10.6092/issn.2531-7342/16813
The study investigated fungal pathogens associated with spoilage of five indigenous fruits (Persia americana, Citrus sinensis, Carica papaya, Annona muricata and Solanum lycopersicum) in Akwa Ibom State, Nigeria. Diseased fruit samples were purchased from nine markets located in three senatorial districts of the State. The phenotypic and genotypic identifications of the fungal pathogens isolated from spoilt fruit samples were carried out using standard cultural, morphological and molecular methods, respectively. The phylogenetic relationship among the fungal species was also constructed using neighbor-joining phylogenetic tree generated based on modified Rogers' genetic distance matrix. The fourteen fungal genera identified (and classified into twenty-two species) were Talaromyces, Lasiodiplodia, Trichoderma, Penicillium, Pichia, Rhizopus, Aspergillus, Fusarium, Moniliella, Mucor, Geotrichum, Candida, Absidia and Purpureocillium. Pathogenicity tests revealed that the fungal isolates were able to cause rots with a range of severity. The most rapid rots were caused by Rhizopus oligosporous, Pichia kudriavzevii and Aspergillus niger within 24 h, while Aspergillus aculeatus and Moniliella suaveolens were slower in initiating rots with earliest being after 48 h. The identification of fungi related with fruit rots and data coming from pathogenicity test are crucial information in order to plan and to apply control strategies during postharvest storage.
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Italian Journal of Mycology
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Isolation, characterization and pathogenicity of fungal pathogens  from indigenous postharvest fruits in Akwa Ibom state, Nigeria
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Zeilinger S, Gupta VK, Dahms TES, Silva RN, Singh HB, Upadhyay RS, Gomes EV, Tsui CK, Nayak SC (2016) Friends or foes? Emerging insights from fungal interactions with plants. FEMS Microbiology Review 40:182–207. https://doi.org/10.1093/femsre/fuv045
Isolation, characterization and pathogenicity of fungal pathogens  from indigenous postharvest fruits in Akwa Ibom state, Nigeria
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                     IJM - Italian Journal of Mycology
                     ISSN 2531-7342 - Vol. 53 (2024): 29 - 44
                     Journal homepage: https://italianmycology.unibo.it/

Research article


        Isolation, characterization and pathogenicity of fungal
     pathogens from indigenous postharvest fruits in Akwa Ibom
                             State, Nigeria
           Ofon-Mbuk Divine-Anthony1, Anthony Ayodeji Adegoke1,2, Olusola T. Oduoye3,
                                   Oghenerobor B. Akpor4

1
  Department of Microbiology, University of Uyo, Uyo, Akwa Ibom State, Nigeria.
2
  Department of Community Health Studies, Faculty of Health Sciences, Durban University of Technology (DUT),
Durban, South Africa
3
  Biotechnology Department, National Centre for Genetic Resources and Biotechnology, Ibadan, Nigeria.
4
  Department of Biological Sciences, Afe Babalola University, Ado-Ekiti, Ekiti State, Nigeria.

Corresponding author e-mail: anthonyadegoke@uniuyo.edu.ng - aayodegoke@gmail.com
______________________________________________________________________________

ARTICLE INFO
Received 23/04/2023; accepted 01/11/2023
https://doi.org/10.6092/issn.2531-7342/16813

Abstract
The study investigated fungal pathogens associated with spoilage of five indigenous fruits (Persia americana, Citrus
sinensis, Carica papaya, Annona muricata and Solanum lycopersicum) in Akwa Ibom State, Nigeria. Diseased fruit
samples were purchased from nine markets located in three senatorial districts of the State. The phenotypic and genotypic
identifications of the fungal pathogens isolated from spoilt fruit samples were carried out using standard cultural,
morphological and molecular methods, respectively. The phylogenetic relationship among the fungal species was also
constructed using neighbor-joining phylogenetic tree generated based on modified Rogers' genetic distance matrix. The
fourteen fungal genera identified (and classified into twenty-two species) were Talaromyces, Lasiodiplodia, Trichoderma,
Penicillium, Pichia, Rhizopus, Aspergillus, Fusarium, Moniliella, Mucor, Geotrichum, Candida, Absidia and
Purpureocillium. Pathogenicity tests revealed that the fungal isolates were able to cause rots with a range of severity. The
most rapid rots were caused by Rhizopus oligosporous, Pichia kudriavzevii and Aspergillus niger within 24 h, while
Aspergillus aculeatus and Moniliella suaveolens were slower in initiating rots with earliest being after 48 h. The
identification of fungi related with fruit rots and data coming from pathogenicity test are crucial information in order to
plan and to apply control strategies during postharvest storage.

Keywords
Fungal pathogens, fungal rot, diseased fruits, phylogenetic relationship, molecular identification

________________________________________________________________________________

Introduction

The relationship among fruits, microorganism and humans have been long and interesting and may
have developed before recorded history (Willey et al., 2008; Balali et al., 2020). Nutrients needed for
                                                      ______________
    Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
    The journal is hosted and maintained by AlmaDL. © 2024 The Authors. This work is licensed under CC BY 4.0
Divine-Anthony et al.
https://doi.org/10.6092/issn.2531-7342/16813                                              Italian Journal of Mycology 53 (2024): 29 - 44
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growth, repair and control of body processes are usually obtained from fruits since they possess
minerals like calcium, potassium, magnesium, as well as dietary fiber, vitamins and sugar
(Hawksworth, 2004; Yahia et al., 2019). It is normal for these fruits to be consumed raw as this is the
best way of obtaining their valuable nutrients. The fruits could be used for making of juices, wine,
marmalades, jams, and salads (Isitua and Ibeh, 2010; Awe, 2011). There are also medicinal properties
attributed to some fruits. According to Nakamura and Miyoshi (2006), eating Carica papaya L. may
reduce the risk of some types of cancer, while Eno et al. (2000) demonstrated that the fruit juice of
C. papaya can decrease the blood pressure in the study carried out with mice. Citrus sinensis L. when
taken as an infusion has shown the ability to lower fevers, stop headaches and stabilize heart
palpitations. The juice from C. sinensis hastens removal of metabolic waste from the body. Vitamin
C, a major component found in C. sinensis helps to boost the body immune system thus helping the
body to fight infections (Etebu and Nwauzoma, 2014). Fruits like Persia americana P. Mill, C.
sinensis and C. papaya contain several biologically active compounds known for antisickling
properties (Iweala et al., 2010; Naiho et al., 2015; Nurain et al., 2016). They are consumed both as
food as well as integrative medicine (Naiho et al., 2015; Nurain et al., 2016). These fruit crops
represent an important economic incoming for local farmers and consequently, the fruit spoilage may
contribute to poverty in those communities.
       The postharvest diseases usually originate from infections that occurred during and after harvest
(Tripathi et al., 2021). Often these infections occur through surface wounds created by mechanical or
insect injury. Secondary fungal pathogens are those fungi which may not invade a healthy tissue but
can cause spoilage after the tissue has been damaged by some physical or physiological causes.
Entrance of fungal pathogens does not depend on the size of the wounds, even a microscopic size
wound may be colonized. Microorganisms can affect the fruit quality as well as human health
(Adeoye et al., 2009; Kiaya, 2014). This is facilitated by washing healthy fruit with sewage
contaminated groundwater (Adegoke and Stenstrom, 2019; Nzima et al., 2020). The main fungal
pathogens involved in post-harvest diseases are Botrytis cinerea Pears., Penicillium spp., Monilinia
spp., Rhizopus stolonifer (Ehrenb.) Vuill., Aspergillus parasiticus Speare, Fusarium spp., Candida
tropicalis (Castell.) Berkhout (Nweke and Ibiam, 2012; Spolti et al., 2012; Sutton et al., 2014).
       Earlier study classified B. cinerea as well as Magnaporthe oryzae B.C. Couch, the most
aggressive plant pathogens able to cause gray mold disease for horticultural crops (Dean et al., 2012).
Their contamination may be aided by irrigation with sewage contaminated water (Adegoke and
Stenstrom, 2019; Nzima et al., 2020) Fungal infections make difficult the cultivation of the crop,
decrease the quality of yield and increase problems during storage (Kebede et al., 2021), with
economic impact (Dean et al., 2012; Drenth and Guest, 2016). In addition to causing quality
deterioration and economic losses, some postharvest fungi also pose threat to human health, due to
the production and the release of mycotoxins (Li et al., 2015; Sanzani et al., 2016).
       Huge economic loss can be ascribed specifically to R. stolonifer, which is an opportunistic
fungal pathogen isolated and identified in fruit samples by Agbabiaka et al. (2015). It had earlier been
reported that R. stolonifer is a soft rot pathogen of tomato in Nigeria, able to colonize damaged fruits
(Mahovic et al., 2009). Some strains of Geotrichum candidum Link were reported to possess
proteolytic, peptidolytic and lipolytic activities, and it is often use in food industry (Kamilari et al.,
2023). Lasiodiplodia theobromae (Pat.) Griffon & Maubl. causes damage to both postharvest fruit
and food crops (Sandlin and Ferrin, 1992; Denman et al., 2000; Muniz et al., 2011). It plays significant


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role in root rot of Brachychiton populneus (Schott & Endl.) R.Br. seedlings (Sandlin and Ferrin,
1992), leaf necrosis, and stem cankers on Proteas (Protea magnifica Link) (Denman et al., 2000),
gummosis of cashew in Brazil (Muniz et al., 2011). Aim of this study was to isolate and characterize
fungal pathogens associated to fruit rots, collected in local fruit market in Uyo (Akwa Ibom State,
Nigeria).

Materials and Methods

Fruit samples
Diseased samples of C. papaya, P. americana, Solanum lycopersicum L., Annona muricata L. and
C. sinensis were randomly purchased from nine local fruit markets located in each Senatorial Districts
of Akwa Ibom State (Nigeria). The 225 fruit samples, stored in sterile plastic bags were transported
to Microbiology Laboratory for mycological analyses

Isolation and morphological characterization of fungal pathogens
Media were prepared according to manufacturer’s specifications. For growth, both potato dextrose
agar (PDA) and potato dextrose broth (PDB) were used. Following sterilization, media were cooled
to 50 °C and 1000 mg L-1 chloramphenicol and 160 mg L-1 gentamycin were added, respectively, to
inhibit potential bacterial contamination. All media and glassware were sterilized using an autoclave
at 121 °C, at about 1 bar for 15 min. Isolation and identification of fungal pathogens were done based
on morphological characteristics. Fungal isolates were obtained from diseased fruits by first washing
with sterile distilled water and surface decontamination with 10% hypochlorite solution for 5 min
(Vose, 1980).
      Sterile scalpel was used to cut 3 × 3 mm sections of the fruits, moving from healthy portions to
the diseased portions where the fungi were likely to be more active, and aseptically plating on PDA
medium and incubating at room temperature (28 °C ± 2 °C). The fungal isolates were repeatedly
transferred into new PDA plates in order to obtain pure cultures, which were submitted to a
preliminary morphological identification by microscope examination according to the protocol
proposed by Samson et al. (1988) and Barnett and Hunter (1987).

Molecular characterization
ZymoBIOMICS™ DNA Miniprep Kit (Zymoresearch, USA) was used for fungal DNA extraction
and purification, in accordance with the manufacturer’s instruction. After the final purification and
elution, the quality of the extracted DNA was determined using Nanodrop spectrophotometer
(Thermo Fisher Scientific, USA).
      PCR amplification of the extracted DNA was carried out in a total of 25 µl mixture containing
12.5 µl of Mastermix 2x (DreamTag MM ThermoFisher, USA), 5 µM of each forward and reverse
primers (Inqaba Biotechnology, South Africa) and 10 ng µl-1 template DNA. The forward and reverse
primers were ITS4 and ITS5 (White et al., 1990; Vongphachanh et al., 2016). The amplification
program consisted in initial denaturation and enzyme activation at 94 °C for 5 min; followed by 36
cycles at 94 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 45 s; and final extension
at 72 °C for 7 min and hold temperature at 4 °C. Gel electrophoresis using 1.5% agarose gel in tris-
boric acetate was used to separate the base pairs. The expected base pair of the amplicons was around
650 bp. The PCR products was purified using ethanol precipitation method as described by Al-Saad


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and Al-Zaalan (2019). The purified PCR products were sequenced by Inqaba Biotechnology (South
Africa) and carried out Blast analysis to determine the identity of the fungal isolates. Sequencing was
done for some fungi representative of phenotypic characteristics.
      The evolutionary distances were computed using the Maximum Composite Likelihood method
and were in the units of the number of base substitutions per site. Empirical base frequencies ranged
from 0.27030 to 0.29889 where G/C ratio was ~0.93345. All positions containing gaps and missing
data were eliminated and there was transition/transversion ratio of 2.00000. The optimal tree with the
branch length that ranged from 0.00006 to 0.34833 was taken and the analysis involved 20 nucleotide
sequences. Neighbor-Joining phylogenetic tree of 20 isolates and similar described strains were used.
This neighbor-joining tree was generated based on modified Rogers' genetic distance matrix using
Mega 7 software, version 7.0.63 (Tamura et al., 2021).
      Novel sequences were deposited in the GenBank database of the National Center for
Biotechnology Information (NCBI) along with their distinct identifiers and accession numbers in line
with standard approach (Adegoke et al., 2022) (Supplementary Table S1).

Pathogenicity tests
The procedure described by Agrios (2005) was followed. Ninety healthy fruits were surface-sterilized
with 70% ethyl alcohol and wound created using a sterile scalpel. Disc of pathogens listed in Tables
2 to 5 previously grown on PDA, identified by morphological and molecular tools, were aseptically
transferred into fruit of P. americana, C. sinensis, C. papaya, A. muricata and S. lycopersicum and
sealed with vaseline to prevent the entry of contaminant microorganisms. The inoculated fruits were
placed in sterile polyethylene bags with moist cotton wool placed inside to create a conducive
environmental condition for the pathogens. The set up was incubated at room temperature of 28 °C ±
2 °C for five days. The incubated fruits were observed daily to monitor any symptom of rot.

Results

The mycological approach allowed to isolate and identify according to morphological criteria 510
fungal pathogens from 225 natural infected fruits. The isolated pathogens belonging to 14 genera
(Talaromyces, Lasiodiplodia, Trichoderma, Penicillium, Pichia, Rhizopus, Aspergillus, Fusarium,
Moniliella, Mucor, Geotrichum, Candida, Absidia and Purpureocillium), classified into 22 species
(Table 1) able to cause symptoms of rots on P. americana, C. sinensis, C. papaya, A. muricata and
S. lycopersicum (Fig. 1).
      Eighteen different identified fungal isolates with varied percentages of occurrences per sample
were obtained from the diseased P. americana fruit samples with R. oligosporus having the highest
occurrence (Table 1). Only one Talaromyces verruculosus strain DIV-20 (accession number
PP249671) was found in 113 samples of P. americana, to give an approximate frequency of 0.9 (i.e.,
1/113*100). From the diseased C. sinensis fruit samples, 19 fungal species were isolated with L.
theobromae strain DIV-19 (PP249672) having the highest percentage of occurrence. Seventeen of
the fungal species were isolated from the diseased C. papaya fruit samples with the highest frequency
of occurrence shown to be P. citrinum strain DIV-17 (PP249674) and A. nomius strain DIV-04
(PP249686). From diseased A. muricata, 18 fungal species were isolates and L. theobromae showing
the highest percentage occurrence. From the diseased S. lycopersicum fruits, 14 different fungal
species were isolated with the highest and lowest % occurrence observed in G. candidum and L.


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theobromae, respectively (Table 1). Supplementary Figure S2 shows the morphology of the main
fungal isolates.




      Fig. 1 – Symptoms of rot recorded on (a) C. papaya, (b) A. muricata, (c) S. lycopersicum, (d) A. muricata (e)
      C. sinensis, (f) P. americana.



Molecular characterization
The DNA extracted from the fungal isolates was of good quality and integrity (Supplementary Fig.
S1a) and the results of PCR amplification of the fungal isolates allowed to yield a specific band
around 650 bp (Supplementary Fig. S1b).
       The following nucleotide sequencing of specific amplicon allowed to generally corroborate the
identification of fungal pathogens carried out according to morphological criteria. There were some
exceptions represented by Aspergillus, which is a complex genus difficult to be identified only by
morphological tools. The nucleotide sequences were used also to analyze the phylogenetic
relationship among the fungal isolates. There were more similar clusters among organisms of the
same genus and the sequences of Lasiodiplodia, Penicillium and Aspergillus showed this expected
close relationship (Supplementary Fig. S3). Candida tropicalis strain DIV-08 with the accession
number PP256043 appears distantly related from all the other fungal isolates.

Pathogenicity of the fungal pathogens on post- harvest fresh healthy fruits
The results of the pathogenicity tests indicated that the selected fungal isolates (T. verruculosus strain
DIV-20, T. verruculosus strain, DIV-2, L. theobromae strain DIV-1, T. koningiopsis strain DIV-18,
G. candidum strain DIV-9 and the other isolates) were evidently pathogenic, owing to their
capabilities to produce similar spoilage signs on fresh healthy fruits of P. americana within 5 Dots



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           Table 1 - Fungal pathogens and % occurrence in the disease fruits.
           Fungal Isolates                                                                         Fruits                                                                                           Total (%)
                                                                                                   P. americana         C. sinensis       C. papaya       A. muricata       S. lycopersicum
                                                                                                   N. (%)               N. (%)            N. (%)          N. (%)            N. (%)
            Talaromyces verruculosus (Peyronel) Samson, N. Yolmaz, Frisvad                         1 (0.9)              1 (1.4)           0 (0.0)         0 (0.0)           0 (0.0)                 2(0.4)
            & Seifert
            Lasiodiplodia theobromae (Pat.) Griffon & Maubl.                                       13 (11.5)            8 (9.2)           9 (8.8)         20 (16.8)         1 (1.1)                 51 (10.0)
            Trichoderma koningiopsis Samuels, Carm. Suárez & H.C. Evans                            1 (0.9)              0 (0.0)           0 (0.0)         0 (0.0)           0 (0.0)                 1 (0.2)
            Penicillium citrinum Thom                                                              4 (3.5)              4 (4.6)           12 (11.8)       7 (5.9)           3 (3.4)                 30 (5.9)
            Pichia kudriavzevii Boidin, Pignal & Besson                                            6 (5.3)              5 (5.7)           1 (0.9)         5 (4.2)           5 (5.6)                 22 (4.3)
            Aspergillus niger Tiegh.                                                               5 (4.2)              4 (4.6)           10 (9.8)        4 (3.7)           6 (6.7)                 29 (5.7)
            Fusarium culmorum (Wm.G. Sm.) Sacc.                                                    7 (6.2)              6 (6.9)           5 (4.9)         4 (3.7)           4 (4.5)                 26 (5.1)
            Fusarium solani (Mart.) Sacc.                                                          6 (5.3)              4 (4.6)           4 (3.9)         5 (4.2)           3 (3.4)                 22 (4.3)
            Aspergillus carbonarius (Bainier) Thom                                                 5 (4.2)              6 (6.9)           2 (2.0)         6 (5.0)           2 (2.2)                 21 (4.1)
            Mucor racemosus Fresen                                                                 5 (5.2)              7 (8.0)           6 (5.9)         5 (4.2)           2 (2.2)                 25 (4.9)
            Geotrichum candidum Link                                                               10(8.8)              6 (6.9)           9 (8.8)         8 (6.7)           18 (20.2)               51 (10.0)
            Candida tropicalis (Casttell) Berkhout                                                 6 (5.3)              4 (4.6)           3 (2.9)         7 (5.9)           14 (15.7)               34 (6.6)
            Absidia corymbifera (Cohn) Sacc. & Trotter                                             5 (4.2)              2 (2.3)           4 (3.9)         3 (2.5)           12 (13.5)               26 (5.1)
            Candida utilis Ladder & Kreger-van Rij                                                 5 (4.2)              5 (5.7)           3 (2.9)         5 (4.2)           0 (0.0)                 18 (3.5)
            Aspergillus aculeatus Iizuka                                                           5 (4.2)              6 (6.9)           5 (4.9)         14 (11.8)         0 (0.0)                 30 (9.5)
            Aspergillus nomius Kurtzman, B.W. Horn & Hesselt                                       8 (7.1)              5 (5.7)           12 (11.8)       9 (7.6)           5 (5.6)                 39 (7.6)
            Candida pseudotropicalis (Castell.) Basgal                                             4 (3.5)              0 (0.0)           2 (2.0)         4 (3.7)           11 (12.4)               21 (4.1)
            Rhizopus microsporus var. oligosporus (Saito) Schipper & Stalpers                      17 (15.0)            5 (5.7)           10 (9.8)        4 (3.7)           3 (3.4)                 39 (7.6)
            Aspergillus parasiticus Speare                                                         0 (0.0)              5 (5.7)           0 (0.0)         5 (5.4)           0 (0.0)                 10 (2.0)
            Aspergillus sclerotiorum G.A. Huber                                                    0 (0.0)              3 (3.4)           0 (0.0)         0 (0.0)           0 (0.0)                 3 (0.6)
            Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken                                0 (0.0)              0 (0.0)           5 (4.9)         4 (3.7)           0 (0.0)                 9 (1.8)
            Hywelm-Jones & Samson
            Moniliella suaveolens (Lindner) Arx                                                    0 (0.0)              1 (1.4)           0 (0.0)         0 (0.0)           0 (0.0)                 1 (0.2)
            Total                                                                                  113 (100)            87 (100)           102 (100)       119 (100)        89 (100)                 510 (100)



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Table 2 - Pathogenicity of the fungal isolates on P. americana fruit samples.
 Fungal Isolates        Macroscopic observations                                                                                                                                                          Inference
                              Day 1                      Day 2                       Day 3                                  Day 4                              Day 5
 T. verruculosus              No spoilage                No spoilage                 Small white mycelia growth             White mycelia growth on            Whole fruits soft & darkened.              Spoilage started
                                                                                     at point of inoculation                fruits. Fruits soft at point       More mycelia growth                        on 3rd day
                                                                                                                            of inoculation

 L. theobromae                No spoilage                No spoilage                 White mycelia growth                   White mycelia growth               Fruits turned black & soft from            Spoilage started
                                                                                     appears on fruits                      spread out from point of           point of inoculation outwards              on 3rd day
                                                                                                                            inoculation

 T. koningiopsis              No spoilage                No spoilage                 Brownish discolouration at             Internal damage &                  Internal damage &                          Spoilage started
                                                                                     inoculation site                       discolouration                     discolouration                             on 3rd day


 R. oligosprous               Fruits firm except         Soft areas increase         White mycelia growth at                Black spores appeared on           Fruits totally soft with much              Spoilage started
                              at site of                 but no exudates             inoculation site                       the white mycelia. Fruits          mycelia growth & black spores              on 1st day
                              inoculation                                                                                   all soft

 G. candidum                  No spoilage. Both          Fruits getting soft by Mucoid yeast-like growth at                 Mucoid growth increases. Mucoid growth slight offensive                       Spoilage started
                              fruits are firm            ripening process       point of inoculation                        Inoculation area very soft odour. Fruits very soft                            on 3rd day



 C. tropicalis                No spoilage, fruits        Small patches of            Mat-like growth increasing             Mat-like growth                    Increased mat-like growth                  Spoilage started
                              are firm                   white mat-like              in area                                spreading all over the             spreading all over the fruits.             on 3rd day
                                                         growths observed.                                                  fruits. Fruits soften              Fruits soften

 A. aculeatus                 No spoilage                No spoilage                 No spoilage                            No spoilage                        Little white growth with brown             Spoilage started
                                                                                                                                                               spores. Inoculation site soft              on 5th day


 A. nomius                    No spoilage                No spoilage                 Moderate cottony white                 Extensive white cottony            Whole fruits having cottony                Spoilage started
                                                                                     mycelial growth at                     growth taking over the             white growth (in tufts). Fruit             on 3rd day
                                                                                     inoculation                            whole fruits                       soft & spongy

 Uninoculated fruit           No spoilage                No spoilage                 No spoilage                            No spoilage                        No spoilage                                No spoilage


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   Table 3 - Pathogenicity of the fungal isolates on C. sinensis fruit samples.
   Fungal Isolates                                                              Macroscopic observations                                                                                           Inference
                                  Day 1                        Day 2                              Day 3                         Day 4                            Day 5
     T. verruculosus               No spoilage                  No spoilage                        Point of inoculation          Water-soaked area                Whole fruit looking               Spoilage started on
                                                                                                   looks water-soaked            increasing & fruits              brownish &                        3rd day
                                                                                                                                 softening                        watersoaked
     L. theobromae                 No spoilage                  Presence of watersoaked            Increased                     Extensive brown                  Extensive rot &                   Spoilage started on
                                                                appearance at site of              watersoaked                   discolouration &                 offensive odour                   2nd day
                                                                inoculation                        appearance &                  offensive odour
                                                                                                   discolouring
     A. niger                      No spoilage                No spoilage                          Water-soaked                  Water-soaked, white              Much rottenness. Black            Spoilage started on
                                                                                                   appearance around             mycelia growth & black           spores on white mycelia           3rd day
                                                                                                   inoculation sites             spores
     F. culmorum                   No spoilage                No spoilage                          Water-soaked                  Cottony white growths            Inoculation sites turn            Spoilage started on
                                                                                                   appearance at                 at site of inoculation           brownish with white               3rd day
                                                                                                   inoculation site                                               cottony growths

     M. suareolens                 No spoilage                No spoilage                          No spoilage                   No spoilage                      Massive discolouring              Spoilage started on
                                                                                                                                                                  with white dry mycelia            5th day
                                                                                                                                                                  growth

     M. racemosus                  No spoilage                  Water-soaked appearance            Increased                     Small amounts of white           Small amounts of white            Spoilage started on
                                                                at inoculation site                watersoaked                   mycelia                          mycelia                           2nd day
                                                                                                   appearance

     G. candidum                   No spoilage                No spoilage                          Water-soaked                  Larger water-soaked              Increased water-soaked            Spoilage started on
                                                                                                   appearance at                 areas, exudates &                area, exudates &                  3rd day
                                                                                                   inoculation site              offensive odour                  offensive odour
     A. aculeatus                  No spoilage                  Water-soaked appearance            Increased                     White mycelia with               White mycelia growth              Spoilage started on
                                                                at site of inoculation             watersoaked                   brown spores on                  with brown spores &               2nd day
                                                                                                   appearance at                 inoculation site                 offensive odour
                                                                                                   inoculation site
     Uninoculated fruit            No spoilage                  No spoilage                        No spoilage                   No spoilage                      No spoilage, just                 No spoilage
                                                                                                                                                                  ripening




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      Table 4 - Pathogenicity of the fungal isolates on C. papaya fruit samples.
      Fungal isolates                                                          Macroscopic observations                                                                                            Inference
                                   Day 1                   Day 2                              Day 3                             Day 4                           Day 5
        L. theobromae              No spoilage             No spoilage                        No spoilage                       Water-soaked                    Extensive white mat-like           Spoilage started
                                                                                                                                appearance mycelia              growth from inoculation            on 4th day
                                                                                                                                growth                          site
        F. culmorum                 No spoilage             Small white mycelia at            Increased white mycelia            Massive fungal growth           Whole fruit covered with Spoilage started
                                                            point of inoculation              growth with fruit                  on fruits. Whole fruit          fungal growth            on 2nd day
                                                                                              softened                           softened                        Whole fruit softened.
        R. oligosprous              No spoilage             Fruits firm fruits at             Water-soaked & soft                Little white floccose           Little mycelial growth            Spoilage started
                                                            inoculation site which            areas around inoculation           mycelia seen                    with presence of black            on 2nd day
                                                            appeared water-soaked             site                                                               spores
        A. niger                    No spoilage             No spoilage                       Little white mycelial              Little white mycelial           Extensive mycelial                Spoilage started
                                                                                              growth seen                        growth with no spores           growth & black spores,            on 3rd day
                                                                                                                                                                 rot & exudates
        M. racemosus                No spoilage             White cottony mycelia             Increased white cottony            Cottony mycelia, grey-          Cottony mycelia, grey-            Spoilage started
                                                            growing out of                    mycelia & presence of              black spores & fruits           black spores & fruits             on 2nd day
                                                            inoculation site                  grey-black spores                  softness                        softness on whole fruits

      G. candidum                   No spoilage             No spoilage                       Mycelia &water-soaked              Increased mycelia &             Extensive mycelia &     Spoilage started
                                                                                              appearance at                      water-soaked                    water-soaked appearance on 3rd day
                                                                                              inoculation site                   appearance
        A. nominus                  No spoilage             Moderate cottony white            Extensive cottony                  White cottony growth            Whole fruits having               Spoilage started
                                                            mycelia at inoculation            growth on whole fruits             developed white spores          cottony growth with               on 2nd day
                                                            site                                                                                                 yellow-green spores
        P. lilacinum                No spoilage             Slight softness at                Slight softness at                 Presence of white felt-         Spread of mycelia with            Spoilage started
                                                            inoculation site                  inoculation site                   like mycelia at                 whole fruits becoming             on 2nd day
                                                                                                                                 inoculation site                soft to touch
        Uninoculated fruit          No spoilage             No spoilage                       No spoilage                        No spoilage                     No spoilage                       No spoilage




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    Table 5 - Pathogenicity of the fungal isolates on A. muricata fruit samples.
          Fungal isolates                                                        Macroscopic Observations                                                                                               Inferences
                                         Day 1                       Day 2                     Day 3                                              Day 4                       Day 5
     L. theobromae              No spoilage               No spoilage                Fruits turning brown at                               Increased brown              Extensive white            Spoilage started
                                                                                     point of inoculations                                 points & white               mycelial growth &          on 3rd day
                                                                                                                                           mycelial growth              browning
      R. oligosprous                   No spoilage                     Little white mycelial             Increased mycelial                Increased black              Extensive black            Spoilage started
                                                                       growth at inoculation site        growth & presence of              spores & white               spores on fruits &         on 2nd day
                                                                                                         black spores                      mycelial growth              softened fruits
      A. parasiticus                   No spoilage                     No spoilage                       No spoilage                       Presence of white            Extensive white            Spoilage started
                                                                                                                                           mat-like mycelia &           mat-like mycelia &         on 3rd day
                                                                                                                                           browning                     brownish area
      A. corymbifera                   No spoilage                     No spoilage                       Site of inoculation               Small white mycelia          Increased growth           Spoilage started
                                                                                                         becoming soft                     at inoculation site          of fluffy white            on 3rd day
                                                                                                                                                                        mycelia
      C. utilis                        No spoilage                     No spoilage                       Softening at inoculation          Increased softening          Extensive damage           Spoilage started
                                                                                                         site & fungal growth              & fungal growth              at site of                 on 3rd day
                                                                                                                                                                        inoculation
      A. aculeatus                     No spoilage                     No spoilage                       Presence of white                 Increased softening          Extensive softening Spoilage started
                                                                                                         mycelia & softening               & white mycelia              & white mycelia     on 3rd day


      A. nomius                        No spoilage                     No spoilage                       Site of inoculation               Increased darkening          Extensive                  Spoilage started
                                                                                                         darkened                          & fungal growth              darkening & fungal         on 3rd day
                                                                                                                                                                        growth
      P. lilacinum                     No spoilage                     No spoilage                       No spoilage                       Slight mycelial              Extensive mycelial         Spoilage started
                                                                                                                                           growth &                     growth &                   on 4th day
                                                                                                                                           discolouration               darkening
      Uninoculated fruits              No spoilage                     No spoilage                       No spoilage                       No spoilage                  No spoilage                No spoilage




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   Table 6 - Pathogenicity of the fungal isolates on S. lycopersicum fruit samples.
    Fungal isolates                                                                 Macroscopic Observations                                                                                       Inference
                                         Day 1                     Day 2                        Day 3                                            Day 4                         Day 5
    P. kudriavzevii             Swelling at site of        Softening at site of     White yeast-like growth                               Increased yeastlike         White yeast-like growth Spoilage started
                                inoculation                inoculation soft         with watery exudates from                             growth & fruit              with total fruit collapse on 1st day
                                                                                    site of inoculation                                   collapse
     A. niger                          Water-soaked                   Heavy black spores at          Heavy black spores.                  Heavy black spores          Total fruits collapse,          Spoilage started
                                       appearance at site of          site of inoculation &          Inoculation site become              & fruit collapse            watery exudates, heavy          on 1st day
                                       inoculation                    white mycelia                  sunken & water soaked                                            black spores

     F. culmorum                      No spoilage                    Softening at inoculation Softening & white mycelia                   Increased white             Extensive white                 Spoilage started
                                                                     site                     growth at inoculation sites                 mycelia growth &            mycelia growth & total          on 2nd day
                                                                                                                                          fruit collapse              fruit collapse
     G. candidum                      No spoilage                     No spoilage. Fruits            Dirty mucoid growth on the Increased mucoid                      Total fruit collapse &          Spoilage started
                                                                      still firm                     fruits. Sunken spots       growth sunken                         exudates                        on 2nd day
                                                                                                                                spots
     M. racemosus                     Water-soaked                    White mycelia from             White fluffy mycelia                 White fluffy                White fluffy mycelia            Spoilage started
                                      appearance at                   point of inoculation           increased                            mycelia with black          abundant. Black spores          on 1st day
                                      inoculation site                                                                                    spores                      abundant
     C. tropicalis                    No spoilage                     No spoilage                    Water-soaked appearance              Water-soaked                Total fruit collapse &          Spoilage started
                                                                                                     at inoculation site                  appearance &                watery exudates                 on 3rd day
                                                                                                                                          watery exudates

     C. utilis                        No spoilage                     No spoilage                    White mycelial growth on             White mycelia               Total fruit collapse,           Spoilage started
                                                                                                     fruits & water soaked-               growth & soft               watery milky exudates           on 3rd day
                                                                                                     appearance                           inoculation site
     Rhizopus spp.                    Water-soaked                   White mycelia seen              White fluffy mycelia                 Increased white             Extensive fluffy white          Spoilage started
                                      appearance                                                                                          fluffy mycelia &            mycelia with black              on 1st day
                                                                                                                                          black spores                spores
     Uninoculated fruits               No spoilage                    No spoilage                    No spoilage                          No spoilage                 No spoilage                     No spoilage




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https://doi.org/10.6092/issn.2531-7342/16813                                              Italian Journal of Mycology 53 (2024): 29 - 44
___________________________________________________________________________________________________________________________________________________

Per Inch (dpi) at 28 ± 2 °C. Rhizopus oligosporus and A. aculeatus elicited spoilage on healthy P.
americana fruit within 24 h after inoculation and 5 dpi, respectively (Table 2).
       In the case of the fresh healthy C. sinensis fruit samples artificially inoculated with L.
theobromae, M. racemosus and A. aculeatus, spoilage was recorded after two days of post
inoculation, displaying water-soaked appearance at the inoculation site. For fruit samples inoculated
with T. verruculosus, F. culmorum and G. candidum, spoilage started on the third day after
inoculation while fruit samples inoculated with M. suaveolens did not initiate spoilage until the fifth
day (Table 3).
       For the healthy C. papaya fruits, when inoculated with F. culmorum, R. oligosprous, M.
racemosus and A. nominus, spoilage was observed from the second day after inoculation while A.
niger and G. candidum elicited spoilage from the third day. When inoculated with L. theobromae and
P. lilacinum, spoilage of C. papaya fruit samples started 3 and 4 days after inoculation, respectively
(Table 4). Annona muricata inoculated with L. theobromae, A. conymbifera, C. utilis, A. aculeatus
and A. nominus started to spoil on the third day after inoculation while those inoculated with P.
lilacinum started to spoil on the fourth day (Table 5).
       In the case of the fresh healthy S. lycopersicum fruit samples, when inoculated with P.
kudriavzevii, A. niger, F. culmorum, G. candidum, C. tropicalis, C. utilis, Rhizopus sp. and M.
racemosus, spoilage was observed 5 days post inoculation. For the fruits inoculated P. kudriavzevii,
A. niger, Rhizopus sp. and M. racemosus, spoilage was elicited within 24 h. Spoilage was observed
48 h post inoculation for fruits when inoculated with F. culmorum and G. candidum, while those
inoculated with C. tropicalis and C. utilis showed spoilage about 72 h after inoculation (Table 6).

Discussion

The roles of fungal pathogens in the rot of fruits have been established in several previous research
(Moss, 2008; Whitehead et al., 2015; Triest and Hendrickx, 2016; Zeilinger et al., 2016) and has been
shown in this study carried out in Akwa Ibom State, Nigeria. The morphological approach can be a
preliminary approach, but the use of molecular analysis, sequencing of the ITS region of fungi proved
to be very useful. During the research, fungal samples initially classified as A. flavus were molecularly
identified as A. nomius strain DIV-4. Those identified as A. ochraceous were molecularly classified
as A. sclerotiorum and A. tamari. In particular, A. nomius are often misidentified as A. flavus (Tam et
al., 2014). The indeterminate nature of the sclerotia would have been a vital divergence between the
A. nomius and A. flavus but not all strains of either A. nomius or A. flavus produce sclerotia. Only
molecular characterization can give a clear distinction between A. nomius, A. flavus and A. tamari.
       The prevalent isolated fungi, including T. verruculosus strain DIV-20, T. verruculosus strain
DIV-2, L. theobromae strain DIV-19, L. theobromae strain DIV-1, T. koningiopsis strain DIV-18, P.
citrinum strain DIV-17, P. kudriavzevii strain DIV-16, A. niger strain DIV-14, G. candidum strain
DIV-9 and the other isolates (Table 1, Supplementary Table S1), were in accordance with the list of
fungal species reported by Udoh et al. (2015). They were isolated in completely different
environmental conditions, demonstrating the ability of fungi to cause spoilage of fruits (Egbuta et al.,
2016). The inoculum source might start from farm through to washing with sewage contaminated
groundwater or surface water containing fungal pathogens (Adegoke and Stenstrom, 2019; Nzima et
al., 2020) or cross contamination from currency notes paid while going through supply chains
(Adegoke and Okoh, 2011).

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       The deterioration observed in our studies revealed that in a period of 5 days of storage, even
the weakest fungi would have done meaningful damage to the post-harvested fruits, causing economic
losses (Dean et al., 2012; Drenth and Guest, 2016). In addition, some postharvest fungi also constitute
serious threat to human health, because of mycotoxigenic potentials of some fungal genera, including
those isolated in our study like Penicillium and Fusarium, with high toxicity to consuming humans
and animals (Li et al., 2015; Sanzani et al., 2016). This can be exemplified by the Fusarium species
which are primary plant pathogens characterized by being septate, fusiform to crescent shaped
macroconidia, and may contain microconidia (Leslie and Summerell, 2006). They are proven
phytopathogens as well as aetiological agents of diseases and infections in humans and animals
(Nucci and Anaissie, 2007). Like many pathogens, their contamination of these fruits is made easier,
when contamination waters are used to wash the fruit (Adegoke and Stenstrom, 2019); the error that
comes with severe human health risks. The health risk may include toxin productions. Fusarium
species produce several types of toxins that affect both humans and animals (Jimenez-Garcia et al.,
2018). Naturally, most postharvest pathogenic fungi are known for necrotrophic attributes, in which
their secreted enzymes or toxins cause cytocidal while the fungi then live saprophytically on the dead
cells (Tian et al., 2016).
       This study established the array of fungal pathogens associated with post-harvest fruits rots in
Akwa Ibom State. the data coming from this study allow to start to plan specific control strategies
against the main post-harvest fungi starting from the field.

Authors’ contributions
Divine-Anthony O: Conceived and designed the experiments, performed the experiments, analyzed and interpreted the
data, contributed reagents, materials, analysis of data and contributed to the writing of the manuscript first draft. Adegoke
AA: redrafted the manuscript, analyzed and interpreted the data, did extensive editing/revision of the manuscript,
contributed reagents, material s and analysis of data. Oduoye OT: performed the experiment, analyzed and interpreted
the data, contributed reagents, materials and analysis of data. Akpor OB: analyzed and interpreted the data and wrote the
manuscript first draft



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