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SUSCEPTIBILITY OF Lymantria monacha AND L. dispar TO THE ENTOMOPATHOGENIC FUNGUS Isaria fumosorosea WIZE
Podložnost Lymantria monacha i L. dispar na entomopatogenu gljivu Isaria fumorosea Wize
Manana Kereselidze, Slavimira Draganova, Daniela Pilarskap>, Andreas Linde
Summary
Isaria fumosorosea is a cosmopolitan fungal species with a large host range including insects which are economically important pests in agriculture and forestry. In the current study the susceptibility of two forest pests Lymantria monacha and L. dispar to an isolate of the fungus Isaria fumosorosea obtained from Hyphantria cunea and re-isolates from L. dispar, L. monacha and Dendrolimus pini was investigated under laboratory conditions. Newly molted third instar larvae of L. monacha and newly molted second, third and fourth instar larvae of L. dispar were inoculated with fungal conidia by various methods: Larvae of L. dispar were either dipped directly into the conidia suspension (1×108 conidia/ml), or indirect methods were applied – by surface contact of larvae with conidial suspensions (1×108, 1×109, 3×107, 3×108, or 4×108 conidia/ml) placed on filter paper discs in Petri dishes or by contact with oak leaves or larch needles dipped in conidia suspension. Larvae in control variants were treated with water. Mortality of larvae was checked daily for 20 days and the efficacy of the fungus was corrected with mortality in the control treatments. It was found that larvae of both Lymantria – species can be infected experimentally with Isaria fumosorosea. Similar corrected efficacy of Isaria fumosorosea for the third instars larvae of L. dispar (12.37 %) and for L. monacha (12.66 %) was found when 1x108 conidia/ml of the isolate from H. cunea was applied on filter paper. The highest corrected efficacy of Isaria fumosorosea for L. dispar larvae was 60.0 % when 1x109 conidia/ml of the isolate from H. cunea was applied on filter paper. A corrected efficacy of 27.85 % was recorded for L. monacha when 4x108 conidia/ml of re-isolate from L. dispar were applied on larch needles. Our results show that L. dispar and L. monacha larvae are within the psihological host range of the used Isaria fumosorosea isolate from H. cunea and re-isolates obtained from infected larvae of D. pini, L. monacha and L. dispar, however their susceptibility is low. Indirect treatment by surface contact of host larvae with fungal conidia caused higher efficacy of mycosis than dipping into the suspension.
Key words: Isaria fumosorosea, Lymantria monacha, Lymantria dispar, bioassays

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Introduction
Uvod
Isaria fumosorosea Wize is a well-known entomopathogenic fungus with a worldwide distribution and a relatively wide host range which makes it an interesting agent for the development of biocontrol methods (Zimmermann 2008; Hunter et al. 2011). For more than 30 years, it was named Paecilomyces fumosoroseus and recently transferred to the genus Isaria (Samson 1974; Luangsa-ard et al. 2004, 2005; Gams et al. 2005; Hodge et al. 2005; Sung et al. 2007). Isaria fumosorosea has been isolated from many arthropods, mainly Lepidoptera, from air, water, plants, and often from soil samples (Meyer et al. 2008; Zimmermann 2008; Tkaczuk et al. 2011). In the catalogue of the USDA-ARS Collection of Entomopathogenic Fungal Cultures (ARSEF) (Humber and Hansen 2005) strains of Isaria fumosorosea are listed from 27 different countries, comprising North America, Central America, South America, Europe, Africa, Australia, and Asia. Isaria fumosorosea is regarded as a species complex, and various strains are successfully used for biocontrol of several pest insect species, for example whiteflies, thrips, aphids, and spider mites. It´s application for whitefly control, for example, started in 1990 with an isolate from Apopka, Florida (later named PFR 97, Apopka strain). This strain was also highly virulent to sweet potato whitefly and other pests (Osborne and Landa 1992, 1994; Landa et al. 1994; Stauderman et al. 2012). De Faria and Wraight (2007) assembled information about mycopesticides and disclosed that the most common among the presented 171 products were mycoinsecticides and mycoacaricids based on Beau­veria bassiana (33.9 %), Metarhizium anisopliae (33.9 %), Isaria fumosorosea (5.8 %), and B. brongniartii (4.1 %).
Because of the high interest for Isaria fumosorosea, it´s biology, ecology, natural occurrence and geographical distribution, host range, production of metabolites and effects of abiotic and biotic factors on the fungus are well studied (Avery et al. 2010). Furthermore, the use of this species in biocontrol in laboratory and field experiments (Feng et al. 2004; Pineda et al. 2007; Daniel and Wyss 2009), including their effects on non-target organisms (Tounou et al. 2003), were investigated by different authors and discussed by Zimmermann (2008).
The aim of our study was to determine the efficacy of an isolate of Isaria fumosorosea, isolated from pupae of Hyphantria cunea Drury in Georgia and re-isolates from Dendrolimus pini L., Lymantria monacha L. and L. dispar L. and to evaluate their potential as biological control agents of the forest pest insects L. monacha and L. dispar under laboratory conditions.
Materials and Methods
Materijali i metode
Lymantria monacha and L. dispar larvae were used for the experiments. First instar L. monacha larvae were collected in May 2013 from pine trees in the vicinity of Biebersdorf (region of Forest District Lieberose, Southern Brandenburg) and second and third instar larvae were collected in June in the vicinity of Staakow (region of Forest District Lieberose, Southern Brandenburg). Material was transferred to the laboratory of the University for Sustainable Development at Eb­erswalde. L. dispar larvae originated from a laboratory strain of the insect (New Jersey standard-strain from USA).
Four isolates of Isaria fumosorosea were used in the bio­assays: One original isolate of Isaria fumosorosea from Hyphantria cunea found in Georgia (ARSEF access no. 10244), and three re-isolates from D. pini, L. monacha and L. dispar. Re-isolates were obtained after contamination of D. pini, L. monacha and L. dispar larvae with conidia of the original isolate of Isaria fumosorosea in the laboratory and isolation of new fungal isolates from dead hosts into pure cultures

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on SDAY. The isolates were cultured for 15 days on slopes of SDAY in tubes and on Petri dishes at 22°C, and obtained conidia were washed down with sterilized water. The concentrations of conidia were determined by enumeration in a Thoma chamber. Insects were inoculated with conidia using several methods (Table 1). Larvae of L. dispar were either dipped directly into the conidia suspension (1x108 conidia/ml), or indirect methods were applied: a) surface con­tact of larvae with 1 ml of conidial suspensions (1x108, 1x109, 3x107, 3x108, or 4x108 conidia/ml) placed on filter paper discs (90 mm in diameter) in Petri dishes (Draganova and Staneva 1990) or b) contact with oak leaves or larch needles dipped in conidia suspension. Larvae in control variants were treated with water. Second, third and fourth instar larvae of L. monacha and L. dispar were used in the experiments. In total, 11 variants were performed with 20 larvae per repetition, with 4 repetitions in each variant. The acronyms for variants are presented in Table 1. All larvae were kept under laboratory conditions at 25 ± 2 °C, 60 ± 5 % R.H and 12:12 h L: D in a climate chamber (Percival Inc.). Lymantria monacha larvae were fed with larch needles. Larvae of Lymantria dispar were reared on artficial diet of Bell et al. (1981) or fed with oak leaves in case of treatment of oak leaves. Mortality of larvae was checked daily for 20 days and the efficacy of the fungus was corrected with mortality in the control treatments and calculated according to Schneider-Orelli’s formula (Püntener 1981).
Results and Discussion
Rezultati i rasprava
The results of the conducted studies are shown in Fig. 1, 2, 3 and 4.
It is evident that L. dispar and L. monacha larvae are within the host range of the used Isaria fumosorosea isolate from H. cunea and the re-isolates obtained from infected larvae of D. pini, L. monacha and L. dispar, but their susceptibility is rather low. The mortality due to mycoses showed a slow increase in all variants and was below 20 % with the exception of the mortality in the variant Ld-v-3 (Fig.1). The cu­mulative daily mortality due to mycosis in this variant in­creased to 41.25 % ± 12.26 on the 4th day and to 60.00 % ± 12.26 on the 11th day after the inoculation with 1x109 conidia/ml of the Georgian isolate of Isaria fumosorosea.
Although insects were inoculated with conidial suspensions with very high concentrations (1x 109 conidia/ml), the mortality 20 dpi didn’t exceed 60 % (Fig. 1, 2). In experiments with third instar L. dispar larvae an increase of the concentration of the conidial suspensions of the same isolate (Georgian isolate of Isaria fumosorosea isolated from H. cunea) from 1x108 to 1x109 conidia/ml resulted in higher efficacy – from 12.37 % ± 4.40 in the variant Ld-v-2 to 60.00 % ± 12.26 in the variant Ld-v-3 (Fig. 2).
According to Keller and Zimmermann (1989) the concentration of infective material necessary to initiate infection depends largely on the host and the pathogen. In our study we observed that larvae still living after 20 days post inoculation in all variants were not infected. They successfully completed their metamorphosis and turned into pupae. This is an evidence of low susceptibility of both Lymantria species to the Georgian isolate and three re-isolates of Isaria fumosorosea.

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Like the other entomopathogenic fungi, Isaria fumosorosea infects its host mainly through the external cuticle penetrating by mechanic pressure of germ tubes and releasing hydrolytic enzymes (St. Leger et al. 1986; Draganova 1988; Hajek and St. Leger 1994; Ali et al. 2010; Augustyniuk-Kram 2011; Augustyniuk-Kram and Kram 2012).
As the aim of this part of the study was to investigate how different modes of exposure of host larvae to fungal conidia affected the mortality caused by the mycosis, larvae were dipped into the suspension, or they were made to walk on filter paper discs in Petri dishes soaking with conidial suspension, or they were made to walk on oak leaves or larch needles sprayed with suspension. The dipping of L.dispar larvae into the suspension (variant Ld-v-4) resulted in the lowest efficacy of 3.75 % ± 1.76 in comparison to the variants with the other modes of inoculation where efficacy was 12.37 % ± 4.40 in the variant Ld-v-2 (indirect treatment by contact with conidia on filter paper discs) and 13.04 % ± 4.93 in the variant Ld-v-6 (indirect treatment by contact with conidia on oak leaves), respectively (Fig. 2). We conclude that indirect exposure of larvae through surface contact of host with conidia caused higher efficacy of mycosis.
According to Dunlap et al. (2007) susceptible insects exposed to blastospores and conidia of Isaria fumosorosea showed declined growth and high levels of mortality. We found that the two lepidopteran hosts species were not susceptible to the examined Isaria fumosorosea isolates. Furthermore, although the larvae were inoculated with highly concentrated suspensions placed on filter papers, the efficacy was rather low.
Bioassays with Beauveria bassiana (Bals. – Criv.) Vuill. and larvae of different insect pests (Ostrinia nubilalis Hb, Lepidoptera; Leptinotarsa decemlineata Say, Coleoptera) showed that the age of the inoculated larvae is of importance for the efficacy (Feng et al. 1985; Draganova 2000). Contrary to the expectation that the susceptibility of larvae will decrease with age as described by Keller and Zimmermann (1989), in our experiments younger instars of L. dispar were more tolerant to mycosis caused by Isaria fumosorosea isolates (variants Ld-v-1 vs Ld-v-2 and Ld-v-5 vs Ld-v-6). Similar observations were made by Ferron (1967) who showed that larvae of Melolontha melolontha F. were more sensitive to Beauveria brongniartii (Saccardo) Petch with increased age.
The bioassays with L. monacha show that 3rd instar larvae have a low susceptibility to mycosis caused by Isaria fumosorosea isolated from H. cunea and to two other re-isolates of the fungus (from L. monacha and from L. dispar (Fig. 3)). The development of the infection was slow in the variant Lm-v-1 with a small increase in efficacy on dpi 7. In contrast, the mortality in the variants Lm-v-2 and Lm-v-3 increased from 14.12 % ± 7.16 and 13.92 % ± 10.04 on dpi 5 to 21.80 % ± 7.16 and 22.78 % ± 10.04 on dpi 7, respectively.
According to Goettel et al. (1990) and Lecheva and Draganova (1998), fungal isolates are more virulent to their initial hosts. In our experiments, discrepant results concerning the initial host and the virulence of the isolates to L. dispar and L. monacha larvae were obtained (Fig. 2, 4). A comparison of the variants Lm-v-2 (inoculation with Isaria fumosorosea isolated from L. monacha larvae) vs Lm-v-1 (inoculation with Isaria fumosorosea isolated from H. cunea) (Fig. 4) and the variants Ld-v-5 (inoculation with Isaria fumosorosea isolated from L. dispar larvae) vs Ld-v-1 (inoculation with Isaria fumosorosea isolated from H. cunea) (Fig. 2) confirms the findings of the cited authors. The efficacy in the variants reached values of 25.65 % ± 7.16 vs 12.66 % ± 5.52 and 3.75 % ± 1.41 vs 1.25 % ± 0.63, respectively. However, when comparing the efficacy in the

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variants Lm-v-2 vs Lm-v-3, the results are contrary. The efficacy was minor in inoculation of L. monacha larvae with conidia of a re-isolate obtained from the initial host (25.65 % ± 7.16 vs 27.85 % ± 10.04).
In bioassays with different isolates of B. bassiana against L. dispar larvae, Draganova et al. (2013) describe that host larvae were tolerant to mycosis caused by the tested isolates, which is in accordance with the findings reported here.
We found that the mycoses caused by Isaria fumosorosea or Isaria farinosa show a remarkably lower lethal effect to forest pests compared to mycoses caused by B. bassiana or M. anisopliae, as has been shown in laboratory studies of Nedveckyte et al. (2011) with larvae of Bupalus piniaria L. (Lepidoptera: Geometridae) and the entomopathogenic fungi Isaria farinosa, B. bassiana and M. anisopliae. Although some hyphomycete species were found in natural popu­lations of L. dispar, and Paecilomeces farinosus was the most common (however in very low infection levels of 4.6 % to 12.2 %), the fungal pathogen with the highest virulence for L. dispar larvae is the entomophthoralean fungus Entomophaga maimaiga Humber, Shimazu et Soper (Hajek et al. 1997). E. maimaiga is a pathogen with high specificity, and its life cycle is perfectly synchronized with the life cycle of its insect host (Hajek 1999).
According to Zimmerman (2008), Isaria fumosorosea should be applied in combination with other entomopathogenic fungi, such as Lecanicillium and Beauveria. This suggestion will be considered in our future experiments. Due to the capacity of Isaria fumosorosea to cause natural epizootics and the rising commercial demand for bioproducts based on this fungus (Zimmermann 2008), further experiments should be directed to the development of effective laboratory trials.
Conclusions
Zaključak
Our results show that L. dispar and L. monacha larvae are within the physiological host range of the used Isaria fumosorosea isolate from H. cunea and re-isolates obtained from infected larvae of D. pini, L. monacha and L. dispar, however their susceptibility is low. Indirect exposure through surface contact of host larvae with fungal conidia caused higher efficacy of mycosis.
Acknowledgments
Zahvale
The authors are grateful to the German Academic Exchange Service (DAAD) and to the staff of the Landeskompetenzzentrum Forest Eberswalde (LFE) for supporting this study.
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Isaria fumosorosea je kozmopolitska vrsta gljive s velikim brojem domaćina, a među njima se nalaze insekti koji imaju veliko ekonomsko značenje kao važni štetnici za poljoprivredu i šumarstvo. U ovome istraživanju podložnost prema izolatima gljive Isaria fumosorosea istraživana je pod laboratorijskim uvjetima na dvije vrste šumskih štetnika, Lymantria monacha i L. dispar, a izolati su dobiveni iz vrste Hyphantria cunea te iz reizolata od L. dispar, L. monacha i Dendrolimus pini. Zaraza je obavljena na tek presvučenom III. larvalnom stadiju vrste L. monacha i na tek presvučenim II., III., i IV. larvalnom stadiju vrste L. dispar. Inokulacija s konidijama gljive odrađena je različitim metodama: larve vrste L. dispar su izravno umočene u suspenziju konidija (1×108 konidija/ml) ili su korištene neizravne metode – površinski kontakt larvi sa suspenzijom konidija (1×108, 1×109, 3×107, 3×108 ili 4×108 konidija/ml) stavljenih na filter papir u Petrijeve posude, ili kontakt s lišćem hrasta ili iglica ariša umočenih u suspenziju konidija. Larve u kontrolnom tretmanu tretirane su s običnom vodom. Mortalitet larvi je svaki dan provjeravan u razdoblju od 20 dana, a uspješnost gljive korigirana je s mortalitetom u kontrolnom tretmanu. Pokusi zaraze vrstom Isaria fumosorosea na obje vrste iz roda Lymantria, utvrdili su da postoji mogućnost zaraze ovom gljivom. Slična korigirana uspješnost vrste Isaria fumosorosea pronađena je kada su III. larvalni stadiji vrsta L. dispar (12,37 %) i L. monacha (12,66 %) bili stavljeni na filter papir sa suspenzijom vrijednosti 1×108 konidija/ml iz vrste H. cunea. Najveća korigirana uspješnost (60 %) bila je kada je izolat iz H. cunea na filter papiru bio korišten za larve L. dispar u suspenziji od 1×109 konidija/ml. Za vrstu L. monacha korigirana uspješnost od 27,85 % zabilježena je kada je korištena iglica ariša umočena u suspenziju od 4×108 konidija/ml iz reizolata L. dispar. Rezultati ovoga istraživanja pokazuju da larve vrsti L. dispar i L. monacha pripadaju među moguće domaćine izolata gljive Isaria fumosorosea dobivenih iz H. cunea i reizolata dobivenih iz zaraženih larvi vrsta D. pini, L. monacha i L. dispar, iako je njihova podložnost vrlo niska. Neizravni tretmani površinskim kontaktom larvi s konidijama gljive, uzrokovoli su veću uspješnost razvoja mikoze, za razliku od umakanja u suspenziju.
Ključne riječi: Isaria fumosorosea, Lymantria monacha, Lymantria dispar, biološka ispitivanja