Plant Pathology (2019) Doi: 10.1111/ppa.

13005

Identification and characterization of Botrytis medusae, a

novel cryptic species causing grey mould on wine grapes in
Australia

L. A. Harper† , M. C. Derbyshire† and F. J. Lopez-Ruiz*

School of Molecular and Life Sciences, Centre for Crop and Disease Management, Curtin University, Bentley, Western Australia, 6102,
Australia

In a collection of 735 Botrytis isolates sampled from Australian wine grape-growing regions, a single isolate from clade
I and group I (based on Bc-hch RFLP analysis) was found. As many Botrytis species are known to live sympatrically, it
was hypothesized that this isolate might be a new Botrytis species. After phenotypic and molecular assays supported
this hypothesis, the species was designated B. medusae. Phylogenetic analyses using the nuclear genes G3PDH, HSP60,
RPB2, NEP1 and NEP2 consistently placed B. medusae in an early-diverging clade I Botrytis spp. lineage. Botrytis
medusae produced white aerial mycelium, grew faster at 30 °C and produced long-branched conidiophore extensions,
compared with B. cinerea and B. pseudocinerea. Botrytis medusae was only able to infect wounded grape leaves and
was significantly less virulent on wounded leaves and berries than B. cinerea. Botrytis medusae also lacked villiform
appendages on the conidial surface and long conidiophores, which are defining features of B. sinoviticola and B. cali-
fornica, respectively. Identification and characterization of new cryptic Botrytis species living in sympatry on grapevines
could potentially provide information to assist disease management strategies for B. cinerea.

Keywords: Botrytis, cryptic species, grey mould, phylogeny, speciation, species complex

respect to a particular disease. Initially, two groups of

Introduction
B. cinerea were reported on a range of hosts including
Grey mould is caused by Botrytis cinerea, anamorph of grapevine, and were distinguished by the presence or
Botryotinia fuckeliana, and has a significant economic absence of the transposable elements Boty and Flipper
impact on grape yields worldwide. There is currently a (Giraud et al., 1997). Subsequently, two Botrytis groups,
scarcity of scientific literature reporting crop losses group I and group II, were shown to exist based on a
caused by B. cinerea (Carisse, 2016). However, global PCR-RFLP test using a fragment of the B. cinerea hch
yield losses of US$2 billion per annum have been locus (Bc-hch) and analysis of nuclear gene sequences
reported (Elmer & Michailides, 2007). (Albertini et al., 2002; Fournier et al., 2003, 2005). The
Several studies have shown that B. cinerea exists Bc-hch locus is the homologue of the Neurospora crassa
within a morphologically cryptic species complex (Gir- het-c gene, which is involved in vegetative incompatibil-
aud et al., 1997, 1999; Albertini et al., 2002; Fournier ity (Fournier et al., 2003). These initial isolates in group
et al., 2003, 2005; Walker et al., 2011; Lorenzini & I were further distinguished from those in group II by
Zapparoli, 2014; Liu et al., 2016; Garfinkel et al., exhibiting natural tolerance to the hydroxyanilide fungi-
2017). Distinguishing different Botrytis species in sympa- cide, fenhexamid (Fournier et al., 2003; Walker et al.,
try and defining their contributions to disease could 2011).
assist in adapting control strategies (Walker, 2016). Grey Using phylogenetic and inter- and intraspecies cross-
mould management could be simplified if only a single ing, group I isolates were initially designated as a single
species was the target for control (Walker et al., 2011). new species, B. pseudocinerea (Walker et al., 2011). Fur-
Furthermore, Stergiopoulos & Gordon (2014) suggest ther studies, using phylogenetic and morphological tech-
that all host–pathogen interactions across the spectrum niques, have characterized three more group I species.
(e.g. parasitism, mutualism and commensalism) should Botrytis calthae (Hennebert & Groves, 1963) was char-
be considered when adjusting management practices with acterized as a group I species using the RFLP Bc-hch test
(Plesken et al., 2015a). Botrytis sinoviticola was identi-
fied in China from table grapes, and B. californica was
*E-mail: [email protected] characterized in the USA from table grapes and blueber-
† ries. Multigene phylogenies support B. sinoviticola and
These authors contributed equally.

ª 2019 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited and
is not used for commercial purposes. 1
2 L. A. Harper et al.

B. californica as new species (Zhou et al., 2014; Saito

Morphological analysis
et al., 2016). Botrytis sinoviticola is morphologically dis-
tinguished from B. cinerea, B. pseudocinerea and B. cali- To compare their morphological characteristics, Botrytis isolates
fornica primarily through the presence of villiform (Bc-7, Bc-385, Bc-410, Bp-362 and B-555) were initially grown
appendages on the conidial surface (Zhou et al., 2014). on potato dextrose agar (PDA; Oxoid) at room temperature for
Botrytis californica is morphologically distinguished from 2 days. Subsequently, 4 mm plugs from actively growing mar-
gins of colonies were placed on fresh PDA plates. Mycelial
B. cinerea, B. pseudocinerea and B. sinoviticola by the
growth rates were assessed by incubating all Botrytis isolates on
formation of long conidiophores (Saito et al., 2016). PDA at 0, 5, 10, 15, 20, 25, 30 and 35 °C, with five replicates
Botrytis sinoviticola and B. californica appear to be sen- per isolate per temperature treatment (Saito et al., 2016). For
sitive to fenhexamid, as they only have slightly higher the 10–35 °C treatments, measurements were recorded after 1
fenhexamid EC50 values than B. cinerea. Botrytis species and 2 days. For 0 and 5 °C, measurements were recorded after
have been previously divided into two clades, clade I and 4 and 8 days due to relatively slow growth rates. Growth rates
clade II. Clade I species exclusively infect eudicots, while were calculated as described by Zhang et al. (2010).
clade II species infect monocots and eudicots (Staats The size and number of sclerotia were assessed by incubating
et al., 2005). Clade I includes the isolates from group I three replicates of all Botrytis species at 20 °C for 14 days, with
and II species B. californica, B. calthae, B. cinerea, 30 sclerotia randomly chosen for measurement and the number
of sclerotia per plate recorded (Zhou et al., 2014). To stimulate
B. eucalypti, B. fabae, B. pelargonii, B. pseudocinerea
sporulation, strains were incubated on PDA for 14 days at room
and B. sinoviticola. Recently, a new clade II Botrytis spe- temperature, under 12 h near-UV light/12 h dark conditions.
cies, B. euroamericana, was found on grapevine in Italy Conidia were harvested by flooding each plate with 20 mL of
(Lorenzini & Zapparoli, 2014; Garfinkel et al., 2017). sterile Milli-Q H2O and scraping with a sterile glass spreader.
Recently, 735 Botrytis isolates were collected from The suspension was then filtered through a 10 mL sterile syringe
wine grapes across Australia for fungicide resistance containing glass wool and centrifuged at 3220 g for 10 min at
screening (authors’ unpublished data). Fifteen of these 4 °C. Following removal of the supernatant, the pellet was
isolates exhibited growth on a discriminatory dose of washed in 5 mL sterile Milli-Q H2O and again centrifuged at
fenhexamid (1 lg mL
1), out of which a single isolate 3220 g for 10 min at 4 °C. This wash step was repeated twice.
showed significantly reduced growth on fenhexamid in The pellet was resuspended in 1 mL sterile Milli-Q H2O and
conidial density was adjusted to 107 spores mL
1 using a
comparison to all other fenhexamid-resistant isolates.
haemocytometer. Samples of conidia were observed under the
The aim of the present study was to identify and charac- microscope (9100), photographs were taken and 100 conidia
terize this isolate fully and to determine whether it repre- per isolate were analysed using IMAGEJ v. 1.51 software
sented a new Botrytis species. (National Institutes of Health). To measure conidiophore length,
conidiophores from 7-days-old sporulating PDA cultures were
attached to clear adhesive tape and then placed onto microscope
Materials and methods slides containing Milli-Q water. Samples were observed under
the microscope (910), photographs were taken and 50 conidio-
Fungal isolations phores per isolate were analysed in IMAGEJ v. 1.51 software.
The isolate B-555 was collected from wine grapes in 2015 from Low strength (25%) PDA was used to reduce fungal growth to
Margaret River, WA (Table 1). Three comparative B. cinerea acquire images of individual conidiophores from 7-days-old
isolates (Bc-7, Bc-385 and Bc-410) were collected from wine sporulating plates under the microscope (910).
grapes from three Australian states (Table 1). A single-spore Scanning electron microscopy (SEM) was used to investigate
Botrytis isolate (#18, DAR45959) was provided by Kim Plum- the morphological features of conidia from isolate B-555, a
mer (La Trobe University, Melbourne, Australia), which was comparative B. cinerea isolate Bc-7, and the B. pseudocinerea
later characterized as B. pseudocinerea (Bp-362) and had been isolate Bp-362. To produce conidia, sporulation was induced for
collected in the Hunter Valley, NSW in 1980 (Table 1). B-555 the three isolates as described above, with modified peanut oat-
and the comparative Botrytis strains were all single-spored and meal agar (Speakman & Pommer, 1986) used instead of PDA;
maintained as mycelial plugs on yeast soluble starch (YSS) med- this medium consistently produced more abundant conidia from
ium (Fillinger et al., 2008) in distilled water at 4 °C. B-555 and B. pseudocinerea. To prepare coverslip samples, two

Table 1 The origin and GenBank accession numbers of partial sequences of the five genes used for phylogenetic analysis of Botrytis medusae,
B. cinerea and B. pseudocinerea.

GenBank accession no.

Species Isolate Origina RPB2 HSP60 G3PDH NEP1 NEP2

B. medusae B-555 Margaret River, WA, 2015 MH732870 MH732866 MH732861 MK211250 MK211255
B. cinerea Bc-7 Margaret River, WA, 2013 MH732872 MH732867 MH732862 MK211247 MK211252
Bc-385 McLaren Flat, SA, 2014 MH732873 MH732868 MH732863 MK211248 MK211253
Bc-410 Dixons Creek, VIC, 2015 MH732874 MH732869 MH732864 MK211249 MK211254
B. pseudocinerea Bp-362 Hunter Valley, NSW, 1980 MH732871 MH732865 MH732860 — MK211256

a
WA, Western Australia; SA, South Australia; VIC, Victoria; NSW, New South Wales.

Plant Pathology (2019)

 A new cryptic Botrytis species 3

drops of poly-L-lysine (Sigma-Aldrich) were placed on 10-mm 555 and to validate whether B-555 and B. pseudocinerea
glass coverslips and allowed to dry overnight. Conidia from infected uninjured berries, isolates were recovered from infected
each isolate were then mounted on the poly-L-lysine coated cov- berries. Isolates were reisolated from three injured berries
erslips by pressing the coated side onto the sporulating area of infected with B-555 and three uninjured berries infected with B-
the peanut oatmeal agar plates. The conidia were fixed in 2.5% 555 and B. pseudocinerea. Species validation of these isolates
glutaraldehyde solution in sodium phosphate buffer (0.05 M, pH was carried out via PCR-RLFP of the Bc-hch gene and growth
7.0) at 4 °C for 16 h. Samples were rinsed in sodium phosphate characteristics on PDA containing 2 lg mL
1 fenhexamid.
buffer (0.05 M, pH 7.0), then progressively dehydrated in a
graded series of ethanol in a Pelco biowave microwave (Ted
Pella Inc.), before critical-point drying (Polaron K3000 critical Bc-hch haplotyping
point dryer; Quorum Technologies Ltd) and mounted on stubs. To extract DNA, sporulation of all isolates was induced on
Samples were coated with 3 nm platinum in a Polaron SC 7640 PDA as described above. Spores and hyphae were scraped off
sputter coater (Quorum Technologies Ltd) and imaged using a each plate using a sterile scalpel and placed in a 1.5 mL micro-
Supra FESEM (Zeiss) at 3 kV. centrifuge tube. Each sample was frozen in liquid nitrogen and
then ground using a sterile pestle. Genomic DNA was isolated
using a BioSprint 15 instrument and BioSprint DNA Plant kit
Assay of fenhexamid sensitivity
(QIAGEN) according to the manufacturer’s instructions. PCR
Analysis of fenhexamid sensitivity of isolate B-555, B. cinerea was performed as described by Fournier et al. (2003), with some
isolates Bc-7, Bc-385 and Bc-410 and B. pseudocinerea isolate modifications. Due to failure to amplify a single band with the
Bp-362 was carried out as described by Zhou et al. (2014). 262/520L primer set, a new primer (hch1F, 50 -
Briefly, PDA plates were amended with technical grade fenhex- GTTTCGACCGGCTTTTACGG-30 ) was designed to replace
amid (Bayer) to final concentrations of 0.005, 0.0075, 0.01, primer 520L. This new primer added only 16 bp to the frag-
0.02, 0.05, 0.1, 1 and 2 lg mL
1 for isolates of B-555 and ment, so had a negligible effect on the restriction profile. The
B. cinerea and to 2, 5, 10, 20, 50 and 75 lg mL
1 for B. pseu- PCR mixture (in 25 lL) contained 40 ng DNA, 5 lL of 109
docinerea. One mycelial plug of each Botrytis species was placed MyTaq premix (Bioline), 0.5 lM of each primer and 1.25 U
on 90 mm Petri dishes containing each of the respective concen- MyTaq polymerase (Bioline). The subsequent thermal profile
trations, with a total of five replicates per concentration for each was as follows: initial denaturation at 94 °C for 2 min; followed
isolate. Plates were incubated in darkness at 20 °C and colony by 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for
diameters were measured at 24 and 48 h. The experiment was 90 s; and a final extension at 72 °C for 4 min. Five microlitres
performed twice. The mycelial growth rate was then used to cal- of each amplicon was digested with HhaI (New England Bio-
culate EC50 values as previously described by Leroux et al. labs) at 37 °C for 1.5 h as described by Fournier et al. (2003).
(1999).

Phylogenetic analysis
Virulence testing
To amplify the partial sequences of G3PDH, RPB2, HSP60,
A detached leaf assay (DLA) was carried out to investigate viru- NEP1 and NEP2 genes from the isolates, primer pairs
lence of B-555, B. cinerea and B. pseudocinerea on young grape G3PDHfor/G3PDHrev, RPB2for/RPB2rev, HSP60for/HSP60rev,
leaves harvested in spring, as previously described by Zhou NEP1for/NEP1revB and NEP2forD/NEP2revD, respectively
et al. (2014) but with a number of modifications. Wine grape were used (Staats et al., 2005, 2007). PCR amplification was
leaves (Vitis vinifera ‘Muscat’) were used instead of table grape carried out as described by Staats et al. (2005). Amplified gene
leaves and the leaves were placed in plastic trays instead of products were confirmed on a 1% agarose gel and then sent to
enamelware. Each tray had either injured or uninjured leaves Macrogen Inc. (Seoul, Korea) for sequencing.
and incubation was at room temperature instead of 20 °C. Partial sequences of the five genes from numerous Botrytis
When recording disease incidence, only complete diseased haloes species were also downloaded from GenBank release 219 (Ben-
were scored as disease. son et al., 2013). The species, accessions and references for these
A detached berry assay (DBA) was carried out to investigate sequences are detailed in Table S1. The genomes of isolates B-
virulence of B-555, B. cinerea and B. pseudocinerea on wine 555 and Bp-362 (B. pseudocinerea) were previously sequenced
grape berries. Ripe wine grape berries (V. vinifera ‘Cabernet and assembled using PacBio long read technology (authors’
Sauvignon’) were harvested from the Curtin University experi- unpublished data). The partial sequences of G3PDH, RPB2,
mental vineyard and disinfected for 5 min in a solution contain- HSP60, NEP1 (NEP1 was not retrievable from the B. pseu-
ing 0.625% sodium hypochlorite and 10% ethanol. The berries docinerea genome) and NEP2 were extracted from these gen-
were then rinsed five times in dH2O and allowed to air dry. Ber- ome assemblies (authors’ unpublished data). To identify the
ries were attached to autoclave tape in sterile Petri dishes (six housekeeping gene fragments in these genomes of the Botrytis
berries per plate) before inoculation. spp., all downloaded sequences of each gene fragment were
To produce inoculum, conidia were harvested from peanut aligned to the assembly using EXONERATE v. 2.2.0 (Slater & Bir-
oatmeal agar as described above. Twelve berries per isolate were ney, 2005). The alignment was parsed using a Bash script and
inoculated with a 10 lL aliquot containing approximately 50 the coordinates and strand information from the alignments
conidia (5 9 103 conidia mL
1 in H2O containing 0.03% were used to extract the corresponding sequences from the
Tween 20), with six berries injured with an 18G sterile needle Botrytis sp. assembly using BEDTOOLS v. 2.25.0 (Quinlan &
at the site of inoculation and six uninjured. Six injured and Hall, 2010).
uninjured berries were inoculated with 10 lL of H2O (contain- G3PDH, HSP60, RPB2, NEP1 and NEP2 gene sequences were
ing 0.03% Tween 20) as controls. All berries were incubated as aligned separately using MAFFT v. 6.42 (Katoh et al., 2002) with
described above for the DLA, except that the berries were incu- default settings. Poor quality regions of these alignments were
bated for 7 days. To fulfil Koch’s postulates (Koch, 1891) for B- removed using TRIMAL v. 1.2 (Capella-Guti
errez et al., 2009). The

Plant Pathology (2019)

4 L. A. Harper et al.

trimmed alignments were then further trimmed using JALVIEW based on all five genes was constructed due to the low number of
(Clamp et al., 2004). This ensured that all sequences in the align- NEP1 and NEP2 sequences available.
ments were of equivalent length. The MAFFT alignments were then Trees were plotted using FIGTREE v. 1.4.3 (http://tree.bio.e
analysed with JMODELTEST v. 2.1.10 (Posada, 2009). The best d.ac.uk/software/figtree/) and manually coloured and annotated
nucleotide substitution model for each alignment was selected in INKSCAPE v. 0.17. To infer species clades, trees were assessed
based on the Bayesian information criterion test. Maximum-likeli- for concordance in topology in relation to B. medusae.
hood phylogenies were constructed for each of these alignments HSP60 sequences from this study, from endophytic Botrytis iso-
using PHYML v. 3.1 (Guindon et al., 2010). The settings used lates (Shipunov et al., 2008) and a number of B. cinerea, B. pseu-
were ‘-d nt -n 1 -b 1000 –run_id <run id> -m <model> -f m -v e -c docinerea and B. californica comparative isolates (Table S2) were
4 -a e –no_memory_check –r_seed 12345 -o tlr -s BEST –print_- aligned and a neighbour-joining consensus tree was constructed in
trace’ where ‘<model>’ represents the model code for the nucleo- GENEIOUS v. 6.1.8 software. The following parameters were used in
tide substitution model identified by JMODELTEST and ‘<run id>’ is the alignment; cost matrix: 65% similarity (5.0/
4.0), gap pen-
a file identifier based on the name of the model. The setting ‘-b alty: 12, gap extension penalty: 3. The neighbour-joining consen-
1000’ was to generate bootstrap values from 1000 randomly sam- sus tree used the Tamura–Nei genetic distance model. The most
pled trees. In addition to the maximum-likelihood trees, a Baye- distant endophytic Botrytis sp. sequence (EU386596) was used as
sian tree was also produced based on a concatenated alignment of an out-group and bootstrap values less than 50% are not shown.
the G3PDH, HSP60 and RPB2 genes using MRBAYES v. 3.2.6
(Huelsenbeck & Ronquist, 2001). The concatenated alignment
was partitioned based on the sequences included (G3PDH, RPB2
Statistical analysis
or HSP60) and the nucleotide substitution model was allowed to All statistical analyses were conducted in SPSS software (IBM). Fen-
vary between each of the partitions. No concatenated alignment hexamid sensitivity, number of sclerotia per plate, conidiophore

Table 2 Mycelial growth and morphological characteristics of Botrytis medusae, B. cinerea and B. pseudocinerea on potato dextrose agar at 20 °C.

Sclerotia Conidiophores Conidia

Mean
Species and Growth (mm Mean no. length Mean Length:
isolate per day) Size (mm) per dish Size (lm) (lm) Size (lm) (lm) width

B. medusae
B-555 14.0 a 1.5–9.5 9 1.3–3.9 82 c 968–2040 9 10–20 1506 b 6.5–15.8 9 4.6–11.0 10.2 9 7.9 1.29
B. cinerea
Bc-7 14.9 b 1.6–9.0 9 1.7–4.0 42 b 1120–2324 9 12–21 1634 b 7.2–16.4 9 5.8–12.2 10.5 9 8.5 1.24 ns
Bc-385 17.3 d 2.4–9.9 9 1.2–5.2 36 b 1126–2169 9 12–22 1642 b 6.4–14.2 9 4.5–9.9 9.3 9 7.1 1.32 ns
Bc-410 15.8 c 1.5–8.0 9 1.0–4.1 26 a 898–2599 9 13–25 1599 b 6.3–15.7 9 5.0–10.7 10.9 9 7.9 1.38 ns
B. pseudocinerea
Bp-362 16.2 c 1.4–9.5 9 1.3–9.5 52 d 766–1745 9 10–18 1277 a 7.2–20.6 9 5.8–12.1 11.6 9 8.5 1.36 ns

Mean values within the same column followed by the same letter are not significantly different according to Waller–Duncan t-test (P = 0.05).
ns, no significant difference according to Student’s t-test (P > 0.05) when compared to B. medusae.

20
B. cinerea
18
B. pseudocinerea
Radial growth rate (mm day–1)

16
B. medusae *
14
*
12

10 Figure 1 Effect of temperature on mycelial

* growth of Botrytis cinerea (Bc-7, Bc-385, Bc-
8 410), B. pseudocinerea (Bp-362) and

6 * B. medusae (B-555). The mean from 15

* replicates was taken for B. cinerea, and 5
4 replicates for B. pseudocinerea and
B. medusae. Vertical bars represent
*
2 standard errors of the means. * indicates
* significant difference when comparing B.
0 medusae with B. cinerea or B.
0 5 10 15 20 25 30 35
pseudocinerea according to a Mann-Whitney
Temperature (⁰C) U-test (P < 0.05).

Plant Pathology (2019)

 A new cryptic Botrytis species 5

(a) (b) (c)

(d) (e) (f)

Figure 2 Colony characteristics and conidial (g) (h) (i)

morphology of three Botrytis species,
B. cinerea (Bc-7; a, d, g), B. pseudocinerea
(Bp-362; b, e, h) and B. medusae (B-555; c,
f, i). Black bars (d–f) = 40 lm. White bars
(g–i) = 4 lm.

c Results

Analysis of morphology shows that isolate B-555

exhibits cryptic phenotypes different to those of
B. cinerea and B. pseudocinerea
EC50 (μg mL–1)

b Isolate B-555 was not easily distinguishable from other

known Botrytis species macroscopically. However, it was
hypothesized that it may exhibit cryptic phenotypic dif-
a a a
ferences in response to its environment. To assess one
aspect of its cryptic phenotype, B-555, three B. cinerea
strains and one B. pseudocinerea strain were grown at
different temperatures. At 20 °C, B-555 grew at
14  0.2 mm per day on average, while B. cinerea and
Bc-7 Bc-385 Bc-410 B-555 Bp-362 B. pseudocinerea both grew at an average of
Isolate 16  0.2 mm per day (Table 2; Fig. 1). Isolate B-555
had a lower mycelial growth rate at 0–25 °C than
Figure 3 Sensitivity of Botrytis cinerea (Bc-7, Bc-385, Bc-410), B. cinerea and B. pseudocinerea, with the growth rates
B. pseudocinerea (Bp-362) and B. medusae (B-555) to fenhexamid. being significantly different for all temperatures except
Columns with the same letter indicate no significant difference between 0 °C (P < 0.05; Fig. 1). At 30 °C and 35 °C, B-555 grew
means according to Waller–Duncan t-test (P = 0.05).
at significantly higher average growth rates of 4.5  0.2
and 0.6  0.1 mm per day, respectively, whereas, at
length and radial growth rate at 20 °C were analysed for all 30 °C, B. cinerea and B. pseudocinerea strains grew at
comparative isolates with one-way ANOVA, with means sepa- average rates of 1.9  0.1 and 0.03  0.02 mm per day,
rated by a Waller–Duncan t-test (P = 0.05). To account for respectively and at 35 °C, they grew at 1.3  0.1 and
heteroscedasticity, all fenhexamid sensitivity data were log 0.05  0.05 mm per day, respectively (P < 0.01; Fig. 1).
transformed before analysis. To compare mean values for myce- All Botrytis species investigated formed grey, whitish
lial growth rates at 0-35 °C, means were analysed using a colonies and similar sized sclerotia on PDA at 20 °C
Mann–Whitney U-test (P = 0.05). For each comparative isolate, (Table 2). Isolate B-555 produced a significantly
injured and uninjured treatments for leaf disease incidence and
(P < 0.05) higher number of sclerotia (mean = 82 per
lesion size were analysed using Student’s t-test (P = 0.01 or
plate) than B. cinerea and B. pseudocinerea isolates
P = 0.05).

Plant Pathology (2019)

6 L. A. Harper et al.

(means = 35 and 52 per plate, respectively; Table 2). In after 7 days of incubation on PDA (25% strength) under
morphological comparison with the group I species, sporulating conditions (Fig. 2d–f). The average conidio-
B. sinoviticola and B. californica, isolate B-555 had a phore lengths were 1506, 1625 and 1277 lm for B-555,
lower sclerotia count (P < 0.05) compared to those B. cinerea and B. pseudocinerea, respectively (Table 2).
found in B. sinoviticola isolates (mean range 179–266; Comparison of the conidial surfaces of Bc-7, Bp-362 and
Zhou et al., 2014) and in B. californica isolates (mean B-555 revealed no obvious differences (Fig. 2g–i).
range 204–790; Saito et al., 2016). In the assay of fenhexamid sensitivity, the fenhexamid
To investigate the morphology of each species under EC50 values for B-555, B. cinerea (average) and B. pseu-
sporulating conditions, each species was grown on PDA docinerea isolates were 0.029, 0.014 and
for 2 days at room temperature under a 12 h near-UV 10.55 lg mL
1, respectively (Fig. 3).
light/12 h dark cycle. Under these conditions, B-555
produced fluffy aerial mycelia, whereas B. cinerea and
Isolate B-555 is less virulent on grape leaves and
B. pseudocinerea did not (Fig. 2a–c). The average length:
berries than B. cinerea but similar to B. pseudocinerea
width ratio of conidia of B-555 was 1.29, compared
with 1.31 and 1.36 for B. cinerea and B. pseudocinerea, To test whether the virulence of B-555 was different to
respectively, and was not significantly different (P > 0.05, that of isolates of the known species B. cinerea and
Table 2). Unlike B. cinerea and B. pseudocinerea, B-555 B. pseudocinerea, virulence assays were conducted on
produced conidiophores with long branched extensions wine grape leaves and wine grapes (Fig. 4a–d).

Bc-7 Bp-362 B-555

(a)
U ninjured
Injured

(b)
Uninj.
Inj.

Figure 4 Infection of (a) wine grape leaves

(Vitis vinifera ‘Muscat’) and (b) wine grape
berries (V. vinifera ‘Cabernet Sauvignon’) by
(c) (d) Botrytis cinerea (Bc-7), B. pseudocinerea
(Bp-362) and B. medusae (B-555); (c)
100 Injured 20 Injured disease incidence (%) caused by B. cinerea
(Bc-7, Bc-385, Bc-410), B. pseudocinerea
Lesion diameter (mm)
Disease incidence (%)

80 Uninjured Uninjured
** ** 15 (Bp-362) and B. medusae (B-555); (d) mean

60
** lesion diameter (mm) from leaf infection
10 caused by B. cinerea (Bc-7, Bc-385, Bc-
40 ** ** 410), B. pseudocinerea (Bp-362) and
B. medusae (B-555). Mean values were
5
20 calculated using only measurements from
complete disease haloes. ** indicate
0 0 significant difference at P < 0.01 using
Bc-7 Bc-385 Bc-410 Bp-362 B-555 Bc-7 Bc-385 Bc-410 Bp-362 B-555
Student’s t-test.
Isolate Isolate

Plant Pathology (2019)

 A new cryptic Botrytis species 7

Virulence of B. medusae, B. pseudocinerea and one

Phylogenetic analysis delineates a new species in the
B. cinerea isolate (Bc-385) on leaves differed signifi-
Botrytis genus, B. medusae
cantly from that of two other B. cinerea isolates, Bc-7
and Bc-410 (P < 0.01, Fig. 4c,d). Isolates B-555, Bp- In the HSP60, RPB2, NEP1, NEP2 and G3PDH+
362 and Bc-385 had mean injured leaf disease inci- HSP60+RPB2-based phylogenies, B-555 was the sole
dences of 63%, 70% and 73%, respectively, compared representative of an early-diverging sister lineage to
with isolates Bc-7 and Bc-410, which had averages of Botrytis clade I, which contains B. pseudocinerea, B. eu-
disease incidence of 100% and 98% (Fig. 4c). Isolates calypti, B. sinoviticola, B. cinerea, B. pelargonii,
B-555, Bp-362 and Bc-385 had average injured leaf B. calthae and B. fabae (Figs 6, 7, 8, 9 & 10). Bootstrap
lesion sizes of 7.2  0.5, 6.6  0.6 and 11.1  1.2 mm supports for B-555 appearing as a sister clade in the
diameter, respectively, and no complete lesion haloes HSP60, RPB2 and NEP2-based maximum-likelihood
were found on uninjured leaves infected with isolates B- trees were 90.8%, 98.9% and 99.8%, respectively
555 and Bp-362 (Fig. 4c). Isolates Bc-7 and Bc-410 had (Figs 6, 7 & 9). In the NEP1-based maximum-likelihood
average injured leaf lesion sizes of 16.5  0.5 and tree, B-555 was also placed in a lineage divergent from
16.1  0.6 mm diameter, respectively (Fig. 4d). Viru- clade I, but again, bootstrap support for this topology
lence testing on grapes (Fig. 4b) showed that Koch’s was lower than 90% (Fig. 8). In the
postulates were fulfilled for B-555 as it was reisolated G3PDH+RPB2+HSP60 Bayesian tree, the posterior
from three injured and three uninjured berries and vali- probability of this taxonomic placement was 1, indicat-
dated through Bc-hch RFLP and fenhexamid sensitivity ing a high confidence in the tree topology (Fig. 10). In
tests (data not shown). the G3PDH-based maximum-likelihood tree, B-555
formed a sister clade to B. californica (Fig. 11). How-
ever, bootstrap support for this placement was lower
Bc-hch haplotyping places B-555 within Botrytis
than 90% (Fig. 11). Thus, it seems likely that B-555 is
group I
the sole representative of an early-diverging Botrytis lin-
For each isolate, the 1187 bp Bc-hch fragment was eage, which is sister to previously described clade I taxa.
amplified and digested with HhaI (Fig. 5). Like B. pseu- This new Botrytis species was designated B. medusae.
docinerea, B-555 showed a restriction profile consistent In all trees that included them (NEP1 was not retriev-
with a group I haplotype, with the presence of a 601 bp able from the B. pseudocinerea genome), the B. cinerea
band compared to the 517 bp band in B. cinerea and B. pseudocinerea isolates used for comparison in this
(Fig. 5). study fell within clades containing other B. cinerea and
B. pseudocinerea isolates, respectively. This confirms

M Group II Group I

700 bp-

500 bp-

300 bp-

100 bp-
Figure 5 Restriction profiles of Botrytis
cinerea (Bc-7, Bc-385, Bc-410),
B. pseudocinerea (Bp-362) and B. medusae
(B-555) isolates after digestion of the Bc-hch
gene amplicons with the enzyme HhaI,
indicating the Botrytis group (I, II) to which
they belong. M = DNA marker. Isolate

Plant Pathology (2019)

8 L. A. Harper et al.

their taxonomic placement and utility as controls for the Whitish colonies produced on PDA, with mycelial
morphological assays. growth on PDA optimal at 25 °C, growing at a mean
increase in diameter of 15.65 mm per day (Fig. 1).
Abundant grey to black sclerotia produced on PDA at
Taxonomy of B. medusae
20 °C with sclerotia singular or aggregated, irregular,
Botrytis medusae, L. A. Harper, M. C. Derbyshire, F. J. spherical to elliptical, 1.5–9.5 9 1.3–3.9 mm
Lopez-Ruiz, sp. nov., Figs 1–4. Mycobank number: (Table 2). White aerial mycelia produced among coni-
MB827445. Ex-type culture = DAR83531 (NSW DPI, diophores under sporulating conditions on PDA at
Australia). room temperature (Fig. 2c). Conidiophores alternately
Etymology: refers to the unique formation of long branched at top, straight, septate, brown, 968–
branched conidiophore extensions on 25% strength PDA 2040 9 10–20 lm (Table 2). On 25% strength PDA,
at room temperature under a 12 h/12 h near-UV light/ produces long branched conidiophore extensions
dark cycle for 7 days. (Fig. 2f). Conidia in botryose clusters, elliptical to

Figure 6 Molecular phylogeny of Botrytis species, using Monilinia fructigena and Sclerotinia sclerotiorum as out-groups, in a maximum-likelihood
tree inferred from the dataset based on the partial DNA sequences of HSP60. The numbers at each node indicate the bootstrap percentage
(n = 1000). Bootstrap support values less than 90% are not shown. The isolates B-555 (B. medusae), Bp-362 (B. pseudocinerea) and Bc-7, Bc-410
and Bc-385 (B. cinerea) are shown in blue, green and purple, respectively.

Plant Pathology (2019)

 A new cryptic Botrytis species 9

ovoid, unicellular, hyaline to pale brown, mean isolate of B. medusae was assessed. Seifert & Rossman
10.2 9 7.9 lm (Table 2). (2010) proposed that authors could potentially make a
Holotype: AUSTRALIA, WESTERN AUSTRALIA, strong case for describing a new fungal species if there is
Margaret River, conidia stored in glycerol at
80 °C of strong evidence but limited biological material.
isolate B-555 from diseased berry of Vitis vinifera Botrytis medusae had a higher mycelial growth rate at
‘Cabernet Sauvignon’, April 2015, L. A. Harper, M. C. 30 °C (>4 mm per day; P < 0.01) than the other Botrytis
Derbyshire, F. J. Lopez-Ruiz. species (≤2 mm per day), B. cinerea, B. pseudocinerea,
B. sinoviticola (Zhou et al., 2014) and B. californica
(Saito et al., 2016). This higher mycelial growth rate
Discussion
could be an adaption to the southwestern Australian cli-
In this study, a new Botrytis species found on wine mate. Recently, another member of the Botrytis cryptic
grapes is proposed, Botrytis medusae. The major limiting species complex (BCSC) has been identified, B. eucalypti
factor in pronouncing a new species here is that only one (Liu et al., 2016). However, unlike B. medusae,

Figure 7 Molecular phylogeny of Botrytis species, using Monilinia fructigena and Sclerotinia sclerotiorum as out-groups, in a maximum-likelihood
tree inferred from the dataset based on the partial DNA sequence of RPB2. The numbers at each node indicate the bootstrap percentage
(n = 1000). Bootstrap support values less than 90% are not shown. The isolates B-555 (B. medusae), Bp-362 (B. pseudocinerea) and Bc-7, Bc-410
and Bc-385 (B. cinerea) are shown in blue, green and purple, respectively.

Plant Pathology (2019)

10 L. A. Harper et al.

B. eucalypti could not grow on PDA at 5 °C and, also in appear different from that reported for B. sinoviticola
contrast to B. medusae, B. eucalypti did not have a (Zhou et al., 2014), but did appear lower than has been
mycelial growth rate of >4 mm per day on PDA at found for conidia of B. californica (Saito et al., 2016)
30 °C (Liu et al., 2016). Botrytis medusae had a higher and B. calthae (Plesken et al., 2015a). Botrytis medusae
sclerotia count than B. cinerea and B. pseudocinerea does not have villiform appendages on the surface of the
(P < 0.05) and a lower sclerotia count (P < 0.05) com- conidia that have been found in B. sinoviticola isolates
pared to those found in B. sinoviticola isolates (mean (Zhou et al., 2014) and has normal length conidiophores
range = 179–266; Zhou et al., 2014) and B. californica (mean = 1506 lm) compared with the long conidio-
isolates (mean range = 204—790; Saito et al., 2016). In phores (mean = 7455 lm) found in B. californica iso-
contrast to B. calthae, B. medusae did not produce scle- lates (Saito et al., 2016). On PDA, B. medusae uniquely
rotia within agar or at the bottom of the Petri dish (Hen- produced conidiophores with long branched extensions.
nebert & Groves, 1963). The conidial length:width ratio Botrytis medusae was distinguished phenotypically
of B. medusae was not significantly different from those from B. pseudocinerea in its sensitivity to fenhexamid.
of B. cinerea, B. pseudocinerea (P < 0.05), nor did it Natural resistance to fenhexamid is very significant in

Figure 8 Molecular phylogeny of Botrytis species, using Sclerotinia sclerotiorum as an out-group, in a maximum-likelihood tree inferred from the
dataset based on the partial DNA sequence of NEP1. The numbers at each node indicate the bootstrap percentage (n = 1000). Bootstrap support
values less than 90% are not shown. The isolates B-555 (B. medusae), and Bc-7, Bc-410 and Bc-385 (B. cinerea) are shown in blue and purple,
respectively.

Plant Pathology (2019)

 A new cryptic Botrytis species 11

B. pseudocinerea, but other group I species such as B. me- study, mycelial rather than conidial inoculum was used,
dusae, B. sinoviticola and B. californica have EC50 values and table grapes instead of wine grapes.
within the sensitive range found in B. cinerea (Leroux Botrytis medusae was distinguishable from related
et al., 1999; Zhou et al., 2014; Saito et al., 2016). Botry- Botrytis species based on phylogenetic analysis involving
tis cinerea isolates that are sensitive to fenhexamid have five nuclear genes. This analysis gave clear evidence for
been found to have an EC50 value within the range of B. medusae being positioned in its own unique clade.
0.008–0.129 lg mL
1 (Leroux et al., 1999; Ma & Botrytis medusae is a sister lineage to Botrytis clade I, as
Michailides, 2005; Saito et al., 2014). As only one type defined by Staats et al. (2005), which includes B. califor-
strain has been identified in this study, it is not possible to nica, B. calthae, B. cinerea, B. eucalypti, B. fabae,
extrapolate the moderate decrease in fungicide sensitivity B. pelargonii, B. pseudocinerea and B. sinoviticola. The
to the entire species without further experimentation. placement of B. medusae in a lineage related to clade I
In tests of virulence, B. medusae caused disease on indicates it is unlikely to be the same species as a novel
uninjured grape berries. In contrast, investigations by clade II endophytic Botrytis sp. found on dandelion by
Zhou et al. (2014) showed that B. sinoviticola could not Shaw et al. (2016). Walker et al. (2011) confirmed that
cause disease on uninjured grapes; however, in that a reproductive barrier between B. cinerea (group II) and

Figure 9 Molecular phylogeny of Botrytis species, using Sclerotinia sclerotiorum as out-group, in a maximum-likelihood tree inferred from the
dataset based on the partial DNA sequence of NEP2. The numbers at each node indicate the bootstrap percentage (n = 1000). Bootstrap support
values less than 90% are not shown. The isolates B-555 (B. medusae), Bp-362 (B. pseudocinerea) and Bc-7, Bc-410 and Bc-385 (B. cinerea) are
shown in blue, green and purple, respectively.

Plant Pathology (2019)

12 L. A. Harper et al.

B. pseudocinerea (group I) exists by investigating crosses plants (Barnes & Shaw, 2003; Sowley et al., 2009; Grant-
of the two species. It remains to be determined if this Downton et al., 2014; Shaw et al., 2016). Shaw et al.
barrier exists between other group I species and (2016) found B. cinerea, B. pseudocinerea, B. mali iso-
B. cinerea, or whether these barriers exist between group lates and an undescribed Botrytis sp. existing as cryptic
I species. However, because B. medusae was living in infections in dandelion. An alignment of partial HSP60
apparent sympatry with B. cinerea and is unlikely to be sequences from reported endophytic Botrytis spp. identi-
a hybrid between B. cinerea and another species based fied in spotted knapweed (Centaurea stoebe; Shipunov
on multiple gene phylogenies, it is probable that this et al., 2008) with B. medusae, B. californica, B. pseu-
reproductive barrier exists. docinerea and B. cinerea partial HSP60 sequences show
Recently, several Botrytis species were shown to exhibit that these Botrytis spp. are related to endophytic species
endophytic stages in their lifecycles in a range of flowering (Fig. S1, Table S2). Thus, B. medusae and, potentially,

Figure 10 Molecular phylogeny of Botrytis species, using Monilinia fructigena and Sclerotinia sclerotiorum as out-groups, in a Bayesian tree inferred
from the dataset based on the concatenated partial DNA sequences of G3PDH, RPB2 and HSP60. The numbers at each node indicate the posterior
probability for tree topology. The isolates B-555 (B. medusae), Bp-362 (B. pseudocinerea) and Bc-7, Bc-410 and Bc-385 (B. cinerea) are shown in
blue, green and purple, respectively. Coloured boxes indicate different species of Botrytis. This tree exhibited the least discrepancy of all trees,
generally grouping Botrytis species into distinct clades.

Plant Pathology (2019)

 A new cryptic Botrytis species 13

other group I species including B. pseudocinerea and Nicot et al., 2011; van Lenteren et al., 2018). For exam-
B. californica, may be strong candidates for further inves- ple, in grapes, management strategies could potentially
tigation of the endophytic stages of Botrytis species. involve ways to increase the frequency of Botrytis species
Understanding the contributions of cryptic Botrytis in low abundance (e.g. B. pseudocinerea, B. sinoviticola,
species living in sympatry could provide information for B. californica, B. euroamericana, B. medusae and
improvement of disease management strategies (Walker, B. prunorum) as a means to induce host resistance at
2016). Numerous microorganisms have been tested as flowering or competitively exclude the highly aggressive
biocontrol agents against B. cinerea, with a small num- and fungicide-resistance-prone B. cinerea. In vineyards,
ber of these currently commercially available (Nicot B. pseudocinerea is common at flowering but not late in
et al., 2011; van Lenteren et al., 2018). Some of these the season, which appears to be due to ecological niche
microorganisms can act as inducers of host resistance or factors and the impact of fungicide application (Walker
as natural competitors, which can lower disease suscepti- et al., 2011; Johnston et al., 2014; Plesken et al.,
bility of the host (Pal & McSpadden Gardener, 2006; 2015b). Furthermore, B. pseudocinerea is hypersensitive

Figure 11 Molecular phylogeny of Botrytis species, using Monilinia fructigena and Sclerotinia sclerotiorum as out-groups, in a maximum-likelihood
tree inferred from the dataset based on the partial DNA sequence of G3PDH. The numbers at each node indicate the bootstrap percentage
(n = 1000). Bootstrap support values less than 90% are not shown. The isolates B-555 (B. medusae), Bp-362 (B. pseudocinerea) and Bc-7, Bc-410
and Bc-385 (B. cinerea) are shown in blue, green and purple, respectively.

Plant Pathology (2019)

14 L. A. Harper et al.

to some fungicides in vitro and development of resistance Fournier E, Giraud T, Albertini C, Brygoo Y, 2005. Partition of the
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application if necessary, with a low likelihood of devel- show genetic recombination in Botryotinia fuckeliana (Botrytis
opment of resistance. Other cryptic sympatric species of cinerea) and transposable elements reveal two sympatric species.
Molecular Biology and Evolution 14, 1177–85.
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Giraud T, Fortini D, Levis C et al., 1999. Two sibling species of the
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