Research Article
Received: 4 August 2015
Revised: 1 February 2016
Accepted article published: 12 February 2016
Published online in Wiley Online Library: 17 March 2016
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7667
Quantification and identification of
microorganisms found on shell and kernel of
fresh edible chestnuts in Michigan
Irwin R Donis-González,a* Daniel E Guyera and Dennis W Fulbrightb
Abstract
BACKGROUND: Chestnut is a relatively new cultivated crop for Michigan, and postharvest loss due to decay has been problematic
as production has increased each year. In 2007, more than 25% of chestnuts were lost to postharvest decay, equivalent to
approximately 5300 kg of fresh product. To determine the organisms responsible for decay, a microbiological survey was
performed in 2006 and 2007 to identify microorganisms involved in postharvest shell (external surface) mold and internal kernel
(edible portion) decay of chestnuts.
RESULTS: Filamentous fungi including Penicillium expansum, Penicillium griseofulvum, Penicillium chrysogenum, Coniophora
puteana, Acrospeira mirabilis, Botryosphaeria ribis, Sclerotinia sclerotiorum, Botryotinia fuckeliana (anamorph Botrytis cinerea)
and Gibberella sp. (anamorph Fusarium sp.) were the predominant microorganisms that negatively impacted fresh chestnuts.
Populations of microorganisms varied between farms, harvesting methods and chestnut parts.
CONCLUSION: Chestnuts harvested from the orchard floor were significantly (P < 0.05) more contaminated than chestnuts
harvested directly from the tree, by more than 2 log colony-forming units (CFU) g−1 . In addition, a significant difference (P <
0.05) in the microbial population was seen between chestnuts submitted by different growers, with average count ranges of
fungi, mesophilic aerobic bacteria (MAB) and yeasts equal to 4.75, 4.59 and 4.75 log CFU g−1 respectively.
© 2016 Society of Chemical Industry
Keywords: postharvest; molds; storage; yeasts; mesophilic aerobic bacteria (MAB)
INTRODUCTION
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Chestnut (Castanea spp.) is considered to be the most popular
nut-bearing tree among the Mediterranean countries of Europe
and many countries in Asia, with new production now starting
in Australia, New Zealand and Chile.1 – 5 The increased market for
chestnuts is partially due to their nutritional value, low lipid content and antioxidant properties.6 – 8 Edible sweet chestnut species
include Castanea dentata Borkh., Castanea mollissima BI., Castanea
crenata Sieb. and Zucc., Castanea sativa Mill., Castanea seguinii
Dode, Castanea pumila Mill. and Castanea henryi Rehd. and Wils.,
among others.1,9,10 Commercial orchards have been established in
various locations in the USA and have been increasing over the
years.1,11 The USA has more than 1500 ha of chestnut tree plantations. Of these, approximately 600 ha of young (less than 10 years
old) chestnut trees are distributed among Michigan, California,
Oregon, Washington and Ohio.11 Commercial plantations are primarily composed of the cultivar ‘Colossal’ (European × Japanese
hybrid C. sativa × C. crenata). Other cultivars planted are composed
predominately of Chinese chestnuts owing to the naturally occurring chestnut blight resistance in this species.1,10 Mature seedling
Chinese chestnut trees (non-grafted trees) also have been planted
in Midwest and Eastern states, with some orchards of seedling
European chestnuts.
North American chestnuts are typically harvested from late
September to the first week of November, with most chestnuts
sold from Thanksgiving through Christmas to specialty ethnic
J Sci Food Agric 2016; 96: 4514–4522
markets, retail stores, food processors, restaurants, holiday festivals, farmers’ markets and individual consumers. In Michigan, fresh
chestnuts and frozen, peeled chestnuts comprise up to 60 and
30% of outlet sales respectively. The remainder is sold dehydrated
as flour, breading and a new product called chestnut slices.12
Since freshness and microbial quality are factors in chestnut
marketing, domestic production has a definite advantage over
imported chestnuts. In Michigan, production is limited primarily
owing to the young nature of the industry and microbial decay.
Nut rot pathogens initiating infection in the field as well as postharvest decay fungi are known to cause severe losses, leading to completely unmarketable chestnuts.
Worldwide, there are two important chestnut kernel pathogens
contributing to kernel decay – both fungal in nature. By pathogen,
we mean a species that can infect a developing or mature healthy
kernel by following evolved mechanisms of pathogenesis. One
∗
Correspondence to: IR Donis-González, Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824, USA.
E-mail: [email protected]
a Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824, USA
b Department of Plant, Soil and Microbial Sciences, Michigan State University,
East Lansing, MI 48824, USA
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© 2016 Society of Chemical Industry
Microorganisms on edible chestnuts in Michigan
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chestnut kernel pathogen, Sclerotinia pseudotuberosa,13 causes
black rot and has been a serious disease of chestnut kernels
in Europe. The other pathogen, Gnomonipsis smithogilvyi, causes
brown rot and has been found in Europe, Australia, New Zealand
and India.14 – 18 Of these two, brown rot is considered to be an
extremely serious threat to chestnut cultivation.19
Several ‘molds’ or fungal species are known to cause severe
postharvest decay and disease problems in chestnuts worldwide.
Among these, Penicillium sp., Aspergillus sp., Fusarium sp., Phomopsis castanea, Acrospeira mirabilis and S. pseudotuberosa (syn. Ciboria
batschiana, S. batschiana; anamorphic from Rhacodiella castanea,
syn. Myrioconium castanea) have been repeatedly identified in
France, Italy, Australia, Chile and the USA.13,20 – 27
Our studies were designed to provide a better understanding of
the impact and possible effect of microorganisms on postharvest
chestnut quality in Michigan. Our goals were to identify microorganisms involved in shell mold and kernel decay on chestnuts,
evaluating the effect of pre- or postharvest methods on microbial
populations, and assess shell mold severity and incidence of kernel
decay of chestnuts collected from different farms in Michigan. This
study was important for two reasons. First, since chestnut cultivation is still a relatively young industry in Michigan, these studies
may act a ‘time zero’ for future studies on kernel pathogens as the
industry matures; and second, to help the young industry cope
with the enormous task of managing postharvest decay. Portions
of this paper were published elsewhere.28
MATERIALS AND METHODS
Collection of chestnut samples and storage
All harvested chestnuts used in this study were collected from the
cultivar ‘Colossal’, a C. sativa × C. crenata (European × Japanese)
hybrid chestnut.1 This is a very popular cultivar in Michigan and
common among Michigan chestnut farms. These nuts were collected either from a single farm in Livingston County, Michigan
during the 2006 and 2007 growing seasons (single-farm study)
or from seven farms scattered throughout Michigan (seven-farm
study). Before long-term storage of chestnuts collected for these
studies, each chestnut sample was transferred to a mesh bag and
then randomly placed within a 2000 kg bin of commercial chestnuts in a cooler at 4 ∘ C located at Michigan State University (East
Lansing, MI, USA). Samples were separately placed on racks to prevent cross-contamination.
J Sci Food Agric 2016; 96: 4514–4522
Identification of microorganisms found on chestnuts
in Michigan
A total of 200 individual ‘Colossal’ chestnuts per growing season
(2006 and 2007) were collected at a chestnut receiving station from
seven different farms. Samples were randomly collected twice during storage (time 0, arrival to 120 days post-arrival at receiving station) to identify the organisms that colonized the chestnuts. Chestnuts showing shell mold or kernel decay symptoms were sorted for
isolation and identification of the causal agents. Before identification, the observed symptoms were described, photographed and
classified.
Identification of microorganisms – MAB and fungi
All microorganisms isolated from chestnuts, whether from shell or
kernel, were selected and subsequently purified after isolation by
repeated transfer on culture media. MAB and fungi were identified
by sequencing rDNA subunits.30
Genomic DNA was extracted from molds using a QIAquick
Gel Extraction Kit (Qiagen, Valencia, CA, USA) as recommended
by the manufacturer. A 0.5–1 mg sample of mycelia taken
from 5–7-day-old molds grown on potato dextrose agar was
immediately ground in a sterile mortar and pestle containing
500 μL of cetyltrimethylammonium bromide (CTAB) buffer (pH
8.3) (Qiagen). After this the DNA was extracted followed the
procedure proposed by Hamelin et al.31 The region including
the two spacers (ITS4 and ITS6) was amplified. A total volume of 25 μL for each reaction contained 60 ng of DNA, 23
μL of AFLP™ amplification core mix (Applied Biosystems, Foster
City, CA, USA), 2.5 units of AmpliTaq Gold DNA polymerase
(Applied Biosystems) and 10 pmol L−1 of each primer. The
primers used, ITS4 (5′ -TCCTCCGCTTATTGATATGC-3′ ) and ITS6
(5′ -GAAGGTGAAGTCGTAACAAGG-3′ ), are complementary to the
final portion of 18S rDNA adjacent to ITS3 and to the initial portion
of 25S rDNA adjacent to ITS2 respectively.30,32
Bacterial genomic DNA was extracted using the protocol
proposed by Jacobs et al.33 The region including the two
spacers (UFLP and URPL) was amplified using the method of
LiPuma et al.34 A total volume of 25 μL for each reaction contained 60 ng of DNA, 23 μL of AFLP™ amplification core mix
(Applied Biosystems), 2.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems) and 10 pmol L−1 of each primer.
The primers UFPL (5′ -AGTTTGATCCTGGCTCAG-3′ ) and URPL
(5′ -GGTTACCTTGTTACGACTT-3′ ) were used for targeting the 16S
rDNA region from the kingdom Procaryotae (bacteria).34
For molds and bacteria, the extracted DNA region was amplified in a 2720 Thermal Cycler (Applied Biosystems). The program
included a cycle at 95 ∘ C for 2 min, 30 cycles of 95 ∘ C for 30 s, 55
∘ C for 30 s and 71 ∘ C for 1 min, and a final elongation at 71 ∘ C for
10 min. The polymerase chain reaction (PCR) product was placed
in wells on agarose gel (12.5 g L−1 ) and electrophoresed (Gel Electrophoresis Power Supply Model 250, Life Technologies, Grand
© 2016 Society of Chemical Industry
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Determination of microbial populations – shell
The following methods were used to determine the microorganisms found on chestnuts regardless of the study (single-farm or
seven-farm study). To determine the microorganisms found on
shells, 25 g chestnut samples were placed in a sterile stomacher
bag (Whirl-Pak, Fisher Scientific, Pittsburgh, PA, USA), diluted 1:5 in
phosphate buffer solution (PBS) (pH 7.4) and agitated for 1 min in
a pulsifier (Filtaflex Ltd, Almonte, ON, Canada). After serial dilution,
100 μL aliquots were spread on trypticase soy agar (Becton and
Dickinson, Baltimore, MD, USA) containing 6 g L−1 Bacto™ yeast
extract (Becton and Dickinson) and 100 μg mL−1 cycloheximide
(Sigma-Aldrich, St Louis, MO, USA) for quantification of mesophilic
aerobic bacteria (MAB), and on potato dextrose agar (Becton and
Dickinson) containing 20 μg mL−1 streptomycin (Sigma-Aldrich)
and 50 μg mL−1 ampicillin (Sigma-Aldrich) for enumeration of
yeasts and molds. The populations of MAB were determined after
48 h at 25 ± 3 ∘ C. Mold and yeast plates were counted after 72 h of
incubation at 25 ± 3 ∘ C.29
Determination of microbial populations – kernel
To determine the microorganisms found on kernels, chestnuts
were flame sterilized after dipping in ethanol (99 mL of 200
proof ethanol in 1 mL of distilled water). After manually removing
the shell, each kernel sample was weighed, placed in a sterile
stomacher bag (Whirl-Pak, Fisher Scientific), diluted 1:5 in PBS and
homogenized for 1 min (normal speed) in a stomacher (Model 400,
Seward, Worthing, UK). The suspension was serially diluted and
quantitatively examined for MAB, yeasts and molds as previously
described.29
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Island, NY, USA) for 3 h at 70 kg m2 s−3 A−1 . After electrophoresis,
the amplified region was purified using a QIAquick PCR Purification
Kit (Qiagen). The PCR product was sequenced in both directions
using moderate-throughput sequencing (Research Technology
and Support Facility, MSU, East Lansing, MI, USA). Sequences were
deposited in Lasergene software (DNASTAR, Madison, WI, USA)
and used as queries for similarity searches in the NCBI nucleotide
database.35 Species reported had a 98–100% match in both directions.
Identification of microorganisms – yeasts
Yeasts were inoculated on potato dextrose agar, incubated for 72
h at 25 ± 3 ∘ C and then sent to the Diagnostic Center for Population and Animal Health (MSU, East Lansing, MI, USA) for further
identification. Yeasts were identified using the API 20C yeast identification system together with microscopic morphology determinations. This computer-assisted system for rapid identification of
yeasts provides results comparable to those obtained by conventional morphological methodologies.36
Quantifying microorganisms from a single farm comparing
nuts collected from ground versus sampled from tree
Mature chestnuts were sampled using a stratified sampling
method with two levels. The first level consisted of nine chestnut
samples (∼1 kg each) taken from the ground after less than 24 h of
contact. The second level was composed of nine chestnut samples
(∼1 kg each) hand-harvested from varying tree heights, in mature
burs. Each sample was placed in a plastic bag and transported in
a portable cooler to the storage facility. Microbial populations on
chestnut shells and kernels over two consecutive growing seasons
(2006 and 2007) were assessed after harvest and at 30, 60, 90 and
120 days of storage.
Microbial mold severity and kernel decay incidence analysis
on chestnuts collected from seven farms
This seven-farm study consisted of evaluating differences between
shell mold severity and kernel decay incidence (microbial quality)
among farms in Michigan. To do so, three 0.8 kg samples of ‘Colossal’ chestnuts per farm were randomly collected before storage
during the 2007 growing season. Each sample was stored in mesh
bags. For consistency, a subsample consisting of three randomly
chosen stored chestnuts collected from each of seven farms was
assessed for severity of shell mold and incidence of kernel decay
after harvest and at 30 and 60 days of storage. Shell mold severity
was calculated as the mean number of a five-point visual qualitative measurement of decay, where 0 = 0% decay, 1 = 1–25% decay,
2 = 26–50% decay, 3 = 51–75% decay and 4 = 76–100% decay.
Incidence of decayed kernels was calculated as the mean number
of chestnuts showing symptoms of decay.
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Statistical analysis
One-factor and repeated measurement designs with analysis of
variance (ANOVA) were done on all microbial count and microbial qualitative assessment data obtained from fresh chestnuts.
Since all chestnuts were randomly assigned to the different treatment conditions, sphericity can be assumed, making multivariate as well as degrees of freedom corrections unnecessary.37,38
Multivariate ANOVA (MANOVA) was also used to determine differences between the microbial populations in chestnuts from different farms. Significance between means was determined using
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IR Donis-González, DE Guyer, DW Fulbright
the Tukey post hoc multiple comparisons of means test at the
95% family-wise confidence level (P = 0.05). Calculations were performed using the statistical package ‘R: a language and environment for statistical computing’ (R Development Core Team,
2007).38
RESULTS
Identification of microorganisms colonizing the shell
of chestnuts in Michigan and their associated symptoms
Chestnuts (from ‘Colossal’ trees pollinized with unknown pollen)
harvested by growers from seven different farms and taken to
a receiving station were colonized by a broad range of microorganisms (Table 1), not all of which were thought to cause shell
mold. Fungi that infested and caused shell mold on fresh chestnuts
were mainly composed of three species of Penicillium (P. expansum, P. griseofulvum, P. chrysogenum) and Coniophora puteana.
Dark-green to black spots with white mycelia were observed on
chestnuts colonized by Penicillium species. These spots began on
the hilum (Fig. 1a). Once the fungus grew, a mantle of white
mycelia containing dark-green, blue, bluish-green or olive-green
completely covered the shell (Fig. 1b). In extreme conditions the
fungus softened and discolored the shell tissue, subsequently
affecting the quality of the fresh product (Fig. 1c). Symptoms
from Penicillium were first observed after harvest and developed
rapidly during storage. Penicillium species belong to the phylum Deuteromycota, producing green, bluish-green or olive-green
spores asexually. Various species cause blue and green mold rots.
They are the most common and usually the most destructive of
all postharvest diseases, affecting various fruits and vegetables,
including commodities of importance such as apples and citrus.39
In addition to the losses caused, the fungus also produces several
mycotoxins such as patulin and zearalenone.40
Coniophora puteana rarely infected the shell; however, when
present, the organism developed in pockets of white web-like
mycelia around several chestnuts (Fig. 1d), which had apparent high moisture content. After manually removing the fungus,
the appearance and quality of the chestnuts were not affected.
This fungus belongs to the phylum Basidiomycota. It has never
been reported affecting chestnuts or other commodities, but
it can be found affecting indoor wood structures and stored
wood, causing wet brown rot, significant decay and economic
damage.41
Identification of microorganisms colonizing the kernel
of chestnuts in Michigan and their associated symptoms
Microorganisms infecting chestnut kernels primarily included
Penicillium spp. (P. griseofulvum, P. expansum, P. chrysogenum),
A. mirabilis, Botryosphaeria ribis, Sclerotinia sclerotiorum, Botryotinia fuckeliana (anamorph Botrytis cinerea) and Gibberella sp.
(anamorph Fusarium sp.). Penicillium spp. (P. griseofulvum, P. expansum, P. chysogenum) produced white mycelia and dark-green,
blue, bluish-green or olive-green spores (Fig. 2a). Penicillium
spp. sometimes penetrated deep into the kernel, resulting in
extensive kernel rot or complete kernel decay (Fig. 2b). Acrospeira
mirabilis appeared as dark-brown spots (conidia) filling the
space between the kernel cotyledons and kernel cracks. Whitish,
web-like mycelia developed around these spots. Brown necrotic
spots were observed around the colonies, which sometimes
enlarged, turned light-brown and finally dark-brown. The infection sometimes penetrated deep into the kernel, softening the
tissue and resulting in opaque brownish kernel decay (Fig. 2c).
© 2016 Society of Chemical Industry
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Microorganisms on edible chestnuts in Michigan
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Table 1. Microorganisms isolated from stored chestnut shell and kernel during 2006 and 2007 growing seasons in Michigan
Symptom
Organism
Fungi
Species
Penicillium sp.
Bacteria
Kernela
appearanceb
Identificationc
F
F
BS, DS
18S (99/99)-M
Penicillium expansum
F
F
BS, DS
18S (99/98)
P. griseofulvum
F
F
BS, DS
18S (99/98)
Comments and references
Genus has been reported to cause mold and decay in
chestnuts22 – 25,43,45,50 and other commodities of economic
importance39
P. chrysogenum
F
F
BS, DS
18S (99/98)
Coniophora puteana
O
NF
DS
18S (100/98)
Not reported in chestnuts before. Fungus of importance causing
indoor wood decay43
Botryosphaeria ribis
(anamorph Fusicoccum
ribis)
NF
O
DS
18S (100/98)
Not reported in chestnuts before. Fungus of importance infecting
many trees, including olive, pecan, pistachio, kiwi, apple and citrus.
It causes cankers, dieback, death and black fruit rots51 – 53
B. tsugae
NF
O
DS
18S (99/99)
Trichoderma viride
NF
R
BS, DS
18S (100/99)
In this study it was found in chestnuts directly harvested from the tree
and during storage. It has never been reported in chestnuts before
and does not appear to cause any postharvest disease. Has been
used as a biological control, belonging to the group of
antibiotic-producing microorganisms54
Botryotinia fuckeliana
(anamorph Botrytis
cinerea)
NF
R
DS
18S (99/99)
Genus has been reported to cause mold and decay in chestnuts25 and
other commodities of economic importance, including berries,
apples and grapes, during storage39
Phoma sp.
NF
R
DS
18S (100/99)
Genus has been reported to cause significant decay in chestnuts25
and other commodities of economic importance, including papayas
and kiwis53,55
Sclerotinia sclerotiorum
NF
R
BS, DS
18S (99/99)
Has never been previously reported on chestnuts, but symptoms and
pathogenesis may be similar to those of S. pseudotuberosa, causal
agent of chestnut black rot.13 Pathogen that significantly affects all
annual vegetables, ornamentals and field crops39
Acrospeira mirabilis
NF
F
BS, DS
M
Gibberella sp. (anamorph
Fusarium sp.)
R
O
DS
18S (100/99)-M
G. moniliformis (anamorph F.
verticillioides)
R
O
DS
18S (100/99)
G. zeae (anamorph F.
culmorum)
R
O
DS
18S (100/99)
G. graminearum (anamorph
F. graminearum)
R
O
DS
M
NF
R
DS
18S (99/99)
Candida sp.
F
F
NS
API 20C
C. guilliermondii
O
O
NS
API 20C
Cryptococcus sp.
F
F
NS
API 20C
Cr. luteolus
O
O
NS
API 20C
Cr. laurentii
O
O
NS
API 20C
Rahnella sp.
O
O
NS
16S (99/99)
Rahnella aquatilis
O
O
NS
16S (98/97)
Bacillus sp.
NF
R
NS
16S (99/99)
Methylobacterium sp.
O
O
NS
16S (99/99)
Discula campestris
Yeasts
Shella
Has been reported to cause significant decay in chestnuts after
harvest and during storage20
Genus has been reported to cause mold and decay in chestnuts,
especially during storage,22 – 25,43,45,50 and other commodities of
economic importance, causing postharvest pink or yellow molds on
vegetables, ornamentals, root crops, tubers, tomatoes and bulbs39
Has never been previously reported on chestnuts. Fungus of
importance causing anthracnose in sugar maple, especially in
seedlings56
In this study, these microorganisms did not appear to cause
postharvest decay in fresh chestnuts. Other microflora survey
studies indicated that after harvest a wide range of economically
important vegetables and other commodities are often colonized
but usually spoiled when they are processed, including chestnuts,
sliced onions and shredded lettuce. Sometimes they are also used
as antagonist microorganisms against certain postharvest
diseases.25,57 – 60 The majority of these microorganisms, including
Rahnella sp., Candida sp. and Cryptococcus sp., are widely
distributed in nature61,62
F, frequent (identified on ≥80% of affected samples); O, occasional (identified on ≤25% of affected samples); R, rare (identified on ≤5 % of affected samples); NF, not found.
BS, symptom appearance before storage; DS, symptom appearance during storage; NS, no symptoms.
c 18S (% match forward/reverse)-M, fungus identified using sequencing of 18S-rDNA subunit and microscopic morphology; 18S (% match forward/reverse), fungus
identified using sequencing of 18S-rDNA subunit; M, microscopic morphology identification; 16S (% match forward/reverse), bacterium identified using sequencing of
16S-rDNA subunit; API 20C, yeast identified using API 20C yeast identification system and microscopic morphology.
a
b
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IR Donis-González, DE Guyer, DW Fulbright
(a)
(b)
(c)
(d)
Figure 1. Chestnuts showing shell mold due to (a–c) Penicillium spp. (P. griseofulvum, P. expansum and P. chrysogenum) and (d) Coniophora puteana.
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Belonging to the phylum Deuteromycota, this fungus causes
considerable losses in chestnuts during storage. It lacks fruiting
bodies, producing conidia asexually. Conidia are produced coiled,
with two pale cells and one dark-brown, verrucose, globose
terminal cell (20–30 μm diameter).20
Botryosphaeria ribis and S. sclerotiorum were isolated from only
a few kernels that presented extreme dark-black decay (Fig. 2d).
In some situations, infected tissue became hard and completely
mummified. Botryosphaeria ribis and S. sclerotiorum belong to
the phylum Ascomycota. Botryosphaeria ribis produces conidia in
pycnidia, while the fungus S. sclerotiorum overwinters as sclerotia
on or within infected tissues and in the ground. In spring or early
summer the sclerotia germinate and produce apothecia, which
contain asci, producing ascospores.39
Botryotinia fuckeliana (anamorph B. cinerea) was isolated from
grayish, completely decayed kernels. The mycelium appeared to
rapidly invade the whole kernel, which became covered with
a whitish-gray, cobweb-like mold. Infected kernels became soft,
watery and acquired a dark-gray to black coloration (Fig. 2e).
Depending on its sexual stage, this fungus belongs to the phylum Ascomycota or Deuteromycota. It causes gray molds or rots
of fruits and vegetables both in the field and during storage. It
produces abundant gray mycelia and long branches of conidiophores, which contain rounded apical cells bearing colorless or
gray, one-celled, ovoid conidia.39
Gibberella sp. (anamorph Fusarium sp.) caused white mold
decay. Affected kernels appeared dry, whitish to light-brown and
extremely pale. A whitish web-like mycelium could be observed
around decayed tissue, usually in the cracks (Fig. 2f ). Depending
on its sexual stage, this fungus belongs to the phylum Ascomycota
or Deuteromycota. The fungus usually produces colorless mycelia
at first, but with age it becomes cream-colored, pale-yellow,
pale-pink or purplish. It produces three kinds of asexual spores,
microconidia, macroconidia and chlamydospores, but also produces sexual spores, through perithecia, during its sexual phase.39
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In this study, this microorganism was usually mistaken for early
infection by Penicillium spp., and usually both were present.
Influence of chestnut harvesting method on microbial
populations
Non-decaying chestnuts yielded low populations of MAB, molds
and yeasts on shells and in kernels when collected on day 0 from
trees and the ground. The microbial population increased significantly during storage (P < 0.01) (Figs 3–5). Microbial populations
at day 0 ranged from 1.8 to 4.61 log colony-forming units (CFU)
g−1 depending on the portion of the chestnut examined (shell
or kernel) and the harvesting method (ground or tree). The shell
yielded significantly higher (P < 0.01) microbial populations compared with the kernel.
In general, populations of molds and MAB tended to increase
during 60–90 days of storage regardless of the harvest procedure
(Figs 3 and 4). Populations of microorganisms were generally
highest in 90-day-old chestnuts at the time of marketing (60–90
days after harvest) (Figs 3 and 4). In the case of molds and MAB,
microbial populations increased to 8 and 8.3 log CFU g−1 by day
60 and then decreased to 6.1 and 6.7 log CFU g−1 after 120 days
of storage respectively. During storage, mold (P < 0.01) and MAB
(P < 0.01) populations of chestnuts harvested directly from the tree
were significantly lower than those of chestnuts harvested from
the ground (Figs 3 and 4).
An irregular growth rate and significant population increase
(P < 0.01) were observed in yeasts colonizing the shell. The yeast
population on chestnuts collected from the orchard ground
increased significantly (P < 0.01) to 8.5 log CFU g−1 during 120
days of storage. Meanwhile, yeast populations from the shell of
chestnuts collected directly from the tree were more variable,
with a decline in population from 4.7 log CFU g−1 on day 30 to 2.3
log CFU g−1 90 days later. Nevertheless, after 120 days, a significant increase (P < 0.01) to 6.7 log CFU g−1 was observed. During
storage, yeast populations on the shell were significantly higher
© 2016 Society of Chemical Industry
J Sci Food Agric 2016; 96: 4514–4522
Microorganisms on edible chestnuts in Michigan
www.soci.org
(a)
(b)
(c)
(d)
(e)
(f)
Figure 2. Chestnuts showing kernel decay due to (a, b) Penicillium spp. (P. griseofulvum, P. expansum and P. chrysogenum), (c) Acrospeira mirabilis, (d)
Botryosphaeria ribis and Sclerotinia sclerotiorum, (e) Botryotinia fuckeliana (anamorph Botrytis cinerea) and (f ) Gibberella sp. (anamorph Fusarium sp.).
Figure 3. Mean mold counts on shells and in kernels of fresh chestnuts
during 120 days of storage at 4 ∘ C. 1 Data points followed by the same
lowercase letter within the same day are not significantly different at
P < 0.05 (ANOVA with post hoc Tukey multiple comparison of means).
2 Overall data points followed by the same capital letter within chestnut
part-harvesting method are not significantly different at P < 0.05 (repeated
measurement design – ANOVA with post hoc Tukey multiple comparison
of means). Error bars indicate standard deviation.
(P < 0.01) than those in the kernel. Populations of yeasts on kernels
collected from the tree or ground remained relatively constant
during storage and were not significantly different (Fig. 5).
J Sci Food Agric 2016; 96: 4514–4522
Populations on shell varied from farm to farm, with MAB, molds
and yeasts ranging from 4.55 to 8.44, 3.94 to 7.74 and 4.75 to 8.28
log CFU g−1 respectively (Fig. 6). A similar scenario was observed
for kernels, with MAB, molds and yeasts ranging from 2.34 to 7.63,
1.99 to 7.69 and 2.68 to 7.24 log CFU g−1 respectively (Fig. 7). Significant differences were observed in shell mold (P < 0.01), MAB
(P < 0.01) and yeast (P < 0.01) populations (Fig. 6) and in kernel
MAB (P < 0.01) and yeast (P < 0.01) populations (Fig. 7). MANOVA
(Wilks’ test) showed that microbial populations differed significantly (P < 0.01) between some farms. Chestnuts from farm C had
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4519
Farm influence on microbial populations on shell and in
kernel of chestnuts
To determine the effect of different growing and harvesting
conditions on postharvest microbial populations of chestnuts on
various farms in Michigan, chestnuts were collected from seven
different growers at Chestnut Growers, Inc. on the day of delivery.
Figure 4. Mean MAB counts on shells and in kernels of fresh chestnuts
during 120 days of storage at 4 ∘ C. 1 Data points followed by the same
lowercase letter within the same day are not significantly different at
P < 0.05 (ANOVA with post hoc Tukey multiple comparison of means).
2 Overall data points followed by the same capital letter within chestnut
part-harvesting method are not significantly different at P < 0.05 (repeated
measurement design – ANOVA with post hoc Tukey multiple comparison
of means). Error bars indicate standard deviation.
www.soci.org
Figure 5. Mean yeast counts on shells and in kernels of fresh chestnuts
during 120 days of storage at 4 ∘ C. 1 Data points followed by the same
lowercase letter within the same day are not significantly different at
P < 0.05 (ANOVA with post hoc Tukey multiple comparison of means).
2 Overall data points followed by the same capital letter within chestnut
part-harvesting method are not significantly different at P < 0.05 (repeated
measurement design – ANOVA with post hoc Tukey multiple comparison
of means). Error bars indicate standard deviation.
Figure 6. Total MAB, mold and yeast counts from shells of fresh chestnuts
after harvest from seven Michigan farms. 1 Values followed by the same
letter within organisms are not significantly different at P < 0.05 (ANOVA)
(Tukey multiple comparison of means). Error bars indicate standard
deviation.
significantly higher microbial populations than chestnuts from
farm D, with chestnuts from other farms showing microbial populations between those of farms C and D (Fig. 7).
4520
Farm influence on microbial quality of chestnuts
To determine the effect of different growing and harvesting conditions on the severity of postharvest mold and kernel decay,
chestnuts were collected from the seven different growers when
received at Chestnut Growers, Inc. On receiving day (day 0), significant differences in mold severity (P < 0.05) and kernel decay
incidence (P < 0.05) were observed between growers (Fig. 8a).
Overall, during 60 days of storage, a significant difference in shell
mold severity (P < 0.01) but not in kernel decay incidence was
observed between growers (Fig. 8b). Farms C, B and E showed
significantly higher shell mold severity compared with other farms,
with values of 2.8, 1.5 and 1.9 log CFU g−1 respectively.
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IR Donis-González, DE Guyer, DW Fulbright
Figure 7. Total MAB, mold and yeast counts from kernels of fresh chestnuts after harvest from seven Michigan farms. 1 Values followed by the
same letter within organisms are not significantly different at P < 0.05
(ANOVA) (Tukey multiple comparison of means). Minimum detectable
level (MDL) = minimum possible count (0.5) × minimum dilution factor × inoculated aliquot (100 μL). Error bars indicate standard deviation.
CONCLUSION
Postharvest shell mold and kernel decay affect a significant portion
of chestnuts worldwide and about 20% of the chestnuts produced
in Michigan. Results showed that fungi, bacteria and yeasts
increased significantly during storage. The extent of increase was
dependent on the part of the chestnut (shell or kernel), the collecting method (tree or ground) and the farm from which is was harvested. Highest populations were found on the shells of chestnuts
collected from the ground. These results are not surprising as the
shell is continually exposed to the environment while the kernel is
protected by the shell and pellicle. Molds were the most noticeable
organisms associated with the shell and kernel decay of chestnuts
during refrigerated storage (4 ∘ C). Similar problems related to
mold decay have also been reported in Asia, Europe, South America and North America.10,13,21 – 23,26,42,43 The fungal flora responsible
for this infestation is diverse and appears to be strongly influenced
by preharvest, harvest and storage conditions. With the traditional
method of harvest, chestnuts that have fallen to the ground are
collected by workers or mechanical harvesters.44 In Italy and
France, attempts have been made to collect chestnuts in nets
either on the ground or suspended above the ground, mainly to
ease chestnut collection45,46 and reduce molding, especially by
S. pseudotuberosa.45 However, a recent study indicated that suspended nets did not reduce chestnut mold after harvest or during
storage.25 Regardless, in the present study, chestnuts harvested
from the orchard floor showed more contamination measured
during storage than those harvested directly from the tree.
Microbial populations were much higher on chestnut shells than
in kernels. In general, only two fungal genera caused obvious mold
symptoms with chestnut shells after harvest and during storage.
In order of importance, these were Penicillium spp. (P. expansum,
P. griseofulvum, P. chrysogenum) and C. puteana. However, at least
five different genera of filamentous fungi were associated with
infected chestnut kernels. These microorganisms, which included
Penicillium spp. (P. griseofulvum, P. expansum, P. chrysogenum),
A. mirabilis, B. ribis, S. sclerotiorum, B. fuckeliana (anamorph B.
cinerea) and Gibberella sp. (anamorph Fusarium sp.), caused the
largest amount of loss. Other organisms might be associated with
chestnut shell mold and kernel decay in Michigan, but during this
two-season study, only these species were repeatedly associated
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J Sci Food Agric 2016; 96: 4514–4522
Microorganisms on edible chestnuts in Michigan
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Figure 8. Shell mold severity and kernel decay incidence from seven Michigan farms during (a) 0 day storage at 4 ∘ C and (b) 60 day storage at 8 ∘ C.
1 Values followed by the same letter within quality index are not significantly different at P < 0.05 (repeated measurement design – ANOVA) (Tukey multiple
comparison of means). Error bars indicate standard deviation.
with postharvest shell mold and kernel decay. Similar findings
have been reported for chestnuts harvested in France, Italy,
Australia, Chile, the USA and other countries.13,20 – 24,26,27
This was the first survey to associate C. puteana, B. ribis and S. sclerotiorum with chestnut shell mold or kernel decay. Furthermore, S.
sclerotiorum, which was found infrequently in our survey, has never
been previously reported on chestnuts, but symptoms and pathogenesis may be similar to those of S. pseudotuberosa, the mold
responsible for chestnut black rot, which is one of the most important postharvest diseases of acorns (Quercus spp.) in Europe.21,26
Findings concerning differences between mold severity in shells
and microbial populations on chestnut kernels among farms
demonstrated that location, preharvest and harvesting conditions
played an important role in quality and decay of chestnuts, as discussed for other commodities.25,47,48 Variation in microbial populations among farms, increases in populations during storage and
variability of populations within the same field during storage suggest multiple preharvest sources of contamination, including soil,
feces, irrigation water, water used to apply fungicides and insecticides, insects, inadequately composted manure, wild animals and
human handlers.49 Therefore complementary studies on the efficacy of preharvest management, preharvest fungicide applications and path of produce contamination, as well as harvesting
methods and storage conditions, must be considered. Furthermore, the interactions between climate and epidemiology of the
fungal flora in the field also need to be better understood. Currently, all these factors must be taken into consideration and confirm the need to control the organisms prior to storage to avoid or
reduce postharvest mold and decay during storage. These efforts
must be in concert with a plan to efficiently identify and manage
affected products during harvest and storage.
Significantly, no major chestnut shell or kernel pathogens were
found during this study. Neither S. pseudotuberosa13 (black rot) nor
G. smithogilvyi17 (brown rot) pathogens were detected. Sclerotinia
sclerotiorum, a close relative of S. pseudotuberosa, was detected
in this study, but it has not been linked to a serious disease of
chestnut kernels.
ACKNOWLEDGEMENTS
J Sci Food Agric 2016; 96: 4514–4522
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