scholarly journals A New Accurate Genotyping HRM Method for Alternaria Species Related to Fruit Rot Diseases of Apple and Pomegranate

2016 ◽  
Vol 4 (3) ◽  
pp. 159-165 ◽  
Author(s):  
Antonios Zambounis ◽  
Aliki Xanthopoulou ◽  
George Karaoglanidis ◽  
Athanasios Tsaftaris ◽  
Panagiotis Madesis
Keyword(s):  
Morphological Characteristics ◽  
Curve Analysis ◽  
Fruit Rot ◽  
Nucleotide Polymorphisms ◽  
Postharvest Diseases ◽  
Accurate Identification ◽  
Complex Species ◽  
Alternaria Species ◽  
Species Specific ◽  
Pomegranate Fruit

Alternaria core rot and Alternaria black heart rot of apple and pomegranate fruit, respectively, are major pre- and postharvest diseases worldwide. However, it is very difficult to differentiate the rot related Alternaria species in the Alternaria complex as they are not always correlate to species-groups based upon morphological characteristics and due to the limited genetic variation these species exhibit among each other. Therefore, it is crucial to exploit novel assays towards the accurate identification and differentiation of these Alternaria species. We have developed, a real-time PCR assay [using species specific primers targeting the endopolygalacturonase (EndoPG) gene] combined with a high-resolution melting (HRM) curve analysis for discrimination of the 14 single nucleotide polymorphisms (SNPs)-based Alternaria haplotypes, which were assigned based on the aligned sequence profiles of 138 Alternaria spp. strains previously isolated from apple and pomegranate rotted fruit. This analysis specifically generated 14 unique HRM curve haplotype profiles among the Alternaria complex species tested. The results showed that HRM curve analysis allows the rapid and adequate identification and genotyping of the three Alternaria species (A. alternata, A. tenuissima and A. arborescens) responsible mostly for the apple and pomegranate fruit rot diseases.

Phylogenetic, Morphological, and Pathogenic Characterization of Alternaria Species Associated with Fruit rot of Mandarin in California

Plant Disease ◽  
2020 ◽  
Author(s):  
Fei Wang ◽  
Seiya Saito ◽  
Themis Michailides ◽  
Chang-Lin Xiao
Keyword(s):  
Phylogenetic Analysis ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Phylogenetic Analyses ◽  
Morphological Characteristics ◽  
Central Valley ◽  
Fruit Rot ◽  
Postharvest Diseases ◽  
Plasma Membrane Atpase ◽  
Alternaria Species

Alternaria rot caused by Alternaria species is one of the major postharvest diseases of mandarin fruit in California. The aims of this study were to identify these Alternaria species using phylogenetic analyses and morphological characteristics and test their pathogenicity to mandarin. Decayed mandarin fruit exhibiting Alternaria rot symptoms were collected from three citrus fruit packinghouses in the Central Valley of California. In total, 177 Alternaria isolates were obtained from decayed fruit and preliminarily separated into three groups representing three species (A. alternata, A. tenuissima and A. arborescens) based on the colony characterization and sporulation patterns. To further identify these isolates, phylogenetic analysis was conducted based on DNA sequences of the second largest subunit of RNA polymerase II (RPB2), plasma membrane ATPase (ATPase) and Calmodulin gene regions in combination with morphological characters. Of the 177 isolates, 124 isolates (70.1%) were identified as A. alternata and 53 isolates (29.9 %) were A. arborescens. The isolates initially identified as A. tenuissima based on the morphological characteristics could not be separated from those of A. alternata in phylogenetic analysis and thus considered A. alternata. Pathogenicity tests showed that both Alternaria species were pathogenic on mandarin fruit at both 5°C and 20°C. Our results indicated that two Alternaria species, A. alternata and A. arborescens, were responsible for Alternaria rot of mandarin fruit in California with A. arborescens causing fruit rot on mandarin being reported for the first time.

scholarly journals Identification of Lasiodiplodia pseudotheobromae Causing Fruit Rot of Citrus in China

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 202
Author(s):  
Jianghua Chen ◽  
Zihang Zhu ◽  
Yanping Fu ◽  
Jiasen Cheng ◽  
Jiatao Xie ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Elongation Factor ◽  
Morphological Characteristics ◽  
Citrus Fruit ◽  
Economic Loss ◽  
Fruit Rot ◽  
Post Inoculation ◽  
Citrus Reticulata ◽  
Postharvest Diseases ◽  
Citrus Reticulata Blanco

Considering the huge economic loss caused by postharvest diseases, the identification and prevention of citrus postharvest diseases is vital to the citrus industry. In 2018, 16 decayed citrus fruit from four citrus varieties—Satsuma mandarin (Citrus unshiu), Ponkan (Citrus reticulata Blanco cv. Ponkan), Nanfeng mandarin (Citrus reticulata cv. nanfengmiju), and Sugar orange (Citrus reticulata Blanco)—showing soft rot and sogginess on their surfaces and covered with white mycelia were collected from storage rooms in seven provinces. The pathogens were isolated and the pathogenicity of the isolates was tested. The fungal strains were identified as Lasiodiplodia pseudotheobromae based on their morphological characteristics and phylogenetic analyses using the internal transcribed spacer regions (ITS), translation elongation factor 1-α gene (TEF), and beta-tubulin (TUB) gene sequences. The strains could infect wounded citrus fruit and cause decay within two days post inoculation, but could not infect unwounded fruit. To our knowledge, this is the first report of citrus fruit decay caused by L. pseudotheobromae in China.

scholarly journals Accurate Identification of Closely Related Mycobacterium tuberculosis Complex Species by High Resolution Tandem Mass Spectrometry

2021 ◽  
Vol 11 ◽  
Author(s):  
Amol O. Bajaj ◽  
Suraj Saraswat ◽  
Juha E. A. Knuuttila ◽  
Joanna Freeke ◽  
J. Benjamin Stielow ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mycobacterium Tuberculosis ◽  
Protein Profiling ◽  
Resolving Power ◽  
Accurate Identification ◽  
Complex Species ◽  
Characterization Methods ◽  
As Species ◽  
Mass Spectrometric Approach ◽  
Species Specific

Rapid and accurate differentiation of Mycobacterium tuberculosis complex (MTBC) species from other mycobacterium is essential for appropriate therapeutic management, timely intervention for infection control and initiation of appropriate health care measures. However, routine clinical characterization methods for Mycobacterium tuberculosis (Mtb) species remain both, time consuming and labor intensive. In the present study, an innovative liquid Chromatography-Mass Spectrometry method for the identification of clinically most relevant Mycobacterium tuberculosis complex species is tested using a model set of mycobacterium strains. The methodology is based on protein profiling of Mycobacterium tuberculosis complex isolates, which are used as markers of differentiation. To test the resolving power, speed, and accuracy of the method, four ATCC type strains and 37 recent clinical isolates of closely related species were analyzed using this new approach. Using different deconvolution algorithms, we detected hundreds of individual protein masses, with a subpopulation of these functioning as species-specific markers. This assay identified 216, 260, 222, and 201 proteoforms for M. tuberculosis ATCC 27294™, M. microti ATCC 19422™, M. africanum ATCC 25420™, and M. bovis ATCC 19210™ respectively. All clinical strains were identified to the correct species with a mean of 95% accuracy. Our study successfully demonstrates applicability of this novel mass spectrometric approach to identify clinically relevant Mycobacterium tuberculosis complex species that are very closely related and difficult to differentiate with currently existing methods. Here, we present the first proof-of-principle study employing a fast mass spectrometry-based method to identify the clinically most prevalent species within the Mycobacterium tuberculosis species complex.

scholarly journals Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2134 ◽  
Author(s):  
Pureum Noh ◽  
Wook Kim ◽  
Sungyu Yang ◽  
Inkyu Park ◽  
Byeong Moon
Keyword(s):  
Herbal Medicines ◽  
Morphological Characteristics ◽  
Pcr Amplification ◽  
Its Region ◽  
Sequencing Analysis ◽  
Accurate Identification ◽  
Republic Of China ◽  
Sequence Characterized Amplified Region ◽  
Angelicae Dahuricae Radix ◽  
Species Specific

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

scholarly journals Monilinia Species Associated with Brown Rot of Cultivated Apple and Pear Fruit in China

Plant Disease ◽  
2016 ◽  
Vol 100 (11) ◽  
pp. 2240-2250 ◽  
Author(s):  
Xiao-Qiong Zhu ◽  
Cheng-Wang Niu ◽  
Xiao-Yu Chen ◽  
Li-Yun Guo
Keyword(s):  
Morphological Characteristics ◽  
Brown Rot ◽  
Conventional Polymerase Chain Reaction ◽  
Polymerase Chain Reaction Method ◽  
Monilinia Fructicola ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
The North ◽  
Monilia Polystroma ◽  
Species Specific

Monilinia isolates were collected from major apple and pear production regions in China from 2004 to 2011 and identified based on their morphological characteristics and three highly conserved loci. The 247 isolates belonged to three species: Monilinia fructicola, Monilia yunnanensis, and Monilia polystroma. M. yunnanensis was the most prevalent (77%), followed by M. polystroma (20%) and Monilinia fructicola (3%). Monilia yunnanensis is primarily distributed in the south, north, and west of China; M. polystroma is limited to the north and east; and Monilinia fructicola was detected only from a few samples from the north and east. Phylogenetic analysis based on internal transcribed spacer, β-tubulin, and laccase (lcc2) genes suggested that Monilia yunnanensis, M. polystroma, and Monilinia fructigena are closely related, and Monilia yunnanensis is more distantly related. We also found that these three species do not show consistent differences in morphological characteristics, including colony morphology, colony expansion rate, conidial characteristics, and the amount of stroma produced in culture. Thus, these three species are more like phylogenetic species in the process of speciation. In addition, a set of species-specific primers based on single-nucleotide polymorphisms and deletions in the lcc2 gene region were designed and a conventional polymerase chain reaction method successfully developed for differentiating Monilinia fructicola, Monilia yunnanensis, M. polystroma, and Monilinia laxa from the other species.

scholarly journals Gene Genealogies Reveal High Nucleotide Diversity and Admixture Haplotypes Within Three Alternaria Species Associated with Tomato and Potato

2020 ◽  
Vol 110 (8) ◽  
pp. 1449-1464
Author(s):  
Tika B. Adhikari ◽  
Thomas Ingram ◽  
Dennis Halterman ◽  
Frank J. Louws
Keyword(s):  
Nucleotide Diversity ◽  
Genetic Relationships ◽  
Haplotype Diversity ◽  
Population Expansion ◽  
Its Region ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Alternaria Species ◽  
Neutrality Tests ◽  
Species Specific

Early blight (EB) and leaf blight are two destructive diseases of tomato in North Carolina (NC), caused by Alternaria linariae and A. alternata, respectively. During the last decade, EB caused by A. solani has increased in potato-producing areas in Wisconsin (WI). We collected 152 isolates of three Alternaria spp. associated with tomato and potato in NC and WI and used the gene genealogical approach to compare the genetic relationships among them. Two nuclear genes: the glyceraldehyde-3-phosphate dehydrogenase (GPDH), RNA polymerase second largest subunit (RPB2), and the rDNA internal transcribed spacer (ITS) region of these isolates were sequenced. Besides, sequences of the GPDH locus from international isolates described in previous studies were included for comparison purposes. A set of single nucleotide polymorphisms was assembled to identify locus-specific and species-specific haplotypes. Nucleotide diversity varied among gene sequences and species analyzed. For example, the estimates of nucleotide diversity and Watterson’s theta were higher in A. alternata than in A. linariae and A. solani. There was little or no polymorphisms in the ITS sequences and thus restricted haplotype placement. The RPB2 sequences were less informative to detect haplotype diversity in A. linariae and A. solani, yet six haplotypes were detected in A. alternata. The GPDH sequences enabled strongly supported phylogenetic inferences with the highest haplotype diversity and belonged to five haplotypes (AaH1 to AaH5), which consisted of only A. alternata from NC. However, 13 haplotypes were identified within and among A. linariae and A. solani sequences. Among them, six (AsAlH1 to AsAlH6) were identical to previously reported haplotypes in global samples and the remaining were new haplotypes. The most divergent haplotypes were AaH1, AsAlH2/AsAlH3, and AsAlH4 and consisted exclusively of A. alternata, A. linariae, and A. solani, respectively. Neutrality tests suggested an excess of mutations and population expansion, and selection may play an important role in nucleotide diversity of Alternaria spp.

scholarly journals A Postharvest Fruit Rot of Apple Caused by Lambertella corni-maris in Washington State

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 201-206 ◽  
Author(s):  
M. S. Wiseman ◽  
F. M. Dugan ◽  
Y. K. Kim ◽  
C. L. Xiao
Keyword(s):  
Ribosomal Subunit ◽  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
The United States ◽  
Skin Lesions ◽  
Apple Fruit ◽  
Washington State ◽  
Fruit Rot ◽  
Postharvest Diseases ◽  
Diseased Fruit

During surveys for postharvest diseases of apple conducted in Washington State, an unknown fruit rot was observed on stored apple fruit collected from commercial fruit packinghouses. This disease was present in 66 of the 179 grower lots sampled, accounting for an average 1 to 3% of the total decayed fruit sampled. The disease appeared to originate from infection of wounds on the fruit skin. Lesions were brown and decayed tissues were spongy. A Lambertella sp. was consistently isolated from the decayed fruit. Sequences of the fungus and those of Lambertella corni-maris in GenBank differed by 0 to 4 bp across the combined small ribosomal subunit + internal transcribed spacer + large ribosomal subunit regions with a maximum identity ranging from 99 to 100%. The fungus grew at 0 to 20°C and formed apothecia on artificial media after 8 to 24 weeks. On potato dextrose agar under a 12-h photoperiod, apothecial dimensions were variable, ranging from 1 to 6 mm in diameter with stipes of 1 to 4 by 0.5 mm. Asci were 76 to 125 by 3.5 to 5.5 μm, inoperculate, eight-spored, clavate, and narrowed at the base. Ascospores were aseptate, 7 to 10 by 2.5 to 4.5 μm, uniseriate to biseriate, and orange-brown at maturity in the ascus. Colony characteristics included little or no aerial mycelium, dark-yellow to gray-black mycelium, gray-black pseudosclerotia, and yellow pigmentation in the agar. Morphological characteristics of the fungus overlapped with the description of L. corni-maris. ‘Fuji’ apple fruit that were wounded, inoculated with representative isolates, and incubated at 0°C yielded the same symptoms as seen in packinghouses, and the fungus was reisolated from the diseased fruit. This is the first report of a fruit rot in stored apple caused by L. corni-maris in the United States. We propose Lambertella rot as the name of this disease.

scholarly journals First Report of Anthracnose Fruit Rot Caused by Colletotrichum acutatum on Strawberry in Denmark

Plant Disease ◽  
2005 ◽  
Vol 89 (4) ◽  
pp. 432-432 ◽  
Author(s):  
T. Sundelin ◽  
M. Schiller ◽  
M. Lübeck ◽  
D. F. Jensen ◽  
K. Paaske ◽  
...  
Keyword(s):  
Morphological Characteristics ◽  
Aerial Mycelium ◽  
Fruit Rot ◽  
Colletotrichum Acutatum ◽  
Enzyme Linked Immunosorbent Assay ◽  
First Report ◽  
Small Fruit ◽  
Quarantine Pathogen ◽  
Species Specific ◽  
Pointed End

Strawberry (Fragaria × ananassa) is the most important small fruit crop in Denmark. The quarantine pathogen Colletotrichum acutatum was detected for the first time in June 2000 in Denmark in a production field on the island of Falster. Strawberry plants of cv. Kimberly showed typical symptoms of anthracnose fruit rot. On mature fruits, brown-to-black lesions with spore masses that were orange to salmon in color were observed. Mummified berries were also observed. The fungus was isolated and identified on the basis of morphological characteristics, and identification was confirmed using enzyme-linked immunosorbent assay at the Central Science Laboratory, York, U.K. Species-specific polymerase chain reaction with the C. acutatum-specific primer pairs acut1/col2 (1) and CaInt2/ITS4 (3) also supported the identification. Additionally, the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal DNA were sequenced in both directions (GenBank Accession No. AY818361). Homology searches with this sequence using BLAST also confirmed the identity. Colonies grown on potato dextrose agar developed white-to-grey aerial mycelium with salmon-colored spore masses, and were beige to black on the reverse side. Conidia were 11.3 (7.3 to 16.6) μm × 3.9 (2.5 to 5.2) μm, hyaline, cylindrical with at least one pointed end, and aseptate. Mycelial growth rate was 8.4 mm per day at 25°C which is similar to earlier reports (2). Spray-inoculated (106 conidia per ml) strawberry fruits cv. Elsanta developed brown, sunken, irregular lesions with salmon-colored acervuli after 2 to 5 days at 25°C. Koch's postulates were fulfilled since the reisolated fungus from these lesions developed the same morphological characteristics as described above. To our knowledge, this is the first report of C. acutatum in Denmark. References: (1) P. V. Martinez-Culebras et al. J. Phytopathol. 151:135, 2003. (2) B. J. Smith et al. Plant Dis. 74:69, 1990. (3) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996.

Diversity and cross-infection potential of Colletotrichum causing fruit rots in mixed-fruit orchards in Kentucky

Plant Disease ◽  
2020 ◽  
Author(s):  
Madison Julia McCulloch ◽  
Shanice Edwards ◽  
Harrison Inocencio ◽  
Franklin Machado ◽  
Etta Nuckles ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Single Gene ◽  
Cross Infection ◽  
Fruit Rot ◽  
Gene Trees ◽  
Accurate Identification ◽  
Factors Affecting ◽  
Colletotrichum Species ◽  
Species Specific ◽  
Fruit Orchards

Fungi in the genus Colletotrichum cause apple, blueberry, and strawberry fruit rots, which can result in significant losses. Accurate identification is important because species differ in aggressiveness, fungicide sensitivity, and other factors affecting management. Multiple Colletotrichum species can cause similar symptoms on the same host, while more than one fruit type can be infected by a single Colletotrichum species. Mixed-fruit orchards may facilitate cross-infection, with significant management implications. Colletotrichum isolates from small fruits in Kentucky orchards were characterized and compared with apple isolates by using a combination of morphotyping, sequencing of voucher loci and whole genomes, and cross-inoculation assays. Seven morphotypes representing two species complexes (C. acutatum and C. gloeosporioides) were identified. Morphotypes corresponded with phylogenetic species C. fioriniae, C. fructicola, C. nymphaeae, and C. siamense, identified by TUB2 and GAPDH barcodes. Phylogenetic trees built from nine single gene sequences matched barcoding results with one exception, later determined to belong to an undescribed species. Comparison of single gene trees with representative whole genome sequences revealed that CHS and ApMat were the most informative for diagnosis of fruit rot species and individual morphotypes within the C. acutatum or C. gloeosporioides complexes, respectively. All blueberry isolates belonged to C. fioriniae, and most strawberry isolates were C. nymphaeae, with a few C. siamense and C. fioriniae also recovered. All three species cause fruit rot on apples in Kentucky. Cross-inoculation assays on detached apple, blueberry, and strawberry fruits showed that all species were pathogenic on all three hosts, but with species-specific differences in aggressiveness.

scholarly journals A New Postharvest Fruit Rot in Apple and Pear Caused by Phacidium lacerum

Plant Disease ◽  
2016 ◽  
Vol 100 (1) ◽  
pp. 32-39 ◽  
Author(s):  
M. S. Wiseman ◽  
Y. K. Kim ◽  
F. M. Dugan ◽  
J. D. Rogers ◽  
C. L. Xiao
Keyword(s):  
Phylogenetic Analysis ◽  
Ribosomal Subunit ◽  
Morphological Characteristics ◽  
Apple Fruit ◽  
Fruit Rot ◽  
Morphological Data ◽  
Postharvest Diseases ◽  
Pear Fruit ◽  
Diseased Fruit ◽  
Molecular Phylogenetic

During surveys for postharvest diseases of apple and pear, an unknown postharvest fruit rot was observed in Washington State. The disease appeared to originate from infection of the stem and calyx tissue of the fruit or wounds on the fruit. An unknown pycnidial fungus was consistently isolated from the decayed fruit. Isolates from apple and pear were characterized and identified by molecular phylogenetic analysis and morphology. Pathogenicity of representative isolates on apple and pear fruit was tested under laboratory or field conditions. A BLAST search in GenBank showed that isolates differed from Phacidium lacerum and its synonym, Ceuthospora pinastri, by only 0 to 4 bp in sequences within part of the combined large ribosomal subunit + internal transcribed spacer + small ribosomal subunit regions. The phylogenetic analysis confirmed the taxonomic placement of the unknown fungus in the genus Phacidium, with the highest match being C. pinastri (formerly anamorphic P. lacerum) and with closely related taxa from GenBank forming congeneric clades. The fungus grew at 0 to 30°C and formed unilocular to multilocular pycnidial conidiomata on artificial media after approximately 5 to 7 days at room temperature. On potato dextrose agar incubated for a 12-h photoperiod, semi-immersed globose to subglobose pycnidial conidiomata were 250 to 1,000 μm in diameter (mean = 350), with 1 to 3 nonpapillate to slightly papillate ostioles and a buff conidial matrix. Conidia produced on phialides were 8 to 13 by 1.5 to 2.5 μm, hyaline, aseptate, cylindrical, with an abruptly tapered, typically slightly protuberant base, 2 to 3 guttules, and sometimes with a mucilaginous, flexuous, unbranched appendage which is attached to the apex of the conidium and disappears with age. Conidiogenous cells were flask shaped and 6 to 15 ×1.5 to 3 μm. Colony characteristics included felt-like aerial white mycelium, gray olivaceous at the center becoming greenish to colorless toward the margin, in concentric rings, with pycnidia forming in 5 to 7 days originating from the center of the plate. Morphological characteristics of the fungus had the greatest conformity with the description for C. pinastri. Based on molecular and morphological data, the fungus is identified as P. lacerum. ‘Fuji’ apple fruit and ‘d’Anjou’ pear fruit that were wounded, inoculated with representative isolates, and incubated at 0°C yielded the same symptoms as seen on decayed fruit collected from commercial fruit packinghouses. Stem-end rot, calyx-end rot, and wound-associated rot developed on fruit inoculated in the orchard after 3 months of cold storage. The fungus was reisolated from the diseased fruit. This is the first report of a fruit rot in apple and pear caused by P. lacerum. We propose Phacidium rot as the name of this disease.

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