OPEN ACCESS
Asian Journal of Plant Pathology
ISSN 1819-1541
DOI: 10.3923/ajppaj.2018.27.37
Research Article
A Novel Isolate of Phyllosticta capitalensis Causes Black Spot
Disease on Guava Fruit in Egypt
Khaled Arafat
Department of Plant Pathology, Faculty of Agriculture, New Valley University, Egypt
Abstract
Background and Objective: Guava black spot (GBS) disease is a quiescent infection, that infect immature fruit prior to harvest. Visible
symptoms of the disease on guava fruit showed sunken lesions with concentric development, variation in color ranging from greenish
black to black and spread in severity affected fruit. An unrecorded disease of guava fruit (Psidium guajava L.) cv. White Balady, was
observed in this study during postharvest disease survey in Egypt. Materials and Methods: Tissues of guava fruit spot used to isolate the
pathogenic fungal. To perform the phylogenetic analysis, the internal transcribed spacer (ITS) region amplified by Polymerase chain
reaction (PCR). To amplify the ITS, the primer ITS-1 and reverse primer ITS-4 used to amplify rDNA-ITS regions of the fungus. The fungal
identification was done by molecular analysis as Phyllosticta capitalensis novel isolate ARAFAT-GF5 according to the GenBank (Accession
number‒LC269950.1; GI: 119461242) with the synonym: Guignardia mangiferae. Results: The isolate ARAFAT-GF5 (626 bootstrap) used
and the Basic Local Alignment Search Tool (BLAST) program used to search for nucleotide sequence homology in GenBank. The
computational analysis of the synonymous DNA sequence was useful for predicting the codon profiling. Pathogenicity test performed
to complete Kochʼs postulates. Typical black spot symptoms developed and the pathogen recovered from the inoculated fruit after 10
days and found as P. capitalensis. Conclusion: This is the first report of black spot disease on guava fruits in Egypt, caused by a novel
isolate of P. capitalensis. The results presented here may enable enhancements in the program of integrated disease management.
Key words: Guava fruits, black spot disease, Phyllosticta capitalensis, Guignardia mangiferae, codon profiling
Citation: Khaled Arafat, 2018. A Novel isolate of Phyllosticta capitalensis causes black spot disease on guava fruit in Egypt. Asian J. Plant Pathol., 12: 27-37.
Corresponding Author: Khaled Arafat, Department of Plant Pathology, Faculty of Agriculture, New Valley University, Egypt Tel: +20 1,062,388,723
Copyright: © 2018 Khaled Arafat. This is an open access article distributed under the terms of the creative commons attribution License, which permits
unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.
Competing Interest: The author have declared that no competing interest exists.
Data Availability: All relevant data are within the paper and its supporting information files.
Asian J. Plant Pathol., 12 (1): 27-37, 2018
INTRODUCTION
MATERIALS AND METHODS
Guava fruit (Psidium guajava L.) cultivated and
widespread in many tropical and subtropical regions. Egypt is
a subtropical country which existing between 22E and 32E
north latitude. In recent years, several studies have focused on
the incidence of postharvest diseases that decrease the value
of the fruits and alter their physical and chemical properties
and contribute to their reduced shelf life1-3. The major
postharvest diseases of guava are including anthracnose,
black spot and astylar end rot3,4. The GBS disease is a quiescent
infection, that infect immature fruit prior to harvest. Fruits with
quiescent infections stay asymptomatic until maturity, when
structural and physiological changes trigger the onset of the
disease5. However, there have been few studies in the
literature reporting GBS disease. The G. psidii reported in the
first study, which causing black spot on guava fruits, during in
the field and transportation in India6. While G. pisidii infection
occurs in young fruits and stays quiescent until maturity in
Brazil7. Similarly, P. psidiicola, reported as a potential cause of
GBS disease in Taiwan8 and in Venezuela9. The results to date,
has been extensive experiments on Guignrdia species, where
10 isolated from asymptomatic tissues on different hosts, that
were G. mangiferae classification by rDNA ITS1-5.8S-ITS2
sequence methods, caused symptoms in guava fruits10. Visible
symptoms of the disease showed sunken lesions with
concentric development, variation in color ranging from
greenish black to black and spread on severity affected fruit9.
The genus Phyllosticta Pers. Ex Desm. confirmed11. It includes
endophytes, plant pathogens and saprobes12-17. Species in the
genus Phyllosticta are mostly plant pathogens of a wide range
of hosts and responsible for diseases, including black spots on
leaf and fruits13,18-24. Phyllosticta species are also potential
biocontrol agents25 and has reported to produce novel
mycotoxin viz. phyllostine and phyllostoxin26. Recently, the
name Phyllosticta Pers. Ex. Desm. (asexual state) and
Guignardia Viala and Ravaz (sexual state) have used
separately following the dual classification system used by
mycologist over several decades27-30. ITS rDNA sequences often
used to infer phylogeny relationships in many groups of fungi,
including Phyllosticta10,14,15,31. Nevertheless, researchers using
molecular methods suggested that the fungi isolates found as
G. psidii could be in fact G. mangiferae or also could be
conspecific to this cosmopolitan species32. The objective of
this study was to identify and characterization of
P. capitalensis novel isolate ARAFAT-GF5 associated with GBS
a new disease on guava fruit in Egypt. Identification of the
isolate performed using DNA sequence data of the rDNA
ITS1-5.8S-ITS2-28S.
Samples collection and isolation of the pathogenic fungal:
P. capitalensis obtained from naturally guava fruit (Psidium
guajava L.) cv. White Balady at an immature and mature stage.
Fruits collected for each three seasons (2015-2017) from
different local markets in El-Kharga city (25.4390 N, 30.5586 E),
New Valley Governorate, Egypt. The collected samples kept in
sterilized polyethylene bags and brought to the laboratory of
the Plant Pathology Department, Faculty of Agriculture, New
Valley University, Egypt. Fruit samples cut into (5 mm) and
immersed in NaOCl (0.5%) for 5 min rinsed in sterile
distilled water, then transfer to blotted dry in sterile paper
towels for drying. Samples transferred into 9 cm Petri dishes
containing potato dextrose agar (PDA) and incubation at
25±3EC under a 12 h photo period for 10-15 days33. The
fungus characterized by initially gray and turned black with
overripe.
Identification of pathogenic fungus: Identification of
pathogenic fungus based on morphological methods, using
characters of the phenotype of the fungus culture, i.e., colony
or hyphae, the characters of the spore or reproductive
structure if these features were discernible 34, 35.
Molecular characterization of pathogenic fungus: The
fungus was grown in the cultivation media and incubated at
25EC for 15 days, then the growth of fungal was scraped and
suspended in 100 :L of distilled water and boiled at 100EC for
15 min and stored at -80EC. DNA was extracted from fungal
cultures using the genomic DNA Prep kit (SolGent, Daejeon,
Korea) according to SDS/CTAB lysis and phenol/chloroform
extraction method15. The ITS region, including ITS1, 5.8S and
ITS4, 28S rRNA amplified via PCR using primer pair ITS1
(5'‒CTTGGTCATTTAGAGGAAGTAA-3') and ITS4 (5'
TCCTCCGCTTATTGATATGC-3')36. The ITS sequence obtained
through the commercial service offered by Macrogen
(Macrogen Comp., South Korea). The sequence compared with
known homologous sequences of Phyllosticta and Guignardia
in databanks (National Center for Biotechnology Information
(NCBI)-(http: //www.ncbi.nlm.nih.gov/Genbank) and The
European Molecular Biology Laboratory (EMBL)
(https://www.ebi.ac.uk/) by the BLSAT program. DNA
sequences deposited in the GenBank database (GenBank
Accession No. LC269950).
Sequence analysis: The BLAST search program at NCBI
(http://blast.ncbi.nlm.nih.gov/BLAST.cgi) used to analyze the
obtained sequence. CLUSTALW program (http://clustalw.ddbj.
28
Asian J. Plant Pathol., 12 (1): 27-37, 2018
concentric development, variation in color ranging from
nig.ac.jp/top-ehtml) applied to achieve the sequence
alignment and phylogeny. Phylogenetic analysis performed by
a neighbor joining method to infer the relationships between
the fungus isolate and sequences available for Phyllosticta and
Guignardia in the NCBI and The EMBL nucleotide databases
using Kimura 2-parameter distances37. For analysis, 100 and 33
bootstrap replicates performed to assess the statistical
support for the tree.
greenish black to black and spread in severity affected fruit.
As shown before, these symptoms showed that, a strong
relationship between symptoms and P. capitalensis as the
pathogenic fungal of GBS disease of guava fruit.
Identification of the pathogenic fungus: Morphological
characters of the isolated fungus from guava fruit showed
that closed to P. capitalensis in colony appearance, although
Nucleotide distributions and frequencies: The DNA
the hyphal growth of PDA culture, produced gray mycelium at
sequence analysis used free software (http://www.
bioinformatics.org/sms2/index.html) usefully to attain the
coding usage of DNA stats. DNA stats returns the number of
occurrences of each residue in the sequence entered. Codon
usage accepts a DNA sequence and returns the number and
frequency of each codon type. Since the program also
compares the frequencies of codons that code for the same
amino acid (synonymous codons). So, it used to assess
whether a sequence shows a preference for certain
synonymous codons38.
the early stage of growth followed by black colored conidia
(Fig. 2). Conidia is hyaline, unicellular, obovate, ranged 6-11 X
5-7 µm (Fig. 3). The pathogen found associated with the GBS
disease based on morphological features identified as
Phyllosticta capitalensis.
Molecular characterization of pathogenic fungus: The
fungus found by molecular analysis as P. capitalensis
according to the GenBank (Accession number-LC269950.1; GI:
119461242) with synonym: Guignardia mangiferae. Analysis of
ITS1, 5.8S rRNA, ITS2, 28S rRNA, partial and complete
Pathogenicity test: Guava fruits obtained from a local
sequence. The DNA from P. capitalensis from guava fruit
supermarket and immediately transferred into mycological
laboratory. Fruits in similar shape and size certain and treated
with 96% ethanol, soaked with sterilized distilled water and
drained at room temperature 25+2EC. Two wounds (5 mm
diameter and 3 mm deep) made through at different
equatorial lines of each fruit using the tip of a sterile corkborer. Each one of guava fruits inoculated with a mycelial plug
(5 mm in diameter) of the fungus culture into each wound.
Other guava fruits having artificial wounds of only plugs of
PDA culture used as control. Twenty-five fruits used for each
treatment and then the fruits air dried and placed in the
plastic boxes (with wetted sterilized cotton pieces to maintain
high-level of humidity). The experiment frequent twice. The
virulence of the tested fungus identified by observing the
development of GBS disease, after 10 days on infested guava
fruits39.
amplified when the PCR region performed using primers ITS1
and ITS4. The corresponding PCR region amplified the ITS
rDNA sequence of region 1 and 4, which also include 5.8S
rRNA and 28S rRNA gene. The PCR produce was 626 bp.
Sequence analysis: ITS sequence of P. capitalensis isolate
(ARAFAT-GF5) aligned with different Phyllosticta isolates
available from the GenBank nucleotide database. The aligned
sequences visually inspected and minor adjustments made to
improve alignment. Phylogenetic analysis performed by a
neighbor joining method to infer the relationships between
the Phyllosticta isolate (ARAFAT-GF5) and sequences available
for
Phyllosticta
and
Guignardia
in
the
NCBI
(https://www.ncbi.nlm.nih.gov/nuccore/?cmd=historysearc
h&querykey=1) and EMBL-EBI (https://www.ebi.ac.uk/ena/
data/view/LC269950) nucleotide databases (Table 1) using
RESULTS
Kimura 2-parameter distances. For analysis, 100 bootstrap
Samples collection and isolation of the pathogenic fungal:
tree. Phyllosticta capitalensis (LC 269950.1) sequence No. 1
replicates performed to assess the statistical support for the
homologous with all sequences, ranged score between (98.08-
Samples of guava fruit at immature and mature stage,
collected according to GBS disease symptoms, from the
different local market in El-Kharga city, New Valley
Governorate, Egypt. The first visible symptoms of the infected
guava fruit were small, slightly sunken on mature fruits.
Symptom developer showed in Fig. 1, sunken lesions with
100%). Furthermore, the isolate LC269950 compared with the
other sequences (33 bootstrap) published for Phyllosticta and
Guignardia isolates obtained in the NCBI. Alignment of the
available sequences of Phyllosticta and Guignardia exposed
both similarity in the ITS sequences (Table 2).
29
Asian J. Plant Pathol., 12 (1): 27-37, 2018
Table 1: Nucleotide length of 100 isolates compared with P. capitalensis and GenBank accession numbers of their characters data
Sequence No.
GenBank (Accession number)
Bootstrap (bp)
Isolates
Pairwise alignments
1
*LC269950.1
626 bp
Sequences 1
2
KR016633.1
626 bp
3
KR015491.1
626 bp
4
KR015441.1
626 bp
5
HM537040.1
626 bp
6
GU066692.1
626 bp
7
GU066670.1
626 bp
8
GQ352495.1
626 bp
9
EU686803.1
626 bp
10
EU167584.1
626 bp
11
EU273524.1
626 bp
12
AM403717.1
626 bp
13
AY601899.1
626 bp
14
NR̲147316.1
625 bp
15
KR015859.1
625 bp
16
AB731125.1
625 bp
17
AB454332.1
625 bp
18
AB454307.1
625 bp
19
AB454279.1
625 bp
20
AB454270.1
625 bp
21
KR016813.1
624 bp
22
MF076618.1
627 bp
23
KR015511.1
623 bp
24
KP743018.1
626 bp
25
JN791606.1
626 bp
26
JN791605.1
626 bp
27
FR863606.1
626 bp
28
HM807531.1
626 bp
29
HM537020.1
626 bp
30
FJ462743.1
626 bp
31
GU066668.1
626 bp
32
GQ352496.1
627 bp
33
AB454364.1
625 bp
34
AB454264.1
625 bp
35
AB454263.1
625 bp
36
AB454262.1
625 bp
37
KP743020.1
626 bp
38
KC218454.1
627 bp
39
JQ809680.1
627 bp
40
JQ086349.1
626 bp
41
HM537060.1
626 bp
42
EU747726.1
627 bp
43
EU747725.1
627 bp
44
KJ883595.1
619 bp
45
KF435651.1
619 bp
46
AB731124.1
622 bp
47
GU066689.1
622 bp
48
AB454315.1
625 bp
49
AB454291.1
625 bp
50
DQ377879.2
619 bp
51
KP998485.1
628 bp
52
GU066723.1
618 bp
53
GU066719.1
621 bp
54
GU066675.1
628 bp
55
GU066669.1
618 bp
56
FJ037766.1
618 bp
57
AY816311.1
621 bp
58
KR015490.1
617 bp
Phyllosticta capitalensis
Fungal endophyte
Fungal endophyte
Fungal endophyte
Fungal endophyte
Guignardia vaccinii
Guignardia sp.
Guignardia sp.
Fungal endophyte
Phyllosticta elongata
Guignardia mangiferae
Guignardia mangiferae
Fungal endophyte
Phyllosticta fallopiae
Fungal endophyte
Guignardia mangiferae
Guignardia mangiferae
Phyllosticta fallopiae
Guignardia sp.
Guignardia philoprina
Fungal endophyte
Phyllosticta elongata
Fungal endophyte
Phyllosticta capitalensis
Guignardia mangiferae
Guignardia mangiferae
Uncultured fungus
Guignardia mangiferae
Fungal endophyte
Guignardia camelliae
Guignardia camelliae
Guignardia sp.
Phyllosticta sp.
Guignardia alliacea
Guignardia alliacea
Guignardia philoprina
Phyllosticta capitalensis
Guignardia sp.
Guignardia sp.
Guignardia camelliae
Fungal endophyte
Guignardia mangiferae
Guignardia mangiferae
Phyllosticta capitalensis
Fungal endophyte
Guignardia mangiferae
Guignardia sp.
Guignardia mangiferae
Phyllosticta miurae
Guignardia sp.
Phyllosticta capitalensis
Guignardia sp.
Guignardia camelliae
Guignardia sp.
Guignardia sp.
Guignardia sp.
Guignardia mangiferae
Fungal endophyte
30
Score
-
Sequences (1:2)
100.00
Sequences (1:3)
100.00
Sequences (1:4)
100.00
Sequences (1:5)
100.00
Sequences (1:6)
100.00
Sequences (1:7)
100.00
Sequences (1:8)
100.00
Sequences (1:9)
100.00
Sequences (1:10)
100.00
Sequences (1:11)
100.00
Sequences (1:12)
100.00
Sequences (1:13)
100.00
Sequences (1:14)
100.00
Sequences (1:15)
100.00
Sequences (1:16)
100.00
Sequences (1:17)
100.00
Sequences (1:18)
100.00
Sequences (1:19)
100.00
Sequences (1:20)
100.00
Sequences (1:21)
100.00
Sequences (1:22)
99.20
Sequences (1:23)
100.00
Sequences (1:24)
99.84
Sequences (1:25)
99.84
Sequences (1:26)
99.84
Sequences (1:27)
99.84
Sequences (1:28)
99.84
Sequences (1:29)
99.84
Sequences (1:30)
99.84
Sequences (1:31)
99.84
Sequences (1:32)
99.20
Sequences (1:33)
99.84
Sequences (1:34)
99.84
Sequences (1:35)
99.84
Sequences (1:36)
99.84
Sequences (1:37)
99.68
Sequences (1:38)
99.04
Sequences (1:39)
99.36
Sequences (1:40)
99.68
Sequences (1:41)
99.68
Sequences (1:42)
99.04
Sequences (1:43)
99.04
Sequences (1:44)
100.00
Sequences (1:45)
100.00
Sequences (1:46)
99.83
Sequences (1:47)
99.83
Sequences (1:48)
99.68
Sequences (1:49)
99.68
Sequences (1:50)
100.00
Sequences (1:51)
99.04
Sequences (1:52)
100.00
Sequences (1:53)
99.83
Sequences (1:54)
98.56
Sequences (1:55)
100.00
Sequences (1:56)
100.00
Sequences (1:57)
99.83
Sequences (1:58)
100.00
Asian J. Plant Pathol., 12 (1): 27-37, 2018
Table 1: Continue
Sequence No.
GenBank (Accession number)
Bootstrap (bp)
Isolates
Pairwise alignments
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
KC816052.1
GU066700.1
AY277709.1
KX908973.1
KR056285.1
JQ759968.1
JQ759953.1
JQ759952.1
AY277712.1
KU671305.1
KR015693.1
KF435717.1
KC686598.1
KF381072.1
JX436789.1
JQ936158.1
GQ352474.1
DQ377880.2
EF419973.1
AY277714.1
KX424992.1
KR016683.1
KR016182.1
KR015353.1
KR014948.1
KF435727.1
HQ622105.1
HM595514.1
EU821358.1
EU821356.1
AY277716.1
AY277713.1
AY277711.1
KU663502.1
KR016814.1
KR016812.1
KR016695.1
KR015487.1
KR056282.1
KF128847.1
JQ759989.1
JQ759948.1
626 bp
620 bp
617 bp
616 bp
616 bp
616 bp
616 bp
616 bp
619 bp
615 bp
618 bp
615 bp
628 bp
625 bp
615 bp
618 bp
621 bp
622 bp
615 bp
618 bp
619 bp
617 bp
614 bp
618 bp
620 bp
614 bp
614 bp
620 bp
614 bp
614 bp
617 bp
617 bp
621 bp
613 bp
613 bp
613 bp
613 bp
613 bp
613 bp
619 bp
616 bp
616 bp
Guignardia mangiferae
Guignardia sp.
Guignardia mangiferae
Dothideomycetes sp.
Phyllosticta capitalensis
Dothideomycetes sp.
Dothideomycetes sp.
Dothideomycetes sp.
Guignardia mangiferae
Phyllosticta capitalensis
Fungal endophyte
Fungal endophyte
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia vaccinii
Guignardia sp.
Guignardia sp.
Fungal endophyte
Guignardia mangiferae
Phyllosticta elongata
Fungal endophyte
Fungal endophyte
Fungal endophyte
Fungal endophyte
Fungal endophyte
Guignardia sp.
Phyllosticta sp.
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Phyllosticta capitalensis
Fungal endophyte
Fungal endophyte
Fungal endophyte
Fungal endophyte
Phyllosticta capitalensis
Guignardia sp.
Dothideomycetes sp.
Dothideomycetes sp.
Sequences (1:59)
Sequences (1:60)
Sequences (1:61)
Sequences (1:62)
Sequences (1:63)
Sequences (1:64)
Sequences (1:65)
Sequences (1:66)
Sequences (1:67)
Sequences (1:68)
Sequences (1:69)
Sequences (1:70)
Sequences (1:71)
Sequences (1:72)
Sequences (1:73)
Sequences (1:74)
Sequences (1:75)
Sequences (1:76)
Sequences (1:77)
Sequences (1:78)
Sequences (1:79)
Sequences (1:80)
Sequences (1:81)
Sequences (1:82)
Sequences (1:83)
Sequences (1:84)
Sequences (1:85)
Sequences (1:86)
Sequences (1:87)
Sequences (1:88)
Sequences (1:89)
Sequences (1:90)
Sequences (1:91)
Sequences (1:92)
Sequences (1:93)
Sequences (1:94)
Sequences (1:95)
Sequences (1:96)
Sequences (1:97)
Sequences (1:98)
Sequences (1:99)
Sequences (1:100)
Score
99.52
99.83
100.00
100.00
100.00
100.00
100.00
100.00
99.83
100.00
99.83
100.00
98.08
99.04
100.00
99.83
99.67
98.87
100.00
99.83
98.54
99.18
100.00
99.02
98.70
100.00
100.00
99.35
100.00
100.00
99.83
99.83
98.87
100.00
100.00
100.00
100.00
100.00
100.00
99.19
99.83
99.83
Fig. 2: Hyphal growth of P. capitalensis on PDA medium
Fig. 1: Natural GBS disease symptoms of guava fruits
31
Asian J. Plant Pathol., 12 (1): 27-37, 2018
Table 2: Nucleotide length of 33 isolates of Phyllosticta or Guignardia compared with P. capitalensis and GenBank accession numbers of their characters data
Sequence No.
GenBank (Accession number)
Bootstrap (bp)
Isolates
Pairwise alignments
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
*LC269950.1
KU671305.1
KU663502.1
KP998485.1
KR056285.1
KR056282.1
KP743020.1
AM403717.1
AY816311.1
EU273524.1
AY277709.1
AY277712.1
AY277711.1
AY277714.1
AY277716.1
EU747726.1
EU747725.1
EU821358.1
EU821356.1
JN791605.1
AB454332.1
AB454315.1
HM807531.1
JN791606.1
KF381072.1
KC816052.1
JX436789.1
AB731125.1
AB731124.1
KC686598.1
KP743018.1
AY277713.1
KJ883595.1
626 bp
615 bp
613 bp
640 bp
641 bp
623 bp
639 bp
638 bp
632 bp
663 bp
624 bp
625 bp
630 bp
625 bp
626 bp
643 bp
642 bp
635 bp
637 bp
663 bp
1207 bp
1207 bp
641 bp
663 bp
646 bp
639 bp
658 bp
628 bp
624 bp
882 bp
639 bp
625 bp
655 bp
Phyllosticta capitalensis
Phyllosticta capitalensis
Phyllosticta capitalensis
Phyllosticta capitalensis
Phyllosticta capitalensis
Phyllosticta capitalensis
Phyllosticta capitalensis
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Guignardia mangiferae
Phyllosticta capitalensis
Guignardia mangiferae
Phyllosticta capitalensis
Sequences 1
Sequences (1:2)
Sequences (1:3)
Sequences (1:4)
Sequences (1:5)
Sequences (1:6)
Sequences (1:7)
Sequences (1:8)
Sequences (1:9)
Sequences (1:10)
Sequences (1:11)
Sequences (1:12)
Sequences (1:13)
Sequences (1:14)
Sequences (1:15)
Sequences (1:16)
Sequences (1:17)
Sequences (1:18)
Sequences (1:19)
Sequences (1:20)
Sequences (1:21)
Sequences (1:22)
Sequences (1:23)
Sequences (1:24)
Sequences (1:25)
Sequences (1:26)
Sequences (1:27)
Sequences (1:28)
Sequences (1:29)
Sequences (1:30)
Sequences (1:31)
Sequences (1:32)
Sequences (1:33)
Score
100.00
100.00
99.04
98.40
98.39
99.68
100.00
99.04
100.00
98.87
98.88
98.08
98.72
98.40
99.04
99.04
98.08
98.08
99.84
99.84
99.52
99.84
99.84
98.88
99.52
98.24
99.84
99.51
98.08
99.84
98.56
98.88
highest bases were (AA and GC) repeated 48 and 45 times
with 7.68 and 7.20%, respectively. Moreover, G, C and A, T
dinucleotide bases found repeated 317 and 309 times with
50.64 and 49.36%, respectively. Trinucleotide frequencies
found the highest bases (GAA) repeated 16 times with 2.56%
and the latest trinucleotide was (CAC) repeated 3 times with
0.48% (Table 3).
Codon usage: The analysis and simulations of P. capitalensis
results for 626 sequences indicated that prediction of
amino acids was the highest (leucine) frequency 26 times
with 124.99 times/1000, followed by (serine, arginine,
alanine, glycine, cysteine, isoleucine, asparagine, Valine,
phenylalanine, glutamine, throine, proline, Tyrosine, glutamic
acid, lysine, aspartic acid, tryptophane, histidine and
methionine) frequencies “26, 18, 16, 15, 14, 11, 11, 11, 11, 9, 9,
9, 8, 7, 6, 6, 5, 5, 4 and 0" with “107.02, 76.93, 72.12, 67.32,
52.88, 52.88, 52.89, 79.86, 43.27, 28.85, 76.67, 38.48, 28.75,
28.85, 28..84, 24.04, 17.57 and 19.23/1000", respectively
(Table 4).
Fig. 3: Conidia spores of P. capitalensis (X400)
Nucleotide distributions and frequencies: The results of
P. capitalensis (LC 269950.1) showed that sequences length,
consisting of 626 bases. Distributions and frequencies of bases
found (C) base repeated 160 times with 25.56%, followed by
(G, T, A) bases repeated 157, 157, 152 times with 25.08, 25.08,
24.28%, respectively. Dinucleotide frequencies found the
32
Asian J. Plant Pathol., 12 (1): 27-37, 2018
Table 3: Frequencies and percentage nucleotides of P. capitalensis
Codon
Frequencies
Percentage
G
A
T
C
Dinucleotide
157
152
157
160
25.08
24.28
25.08
25.56
GG
GA
GT
GC
AG
AA
AT
AC
TG
TA
TT
TC
CG
CA
CT
CC
Trinucleotide
40
41
30
45
32
48
41
31
41
31
44
41
44
32
42
42
6.40
6.56
4.80
7.20
5.12
7.68
6.56
4.96
6.56
4.96
7.04
6.56
7.04
5.12
6.72
6.72
AAA
AAC
AAG
AAT
ACA
ACC
ACG
ACT
AGA
AGC
AGG
AGT
ATA
ATC
ATG
ATT
CAA
CAC
CAG
CAT
CCA
CCC
CCG
CCT
CGA
CGC
CGG
CGT
CTA
CTC
CTG
CTT
GAA
GAC
GAG
GAT
GCA
GCC
GCG
10.00
12.00
12.00
14.00
5.00
9.00
9.00
8.00
5.00
8.00
10.00
9.00
9.00
11.00
7.00
14.00
11.00
3.00
9.00
9.00
6.00
10.00
11.00
15.00
12.00
12.00
15.00
5.00
6.00
11.00
12.00
13.00
16.00
11.00
6.00
8.00
7.00
15.00
14.00
1.60
1.92
1.92
2.24
0.80
1.44
1.44
1.28
0.80
1.28
1.60
1.44
1.44
1.76
1.72
2.24
1.76
0.48
1.44
1.44
0.96
1.60
1.76
2.40
1.92
1.92
2.40
0.80
0.96
1.76
1.92
2.08
2.56
1.76
0.96
1.28
1.12
2.40
2.24
Table 3: Continue
Codon
GCT
GGA
GGC
GGG
GGT
GTA
GTC
GTG
GTT
TAA
TAC
TAG
TAT
TCA
TCC
TCG
TCT
TGA
TGC
TGG
TGT
TTA
TTC
TTG
TTT
Nucleotide
Frequencies
9.00
11.00
14.00
5.00
9.00
11.00
6.00
7.00
6.00
11.00
5.00
5.00
10.00
14.00
7.00
10.00
10.00
13.00
11.00
10.00
7.00
5.00
13.00
15.00
11.00
Percentage
1.44
1.76
2.24
0.80
1.44
1.76
0.96
1.12
0.96
1.76
0.80
0.80
1.60
2.24
1.12
1.60
1.60
2.08
1.76
1.60
1.12
0.80
2.08
2.40
1.76
Fig. 4: Artificial GBS disease symptoms of guava fruit
inoculated with P. capitalensis
Taxonomy
Phyllosticta capitalensis ( Guignardia mangiferae):
Eukaryota, Fungi, Dikarya, Ascomycota, Pezizomycotina,
Dothideomycetes, Dothideomycetes incertae sedis,
Botryosphaeriales, Phyllostictaceae, Phyllosticta.
Pathogenicity tests: Pathogenicity of the characteristic
P. capitalensis novel isolate ARAFAT-FG5 confirmed by
inoculating guava fruits at mature stage. The artificially
inoculated guava fruits developed black spot symptoms after
10 days of the inoculation. These symptoms similar to those of
the naturally infected guava fruits (Fig. 4). All control fruits
33
Asian J. Plant Pathol., 12 (1): 27-37, 2018
Table 4: Frequencies amino acids probability of P. capitalensis
Amino acid
Codon
Ala
Ala
Ala
Ala
Cys
Cys
Asp
Asp
Glu
Glu
Phe
Phe
Gly
Gly
Gly
Gly
His
His
Ile
Ile
Ile
Lys
Lys
Leu
Leu
Leu
Leu
Leu
Leu
Met
Asn
Asn
Pro
Pro
Pro
Pro
Gln
Gln
Arg
Arg
Arg
Arg
Arg
Arg
Ser
Ser
Ser
Ser
Ser
Ser
Thr
Thr
Thr
Thr
Val
Val
Val
Val
Trp
Tyr
Tyr
GCG
GCA
GCT
GCC
TGT
TGC
GAT
GAC
GAG
GAA
TTT
TTC
GGG
GGA
GGT
GGC
CAT
CAC
ATA
ATT
ATC
AAG
AAA
TTG
TTA
CTG
CTA
CTT
CTC
ATG
AAT
AAC
CCG
CCA
CCT
CCC
CAG
CAA
AGG
AGA
CGG
CGA
CGT
CGC
AGT
AGC
TCG
TCA
TCT
TCC
ACG
ACA
ACT
ACC
GTG
GTA
GTT
GTC
TGG
TAT
TAC
Number
11
10
16
10
14
12
17
8
6
8
10
8
14
13
10
11
7
4
8
10
13
9
9
10
7
13
7
9
13
10
7
16
8
6
6
6
19
10
9
3
11
7
6
8
11
11
10
12
15
8
12
14
15
7
10
11
15
14
11
8
10
Sum
47
26
25
14
18
48
11
31
18
59
10
23
26
29
44
67
48
50
11
18
Per thound
Sum
17.57
15.97
25.56
15.97
22.36
19.17
27.16
12.78
9.58
12.78
15.97
12.78
22.36
20.77
15.97
17.57
11.18
9.58
12.79
15.97
20.77
14.38
14.38
15.97
11.18
20.77
11.18
14.38
20.77
15.97
11.18
25.56
12.78
9.58
9.58
9.58
30.35
15.97
14.38
4.79
17.57
11.18
9.58
12.78
17.57
17.57
15.97
19.17
23.96
12.78
19.17
22.36
23.96
11.18
15.97
17.57
23.96
22.36
17.57
12.78
15.97
75.07
persisted healthy. The pathogen of the inoculated fruit was reisolated, cultivated and confirmed as ARAFAT-FG5 isolate
based on fungal morphology. Pathogenicity tests revealed the
presence of P. capitalensis as the pathogen for GBS in
El-Kharga city, New Valley Governorate, Egypt. The
morphological characteristic identification of the pathogen
confirmed with a molecular and phylogenetic approach.
41.53
22.36
DISCUSSION
28.75
This was the first study of the GBS disease in Egypt, with
76.67
P. capitalensis novel isolate ARAFAT-GF5 of guava fruit. The
postharvest diseases caused by fungi handle biodeterioration
of tropical fresh fruits pulp40,41. Postharvest fungal pathogens
cause severe losses on guava during postharvest storage and
marketing. The most aggressive pathogen is P. capitalensis on
guava fruits under environmental conditions in El-Kharga city,
New Valley Governorate-Egypt. The presence GBS disease had
received little attention and not well documented in Egypt,
hence, this study focused more attention to this disease.
During investigation of postharvest fungal diseases, GBS
disease of novel symptoms seen, comprehension of disease
symptoms on plant hosts is important for field identification
by taxonomists as well as a plant pathologist interested in
disease incidence, management and distribution42. After
infection by P. capitalensis the guava mature fruit may
become sunken lesions with concentric development,
variation in color ranging from greenish black to black and
spread on severity affected fruit and pycnidia on fruits is
usually black. The fungus isolated and found using a
combination of morphological and molecular (ITS region
sequences) methods. The morphological characteristics of the
fungus P. capitalensis isolated from guava fruit, helped to
show the fungus on PDA medium16,42,43. In the recent decade,
results of molecular biology have progressed the systematic
classifications of different multiplex groups of plant
pathogenic fungi, including Phyllosticta species that have
helped to facilitate the identification of species and resolution
of species complexʼs44-46. The ITS phylogram supported the
identify of P. capitalensis (or Guignardia mangiferae) as a
common foliar endophyte and pathogen with wide range of
hosts15,47. Most of endophytic fungi belong to the ascomycetes
and asexual fungi48. P. capitalensis was recorded 1543 times
in GenBank to 31 August 2018 (https://www.ncbi.nlm.nih.gov/
Taxonomy/Browser/wwwtax.cgi?id=121624). However, there
are powerful proofs that G. psidii and G. mangiferae are either
the same species10. Moreover, no data available about the
biology and ecology of the G. endophytes except for the
production of chemically novel and pharmaceutically useful
20.76
49.53
28.76
94.25
15.97
36.74
41.52
46.32
70.28
107.02
76.67
79.86
17.57
28.75
34
Asian J. Plant Pathol., 12 (1): 27-37, 2018
secondary metabolism of some isolates in Brazil49. Codon
usage plays a significant role in the efficiency of the gene
expression system. Studies have shown that the presence of
rare codons influences gene expression levels and the
solubility and amount of the expressed protein50,51. Therefore,
synonymous codons not only specify protein sequences and
translation dynamics, but also help determine gene expression
levels52. However, this review emphasizes the significant role
of determining codon usage to gene expression levels.
7.
8.
9.
CONCLUSION
10.
The novel isolate of P. capitalensis which isolated from
guava fruit, found caused GBS as a new disease in Egypt. This
is the first report of P. capitalensis causing GBS in Egypt. The
results presented here may enable enhancements in the
program of integrated disease management.
11.
SIGNIFICANCE STATEMENT
12.
This study discover the Phyllosticta capitalensis that can
be beneficial for identification the causal agent of guava black
spot disease This study will help the researcher to uncover the
critical areas of postharvest diseases that many researchers
were not able to explore. Thus a new theory on fungal
taxonomy may be arrived at.
13.
14.
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