Australasian Plant Pathol.
(2017) 46:397–405
DOI 10.1007/s13313-017-0502-3
ORIGINAL ARTICLE
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 in Oceania (Australia, New Zealand), and Europe
(Italy, France, Switzerland, United Kingdom). The causal
agent has been identified as the fungus Gnomoniopsis
smithogilvyi (Gnomoniaceae, Diaporthales). Koch’s
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
were pathogenic on all three varieties, however the Italian
isolate produced smaller lesions on all three, and was only
mildly pathogenic on Decoppi Marone. Infection of chestnut
floral and vegetative tissues was investigated during the chest-
nut phenological stages of flowering (sampled twice over two
years), immature nuts and burrs, mature nuts and burrs, and
tree dormancy (immature nuts and burrs, mature nuts and
burrs, and tree dormancy were sampled once in one year).
Gnomoniopsis smithogilvyi was present in all the sampled
phenological stages, most frequently from female flowers dur-
ing the flowering period. In the first year, the fungus infected
82% of female flowers, while in the second year only 10%
were infected. Ascospore trapping in a laboratory chamber
experiment confirmed ascopores are released in to the air from
infected burrs. Ascospore trapping in an Australian chestnut
orchard during the flowering period also showed ascospores
are released in to the air, with peak trapping times at sunset
and the hours following sunset (8-11 pm), and the hours fol-
lowing sunrise (7-9 am). These experiments support the hy-
pothesis that ascospores of G. smithogilvyi infect chestnut
* D. I. Guest
david.guest@sydney.edu.au
1
Sydney Institute of Agriculture, University of Sydney, 1 Central
Avenue, Australian Technology Park, Eveleigh, Sydney, NSW 2015,
Australia
trees, particularly the flowers. The fungus then exists as a
latent pathogen in reproductive and vegetative tissues, leading
to the development of the disease during nut maturity.
Keywords Disease cycle . Koch’s postulates . Spore trapping
Introduction
Chestnut rot is an important disease of chestnut trees and has
been reported on Castanea sativa, and C. crenata × C. sativa
hybrids in Oceania (Australia, New Zealand) where they are
grown as a tree crop, and on C. sativa in Europe (France, Italy,
Switzerland, United Kingdom) where they are grown as tree
crops, as ornamental trees, and occur as components of native
forests (Shuttleworth et al. 2012a, b; Visentin et al. 2012;
Dennert et al. 2015; Lione et al. 2015; EPPO 2016). In addi-
tion to Castanea spp., Gnomoniopsis smithogilvyi has recently
been reported as an associate of branch diseases of Corylus
avellana (hazelnut) in Italy (Linaldeddu et al. 2016).
Chestnut rot affects the kernel of the nuts, resulting in
browning and necrosis of the endosperm and embryo
(Shuttleworth et al. 2012a, b). The disease is mainly
expressed post-harvest, however reports by Australian
growers show the disease can occur on nuts still attached
to the tree. The disease is cryptic, where healthy looking
nuts on the surface are rotten internally. In Australia,
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
Switzerland up to 21% (Dennert et al. 2015).
Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales)
has been identified as the main causal agent (Shuttleworth
et al. 2015, 2012b). In addition to chestnut rot,
G. smithogilvyi has been reported to cause canker of
398
C. sativa in India (Dar and Rai 2015), and canker of grafting
scions in Switzerland (Pasche et al. 2016). The fungus is also
associated with galls caused by the chestnut gall wasp,
Dryocosmus kuriphilus in Italy and Switzerland (Vinale
et al. 2014; Meyer et al. 2015), and has been reported to reduce
the abundance of Cryphonectria parasitica associated with
chestnut galls (Meyer et al. 2015).
The infection process of chestnut rot is still being
unravelled. Asymptomatic infection of male and female
flowers, nuts, burrs and branches has been reported in
Australia (Shuttleworth et al. 2012a), from nuts, flowers and
branches in Italy (Visentin et al. 2012), and nuts, branches,
and leaves in Switzerland (Pasche et al. 2016; Dennert et al.
2015). As the infection was asymptomatic, it was assumed
that the fungus colonised chestnut tissues as an endophyte,
becoming pathogenic as the nuts ripened. However, Ogilvy
(1998) reported a correlation between rainfall during the
flowering period and increased chestnut rot incidence, sug-
gesting a latent pathogenic infection of flowers. Smith &
Ogilvy (2008) found that ascospores released from dead
chestnut litter on the orchard floor was the primary inoculum
initiating chestnut rot. Visentin et al. (2012) also reported that
25% of artificially infected chestnut flowers developed chest-
nut rot. Therefore the role of endophytic colonisation in the
development of nut rot remains unclear.
The current study had three aims. The first was to complete
pathogenicity testing on nuts from Australian chestnut varie-
ties with isolates of G. smithogilvyi from Australia, New
Zealand and Italy. The second was to further investigate the
infection process through isolations from chestnut floral and
vegetative tissues representing four phenological stages of the
trees, the third was to further investigate the hypothesis of
floral infection via air-borne ascospores both in the laboratory
and in the field.
Materials and methods
Experiment 1. Pathogenicity testing of G. smithogilvyi
isolates from Australia, New Zealand and Italy on nuts
from Australian chestnut varieties
Nuts of three commercial Australian chestnut varieties were
tested, Red Spanish, Decoppi Marone and Purton’s Pride. The
shell and pellicle were removed from nuts, and immersed in
10% NaOCl for 3 min, 70% ethanol for 1 min, and air-dried
on lint-free tissue. The stylar end of each nut was asceptically
cut to expose a 5 mm2 surface on to which the prepared co-
nidial suspensions were applied. Three isolates of
G. smithogilvyi were tested on each variety, one from
Australia (CBS130190 ex-type; CBS = Centraalbureau voor
Schimmelcultures, Utrecht, the Netherlands), one from New
Zealand (MUT411; MUT = Mycotheca Universitatis
Shuttleworth L.A., Guest D.I.
Taurinensis, University of Turin, Italy) and one from Italy
(MUT401). Conidia were produced from one-week old cul-
tures grown on malt extract agar (MEA) at room temperature
under 12 h light/dark cycles. The plates were flooded with
sterile water and stirred manually with a sterile glass rod.
Conidia numbers were quantified with a haemocytometer.
Ten μL sterile distilled water was applied to each of the nuts
in the control treatment. Twenty nuts were inoculated for each
isolate, 50 conidia per nut. After application, the nuts were
wrapped individually in Parafilm to prevent contamination
from opportunistic microbes.
One week after inoculation, nuts were cut longitudinally
from the stylar to hylum end and the lesion lengths were
measured. For standardisation, if a nut had a lesion along its
entire length it was recorded as 23 mm, as this was the mean
total length of nuts used in the experiment. An analysis of
variance (ANOVA, p< 0.05) was completed in Genstat
Version 17 (VSN International Ltd., United Kingdom) to test
if lesion lengths of each isolate tested on each variety were
significantly different to the control.
Experiment 2. Isolation of G. smithogilvyi
from asymptomatic floral and vegetative tissues
Various asymptomatic floral and vegetative tissues of
chestnut trees were collected from Mullion Creek, New
South Wales on a single day between the 1st-3rd of
each month in December 2008 and February, April,
August and December 2009. These months represent
various phenological stages of chestnut trees including
flowering, immature nuts and burrs, mature nuts and
burrs, and tree dormancy. Fifteen trees were selected,
three varieties (Red Spanish, Decoppi Marone, Purton’s
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).
Sections of the various tissues were cut 5 mm 3 to
1 cm3 in size, except female flowers and dormant buds,
which were kept whole. The time of surface-
disinfestation depended on the tissue type. Softer tissues
such as female and male flowers, peduncles, the various
leaf structures (petioles, mid-veins, margins) and current-
year branches were added to 95% ethanol for 30 s, 10%
NaOCl for 1 min, rinsed with sterile deionised water,
added to 95% ethanol for 30 s, and then air-dried on
lint-free tissue. Samples were then cut into quarters and
plated on to 2% MEA. Isolation percentage for each tis-
sue type was then calculated. A selection of the isolates
from female and male flowers, terminal leaf margin, first
and second year branches were identified phylogenetical-
ly and morphologically as G. smithogilvyi (Shuttleworth
et al. 2015, 2012b, see also MycoBank record MB
800259). The remaining colonies that were not sequenced
The infection process of chestnut rot
were morphologically identified with reference to the ex-
type culture of G. smithogilvyi CBS 130190
(Shuttleworth et al. 2012b).
Isolations of G. smithogilvyi from female flowers in 2008
was compared to rot incidence in 2009, and the isolation of
G. smithogilvyi from female flowers in 2009 was compared to
rot incidence in 2010. Chestnut rot incidence (%) was deter-
mined using the dissection and visual assessment protocols
described in Shuttleworth et al. (2012a). In 2009, 293 chest-
nuts were sampled from three varieties, Decoppi Marone
(n = 107), Purton’s Pride (n = 93) and Red Spanish (n = 93).
In 2010, 288 chestnuts were sampled from the same varieties,
RS (n = 100), DM (n = 86), and PP (n = 102). The results were
pooled and chestnut rot incidence (%) was determined.
Experiment 3. Capturing G. smithogilvyi ascospores
in the laboratory
A chamber experiment was designed to capture ascospores
from infected burrs in the laboratory. Four dead chestnut burr
fragments, variety Red Spanish, were collected from Mullion
Creek, NSW. Burrs were placed in a 35 cm × 30 cm × 20 cm
rectangular plastic container with a ‘Cool Master’ computer fan
(model: A12025-12CB-3BN-F1. DC1 2 V, 0.16A) on one side,
and 6.5 cm diameter agar plates containing 2% potato dextrose
agar (PDA) on the other side from the burrs. Before placing the
burrs into the container, they were sprayed with sterile water
until saturation. To increase humidity, tissues were soaked with
sterile water and placed in each corner of the container, and
three 5.5 cm diam agar plates were filled with sterile water
and positioned in front of the fan. The lids of agar plates con-
taining agar were not removed until the fan, burrs, moistened
tissues and water in the agar plates were in position. This was to
prevent contamination of the agar by physical contact with
objects. The agar plate lids were removed, and the top of the
container was sealed with plastic cling wrap and taped closed to
stop external airborne spores contaminating the agar plates. The
burrs were incubated for 2 × 1 week intervals under 12 h l/d
23 °C. The agar plates were observed at the end of the first week
to see if they were positive for chestnut rot morphotype colo-
nies. After one week PDA plates were removed and fresh PDA
plates were placed inside the container for the second week.
Isolates captured in the laboratory experiment, morphology
and culture deposit
Single-spore cultures were produced and transferred to
2% MEA, MYA (2% MEA supplemented with 0.3%
yeast extract), and 2% potato dextrose agar (PDA; Fig.
2). Cultures were compared to the morphological de-
scription of the ex-type of G. smithogilvyi (Shuttleworth
et al. 2012b). A representative culture was deposited at
Centraalbureau voor Schimmelcultures, Utrecht,
399
The Netherlands, as CBS 133189, with a duplicate de-
posited at the Royal Botanic Garden, Sydney as RBG
5715.
Sequencing, phylogenetic analyses and GenBank accessions
Individual locus phylogenies were inferred and genealog-
ical concordance phylogenetic species recognition
(GCPSR; Taylor et al. 2000) used to determine the phy-
logenetic identity of one of the captured isolates. The
internal transcribed spacer regions of rDNA including
5.8S (ITS), partial translation elongation factor 1-alpha
(TEF1-α), and beta-tubulin (β-tubulin) were used. PCR
conditions were identical to Walker et al. (2010). The
primers used to amplify and sequence for ITS were
ITS5/ITS4 (White et al. 1990), for TEF1-α they were
EF1-728F (Carbone and Kohn 1999)/EF1-1567R
(Rehner 2001), and for β-tubulin they were T1/T2
(O'Donnell and Cigelnik 1997). Both the forward and re-
verse strands were sequenced to reduce the presence of
ambiguous nucleotides. Sanger sequencing was performed
by the Ramaciotti Centre, University of New South
Wales. Sixteen reference taxa from the Gnomoniaceae
were used in the phylogenetic analyses including the ex-
type culture of G. smithogilvyi (CBS 130190), and
outgroups Ophiognomonia setacea CBS 128354 and
Plagiostoma sp. CBS 128351. Sequences for the
reference taxa were sourced from Walker et al. (2010)
and Walker et al. (2012). Gnomoniopsis guttulata (MS
0312) was excluded from the TEF1-α and β-tubulin anal-
yses as there were no sequences available on GenBank.
Sequences were aligned using the MAFFT option of
Geneious Version 7 Geneious® R7 v7.1.2 (Biomatters
Ltd., New Zealand) with default settings applied.
Consensus sequences were trimmed and edited manually
where necessary. The sequence length for each alignment
after trimming was ITS: 503 bp, TEF1-α: 425 bp and β-
tubulin: 407 bp. Maximum parsimony (MP) and Bayesian
analyses were used to infer phylogenetic trees. MP trees
were calculated using MEGA 7 (Kumar et al. 2016) with
all sites and gaps included. The Tree-Bisection-
Reconnection (TBR) option was used with the number
of initial trees (random addition) set at 10 and the maxi-
mum number of trees to retain set at 100. Branch support
was determined using maximum parsimony bootstrap
(MPBS) resampling with 1000 replicates (Felsenstein
1985). Strong branch support for a species was deter-
mined as MPBS ≥70%. Bayesian analyses were per-
formed with the MRBAYES (Huelsenbeck and Ronquist
2001) plugin of Geneious. jModeltest 0.1.1 (Darriba et al.
2012) was used to estimate the model that best fit the
data. The resulting models that best fit the data for the
ITS and β-tubulin datasets were GTR + I + G, and for the
400
TEF1-α dataset GTR + I. One million generations were run
with a sampling frequency every 2000 generations. The first
100,000 generations were eliminated as burn-in. A threshold
of ≥0.95 of branches was used for determining strong
Bayesian posterior probability support (BP). The sequences
generated for the culture CBS 1383189 were deposited in
GenBank under accessions ITS: KY952223, TEF1-α:
KY952224, and β-tubulin: KY952225.
Experiment 4. Trapping G. smithogilvyi ascospores in an
Australian chestnut orchard
A Burkard 7 day recording volumetric spore-trap (BVST)
(Burkard Manufacturing Co. Ltd. UK. http://www.
burkard.co.uk/7dayst.htm) was set up at Brittle Jacks
orchard, Mullion Creek, NSW in December 2010,
following the manufacturers instructions. The BVST was
set up on a trolley 1 m above the orchard floor. Silicon gel
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
drum rotates at a speed of 2 mm per hour. The BVST was
operated during the flowering period from 1 pm on 11/12/
2009 to 1 pm on 18/12/2009. After this period the tape
was cut into sections corresponding to each day, and
mounted onto microscope slides and viewed with the
400X magnification under an Olympus CX40 compound
microscope with the Olympus AnalySIS 5® Soft Imaging
System©. Ascospores fitting the description of G.
smithogilvyi were counted each hour (1 pm, 2 pm, 3 pm
etc.) (Shuttleworth et al. 2012b).
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
ANOVA are presented above 12
error bars. CBS = Centraalbureau
10
voor Schimmelcultures, Utrecht,
the Netherlands;
MUT = Mycotheca Universitatis
Taurinensis, University of Turin,
Italy
Shuttleworth L.A., Guest D.I.
Results
Experiment 1. Pathogenicity testing of G. smithogilvyi
isolates from Australia, New Zealand and Italy on nuts
from Australian chestnut varieties
The isolate from Australia produced similar lesion lengths
(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
lengths ranging from 18 mm in Decoppi Marone, to 20 mm
in Red Spanish and Purton’s Pride. All three lesion lengths
produced with the New Zealand isolate were significantly dif-
ferent to the control (p < 0.05). The Italian isolate produced
lesions on Red Spanish and Purton’s Pride that were shorter
in length compared to the other isolates, but were significantly
different to the control (0.016 and <0.001 respectively).
However, the Italian isolate did not induce lesions on Decoppi
Marone significantly longer than the control (p = 0.517).
Experiment 2. Isolation of G. smithogilvyi as a latent
pathogen from floral and vegetative tissues
Gnomoniopsis smithogilvyi was isolated from reproductive
and vegetative tissues in all five of the sampled time periods
that represented four chestnut phenological stages (Table 1).
The fungus was most frequently isolated from flower and nut
tissues, as well as from young leaves. It was rarely isolated
from woody tissues. The fungus was isolated from 82% of
female flowers sampled in December 2008 and 10.6% of nuts
originating from this flowering developed chestnut rot in April
* *
* * *
* *
Variety
*
Red Spanish
Decoppi Marone
Purton's Pride
8 *
6 to the control
NS (p≤0.05)
4 NS
0
CBS130190 MUT411 MUT401 Control
Australia New Zealand Italy
Isolate
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
in the laboratory
Morphology
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-
tion. Morphological characters of the asexual culture pro-
duced by the ascospores were within the ranges of those of
the ex-type culture of G. smithogilvyi (Fig. 2).
Phylogenetic analyses
The sequences from the culture produced from the captured
ascospore (CBS 1383189) grouped with the ex-type of
G. smithogilvyi with strong branch support in all three indi-
vidual locus phylogenies. On ITS and β-tubulin branch sup-
ports were 100% MPBS and 1.0 BP, and on TEF1-α they
were 96% MPBS and 0.98 BP (Fig. 3).
the day and at night with peak periods between 8 and 11 pm
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.
Discussion
Our findings provide further evidence that G. smithogilvyi is the
main causal agent of chestnut rot, and infection by ascospores is
key to the infection of flowers leading to the disease. Isolates
from Australia and New Zealand were both virulent pathogens
on all three of the Australian chestnut varieties, while the Italian
isolate was less virulent, and did not produce significant lesions
on Decoppi Marone. The pathogen was shown to colonise floral
and vegetative tissues asymptomatically in all five of the sampled
time periods. The highest percentage was 82% from female
flowers, supporting the floral infection hypothesis of Ogilvy
(1998) and Smith and Ogilvy (2008). The fact that isolation from
female flowers for example changed from 82% to 10% in the
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
Australian chestnut growers that the disease changes each year.
The isolation percentage from female flowers in both of the
sampled years also corresponded to a decreased rot incidence
the following year and suggests when infection of female flowers
is lower, rot incidence the following year is lower. The 3 and
4 year-old branches showed a relatively low or absent isolation of
G. smithogilvyi. This may be due to the bark providing a physical
barrier to ascopores from entering the trees from the orchard
atmosphere. It also suggests that movement of the endophyte
throughout the chestnut tree from one tissue type to another
may not be as important as colonisation from airborne propa-
gules entering externally.
The ascospore trapping experiments both supported the hy-
pothesis of Smith and Ogilvy (2008) that the primary source of
infection is via ascospores released from dead litter on the or-
chard floor. In the laboratory experiment, ascospores were cap-
tured in the second week of the experiment. The isolate that was
sequenced grouped with the ex-type of G. smithogilvyi in all
three individual phylogenies with strong branch supports indi-
cating it is G. smithogilvyi. In the field experiment, ascospores
were trapped from the atmosphere during the day and at night
with peak periods at sunset and the hours following sunset
peaking at 10 pm, and the hours following sunrise peaking at
8 am. Precipitation events have been reported to initiate spore
release in other members of the Diaporthales including
Anisogramma anomala, the cause of eastern filbert blight
(Pinkerton et al. 1998), and Diaporthe sp., one of the causal
agents of branch canker of avocado (Eskalen et al. 2013). No
rain fell during the experiment, which indicates ascospores are
not released immediately after a rain event. In the laboratory
experiment, ascospores were captured one week after being
soaked with water, which confirms the field observation that
ascospore release may take time to occur. In the field experi-
ment, rain may have fallen in the week prior to the experiment,
which inititated ascospore release during the sampling period.
Floral infection by fungi may occur directly through pene-
tration of one or more floral tissues or indirectly through sys-
temic infection of the apical meristem (Ngugi and Scherm
2006). In regard to primary infection occurring via ascospores
externally, Smith and Ogilvy (2008) explained that the presence
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
77/1.0
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

96/0.97
G. tormentillae CBS 904.79
G. macounii CBS 121468 G. tormentillae CBS 904.79
97/1.0
G. comari CBS 806.79
-/0.98
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

92/-
G. sanguisorbae CBS 858.79

CBS 133189 captured isolate

G. comari CBS 806.79 G. idaeicola CBS 125676
100/1.0

78/- CBS 133189 captured isolate CBS 133189 captured isolate

G. smithogilvyi CBS 130190 96/0.98 100/1.0
81/1.0
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

99/1.0
Plagiostoma sp. CBS 128351 Plagiostoma sp. CBS 128351
100/1.0
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
180
flowering period in Mullion
Creek, NSW. Error bars indicate 160
standard error of the mean
140

120

100

80

60

40

20

pm

am
m

m
pm

am

m
am

pm
1p

2p

3p

4p

5p

6p

7p

8p

9p

1a

2a

3a

4a

5a

6a

7a

8a

9a
11

11
10

12

10

12
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
embryo
and endosperm Healthy creamy
yellow embryo and
endosperm

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

Terminal
Asexual phase Male
leaf Sexual phase
flowers

Female
flowers
Current year
branch

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
burr
containing
asci and
ascospores

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

occurred in June (though the overall figures seem low), which
corresponds to the chestnut flowering period in north-west
Italy (Botta et al. 1995), and supports the hypothesis of rainfall
during flowering increasing rot incidence at chestnut matura-
tion. A comprehensive disease cycle for chestnut rot is pre-
sented in Fig. 6. Aspects of this disease cycle likely overlap
with chestnut canker caused by G. smithogilvyi.
The role of the asexual phase in Australian orchards has not
been extensively observed. In Switzerland, the asexual morph
of G. smithogilvyi has been reported from cankers of C. sativa
(Pasche et al. (2016), and the asexual morph of Gnomoniopsis
comari, the cause of fruit rot and leaf blotch of strawberry, is
reported to be dispersed by rain-splashed conidia (Reid 2015).
Rain-splash has been suggested as a source of infection of
spores in the production of chestnut rot (Ogilvy 1998).
Ogilvy et al. (1998) also reported chestnut flowers likely have
a critical period of receptivity to spores suggested as days 8–
17 of flowering (day one is considered when the flowers start
to elongate). In Australia, chestnut trees flower in approxi-
mately a month long period in December and early January
(Smith & Ogilvy 2008) depending on location and variety.
Therefore flower receptivity to airborne ascospores would be
different for each variety, and for each year, with the orchard
microclimate affecting the flowering time of each variety. This
variation in flowering times and timing of ascospore release
are explanations of the variation in rot incidence each year.
The results of this study aids our understanding of
the infection process and disease cycle of chestnut rot
in Oceania and Europe. It gives insight in to the bio-
logical mechanisms that G. smithogilvyi exploits in or-
der to infect its chestnut host. The results will assist
chestnut growers to target disease management strate-
gies, particularly litter on the orchard floor as the pri-
mary source of infection. Targeted disease management
will help decrease the incidence of chestnut rot down to
the Australian industry standard of less than 1%.
The infection process of chestnut rot
Acknowledgements We thank the University of Sydney, Horticulture
Innovation Australia Ltd. and Chestnuts Australia Inc. for financial sup-
port. We thank the Royal Botanic Garden, Sydney and Mycotheca
Universitatis Taurinensis, University of Turin, Italy, for providing cul-
tures. Thank you to Mr. David Ogilvy for the use of his orchard for the
isolation and spore trapping experiments, and to Simone Fouche, Forestry
and Agricultural Biotechnology Institute (FABI), University of Pretoria
for assisting with the pathogenicity testing.
References
Botta R, Vergano G, Me G, Vallania R (1995) Floral biology and embryo
development in chestnut (Castanea sativa Mill.) Hortscience 30:
1283–1286. http://hortsci.ashspublications.org/content/30/6/1283.
full.pdf
Carbone I, Kohn LM (1999) A method for designing primer sets for
speciation studies in filamentous ascomycetes. Mycologia 91:553–
556
Dar MA, Rai MK (2015) Gnomoniopsis smithogilvyi a canker causing
pathogen on Castanea sativa: first report. Mycosphere 6:327–336
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more
models, new heuristics and parallel computing. Nat Methods 9:772
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
EPPO (2016) First report of Gnomoniopsis smithogilvyi in the United
Kingdom. European and Mediterranean Plant Protection
Organization. EPPO Global Database. No. 11, 2016, article
2016/214. https://gd.eppo.int/reporting/article-5959. Accessed Mar
2017
Eskalen A, Faber B, Bianchi M (2013) Spore trapping and pathogenicity
of fungi in the Botryosphaeriaceae and Diaporthaceae associated
with avocado branch canker in California. Plant Dis 97:329–332
Felsenstein J (1985) Confidence limits on phylogenies: an approach using
the bootstrap. Evolution 6:227–242
Huang HC, Kokko EG, Erickson RS (1999) Infection of alfalfa pollen by
Botrytis cinerea. Botanical Bulletin of Academia Sinica 40:101–106
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics 17:754–755
Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary
genetics analysis Version 7.0 for bigger datasets. Mol Biol Evol 33:
1870–1874
Linaldeddu BT, Deidda A, Scanu B, Franceschini A, Alves A,
Abdollahzadeh J, Phillips AJL (2016) Phylogeny, morphology and
pathogenicity of Botryosphaeriaceae, Diatrypaceae and
Gnomoniaceae associated with branch diseases of hazelnut in
Sardinia (Italy). Eur J Plant Pathol 144:1–21
Lione G, Giordano L, SIllo F, Gonthier P (2015) Testing the effects of
climate on the incidence of the emergent nut rot agent of chestnut
Gnomoniopsis castanea. Plant Pathol 64:852–863
Meyer JB, Gallien L, Prospero S (2015) Interaction between two invasive
organisms on the European chestnut: does the chestnut blight fungus
benefit from the presence of the gall wasp? FEMS Microbiol Ecol
91(11):fiv122
Ngugi HK, Scherm H (2004) Pollen mimicry during infection of blue-
berry flowers by conidia of Monilinia vaccinii-corymbosi. Physiol
Mol Plant Path 64:113–123
Ngugi HK, Scherm H (2006) Annual review of phytopathology. Annual
Reviews 44:261–282
405
O'Donnell K, Cigelnik E (1997) Two divergent intragenomic rDNA ITS2
types within a monophyletic lineage of the fungus Fusarium are
nonorthologous. Mol Phylogenet Evol 7:103–116
Ogilvy D (1998) Phomopsis when does it strike? The Australian
Nutgrower 12:16–18
Pasche S, Calmin G, Auderset G, Crovadore J, Pelleteret P, Mauch-Mani
B, Barja F, Paul B, Jermini M, Lefort F (2016) Gnomoniopsis
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
the release of ascospores of Anisogramma anomala. Phytopathology
88:122–128
Rehner S (2001) Primers for elongation factor 1–α (EF1–a) www.aftol.
org/pdfs/EF1primer.pdf
Reid A (2015) Gnomoniopsis fruit rot and leaf blotch of strawberries.
Department of Agriculture and Food. Government of Western
Australia. https://www.agric.wa.gov.au/strawberries/gnomoniopsis-
fruit-rot-and-leaf-blotch-strawberries
Shuttleworth LA, Liew ECY, Guest DI (2012a) Survey of the incidence
of chestnut rot in south-eastern Australia. Australas Plant Pathol 42:
63–72. Published online 11 December 2012
Shuttleworth LA, Liew ECY, Guest DI (2012b) Fungal Planet description
sheet 108: Gnomoniopsis smithogilvyi L.A. Shuttleworth, E.C.Y.
Liew & D.I. Guest, sp. nov. Persoonia 28:142–143 http://www.
fungalplanet.org/content/pdf-files/FungalPlanet108.pdf
Shuttleworth LA, Walker DM, Guest DI (2015) The chestnut pathogen
Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) and its
synonyms. Mycotaxon 130:929–940
Smith HC, Ogilvy D (2008) Nut rot in chestnuts. The Australian
Nutgrower 22:10–15
Stelfox D, Williams JR, Soehngen U, Topping RC (1978) Transport of
Sclerotinia sclerotiorum ascospores by rapeseed pollen in Alberta.
Plant Dis Rep 62:576–579
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS,
Fisher MC (2000) Phylogenetic species recognition and species
concepts in fungi. Fungal Genet Biol 31:21–32
Tudzynski P, Scheffer J (2004) Claviceps purpurea: molecular aspects of
a unique pathogenic lifestyle. Mol Plant Pathol 5:377–388
Vinale F, Ruocco M, Manganiello G, Guerrieri E, Bernardo U, Mazzei P,
Piccolo A, Sannino F, Caira S, Woo SL, Lorito M (2014)
Metabolites produced by Gnomoniopsis castanea associated with
necrosis of chestnut galls. Chem Biol Technol 1:8
Visentin I, Gentile S, Valentino D, Gonthier P, Tamietti G, Cardinale F
(2012) Gnomoniopsis castanea sp. nov. (Gnomoniaceae,
Diaporthales) as a causal agent of nut rot of sweet chestnut. J Plant
Pathol 94:411–419
Walker D, Castlebury LA, Rossman AY, Sogonov MV, White JF (2010)
Systematics of genus Gnomoniopsis (Gnomoniaceae, Diaporthales)
based on a three gene phylogeny, host associations and morphology.
Mycologia 102:1479–1496
Walker DM, Castlebury LA, Rossman AY, White JF (2012) New molec-
ular markers for fungal phylogenetics: Two genes for species-level
systematics in the Sordariomycetes (Ascomycota). Mol Phylogenet
Evol 64:500–512
White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics. In:
Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a
guide to methods and applications. Academic Press, San Diego, pp
315–322