Combined effects of thinning and decline on fine root dynamics in a Quercus robur L. forest adjoining the Italian Pre-Alps.

Aims: Oak decline is a complex phenomenon, characterized by symptoms of canopy transparency, bark cracks and root biomass reduction. Root health status is one of the first stress indicators, and root turnover is a key process in plant adaptation to unfavourable conditions. In this study, the combined effects of decline and thinning were evaluated on fine root dynamics in an oak forest adjoining the Italian Pre-Alps by comparison of acute declining trees with non-declining trees, both with and without thinning treatment of surrounding trees. Methods: Dynamics of volumetric root length density (RLD V ) and tip density (RTD V ), root tip density per unit length of root (RTD L ), diameter, branching index (BI) and mycorrhizal colonization were monitored by soil coring over 2 years as possible descriptors of decline. Key Results: At the beginning of the experiment, the relationship between canopy transparency and root status was weak, declining trees having slightly lower RLD V (-20 %) and RTD V (-11 %). After a 1 year lag, during which the parameters were almost unaffected, BI and RLD V , together with tip density, tip vitality and mycorrhizal colonization, became the descriptors most representative of both decline class and thinning. Thinning of declining trees increased RLD V (+12 %) and RTD V (+32 %), but reduced tip mycorrhizal colonization and vitality over time compared with non-thinned trees, whereas the opposite occurred in healthy trees, together with a marked decrease in branching. After thinning, there was an initial reduction in the structure of the ectomycorrhizal community, although recovery occurred about 10 months later, regardless of decline severity. Conclusions: Decline causes losses of fine root length, and a moderate recovery can be achieved by thinning, allowing better soil exploration by oak roots. The close correlation between root vitality and mycorrhizal colonization and their deterioration after thinning indicates that decline does not benefit from reduced root competition, excluding the hypothesis of limited water and nutrient availability as a possible cause of the syndrome in this forest.

First Record of Thousand Cankers Disease Fungal Pathogen Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans regia in Europe.

Thousand cankers disease (TCD) is a disease complex caused by the fungus Geosmithia morbida Kolarik (Ascomycota, Hypocreales) and its vector Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae; walnut twig beetle, WTB). Since the mid-1990s, the disease was responsible for widespread mortality of many walnut species in the United States (4). After the first detection of TCD on black walnut (Juglans nigra L.) in Italy (3), an extensive survey was activated in cooperation with the Regional Phytosanitary Service. In May 2014, early TCD symptoms (4) were observed on English walnuts (J. regia L.). Canopies showed yellowing, wilting, and dieback of the youngest twigs, and a number of small brown cankers. Longitudinal and radial sections sampled through the cankers revealed gray to brown discoloration of both phloem and bark, and the presence of bark beetle galleries. Xylem discoloration was never observed. From one ~20-year-old European walnut growing in a garden neighboring an infected black walnut plantation (Santorso, Vicenza, 45°72' N, 11°40' E), a number of 1- to 2.5-cm-diameter twigs showing cankers up to 2 cm long surrounding bark beetle holes were collected. Whitish mycelium producing verticillate conidiophores was detected inside the insect galleries. From the necrotic margin of eight cankers previously surface-sterilized with 3% sodium hypochlorite, two 4-mm-wide chips per canker were placed on potato dextrose agar and incubated at 28 ± 1°C in the dark. Slow growing lobate, plane, yellowish-ocher colonies with hyaline mycelium appeared in 5 days. After subculturing to the same medium, growth features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolarik's description of G. morbida (2) were observed. The ITS region of rDNA from the fungus strain LM14GM001-JR was amplified by using ITS1F and ITS4 primers and sequenced obtaining a 387-bp gene fragment. BLAST analysis showed 99% identity to the G. morbida strain U19 (GenBank Accession No. KF808301.1) for 384 bp, and 99% identity to the G. morbida strain LM13GM001-JN previously isolated from J. nigra in Italy (3). From the same samples, two emerging beetles were collected and identified as P. juglandis both morphologically (5) and genetically by DNA extraction following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxidase I was then amplified by using universal primers (1) and sequenced to obtain a 614-bp fragment of the gene. BLAST analysis showed 100% identity to P. juglandis based on comparison with KJ451422. A few other English walnuts with both the fungus and WTB were also found close to other infected black walnut plantations. To our knowledge, this is the first record of G. morbida and P. juglandis on J. regia in Europe, where the tree is cultivated for both fruit and timber production, as well as a traditional landscape tree. Voucher specimens are stored in the TeSAF herbarium and in the DAFNAE insect collection. References: (1) O. Folmer et al. Mol. Marine Biol. Biotechnol. 3:294, 1994. (2) M. Kolarik et al. Mycologia 103:325, 2011. (3) L. Montecchio and M. Faccoli. Plant Dis. 98:696, 2014. (4) S. J. Seybold et al. USDA Forest Service, NA-PR-02-10, 2013. (5) S. L. Wood. Great Basin Naturalist Memoirs 6:1123, 1982.

First Record of Thousand Cankers Disease Geosmithia morbida and Walnut Twig Beetle Pityophthorus juglandis on Juglans nigra in Europe.

Thousand cankers disease (TCD) of walnut is responsible for widespread mortality of black walnut (Juglans nigra L.) in the United States since the mid-1990s (2). The disease is caused by the fungus Geosmithia morbida Kolarik (Ascomycota, Hypocreales), vectored by the walnut twig beetle Pityophthorus juglandis Blackman 1928 (Coleoptera, Scolytinae). In September 2013, TDC was observed in northeastern Italy (Bressanvido, Vicenza, 45°39' N, 11°38' E) in black walnuts of different ages: ~80-year-old plants growing in a garden and 17-year-old trees belonging to a nearby walnut plantation for timber production. Main symptoms were yellowing, wilting, twig and branch dieback, and a high number of small bark cankers (3). Longitudinal and radial sections collected through the cankers revealed gray to brown discoloration of both phloem and outer bark, and the presence of bark beetle galleries radiating from the mating chamber and developing horizontally (across the wood grain), and vertical (along the grain) larval galleries. Occasionally, discoloration involved the outward xylematic tissues. Fungal fruiting bodies were not found on or near the cankers. Whitish mycelium, sometimes producing verticillate conidiophores, was frequently detected inside galleries. A number of 1- to 3-cm diameter twigs showing cankers up to 2 cm long surrounding bark beetle penetration holes were randomly collected. From samples, emerging beetles were identified as P. juglandis both morphologically (4) and genetically. DNA extraction was carried following a standard salting out protocol. The barcode region of the mitochondrial gene cytochrome oxydase I was then amplified using universal primers (1) and sequenced, obtaining 627 bp. BLAST analysis showed 100% identity to P. juglandis. Sequences were finally deposited in the BoldSystem database (GenBank Accession No. KF725084). From the necrotic margin of six cankers previously surface-sterilized with 3% sodium hypochlorite, two 3-mm-wide chips per canker were placed on potato dextrose agar and incubated at 23 ± 1°C in the dark. Among a variety of microorganisms, slow growing lobate, plane, yellowish-ochre colonies with hyaline mycelium appeared in 6 days. After subculturing to the same medium, growing features, mycelium, conidiophores, and conidia with morphological characteristics matching Kolarik's description of G. morbida (2) were observed. Same result was obtained culturing the mycelium growing inside the galleries. The ITS region of rDNA was amplified using ITS1F and ITS4 primers and sequenced, obtaining 597 bp. BLAST analysis showed 100% identity to G. morbida strain U173 (HF546283.1) for 558 bp. To our knowledge, this is the first record of TCD and P. juglandis to Europe, where walnut species (mainly J. regia, J. nigra, and their hybrids) are intensively cultivated for timber production. This finding is therefore of particular importance. An intensive survey of the disease is suggested, both to assess fungus/beetle presence and to verify possible pathways of introduction, likely associated to importation of infested/infected timber from native Nearctic regions. Voucher specimens are stored in the TeSAF herbarium (fungus) and in the DAFNAE insect collection. References: (1) O. Folmer et al. Mol. Marine Biol. Biotechnol. 3:294, 1994. (2) M. Kolarik et al. Mycologia 103:325, 2011. (3) C. Nischwitz and M. Murray, Utah Pests Fact Sheet, PRP-015pr, 2011. (4) S. L. Wood. Great Basin Naturalist Memoirs 6:1123, 1982.

First Report of Chalara fraxinea on Common Ash in Italy.

In many European countries, the anamorphic Chalara fraxinea Kowalski (teleomorph Hymenoscyphus albidus [Roberge ex Desm.] Phillips; 1-3) is responsible for a severe and rapidly spreading dieback of common ash (Fraxinus excelsior L.) since it was first reported in Poland. Recently, this disease was added to the EPPO Alert List and the NAPPO Phytosanitary Alert System. Symptomatic trees were observed in a 1.8-ha ash-maple forest in northeastern Italy (Fusine, UD; 46°30'N, 13°37'E; 782 m above sea level) along the Italo-Slovenian border in July 2009. Symptoms were found on approximately 10% of mature common ash and 70% of seedlings. Main symptoms were shoot, twig, and branch dieback, wilting, and bark cankers (1). Fungal fruiting bodies were not found on or near the canker surface. Furthermore, longitudinal and radial sections through the cankers revealed gray-to-brown xylem discoloration. One symptomatic 3-year-old plant was randomly selected and from the necrotic margin of one canker previously surface-sterilized with 3% sodium hypochlorite and rinsed, four 2-mm-wide chips were placed on malt extract agar (MEA) and incubated at 21 ± 1°C in the dark. Among a variety of microorganisms, after 19 days, slow-growing colonies (mean radius of 12 mm) appeared that were effuse, cottony, and often fulvous brown but sometimes dull white with occasional gray-to-dark gray patches. The purified isolate was then transferred to the same medium at 4 ± 1°C in the dark, and after 11 days, hyaline-to-dark gray phialides were observed producing numerous conidia in slimy droplets and sometimes in chains. Phialophores measured 8.6 to 21.0 (15.1) µm long (n = 20), 4.2 to 13.4 (8.8) × 3.6 to 5.5 (4.7) µm at the base, and 5.2 to 8.7 (6.5) × 2.5 to 3.1 (2.8) µm at the collarette; conidia measured 2.8 to 4.2 (3.4) × 1.9 to 2.5 (2.2) µm (n = 40); and first formed conidia measured 5.5 to 6.5 (5.9) × 1.8 to 2.5 (2.1) µm (n = 20). These morphological characteristics matched Kowalski's (1) description of C. fraxinea. In August of 2009, the fungal isolate was used to test pathogenicity with current year shoots of 25 6-year-old (150 to 210 cm high) asymptomatic common ash trees under quarantine conditions (Slovenian Forestry Institute's experimental plots). For every plant, the bark of the main shoot (10 to 13 mm in diameter) was wounded with a 6-mm-diameter cork borer. Twenty saplings were inoculated with one 6-mm-diameter mycelial plug obtained from the margin of a 26-day-old culture (MEA), while five saplings were inoculated with sterile MEA plugs. All wounds were sealed with Parafilm and aluminum foil. After 28 days, all plants inoculated with the C. fraxinea showed bark lesions (2 to 39 mm long, mean 7 mm) and wood discoloration (6 to 85 mm long, mean 22 mm) from which the pathogen was reisolated. These symptoms were absent from controls and the pathogen was never reisolated. To our knowledge, this is the first report of C. fraxinea in Italy. Investigations on its presence in all Fraxinus species naturally growing in the investigated area and in the nearest regions are in progress. The obtained isolate is preserved in both Padova and Ljubljana herbaria as CFIT01. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:304, 2009.

First Report of Damping-Off of Common Oak Plantlets Caused by Cylindrocladiella parva in Italy.

In April 2006, damping-off of common oak (Quercus robur L.) plantlets as much as 3-years-old was observed in a typically declining Q. robur L. forest located in northeastern Italy (Cessalto, VE). Cotyledons on seedlings were chlorotic and wilted and occasionally desiccated. Stems were stunted and discolored with yellowish brown longitudinal streaks starting from the collar. Rootlets were partly rotten and their apices frequently dead (approximately 65%). There were no fruiting bodies on or near the damaged areas. Longitudinal stem sections showed dark brown streaks in the vascular tissue, and microscopic examination revealed that vessels frequently contained mycelium. Twenty plants with these symptoms were selected, and from each of them, isolations were made from surface-sterilized (1% sodium hypochlorite) necrotic margins of stems. Lesions were cut lengthwise into two parts approximately 5 mm long, placed on potato dextrose agar, and incubated at 22 ± 1°C for 5 days in the dark. Among a variety of microorganisms, Cylindrocladiella parva (P.J. Anderson) Boesewinkel was isolated from 12 plants. The epidermis on the collars of 21-day-old, container-grown Q. robur asymptomatic seedlings was surface sterilized with 1% sodium hypochlorite, rinsed in water, and gently scraped (wounded) with a sterile scalpel. After masking the remainder of the plant with a plastic sheet, the wounds were sprayed with a conidial suspension in water (103 conidia/ml) and sealed with Parafilm. Controls were treated the same way but sprayed only with sterile water. Each treatment was replicated on 10 seedlings and incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12 h of natural light per day). After 21 days, wounds treated with C. parva developed necrotic lesions that turned into small patches of dead epidermis forming discolored streaks. Radial sections through the stem 15 mm above the inoculation site from five plants showed the presence of mycelium in the vessels from which the fungus was reisolated. Thirty-five days after inoculation, the remaining five plants showed the same symptoms as those observed in the forest and microscopic observations confirmed the presence of the fungus. No disease symptoms or mycelium in the xylem tissue were observed in the control plants. The pathogenicity test was repeated twice with the same results. C. parva is the known causal agent of seedling blight, damping-off, and root rots on a broad host range of monocots and dicots. Detailed information on taxonomic and epidemiological features is available (1). To our knowledge, this is the first report of the disease in Italy where common oak decline is widespread (2). The isolate is preserved in the Centraalbureau voor Schimmelcultures (Baarn, the Netherlands) collection (No. 114857). References: (1) P. W. Crous and M. J. Wingfield. IMI Descriptions of Fungi and Bacteria. 116:1160, 1993. (2) A. Ragazzi and I. Dellavalle, eds. Decline of Oak Species in Italy. Problems and Perspectives. Accademia Scienze Forestali, Firenze, Italy, 2000.

Damping-Off of Beech Seedlings Caused by Fusarium avenaceum in Italy.

During May of 2004, damping-off of an estimated 70% of beech (Fagus sylvatica L.) seedlings was observed in a bare-root forest nursery located in northern Italy. Twenty-eight days after sowing, cotyledonary leaves were chlorotic, wilted, and occasionally desiccated; stem and collars appeared stunted and discolored with yellowish gray-to-brownish longitudinal streaks arising from the foot region, and rootlets were partially to completely rotten. No fruiting bodies were present on or near the damaged regions. Seedling establishment was poor within the disease foci that gradually increased in size. Longitudinal sections through the damaged stems showed dark brown streaks in the vascular tissue, and microscopic examination revealed that vessels frequently contained mycelium. Ten symptomatic plants were selected, and isolations were made from the necrotic margins of stems previously surface sterilized with 1% sodium hypochlorite and thoroughly rinsed, longitudinally cut into two parts 5-mm long, placed on potato dextrose agar (PDA), and incubated at 22 ± 1°C for 5 days in the dark. Although a variety of microorganisms were isolated, Fusarium avenaceum (Fr.:Fr.) Sacc. (1) was always recovered. Artificial inoculations with the fungus were made on 20-day-old, container-grown beech seedlings. The epidermis surrounding the collar was surface sterilized with 1% sodium hypochlorite, rinsed, and gently scraped with a sterile scalpel. After masking the rest of the plant with a plastic sheet, the wound was sprayed with a conidial suspension containing 103 macroconidia per cm3 in water and sealed with Parafilm. Controls were treated the same way but with sterile water. Each treatment was applied to 10 seedlings and incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12 h of natural light per day). After 20 days, wounds treated with F. avenaceum showed necrotic lesions that developed into small patches of dead epidermis. Radial sections through the stem 2 cm above the inoculation site from five plants showed the presence of mycelium in the vessels, from which the fungus was reisolated. Thirty days postinoculation, the remaining five plants showed the same symptoms observed in the nursery, and microscopic observations confirmed the presence of the fungus. No disease symptoms or mycelium were observed in the inner tissues of control plants. The pathogenicity test was repeated twice with the same results. The fungus was not detected by culturing 100 surface-sterilized seeds from the same stock that had been sown in the nursery. F. avenaceum has a broad host range including angiosperms and gymnosperms, and the described symptoms are fairly typical (2). To our knowledge, this is the first report of this disease in Italy. Further research on fungal survivability in nursery soil and plant debris is in progress. The isolate is preserved in the Centraalbureau voor Schimmelcultures (NL) collection as no. 115957. References: (1) C. Booth. Fusarium. Commonwealth Mycological Institute, Kew, UK, 1977. (2) H. Butin. Tree Diseases and Disorders. Oxford University Press, New York, 1995.

A Twig Canker on Russian Olive Caused by Phomopsis arnoldiae in Italy.

In June 2002, open, irregularly shaped cankers on stems and twigs of Russian olive (Eleagnus angustifolia L.) were observed in central Italy in two neighboring experimental walnut timber plantations (i.e., Juglans regia L. or J. nigra L. grown with Alnus glutinosa L. and E. angustifolia as nitrogen-fixing plants). Foliage distal to the cankers appeared chlorotic and wilted and occasionally desiccated. No fungal, fruiting bodies were present on or near the canker surface, nor were symptoms were observed on root collars or roots. Radial sections through the cankers revealed dark brown discoloration of xylem, and microscopic examination showed that vessels frequently contained tyloses and mycelium. Four symptomatic plants were selected, and from each of these plants, isolations were made from one canker. From the necrotic margin of each canker previously surface-sterilized with 2% sodium hypochlorite and rinsed, two chips, 3-mm-wide, were placed on potato dextrose agar (PDA) and incubated at 20 ± 1°C for 8 days in the dark. Among a variety of microorganisms isolated were Coniothyrium fuckelii Sacc., Penicillium spp., and Phomopsis arnoldiae B. Sutton (3). Artificial inoculations were made on 3-year-old, container-grown E. angustifolia seedlings using two isolates each of the three fungi. Where the stems measured 5 mm in diameter, the bark was surface sterilized with 2% sodium hypochlorite, rinsed, wounded with a 3-mm-diameter cork borer, inoculated with a PDA disk containing mycelium and spores, and the wound sealed with Parafilm. Controls were treated the same way but with sterile disks of PDA. Each treatment was replicated with 10 seedlings and incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12 h of natural light per day) for 60 days. After 30 days, wounds treated with P. arnoldiae showed necrotic lesions that developed into small patches of dead bark that cracked forming cankers. Radial sections through the stem at the canker site from 10 plants (five per isolate) showed the presence of mycelium in the vessels, from which P. arnoldiae was reisolated. After 60 days, the cankers on the remaining 10 plants measured 8 to 14 mm long, and microscopic observations confirmed the presence of the fungus. No disease symptoms or mycelium in the inner tissues were observed in the control plants or in the plants inoculated with the other fungi. The pathogenicity test was repeated twice with the same results. Detailed descriptions of both fungal features in vitro (1,3) and symptoms on larger plants are available (2). To our knowledge, this is the first report of this disease in Italy. Further research is in progress since Russian olive in Italy is frequently grown in the nursery for agronomic purposes because of its nitrogen-fixing ability. Cultures of our isolates of P. arnoldiae, the identification of which was confirmed by the Centraal Bureau voor Schimmelcultures (Utrecht, the Netherlands), are preserved in the herbarium L. Montecchio (LMPa1 and LMPa2) in Padova. References: (1) R. H. Arnold and J. C. Carter. Mycologia 66:191, 1974. (2) W. A. Sinclair et al. Diseases of Trees and Shrubs. Cornell University Press, Ithaca, NY, 1987. (3) B. C. Sutton. The Coelomycetes. Commonwealth Mycological Institute, Kew, UK, 1980.

A Twig Canker on English Hawthorn Caused by Coniothyrium sporulosum in Italy.

Large, open, irregularly shaped cankers on trunks and twigs of 4- to 12-year-old Crataegus monogyna Jacq. in a mixed oak forest in northern Italy were observed in 2001. Radial sections through the cankers, which often occurred at sites of hail damage from the previous year, showed a brown discoloration in the xylem. Foliage distal to the cankers became chlorotic, wilted, and finally died. When the disease was severe, the entire top of the plant died, and epicormic shoots developed below the cankers. Fruiting bodies were not present on the canker surface, and symptoms were not observed on root collars or roots. Microscopic examination of cankers showed that vessels frequently contained mycelium. Five symptomatic plants were selected, and from each of these plants isolations were made from one canker. Two chips, 3 mm wide, were cut from the necrotic margin of each canker, superficially sterilized with sodium hypochlorite, rinsed, plated on potato dextrose agar (PDA), and incubated at 20 ± 1°C for 8 days in the dark. Among a variety of microorganisms isolated from the necrotic tissues, Fusarium solani (Mart) Sacc. and Coniothyrium sporulosum (Gams & Domsch) van der Aa (1,2) were isolated from 70 and 100% of the chips, respectively. Artificial inoculations were made on 3-year-old, container-grown Crataegus monogyna seedlings obtained from the same seed stock using two isolates each of the two fungi. Where the stems measured 5 mm in diameter, the bark was surface sterilized with sodium hypochlorite, rinsed, wounded with a 2-mm-diameter cork borer, inoculated with a PDA disk containing mycelium and spores, and the wound sealed with Parafilm. Controls were treated the same but using sterile discs of PDA. Each treatment was replicated on five seedlings, incubated in the greenhouse (20 ± 2°C, 80% relative humidity, and 12-h natural light per day) for 60 days. After 20 days, the 10 plants treated with C. sporulosum showed small necrotic lesions, which developed into small patches of dead bark that cracked and formed spindle-shaped cankers. Radial sections through the stem at the canker site from four of the inoculated plants showed the presence of mycelium in the vessels, and C. sporulosum was reisolated from the infected tissue. After 60 days, the cankers measured as much as 22 mm, and the microscopic observations on the remaining six plants confirmed the presence of the fungus. No disease symptoms or mycelium in the inner tissues were observed for control plants, or plants inoculated with Fusarium spp. The present work adds the genus Crataegus to the wide list of hosts (e.g., Rubus, Malus, Quercus, Picea, Taxus, Juniperus, Chamaecyparis, Cupressocyparis, and Mahonia) susceptible to C. sporulosum, which may act as an important pathogen under forest conditions, in orchards, and in breeding programs. References: 1) W. Gams and K. H. Domsch. Nova Hedwigia, 18:1, 1969. 2) I. Vegh and A. Le Berre. Pepinieristes Hort. Maraich. Rev. Hortic. 331:11, 1992.