Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens

Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens

Accepted Manuscript Emerging new crown symptoms on Castanea sativa (Mill.): attempting to model interactions among pests and fungal pathogens Andrea V...

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Accepted Manuscript Emerging new crown symptoms on Castanea sativa (Mill.): attempting to model interactions among pests and fungal pathogens Andrea Vannini, Carmen Morales-Rodriguez, MariaPia Aleandri, Natalia Bruni, Matteo Dalla Valle, Tommaso Mazzetto, Diana Martignoni, AnnaMaria Vettraino PII:

S1878-6146(18)30100-4

DOI:

10.1016/j.funbio.2018.05.006

Reference:

FUNBIO 933

To appear in:

Fungal Biology

Received Date: 23 January 2018 Revised Date:

7 May 2018

Accepted Date: 22 May 2018

Please cite this article as: Vannini, A., Morales-Rodriguez, C., Aleandri, M., Bruni, N., Valle, M.D., Mazzetto, T., Martignoni, D., Vettraino, A., Emerging new crown symptoms on Castanea sativa (Mill.): attempting to model interactions among pests and fungal pathogens, Fungal Biology (2018), doi: 10.1016/j.funbio.2018.05.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Emerging new symptoms on chestnut

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1 Emerging new crown symptoms on Castanea sativa (Mill.): attempting to model interactions among

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pests and fungal pathogens

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Andrea Vannini, Carmen Morales-Rodriguez, MariaPia Aleandri, Natalia Bruni, Matteo Dalla Valle, Tommaso

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Mazzetto, Diana Martignoni, AnnaMaria Vettraino

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DIBAF – University of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo (Italy)

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Abstract

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In the 2015-2016 growing seasons, two novel symptoms were assessed on the crown of trees in orchards

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and coppices of chestnut groves in Central Italy. The first symptom was flagging of annual shoots with

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green leaves undergoing sudden wilt and turning brown later in the season. The second symptom consisted

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of leaves on annual shoots turning yellow before wilting in absence of flagging represented the second

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symptom. Samples were collected along transects in early summer, late summer and winter, and processed

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in the laboratory. The flagging symptom was associated in early summer with the presence of C. parasitica

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in cryptic dried buds on stems from the previous year's growth. The pathogen was also found in dormant

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buds in winter, suggesting that the infection could take place in summer during the Chinese gall wasp

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oviposition period. Cryphonectria parasitica was also isolated from abandoned galls in winter supporting

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the hypothesis that galls are a potential source of inoculum for crown infections. Aetiology of yellowing was

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not clarified and no fungal taxa were specifically associated with this symptom. Gnomoniopsis castanea,

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Cryphonectria parasitica and, in early summer, Colletotrichum acutatum were the most abundant fungal

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taxa isolated from chestnut shoots and buds.

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Keywords: chestnut diseases; flagging; yellowing; Cryphonectria parasitica; Gnomoniopsis castanea;

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Colletothricum acutatum

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Introduction

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European chestnut (Castanea sativa Mill.) is a multipurpose tree species of the Fagaceae family with a

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distribution range in all the countries facing the Mediterranean basin. Also present in the southern United

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Kingdom, Germany, Portugal, it forms vast pure natural forests. Sometimes these forests are maintained

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for enhanced fruit production or it is cultivated in orchards dedicated to fruit production. During the last

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two centuries, C. sativa was challenged by a number of invasive pests and diseases introduced through

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global trade of wood and non-wood products, including ink disease (Phytophthora spp.), chestnut blight

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(Cryphonectria parasitica (Murr.) Barr.) and, more recently, the Chinese gall wasp, Dryocosmus kuryphilus

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Yasumatsu (Conedera et al., 2016).

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European chestnut is also susceptible to many native and cryptic pests and pathogens mostly causing

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damages to fruits and other organs. Among these, the fruit moths (Pammene fascianav L., Cydia splendana

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(Hubner) and C. fagiglandana (Zeller)); the chestnut weevil, Curculio elephas Gyllenhaal; the fruit brown rot

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fungus Gnomoniopsis castanea G. Tamietti; and the chestnut leaf spot fungus, Mycosphaerella

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maculiformis (Pers.) J. Schröt (Conedera et al., 2016).

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In a general context of global changes, the interaction among native and exotic pests and pathogens

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increase the deleterious effects on an ecosystem, such as the domesticated chestnut groves, where the

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deployment of selected cultivars, the use of pesticides and the pressure of a changing climate sensibly

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decrease their resilience. For instance, G. castanea, a common endophyte of chestnut, is currently severely

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affecting fruit production, causing brown rot of kernels in post-harvest with incidence reaching 60% of the

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total production (Vannini et al., 2017). Its massive presence in chestnut groves was supposed to be

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associated with the Chinese gall wasp infestation (Vannini et al., 2017), specifically to the production of

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secondary inoculum on colonized galls (Maresi et al., 2013). Similarly, Meyer et al. (2015) proposed an

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interaction between the gall wasp infestation and C. parasitica isolated from abandoned galls.

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In such an ecological context, in the 2015-2016 seasons, two previously undescribed symptoms have been

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recorded in Central Italy on the crown of adult chestnut trees, in areas heavily infested by Chinese gall

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wasp. The first symptom concerns the flagging of current year green shoots scattered around the crown

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(Figure 1A). The symptom appears early in the season (May-June), after bud break. Green shoots suddenly

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wilt without discolouration assuming a typical flagging appearance (Figure 1B). At closer observation,

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cryptic dried buds on the previous year's stems are constantly associated with flagging (Figure 1C).

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In July-August, the shoots turn necrotic and appear brown and withered. The leaves remain on the tree

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even after leaf shading, remaining visible on the crown during the winter (Figure 1A). Cankers caused by C.

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parasitica are sometime present on the growth of the previous year's stems. This symptom does not

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resemble those reported in Switzerland by Prospero and Forster (2011) and Pasche et al. (2016) which was

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attributed to C. parasitica and G. castanea, respectively.

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The second symptom can be noticed in May-June as well, and includes a rapid discolouration of leaves on

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the shoot turning yellow but maintaining turgor (Figure 2). Later in the season, the leaves withered on the

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crown.

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The aims of this work were to study the aetiology of these novel symptoms, to clarify the epidemiological

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implications, and discover possible associations with other factors challenging the chestnut ecosystem.

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Materials and methods

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Sampling of plant material

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The study was carried out in 2015 and 2016, in one of the largest chestnut districts in Central Italy, the

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Monti Cimini area (province of Viterbo). Highly productive, managed orchards and coppices characterize

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this chestnut growing area. Dryocosmus kuriphilus has been present since 2006 causing severe infestations

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and damage (Vannini et al., 2017).

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Sampling was conducted along two linear transects, about 2 km in length. Transect 1 (T1) was located

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north-west of the volcanic cone of Vico Lake (42°21'36"N 12°07'28"E) in the municipality of Viterbo.

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Transect 2 (T2) was located at a location north of the volcanic cone of Vico Lake (42°21'48"N 12°12'00"E) in

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the municipality of Canepina. A minimum of twenty adult trees were tagged along each transect to

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uniformly cover the 2 km distance (Table 1).

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The first sampling was conducted along T1 on September 26, 2015. At this period of the season, the

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flagging symptoms were wilted and brown. Three typologies of samples were collected (20 samples for

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each category and tree): i) flagging shoots without visible cankers on the previous year growth (FLG-NC); ii)

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flagging shoots with cankers visible on the previous year growth (FLG-C); iii) healthy shoots (HLS) without

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evident symptoms. For each sample, the following sub-samples were considered for fungal detection: a)

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the bark at the interface between healthy and necrotised tissue, and b) the leaf blade including the main

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leaf vein.

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The second sampling was conducted along T1 on January 13, 2016 during dormancy. Four typologies of

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samples were collected (25 samples for each category): i) abandoned galls of Chinese gall wasp (GLH) and

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dormant buds (BDH) on healthy twigs; ii) abandoned galls (GLC) and dormant buds (BDC) on twigs with

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active cankers at the base of the twig.

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The third sampling was conducted along T2 on June 16, 2016 about one month after bud break. Three

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typologies of samples were collected from each of ten trees along the transect: i) FLG-NC; ii) yellowing

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shoots in absence of flagging (YLS); iii) HLS. For each sample, the following sub-samples were considered: a)

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the bark at the interface between healthy and necrotized tissue, b) galls present on the sample, and c)

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cryptic dried buds on the previous year's growth.

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Fungal isolation

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Each plant sample/sub-sample was cut in seven fragments not exceeding 5 x 5 mm. Each fragment was

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surface sterilized with 60% ethanol for 30 s, rinsed three times in sterile distilled-H2O and dried on filter

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paper. Fragments were placed onto Petri dishes containing potato dextrose agar (PDA, Oxoid, UK, 39 g l-1)

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amended with streptomycin sulphate (0,06 g l-1), and incubated at 20±2 °C. After 7 d of incubation, plates

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were observed for the presence of fungal colonies. Identification was based on colony and reproductive

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structure morphology, and molecular traits (see below).

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Molecular identification of fungi

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DNA was extracted from fresh mycelium grown in PDB (potato dextrose broth) with the Nucleo- Spin Plant

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II mini kit (Mackery Nagel, Germany) following the manufacturers’ instructions. DNA concentration was

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assessed by gel electrophoresis and DNA was diluted 1:10 to perform PCR. Amplification of EF 1-a, RPB II, β-tubulin (only for G. castanea) and ITS (for all morphotypes) was carried out

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using the primers designed for other studies (Table S1, Supplementary material)).

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For each PCR reaction, the master mix consisted of 2X MyTaq MIX (Bioline, UK), 0.50 μM of each primer

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and approximately 5–20 ng DNA in a final reaction volume of 25 μL. Cycling conditions consisted of an

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initial denaturation of 3 min at 95°C, followed by 35 cycles consisting of 15 sec at 94°C, 15 sec at the

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respective annealing temperature and 10 sec at 72°C, followed by a final elongation of 5–10 min at 72°C.

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Amplicons were purified with NucleoSpin Gel and PCR Clean-up (Mackery Nagel). Sequencing reactions

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were performed by Macrogen Europe Laboratory (Amsterdam, The Netherlands) and forward and reverse

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sequences were assembled and edited using BioEdit (Ibis Bioscience) and compared to NCBI database

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(https://blast.ncbi.nlm.nih.gov/ Blast.cgi?PAGE_TYPE=BlastSearch).

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For phenotype assessment, field isolates of C. parasitica were grown on PDA for 7 days in the dark,

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followed by 7 days in daylight at room temperature. Hypovirus infected isolates were identified by their

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white culture morphology (Bissegger, Rigling and Heiniger, 1997).

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The VC type of the C. parasitica cultures was assessed according to the merging/barrage response with C.

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parasitica cultures belonging to known VC groups (Anagnostakis 1987; Bissegger, Rigling and Heiniger,

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1997). Cultures were paired on PDA with a tester strain of the most common VC types in Italy (EU-1, EU-2,

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EU-5, EU-12) (Cortesi et al., 1998). Each test for each isolate was performed in triplicate.

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Statistical analysis

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Selection of appropriate statistical analysis depended on the type of data obtained. Some tissue/symptom

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variable resulted in no-isolation of specific fungal taxa from any of the replicates. These data with zero

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mean value and zero variance, could not be considered in the dataset foreseen for multivariate analysis

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(e.g. Two-way ANOVA). In such cases simpler datasets had to be considered and analyzed with univariate 5

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One-way ANOVA, and Unpaired t-test (with or without Welch correction). Tukey’s multiple comparison

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tests was used as post-test. All the analyses, including linear regression, were carried out with Graphpad

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Prism version 7.00 (GraphPad Software, San Diego California USA).

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Results

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Samplings

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Results of the first sampling (September 2015) are illustrated in Figure 3. A total of 259 isolates of G.

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castanea were obtained. Gnomoniopsis castanea was abundantly isolated from leaf blades and, to a lesser

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extent, from lignified shoot tissues.

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Not significant differences were found between HLS, FLG-NC and FLG-C in frequency of isolation of G.

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castanea from leaf tissues (One-way ANOVA F=0.49, P>0.05). Gnomoniopsis castanea was not isolated from

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bark of FLG-C. No difference in frequency of isolation from bark was found between HLS and FLG-NC

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(Unpaired t-test, P>0.05) (Figure 3A). Significant differences were found between leaf and bark of HLS

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(Unpaired t-test, P<0.05) and FLG-NC (Unpaired t-test, P<0.0001) (Figure 3A).

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A total of 105 isolates of C. parasitica were obtained. Cryphonectria parasitica was not isolated from HLS

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samples (leaf and bark). The pathogen was isolated from bark tissues of FLG-NC and FLG-C, while frequency

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of isolation from leaf blades of FLG-C and FLG-NC was negligible (0.04) and null respectively (Figure 3B). No

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significant difference was found in frequency of isolation of C. parasitica between bark samples of FLG-NC

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and FLG-C (Unpaired t-test, P>0.05), while difference between leaf and bark of FLG-C was significant

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(Unpaired t-test, P<=0.0013) (Figure 3B). No further fungal taxa were investigated in this experiment.

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Results of the second sampling (January 2016) are illustrated in Figure 4. Gnomoniopsis castanea (348

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isolates) and C. parasitica (53 isolates) were the most frequent fungal taxa. Gnomoniopsis castanea was

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isolated at the highest frequencies from all categories, dormant buds and abandoned galls with and

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without active cankers on the shoots. No statistical effect of the variables ‘tissue’ and ‘symptom’ was found

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(P>0.05) (Figure 4A). Cryphonectria parasitica was also isolated from all the categories. Two-Way ANOVA

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suggested an overall weak effect of the variable ‘tissue’ on C. parasitica isolation, explaining the 5.1% of 6

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total variance (P<0.02). However no significant differences were found between tissues within each

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symptom category at the Tukey’s multiple comparison test (P>0.05) (Figure 4B). There was no interaction

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between variables (P>0.05).

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Linear regression analysis comparing the isolation frequency of G. castanea vs. C. parasitica resulted in

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significant R squares for both abandoned galls and dormant buds (R square 0.52, F 24,6, P<0.0001; R square

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0.35, F 12.6, P<0.0017 for abandoned galls and dormant buds, respectively) (Figure 5). Based on these

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results, the presence of G. castanea was inversely related to the presence of C. parasitica in the tissues.

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Additional taxa were isolated from tissues, Colletothricum acutatum (14 isolates); Botryosphaeria dothidea

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(9 isolates); Fusarium spp (4 isolates); Sordaria sp. (2 isolates); Penicillium sp. (2 isolates).

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Results of the third sampling (June 2016) revealed the presence of three main fungal taxa associated with

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chestnut tissues, Gnomoniopsis castanea (477 isolates), Cryphonectria parasitica (79 isolates), and

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Colletotothricum acutatum (64 isolates). Additional taxa were isolated from FLG-NC and YLS, but HLS, at

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frequencies below 0.05%. They included Fusarium sp. (10 isolates); Botryosphaeria dothidea (3 isolates);

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Aureobasidium sp. (3 isolates); Sordaria sp. (1 isolate), Penicillium sp. (2 isolates).

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In Figure 6, the frequency of isolation of the three main taxa from healthy control, flagging and yellowing

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symptoms is shown. Cryphonectria parasitica was associated with all the FLG-NC samples processed.

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Cryphonectria parasitica was not isolated from controls while frequency of isolation from yellowing

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samples was very low (0.003; one bark sample positive out of 44 processed). Gnomoniopsis castanea was

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the most abundant taxa isolated, while the frequency of isolation of C. acutatum was always below 0.2

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(Figure 6).

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In Figure 7, the isolation frequencies of the three taxa are reported per tissue and symptom category.

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Frequencies of isolation of G. castanea from bark (ANOVA, F=1.9, P>0.05) and galls (ANOVA, F=0.18,

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P>0.05) were not significantly different between healthy controls (HLS), flagging (FLG-NC) and yellows (YLS)

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(Figure 7A). Presence of G. castanea in cryptic dried buds was significantly higher in yellows than in flagging

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(Unpaired t-test, P=0.0024). Cryptic dried buds were not present on healthy controls. Within flagging

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symptoms, frequency of G. castanea was significantly lower in cryptic dried buds compared to gall and bark

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(Tukey’s multiple comparison test, P<0.0001). (Figure 7A).

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Cryphonectria parasitica was not isolated from healthy controls. Its frequency of isolation was also

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negligible from yellows, where the pathogen was isolated at very low frequency from bark tissues (0.007).

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A significant effect of tissue type was found on isolation of C. parasitica (ANOVA, F=68.39, P<0.0001).

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Frequency of isolation of C. parasitica was significantly lower in galls compared to both cryptic dried buds

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and bark (Tukey’s multiple comparison test, P<0001).

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No effects of tissues and symptoms were found on isolation frequency of C. acutatum

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Cryphonectria parasitica isolates

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All but three isolates of C. parasitica recovered (237) showed an orange phenotype with abundant

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production of reproductive asexual fruiting bodies on stromata, suggesting absence of Hypovirus infection.

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VC types of the field isolates is reported in Figure 8. The four most frequent types in Italy, EU-1, EU-2, EU-5

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and EU-12 (Cortesi et al., 1998), accounted for 82% of the isolates. Noticeable, the isolates of C. parasitica

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from the dried bud and the bark of the same FLG-NC sample belonged to the same VC type.

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Discussion

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In the present study, two new crown symptoms of European chestnut in Italy have been investigated about

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their aetiology and epidemiology. Growers first recorded both symptoms during the 2014 growing season

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corresponding to the peak of Chinese gall wasp infestation and impact in Central Italy (Vannini et al., 2017).

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Flagging

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Flags are common crown symptoms of C. parasitica, being the consequence of necrotic lesions and cankers

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on branches (Rigling and Prospero, 2018). Any fresh lesion or crack on branches caused by different agents

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(wind, fire, drought, and artificial wounds) serve as entry points for the fungus. The fungus colonizes the

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bark tissues and meristems causing necrosis of the branch and the wilt of clusters of annual shoots, in the

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current season or after 1-2 years, depending on age and size of the branch.

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The flagging symptoms reported here differ from this well-known classical flagging. The point of departure

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between the classical flagging symptom and the new flagging symptoms described here mainly concerns

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the sudden wilt in early summer (May-June) of single current year shoots scattered around the crown

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(Figure 1A). Furthermore, it is suggested that the presence of cankers infected with C. parasitica on the

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previous year's stem growth, as well as the presence of abandoned Chinese gall wasp galls, are not

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preconditions for development of the symptom. This newly described flagging is, instead, associated with

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the colonization of C. parasitica from cryptic dried buds. Necrotrophic and toxic activity associated with C.

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parasitica infections, (Zhang et al., 2013) even on green tissues (Newhouse et al., 2014), might account for

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the collapse and wilt of annual shoots in early summer in the absence of classical cankers on older stem

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growth.

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In such context, the understanding of the time of bud infection is a major issue in order to clarify the cycle

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of the disease. According to what suggested by Prospero and Foster (2011), C. parasitica can colonize

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tissues by endophytic inoculum or by external inoculum through fresh wounds. The endophytic presence of

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C. parasitica in healthy chestnut tissues is well known since long time in literature (Bissegger and

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Sieber,1994) while this flagging symptom was firstly noticed in 2014, eight years after the first record of the

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Chinese gall wasp in the area.

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An alternative hypothesis is that the Chinese gall wasp provides the fungus appropriate penetration sites to

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infect buds. Chestnut starts to differentiate new axillary buds in late May. These buds enter dormancy

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during fall until bud break the following spring. Chinese gall wasp oviposition activity takes place in July-

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August and causes micro-wounds on the buds. In highly infested areas, more than one wasp may oviposit in

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the same bud (Kato and Hijii, 2001). Cryphonectria parasitica requires fresh wounds to establish the

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infection (Rigling and Prospero, 2018). Thus, the infection might take place at the time of oviposition (July-

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August) by means of vectored inoculum. As evidenced in the present study, and reported by Prospero and

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Foster (2011) and Mayer et al.(2015), abandoned galls represent an optimal source of C. parasitica

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inoculum at the distal part of the crown, that can be easily spread by wind, insects and water (depending

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on the type of inoculum, conidia or ascospores). The isolation of a majority of Hypovirus free C. parasitica

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isolates belonging to the most diffused VC types of the area (Pasquini et al., 2009) from flagging symptoms

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supports the hypothesis of Meyer et al. (2015), that airborne ascospores are mostly involved in the

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infection processes at the distal part of the crown. In fact, during Chinese gall wasp oviposition in July-

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August, C. parasitica ascospores are still discharged from perithecia in stromata (Guerin et al., 2001) and

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commonly dispersed by wind or possibly insects. However, there is no evidence that D. kuriphilus adults

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carry C. parasitica inoculum to buds. Cryphonectria parasitica has been isolated from dormant buds in

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winter suggesting that the fungus also overwinters in the buds. Guerin and Robin (2003) demonstrated that

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C. parasitica infections result in null or poor lesion development from summer throughout the fall and

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winter season. Therefore, it can be assumed that after bud infection in summer, C. parasitica remains in

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latency or expresses a very limited amount of tissue colonization. In spring is considered the best season for

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C. parasitica to develop lesions as it was determined that the fungus colonizes and kills the tissues before

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bud break (Guerin and Robin, 2003).

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Studies on the interaction between Chinese gall wasp and C. parasitica have been previously reported. For

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example, Prospero and Foster (2011) observed dieback on first and second season stems and the presence

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of abandoned galls and cankers on the stems. The authors speculated that the abandoned galls might

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function as a reservoir of C. parasitica inoculum for disease development.

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Additional taxa were found associated with flagging, as well as with the yellowing symptoms. Gnomoniopsis

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castanea was the taxon most frequently isolated from healthy and symptomatic tissues. The massive

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endophytic colonization of chestnut tissues and organs by G. castanea has been reported by several studies

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(Shuttleworth et al. 2012; Visentin et al. ,2012; Vannini et al., 2017). In the present study, G. castanea

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showed a preference for green tissues (leaves?) compared to bark tissues, while C. parasitica was the

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opposite. Such differences probably account for the low frequency of isolation of G. castanea from cryptic

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dried buds colonized by C. parasitica. The pathogenic nature of G. castanea on several organs of chestnut is

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also well known. The fungus is recognized as the agent of brown rot of kernels (Smith & Ogilvy 2008;

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Visentin et al. 2012; Shuttleworth et al. 2013; Dennert et al. 2015), of twig cankers in India and Europe (Dar

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& Rai 2015; Pasche et al.. 2016), and of gall necrosis (Magro et al., 2010; Seddaiu et al., 2017). In the

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present study, G. castanea was not statistically associated with any of these symptoms. The frequency of

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other taxa, such as C. acutatum, increased from both healthy and symptomatic tissues in early summer,

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however, its presence was not statistically associated with any symptom category. Meyer et al. (2015) and

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Gaffuri et al. (2015) have reported C. acutatum as a frequent colonizer of abandoned galls. Gaffuri et al.

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(2017) also reported C. acutatum as the agent of pink rot of chestnut kernels. Colletotrichum acutatum is a

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cosmopolitan fungal species associated with hundreds of woody and herbaceous hosts, on which it can

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cause anthracnose, canker, leaf spot, shepherd's crook, fruit rot, seedling damping off and blight, and

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cutting dieback (Farr and Rossman, 2018).

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Yellowing

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Aetiology of this symptom is difficult to determine at this time. Fungal community isolated from the

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symptom was not different from that isolated from healthy controls. The taxa isolated at higher

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frequencies were G. castanea and C. acutatum while C. parasitica was extremely rare. A chestnut yellow

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disease was previously described on Castanea crenata by Jung et al. (2002) in Korea and by Mittempergher

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and Sfalanga (1998) and Vettraino et al. (2005) on Castanea sativa in Italy. The Asiatic record was

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associated with ‘Candidatus Phytoplasma castaneae’, while no phytoplasmas or viruses were detected from

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the chestnut yellows in Italy. Symptoms described in the present study are somehow different from those

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recorded by Mittempergher and Sfalanga (1998) and Vettraino et al. (2005), where, in addition to yellowing

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and distortion, the leaves appeared smaller and greenish until late in the season. Furthermore, such

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symptomatology was diffuse on the whole crown and limited to the Marrone variety. A similar symptom

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on leaf blade is produced by G. castanea colonizing galls differentiated on the leaf blade. However, at the

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moment any aetiological explanation must be considered speculative.

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In conclusion, the ‘Chinese gall wasp’ caused a tremendous impact in natural and domesticated chestnut

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ecosystems. Direct damage by the insect to tree vegetative vigour and physiology are known and described

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(Battisti et al., 2014). Furthermore, the invasion by this alien insect determined qualitative and quantitative

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changes in the chestnut ecosystem resulting in a general decrease of resilience and changes in population

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dynamics and growth of some microorganisms (e.g. G. castanea)(Vannini et al., 2017), and changes in the

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biological and disease cycle of primary pathogens as C. parasitica (Meyer et al., 2015). Impact of alien

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invasive pests is frequently based on the direct damage to the main host(s), while the interaction of the

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invader with other components of the biome, and the resulting risk of unexpected synergies, should be

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equally considered and investigated.

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Acknowledgment

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A special thanks to Fabrizio Pini, grower and president of the Agriculture Italian Confederation for the

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Province of Viterbo, for providing the first record of the symptoms described.

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oxidase transgene expression reduces Cryphonectria parasitica-induced necrosis in a transgenic

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American chestnut (Castanea dentata) leaf bioassay. Transgenic Research 22, 973-982.

360 Figure captions

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Figure 1. Symptom of flagging: (A) appearance of flagging on the chestnut crown in winter; (B) appearance

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of symptom of flagging in early summer; (C) cryptic dried bud at the base of flagging with evident necrosis

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of tissues

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Figure 2. Symptom of yellowing on chestnut crown in early summer

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Figure 3. Isolation frequency of Gnomoniopsis castanea (A) and Cryphonectria parasitica (B) from leaves

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and bark of healthy controls (HLS), flagging shoots without (FLG-NC) and with (FLG-C) an obvious canker at

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the base of the shoot. Results refer to the September 2015 sampling. Vertical bars represent SE. Same

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upper-case letter indicates no significant differences between symptoms (HLS, FLG-NC, FLG-C) within the

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same tissue. Same lower-case letter indicates no significant differences between tissues (leaf and bark)

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within the same symptom. P>0.05 at Unpaired t-test or Tukey’s multiple comparison test (refer to Results

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section). NI= Taxa not Isolated.

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Figure 4. Isolation frequency of Gnomoniopsis castanea (A) and Cryphonectria parasitica (B) from dormant

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buds and abandoned galls in winter (second sampling, January 2016). Dormant buds with (BDC) and

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without (BDH) a canker on the shoot; abandoned galls with (GLC) and without (GLH) a canker on the shoot.

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Vertical bars represent SE. Same upper-case letter indicates no significant differences between symptoms

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(canker, no-canker) within the same tissue. Same lower-case letter indicates no significant differences

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between tissues (buds and galls) within the same symptom. Tukey’s multiple comparison test (P>0.05).

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Figure 5. Linear regression between isolation frequencies of Gnomoniopsis castanea and Cryphonectria

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parasitica from abandoned galls (red line) and dormant buds (black line). Dotted lines represent the 95%

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confidence level (R square 0.52, F 24,6, P<0.0001; R square 0.35, F 12.6, P<0.0017 for abandoned galls and

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dormant buds, respectively)

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Figure 6. Frequency of isolation of the three main fungal taxa from healthy control (A), flagging (B) and

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yellowing (C) symptoms. Results refer to the third sampling (June 2016). Vertical bars represent SE. Same

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letter indicates no significant differences between means at the Unpair t-test or Tukey’s multiple

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comparison test (P>0.05) (refer to Results section). NI= Taxa not isolated.

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Figure 7. Isolation frequencies of the three main fungal taxa from bark, and galls of healthy controls (HLS),

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flagging shoots without an obvious canker at the base (FLG-NC), and yellowing symptom (YLS). Vertical bars

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represent SE. Same upper-case letter indicates no significant differences between symptoms (HLS, FLG-NC

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and YLS) within the same tissue. Same lower-case letter indicates no significant differences between bark

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and galls within the same symptom. P>0.05 at Unpaired t-test or Tukey’s multiple comparison test (refer to

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Results section). NI= Taxa not Isolated.

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Figure 8. Composition among VC groups of the Cryphonectria parasitica population isolated from flagging

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symptoms in 2015-2016 compared with that reported in 2005 by Pasquini (2009) (up right)

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Sampling

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orchard

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42°21'48"N 12°12'00"E

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