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In July 2010, extensive decline of English ash (Fraxinus excelsior L.) was observed near Chiusi della Verna, Tuscany, in central Italy (altitude 956 m a.s.l.; lat. 43° 41′ 54″ N, long. 11° 56′ 9″E). The symptoms on the tree trunks and leaves included lengthwise bark cracks, detached bark, withering of the crown where the bark was detached, and extensive microphyllia. In September 2010, perithecial stromata were observed in the parts of the tree that had lost their bark. They were applanate and black and fruiting bodies were recognized as Biscogniauxia mediterranea (De Not.) Kuntze (basionym Sphaeria mediterranea (De Not.), Ascomycotina, Xylaraceae), a fungus causing charcoal canker that attacks oaks, Acer spp., Castanea sativa Miller, Fagus sylvatica L., and Platanus acerifolia Willd. The biometric characteristics of 30 stromata were examined. The stromata were slightly convex, ellipsoid, and elongate, 7.2 to 20.5 × 3.5 to 4.2 cm. The perithecia were ovoid to tubular, 0.74 to 0.80 × 0.12 to 0.15 mm; the asci were short and stipitate, 7.9 to 10.0 × 120.2 to 170.4 μm. The ascospores were ovoid, brownish-black, with narrowed and roundish ends, 6.9 to 9.1 × 14.6 to 20.0 μm. Colonies grown on PDA at 25°C for 5 days were grey viewed from the top and black viewed from the dish underside. A comparison with the data in the literature (1) confirmed the macro- and microscopic identification. Traditional identification was further confirmed by sequence information from the rDNA ITS region. A BLAST search of the ITS sequence of our B. mediterranea isolate (GenBank Accession No. JX262798) revealed an exact match (100%) with several reference sequences of the fungus present in the database, mainly from oak hosts. Four branches each of five English ash trees growing in a natural environment were inoculated at the trunk junction with a 10–5 ml ascospore suspension. Control consisted of a branch per tree inoculated with an identical volume of sterile water only. After 25 days, the bark became detached and after a further 15 days the typical black stromata appeared. The pathogen was reisolated from the lesions, confirming Koch's postulates. No symptoms were observed on control branches, which presented healed wounds. B. mediterranea was first detected in 1986 on Quercus cerris L. and Q. pubescens Willd. in the Circeo natural park in central Italy (altitude 50 m a.s.l; lat. 41° 27′ 55″44 N, long. 12° 53′ 53″52 E). From there it spread north to other parts of the country. All oak species in Italy appear to be susceptible. Its northward expansion is likely associated with the high temperature and water stress that have been affecting the Italian peninsula for the last few years (4). Most recently, B. mediterranea has also been reported on Q. cerris in the Karst region of Slovenia (lat. 45° 43′ 03″7 N, long. 13° 45′ 20″ 4 E). This confirms its current spread to the more northerly territories, most likely because of ongoing changes in the climate that are creating optimal conditions for its survival in areas that were previously unsuitable to it. References: (1) P. Capretti and L. Mugnai, Inform. Fitopatol. 37:39, 1987. (2) D. Jurc and N. Ogris. Plant Pathol. 55:299, 2006. (3) A. Ragazzi. Micologia Italiana 1:29, 2009. (4) A. Vannini and R. Valentini. Tree Physiol. 14:129, 1994.

High mortality was noticed in a 10-year-old stand of Pinus pinea in the Alberese area (Grosseto, central Italy, elev. 40 m, 42° 39′ 46″ N, 11° 06′ 25″ E) in July 2010. Aerial symptoms of trees included chlorosis, crown thinning, stunted growth, bark lesions at the stem base with resinous exudations, and extensive necroses of the underlying xylem tissue. Woody roots of two uprooted trees exhibited bark necroses and a high proportion of fine roots was destroyed. Soil around necrotic roots was baited using apple fruits (cv. Gala). After 1 week of incubation at 24°C, typical firm fruit rot developed and small tissue samples were transferred to clarified V8 agar (V8A) amended with 5 ml/l PARPNH and incubated at 24°C. After 7 days, stellate to rosaceous, finely lobed cottony colonies arose that were transferred to FPM medium and incubated at 24°C. Within 7 days, spherical oogonia with a smooth surface and predominantly paragynous antheridia formed; sporadic amphyginous antheridia could be observed. Colony squares (1 cm2) were then placed in filtered and sterilized pond water. After 48 h, ovoid, obpyriform, or clavate, nonpapillate, persistent sporangia with internal nested and extended proliferation were formed. Fifty oogonia and 30 sporangia were measured. The diameter of the 50 spherical oogonia varied from 33.6 to 44.9 μm (avg. 39 μm); dimensions of the 30 sporangia were 42.6 to 59.8 × 28.9 to 47.8 μm (avg. 52.95 × 38.98 μm; 1:b ratio 1.37). The isolate was identified as Phytophthora humicola W. H. Ko & Ann on the basis of colony type, size and morphology of oogonia and sporangia, average length/width ratio of sporangia, the homothallic formation of oogonia (4), and ITS rDNA sequence information (GenBank Accession No. JQ757060). A BLAST search of the ITS sequence of P. humicola isolate B33 revealed a 99% identity with the Phytophthora ITS Clade 6 species P. humicola and P. inundata (2). This latter species could be ruled out, however, since it is self-sterile, whereas our isolate B33 was self-fertile (3). A strain of P. humicola was deposited in the CBS-KNAW Fungal Biodiversity Centre, strain no. CBS129249. Pathogenicity tests were conducted on 10 one-year-old twigs of Pinus pinea. A bark portion was removed aseptically and a V8A disc (0.5 cm diam.) of P. humicola mycelium was placed on the wound. Control twigs (3) received sterile V8A discs. Inoculated and control twigs were incubated at 20°C in the dark. Clearly noticeable necrotic lesions (avg. length 2.2 × 0.68 cm) were observed after 15 days on inoculated twigs. Control twigs showed no symptoms. Reisolations on selective V8-PARPNH-agar confirmed P. humicola as the causal agent. P. humicola is mainly associated with woody horticultural crops (1, 3), while the other taxa grouped with this species in Clade 6 are mainly found in forest and riparian ecosystems (1). These aquatic Phytophthora species normally have a saprophytic lifestyle, but under favourable environmental circumstances can act as opportunistic pathogens, attacking susceptible trees and causing scattered mortality in forest stands and natural ecosystems (3). To our knowledge, this is the first report of P. humicola from a pine stand. It is supposed that the pathogen reached the stand through infected plant material or infested soil introduced into the stand. References: (1) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (2) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (3) T. Jung et al. Persoonia 26:13, 2011. (4) W. H. Ko and P. J. Ann. Mycologia 77:631, 1985.

In spring 2013, pot-grown Viburnum tinus plants shipped to an ornamental nursery in Pescia (Pistoia, central Italy, 287 m a.s.l., 43°54′0″ N, 10°41′0″ E) from another local nursery were found to bear disease symptoms. Symptoms included brown to black foliar lesions, later expanding into larger blotches; necrosis of the petioles; shoot wilting and folding; browning of the stems; and necrosis of the cambium. Infected leaves, shoots, and entire plants eventually died. Tissue samples (2 mm2) were cut at the edge of active lesions from tissue of the phloem, the xylem, and the leaves and plated on selective PARPNH V8 agar (V8A) (1). Rose-shaped and finely lobed cottony colonies arose in 2 to 3 days. Mono-hyphal colonies were isolated and transferred to V8A. Square colony pieces (1 cm2) from isolates SB05a and SB05b were placed in filtered pond water after 5 to 7 days. Semipapillate, caducous sporangia with a rounded or conical base were produced within 24 h, individually or in pairs, on each sporangiophore. Sporangia (n = 30 per isolate) were examined: they were 56.2 ± 9.5 × 29.3 ± 4.3 μm (l:b ratio 1.9 ± 0.3). Exit pores averaged 7.0 ± 1.0 μm. Sporangia were ellipsoid (30%), lemon-shaped (28.3%), ovoid (20%), obovoid (16.7%), ampulliform (3.3%), or “peanut-like” (1.7%). Globose chlamydospores, borne intercalarly or terminally, were abundant on both V8A and carrot agar (CA), and were on average 54.7 ± 8.5 μm. Mono-hyphal isolates incubated for 7 days at 23°C were also transferred to CA, corn meal agar (CMA), malt extract agar (MEA), potato dextrose agar (PDA), and V8A. Colonies on these media were identical in shape and appearance to those described in previous reports (2,4). Isolates were identified as Phytophthora ramorum Werres, De Cock & Man in't Veld (4) on the basis of colony type; size, the average l:b ratio and shape of sporangia; and the type and size of the chlamydospores. Isolates were found to be the A1 mating type by pairing them with P. cryptogea BBA 63651 (mating type A2). PCR-amplification of the rDNA ITS region with specific primers Ph1/Ph4 (3) gave fragments of the expected size (GenBank Accession Nos. KF181162 and KF181163). A BLAST search of these ITS sequences in the database found that isolates of P. ramorum were the closest phylogenetically with 100% homology (YQ653034 and HM004221). Pathogenicity tests were conducted on 16 detached V. tinus leaves. A small cut was made aseptically on each of the leaf surfaces and a V8A disc (0.5 cm Ø) with mycelium was placed over the wounds. Control leaves received only sterile V8A discs. Inoculated and control leaves were incubated at 23°C in the dark. Necrotic areas (average 3.5 ± 1.3 cm2) arose on inoculated leaves after 6 days. Control leaves had no symptoms. Re-isolations on PARPNH V8A confirmed P. ramorum as the causal agent. P. ramorum was reported in Italy in 2003 on the exotic Rhododendron yakushimanum (2). This is the first report of the pathogen on a native species (V. tinus) in this country. The Pistoia area is important for nursery gardens and flowers. P. ramorum, which probably arrived on infected plant material, could compromise the export/import trade in stock plants. For this reason, the plant protection services were promptly alerted and the infected plants were destroyed. References: (1) Y. Balci et al. Plant Dis. 91:705, 2007. (2) C. Gullino et al. Inf. Agrar. 19:87, 2003. (3) K. J. Hayden et al. Phytopathology 94:1075, 2004. (4) S. Werres et al. Mycol. Res. 105:1155, 2001.

Cork oak (Quercus suber) forests are economically and culturally intertwined with the inhabitants of the Mediterranean basin and characterize its rural landscape. These forests cover over two million hectares in the western Mediterranean basin and sustain a rich biodiversity of endemisms as well as representing an important source of income derived from cork production. Currently cork oak forests are threatened by several factors including human-mediated disturbances such as poor or inappropriate management practices, adverse environmental conditions (irregular water regime with prolonged drought periods), and attacks of pathogens and pests. All these adverse factors can interact, causing a complex disease commonly known as “oak decline.” Despite the numerous investigations carried out so far, decline continues to be the main pathological problem of cork oak forests because of its complex etiology and the resulting difficulties in defining suitable control strategies. An overview of the literature indicates that several pathogenic fungi and oomycota can play a primary role in the etiology of this syndrome. Therefore, the aim of this review is to analyze the recent advances achieved regarding the bio-ecology of the endemic and emerging pathogens that threaten cork oak trees with particular emphasis on the species more directly involved in oak decline. Moreover, the effect of climate change on the host-pathogen interactions, a task fundamental for making useful decisions and managing cork oak forests properly, is considered.

Findings of a study extending over a number of years on some key aspects of the biology and ecology of Phomopsis quercina in Mediterranean oak forests are reported. The main biometric parameters and physiological requirements of this significant endophytic fungus were determined in the laboratory. The microorganism was also studied in the field to explore its lifestyle in natural oak stands. The isolation frequencies of the fungus in various oak species were also related to the vitality of the trees (healthy or declining) and this showed that the fungus is involved in the widespread phenomenon of oak decline. In planta studies have proved the pathogenic activity of P. quercina in oak, and have found that it remains latent for a long time in the inner wood of oaks, but may turn into an aggressive and harmful coloniser of the tree if this is weakened by some stress factors. An important part of the study concerns an examination of the antagonism that some other endophytes, which colonise the same oak tissues and organs as P. quercina, display against this fungus. The increasing presence and abundance of P. quercina in declining (and especially drought-stressed) oaks suggests that climate change is having a significant role in destabilising the precarious balance between the host tree and the fungus, to the benefit of the latter. Lastly, some practical suggestions are offered on ways to counter the epidemic spread of this latent pathogen.

The present-day phytosanitary disasters caused by biological invasions are afflicting urban and peri-urban forest stands worldwide, as well as the varied services they normally provide. In Europe, we are witnessing an alarming situation due to an increasing introduction of infectious diseases and pests. The authors present an up-to-date list of alien microbial pathogens and insect pests affecting urban greening that have been accidentally imported in Italy or that are likely to be introduced. Information about the biology, epidemiology, ethology, and control of these invasive organisms is provided. For each species, the current geographical distribution, including newly-colonized areas, is also given, as well as the chronological progression of its occurrence. Particular detail is used for describing symptoms—the key diagnostic elements for appropriate and timely phytosanitary management. This paper will benefit urban forest management, which is a crucial factor in maintaining the social and ecological viability of urban green spaces, as well as ecosystem services. The importance of engaging citizens in community-based monitoring of urban greenspaces for tracking the location, abundance, and pathways of invasive pathogens and pests will also be touched upon.

Many biotic and abiotic factors contribute to the onset of oak decline. Among biotic agents, a variety of fungi and insects cause extensive disease and insect outbreaks in oak forests. To date, research on fungus-insect interactions in Mediterranean forest ecosystems is still scarce and fragmentary. In this study, we investigated the assemblage of endophytic mycobiota and insect pests occurring in a declining oak stand, with the aim to explore if, and to what extent, the insect species were active vectors of fungal propagules. It emerged that some known latent pathogens of the Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, Diplodia seriata, Dothiorella sarmentorum, and Neofusicoccum parvum were isolated at high frequency from physiologically-impaired trees. In addition, propagules of these fungi were isolated from five insects, two of which (Cerambyx welensii and Coraebus fasciatus) are main oak pests. The life-history strategies of these fungi and those of wood-boring beetles were strikingly interconnected: both the fungi and beetles exploit drought-stressed trees and both occur at high frequency during hot, dry periods. This synchronicity increased their chance of co-occurrence and, consequently, their probability of jointly leading to oak decline. If these interactions would be confirmed by future studies, they could help to better understand the extensive decline/dieback of many Mediterranean forest ecosystems.

I molteplici servizi svolti dalle foreste (conservazione della biodiversita, del suolo e delle risorse idriche, mitigazione dei cambiamenti climatici, purificazione dell’aria e dell’acqua, produzione, funzione paesaggistica e ricreativa) dipendono fortemente dallo stato di salute degli ecosistemi forestali. In questo contributo si riassumono gli spunti principali emersi durante la sessione «Selvicoltura, Protezione delle Foreste» del III Congresso Nazionale di Selvicoltura, Taormina, 16 - 19 Ottobre 2008, in merito alle interazioni tra selvicoltura e protezione delle foreste in Italia.

The multiple services provided by forests (conservation of: biodiversity, soil and water resources, mitigation of climate change, cleaning of air and water, timber production, landscape and recreation) are strongly dependent on forest health. In this paper we synthesize the main outcomes of the session on ‘Silviculture, Forest Protection’ held within the III Italian National Congress of Silviculture, Taormina, 16-19 October 2008

During the early part of the 1980s, a major project called Parco Nord was undertaken by the Lombardia Region to establish forest resources within an industrial area located in the northern part of the city of Milan. Since 1983, more than 60 ha of formerly industrial land has been converted into urban forest plantations, thus creating large patches of trees with the potential to sustain a wide range of functions and services. This paper describes an integrative study aimed to assess the current status of forest resources in Parco Nord. It focuses on the actions taken to determine whether forest resources significantly changed their status 20 years after their establishment, considering historical field data and records of management practices. Analyses have been conducted at both stand and tree level by collecting quantitative and qualitative parameters. Stand-level analysis gave a quantitative estimation of the response of species to ecological conditions and management practices while tree-level analysis provided evidence of species renovation after thinning operations.

Aims: To assess the effect of different concentrations of gossypol (0, 2, 4, 10 and 20 mg l–1) in combination with NaCl and Na2SO4 (20 mS cm–1) on the conidial germination and viability of Fusarium oxysporum f.sp. vasinfectum (Fov). Methods and Results: A multinomial logistic model was developed to estimate the germination probability of Fov. The inhibitory effect was markedly evident at the two highest concentrations of gossypol; it varied among the isolates tested and with time, and it was attenuated by the presence of sodium salts. The inhibition was temporary as the germination probability increased after 8 h. Fluorescent staining revealed that gossypol either killed the conidia or retarded the elongation of the germ tubes. Conclusion: Fov showed the ability to overcome gossypol inhibition over time, and the inhibitory effect is reduced under saline conditions. Differential responses among Fov isolates to the presence of gossypol suggest that gossypol tolerance is genetically determined in the pathogen. Significance and Impact of the Study: This study suggests that selecting for high plant gossypol cultivars would have minimal effect on the overall Fov resistance of cotton. A new statistical model was developed to explore the statistical significance of plant–pathogen interactions.

Investigation of the extracts of a culture of Cladosporium tenuissimum , a known hyperparasite of several rust fungi, gave rise to the isolation of cladosporols B–E ( 2 – 5 ). Their structure and stereochemistry were elucidated on the basis of 1 H and 13 C NMR evidence and CD measures. Cladosporols 1 – 5 were active in inhibiting the urediniospore germination of the bean rust agent Uromyces appendiculatus .

Interactions between the mycoparasite Cladosporium tenuissimum and the bean rust Uromyces appendiculatus were studied through light and electron microscopy in vitro at the host-parasite interface. Urediniospore germination decreased on contact with ungerminated C. tenuissimum conidia, possibly due to antibiosis mechanisms. C. tenuissimum grew towards the bean rust spores and coiled around their germ tubes. Penetration of the urediniospores occurred either enzymatically and/or mechanically, through appressorium or infection cushion structures, from which a thin penetrating hypha was generated. Enzyme production by the mycoparasite was suggested by the loosening of the matricial components of the spore wall, which sometimes left chitin fibrils visible. Mycoparasite hyphae grew within the host spore, emptied its content, and emerged profusely forming conidiophores and conidia. C. tenuissimum was able to grow on media containing laminarin, suggesting the ability of producing glucanases, but not when chitin was used as the sole carbon source. Conidia that had been grown on a sugar-rich medium, filtered, and extracted with organic solvents, were found to contain cladosporol and related compounds. Complete control of the bean rust disease was achieved by application of C. tenuissimum culture filtrates but not by conidial suspensions. This is the first report of parasitism by C. tenuissimum on U. appendiculatus. These investigations provide additional observations on a genus besides Melampsora and Cronartium from which this fungus has been isolated and tested to date. The possible role of environmental factors for the exploitation of this organism as a biocontrol agent is also mentioned.

Summary The composition of the endophytic fungal assemblages in Quercus cerris, Q. pubescens and Q. robur, three oak species most susceptible to decline, was investigated in Fagare ` (Padova), Ulignano (Pisa), and Radda in Chianti (Firenze). The endophytic assemblages were studied as a function of health status of the whole tree and of twigs and leaves. The effect of station altitude was tested on Q. cerris. Samples were gathered from healthy and declining trees. In April (bud opening time) 20 twigs and 10 leaves were collected from each tree. Fungi were isolated from twig segments and leaf portions. A total of 23 fungal genera with 27 different species were isolated, in addition to a few sterile mycelia. Eleven species were found to be common to all three oak species, while other species were found only on one oak species, suggesting host specificity. Among the fungal species isolated, some were known to be pathogenic (Apiognomonia quercina, Colpoma quercinum, Diplodia mutila, Phomopsis quercina). The colonization frequency (CF) of pathogenic species varied between 0.9% for A. quercina in Q. cerris and 60.2% for P. quercina in Q. robur. Analysis of variance showed a statistically significant difference among the oak species tested. The CF was higher in declining trees in comparison with healthy trees, and also in twigs vs. leaves. The CF was found to be higher for Q. cerris trees growing at sea level as compared with those situated at an elevation of 350‐400 m. In stands where, on account of particular ecological conditions, pathogenic behaviour is displayed simultaneously by more than one endophytic fungal species, trees accentuate their decline.

Summary Soil-borne species of Phytophthora were isolated from 19 of 30 examined oak forest areas in Italy. The frequency of isolated Phytophthora spp. (35.2%) was significantly correlated with soil pH and longitude of the sites. Eleven Phytophthora species were detected. Phytophthora cambivora, P. cinnamomi and P. cactorum were recovered from sites in central and southern Italy whereas P. quercina was isolated in the northern and central part of the country. Phytophthora citricola occurred all over Italy. Phytophthora quercina was the only species significantly associated with declining oak trees.

A severe dieback of Acer pseudoplatanus trees was noticed in planted forest stands in northern Italy in 2010. Affected trees showed collar rot and aerial bleeding cankers along the stems, leading to crown dieback and eventually death. An unknown Phytophthora species was consistently isolated from necrotic bark and xylem tissue and from rhizosphere soil. Based on its unique combination of morphological and physiological characters and phylogenetic analysis, this new taxon is here described as Phytophthora acerina sp. nov. Phylogenetic analysis of ITS, cox1 and beta-tubulin gene regions demonstrated that P. acerina is unique and forms a separate cluster within the 'P. citricola complex', closely related to P. plurivora. Phytophthora acerina is homothallic with smooth-walled oogonia, thick-walled, mostly aplerotic oospores with a high abortion rate, paragynous antheridia, and persistent, morphologically variable semipapillate sporangia. Four to 5-week-old cultures produced globose to subglobose, appressoria-like and coralloid hyphal swellings and characteristic stromata-like hyphal aggregations. Optimum and maximum temperatures for growth are 25 degrees C and 32 degrees C, respectively. Genetic uniformity of all 15 studied isolates and the apparent absence of this species in the extensive surveys of nurseries, forests and seminatural ecosystems conducted in the previous two decades across Europe indicate a recent clonal introduction to northern Italy. Under-bark inoculation tests demonstrated high aggressiveness of P. acerina to A. pseudoplatanus indicating that this pathogen might be a serious risk to maple plantations and forests in Europe. Italia Nostra Onlus - Centro della Forestazione Urbana (CFU); Scottish Government info:eu-repo/semantics/publishedVersion

Fusarium eumartii is a fungus associated with declining Quercus robur, in which it is found in the vessels. The response of oak species to infection is known to vary: Q. robur is susceptible, but Quercus cerris and Quercus pubescens are resistant. An experiment was carried out in 1996 and repeated in 1997, to examine how F. eumartii colonization differed in oak species that were susceptible or resistant to the fungus by counting the number of vessels with mycelium at various distances from the inoculation site in infected seedlings and by determining the amount of viable fungus in infected tissue. Infected vessels with mycelium were counted on sections (10 μm thick) cut at 0, 2, 4, 6, 8 and 10 cm from the inoculation site on 1-year-old inoculated seedlings as well as on sections cut every 2 cm to the seedling tip. The amount of viable fungus was determined by counting the colony forming units (CFUs) in stem segments from the same seedlings. Quercus robur seedlings had the greatest number of infected vessels and the greatest number of CFUs. Forty days after inoculation, the extent of vertical fungal spread was 28.12 cm in Q. robur, 3.15 cm in Q. cerris and 3.00 cm in Q. pubescens. The greatest number of CFUs was found in Q. robur at day 5 after inoculation. Analysis of variance confirmed the results.

Axenic cultures of the aspen rust Melampsora larici-tremulae were established for the first time by seeding aeciospores artificially produced on larch onto three chemically defined media. Cultures were maintained for 9 months and evaluated for their appearance, growth rates and sporulation ability. Two types of colonies were observed. One, white and fluffy tending to felty, did not sporulate in culture. The other, white-orange with a more compact aerial mycelium, sporulated profusely producing various spore like bodies and other unusual structures. An initial lag phase, with reduced growth, characterised primary rust cultures in the first 4–5 weeks. After about one month, cultures seemed adapted to utilising exogenous nutrients for sustained growth and their development rate increased substantially. As a result of selection under unusual growing conditions, fast-growing genetic variants were also observed in the nutrient agar. Growth on all the three test media, containing different organic and inorganic constituents, suggested that nutritional requirements were non-specific. The importance of axenic cultures in the study of the nutritional requirements of aspen rust and the potential of these investigations to elucidate some physiological and genetic aspects of the host-parasite relationship are discussed.

The presence of Discula quercina on dormant buds and twigs of declining Quercus cerris was studied on two test sites. A number of fungi, including some endophytic ones, were isolated in association with D. quercina as part of the total oak mycoflora: Acremonium spp., Diplodia mutila, Phomopsis quercina, Hypoxylon mediterraneum, Cylindrocarpon sp., Phoma cava and Trichoderma viride plus some less frequent species. The distribution of these fungi did not appear to be related to the altitude of the test sites studied: sea level and 600 m a.s.l., nor to the differing composition of the site vegetation. Discula quercina was isolated both from the twigs (18.02% from woody tissue; 5.82% bark tissue) and from the buds (11.62% embryo; 14.55% bud scales). The results indicate that D. quercina is able to live in symbiosis with its host. Although the colonization of the aerial parts of Q. cerris is possible through infection with wind-borne inoculum, leaf infection can also occur through overwintering mycelium migrating to the leaves mainly from the buds, but also to a lesser extent from the twigs. Zusammenfassung Discula quercina als Endophyt absterbender Exemplare von Quercus cerris An zwei Standorten wurde untersucht, ob Discula quercina in ruhenden Knospen und Zweigen absterbender Zerr-Eichen (Quercus cerria) vorkommt. Dabei wurden neben D. quercina verschiedene Pilze isoliert - darunter einige Endophyten -, die ebenfalls zur Eichen-Mykoflora gehoren:Acremonium spp., Diplodia mutila, Phomopsis quercina, Hypoxylon mediterraneum, Cylindrocarpon sp., Phoma cava und Trichoderma viride, auserdem einige seltener vorkommende Arten. Die Verteilung dieser Pilze hing offenbar weder von der Hohenlage der Versuchsstandorte (Meereshohe und 600 m uber N.N.) noch von der unterschiedlichen Zusammensetzung der Vegetation der Versuchsstandorte ab. D. quercina wurde sowohl aus den Zweigen 18,02% aus Holzgewebe; 5,82% aus Rindengewebe als auch aus den Knospen (11,62% Embryo;14,55% Knospenschuppen) isoliert. Die Ergeb- nisse zeigen, dasD. quercina in Symbiose mit der Wirtspflanze leben kann. Die Besiedlung der oberirdischen Teile von Q. cerris ist durch eine Infektion mit windburtigem Inokulum moglich, doch eine Infektion der Blatter kann auch durch uberwinterndes Myzel erfolgen, das hauptsachlich von den Knospen, in geringerem Mase aber auch von den Zweigen zu den Blattern gelangt.

Some experiments were conducted (in duplicate) to determine the effects of the osmotic potential on the growth of Diplodia mutila, and the effects of the pre-dawn water potential on the development of cankers by D. mutila on Quercus robur. Mycelium of D. mutila was grown on liquid medium enriched with various amounts of polyethylene glycol so as to give osmotic potential values of -0.35, -1.18, -1.75, -3.00 and -3.98 MPa. At the two lowest MPa values (-3.00 and -3.98 MPa) D. mutila growth was slower in relation to the fresh weight and the dry weight of the mycelium. To determine the pre-dawn water potential 30 3-year-old Q. robur seedlings were inoculated with D. mutila mycelium. Water-stressed seedlings had lower pre-dawn water potential values (MPa -2.92) and longer cankers than the unstressed seedlings (MPa -0.79). It is concluded that Q. robur, a highly hygrophilous oak, suffers from water stress to such an extent that its structural and chemical defences are overcome and the seedlings are seriously damaged. A similar situation is observed in nature, where frequent and prolonged droughts may induce states of water stress that account for the spread and the incidence of D. mutila in Italy. Zusammenfassung Wasserstres und Entwicklung von Diplodiamutila-Rindennekrosen bei Quercus robur Verschiedene Experimente wurden (zweifach) durchgefuhrt, um den Einflus des osmotischen Potentials auf das Wachstum von Diplodia mutila sowie die Wirkungen des vor Tagesanbruch herrschenden Wasserpotentials auf die Entwicklung der durch Diplodia mutila verursachten Rindennekrosen bei Quercus robur zu untersuchen. Myzel von D. mutila wurde auf Flussigmedium angezogen, das unterschiedliche Mengen PEG enthielt, wodurch die Werte des osmotischen Potentials bei –0,35, –1,18, –1,75, –3,00 und –3,98 MPa lagen. Bei den beiden niedrigsten MPa-Werten (–3,00 und –3,98 MPa) wuchs D. mutila langsamer, was anhand des Frischgewichts und des Trockengewichts des Myzels ermittelt wurde. Zur Bestimmung des vor Tagesanbruch herrschenden Wasserpotentials wurden 30 drei Jahre alte Samlinge von Q. robur mit Myzel von D. mutila inokuliert. Unter Wasserstres leidende Samlinge wiesen vor Tagesanbruch niedrigere Wasserpotentiale (MPa –2,92) und langere Rindeneekrosen auf als die nicht gestresten Samlinge (MPa –0,79). Es wird gefolgert, dasQ. robur als Eiche, die viel Feuchtigkeit benotigt, unter Wasserstres so sehr leidet, das ihre strukturellen und chemischen Abwehrmechanismen nicht ausreichend wirksam sind und die Samlinge von D. mutila stark geschadigt werden. Eine ahnliche Situation wird in der freien Natur beobachtet–die Ausbreitung und die Befallshaufigkeit von D. mutila in Italien gehen vermutlich auf haufige und lange andauernde Trockenperioden zuruck.

Ten-year-old field-grown Quercus pubescens Willd. and Quercus cerris L. individuals were inoculated with Diplodia mutila (teleomorph Botryosphaeria stevensii Shoe-maker) to observe symptom evolution over 2 years. The experiments were carried out for the 3 years: 1991, 1992 and 1993. At the same time, the effect of D. mutila on leaf water potential and peroxidase activity was assessed on the leaves of 2-year-old seedlings from the same two species growing under controlled conditions (18 h day, relative humidity 75%), temperature 24/15°C day/night). Quercus pubescens was more susceptible than Q. cerris, and frequently exhibited dieback of the branch above the inoculation site. On the seedlings, the visible symptoms of infection were much more severe than on the older trees. The infected seedlings showed a lower leaf water potential than healthy seedlings, particularly those of Q. pubescens after the third week. The peroxidase activity was increased in infected Q. pubescens seedlings compared with both groups of control seedlings (wounded but not inoculated, and neither wounded nor inoculated) 14 and 21 days after inoculation. Leaf water potential and peroxidase activity are known indicators of stress which appear even before any visible symptoms; they could therefore serve as early indicators of D. mutila infection. Zussamenfassung Zehn Jahre alte, im Feld wachsende Quercus cerris- und Quercus pubescens-Baume wurden mit Diplodia mutila (Teleomorph Botryosphaeria stevensii Shoemaker) inokuliert, um die Befallssymptomentwicklung uber einen Zeitraum von 2 Jahren beobachten zu konnen. Die Versuche wurden in den Jahren 1991, 1992 und 1993 durchgefuhrt. Zur gleichen Zeit wurde der Einflus einer D. mutila-Infektion auf das Wasserpotential sowie die Peroxidasaktivitat in den Blattern von 2 Jahre alten Samlingen der gleichen Quercus-Spezies ermittelt, die unter kontrollierten Bedingungen (18 h Tag, RH75%, Temperature 24/15°C Tag/Nacht) wuchsen. Festgestellt werden konnte, dasQ. pubescens anfalliger als Q. cerris ist, ein Absterben der Zweige oberhalb des Inokulationspunktes wurde haufig beobachtet. Die sichtbaren Befallssymptome waren deutlich starker bei Samlingen als bei den alteren Baumen. Infizierte Samlinge hatten ein niedrigeres Blattwasserpotential als gesunde Samlinge, besonders bei Q. pubescens drei Wochen nach der Inokulation. 14 und 21 Tage nach der Inokulation war die Peroxidaseaktivitat in infizierten Q. pubescens-Samlingen erhoht, verglichen mit den Kontrollen (verletzte aber nicht inokulierte sowie weder verletzte noch inokulierte Samlinge). Anderungen des Blattwasserpotentials und der Peoxidaseaktivitat sind bekannte Stresindikatoren, die mesbar sind, bevor Symptome eines Befalls erscheinen. Von daher konnten sie als ein fruher Nachweis einer D. mutila-Infektion dienen.

Two rapid, independent molecular assays have been developed for discriminating between the closely related rusts of hard pines, Cronartium flaccidum and Peridermium pini . A portion of the ITS region and a portion of the IGS region from the ribosomal RNA operon of the two organisms were amplified with the PCR. Amplifications were made using DNA extracted from aeciospores collected from unopened aecia, taking a small number of spores as a source of template DNA. Amplified fragments were subjected to RFLP or to SSCP analysis. Digests of the amplified products from the IGS1 region were electrophoresed on polyacrylamide gel and stained with ethidium bromide. Hinf I digestion of these fragments created polymorphic restriction profiles which allowed differentiation of the autoecious P. pini from the heteroecious C. flaccidum . Similar-sized DNA fragments representing the ITS2 region of the two rusts were denatured, subjected to electrophoresis as single strands on polyacrylamide gel under non-denaturing conditions and ‘silver stained’. The different mobility displayed by these short fragments revealed sequence polymorphism in the examined portion of the ITS region. This technique therefore represents an accurate and sensitive method for detection of base changes in given sequences of genomic DNA. A high level of homology was found between the two biotroph organisms in the loci screened. Results obtained in this trial indicate that PCR-RFLP and PCR-SSCP can be used as simple, speedy taxonomic tools for elucidating relationships among related organisms.

Summary Forty isolates of Diplodia mutila, a dieback agent on oaks were characterized by their capacity to produce heterokaryons, by their vegetative compatibility, and by their pathogenicity tests on Quercus cerris. The forty isolates were collected from Quercus cerris, Quercus frainetto, Quercus ilex, Quercus pubescens, Quercus robur and Quercus suber in different regions in Italy. Three nit mutants (nitt, nit and nitM) were identified with different nitrogen sources: sodium nitrite, sodium nitrate, uric acid, ammonium tartrate and hypoxanthine. On the two nutrient media employed, a potato dextrose agar (PDA) medium with chlorate (PDC), and a minimal agar medium with chlorate (MMC), isolates obtained from Q. cerris yielded the greatest number of mutants: 254 on PDC (out of a total of 583) and 190 on MMC (out of a total of 440). Nine vegetative compatibility groups (VCGs) were identified. There was a strong association between these VCGs and host species. All isolates tested except one from Q. cerris were pathogenic.

Summary Tests in vitro and in situ showed that both mycelial and conidial suspensions of Acremonium mucronatum were antagonistic for Diplodia mutila, a common pathogen on the apical twigs of declining Quercus cerris, Quercus frainetto, Quercus pubescens and Quercus robur. Antagonism was greater with the mycelium than with the conidial suspensions, and was strongest at a test temperature of 20°C. The results suggest that pathogenicity of D. mutila on declining trees may be significantly reduced by the presence of A. mucronatum.

Since virulence ofFusarium oxysporum f.sp.vasinfectum (FOV) on cotton (Gossypium hirsutum) is enhanced when the fungus is cultivated in a saline environment, excessively saline water must not be used for the irrigation of cotton. However, the limitations thus placed on the available water resources may lead to conditions of enforced water stress for the plant. The present study investigated whether water stress affects the susceptibility of cotton to FOV. Groups of 2-month-old cotton plants of theFusarium-susceptible Coker 304 and the moderately resistant GSC 20 varieties were maintained without watering for varying periods immediately before or after being inoculated with FOV (15 plants per group, two replications). Watering was suspended for 3, 6, 12 or 24 days before inoculation, and for 3, 6, 12 or 15 days after inoculation. After inoculation the plants were maintained in a controlled environment with a 15,000 lux, 12-h photoperiod, at 28°/24°C D/N, 20% r.h. Xylem water potential was determined in a pressure chamber. Percent infected leaf area and date of onset of wilt were the parameters used to define severity of FOV infection. There was a consistent relation between low water potential in the xylem (-7 and -20 MPa) and severity of infection, particularly when the dry period occurred after inoculation. After exposure to the lowest post-inoculation water potentials, even variety GSC 20, which is normally moderately resistant, exhibited a fairly high percent infected leaf area. This should be taken into account when the cotton grower is faced with water shortages, especially during the period from branching to flower bud break.

Summary The callus-fungal method was employed to test the response to C. flaccidum of the highly susceptible P. nigra var. laricio and the resistant P. sylvestris, and to ascertain whether results obtained with this method matched in planta observations. Calli were inoculated with axenie cultures of C. flaccidum obtained by incubating basidiospores on modified Schenk and Hildebrandt medium. Several parameters were evaluated. Colony growth was more rapid on P. nigra var. laricio. Colonies were dense on P. nigra var. laricio, but sparse on P. sylvestris. Aerial hyphae growth was abundant on P. nigra var. laricio, but less frequent on P. sylvestris. Hyphal branching began after 18 h on P. nigra var. laricio and after 45 h on P. sylvestris. Necrosis of the host cells set in after 24 h on P. nigra var. laricio, and after 70 h on P. sylvestris. The number of cells with plasmolysis was much larger in P. nigra var. laricio than in P. sylvestris. These results were consistent with the known resistance of the two species on whole plants.

This chapter summarises research carried out on the biology, ecology and the impact of the holomorph Apiognomonia quercina/Discula quercina in oak forests. The major life-history traits and aspects of the epidemiology (isolation, survival, reproduction, dispersal, host selectivity) and control of the oak anthracnose agent are elucidated. The role of weather patterns in disrupting the delicate interaction between the host tree and the microorganism is outlined. The evidence suggests that changes in the climate profoundly alter the plant-endophyte symbiosis, generating conflicts of interest between the partners in the interaction. When such competing interests arise, the survival and reproduction of one member of the interaction do not conform with that of the partner. The interaction becomes thus disadvantageous and harmful to one of the organisms and the symbiosis from mutualistic or neutral turns antagonistic. The fungal partner, which under normal conditions survives in quiescence, with a low biomass, resumes growth but now switches from a latent, asymptomatic occupier of inner oak tissues to an aggressive coloniser that sporulates profusely over the tree surface. The importance of investigating the functioning and the role of the plant-endophyte symbiosis in perennial host trees in natural forests is stressed.

In the last 30 years, the spread of emerging and invasive fungal pathogens has had a profound impact on plants, animals and humans, causing crop losses and damaging their health with economic and social impact. Awareness of the biodiversity of these pathogens is essential for their detection, identification and control. In this article, their diversity in Italy is reviewed taking into consideration the international context. Fungal endophytes, important ecological agents whose lifestyle can be switched by stochastic events from beneficial/neutral to pathogenic, are included in this review.

Two strains of a species of Colletotrichum were obtained from infected olives in India. Their morphological characteristics and pathogenicity to various fruits were compared with those of isolates of C. acutatum, C. gloeosporioides, and C. musae. Despite their morphological variability, the two isolates were referred to C. gloeosporioides, the anamorph of Glomerella cingulata. For one isolate, the presence of ascocarps was recorded in culture.

The penetration of Cronartium flaccidum into the cotyledons, primary needles and secondary needles of Pinus pinaster and P. nigra var, laricio was investigated. SEM examination showed that the fungus did not form appressoria and that the germ tube penetrated directly into the stomata.

Fungal endophytes that colonize forest trees are widespread, but they are less well known than endophytes infecting grasses. The few studies on endophytes in trees mainly concern the tropical areas and the northernmost latitudes, while similar investigations in the Mediterranean region have so far been scarce and incidental. Endophytes are studied mostly in economically important forests suffering from diseases, such as oak forests. One common endophyte that has received some study on oak is the mitosporic Discula quercina. This paper, after first addressing some basic problems on tree endophytes, examines the ecology of D. quercina in Mediterranean oak stands. D. quercina is usually viewed as a symptomless colonizer of healthy Quercus cerris, infecting new leaves early in the growing season, in an unstable equilibrium between transient mutualism/neutralism and latent pathogenesis. It is postulated here that climatic factors can change the endophytic nature of D. quercina, turning it into a weak pathogen or an opportunistic invader of senescing and indeed healthy trees. It is argued more generally that stochastic events can cause the lifestyle of an endophyte to switch from beneficial/neutral to pathogenic, transforming the tree–endophyte interaction, an interaction that depends in part on the matching genomes of the tree and endophyte, and on the environmental context.

This chapter reviews strategies in rust control, with a special emphasis on biological control, in the light of evidence produced in recent years showing that plant disease control is most effective when an integrated management approach is followed. A survey of the fungal antagonists (hyperparasites) most effective against rust pathogens is given. The mode of action of these antagonists is described, and the main problems concerning biological control are discussed, on the basis of the optimal characteristics of an antagonist or biocontrol agent. The value and limitations of other control measures besides biological control (eradication, definition of hazard areas, quarantine, cultural practices, chemical treatments, and plant breeding for disease resistance) are also outlined. A consideration of all control measures suggests that crop protection requires a holistic approach integrating a broad range of control techniques.

The synthesis of dehydrin-like proteins in 24-month-old Quercus cerris and Q. ilex seedlings subjected to water stress and/or infection with Phytophthora cinnamomi , was investigated. An estimated 50 kDa dehydrin-like protein was observed in leaves of symptomless Q. ilex (drought tolerant) in control, as well as in seedlings infected with P. cinnamomi , throughout the experiment. Water stress, with a midday stem water potential value lower than −2.7 MPa (up to 8 weeks), increased band intensity. Two new polypeptides with a molecular weight of about 60 and 63 kDa, respectively, were detected in Q. ilex , under enhanced water stress conditions (≤−4.0 MPa). No dehydrin-like proteins were observed in leaves of symptomless Q. cerris (drought susceptible) neither in control nor in infected treatments. With a midday stem water potential lower than −3.8 MPa (up to 7 weeks), water stress induced the synthesis of a protein with an approx. molecular weight of 43 kDa, while a band of about 36.5 kDa was detected for both water stress and infection treatments. These results suggest the involvement of dehydrin-like proteins in the response of Q. ilex and Q. cerris seedlings to different stresses.

The quarantine pathogen Phytophthora ramorum has recently been found on dying Viburnum tinus in the nursery area of Pistoia, central Italy (43°56′0″ N, 11°1′0″ E) (3). As part of a surveillance program aimed at detecting P. ramorum in this area, the Phytophthora taxon Pgchlamydo was consistently found associated with symptomatic V. tinus. The crowns of these plants were wilted, and some plants also showed root and collar rot and underbark necrosis. Water courses adjacent to the nursery with the infected V. tinus were tested for the pathogen. Samples from seven symptomatic plants were placed on a selective V8A-PARPNH medium within 24 h from sampling. Tissue pieces (2 mm2) of 12 baits (apple fruits) exposed for a week in water bodies were plated on the same medium. Cottony colonies arose after 2 to 3 days of incubation at 23°C in the dark and were transferred to potato dextrose agar (PDA) in purity. Mycelial DNA was extracted with a commercial kit (Sigma-Aldrich). The rDNA ITS region and a portion of the mtDNA cox1 gene were PCR-amplified and the amplicons digested with the restriction enzymes MspI and AluI (for the ITS region) and RsaI (for the cox1 gene region). Isolates R7 from V. tinus, and ES2M5, ES2M11, and ES1M12 from the water bodies belonged to the same taxon based on restriction analysis of both DNA regions coupled with ITS-rDNA sequence homology (GenBank Accession Nos. KJ396773 to 76). A BLAST search in GenBank found that all isolates had a 99% identity in the ITS-rDNA with the Phytophthora ITS Clade 6 member P. taxon Pgchlamydo. Sporangia produced after incubation in filtered pond water for 24 h were mostly ovoid (sometimes obpyriform), non-papillate, non-caducous. Some sporangia were emptied with external proliferation and had hyphal swellings. Thirty sporangia were measured and averaged 42.4 ± 6.2 × 29.9 ± 3.5 μm (range 30.0 to 56.1 × 22.5 to 38.0), with a length/width ratio of 1.4 ± 0.2 (1.2 to 2.0), and exit pores of 11.7 ± 1.5 μm (9.0 to 14.6). Optimum colony growth on V8A at 30°C was 4.4 ± 0.4 mm day–1, and the maximum temperature for growth was 32°C. Inoculation on twigs of Fagus sylvatica and V. tinus produced necrotic lesions of 2.6 ± 0.5 cm (2.1 to 3.5) and 4.7± 0.5 cm (3.8 to 5.6) respectively after 3 weeks of incubation at 23°C in the dark. Inoculation on V. tinus leaves resulted in lesions averaging 3.3 ± 1.1 × 2.1 ± 0.6 cm (range 2 to 5 × 1.5 to 3) after 2 weeks of incubation at 23°C in the dark. Control plant material showed no symptoms.The Phytophthora taxon Pgchlamydo has been reported on several ornamental and woody species, including Arctostaphylos sp., Camellia spp., Laurus nobilis, Buxus sempervirens, Rhododendron sp., Arbutus unedo, Prunus sp., Pseudotsuga sp., and Sequoia sempervirens, in North America and Europe (1,2). This is the first report, to our knowledge, of this taxon on V. tinus in Italy. V. tinus is widely sold in European nurseries, and it is also one of the most common hosts of P. ramorum (4). The fact that V. tinus is a host for both oomycetes, and the two microorganisms induce a similar symptomology (wilt), might complicate the control efforts of the phytosanitary inspection services aimed at restricting P. ramorum foci in Europe. References: (1) C. L. Blomquist et al. Plant. Dis. 96:1691, 2012. (2) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (3) B. Ginetti et al. Plant. Dis. 98:423, 2104. (4) S. Prospero et al. Plant Pathol. 62:1063, 2013.

The park Boscoincittà, Milan, North Italy (136 m a.s.l., 45°29′06″ N, 9°5′32″ E), has an area of 110 ha and includes tree stands, wood clearings, trails, and watercourses. Recently, common walnut (Jugland regia) trees in the park have begun to suffer from a progressive dieback that has caused roughly 90% mortality. Aerial symptoms were stunted growth, loss of vigor, crown thinning, and bark cankers with tarry exudates on the lower stem. The xylem tissue of trees showed large necrosis and flame-shaped discoloration below the bark. Since the dieback seemed caused by Phytophthora, samples were taken from three symptomatic trees and, by baiting, from the nearby soil and watercourses. Isolations from apple baits were carried out after a week. Isolations taken from tissue at the edge of active lesions of the trees were transferred on the selective medium V8A-PARPNH (1) and incubated at 24°C. Cottony colonies appeared after 3 days and single hyphal tip derivatives were transferred to V8A for a further 4 to 7 days. Fragments (1 cm2) of mycelium of the subcultured colonies were then placed in filtered (Ø 0.22 μm nitrocellulose filters, Millipore) pond water. Three isolates were retrieved within 24 h, two from tree tissue and one from water. These produced ovoid, non-papillate sporangia, of which 30 per isolate were measured. Sporangia averaged 52.5 ± 9.6 × 32.9 ± 4.7 μm (range 30.8 to 67 × 22.5 to 42.8) with a l:b ratio of 1.59 ± 0.19 (range 1.27 to 2.05), and exit pores of 11.1 ± 1.7 μm (range 7.31 to 14.21). External proliferation from previously emptied sporangia and hyphal swellings were observed on V8A. On V8A, colonies had optimal growth at 32°C (5.7 ± 0.8 mm d–1) with a maximum at 37°C. Colonies had a chrysanthemum-shaped, scanty fluffy aspect on PDA. Isolates were identified as Phytophthora taxon walnut on the basis of macro- and micro-morphology and sequence information from the ITS-rDNA region, that was amplified with primers ITS6 and ITS4 (3) after DNA extraction with a commercial kit (Sigma Aldrich). A region of the cox1 gene of isolate B164 was also amplified with primers OomCoxILevup and Fm85mod (4) and sequenced (GenBank Accession No. KC291584), but this was irrelevant for identification purposes because that gene region has not been sequenced for other isolates of this taxon. A BLAST search in GenBank and the Phytophthora database revealed a 99% identity of the ITS-rDNA from our isolate B164 (KC291550) with the P. taxon walnut isolate P532 (AF541910) (2). Inoculation trials were conducted on 10 detached leaves. A little lesion was produced with a sterile scalpel on the lower leaf surfaces and a 0.5 cm Ø agar plug was placed over the wounds. Necrotic lesions averaged 3.7 ± 1.6 × 2.0 ± 0.5 cm after 1 week of incubation at 20°C in the dark. Control leaves showed no symptoms. Re-isolations on V8-PARPNH agar confirmed Phytophthora taxon walnut as the causal agent. Members of the Phytophthora genus grouping with the Phytophthora taxon walnut in clade 6 are mainly reported as saprophytes or pathogens from riparian ecosystems and forests (2). To our knowledge, this is the first report of Phytophthora taxon walnut from Italy. Since the oomycete proved in our growth trial to be distinctly thermophilic, we hypothesize that its spread is being favored by the rising temperatures observed during the last decades in the area. References: (1) Y. Balci et al. Plant Dis. 91:705, 2007. (2) C. M. Brasier et al. Mycol. Res. 107:277, 2003. (3) D. E. L. Cooke et al. Fungal Gen. Biol. 30:17, 2000. (4) G. P. Robideau et al. Bioinformatics 19:1572, 2011.

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 × 107 conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4°C. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wall and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host cell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known β-1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

Between 2007 and 2011, Acer pseudoplatanus and Quercus robur trees declined in the North Park and the Boscoincittà Park in Milan, Italy (lat. 45° 27′ 47″ N, long. 09° 11′ 16″ E, elev. 121 m). Symptoms included extensive lengthwise bark cracks, with necrosis of the wood tissue underneath. Isolations were conducted from bark and infected wood after surface sterilization with 10% H2O2 for 10 min, followed by five washings in sterilized water. Tissue was placed on potato dextrose agar (PDA) in petri dishes amended with 0.06 g/l streptomycin at pH 6.5. The dishes were incubated in the dark at 24°C. Colonies reached a diameter of 7 to 8 cm in 7 days. They were whitish grey on the upper surface and blackish at the bottom, and consisted of dense aerial mycelia. The conidia were hyaline, ellipsoid, smooth, non-septate, thin-walled, with their upper end wider than their lower end, and measured 14 to 20 × 4.5 to 7 μm. The causal organism was identified as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips (basionym Fusicoccum parvum Pennycook & Samuels; teleomorph Botryosphaeria parva Pennycook & Samuels) based on biometric characteristics, common taxonomic keys, and the sequence information from the rDNA ITS region and the elongation factor 1-alpha (EF1-α) gene (GenBank Accession Nos. HQ893535 and HQ893537, respectively). All morphological traits corresponded to those of the holotype (1), while BLAST searches of the GenBank database revealed for both loci 100% homology with reference sequences. To confirm the involvement of N. parvum in the tree death, and fulfil Koch's postulates, 20 2-year-old seedlings of A. pseudoplatanus and Q. robur (10 each) were artificially infected in May with N. parvum in 2008 and in 2009. A bark portion was removed aseptically and a PDA disc (0.5 cm diameter) of N. parvum mycelium was placed on the wound. Control seedlings (3 per species) received sterile PDA discs. The inoculation site was wrapped in Parafilm for 10 days. After 3 weeks, infected seedlings showed sunken bark lesions associated with necrosis and bark cracks. When the bark was stripped, the wood below these lesions was also necrotic. Control seedlings developed no symptoms. Mycelium of N. parvum was successfully reisolated from the infected tissue. N. parvum is an important member of the Botryosphaeriaceae, a family of cosmopolitan endophytes that have been known for many years (2). The pathogen has been in recent years linked to complex syndromes, in which a prominent symptom is the formation of bark cankers. Although common on fruit crops, it has increasingly been reported on forest trees (3,4). References: (1) P. Crous et al. Stud. Mycol. 55:248, 2006. (2) P. A. Saccardo. Michelia 1:1, 1877. (3) M. E. Sánchez et al. Plant Dis. 87:1515, 2003. (4) B. Slippers and M. J. Wingfield. Fung. Biol. Rev. 21:90, 2007.

The Parco Nord Milano, Italy is a 600-ha green area (45°53′71″N, 9°20′97″E). Since the spring of 2002, extensive and unknown decline was observed on 20-year-old sycamore (Acer pseudoplatanus), red oak (Quercus rubra), and English oak (Q. robur) plantations. The trees showed branch and twig dieback associated with bark cankering along the whole stem and an irregular to wilted crown. A closer inspection of the inner part of the symptomatic trunk and roots revealed a dark bluish tissue discoloration. From the symptomatic trunk and root tissue, a dark gray abundant mycelium was consistently isolated on potato dextrose agar (PDA). The sequenced internal transcribed spacer (ITS) regions of rDNA (GenBank Accession Nos. DQ198265, DQ198266, and DQ198267) showed 99% identity to Botryosphaeria dothidea strain CBS110302 (GenBank Accession No. AY259092). A Fusicoccum sp. with black pycnidia was consistently collected from the cankers. The conidia were hyaline, aseptate, fusiform to narrowly ellipsoidal, with a truncate base (22 × 4.5 μm), referred to as Fusicoccum aesculi (CBS identification), the anamorph of B. dothidea (2). Pathogenicity tests were conducted for all three hosts by stem inoculation on 18-month-old seedlings growing in plastic pots containing a 2:1 turf/sand mixture. Two experiments were conducted using two inoculation techniques. In the first trial, 6-mm-diameter mycelial plugs of B. dothidea were applied to 6-mm-long bark wounds. The same inoculation method was used for application to bark without wounding. Control seedlings were inoculated with sterile agar plugs in a similar fashion as above. Inoculated and control seedlings were kept in a growth chamber and watered once per week. In the second trial, segments of branches 15-cm long were inoculated with 6-mm-diameter B. dothidea plugs (1), with and without any wounding. Control segments of branches were treated with sterile agar plugs in place of fungal mycelium. The branches were incubated at 23°C in moist chambers. For both experiments, the inoculated stem portions were wrapped with Parafilm to prevent desiccation. There were three replicate seedlings per inoculation technique, experiment, and plant species. After 2 months, all seedlings showed bark cankers and pycnidial formation, while the controls were symptomless. An inner dark bluish stem tissue discoloration was observed. The symptoms were more abundant on the segment of branches where the inoculum was applied to the wounded bark. B. dothidea was successfully reisolated, confirming Koch's postulates. To our knowledge, although it is accepted as synonymous of B. berengeriana (2), this is the first report of B. dothidea on sycamore, red oak, and English oak in Italy. The fungus was previously reported in Italy to cause canker on Platanus spp. References: (1) M. E. Sanchez et al. Plant Dis. 87:1515, 2003. (2) B. Slippers et al. Mycologia 96:83, 2004.