First Report of Xiphinema rivesi (Nematoda, Longidoridae) in Washington State

Dagger nematode, Xiphinema rivesi Dalmaso, 1969 reportedly transmits several viruses in North America and Europe (2) leading to severe yield reduction in crops. Soil samples were collected in March 2013 during a survey of cherry orchards in Chelan County, WA; these historically suffer from cherry rasp leaf disease, caused by Cherry rasp leaf virus (CRLV) (genus Cheravirus). Soil samples were transported to the WSDA nematology laboratory in Prosser, WA, where 250-cc subsamples were processed using sucrose centrifugal flotation (1). Dagger nematodes were hand-picked and stored in 0.1% sodium chloride before being sent to the USDA-ARS Nematology Laboratory in Beltsville, MD, for morphological and molecular identification. The morphological and molecular analysis of adult females identified the dagger nematode species as Xiphinema rivesi Dalmaso, 1969 (4). Morphological characters used for identification included female body, and total stylet length (odontostyle and odontophore), location of guiding ring from oral aperture, head and tail shape, various tail measurements, and vulva percentage in relation to body length. Measurements of females (n = 10) include a mean body length of 1,902 ± 162.4 (1,832 to 2,203) μm, odontostyle 83 ± 3.5 (80 to 90) μm, odontophore 54.8 ± 4.2 (50 to 65) μm, total stylet 137.8 ± 4.2 (130 to 145) μm, guiding ring from oral aperture 70 ± 5.1 (60 to 75) μm, tail 30.8 ± 2.5 (27.5 to 35.0) μm, body diameter at anus 24.7 ± 1.7 (22 to 28) μm, J (hyaline portion of tail) 6.0 ± 0.9 (5.0 to 7.5) μm, body diameter at beginning of J 8.5 ± 1.0 (7.5 to 10.5) μm, body diameter at 5 μm from tail terminus 7.5 ± 0.2 (7.0 to 8.0) μm, and V% 52.2 ± 1.8 (49.4 to 55.0) μm. Molecular diagnosis of X. rivesi was confirmed after DNA was extracted from two individual nematodes by mechanical disruption with a micro knife in 20 μl worm lysis buffer containing 500 mM KCl, 100 mM Tris-Cl (pH8.3), 15 mM MgCl2, 10 mM dithiothreitol (DTT), 4.5% Tween 20, and 0.1% gelatin. DNA extracts were stored at –80°C until needed, then thawed, 1 μl proteinase K (from 2 mg/ml stock) was added, and the tubes were incubated at 60°C for 60 min, followed by 95°C for 15 min. The 28S large ribosomal D2-D3 expansion segment was amplified with D2A (5′-ACAAGTACCGTGAGGGAAAGTT-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′), and the internal transcribed spacer (ITS) region was amplified with primers TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′), as previously described (3). To verify the identity of the sequences generated from PCR, sequenced products were subjected to a database search using BLAST. Sequences from the 28S region were >99% identical to several sequences of X. rivesi sampled from Spain (GenBank Accessions JQ990038, JQ990039, HM921357, and HM921358). Sequences from the ITS region were 97 to 98% identical to X. rivesi sequences (FR878063 to FR878066) obtained from the host Vitis vinifera from Italy. To the best of our knowledge, this is the first report of this nematode from the Washington. The quick and persistent spread of CRLV in most of the orchards visited calls for concern and there is need for urgent control measures against this vector nematode. References: (1) W. R. Jenkins. Plant Dis. Rep. 48:692, 1964. (2) S. Sirca et al. Plant Dis. 91:770, 2007. (3) Skantar et al. J. Nematol. 44:58, 2012. (4) M. R. Wojtowicz. et al. J. Nematol. 14:511, 1982.

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First Report of Longidorus breviannulatus Associated with Damage on Creeping Bentgrass Golf Greens in Québec, Canada

Plant Disease ◽

10.1094/pdis-93-8-0846c ◽

2009 ◽

Vol 93 (8) ◽

pp. 846-846 ◽

Cited By ~ 2

Author(s):

L. Simard ◽

G. Bélair ◽

S. Miller

Keyword(s):

Management Practices ◽

Creeping Bentgrass ◽

Soil Samples ◽

Parasitic Nematodes ◽

Morphological Examination ◽

Oral Aperture ◽

Golf Greens ◽

First Report ◽

Dry Soil ◽

Plant Parasitic

Creeping bentgrass, Agrostis stolonifera L., is the most important turfgrass species cultivated on golf greens in Canada. The needle nematode, Longidorus breviannulatus Norton & Hoffman, has several plant hosts including Gramineae such as corn, Zea mays L. (3), and creeping bentgrass (1). This large, plant-parasitic nematode is found most frequently in sandy soils and is encouraged by irrigation (2). In 2006, irregular, yellowish/chlorotic, and dead turfgrass patches were observed for the first time on several sand-based creeping bentgrass cv. Penncross greens on a golf course in Bromont, Québec (45°19′N, 72°39′W). Furthermore, a noticeable decline of the turfgrass root system was observed. Creeping bentgrass was grown with the following management practices: mowing height 3.18 mm, fertilization 2.27 kg N/0.45 kg P2O5/3.18 kg K2O/92.9 m2/year, aeration two times per year with 9.53-mm-diameter hollow core. On 5 July 2006, soil (0.5 kg) was sampled from two damaged areas of green no. 11. Three plugs (5-cm diameter × 15 cm deep) were taken from each area with a soil probe and pooled to form two separate samples. Another set of soil samples was collected on 12 July from three golf greens (nos. 10, 11, and 16). One sample was taken from each of three damaged areas and two healthy areas of each green. Plant-parasitic nematodes were extracted from 100-ml volumes of each soil sample by the Baermann pan and funnel extraction methods. Numbers of L. breviannulatus from the soil sampled on 5 July were counted with a stereo-microscope after 4 days of extraction, while numbers of L. breviannulatus in the rest of the samples were counted after 7 days of extraction. Identification was determined by morphological examination of a small number of adult female nematodes (n = 7). Characteristics distinguishing these specimens as L. breviannulatus include: amphidial pouches (bilobed and extending to the guiding ring), length 5,115 μm (4,780 to 6,230 μm), distance of the guiding ring from the oral aperture 26 μm (24 to 30 μm), odontostyle length 83 μm (78 to 90 μm), and tail length 42 μm (37 to 50 μm). In the two soil samples collected on 5 July, 0 and 183 juveniles per kilogram of dry soil were recovered with the Baermann pan method. In samples collected on 12 July from damaged areas of three greens, averages of 16 (min 0 and max 60) and 22 (min 0 and max 80) juveniles per kilogram of dry soil were obtained with the Baermann pan and funnel methods, respectively. No L. breviannulatus was found in any sample from healthy areas. Although no damage threshold has been established for L. breviannulatus in creeping bentgrass, low numbers of needle nematodes can be damaging in other crops such as corn. In our study, the occurrence of the damage was related to populations of the needle nematode, and therefore, the nematode was the likely cause of the damage. In Canada, L. breviannulatus is reported from Ontario (4). To our knowledge, this is the first report of the occurrence of L. breviannulatus in Québec. References: (1) L. B. Forer. Plant Dis. Rep. 61:712, 1977. (2) R. B. Malek et al. Plant Dis. 64:1110, 1980. (3) D. C. Norton and J. K. Hoffmann. J. Nematol. 7:168, 1975. (4) W. Ye and R. T. Robbins. J. Nematol. 36:220, 2004.

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First Report of Anthracnose of Onion Caused by Colletotrichum coccodes in Ohio

Plant Disease ◽

10.1094/pdis-01-14-0032-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1271-1271 ◽

Cited By ~ 2

Author(s):

F. Baysal-Gurel ◽

N. Subedi ◽

D. P. Mamiro ◽

S. A. Miller

Keyword(s):

Leaf Tissue ◽

Its Region ◽

The United States ◽

Tween 20 ◽

Colletotrichum Coccodes ◽

Academic Press ◽

Conidial Suspension ◽

First Report ◽

Allium Cepa L ◽

Total Dna

Dry bulb onion (Allium cepa L. cvs. Pulsar, Bradley, and Livingston) plants with symptoms of anthracnose were observed in three commercial fields totaling 76.5 ha in Huron Co., Ohio, in July 2013. Symptoms were oval leaf lesions and yellowing, curling, twisting, chlorosis, and death of leaves. Nearly half of the plants in a 32.8-ha field of the cv. Pulsar were symptomatic. Concentric rings of acervuli with salmon-colored conidial masses were observed in the lesions. Conidia were straight with tapered ends and 16 to 23 × 3 to 6 μm (2). Colletotrichum coccodes (Wallr.) S. Hughes was regularly isolated from infected plants (2). Culturing diseased leaf tissue on potato dextrose agar (PDA) amended with 30 ppm rifampicin and 100 ppm ampicillin at room temperature yielded white aerial mycelia and salmon-colored conidial masses in acervuli. Numerous spherical, black microsclerotia were produced on the surface of colonies after 10 to 14 days. To confirm pathogen identity, total DNA was extracted directly from a 7-day-old culture of isolate SAM30-13 grown on PDA, using the Wizard SV Genomic DNA Purification System (Promega, Madison, WI) following the manufacturer's instructions. The ribosomal DNA internal transcribed spacer (ITS) region was amplified by PCR using the primer pair ITS1 and ITS4 (2), and sequenced. The sequence, deposited in GenBank (KF894404), was 99% identical to that of a C. coccodes isolate from Michigan (JQ682644) (1). Ten onion seedlings cv. Ebenezer White at the two- to three-leaf stage of growth were spray-inoculated with a conidial suspension (1 × 105 conidia/ml containing 0.01% Tween 20, with 10 ml applied/plant). Plants were maintained in a greenhouse (21 to 23°C) until symptoms appeared. Control plants were sprayed with sterilized water containing 0.01% Tween 20, and maintained in the same environment. After 30 days, sunken, oval lesions each with a salmon-colored center developed on the inoculated plants, and microscopic examination revealed the same pathogen morphology as the original isolates. C. coccodes was re-isolated consistently from leaf lesions. All non-inoculated control plants remained disease-free, and C. coccodes was not re-isolated from leaves of control plants. C. coccodes was reported infecting onions in the United States for the first time in Michigan in 2012 (1). This is the first report of anthracnose of onion caused by C. coccodes in Ohio. Unusually wet, warm conditions in Ohio in 2013 likely contributed to the outbreak of this disease. Timely fungicide applications will be necessary to manage this disease in affected areas. References: (1) A. K. Lees and A. J. Hilton. Plant Pathol. 52:3. 2003. (2) L. M. Rodriguez-Salamanca et al. Plant Dis. 96:769. 2012. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First Report of Leaf Spot Caused by Corynespora cassiicola on Baphicacanthus cusia in China

Plant Disease ◽

10.1094/pdis-10-12-0921-pdn ◽

2013 ◽

Vol 97 (5) ◽

pp. 690-690

Author(s):

Q.-L. Li ◽

S.-P. Huang ◽

T.-X. Guo ◽

Z.-B. Pan ◽

J.-Y. Mo ◽

...

Keyword(s):

Leaf Spot ◽

Ipomoea Batatas ◽

Its Region ◽

Tween 20 ◽

Herbaceous Plant ◽

Commonwealth Mycological Institute ◽

Corynespora Cassiicola ◽

Single Spore ◽

First Report ◽

Perennial Herbaceous Plant

Baphicacanthus cusia is a perennial herbaceous plant in the family Acanthaceae that is native to China, where it grows in warm temperate mountainous or hilly regions. It is commonly used as a Chinese herbal medicine. In March 2012, symptoms of leaf spot were observed on leaves of B. cusia in Long'an County, Guangxi, China, where this plant is extensively cultivated. Symptoms were initially small brown dots which developed into irregular to circular leaf spots. These spots enlarged and overlapped, extending until the 7- to 9-cm-long and 3- to 4-cm-wide leaves withered entirely, mostly within 2 months. On potato dextrose agar (PDA), the same fungus was cultured from 92% of 75 symptomatic leaf samples that had been surface sterilized in a 45-second dip in 0.1% mercuric chloride. Fungal structures were observed on diseased leaves: conidiophores (85 to 460 × 4 to 8 μm) were erect, brown, single or in clusters, and conidia (36 to 90 × 5 to 16 μm) were single or in chains of two to four, brown, cylindrical or obclavate, straight or slightly curved, with 3 to 18 pseudosepta and a conspicuous hilum. Three single-spore isolates were identified as Corynespora cassiicola (Berk & Curt.) Wei based on morphological and cultural characteristics (1). The rDNA internal transcribed spacer (ITS) region of one isolate, ZY-1, was sequenced (GenBank Accession No. JX908713), and it showed 100% identity to C. cassiicola, GenBank FJ852716, an isolate from Micronesia cultured from Ipomoea batatas (2). Pathogenicity tests were performed with each of the three isolates by spraying conidial suspensions (5 × 104 conidia/ml) containing 0.1% Tween 20 onto the surfaces of leaves of 60-day-old, 20-cm tall plants. For each isolate, 30 leaves from five replicate plants were treated. Control plants were treated with sterilized water containing 0.1% Tween 20. All plants were incubated for 36 h at 25°C and 90% relative humidity in an artificial climate chamber, and then moved into a greenhouse. Seven days after inoculation, dark brown spots typical of field symptoms were observed on all inoculated leaves, but no symptoms were seen on water-treated control plants. Koch's postulates were fulfilled by reisolation of C. cassiicola from diseased leaves. To our knowledge, this is the first report of C. cassiicola infecting B. cusia worldwide. References: (1) M. B. Ellis. Dematiaceous Hyphomycetes. Commonwealth Mycological Institute: Kew, Surrey, England, 1971. (2) L. J. Dixon et al. Phytopathology 99:1015, 2009.

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First report of the cactus cyst nematode, Cactodera cacti, infecting Cereus jamacaru (Cactaceae) in Brazil

Plant Disease ◽

10.1094/pdis-09-21-1924-pdn ◽

2021 ◽

Author(s):

Francisco Bruno da Silva Café ◽

Rhannaldy Benício Rebouças ◽

Juvenil H. Cares ◽

Cristiano Souza Lima ◽

Francisco de Assis Câmara Rabelo Filho ◽

...

Keyword(s):

Body Length ◽

Morphological Characteristics ◽

Its Region ◽

The Body ◽

Control Treatment ◽

Molecular Characteristics ◽

Width Ratio ◽

Morphometric Characteristics ◽

First Report ◽

Second Stage

During a survey in 2018 for plant nematodes associated with roots and soil in cactus cultivation areas in Ceará State (3°44'48"S, 38°34'29"W), cysts were found on roots of mandacaru, Cereus jamacaru DC. This cactus is native to Brazil, can grow to 6-10 meters in height, and is widely distributed in the Northeast region (Romeiro-Brito et al. 2016) where it is used in construction, in disease remedies, as forage, and as an ornamental (Sales et al. 2014). Several cysts, second-stage juveniles (J2) and eggs extracted from the soil and roots, using sucrose centrifugation, were examined by scanning electron microscopy (SEM) and light microscopy (LM) to determine morphological and morphometric characteristics. Molecular characteristics were determined by DNA extraction from J2 and embryonated eggs using a protocol specific for Heteroderidae (Subbotin et al., 2018). The internal transcribed spacer sequence (ITS) region of the rDNA and D2-D3 regions of the 28S rDNA were amplified using the universal primers TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′), D2A(5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B(5′-TCGGAAGGAACCAGCTACTA-3′), respectively. To confirm that mandacaru is a host for C. cacti, six plantlets of mandacaru were inoculated with 1,800 eggs of the nematode, and kept in a greenhouse at 31 ± 3 ºC and irrigated daily. Six non inoculated mandacaru plantlets served as control treatment. Morphometric characteristics of cysts (n=35) were body length, excluding neck, 555.8 ± 87.8 (354,9 - 727,6) μm, body width 392.1 ± 63.4 (297.9 - 553.7) μm, neck length 63.5 ± 25.8 (49.8-105.0) μm, length to width ratio 1.4 ± 0.2 (1.0-1.8) μm and vulval cone length 48.4 ± 15.2 (40.7 –53.6) μm. Cysts had a rough surface, were lemon-shaped to rounded and had a zigzag cuticular pattern with a protruding vulval cone. They were circumfenestrate without underbridge and bullae, but with the presence of vulval denticles. Measurements of second-stage juveniles (n = 13) included the body length 511.2 ± 33.7 (452.7 - 551.5) μm, stylet length 28.0 ± 2.8 (25.4 - 34.0) μm, tail length 50.7 ± 5.1 (40.6 - 57.4) μm, tail hyaline region 22.7 ± 2.2 (18.9 – 27.1), with a = 20.9 ± 2.2 (17.7-24.3) μm, b = 5.4 ± 0.4 (5.1-5.8) μm, b'= 3.4 ± 0.4 (3.1-3.9) μm, c = 10.2 ± 1.3 (8.9-13.3) μm and c' = 3.8 ± 0.4 (3.0-4.5) μm. The observations of essential morphological characteristics for identification indicated that the species found on C. jamacaru was Cactodera cacti (Filipjev & Schuurmans-Stekhoven, 1941) Krall & Krall, 1978. The sequences of the studied rDNA regions were submitted to GenBank (ITS: MW562829 and D2–D3 regions of 28S: MW562830). The samples used for molecular analysis showed a high degree of sequence identity (99.59%) with C. cacti, from China, Iran and USA for the ITS region. The identity of the D2-D3 regions of 28S sequence was 99.54% with C. cacti isolates from Germany and 99.41% with isolates from USA. Phylogenetic analyses were performed using Maximum likelihood (ML) method for both individual loci, confirming the species as Cactodera cacti. All inoculated mandacaru plantlets showed C. cacti cysts on the roots after 60 days, confirming that mandacaru is a host for C. cacti. This species was reported in São Paulo State, in 2001, associated with ornamental cactus cultivated in pots, but plant species were not identified (Santos et al., 2001). The second report in Brazil was to Schlumbergera sp., an ornamental plant (Oliveira et al. 2007). In both studies, the nematode was not morphologically nor molecularly characterized. Cactodera cacti has been commonly associated with cactus worldwide (Esser, 1992). It has been reported in association with C. jamacaru was first reported in 2011 in China (Duan et al. 2012). This is the first report of the occurrence of C. cacti on C. jamacaru in field conditions in Brazil, and its presence in cactus cultivation areas with agricultural importance represents a threat to cactus production in the country.

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First Report of Powdery Mildew of Platanus occidentalis Caused by Erysiphe platani in Korea

Plant Disease ◽

10.1094/pdis-10-12-0982-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 843-843 ◽

Cited By ~ 2

Author(s):

Y. J. La ◽

S. E. Cho ◽

H. D. Shin

Keyword(s):

North America ◽

Powdery Mildew ◽

Its Region ◽

The United States ◽

Control Measures ◽

Base Substitution ◽

Width Ratio ◽

First Report ◽

Platanus Occidentalis ◽

Young Leaves

Platanus occidentalis L., called American sycamore or American plane, is native to North America. The trees are commonly planted throughout the world on the sides of roads and in parks. In June 2012, diseased leaves exhibiting signs of powdery mildew from a park in Daegu City of Korea were sent to Plant Clinic of Seoul National University for diagnosis. Our observations in Daegu City during September and October 2012 showed that nearly 99% of the approximately 1,000 trees surveyed were infected with a powdery mildew. Voucher specimens (n = 6) were deposited at the Korea University Herbarium (KUS). Symptoms were characterized by chlorosis, distortion, or cupping of young leaves. White superficial colonies developed amphigenously on leaves. Hyphae were flexuous to straight, branched, septate, 4 to 7 μm wide, and had lobed appressoria. Conidiophores were 120 to 350 × 5 to 7.5 μm and produced conidia singly. Foot-cells of conidiophores were straight, cylindric, and 115 to 200 μm long. Conidia were hyaline, ellipsoid-ovoid, measured 33 to 47.5 × 17.5 to 29 μm with a length/width ratio of 1.5 to 2.0, lacked distinct fibrosin bodies, and showed reticulate wrinkling of the outer walls. Germ tubes were produced on the subterminal position of conidia. No chasmothecia were observed. The structures and measurements were compatible with those of the anamorphic state of Erysiphe platani (Howe) U. Braun & S. Takam. (1). To confirm the identification, the complete internal transcribed spacer (ITS) region of the rDNA from isolate KUS-F26959 was amplified with nested PCR and sequenced. The resulting sequence of 625 bp was deposited in GenBank (Accession No. JX997805). A GenBank BLAST search of this sequence showed only one base substitution with the four sequences (JQ365940 to JQ365943) of E. platani on Platanus spp. Pathogenicity was confirmed through inoculation tests by gently pressing diseased leaves onto young leaves of three 2-year-old disease-free seedlings. Three non-inoculated plants were used as control. Plants were maintained in a greenhouse at 24 to 30°C. Inoculated leaves developed symptoms after 7 days, whereas the control plants remained symptomless. The fungus present on the inoculated leaves was morphologically identical to that observed on the original diseased leaves, fulfilling Koch's postulates. Since E. platani first was recorded in the United States in 1874, it has been regarded as endemic in North America. From the second half of the 20th century, introduction and expansion of the range of this fungus to South America, South Africa, Australia and New Zealand, Europe, and Asia have been reported (1,2). To our knowledge, this is the first report of E. platani infections of P. occidentalis in Korea. This species was recorded on P.× hispanica from Japan in 1999 (4) and on P. orientalis from China in 2006 (3), suggesting invasive spread of the sycamore powdery mildew in East Asia. Since American sycamores are widely planted in Korea, control measures should be made to prevent further spread of the disease. References: (1) U. Braun and R. T. A. Cook. Taxonomic Manual of the Erysiphales (Powdery Mildews), CBS Biodiversity Series No.11. CBS, Utrecht, 2012. (2) D. F. Farr and A. Y. Rossman. Fungal Databases. Syst. Mycol. Microbiol. Lab., Online publication, ARS, USDA, Retrieved October 22, 2012. (3) C. Liang et al. Plant Pathol. 57:375, 2008. (4) S, Tanda. J. Agric. Sci., Tokyo Univ. Agric. 43:253, 1999.

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First Report of the Apple Root-Knot Nematode, Meloidogyne mali, on Maple Trees in Republic of Korea

Plant Disease ◽

10.1094/pdis-09-21-2121-pdn ◽

2021 ◽

Author(s):

Heonil Kang ◽

Jongmin Seo ◽

Hyoung-Rai Ko ◽

Sohee Park ◽

Nam-Sook Park ◽

...

Keyword(s):

Body Length ◽

National Park ◽

Its Region ◽

28S Rdna ◽

Republic Of Korea ◽

Egg Mass ◽

Lateral Field ◽

First Report ◽

Future Production ◽

Stylet Length

Meloidogyne mali was originally described in Japan on roots of an apple rootstock (Malus prunifolia) (Itoh et al. 1969) and found on elm trees in Italy, Netherlands, Belgium, France and United Kingdom, and euonymus in the USA (EPPO 2018; Prior et al. 2019). In Italy, the nematode was initially described as a new species, Meloidogyne ulmi, but was later synonymized with M. mali (Ahmad et al., 2013). During the study of Meloidogyne species in Republic of Korea, galled roots were found on Acer palmatum collected in Naejangsan National Park, Republic of Korea located at 35°29'29.1"N, 126°55'42.7"E, altitude 147.8 m. Morphologically, the perineal patterns of the females was very similar to M. mali due to rounded dorsal arch and smooth, finely-spaced, indistinct striae. Lateral field shallow, narrow, and faint. Phasmids large, very distinct. Head region of second–stage juveniles flattened anteriorly to hemispherical, slightly set-off from body, without annulations, low head cap. Stylet slender, sharply pointed cone, cylindrical shaft with rounded knob sloping posteriorly. Tail conoid with irregular, and rounded end. Rectum undilated. Several micrographs were made from 25 J2s and females for mean, standard deviation and range. J2s were measured with a body length: 408.2 ± 25.1 (366-449) µm, maximum body width: 15.9 ± 1.0 (14.1-17.9) µm, stylet length: 14.1 ± 0.5 (13.1-15.3) µm, hyaline tail terminus: 10.0 ± 0.9 (8.3-11.0) µm and tail length: 31.7 ± 3.0 (26.0-36.1) µm. Females (n=25) were characterized by a body length: 656.7 ± 102.7 (516-947) µm, a stylet length: 16.4 ± 2.2 (13.9-19.0) µm, a vulval slit length: 22.2 ± 1.8 (19.8-25.7) µm, and a vulva-anal distance: 20.2 ± 2.4 (17.1-25.4) µm. Morphological measurements and configuration of perineal patterns (Fig. 1S) were comparable to M. mali (Itoh et al. 1969; Ahmed et al. 2013; Gu et al. 2020). To confirm pathogenicity, a modified version of Koch’s postulates was conducted in the greenhouse by inoculating 300 eggs from a single egg mass onto each of three, two-year-old A. palmatum plants, grown in sterilized sandy soil. After about one year, symptoms developed on the maple tree roots, with numerous galls containing females and egg masses by visual inspection. In addition, PCR was performed for the 28S rDNA D2-D3 segment and ITS region using the primers D2A, D3B, TW81 and AB28. The resulting sequences (MW522548, MW522549, MW523004 and MW523005) were at least 99% identical to other 28S rDNA D2-D3 segment and ITS region sequences on Genbank (MT406757 and JX978229). The molecular phylogenetic relationships of this species strongly supports M. mali (Fig. 2S). To the best of our knowledge, this is the first report of M. mali in Republic of Korea. The host range of M. mali includes many species which are of economic importance in fruit trees (e.g. apple, chestnut, fig, mulberry), forestry trees (e.g. elm, maple, oak, Yew), and vegetable crops (e.g. cabbage, carrot, cucumber, eggplant, soybean, watermelon). The potential danger to these economically important plants caused M. mali to be added the EPPO Alert List and also the Quarantine List of the Korean Animal and Plant Quarantine Agency. Additionally, in our survey around the Naejangsan National Park, M. mali was not found on other economically important host crops, such as grapes. Although this nematode was not detected other crops, it requires regular monitoring because it poses a serious threat to the future production of these crops.

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First report of Heterodera filipjevi on winter wheat from Hebei Province in North China

Plant Disease ◽

10.1094/pdis-11-20-2519-pdn ◽

2021 ◽

Author(s):

Jiang-kuan Cui ◽

Haohao Ren ◽

Kunyuan Chen ◽

Bo Zhou ◽

Deliang Peng ◽

...

Keyword(s):

Winter Wheat ◽

Body Length ◽

North China ◽

Soil Samples ◽

Hebei Province ◽

Central China ◽

Effective Control ◽

Heterodera Avenae ◽

Cyst Nematodes ◽

First Report

Three of the cereal cyst nematodes, Heterodera avenae, H. filipjevi and H. latipons are considered to be the most economically important cyst nematodes that affect cultivated cereals around the world. H. filipjevi was first detected in China from Xuchang, Henan Province in 2010 (Peng et al. 2010) and now has been recorded in the Central China of Henan, Shandong and Anhui provinces and the Northwest China of Xinjiang Uygur Autonomous Region (Cui et al. 2020). In June 2019, 42 samples consisting of roots and soil were collected from winter wheat fields in Hebei Province of North China. Cysts were detected in 37 soil samples with a mean of 6.4 ± 1.67 cysts per 100 ml of soil. Cysts and second-stage juveniles (J2s) were extracted from root and soil following Cobb's sieving gravity method. Morphological and molecular studies of J2s and cysts confirmed its identity with H. filipjevi in 5 samples from Handan (N36°10'052" and E114°35'056"; N36°37'054" and E114°22'052"), Xingtai (N36°53'060" and E114°30'011") and Shijiazhuang (N 37°26'048" and E 116°05'039") in Hebei Province, China. Morphologically, the cysts are lemon-shaped, light or dark brown in color. The vulval cone is bifenestrate with horseshoe-shaped semifenestrae, strongly globular bullae, and well-developed underbridge. Measurements (mean +_ sd (range)) of cysts (n=10), body length not including neck is 743.0 ± 36.1 μm (665 - 780 μm), body width is 559.0 ± 50.0 μm (455 - 639 μm), length / width ratio is 1.33 ± 0.07 (1.20 - 1.46); neck length is 99.3 ± 8.8 μm (85 - 122 μm); fenestrae length is 56.8 ± 5.0 μm (49 - 65 μm) and width is 25.5 ± 1.8 μm (21.1 - 27.8 μm); underbridge length is 84.0 ± 8.1 μm (62 - 93 μm); and vulval slit length is 8.6 ± 0.5 μm (7.2 - 9.1 μm). Measurements of J2s (n = 12), body length is 541 ± 11.4 μm (490 - 578 μm); stylet length is 22.3 ± 0.5 μm (22.0 - 25.0 μm) with anchor-shaped basal knobs; tail length is 57.7 ± 3.7 μm (52.7 - 65.2 μm), and hyaline tail terminal length is 36.5 ± 2.8 μm (32 - 39.8 μm). The tail had a sharp terminus. Morphology of the cysts and J2s were consistent with the record of H. filipjevi (Peng et al. 2010; Subbotin et al. 2010). The amplifications of rDNA-internal transcribed spacer (ITS) fragments were generated with a PCR fragment of 1054 bp from single cysts of each population, using primers TW81 and AB28 (Joyce et al. 1994). The PCR tests for each sample were repeated five times. The PCR product was purified and sequenced. All nucleotide sequences of ITS-rDNA were submitted to GenBank under accession numbers MW282843-6. Sequences from the ITS region were more than 99.5% identical to those of H. filipjevi from Egypt (KF225725), Turkey (KR704308, KR704293 and MN848333) and China (KT314234, MT254744 and KY448473). These results from ITS supported its identity as H. filipjevi. The results were also confirmed by species specific sequence characterized amplified region primers of H. filipjevi (Peng et al. 2013). Pathogenicity of the H. filipjevi was confirmed by infection of winter wheat (Triticum aestivum L cv. ‘Aikang58’) and examination of the nematode development and reproduction. Wheat seeds were germinated in petri dishes and then transplanted into five polyvinyl chloride tubs (3 cm in diameter, 25 cm in length) that contained 150 cm3 of a sterile soil mixture (loamy soil: sand = 1:1), each with 5 cysts (mean of 252.0 eggs/cyst). Plants were grown in an artificial climate box for one week at 14/18°C, two weeks at 16/20°C, five weeks at 18/25°C and two weeks at 22/30°C, under 8 h of darkness/16 h light and normal culturing practices (Cui et al. 2015). The parasitic J2s, third and fourth-stage juveniles, and adult females were observed in roots stained with acid fuchsin at 10, 20, 30, and 50 days after inoculation (DAI), and an average of 32.0 cysts per tubes were extracted 70 DAI. The new cyst’ morphological and molecular characteristics were identical to the H. filipjevi cysts from the original soil samples. Three other tubes without cysts were set as control and there were no newly formed cysts. Heterodera avenae and H. filipjevi had been detected in a total of 16 wheat-producing provinces in China, which resulted in losses of 1.9 billion CNY year-1 (Cui et al. 2015). To our knowledge, this is the first report of H. filipjevi in Hebei Province of North China. Cereal cyst nematodes are easily transferred to non-infested areas by many avenues, resulting in increased species and pathotype complexity (Cui et al. 2020). Once H. filipjevi continues to spread in main wheat producing area of China, it could become be a new threat to cereals production. It is time to take effective control methods to prevent H. filipjevi further dispersal, especially through the farming machinery transmission. Hebei Province is one of the most important major grain-producing areas, our findings will be very beneficial for H. filipjevi management and further research on winter wheat in Hebei Province, North China.

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First Report of Alternaria alternata f. sp. cucurbitae Causing Alternaria Leaf Spot of Melon in the Mid-Atlantic Region of the United States

Plant Disease ◽

10.1094/pdis-92-4-0652b ◽

2008 ◽

Vol 92 (4) ◽

pp. 652-652 ◽

Cited By ~ 2

Author(s):

X. G. Zhou ◽

K. L. Everts

Keyword(s):

United States ◽

Disease Severity ◽

Body Length ◽

Alternaria Alternata ◽

Leaf Spot ◽

The United States ◽

Tween 20 ◽

Conidial Suspension ◽

First Report ◽

Alternaria Leaf Spot

Alternaria alternata f. sp. cucurbitae, the casual agent of Alternaria leaf spot, was first described in Greece where it caused severe losses to greenhouse-grown cucumbers (Cucumis sativus) (3,4). The fungus also attacks melon (C. melo) and watermelon (Citrullus lanatus) (1–3). In late June of 2006, following a period of windy and rainy days, numerous dark brown, circular lesions, 0.5 to 1 mm in diameter, were observed on leaves of melons in a field in Wicomico County, Maryland. The lesions gradually enlarged and coalesced into large, nearly circular, or irregularly shaped lesions that could be as long as 3 cm. The center of the lesions was light tan, surrounded by a dark brown ring and a chlorotic halo, and tended to split in the later development stages. Most of the lesions appeared on the edge of the leaves and no lesions developed on the stems and fruit. Lesions first started on old leaves and then developed on leaves in the middle part of the canopy. Leaf lesions were observed on melon cvs. Ananas, Honeydew Greenflesh, and Israeli. Disease severity ranged from 3 to 20% of the leaf area affected. Small pieces (3 × 3 mm) of tissue removed from the margin between healthy and diseased tissue were surface disinfected in 0.5% NaOCl for 2 min and plated on acidified, ¼-strength potato dextrose agar. Isolations made from diseased tissue frequently (61%) yielded fungal colonies with morphological features and spore dimensions that were consistent with the description of A. alternata f. sp. cucurbitae (1,3). Fungal isolates were characterized by small, short-beaked, multicellular conidia. Conidia were ovoid, obclavate, and sometimes ellipsoidal with the average overall body length of 39 μm (range, 17 to 80 μm) and width of 14 μm (range, 7 to 20 μm). Conidia were produced on short conidiophores in chains. The beaks were short (often less than one-third the body length) and conical or cylindrical. Pathogenicity of six single-spore isolates was determined on four melon cultivars (Honeydew Greenflesh, Israeli, Tam Dew, and Topmark) and one watermelon cultivar (Sugar Baby) in a greenhouse. Twenty plants of each cultivar at the one-true-leaf stage were sprayed with a conidial suspension (106 conidia/ml) of each isolate amended with 0.1% (vol/vol) of Tween 20 until runoff (1.5 to 2 ml per plant). Inoculation with sterile distilled water amended with 0.1% Tween 20 served as controls. The plants were placed in a dew growth chamber for 48 h at 24°C and subsequently maintained in a greenhouse at 21 to 29°C. At 4 to 5 days after inoculation, each isolate induced leaf lesions on each inoculated cultivar similar to typical lesions observed in the field. There was no significant difference in disease severity among the cultivars tested or between melon and watermelon. Control plants remained symptomless. The fungus was readily reisolated from symptomatic tissues. To our knowledge, this is the first report of A. alternata f. sp. cucurbitae causing Alternaria leaf spot of melon in the Mid-Atlantic United States and the only report outside Georgia in the southern region of the United States (D. B. Langston, personal communication) and Greece. References: (1) D. L. Vakalounakis. Plant Dis. 74:227, 1990. (2) D. L. Vakalounakis. Ann. Appl. Biol. 117:507, 1990. (3) D. L. Vakalounakis. Alternaria leaf spot. Page 24 in: Compendium of Cucurbit Diseases. T. A. Zitter et al., eds. The American Phytopathological Society, St. Paul, MN, 1996. (4) D. L. Vakalounakis and N. E. Malathrakis. J. Phytopathol. 121:325, 1988.

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First Report of Cherry Stem Rot and Leaf Necrosis Disease Caused by Phytophthora nicotianae in Yantai, China

Plant Disease ◽

10.1094/pdis-05-14-0489-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 284-284

Author(s):

X. L. Yu ◽

X. Q. Liu ◽

P. S. Wang ◽

Y. Z. Wang

Keyword(s):

Average Length ◽

Morphological Characteristics ◽

Its Region ◽

Phytophthora Species ◽

Phytophthora Nicotianae ◽

Control Measures ◽

Stem Rot ◽

Economic Losses ◽

First Report ◽

Leaf Necrosis

Cherry (Cerasus avium (Linn.) Moench) is the third most economically important fruit in Yantai, Shandong Province, China. In August 2012, brown spots or necrosis on cherry seedling leaves, with an incidence of 8.2 to 34.3%, were observed in some fields of cherry seedlings in Yantai. Our survey indicated that the economic losses could reach up to 15.3% if disease conditions, such as a cool rainy summer season, were favorable. Conspicuous watery lesions on the stems turned to brown streaks; the leaves all wilted; and finally the plants collapsed. Diseased stem and leaf samples were surface-disinfected in 1% sodium hypochlorite for 1 min, rinsed three times in sterile water, which was absorbed with filter paper, and then transferred to 10% V8 juice agar medium containing 50 μg/ml ampicillin and 5 μg/ml carbendazim (1). The plates were incubated at 22°C in the dark for 5 days. The colonies consisted of white, loose, fluffy aerial mycelia. Eight isolates were obtained, and all were identified as Phytophthora nicotianae based on morphological characteristics and the sequence of the internal transcribed spacer (ITS) region of rDNA. The sporangia were ovoid/spherical, obturbinate with rounded bases and prominent papillae that were 37.5 to 62.5 × 30 to 50 μm (average 46.4 × 37.8 μm, n = 100) in size, with an average length-to-breadth ratio of 1.2. Chlamydospores were terminal, intercalary, and measured 19 to 42 μm (average 30.4 μm), which is typical of P. nicotianae (2). The genomic DNA of the eight isolates was extracted from mycelia. The ITS region of all eight isolates was amplified using primers ITS1 and ITS4, producing specific products that were directly sequenced. The sequence of a representative isolate P1401 (895 bp) was submitted to GenBank (Accession No. KJ754387). It was 100% similar to P. nicotianae strains NV-20T and TARI 22073 (KC768775 and GU111667). To confirm the pathogenicity, at least 10 cherry leaves and new stems were inoculated with mycelial plugs (5 × 5 mm) from each isolate. Necrosis of leaves and stems was observed 4 and 7 days after inoculation, respectively. No symptoms were observed on the control leaves and stems that were inoculated with blank agar plugs. P. nicotianae was re-isolated from the infected leaves, and the ITS sequence was analyzed to confirm its identity. Phytophthora species, such as P. cambivora, P. megasperma, and P. drechsleri, had been previously isolated from cherry (3), but to the best of our knowledge this is the first report of stem rot and leaf necrosis disease caused by P. nicotianae on cherry. Since the economic loss caused by this disease could reach 15% if an outbreak occurred in a rainy summer, control measures should be implemented. References: (1) Y. Balci et al. Mycol. Res. 112:906, 2008. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St Paul, MN, 1996. (2) S. M. Mircetich and M. E. Matheron. Phytopathology 66:549, 1976.

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First Report of Boeremia Blight Caused by Boeremia exigua var. exigua on Pyrethrum in Australia

Plant Disease ◽

10.1094/pdis-05-11-0424 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1478-1478 ◽

Cited By ~ 5

Author(s):

S. J. Jones ◽

F. S. Hay ◽

T. C. Harrington ◽

S. J. Pethybridge

Keyword(s):

Disease Incidence ◽

Elongation Factor ◽

Its Region ◽

Tween 20 ◽

Width Ratio ◽

Malt Extract ◽

Base Pairs ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Surface Sterilization

Pyrethrum (Tanacetum cinerariifolium) is produced for extraction of insecticidal compounds from the flower achenes. In 2004 and 2006, isolations from necrotic lesions on stems and leaves in three fields in northern Tasmania, Australia yielded four unidentified fungal isolates. Leaf lesions were medium brown and circular (2 to 4 mm in diameter) or irregular in shape (2 to 5 mm long). Stem lesions were irregular, necrotic spots, 5 to 15 mm below the flower peduncle, medium brown, 2 to 4 mm long, and 1 to 2 mm wide. Isolations were conducted on water agar following surface sterilization. Isolates were identified by colony characteristics and the presence of metabolite ‘E’ (1). On oatmeal agar (OA), colonies had irregular margins, were greenish olivaceous-to-olivaceous gray with sparse, white, floccose, aerial mycelia. On malt extract agar (MEA), cultures were variable in color with olivaceous black centers with soft, dense, aerial mycelia. Conidia were hyaline, ellipsoidal to oblong, mainly aseptate, but occasionally 1-septate with dimensions ranging from 2.5 to 7.5 × 1.8 to 3.8 μm (length/width ratio = 1.7 to 2.1). All isolates had moderate reactions to the NaOH test for metabolite ‘E’. DNA was extracted from all four isolates with a DNeasy Plant Mini Kit (QIAGEN Inc., Valencia, CA). For identification, the internal transcribed spacer region (ITS1, 5.8s, and ITS2) and part of the translation elongation factor (TEF) region were amplified and sequenced. Primers ITS1 and ITS4 (2) were used for the ITS region and primers EFCF1 (5′-AGTGCGGTGGTATCGACAAG) and EFCF6 (3′-CATGTCACGGACGGCGAAAC) were used for the TEF. Amplicons were sequenced in both directions and consensus sequences assembled. The ITS sequence was 100% identical to Boeremia exigua var. exigua (GenBank Accession No. GU237715). Base pairs 413 to 1,214 of the TEF sequence from the pyrethrum isolates matched base pairs 1 to 802 (799 of 802 identities) of B. exigua var. exigua (GenBank Accession No. GU349080). All isolates were confirmed as B. exigua var. exigua using morphology and sequencing. Pathogenicity tests were conducted three times in separate glasshouse trials for two of the four isolates. For each isolate, conidial suspensions in water (3 ml/plant) from MEA, adjusted to 5 × 105/ml were applied with Tween 20 (1 drop per 100 ml of water) to 8-week-old pyrethrum plants (five pots per isolate with four plants per pot) using a hand-held spray bottle. Twenty plants were sprayed with water and Tween 20 as nontreated controls. Plants were covered with plastic bags for 48 h after inoculation and examined for symptoms after 15 days at 20°C. Disease incidence (number of symptomatic leaves affected per total number of leaves) of the inoculated plants varied from 7.5 to 9.4%. Noninoculated plants did not develop symptoms. Isolations resulted in cultures morphologically identical on MEA and OA to those inoculated. To our knowledge, this is the first report of B. exigua var. exigua causing disease in pyrethrum. Cultures were deposited in the New South Wales Department of Agriculture collection (DAR79101 to 79104) and TEF and ITS sequences for DAR79101 in GenBank (Accession Nos. JF925328 and JF925329, respectively). Boeremia blight is likely to contribute to the fungal disease complex causing reductions in green leaf area in Australian pyrethrum production. References: (1) M. M. Aveskamp et al. Stud. Mycol. 65:1, 2010. (2) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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