First Report of Colletotrichum siamense causing Anthracnose on White Frangipani (Plumeria alba L.) in Malaysia

Plumeria alba L. is a flowering plant in the family Apocynaceae and widely cultivated in Malaysia as a cosmopolitan ornamental plant. In January 2020, anthracnose lesions were observed on leaves of Plumeria alba planted in Agricultural Farm, Universiti Putra Malaysia, in Selangor state, Malaysia. The disease mainly affected the leaves with symptoms occurring with approximately a 60% disease incidence. Ten symptomatic leaves were sampled from 3 different trees in the farm. Symptoms initiated as small circular necrotic spots that rapidly enlarged into black lesions with pale brown borders. Diseased tissues (5×5 mm) were surface-sterilized with 70% ethanol for 1 min, rinsed three times with sterile distilled water, dried on sterile filter papers, plated on PDA and, incubated at 25 °C with a 12-h photoperiod. A total of seven single-spore isolates with similar colony morphologies were obtained from tissue samples. After 7 days, the colonies raised the entire margin and showed white-to-gray aerial mycelium, orange conidial masses in the center and appeared dark brown at the center of the reverse view. The conidia were 1-celled, hyaline, smooth-walled, cylindrical with narrowing at the center, averaged (13-15 μm × 3 - 4 μm) (n=40) in size. Morphological characteristics of the isolates were similar to those detailed in taxonomic description of Colletotrichum sp. (Prihastuti et al. 2009). For molecular identification, genomic DNA of two representative isolates, PL3 and PL4 was extracted from fresh mycelium using DNeasy Plant Mini Kit (Qiagen, USA). The internal transcribed spacer (ITS) region, actin (ACT) and calmodulin (CAL) genes were amplified using ITS5/ITS4 (White et al. 1990), ACT-512F/783R (Carbone and Kohn 1999) and CL1C/CL2C primer sets (Weir et al. 2012). A BLAST nucleotide search of GenBank using ITS sequences showed 100% identity to Colletotrichum siamense ex-type culture ICMP 18578 (GenBank accession no. JX010171). ACT and CAL sequences showed 100% identity with C. siamense ex-type isolate BPD-I2 (GenBank accession no. FJ907423 and FJ917505). The sequences were deposited in GenBank (ITS: accession nos. MW335128, MT912574), ACT: accession nos. MW341257, MW341256, CAL: accession nos. MW341255 and MT919260). Based on these morphological and molecular characteristics, the fungus was identified as C. siamense. Pathogenicity of PL3 and PL4 isolates was verified using four healthy detached leaves of Plumeria alba. The leaves were surface-sterilized using 70% ethanol and rinsed twice with sterile water before inoculation. The leaves (three inoculation sites/leaf) were wounded by puncturing with a sterile needle through the leaf cuticle and inoculated in the wound site with 10-μl of conidial suspension (1×106 conidia/ml) from 7-days-old culture on PDA. Four leaves were used as a control and were inoculated only with 10-μl of sterile distilled water. Inoculated leaves were kept in humid chambers for 2 weeks at 25 °C with 98% relative humidity on a 12-h fluorescent light/dark period. The experiment was repeated three times. Anthracnose symptoms were observed on all inoculated leaves after 3 days, whereas controls showed no symptoms. Fungal isolates from the diseased leaves showed the same morphological characteristics as isolates PL3 and PL4, confirming Koch’s postulates. C. siamense has been reported causing anthracnose on rose (Rosa chinensis) in China (Feng et al. 2019), Coffea arabica in Thailand (Prihastuti et al. 2009) and mango leaf anthracnose in Vietnam (Li et al. 2020). To our knowledge, this is the first report of Colletrotrichum siamense causing leaf anthracnose on Plumeria alba in Malaysia. Accurate identification of this pathogen provides a foundation in controlling anthracnose disease on Plumeria alba.

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First Report of Anthracnose Caused by Colletotrichum brasiliense on Violet Passion Fruit in China

Plant Disease ◽

10.1094/pdis-11-20-2485-pdn ◽

2021 ◽

Author(s):

G. Y. Shi ◽

Quan Zeng ◽

Y. W. Wei ◽

Chun Jin Hu ◽

X. L. Ye ◽

...

Keyword(s):

Large Scale ◽

Disease Incidence ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Passion Fruit ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms

Violet passion fruit (Passiflora edulis Sims) is an important tropical and subtropical perennial evergreen vine with large-scale cultivation in Guangxi, China. Between May and September 2020, anthracnose symptoms occurred on passion fruit (cultivar Tainong No. 1) in Xingye county (22°77′13″N, 110°07′80″E) in Guangxi province, China. The disease incidence varied from 25 to 60% in different orchards. Initial symptoms on young fruits appeared as multiple tiny water-soaked, oval to irregular pale greenish spots. As the disease progressed, the lesions became medium brown, with sunken cavities. Under humid conditions, acervuli containing masses of conidia and dark setae were found on the lesions. The affected fruits became shriveled. Tissue pieces (5 × 5 mm) were cut out from infected fruits, surface sterilized in 75% ethanol for 15 s and 0.1% HgCl2 for 2 min, washed three times with sterile water, placed onto potato dextrose agar (PDA), and incubated at 28 °C for three days. Of the 29 Colletotrichum isolates obtained , the isolate B13 was selected for morphological characterization. B13 was purified by single spore isolation and incubated on PDA at 25°C under continuous fluorescent light irradiation, producing white to pale yellow colonies with dense aerial mycelia. The reverse side of the colony was pale yellowish to olive. Conidia were hyaline, unicellular, straight, cylindrical, with both ends slightly round or one end round and the other slightly pointed, measuring 10.5 to 18.8 (average 16.4) × 5.4 to 7.2 (average 6.3) µm (n = 50). Appressoria were light brown to dark black, smooth-walled, lobed, often with a roundish outline, sometimes also triangular, 7.2 to 10.9 (average 9.1) × 6.8 to 9.2 (average 8.2) µm (n = 50). Morphological characteristics of the isolate matched those of Colletotrichum brasiliense (Damm et al. 2012). The internal transcribed spacers (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-tubulin (TUB2) genes of strain B13 were sequenced using the method and primers of Damm et al. (2012). Sequences of the amplified DNA regions were submitted to GenBank (ITS: MW198820; ACT: MW266083; GAPDH: MW266084; and TUB2: MW266085). A concatenated maximum likelihood phylogenetic tree was built using MEGA 7.0.21 in which B13 clustered with C. brasiliense and clearly separated from other Colletotrichum spp. Pathogenicity of B13 was assayed using one-year-old plants of violet passion fruit cultivar ‘Tainong No. 1’. Conidial suspensions were prepared from 7-day-old cultures grown on PDA at 28°C Sterile distilled water was used to dislodge conidia from the culture dish and the conidial concentration was adjusted to 1 × 106 spores mL-1 using a haemocytometer. Fruits were rinsed with sterilized water and wounded with a sterile needle at three locations. Three fruits were inoculated by spraying with 20 mL of the conidial suspension. Control fruits were sprayed with distilled water. Fruits were then covered with plastic bags to maintain high relative humidity . After 9 days, all inoculated fruits developed brown spots with sunken cavities, resembling symptoms observed in the field, and controls remained symptomless. Fungal cultures with phenotypic features similar to C. brasiliense were re-isolated from the symptomatic fruits, verifying C. brasiliense as the causal agent of the disease based on Koch’s postulates. C. boninense, C. gloeosporioides, C.queenslandicum, C. brevisporum, and C. karstii were reported as causal agents of anthracnose on passion fruit (Júnior et al.2010; Power et al. 2010; James et al.2014; Du et al.2017; Ran et al.2020). To the best of our knowledge, this is the first report of C. brasiliense causing anthracnose on passion fruit in China.

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First Report of Colletotrichum siamense Causing Anthracnose on Zinnia elegans Jacq. in China

Plant Disease ◽

10.1094/pdis-04-20-0803-pdn ◽

2020 ◽

Author(s):

Wen Li ◽

Yue-qiu He ◽

Tao Fu ◽

Li Lin ◽

Feng Liu ◽

...

Keyword(s):

Phylogenetic Trees ◽

Colletotrichum Gloeosporioides ◽

Disease Incidence ◽

Morphological Characteristics ◽

Zinnia Elegans ◽

Likelihood Method ◽

Distilled Water ◽

First Report ◽

Colletotrichum Siamense ◽

Leaf Tissues

Zinnia elegans (syn. Zinnia violacea), known as common zinnia, is one of the most spectacular ornamental plants in the family Asteraceae. Zinnia plants are widely cultivated in China for their impressive range in flower colours and profuse bloom over a long period. In April 2019, Zinnia plants grown in Ningbo Botanical Garden (29°56′57″N, 121°36′20″E) were found to have many circular necrotic lesions. In the early infection stage, the lesions appeared as small circular specks which developed later into large spots (15 to 32 mm diameter). Typical symptoms appeared to be grayish white centers with a chlorotic edges and disease incidence reached approximately 80% of plants in the affected field. Moreover, the growth of Zinnia plants was seriously affected by the disease. To identify the causative pathogen associated with the disease, 10 symptomatic leaves were collected from ten different Zinnia plants. Leaf tissues were cut from the lesion margins, surface sterilized with 75% ethanol for 30 seconds and rinsed three times in sterile distilled water. The leaf tissues were then dipped into 10% sodium hypochlorite for 2-3 minutes, washed three times in distilled water and dried on a sterile filter paper. After drying, the surface-sterilized leaf discs were transferred to potato dextrose agar (PDA) plates and incubated at 28°C for 2 to 3 days under the 12 h photoperiod. A total of ten pure fungal isolates were obtained and all the isolates displayed the same colony structure. Afterwards, three pure strains were randomly selected (F1, F3 and F5) for further study. The fungal colonies showed gray to brownish aerial mycelia with pink-colored masses of conidia. Conidia were one-celled, hyaline, cylindrical to subcylindrical, spindle-shaped with obtuse ends, measuring from 15.6 to 17.3 × 4.6 to 5.1 μm with both ends rounded. These morphological characteristics were consistent with the description of Colletotrichum gloeosporioides complex (Weir et al. 2012). The identity of a representative isolate, F3, was confirmed by a multilocus approach. Genomic DAN of isolate F3 was extracted and partial sequences of actin (ACT), chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal internal transcribed spacer (ITS), manganese-superoxide dismutase (SOD2) , glutamine synthatase (GS), beta-tubulin (TUB2) and calmodulin (CAL) were amplified and sequenced as previously described (Weir et al. 2012). These nucleotide sequences were deposited in GenBank (accession MN972436 to MN972440, and MT266559 to MT266561; all sequences in FASTA format are shown (Supplementary S1). BLAST analysis of ITS, ACT, CHS, GAPDH and GS sequences from the F3 isolate revealed similarity to C. gloeosporioides voucher strain ZH01 with 100%, 100%，99%, 99% and 99% identity, respectively. SOD, TUB2 and CAL sequences showed similarity to C. siamense with 100%, 100% and 100% identity, respectively. The phylogenetic trees were constructed by Maximum Likelihood method (ML) using JTT model implemented in the MEGA 7. Results inferred from the concatenated sequences (ACT, CHS, GAPDH, ITS, SOD, GS, TUB2 and CAL) placed the isolate F3 within the C. siamense cluster (Supplementary S2). To confirm pathogenicity of the fungus, Koch’s postulates were conducted by spraying 20 Zinnia plants (60-day-old) with a 1 × 106 conidia/ml suspension. Plants were maintained in the growth chamber at 25°C and 85% relative humidity. After 10 to 15 days, symptoms were observed on all inoculated leaves and resembled those observed in the field, whereas the control plants remained asymptomatic. Here, C. siamense was isolated only from the infected Zinnia leaves and identified by morphological and gene sequencing analyses. C. siamense has been reported in many crops in China (Yang et al. 2019; Chen et al. 2019; Wang et al. 2019). However, to our knowledge, this is the first report of anthracnose caused by C. siamense on Zinnia elegans in China. References Chen, X., Wang, T., Guo, H., Zhu, P. K., and Xu, L. 2019. First report of anthracnose of Camellia sasanqua caused by Colletotrichum siamense in China. Plant Dis. 103:1423-1423. Wang, Y., Qin, H. Y., Liu, Y. X., Fan, S. T., Sun, D., Yang, Y. M., Li, C. Y., and Ai, J. 2019. First report of anthracnose caused by Colletotrichum siamense on Actinidia arguta in China. Plant Dis. 103:372-373. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud. Mycol. 73: 115-180. Yang, S., Wang, H. X., Yi, Y. J., and Tan, L. L. 2019. First report that Colletotrichum siamense causes leaf spots on Camellia japonica in China. Plant Dis. 103:2127-2127.

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First Report of Brown Blight Disease Caused by Colletotrichum gloeosporioides on Camellia sinensis in Anhui Province, China

Plant Disease ◽

10.1094/pdis-08-13-0896-pdn ◽

2014 ◽

Vol 98 (2) ◽

pp. 284-284 ◽

Cited By ~ 16

Author(s):

M. Guo ◽

Y. M. Pan ◽

Y. L. Dai ◽

Z. M. Gao

Keyword(s):

Camellia Sinensis ◽

Colletotrichum Gloeosporioides ◽

Tea Plant ◽

Anhui Province ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Twig Blight

Yellow Mountain fuzz tip, a cultivar of Camellia sinensis (L.) Kuntze, is commonly grown in the Yellow Mountain region in Anhui Province of China. During 2011 to 2012, leaf and twig blight on tea plants occurred from July to September in growing regions. Symptoms of blight on leaves of infected plants were detected in 30 to 60% of the fields visited and up to 500 ha were affected each year. Symptoms began as small, water-soaked lesions on young leaves and twigs and later became larger, dark brown, necrotic lesions, 1 to 3 mm in diameter on leaves and 2 to 5 mm long on twigs. To determine the causal agent, symptomatic leaf tissue was collected from plants in Gantang and Tangkou townships in September 2012. Small pieces of diseased tea leaves and twigs were surface-disinfested in 2% NaClO for 3 min, rinsed twice in distilled water, plated on potato dextrose agar, and incubated at 28°C for 5 days. Eleven isolates were recovered and all cultures produced white-to-gray fluffy aerial hyphae and were dark on the reverse of the plate. The hyphae were hyaline, branching, and septate. Setae were 2- to 3-septate, dark brown, acicular, and 78.0 to 115.0 μm. Conidiogenous cells were hyaline, short, branchless, cylindrical, and 11.3 to 21.5 × 4.2 to 5.3 μm. Conidia were hyaline, aseptate, guttulate, cylindrical, and 12.5 to 17.3 × 3.9 to 5.8 μm. Appresoria were ovate to obovate, dark brown, and 8.4 to 15.2 × 7.8 to 12.9 μm. DNA was amplified using the rDNA-ITS primer pair ITS4/ITS5 (3), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) primer pair GDF/GDR (2) and beta-tubulin 2 gene (Tub2) primer pair Btub2Fd/Btub4Rd (4). Sequences (GenBank Accession Nos. KC913203, KC913204, and KC913205) of the 11 isolates were identical and revealed 100% similarity to the ITS sequence of strain P042 of Colletotrichum gloeosporioides (EF423527), 100% identity to the GAPDH of isolate C07009 of C. gloeosporioides (GU935860), and 99% similarity to Tub2 of isolate 85 of C. gloeosporioides (AJ409292), respectively. Based on the above data, the 11 isolates were identified as C. gloeosporioides (Penz.) Penz. & Sacc. To confirm pathogenicity, Koch's postulate was performed and 4 ml of conidial suspension (1 × 105 conidia/ml) of each of the 11 isolates was sprayed on five leaves and five twigs per plant on four 12-month-old Yellow Mountain fuzz tip plants. Control plants were sprayed with distilled water. The inoculated plants were maintained at 28°C in a greenhouse with constant relative humidity of 90% and a 12-h photoperiod of fluorescent light. Brown necrotic lesions appeared on leaves and twigs after 7 days, while the control plants remained healthy. The experiments were conducted three times and the fungus was recovered and identified as C. gloeosporioides by both morphology and molecular characteristics. Tea plant blight caused by C. gloeosporioides was identified in Brazil (1), but to our knowledge, this is the first report of C. gloeosporioides causing tea leaf and twig blight on Yellow Mountain fuzz tip plants in Anhui Province of China. References: (1) M. A. S. Mendes et al. Page 555 in: Embrapa-SPI/Embrapa-Cenargen, Brasilia, 1998. (2) M. D. Templeton et al. Gene 122:225, 1992. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990. (4) J. H. C. Woudenberg et al. Persoonia 22:56, 2009.

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First Report of Alternaria Blight of Potato Caused by Alternaria tenuissima in China

Plant Disease ◽

10.1094/pdis-08-12-0763-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1246-1246 ◽

Cited By ~ 7

Author(s):

H. H. Zheng ◽

X. H. Wu

Keyword(s):

Solanum Tuberosum L ◽

Histone Gene ◽

Gansu Province ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Distilled Water ◽

Conidial Suspension ◽

Species Identity ◽

First Report ◽

Alternaria Blight

Potato (Solanum tuberosum L.) is grown worldwide as a major food crop. Potato early blight is an important disease caused by Alternaria solani (4). In 2011, diseased potato leaves with blight symptoms were collected from 21 sites (incidence averaged 60% for about 2,000 ha of potato fields examined) in Gansu Province, northwest China. Small pieces of tissue taken from the margin between healthy and diseased tissues were surface-disinfected in 0.3% NaOCl for 2 min, rinsed with sterilized, distilled water, then placed on potato dextrose agar (PDA) at 25°C in the dark. Two of 24 Alternaria isolates from single-spore cultures were identified preliminarily as A. tenuissima, and the remaining isolates as A. solani or A. alternata, based on morphological traits. Colony appearance on potato carrot agar (PCA) was loosely cottony under a day/night cycle of 8 h fluorescent light/16 h dark at 22°C for 7 days (3). The isolates were characterized by formation of unbranched conidial chains up to 12 conidia in length, with one or two lateral branches forming occasionally. Conidia were typically ovoid to obclavate, and ranged from 20.4 to 42.4 × 7.7 to 13.2 μm. Transverse septa and longitudinal septa of conidia varied from 1 to 6 and 0 to 2, respectively. Short conidiophores arose singly and were 15.1 to 76.8 μm long by 2.4 to 6.2 μm wide. The internal transcribed spacer (ITS) region of rDNA and partial coding sequence of a histone gene were amplified from genomic DNA of the two A. tenuissima isolates using the ITS1/ITS4 and H3-1a/H3-1b primers (2), respectively. The ITS sequences of the two isolates (GenBank Accession Nos. JX495165 and JX495166) were 100% identical to those of A. tenuissima strains sdau 07-100 and BL08-3 (GQ871507 and AB470887), as well as to other Alternaria species, but the partial histone gene sequences (JX495167 and JX495168) were 99% identical to those of A. tenuissima isolates CR27, MA1, MA6, and CN-L-01 (AF404622, AF404633, AF404634, and EF371552, respectively) with less similarity to those of other Alternaria spp. Therefore, the isolates were identified as A. tenuissima based on morphological and molecular characteristics. Pathogenicity tests were conducted by inoculating detached leaves (30 per isolate) from 45-day-old plants of potato cv. Favorita with 20 μl drops (one drop per leaf) of a conidial suspension containing 106 conidia/ml in sterilized, distilled water. Thirty control leaves were inoculated similarly with sterilized, distilled water. Inoculated leaves were incubated in chambers at 25°C and 90% RH with a 12-h photoperiod/day. After 7 days, symptoms on the inoculated leaves were similar to those naturally occurring on the original plants, and the two cultures were reisolated consistently from those leaves, and the species identity was confirmed by morphological and molecular characteristics, fulfilling Koch's postulates. The control leaves remained asymptomatic and Alternaria was not isolated from those leaves. Alternaria blight of potato caused by A. tenuissima was previously detected in Iran (1). To our knowledge, this is the first report of A. tenuissima causing blight on potatoes in China. References: (1) S. T. Ardestani et al. Iran. J. Plant Pathol. 45:83, 2010. (2) N. L. Glass and G. C. Donaldson. Appl. Environ. Microbiol. 61:1323, 1995. (3) E. G. Simmons. Alternaria. An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, the Netherlands, 2007. (4) J. E. van der Waals et al. Plant Dis. 88:959, 2004.

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First report of leaf spot disease caused by Stemphylium eturmiunum on American sweetgum (Liquidambar styraciflua L.) in China

Plant Disease ◽

10.1094/pdis-09-20-1877-pdn ◽

2021 ◽

Author(s):

Yun-fei Mao ◽

Li Jin ◽

Huiyue Chen ◽

Xiang-rong Zheng ◽

Minjia Wang ◽

...

Keyword(s):

Leaf Spot ◽

Disease Incidence ◽

Morphological Characteristics ◽

Liquidambar Styraciflua ◽

Leaf Spot Disease ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Wood Processing ◽

Light Yellow

American sweetgum (Liquidambar styraciflua L.) is an important tree for landscaping and wood processing. In recent years, leaf spots on American sweetgum with disease incidence of about 53% were observed in about 1200 full grown plants in a field (about 8 ha) located in Pizhou, Jiangsu Province, China. Initially, dense reddish-brown spots appeared on both old and new leaves. Later, the spots expanded into dark brown lesions with yellow halos. Symptomatic leaf samples from different trees were collected and processed in the laboratory. For pathogen isolation, leaf sections (4×4mm) removed from the lesion margin were surface sterilized with 75% ethanol for 20s and then sterilized in 2% NaOCl for 30s, rinsed three times in sterile distilled water, incubated on potato dextrose agar (PDA) at 25 °C in the darkness. After 5 days of cultivation, the pure culture was obtained by single spore separation. 6 isolate samples from different leaves named FXA1 to FXA6 shared nearly identical morphological features. The isolate FXA1 (codes CFCC 54675) was deposited in the China Center for Type Culture Collection. On the PDA, the colonies were light yellow with dense mycelium, rough margin, and reverse brownish yellow. Conidiophores (23–35 × 6–10 µm) (n=60) were solitary, straight to flexuous. Conidia (19–34 × 10–21 µm) (n=60) were single, muriform, oblong, mid to deep brown, with 1 to 6 transverse septa. These morphological characteristics resemble Stemphylium eturmiunum (Simmons 2001). Genomic DNA was extracted from mycelium following the CTAB method. The ITS region, gapdh, and cmdA genes were amplified and sequenced with the primers ITS5/ITS4 (Woudenberg et al. 2017), gpd1/gpd2 (Berbee et al. 1999), and CALDF1/CALDR2 (Lawrence et al. 2013), respectively. A maximum likelihood phylogenetic analysis based on ITS, gapdh and cmdA (accession nos. MT898502-MT898507, MT902342-MT902347, MT902336-MT902341) sequences using MEGA 7.0 revealed that the isolates were placed in the same clade as S. eturmiunum with 98% bootstrap support. All seedlings for pathogenicity tests were enclosed in plastic transparent incubators to maintain high relative humidity (90%-100%) and incubated in a greenhouse at 25°C with a 12-h photoperiod. For pathogenicity, the conidial suspension (105 spores/ml) of each isolate was sprayed respectively onto healthy leaves of L. styraciflua potted seedlings (2-year-old, 3 replicate plants per isolate). As a control, 3 seedlings were sprayed with sterile distilled water. After 7 days, dense reddish-brown spots were observed on all inoculated leaves. In another set of tests, healthy plants (3 leaves per plant, 3 replicate plants per isolate) were wound-inoculated with mycelial plugs (4×4mm) and inoculated with sterile PDA plugs as a control. After 7 days, brown lesions with light yellow halo were observed on all inoculation sites with the mycelial plugs. Controls remained asymptomatic in the entire experiment. The pathogen was reisolated from symptomatic tissues and identified as S. eturmiunum but was not recovered from the control. The experiment was repeated twice with the similar results, fulfilling Koch’s postulates. S. eturmiunum had been reported on tomato (Andersen et al. 2004), wheat (Poursafar et al. 2016), garlic (L. Fu et al. 2019) but not on woody plant leaves. To our knowledge, this is the first report of S. eturmiunum causing leaf spot on L. styraciflua in the world. This disease poses a potential threat to American sweetgum and wheat in Pizhou.

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First Report of Coffee Canker Disease Caused by Ceratocystis fimbriata in China

Plant Disease ◽

10.1094/pdis-08-21-1808-pdn ◽

2022 ◽

Author(s):

Kecheng Xu ◽

Ruiqi Zhang ◽

Haixia Lu ◽

Jinglong Zhang ◽

Jing Yang ◽

...

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Molecular Characteristics ◽

Outer Cell ◽

Leaf Margin ◽

Malt Extract ◽

Distilled Water ◽

Ceratocystis Fimbriata ◽

Canker Disease ◽

First Report

Coffee (Coffea arabica L.) is one of the most important agricultural commodities in the world market. As an important cash crop in China, coffee is cultivated mainly in Yunnan and Hainan provinces. During October 2013 and September 2020, coffee trees showing typical dieback and wilt symptoms were found in Nanping town (N 22° 38', E 101° 0'), Pu’er, and Puwen town (N 22° 32', E 101° 4'), Xishuangbanna in Yunnan province, China. Symptomatic trees initially exhibited yellowing of foliage, expanding in size along the leaf margin, then became blighted and dry, and the internal xylem in main stem discolored brown to black. Infected trees eventually developed dieback and wilt. Disease incidence ranged from 10% to 22% and 25% to 40% of crown symptoms in the affected coffee trees. In extreme cases, 50% out of 380 trees were affected. The stems of coffee trees with typical symptoms were collected, and then the diseased 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, fungal mycelium was observed growing from the tissue. Three isolates (C3-1, C3-2, and C3-2-1) were obtained by picking spore masses from the apices of perithecia and transferring them to malt extract agar (MEA) medium and incubated at 25°C for 10 days to observe the cultural features. In culture, colonies reaching 65 mm within 10 days, mycelium initially white, then becoming light blue-green. After 6 days of formation, perithecia were black, globose (123.8 - 173.4 μm × 138.2 - 180.6 μm), and showed a long black neck (414.2 - 650.0 μm). Ascospores with outer cell wall forming a brim, hat-shaped, accumulating in a mucilaginous mass at the tips of ostiolar hyphae (4.3 μm × 6.0 μm). Cylindrical endoconidia (14.1 - 45.2 μm × 3.5 - 5.7 μm) were hyaline. Chain of barrel-shaped conidia (6.6 - 10.2 μm × 6.8 - 8.8 μm) were found. Aleuroconidia (10.8 - 16.9 μm × 9.1 - 13.0 μm) were olive-brown, ovoid or obpyriform, and smooth. Morphological characteristics of the fungus were consistent with the description of Ceratocystis fimbriata Ellis & Halst. (Engelbrecht and Harrington 2005). The three isolates were used for molecular identification, and their genomic DNA was extracted using the chelex-100 method (Xu et al. 2020). The internal transcribed spacer (ITS) region of rDNA was sequenced using the procedures of Thorpe et al. (2005). Analysis of the ITS sequence data (GenBank accessions KY580836, KJ511480, and KJ511479) showed that the isolates were 100% homologous to isolates of C. fimbriata from Punica granatum, Camellia sinensis, and Cucumis sativus in China (GenBank accessions KY580891, KY580870, and MH535909, respectively) by BLAST analysis. Neighbor-joining (NJ) phylogenetic analysis was performed using MEGA 6.06 based on the ITS sequences. The three isolates were clustered on the same clade with other C. fimbriata isolates with a high bootstrap value (90%). Therefore, the fungus was identified as C. fimbriata based on both morphological and molecular characteristics. Pathogenicity of the three isolates was tested by inoculating one-year-old pot grown coffee seedlings (C. arabica) through drenching the loams with 30 ml spore suspension (1 × 106 spores/ml). Control plants were inoculated with 30 ml of sterile distilled water. The trees were kept in a controlled greenhouse at 25°C and watered weekly. One month after inoculation, all inoculated plants produced typical dieback and wilt symptoms, whereas the control trees showed no symptoms. The same fungus was isolated from the inoculated trees on PDA and identified as C. fimbriata according to the methods described above, and no fungal growth was observed in the controls, thus fulfilling the Koch's postulates. Coffee canker disease caused by C. fimbriata has been reported in Indonesia and Colombia (Marin et al. 2003). To our knowledge, this is the first report of C. fimbriata causing canker disease of coffee trees in China.

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First report of leaf spot disease caused by Colletotrichum siamense on Cornus hongkongensis (Hemsl.) in China

Plant Disease ◽

10.1094/pdis-11-20-2525-pdn ◽

2021 ◽

Author(s):

Qing-Hai Wang ◽

Yang Zhang ◽

Yu-tong Zhang ◽

Dong Li ◽

Xiao-li Lin ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Disease Incidence ◽

Morphological Characteristics ◽

Control Measures ◽

Bootstrap Support ◽

Leaf Spot Disease ◽

Pathogenicity Test ◽

Accurate Identification ◽

First Report ◽

Colletotrichum Siamense

Cornus hongkongensis (Hemsl.) is an excellent ornamental tree species in China and elsewhere. In 2019, C. hongkongensis anthracnose was firstly observed at the campus of Jiangxi Agricultural University (JXAU) (28°45′56″N, 115°50′21″E), then found in parks, Nanchang, China. In early August, the disease appeared and lasted until the leaves dropped (November). The disease incidence was above 60%, and the diseased leaf rate was above 70%. The lesions mostly appeared along the leaf edges. Some small round to irregular lesions also developed in other parts of the leaves. These diseased leaves had circular or irregularly shaped spots with gray-white color in the center and dark brown on the edge of the lesions. Later, the lesions became necrotic and shriveled. As the disease progressed, the spots coalesced so that affected leaves appeared blighted (Supplementary Figure 1 A-C). To identify the pathogen, leaves with typical symptoms from the campus of JXAU were collected and small pieces (5 × 5 mm) from the lesion borders were surfaced sterilized in 70% ethanol for 30 s, followed by 1 min in 3% NaOCl, and then rinsed with sterile distilled water three times. Leaf pieces were placed on potato dextrose agar (PDA) and incubated at 25 °C under a 12-h light/dark cycle (3000 lx). Pure cultures were obtained from individual conidia by single spore isolates. For studies of microscopic morphology, a representative isolate JX-S4 was subcultured on PDA. The colony of JX-S4 was white and turning gray and light gray on the reverse side, producing dark-green pigmentation near the center (Supplementary Figure 1 D). The conidia were one-celled, straight, hyaline, subcylindrical with rounded ends and 16.9 ± 1.6 × 6.0 ± 0.6 µm (n = 50) in size. Appressoria were one-celled, pale brown, thick-walled, ellipsoidal, and measured 8.7 ± 1.7 × 6.4 ± 0.8 µm (n = 50) (Supplementary Figure 1 E, F). The morphological characteristics of JX-S4 matched those of the Colletotrichum siamense species (Weir et al. 2012). For accurate identification, the internal transcribed spacer (ITS) and the genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS-I), beta-tubulin 2 (TUB2), and calmodulin (CAL) were respectively amplified with primers ITS1/ITS4, GDF/GDR, CHS-79F/CHS-345R, βt2a/βt2b, and CL1/CL2. The sequences were deposited in GenBank (Accession nos. MT587807, MT628710, MT628709, MT628711, and MT628708). Phylogenetic analysis was calculated with concatenated sequences (ITS, GAPDH, CHS-I, CAL, and TUB2) using MEGA 7. In the maximum likelihood phylogenetic tree, Isolate JX-S4 was clustered with C. siamense with 93% bootstrap support (Supplementary Figure 2). Based on the morphological characteristics and phylogenetic analysis, JX-S4 was identified as C. siamense. Pathogenicity test of JX-S4 was verified on 45 attached healthy leaves from three C. hongkongensis plants (10-year-old) at the campus of JXAU inoculated with mycelial plugs (φ=5 mm) from the culture edge (6-day-old) on PDA. And an additional 45 healthy leaves were inoculated with PDA plugs as controls. The leaves were wounded with a red-hot needle (φ=0.5 mm). All treatment and control leaves were wrapped up with black plastic bags to keep them moist for 2 days. The pathogenicity tests were repeated twice. Within 7 days, all the inoculated leaves developed the lesions, which were similar to those observed in the field. Control leaves were asymptomatic (Supplementary Figure 1 G, H). The same fungus was re-isolated from the symptomatic tissues, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. siamense causing C. hongkongensis anthracnose. This finding provides crucial information for managing this disease. For example, when diagnosing Cornus anthracnose, C. siamense needs to be looked out for and appropriate control measures implemented.

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First report that Colletotrichum aenigma causes leaf spots on Aquilaria sinensis in China

Plant Disease ◽

10.1094/pdis-11-20-2381-pdn ◽

2021 ◽

Author(s):

Cong Li ◽

Jun Ang Liu ◽

Guo ying Zhou

Keyword(s):

Control Plant ◽

Central Area ◽

Molecular Characteristics ◽

Fluorescent Light ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

Aquilaria Sinensis ◽

First Report ◽

Leaf Spots

Aquilaria sinensis (Lour.) Spreng, also known as eaglewood, belongs to the Thymelaeaceae family and has a considerably high medicinal value. It has been enlisted as the class II national key protective plant. In June 2019, about 15 percent of A. sinensis treelets in a forest area of China's Hainan province were observed to have the anthracnose symptoms. The diseased spots on leaves of A. sinensis treelets were usually round or irregular with pale yellow edges. The color of the center of the lesion was firstly light brown and then black or yellowish-brown. Small pieces of tissue from the edge of the leaf spots were surface sterilized in 75% alcohol for the 60s, washed twice with sterile distilled water, and then cultivated at 28 °C in darkness on potato dextrose agar (PDA) medium. One fungus was systematically isolated to get pure cultures. The culturing of the three isolates was carried out in PDA media at 28 °C for a week. The average diameter of the collateral colony was 6.80 ±0.60 cm. Initially, the fungal colonies were white aerial mycelium and the central area of the colonies slowly turned jacinth. After seven days, the central mycelium turns grayish-green and the colonies’ undersurfaces were grey to white. The colony's surfaces were fluffy and round with smooth edges. Conidia were cylindrical, smooth, and transparent, with a slight indentation in the middle and uneven distribution of small particles inside, 12.5–20.6×3.5–6.8 µm (ave=15.9±1.40×5.18±1.07, n=50). Appressoria were typically elliptic or irregular and brown to dark brown. The isolates were characterized as Colletotrichum gloeosporioides species complex on the basis of the conidial morphology and culture representation, (Deng et al. 2017; Weir et al. 2012). To further verify the identification of the species, CX-0301, the isolated representative strains were extracted for genomic DNA. mating type 1-2-1 (Mat-1-2-1) ApMat, actin (ACT) gene, chitin synthase (CHS), and beta-tubulin (TUB2) gene were amplified using the primer pairs VcaMat-5F/VcaMat-5R, ACT-512F/ACT-783R, CHS-1-79F/CHS-1-354R, and TUB2-T1/Bt2b, respectively (Damm et al. 2012; Du et al. 2005). The homologous sequences of MN310694, MN310693, MN310692, and MN310691 were submitted to GenBank. These genes have ≥a 97% sequence similarity to the genes of Colletotrichum aenigma (MG717319.1, MG717317.1, MH476565.1, MH853679.1, respectively) in GenBank. These morphological and molecular characteristics identified that the pathogen is C. aenigma. (Weir et al. 2012). To further verify the isolated pathogen, the pathogenicity test was performed on uninfected healthy 2-year-old eaglewood seedlings. The conidial suspension (1×106 conidia/ml) of 5ml was sprayed on both surfaces of 10 leaves of plants of the same age and height and the controls were treated solely with distilled water (Deng et al. 2017). Upon completion of inoculation, plants were kept under greenhouse conditions with an assigned temperature of 28 ± 2°C while keeping relative humidity to 90% on a 12-h fluorescent light/dark regime. Anthracnose-like symptoms were observed 6 days postinoculation. The control plant tissues remained healthy. Follow up reisolation of C. enigma culture was obtained in PDA agar plates from leaf infected lesions, and the morphological features were found to be consistent with that of CX-0301 isolate, satisfying Koch's postulates. Based on the characterized information, it is the first report of Colletotrichum aenigma responsible for causing leaf spots on Aquilaria sinensis in China. Thereby, this provides a theoretical reference for the research and control of anthracnose on A. sinensis.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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First report of Colletotrichum siamense Causing Blossom Blight on Thai basil (Ocimum basilicum L.) in Malaysia

Plant Disease ◽

10.1094/pdis-06-20-1371-pdn ◽

2020 ◽

Author(s):

Siti Izera Ismail ◽

Nur Adlina Rahim ◽

Dzarifah Zulperi

Keyword(s):

Disease Incidence ◽

Its Region ◽

Ocimum Basilicum ◽

Distilled Water ◽

Single Spore ◽

First Report ◽

Plastic Bags ◽

Colletotrichum Siamense ◽

Primer Sets ◽

Acca Sellowiana

Thai basil (Ocimum basilicum L.) is widely cultivated in Malaysia and commonly used for culinary purposes. In March 2019, necrotic lesions were observed on the inflorescences of Thai basil plants with a disease incidence of 60% in Organic Edible Garden Unit, Faculty of Agriculture in the Serdang district (2°59'05.5"N 101°43'59.5"E) of Selangor province, Malaysia. Symptoms appeared as sudden, extensive brown spotting on the inflorescences of Thai basil that coalesced and rapidly expanded to cover the entire inflorescences. Diseased tissues (4×4 mm) were cut from the infected lesions, surface disinfected with 0.5% NaOCl for 1 min, rinsed three times with sterile distilled water, placed onto potato dextrose agar (PDA) plates and incubated at 25°C under 12-h photoperiod for 5 days. A total of 8 single-spore isolates were obtained from all sampled inflorescence tissues. The fungal colonies appeared white, turned grayish black with age and pale yellow on the reverse side. Conidia were one-celled, hyaline, subcylindrical with rounded end and 3 to 4 μm (width) and 13 to 15 μm (length) in size. For fungal identification to species level, genomic DNA of representative isolate (isolate C) was extracted using DNeasy Plant Mini Kit (Qiagen, USA). Internal transcribed spacer (ITS) region, calmodulin (CAL), actin (ACT), and chitin synthase-1 (CHS-1) were amplified using ITS5/ITS4 (White et al. 1990), CL1C/CL2C (Weir et al. 2012), ACT-512F/783R, and CHS-79F/CHS-345R primer sets (Carbone and Kohn 1999), respectively. A BLAST nucleotide search of ITS, CHS-1, CAL and ACT sequences showed 100% similarity to Colletotrichum siamense ex-type cultures strain C1315.2 (GenBank accession nos. ITS: JX010171 and CHS-1: JX009865) and isolate BPDI2 (CAL: FJ917505, ACT: FJ907423). The ITS, CHS-1, CAL and ACT sequences were deposited in GenBank as accession numbers MT571330, MW192791, MW192792 and MW140016. Pathogenicity was confirmed by spraying a spore suspension (1×106 spores/ml) of 7-day-old culture of isolate C onto 10 healthy inflorescences on five healthy Thai basil plants. Ten infloresences from an additional five control plants were only sprayed with sterile distilled water and the inoculated plants were covered with plastic bags for 2 days and maintained in a greenhouse at 28 ± 1°C, 98% relative humidity with a photoperiod of 12-h. Blossom blight symptoms resembling those observed in the field developed after 7 days on all inoculated inflorescences, while inflorescences on control plants remained asymptomatic. The experiment was repeated twice. C. siamense was successfully re-isolated from the infected inflorescences fulfilling Koch’s postulates. C. siamense has been reported causing blossom blight of Uraria in India (Srivastava et al. 2017), anthracnose on dragon fruit in India and fruits of Acca sellowiana in Brazil (Abirami et al. 2019; Fantinel et al. 2017). This pathogen can cause a serious threat to cultivation of Thai basil and there is currently no effective disease management strategy to control this disease. To our knowledge, this is the first report of blossom blight caused by C. siamense on Thai basil in Malaysia.

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