Forster, B.; M.; Grodzki, W. (eds.) 1999: Methodology of Forest Insect and Disease Survey in Central Europe. Proceedings of the Second Workshop ofthe IUFRO WP 7.03.10, April20-23, 1999, Sion-Chateauneuf, Switzerland. Birmensdorf, Swiss Federal Institute for Forest, Snow and Landscape Research (WSL) 98-101. XYLOSANDRUS GERMANUS AND WALNUT DISEASE: AN ASSOCIATION NEW TO EUROPE Frigimelica G.1, Stergulc, F.\ Zandigiacomo P.2, Faccoli M.1 & Battisti A.1 1) Institute of Agricultural Entomology, University ofPadua 16/a via Rornea, 35020 -Legnaro (PD)-Italy 2) Department of Plant Protection, University ofUdine, Italy 208, Via delle Scienze, 33100 Udine, Italy Introduction The forest service of the Friuli Venezia Giulia district (north-eastern Italy) pays a great attention to the sanitary conditions of both forests and plantations (Stergulc et al., 1999). The damage caused by pests and diseases are the object of a permanent monitoring net, called "BAUSINVE-Forest Pest and Disease Inventory", in which any kind of biotic damage occurring in any part of the district, is registered. Foresters were provided with an identification handbook of both pests and diseases (Stergulc & Frigirnelica, 1996). At the beginning of the summer of 1998, several trees in young plantations of European walnut (Juglans regia L.), situated in this district, showed a series of disease symptoms, such as wilting, dieback, stern cankers and production of sprouts near the ground. On the bark of diseased trees either pink or whitish sporodochia were visible. A large part of diseased trees were also colonised by the ambrosia beetle Xylosandrus germanus (Blandford) (=Xyleborus germanus, Coleoptera: Scolytidae), a species new to Italy (Stergulc et al., 1999). A similar situation was observed in Northern America by several authors who reported the association between X germanus and fungi of the genus Fusarium, as involved in the American black walnut (Juglans nigra L.) disease (Hoffrnan, 1941; Kessler, 1974; Anderson & Hoffard, 1978; Norris, 1979; Weber & McPherson, 1983; Weber & McPherson, 1984). The female of X germanus introduces pathogenic fungi into the tree as she excavates her tunnel into wood. This fungi may cause a cankered area in the stern, usually causing top or branch dieback and resprouting from the base of the tree (Weber, 1982). Xylosandrus germanus is an ambrosia beetle, around 2.5 mm long, with world-wide distribution (Nobuchi, 1981 ). It attacks a wide range of both living plants and timber of deciduous and coniferous species (Weber & McPherson, 1984). As with all arnbrosia beetles, X germanus derives its nourishment during the larval and adult stage from an "arnbrosia" fungus that the female adults carry in rnycangia and introduce into host plants during gallery formation (Weber & McPherson, 1984). The arnbrosia fungus was identified as Ambrosiella hartigii (Batra) (=Monilia candida Hartig (Batra, 1967)) (Francke-Grosrnann, 1967; Weber & McPherson, 1984). During the gallery formation the frass pushed out by the female often extends from the entrance hole forming a long, easily visible clear cylinder. Adults of X germanus usually overwinter in galleries at the base of the attacked trees (Gauss, 1960; Weber & McPherson, 1983). In spring the adult flight begins during early to mid May. Usually, there are two generations per year. The aim of this paper is to contribute to the knowledge of this association in Europe. As a first contribution, attention was paid mostly to the identification of the organisms involved and to the estimation of the degree of attack in plantations for wood production. 98 Materials and methods Two plantations that were severely affected by both beetle and fungi were studied. The first plantation (Treppo Grande, Udine) was characterised by 4 to 7 year-old trees, with a density of about 500 trees/ha and a total extension of 0.8 ha (409 trees). The second plantation (Tavagnacco, Udine) was constituted by 200 trees 8-year-old, growing at a density of 280 trees/ha on a total area of 0.7 ha. The degree of attack to the trees was estimated as the percentage of trees infected by fungi and/or colonised by X germanus. The symptoms associated with the infections of fungi and/or with the attack of ambrosia beetles (holes ofX germanus, sprouts at tree base, crown dieback, stem cankers, sporodochia on the bark) were also recorded. Other plantations were surveyed to detect the disease, but they were not considered for the estimation of the damage. Several samples of bark were collected from the margins of the cankers on diseased walnut trees. Samples of dying trunks, branches and twigs were also collected. Fungi and bacteria were isolated from small pieces of inner bark tissue, excised from the margin of the necrotic area after removing the upper layers of bark with a sterile lancet. Excised samples were then plated on Difco PDA (Potato Dextrose Agar). The emerging colonies of both fungi and bacteria were subcultured on the same substrate. Isolates of fungi were also obtained from conidia drawn from sporodochia. Isolates were identified by the Centraalbureau voor Schimmelcultures (Baarn, The Netherlands). Branches showing holes of X germanus were put in rearing chambers at room temperature to obtain adult emergence. Beetles were collected daily until the end of emergence. Results The most frequently isolated fungi from bark samples were Fusarium spp. Conidial isolates originated from sporodochia also provided Fusarium spp. colonies. The most abundant Fusarium species was F. merismoides Corda; other less frequent species were F. lateritium var. majus Wollenweber and F. oxysporum Schlechtendahl: Fries. Isolations from inner bark tissues yielded also a few saprophytic fungi, as Trichoderma sp., Penicillum sp. and Gliocladium sp. Bacterial colonies were obtained in a considerable number from dead bark and wood tissues. They were characterised as fast growing on PDA, Gram negative, oxidase negative, weakly fluorescent and rod shaped. Insects emerging from infested branches were almost exclusively females of X germanus, a species unknown to Italy. A few females of Xyleborus dispar (Fabricius) and Xyleborus saxesenii (Ratzeburg) were also collected from the infested wood both in the laboratory and in the field. The nitidulid beetle Gliscrochilus quadrisignatus (Say) was also found in the field associated with holes and wounds emitting a blackish fluid. The cerambycid beetles Neoclytus acuminatus (Fabricius) and Xylotrechus stebbingi Gahan were observed to lay eggs into dying trees showing evident symptoms of the disease. Trees free from both insect holes and fungal fruiting bodies represented the 35.2% at the first plantation and the 65% at the second. Trees showing both insect and fungus were the 25.9% and 6.5%, respectively, a similar rate to that observed for trees being attacked only by insects (25.9% and 9.0%, respectively). The fungus alone was detected with higher frequency in the second plantation (13.0% and 19.5%). The application of they} test to the above data showed that the number of cases of association between insect and fungus is significantly higher than that expected if the two types of symptoms were independent at both plantations (first plant. x2 = 22.9, p<O.OOI, second plant. x2 = 4.8, p<0.05). 99 In summer 1998 other eleven young plantations growing in the same district showed signs of disease and/or pest attack. Two plantations showed the association of insect and fungus, in seven plantations only X germanus was detected while dieback caused by Fusarium alone occurred in two plantations. Discussion The association between X germanus and Fusarium spp. seems to be present also in Italy on Juglans regia, with about the same appearance shown in North America on Juglans nigra. Even if we did not demonstrate that X germanus may carry the propagules of the fungus on its body, it is a likely potential vector of the fungi. In fact, besides at A. hartigii, also pathogenic fungi including Fusarium spp. (Kessler, 1974; Anderson & Hoffard, 1978), F. lateritium Nees, F. oxysporum Schlechter and Ceratocystis ulmi (Buisman) (Norris, 1979), F. moniliforme var. subglutinans Wollenweber & Reinking, F. sambucinum Fuckel and F. so/ani (Martius) (Weber & McPherson, 1984), were isolated from both adults and galleries in North America. Cankers caused by Fusarium spp. on Juglans nigra were often reported for American plantations. Multiple cankers both on stems and branches caused by Fusarium solani were noticed in a 7-year-old plantation in Kansas (Tisserat, 1987); F. sporotrichioides caused stem cankers in Wisconsin and in Minnesota (Cummings & Kuntz, 1985); Carlson et al. (1993) found Fusarium solani as the most frequent Fusarium species isolated from cankers in black walnut plantations in five central states (Illinois, Iowa, Minnesota, Missouri and Wisconsin). About the 30% of these cankers were associated with non-specific wounds while only 1.6% with attacks of an unidentified ambrosia beetle. Trials carried out with isolates from cankers occurred on J nigra stems in a young plantation in southern Illinois, demonstrated that F. lateritium and F. oxisporum were capable to induce canker development when introduced into healthy walnut stems (Kessler, 1974). Fusarium merismoides is reported to be an ubiquitous fungus, occasionally causing disease in plants. The only report of Fusarium merismoides as an agent of disease on trees concerns mimosa trees, as reported by Hepting (1936) in the Carolinas islands. This fungus was associated with diseases of agricultural crops (Fletcher & Lord, 1985). The effective role of F. merismoides on walnut disease requires further research. A strong correlation among Fusarium solani cankers, X germanus and Xyleborus sayi was also found in a 6-year-old tulip poplar (Liriodendron tulipifera L.) plantation in Ohio (Anderson & Hoffard, 1978). The disease course observed in tulip poplar trees was very similar to that described for the black walnut. This fact suggests that the association Fusarium - ambrosia beetle is likely not to be confined to Juglans spp., but it could also extend to other trees, as recently observed on young dying chestnut trees in north-eastern Italy.