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Australasian Plant Pathol.

(2017) 46:397–405
DOI 10.1007/s13313-017-0502-3


The infection process of chestnut rot, an important disease caused

by Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales)
in Oceania and Europe
L. A. Shuttleworth 1 & D. I. Guest 1

Received: 30 October 2016 / Accepted: 6 June 2017 / Published online: 3 July 2017
# Australasian Plant Pathology Society Inc. 2017

Abstract Chestnut rot is an important disease of chestnut trees, particularly the flowers. The fungus then exists as a
trees in Oceania (Australia, New Zealand), and Europe latent pathogen in reproductive and vegetative tissues, leading
(Italy, France, Switzerland, United Kingdom). The causal to the development of the disease during nut maturity.
agent has been identified as the fungus Gnomoniopsis
smithogilvyi (Gnomoniaceae, Diaporthales). Koch’s Keywords Disease cycle . Koch’s postulates . Spore trapping
Postulates was demonstrated on nuts from three Australian
chestnut varieties with G. smithogilvyi from Australia, New
Zealand and Italy. The Australian and New Zealand isolates Introduction
were pathogenic on all three varieties, however the Italian
isolate produced smaller lesions on all three, and was only Chestnut rot is an important disease of chestnut trees and has
mildly pathogenic on Decoppi Marone. Infection of chestnut been reported on Castanea sativa, and C. crenata × C. sativa
floral and vegetative tissues was investigated during the chest- hybrids in Oceania (Australia, New Zealand) where they are
nut phenological stages of flowering (sampled twice over two grown as a tree crop, and on C. sativa in Europe (France, Italy,
years), immature nuts and burrs, mature nuts and burrs, and Switzerland, United Kingdom) where they are grown as tree
tree dormancy (immature nuts and burrs, mature nuts and crops, as ornamental trees, and occur as components of native
burrs, and tree dormancy were sampled once in one year). forests (Shuttleworth et al. 2012a, b; Visentin et al. 2012;
Gnomoniopsis smithogilvyi was present in all the sampled Dennert et al. 2015; Lione et al. 2015; EPPO 2016). In addi-
phenological stages, most frequently from female flowers dur- tion to Castanea spp., Gnomoniopsis smithogilvyi has recently
ing the flowering period. In the first year, the fungus infected been reported as an associate of branch diseases of Corylus
82% of female flowers, while in the second year only 10% avellana (hazelnut) in Italy (Linaldeddu et al. 2016).
were infected. Ascospore trapping in a laboratory chamber Chestnut rot affects the kernel of the nuts, resulting in
experiment confirmed ascopores are released in to the air from browning and necrosis of the endosperm and embryo
infected burrs. Ascospore trapping in an Australian chestnut (Shuttleworth et al. 2012a, b). The disease is mainly
orchard during the flowering period also showed ascospores expressed post-harvest, however reports by Australian
are released in to the air, with peak trapping times at sunset growers show the disease can occur on nuts still attached
and the hours following sunset (8-11 pm), and the hours fol- to the tree. The disease is cryptic, where healthy looking
lowing sunrise (7-9 am). These experiments support the hy- nuts on the surface are rotten internally. In Australia,
pothesis that ascospores of G. smithogilvyi infect chestnut chestnut rot incidence up to 72% has been found
(Shuttleworth et al. 2012b), in north-west Italy up to
93.5% (Lione et al. 2015; Visentin et al. 2012) and in
* D. I. Guest
Switzerland up to 21% (Dennert et al. 2015).
Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales)
Sydney Institute of Agriculture, University of Sydney, 1 Central
has been identified as the main causal agent (Shuttleworth
Avenue, Australian Technology Park, Eveleigh, Sydney, NSW 2015, et al. 2015, 2012b). In addition to chestnut rot,
Australia G. smithogilvyi has been reported to cause canker of
398 Shuttleworth L.A., Guest D.I.

C. sativa in India (Dar and Rai 2015), and canker of grafting Taurinensis, University of Turin, Italy) and one from Italy
scions in Switzerland (Pasche et al. 2016). The fungus is also (MUT401). Conidia were produced from one-week old cul-
associated with galls caused by the chestnut gall wasp, tures grown on malt extract agar (MEA) at room temperature
Dryocosmus kuriphilus in Italy and Switzerland (Vinale under 12 h light/dark cycles. The plates were flooded with
et al. 2014; Meyer et al. 2015), and has been reported to reduce sterile water and stirred manually with a sterile glass rod.
the abundance of Cryphonectria parasitica associated with Conidia numbers were quantified with a haemocytometer.
chestnut galls (Meyer et al. 2015). Ten μL sterile distilled water was applied to each of the nuts
The infection process of chestnut rot is still being in the control treatment. Twenty nuts were inoculated for each
unravelled. Asymptomatic infection of male and female isolate, 50 conidia per nut. After application, the nuts were
flowers, nuts, burrs and branches has been reported in wrapped individually in Parafilm to prevent contamination
Australia (Shuttleworth et al. 2012a), from nuts, flowers and from opportunistic microbes.
branches in Italy (Visentin et al. 2012), and nuts, branches, One week after inoculation, nuts were cut longitudinally
and leaves in Switzerland (Pasche et al. 2016; Dennert et al. from the stylar to hylum end and the lesion lengths were
2015). As the infection was asymptomatic, it was assumed measured. For standardisation, if a nut had a lesion along its
that the fungus colonised chestnut tissues as an endophyte, entire length it was recorded as 23 mm, as this was the mean
becoming pathogenic as the nuts ripened. However, Ogilvy total length of nuts used in the experiment. An analysis of
(1998) reported a correlation between rainfall during the variance (ANOVA, p< 0.05) was completed in Genstat
flowering period and increased chestnut rot incidence, sug- Version 17 (VSN International Ltd., United Kingdom) to test
gesting a latent pathogenic infection of flowers. Smith & if lesion lengths of each isolate tested on each variety were
Ogilvy (2008) found that ascospores released from dead significantly different to the control.
chestnut litter on the orchard floor was the primary inoculum
initiating chestnut rot. Visentin et al. (2012) also reported that Experiment 2. Isolation of G. smithogilvyi
25% of artificially infected chestnut flowers developed chest- from asymptomatic floral and vegetative tissues
nut rot. Therefore the role of endophytic colonisation in the
development of nut rot remains unclear. Various asymptomatic floral and vegetative tissues of
The current study had three aims. The first was to complete chestnut trees were collected from Mullion Creek, New
pathogenicity testing on nuts from Australian chestnut varie- South Wales on a single day between the 1st-3rd of
ties with isolates of G. smithogilvyi from Australia, New each month in December 2008 and February, April,
Zealand and Italy. The second was to further investigate the August and December 2009. These months represent
infection process through isolations from chestnut floral and various phenological stages of chestnut trees including
vegetative tissues representing four phenological stages of the flowering, immature nuts and burrs, mature nuts and
trees, the third was to further investigate the hypothesis of burrs, and tree dormancy. Fifteen trees were selected,
floral infection via air-borne ascospores both in the laboratory three varieties (Red Spanish, Decoppi Marone, Purton’s
and in the field. Pride), and 5 trees per variety. Five branches were col-
lected per tree, 75 branches in total (except December
2009 when 30 branches in total were collected).
Materials and methods Sections of the various tissues were cut 5 mm 3 to
1 cm3 in size, except female flowers and dormant buds,
Experiment 1. Pathogenicity testing of G. smithogilvyi which were kept whole. The time of surface-
isolates from Australia, New Zealand and Italy on nuts disinfestation depended on the tissue type. Softer tissues
from Australian chestnut varieties such as female and male flowers, peduncles, the various
leaf structures (petioles, mid-veins, margins) and current-
Nuts of three commercial Australian chestnut varieties were year branches were added to 95% ethanol for 30 s, 10%
tested, Red Spanish, Decoppi Marone and Purton’s Pride. The NaOCl for 1 min, rinsed with sterile deionised water,
shell and pellicle were removed from nuts, and immersed in added to 95% ethanol for 30 s, and then air-dried on
10% NaOCl for 3 min, 70% ethanol for 1 min, and air-dried lint-free tissue. Samples were then cut into quarters and
on lint-free tissue. The stylar end of each nut was asceptically plated on to 2% MEA. Isolation percentage for each tis-
cut to expose a 5 mm2 surface on to which the prepared co- sue type was then calculated. A selection of the isolates
nidial suspensions were applied. Three isolates of from female and male flowers, terminal leaf margin, first
G. smithogilvyi were tested on each variety, one from and second year branches were identified phylogenetical-
Australia (CBS130190 ex-type; CBS = Centraalbureau voor ly and morphologically as G. smithogilvyi (Shuttleworth
Schimmelcultures, Utrecht, the Netherlands), one from New et al. 2015, 2012b, see also MycoBank record MB
Zealand (MUT411; MUT = Mycotheca Universitatis 800259). The remaining colonies that were not sequenced
The infection process of chestnut rot 399

were morphologically identified with reference to the ex- The Netherlands, as CBS 133189, with a duplicate de-
type culture of G. smithogilvyi CBS 130190 posited at the Royal Botanic Garden, Sydney as RBG
(Shuttleworth et al. 2012b). 5715.
Isolations of G. smithogilvyi from female flowers in 2008
was compared to rot incidence in 2009, and the isolation of Sequencing, phylogenetic analyses and GenBank accessions
G. smithogilvyi from female flowers in 2009 was compared to
rot incidence in 2010. Chestnut rot incidence (%) was deter- Individual locus phylogenies were inferred and genealog-
mined using the dissection and visual assessment protocols ical concordance phylogenetic species recognition
described in Shuttleworth et al. (2012a). In 2009, 293 chest- (GCPSR; Taylor et al. 2000) used to determine the phy-
nuts were sampled from three varieties, Decoppi Marone logenetic identity of one of the captured isolates. The
(n = 107), Purton’s Pride (n = 93) and Red Spanish (n = 93). internal transcribed spacer regions of rDNA including
In 2010, 288 chestnuts were sampled from the same varieties, 5.8S (ITS), partial translation elongation factor 1-alpha
RS (n = 100), DM (n = 86), and PP (n = 102). The results were (TEF1-α), and beta-tubulin (β-tubulin) were used. PCR
pooled and chestnut rot incidence (%) was determined. conditions were identical to Walker et al. (2010). The
primers used to amplify and sequence for ITS were
Experiment 3. Capturing G. smithogilvyi ascospores ITS5/ITS4 (White et al. 1990), for TEF1-α they were
in the laboratory EF1-728F (Carbone and Kohn 1999)/EF1-1567R
(Rehner 2001), and for β-tubulin they were T1/T2
A chamber experiment was designed to capture ascospores (O'Donnell and Cigelnik 1997). Both the forward and re-
from infected burrs in the laboratory. Four dead chestnut burr verse strands were sequenced to reduce the presence of
fragments, variety Red Spanish, were collected from Mullion ambiguous nucleotides. Sanger sequencing was performed
Creek, NSW. Burrs were placed in a 35 cm × 30 cm × 20 cm by the Ramaciotti Centre, University of New South
rectangular plastic container with a ‘Cool Master’ computer fan Wales. Sixteen reference taxa from the Gnomoniaceae
(model: A12025-12CB-3BN-F1. DC1 2 V, 0.16A) on one side, were used in the phylogenetic analyses including the ex-
and 6.5 cm diameter agar plates containing 2% potato dextrose type culture of G. smithogilvyi (CBS 130190), and
agar (PDA) on the other side from the burrs. Before placing the outgroups Ophiognomonia setacea CBS 128354 and
burrs into the container, they were sprayed with sterile water Plagiostoma sp. CBS 128351. Sequences for the
until saturation. To increase humidity, tissues were soaked with reference taxa were sourced from Walker et al. (2010)
sterile water and placed in each corner of the container, and and Walker et al. (2012). Gnomoniopsis guttulata (MS
three 5.5 cm diam agar plates were filled with sterile water 0312) was excluded from the TEF1-α and β-tubulin anal-
and positioned in front of the fan. The lids of agar plates con- yses as there were no sequences available on GenBank.
taining agar were not removed until the fan, burrs, moistened Sequences were aligned using the MAFFT option of
tissues and water in the agar plates were in position. This was to Geneious Version 7 Geneious® R7 v7.1.2 (Biomatters
prevent contamination of the agar by physical contact with Ltd., New Zealand) with default settings applied.
objects. The agar plate lids were removed, and the top of the Consensus sequences were trimmed and edited manually
container was sealed with plastic cling wrap and taped closed to where necessary. The sequence length for each alignment
stop external airborne spores contaminating the agar plates. The after trimming was ITS: 503 bp, TEF1-α: 425 bp and β-
burrs were incubated for 2 × 1 week intervals under 12 h l/d tubulin: 407 bp. Maximum parsimony (MP) and Bayesian
23 °C. The agar plates were observed at the end of the first week analyses were used to infer phylogenetic trees. MP trees
to see if they were positive for chestnut rot morphotype colo- were calculated using MEGA 7 (Kumar et al. 2016) with
nies. After one week PDA plates were removed and fresh PDA all sites and gaps included. The Tree-Bisection-
plates were placed inside the container for the second week. Reconnection (TBR) option was used with the number
of initial trees (random addition) set at 10 and the maxi-
Isolates captured in the laboratory experiment, morphology mum number of trees to retain set at 100. Branch support
and culture deposit was determined using maximum parsimony bootstrap
(MPBS) resampling with 1000 replicates (Felsenstein
Single-spore cultures were produced and transferred to 1985). Strong branch support for a species was deter-
2% MEA, MYA (2% MEA supplemented with 0.3% mined as MPBS ≥70%. Bayesian analyses were per-
yeast extract), and 2% potato dextrose agar (PDA; Fig. formed with the MRBAYES (Huelsenbeck and Ronquist
2). Cultures were compared to the morphological de- 2001) plugin of Geneious. jModeltest 0.1.1 (Darriba et al.
scription of the ex-type of G. smithogilvyi (Shuttleworth 2012) was used to estimate the model that best fit the
et al. 2012b). A representative culture was deposited at data. The resulting models that best fit the data for the
Centraalbureau voor Schimmelcultures, Utrecht, ITS and β-tubulin datasets were GTR + I + G, and for the
400 Shuttleworth L.A., Guest D.I.

TEF1-α dataset GTR + I. One million generations were run Results

with a sampling frequency every 2000 generations. The first
100,000 generations were eliminated as burn-in. A threshold Experiment 1. Pathogenicity testing of G. smithogilvyi
of ≥0.95 of branches was used for determining strong isolates from Australia, New Zealand and Italy on nuts
Bayesian posterior probability support (BP). The sequences from Australian chestnut varieties
generated for the culture CBS 1383189 were deposited in
GenBank under accessions ITS: KY952223, TEF1-α: The isolate from Australia produced similar lesion lengths
KY952224, and β-tubulin: KY952225. (mean 19 mm) on all three chestnut varieties and were all sig-
nificantly different to control nuts (p < 0.05) (Fig. 1). The iso-
late from New Zealand also produced similar mean lesion
Experiment 4. Trapping G. smithogilvyi ascospores in an lengths ranging from 18 mm in Decoppi Marone, to 20 mm
Australian chestnut orchard in Red Spanish and Purton’s Pride. All three lesion lengths
produced with the New Zealand isolate were significantly dif-
A Burkard 7 day recording volumetric spore-trap (BVST) ferent to the control (p < 0.05). The Italian isolate produced
(Burkard Manufacturing Co. Ltd. UK. http://www. lesions on Red Spanish and Purton’s Pride that were shorter was set up at Brittle Jacks in length compared to the other isolates, but were significantly
orchard, Mullion Creek, NSW in December 2010, different to the control (0.016 and <0.001 respectively).
following the manufacturers instructions. The BVST was However, the Italian isolate did not induce lesions on Decoppi
set up on a trolley 1 m above the orchard floor. Silicon gel Marone significantly longer than the control (p = 0.517).
was used as the adhesive on the plastic tape attached to
the internal drum. Air is drawn into the BVST at 10 L per
min over silicon-coated tape mounted on the drum. The Experiment 2. Isolation of G. smithogilvyi as a latent
drum rotates at a speed of 2 mm per hour. The BVST was pathogen from floral and vegetative tissues
operated during the flowering period from 1 pm on 11/12/
2009 to 1 pm on 18/12/2009. After this period the tape Gnomoniopsis smithogilvyi was isolated from reproductive
was cut into sections corresponding to each day, and and vegetative tissues in all five of the sampled time periods
mounted onto microscope slides and viewed with the that represented four chestnut phenological stages (Table 1).
400X magnification under an Olympus CX40 compound The fungus was most frequently isolated from flower and nut
microscope with the Olympus AnalySIS 5® Soft Imaging tissues, as well as from young leaves. It was rarely isolated
System©. Ascospores fitting the description of G. from woody tissues. The fungus was isolated from 82% of
smithogilvyi were counted each hour (1 pm, 2 pm, 3 pm female flowers sampled in December 2008 and 10.6% of nuts
etc.) (Shuttleworth et al. 2012b). originating from this flowering developed chestnut rot in April

Fig. 1 Mean lesion length * *

20 * * *
produced on chestnut kernels of * *
three Australian chestnut varieties 18
after inoculation with
G. smithogilvyi isolates from 16
Australia, New Zealand and Italy.
Mean lesion length (mm)

Error bars represent standard error 14
of the mean. Results of the Red Spanish
ANOVA are presented above 12
Decoppi Marone
error bars. CBS = Centraalbureau Purton's Pride
voor Schimmelcultures, Utrecht,
the Netherlands; 8 *
MUT = Mycotheca Universitatis
Taurinensis, University of Turin, 6 to the control
NS (p≤0.05)
4 NS

CBS130190 MUT411 MUT401 Control
Australia New Zealand Italy
The infection process of chestnut rot 401

Table 1 Isolation frequency (%) of Gnomoniopsis smithogilvyi from represent the various phenological stages of the trees, early
asymptomatic chestnut tissues collected in Mullion Creek, NSW. Dec = flowering, early Feb = immature nuts and burrs, early
Samples were collected on a single day between the 1st-3rd of Apr = mature nuts and burrs, early Aug = tree dormancy. - = tissue type
December 2008, and 1st-3rd February, April, August 2009. These periods not available due to phenological stage

Tissue type Early Dec 2008 Early Feb 2009 Early Apr 2009 Early Aug 2009 Early Dec 2009

Female flowers 82 - - - 10
Male flowers (dead in Feb & Apr) 59 (n = 30) 28 (n = 30) 53 (n = 30) - 3
Dead styles - 47 40 - -
Pedicels 28 32 60 - 0
Burr equators - 36 65 - -
Shell equators - 3 21 - -
Kernels - 0 16 - -
Terminal leaf petioles 9 9 17 - 0
Terminal leaf mid-veins 9 21 25 - 0
Terminal leaf margin 33 39 52 - 0
Current year branches 17 21 16 25 10
2 year-old branches 8 5 1 7 20
3 year-old branches
- bark & vascular cambium 3 0 NS 0 0
- heartwood & pith 0 0 NS 0 0
4 year-old branches
- bark & vascular cambium 3 0 NS 1 0
- heartwood & pith 0 0 NS 0 0
Dormant terminal buds - - - 41 -

NS = not sampled. n = 75/tissue type unless otherwise labelled

2009. In December 2009, 10% of female flowers were infect- Experiment 4. Trapping G. smithogilvyi ascospores in an
ed, leading to 6% nut rot incidence in April 2010. Australian chestnut orchard

Ascospores were trapped from the orchard atmosphere during

Experiment 3. Capturing G. smithogilvyi ascospores
the day and at night with peak periods between 8 and 11 pm
in the laboratory
and 7–9 am (Fig. 4). The highest mean hourly frequency of
ascospores was 165 ascospores per m3 of air, occurring at
10 pm. No rain fell during the sampling period.
Three colonies of the G. smithogilvyi asexual phase grew on
one of the agar plates in the second week of the experiment.
No evidence of mites or other arthropods were observed on
the plates, which excludes them as possible source of infec-
Our findings provide further evidence that G. smithogilvyi is the
tion. Morphological characters of the asexual culture pro-
main causal agent of chestnut rot, and infection by ascospores is
duced by the ascospores were within the ranges of those of
key to the infection of flowers leading to the disease. Isolates
the ex-type culture of G. smithogilvyi (Fig. 2).
from Australia and New Zealand were both virulent pathogens
on all three of the Australian chestnut varieties, while the Italian
Phylogenetic analyses isolate was less virulent, and did not produce significant lesions
on Decoppi Marone. The pathogen was shown to colonise floral
The sequences from the culture produced from the captured and vegetative tissues asymptomatically in all five of the sampled
ascospore (CBS 1383189) grouped with the ex-type of time periods. The highest percentage was 82% from female
G. smithogilvyi with strong branch support in all three indi- flowers, supporting the floral infection hypothesis of Ogilvy
vidual locus phylogenies. On ITS and β-tubulin branch sup- (1998) and Smith and Ogilvy (2008). The fact that isolation from
ports were 100% MPBS and 1.0 BP, and on TEF1-α they female flowers for example changed from 82% to 10% in the
were 96% MPBS and 0.98 BP (Fig. 3). second year of sampling indicates the infection is dynamic and
402 Shuttleworth L.A., Guest D.I.

Fig. 2 Asexual morph of

G. smithogilvyi produced by a
captured ascospore in the
laboratory chamber experiment.
a = culture on PDA, b = conidia.
Scale a = 500 μm, b = 10 μm

changes from year to year. This helps explain the observation by were trapped from the atmosphere during the day and at night
Australian chestnut growers that the disease changes each year. with peak periods at sunset and the hours following sunset
The isolation percentage from female flowers in both of the peaking at 10 pm, and the hours following sunrise peaking at
sampled years also corresponded to a decreased rot incidence 8 am. Precipitation events have been reported to initiate spore
the following year and suggests when infection of female flowers release in other members of the Diaporthales including
is lower, rot incidence the following year is lower. The 3 and Anisogramma anomala, the cause of eastern filbert blight
4 year-old branches showed a relatively low or absent isolation of (Pinkerton et al. 1998), and Diaporthe sp., one of the causal
G. smithogilvyi. This may be due to the bark providing a physical agents of branch canker of avocado (Eskalen et al. 2013). No
barrier to ascopores from entering the trees from the orchard rain fell during the experiment, which indicates ascospores are
atmosphere. It also suggests that movement of the endophyte not released immediately after a rain event. In the laboratory
throughout the chestnut tree from one tissue type to another experiment, ascospores were captured one week after being
may not be as important as colonisation from airborne propa- soaked with water, which confirms the field observation that
gules entering externally. ascospore release may take time to occur. In the field experi-
The ascospore trapping experiments both supported the hy- ment, rain may have fallen in the week prior to the experiment,
pothesis of Smith and Ogilvy (2008) that the primary source of which inititated ascospore release during the sampling period.
infection is via ascospores released from dead litter on the or- Floral infection by fungi may occur directly through pene-
chard floor. In the laboratory experiment, ascospores were cap- tration of one or more floral tissues or indirectly through sys-
tured in the second week of the experiment. The isolate that was temic infection of the apical meristem (Ngugi and Scherm
sequenced grouped with the ex-type of G. smithogilvyi in all 2006). In regard to primary infection occurring via ascospores
three individual phylogenies with strong branch supports indi- externally, Smith and Ogilvy (2008) explained that the presence
cating it is G. smithogilvyi. In the field experiment, ascospores of multiple-embryo chestnuts, where one embryo is rotten and

ITS TEF1-α β-tubulin

G. occulta CBS 125678
G. occulta CBS 125678 G. occulta CBS 125678

81/- 79/0.99 G. alderdunensis CBS 125680

G. chamaemori CBS 804.79
G. chamaemori CBS 804.79
G. guttulata MS 0312 G. chamaemori CBS 804.79
G. alderdunensis CBS 125680

G. alderdunensis CBS 125680 G. macounii CBS 121468

G. clavulata CBS 121255 94/0.97

G. fructicola CBS 208.34

G. racemula CBS 121469
G. sanguisorbae CBS 858.79
G. racemula CBS 121469
G. idaeicola CBS 125676 G. sanguisorbae CBS 858.79

G. tormentillae CBS 904.79
G. macounii CBS 121468 G. tormentillae CBS 904.79
G. comari CBS 806.79
G. racemula CBS 121469 93/1.0 G. comari CBS 806.79
G. macounii CBS 121468

G. tormentillae CBS 904.79 G. fructicola CBS 208.34

G. sanguisorbae CBS 858.79

CBS 133189 captured isolate

G. comari CBS 806.79 G. idaeicola CBS 125676

78/- CBS 133189 captured isolate CBS 133189 captured isolate

G. smithogilvyi CBS 130190 96/0.98 100/1.0
91/0.99 92/1.0
-/1.0 G. smithogilvyi CBS 130190
G. paraclavulata CBS 123202 G. smithogilvyi CBS 130190

G. clavulata CBS 121255 G. paraclavulata CBS 123202 G. clavulata CBS 121255

G. idaeicola CBS 125676 G. fructicola CBS 208.34 G. paraclavulata CBS 123202

Plagiostoma sp. CBS 128351 Plagiostoma sp. CBS 128351
Plagiostoma sp. CBS 128351

Ophiognomonia setacea CBS 128354 Ophiognomonia setacea CBS 128354

Ophiognomonia setacea CBS 128354

Fig. 3 Individual ITS, TEF1-α and β-tubulin phylogenies with CBS 133189, the culture produced from a captured ascospore in the laboratory chamber
experiment. Maximum parsimony bootstrap values and Bayesian posterior probabilities are located on branch nodes
The infection process of chestnut rot 403

Fig. 4 Mean hourly ascospore 200

Mean ascospore frequency per m3 of air per hour

frequency during the chestnut
flowering period in Mullion
Creek, NSW. Error bars indicate 160
standard error of the mean

































Time of day

the others are healthy (Fig. 5), would not likely occur if the In alfalfa, Botrytis cinerea has been found to infect via direct
infection process was purely systemic. The evidence from the penetration of pollen cell walls, with penetration most frequent
current study suggests that G. smithogilvyi infects via the through the pollen germ pores (Huang et al. 1999).
stigma-style (gynoecial) pathway and not via active penetration The infection of chestnut flowers by ascospores is likely to
of the ovary wall. Multiple embryo chestnuts where one is be affected by several abiotic and biotic factors. Abiotic fac-
rotten and the other healthy, suggests the pellicle layer sur- tors include temperature, rainfall, rain-splash, relative humid-
rounding each embryo is impermeable to the fungus if one of ity and wind in the orchard microclimate. These changes oc-
the embryos is infected and prevents infection of the other cur at the scale of individual trees and even individual flowers.
embryo. Monolinia vaccinii-corymbosi, the causal agent of Biotic factors include timing of flowering of each chestnut
mummy berry disease of blueberry and Claviceps purpurea variety, the release of spores by the fungus, and the transmis-
the cause of ergot of rye are other important examples of fungi sion of spores between flowers and trees by insects, for exam-
adapted to infecting via the gynoecial pathway (Ngugi and ple, bees, beetles and earwigs (Shuttleworth et al. 2012a). In
Scherm 2004; Tudzynski and Scheffer 2004). Pollen is another Australia and New Zealand, rainfall during the flowering pe-
method whereby G. smithogilvyi may gain access to female riod has been reported to increase the incidence of chestnut rot
flowers and infection of pollen grains can be active or passive. (Ogilvy 1998, Smith and Ogilvy 2008). In north-west Italy,
Ascospores of Sclerotinia sclerotiorum for example, are report- Lione et al. (2015) reported temperature was more important
ed to be transported passively from flower to flower on the for chestnut rot development than rainfall. Interestingly the
surface of contaminated rapeseed pollen (Stelfox et al. 1978). rainfall graph in that paper showed the highest average rainfall

Fig. 5 Chestnut containing two

embryos, one rotten and the other
healthy. Scale = 1 cm

Diseased brown
and endosperm Healthy creamy
yellow embryo and

Pellicle surrounding
healthy embryo and
404 Shuttleworth L.A., Guest D.I.

Asexual phase Male
leaf Sexual phase

Current year

Secondary infection Primary infection

Conidia transported by rainsplash, Infected flowers (catkins), leaves and Ascospores overwintered on dead litter on the orchard floor are
wind and insects infect flowers, branches (latent phase) continues in to transported up to flowers, leaves and branches by wind and insects,
leaves and branches chestnut maturity and tree dormancy particularly after rain. Infection depends on timing of flowering
and ascospore release

Chestnut rot symptoms at nut

maturity (pathogenic phase) Perithecia on
asci and

Conidiomata containing conidia

At maturity infected chestnuts and burrs fall to the

orchard floor providing the source of primary inoculum.
Infected leaves also fall in autumn/winter. Buds and
branches infected (latent) in winter

Fig. 6 The disease cycle of chestnut rot caused by G. smithogilvyi. Line 10 μm, conidioma without conidia 500 μm, conidioma with conidia
drawings of G. smithogilvyi courtesy of Catherine Wardrop, Royal 500 μm. Scale for the sexual phase line illustration represents (clockwise
Botanic Garden, Sydney. Photo of diseased chestnut and chestnut tree from top left) dead burr with perithecia attached 3 cm, ascospores and
from Shuttleworth et al. (2012b). Photo of chestnut flowers Lucas ascus 10 μm, perithecia close up 200 μm, diversity of form of perithecia
Shuttleworth. Scale for the asexual phase line illustration represents 600 μm, perithecia in dead burr tissue 750 μm
(clockwise from top left) conidiomata with conidia 750 μm, conidia

occurred in June (though the overall figures seem low), which to elongate). In Australia, chestnut trees flower in approxi-
corresponds to the chestnut flowering period in north-west mately a month long period in December and early January
Italy (Botta et al. 1995), and supports the hypothesis of rainfall (Smith & Ogilvy 2008) depending on location and variety.
during flowering increasing rot incidence at chestnut matura- Therefore flower receptivity to airborne ascospores would be
tion. A comprehensive disease cycle for chestnut rot is pre- different for each variety, and for each year, with the orchard
sented in Fig. 6. Aspects of this disease cycle likely overlap microclimate affecting the flowering time of each variety. This
with chestnut canker caused by G. smithogilvyi. variation in flowering times and timing of ascospore release
The role of the asexual phase in Australian orchards has not are explanations of the variation in rot incidence each year.
been extensively observed. In Switzerland, the asexual morph The results of this study aids our understanding of
of G. smithogilvyi has been reported from cankers of C. sativa the infection process and disease cycle of chestnut rot
(Pasche et al. (2016), and the asexual morph of Gnomoniopsis in Oceania and Europe. It gives insight in to the bio-
comari, the cause of fruit rot and leaf blotch of strawberry, is logical mechanisms that G. smithogilvyi exploits in or-
reported to be dispersed by rain-splashed conidia (Reid 2015). der to infect its chestnut host. The results will assist
Rain-splash has been suggested as a source of infection of chestnut growers to target disease management strate-
spores in the production of chestnut rot (Ogilvy 1998). gies, particularly litter on the orchard floor as the pri-
Ogilvy et al. (1998) also reported chestnut flowers likely have mary source of infection. Targeted disease management
a critical period of receptivity to spores suggested as days 8– will help decrease the incidence of chestnut rot down to
17 of flowering (day one is considered when the flowers start the Australian industry standard of less than 1%.
The infection process of chestnut rot 405

Acknowledgements We thank the University of Sydney, Horticulture O'Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2
Innovation Australia Ltd. and Chestnuts Australia Inc. for financial sup- types within a monophyletic lineage of the fungus Fusarium are
port. We thank the Royal Botanic Garden, Sydney and Mycotheca nonorthologous. Mol Phylogenet Evol 7:103–116
Universitatis Taurinensis, University of Turin, Italy, for providing cul- Ogilvy D (1998) Phomopsis when does it strike? The Australian
tures. Thank you to Mr. David Ogilvy for the use of his orchard for the Nutgrower 12:16–18
isolation and spore trapping experiments, and to Simone Fouche, Forestry Pasche S, Calmin G, Auderset G, Crovadore J, Pelleteret P, Mauch-Mani
and Agricultural Biotechnology Institute (FABI), University of Pretoria B, Barja F, Paul B, Jermini M, Lefort F (2016) Gnomoniopsis
for assisting with the pathogenicity testing. smithogilvyi causes chestnut canker symptoms in Castanea sativa
shoots in Switzerland. Fungal Genet Biol 87:9–21
Pinkerton JN, Johnson KB, Stone JK, Ivors KL (1998) Factors affecting
References the release of ascospores of Anisogramma anomala. Phytopathology
Rehner S (2001) Primers for elongation factor 1–α (EF1–a) www.aftol.
Botta R, Vergano G, Me G, Vallania R (1995) Floral biology and embryo org/pdfs/EF1primer.pdf
development in chestnut (Castanea sativa Mill.) Hortscience 30: Reid A (2015) Gnomoniopsis fruit rot and leaf blotch of strawberries.
1283–1286. Department of Agriculture and Food. Government of Western
full.pdf Australia.
Carbone I, Kohn LM (1999) A method for designing primer sets for fruit-rot-and-leaf-blotch-strawberries
speciation studies in filamentous ascomycetes. Mycologia 91:553– Shuttleworth LA, Liew ECY, Guest DI (2012a) Survey of the incidence
556 of chestnut rot in south-eastern Australia. Australas Plant Pathol 42:
Dar MA, Rai MK (2015) Gnomoniopsis smithogilvyi a canker causing 63–72. Published online 11 December 2012
pathogen on Castanea sativa: first report. Mycosphere 6:327–336
Shuttleworth LA, Liew ECY, Guest DI (2012b) Fungal Planet description
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more
sheet 108: Gnomoniopsis smithogilvyi L.A. Shuttleworth, E.C.Y.
models, new heuristics and parallel computing. Nat Methods 9:772
Liew & D.I. Guest, sp. nov. Persoonia 28:142–143 http://www.
Dennert FG, Broggini GAL, Gessler C, Storari M (2015) Gnomoniopsis
castanea is the main agent of chestnut rot in
Switzerland. Phytopathol Mediterr 54:199–211 Shuttleworth LA, Walker DM, Guest DI (2015) The chestnut pathogen
EPPO (2016) First report of Gnomoniopsis smithogilvyi in the United Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) and its
Kingdom. European and Mediterranean Plant Protection synonyms. Mycotaxon 130:929–940
Organization. EPPO Global Database. No. 11, 2016, article Smith HC, Ogilvy D (2008) Nut rot in chestnuts. The Australian
2016/214. Accessed Mar Nutgrower 22:10–15
2017 Stelfox D, Williams JR, Soehngen U, Topping RC (1978) Transport of
Eskalen A, Faber B, Bianchi M (2013) Spore trapping and pathogenicity Sclerotinia sclerotiorum ascospores by rapeseed pollen in Alberta.
of fungi in the Botryosphaeriaceae and Diaporthaceae associated Plant Dis Rep 62:576–579
with avocado branch canker in California. Plant Dis 97:329–332 Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,
Felsenstein J (1985) Confidence limits on phylogenies: an approach using Fisher MC (2000) Phylogenetic species recognition and species
the bootstrap. Evolution 6:227–242 concepts in fungi. Fungal Genet Biol 31:21–32
Huang HC, Kokko EG, Erickson RS (1999) Infection of alfalfa pollen by Tudzynski P, Scheffer J (2004) Claviceps purpurea: molecular aspects of
Botrytis cinerea. Botanical Bulletin of Academia Sinica 40:101–106 a unique pathogenic lifestyle. Mol Plant Pathol 5:377–388
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of Vinale F, Ruocco M, Manganiello G, Guerrieri E, Bernardo U, Mazzei P,
phylogenetic trees. Bioinformatics 17:754–755 Piccolo A, Sannino F, Caira S, Woo SL, Lorito M (2014)
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary Metabolites produced by Gnomoniopsis castanea associated with
genetics analysis Version 7.0 for bigger datasets. Mol Biol Evol 33: necrosis of chestnut galls. Chem Biol Technol 1:8
1870–1874 Visentin I, Gentile S, Valentino D, Gonthier P, Tamietti G, Cardinale F
Linaldeddu BT, Deidda A, Scanu B, Franceschini A, Alves A, (2012) Gnomoniopsis castanea sp. nov. (Gnomoniaceae,
Abdollahzadeh J, Phillips AJL (2016) Phylogeny, morphology and Diaporthales) as a causal agent of nut rot of sweet chestnut. J Plant
pathogenicity of Botryosphaeriaceae, Diatrypaceae and Pathol 94:411–419
Gnomoniaceae associated with branch diseases of hazelnut in Walker D, Castlebury LA, Rossman AY, Sogonov MV, White JF (2010)
Sardinia (Italy). Eur J Plant Pathol 144:1–21 Systematics of genus Gnomoniopsis (Gnomoniaceae, Diaporthales)
Lione G, Giordano L, SIllo F, Gonthier P (2015) Testing the effects of based on a three gene phylogeny, host associations and morphology.
climate on the incidence of the emergent nut rot agent of chestnut Mycologia 102:1479–1496
Gnomoniopsis castanea. Plant Pathol 64:852–863 Walker DM, Castlebury LA, Rossman AY, White JF (2012) New molec-
Meyer JB, Gallien L, Prospero S (2015) Interaction between two invasive ular markers for fungal phylogenetics: Two genes for species-level
organisms on the European chestnut: does the chestnut blight fungus systematics in the Sordariomycetes (Ascomycota). Mol Phylogenet
benefit from the presence of the gall wasp? FEMS Microbiol Ecol Evol 64:500–512
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
Ngugi HK, Scherm H (2004) Pollen mimicry during infection of blue-
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
berry flowers by conidia of Monilinia vaccinii-corymbosi. Physiol
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a
Mol Plant Path 64:113–123
guide to methods and applications. Academic Press, San Diego, pp
Ngugi HK, Scherm H (2006) Annual review of phytopathology. Annual
Reviews 44:261–282