Insect-truffle interactions – potential threats to emerging industries?

Fungal Ecology 25 (2017) 59e63

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Commentary

Insect-truffle interactions e potential threats to emerging industries? Aleksandra Rosa-Gruszecka a, Alan C. Gange b, *, Deborah J. Harvey b, Tomasz Jaworski a,
ska d
ski a, Radosław Plewa a, Szymon Konwerski c, Dorota Hilszczan Jacek Hilszczan Department of Forest Protection, Forest Research Institute, Se˛ kocin Stary, Braci Le
snej 3, 05-090 Raszyn, Poland School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK c
, Poland Natural History Collections, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan d Department of Forest Ecology, Forest Research Institute, Se˛ kocin Stary, Braci Le
snej 3, 05-090 Raszyn, Poland a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 June 2016 Received in revised form 26 September 2016 Accepted 12 October 2016 Available online 16 November 2016

Truffle harvests are declining in Europe, due to droughts, and this offers an opportunity for production to be developed in countries such as the UK and Poland, where rainfall tends to be higher. Drier Medi
rigord truffle (Tuber terranean summers seem to be associated with a decrease in the harvest of the Pe melanosporum) in Spain, France and Italy. However, other species, for example the Burgundy truffle (Tuber aestivum) offer opportunities for production in the more temperate environments north of the Alps. Truffles across Europe can be infested by insect larvae, seriously reducing their economic and culinary quality. Here, using a combination of literature sources and a field survey, we present a commentary on insects attacking truffles, aiming to highlight those species that could be potential pests in the British and Polish emergent industries. There is a remarkable disparity in coincidence of records of insects and truffles in these countries, yet a survey in Poland confirms that insects can be abundant. We discuss reasons for this disparity and suggest that biochemical methods could easily be developed for detection of the truffles and their attackers. © 2016 Elsevier Ltd and British Mycological Society. All rights reserved.

Corresponding Editor: Nicolai Vitt Meyling Keywords: Coleoptera Diptera Hypogeous fungi Insect Tuber Volatiles

1. Introduction Truffles are hypogeous fungi belonging to the Pezizales, mostly in the genus Tuber, and comprise a large group of ectomycorrhizal fungi growing in symbiosis with the roots of several vascular plant species (angiosperms and gymnosperms). The fruit body of these fungi is a subterranean complex apothecium, commonly known as the truffle. The geographic distribution of truffles mainly covers the temperate zones of the Northern Hemisphere, with at least three areas of genetic differentiation in Europe, South East Asia and North America (Pomerico et al., 2006). So far, seven species of Tuber have been reported from Poland, namely Tuber mesentericum (Ławrynowicz, 1999), Tuber aestivum, Tuber excavatum, Tuber rufum
ska et al., 2008), Tuber maculatum (Ławrynowicz, 2009), (Hilszczan
ska et al., 2013) and Tuber brumale Tuber macrosporum (Hilszczan
nyi et al., 2014). According to the Fungal Records Database of (Mere the British Isles (http://www.fieldmycology.net/FRDBI/FRDBI.asp) all 7 species have been recorded in the UK, though none have many

* Corresponding author. E-mail address: [email protected] (A.C. Gange). http://dx.doi.org/10.1016/j.funeco.2016.10.004 1754-5048/© 2016 Elsevier Ltd and British Mycological Society. All rights reserved.

attributed records. That with the most is T. aestivum with 110 records, but to put this into perspective, the fungal species with the most records (Hypholoma fasciculare) has 16,259 (as of 21 September 2016). Economically, truffles are the most valuable non-timber products of forest ecosystems, and are highly prized for their culinary qualities in countries such as France, Italy and Spain. Highly desirable truffles (i.e. Tuber magnatum (white) or Tuber melanosporum (black)) may attract remarkable prices, of around V2000eV3000 kg
1, with the industry in Italy worth around V400 million per annum (Büntgen et al., 2012; Pieroni, 2016). This may be the primary reason why a truffle industry is emerging in countries such as the UK and Poland. However, it may also be due to the decline of harvests of the highly-prized black truffle (T. melanosporum) in its main habitats due to increased frequency of droughts (Büntgen et al., 2011, 2012, 2015). Although neither T. magnatum nor T. melanosporum have been found in the UK or Poland, two other species (T. aestivum and T. brumale) are commercially traded in countries such as Spain and Hungary (Stobbe et al., 2013; Martin-Santafe et al., 2014). Indeed, recent evidence suggests that T. aestivum in particular may be found in suitable areas north of the Alps, such as Germany, and even as far

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north as southern Sweden and Finland (Stobbe et al., 2012, 2013). The first cultivated specimen of T. aestivum was found in England in March 2015 (http://www.bbc.co.uk/news/science-environment31826764) and in Wales in July 2016 (http://www.itv.com/news/ wales/2016-07-25/first-ever-cultivated-truffle-harvested-inwales/). In Poland, three truffle orchards (with T. aestivum) have been established and maintained by the Forest Research Institute,
ska et al., 2008; Hilszczan
ska one of which is productive (Hilszczan
ska, 2016). Therefore, there is great and Sierota, 2010; Hilszczan economic potential for the emergent industries in more northerly countries to fill gaps in the European market, and it is timely to identify any problems that might reduce their potential in future. Ecologically, these fungi are of considerable importance because of the benefits of the mutualistic association they provide to their host plants (Pacioni and Comandini, 1999). In addition, the relatively long-lived fruit body provides a food source for invertebrates and vertebrates (Johnson, 1996; Blackwell, 2005). Some species of truffles, e.g. T. magnatum, T. melanosporum and T. aestivum, have a high culinary value because of their aroma (Mello et al., 2006) and in natural habitats, the volatiles produced are essential for attracting animals that spread the spores (Fogel and Trappe, 1978). However, some animals have evolved a capacity for feeding on truffle sporophores. These belong to various taxonomic groups and are termed ‘hydnophagous’ (Pacioni, 1989), from Greek ‘hydnon’, truffle, and ‘phagous’, eating. For certain animals, such as mammals (rodents, deer, boars), birds and slugs, truffles are a valuable part of their diet (Johnson, 1996; Vernes et al., 2015), yet for several species or genera of arthropods, mainly in the Coleoptera and Diptera, the fungi may represent their complete diet (Pacioni et al., 1995; Di Santo, 2013). Indeed, there is much anecdotal evidence that truffles may be discovered by searching for the flies that oviposit within the fruit body, though this is a laborious and unpredictable procedure (Blackwell, 2005). Scattered through the literature are occasional reports of truffle sporocarps being infested by insect larvae, thus reducing their marketable value considerably (Ciampolini and Suss, 1982; Martin-Santafe et al., 2014). Our aim here is to provide a survey of the insects that may be associated with truffles in two countries, which as well as being ecologically interesting, highlights potential problems for the emerging truffle industry. 2. Survey methods We present a survey of known host associations in each country, using databases and field sampling. The UK insect fauna is relatively well recorded and we used national distribution data, available through the National Biodiversity Network Gateway (https://data. nbn.org.uk/). The UK National Grid divides the country into 2500 (10 km  10 km) squares and records are provided at this scale. We extracted a list of the 10 km  10 km squares in which T. aestivum has been recorded and compared these with 10 km  10 km records of the principal insect species associated with truffles (described below). National record data for Poland are far less developed, but we extracted data for fungi and insects from the Universal Transverse Mercator (UTM) 10 km  10 km grid (http://baza.biomap.pl/pl/db). The Polish UTM divides the country into 3384 (10 km  10 km) squares (Iwan et al., 2012). These were supplemented by surveys in
rz Upland, four geographical regions in Poland: Nida Basin, Przedbo
w Upland and Chełm Hills (Table 1). Records were obtained Miecho using two methods of collecting insects associated with truffles. In 2012e2014 Tuber spp. fruit bodies inhabited by adults and larvae were collected and larvae reared to adult. Information on sampling effort is given in Table 2. In addition, insects were also collected in two adjacent regions


w Upland with traps installed in natural of Nida Basin and Miecho habitats of T. aestivum. A total of 24 modified funnel traps were used in both localities. To minimize collection of non-target invertebrates (e.g. Carabidae, Silphidae) or small vertebrates (lizards, mice), traps were buried in soil with the upper edge of the funnel left a few centimeters above the ground, and were covered with a plastic roof placed 2 cm above the funnel. To preserve collected insects, a container filled with 200 ml of ethylene glycol was put underneath the funnel. Each trap was baited using a small piece of T. aestivum mature fruit body, placed inside a 2 ml perforated container that was installed under the trap cover. Traps were emptied every third week from mid-July to early October 2012. Beetles were identified by S. Konwerski and flies (Diptera) were identified by A. Wo
znica. 3. Truffle insects and their distributions The coleopteran fauna associated with truffles is mainly represented by the beetle Leiodes cinnamomea (Coleoptera: Staphylinoidea) (Arzone, 1970, 1971). Adult females of the species are attracted by truffle volatiles in its early stage of growth, but not when the fruit body is mature (Hochberg et al., 2003). The beetle appears to be specific to the genus Tuber, particularly T. melanosporum, with some records from T. aestivum and T. excavatum (Fogel and Peck, 1975; Pacioni et al., 1991; Bratek et al., 1993) and completes its life cycle in or adjacent to the fruit body (Arzone, 1970; Newton, 1984). Nevertheless it can cause extensive damage to the truffle (Fig. S1). In the UK, L. cinnamomea is considered hard to find (Blackwell, 2005) and is designated as Nationally Notable, having been recorded in only 25 of the 2500 10 km  10 km squares of the National Grid (https://data.nbn.org. uk/(accessed 21/9/2016)). Its most likely host in the UK, T. aestivum, has been recorded from 40 of the 10 km  10 km squares, yet in only 3 squares are there coincidental records of beetle and truffle. This represents just 12% of the recorded beetle distribution and 7.5% of squares with truffle records. Given that the insect is host specific, one would expect that all beetle records would coincide with those of the truffle, but instead there is a great discrepancy in records. This is likely due to the well-known bias that exists within such data bases (e.g. Ward, 2014), but with truffles in a country like the UK it is likely to be particularly acute. As the fungus is highly prized, locality records are very unlikely to be advertised by those seeking the fruit bodies for commercial purposes, creating a highly biased distribution. Entomologists are more likely to present their records, as they are far less likely to have a vested interest in truffle collection. Thus, the available data suggest that the insect may have little impact on the industry, but this is subject to the serious bias aforementioned. It is likely that L. cinnamomea is considerably more common than thought and should not be dismissed as a potential pest species. In Poland L. cinnamomea is known from 15 squares of the UTM 10 km  10 km grid (http://baza.biomap.pl/pl/db (accessed 21/9/ 2016)). However, none of these localities are coincident with known Tuber spp. sites. The field survey (Table 1) showed that larvae of this beetle can infest ascocarps of T. aestivum and T. exacavatum. However, considerably fewer larvae were found in T. excavatum. Along with L. cinnamomea another leiodid beetle, Leiodes oblonga, was found, both as adults and larvae, in T. aestivum fruit bodies. Other hosts of this species' larvae were T. excavatum and T. rufum (Table 1). Koch (1991) reported L. oblonga from truffles, but without distinguishing the fungal species. L. oblonga has been recorded from 16 (10 km  10 km) squares in the UK, yet only one of these coincides with T. aestivum. The results here seem to be the first report on L. oblonga associations with truffles. L. oblonga is very similar to L. cinnamomea but correct identification of both species is

A. Rosa-Gruszecka et al. / Fungal Ecology 25 (2017) 59e63

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Table 1 Insects associated with Tuber spp. in Poland (asterisks indicate number of specimens reared from fruit bodies of Tuber, data without asterisks show number of specimens caught in traps). Insect species Diptera Heleomyzidae Suillia affinis (Meigen, 1830) Coleoptera Bolboceratidae Odonteus armiger (Scopoli, 1772) Leiodidae Anisotoma orbicularis (Herbst, 1792) Apocatops nigrita (Erichson, 1837) Colenis immunda (Sturm, 1807) Fissocatops westi (Krogerus, 1931) Leiodes cinnamomea (Panzer, 1793) Leiodes oblonga (Erichson, 1845)

Leiodes polita (Marsham, 1802) Nargus velox (Spence, 1815) Ptomaphagus sericatus (Chaudoir, 1845) Ptomaphagus varicornis (Rosenhauer, 1847) Sciodrepoides fumatus (Spence, 1815) Sciodrepoides watsoni (Spence, 1815) Phalacridae Stilbus testaceus (Panzer, 1797) Staphylinidae Atheta dilaticornis (Kraatz, 1856)

Tuber species

Nida Basin


w Upland Miecho


rz Upland Przedbo

T. aestivum

2635

1875

3*

T. aestivum T. T. T. T. T. T. T. T. T. T. T. T. T. T. T.

aestivum aestivum aestivum aestivum aestivum excavatum aestivum excavatum rufum aestivum aestivum aestivum aestivum aestivum aestivum

Chełm Hills

1 1 1 4* 6 5* 14 þ 1* 1*

1 5 33*

25*

10

22* 7*

2 1 141 1 2 5

3 1 4

T. aestivum

7*

T. aestivum

1*

Table 2 Number of Tuber spp. fruit bodies collected in Poland in 2012e2014. Tuber species

Year

Region

T. aestivum

2012 2013 2014 2012 2013 2014 2012 2013 2014

35 125 484 236 427 291 1

3 166 368

1599

537

Nida basin

T. excavatum

T. rufum

Total

possible based on the analysis of the male genitalia (Nunberg, 1987). Therefore, further studies on Leiodidae associated with truffles should take into consideration the problem of similarity of both species and molecular methods may be best employed to separate them. Other species of beetle in the same family as L. cinnamomea in the UK include Agaricophagus cephalotes and Colenis immunda (Horsfield, 2002). While these species are known to attack T. aestivum elsewhere (Bratek et al., 1993, 2010), there is no information available on their biology in the UK. A. cephalotes is very rare (just seven (10 km  10 km) records), while C. immunda is more common (57 (10 km  10 km) records, of which three coincide with T. aestivum). C. immunda was also reared from fruit bodies in the Polish field survey (Table 1). However, this beetle appears to have many above-ground fungal hosts (Schigel, 2011), and so is likely to pose little threat to truffle cultivation. The fruit bodies of truffles are also commonly inhabited by various species of Diptera, mainly of the genus Suillia (Ciampolini and Suss, 1982; Krivosheina, 2008). The adult females fly close to the soil surface and lay their eggs on the ground, above the truffle fruit bodies, so that the larvae can easily reach them on hatching (Talou et al., 1990). Larval feeding can cause extensive damage


w upland Miecho


rz upland Przedbo

Chełm hills

17

5 22

(Fig. S2). Martin-Santafe et al. (2014) and Duaso (2012) report infestation of fruit bodies of T. aestivum and T. melanosporum by another fly, Helomyza tuberivora but this species does not seem to occur in the UK. Instead, Suillia affinis (which appears to be a senior synonym of Helomyza affinis) and Suillia pallida do occur, S. affinis having been recorded from 99 of the 10 km  10 km squares and S. pallida from 58. Of the S. affinis squares, just four are coincidental with T. aestivum, suggesting that either the same biased recording problem exists or that the fly is not host specific (Baehrmann and Adaschkiewitz, 2003). Notwithstanding the recording problem, the four squares represent 5% of fly squares and 12.5% of truffle squares. A similar situation exists with S. pallida where the figures are 3.4% and 5%, respectively. Thus neither species may pose much of a threat to UK truffle species such as T. aestivum, even though they infest these elsewhere (Papp, 1994; Baehrmann and Adaschkiewitz, 2003). As in UK, the truffle fly Suillia gigantea has not been recorded in Poland, in contrast to S. affinis which was reported from the
w National Park) (Wo
southern part of the country (Ojco znica and Klasa, 2009). Soils in this part are calcareous and conducive to truffle development, so T. aestivum could be a potential host species. The fly was the most numerous of all trapped insects (Table 1).

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Based on research of Lo Giudice and Wo
znica (2013) we can also speculate that since localities of S. gigantea and S. affinis overlap in such regions as Tuscany, Lombardia and Umbria, S. affinis may be an indicator of truffle presence and a potential pest in Poland. Chandler (2010) reported the association with Tuber for Diptera of the genus Cheilosia (Syrphidae) and one species (Cheilosia soror) has been bred from truffles (Falk, 1991). This fly has been recorded from 173 of the 10 km  10 km squares in the UK. Perhaps of most interest is that this species shows a very different coincidence with T. aestivum compared with the previous insect species: 35% of the truffle squares also possess a record of this fly. While there is virtually no data available on the biology of this species, it would certainly merit investigation as a species of potential pest concern. 4. Truffle biochemistry The chemicals given off by truffles contribute to their characteristic aroma and give them their high monetary value (Costa et al., 2015). This suite of volatiles gives each a species-specific profile, which changes over time. This has been linked to maturation of the fruit body, as well as environmental factors and more recently to genetic variability (Splivallo et al., 2012). A single fruit body pro
et al., 2010; duces 20e50 volatile organic compounds (Cullere Splivallo et al., 2011), but those considered to be biologically important include dimethyl sulphide, which attracts dogs and pigs, and eight carbon-containing volatiles, which attract the two main insect species associated with truffles, L. cinnamomea and S. pallida (Talou et al., 1990; Splivallo et al., 2012). Demand for such a rare and expensive crop in the UK and Poland can only be realised if the truffles are both of good quality and pestfree. Recent chemoecological research has, therefore, centred around identification of the exact chemical profiles emitted by the fruit bodies and how they change over time. This information can then be used in the development of devices to monitor both the quality of the truffles i.e. electronic noses (Pennazza et al., 2013; Costa et al., 2015) and pest control in truffle harvests (Hochberg et al., 2003). The use of volatiles given off by the fruit bodies of truffles to reduce infestation by insect species has engendered remarkably little interest. To date, this work has focused on L. cinnamomea, and its attraction to both the volatiles produced by truffles and those produced by the beetle itself (Hochberg et al., 2003). These authors showed that neither sex is attracted by ripe truffle odours, but that females are attracted to immature truffles and males to pheromones produced by females. To date, these observations have never been examined experimentally. It could be critical in aiding the emergent industries to protect their harvests against pest insects through the development of attractant traps that would divert the beetles from the fruit bodies. However, this presents an intriguing philosophical dilemma; if the beetles are as rare as national records suggest, then one could question the ethics of developing traps that kill such rare, pest insects. There is a clear need to develop traps that catch live insects, in order to accurately determine population sizes and distributions and thereby address this dilemma. 5. Conclusions and future perspectives Truffles are highly prized and their economic value is dependent not only on the aromas they emit, but also on the fruit bodies being free of insect larvae. Truffle harvests have shown notable declines in parts of Europe, and this offers important economic opportunities in countries such as the UK and Poland to fill market gaps. It is important that these emergent industries do not fail due to poor material that is infested with insects. Here, we have tried to highlight those species of insect most likely to become pests in this

industry. Understanding their ecology will enable us to determine whether the disparity in national records of insects and their hosts is real or due to recorder bias. Prevention of attack by insects is important for a truffle producer, but presents an ethical dilemma of whether such rare insects should be trapped and killed. The development of electronic noses could aid truffle harvesters, since these will be consistent and immortal, unlike a pig or a dog. Acknowledgments We are deeply grateful to Dr Domizia Donnini (University of Perugia, Italy) for help with specimens of Leiodes cinnamomea, Dr Andrzej Wo
znica (Wrocław University of Environmental and Life Sciences, Poland), Dr Cezary Bystrowski (Forest Research Institute,
ski Poland) for identification of Suilla affinis and Karol Komosin (University of Warmia and Mazury, Poland) for identification of Atheta dilaticornis. We also thank Prof. Jerzy Borowski (Warsaw University of Life Sciences, Poland) for providing literature. This work was supported by State Forest Holding, project No. OR-2717/19/11, Forest Research Grant No. 260102 and by the Polish Ministry of Science and Higher Education, project No. 240309. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.funeco.2016.10.004. References Arzone, A., 1970. Reperti ecologici ed etologici di Leiodes cinnamomea Panzer vivente su Tuber melanosporum Vittadini (Coleoptera Staphylinoidea). Annali  Sci. Agrar. della Univ. Degli Studi Torino 5, 317e357 ([in Italian]). della Facolta Arzone, A., 1971. Nuovi reperti sulla biologia di Leiodes cinnamomea Panzer in Tuber magnatum Pico (Coleoptera, Staphylinoidea). Allionia Boll. dell'Instituto Orto  Torino 17, 121e129 ([in Italian]). Bot. dell'Universitaa Baehrmann, R., Adaschkiewitz, W., 2003. Beitrag zur Oekologie und Fauna der Heleomyzidae Mitteldeutschlands (Insecta: Diptera). Faun. Abh. Dresd. 24, 185e204 [in German]. Blackwell, T., 2005. Discovering discos and other ascomycetes. Field Mycol. 6, 15e21.
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