First Report of Sheath Blight Caused by Waitea circinata Affecting Foxtail Millet (Setaria italica) in China

Foxtail millet (Setaria italica) is planted widely in northern China, especially in Hebei, Shanxi, Shandong, and Henan provinces. Although several diseases reduce production of this important crop species, sheath blight is considered one of the important diseases of foxtail millet in China. Sheath blight is caused by a soil-borne pathogen and is difficult to control. Epidemics are most common at the late growth stage of foxtail millet. In August 2013, an outbreak was recorded in Shijiazhuang city, Hebei, with an incidence of about 60%. Typical disease symptoms consisted of large, irregular lesions with reddish-brown margin and as the disease progressed, the plants lodge. Three representative sheath fragments (each 1 cm long) were collected from diseased plants during that outbreak. The samples were disinfected with 0.5% (v/v) sodium hypochlorite, rinsed with sterile water, placed on a water agar plate, and then incubated at 26°C in the dark for two days. After the hyphae appeared, ~3-mm-long hyphal tips from typical colonies were excised and transferred to potato dextrose agar (PDA) plates. Three isolates were obtained and all showed typical features of Rhizoctonia-like fungus. Each isolate occupied its whole plate within 5 days of incubation at 26°C in the dark, and abundant aerial mycelia were produced. The color of all colonies was first orange, turning a salmon color when the mycelia matured. Orange sclerotia appeared after 2 weeks of incubation. The nuclei were stained with DAPI (2-(4-amidinophenyl)-1H-indole-6-carboxamidine) and observed under a fluorescent microscope. The hyphal cells were multinucleate and the mycelia branched at a right angle. For molecular identification of the pathogen, mycelia of each isolate were cultured in potato dextrose broth at 26°C for a week, and genomic DNA was extracted from mycelia and used as a template for PCR amplification. The primers set of ITS1 and ITS4 was used for amplification of rDNA-ITS from these isolates and the amplified rDNA-ITS regions of all isolates (GenBank Accession Nos. KJ765700, KJ765701, and KJ765702, respectively) were 99% identical to other Waitea circinata deposited in GenBank (1,2). To further confirm the pathogenicity of the isolates, freshly collected PDA plugs were inoculated on the lower leaf blades of 8-week-old seedlings of the foxtail millet variety Yugu 1. PDA plugs without the isolate were used as a negative control. Five plants were used for each isolate and negative control. After inoculation, pots were placed together in a moist chamber at 26°C. No symptoms developed on the control plants, while obvious lesions appeared on the sheaths of tested plants at 5 days post inoculation and later the plants were lodging. The fungus was re-isolated from diseased plants and confirmed to be W. circinata based on morphological characteristics and sequence analysis as previously described, completing Koch's postulates. Further, on the basis of morphological tests, pathogenicity assays, and molecular analyses, the pathogen of foxtail millet sheath blight was identified as W. circinata (4). Although Rhizoctonia solani AG-1, AG-4 has been reported in earlier studies as the pathogen causing foxtail millet sheath blight, there has been no previous report of the disease caused by W. circinata (3). To our knowledge, this is the first report of foxtail millet sheath blight caused by W. circinata in China. With the spread of high millet plant density and fertilizer application, this disease may become a major threat to foxtail millet; therefore, W. circinata should be taken into account when designing measures for disease control in foxtail millet. References: (2) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (1) M. Fiers et al. Eur. J. Plant. Pathol. 128:353, 2010. (4) W. D. Gao. Acta Phytopathol. Sinica 17:247, 1987. (3) T. Toda et al. Plant Dis. 89:536, 2005.

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First Report of Pythium Root and Stalk Rot of Forage Corn Caused by Pythium arrhenomanes in Japan

Plant Disease ◽

10.1094/pdis-01-14-0059-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1155-1155 ◽

Cited By ~ 2

Author(s):

T. Tsukiboshi ◽

K. Sugawara ◽

A. Masunaka

Keyword(s):

Agar Plate ◽

Its Region ◽

Barley Grain ◽

Field Inoculation ◽

Potato Dextrose Broth ◽

First Report ◽

Stalk Rot ◽

Pythium Arrhenomanes ◽

Rdna Its ◽

Corn Plants

Corn (Zea mays L.) is the most important forage crop in Japan. It was cultivated on 92,000 ha in 2011 and was mainly used as whole crop silage for cattle feed. In September 2009, a root and stalk rot disease was detected on corn plants cultivated in Tochigi, located in the central region of Japan. The symptoms of the disease included wilting of whole plants after the R5 (dent) stage (2) with drooping ears. Roots turned black and their number decreased. Further, the stalks became hollow and soft and harbored white hyphae. This tissue deterioration made machine harvest difficult. We obtained seven isolates of a Pythium-like organism by single hypha isolation from surface-sterilized pieces of diseased roots and stems on water agar and deposited one of the isolates at the NIAS genebank, Japan, under the accession no. MAFF511547. The isolate was grown in the dark on V8 juice agar medium for 10 days to produce oogonia. The oogonia were globose, light brown to yellow, smooth, 23.9 to 30.5 μm in size, and had 1 to 8 antheridia. Oospores were mostly plerotic, and oogonia walls were 1.3 to 2.7 μm thick. The morphology of the isolates was similar to that of Pythium arrhenomanes Drechsler and consistent with the species description (3). We analyzed the rDNA-ITS region sequences of the isolate as described by Kageyama et al. (1). The sequence (GenBank Accession No. AB903904) showed 99.1% (783/790 bp) similarity with that of P. arrhenomanes (AY598628). On the basis of morphological and rDNA sequence similarities, we identified the isolates obtained from corn as P. arrhenomanes. The pathogenicity of the isolate was confirmed by planting corn seedlings of the commercial Pioneer Brand hybrid 36B08 immediately after germination in five replicate pots containing soil mixed with 5% boiled barley grain by weight, incubated with or without the isolate for 7 days. After 10 days of incubation in a greenhouse at 20 to 25°C, only the inoculated plants exhibited symptoms of root and stalk rot. Since the inoculated organism was readily re-isolated from the diseased stems and roots, the pathogenicity of the isolate was confirmed. For field observation, the same hybrid of forage corn was sown in the fields in Nasushiobara, Tochigi, on 16 May 2011. The hybrid was sown in a row of 2 m, with 20 seeds planted at a distance of 10 cm with two replicates. For inoculum, the isolate was cultured on 5-cm-long wooden toothpicks, previously soaked in potato dextrose broth and placed on a V8 agar plate for 7 days at 25°C in the dark until covered by hyphae. The toothpicks were pierced into wounds made on the stems of corn plants, approximately 10 cm above the ground, using a thin iron needle. The wounds were about 2 mm in diameter and 2 cm deep. Field inoculation was conducted in late July at the R1 (silking) growth stage. Disease symptoms were observed in mid-September at R5, and only those plants that were inoculated with the toothpicks harboring the hyphae exhibited the typical stem rot symptoms. To our knowledge, this is the first report of root and stalk rot caused by P. arrhenomanes in forage corn in Japan. References: (1) K. Kageyama et al. J. Phytopathol. 151:485, 2003. (2) S. W. Ritchie et al. Spec. Rep. 48. Iowa State Univ. Coop Ext. Serv., Ames, 1993. (3) A. J. Van der Plaats-Niterink. Stud. Mycol. 21:1, 1981.

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First Report of Sweet potato leaf curl Georgia virus on Sweet Potato in China

Plant Disease ◽

10.1094/pdis-10-12-0967-pdn ◽

2013 ◽

Vol 97 (10) ◽

pp. 1388-1388 ◽

Cited By ~ 2

Author(s):

Y. Qin ◽

Z. Zhang ◽

Z. Qiao ◽

Q. Qiao ◽

D. Zhang ◽

...

Keyword(s):

Genetic Diversity ◽

Nucleotide Sequence ◽

Sweet Potato ◽

Leaf Growth ◽

Negative Control ◽

Post Inoculation ◽

Natural Occurrence ◽

Potato Leaf ◽

First Report ◽

Leaf Curl

Begomoviruses infecting sweet potato (Ipomoea batatas) are phylogenetically distinct from other members of the genus Begomovirus, and have been named “sweepoviruses” (1). Sweepoviruses cause sweet potato yield losses and cultivar decline, and have been found in China (1,3). In 2011, a survey was conducted to determine the incidence, genetic diversity, and distribution of sweepoviruses in China. Thirty sweet potato cuttings showing upward leaf curl, leaf roll, chlorosis, and stunting were collected from fields in Jiangsu, Guangxi, Guizhou, Shanxi, Henan, and Hebei Provinces. Five-leaf growth stage I. setosa plants were inoculated by side-grafting with scions from these samples, and grown in an insect-proof greenhouse in 20-cm-diameter clay pots. Each sample was grafted onto three replicate plants. Healthy, non-grafted I. setosa plants were used as the negative control treatment. Total nucleic acids were extracted from 100 mg fresh leaves harvested 30 days post-inoculation (dpi) from symptomatic and negative control plants using the Universal Genomic DNA Extraction Kit (TaKaRa, Dalian, China). Universal primers for amplification of Geminiviruses (BM-V [5′-KSGGGTCGACGTCATCAATGACGTTRTAC-3′] and BM-C [5′-AARGAATTCATKGGGGCCCARARRGACTGGC-3′]) (2) were used to amplify the begomovirus A component by PCR assay. A DNA fragment of the expected size (2.8 kb) was obtained from grafted leaf samples of the Hebei Province plant, and was cloned into the pMD-19T vector (TaKaRa). The recombinant plasmid was transformed into competent cells of Escherichia coli strain JM109, and the inserted fragment sequenced. The nucleotide sequence obtained (GenBank Accession No. JX448368) was 2,785 nt long, and contained two open reading frames (ORFs) in the virion sense, and four ORFs in the complementary sense, similar to other monopartite begomoviruses (1). The sequence was compared with sequences in GenBank using BLAST. The results revealed the greatest nucleotide sequence identity, 90.8%, with that of the Sweet potato leaf curl Georgia virus (SPLCGV) from Georgia, United States (AF326775). The sequence also shared identities of <89% with other sweepoviruses, and was therefore designated SPLCGV-China: Hebei: 2011. Comparison of the complete genome sequence of SPLCGV-China: Hebei: 2011 with SPLCGV revealed an 18 nucleotide insertion between AV-1 and AC-3. The results confirmed that the sweet potato sample from which SPLCGV-China: Hebei: 2011 was obtained was infected with SPLCGV. To our knowledge, this is the first report of the natural occurrence of SPLCGV in China. This study will assist with understanding the presence of this virus and genetic diversity of sweepoviruses in China. References: (1) H. P. Bi and P. Zhang. Arch. Virol. 157:441, 2012. (2) R. W. Briddon and P. G. Markham. Mol. Biotechnol. 1:202, 1994. (3) Y. S. Luan et al. Virus Genes 35:379, 2007.

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First Report of Fusarium falciforme (FSSC 3+4) Causing Rot of Industrial Hemp (Cannabis sativa) in California

Plant Disease ◽

10.1094/pdis-08-21-1640-pdn ◽

2021 ◽

Author(s):

Kelley Rose Paugh ◽

Johanna Del Castillo Múnera ◽

Cassandra L Swett

Keyword(s):

Root Rot ◽

Cannabis Sativa ◽

Negative Control ◽

Stem Rot ◽

Tween 20 ◽

Post Inoculation ◽

First Report ◽

Fusarium Falciforme ◽

Stem Lesions ◽

Industrial Hemp

Industrial hemp (Cannabis sativa) is a newly legal crop in California that is grown for cannabidiol oil, fiber and seed. In August 2019, whole plant decline and root rot were observed affecting <5% of plants in two industrial fields in Fresno County, CA. Symptoms included chlorotic, collapsed foliage, stem vascular discoloration, and root rot with abundant mycelial growth. Stem and root segments (1-2 cm) from three to five diseased plants were agitated in 0.1% tween-20 and soaked in 70% ethanol for 30 s and 1% NaOCl for 2 min. After incubating for 5 to 7 days on 1:10 potato dextrose agar (PDA) amended with tetracycline, Fusarium selective medium (FSM), and PARP (pimaricin + ampicillin + rifampicin + pentachloronitrobenzene [PCNB] agar) medium, white to pale cream aerial mycelium emerged from tissue of all plants on PDA and FSM but not PARP. Isolates cultured on 0.1% potassium chloride agar formed heads of microconidia on long monophialides consistent with the Fusarium solani species complex (FSSC) (Leslie and Summerell 2008). To obtain pure cultures of two isolates (CS529 and CS530), a single-hyphal tip was excised and grown on PDA. DNA was extracted from actively growing mycelium (PrepMan Ultra kit). The translation elongation factor gene (EF-1α) was amplified via PCR using EF1/EF2 primers (O’Donnell et al. 1998). Sequences of the two isolates were identical and deposited under accession number MW892973 in GenBank. The 599 bp sequence was 99.33% identical to FSSC 3 + 4 (Fusarium falciforme) accessions FD_01443_EF-1a based on FUSARIUM-ID BLAST analysis. To evaluate pathogenicity, stems of hemp plants (cv. ‘Berry Blossom’; n=8 plants per isolate) were wounded by penetrating the epidermis in an area about 0.5-cm square by 1-mm deep and 8-inches above the soil line. A 0.5 cm-diameter plug of 7-day old F. falciforme-colonized PDA was placed against the wound. Inoculation sites were loosely wrapped with parafilm for 2 days. A negative control consisted of a sterile PDA plug (n=3). Treatments were arranged in a completely randomized design in a greenhouse. The experiment was conducted once, due to regulatory restrictions at campus facilities. At 61 days post-inoculation, external stem lesions were significantly larger in diameter (P < 0.05; Tukey’s HSD) in plants inoculated with CS529 (8 ± 1 mm) compared to the control (2 ± 0 mm), and larger but not significant for CS530 (6 ± 1 mm). Internal stem lesions (i.e., rot in stele) were observed in plants inoculated with CS529 (9 ± 3 mm); stem rot was very minor in plants treated with CS530 (1 ± 1 mm) and nonexistent for control plants. No other disease symptoms were observed. F. falciforme was isolated from stems of CS529- and C530-inoculated plants. Sequences of re-isolates matched 100% with accession MW892973. These results suggest that F. falciforme causes rot in hemp in California. These studies specifically confirm stem rot abilities; field observations of root rot indicate root rotting abilities, but further tests are needed for confirmation. This is the first report of F. falciforme causing disease in industrial hemp. FSSC was described as causing foot rot in hemp in Italy (Sorrentino et al. 2019), but these isolates belonged to phylogenetic species 5 (F. solani) not F. falciforme. In addition, F. falciforme was reported as causing root rot in hydroponically grown cannabis (Punja and Rodriguez 2018). These studies provide the foundation for development of management tools for hemp disease.

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First Report of Bacterial Canker Caused by Pseudomonas syringae pv. morsprunorum Race 1 on Cherry in Chile

Plant Disease ◽

10.1094/pdis-11-20-2524-pdn ◽

2021 ◽

Author(s):

Hector Garcia ◽

Elisa Miranda ◽

Miguel Abelardo López ◽

Samuel Parra ◽

Carlos Rubilar ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Prunus Avium ◽

Disease Incidence ◽

Agar Plate ◽

Bacterial Suspension ◽

Post Inoculation ◽

Accession Number ◽

First Report ◽

Race 1 ◽

Necrotic Spots

Chile is the main exporter of sweet cherries (Prunus avium), with a total of 228.6 thousand tons exported in the 2019-20 season, and a production from the Coquimbo to the Aysén region (http://www.iqonsulting.com/yb/). In January 2019, cherry trees from a commercial orchard located near Osorno city (40°37'S, 72°54'W), Region de Los Lagos, Chile, showed symptoms such as the presence of wood cankers, necrotic spots in leaves, and premature defoliation, with a mean disease incidence near 40%. Symptomatic leaves with necrotic spots were collected for analysis, from which all the necrotic spots were extracted by incision with a sterile scalpel, macerated in 30 mL of AFT buffer and subsequently, 100 µL of the suspension was plated on King’s B (KB) agar and incubated for 48 to 72 h at 27°C, obtaining a total of two bacterial colonies identified as 7684.1 and 7684.2. Afterward, each colony was stroked in a new KB agar plate, incubated for 16 h at 27°C, and the obtained biomass was used in subsequent experiments. In KB agar, both colonies exhibited fluorescence under UV light and, according to the LOPAT method (Lelliott et al., 1966), they were gram negative, positive to levan and tobacco hypersensitivity tests and negative to oxidase, potato soft rot, arginine dihydrolase and gelatin tests, and were confirmed as Pseudomonas syringae. Then, the 16s and gyrB genes of each isolate were amplified by PCR, sequenced, and compared with the NCBI Genbank database (Weisburg et al., 1991; Sarkar and Guttman, 2004), finding a 99,93% genetic similarity (1064/1065) with a previously reported 16s sequence of a Pseudomonas syringae pv. morsprunorum (Psm) isolate (accession number CP026558.1), and a 99,69% (636/638) with a previously reported gyrB gene of Psm (accession number LC364094.1), respectively. Additionally, the closest pathovar different to morsprunorum aligned with our gyrB sequence was P. syringae pv. aesculin, with 97,8% of identity (624/638). Our sequences were deposited in Genbank with the accession numbers MN528473 (16s), MN535696 (gyrB) for 7684.1, and MN528474 (16s), MN535697 (gyrB) for 7684.2. To identify if the isolates correspond to Psm races 1 (Psm1) or 2 (Psm2), race-specific conventional PCRs and qPCRs assays were carried out using the specific primers described by Kaluzna et al., (2016), showing that the two isolates were positive to Psm1 in both PCR assays. Pathogenicity was tested by inoculating immature cherry fruitlets (cv. Sweetheart) with bacterial suspension at 108 CFU/mL. For each strain, ten fruitlets were inoculated by pricking with a sterile needle previously immersed in the bacterial suspension (Ruinelli et al., 2019). Sterile distilled water was used as negative control. Seven to fourteen days post-inoculation, necrotic and water-soaked brown lesions with yellow margins were observed on the fruits inoculated with bacterial strains. The pathogen was reisolated and confirmed as Pseudomonas syringae pv. morsprunorum by 16s and gyrB sequencing, and as race 1 by race-specific PCRs. Our results were confirmed by the National Plant Protection Organization, (Servicio Agrícola y Ganadero de Chile, SAG), generating the first report of Psm race 1 in Chile. Thus, SAG established new protocols for quarantine of absent pests in the national territory (Resol. N°3080, SAG, Chile), and an immediate phytosanitary program for Psm (Resol. Exenta N°8948/2019, SAG, Chile). In conclusion, our discovery contributes to the monitoring and control of the disease in Chile.

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First Report of Rhexocercosporidium panacis Causing Rusty Root of Panax ginseng in Northeastern China

Plant Disease ◽

10.1094/pdis-01-14-0082-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1580-1580 ◽

Cited By ~ 4

Author(s):

X. H. Lu ◽

A. J. Chen ◽

X. S. Zhang ◽

X. L. Jiao ◽

W. W. Gao

Keyword(s):

Panax Ginseng ◽

Agar Plate ◽

Its Region ◽

Culture Collection ◽

Northeastern China ◽

Post Inoculation ◽

Academic Press ◽

First Report ◽

Irregular Shapes ◽

Its Sequence Analysis

In northeastern China, Asian ginseng (Panax ginseng) roots exhibited reddish brown lesions of various sizes, irregular shapes, and diffuse margins, typical of rusty root disease. The lesions remain superficial, smooth, and limited to the epidermal and peridermal tissues. In September 2013, 10 symptomatic roots were collected from each of three fields in Jilin and Heilongjiang provinces. One piece of symptomatic skin tissue from each root was excised, surface-disinfested in 1% NaClO for 3 min, rinsed three times with sterile water, and then placed on tetracycline-amended (50 μg/ml) potato dextrose agar. After incubation at 22 ± 1°C in the dark for a week, small olivaceous black colonies developed from the symptomatic tissue from five of the 30 samples. No spores were observed. A single hyphal tip of each colony was transferred to a fresh V8 agar plate to purify the culture. Two-week-old colonies on V8 agar were olivaceous gray, and 42 to 46 mm in diameter with an outer white margin (3 to 5 mm wide). Conidia produced in V8 broth after 3 weeks with a 12-h photoperiod were straight and hyaline, cylindrical or subcylindrical with no or one septum. Mature conidia were 12.8 to 21.8 × 2.2 to 4.5 μm (mean 18.2 × 3.0 μm, n = 100 conidia for each of three isolates). Three isolates selected randomly were further identified by analyzing the partial sequences of the ITS region of rDNA with primers ITS4 and ITS5 (5), and partial sequences of β-tubulin with the primers tub2F and tub2R (1). Sequences of the three isolates (GenBank Accession Nos. KJ149287, KJ149288, and KJ149290 to 93) showed 99% to 100% homology with previously identified and deposited Rhexocercosporidium panacis isolates (DQ2499992 and DQ457119) for both loci (3). Therefore, the three isolates were identified as R. panacis and deposited in China General Microbiological Culture Collection Center (CGMCC3.17259 to 61). Pathogenicity of R. panacis in Asian ginseng was investigated using these three isolates as described previously with slight modifications (4). Bare roots of 3-year-old Asian ginseng were surface-disinfested as described above, and inoculated with mycelial plugs (4 mm diameter) cut from the margin of actively growing colonies of the isolates on V8 agar. Three mycelial plugs were placed on each root at 3-cm intervals and four roots (replicates) were inoculated for each isolate. Four additional roots were inoculated with non-colonized agar plugs as control. The treated roots were placed on moist filter paper in an enamel tray. The plates were sealed with plastic wrap to prevent desiccation and incubated in the dark at 18 ± 1°C. Four weeks post inoculation, all the inoculated ginseng roots showed red-brown lesions, which turned to dark red or black over time. R. panacis was recovered from symptomatic roots for all isolates and confirmed by ITS sequence analysis. The mock-inoculated control roots remained symptomless and no R. panacis was isolated. The inoculation experiment was repeated and showed the same results. R. panacis was reported in 2006 to infect roots of Panax quinquefolius (2,3,4). To our knowledge, this is the first report of R. panacis causing rusty root of P. ginseng. References: (1) P. R. Hirsch et al. Mycol. Res. 104:435, 2000. (2) Z. K. Punja et al. Can. J. Plant Pathol. 35:503, 2013. (3) R. D. Reeleder. Mycologia. 99:91, 2007. (4) R. D. Reeleder et al. Phytopathology 96:1243, 2006. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Barley virus G in Foxtail Millet (Setaria italica) in Korea

Plant Disease ◽

10.1094/pdis-01-17-0036-pdn ◽

2017 ◽

Vol 101 (6) ◽

pp. 1061 ◽

Cited By ~ 3

Author(s):

J. Oh ◽

C. Y. Park ◽

H.-G. Min ◽

H.-K. Lee ◽

Y.-A. Yeom ◽

...

Keyword(s):

Foxtail Millet ◽

Setaria Italica ◽

First Report

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First Report of Colletotrichum acutatum Associated with Stem Blight of Blueberry Plants in China

Plant Disease ◽

10.1094/pdis-08-12-0738-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 422-422 ◽

Cited By ~ 3

Author(s):

C.-N. Xu ◽

Z.-S. Zhou ◽

Y.-X. Wu ◽

F.-M. Chi ◽

Z.-R. Ji ◽

...

Keyword(s):

Vaccinium Corymbosum ◽

Colletotrichum Acutatum ◽

Post Inoculation ◽

Pathogenicity Test ◽

Distilled Water ◽

Conidial Suspension ◽

First Report ◽

Rdna Its ◽

Its Sequence Analysis ◽

Species Specific

An anthracnose disease was observed on stems of high-bush blueberry plants (Vaccinium corymbosum L.) in Liaoning Province, China in 2012. The typical symptoms consist of sudden wilting and dieback of stems during the growing season. Dark brown lesions originate from infected buds and kill portions of the stems. Lesions have grayish white centers, with the necrotic areas becoming 6 to 8 cm in length. Disinfected stem pieces were placed on potato dextrose agar (PDA) and incubated at 28°C for 5 to 7 days, after which the emerging colonies were transferred to fresh PDA. All isolates initially produced white growth, but turned pink after 7 days before becoming blackish green. The average colony diameter was 65.5 to 75.0 mm after 7 days. Conidia were aseptate, hyaline, fusiform to ellipsoid, 8.5 to 16.5 × 2.5 to 4.0 μm in size and single celled with two to seven oil globules. Setae were not found on the acervuli. These characteristics matched published descriptions of Colletotrichum acutatum (1) (teleomorph Glomerella acutata). Pathogenicity test was confirmed in 15 2-year-old healthy potted plants of cv. Berkeley. Stems of 10 plants were punctured with flamed needles and sprayed with 5 ml of conidial suspension (106 conidia per ml in sterile distilled water) of isolate LNSW1. Five control plants were inoculated with sterile distilled water. Seven days after inoculation, eight of the 10 blueberry plants exhibited stem lesions, leaf chlorosis, followed by branch dieback 15 days post-inoculation. The symptoms were similar to those observed on diseased plants in the field, and no lesions were observed on control plants. The pathogen was reisolated from the margin of lesions and identified by colony growth characteristics on PDA. PCR amplification of one isolate (LNSW1) was carried out by utilizing the universal rDNA-ITS primer pair ITS1/ITS4. The sequence (557 bp) of isolate LNSW1 (GenBank Accession No. JX392857) showed 99% identity to G. acutata (AB443950) and C. acutatum (AJ749672) in a BLAST search. An approximately 490-bp fragment was amplified from LNSW1 by the species-specific primer pair CaInt2/ITS4 (2). The pathogen was identified as G. acutata (asexual stage: C. acutatum J.H. Simmonds) on the basis of morphological characters, rDNA-ITS sequence analysis, and a PCR product with species-specific primers. To our knowledge, this is the first report of C. acutatum in high-bush blueberry plants in China. References: (1) C. Lei et al. Fungal Diversity 12:183, 2009. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996

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First Report of Dwarf Disease in Foxtail Millet (Setaria italica) Caused by Barley Yellow Striate Mosaic Virus in China

Plant Disease ◽

10.1094/pdis-10-19-2056-pdn ◽

2020 ◽

Vol 104 (4) ◽

pp. 1262-1262

Author(s):

H. R. Shen ◽

Z. P. Dong ◽

Y. F. Wang ◽

J. Z. Quan ◽

H. Bai ◽

...

Keyword(s):

Mosaic Virus ◽

Foxtail Millet ◽

Setaria Italica ◽

First Report ◽

Dwarf Disease

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First Report of Colletotrichum aenigma Causing Apple Glomerella Leaf Spot on the Granny Smith Cultivar in China

Plant Disease ◽

10.1094/pdis-10-20-2298-pdn ◽

2021 ◽

Author(s):

Zhaohui Zhang ◽

Mingjie Yan ◽

Wenwen Li ◽

Yunzhong Guo ◽

Xiaofei Liang

Keyword(s):

Leaf Spot ◽

Manganese Superoxide Dismutase ◽

Negative Control ◽

Post Inoculation ◽

Pathogenicity Test ◽

Conidial Suspension ◽

Species Identity ◽

First Report ◽

Leaf Tissues ◽

Glomerella Leaf Spot

Glomerella leaf spot (GLS) is a devastating fungal disease causing pre-mature defoliation on apple (Malus domestica). It was first reported in 1970s and since then has been reported in North America, South America and Asia. GLS disease is caused by Colletotrichum fungi and the pathogens are genetically diverse, encompassing at least nine species belonging to three species complexes (Velho et al. 2018). In August 2018, disease with sudden leaf necrosis symptom, typical of GLS symptom appearance, occurred in a Granny Smith orchard in Wugong county, China, over 70% tree leaves bared brown and necrotic lesions. Small leaf tissues (3-4 mm2) cut from lesion margins were surface sterilized for 30 s in 3% NaClO and 30 s in 75% ethanol, followed by rinsing three times in sterile water before transferring onto potato dextrose agar (PDA) plates (25 ± 2°C). Seven isolates were obtained, all producing round cottony colonies on PDA, being white to pale on the upper side and dark green on the reverse side. Conidia were single-celled, cylindrical and transparent (17.33 ± 1.29 × 5.11 ± 0.77 μm, n=50). Appressoria were single-celled, thick-walled, dark brown, oval or irregular shaped, sometimes lobed (9.07 ± 0.88 × 6.66 ± 0.33 μm, n=50). The morphological and cultural characteristics of the fungal isolates matched the descriptions of Colletotrichum aenigma (Weir et al. 2012). To confirm the species identity, genomic DNAs were isolated from two representative isolates (QSG1 and QSY1), and the internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-tubulin (TUB2), manganese-superoxide dismutase (SOD2), chitin synthase (CHS), and calmodulin (CAL) regions were amplified by PCR using reported primers (Weir et al. 2012). The sequences were deposited in GenBank (Accession Nos. MT872061, MT873580, MT873581, MT888183, MT888185, MT888187 and MT888189 for QSG1, and MT834933, MT835166, MT873579, MT888182, MT888184, MT888186 and MT888188 for QSY1). BLASTn search against GenBank nr database showed that ITS sequences of the two strains showed high nucleotide identity (over 99%) to sequences derived from the Colletotrichum gloeosporioides species complex (CGSC). Further concatenated phylogenetic analysis with reported CGSC strains (Weir et al. 2012) placed QSG1 and QSY1 in the clade of C. aenigma. To fulfill Koch’s postulates, field pathogenicity test was performed. The experiment was performed in an orchard located in Yangling in September 2020, with the daily average temperature ranging between 15 - 20 ℃. Healthy ‘Granny Smith’ leaves were surface sterilized with 70% alcohol and inoculated with conidial suspension (106 conidia/mL) using cotton swabs. For each isolate, 10 leaf inoculations were performed. Inoculation with distilled water served as a negative control. Inoculated leaves were covered with plastic bags to maintain high humidity and the bags were removed at 48 hours post inoculation (hpi). Conidium-inoculated leaves started to exhibit GLS-resembling necrotic lesions from 5 dpi onward. The lesion extent, however, varied among inoculated leaves, ranging from blurry, small-sized lesions to blight of entire leaf. In contrast with conidium inoculations, water-inoculated leaves remained asymptomatic until 14 dpi. Re-isolated fungi from the symptomic leaf tissues were identical to C. aenigma in morphological appearance. Taken together, this is the first report of GLS on ‘Granny Smith’ apple, and the first report of C. aenigma causing apple GLS in China. This information should provide important guideline for developing field control practices of GLS.

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The First Report of Cucumber Mosaic Virus Infecting Water Chestnut in China

Plant Disease ◽

10.1094/pdis-06-13-0663-pdn ◽

2014 ◽

Vol 98 (1) ◽

pp. 164-164 ◽

Cited By ~ 2

Author(s):

J. Liu ◽

Y. F. Wang ◽

N. Hong ◽

G. P. Wang ◽

L. P. Wang

Keyword(s):

Cucumber Mosaic Virus ◽

Mosaic Virus ◽

Negative Control ◽

Post Inoculation ◽

Flanking Sequence ◽

Nicotiana Glutinosa ◽

First Report ◽

Water Chestnut ◽

Pcr Product ◽

Commercial Kits

Water chestnut (Eleocharis dulcis), which is cultivated worldwide today, first originated in India and China. It is a popular seasonal aquatic vegetable valuable to people for its sweet crisp taste and rich nutrition. In October 2012, field-grown water chestnut seedlings (E. dulcis) showing mosaic, chlorotic, dwarfing, and malformed symptoms were observed in Fanggaoping Town, Tuanfeng County, Hubei Province, China. Sap from leaf-like stems of two symptomatic seedlings (BQ6 and BQ7) were mechanically inoculated onto Nicotiana glutinosa plants using 0.01 M phosphate buffer (pH 7.4) to investigate whether viral etiology was responsible for the disease. Typical symptoms of chlorosis and systemic mosaic similar to that inflicted by Cucumber mosaic virus (CMV) were observed on inoculated N. glutinosa leaves 13 days post inoculation, whereas mock inoculated seedlings remained symptomless. Three naturally field-grown symptomatic water chestnut and the inoculated N. glutinosa seedlings, together with a healthy water chestnut plant as negative control, were sampled. Double-antibody sandwich (DAS)-ELISA with antisera against CMV using commercial kits (Agdia, Elkhart, IN) was carried out to detect and confirm the presence of CMV. The symptomic water chestnut and inoculated N. glutinosa seedlings tested positive for CMV. Total RNAs were extracted using the SDS column isolation method from leaves of the inoculated N. glutinosa and stems of 13 field-grown symptomatic water chestnuts. The extracted RNAs were subjected to reverse transcription. The first-round PCR was carried out using the obtained cDNAs as template with the CMV specific primer set CMV-3F (5′-GCGATGYCGTGTTGAGAAG-3′) and CMV-3R (5′-TTTAGCCGTAAGCTGGATGGA-3′) targeting a 983-bp fragment covering 657 nt of the whole CP and partial flanking sequence within RNA3 referred as ‘Fny’ strain in GenBank (Accession No. D10538). The resulting amplicons were diluted 1:20 and further amplified with the nested-primer set CMV-P1 (5′-ATGGACAAATCTGAATCAACC-3′) and CMV-P2 (5′-TAAGCTGGATGGACAACCCGT-3′) targeting a fragment of 777 bp corresponding to the complete CP followed by part of 3′-UTRs of RNA3 (1). The amplicons of the expected size of ~777-bp were consistently amplified from 13 naturally infected water chestnuts and inoculated N. glutinosa. The PCR product derived from BQ6 isolate was cloned and three clones sequenced in both directions. The sequence (GenBank Accession No. KF268463) was analyzed by MEGA5 software (3). Sequence comparison of the complete CP gene of BQ6 isolate showed 98% nt and 99% amino acid (aa) identity with CMV isolate RP6 from South Korea (GenBank Accession No. KC527735) in subgroup I and had low similarities of 76% nt and 80% aa to that of CMV isolate infecting Trifolium from Hungary (GenBank Accession No. L15336) belonging to subgroup II of CMV. Phylogenetic analysis showed BQ6 isolate was more closely related to the isolates belonging to IB subgroup of CMV (GenBank Accession Nos. EF153739, DQ302715, and KC576805) (2). To our knowledge, this is the first report of CMV infecting water chestnut (E. dulcis) in China. CMV infection may pose a significant threat to water chestnut production. This result provide information to the producer that the CMV-free seedlings should be chosen for cultivation of water chestnut. References: (1) P. Palukaifis et al. Adv. Virus Res. 41:281, 1992. (2) S. K. Raj et al. Plant Dis. 92:171, 2008. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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