Scientia Horticulturae 257 (2019) 108737

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Pre- and post-harvest factors that affect the quality and commercialization of T
the Tahiti lime
⁎
Blanca Lucia Botina A, María Cristina García M , Yajaira Romero B
Corporación Colombiana de Investigación Agropecuaria-Agrosavia, Centro de Investigación Tibaitata- Km 14 vía Mosquera, Bogotá, Colombia

A R T I C LE I N FO A B S T R A C T

Keywords: Colombia has an important Tahiti lime production, but despite the adequate edaphoclimatic and seasonal ad-
Rootstock vantages for its production, its competitiveness is limited by appearance affectations and the short useful post-
Surface damage harvest life caused by inadequate handling. Different symptomatology can be identified in the external ap-
Diseases pearance of the fruit that can be triggered by either preharvest, or postharvest factors. In the current study, the
Storage
effect of the location (Lebrija and Villavicencio), the rootstocks (Citromelo, Kryder and Volkameriana), the crop
Postharvest injuries
Postharvest losses
season (dry and rainy seasons) and the storage conditions (temperature and disinfection) on fruit quality were
Environmental conditions assessed. The relationship between the damage affecting the appearance and the evaluation factors were
identified using a Pearson Chi-square statistical analysis. The best quality was observed in fruit from Lebrija,
harvested in the dry season, disinfected, and stored at 10 °C. In the identification of the biological factors that
affect the appearance of the Tahiti lime fruit, fungi developed during pre- and post-harvest phase were isolated,
and strains of the genera Colletotrichum spp., Fusarium spp., Alternaria spp., Penicillium spp., Acremonium spp.,
Trichoderma spp., Curvularia spp., Phoma spp., Stachybotrys spp., and Ulocladium spp.,were identified.

1. Introduction as conditioning (selection, washing, disinfection, waxing, sorting, and

Tahiti lime (Citrus latifolia Tanaka) is a citrus fruit worldwide
commercialized. Imports of this fruit to European countries and the
United States have increased due to the growing use of fresh limes in
the preparation of food and beverages. The leading supply comes from
Mexico and Brazil; however, other developing countries such as
Colombia, Vietnam, Guatemala, and Peru are emerging as exporters
(CBI, 2018).
This fruit crop is grown in 21 departments of Colombia in orchards
and commercial plantations, due to the favorable environmental con-
ditions of the country that allows its production when the international
market shows low supply periods. In 2016, Colombia had a production
of 79,200 t of lime (Agronet, 2018). However, only 12% of the pro-
duction was exported (Legiscomex, 2018). Crop management and
maintenance of the external fruit quality are some of the limitations
that prevent an increase in the participation of the country in the world
market (León et al., 2009).
There are several factors along the Tahiti lime chain that determine
the final quality and safety of the fruit. In the pre-harvest or production
cycle, crop location, soil and climate conditions (Castle, 1995), the
rootstock, the cultural activities carried out, and the presence of pests
and diseases, affect fruit quality. Likewise, post-harvest operations such
packaging), transportation, and storage also affect the maintenance of
fruit appearance, its shelf life, quality, and market value.
Post-harvest diseases causes significant economic losses, and one of
the main problems associated with this is that symptoms are not
identified in the early stages. For this reason, fruit that are already
infested from cropping remain in the conditioning line, quickly dis-
persing the disease to the rest of the fruit, and causing important fruit
loss. Usually, filamentous fungi cause diseases that generate rots in the
fruit, and the symptoms depend on the causative pathogen and the
saprophytic fungi that are present on the fruit surface (Pássaro et al.,
2012).
The generation of strategies to minimize fruit damage and maintain
its quality for longer periods is crucial to increase the international
market competitiveness of the chain. Hence, to identify the causes that
affect fruit quality, especially regarding its appearance, is a clue factor
to reduce postharvest losses and increase the acceptance and value of
Tahiti lime in the international market. In view of that, the aim of this
study was to assess the effect of the location (associated with edapho-
climatic conditions), the rootstock, the growing season, and the storage
conditions on the final appearance of Tahiti lime fruit (main quality
parameter judged by the market) for international market commercia-
lization.

⁎
Corresponding author.
E-mail address: mcgarcia@agrosavia.co (M.C. García M).

https://doi.org/10.1016/j.scienta.2019.108737
Received 30 May 2019; Received in revised form 31 July 2019; Accepted 31 July 2019
Available online 14 August 2019
0304-4238/ Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
B.L. Botina A, et al.
2. Materials and methods
2.1. Plant material
Tahiti lime (Citrus latifolia Tanaka) fruit from the Volkameriana,
Citromelo and Kryder rootstocks were obtained from two locations: the
producing farm La Bodega (Department of Santander, municipality of
Lebrija, Vereda El Puente), located at 7° 3′ 17.2′' N and 0.73° 13′ 21.5′'
W, at an altitude of 1138 m above the sea level, with an average tem-
perature of 22.7 °C, average precipitation of 1185 mm, and relative
humidity of 75.2%; and the second one was the Research Center "La
Libertad" of AGROSAVIA (Department of Meta, municipality of
Villavicencio), located at 4° 03′ N, and 73° 29′ W, at an altitude of
336 m.a.s.l., with an average temperature of 26 °C, and precipitation of
2918 mm.
2.2. Experimental design and treatments
Treatments were arranged in a three × two × two factorial (three
rootstocks, two locations and two season) with four replications ar-
ranged in a completely randomized block design with 18-tree plots, (six
trees per every rootstocks). 1015 and 2243 lime fruit were harvested in
dry and rainy season respectively, refrigerated and transported to the
Analytical Chemistry Laboratory of the Tibaitatá Research Center of
AGROSAVIA located in Mosquera, Cundinamarca, where they under-
went three different storage conditions: Treatment T1 (fruit stored at
10 °C), treatment T2 (fruit disinfected by immersion in 250 ppm of so-
dium hypochlorite for 4 min, rinsed, drained and stored at 10 °C); and
the control treatment T3 (fruit stored at room temperature, i.e., 18 °C).
Quality of lime fruit was measure in a relative approach, according to
the methodology used by Rodríguez et al. (2001). The evaluation of the
effect of each factor on the quality of Tahiti Lime was quantified ac-
cording to formula like this, where healthy fruit was used as an ex-
ample.
Number of healthy fruit
% healthy lime = x 100
Number of total fruit evaluated
A similar formula was used for quantifying the different type of
preharvest and postharvest damage. The quality criteria chosen was
appearance due to for international market this trait is the most im-
portant criteria for quality in Tahiti lime.
2.3. Classification of superficial fruit damage
The lime fruit was evaluated after five-week storage period and then
the damages observed on the surface were identified and classified, not
only for its origin but also for the type. Thus, Tahiti lime showing a
healthy appearance and suitable for marketing was classified in the first
group, healthy fruit. Fruit with sunspots, scars or damage caused by
pests were classified as fruit with pre-harvest damage. Fruit with al-
terations such as loss of green color and the appearance of yellow tones,
dehydrated or dryness fruit, brown fruit not associated to biological
damages, mechanical damages, and damages by microbiological agents
were classified as lime with postharvest damage. These damages were
typified and quantified by season, location, rootstocks and storage
treatment.
2.4. Isolation and identification of fungi
The lime fruit selected for the isolation of microorganisms that de-
veloped damage during storage and those that were initially collected
from the culture of healthy characteristics or with some superficial
condition were subjected to disinfection being rinsed in 75% alcohol
and 1% sodium hypochlorite for one minute, interspersing rinses with
sterile distilled water between disinfectant solution cleansings. Later
fruit was incubated in humid chambers at 19 °C for five weeks. At the
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Scientia Horticulturae 257 (2019) 108737
end of the incubation period the microorganisms were isolated in
Potato Dextrose Agar (PDA Oxoid) with Chloramphenicol 0.05%
(Sigma®) and incubation at 25 °C for seven days.
The macroscopic and microscopic morphological characterization of
each of the isolated fungi was carried out based on the key proposed by
Barnett and Hunter (2003). The observation of the fungal structures
was carried out through slides or micro-cultures carried out in PDA
(Oxoid) at pH 5, Water Agar (Oxoid) and Malt Agar (Merck).
2.5. Statistical analysis
A simple correspondence analysis was applied to evaluate the effect
of the variation source, such as the season, location, rootstock, and
storage treatment, on the susceptibility of the external fruit damages,
using the Chi-square statistic and calculating relative frequencies; the
statistical program SAS version 9.4 was used.
3. Results
3.1. Factors related to fruit damage
The effect of the location, the harvest season and the rootstock on
fruit quality are presented in Fig. 1(a–c). During the rainy and dry
season, and after five-week of storage, only 10 and 13% of Tahiti Lime
maintained its quality, and therefore, the remaining fruit, showed da-
mage in rainy and dry season 87 and 90%, respectively. The causes of
damage in the two seasons were different, and according to the simple
correspondence analysis, there is a relationship between the harvest
season and the type of damage shown by the fruit (P-value < 0.0001,
Chi square 239.0025 with df 2). In the rainy season at the end of the
storage period, 67% of the limes harvested in the two regions presented
damages originated during post-harvest; meanwhile, in the dry season,
48% of the fruit presented damage originated during pre-harvest and
42% in post-harvest, Fig. 1a. Regarding the locality and the effect on
the causes of fruit damage, there is a relationship between these two
variables (P-value < 0.0001) according to the simple correspondence
analysis using Pearson's Chi-Square test. Fig. 1b shows fruit from Leb-
rija reported 15% healthy fruit at the end of the storage period, com-
pared to the only 2% reported for fruit from Villavicencio. In the case of
Lebrija, post-harvest factors (50%) were the major causes of fruit da-
mage; with also a considerable participation (35%) of pre-harvest fac-
tors. The causes of fruit damage coming from Villavicencio were due to
both pre- and post-harvest factors, without presenting significant dif-
ferences between them.
Finally, Fig. 1c describes the response of each of the rootstocks to
different damage factors during pre- and post-harvest. Although this
figure allows observing a higher percentage of post-harvest damage
compared to the pre-harvest damage suffered by fruit from different
rootstocks, the statistical analysis did not find a relationship between
these two variables (P-value > 0.0650), concluding that the pre- and
post-harvest damage of the fruit does not depend or is not associated
with the rootstocks.
3.2. Types of pre-harvest damage
Fig. 2, shows different preharvest damage symptoms found in the
samples and used to typify this kind of damage. The most common were
sunburn (Fig. 2a), which looks like a dicoloration of peel fruit; scars
(Fig. 2b), caused by scratches, cuts, punctures and scrasping or rubbing
with the branches; and damage caused by pest (Fig. 2c). Damage caused
by insects were evidenced by fruit showing a cork-like bark, spots,
scarred stings or rough texture of a dark brown or gray color, scar-
ification of the fruit peel of a silvery color, or chlorotic stippling and
destruction of sub-epidermal cells (galleries) by excavation (Mangione,
2016); as observed in Fig. 2c.
B.L. Botina A, et al.
Fig. 1. Effect of: a) Growing season (rainy and dry seasons), b) Production
location (Lebrija and Villavicencio), and c) Rootstock (Citromelo, Kryder and
Volkameriana), on the Tahiti Lime quality.
3.3. Preharvest damage, growing season, location and rootstocks
The incidence of pre-harvest damage and its relationship with the
growing season, the location, and the rootstock can be seen in Fig. 3.
The statistical analysis indicated that there is a relationship between the
growing season and pre-harvest damage factors (P-value < 0.0001).
Fig. 3a shows that the damage by scars as well as by sunburn is more
common in the rainy season than in dry season. Conversely to those
damage by pests that are more common in the dry season, which is in
concordance with reported by Departamento Administrativo Nacional
de Estadística, DANE (2015).
According to the statistical analysis, a relationship between pre-
harvest damage and location was also found (P-value < 0.0001).
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Scientia Horticulturae 257 (2019) 108737
Fig. 3b shows how damage by pests is the primary cause of loss in Tahiti
lime fruit from Villavicencio; whilst, in Lebrija, the main cause of loss
was sun damage; although damages by scars were also found in higher
proportion in fruit coming from this location.
Regarding the rootstocks, the statistical analysis indicates that there
is a relation between rootstocks and the pre-harvest damages (P-
value < 0.0003). The rootstocks Citromelo (42%) and Volkameriana
(46%) are more susceptible to damage by pests but less susceptible to
damage by scars (Fig. 3c). Regarding damage by sunburns, no sig-
nificant differences were observed.
3.4. Types of post-harvest damage
Fig. 4 illustrates the different types of damage that fruit may show in
post-harvest and that were taken as a reference to establish the damage
typology in this study.
Dehydration (Fig. 4a) is one of the most common damage in post-
harvest due to its handling and storage under low relative humidity
conditions, usually below the recommended of 90%. In this study, lime
with different degradation of chlorophyll refers to the fruit that lost its
green color and showed yellow tones in different degrees as is depicted
by the Fig. 4b; due to chlorophyll degradation during postharvest sto-
rage, but not for ripeness process as Tahiti lime is a non-climateric fruit.
Oil spotting or oleocellosis is one of the most typical damage in citrus
and it is caused by the rupture of the oils glands, releasing oil that has a
phytotoxic action on the surrounding cells, generating a damage as that
illustrated in the Fig. 4c. Despite oleocellosis can be due to different
causes, this damages was classified as a mechanical damage, con-
sidering that bruises is one of the most common cause of the breakage
of oil cells, in the flavedo. Brown color, Fig. 4d, can have different
causes, but one of most known is by chilling injury, and among their
symptoms can be cited pitting and brown discoloration. Mechanical
injuries, Fig. 4e. is a common damage and also is directly related to
microbial damage, Fig. 4f., as spores or other forms of microbial in-
fection use this injuries to penetrate or colonize the fruit.
The fruit in post-harvest phase were classified in five categories:
healthy fruit, and four additional categories according to the type of
damage (yellow color, brown color, mechanical and microbiological
damage).
The statistical analysis for post-harvest damage variables and sto-
rage treatments indicates that there is a relation between these two
factors with a P-value < 0.0001. In Fig. 5, storage treatments T1 and T2
show a substantial difference in terms of percentage of healthy fruit (43
and 45%, respectively) compared to the control T3 (13%).
3.4.1. Yellowing
Treatments under refrigeration although reduced the loss of green
color, did not control the discoloration of the lime fruit caused by
chlorophyll oxidation (Arias and Toledo, 2000). Lime fruit under room
temperature presented 79% of fruit with yellow color compared to 56
and 52% of the fruit subjected to T1 and T2 respectively.
3.4.2. Dehydration
In the control treatment (T3) most of the fruit turned from green to
yellow color; but also 93% of this yellow fruit showed marked dehy-
dration symptoms. 7% showed only dehydration symptoms keeping its
green color along storage.
3.4.3. Brown spot
The second most frequent fruit damage during storage were brown
spots or brown pigmentation on the fruit peel with values of 17%, 19%,
and 13% for treatments T1, T2, and T3, respectively. This type of da-
mage has different causes; thus, its identification depends on the ad-
ditional symptomatology evidenced. It can be generated by chilling
injury, diseases or by the normal senescence process.
B.L. Botina A, et al.
Fig. 2. Physical alterations generated throughout pre-harvest stage in Tahiti lime fruit: a) Sunburn or sunspot; b) Scar on the fruit surface; and c) Damage caused by
pests.
3.4.4. Mechanical damages
Injuries such as bruises, cuts, and abrasion were a constant in all
storage treatments (Fig. 5), being lower in fruit under storage at room
temperature.
3.4.5. Microbiological damage
This type of damage was identified in all treatments affecting less
than 4% of the storage fruit.
3.5. Fungi isolation
The aim of this phase was to identify the types of pathogenic or
saprophytic microorganisms present in the fruit that could growth and
proliferate it they found appropriate conditions. Among the fruits se-
lected for the microbiological analysis, 88 strains were identified
(Table 1). According to the morphological characteristics of the colony
and the microscopic structures of the strains, the most common genera
identified were the following: Colletotrichum spp., Fusarium spp., Peni-
cillium spp., Acremonium spp., Trichoderma spp., Curvularia spp., Phoma
spp., Alternaria spp., Stachybotrys spp., and Ulocladium spp. The de-
scription of each genus identified coincided with the one mentioned by
Barnett and Hunter (2003).
The genera recognized as citrus phytopathogens Colletotrichum spp.,
Fusarium spp. Penicillium spp., and Alternaria spp. were identified in
fruit both, under or without post-harvest treatment, Fig. 6. From all the
Tahiti lime analyzed, 20 isolates of Colletotrichum spp., 41 strains of
Fusarium spp., seven of Penicillium spp., and only one accession of Al-
ternaria spp. were identified.
Isolates of Colletotrichum spp. (Fig. 6a) were obtained from fruit
with brown lesions and sunken necrotic tissue (Fig. 6b), typical symp-
toms of anthracnose (Peres et al., 2002).
Isolates identified as Penicillium spp. (Fig. 6c) were obtained from
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Scientia Horticulturae 257 (2019) 108737
fruit that had soft, watery areas, whitish mycelium, and green spores. In
Fig. 6d, the external appearance of the fruit affected by this pathogen
can be observed. This genus can invade and colonize wounds and plant
tissue (Fig. 6e), as was found in this study. Species of the genus Alter-
naria (Fig. 6f) as A. citri and A. alternata are recognized as the causal
agents of black rot, brown spot or dark green peduncle rot in all citrus
species. The spores of the fungus produce a latent infection that is de-
velops when the fruit mature (Fig. 6g); the disease occurs in the inner
part of the fruit with a dry and black rot as shown in Fig. 6h (Ohtani
et al., 2009; Yahia, 2011).
The genera Curvularia spp. and Phoma spp. were isolated from fruit
that had been stored and developed mycelium on the surface of the
fruit. Isolates of Ulocladium spp. and Stachybotrys spp. were obtained
from the mycelium developed in the peduncle of overgrown lime fruit
that was not subject to storage treatments.
4. Discussion
The storage period allowed the expression and categorization of
different types of damages on the Tahiti lime fruit, and from this point
onwards, recommendations or strategies for its control can be gener-
ated.
The growing season and the harvest operation make a critical dif-
ference in fruit susceptibility to different causes of damage. In this
sense, in the rainy season, the fruit is more susceptible to post-harvest
damage compared to the dry season. These results can be explained by
the fact that citrus fruit has tolerance to drought, but are affected by
excess moisture (Hernández et al., 2015). In the rainy season, the fruit
reaches senescence faster than in dry season, becoming its skin yellow
and soft. This makes the fruit more susceptible to mechanical damage,
such as bruises, cuts, and wounds (Landanya, 2008), increasing the risk
of deterioration during its post-harvest handling (Tyagi et al., 2017).
B.L. Botina A, et al.
Fig. 3. Incidence of preharvest damage (sunburn, scars, and pests) on Tahiti
Lime quality, in relation to a) Growing season (rainy and dry season), b)
Production locality (Lebrija and Villavicencio), and c) Rootstock (Citromelo,
Kryder and Volkameriana).
Moreover, in this season, the humidity increases, supporting conditions
for the propagation of fungal, bacterial and viral diseases which are
expressed later on during post-harvest phase (Fischer and Orduz-
Rodríguez, 2012). Therefore, the post-harvest require more demanding
and careful handling during rainy season compared to the dry season, to
keep Tahiti lime quality and increase its shelf life.
Regarding the location, the best fruit quality obtained at the end of
storage was that brought from Lebrija. It can be explained by the fact
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Scientia Horticulturae 257 (2019) 108737
that regions, with a more temperate climate, just like Lebrija (22.7 °C
average temperature), are more suitable for Tahiti lime crop as the fruit
is less susceptible to damage compared to warmer regions such as
Villavicencio, whose average temperature is 26 °C (Hernández et al.,
2015). Concerning the rootstock, although there was not a clear rela-
tion with the different types of damage, it is important to select the
most appropriate rootstock taking into account the genotype-environ-
ment relationship. The edaphoclimatic conditions, the presence of the
most common plagues and diseases in the region and the resistance of
the rootstock to these diseases, as well as particular fruit features sought
(e.g., color, size, thickness of the fruit peel, content of juice, con-
centration of acids and sugars) are factors that must be considered to
choose the most suitable rootstocks to fulfill the market requirements,
(Yahia, 2011; Orduz and Mateus, 2012). Therefore, it is important to
take these factors into account when deciding cultivate this citrus. For
example if the region where is expected to grow lime trees is known
because of its strong wind currents; then Kryder rootstock would not be
appropriate to grow there, as this rootstock was more susceptible to
scars damages than Volkameriana or Citrumelo. In case of high pre-
sence of pests in the region where Tahiti Lime will be crop, probably
Kryder should be more suitable than Volkameriana or Citrumelo as it
was less susceptible to this kind of damage.
4.1. Pre harvest damage
4.1.1. Sunburn
Regarding the damages originated in this phase, one of the most
common damages is sunburn or sunspots, which restrict the entry of
lime tahiti to international markets. This damage seems to be caused by
excess of exposition of the fruit to solar radiation, while the fruit is
attached at the tree; but also while the harvested fruit is left without
any protection under direct sunlight exposition. Sunlight raises the
temperature of the surface of the fruit generating changes in cell pig-
ments that give rise to yellow or pale green tones that affect the color
homogeneity of the fruit. In extreme cases, cell death can occur, which
results in necrotic areas of variable diameter and a dry appearance with
hard texture (Chabbal et al., 2014). This type of damage generates
economic losses between 6 and 50% of the production (Guerrero,
2014). Hence, to reduce its incidence, an adequate pruning to manage
the architecture of the tree could protect the fruit in the course of
cropping. On the other hand, transitory storage of the harvested fruit
under protected conditions, could help to reduce sunspot damage.
Damages by scars are more complex factors to control since many
of them are caused by climatic conditions, combined to the character-
istics of the Lime tree. Meteorological phenomena such as hailstorms,
rains and high wind speed currents generates wounds due to the strong
contact of the thorns and branches with the fruit. Small fruit are highly
susceptible to suffer this type of injury (Fischer and Orduz-Rodríguez,
2012), and when the fruit grows the scar becomes more evident af-
fecting the appearance of the fruit.
Results showed that scars and sunspots damage are higher in rainy
season than in dry season probably due to the fact that storms and
hailstorms are more frequently in that season. Moreover, the residual
water on the lime surface increase the effect of sunspots when the fruit
is expose to direct sunlight (Departamento Administrativo Nacional de
Estadística and DANE, 2015).
Concerning to location, the higher damage generated by scars in
lime fruit from Lebrija can be explained considering the higher wind
speed found in Lebrija (8.5 km h−1) compared to average wind speed in
Villavicencio, which does not exceed 2 km h−1 (Hernández et al.,
2015). To reduce the scar damage caused by the wind and the hail-
storms is recommended the use of curtains or natural barriers; espe-
cially in places where the average wind speed exceeds 20 km h−1
(Rodriguez, 2002).
B.L. Botina A, et al.
Fig. 4. Physical alterations developed during post-harvest in Tahiti lime fruit: a) Dehydrated fruit; b) Lime with different degradation of chlorophyll; c) oil sporting or
oleocellosis; d) Brown colored fruit; e) Fruit with mechanical damage; f) Fruit with microbiological damage.
4.1.2. Insects plague
Another main limitation in production and commercialization of
citrus fruit is the damage caused by insects plague (León and Kondo,
2017). The presence of thrips, mites or leaf miners in citrus crops is
widespread and was corroborated in this study. Symptoms depend on
the insect that causes the damage and the degree of damage. This cause
of damage is also one of the most common in Tahiti lime that dete-
riorates its appearance and reduce its export possibilities. In Colombia,
the main pests affecting citrus fruit are the citrus rust mite (Phyllocop-
truta oleivora), aphids (brown or black citrus aphids; Toxoptera ci-
tricidus), scale insects or citrus mussel scale (Lepidosaphes beckii), citrus
mealybug (Planococcus citri.) and citrus orthezia (Orthezia praelonga)
(DANE, 2015).
Rootstocks were susceptible to pests damage more than scars. This
was expected considering that one of the most common traits looked for
in a rootstocks is its resistance or tolerance to most important diseases
or plagues. Although skin thickness can be associated to higher fruit
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Scientia Horticulturae 257 (2019) 108737
resistance to mechanical injuries and thus, less susceptibility to scars,
skin thickness has been not completely related to rootstocks.
4.2. Postharvest damage
4.2.1. Yellowing
This is one of the causes for which the fruit is rejected in the in-
ternational market. Refrigeration contributed to slows down the speed
of lime fruit degreening comparing to storage at room temperature, but
it was not enough to control the degradation of the chlorophyll, and
therefore, the fruit took a yellow coloration. Thus, refrigeration re-
quires a combination with other conservation techniques such as
ethylene control in order to avoid the degreening of the fruit.
Dehydration or dryness was present in almost all fruit stored at
room temperature. However it is important to mention that relative
humidity was not controlled. It oscillated around 65% in the day and
85% at night, conditions that increase fruit transpiration through its
Fig. 5. Tahiti Lime quality and main postharvest damage
identified (yellow color, brown color, mechanical damage,
microbial damage) after 5-week storage under three treat-
ments, T1: Storage at 10 °C, T2: Fruit disinfected with sodium
hypochlorite (200 ppm) and storage at 10 °C, and T3: Control
treatment with storage at 18 °C (For interpretation of the re-
ferences to colour in this figure legend, the reader is referred
to the web version of this article).
B.L. Botina A, et al. Scientia Horticulturae 257 (2019) 108737

Table 1 be observed (Arias and Toledo, 2000). Thus, it is essential to use a

Number of fungi strains per genera identified in healthy fruit, in fruit with protective film, either packaging with a plastic film or waxing to protect
symptoms, and in fruit subjected to three storage treatments (T1: Storage at fruit from moisture loss, along the commercialization chain. However,
10 °C, T2: Fruit disinfected with sodium hypochlorite (200 ppm) and storage at this conditions could be complemented with control of the relative
10 °C, and T3: Control, storage at 18 °C).
humidity around 90% in storage and transport facilities.
Isolates Healthy fruit Fruit with Treatments for stored fruit
symptoms
T1 T2 T3 4.2.2. Brown colorations
Another important damage identified in the study were the
Acremonium spp. 2 2 browning. Although it was not possible to identify the cause of this
Alternaria spp. 1
change of color with certainty, low temperature could have contributed
Colletotrichum spp. 5 2 2 8 3
Curvularia spp. 1 to this damage. However, statistical analysis did not show any differ-
Fusarium spp. 21 6 13 1 ences among the refrigerated treatments and room temperature storage.
Penicillium spp. 1 4 2 Then brown color could be due to the oxidation of chlorophyll gives rise
Phoma spp. 1 to pheophytin or a chlorophyllin that later releases a pheophorbide
Stachybotrys spp. 1
characterized by its brown or brownish color as observed in Tahiti lime
Trichoderma spp. 1 10
Ulocladium spp. 1 fruit (Hörtensteiner, 2013; and Hörtensteinera and Kräutlerb, 2011).
Therefore, to avoid this coloration, work should be done to control the
oxidation process of chlorophyll, by controlling enzymes activity.
stomata. As a result, fruit lost high amount of moisture brought dry and
hard appearance to most of lime fruit. This fact confirmed results found
4.2.3. Mechanical damage
by Castellano et al. (2016) who identified the increase in fruit firmness
Although bruises are one of the most frequent causes of damage in
stored at 18 and 30 °C. In some cases, signs of wilting and wrinkling can
post-harvest, in this case, the damages due to mechanical causes were

Fig. 6. a) An acervulus of Colletotrichum spp. in 40x; b) External appearance of lime fruit affected by Colletotrichum spp.; c) Conidia of Penicillium spp. in 40x; d)
External and e) internal appearance of lime fruit contaminated by Penicillium spp.; f) A conidia of Alternaria spp. in 40x; g) External and h) internal appearance of lime
fruit contaminated by Alternaria spp.

7
B.L. Botina A, et al.
minimal; especially for those fruit storage at room temperature, prob-
ably due to the reduced handling that this fruit was subjected. Although
all fruit was carefully handled, fruit driven to refrigeration and disin-
fection underwent additional unit operations that those taken into room
temperature storage. Although mechanical damage is the main cause of
oil spotting or oleocellosis in lime Tahiti; in the present study this type
of damage was irrelevant. Some of the main causes of oleocellosis are
mechanical injuries such as bumps and bruises, other causes can gen-
erate this same effect, such as differential water stress through the skin,
a high degree of fruit maturity, damage by pests and nutritional im-
balances. In all cases, oleocellosis can manifest itself after several days
of having received the lesion (Zheng et al., 2010).
4.2.4. Microbial damage
Pathogenic and saprophytic fungi are the primary causal agents of
microbiological damage in the Tahiti lime fruit and the manifestation of
decay (Photita et al., 2005). According to Alarcón et al. (2012) some
fungi can be found on the surface of the fruit in a latent form, without
showing any symptoms; but when they find appropriate conditions for
growing and multiplication, they colonize the plant tissue through
specific structures, expression of enzymes and releasing metabolites
that generate undesirable changes in the fruit (Centis et al., 1997).
Other source of contamination are the spores of these microorganisms
that are transported through water, wind; and from contaminated tools
and elements used during harvest, transport, and storage. Thus, fruit
with physical injuries such as bruise, abrasion, wounds, senescence or
caused by pests are more susceptible to damage by fungi, as the spores
can find the right conditions for their development on this injuries.
Thus, this infection systems obligate to carry out disinfection process
for both, fruit but also for facilities and tools used for its manipulation.
Although different genera were identified in this study, it was inter-
esting that the genus Penicillium spp. was not the most common as ex-
pected according to what was reported in the literature (Guerrero et al.,
2007; Sukmawati and Miarsyah, 2017).
4.3. Fungal isolation
The genera Colletotrichum spp., Fusarium spp. Penicillium spp., and
Alternaria spp., were identified in fruit just harvested without storage
and also in fruit after underwent storage. This allows state that these
genera come from the crop and remains in a latent state waiting for the
right conditions to colonize the fruit; but also from the storage facilities
when the cleaning practices of these places are not appropriate.
Although these fungi are recognized as citrus phytopathogens, they also
have the capacity to be secondary or saprophytic microorganisms
(Peres et al., 2002; Duran and Moreno, 2000; Guerrero et al., 2007;
Timmer et al., 2003; Perfect et al., 1999; Abd-Elsalam et al., 2015;
Gutiérrez et al., 2015).
Colletotrichum spp. has been isolated as an endophyte or phyto-
pathogen, and it can reach the fruit through water or the dispersion of
conidia present in the environment. The isolates were acquired from
lime fruit that showed typical symptoms of anthracnose as those re-
ported by Peres et al. (2002). Strains such as C. gloesporioides and C.
acutatum keep a quiescent infection by means of the conidia that ger-
minate on the surface, forming an appressorium, (Peres et al., 2002;
Alarcón et al., 2012). Therefore, only when environmental conditions
concerning to temperature, relative humidity and nutrients are suitable
for the fungi development the fruit deterioration process begins. Thus,
this could explain the presence of mycelium corresponding to strains of
Colletotrichum spp., in apparent healthy fruit.
In countries that produce commercial citrus species, anthracnose
can cause losses of up to 90% in the production (Osorio and Ospina,
2001). In Brazil, Colletotrichum acutatum Simmonds has been identified
as the causative agent of the "premature fruit fall" disease, recognized as
one of the most severe that affects true lemons [Citrus limon (Linn.)
Burm.], Tahití lime (C. latifolia Tanaka), lime or sour lime (C.
8
Scientia Horticulturae 257 (2019) 108737
aurantifolia Swingle) and sweet orange (C. Sinensis Osbeck) (Arias et al.,
2006). In Colombia, anthracnose has been reported in different citrus
producing areas and is recognized as a limiting disease that affects the
production and post-harvest quality of numerous fruit crops (Osorio
and Ospina, 2001; Sanabria et al., 2010). The genus Fusarium spp. be-
longs to the group of pathogens that produce the most predominant
post-harvest rots in citrus together with Penicillium digitatum and As-
pergillus niger (Abd-Elsalam et al., 2015). Symptoms include different
brown lesions and superficial depressions of the skin, which can ori-
ginate in the peduncle (Castro et al., 2000). The presence of Fusarium
spp. in stored fruit and vegetables is a potential risk to consumers, due
to its ability to produce mycotoxins under appropriate conditions (Abd-
Elsalam et al., 2015). The diseases called green rot, and blue rot are
caused by the species P. digitatum and P. italicum, respectively. These
phytopathogens generate great quality problems in citrus fruit
throughout the marketing cycle (Guerrero et al., 2007), and are re-
cognized as responsible for more than 60% of the losses caused by fungi
in fruit preserved under refrigeration (Sukmawati and Miarsyah, 2017).
However, the isolates obtained from Fusarium spp. and Colletotrichum
spp., exceeded the number of isolations of this Penicilliun spp. pathogen.
This result can be explained in the fact that the main source of this fungi
is the lack of cleaning and disinfection of postharvest facilities (Arias
and Toledo, 2000); and this causes were controlled in this study, as
special care was taken in the adequate cleaning and disinfection pro-
cedures of the refrigeration equipment and areas where the fruit was
handled and storage, reducing the presence of this Penicillium spp. The
rotting produced by Alternaria spp. is a disease that can be observed in
lemon and orange stored at low temperatures for long periods. Fruit
with cuticle injuries and sunburn damage are the most susceptible.
Although the external symptoms are not very evident, the fruit affected
in the orchard can be identified by observing a darker skin color in
diseased fruit and a softer consistency than normal in the edges. The
symptoms of this disease are sometimes confused with those of an-
thracnose (Castro et al., 2000; Ohtani et al., 2009). Species such as A.
citri and A. alternata are recognized as the causal agents of black rot,
brown spot or dark green peduncle rot in all citrus species (Timmer
et al., 2003). The symptoms are recognized by the dry and black rot of
the fruit (Ohtani et al., 2009; Yahia, 2011) as that observed in the
present study. This phytopathogen is also recognized as the cause of the
leaf spot and the brown spot in tangerines (Timmer et al., 2003).
So far no strains of the phytopathogenic fungi Acremonium spp. and
Trichoderma spp. have been reported in the literature for Tahiti lime.
However, in the current study isolates that belonged to these genera
were identified. Acremonium spp. is recognized as a microorganism that
belongs to the microbiome of the fruit surface of jujube (Singh and
Sumbali, 1998) and mango (Thiyam and Sharma, 2013). This genus can
produce enzymes that degrade plant tissue such as amylases and cel-
lulases, and therefore, it is also recognized as a saprophyte (Thiyam and
Sharma, 2013).
The genus Trichoderma spp., is found particularly in soil, root and
foliar environment. It is an ubiquitous microorganism that grows ra-
pidly and requires minimal nutritional components. Its behavior is sa-
prophytic and can grow on organic waste or as a parasite of other fungi
(Kubicek and Harman, 2002; Cepero et al., 2015). The presence of this
genus in the analyzed limes can be explained on the fact that this fungi
can be obtained from air isolations, seeds, dead plant material, insects
and fruit surfaces (Kubicek and Harman, 2002; Hernández et al., 2015).
On the other hand, it is important to mention that Trichoderma spp.
has the capacity to produce enzymes of commercial interest and to be
an antagonist of phytopathogens of crops and fruits in post-harvest
(Samuels, 1996; Kubicek and Harman, 2002; Hernández et al., 2015).
This microorganism can use different mechanisms for biological control
such as competition for space and nutrients, mycoparasitism, antibiosis
and the induction of systemic or localized resistance in plants (Monte,
2001). Previous studies mention that genera such as Rhizoctonia, Col-
letotrichum, Sclerotinia sclerotiorum, S. minor, Fusarium oxysporum,
B.L. Botina A, et al.
Phytophtora, Botrytis, Diaporthe, Phoma, Phytium, Rhizopus, among
others, are susceptible genera to the presence of Trichoderma spp.
(Samuels, 1996).
The genera Curvularia spp. and Phoma spp. were isolated from fruit
that had been stored and developed mycelium on the surface of the
fruit. Both genera are ubiquitous, saprophytic and phytopathogenic in
some crops. Curvularia spp. causes leaf spots on young leaves of grasses,
cereals, and corn (Holliday, 1995). In post-harvest, it has been identi-
fied as the cause of dark necrotic lesions and dry brown spots in mango,
and brown spots in Phyllanthus emblica (Thiyam and Sharma, 2013).
Conversely, Phoma spp. is a parasitic, slightly opportunistic and pa-
thogenic microorganism of plants such as tomato (Chen et al., 2015).
The genera Ulocladium spp. and Stachybotrys spp., are commonly
found as saprotrophs and are isolated from decomposing plant material
(Pinruan et al., 2004; Runa et al., 2009; Cepero et al., 2015). A few
species of the genus Ulocladium spp. have been reported as phyto-
pathogens of pumpkin leaves and fruit in which a deep black rot is
generated (Auger et al., 2006).
In general, all the fungi genera identified in the current research can
have its origin in the crop, because these are characterized by being
ubiquitous and found in different niches, including land, water or air.
The latent infection present on the surface of contaminated fruit only
manifested itself during post-harvest or when the fruit round the sui-
table conditions, such as humid chambers that stimulated and allowed
the development of the structures that colonize plant tissue. Therefore,
disinfecting treatments both for the fruit and for the facilities and ele-
ments in contact with the fruit should be a permanent practice to
achieve a reduction in post-harvest losses and attain long shelf life.
5. Conclusions
• Fruit harvested during the rainy season show sunspots and mainly
post-harvest damage; meanwhile, the damages of cropping origin
are more common in fruit harvested during the dry season, espe-
cially those due to affections generated by plague insects.
• The conditions of Lebrija, with an average temperature of 22.7 °C,
and average precipitation of 1185 mm, led to obtaining better fruit
quality compared to fruit from Villavicencio, a region characterized
by a higher average temperature of 26 °C and precipitation of
2918 mm, although the latter location shows lower edah speed.
• The damage showed by the Tahiti lime fruit is not dependent on the
rootstock, although Citromelo achieved a slightly lower percentage
of good quality fruit at the end of the storage period and the highest
percentage of crop origin damage.
• Most of the diseases found in post-harvest have their origin during
cropping and are only expressed until they find suitable conditions
for their development. This was the case of the fungi genera
Colletotrichum spp., Fusarium spp., Alternaria spp., Penicillium spp.,
Acremonium spp., Trichoderma spp., Curvularia spp., Phoma spp.,
Stachybotrys spp., and Ulocladium spp, which were identified under
cultivation and storage.
Funding
This work was supported by Ministerio de Agricultura y Desarrollo
Rural de Colombia (MADR).
Declaration of Competing Interest
None.
Acknowledgments
The authors wish to thank Corporación Colombiana de
Investigación Agropecuaria – AGROSAVIA that implemented the
Project from which this study was developed
9
Scientia Horticulturae 257 (2019) 108737
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