scholarly journals First Report of Xanthomonas fragariae strain YL19 causing crown infection pockets in strawberry in Liaoning Province, China

Plant Disease ◽  
2021 ◽  
Author(s):  
Jiayue Feng ◽  
Yu Lian Li ◽  
Dan Juan Wang ◽  
YangYang Ma ◽  
XiaoLin Cai ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Leaf Surface ◽  
Causal Agent ◽  
Angular Leaf Spot ◽  
Liaoning Province ◽  
Bacterial Suspension ◽  
Bacterial Disease ◽  
Sterile Water ◽  
Data Set ◽  
Initial Symptoms

Strawberry (Fragaria × ananassa Duch.) is an important fruit crop in China. Typical crown infection pockets symptoms were observed on the infected strawberry in Liaoning province, China (121°60′E, 38°90′N) in the autumn of 2017. The disease incidence was estimated to be around 5 to 10 %, but could reach 30 to 40 % in some heavily affected plastic tunnels. The infected plants early displayed water-soaked symptoms on the abaxial leaf surface and subsequently developed reddish-brown shaped stripes and coalesced lesions on the adaxial leaf surface around the main veins (Fig. 1-A, 1-B). Several variable-size (0.3-0.8 mm in diameter) pockets were observed inside the crown tissues after dissection (Fig. 1-C). The diseased plants rarely reached fruiting and were easily broken between the crown tissue and the stem, and would eventually die. To identify the causal agent of this disease, the several surface-disinfested infected main veins and crown tissues were individually ground in sterile water and plated on sucrose peptone agar(SPA) medium (Hayward 1960) with 10-fold serial dilutions and incubated at 25℃. A number of yellow colonies grew on the medium at the 10-4 dilution 7 days after plating (Fig. 1-D) in all specimens. The colonies were aerobic, yellow, viscous, smooth, and gram-negative, which is a typical characteristic of Xanthomonas. To confirm identity of the causal bacteria, 18 colonies selected randomly were subjected to polymerase chain reactions (PCR) for the amplification of the cpn60 (Sahin et al. 2010), gyrB, rpoD, and fyuA (C Manceau et al. 2011), respectively. The results showed that the 18 colonies are identical. The cpn60, gyrB, rpoD, and fyuA sequences of this isolate were deposited in GenBank with accession numbers MT513132.1, MW233896, MW233897, and MW233895, respectively. BLAST searches with sequences of this isolate cpn60, gyrB, rpoD, and fyuA revealed 97.7%, 96.4%, 97.8%, and 97.3% similarity with the corresponding sequences of X. fragariae strain NBC2815 (LT853880.1), respectively. The resulting concatenated data set of cpn60-gyrB-rpoD-fyuA was used to build a Multilocus Sequence Analysis (MLSA) by maximum likelihood criteria (Fig. 2). The cpn60-gyrB-rpoD-fyuA sequences of the isolate from Liaoning clustered in the clade containing the type strain of X. fragariae NBC2815, indicating that it belongs to X. fragariae. Thus, the bacterial strain from Liaoning was designated as X. fragariae strain YL19. To fulfill Koch’s postulates, the base of leaf petioles of disease-free strawberry plants were syringe-infiltrated inoculated with bacterial suspension (2×108 CFU) prepared from colonies of X. fragariae YL19 washed from SPA plates. The inoculated and control (treated with sterile water) were placed in a chamber (25/20℃day/night,≥90% relative humidity(RH), 12/12 h photoperiod) for three months. After one month, water-soaked symptoms were observed in the crown tissues of all X. fragariae YL19-inoculated plants. Two months after inoculation, a significant crown pocket similarly to initial symptoms observed in the field was developed on all inoculated plants. No symptoms were observed in the control plants. The bacteria were re-isolated from the symptomatic leaves, petioles and crowns, and confirmed as X. fragariae YL19 by the above mentioned morphological and molecular analyses. Pathogenicity tests were conducted three times and the same results were obtained. It was reported that X. fragariae usually causes angular leaf spot, a serious bacterial disease in many strawberry production regions worldwide. The typical symptoms of angular leaf spot caused by X. fragariae include reddish-brown, irregular spots on the upper leaf surface, water-soaked lesions developed along leaf veins. Although angular leaf spot caused by X. fragariae has been reported in Tianjin and Taiwan province, China (Wang et al. 2017; Wu et al. 2020), there is no report about the symptoms that infection pockets on crowns caused by X. fragariae strain YL19 as described above. This result indicated that YL19 is different from the other two X. fragariae strains reported in China or the disease caused by YL19 could be a severe case of angular leaf spot and vascular decline or collapse in strawberry (Bradbury, 1977). . To the best of our knowledge, these results showed a previously unreported new strain YL19 of X. fragariae is the causal agent of crown infecton pocket in strawberry in China, it may lead to serious losses to the local strawberry industry. This report will assist in developing management measures for this disease promptly.

scholarly journals First Report of Alternaria tenuissimain Causing Leaf Spot of Celery (Apium graveolens) in China

Plant Disease ◽  
2020 ◽  
Author(s):  
Jianqiang Zhang ◽  
Kangli Wu ◽  
Xiaomeng Zhang ◽  
Jiajia Li ◽  
Abdramane salah zene ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Leaf Spot ◽  
Leaf Age ◽  
Apium Graveolens ◽  
Control Group ◽  
Sterile Water ◽  
Internal Transcribed Spacer Region ◽  
Data Set ◽  
First Report ◽  
Initial Symptoms

Celery (Apium graveolens) is one of the most widely grown vegetables in the world. A survey in Anding District of Gansu Province in 2019 showed that the incidence of celery leaf spot was 25%-45%. The disease mainly occurs in late June and July. The leaf spot is conducive to the onset at high temperature and humidity environment. The initial symptoms were many small light brown, irregular-shaped on the leaves. The lesions gradually enlarged in the later stage of the disease, and multiple lesions coalesced to form large irregular brown spots, eventually the whole leaves died. A 3~4mm leaf tissue was cut from the junction of the diseased leaf and the healthy area, the leaf tisse was surface-sterilized in 1.5% NaClO for 1 min and washed with sterile water. Then, it was incubated on potato dextrose agar (PDA) and obtained the pure culture (Q1). After 5 days of cultivation at 25°C, the fungal colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. The conidia were obclavate or ellipsoid, pale brown, with 3~4 longitudinal septa and 2~7 transverse septa, and measured 20.0 to 50.0 × 3.5 to 14.0μm (n=50). Conidiophores were septate, arising singly, and measured 3.5 to 40.0 × 2.5 to 4.5 μm (n=50). Based on morphological characteristics, the fungus was preliminarily identified as A.tenuissima (Simmons 2007). To further confirm the identification, the internal transcribed spacer region (ITS), translation elongation factor 1-α gene (TEF), RNA polymerase II second largest subunit (RPB2), major allergen Alt a 1 gene (Alt a 1), endopolygalacturonase gene (endoPG), anonymous gene region (OPA10-2) and glyceraldehyde 3-phos-phatedehydrogenase (GAPDH) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004), EF1-728F/EF1-986R (Carbone et al. 1999), RPB2-5F2/RPB2-5R (Sung et al. 2007), Alt-for/Alt-rev (Hong et al. 2005), EPG-specific/EPG-3b (Peever et al. 2004), OPA10-2R/OPA10-2L (Peever et al. 2004) and Gpd1/Gpd2 (Berbee et al. 1999) (GenBank accession no.MN046364, MW016001, MW016002, MW016003, MW016004, MW016005, MW016006). DNA sequences of TEF, RPB2, endoPG, OPA10-2 and GAPDH were 100% identical to those of A. tenuissima (MN256108, MK605866, KP789503, JQ859829 and MK683802), but ITS and Alt a 1 were 100% similarity with A. tenuissima (MN615420, JQ282277) and A. alternate (MT626589, KP123847). The ITS and Alt a 1 sequence did not distinguish A. tenuissima from the A. alternate complex. Maximum likelihood phylogenetic analyses were performed for the combined data set with TEF, RPB2, and endoPG using MEGA6 under the Tamura-Nei model (Kumar et al. 2016). The isolate Q1 clustered with type strain A. tenuissima CBS 918.96. The 20 celery plants of 4-7 leaf age were used test the pathogenicity of Q1, the ten plants were sprayed with 20ml of spore suspension (1×105 spores/ml), the control was sprayed with 20mL sterile water, which were placed in a growth chamber (25℃, a 14h light and 10h dark period, RH > 80%). Eight days after inoculation, 40% of the leaves formed lesions, which were consistent with the field observation，the control group was asymptomatic. The pathogen was reisolated from infected leaves to fulfill Koch’s postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot on celery in China.

scholarly journals First Report of Pseudomonas syringae pv. aptata Causing Bacterial Leaf Spot on Sugar Beet in Serbia

Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 281-281 ◽  
Author(s):  
V. Stojšin ◽  
J. Balaž ◽  
D. Budakov ◽  
Slaviša Stanković ◽  
I. Nikolić ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Pseudomonas Syringae ◽  
Leaf Spot ◽  
Leaf Surface ◽  
Negative Control ◽  
Gyrb Gene ◽  
Bacterial Suspension ◽  
Bacterial Strains ◽  
Gene Sequences ◽  
Bacterial Leaf Spot

A severe bacterial leaf spot was observed during June and July 2013 on commercial cultivars of sugar beet (Beta vulgaris var. saccharifera) in the Vojvodina Province of Serbia. Serbia is a major sugar beet production area in southeastern Europe, with 62,895 ha and 3 million tons of sugar beet yield in 2013. A foliar leaf spot observed in 25 commercial sugar beet fields surveyed ranged from 0.1 to 40% severity. Symptoms were characterized as circular or irregular, 5- to 20-mm diameter, white to light brown necrotic spots, each with a dark margin. Diseased leaves were rinsed in sterilized, distilled water (SDW) and dried at room temperature, and leaf sections taken from the margin of necrotic tissue were macerated in SDW. Isolations from 48 symptomatic leaves onto nutrient agar with 5% (w/v) sucrose (NAS) produced bacterial colonies that were whitish, circular, dome-shaped, and Levan-positive. Representative isolates (n = 105) were Gram negative; aerobic; positive for catalase, fluorescence on King's medium B, and tobacco hypersensitivity; and negative for oxidase, potato rot, and arginine dehydrolase. These reactions corresponded to LOPAT group Ia, which includes Pseudomonas syringae pathovars (2). Repetitive extragenic palindromic sequence (rep)-PCR was used for genetic fingerprinting the isolates using the REP, ERIC, and BOX primers. Twenty-five different profiles were obtained among the strains. From each profile group, one representative strain was sequenced for the gyrB gene (1). Four heterogenic groups were observed, and representative gyrB gene sequences of each group were deposited in the NCBI GenBank (Accession Nos. KJ950024 to KJ950027). The sequences were compared with those of pathotype strain P. syringae pv. aptata CFBP 1617 deposited in the PAMDB database; one strain was 100% homologous, and the other three were 99% homologous. To fulfill identification of the Serbian sugar beet isolates, gltA and rpoD partial gene sequences were determined (1), and the sequences were deposited as Accession Nos. KM386838 to KM386841 for gltA and KM386830 to KM38683033 for rpoD. The sequences were 100% homologous with those of pathotype strain CFBP 1617. Pathogenicity of each of four representative bacterial strains was tested on 3-week-old plants of the sugar beet cultivars Marinela, Serenada, and Jasmina (KWS, Belgrade, Serbia) and Lara (NS Seme, Novi Sad, Serbia) by atomizing a bacterial suspension of ~106 CFU/ml of the appropriate isolate onto the abaxial leaf surface of three plants per cultivar until water-soaking of the leaf surface was observed. Three plants of each cultivar atomized similarly with P. syringae pv. aptata CFBP 2473 and SDW served as positive and negative control treatments, respectively. Inoculated plants were kept in a clear plastic box at 80 to 100% RH and 17 ± 1°C and examined for symptom development over 3 weeks. For all test isolates and the control strain, inoculated leaves first developed water-soaked lesions 7 days after inoculation (DAI). By 10 to 14 DAI, lesions were necrotic and infection had spread to the petioles. By 21 DAI, wilting was observed on more than 50% of inoculated plants. Negative control plants were symptomless. Bacteria re-isolated onto NAS from inoculated leaves had the same colony morphology, LOPAT results, and gyrB partial gene sequences as described for the test strains. No bacteria were re-isolated from negative control plants. Based on these tests, the pathogen causing leaf spot on sugar beet in Serbia was identified as P. syringae pv. aptata. References: (1) P. Ferrente and M. Scortichini. Plant Pathol. 59:954, 2010. (2) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966.

scholarly journals Angular Leaf Spot: A Bacterial Disease in Strawberries in Florida

EDIS ◽  
1969 ◽  
Vol 2005 (1) ◽  
Author(s):  
Natalia A. Peres ◽  
Silvia I. Rondon ◽  
James F. Price ◽  
Daniel J. Cantliffe
Keyword(s):  
Leaf Spot ◽  
Gray Mold ◽  
Angular Leaf Spot ◽  
Publication Date ◽  
Bacterial Disease ◽  
University Of Florida ◽  
Pythium Root Rot ◽  
Xanthomonas Fragariae ◽  
Rapid Spread ◽  
Sphaerotheca Macularis

Angular Leaf Spot (ALS) is a bacterial disease caused by Xanthomonas fragariae Kennedy & King, a pathogen highly specific to wild and cultivated strawberry, Fragaria x ananassa Duchesne (Legard et al. 2003). ALS is an important disease on winter strawberry production worldwide. In the U.S., it ranks 6th in economic importance after gray mold (Botrytis cinerea L.), verticillium wilt (Verticillium alboatrum Reinke & Berth), powdery mildew (Sphaerotheca macularis L.), anthracnose (Colletotrichum spp.), and pythium root rot (Pythium spp.) (Sorensen et al. 1997). The rapid spread of ALS is influenced by the increasing rate of interchange of plant material. This is document PP-199, a publication of the Plant Pathology Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida. Publication date: June 2004. PP-199/PP120: Angular Leaf Spot of Strawberries (ufl.edu)

scholarly journals First report of Fusarium avenaceum causing Leaf Spot on Angelica sinensis in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Zikun Zhang ◽  
Jianqiang Zhang ◽  
Wanxia Zhang ◽  
Zhian Kou ◽  
Xinfang Wang ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Chinese Herb ◽  
Gansu Province ◽  
Fusarium Avenaceum ◽  
Angelica Sinensis ◽  
Conidial Suspension ◽  
Data Set ◽  
First Report ◽  
Initial Symptoms ◽  
Β Tubulin

Angelica sinensis (Oliv) Diels (Umbelliferae) is a popular Chinese herb that is mainly distributed in Gansu Province, China, accounting for more than 90% of the national output and sales. A survey for diseases of A. sinensis in Gansu Province in August 2019 found foliar disease with an incidence of 60 to100%, and severities ranging from 5 to 15%. The disease mainly occurred in late July and August. The initial symptoms included many light brown, small lesions, round or irregular in shape, which gradually increased in size. White mycelia was visible in the lesions. Severely affected leaves became chlorotic, withered and died. In the Angelica planting area in Weiyuan County (33°26′N, 104°02′E) diseased leaves from 20 plants were collected by the five-point sampling method (Zheng et al. 2018), and small samples (4 × 4 mm2) wee cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol for 30 sec, washed three times with sterilized water and dried on sterilized filter paper, and placed on potato dextrose agar plates. After 5 days at 25°C, five morphologically similar colonies were obtained. Colonies were somewhat round with pink overall and formed abundant fluffy white mycelium in the center. Conidia were solitary, macrospores slender, straight to slightly falcate with 2 to 6 septa, and ranged from 20.0 to 77.6 µm × 2.5 to 3.6 µm (n=50). The microspores were elliptical and ranged from 3.0 to 8.0 µm × 2.5 to 3.0 µm (n=5). The strong pink pigment was observed on the reverse side of the PDA culture. The morphological characteristics were consistent with the description of Fusarium avenaceum (Parikh et al. 2018; Jahedi et al. 2019). To further identify the strains, the internal transcribed spacer (ITS), β-tubulin, translation elongation factor 1α (EF1-α), and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2, and 5f2/7cr (Glass and Donaldson 1995; O’Donnell et al. 2010; White et al. 1990), respectively. The sequences of the five strains were identical, and that of representative strain K0721 were deposited in GenBank (ITS, MZ389899; TUB2, MZ398139; EF1-α, MZ388462; RPB2, MZ394004). BLAST analysis revealed that the ITS, β-tubulin, EF1-α, and RPB2 sequences were 100% (563/563), 100% (423/423), 99% (643/649), and 99% (930/935) homology, with those of F. avenaceum (KP295511.1, KY475586.1, KU999088.1, and MH582082.1), respectively. A multigene phylogenetic tree was inferred by Maximum likelihood phylogenetic analyses based on the combined data set with ITS, EF1-α and RPB2. The strain K0721 was clustered with F. avenaceum. Pathogenicity tests were performed on five 1-month-old healthy plants in plastic pots (20 cm. diam.) with sterilized soil. Each was sprayed with 50 μl of a conidial suspension (1×104 conidia/mL), and 5 healthy plants were sprayed with sterile water as controls. Small lesions were observed after 5 days at 25℃ in a greenhouse. Symptoms were similar to those observed under field conditions. Control plants remained symptomless. Six isolates were reisolated from infected leaves and all confirmed to be F. avenaceum based on morphological observations and molecular identification. To our knowledge, only Septoria anthrisci has been previously reported as a pathogen of A. sinensis leaf spot (Wang et al. 2018), and this is the first report of F. avenaceum causing this disease. This discovery needs to be considered in developing and implementing disease management programs in A. sinensis production.

scholarly journals First Report of Cochliobolus sativus Causing Leaf Spot on Paper Mulberry in China

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1586-1586 ◽  
Author(s):  
P. S. Wu ◽  
K. Chen ◽  
H. Z. Du ◽  
J. Yan ◽  
Q. E. Zhang
Keyword(s):  
Leaf Spot ◽  
Leaf Tissue ◽  
Pathogenic Fungi ◽  
Bipolaris Sorokiniana ◽  
Causal Agent ◽  
Cochliobolus Sativus ◽  
Conidial Suspension ◽  
Sterile Water ◽  
First Report ◽  
Forest Park

Paper mulberry, Broussonetia papyrifera (L.) Vent., is a highly adaptable, fast-growing tree that is native to eastern Asia. Its ability to absorb pollutants makes it ideal for ornamental landscapes, especially in industrial and mining areas. During the summer of 2010, brown lesions were observed on leaves of paper mulberry in Baiwangshan Forest Park, Beijing, China. These lesions were ovoid to fusiform and 4 to 9 × 2 to 4 mm with dark brown centers and light brown irregular edges. Spots on severely infected leaves sometimes coalesced to form long stripes with gray centers. To isolate the causal agent of the lesions, 4-mm2 pieces of diseased leaf tissue from 12 leaves were collected at the lesion margins and surface disinfected in 0.5% NaOCl for 3 min, rinsed three times with sterile water, plated on water agar, and incubated at 25°C with a 12-h photoperiod. After 5 days, the cultures, which became dark brown to black, were observed. Conidiophores (120 to 220 × 4 to 7 μm) were solitary or in groups of two to five, straight or flexuous with swollen bases, and light or dark brown. Conidia were dark olive brown, spindle- or oval-shaped with truncated ends (60 to 120 × 15 to 30 μm), slightly curved, and containing 3 to 12 distoseptate (mostly 6 to 10). Pseudothecia, produced after 14 days in culture, were dark brown to black and flask shaped (420 to 530 μm in diameter with 85 to 100 × 75 to 90 μm ostiolar beaks). Asci were cylindrical (100 to 220 × 30 to 40 μm) and contained eight ascospores. Ascospores were filiform, (150 to 360 × 6 to 9 μm), hyaline, with 6 to 11 septations. Isolates were identified as Cochliobolus sativus (Ito & Kurib.) Drechsler & Dastur (anamorph Bipolaris sorokiniana (Sacc. & Sorok.) Shoem.) on the basis of culture color and dimensions and colors of pseudothecia, asci, ascospores, conidiophores, and conidia (2,3). The identity of one isolate was confirmed by ITS1-5.8S-ITS2 rDNA sequence (GenBank Accession No. HQ 654781) analysis that showed 100% homology to C. sativus listed in Berbee et al. (1). Koch's postulates were performed with six potted 3-month-old paper mulberry plants. An isolate was grown on potato dextrose agar for 14 days to obtain conidia for a conidial suspension (3 × 104 conidia/ml). Three of the potted plants were sprayed with the conidial suspension and three were sprayed with sterile water as controls. Each plant was covered with a plastic bag for 24 h to maintain high humidity and incubated at 25°C with a 12-h photoperiod. After 7 days, the inoculated plants showed leaf symptoms identical to those previously observed on paper mulberry trees in the Baiwangshan Forest Park, while control trees remained symptom free. Reisolation of the fungus from the inoculated plants confirmed that the causal agent was C. sativus. C. sativus is widely distributed worldwide causing a variety of cereal diseases. Wheat and barley are the most economically important hosts. To our knowledge, this is the first report of C. sativus as a pathogen causing leaf spot of paper mulberry in China. References: (1) M. L. Berbee et al. Mycologia 91:964, 1999. (2) M. B. Ellis. Dematiaceous Hyphomycetes. CABI, Oxon, UK, 1971. (3) A. Sivanesan et al. No.701 in: Descriptions of Pathogenic Fungi and Bacteria. CAB, Kew, Surrey, U.K., 1981.

scholarly journals First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China

Plant Disease ◽  
2021 ◽  
Author(s):  
Shucheng Li ◽  
Liuhua Xiao ◽  
Fan Wu ◽  
Yinbao Wang ◽  
Mingshu Jia ◽  
...  
Keyword(s):  
Leaf Spot ◽  
Juglans Regia ◽  
Morphological Characteristics ◽  
Effective Control ◽  
Bootstrap Support ◽  
Sterile Water ◽  
First Report ◽  
Myrica Rubra ◽  
Initial Symptoms ◽  
Colletotrichum Fructicola

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.

scholarly journals First Report of Xanthomonas fragariae Causing Angular Leaf Spot on Strawberry Plants in México

Plant Disease ◽  
2014 ◽  
Vol 98 (5) ◽  
pp. 682-682 ◽  
Author(s):  
S. P. Fernández-Pavía ◽  
G. Rodríguez-Alvarado ◽  
E. Garay-Serrano ◽  
R. Cárdenas-Navarro
Keyword(s):  
Leaf Spot ◽  
Pathogenic Bacteria ◽  
Disease Incidence ◽  
Angular Leaf Spot ◽  
Pcr Analysis ◽  
Bacterial Suspension ◽  
American Phytopathological Society ◽  
First Report ◽  
Leaf Surfaces ◽  
Xanthomonas Fragariae

The state of Michoacán is the most important strawberry producer in México. During January 2007, field-grown strawberry plants cv. Aromas showing vein necrosis were observed in 3 ha in Zamora County, in fruit production fields. The average disease incidence in the field was 80%. Infected plants presented water-soaked lesions limited by veins on the lower leaf surfaces, which enlarged to form angular spots (1). Additionally, most affected plants presented severe necrosis in the main veins and reddish to necrotic lesions on the upper leaf surfaces. Gram-negative bacteria were consistently isolated from leaves with water-soaked lesions. Isolated bacteria produced mucoid, yellow colonies on YDC, grew on tween and nutrient agar (NA), but not on SX media. Strains produced non-fluorescent colonies on King's B media, were positive starch hydrolysis, negative esculin hydrolysis; and produced acid from fructose but not from arabinose, galactose, celobiose, and trehalose. Growth was inhibited by 2% NaCl (3). Indirect ELISA analysis (NEOGEN, Lansing, MI) was conducted using antibodies specific for Xanthomonas fragariae. Conventional PCR assay using the primer pairs 241A/241B was performed (2). The ELISA test was positive. The expected 300- and 550-bp bands were observed in the PCR analysis. The bacteria was identified as X. fragariae Kennedy and King. Pathogenicity tests were conducted twice in a greenhouse (24 ± 4°C) on a total of five strawberry cv. Aromas plants. The main vein of each of three leaves per plant were punctured using sterile needles. Pathogen inoculum was obtained from 6- to 8-day-old NA cultures. Bacteria were applied onto the wounds with a sterile cotton swab dipped into the bacterial suspension (105 CFU/ml). Inoculated plants were covered with plastic bags for 48 h. Symptoms resembling those seen in the field developed on all inoculated plants after 9 days. X. fragariae was re-isolated from the necrotic lesions and identified by PCR. Control plants were similarly inoculated with water but did not develop symptoms. To our knowledge, this is the first report of X. fragariae causing angular leaf spot in strawberry in Michoacán, México. References: (1) J. L. Maas, ed. Compendium of Strawberry Diseases. The American Phytopathological Society, St. Paul, MN, 1998. (2) M. R. Pooler et al. Appl. Environ. Microbiol. 62:3121, 1996. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

scholarly journals First report of Anthracnose Disease on Bletilla striata (Thunb.) Reichb. f. (Baiji) caused by Colletotrichum orchidophilum in Yunnan, China

Plant Disease ◽  
2021 ◽  
Author(s):  
Kuan Yang ◽  
Huiling Wang ◽  
Kunhao Ye ◽  
Hongping Huang ◽  
Zhaoxia Wei ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Disease Incidence ◽  
Control Measures ◽  
Control Group ◽  
Sterile Water ◽  
Data Set ◽  
Bletilla Striata ◽  
First Report ◽  
Anthracnose Disease ◽  
Initial Symptoms

Bletilla striata (Thunb.) Reichb. f. has high ornamental and medicinal value. B. striata has been used in Chinese medicine for thousands of years to treat various diseases, such as peptic ulcers, gastrointestinal diseases, lung diseases, and traumatic bleeding (Jiang et al. 2021). In September 2020, about 30% of B. striata plants in a plantation in Lancang County (Puer City, Yunnan Province, 22°48'17" N, 99°46'58" E) were found with symptoms resembling Bletilla anthracnose disease. In May 2021, symptomatic leaves were observed again with a disease incidence rate reached 54.05%, and were collected. The initial symptoms were the appearance of one or more round light brown spots on the leaves. Subsequently, the lesions became larger, forming oval brown-black spots. On severely affected leaves, several spots coalesced, causing withering and death of the leaves. To isolate the pathogen, 10 symptomatic tissues (5mm2) of 10 infected leaves were disinfected in 75% ethanol for 30s then in 2% sodium hypochlorite for 3 minutes, rinsed with sterile water for 3 times, incubated on potato dextrose agar (PDA) plates at 25℃ for 5 to 7 days with a 12 h light/dark photoperiodic cycle. Finally, ten pathogen strains with similar morphology were isolated and pure cultures were obtained by single spore isolation. The morphology of fungal colonies was round, carpet-like, flat and neat, and the color was gray black. The conidiophores were colorless to brown and oval or stick-shaped. The conidia were cylindrical or oblong, consisted of a single cell with blunt round ends, and were on average 12.92 (8.13–21.21) μm×3.96 (2.55–5.90) μm (n=200) in size. Based on the morphology and conidial characteristics, the pathogen was identified as Colletotrichum sp. (Damm et al. 2012). The total genomic DNA of representative isolates (LCTJ-02, LCTJ-03, LCTJ-04, LCTJ-05 and LCTJ-06) was extracted and the ITS region, GAPDH, ACT and HIS3 gene regions were amplified and sequenced using the primer pairs ITS1/ITS4, GDF1/GDR1, 512F/783R, and CylH3F/CylH3R, respectively (Cai et al. 2009). The sequences were deposited in GenBank (MZ433189 to MZ433193 for ITS, MZ436430 to MZ436434 for GAPDH, MZ436435 to MZ436439 for HIS3 and MZ448474 to MZ448478 for ACT). BLAST search revealed that all sequences showed 98% to 100% homology with the corresponding sequences of C. orchidophilum ex-type (CBS 632.80). Phylogenetic trees were constructed using neighbor-joining and maximum likelihood methods for the combined data set of the ITS, GAPDH, ACT, and HIS3 genes by MEGA-X (Kumar et al.2018). In general, LCTJ-02, LCTJ-03, LCTJ-04, LCTJ-05, LCTJ-06, and C. orchidophilum type strains clustered on the same clade. To confirm pathogenicity, the five isolates of C. orchidophilum were inoculated in healthy leaves of potted B. striata, as described by Cai et al. (2009), with slight modifications. Each leaf was inoculated with three drops(10 µl drop-1) of conidia suspension (106 spores mL-1). The control group was mock inoculated with sterile water the same way. All samples were covered with plastic bags and maintained at 70% to 80% relative humidity for seven days. After this period, all inoculated leaves showed similar lesions, and the symptoms were identical to those observed in the field. The control plants remained healthy. The pathogens were re-isolated from two leaves of each treatment and re-identified as C. orchidophilum. To our knowledge, this is the first report of C. orchidophilum causing anthracnose on B. striata in China. In the future, the occurrence and transmission of this pathogen should be further studied in order to develop reasonable control measures.

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