UNIVERSITÀ DEGLI STUDI DELLA TUSCIA DI VITERBO DIPARTIMENTO DAFNE Corso di Dottorato di Ricerca in Protezione delle Piante – XXIV Ciclo “Chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu): antennal morphology and natural control” AGR/11 Tesi di dottorato di: Tutore Dott.ssa Francesca Riga Prof. Bruno Paparatti Coordinatore del corso Co-tutore Prof. Leonardo Varvaro Prof. Nunzio Isidoro 26/06/2013 Contents 1 General introduction 1 1.1 The genus Castanea 1 1.2 Chestnut cultivation 2 1.2.1 The chestnut orchard in Europe and Italy 2 1.2.2 Chestnut orchards in Marche Region 4 1.3 Cinipid gall wasps 4 1.4 Galls 5 1.5 Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) 7 1.5.1 Origin and diffusion 7 1.5.2 Morphology 10 1.5.3 Life cycle 11 1.5.4 Host plants 11 1.5.5 Symptoms and damages 12 1.5.6 Control measures 12 1.5.6.1 Resistant varieties 12 1.5.6.2 Chemical control 13 1.5.6.3 Agronomical control 13 1.5.7 Preventive measures 13 1.5.8 Native parasitoids 14 1.5.9 Exotic Parasitoid – Torymus sinensis 14 1.5.9.1 - Technical notes on the release of Torymus sinensis in Italy 16 2 Aim 17 3 Antennal morphology 18 3.1 Introduction 18 3.1.1 Antenna 18 3.1.2 Definition of sensillum 18 3.1.3 Type of sensilla 19 3.1.3.1 Aporous sensilla 20 3.1.3.2 Uniporous sensilla 21 3.1.3.3 Multiporous sensilla 21 3.2 Aim 23 3.3 Materials and methods 24 3.3.1 Sample preparation 24 3.3.1.1 Scanning Electron Microscopy 24 3.3.1.2 Transmission electron microscopy 24 3.4 Result and discussion 25 3.4.1 Sensilla Trichoidea (ST) 26 3.4.2 Sensilla chaetica (SCH) 27 3.4.3 Multiporous Plate Sensilla (MPS) 29 4.4.4 Coeloconic Sensilla (CS) 30 4.4.5 Styloconic Sensilla (SS) 32 3.5 Conclusions 33 4 Natural control 34 4.1 Introduction 34 4.1.1 Natural enemies of cynipid gallwasps: chalcid parasitoid wasps 34 4.1.2 Natural control of D. kuriphilus in Asia 36 4.1.3 Natural control of D. kuriphilus in Italy 38 4.1.4 Notes on Calcidoidea families recruited in Italian chestnuts 40 4.2 Aim 42 4.3 Materials and methods 43 4.3.1 Description of sites 43 4.3.2 Collection of D. kuriphilus galls on plants of C. sativa - Year 2010 46 4.3.3 Collection of galls on wild vegetation – Year 2010 47 4.3.4 Collection galls of D. kuriphilus on plants of C. sativa - Year 2011 47 4.3.5 Collection of galls on wild vegetation- Year 2011 47 4.3.6 Vegetational surveys 48 4.3.7 Infestation rate – Year 2011 49 4.3.8 Statistical analysis 50 4.4 Results and discussion 51 4.4.1 Collection of chestnut galls - Year 2010 51 4.4.2 Collection of chestnut galls - Year 2011 52 4.4.3 Collection of galls from wild vegetation - Year 2010 60 4.4.4 Collection of galls from wild vegetation - Year 2011 60 4.4.5 Infestation rate – Year 2011 61 4.4.6 Vegetational surveys 64 4.5 Conclusions 67 5 Appendix 69 6 References 96 Acknowledgments 106 1 General introduction 1.1 The genus Castanea The chestnut tree Castanea sativa Miller is native of South-Eastern Europe and Asia Minor. During the Roman Empire, its cultivation was widespread both for the edible fruits, and for the excellent wood (AA. VV., 2001a). In the Middle Ages, chestnut cultivation has been associated with various monastic orders that have contributed to its spread and care and, even today, the memories of this type of association remain, like a symbiosis between the trees and ancient monasteries. Chestnut distribution affects temperate and warm-temperate mountain areas, below the beech altitudinal range. The altitudinal range of chestnut (Castanetum) is between 200-300 m and 800-1000 m. At lower altitudes the vegetation is characterized by oak woods, while the band above is mainly submontane beech (Fagetum, from 800-900 m, in the Apennines its relevance band goes up to 1000 m). Normally, due to its low tolerance to limestone (the limestone is sustained only with high seasonal rainfall), chestnut grows over arenaceous-pelitic or arenaceous substrates, where they find the conditions of neutrality or sub-acid environment required by the species. Sandy and mineralized soils are appreciated, ensuring ventilation to root system (AA.VV., 2010). The Genus Castanea belongs to the Fagaceae family, and is a monoecious plant with male and female flowers carried by the same plant. This genus includes a dozen species of shrubs and trees; the most important species belonging to the genus Castanea, are the following: • European species: sweet chestnut (C. sativa) (also called "Spanish chestnut" in the US) is the only European species of chestnut, though successfully introduced to the Himalayas and other temperate parts of Asia. • Asiatic species: Castanea crenata (Japanese chestnut), Castanea mollissima (Chinese chestnut), Castanea davidii (China), Castanea henryi (Chinese chinkapin, also called Henry's chestnut – China) and Castanea seguinii (also called Seguin's chestnut - China). • American species: Castanea dentata (American chestnut - Eastern states), Castanea pumila (American- or Allegheny chinkapin, also known as "dwarf chestnut" - Eastern states), Castanea alnifolia (Southern states), Castanea ashei (Southern states), Castanea floridana (Southern states) and Castanea paupispina (Southern states). 1 C. sativa is a medium-sized to large deciduous tree, that reaches a height of 20–35 m. The oblong-lanceolate, boldly toothed leaves are 16–28 cm (centimeters) long and 5–9 cm broad. The flowers of both sexes are borne in 10–20 cm long upright catkins, the male flowers in the upper part and female flowers in the lower part. In the Northern hemisphere, they appear in late June to July, and in autumn the female flowers develop into spiny cupules containing 3-7 brownish nuts that are shed during October. The female flowers eventually form a spiky sheath that deters predators from the seed. Some cultivars produce only one large nut per cupule, rather than the average two to four nuts. The bark often has a net-shaped (retiform) pattern with deep furrows or fissures running spirally in both directions up the trunk. 1.2 Chestnut cultivation 1.2.1 - The chestnut orchard in Europe and Italy Chestnut is economically and environmentally important in Italy. Chestnut orchards are part of traditional farming in many Italian regions and provide a non-negligible additional income to farmers. The widespread distribution of chestnuts, cultivated or naturalized, is crucial for the maintenance of a local biodiversity but also to maintain steep slopes in mountain areas. (Quacchia et al., 2008) Woodland management are mainly chestnut coppice, and meadow chestnut, a sort of high forest with a few scattered individuals (70-150/ha) large and constantly mowed grass undergrowth to make easy fruits collection. The meadow chestnut can be regarded as a kind of orchard, in which cultural practices are typical of this management: pruning, mowing grass, etc.. (AA.VV., 2001a). Chestnut wood represents an excellent building material, while meadow chestnut produces fruits that processed in different ways can provide a wide range of food products (fresh chestnuts, flour, cream, etc...) • Chestnut for fruit production: world production of chestnuts is concentrated in two large areas, Asia and Europe, representing respectively 80% and 16% of world production (FAO, 2008) . It should be stressed that the Asian production is obtained from chestnut species (C. crenata . - Japanese chestnut, C. mollissima - Chinese chestnut, and hybrids thereof) different from European one (C. sativa ) and with different and lower organoleptic characteristics. 2 In Europe (from Turkey to Portugal) production is largely based on European chestnut (Castanea sativa Miller) but in a few areas, such as the South-Western France, also hybrids are cultivated. Italian production is estimated between 50 000 and 70 000 tons. The part of world production has decreased from 11% to 4% due to China increased production. European production, after a drastic decline in the 1960s and 1970s, has reached around 170,000 tons. The main European producers are Italy, Turkey and Portugal, which account for 30%, 29% and 15% of European chestnut Italy is the largest exporter of chestnuts for the quality of trade, while is second for quantity traded; the export price of Italian chestnut is, in fact, higher than that of Asian chestnuts. The ISTAT data of 2007 show that chestnut production is mainly concentrated in the Central and Southern regions, especially in Campania (13.3 thousand ha), Calabria (10.7 thousand ha), Tuscany (7.8 thousand ha) and Lazio (5.2 thousand ha), while in the Northern regions Piedmont (5.4 thousand ha) and Emilia-Romagna (2.2 thousand ha) are the main producers. The Italian chestnut farms are small to medium size. In fact, on average, 80% of the farms and 40% of the area is included within SAU 0-5 ha, while the chestnut fruit average area is about 1 ha. (AA.VV., 2010) • Chestnut for wood production - The number of timber industry In the national scene, where 10.5 out of 30 total million ha are occupied by forests, the fraction invested in chestnut takes a very prominent role reaching 2.62% of the Italian land area and 7.53% of the forest. The extensions of Piedmont, Tuscany and Liguria are no less than 50% of the national heritage. Including the regions that have assets of more than 30,000 ha (Lombardy, Calabria, Campania, Emilia Romagna and Lazio), we get to 90% of the entire national surface, it follows that over 50% of the regions have very low chestnut surface. Overall, over 75% of Italian chestnut is intended for timber production. (AA.VV, 2010) 3 1.2.2 - Chestnut orchards in Marche Region The presence of chestnut in Marche Region is limited by soil characteristics, infact C. sativa is a calcifuge plant. Except of a chestnut area of about 100 hectares is in Pesaro-Urbino province, in particular in high Montefeltro district, we can say that chestnut areas are concentrated in the southernmost provinces of the Region. According to data shown by IPLA (AA.VV.,2001b), more than half of the chestnut areas of Marche are located on the “Monti della Laga”, in high Tronto Valley. In the Piceno district chestnut distribution is further concentrated in the municipalities of Acquasanta Terme, Arquata del Tronto, Montegallo, Montemonaco and Roccafluvione, for about 2100 ha (about 90% ) (AA.VV., 2010). The organoleptic characteristics of the cultivars widespread on the territory make regional chestnuts particularly precious. Chestnut areas in the Piceno district are partly subject to recurrent cultural practices. According to data of "Regional Inventory”, chestnut in Marche Region involves 4.6 ha, 1.8% of the total regional surface. 1.3 – Cinipid gall wasps Cynipid gall wasps consist of six tribes (Tab. 1.1): Aylacini, Diplolepidini, Eschatocerini, Pediaspidini and Cynipini are gall inducers, and Synergini are gall-associated inquilines. They comprise around 1,300 species worldwide, predominantly in temperate regions of the Holarctic.The Eschatocerini are restricted to the Nearctic, but the other five tribes are found in Asia (Abe et al., 2007). The Cynipini includes around 1,000 species, and most of them induce galls on oak Quercus trees. Heterogony, cyclical parthenogenesis occurs in many species of this tribe. Female adults of the bisexual and unisexual generations of the same species differ considerably, not only in their morphology but also in the shape of galls that their offspring induce. Therefore, the two generations of the same species have often been described as different species, which confuses the taxonomy of oak gall wasps (Abe, 2010) 4 Tribes No. of genera No. of Hosts species Aylacini 18 122 Asteraceae, Rosaceae, Lamiaceae Papaveraceae, Apiaceae, Valerianaceae, Brassicaceae, Smilax (Smilaceae) Diplolepidini 2 50 Rosa (Rosaceae) Eschatocerini 1 3 Acacia, Prosopis (Fabaceae) Pediaspidini 3 3 Acer (Aceraceae) Cynipini 27 c. 1,000 Fagaceae (mostly Quercus, also Castanea, Chrysolepis and Lithocarpus) Synergini 8 159 Inquilines in galls induced by other insects Tab. 1.1 - Classification, diversity and host associations of Cynipinae (after Abe et al., 2007) 1.4 - Galls Galls are structures composed of plant tissue within which the insect feeds; they can form on roots, fruits, buds, leaves and flowers (Fig. 1.1). Many groups of insects induce the production of galls in plants. Associations of galling insect an host are often species-specific, with the insect inducing a highly characteristic type of host differentiation. Some such associations are so specific that they have been used as taxonomic criteria for some insects (Hewett, 1977). Insect galls are dramatic examples of extended phenotypes: although composed of host plant tissues, their development is largely controlled by insect genes (Stone and Schönrogge, 2003). It is likely that the altered host differentiation results from a changed metabolism in response to a chemical stimulus from the insect (Hewett, 1977). 5 Fig. 1.1 - Distribution of tissues within galls Diplolepsis rosaefolii (Hymenoptera: Cynipidae) (Stone and Schönrogge, 2003). Three general mutually compatible hypotheses for the adaptive significance of insect galls have been proposed: (a) The nutrition hypothesis, which states that gall inducers regulate the nutritive value of the plant tissues on which they feed to their own benefit; (b) the microenvironment hypothesis, which states that gall occupation protects the gall inducer from external fluctuations in microclimate; and (c) the enemy hypothesis, which states that gall structures have been selected to reduce mortality imposed on the gall inducer by natural enemies (Stone et al., 2002). It’s well known, for example, that the gall can act as a metabolic sink for photosynthate, attracting food for the sedentary parasite. One of the physiological mechanisms is mediated by cytokinin, that have also been extracted from the larvae of D. kuriphilus. This evidence indicates that, although the hormone may arise from the host, it is being concentrated in the body of the insect, then being excreted and having its effect (Hewett, 1977). Another physiological process that occours in galling cynipids is manipulation of tannins, both in the inner nutritive tissue and in the external protective one (Ikai and Hijii, 2007). 6