Filamentous fungi associated with natural infection of noble rot on withered grapes

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International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
Short communication
Filamentous fungi associated with natural infection of noble rot on withered
grapes
M. Lorenzinia, B. Simonatoa, F. Favatia, P. Bernardib, A. Sbarbatib, G. Zapparolia,⁎
a Dipartimento di Biotecnologie, Università degli Studi di Verona,strada Le Grazie 15, 37134 Verona, Italy
bDipartimento di Neuroscienze, Biomedicina e Movimento, Università degli Studi di Verona, piazzale L.A. Scuro 10, 37134 Verona, Italy
A R T I C L E I N F O
Keywords:
Fungi
Noble rot
Withered grapes
Penicillium adametzoides
Cladosporium cladospoirioides
Coniochaeta polymorpha
A B S T R A C T
The effects of noble rot infection of grapes on the characteristics of different types of wine, including Italian
passito wine, are well known. Nevertheless, there is still little information on filamentous fungi associated with
noble-rotten grapes. In this study, withered Garganega grapes for passito wine production, naturally infected by
noble rot, were analyzed and compared to sound grapes. Skin morphology and fungal population on berry
surfaces were analyzed. Scanning electron microscopy analysis revealed microcracks, germination conidia and
branched hyphae on noble-rotten berries. Penicillium, Aureobasidium and Cladosporium were the most frequent
genera present. Analysis of single berries displayed higher heterogeneity of epiphytic fungi in those infected by
noble-rot than in sound berries. Penicillium adametzoides, Cladosporium cladospoirioides and Coniochaeta poly-
morpha were recovered. These, to the best of our knowledge, had never been previously isolated from withered
grapes and, for C. polymorpha, from grapevine. This study provided novel data on noble rot mycobiota and
suggests that fungi that co-habit with B. cinerea could have an important role on grape and wine quality.
1. Introduction
The term “noble rot” indicates the endophytic infection of Botrytis
cinerea on grape berries that occurs under specific enviromental con-
ditions (Ribéreau-Gayon et al., 2006). The development of noble rot on
grapes can occur on-vine or off-vine in the case of post-harvest storage.
This latter occurrence can be observed in withered grapes, carried out
in closed and ventilated rooms called fruttaio (fruit-drying room), used
for the production of Italian passito wines (Mencarelli and Tonutti,
2013). The influence of noble rot on wine aroma of some of these wines
has been analyzed (Fedrizzi et al., 2011). The positive effects of this
fungus on the sensory characteristics of passito sweet wines have en-
couraged controlled botrytization in post-harvest conditions
(Mencarelli and Tonutti, 2013; Tosi et al., 2013).
To date, natural withering is the most common grape dehydration
process for passito wines production and the occurrence of noble rot
infection on grapes is highly variable depending on seasonal and de-
hydration conditions (Mencarelli and Tonutti, 2013). During withering,
winemakers remove damaged and decayed bunches or berries, which
are a potential source of infection and wine defects, leaving only those
that are sound or affected by noble rot. Nevertheless, in favourable
conditions the activation of latent infections on these remaining berries
can cause fungal disease outbreaks. In particular, the switch from noble
rot to gray mould could be rapid, as could be the saprophytic coloni-
zation of berries by other fungi.
Withered grapes are colonized by several fungal species, including
those that have an important pathogenic role (Lorenzini et al., 2016).
Up to now however, the ecology of filamentous fungi in withered
grapes has scarcely been investigated because they do not play a crucial
role on grape fermentation such as yeasts. Non-Botrytis fungal species
can affect the noble rot development and lead to disease occurrence
with negative effects on grape and wine quality (Rousseaux et al.,
2014). However, the fungal consortium associated with noble-rotten
grapes is still largely unknown.
The present study analyzed filamentous fungi associated with in-
fection of noble rot on Garganega grapes that frequently occurs during
natural withering of this local variety that is used for the production of
passito sweet wines such as Recioto and Vin Santo. Skin morphology,
frequency of main fungal families or genera and species identification
of isolates from single berries were performed.
2. Materials and methods
2.1. Grape sampling and berry classification
Grape samples of Garganega variety withered in natural conditions
https://doi.org/10.1016/j.ijfoodmicro.2018.03.004
Received 14 September 2017; Received in revised form 7 March 2018; Accepted 10 March 2018
⁎ Corresponding author.
E-mail address: giacomo.zapparoli@univr.it (G. Zapparoli).
International Journal of Food Microbiology 272 (2018) 83–86
Available online 13 March 2018
0168-1605/ © 2018 Elsevier B.V. All rights reserved.
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with the traditional surmaturation technique, were collected in a fruit-
drying room located in the Soave winemaking area (Italy) after five
months from the harvest (vintage 2015). The incidence of noble-rotten
grapes, estimated by visually inspection, was approximatevly 50–60%.
Berries were classified in three categories (Fig. S1): “sound” when un-
damaged with homogeneus yellowish to amber skin, swollen or par-
tially shrivelled; “noble-rotten” when undamaged with light to dark
brown skin, partially or largely shrivelled (mummy-like); “damaged
and decayed” when even partially rotted, with visible fractures or craks,
partially or totally covered with mycelium. This last category was not
considered in this study since generally they are discarded and not vi-
nificated. Two individual batches of sound and noble-rotten (about
500 g each) were randomly selected from single clusters and aseptically
transferred to laboratory for analysis. The presence of B. cinerea in
noble-rotten berries and its absence in sound berries was confirmed by
performing a species-specific PCR analysis using as template DNA ex-
tracted from an amount of 50 g of berries. DNA extraction was carried
out according to Rezaian and Krake (1987), while PCR assay was per-
formed using Bot-F and Bot-R primers as described by Lorenzini and
Zapparoli (2014) (Fig. S2).
2.2. Scanning electron microscopy analysis of berries
Representative berries of each category (three sound and four noble-
rotten), randomly selected from each batch, were analyzed by scanning
electron microscopy (SEM, ESEM XL30, FEI-Philips, Hillsboro, OR)
after sample preparation. Then, the samples were fixed in 2% w/v
glutaraldehyde in phosphate buffer for 3 h and then dehydrated in
graded acetones. Then the samples were treated by critical point dryer
(CPD 030, Bal-tec, Balzers, Liechtenstein), mounted on metallic speci-
mens stubs, sputter-coated with gold (MED 010 Balzers), and examined
by SEM.
2.3. Determination of water activity of berries
Water activity (aw) was measured in two categories of berries
(sound and noble-rotten) and determination was carried out using a
Hygropalm HC2-AW (Rotronic Italia srl, Milan, Italy) apparatus
equipped with a thermostated stainless steel sample holder (WP-40TH,
Rotronic Italia srl). Sound and noble-rotten berries, randomly selected
from each batch, were placed in disposable supports and aw determi-
nation was carried out at constant temperature of 25 °C. Measurements
for each category were carried out in triplicate and the average aw value
with standard deviation was reported. The t-test was applied to test for
statistical differences between noble-rottenand sound berries.
2.4. Isolation and identification of fungi
A total of 116 berries (49 sound and 67 infected by noble rot),
randomly selected from each batch, were individually used for fungal
isolation. Each berry was directly plated by rolling onto malt extract
agar (MEA, 2% w/v malt extract, 0.1% w/v peptone, 2% w/v dextrose,
1.5% w/v agar) and DG18 agar (22.0% w/v glycerol, 1% w/v dextrose,
0.5% w/v peptone, 0.1% w/v potassium dihydrogen phosphate, 0.05%
w/v magnesium sulfate, 0.01% w/v chloramphenicol, 0.0002% w/v
dichloran, 1.5% w/v agar). After incubation for two–four days at 25 °C,
individual colonies were isolated and purified through repeated
streaking on MEA and DG18 agar. About 550 isolates were stored on
MEA slants at 4 °C.
Classification of isolates grown on MEA, potato dextrose agar
(Difco, Laboratories, Detroit, MI), oatmeal agar and yeast extract su-
crose agar (Samson et al., 2004) was carried out evaluating the colony
morphology and microscopic features according to Pitt and Hocking
(2009) and by comparison with morphological characteristics of strains
from withered grapes previoulsy identified by Lorenzini et al. (2016).
All isolates were classified at family or genus level. A total of 200
isolates were identified at species level and 182 out of them (65 from
sound and 117 from noble-rotten berries) were isolated from a re-
presentative berry sample. This berry sample was costituted by 15
sound and 21 noble-rotten berries (about 30% of 116 berries) selected
according to the number of fungal genera and families recognized on
each berry. Botrytis cinerea, Epicoccum nigrum, Alternaria alternata spe-
cies-group and Aureobasidium pullulans complex were mainly re-
cognized by deeper morphological analysis (e.g. mycelium, vegetative
and/or reproductive structures, conidiophores, conidial patterns, con-
idia). Species attribution of these isolates was confirmed by phyloge-
netic analysis carried out only on representative strains. Classification
at species level of Penicillium, Aspergillus section Nigri, Cladosporium,
Fusarium, Botryosphaeriaceae and Mucoraceae, including isolates be-
longing to other genera, was mainly carried out by phylogenetic ana-
lysis due to high morphological similarity among species of the same
genus.
2.5. DNA amplification and sequence analysis
DNA was extracted from pure culture of isolates as previously de-
scribed (Lorenzini and Zapparoli, 2014). Partial gene or region se-
quences used for phylogenetical analysis were Internal Transcribed
Spacer (ITS) using the primer pairs ITS1/ITS4 (White et al., 1990),
Large Subunit (LSU) with primer pairs LR0R/LR7 (Rehner and Samuels,
1994; Vilgalys and Hester, 1990) and Actin (ACT) with primer pair
ACT-512F/ACT-783R (Carbone and Kohn, 1999).
The amplification conditions were carried out as described by White
et al. (1990) for ITS (ITS1/ITS4), de Gruyter et al. (2009) for LSU
(LR0R/LR7), Carbone and Kohn (1999) for ACT.
The amplification products were visualized by agarose gel electro-
phoresis (1% w/v) and purified using the NucleoSpin gel and PCR
Clean-up kit (Macherey-Nagel, Düren, Germany) according to the
manufacturer's instructions. Sequencing of these products was carried
out in both directions using the same primers as for amplification
(Eurofins Genomics, Ebersberg, Germany).
2.6. Phylogenetic analysis
Phylogenetic analysis was conducted using sequences from the
Clustal W multiple alignment output using neighbor-joining (NJ) sta-
tistical method and maximum composite likelihood (ML) substitution
model in the MEGA 7.0 interface. The phylogeny trees inferred from
each sequence dataset were constructed by the NJ method and in-
dividually tested with a bootstrap (BS) of 1000 replicates to ascertain
the reliability of a given branch pattern in each NJ tree.
For studying phylogenetic relationships, sequences of reference
strains closely related to isolates from withered grapes object of this
study were recovered from the literature (Friebes et al., 2016;
Sandoval-Denis et al., 2016; Visagie et al., 2014).
3. Results and discussion
3.1. SEM and water activity analysis in berries
SEM analysis of berry skin revealed frequent presence of micro-
cracks and wounds, conidia, germination conidia, branched hyphae and
mycelium on noble-rotten berries, while mostly rare on the skin of
sound berries (Fig. 1). Microcracks on skin play an imporant role in
susceptibility to conidia germination and subsequent mycelial growth
(Padgett and Morrison, 1990). This SEM data suggest possible effects of
noble rot infection on the occurrence of microcracking although a
deeper analysis (e.g., determination of frequency of microcraks on
berry cuticular membrane) is recommended.
Water activity values differed significantly (p 80% of berries
(Table 1). Cladosporium was recovered in most of the berries. As ex-
pected, Botrytis was more frequent in noble-rotten berries than in sound
ones. Large differences (> 50% in term of frequency between sound
and noble-rotten berries) were observed for Fusarium, Aspergillus section
Nigri and Epicoccum. Beside the 10 fungal genera or families listed in
Table 1, other fungi such as Trichoderma, Aspergillus section Flavi and
Nigrospora were found in both berry categories but at very low fre-
quency ((Lorenzini et al., 2016) but their presence has already been
reported on fresh grapes (Bejaoui et al., 2006; Briceño and Latorre,
2008; Sage et al., 2004). Moreover, an isolate from a sound berry was
identified as Coniochaeta polymorpha by phylogenetic analysis (Fig. S5)
among fungi belonging to genera not listed in Table 1. Despite no
documentation of C. polymorpha having ever been previously isolated
from grapevine or other plant materials, its isolation in withered grapes
Fig. 1. SEM analysis of skin of sound berries (A) and noble-rotten berries (B-H). Bar is 500 μm in A and B, 200 μm in C, 50 μm in D, G and H, 10 μm in E, 5 μm in F. Arrows show hyphaes
protruding through crevice of a lenticel.
Table 1
Frequency (%) of main fungal genera or families identified in 67 noble-rotten and 49
sound berries.
Noble-rotten Sound
Penicillium 84.9a 95.3
Aureobasidium 89.0 81.4
Cladosporium 72.6 62.8
Botrytis 58.9 34.9
Alternaria 31.5 27.9
Epicoccum 26.0 16.3
Aspergillus section Nigri 16.4 9.3
Fusarium 19.2 7.0
Botryosphaeriaceae 6.8 11.6
Mucoraceae 2.7 4.7
a n. of isolates of each genus or family on total n. of berries (116, 67 noble-rotten and
49 sound) in percentage.
Fig. 2. Percentage of single berries from which different fungal groups (listed in Table 1)
were isolated. Number of berries selected to identify isolates at species level is indicated
on each column.
M. Lorenzini et al. International Journal of Food Microbiology 272 (2018) 83–86
85
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should not be surprising, since Coniochaeta species are usually asso-
ciated with wounded, senescent or dead plant tissues (Damm et al.,
2010). Isolation of C. velutina from fresh grapes was reported by Sage
et al. (2004).
Understanding the spatial heterogenicity in fungal distribution
among berries during withering is epidemiologically and ecologically
important, however withered grapes do not seem to be a homogeneous
habitat for fungi. It has been documented that yeast species distribution
might be subjected to significant intra-vineyard spatial fluctuations due
to variations in inter- and intra-vine microclimates and vine ecosystems
(Setati et al., 2012). It is conceivable that the observed fungal hetero-
genicity among withered grapes might be due to the fact that fruit-
drying rooms contain grapes from different vineyards. Moreover, it is
likely that fungal communties go through further variations during the
withering process as demostrated in yeast populations on Erbaluce
grapes by Rantsiou et al. (2013). The mycobiota on Garganega grapes at
the end of natural withering described in our study is probably the
result of the combination of many pre- and post-harvest factors (e.g.
seasonal conditions, vineyard and withering management) that affect
survival of different species and their interactions. In this specific
ecosystem, the development of noble rot is linked to these factors and
seems to affect the presence of epiphytic fungi on berries.
In conclusion, this study on mycobiota linked to noble-rot of
Garganega grapes for passito wine production suggests that each single
berry can be considered a micro-environment harbouring a complex
microbial community. Such an ecosystem seems to be unstable, since
several factors (i.e. temperature, humidity, withering process and in-
sects) strongly affect the contamination of fungi and their interactions
making different fungal community structures among berries. The en-
dophytic infection of B. cinerea, detected in noble-rotten berries by PCR
assay in this study, appears to be an essential factor when under-
standing the epidemiology of epiphytic fungi in withered grapes. The
effects of noble rot on grape and wine composition are probably attri-
butable to the fungal community associated with noble-rotten berries
rather than only to B. cinerea. Future studies will aim to investigate the
structure of this community in detail and its impact on grape and wine
quality.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.ijfoodmicro.2018.03.004.
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Table 2
Frequency (%) of each species belonging to fungal genera or families listed in Table 1
identified in 182 isolates from 36 berries (21 noble-rotten and 15 sound).
Species Noble-rotten Sound
Penicillium expansum 12.8a 20.0
Penicillium crustosum 11.1 9.2
Penicillium crocicola 1.7 1.5
Penicillium adametzioides 0.0 1.5
Penicillium glabrum 2.6 1.5
Penicillium oxalicum 0.9 0.0
Aureobasidium pullulans complex 14.5 16.9
Cladosporium pseudocladospoirioides 10.3 9.2
Cladosporium halotolerans 8.5 7.7
Cladosporium cladospoirioides 0.9 0.0
Fusarium verticillioides 2.6 3.1
Fusarium equiseti 1.7 0.0
Botrytis cinerea 11.1 9.2
Alternaria alternata species group 7.7 7.7
Aspergillus uvarum 1.7 1.5
Aspergillus welwitschiae 1.7 0.0
Aspergillus tubingensis 0.9 1.5
Epicoccum nigrum 5.1 4.6
Neofusicoccum parvum 0.9 3.1
Botryosphaeria dothidea 1.7 0.0
Rhizopus arrhizus 0.9 1.5
Mucor circinelloides 0.9 0.0
a n. of isolates of each species on total n. of isolates (182, 117 from noble-rotten and 65
from sound berries) in percentage.
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Tássia Nievierowski
Highlight
	Filamentous fungi associated with natural infection of noble rot on withered grapes
	Introduction
	Materials and methods
	Grape sampling and berry classification
	Scanning electron microscopy analysis of berries
	Determination of water activity of berries
	Isolation and identification of fungi
	DNA amplification and sequence analysis
	Phylogenetic analysis
	Results and discussion
	SEM and water activity analysis in berries
	Isolation frequency and species identification
	Supplementary data
	References