Colletotrichum Species Related To “Nam Dork Mai See Tong” Mango In Central Thailand And Risk Detection of Carbendazim-Resistant Strains

Abstract Nam Dork Mai See Tong mango is a deluxe commercial fruit in Thailand, however anthracnose disease becoming a major problem affecting market-driven. Fungicide treatment is the main component in the mango anthracnose management, notwithstanding the appearance of fungal-resistance has been become a factor of fungicide limition and can affect to increasingly higher costs. Thirty-two isolates of the Colletotrichum species complex were obtained from anthracnose-diseased mango cv. Nam Dork Mai See Tong in 3 provinces of Thailand. Fungal identification based on morphological and molecular markers was investigated. All isolates were divided into 3 species: C. asianum , C. gloeosporioides and C. siamense . Pathogenicity tests revealed that all 3 species caused symptoms on artificially unwounded mango fruits and leaves. Only C. gloeosporioides have previously been reported on mango, while C. asianum and C. siamense were first reports associated with mango in Thailand. The responsiveness of Colletotrichum isolates to carbendazim was evaluated using an MIC assay. Nine isolates of C. asianum were highly resistant, while 5 and 8 isolates of C. gloeosporioides were resistant (R) and highly resistant (HR), respectively. Moreover, all isolates of C. siamense were HR. Mutations were detected by PCR of a partial sequence of the β-tubulin gene. The sequence of β-tubulin gene in HR strains showed a single nucleotide transversion of adenine to cytosine, resulting in a substitution at codon 198. However, R strains were found to have only an amino acid change at codon 200. Additionally, PCR-RFLP was applied as a rapid technique to detect carbendazim resistance. The results demonstrated that only the Bsh 1236I restriction enzyme generated two bands (200 and 300 bp) in the highly resistant strain, but these bands were absent in the sensitive (S) and R strains. Hence, PCR-RFLP technique showed the potential to specifically detect benzimidazole fungicide resistance in 3 species of Colletotrichum. Moreover, this technique is accessible and feasible for laboratory assessment

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A single nucleotide polymorphism in the β-tubulin gene distinguishing two genotypes of Erysiphe necator expressing different symptoms on grapevine

Plant Pathology ◽

10.1111/j.1365-3059.2006.01390.x ◽

2006 ◽

Vol 55 (4) ◽

pp. 505-512 ◽

Cited By ~ 19

Author(s):

L. Amrani ◽

M.-F. Corio-Costet

Keyword(s):

Single Nucleotide Polymorphism ◽

Nucleotide Polymorphism ◽

Tubulin Gene ◽

Single Nucleotide ◽

Erysiphe Necator ◽

Β Tubulin

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First Report of Lasidiplodia theobromae and Cryptovalsa ampelina Associated with Grapevine Decline from Castilla y León, Spain

Plant Disease ◽

10.1094/pdis-93-5-0545c ◽

2009 ◽

Vol 93 (5) ◽

pp. 545-545 ◽

Cited By ~ 4

Author(s):

M. T. Martin ◽

L. Martin ◽

M. T. de-Francisco ◽

R. Cobos

Keyword(s):

Dna Sequences ◽

Morphological Characteristics ◽

Tubulin Gene ◽

Current Season ◽

First Report ◽

Grapevine Decline ◽

Eutypa Dieback ◽

Castilla Y Leon ◽

Pathogenicity Tests ◽

Β Tubulin

Symptoms of grapevine decline were surveyed. Samples from mature vines exhibiting external symptoms of Eutypa dieback and Esca were collected, as were young plants with and without external symptoms, and fungal isolations were performed. In 2007, 3-year-old grapevines (cv. Tempranillo grafted onto 110R rootstock) with low vigor, reduced foliage, and vascular streaking in the wood were observed. Small pieces of discolored wood were placed onto malt extract agar supplemented with 0.25 g/liter of chloramphenicol, incubated at 25°C, and resulting colonies were transferred to potato dextrose agar (PDA). Isolates were characterized by abundant aerial and fast-growing mycelium covering the plate surface after 3 days, mycelium became dark green. Pycnidia contained thick-walled, aseptate conidia 15 to 35 × 10 to 15 μm. Lasidiplodia theobromae was identified based on morphological characteristics (3) and confirmed by banding patterns obtained after the digestion of the 1,200-bp amplicon generated with ITS1 and NL4 primers (2) using restriction endonucleases (2). Single-spore cultures were generated and DNA sequences of the rDNA internal transcribed spacer region, partial sequence of the 5′ end of the β-tubulin gene, and a fragment of the elongation factor further confirmed the identification and revealed genetic similarity with other isolates of L. theobromae. A sequence of each fragment was deposited in GenBank with Accession Nos. EU600925, EU597297, and EU597298, respectively. Pathogenicity tests were conducted on four replicate rootstocks (110R) and 15 canes of current-season growth (cv. Tempranillo). Plants were inoculated with an agar plug containing L. theobromae; controls were treated with agar only. Grapevines were maintained in a greenhouse at 20 to 25°C. After 3 months, L. theobromae was reisolated from internal vascular lesions in 100 and 66% of inoculated rootstocks and canes, respectively. Control plants were asymptomatic and L. theobromae was not recovered. Using the same methodology, a fungus identified based on morphological characteristics in culture as Cryptovalsa ampelina (1) was isolated from grapevines (cv. Tempranillo) planted in 1987. Cultures in PDA were white to creamy white and cottony with diffuse margins. Colonies covered the 90-mm-diameter petri dish surface in 5 days. Conidia were 20 to 23 × 1 to 1.5 μm, unicellular, hyaline, and filiform. PCR amplifications of the DNA extracts of C. ampelina with Camp-1 and Camp-2R primers gave a characteristic DNA fragment of 300 bp (3) and DNA sequences of the ITS4-ITS5 amplicons (GenBank Accession No. EU597296) confirmed the identification. For the first time, the 5′ end of the β-tubulin gene was sequenced and deposited in GenBank (Accession No. EU600926). Pathogenicity tests were conducted as described above for L. theobromae. Both pathogens were examined in the same experiment. C. ampelina was reisolated from internal brown streaking lesions in 25% of the rootstocks and 33% of the canes. Control plants exhibited no symptoms. L. theobromae appeared to be a more aggressive pathogen than C. ampelina on grapevine with more internal brown streaking and greater recovery of pathogen from inoculated samples. To our knowledge, this is the first report of L. theobromae and C. ampelina causing grapevine decline in Castilla y León. References: (1) J. Luque et al. Phytopathol. Mediterr. 45:S101, 2006. (2) M. T. Martin and R. Cobos. Phytopathol. Mediterr. 46:18, 2007. (3) D. Pavlic et al. Stud. Mycol. 50:313, 2004.

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A PCR-RFLP marker distinguishing Ophiostoma clavigerum from morphologically similar Leptographium species associated with bark beetles

Canadian Journal of Botany ◽

10.1139/b03-101 ◽

2003 ◽

Vol 81 (11) ◽

pp. 1104-1112 ◽

Cited By ~ 21

Author(s):

Sangwon Lee ◽

Jae-Jin Kim ◽

Simon Fung ◽

Colette Breuil

Keyword(s):

Bark Beetles ◽

Fragment Length Polymorphism ◽

Morphological Characteristics ◽

Tubulin Gene ◽

Reliable Identification ◽

Dendroctonus Jeffreyi ◽

Pcr Rflp ◽

Specific Restriction ◽

Restriction Fragment Length ◽

Β Tubulin

Ophiostoma clavigerum, carried by Dendroctonus ponderosae and Dendroctonus jeffreyi, has morphological characteristics that are similar to other Ophiostoma and Leptographium species. The partial β-tubulin gene of 45 strains belonging to seven species was amplified by PCR and digested by the restriction enzyme HinfI. The specific restriction fragment length polymorphism obtained for O. clavigerum provided the means for its reliable identification. We are also reporting that O. clavigerum ascocarps have short necks; this fact has not been shown previously.Key words: Ophiostoma clavigerum, Leptographium, β-tubulin gene, RFLP, bark beetle.

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PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus

Journal of Microbiological Methods ◽

10.1016/j.mimet.2015.08.007 ◽

2015 ◽

Vol 117 ◽

pp. 144-147 ◽

Cited By ~ 12

Author(s):

Tuba Nasri ◽

Mohammad Taghi Hedayati ◽

Mahdi Abastabar ◽

Alessandro C. Pasqualotto ◽

Mojtaba Taghizadeh Armaki ◽

...

Keyword(s):

Rapid Identification ◽

Tubulin Gene ◽

Important Species ◽

Pcr Rflp ◽

Β Tubulin

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First Report of Soft Rot Caused by Aspergillus niger sensu lato on Mother-in-law’s Tongue in China

Plant Disease ◽

10.1094/pdis-03-20-0678-pdn ◽

2020 ◽

Author(s):

Siliang Huang ◽

Xue Ling Zheng ◽

Di Yang ◽

Jinping An ◽

Lu Wang ◽

...

Keyword(s):

Aspergillus Niger ◽

Phylogenetic Tree ◽

Soft Rot ◽

Leaf Margin ◽

Strict Sense ◽

Tubulin Gene ◽

First Report ◽

Rdna Its ◽

Pathogenicity Tests ◽

Β Tubulin

“Mother-in-law’s tongue” (MLT) [Dracaena trifasciata (Prain) Mabb. (syn. Sansevieria trifasciata Prain.)], also known as “Saint George's sword", “snake plant”, “tiger's tail orchid", etc., is an evergreen perennial ornamental plant grown worldwide. In September 2016, severe soft rot occurred on the leaves of MLT in a flower market in Nanyang city (32º56´N, 112º32´E), Henan province, China with 25% disease incidence (n=100). Water-soaked spots initially appeared on the leaf margin, enlarged rapidly, and became soft rot under excessively watered conditions. A blight zone was visualized at the margin of a developing lesion in backlit conditions. Severely affected leaves folded down from the lesions. Lesion expansion stopped under dry conditions. Grey or dark brown mycelia were frequently seen on the lesions. Tissue pieces (4×4 mm2) at the margin of lesions were cut out, treated with 75% ethanol for 10 s, followed by 70 s in 0.1% HgCl2, rinsed eight times with sterile water, and plated on potato dextrose agar (PDA) medium. Pure Aspergillus cultures were obtained from the surface-disinfected lesions after 4 days of incubation at 26°C. Two single-spore-derived isolates (An-1 and An-2) were randomly selected and used for morphological and molecular identifications as well as pathogenicity tests. The isolates formed round dark brown colonies with a large number of conidia after 5 days of incubation on PDA at 28°C. Conidia were subsphaeroidal or oblate, unicellular, dark brown, 2.9-4.2(3.5) × 1.9-3.4(2.7) μm in size (n=100), developed from a two-series of strigmata born on a conidial head, with ridge or stab-shaped prominences. For pathogenicity tests, the two isolates were separately grown on oatmeal agar and incubated at 30°C for 6 days. Mycelial plugs (5 mm diam.) were inoculated on the scalpel incision X-shaped wounds of surface-disinfected leaves of MLT. The inoculated leaves were kept on a two-layer of wet napkin in a steel basin covered with a plastic film. Soft rot symptoms developed from the wounds 6 days after incubation, similar to those observed on naturally affected leaves. The An-1- and An-2-inoculated unwounded leaves remained symptomless during the pathogenicity tests. Fungal cultures with the same phenotypes as the inocula were consistently reisolated from the lesions of the leaves inoculated by each of the two isolates, verifying the isolates as the causal agent of the disease based on Koch’s postulates. Both β-tubulin gene and rDNA-ITS (internal transcribed spacer) sequences of the two isolates were separately amplified and sequenced. Sequences were submitted to GenBank with accession numbers MN259522 and MN259523 for the β-tubulin gene sequences, and accession numbers MN227322 and MN227324 for the rDNA-ITS sequences of An-1 and An-2, respectively. Both An-1 and An-2 were clustered with members of Aspergillus niger van Tieghem in the phylogenetic tree of rDNA-ITS, clearly separated from other Aspergillus spp. In the phylogenetic tree of β-tublin gene, both An-1 and An-2 formed a subclade inside a large clade consisting of members of A. niger in strict sense. Based on the molecular and morphological results, both An-1 and An-2 clearly separated from other Aspergillus spp. and can be considered as A. niger sensu lato. Foliar diseases of MLT are known to be caused by a few fungal species such as Chaetomella spp. (Li et al. 2014) and Colletotrichum sansevieriae (Nakamura et al. 2006). This is the first report of A. niger sensu lato causing soft rot on MLT in China.

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Phylogenetic Analyses of Phytopathogenic Isolates of Verticillium spp.

Phytopathology ◽

10.1094/phyto-96-0582 ◽

2006 ◽

Vol 96 (6) ◽

pp. 582-592 ◽

Cited By ~ 57

Author(s):

Qing-Ming Qin ◽

Gary E. Vallad ◽

Bo Ming Wu ◽

Krishna V. Subbarao

Keyword(s):

Genetic Relationships ◽

Phylogenetic Analyses ◽

Intergenic Spacer ◽

Tubulin Gene ◽

Sequence Comparisons ◽

Distance Method ◽

Pathogenicity Tests ◽

Sequence Similarities ◽

Β Tubulin ◽

Potential Origin

To better understand the genetic relationships between Verticillium dahliae isolates from lettuce and other phytopathogenic Verticillium spp. isolates from various hosts and geographic locations, the complete intergenic spacer (IGS) region of the nuclear ribosomal RNA gene (rDNA) and the β-tubulin gene were amplified and sequenced. The sequences of the complete IGS region and the β-tubulin gene were used alone and in combination to infer genetic relationships among different isolates of Verticillium with the maximum-likelihood distance method. Phylogenetic analyses set sequences into four distinct groups comprising isolates of V. albo-atrum, V. tricorpus, and V. dahliae from cruciferous and noncruciferous hosts. Within the four Verticillium groups, isolates of V. dahliae from cruciferous hosts displayed the closest affinity to V. dahliae from noncruciferous hosts. Isolates of V. dahliae from noncruciferous hosts could be further divided into four subgroups based on sequence similarities within the IGS region. Cross-pathogenicity tests demonstrated that most Verticillium isolates were as virulent on other hosts as on their hosts of origin. A phenogram based on the cross pathogenicity of individual isolates resembled those derived from the IGS and β-tubulin sequence comparisons. On the basis of the data presented, the potential origin of some isolates of V. dahliae pathogenic on lettuce is proposed.

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The profile of codon 200 β-tubulin gene of Ascaris lumbricoides L. and Trichuris trichiura L. from infected people in Nangapanda Sub-district, East Nusa Tenggara

Medical Journal of Indonesia ◽

10.13181/mji.v27i4.1935 ◽

2018 ◽

Vol 27 (4) ◽

pp. 304-9

Author(s):

Yuliana Yuliana ◽

Yenny Djuardi ◽

Taniawati Supali

Keyword(s):

Reference Sequence ◽

Intestinal Helminth ◽

Nucleotide Polymorphism ◽

Tubulin Gene ◽

Single Nucleotide ◽

Infected People ◽

The People ◽

Polymerase Chain ◽

Β Tubulin

Background: The mass administration of anthelmintic such as albendazole is one of the strategies for eliminating soil-transmitted helminth (STH) infection. The widespread and long-term use of anthelmintics can cause resistance. The research on animals shows that factor that the single-nucleotide polymorphism (SNP) codon 200 β-tubulin gene of the worms is one of the factors that can cause the decreased efficacy of anthelmintics. This study aimed to determine the bases of codon 200 in A. lumbricoides and T. trichiura, which infect the people in Nangapanda, East Nusa Tenggara.Methods: The worm samples were obtained from the intestinal helminth-infected patients from Nangapanda Sub-district. The DNA from the worm tissues were isolated, amplificated by polymerase chain reaction (PCR), and sequenced. The sequencing results were aligned to the reference sequence to obtain the codon bases in the 200 β-tubulin gene.Results: TTC constitute the codon bases in the 200 β-tubulin gene found in two A. lumbricoides and one T. trichiura.Conclusion: The SNP codon 200 β-tubulin gene was absent in A. lumbricoides or T. trichiura worms that were examined in this study.

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