Characterizing the interaction between Botrytis

cinerea and grapevine inflorescences
Z.M. Haile1,2,a, P. Sonego3, K. Engelen3, U. Vrhovsek4, P. Tudzynski5, E. Baraldi2 and C. Moser1
1Genomics and Biology of Fruit Crops Department, Research and Innovation Centre, Fondazione Edmund Mach,
Via E. Mach 1, San Michele all’Adige 38010 (TN), Italy; 2Department of Agricultural Sciences, University of
Bologna, Viale Fanin, 46, 40127, Bologna, Italy; 3Computational Biology Department, Research and Innovation
Centre, Fondazione Edmund Mach, Via E. Mach 1, San Michele all’Adige 38010 (TN), Italy; 4Food Quality and
Nutrition Department, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1, San Michele
all’Adige 38010 (TN), Italy; 5Institute for Biology and Biotechnology of Plants, Westf Wilhelms University,
Schlossplatz 8, D-48143 Muenster, Germany.

Abstract
Botrytis cinerea causes bunch rot in grapevine that affects both fruit quality and
yield. The pathogen is difficult to manage due to asymptomatic infections that often
occur during flowering. It is believed that after early colonization of the flowers, the
fungus enters a quiescent phase until the onset of ripening. At that time, the infection
then becomes active by further colonizing the ripening berry without visible signs and
eventually displaying visible signs of rot. In order to characterize the molecular
mechanisms associated with the grapevine/fungus interaction, inflorescences of Vitis
vinifera (‘Pinot Noir’) were inoculated with B. cinerea at the cap falling stage
(EL25/26). Floral samples were collected at 24 and 96 h post inoculation (hpi) and
either plated on selective medium, or subjected to polyphenol and RNA sequence
analyses. A microscopic examination of the infection process was also conducted. B.
cinerea produced appressoria in grapevine flowers and penetrated the flower cuticle
(gynoecium) within 24 hpi but there was no apparent disease progress afterwards
even though the presence of the pathogen on flowers/fruitlets was confirmed by
plating tissues on selective media. The analysis of phenols indicated a significant
increase of several stilbenoids, including oligomeric ones, in samples that had been
inoculated. A much larger number of genes (1193) appeared to be modulated in the
inoculated samples at 24 hpi than at 96 hpi. Based on GO enrichment and VitisNet
analyses, there were more enriched classes, including those involved in plant-
pathogen interaction, at 24 hpi than at 96 hpi. Additionally, a greater number of B.
cinerea genes were detected at 24 hpi than at 96 hpi. These data suggest that within
96 hpi, the pathogen enters into a quiescent phase.

Keywords: grapevine flower/fruitlet, Botrytis infection, quiescence

INTRODUCTION
Botrytis cinerea Pers.:Fries, a cosmopolitan necrotrophic fungus, causes pre- and
postharvest diseases in a wide range of crops, including grapevine. The pathogen has diverse
hosts that can serve as sources of inoculum, different modes of infection, and the ability to
grow and propagate as mycelia and/or conidia. It can also survive for extended periods as
sclerotia in crop debris (Williamson et al., 2007). Botrytis cinerea can invade young tissues
causing necrosis but in most cases it opportunistically infects wounds or senescing tissue. It
is capable of penetrating host tissues both actively and passively. The pathogen induces
necrosis by producing toxins and reactive oxygen species (ROS) (van Kan, 2005), and may
manipulate a host’s metabolism to facilitate colonization (Choquer et al., 2007; Williamson
et al., 2007). The induction of host cell death resulting in tissue injury culminates as plant or
fruit rot, affecting quality and yield (van Kan, 2005). In vineyards, B. cinerea is part of the
natural microflora and previous season infections are the main source of new infections

a
E-mail: zeraye.haile@fmach.it

  Acta Hortic. 1144. ISHS 2016. DOI 10.17660/ActaHortic.2016.1144.4 29

Proc. III IS on Postharvest Pathology: Using Science to Increase Food Availability
  Eds.: A. Ippolito et al.
(Nair et al., 1995). Primary infection is usually initiated by airborne conidia from
overwintering sources (Elmer and Michailides, 2004). Even though B. cinerea infection of
grapes can occur at any stage of fruit development, it often occurs during bloom (McClellan
and Hewitt, 1973; Nair et al., 1995; Keller et al., 2003). After the initial infection, the
pathogen generally remains quiescent until the onset of fruit ripening and then once again
becomes active when the environment is conducive, causing bunch rot (McClellan and
Hewitt, 1973; Keller et al., 2003; Pezet et al., 2003). Serious levels of infection and rot due to
quiescent infection can also occur after the harvest of apparently healthy berries. What
makes the pathogen stay quiescent until berry ripening is unknown, but the presence of
preformed and inducible antifungal compounds in relatively higher proportion in immature
berries may play a role in pathogen quiescence (Deytieux-Belleau et al., 2009; Goetz et al.,
1999; Keller et al., 2003). The role of other factors responsible for triggering the transition
from a quiescent to an active infection are also poorly understood, though physiological and
biochemical changes associated with a decline in plant defense (Barnes and Shaw, 2002) and
favorable climatic conditions at harvest definitely play a significant part (Nair and Allen,
1993; Sosa-Alvarez et al., 1995). To date, only a few studies have been conducted on
quiescent infections of B. cinerea in grapevine inflorescences (McClellan and Hewitt, 1973;
Nair et al., 1995; Keller et al., 2003; Pezet et al., 2003; Sanzani et al., 2012). These studies
demonstrated that flower infections are responsible for a significant proportion of the bunch
rot that occurs in ripening fruit and inoculum carry over to the subsequent season, which
implies that floral infections are an important stage in the epidemiology of B. cinerea. The
current understanding of the molecular mechanisms associated with B. cinerea quiescent
infection in grapevine flower/fruitlet is very incomplete. Hence, the objective of the present
study was to understand the molecular mechanisms associated with the interaction between
B. cinerea and grapevine inflorescences in order to shed light on the quiescence process.
Combinations of approaches were utilized including, microscopic observation, and
metabolic, and transcriptomic analyses at early stages of the infection process.

MATERIALS AND METHODS

Plant material and Botrytis cinerea inoculation

Flowers were raised from fruiting cuttings of Vitis vinifera (‘Pinot Noir’). Cuttings with
3-4 nodes were first treated with rooting solution, 300 ppm of indol-3-butyric acid, for 15
min and then transferred into a 10-cm diameter pot filled with wet wool rock. The pots were
placed on heat mat in cold room to allow rooting first while the winter buds were dormant.
After five to six weeks, individual rooted cuttings were repotted into a 1.5-L pot using
commercial potting mix and transferred to a greenhouse. Following budburst, the vegetative
apex was removed to promote inflorescence growth. The number of flowers in a cluster
were thinned in order to have a manageable number of flowers per cluster and to insure that
all flowers in a cluster could be inoculated with B. cinerea conidia. A transgenic strain of B.
cinerea (isolate B05.10) expressing green fluorescent protein (GFP) was cultured on potato
dextrose agar (PDA) in Petri dishes and incubated at 25°C. After 10 days, conidia were
harvested by agitating agar bearing mycelia and conidia with distilled water. The suspension
was filtered and the concentration of conidia was determined under light microscope using a
hemocytometer. At anthesis (EL25/26) each flower received about 300 conidia and the
whole cutting was then immediately bagged for 24 h in order to create high humidity, which
is critical for conidial germination.

Microscopic observation and plating out

Thin slices of fertilized gynoecia, infected with GFP labeled B05.10, were manually cut
for microscopic observation. Pictures were taken with a laser scanning confocal microscope
(Leica TCS SP5 - Leica Microsystems, Germany). In addition to the GFP labeled B05.10 being
fluorescent, it is also capable of growing on selective media (resistant to Hygromycin) which
means its presence can be detected when tissues are cultured on selective media containing
hygromycin. Plating of the infected flowers/fruitlets was made on PDA (with Hygromycin B

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at 70 µg mL-1) to check for the presence of quiescent B05.10 at various days after
inoculation. Eight flowers/fruitlets from each of six biological replicates were plated on
selective media. An inflorescence from a fruiting cutting was considered as a biological
replicate.

Polyphenol content and RNA-sequence analyses

Samples of inflorescence from fruiting cuttings that were either mock- or B05.10
conidia inoculated at anthesis, were collected at 24 and 96 h post inoculation (hpi),
immediately frozen in liquid nitrogen, and kept at -80°C until use. Prior to polyphenol and
RNA extraction, the samples were ground in liquid nitrogen. Six biological replicates were
used for polyphenol analysis. Sample preparation and HPLC-DAD-MS analysis were
conducted using the method described in Vrhovsek et al. (2012). RNA was extracted using
Plant Total RNA Kit (Sigma-Aldrich) following the manufacturer’s protocol and was
quantified with a ND-8000 spectrophotometer. Three biological replicates were used for the
transcriptome study. Approximately 20 million strand-specific, 100 bp sequences were
obtained per sample using a Next Generation Sequencing Platform HiSeq 1500 (Illumina,
USA). The quality of the reads was checked using FastQC (version 0.11.2) software
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Illumina paired-end raw
reads were pre-processed for both quality and adapter trimming with qtrim
(https://ccb.jhu.edu/software/fqtrim/index.shtml). The resulting reads were aligned
separately to both the B. cinerea (strain B05.10) (http://www.broadinstitute.org) and grape
(12x v1, http://genomes.cribi.unipd.it/) genomes using the Subread aligner (Liao et al.,
2014). Raw read counts were extracted from the Subread alignments using the featureCount
read summarization program (Liao et al., 2014). Differential expression analysis was
performed taking advantage of the voom method (Law et al., 2014) which estimates the
mean-variance relationship of the log-counts, generating a precision weight for each
observation that is fed into the limma empirical Bayes analysis pipeline (Smyth, 2004).
Genes were considered differentially expressed if they had fold change of ≥1.5 and p-value
<0.05. Gene ontology enrichment was computed using customized annotation and annotated
reference of GO plant slim into the AgriGO analysis tool (http://bioinfo.cau.edu.cn/agriGO/
analysis.php). A GO term was considered significantly enriched, if the FDR was <0.05 and p-
value <0.05 when compared to all gene transcripts annotated in the reference genome. The
grapevine molecular network, VitisNet, (Grimplet et al., 2012) was also used to identify
enriched molecular networks.

RESULTS AND DISCUSSION

Microscopy and culturing on selective media

Flowers were infected at the cap falling stage (EL25/26) with GFP-labelled B05.10
conidia. Confocal microscopy revealed that within 24 h after infection, fungal conidia had
germinated, developed appressoria, and penetrated the flower cuticle, gynoecium (Figure
1A). Even though appressoria formation and penetration was seen within 24 hpi, visual
inspection later revealed no signs and/or symptoms of B. cinerea. The presence of the
pathogen on healthy-looking, inoculated flowers/fruitlets was checked by plating the floral
tissues on selective media. On average, the presence of B. cinerea was confirmed on more
than 90% of the healthy-looking, inoculated flowers/fruitlets (Figure 1B). This suggests that
the pathogen was there but not growing actively enough to cause disease symptoms.

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Figure 1. Confocal microscope observations and culturing of grapevine flowers infected by
Botrytis cinerea (GFP-labelled B05.10). (A) Confocal microscope image of GFP-
labelled B05.10 infecting the cuticle of a grapevine flower 24 h after inoculation.
App, appressoria; white bar represents 10 µm. (B) Culturing of grapevine flowers
inoculated by Botrytis cinerea (GFP-labelled B05.10) at anthesis on selective
media. The presence of quiescent B. cinerea on flowers was checked for 7 days
after infection. Values at each day represent mean proportion of flowers/fruitlets,
taken from 6 biological replicate (eight flowers/fruitlets from each of biological
replicate), showing B. cinerea growth on selective media. Error bars indicate
standard error.

Polyphenol content
The targeted secondary metabolite analysis detected fifty-four polyphenols, which
were placed into five broader categories (Table 1). Several stilbenoids (including oligomeric
ones) and flavonoids, as well as some phenypropanoids (such as caftaric acid) exhibited a
significant elevation in content following B. cinerea infection. Most flavan-3-ols were
significantly higher in the inoculated flowers at 96 hpi but not at 24 hpi. Unlike the other
metabolites, a number of stilbenoids, such as trans-e-viniferin, E-cis- and Z-miyabenol, and
isohopeaphenol, were detected only following infection (data not shown). Previous studies
have reported that constitutive grape berry phenolic compounds have an inhibitory effect on
B. cinerea (Goetz et al., 1999; Keller et al., 2003).

Table 1. Concentration (µg g-1) of polyphenol content in mock and B05.10 infected
flowers/fruitlets at 24 and 96 h post infection (hpi).
Polyphenols 24 hpi 96 hpi
(µg g-1 fresh weight) Control Treatment Control Treatment
Benzoic acids 12.4(±2.4) 13(±1.8) 13.2(±3.0) 20.6(±3.6)
Phenylpropanoids 673.8(±122.8) 769(±206.5) 1073.3(±148.6) 1342.5(±209.2)
Flavan-3-ols 654.7(±131.6) 595.2(±203.4) 997.2(±132.0)b 1790.9(±270.4)a
Flavonols 1050.6(±89.2) 1013.5(±22.0) 675.4(±100.9) 808.7(±120.7)
Stilbenoids 124.9(±18.8) 133.7(±8.5) 47.9(±17.0)b 158.9(±36.7)a
Figures in bracket represent standard errors; Values followed by a different letter are significantly different according to t-test
(P≤0.05).

Transcriptome analysis
The transcriptomic analyses results presented in this paper are preliminary. Only GO
terms of Biological Process for both organisms are shown for illustrative purposes (Figure
2). A total of 1401 unique grapevine genes were differentially expressed in the combined 24
and 96 hpi samples obtained from inoculated flowers. The number of differentially
expressed genes at 96 hpi was less than a fourth of the differentially expressed genes at 24
hpi, indicating that most of the plant response occurs in the first hours post infection. GO

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enrichment analysis revealed that 10, 14, and 2 GO terms from Biological Process, Molecular
Function, and Cellular Component, respectively, were significantly enriched at 24 hpi. In
contrast, only 1 and 3 GO terms (Biological Process and Cellular Component, respectively)
were found to be enriched at 96 hpi. The largest GO Biological Process categories at 24 hpi
were “macromolecule modification”, “secondary metabolic process”, and “catabolic process”
(Figure 2A). Some of these GO terms were also observed in tomato plants infected with B.
cinerea (Smith et al., 2014). Networks for transcriptional factors, defense related plant
phytohormones, oxidative burst, and secondary metabolites were enriched at 24 hpi in the
Molecular Network Enrichment analysis (using VitisNet annotation). In contrast, only the
network for cell wall modification was enriched at 96 hpi (data not shown). The enrichment
analyses suggested that the plant recognizes the invading fungus and establishes an effective
defense within 24 hpi. At 96 hpi, the response is much weaker which may imply that the
plant arrested the pathogen’s progress before this time or that the fungus had gone
quiescent on its own. The number of B. cinerea transcripts reads obtained from inoculated
flowers was very little compared to the number of reads obtained from B. cinerea cultured
on PDB. As a result, it was not possible to determine differential expression of B. cinerea
transcripts. A false discovery rate threshold was set based on B. cinerea transcripts in un-
inoculated grapevine samples. Based on this threshold, a total of 479 genes were detected at
both 24 and 96 hpi. These genes fell into 12 GO terms for Biological Process (Figure 2B).
From the detected genes, only 22 genes were uniquely found at 96 hpi and 160 genes were
shared between the two time points. Interestingly, of the shared genes, about 41% of them
were ribosomal proteins, while among the others a significant number were carbohydrate
active enzymes (CAZymes) which target the cell wall polysaccharide (Blanco-Ulate et al.,
2014). The fact that the number of CAZymes was three times higher at 24 hpi than 96 hpi
suggests that B. cinerea is somehow less active at 96 hpi, and is probably entering a
quiescent phase.

Figure 2. Biological Processes GO slim terms associated with 24 and 96 hours post
inoculation (hpi). (A) Enrichment analysis of biological processes associated with
differentially expressed V. vinifera genes at 24 and 96 hpi. Enriched Biological
Process of upper level ontology terms are shown. (B) Biological Processes GO slim
terms of B. cinerea genes expressed during infection of grapevine flowers at 24
and 96 hpi.

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CONCLUSIONS
Conidia of B. cinerea germinate, produce appressoria, and penetrate cuticle of the
grapevine flowers within 24 h. Following the infection of B. cinerea, the plant reacts by up
regulating innate defense mechanisms to contain the pathogen’s infection. The defense
mechanisms employed from the grapevine flower/inflorescence do not eliminate the
pathogen but rather keep it latent (quiescent phase).

ACKNOWLEDGEMENTS
The research was funded by Fondazione Edmund Mach, San Michele all’Adige, Italy.

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