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

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

⁎
Corresponding author.
E-mail address: [email protected] (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. Scientia Horticulturae 257 (2019) 108737

2. Materials and methods end of the incubation period the microorganisms were isolated in
Potato Dextrose Agar (PDA Oxoid) with Chloramphenicol 0.05%
2.1. Plant material (Sigma®) and incubation at 25 °C for seven days.
The macroscopic and microscopic morphological characterization of
Tahiti lime (Citrus latifolia Tanaka) fruit from the Volkameriana, each of the isolated fungi was carried out based on the key proposed by
Citromelo and Kryder rootstocks were obtained from two locations: the Barnett and Hunter (2003). The observation of the fungal structures
producing farm La Bodega (Department of Santander, municipality of was carried out through slides or micro-cultures carried out in PDA
Lebrija, Vereda El Puente), located at 7° 3′ 17.2′' N and 0.73° 13′ 21.5′' (Oxoid) at pH 5, Water Agar (Oxoid) and Malt Agar (Merck).
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 2.5. Statistical analysis
humidity of 75.2%; and the second one was the Research Center "La
Libertad" of AGROSAVIA (Department of Meta, municipality of A simple correspondence analysis was applied to evaluate the effect
Villavicencio), located at 4° 03′ N, and 73° 29′ W, at an altitude of of the variation source, such as the season, location, rootstock, and
336 m.a.s.l., with an average temperature of 26 °C, and precipitation of storage treatment, on the susceptibility of the external fruit damages,
2918 mm. using the Chi-square statistic and calculating relative frequencies; the
statistical program SAS version 9.4 was used.
2.2. Experimental design and treatments

Treatments were arranged in a three × two × two factorial (three 3. Results

rootstocks, two locations and two season) with four replications ar-
ranged in a completely randomized block design with 18-tree plots, (six 3.1. Factors related to fruit damage
trees per every rootstocks). 1015 and 2243 lime fruit were harvested in
dry and rainy season respectively, refrigerated and transported to the The effect of the location, the harvest season and the rootstock on
Analytical Chemistry Laboratory of the Tibaitatá Research Center of fruit quality are presented in Fig. 1(a–c). During the rainy and dry
AGROSAVIA located in Mosquera, Cundinamarca, where they under- season, and after five-week of storage, only 10 and 13% of Tahiti Lime
went three different storage conditions: Treatment T1 (fruit stored at maintained its quality, and therefore, the remaining fruit, showed da-
10 °C), treatment T2 (fruit disinfected by immersion in 250 ppm of so- mage in rainy and dry season 87 and 90%, respectively. The causes of
dium hypochlorite for 4 min, rinsed, drained and stored at 10 °C); and damage in the two seasons were different, and according to the simple
the control treatment T3 (fruit stored at room temperature, i.e., 18 °C). correspondence analysis, there is a relationship between the harvest
Quality of lime fruit was measure in a relative approach, according to season and the type of damage shown by the fruit (P-value < 0.0001,
the methodology used by Rodríguez et al. (2001). The evaluation of the Chi square 239.0025 with df 2). In the rainy season at the end of the
effect of each factor on the quality of Tahiti Lime was quantified ac- storage period, 67% of the limes harvested in the two regions presented
cording to formula like this, where healthy fruit was used as an ex- damages originated during post-harvest; meanwhile, in the dry season,
ample. 48% of the fruit presented damage originated during pre-harvest and
42% in post-harvest, Fig. 1a. Regarding the locality and the effect on
Number of healthy fruit the causes of fruit damage, there is a relationship between these two
% healthy lime = x 100
Number of total fruit evaluated variables (P-value < 0.0001) according to the simple correspondence
A similar formula was used for quantifying the different type of analysis using Pearson's Chi-Square test. Fig. 1b shows fruit from Leb-
preharvest and postharvest damage. The quality criteria chosen was rija reported 15% healthy fruit at the end of the storage period, com-
appearance due to for international market this trait is the most im- pared to the only 2% reported for fruit from Villavicencio. In the case of
portant criteria for quality in Tahiti lime. Lebrija, post-harvest factors (50%) were the major causes of fruit da-
mage; with also a considerable participation (35%) of pre-harvest fac-
2.3. Classification of superficial fruit damage tors. The causes of fruit damage coming from Villavicencio were due to
both pre- and post-harvest factors, without presenting significant dif-
The lime fruit was evaluated after five-week storage period and then ferences between them.
the damages observed on the surface were identified and classified, not Finally, Fig. 1c describes the response of each of the rootstocks to
only for its origin but also for the type. Thus, Tahiti lime showing a different damage factors during pre- and post-harvest. Although this
healthy appearance and suitable for marketing was classified in the first figure allows observing a higher percentage of post-harvest damage
group, healthy fruit. Fruit with sunspots, scars or damage caused by compared to the pre-harvest damage suffered by fruit from different
pests were classified as fruit with pre-harvest damage. Fruit with al- rootstocks, the statistical analysis did not find a relationship between
terations such as loss of green color and the appearance of yellow tones, these two variables (P-value > 0.0650), concluding that the pre- and
dehydrated or dryness fruit, brown fruit not associated to biological post-harvest damage of the fruit does not depend or is not associated
damages, mechanical damages, and damages by microbiological agents with the rootstocks.
were classified as lime with postharvest damage. These damages were
typified and quantified by season, location, rootstocks and storage 3.2. Types of pre-harvest damage
treatment.
Fig. 2, shows different preharvest damage symptoms found in the
2.4. Isolation and identification of fungi 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
The lime fruit selected for the isolation of microorganisms that de- (Fig. 2b), caused by scratches, cuts, punctures and scrasping or rubbing
veloped damage during storage and those that were initially collected with the branches; and damage caused by pest (Fig. 2c). Damage caused
from the culture of healthy characteristics or with some superficial by insects were evidenced by fruit showing a cork-like bark, spots,
condition were subjected to disinfection being rinsed in 75% alcohol scarred stings or rough texture of a dark brown or gray color, scar-
and 1% sodium hypochlorite for one minute, interspersing rinses with ification of the fruit peel of a silvery color, or chlorotic stippling and
sterile distilled water between disinfectant solution cleansings. Later destruction of sub-epidermal cells (galleries) by excavation (Mangione,
fruit was incubated in humid chambers at 19 °C for five weeks. At the 2016); as observed in Fig. 2c.

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B.L. Botina A, et al. 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
Fig. 1. Effect of: a) Growing season (rainy and dry seasons), b) Production chlorophyll oxidation (Arias and Toledo, 2000). Lime fruit under room
location (Lebrija and Villavicencio), and c) Rootstock (Citromelo, Kryder and temperature presented 79% of fruit with yellow color compared to 56
Volkameriana), on the Tahiti Lime quality. and 52% of the fruit subjected to T1 and T2 respectively.

3.3. Preharvest damage, growing season, location and rootstocks 3.4.2. Dehydration
In the control treatment (T3) most of the fruit turned from green to
The incidence of pre-harvest damage and its relationship with the yellow color; but also 93% of this yellow fruit showed marked dehy-
growing season, the location, and the rootstock can be seen in Fig. 3. dration symptoms. 7% showed only dehydration symptoms keeping its
The statistical analysis indicated that there is a relationship between the green color along storage.
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 3.4.3. Brown spot
common in the rainy season than in dry season. Conversely to those The second most frequent fruit damage during storage were brown
damage by pests that are more common in the dry season, which is in spots or brown pigmentation on the fruit peel with values of 17%, 19%,
concordance with reported by Departamento Administrativo Nacional and 13% for treatments T1, T2, and T3, respectively. This type of da-
de Estadística, DANE (2015). mage has different causes; thus, its identification depends on the ad-
According to the statistical analysis, a relationship between pre- ditional symptomatology evidenced. It can be generated by chilling
harvest damage and location was also found (P-value < 0.0001). injury, diseases or by the normal senescence process.

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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 fruit that had soft, watery areas, whitish mycelium, and green spores. In
Injuries such as bruises, cuts, and abrasion were a constant in all Fig. 6d, the external appearance of the fruit affected by this pathogen
storage treatments (Fig. 5), being lower in fruit under storage at room can be observed. This genus can invade and colonize wounds and plant
temperature. 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
3.4.5. Microbiological damage agents of black rot, brown spot or dark green peduncle rot in all citrus
This type of damage was identified in all treatments affecting less species. The spores of the fungus produce a latent infection that is de-
than 4% of the storage fruit. 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
3.5. Fungi isolation et al., 2009; Yahia, 2011).
The genera Curvularia spp. and Phoma spp. were isolated from fruit
The aim of this phase was to identify the types of pathogenic or that had been stored and developed mycelium on the surface of the
saprophytic microorganisms present in the fruit that could growth and fruit. Isolates of Ulocladium spp. and Stachybotrys spp. were obtained
proliferate it they found appropriate conditions. Among the fruits se- from the mycelium developed in the peduncle of overgrown lime fruit
lected for the microbiological analysis, 88 strains were identified that was not subject to storage treatments.
(Table 1). According to the morphological characteristics of the colony
and the microscopic structures of the strains, the most common genera 4. Discussion
identified were the following: Colletotrichum spp., Fusarium spp., Peni-
cillium spp., Acremonium spp., Trichoderma spp., Curvularia spp., Phoma The storage period allowed the expression and categorization of
spp., Alternaria spp., Stachybotrys spp., and Ulocladium spp. The de- different types of damages on the Tahiti lime fruit, and from this point
scription of each genus identified coincided with the one mentioned by onwards, recommendations or strategies for its control can be gener-
Barnett and Hunter (2003). ated.
The genera recognized as citrus phytopathogens Colletotrichum spp., The growing season and the harvest operation make a critical dif-
Fusarium spp. Penicillium spp., and Alternaria spp. were identified in ference in fruit susceptibility to different causes of damage. In this
fruit both, under or without post-harvest treatment, Fig. 6. From all the sense, in the rainy season, the fruit is more susceptible to post-harvest
Tahiti lime analyzed, 20 isolates of Colletotrichum spp., 41 strains of damage compared to the dry season. These results can be explained by
Fusarium spp., seven of Penicillium spp., and only one accession of Al- the fact that citrus fruit has tolerance to drought, but are affected by
ternaria spp. were identified. excess moisture (Hernández et al., 2015). In the rainy season, the fruit
Isolates of Colletotrichum spp. (Fig. 6a) were obtained from fruit reaches senescence faster than in dry season, becoming its skin yellow
with brown lesions and sunken necrotic tissue (Fig. 6b), typical symp- and soft. This makes the fruit more susceptible to mechanical damage,
toms of anthracnose (Peres et al., 2002). such as bruises, cuts, and wounds (Landanya, 2008), increasing the risk
Isolates identified as Penicillium spp. (Fig. 6c) were obtained from of deterioration during its post-harvest handling (Tyagi et al., 2017).

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B.L. Botina A, et al. 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
Fig. 3. Incidence of preharvest damage (sunburn, scars, and pests) on Tahiti
hailstorms are more frequently in that season. Moreover, the residual
Lime quality, in relation to a) Growing season (rainy and dry season), b)
Production locality (Lebrija and Villavicencio), and c) Rootstock (Citromelo, water on the lime surface increase the effect of sunspots when the fruit
Kryder and Volkameriana). is expose to direct sunlight (Departamento Administrativo Nacional de
Estadística and DANE, 2015).
Concerning to location, the higher damage generated by scars in
Moreover, in this season, the humidity increases, supporting conditions
lime fruit from Lebrija can be explained considering the higher wind
for the propagation of fungal, bacterial and viral diseases which are
speed found in Lebrija (8.5 km h−1) compared to average wind speed in
expressed later on during post-harvest phase (Fischer and Orduz-
Villavicencio, which does not exceed 2 km h−1 (Hernández et al.,
Rodríguez, 2012). Therefore, the post-harvest require more demanding
2015). To reduce the scar damage caused by the wind and the hail-
and careful handling during rainy season compared to the dry season, to
storms is recommended the use of curtains or natural barriers; espe-
keep Tahiti lime quality and increase its shelf life.
cially in places where the average wind speed exceeds 20 km h−1
Regarding the location, the best fruit quality obtained at the end of
(Rodriguez, 2002).
storage was that brought from Lebrija. It can be explained by the fact

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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 resistance to mechanical injuries and thus, less susceptibility to scars,
Another main limitation in production and commercialization of skin thickness has been not completely related to rootstocks.
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 4.2. Postharvest damage
widespread and was corroborated in this study. Symptoms depend on
the insect that causes the damage and the degree of damage. This cause 4.2.1. Yellowing
of damage is also one of the most common in Tahiti lime that dete- This is one of the causes for which the fruit is rejected in the in-
riorates its appearance and reduce its export possibilities. In Colombia, ternational market. Refrigeration contributed to slows down the speed
the main pests affecting citrus fruit are the citrus rust mite (Phyllocop- of lime fruit degreening comparing to storage at room temperature, but
truta oleivora), aphids (brown or black citrus aphids; Toxoptera ci- it was not enough to control the degradation of the chlorophyll, and
tricidus), scale insects or citrus mussel scale (Lepidosaphes beckii), citrus therefore, the fruit took a yellow coloration. Thus, refrigeration re-
mealybug (Planococcus citri.) and citrus orthezia (Orthezia praelonga) quires a combination with other conservation techniques such as
(DANE, 2015). ethylene control in order to avoid the degreening of the fruit.
Rootstocks were susceptible to pests damage more than scars. This Dehydration or dryness was present in almost all fruit stored at
was expected considering that one of the most common traits looked for room temperature. However it is important to mention that relative
in a rootstocks is its resistance or tolerance to most important diseases humidity was not controlled. It oscillated around 65% in the day and
or plagues. Although skin thickness can be associated to higher fruit 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).

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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.

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minimal; especially for those fruit storage at room temperature, prob- aurantifolia Swingle) and sweet orange (C. Sinensis Osbeck) (Arias et al.,
ably due to the reduced handling that this fruit was subjected. Although 2006). In Colombia, anthracnose has been reported in different citrus
all fruit was carefully handled, fruit driven to refrigeration and disin- producing areas and is recognized as a limiting disease that affects the
fection underwent additional unit operations that those taken into room production and post-harvest quality of numerous fruit crops (Osorio
temperature storage. Although mechanical damage is the main cause of and Ospina, 2001; Sanabria et al., 2010). The genus Fusarium spp. be-
oil spotting or oleocellosis in lime Tahiti; in the present study this type longs to the group of pathogens that produce the most predominant
of damage was irrelevant. Some of the main causes of oleocellosis are post-harvest rots in citrus together with Penicillium digitatum and As-
mechanical injuries such as bumps and bruises, other causes can gen- pergillus niger (Abd-Elsalam et al., 2015). Symptoms include different
erate this same effect, such as differential water stress through the skin, brown lesions and superficial depressions of the skin, which can ori-
a high degree of fruit maturity, damage by pests and nutritional im- ginate in the peduncle (Castro et al., 2000). The presence of Fusarium
balances. In all cases, oleocellosis can manifest itself after several days spp. in stored fruit and vegetables is a potential risk to consumers, due
of having received the lesion (Zheng et al., 2010). to its ability to produce mycotoxins under appropriate conditions (Abd-
Elsalam et al., 2015). The diseases called green rot, and blue rot are
4.2.4. Microbial damage caused by the species P. digitatum and P. italicum, respectively. These
Pathogenic and saprophytic fungi are the primary causal agents of phytopathogens generate great quality problems in citrus fruit
microbiological damage in the Tahiti lime fruit and the manifestation of throughout the marketing cycle (Guerrero et al., 2007), and are re-
decay (Photita et al., 2005). According to Alarcón et al. (2012) some cognized as responsible for more than 60% of the losses caused by fungi
fungi can be found on the surface of the fruit in a latent form, without in fruit preserved under refrigeration (Sukmawati and Miarsyah, 2017).
showing any symptoms; but when they find appropriate conditions for However, the isolates obtained from Fusarium spp. and Colletotrichum
growing and multiplication, they colonize the plant tissue through spp., exceeded the number of isolations of this Penicilliun spp. pathogen.
specific structures, expression of enzymes and releasing metabolites This result can be explained in the fact that the main source of this fungi
that generate undesirable changes in the fruit (Centis et al., 1997). is the lack of cleaning and disinfection of postharvest facilities (Arias
Other source of contamination are the spores of these microorganisms and Toledo, 2000); and this causes were controlled in this study, as
that are transported through water, wind; and from contaminated tools special care was taken in the adequate cleaning and disinfection pro-
and elements used during harvest, transport, and storage. Thus, fruit cedures of the refrigeration equipment and areas where the fruit was
with physical injuries such as bruise, abrasion, wounds, senescence or handled and storage, reducing the presence of this Penicillium spp. The
caused by pests are more susceptible to damage by fungi, as the spores rotting produced by Alternaria spp. is a disease that can be observed in
can find the right conditions for their development on this injuries. lemon and orange stored at low temperatures for long periods. Fruit
Thus, this infection systems obligate to carry out disinfection process with cuticle injuries and sunburn damage are the most susceptible.
for both, fruit but also for facilities and tools used for its manipulation. Although the external symptoms are not very evident, the fruit affected
Although different genera were identified in this study, it was inter- in the orchard can be identified by observing a darker skin color in
esting that the genus Penicillium spp. was not the most common as ex- diseased fruit and a softer consistency than normal in the edges. The
pected according to what was reported in the literature (Guerrero et al., symptoms of this disease are sometimes confused with those of an-
2007; Sukmawati and Miarsyah, 2017). 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,
4.3. Fungal isolation 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 genera Colletotrichum spp., Fusarium spp. Penicillium spp., and the fruit (Ohtani et al., 2009; Yahia, 2011) as that observed in the
Alternaria spp., were identified in fruit just harvested without storage present study. This phytopathogen is also recognized as the cause of the
and also in fruit after underwent storage. This allows state that these leaf spot and the brown spot in tangerines (Timmer et al., 2003).
genera come from the crop and remains in a latent state waiting for the So far no strains of the phytopathogenic fungi Acremonium spp. and
right conditions to colonize the fruit; but also from the storage facilities Trichoderma spp. have been reported in the literature for Tahiti lime.
when the cleaning practices of these places are not appropriate. However, in the current study isolates that belonged to these genera
Although these fungi are recognized as citrus phytopathogens, they also were identified. Acremonium spp. is recognized as a microorganism that
have the capacity to be secondary or saprophytic microorganisms belongs to the microbiome of the fruit surface of jujube (Singh and
(Peres et al., 2002; Duran and Moreno, 2000; Guerrero et al., 2007; Sumbali, 1998) and mango (Thiyam and Sharma, 2013). This genus can
Timmer et al., 2003; Perfect et al., 1999; Abd-Elsalam et al., 2015; produce enzymes that degrade plant tissue such as amylases and cel-
Gutiérrez et al., 2015). lulases, and therefore, it is also recognized as a saprophyte (Thiyam and
Colletotrichum spp. has been isolated as an endophyte or phyto- Sharma, 2013).
pathogen, and it can reach the fruit through water or the dispersion of The genus Trichoderma spp., is found particularly in soil, root and
conidia present in the environment. The isolates were acquired from foliar environment. It is an ubiquitous microorganism that grows ra-
lime fruit that showed typical symptoms of anthracnose as those re- pidly and requires minimal nutritional components. Its behavior is sa-
ported by Peres et al. (2002). Strains such as C. gloesporioides and C. prophytic and can grow on organic waste or as a parasite of other fungi
acutatum keep a quiescent infection by means of the conidia that ger- (Kubicek and Harman, 2002; Cepero et al., 2015). The presence of this
minate on the surface, forming an appressorium, (Peres et al., 2002; genus in the analyzed limes can be explained on the fact that this fungi
Alarcón et al., 2012). Therefore, only when environmental conditions can be obtained from air isolations, seeds, dead plant material, insects
concerning to temperature, relative humidity and nutrients are suitable and fruit surfaces (Kubicek and Harman, 2002; Hernández et al., 2015).
for the fungi development the fruit deterioration process begins. Thus, On the other hand, it is important to mention that Trichoderma spp.
this could explain the presence of mycelium corresponding to strains of has the capacity to produce enzymes of commercial interest and to be
Colletotrichum spp., in apparent healthy fruit. an antagonist of phytopathogens of crops and fruits in post-harvest
In countries that produce commercial citrus species, anthracnose (Samuels, 1996; Kubicek and Harman, 2002; Hernández et al., 2015).
can cause losses of up to 90% in the production (Osorio and Ospina, This microorganism can use different mechanisms for biological control
2001). In Brazil, Colletotrichum acutatum Simmonds has been identified such as competition for space and nutrients, mycoparasitism, antibiosis
as the causative agent of the "premature fruit fall" disease, recognized as and the induction of systemic or localized resistance in plants (Monte,
one of the most severe that affects true lemons [Citrus limon (Linn.) 2001). Previous studies mention that genera such as Rhizoctonia, Col-
Burm.], Tahití lime (C. latifolia Tanaka), lime or sour lime (C. letotrichum, Sclerotinia sclerotiorum, S. minor, Fusarium oxysporum,

8
B.L. Botina A, et al. Scientia Horticulturae 257 (2019) 108737

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