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Plant Disease ◽  
2022 ◽  
Author(s):  
Kecheng Xu ◽  
Ruiqi Zhang ◽  
Xue Li ◽  
Jinglong Zhang ◽  
Hang Zhao ◽  
...  

Eucalyptus species are widely planted in the tropics and subtropics, and eucalyptus is among the most important cash crop in Southern China. One of the most important diseases on eucalyptus is Ceratocystis wilt, caused by the fungus Ceratocystis fimbriata Ellis & Halst., and the genus name Chalaropsis has been proposed for anamorphs of Ceratocystis species (de Beer et al. 2014). During April 2018, severely infected Eucalyptus robusta trees were observed in Kunming, Yunnan Province, China. Symptomatic trees initially exhibited yellowing and wilting of foliage on individual branches, then spread to the whole canopy, sometimes followed by death of the whole tree. Reddish-brown to dark-brown discoloration in the woody xylem of affected trees, sometimes a grayish white layer of fungal growth may be seen. The disease was observed on 16% of trees surveyed. The base of trunks with typical symptoms were collected, then the discolored xylem tissues were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) solution for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA) medium, and incubated at 25°C. After 6 days, a fungus was consistently observed growing from the tissue. Three isolates were obtained. In culture, colonies reaching 54mm diam within 15 days, mycelium initially white, then becoming celadon. Endoconidia unicellular, smooth, cylindrical, straight, biguttulate, 11.21 - 32.26 × 4.12 - 5.25 μm. Phialides produced on short, septate, aerial hyphae, lageniform and chain of phialoconidia (3.62 - 5.89 × 31.39 - 65.76 μm) were also observed. Chlamydospores (11.45 - 14.26 × 10.06 - 12.22 μm) were single, dark, thick-walled. Morphological characteristics of the fungus were consistent with the description of Chalaropsis thielavioides (Paulin-Mahady et al. 2002). The two of three isolates were used for molecular identification and genomic DNA was extracted from isolates (EKY2-2-1, EKY2-2-2) using the chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions MW242701, MW242702) showed that the isolates were 99% - 100% homologous to isolates of C. thielavioides from Hevea rubber, Monstera deliciosa L. and ants in China and Rosa sp. in Australia (GenBank accessions KT963172, KJ511482, KT963173 and KX954598) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis were performed using MEGA 6.06 based on ITS sequences (Fig 1), the evolutionary distances were computed using the Maximum Composite Likelihood method. Analyses showed that both isolates (EKY2-2-1, EKY2-2-2) located on the same clade with all C. thielavioides, and clustered with the C. thielavioides strains with high bootstrap support (97% - 100%). Therefore, the fungus was identified as C. thielavioides based on morphology and molecular evidences. Pathogenicity of C. thielavioides was tested by inoculation of six one-year-old pot grown Eucalyptus citriodora seedlings. The sterilized soil of six seedlings was inoculated by drenching with 20 ml spore suspension (2.0 × 106 spores / ml). Control plants were inoculated with 20 ml of sterile distilled water. The seedlings were kept in a controlled greenhouse at 25°C and watered weekly. After one month incubation, all the isolates produced wilt symptoms, whereas control trees showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. thielavioides according to the methods described above, and no fungal growth was observed in the controls, thus satisfying Koch's postulates. Although wilt and canker caused by Ceratocystis fimbriata on eucalyptus have been previously reported in Brazil, Uruguay, Uganda, China and Pakistan (Ferreira et al. 1999; Li et al. 2014; Alam et al. 2017), eucalyptus wilt caused by C. thielavioides has not been reported anywhere. Also, wilt of rubber tree and postharvest rot on carrot caused by C. thielavioides have been reported (Li et al. 2021; Xu et al. 2020). To our knowledge, this is the first report of eucalyptus wilt caused by C. thielavioides in China.


Plant Disease ◽  
2022 ◽  
Author(s):  
Haoyu Wang ◽  
Disen Feng ◽  
Lingqiao Chen ◽  
Junhua Yang ◽  
Xichun Wang ◽  
...  

Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O’Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch’s postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.


Plant Disease ◽  
2022 ◽  
Author(s):  
Kecheng Xu ◽  
Ruiqi Zhang ◽  
Jie Li ◽  
Xue Li ◽  
Jing Yang ◽  
...  

The rubber tree (Hevea brasiliensis) is an important economic resource for the rubber and latex industry. During November 2013 and June 2016, rubber trees showing typical wilt symptoms were found in Mengla, Xishuangbannan, Yunnan, China (N 21° 28', E 101° 33'). Symptomatic trees initially exhibited wilting of foliage on individual branches, then spread to the whole canopy, finally followed by death of the whole tree. Dark-blue to black discoloration was observed in the inner bark and affected xylem, a grayish layer of fungal growth and sporulation occasionally. The disease was detected on 20% of trees surveyed. The diseased tissues of three rubber trees were surface disinfected with 75% ethanol for 30 s and 0.1% mercuric chloride (HgCl2) for 2 min, rinsed three times with sterile distilled water, plated onto potato dextrose agar (PDA), and incubated at 25°C. After 7 days, a fungus was consistently observed growing from the tissue. Three single-spore isolates were obtained. In culture, colonies reaching 69 mm diam within 10 days, mycelium was initially white, then becoming celadon. After 5 days of perithecium formation, observed perithecia were black, globose (173.1 - 237.9 × 175.6 - 217.2 μm) and showed a long black neck (507.3 - 794.1 μm). Ascospore with outer cell wall forming a brim, hat-shaped at the tips of ostiolar hyphae (3.43 × 5.63 μm). Cylindrical endoconidia (10.5 - 39.7 × 3.5 - 6.6 μm) were hyaline. Chain of barrel-shaped conidia (7.2 - 9.5 × 4.1 - 6.2 μm) was found. Aleuroconidia were ovoid or obpyriform, and smooth (10.2 - 14.1 × 8.4 - 10.6 μm). Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata (Engelbrecht and Harrington 2005). The genomic DNA was extracted from isolates (XJm10-2-5, XJm8-2-5, XJm4) using the Chelex-100 method (Xu et al. 2020). The ITS region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of ITS sequence data (GenBank accessions KJ511488, KJ511485, KT963149) showed that the isolates were 100% homologous to those of the isolates on Punica granatum and Colocasia esculenta from China (GenBank accessions KT963152, MH793673) by BLAST analysis. Neighbor-joining phylogenetic analyse were performed using MEGA 6.06 based on ITS sequences (Fig. 1). Analyses showed that all isolates located on the same clade with all C. fimbriata with a high bootstrap support. Therefore, the fungus was identified as C. fimbriata based on morphology and molecular evidences. Pathogenicity of C. fimbriata isolated from this study was tested by inoculation of three one-year-old pot-grown (3L) seedlings of rubber tree. The soil of three seedlings was inoculated by drenching with 30 ml spore suspension (2.0 × 106 spores / ml). Three control plants were inoculated with 30 ml of sterile distilled water. The experiment was repeated three times. The plants were kept in a controlled greenhouse at 25°C and watered weekly. After the inoculation for one month, all the isolates produced typical wilt symptoms, while control plants showed no symptoms. The original fungus was successfully re-isolated from inoculated trees and identified as C. fimbriata according to the methods described above. The pathogenicity assay showed that C. fimbriata was pathogenic to rubber trees. C. fimbriata was first reported on rubber tree in Brazil (Albuquerque et al. 1972; Silveira et al. 1985). To the best of our knowledge, this is the first report of C. fimbriata causing wilt of rubber tree in China. This finding contributes to understanding the diversity of this pathogen, and it appears to be a significant threat to rubber trees in its ecosystem.


2022 ◽  
Vol 82 ◽  
Author(s):  
I. Hussain ◽  
S. A. Mehmood ◽  
S. Ahmed ◽  
M. Salim ◽  
A. Hussain ◽  
...  

Abstract Extensive field surveys were carried out to explore the distribution of Leisler’s Bat Nyctalus leisleri (Kuhl, 1819) in selected area of FATA regions, Pakistan. Specimens of Leisler’s Bat Nyctalus leisleri (Kuhl, 1819) (n5) were collected from Kurram Agency (Shublan) (N33.8229788 E70.1634414) at elevation 1427m and Khyber Agency (Landi Kotel) (N34.0909899 E71.1457517) at elevation 1091m for two years survey extending from May 2013 through August 2015. The mean head and body length, hind foot length, ear length and tail length the Nyctalus leisleri specimens captured from the study area was 65.08 ± 1.58 mm, 44.06 ± 0.52 mm, 8.38 ± 0.60 mm, 13.20 ± 0.99 mm and 39.46 ± 1.46 mm, respectively. For molecular analysis the sequences of COI gene were obtained and analyzed. The mean intraspecific divergences of Nyctalus leisleri was 0.04%. The mean interspecific divergences of Nyctalus noctula and Nyctalus leisleri was 0.2%. The mean concentration of each nucleotides was A = (26.3%), T = (32.8%), G = (15.9%) and C = (25.0%). The mean A+T contents were 59.2%and C+G were 40.9%. In the phylogenetic tree Nyctalus leisleri and Nyctalus noctula clustered with significant bootstrap support value.


Author(s):  
Kirk Broders ◽  
Gloria Iriarte ◽  
Gary Bergstrom ◽  
Emmanuel Byamukama ◽  
Martin Chilvers ◽  
...  

The genus Phyllachora contains numerous obligate fungal parasites that produce raised, melanized structures called stromata on their plant hosts referred to as tar spot. Members of this genus are known to infect many grass species but generally do not cause significant damage or defoliation, with the exception of P. maydis which has emerged as an important pathogen of maize throughout the Americas, but the origin of this pathogen remains unknown. To date, species designations for Phyllachora have been based on host associations and morphology, and most species are assumed to be host specific. We assessed the sequence diversity of 186 single stroma isolates collected from 16 hosts representing 15 countries. Samples included both herbarium and contemporary strains that covered a temporal range from 1905-2019. These 186 isolates were grouped into 5 distinct species with strong bootstrap support. We found three closely related, but genetically distinct groups of Phyllachora are capable of infecting maize in the United States, we refer to these as the P. maydis species complex. Based on herbarium species, we hypothesize that these three groups in the P. maydis species complex originated from Central America, Mexico and the Caribbean. Although two of these groups were only found on maize, the third and largest group contained contemporary strains found on maize and other grass hosts, as well as herbarium specimens from maize and other grasses that include 10 species of Phyllachora. The herbarium specimens were identified based on morphology and host association, but our sequence data indicates some Phyllachora species are capable of infecting a broad range of host species and there may be significant synonymy in the Phyllachora genus and additional work on species delineation and host specificity should be considered.


Plant Disease ◽  
2021 ◽  
Author(s):  
Shucheng Li ◽  
Liuhua Xiao ◽  
Fan Wu ◽  
Yinbao Wang ◽  
Mingshu Jia ◽  
...  

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.


Phytotaxa ◽  
2021 ◽  
Vol 527 (3) ◽  
pp. 177-190
Author(s):  
ZIN HNIN HTET ◽  
AUSANA MAPOOK ◽  
YUSUFJON GAFFOROV ◽  
K.W. THILINI CHETHANA ◽  
SAISAMORN LUMYONG ◽  
...  

Laburnicola is a genus in Didymosphaeriaceae that includes saprobic and endophytic fungal taxa. The current study conducted in the subalpine region of Uzbekistan discovered a new species on a dead stem of a wild rose plant. Maximum likelihood and Bayesian analyses of combined LSU, SSU, ITS, and TEF 1-ɑ dataset confirmed the new species’ taxonomic position in Laburnicola. Our new species, Laburnicola zaaminensis (TASM 6152), was clustered with L. dactylidis (MFLUCC 16-0285) with strong bootstrap support. A detailed description together with illustrations are provided for Laburnicola zaaminensis. Furthermore, an annotated species list, a distribution map, and a taxonomic key for Laburnicola species are provided. This is the first record of Laburnicola from Central Asia.


2021 ◽  
Author(s):  
David Champredon ◽  
Devan G Becker ◽  
Connor Chato ◽  
Gopi Gugan ◽  
Art G Poon

Genetic sequencing is subject to many different types of errors, but most analyses treat the resultant sequences as if they are known without error. Next generation sequencing methods rely on significantly larger numbers of reads than previous sequencing methods in exchange for a loss of accuracy in each individual read. Still, the coverage of such machines is imperfect and leaves uncertainty in many of the base calls. On top of this machine-level uncertainty, there is uncertainty induced by human error, such as errors in data entry or incorrect parameter settings. In this work, we demonstrate that the uncertainty in sequencing techniques will affect downstream analysis and propose a straightforward method to propagate the uncertainty. Our method uses a probabilistic matrix representation of individual sequences which incorporates base quality scores as a measure of uncertainty that naturally lead to resampling and replication as a framework for uncertainty propagation. With the matrix representation, resampling possible base calls according to quality scores provides a bootstrap- or prior distribution-like first step towards genetic analysis. Analyses based on these re-sampled sequences will include a more complete evaluation of the error involved in such analyses. We demonstrate our resampling method on SARS-CoV-2 data. The resampling procedures adds a linear computational cost to the analyses, but the large impact on the variance in downstream estimates makes it clear that ignoring this uncertainty may lead to overly confident conclusions. We show that SARS-CoV-2 lineage designations via Pangolin are much less certain than the bootstrap support reported by Pangolin would imply and the clock rate estimates for SARS-CoV-2 are much more variable than reported.


Phytotaxa ◽  
2021 ◽  
Vol 527 (1) ◽  
pp. 41-50
Author(s):  
LEI WANG ◽  
SHI-TONG LIU ◽  
YUN LIU ◽  
YING-MEI LIANG

Three rust fungi belonging to the genus Hyalopsora occurring on ferns were found in China. Hyalopsora minispora and H. tibetica are described as new species in this paper. Hyalopsora minispora can be distinguished from known species by its relatively small urediniospores (19.5–23.0 × 12.0–19.0 μm) with 4–6 scattered germ pores and amphispores in a common pustule. Hyalopsora tibetica is mainly characterized by urediniospores with 2–6 scattered germ pores and amphispores. Hyalopsora neocheilanthis is described and reported as a new record for China. The phylogenetic relationships of Hyalopsora species and related taxa were examined by comparing the sequences of their internal transcribed spacer 2 (ITS2) barcode and 28S rDNA gene segments. Hyalopsora is supported with strong bootstrap support and it differs from the two other fern-infecting genera viz. Milesina and Uredinopsis.


Plant Disease ◽  
2021 ◽  
Author(s):  
Donghun Kang ◽  
Jungyeon Kim ◽  
Youn Mi Lee ◽  
Balaraju Kotnala ◽  
Yongho Jeon

In September 2020, typical anthracnose symptoms were observed on cotton (Gossypium indicum Lam.) leaves growing in Hahoe village, Andong, Gyeongbuk Province, Korea. The leaves of the infected plants initially showed spots with halo-lesions which became enlarged and spread to the entire leaf surface area. The infected leaves later became yellowish and chlorotic (Fig. 1A). The disease incidence was at least 90% in the field. For pathogen isolation, fresh samples collected from symptomatic leaves were cut into small pieces (4 to 5 mm2), surface-sterilized in 1% sodium hypochlorite for 1 min, rinsed three times, and macerated in sterile distilled water (SDW). They were spread onto potato dextrose agar (PDA) plates and incubated at 25 °C for 5 days under a 12-h photoperiod. Five isolates were recovered from the infected leaves. Purified fungal colonies were initially white, later turned yellow on PDA medium. Conidia were yellow-colored, smooth-walled, aseptate, straight or slightly distorted, and cylindrical with one end slightly acute or with broadly rounded ends, and with size ranges from 15.3 to 17.5 µm (length) × 4.5 to 5.2 µm (width) (Fig. 1B). The morphological characteristics of the present isolates were consistent with those of Colletotrichum gloeosporioides (Weir et al. 2012). A single isolate, ANUK97, was selected for identification. The multilocus sequence analysis (MLSA) of the actin (ACT), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), internal transcribed spacer (ITS) rDNA, and β-tubulin (Tub2) were amplified by PCR with the primer pairs of ACT-521F/ACT-783R, CL1C/CL2C, GDF/GDR, ITS1/ITS4, and T1/T2, respectively (White et al. 1990). The resulting sequences were deposited in GenBank under accession numbers MW580367 (ACT), MW580368 (CAL), MW580369 (GAPDH), MW580370 (ITS), and MW580371 (TUB2). A nucleotide BLAST search revealed that ACT, CAL, GAPDH, ITS, and TUB2 sequences be 99% similar to accession numbers MN307380.1, MH155176.1, MK796226.1, MW580370.1, and JX010377.1, respectively of C. theobromicola. Maximum likelihood (ML) phylogenetic analysis was conducted based on a combined dataset of ACT, CAL, GAPDH, ITS, and TUB2 sequences using MEGA-X 10.1.8. The isolate ANUK97 was clustered with a representative strain C. theobromicola CBS124945 100% bootstrap support (Fig. 2). For the pathogenicity test, two-month-old cotton seedlings (n = 10) were inoculated with conidial suspensions (10⁶ spore/mL) of C. theobromicola obtained from 7-day-old PDA cultures at 25 °C by spray method. Seedlings treated with sterile distilled water served as controls. Inoculated and control cotton plants were incubated in the greenhouse at 25 °C under a 12-h photoperiod. After 7 days, necrotic lesions were observed on the artificially inoculated cotton plants, while control plants did not develop any disease symptoms. The pathogen was re-isolated from infected cotton leaves, but not from control plants to fulfill Koch’s postulates. To our knowledge, this is the first report of anthracnose of cotton caused by Colletotrichum theobromicola in Korea.


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