.

TABNatural Occurrence of Entomophthorales on Aphid

Locality Month Host plant

6 Reykjavik Aug. Capsella bursa-pastoris AcyrRosa spp. MacSalix spp. Cav

Oct. Capsella bursa-pastoris AcyrRosa spp. MacSalix spp. Cav

10 Selfoss Aug. Rosa spp. Mac16 Skaftafell Aug. Alchemilla vulgaris Acyr

Geranium sylvaticum AcyrHieracium spp. NasSalix spp. CavBetula spp. Betu

20 Geysir Aug. Alchemilla vulgaris AcyrSalix spp. Cav

21 Vik Aug. Alchemilla vulgaris AcyrElymus arenarius Schi

22 Eldhraun Aug. Hieracium spp. NasBetula spp. Betu

TABMorphological Features of Neozygites fresenii an

Elatobium abietinum and Ca

Fungal species andhost insect

No. ofisolates

Primaryconidia,

mean length(mm)

Primaryconidia,

mean width(mm)

LMea(prcon

Neozygites freseniiElatobium abietinum 4 20.9–22.3 15.6–17.6 1.2Cavariella aegopodii 2 22.3–22.8 17.6–17.8

Entomophthora planchonianaElatobium abietinum 4 17.4–19.3 15.7–16.7 1.1Cavariella aegopodii 3 19.2–20.3 16.1–17.6 1.1

Note. All counts and measurements are based on 20 objects per isola

El. abietinum may come in contact with the inocu-um either by contact with sporulating aphid cadavers,irectly from the soil (Latteur, 1977; Coremans-elseneer et al., 1983; Nielsen et al., 1998), from air-

borne conidia, or from germinating resting spores(Coremans-Pelseneer et al., 1983; Steinkraus et al.,999). El. abietinum is separated in space from the soilnd other sporulating aphid species since it is the onlypecies from Aphididae feeding on Sitka spruce. El.bietinum may, however, come in contact with inocu-um by falling to the ground. Sopp et al. (1987) found

that 20–50% of a cereal aphid population fell to theground each day. In order to evaluate whether this“falling down behavior” was common among IcelandicEl. abietinum, sticky traps were placed under a Sitkaspruce (C. Nielsen, unpublished). During the test pe-

2eeding on Different Host Plants in Iceland, 1999

Aphid species

No. ofincubated

aphids Co.

obsc

uru

s

En

.pla

nch

onia

na

Pa.

neo

aph

idis

N.f

rese

nii

siphon pisum 10 2iphum rosae ;2500 3 45lla aegopodii 42 1siphon pisum 80 7iphum rosae 416 15lla aegopodii ;200 5iphum rosae ;500 20siphon pelargonii 10 1siphon malvae 3 1via compositellae 120 25lla aegopodii ;500 3 1his quadrituberculata ;200siphon pelargonii 22 11lla aegopodii ;800 1 3 34siphon pelargonii 4 2hid rufula 50 3via compositellae 80 3his quadrituberculata ;100

3ntomophthora planchoniana Originating from

iella aegopodii, Respectively

tiory)

Number ofnuclei pr.conidium

Secondaryconidia,

mean length(mm)

Secondaryconidia,

mean width(mm)

L/WMean ratio(secondary

conidia)

Capilliphoresmean length

(mm)

4 4–5 21.6–27.4 12.8–14.6 1.7–1.9 50.9–59.04–5 24.2–25.8 13.5–13.7 1.8–1.9 52.5–67.9

2 4–9 15.1–17.5 12.7–15.1 1.1–1.2 —2 6–9 15.9–18.3 14.2–16.4 1.1 —

LEs F

thorosariethorosarierosthothoanoarielapthoariethozapanolap

LEd E

var

/Wn raimaidia

–1.1.3

–1.–1.

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e

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78 NIELSEN ET AL.

riod only very few individuals of El. abietinum werecaught on these traps, thus suggesting that the fallingdown behavior was not common for Icelandic El. abi-tinum. It is also unlikely that El. abietinum individ-

uals, which fall to the ground, will be able to reenterthe tree canopy since the aphid is predominantly apter-ous. Living aphids or cadavers falling to the groundmay, however, facilitate the distribution of inoculumfrom El. abietinum to other aphid species. Furtherdistribution of the fungus within other aphid speciessuch as Ca. aegipodii might be expected to be muchmore rapid than distribution within El. abietinum pop-ulations. This is due to the fact that Ca. aegipodii andother Cavariella species on willows are very common inIceland (Ottosson, 1985). In contrast, spruce planta-tions in Iceland are small and isolated, and El. abieti-num is only abundant in peak years which occur onceor twice each decade (Carter and Halldorsson, 1998).Host-alternation between Salix spp. and Umbelliferae,which are common amongst Cavariella spp. (Blackmannd Eastop, 1994), is likely to enhance further theistribution of fungal infection through Cavariella spp.inally, there is the possibility of contact between air-orne conidia and healthy aphids. Both Wilding (1970)nd Steinkraus et al. (1996) found high concentrations

of entomophthoralean conidia in the air over agricul-tural fields during epizootics. Even though the amountof conidia was never measured in this study, it is notbelieved that high densities were common in the sam-pled localities since all localities consisted of only a fewplants of the same species. Epizootics in one of theaphid populations will probably only release a fewconidia to the air compared to several hectares of anagricultural field crop with dense population of in-

FIG. 3. Accumulated mortality over time of Icelandic adults of(ARSEF 5372) of Pandora neoaphidis. The bioassay was carried out

fected aphids. s

Previous studies have shown a difference in suscep-tibility to infection between insect host species(Steinkraus and Kramer, 1987; Hajek et al., 1995) andeven clones of aphids (Milner, 1985). Furthermore, theability of a fungus to infect is highly dependent on theamount of inoculum received by the aphid host (Milnerand Soper, 1981; Feng and Johnson, 1991). In thepresent study the amount of inoculum used in thebioassays was high, and it is doubtful whether El.abietinum was ever exposed to such high doses in thefield. It is therefore likely that El. abietinum was ableto avoid infection from other aphid species in the field.An LT50 value of 3.9 days at 18°C for El. abietinuminfected with Pa. neoaphidis was not longer than ex-pected from earlier records of lethal times of otheraphids infected with this species (Brobyn and Wilding,1977; Milner and Bourne, 1983; Schmitz et al., 1993;

ielsen et al., 2001a). This means that for this systempossibly low susceptibility of El. abietinum to Pa.

eoaphidis was not expressed in the median lethalime. However, in a study comparing ecologically asso-iated hosts of Entomophthora muscae Cohn a pro-onged incubation period was found for nontypical in-ects hosts (Steinkraus and Kramer, 1987; Mullens,989).Based on this study, it is concluded that the best

trategy for enhancing the impact of entomophtho-alean fungi on El. abietinum populations would be toelease some of the species which are not present in El.bietinum populations. For example, En. planchoni-na could be released in the eastern population of El.bietinum and N. fresenii in the western population ofl. abietinum. For successful establishment of the

ungi, the present study clearly pointed out that the

tobium abietinum treated with conidia of a Danish in vitro isolate18°C with 10 aphids in each of two replicates.

Ela

election of fungi for release is very critical. The selec-

t

79ENTOMOPHTHORALES ON Elatobium abietinum

tion of strains for release should mainly be based onstrains infecting El. abietinum under natural condi-ions.

ACKNOWLEDGMENTS

The authors thank Dorthe Britt Jensen, The Royal Veterinary andAgricultural University, for technical assistance and Chris Lomer forthe correction of English phraseology. Mark S. Taylor, IACR-Roth-amsted, UK, is mercifully thanked for verification of the identity ofsome of the aphid specimens. Financial support was given by EUFAIR3-PL96-1792, RESFORAPHID.

REFERENCES

Abbott, W. S. 1925. A method for computing the effectiveness of aninsecticide. J. Econ. Entomol. 18, 265–267.

Austarå, Ø., Carter, C., Eilenberg, J., Halldorsson, G., and Harding,S. 1997. Natural enemies of the green spruce aphid in spruceplantations in maritime North West Europe. Buvısindi 11, 113–124.

Barrachina, M. N. 1999. “Contribuco al Coneixement de les Ento-moftorals Entomopatogenes de la Provincia de Barcelona” (Con-tribution to the Knowledge of Entomopathogenic Entomophtho-rales from the Barcelonean Province“). Treball de recerca [Over-view of the research], Universitat Autonoma de Barlelona (InCatalan).

Bejer-Petersen, B. 1962. Peak years and regulation of numbers inthe aphid Neomyzaphis abietina (Walker). Oikos 13, 155–168.

Blackman, R. L., and Eastop, V. E. 1994. “Aphids on the World9sTrees.” CAB International, University Press, Cambridge, UK.

Brobyn, P. J., and Wilding, N. 1977. Invasive and developmentalprocesses of Entomophthora species infecting aphids. Trans. Br.Mycol. Soc. 69, 349–366.

Byford, W. J., and Reeve, G. J. 1969. Entomophthora species attack-ing aphids in England, 1962–1966. Trans. Br. Mycol. Soc. 52,342–346.

Cameron, P. J., and Milner, R. J. 1981. Incidence of Entomophthoraspp. in sympatric populations of Acyrthosiphon kondoi andAcyrthosiphon pisum alfalfa pests. N. Z. J. Zool. 8, 441–446.

Carter, C. I., and Halldorsson, G. 1998. Origin and background to thegreen spruce aphid in Europe. In “The Green Spruce Aphid inWestern Europe; Ecology, Status, Impacts and Prospects for Man-agement” (K. R. Day, G. Halldorsson, S. Harding, and N. A Straw,Eds.). Forestry Commission, Technical Paper 24, 1–14.

Coremans-Pelseneer, J., Villiers, S., and Matthys, V. 1983. Ento-mophthorales found on wheat aphids, in soil, and air on the samefield. Four years compared results. Med. Fac. Landbouww. Rijk-suniv. Gent. 48, 207–224.

Crute, S., and Day, K. 1990. Understanding the impact of naturalenemies on spruce aphid populations through simulation model-ling. In “Population Dynamics of Forest Insects” (A. D. Watt, S. R.Leather, M. D. Hunter, and N. A. Kidd, Eds.), pp 329–337. Inter-cept, Andover, Hampshire.

Einarsson, M. 1976. “Vedurfar a Islandi” (“Climate in Iceland”). 150pp. Idunn, Reykjavik. (In Icelandic).

Feng, M. G., and Johnson, J. B. 1991. Bioassay of four entomoph-thoralean fungi (Entomophthorales) against Diuraphis noxia andMetopolophium dirhodum (Homoptera: Aphididae). Environ. En-tomol. 20, 338–345.

Hajek, A. E., Butler, L., and Wheeler, M. M. 1995. Laboratory bio-assays testing the host range of the gypsy moth fungal pathogenEntomophaga maimaiga. Biol. Control 5, 530–544.

Hajek, A. E., Butler, L., Walsh, S. R. A., Silver, J. C., Hain, F. P.,

of the gypsy moth (Lepidoptera: Lymantriidae) pathogen Ento-mophaga maimaiga (Zygomycetes: Entomophthorales) in the fieldversus laboratory. Environ. Entomol. 25, 709–721.

Heie, O. E. 1980. The Aphidoidea (Hemiptera) of Fennoscandia andDenmark. I. In “Fauna Entomologica Scandinavica,” Vol. 9. Scan-dinavian Science Press, Klampenborg, Denmark.

Heie, O. E. 1982. The Aphidoidea (Hemiptera) of Fennoscandia andDenmark. II. In “Fauna Entomologica Scandinavica,” Vol. 11.Scandinavian Science Press, Klampenborg, Denmark.

Heie, O. E. 1986. The Aphidoidea (Hemiptera) of Fennoscandia andDenmark. III. In “Fauna Entomologica Scandinavica,” Vol. 17.Scandinavian Science Press, Klampenborg, Denmark.

Heie, O. E. 1992. The Aphidoidea (Hemiptera) of Fennoscandia andDenmark. IV. In “Fauna Entomologica Scandinavica,” Vol. 25.Scandinavian Science Press, Klampenborg, Denmark.

Heie, O. E. 1994. The Aphidoidea (Hemiptera) of Fennoscandia andDenmark. V. In “Fauna Entomologica Scandinavica,” Vol. 28.Scandinavian Science Press, Klampenborg, Denmark.

Humber, R. A. 1989. Synopsis of a revised classification for Ento-mophthorales (Zygomycotina). Mycotaxon 34, 441–460.

Humber, R. A. 1997. Fungi: Identification. In “Manual of Techniquesin Insect Pathology” (L. Lacey, Ed.), pp. 153–186. Academic Press,San Diego.

Keller, S. 1987. Arthropod–pathogenic Entomophthorales of Switzer-land. I. Conidiobolus, Entomophaga and Entomophthora. Sydowia40, 122–167.

Keller, S. 1991. Arthropod-pathogenic Entomophthorales of Switzer-land. II. Erynia, Eryniopsis, Neozygites, Zoophthora, and Tari-chium. Sydowia 43, 39–122.

Keller, S. 1997. The genus Neozygites (Zygomycetes, Entomophtho-rales) with special reference to species found in tropical regions.Sydowia 49, 118–146.

Kristinsson, H. 1987. “Flowering Plants and Ferns of Iceland.” Ornog Orlygur, Reykjavik.

Lambers, D. H. R. 1955. Hemiptera 2. Aphididae. In “The Zoology ofIceland” (A. Fridriksson and S. L. Tuxen, Eds.), Vol. III, Part 52 a.Munksgaard, Copenhagen and Reykjavik.

Latteur, G. 1977. Sur la possibilite d’infection directe d’Aphides parEntomophthora a partir de sols herbergeant un inoculum naturel.C. R. Acad. Sci. Paris 284 (serie D), 2253–2256.

Milner, R. J. 1985. Distribution in time and space of resistance to thepathogenic fungus Erynia neoaphidis in the pea aphid Acyrthosi-phon pisum. Entomol. Exp. Appl. 37, 235–240.

Milner, R. J., and Bourne, J. 1983. Influence of temperature andduration of leaf wetness on infection of Acyrthosiphon kondoi withErynia neoaphidis. Ann. Appl. Biol. 102, 19–27.

Milner, R. J., and Soper, R. S. 1981. Bioassay of Entomophthorafungi against the spotted alfalfa aphid Therioaphis trifolii f. macu-lata. J. Invertebr. Pathol. 37, 168–173.

Milner, R. J., Holdom, D. G., and Glare, T. R. 1984. Diurnal patternsof mortality in aphids infected by entomophthoran fungi. Entomol.Exp. Appl. 36, 37–42.

Mullens, B. A. 1989. Cross-transmission of Entomophthora muscae(Zygomycetes: Entomophthoraceae) among naturally infectedmuscoid fly (Diptera: Muscidae) hosts. J. Invertebr. Pathol. 53,272–275.

Nielsen, C., Hajek, A. E., Humber, R. A., and Eilenberg, J. 1998.Soil—A natural source of entomophthoralean fungi infectingaphids. IOBC/WPRS Bull. 21, 45–48.

Nielsen, C, Eilenberg, J, Harding, S, Oddsdottir, E., and Halldors-son, G. 2000. Entomophthoralean fungi infecting the green spruceaphid (Elatobium abietinum) in the North-western part of Europe.

W

80 NIELSEN ET AL.

Nielsen, C., Dromph, K. M., and Eilenberg, J. 2001a. “Entomophtho-rales on cereal aphids: Characterisation, growth, virulence,epizootiology and potential for microbial control.” Pesticide Re-search No. 53, The Danish Environmental Protection Agency,Copenhagen, Denmark.

Nielsen, C., Sommer, C., Eilenberg, J., Hansen, K. S., and Humber,R. A. 2001b. Characterization of aphid pathogenic species in thegenus Pandora using PCR techniques and digital image analysis.Mycologia 93, 864–874.

Ottosson, J. G. 1985. Islensk skordyr a trjam og runnum (Insects ontrees and shrubs in Iceland). Skogræktarritið [J. Icelandic For.Assoc.] 33–42. (In Icelandic).

Papierok, B., and Hajek, A. E. 1997. Fungi: Entomophthorales. In“Manual of Techniques in Insect Pathology” (L. Lacey, Ed.), pp.187–212. Academic Press, San Diego.

Petursson, J. G. 1999. “Skogræktaroldin, samanteknar tolur ur Ar-sriti Skogræktarfelags Islands” (“The First 100 Years of TreePlanting in Iceland”). Skogræktarritið [J. Icelandic For. Assoc.]49–53. (In Icelandic with an English summary).

Rohel, E., Couteadier, Y, Papierok, B., Cavelier, N., and Dedryver,C. A. 1997. Ribosomal internal transcribed spacer size variationcorrelated with RAPD-PCR pattern polymorphisms in the ento-mopathogenic fungus Erynia neoaphidis and some closely relatedspecies. Mycol. Res. 101, 573–579.

Sigurdsson, V., Halldorsson, G., Sigurgeirsson, A., Thorsson, A. T.,and Anamthawat-Jonsson, K. 1999. Genetic differentiation of thegreen spruce aphid (Elatobium abietinum Walker), a recent in-vader to Iceland. Agric. For. Entomol. 1, 157–163.

Schmitz, V., Dedryver, C. A., and Pierre, J. S. 1993. Influence of anErynia neoaphidis infection on the relative rate of increase of thecereal aphid Sitobion avenae. J. Invertebr. Pathol. 61, 62–68.

Snorrason, A., Jonsson, H. J., Svafarsdottir, K., Gudbergsson, G.,and Traustason. T. 2000. “Rannsoknir a kolefnisbindinguræktaðra skoga a Islandi” (“A study on carbon sequestriation inforest plantations in Iceland”). Skogræktarritið [J. Icelandic For.

Sopp, P. I., Sunderland, K. D., and Coombes, D. S. 1987. Observa-tions on the number of cereal aphids on the soil in relation to aphiddensity in winter wheat. Ann. Appl. Biol. 111, 53–57.

Steenberg, T., and Eilenberg, J. 1995. Natural occurrence of ento-mopathogenic fungi on an agricultural field site. Czech Mycol. 48,89–96.

Steinkraus, D. C. 1998. The Neozygitaceae: Important small Arthro-pod specialists. Proceedings, VII’th International Colloquium onInvertebrate Pathology and Microbial Control, Sapporo, Japan,August 23–28, 1998, pp. 258–262.

Steinkraus, D. C., and Kramer, J. P. 1987. Susceptibility of sixteenspecies of Diptera to the fungal pathogen Entomophthora muscae(Zygomycetes: Entomophthoraceae). Mycopathologia 100, 55–63.

Steinkraus, D. C., Hollingsworth, R. G., and Boys, G. O. 1996. Aerialspores of Neozygites fresenii (Entomophthorales: Neozygitaceae):density, periodicity, and potential role in cotton aphid (Homoptera:Aphididae) epizootics. Environ. Entomol. 25, 48–57.

Steinkraus, D. C., Howard, M. N., Hollingsworth, R. G., and Boys,G. O. 1999. Infection of sentinel cotton aphids (Homoptera: Aphi-didae) by aerial conidia of Neozygites fresenii (Entomophthorales:Neozygitaceae). Biol. Control 14, 181–185.

Straw, N. A., Halldorsson, G., and Benedikz, T. 1998. Damage sus-tained by individual trees: Empirical studies on the impact of thegreen spruce aphid. In “The Green Spruce Aphid in Western Eu-rope; Ecology, Status, Impacts and Prospects for Management”(K. R. Day, G. Halldorsson, S. Harding, and N. A Straw, Eds.).Forestry Commission, Technical Paper 24, 5–31.

Thoizon, G. 1970. Specificite du parasitisme des aphides par lesEntomophthorales. Ann. Soc. Ent. Fr. (N. S) 6, 517–562.

Throne, J. E., Weaver, D. K., Chew, V., and Baker, J. E. 1995. Probitanalysis of correlated data: multiple observations over time at oneconcentration. J. Econ. Entomol. 88, 1510–1512.ilding, N. 1970. Entomophthora conidia in the air-spora. J. Gen.Microbiol. 62, 149–157.

Wilding, N. 1981. Pest control by Entomophthorales. In “MicrobialControl of Pest and Plant Diseases 1970–1980” (H. D. Burges,