First report of leaf blight on Magnolia coco caused by Nothophoma quercina in China

Magnolia coco (Lour.) DC. is an ornamental shrub and widely cultivated in southern China (Nana et al. 2017). In April 2020, leaf blight symptoms were observed on the leaves of M. coco in the Chengdu campus of Sichuan Agricultural University (30°42′19.92″N, 103°51′30.61″E, 493 m) where didn’t have great protection, with roughly 70% leaves per plant were diseased. The initial symptoms presented on the leaf apex, which was manifested as dark brown spots surrounded with obvious yellowish halo (Fig. 1). As the disease progressed, spots gradually enlarged and coalesced covering the leaf, and severe infection finally caused leaf necrosis and plant decline. Four specimens from different diseased plants were used for pathogen isolation and morphological observation. Four fungal isolates were obtained from four specimens, following Chomnunti et al. (2014). Colonies on potato dextrose agar (PDA) medium were initially white and then light brown to dark brown. Pycnidia measured 284-427 × 326-554 μm (x=372.8 μm × 476.1 μm, n=20), and were brownish-black to black, broadly globose to irregular. The pycnidial wall measured 16-27 μm wide (n=20) and was composed of hyaline to brown cells of textura angularis. Conidiophores were absent, and the conidiogenous cells are pear-shaped, colorless, and smooth. Conidia measured 5-8 × 4-6 μm (x=6.5 μm × 4.6 μm, n=50), and were elliptical or subglobose, thick-walled, aseptate, hyaline, smooth, brown. These asexual structures were similar to Nothophoma quercina (Syd. & P. Syd.) Qian Chen & L. Cai described by Chen et al. (2017). The genomic DNA of representative isolate SICAUCC 21-0011 was extracted, and the internal transcribed spacers (ITS), 28S large subunit rDNA (LSU), RNA polymerase II large subunit 2 (RPB2), and beta-tubulin (TUB2) regions were amplified using the primer pairs ITS5/ITS4, LR0R/LR5, FRPB2-5F/FRPB2-7cR, and T1/BT4R, respectively. The accession numbers deposited in GenBank were MW541930 (ITS), MW541934 (LSU), MW883395 (RPB2), and MW883394 (TUB2). Nucleotide BLAST showed high homology with the sequences of N. quercina, viz. GU237900 (ITS, 485/486, 99.79%), EU754127 (LSU, 862/862, 100%), KT389657 (RPB2, 593/596, 99.49%), and GU237609 (TUB2, 333/335, 99.40%). Phylogenetic analyses based on a combined dataset showed 100% bootstrap support values in a clade with N. quercina complexes (Fig. 2). Four healthy potted plants (2-years-old) with 15 to 20 leaves per plant were sprayed with conidial suspension (105 conidia/mL) prepared from 4-week-old cultures of SICAUCC 21-0011, which incubated on PDA at 25℃, onto the wounded sites via pin-prick inoculation described by Desai et al. (2019). Another four plants were sprayed with sterilely distilled water as controls. Inoculated plants were cultured in a growth chamber (25℃, 95% relative humidity, and 12-h photoperiod). About 30 days later, brown spots were found on the inoculated leaves, which were similar to those observed in the field. There were no symptoms on the control plants, and the pathogen was re-isolated from the diseased leaves and characterized morphologically. N. quercina has been reported on Photinia × fraseri Dress, Aucuba japonica, Malus micromalus, and Chaenomeles sinensis (Mohamed et al. 2019, Lv et al. 2020, Zou et al. 2021). To our knowledge, this is the first report of leaf blight on M. coco caused by N. quercina. M. coco is one of the important ornaments in the courtyard, street, and park in China, and the risk of this pathogen needs further exploration and effective control measures should be made. Qian Zeng, Yicong Lv, and Xinyue Li contributed equally to this work.

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First Report of a Leaf Blight Caused by Pyricularia pennisetigena on Cenchrus echinatus in Paraguay

Plant Disease ◽

10.1094/pdis-02-21-0287-pdn ◽

2021 ◽

Author(s):

Cinthia C. Cazal-Martínez ◽

Yessica Magaliz Reyes Caballero ◽

Alice Chávez ◽

Pastor Enmanuel Pérez Estigarribia ◽

Alcides Rojas ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Large Subunit ◽

Fungal Species ◽

Leaf Blight ◽

Internal Transcribed Spacers ◽

Conidial Suspension ◽

Sterile Water ◽

Potential Threat ◽

First Report ◽

Microscopic Features

The genus Pyricularia contains several fungal species known to cause diseases on plants in the Poaceae family (Klaubauf et al. 2014; Wang et al. 2019). While sampling for P. oryzae during March-2015 and April-2018, common weed Cenchrus echinatus L. was observed with leaf lesions in and around experimental wheat fields in the departments of Canindeyú and Itapúa. C. echinatus samples from both locations displayed similar leaf lesions, varying from small light brown pinpoint to elliptical brown lesions with greyish center. Symptomatic leaves were surface disinfested and cultured on potato dextrose agar (PDA) amended with 1% gentamicin at 25°C. Two monosporic isolates were obtained, one from Itapúa (ITCeh117) and the other from Canindeyú (YCeh55). The isolates were subsequently grown on oatmeal agar (OA) and PDA under a 12-h photoperiod at 25°C and evaluated after ten days for colony diameter, sporulation, macroscopic and microscopic features. Colonies on OA reached up to 4.8 cm diameter and were light grey, whereas colonies on PDA reached up to 5.3 cm diameter and were brown with grey centers, with cottony mycelium and broad white rims. Mycelium consisted of smooth, hyaline, branched, septate hyphae 4-4.5 µm diameter. Conidiophores were erect, straight or curved, unbranched, medium brown and smooth. Conidia were solitary, pyriform, pale brown, smooth, granular, 2-septate, 32-33 × 9-10 μm; truncated with protruding hilum and varied in length from 1.0 to 1.5 μm and diameters from 2.0 to 2.2 μm. Both isolates were similar and identified as Pyricularia pennisetigena, according to morphological and morphometric characteristics (Klaubauf et al. 2014). Subsequently, genomic DNA was extracted from each isolate using the primers described in Klaubauf et al. (2014) to amplify and sequence the internal transcribed spacers (ITS), partial large subunit (LSU), partial RNA polymerase II large subunit gene (RPB1), partial actin gene (ACT), and partial calmodulin gene (CAL). Sequences from each isolate (YCeh55/ITCeh117) were deposited in GenBank with the following submission ID for ITS: MN947521/MN947526, RPB1: MN984710/MN984715, LSU: MN944829/MN944834, ACT: MN917177/MN917182, and CAL: MN984688/MN984693. Phylogenetic analysis was conducted using the software Beast v1.10.4. The results obtained from the concatenated matrix of the five loci placed these isolates in the P. pennisetigena clade. To confirm pathogenicity, each isolate was adjusted to 5×104 conidia/ml of sterile water and C. echinatus plants were sprayed with the conidial suspension for isolate YCeh55, ITCeh117 or sterile water using an oilless airbrush sprayer until runoff. The three treatments were kept in the greenhouse at 25-28°C and about 75% relative humidity under natural daylight. Each treatment included three to five inoculated plants and 10 leaves were evaluated per treatment. Symptoms were observed 8-15 days after inoculation and were similar to those originally observed in the field for both isolates, whereas the control plants remained asymptomatic. P. pennisetigena was re-isolated from the inoculated leaves fulfilling Koch’s postulates. To our knowledge, this is the first report of leaf blight on C. echinatus caused by P. pennisetigena in Paraguay. The occurrence of P. pennisetigena in the region and its ability to infect economically important crops such as wheat and barley (Klaubauf et al. 2014; Reges et al., 2016, 2018) pose a potential threat to agriculture in Paraguay.

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First Report of Nigrospora Leaf Blight on Sesame Caused by Nigrospora sphaerica in China

Plant Disease ◽

10.1094/pdis-07-13-0684-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 842-842 ◽

Cited By ~ 3

Author(s):

H. Zhao ◽

H. Y. Liu ◽

X. S. Yang ◽

Y. X. Liu ◽

Y. X. Ni ◽

...

Keyword(s):

Morphological Characteristics ◽

Leaf Blight ◽

Morphological Data ◽

Its2 Region ◽

Oilseed Crop ◽

Conidial Suspension ◽

First Report ◽

Initial Symptoms ◽

Central Regions ◽

Leaf Pathogen

Sesame (Sesamum indicum L.) is an important oilseed crop widely grown in the central regions of China. A new leaf blight has increasingly been observed in sesame fields in Anhui, Hubei, and Henan provinces since 2010. Approximately 30 to 40% of the plants were symptomatic in the affected fields. Initial symptoms were yellow to brown, irregularly shaped lesions. Lesions later expanded and the affected leaves tuned grayish to dark brown and wilted, with a layer of whitish mycelial growth on the underside. Severe blighting caused the center of lesions to fall out, leaving holes in the leaves. Sections of symptomatic leaf tissues were surface-sterilized in 75% ethanol for 30 s, then in 1% HgCl2 for 30 s, rinsed three times in sterile distilled water, and plated onto potato dextrose agar (PDA). The resulting fungal colonies were initially white, and then became grayish-brown with sporulation. Conidia were single-celled, black, smooth, spherical, 14.2 to 19.8 μm (average 17.1 μm) in diameter, and borne on a hyaline vesicle at the tip of each conidiophore. Morphological characteristics of the isolates were similar to those of Nigrospora sphaerica (1). To verify the identification based on morphological features, the ITS1-5.8S-ITS2 region of the ribosomal RNA was amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primers (3), and then sequenced and compared to the GenBank database through a BLAST search. Comparison of the sequence revealed 100% similarity to N. sphaerica (GenBank Accession No. JF817271.1). On the basis of morphological data and the ITS rDNA sequence, the isolate was determined to be N. sphaerica. Pathogenicity tests were conducted using fresh and healthy sesame leaves of 10 plants. A conidial suspension (106 conidia/ml) collected from a 7-day-old culture on PDA was used for inoculation. Leaves of 10 plants were spray-inoculated with the spore suspension at the 6-week-old growth stage, and an additional 10 plants were sprayed with sterile water. Inoculated plants were covered with polyethylene bags to maintain high humidity. Plants were kept at 28°C and observed for symptom every day. Ten to 15 days after inoculation, inoculated leaves developed blight symptoms similar to those observed on naturally infected leaves. No symptoms were observed on the control leaves. N. sphaerica was re-isolated from the inoculated leaves, thus fulfilling Koch's postulates. N. sphaerica has been reported as a leaf pathogen on several hosts worldwide (2). To our knowledge, this is the first report of Nigrospora leaf blight on sesame caused by N. sphaerica in China. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. CMI, Kew, Surrey, UK, 1971. (2) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ . July 01, 2013. (3) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First Report of Leaf Blight in Chinese Hickory (Carya cathayensis) Caused by Alternaria petroselini in China

Plant Disease ◽

10.1094/pdis-02-13-0176-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1253-1253 ◽

Cited By ~ 1

Author(s):

Y. H. Liu ◽

C. Q. Zhang ◽

B. C. Xu

Keyword(s):

Management Practices ◽

Morphological Characteristics ◽

Morphological Characterization ◽

Leaf Blight ◽

Internal Transcribed Spacers ◽

Infected Leaf ◽

First Report ◽

Plastic Bags ◽

Initial Symptoms ◽

Carya Cathayensis

Chinese hickory (Carya cathayensis) is one of the important economic forest crops in Zhejiang and Anhui Provinces, China. In 2012, nearly 40% of hickory orchards and 6.8% of hickory trees were affected by leaf blight in Zhejiang. Initial symptoms consisted of small, brown, water-soaked lesions, which subsequently enlarged and developed a black sporulating necrotic center surrounded by a chlorotic halo. Infected leaf samples collected from 25 different orchards in Lin'an and 13 different orchards in Chun'an were surface sterilized with 1.5% sodium hypochlorite for 1.5 min, rinsed in water, plated on 2% potato dextrose agar (PDA), and incubated at 25°C in the dark for 1 week. Single conidium cultures were consistently isolated and cultured on PDA and V8 agar for morphological characterization (1,3). On both agar media, colonies were dark olive brown with smooth margins and concentric rings of sporulation. Conidia were solitary, darkly pigmented, predominantly ovoid-subsphaeroid, and 23 to 52 × 13 to 23 μm with up to six or seven transepta and one to three longisepta. The ribosomal internal transcribed spacers ITS1 and ITS2 of 10 isolates were amplified using primers ITS1/ITS4 on DNA extracted from mycelium and nucleotide sequences showed 100% similarity to that of A. petroselini (GenBank Accession Nos. AY154685.1 and EU807868.1). To confirm pathogenicity, 10 uninfected leaves from each of 10 C. cathayensis trees were sprayed either with a conidia suspension (105 conidia per ml) or with distilled water only to serve as an un-inoculated control. Leaves were subsequently wrapped in plastic bags to retain moisture, and incubated for 48 h. After 1 week, 8 of 10 isolates caused lesions identical to those initially described whereas no symptoms developed on water inoculated leaves. Cultures reisolated from lesions and cultured on PDA exhibited morphological characteristics identical to A. petroselini (1,2,3), confirming Koch's postulates. To our knowledge, this is the first report of leaf blight in C. cathayensis, and this identification would allow producers to identify for appropriate management practices. References: (1) P. M. Kirk et al. The Dictionary of the Fungi, 10th edition, page 159. CABI Bioscience, UK, 2008. (2) B. M. Pryor et al. Mycologia 94:49, 2002. (3) E. G. Simmons. Alternaria: An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007.

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First Report of Berkeleyomyces basicola Causing Black Root Rot on Lisianthus (Eustoma grandiflorum) in China

Plant Disease ◽

10.1094/pdis-08-21-1724-pdn ◽

2021 ◽

Author(s):

Yishuo Huang ◽

Xuewen Xie ◽

Yanxia Shi ◽

A LI CHAI ◽

Lei Li ◽

...

Keyword(s):

Root Rot ◽

Mangifera Indica ◽

Large Subunit ◽

Morphological Characteristics ◽

Internal Transcribed Spacers ◽

Likelihood Method ◽

Conidial Suspension ◽

Eustoma Grandiflorum ◽

Black Root Rot ◽

First Report

Lisianthus (Eustoma grandiflorum (Raf.) Shinn.) is an important ornamental plant ranking in the top 10 cut flowers worldwide (Xiao et al., 2018). In 2020 and 2021, black root rot was found as a major disease limiting lisianthus production in Yunnan Province, China. Black root rot was first observed in early July 2020 on lisianthus grown in a commercial flower-production plantation, with nearly 60% plants infected. Symptoms appeared as coalescing necrotic lesions leading to black discoloration of the roots. Root damage induced by disease resulted in insufficient water and nutrient uptake by the plant, causing stunting and whole-plant wilting. The pathogen could not infect the intact endodermis, and vascular tissues below the discolored cortical tissue remained healthy. Symptomatic roots were surface sterilized using 1% NaClO for 1 min, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25°C for 7 days in the dark. The morphological characteristics were basically consistent: the colonies were white to gray in color, and the conidiophores were colorless to brown, solitary or clustered. Conidia were single-celled, colorless, rod-shaped, and obtuse at both ends. Chlamydospores were dark brown, clustered or solitary. The morphological characteristics of the pathogen were similar to those of Berkeleyomyces basicola (Berk. & Broome) W.J. Nel, Z.W. de Beer, T.A. Duong & M.J. Wingf. (Nakane et al. 2019). DNA was extracted from mycelia of representative isolate TB using the Plant Genomic DNA Kit (Tiangen, Beijing, China). The internal transcribed spacers (ITS), DNA replication licensing factor (MCM7), ribosomal large subunit (LSU), and 60S ribosomal protein RPL10 (60S) regions were amplified with primer pairs ITS1/ITS4 (Groenewald et al. 2013), MCM7-for/MCM7-rev, LR0R/LR5, and 60S-506F/60S-908R, respectively (Nel et al. 2018). Phylogenetic analysis of multiple genes (Bakhshi et al. 2018) was conducted with the maximum likelihood method using MEGA7. The sequences of our isolate (TB) and three published sequences of B. basicola were clustered into one clade with a 100% bootstrapping value. The accession numbers of B. basicola reference sequences are MF952423 (ITS), MF967079 (MCM7), MF948658 (LSU), and MF967072 (60S) of isolate CMW6714; MF952428 (ITS), MF967088 (MCM7), MF948661 (LSU), and MF967073 (60S) of isolate CMW25440; MF952429 (ITS), MF967102 (MCM7), MF948659 (LSU), and MF967075 (60S) of isolate CMW49352. The sequences of TB have been deposited in GenBank with accession numbers MZ351733 for ITS, MZ695817 for MCM7, MZ695816 for LSU, and MZ695815 for the 60S region. To verify the pathogenicity of the fungus, inoculations were performed on ten 2-month-old potted lisianthus plants by dipping the roots into a conidial suspension (105 spores/ml) for 2 h. Ten plants were mock inoculated with distilled water as a control. Symptoms of black root rot were observed 30 days after inoculation, whereas the control roots remained healthy. The causal fungus has a host range of over 230 species and is a destructive pathogen of many crops and ornamental plants, including cotton (Gossypium barbadense L.), tobacco (Nicotiana tabacum L.) and mango (Mangifera indica L.) (Shukla et al. 2021; Toksoz and Rothrock 2009). This is the first report worldwide of B. basicola infecting lisianthus. This discovery is of great importance for Chinese flower growers because this fungus is well established in the observed area, and effective measures are needed to manage this disease.

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First Report of Curvularia richardiae Causing Leaf Blight on Richardia brasiliensis in Brazil

Plant Disease ◽

10.1094/pdis-01-14-0072-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1740-1740

Author(s):

W. S. Lisboa ◽

L. L. Duarte ◽

R. W. Barreto

Keyword(s):

Large Subunit ◽

Culture Collection ◽

Leaf Blight ◽

The Other ◽

Conidial Suspension ◽

Weed Species ◽

First Report ◽

Plant Press ◽

Agricultural Weed ◽

The Tropics

Richardia brasiliensis (Rubiaceae), also known as white eye or ‘poaia-branca’ in Brazil, is an important agricultural weed in the tropics (2). Relatively little is known about diseases affecting this species. In March 2013, all of the plants of this weed species invading an orchid plantation in Nova Friburgo (State of Rio de Janeiro) and a private orchard at Viçosa (State of Minas Gerais) in Brazil were found to bear intense leaf blight symptoms. Lesions were circular to elliptical, 1.4 to 10.5 mm in diameter, grayish to pale brown, and coalesced leading to necrosis of large areas of the leaves. Leaf samples were collected, dried in a plant press, and representative specimens deposited in the local herbarium at the Universidade Federal de Viçosa (Accession Nos. VIC 39759 and VIC 39760). A fungus found in association with diseased tissues was isolated by directly transferring conidia from infected leaves onto PDA plates, and two isolates were deposited in a local culture collection (COAD Accession Nos. 1335 and 1443). Conidia were removed from infected leaves using a scalpel, and mounted in lactophenol and lactofuchsin for observation with a light microscope (Olympus BX 51). Conidiophores were epiphylous, isolated, subcylindrical, straight to slightly curved, 97.5 to 170.0 × 5.0 to 8.0 μm, 2 to 6 septate, unbranched, pale brown and paler towards the apex, and smooth. Conidia were straight to slightly curved, pyriform to obovoid, 35.5 to 43.5 × 12.5 to 25.0 μm, with the apex rounded and the base subacute, 1 to 3 distoseptate, the subterminal cell often dark brown and larger than the other cells (sometimes leading to the distortion and curving of conidia); the other cells were golden brown and the conidia were smooth. The morphology of the fungus on R. brasiliensis was equivalent to that described for Curvularia richardiae (1). Genomic DNA was extracted from a 7-day-old pure culture of both isolates, and the large subunit (LSU) region of ribosomal DNA (rDNA) was amplified with the primers LR0R/LR5 (3). The resulting sequences were deposited in GenBank (KF880800 and KF880801). A BLASTn search revealed 99% similarity of the two isolates from Brazil with the LSU sequence of an isolate of Cochiobolus geniculatus (JN941528). Three healthy, 10-cm-tall R. brasiliensis plants were inoculated with a conidial suspension (1 × 106 conidia/ml) of isolate COAD 1335 until runoff, and the plants kept for 2 days in a dew chamber at 26 ± 3°C. Additionally, two plants were sprayed with distilled water and kept under the same conditions. Six days after inoculation, symptoms appeared on all inoculated plants that were similar to symptoms on plants in the field. Non-treated control plants remained healthy. C. richardiae was isolated from the lesions on inoculated plants. Although there is an incomplete record of a Curvularia sp. associated with seeds of R. brasiliensis in Brazil (4), that record included no description of the fungus or information on a disease caused on the plants. This is the first report of C. richardiae causing a disease on R. brasiliensis in Brazil. Although the fungus was first described in Australia (1), C. richardiae is most likely a native from the neotropics, as is the host plant, R. brasiliensis. The fungus was probably introduced accidentally into Australia on the weedy host but has remained unnoticed in the native range until now. References: (1) J. L. Alcorn. Trans. Brit. Mycol. Soc. 56:155, 1971. (2) R. R. Rosseto et al. Planta Daninha 15:25, 1997. (3) R. Vilgalys et al. J. Bacteriol. 172:4239, 1990. (4) C. Yamashita et al. Fitopatol. Bras. 13:122, 1988.

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First report of Plectosphaerella cucumerina causing root rot on fennel in China

Plant Disease ◽

10.1094/pdis-01-21-0166-pdn ◽

2021 ◽

Author(s):

Fengfeng Cai ◽

Chengde Yang ◽

Ting Ma

Keyword(s):

Root Rot ◽

Morphological Characteristics ◽

Control Measures ◽

Effective Control ◽

Herbaceous Plant ◽

Conidial Suspension ◽

Foeniculum Vulgare ◽

Medicinal Uses ◽

First Report ◽

Plectosphaerella Cucumerina

Fennel (Foeniculum vulgare Mill.) is herbaceous plant commonly cultivated for culinary and medicinal uses in China (Shi et al. 2016 ). In May 2019, disease of fennel was observed in Yumen City, Gansu Province, China (N 40°28'/E 97°05'). The incidence across the fields (about 0.23 hectare) was about 4.5%. The outer leaves of diseased fennel wilted, the rhizome changed color from brown to dark brown，necrosis and rot symptoms developed on the root. Finally, the whole plant wilted and died. When pulling up, it was easy to break the root. To identify the pathogen, 15 samples of diseased plants were collected and symptomatic rhizome tissues were surface disinfected with 0.1% HgCl solution for 30 s, rinsed in sterilized water 3 times, placed on potato dextrose agar (PDA), and incubated at 25℃ in the dark. About 7 days, hyphal tips from the tissue edge were transferred to a new PDA for purification. Three isolates obtained were named as hxa, hxb and hxc. To confirm their pathogenicity, two-month old fennel seedings planted in pots, three seedings per pot, with sterilized nutrient soil were inoculated by pouring 50 ml of conidial suspension (107 conidium/mL) produced from the three isolates. Plants inoculated with sterilized water only were included as controls. Six pots of inoculated plants were maintained in climatic cabinet / chamber (> 85% RH, 25°C). The pathogenicity tests were conducted twice. After 7 days, the plants inoculated with conidial suspension of hxa developed brown necrosis and wilt symptoms resembling those originally observed in the field, whereas the controls and the plants inoculated with the other two isolates had no symptoms. Furthermore, hxa was reisolated from rhizome of these diseased plants. The results indicated that isolate hxa was the pathogen causing root rot of fennel. The colonies of hxa on PDA were white-to-cream, slimy, mycelium appressed, aerial mycelium absent. Mycelium was hyaline, septate and formed hyphal coils. Conidiophores were solitary, hyaline, sometimes crooked or sinuous, widest at the base, gradually tapering to the apex. Conidia were smooth, hyaline, aseptate, elliptical and ovoid, measuring 5.97 to 9.51 × 2.13 to 3.58 um (avg. 7.58×2.78, n=100). These morphological characters of the fungal isolates were identical to those of Plectosphaerella cucumerina (Carlucci et al. 2012). For molecular identification, genomic DNA was extracted from the mycelium, and the rDNA internal transcribed spacer (ITS) region, portions of the 28S ribosomal RNA (LSU), calmodulin (CaM) and translation elongation factor 1α (Ef‐1α) gene were amplified using primer pairs ITS1/ITS4, LROR/LR5, CMD5/CMD6 and 688f/1251R (White et al., 1990; Hopple et al., 1999; Hong et al., 2005; Alves et al., 2008), respectively. The sequences of these genes were deposited in GenBank (accessions: ITS as MW426266, LSU as MW433724, CaM as MW448071 and EF-1a as MW459981) and used in analysis to generate a phylogenetic tree. These sequences showed 100, 100, 96 and 97.32% homology to the sequences of P. cucumerina (GenBank accession no. EU594566, MH867359, KY416911 and KY964491), respectively. According to morphological characteristics and phylogenetic analysis, isolate hxa was identified as P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing root rot of fennel in China as well as worldwide. This finding may help to take effective control measures of root rot on fennel.

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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 Leaf Blight of Japanese Yew Caused by Pestalotiopsis microspora in Korea

Plant Disease ◽

10.1094/pdis-08-13-0821-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 691-691 ◽

Cited By ~ 2

Author(s):

Y. H. Jeon ◽

W. Cheon

Keyword(s):

Dna Sequences ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Blight ◽

Economic Damage ◽

Leaf Margin ◽

Conidial Suspension ◽

First Report ◽

Pestalotiopsis Microspora ◽

Large Trees

Worldwide, Japanese yew (Taxus cuspidata Sieb. & Zucc.) is a popular garden tree, with large trees also being used for timber. In July 2012, leaf blight was observed on 10% of Japanese yew seedling leaves planted in a 500-m2 field in Andong, Gyeongsangbuk-do Province, South Korea. Typical symptoms included small, brown lesions that were first visible on the leaf margin, which enlarged and coalesced into the leaf becoming brown and blighted. To isolate potential pathogens from infected leaves, small sections of leaf tissue (5 to 10 mm2) were excised from lesion margins. Eight fungi were isolated from eight symptomatic trees, respectively. These fungi were hyphal tipped twice and transferred to potato dextrose agar (PDA) plates for incubation at 25°C. After 7 days, the fungi produced circular mats of white aerial mycelia. After 12 days, black acervuli containing slimy spore masses formed over the mycelial mats. Two representative isolates were further characterized. Their conidia were straight or slightly curved, fusiform to clavate, five-celled with constrictions at the septa, and 17.4 to 28.5 × 5.8 to 7.1 μm. Two to four 19.8- to 30.7-μm-long hyaline filamentous appendages (mostly three appendages) were attached to each apical cell, whereas one 3.7- to 7.1-μm-long hyaline appendage was attached to each basal cell, matching the description for Pestalotiopsis microspora (2). The pathogenicity of the two isolates was tested using 2-year-old plants (T. cuspidata var. nana Rehder; three plants per isolate) in 30-cm-diameter pots filled with soil under greenhouse conditions. The plants were inoculated by spraying the leaves with an atomizer with a conidial suspension (105 conidia/ml; ~50 ml on each plant) cultured for 10 days on PDA. As a control, three plants were inoculated with sterilized water. The plants were covered with plastic bags for 72 h to maintain high relative humidity (24 to 28°C). At 20 days after inoculation, small dark lesions enlarged into brown blight similar to that observed on naturally infected leaves. P. microspora was isolated from all inoculated plants, but not the controls. The fungus was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spaces (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures, and amplified with the ITS1/ITS4 primers and sequenced as previously described (4). Sequences were compared with other DNA sequences in GenBank using a BLASTN search. The P. microspora isolates were 99% homologous to other P. microspora (DQ456865, EU279435, FJ459951, and FJ459950). The morphological characteristics, pathogenicity, and molecular data assimilated in this study corresponded with the fungus P. microspora (2). This fungus has been previously reported as the causal agent of scab disease of Psidium guajava in Hawaii, the decline of Torreya taxifolia in Florida, and the leaf blight of Reineckea carnea in China (1,3). Therefore, this study presents the first report of P. microspora as a pathogen on T. cuspidata in Korea. The degree of pathogenicity of P. microspora to the Korean garden evergreen T. cuspidata requires quantification to determine its potential economic damage and to establish effective management practices. References: (1) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) S. S. N. Maharachchikumbura. Fungal Diversity 50:167, 2011. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

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First report of Coniella castaneicola causing leaf blight on blueberry (Vaccinium virgatum) in China

Plant Disease ◽

10.1094/pdis-05-21-1122-pdn ◽

2021 ◽

Author(s):

Jiahao Lai ◽

Guihong Xiong ◽

Bing Liu ◽

Weigang Kuang ◽

Shuilin Song

Keyword(s):

Molecular Identification ◽

Morphological Characteristics ◽

Leaf Blight ◽

Yield Potential ◽

Effective Control ◽

Economic Losses ◽

Distilled Water ◽

First Report ◽

Fungal Isolates ◽

Blight Disease

Blueberry (Vaccinium virgatum), an economically important small fruit crop, is characterized by its highly nutritive compounds and high content and wide diversity of bioactive compounds (Miller et al. 2019). In September 2020, an unknown leaf blight disease was observed on Rabbiteye blueberry at the Agricultural Science and Technology Park of Jiangxi Agricultural University in Nanchang, China (28°45'51"N, 115°50'52"E). Disease surveys were conducted at that time, the results showed that disease incidence was 90% from a sampled population of 100 plants in the field, and this disease had not been found at other cultivation fields in Nanchang. Leaf blight disease on blueberry caused the leaves to shrivel and curl, or even fall off, which hindered floral bud development and subsequent yield potential. Symptoms of the disease initially appeared as irregular brown spots (1 to 7 mm in diameter) on the leaves, subsequently coalescing to form large irregular taupe lesions (4 to 15 mm in diameter) which became curly. As the disease progressed, irregular grey-brown and blighted lesion ran throughout the leaf lamina from leaf tip to entire leaf sheath and finally caused dieback and even shoot blight. To identify the causal agent, 15 small pieces (5 mm2) of symptomatic leaves were excised from the junction of diseased and healthy tissue, surface-sterilized in 75% ethanol solution for 30 sec and 0.1% mercuric chloride solution for 2 min, rinsed three times with sterile distilled water, and then incubated on potato dextrose agar (PDA) at 28°C for 5-7 days in darkness. Five fungal isolates showing similar morphological characteristics were obtained as pure cultures by single-spore isolation. All fungal colonies on PDA were white with sparse creeping hyphae. Pycnidia were spherical, light brown, and produced numerous conidia. Conidia were 10.60 to 20.12 × 1.98 to 3.11 µm (average 15.27 × 2.52 µm, n = 100), fusiform, sickle-shaped, light brown, without septa. Based on morphological characteristics, the fungal isolates were suspected to be Coniella castaneicola (Cui 2015). To further confirm the identity of this putative pathogen, two representative isolates LGZ2 and LGZ3 were selected for molecular identification. The internal transcribed spacer region (ITS) and large subunit (LSU) were amplified and sequenced using primers ITS1/ITS4 (Peever et al. 2004) and LROR/LR7 (Castlebury and Rossman 2002). The sequences of ITS region (GenBank accession nos. MW672530 and MW856809) showed 100% identity with accessions numbers KF564280 (576/576 bp), MW208111 (544/544 bp), MW208112 (544/544 bp) of C. castaneicola. LSU gene sequences (GenBank accession nos. MW856810 to 11) was 99.85% (1324/1326 bp, 1329/1331 bp) identical to the sequences of C. castaneicola (KY473971, KR232683 to 84). Pathogenicity was tested on three blueberry varieties (‘Rabbiteye’, ‘Double Peak’ and ‘Pink Lemonade’), and four healthy young leaves of a potted blueberry of each variety with and without injury were inoculated with 20 μl suspension of prepared spores (106 conidia/mL) derived from 7-day-old cultures of LGZ2, respectively. In addition, four leaves of each variety with and without injury were sprayed with sterile distilled water as a control, respectively. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH greater than 80%). After 4 days, all the inoculated leaves started showing disease symptoms (large irregular grey-brown lesions) as those observed in the field and there was no difference in severity recorded between the blueberry varieties, whereas the control leaves showed no symptoms. The fungus was reisolated from the inoculated leaves and confirmed as C. castaneicola by morphological and molecular identification, fulfilling Koch’s postulates. To our knowledge, this is the first report of C. castaneicola causing leaf blight on blueberries in China. The discovery of this new disease and the identification of the pathogen will provide useful information for developing effective control strategies, reducing economic losses in blueberry production, and promoting the development of the blueberry industry.

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First Report of Black Stem of Avicennia marina Caused by Fusarium equiseti in China

Plant Disease ◽

10.1094/pdis-08-13-0873-pdn ◽

2014 ◽

Vol 98 (6) ◽

pp. 843-843 ◽

Cited By ~ 6

Author(s):

N.-H. Lu ◽

Q.-Z. Huang ◽

H. He ◽

K.-W. Li ◽

Y.-B. Zhang

Keyword(s):

Morphological Characteristics ◽

Its Region ◽

Avicennia Marina ◽

Optical Microscope ◽

Ethanol Solution ◽

Morphological Observation ◽

Fusarium Equiseti ◽

First Report ◽

Initial Symptoms ◽

Β Tubulin

Avicennia marina is a pioneer species of mangroves, a woody plant community that periodically emerges in the intertidal zone of estuarine regions in tropical and subtropical regions. In February 2013, a new disease that caused the stems of A. marina to blacken and die was found in Techeng Island of Zhanjiang, Guangdong Province, China. Initial symptoms of the disease were water-soaked brown spots on the biennial stems that coalesced so whole stems browned, twigs and branches withered, leaves defoliated, and finally trees died. This disease has the potential to threaten the ecology of the local A. marina community. From February to May 2013, 11 symptomatic trees were collected in three locations on the island and the pathogen was isolated as followed: tissues were surface disinfected with 75% ethanol solution (v/v) for 20 s, soaked in 0.1% mercuric chloride solution for 45 s, rinsed with sterilized water three times, dried, placed on potato dextrose agar (PDA), and incubated for 3 to 5 days at 28°C without light. Five isolates (KW1 to KW5) with different morphological characteristics were obtained, and pathogenic tests were done according Koch's postulates. Fresh wounds were made with a sterile needle on healthy biennial stems of A. marina, and mycelial plugs of each isolate were applied and covered with a piece of wet cotton to maintain moisture. All treated plants were incubated at room temperature. Similar symptoms of black stem were observed only on the stems inoculated the isolate KW5 after 35 days, while the control and all stems inoculated with the other isolates remained symptomless. An isolate similar to KW5 was re-isolated from the affected materials. The pathogenic test was repeated three times with the same conditions and it was confirmed that KW5 was the pathogen causing the black stem of A. marina. Hyphal tips of KW5 were transferred to PDA medium in petri dishes for morphological observation. After 48 to 72 h, white, orange, or brown flocculence patches of KW5 mycelium, 5.0 to 6.0 cm in diameter, grew. Tapering and spindle falciform macroconidia (11 to 17.3 μm long × 1.5 to 2.5 μm wide) with an obviously swelled central cell and narrow strips of apical cells and distinctive foot cells were visible under the optical microscope. The conidiogenous cells were intertwined with mycelia and the chlamydospores were globose and formed in clusters. These morphological characteristics of the isolate KW5 are characteristic of Fusarium equiseti (1). For molecular identification, the ITS of ribosomal DNA, β-tubulin, and EF-1α genes were amplified using the ITS4/ITS5 (5), T1/T2 (2), and EF1/EF2 (3) primer pairs. These sequences were deposited in GenBank (KF515650 for the ITS region; KF747330 for β-tubulin region, and KF747331 for EF-1α region) and showed 98 to 99% identity to F. equiseti strains (HQ332532 for ITS region, JX241676 for β-tubulin gene, and GQ505666 for EF-1α region). According to both morphological and sequences analysis, the pathogen of the black stem of A. marina was identified as F. equiseti. Similar symptoms on absorbing rootlets and trunks of A. marina had been reported in central coastal Queensland, but the pathogen was identified as Phytophthora sp. (4). Therefore, the disease reported in this paper differs from that reported in central coastal Queensland. To our knowledge, this is the first report of black stems of A. marina caused by F. equiseti in China. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual, 1st ed. Wiley-Blackwell, Hoboken, NJ, 2006. (2) K. O'Donnell and E. Cigelnik. Mol. Phylogenet. Evol. 7:103, 1997. (3) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA. 95:2044, 1998. (4) K. G. Pegg. Aust et al. Plant Pathol. 3:6, 1980. (5) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

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