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The small spruce bark beetle Ips amitinus (Eichhoff, 1871) is taxonomically placed in Coleoptera, Curculionidae, Scolytinae. I. amitinus is predominantly found in the spruce forests in mountainous areas of Central Europe (Jurc & Bojović 2004). Principal hosts are Picea spp. and Pinus spp. (Cognato 2015), most frequently Norway spruce (Picea abies (L.) Karst.) and Scots pine (Pinus sylvestris L.). Occasionally, other pines (P. cembra L., P. mugo Turra), silver fir (Abies alba Mill.) and European larch (Larix decidua Mill.) are also attacked. This bark beetle often remains undetected because it is confused with other, more common bark beetle species with which it often co-occurs, such as Ips typographus L. (Knížek et al. 2001, Holuša et al. 2012, Jurc & Bojović 2004). I. amitinus is distinguished from other Eurasian Ips spp. by its straight antennal club sutures. The species is a secondary pest, primarily colonizing recently dead or weakened trees. Nevertheless, under favourable weather and trophic conditions it can become dangerous, particularly for younger trees and plantations (Jurc & Bojović 2004, Okland & Skarpaas 2008).
In Slovenian forests some species that had not been economically important in the past might now, due to climate changes, cause damages in forests (Jurc & Bojović 2004). Among them is I. amitinus. Data on the location of specimens in the collection of the Natural History Museum of Slovenia showed an I. amitinus location in Pohorje (coll. Peyer) and a location in Peca (coll. Pavlin), in addition to Košenjak (Jurc 2003, Jurc & Bojović 2004). A gradation of I. amitinus infestation appeared from 2002-2003 where there were stands of 70- to 80-year-old Norway spruce in the Alpine region of Slovenia, at an altitude of 1270-1500 m above sea level, where snow breakage, extreme drought and warm weather were recorded in the years prior to attack (Jurc & Bojović 2004). I. amitinus are still present at this location but at a low population level (Ribič 2007).
I. amitinus, like other bark beetles, are vectors of various fungi. The fungal spore attaches to the bark beetles bodies’ and are dispersed to new host plants (Harrington 1993). Known fungal associates of the I. amitinus beetle are several genera from ascomycetes, mostly known as ophiostomatoid fungi. Ophiostomatoid fungi cause considerable economic losses in the forestry and timber production due to intensive discolorations or sap staining and vascular wilt diseases (Gibbs 1993, Harrington 1993). However, information regarding the basic ecological characteristics of I. amitinus is scarce (Holuša et al. 2012). The association of I. amitinus with ophiostomatoid fungi is equally poorly researched (Grosmann 1931, Kirisits et al. 2000).
Because environmental changes can influence bark beetle distribution as well as the distribution of its associated fungi, research on different bark beetle-fungi associations is essential (Linnakoski et al. 2010, Rice et al. 2008). Thus,the aim of this study was to obtain a better understanding of the association between the ophiostomatoid fungi assemblage connected with I. amitinus in Norway spruce. Therefore, we investigated 1) species composition of fungi associated with I. amitinus and we define their pathogenicity for the host tree, 2) the association between fungi and the I. amitinus development stage, 3) the influence of colonization time and 4) the influence of colonization position within the Norway spruce.
MATERIALS AND METHODS
Materijali i metode
Sample collection – Uzimanje uzoraka
Material for this study was collected in Dravograd, in north Slovenia (Koroška, 46º38´45˝N 15º2´10˝ E, altitude 1270 m a.s.l.) where Norway spruce trees were felled during the winter 2010 windthrow. They were naturally infested by the bark beetle I. amitinus in spring 2010. 204 beetles, 40 larvae, and 40 pupae were collected and placed individually in sterile tubes. Wood discs were cut from two trees at 0.5 m, 6 m and 15 meters above the stump (6 wood discs in total) and transported to the laboratory. Material was collected at the beginning of June 2010, immediately after the first colonization of bark beetles, and one month later in July 2010, when the first beetle generation had already built their galleries and the young adults were not emerging from the wood yet.
Fungal isolation – Izolacija gljiva
Fungi were isolated from sapwood of steam discs following a methodology similar to Solheim (1992a, 1992b) and Kirisits (2010). All the laboratory work was done in the laboratory of Forestry Institute of Slovenia in the Department of Forest Protection. Stem discs were split longitudinally in the laboratory one day after they were cut in the field. Three circular sections from each disc were taken, 18 total sections, and 158 wood chips in total were placed into a growing medium of 2% malt extract agar (MEA; 2% Bacto™ Malt Extract, 1.5% Difco™ Agar Technical; Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA). Pieces of wood were taken from under female galleries at 2 mm, 5 mm and at every subsequent 5 mm into the sapwood to a depth of 35 mm using sterile technique used by Haberkern et al. (2002), Solheim (1992a; 1992b) and Kirisits (2009). Petri dishes were incubated in the dark at 22°C to 25°C till the mycelia started to produce.
Insect-associated fungi were gathered from the beetles. Each adult beetle (as well as each pupa and larva) was placed directly on to the MEA plates and smashed. Petri dishes were stored in the dark at 22°C to 25°C till the mycelia started to produce. They were inspected every day for fungal