Vitalnost spora mikrosporidija izoliranih iz gusjenica gubara (Lymantria dispar) nakon dugotrajne pohrane u tekućem dušiku

Data on the viability of microsporidian isolates from Lymantria dispar after long-term storage in liquid nitrogen are presented. Eight microsporidian isolates from L. dispar were tested for their infectivity against L. dispar larvae: ­Vairimorpha disparis, Nosema lymantriae, Nosema portugal, Nosema sp. (Poland), Nosema sp. (Ebergassing), Nosema sp. (Germany), Nosema sp. (Schweinfurt) and Nosema sp. (Veslec). The survival of spores in liquid nitrogen was ­studied in detail for N. portugal and Nosema sp. (Ebergassing) which had been stored in liquid nitrogen almost 19 years and used for individual per oral infections while the other six isolates were used only in surface contamination per oral experiments. Our study confirms that storage in liquid nitrogen is a suitable option for long-term storage of Nosema and Vairimorpha species from lepidopteran hosts. Spores survived for up to 19 years; however, the experiments show that there is a significant loss of viability. In some cases, spores had lost viability already after 7 years in liquid nitrogen. We recommend producing fresh material every 5 years to maintain collections in liquid nitrogen. No material that had been stored in liquid nitrogen for extended periods should be used for infection experiments.U radu se prikazuju rezultati preživljenja mikrosporidija izoliranih iz gubara (Lymantria dispar) nakon dugotrajne pohrane u tekućem dušiku. Infektivnost osam mikrosporidijskih L. dispar izolata testirano je na ličinkama gubara: Vairimorpha disparis, Nosema lymantriae, Nosema portugal, Nosema sp. (Poljska), Nosema sp. (Ebergassing), Nosema sp. (Njemačka), Nosema sp. (Schweinfurt) and Nosema sp. (Veslec). Preživljenje spora u tekućem dušiku detaljno je praćeno kod N. portugal i Nosema sp. (Ebergassing) koje su tako čuvane skoro 19 godina i aplicirane oralnom infekcijom i ponaosob na svaku pojedinu ličinku gubara. Ostalih 6 izolata aplicirano je površinskom kontaminacijom hranjivog supstrata, također oralnim infekcijskim putem. Od 8 mikrosporidijskih izolata apliciranih površinskom kontaminacijom samo kod 4 izolata, Vairimorpha disparis, Nosema lymantriae, Nosema sp. (Ebergassing) i Nosema sp. (Poljska), došlo je do uspješne infekcije ličinki gubara. Dok je kod svih testiranih ličinki (100%) uspješno realizirana infekcija sporama prve tri mikrosporidije, tek 21,1% tretiranih ličinki uspješno je inficirano sporama Nosema sp. (Poljska). Na kraju inkubacijskog razdoblja, kod svih ličinki inficiranih sa sva 4 izolata disekcijom je utvrđen visok stupanj zaraze; tkiva su bila ispunjena sporama. Mikroskopska pretraga ličinki L. dispar individualno tretiranih sporama Nosema portugal uz tri različite doze (100, 1000 i 10000 spora) pokazala je da su spore N. portugal izgubile infektivnost nakon 19 godina pohrane u tekućem dušiku; niti jedna od testiranih ličinka nije bila zaražena. Naprotiv, spore Nosema sp. (Ebergassing) zadržale su infektivnost nakon istog razdoblja pohrane u tekućem dušiku od 18,75 godina. Niti jedna od testiranih ličinki nije zaražena nakon oralnog unosa od 100 spora. Oralna aplikacija od 1000 spora po ličniki rezultirala je ukupnom uspješnom infekcijom 4,1% ličinki, a aplikacija od 10000 spora s 68,8% inficiranih ličinki. U svim uspješnim slučajevima uspješno zaraženih ličinki, na kraju inkubacijskog razdoblja infekcija je bila dobro razvijena, a masno tijelo prepuno spora. Istraživanje je potvrdilo da je pohrana mikrosporidija Nosema i Vairimorpha vrsta i domaćina iz reda leptira prikladna opcija za dugotrajno čuvanje izolata. Spore su preživjele i do 18 i pola godina, iako je tijekom eksperimenta zamijećen i značajan pad njihove infektivnosti. U pojedinim slučajevima spore su odumrle već nakon 7 godina pohrane u tekućem dušiku. Preporuka je da prilikom skladištenja i pohrane mikrosporidijskih izolata u tekućem dušiku svakih 5 godina repozitorij obnavlja svježim izolatima. Materijal koji se dulje vrijeme skladišti u tekućem dušiku nije prikladan i ne bi se smio koristiti u infektološkim testovima

Potential impact of Entomophaga maimaiga Humber, Shimazu, and Soper (Entomophthorales Entomophthoraceae) on the lepidopteran fauna inhabiting cork forests in Sardinia (Italy).

Periodic outbreaks of forest defoliators like the gypsy moth cause severe impact to the forest ecosystem, which is normally counterbalanced by the action of their natural enemies, including predators, parasitoids, and entomopathogens. Among the latter, the host-specific fungus Entomophaga maimaiga can be very effective under favourable conditions. Whilst its close evolutionary relationship with gypsy moth, this entomopathogen has never been detected in certain forest areas where L. dispar is a common pest. The results of three years laboratory assays with two different strains of E. maimaiga from Bulgaria and Croatia against Lepidopteran species inhabiting cork oak forests in Sardinia are reported. Significant toxicity and virulence against gypsy moth larvae exposed to soil contaminated with resting spores of the fungus was detected for both strains, even if the strain from Bulgaria was significantly more effective. Significant lethal effects were observed also on M. neustria larvae, but a successful development and reproduction of the fungus within insect cadavers was detected only in the gypsy moth. No significant effects were observed on other Lepidopteran species.  Given a proper choice of candidate strains, the introduction of E. maimaiga in Sardinia, to manage the disruptive action of the gypsy moth would be desirable

PODLOŽNOST Lymantria monacha I L. dispar NA ENTOMOPATOGENU GLJIVU Isaria fumorosea WIZE

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.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

Ефект на Beauveria bassiana (щам ATCC 74040) върху два вида листояди вредители по царевицата в лабораторни условия

In some European countries, the Western corn rootworm, Diabrotica virgifera virgifera, and the cereal leaf beetle, Oulema melanopus, are present in maize stands in high population density, occasionally causing damage of the crops. Susceptibility of adults of these species and larvae of the cereal leaf beetle to the commercially available product Naturalis® based on Beauveria bassiana strain ATCC 74040 was explored in laboratory conditions. The results of the bioassays showed that the fungal strain caused the highest corrected mortality on O. melanopus larvae with average values above 95% for all conidia concentrations (from 2.3 × 102 to 2.3 × 107 conidia/ml) tested. For the adults of the two pests, the average mortality increased positively with concentration of conidia and the values ranges from 9.5% to 94.2% for O. melanopus (concentrations from 2.3 × 102 to 2.3 × 107 conidia/ml) and from 19.3% to 92.4% for D. v. virgifera (concentrations from 2.3 × 103 to 2.3 × 107 conidia/ml). Accordingly, the LC50 values for O. melanopus larvae and adults and D. v. virgifera adults were determined as 4.6, 8.3 × 104 and 4.3 × 105 conidia/ml, respectively. Further studies to confirm the susceptibility of the larvae of the cereal leaf beetle to Naturalis® under field conditions should be conducted.В някои европейски страни западният царевичен коренов червей Diabrotica virgifera virgifera и обикновената житна пиявица Oulema melanopus присъстват в царевичните площи във висока плътност, нанасяйки щети на културите. В лабораторни условия беше изследвана чувствителността на възрастните индивиди от тези видове и ларвите на обикновената житна пиявица към търговския продукт Naturalis®, който съдържа Beauveria bassiana (щам ATCC 74040). Резултатите от опитите показват, че гъбният щам причини най-високата коригирана смъртност на ларвите на O. melanopus със средни стойности над 95% за всички изпитани концентрации на конидиите (от 2.3 × 102 до 2.3 × 107 конидии/ml). За възрастните на двата вида вредители средната смъртност нараства с увеличаване на концентрацията на конидиите и стойностите варират от 9.5% до 94.2% за O. melanopus (концентрации от 2.3 x 102 до 2.3 × 107 конидии/ml) и от 19.3% до 92.4% за D. v. virgifera (концентрации от 2.3 × 103 до 2.3 × 107 конидии/ml). Cтойностите на LC50 за ларвите и възрастните на O. melanopus и възрастните индивиди на D. v. virgifera бяха съответно 4.6, 8.3 × 104 и 4.3 × 105 конидии/ml. Необходимо е да се проведат допълнителни изследвания за потвърждаване на високата чувствителност на ларвите на обикновената житна пиявица към Naturalis® в полеви условия

PRVI NALAZ ENTOMOPHAGA MAIMAIGA (ENTOMOPHTHORALES: ENTOMOPHTHORACEAE) U POPULACIJAMA LYMANTRIA DISPAR U GRČKOJ I REPUBLICI MAKEDONIJI

The entomopathogenic fungus Entomophaga maimaiga Humber, Shimazu & Soper (Entomophtorales: Entomophtoraceae) was found for first time in populations of gypsy moth, Lymantria dispar (L.) (Lepidoptera: Erebidae), in Greece and the Former Yugoslavian Republic of Macedonia (FYROM) after its introduction in Bulgaria in 1999. Monitoring studies were conducted in 2012 in oak stands in three sites in the Xanthi region in Greece, and in three sites in FYROM in the Prilep region. Gypsy moth larvae, predominately in fourth to sixth instar, were collected in May and June. During laboratory rearing, mortality of gypsy moth larvae collected in two sites in Greece ranged from 36.4–89.3%. Larval mortality of L. dispar in the three sites in FYROM ranged from 16.7–87.8%. Dead larvae were analysed under light microscopy for presence of E. maimaiga and other entomopathogens. E. maimaiga was recorded from one site in Greece (Kidaris vill.), and in all study sites in FYROM (Toplica, Belovodica and Krushevo vill.). Azygospores of E. maimaiga were found in the bodies of 78.6% of gypsy moth larvae from Kidaris, and in 8.3–16.3% of the larvae from sites in FYROM. Recent records of E. maimaiga in Serbia and the European part of Turkey, and present findings in Greece and FYROM, indicate that the fungus most probably has invaded gypsy moth populations in other parts of Balkan Peninsula.Entomopatogena gljiva Entomophaga maimaiga Humber, Shimazu & Soper (Entomophtorales: Entomophtoraceae) prvi je puta utvrđena u populacijama gubara, Lymantria dispar (L.) (Lepidoptera: Erebidae) u Grčkoj i Republici Makedoniji nakon introdukcije ovog patogena u Bugarsku 1999. godine. Tijekom 2012. godine obavljen je pregled hrastovih sastojina na tri lokacije u području Xanthi u Grčkoj i na tri lokacije u okolici Prilepa u Makedoniji. Gusjenice gubara sakupljane su većinom u četvrtom do šestom larvalnom stadiju u razdoblju svibanj–lipanj. Tijekom laboratorijskog uzgoja smrtnost gusjenica sakupljenih u Grčkoj iznosila je od 36,4 do 89,3 %. Smrtnost gusjenica s lokaliteta u Makedoniji bila je između 16,7 i 87,8 %. Uginule gusjenice analizirane su pomoću svjetlosnog mikroskopa na prisutnost E. maimaiga i ostalih patogena. Patogena gljiva E. maimaiga utvrđena je na uzorku s jednog lokaliteta u Grčkoj (Kidaris) i na uzorcima svih lokaliteta u Makedoniji (Toplica, Belovodica i Kruševo). Azigospore E. maimaiga utvrđene su u 78,6 % uginulih gusjenica s lokaliteta Kidaris i između 8,3 i 16,3 % uginulih gusjenica s tri lokaliteta u Makedoniji. Nedavni nalazi E. maimaiga u Srbiji i europskom dijelu Turske te najnoviji nalazi u Grčkoj i Republici Makedoniji ukazuju na vjerojatnost da je ovaj patogen već inficirao populacije gubara u ostalim dijelovima balkanskog poluotoka

FIRST RECORD OF GYPSY MOTH ENTOMOPATHOGENIC FUNGUS Entomophaga maimaiga Humber, Shimazu & R.S. Soper IN BOSNIA AND HERZEGOVINA

Nedavna otkrića gljivičnog patogena Entomophaga maimaiga Humber, Shimazu & R.S. Soper u susjednim istočnim i sjevernim zemljama Europe, posebice u Hrvatskoj, te pojavnost progradacija populacija gubara (Lymantria dispar L.) u pojedinim područjima Bosne i Hercegovine, privukla su pozornost istraživača na mogućnost prisutnosti ovog patogena u lokalnim populacijama gubara. Interes istraživača usmjerio se na lokacije zabilježenih mjesta progradacije gubara koja se nalaze u sjevernim dijelovima Bosne i Hercegovine. S obzirom na malu zračnu udaljenost tih lokacija s lokacijama masovne pojavnosti patogena E. maimaiga u Republici Hrvatskoj i Srbiji, hipoteza je bila provjeriti mogućnost prelaska patogena duž graničnog područja rijeke Save i Drine te ulazak u Bosnu i Hercegovinu.Istraživanje je obavljeno početkom srpnja 2013. godine. Odabrano je pet lokacija u kojima je zabilježeno stanje progradacije populacije gubara, te uočen značajni mortalitet larvi gubara od nepoznatog uzročnika. Uzorci larvalnih kadavera sakupljeni su sa stabala na visini od 0,5–1,5 m iznad razine tla. Sakupljani su samo stariji larvalni stadiji (L4 – L6) zbog kasnijeg vremenskog razdoblja uzorkovanja, te analizirani u Laboratoriju za patologiju drveća Šumarskog fakulteta Sveučilišta u Zagrebu.Kod svih pet uzoraka tkiva uginulih larvi s pet različitih lokacija, pronađene su azigospore ili konidije (konidio­spore) i azigospore vrste E. maimaiga. Tip spora koji će se formirati nakon smrti domaćina ovisi o patogenu i načinu infekcije, faktorima vezanim za domaćina i okolišnim uvjetima. Makroskopski simptomi napada gljive E. maimaiga bili su jasno vidljivi na terenu, zajedno s nekim znacima larvalnog mortaliteta od strane poliedrije (LdMNPV), međutim u znatno manjem obujmu. Prisutno je bilo vrlo malo znakova parazitoidnog mortaliteta, ali sa značajnom prisutnošću tipičnih predatora gubara kao što je Calosoma sycophanta L., koji je bio prisutan u sta­diju ličinke i imaga.The fungal pathogen Entomophaga maimaiga Humber, Shimazu & R.S. Soper (Enthomophthorales: Entomophthoraceae) was found in the north-east Bosnia and Herzegovina populations of Lymantria dispar L. (Lepidoptera: Erebidae) in summer 2013 at 5 localities (Figure 1, Table 1). Since the first introduction of this pathogen in Bulgaria in 1999 several southeastern European countries confirmed its presence in subsequent years. Recent findings of this pathogen in the neighbouring countries, especially in Croatia and Serbia, and also regarding to the progradation of L. dispar populations in some parts of Bosnia and Herzegovina, attracted the attention of the researchers on the possibility of finding this highly selective fungal pathogen. The sampling localities were selected based on pre-collected data of reported L. dispar population progradation, but also to the relative geographical vicinity regarding the new Croatian positive sites with E. maimaiga fungal pathogen. Large areas were aerially sprayed with a Bacillus thuringiensis kurstaki – based bacterial insecticide and larval development was closely inspected in the field. In the last 70 years, from the first reported outbreak of L. dispar in Bosnia and Herzegovina, many outbreaks have been reported, causing the supstantial economical and ecological damage in forestry but also in agriculture production. Biological insecticide based on the Bacillus thuringiensis kurstaki, commonly used for the suppression of the local populations of L. dispar, does not always produce the desired impact. Consequently, the highly selective, host specific, fungal pathogen that could be put in use as a biological control agent of the L. dispar populations is found to be very interesting for both science and professional application thus deminishing the economical and ecological negative feedback of this indigenous defoliator. The research was carried out during July 2013 throughout the north-east part of Bosnia and Herzegovina where gypsy moth populations continued an ongoing outbreak. From five selected localities, where excessive mortality was observed, larval cadavers were sampled from tree trunks 0,5 – 1,5 m above the ground. Only older larval stages (L4 – L6) were sampled due to the late sampling period. Cadavers were inspected, and larval tissue samples were prepared under the stereo microscope (LEICA Leitz MZ8) and light stereo microscope (Motic SMZ – 168 TLED). Process was digitaly documented with Olympus SP – 500UZ digital camera equipped with the Olympus QuickPHOTO CAMERA 2.3 digital imaging software. Larval tissue samples were inspected under light microscope (Olympus BX53) and images were recorded by digital camera Motic MoticamPro 252A. Measurements of azygospores and conidia (conidiospores) were made via digital imaging software Motic Images Plus 2.0 and Motic Images Advanced 3.2. associated with a compound microscope. Microscopic analysis of the dead tissue larvae, in all five locations, confirmed both conidia and azygospores or azygospores only, of the E. maimaiga species. Spore dimensions were as follows: pear-shaped conidia crosswise 25,7–35,1 µm and 34,6–43,7 µm lenghtwise; azygospores 32,2–47,9 µm in diameter (Figure 2, Figure 3). The type of spores (conidia or azygospores or both) that will form after host death is determined by the pathogen and the type of host infection, host-related factors and environmental conditions. Macroscopic symptoms of E. maimaiga attack were easily recognizable on the tree trunks along with some signs of larval mortality caused by Lymantria dispar multicapsid nuclear polihedrosis virus (LdMNPV) (Figure 4). There were very few signs of parasitoid mortality, but unlike in the Croatian sites (Hrašovec et al. 2013), with a great abundant presence of gypsy moth predators like Calosoma sycophanta L. (Figure 5), which could be an indicator that the pathogen has emerged when the L. dispar population was already starting its descent into a retrogradation phase. Just like in the Croatian localities where the sampling took place (Hrašovec et al. 2013), dead larvae were hanging from the tree trunks head down all through the sites and no living larva or viable pupa could be found in the area. Based on the field collections and microscopic analysis, entomopathogenic fungus E. maimaiga, a pathogen of L. dispar introduced on the European continent, has been confirmed in Bosnia and Herzegovina. Extensive monitoring of the fungal pathogen in the following years will give us the information on pathogen spatial spread, its speed and the possibility for establishing its permanent position among the other local indigenous species. Nevertheless, there are some concerns whether E. maimaiga may show some direct and indirect impacts on non-targeted and beneficial organisms in the future, or change the basic community structure of folivore insect guilds on oaks, maybe resulting in increasing populations of other defoliating insect groups such as tortricids, geometrids and sawflies. These concerns will demand more scientific attention in the future

First record of the pathogenic fungus Entomophaga maimaiga Humber, Shimazu, and Soper (Entomophthorales: Entomophthoraceae) within an outbreak populations of Lymantria dispar (Lepidoptera: Erebidae) in Croatia

Background and Purpose: The last wave of a gypsy moth (Lymantria dispar) outbreak in Croatia and recent discoveries of its fungal pathogen Entomophaga maimaiga in eastern neighboring countries focused the attention of researchers on the possible presence of this pathogen in local gypsy moth populations. Since the first introduction of E. maimaiga in Bulgaria in 1999, several southeastern European countries confirmed its presence in subsequent years. One unchecked report made by a local forester "of dramatically high mortality in gypsy moth larvae at one locality at the easternmost part of Croatia in early summer 2012" raised the interest in whether E. maimaiga occurred in Croatia even higher. In spring 2013, the gypsy moth outbreak area grew even larger. Large areas were aerially sprayed with a Bacillus thuringiensis kurstaki-based bacterial insecticide and larval development was closely inspected in the field. One of the tasks was to check on the possible presence of E. maimaiga in gypsy moth populations in Croatia, and these results are presented here. Materials and Methods: The research was conducted during June and July 2013 throughout the Eastern part of Croatia where gypsy moth populations entered into or continued an ongoing outbreak. From ten selected localities where excessive mortality was observed by local foresters, larval cadavers were sampled fromtree trunks 0,5 – 1,5mabove the ground. Only older larval stages (L4–L6) were sampled due to the period of sampling. The cadavers were placed in Petri dishes on moistened filter paper discs, after which they were stored in the refrigerator on +4 °C for 48 – 72 h. Larval tissue samples were inspected under the light microscope.During the process many images were recorded by digital camera. Measurements of spores and vegetative stages were made via digital imaging software associated with a compound microscope. Selected samples of field collected dead larvae were stored in ETOH for further DNA molecular analysis. Results: In nine out of ten field samples of dead gypsy moth larvae, either conidia or resting spores of E. maimaiga were confirmed. Depending on collection date, microscopic analysis confirmed both conidia and azygospores or azygospores only. The latter was typically the case when fully dry cadavers were collected towards mid July and later. Spore dimensions were as follows: pear-shaped conidia 24.2–35.8 μm crosswise and 29.5–43.6 μm lengthwise; azygosporees 31.7–47.1 μm diameter. Spore sizes and their general shape varied in accordance with gypsy moth cadaver age in days, time of year when collecting was done and general weather conditions that prevailed immediately prior to sampling.Macroscopic symptoms ofE.maimaiga attackwere already clearly visible in the field, along with some signs of larval mortality caused by ucleopolyhedrovirus (NPV), but to a lesser extent. There were very few signs of parasitoid mortality and no visible presence of typical gypsy moth predators like Calosoma sycophanta. Larval mortality that could be assigned dominantly to E. maimaigawas highest in the easternmost localities. By the beginning of June thousands of larvae were hanging head down on tree trunks and no living larva or viable pupa could be found in the area. Conclusions: Based on the field collections and microscopic analysis, it can be concluded that E. maimaiga, a pathogen of L. dispar introduced on the European continent, has been confirmed in Croatia. On the basis of some reports from the previous year, it is reasonable to assume that E. maimaiga appeared at least one year earlier (2012) but this cannot be proved now, in spite of the fact that the location of its possible presence in 2012was somehowlogical as itwas reported fromthe far eastern borderwith Serbia and E.maimaiga alreadywas known to occur in Serbia. Further targeted research in subsequent years, following the ongoing gypsy moth outbreak, should give a better picture of the spread and efficacy of this pathogen within Europe

Insecticide activity of Greek oregano essential oil and entomopathogenic fungus Metarhizium pemphigi against Diabrotica virgifera virgifera LeConte

The western corn rootworm (WCR), Diabrotica virgifera virgifera, is an invasive alien species and an important pest of maize in Europe. The insecticide potential of Greek oregano, Origanum vulgare subsp. hirtum, essential oil (EO) and the entomopathogenic fungus Metarhizium pemphigi were evaluated against WCR adults in laboratory conditions as alternative control agents. Pure undissolved Greek oregano EO applied at a volume of 3, 5 and 10 µl caused 97–100% mean corrected mortality of the test individuals with a rapid lethal effect. Treatment of WCR with four concentrations of EO (0.01, 0.1, 1 and 10 µl/ml) showed a strong concentration–time effect. Mortality rate increased with increasing concentration and exposure time. The median lethal concentration LC50 was 0.03 µl/ml. The time needed for Greek oregano EO to cause LT50 of D. v. virgifera ranged from 17.95 days for the lowest concentration (0.01 µl/ml) to 90 min for the highest concentration (10 µl/ml). Metarhizium pemphigi, which was tested in five conidia concentrations (2 × 103–2 × 107 conidia/ml), showed relatively lower effectiveness against D. v. virgifera adults. At the end of the bioassay (ten days post-treatment), the mean corrected mortality of the test specimens varied from 14 to 73%. LC50 was 3.3 × 106 conidia/ml. The results showed that Greek oregano EO had a bioinsecticidal effect against WCR adults at low concentrations and they are a basis for further investigations on the EO as an environmentally friendly alternative to chemical insecticides