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. 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