First Report of Phytophthora Rot on Alders Caused by Phytophthora alni subsp. alni in Spain

Phytophthora alni, a soil- and waterborne pathogen, causes aggressive root and collar rot on riparian alder populations (1,2,4). The disease has been described from several European countries with a destructive impact in Great Britain (1,2). All European alder species and the red alder (Alnus rubra) are highly susceptible. P. alni has multiple variants that have been placed in three subspecies: P. alni subsp. alni, P. alni subsp. uniformis, and P. alni subsp. multiformis (1). In July 2009, a survey of symptoms of Phytophthora rot from A. glutinosa at 20 riparian stands along the Avia River in Galicia (northwest Spain) was conducted. Affected trees showed symptoms of Phytophthora rot including abnormally small, sparse, and yellowish foliage, dieback in the canopy, necroses of the inner bark and cambium, and bleeding cankers on the trunks (2,4). Phytophthora spp. were baited from saturated rhizosphere soil and watercourses using oak leaflets (4). Roots and tissue from fresh active inner bark lesions were transferred to selective medium V8-PARPH agar (4) and incubated for 7 days at 22°C in the dark. A Phytophthora sp. was isolated, transferred to carrot agar (CA), and incubated in the dark. Colonies were appressed, often irregular in outline, and with limited aerial mycelium (1). Growth on CA occurred from 4 to 31°C with optimum growth at 23 to 25°C. Chlamydospores were not observed. Ellipsoid, nonpapillate, noncaducous sporangia had a length/breadth average ratio of 1.4. Nesting and extended internal proliferation occurred. Oogonia, antheridia, and oospores were abundantly produced in a single culture. Oogonia with tapered stalks were spherical (mature oogonia 38 to 50 μm in diameter) and some had ornamented walls or bullate protuberances (1,2). Antheridia were large, amphigynous, and predominantly two-celled (23 to 37 × 16 to 23 μm). Oospores were plerotic. Distorted comma-shaped or smaller oogonia and aborted oospores were observed (1). Amplification of DNA was accomplished by using sequence-characterized amplification region-PCR primers (3). The amplicon sizes obtained were identical to P. alni subsp. alni (3). Internal transcribed spacer (ITS)-DNA and nadh1 mitochondrial gene were also amplified. DNA sequences of ITS and mt-DNA regions were deposited in GenBank (Nos. GU108602 and GU108603). Comparison of the sequences showed 100% homology with P. alni subsp. alni (GenBank Nos. FJ746679 and DQ202490). P. alni subsp. alni was recovered from trees at 3 of 20 riparian alder stands with symptoms. Pathogenicity of one representative isolate was confirmed by inoculating 10 3-year-old A. glutinosa seedlings grown in pots. One shallow cut was made into the bark at the collar level. A colonized agar plug, from the margin of an actively growing colony of P. alni subsp. alni, was inserted beneath the flap that was sealed with Parafilm. Five controls seedlings received only sterile CA agar plugs. Plants were incubated at 24°C and 95% humidity for 30 days. On inoculated plants, necroses progressed bidirectionally from the wound, and dead leaves and wilting of shoots were observed. P. alni subsp. alni was recovered from inoculated seedlings, but not from controls. To our knowledge, this is the first report of Phytophthora rot on alder caused by P. alni subsp. alni in Spain. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) J. Gibbs et al. For. Comm. Bull. 126, 2003 (3) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (4) T. Jung et al. Plant Pathol. 53:197, 2004.

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First Report of Root Rot of Pedunculate Oak and Other Forest Tree Species Caused by Phytophthora plurivora in the Czech Republic

Plant Disease ◽

10.1094/pdis-94-2-0272b ◽

2010 ◽

Vol 94 (2) ◽

pp. 272-272 ◽

Cited By ~ 2

Author(s):

M. Mrazkova ◽

K. Cerny ◽

M. Tomsovsky ◽

V. Holub ◽

V. Strnadova ◽

...

Keyword(s):

Czech Republic ◽

Dna Sequences ◽

Tree Species ◽

Root Rot ◽

Root Hairs ◽

Aerial Mycelium ◽

Pedunculate Oak ◽

The Czech Republic ◽

First Report ◽

Agar Plug

From 2006 to 2008, several similar Phytophthora isolates were obtained from roots of mature Quercus robur and other tree species (Acer platanoides, Fraxinus excelsior, Q. rubra, and Tilia cordata) in forests and parks in several areas in the Czech Republic. The trees were characterized by chlorotic and reduced foliage, crown dieback, and reduced root hairs. Several isolates of Phytophthora were obtained from necrotic roots of these trees and identified as Phytophthora plurivora Jung & Burgess (1). Isolated colonies grown on V8A medium were radiate to slightly chrysanthemum shaped with limited aerial mycelium in the center. Optimum growth was at 25°C, minimum at 5°C and maximum at 32°C. Radial growth of colonies averaged 6.4 mm/day at 20°C. The isolates were homothallic and produced abundant smooth-walled, spherical oogonia (23.3 to 29.1 μm in diameter), oospores were nearly plerotic or plerotic (21.8 to 26.9 μm in diameter), and the oospore wall was 1.2 to 1.4 μm thick. Antheridia were usually paragynous and measured 8.4 to 12 × 6.5 to 8 μm, but amphigynous antheridia were occasionally observed. Noncaducous, semipapillate sporangia formed on simple or sympodial sporangiophores, were obpyriform, ovoid, ellipsoid or irregular in shape, and occasionally distorted with more than one apex. Sporangia dimensions were 33 to 65 × 24 to 33 μm; L/B ratio 1.2 to 1.6 (–2.1). Comparison of DNA sequences of internal transcribed spacer (ITS) regions of isolates (representative strain GenBank Accession No. FJ952382) confirmed the 100% identity of P. plurivora (1). The soil infestation test was conducted using a P. plurivora isolate acquired from roots of Q. robur and 20 3-year-old plants of Q. robur. Sterilized millet seeds colonized by pathogen with the method as described (2) were used as inoculation medium and added into sterilized peat substrate at the rate of 0.5% (vol/vol). The plants were cultivated in 5.8-liter pots in a greenhouse (20°C, 16-h/8-h photoperiod). After 4 months, the roots of all plants were washed, dried, and weighed. The root biomass of 20 infected plants was significantly reduced by approximately 25% on average compared with the control 20 plants (P < 0.05, t-test, Statistica 7.1). The pathogen was consistently reisolated from the roots of infected plants but not from control plants. Stem inoculation tests were conducted with 20 replicates in each group of 2-year-old plants of oak, maple, ash, and lime and isolates acquired from the hosts. On each seedling, a 5-mm-diameter bark plug was removed 5 cm above the collar. The inoculum (5-mm-diameter V8A agar plug with actively growing mycelium) was applied to the exposed substrate. The wounds were sealed with Parafilm. Stem necrosis developed in all cases after 1 to 2 weeks, whereas control plants remained healthy. The pathogen was successfully reisolated from necrotic stem tissues. To our knowledge, this is the first report of P. plurivora causing root rot on oak, maple, ash, and lime in the Czech Republic. On the basis of the host range and distribution of P. plurivora in the Czech Republic, it can be assumed that, as elsewhere in Europe (1), this pathogen is widespread and is a common cause of decline of many tree species. References: (1) T. Jung and T. I. Burgess. Persoonia 22:95, 2009. (2) C. Robin et al. Plant Pathol. 50:708, 2001.

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First Report of Dieback on Hybrid Rhododendrons Caused by Neofusicoccum luteum and N. parvum in Spain

Plant Disease ◽

10.1094/pdis-10-10-0723 ◽

2011 ◽

Vol 95 (2) ◽

pp. 221-221 ◽

Cited By ~ 6

Author(s):

C. Pintos Varela ◽

V. Redondo Fernández ◽

J. P. Mansilla Vázquez ◽

O. Aguín Casal

Keyword(s):

Dna Sequences ◽

Uv Light ◽

Morphological Characteristics ◽

Phytophthora Ramorum ◽

Malt Extract ◽

First Report ◽

Agar Plug ◽

Thin Walled ◽

Neofusicoccum Luteum

During the conducting of Phytophthora ramorum surveys at Galician public parks (northwestern Spain) in 2010, established Rhododendron spp. plants were observed to be exhibiting leaf spots and necrosis, shoot blight, and cankers and dieback of shoots and branches. Branches and leaves of affected rhododendrons contained pseudothecia with bitunicate asci and hyaline pseudoparaphyses, and pycnidia were observed within the same stromatic masses. Symptomatic samples were disinfested in 0.5% sodium hypochlorite for 3 min. Tissues were cut from the margin of lesions, placed onto malt extract agar amended with streptomycin (25 μg ml–1), and incubated at 25°C in the dark. Cultures displaying morphological characteristics associated with Botryosphaeriaceae species were subcultured on 2% water agar with sterilized Pinus pinaster needles as a substrate and incubated at 25°C under near-UV light to encourage pycnidial production (1). Single conidial cultures gave rise to two distinct colonies on potato dextrose agar (PDA) at 25°C. In type 1, isolates produced a sparse, aerial mycelium and a characteristic yellow pigment that was more intense after 3 days, thereafter becoming violaceous and gradually turning dark gray. Growth occurred in the range of 4 to 38°C with an optimum at 29°C. Conidia were hyaline, fusiform, aseptate, thin walled, and averaged 21.1 (14.3 to 25.0) × 5.7 (4.3 to 6.8) μm with a length/width (L/W) ratio of 3.7 ± 0.4 (n = 100). On the basis of these characteristics, isolates were identified as Neofusicoccum luteum (1,3). Colonies of type 2 produced a dense, white-to-yellowish mycelium that rapidly became gray followed by marked diurnal zonation. Mycelial growth occurred in the range of 6 to 38°C with an optimum at 29 to 30°C. Conidia were hyaline, elliptical or fusiform, aseptate, thin walled, and averaging 18.3 (14.1 to 20.7) × 5.8 (4.6 to 7.0) μm with a L/W ratio of 3.2 ± 0.4 (n = 100). These isolates were identified as N. parvum (1,2). Identity was confirmed by DNA sequences analysis of internal transcribed spacer (ITS) regions. Comparison of the sequences of type 1 and 2 showed 100% homology with N. luteum and N. parvum (GenBank Accession Nos. EU673311 and GU251146, respectively). Representative sequences were deposited at GenBank (Accession Nos. HQ197352 and HQ197351). Pathogenicity of each isolate of N. luteum and N. parvum was confirmed by inoculating four 3-year-old Rhododendron spp. seedlings grown in pots. Shallow cuts were made in three branches of each plant. A colonized 6-mm agar plug, removed from the margin of an actively growing colony, was inserted beneath the flap and sealed with Parafilm. Four control seedlings received only sterile PDA agar plugs. Plants were maintained at 26°C and 70% humidity for 21 days. Inoculated plants began showing symptoms after 3 days. Necrosis progressed quickly and bidirectionally from the wound, resulting in death of leaves and wilting of shoots. N. luteum and N. parvum were reisolated from all inoculated plants but not from the controls. To our knowledge, this is the first report of N. luteum and N. parvum on Rhododendron spp. in Spain. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) S. R. Pennycook et al. Mycotaxon 24:445, 1985. (3) A .J. L. Phillips et al. Sydowia 54:59, 2002.

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First Report of Fusarium proliferatum Causing Rot of Garlic Bulbs (Allium sativum) in India

Plant Disease ◽

10.1094/pdis-08-11-0649 ◽

2012 ◽

Vol 96 (2) ◽

pp. 290-290 ◽

Cited By ~ 9

Author(s):

N. Ravi Sankar ◽

Gundala Prasad Babu

Keyword(s):

Sodium Hypochlorite ◽

Allium Sativum ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Its Region ◽

Fusarium Proliferatum ◽

Distilled Water ◽

Sequence Comparisons ◽

First Report ◽

Plant Pathol

In September 2009, diseased garlic bulbs (Allium sativum L. cv. Yamuna Safed) were received from producers and exporters in Hyderabad, Andra Pradesh, India. From 2009 to 2010, similar symptoms were observed on stored garlic bulbs (cvs. Yamuna Safed and Agrifound White) in Chittoor, Kadapa, and Hyderabad districts. In some locations, approximately 60% of the garlic bulbs were affected. At first, infected bulbs showed water-soaked, brown spots and then the disease progressed as small, slightly depressed, tan lesions. A total of 120 diseased samples were collected from all localities. Infected tissues were surface sterilized in 1% sodium hypochlorite for 2 min, rinsed three times in sterile distilled water, plated on potato dextrose agar (PDA), and incubated at 25°C for 7 days. Resultant fungal colonies were fast growing with white aerial mycelium and violet to dark pigments. Hyphae were septate and hyaline. Conidiophores were short, simple, or branched. Microconidia were abundant, single celled, oval or club shaped, measuring 4.5 to 10.5 × 1.3 to 2.5 μm, and borne in chains from both mono-and polyphialides. Macroconidia were not produced. On the basis of morphological characteristics, the pathogen was identified as Fusarium proliferatum (Matsushima) Nirenberg (2). Identification was confirmed by amplification of the internal transcribed spacer (ITS) region. Genomic DNA was extracted from pure cultures of an isolate, and the ITS region was amplified using the ITS4/5 primer pair. PCR amplicons of approximately 574 bp were obtained from isolates, and sequence comparisons with GenBank showed 99% similarity with F. proliferatum (Accession No. FN868470.1). Sequence from this study was submitted to GenBank nucleotide database (Accession No. AB646795). Pathogenicity tests were conducted with three isolates of the fungus following the method of Dugan et al. (1). Each assay with an isolate consisted of 10 garlic cloves disinfected in 1% sodium hypochlorite for 45 s, rinsed with sterile distilled water, and injured to a depth of 4 mm with a sterile 1-mm-diameter probe. The wounds were filled with PDA colonized by the appropriate isolate from a 5-day-old culture. Ten cloves for each tested isolate received sterile PDA as a control. The cloves were incubated at 25°C for 5 weeks; tests were repeated once. After 17 days, rot symptoms similar to the original symptoms developed on all inoculated cloves and F. proliferatum was consistently reisolated from symptomatic tissue, fulfilling Koch's postulates. No fungi were recovered from control cloves. F. proliferatum has been reported on garlic in the northwestern United States (1), Serbia (4), and Spain (3). To our knowledge, this is the first report of F. proliferatum causing rot disease on garlic bulbs in India. References: (1) F. M. Dugan et al. Plant Pathol. 52:426, 2003. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006. (3) D. Palmero et al. Plant Dis. 94:277, 2010. (4) S. Stankovic et al. Eur. J. Plant Pathol. 48:165, 2007.

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First Report in China of Soft Rot of Ginger Caused by Pythium aphanidermatum

Plant Disease ◽

10.1094/pdis-01-14-0094-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1011-1011 ◽

Cited By ~ 4

Author(s):

Y. Li ◽

L. G. Mao ◽

D. D. Yan ◽

X. M. Liu ◽

T. T. Ma ◽

...

Keyword(s):

Aerial Mycelium ◽

Zingiber Officinale ◽

Average Diameter ◽

Soft Rot ◽

Pythium Aphanidermatum ◽

Selective Medium ◽

Rrna Gene ◽

Distilled Water ◽

First Report ◽

Pythium Soft Rot

Ginger (Zingiber officinale Roscoe) is an important commercial crop planted on more than 13,000 ha annually in Anqiu city, Shandong Province, China. From 2010 to 2011, the incidence of Pythium soft rot disease on cv. Laiwu Big Ginger reached 40 to 75% in Anqiu and yield losses of up to 60% were observed. The disease symptoms included brown spots on ginger rhizomes followed by soft rot, stems and leaves above ground becoming withered and yellow, and water soaking on the collar region. The soft rot did not produce offensive odors, which is different from bacterial rots (2). Forty symptomatic rhizomes were sampled from eight farms. Martin's method (1) was used to isolate the pathogen. Ten pieces from each rhizome were washed with sterile distilled water for 30 s and plated on Martin's selective medium at 26°C in a chamber without light. Colonies grew with cottony aerial mycelium. Main hyphae were 5.7 to 9.6 μm wide. Globose sporangia consisting of terminal complexes of swollen hyphal branches were 11.4 to 18.3 μm wide. The average diameter of zoospores was 9.2 μm. The oogonia were globose and smooth, with a diameter of 21 to 33 μm. The sequences of the rRNA gene internal transcribed spacer (ITS) regions 1 and 2 and the 5.8S gene of five isolates were amplified using primers ITS1 and ITS4 (4), and the nucleotide sequence was the same as isolate No. 2, which was deposited in GenBank (Accession No. KC594034). A BLAST search showed 99% identity with Pythium aphanidermatum strain 11-R-8 (Accession No. JQ898455.1). Pathogenicity tests of five isolates were carried out in a greenhouse. Sixty plants (cv. Laiwu Big Ginger) were grown for 30 days in plastic pots (diameter 20 cm) in sandy soil (pH 5.48) and inoculated. Ten plants were used as untreated controls. Five isolates were grown on Martin's liquid medium for 72 h and the spores were harvested in sterile distilled water. Aqueous spore suspensions of the five isolates were adjusted with deionized water to 1 × 108 CFU/ml and injected with a syringe into the soil around the rhizome of the plants. Plants were then placed in the greenhouse at 24 to 26°C and assessed for rhizome rot on the 14th day after inoculation. The inoculated isolates were recovered from the diseased rhizomes, confirming their pathogenicity. To our knowledge, this is the first report of ginger Pythium soft rot caused by P. aphanidermatum in China. Ginger Pythium soft rot caused by P. myriotylum is reported in Taiwan (3). References: (1) F. N. Martin. Page 39 in: The Genus Pythium. American Phytopathological Society, St. Paul, MN, 1992. (2) E. E. Trujillo. Diseases of Ginger (Zingiber officinale) in Hawaii, Circular 62, Hawaii Agricultural Experiment Station, University of Hawaii, December 1964. (3) P. H. Wang. Lett. Appl. Microbiol. 36:116, 2003. (4) T. J. White. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

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First Report of Begonia Elatior Wilt Disease Caused by Fusarium foetens in France

Plant Disease ◽

10.1094/pdis-07-12-0659-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 144-144 ◽

Cited By ~ 2

Author(s):

C. Saurat ◽

C. Fourrier ◽

V. Wilson ◽

C. Casset ◽

R. Ioos

Keyword(s):

Fusarium Species ◽

Aerial Mycelium ◽

Selective Medium ◽

Economic Losses ◽

Adhesive Tape ◽

Morphological Examination ◽

Potted Plants ◽

Mother Plants ◽

Fusarium Foetens ◽

Plant Pathol

Fusarium foetens is a destructive vascular pathogen on Begonia, mainly on cultivars of Begonia elatior hybrids (Begonia × hiemalis), which has recently been identified in Europe and Northern America (1,3). This Fusarium species has been responsible for severe damage in the begonia flower industry (1) and is listed as an EPPO A2 quarantine pathogen since 2007. In May 2007, wilted potted plants of B. elatior showing chlorotic leaves and basal stem rot were observed in a nursery located in the west of France (La Flèche, Sarthe). Symptomatic foliar and basal stem pieces were plated on a Fusarium semi selective medium, dichloran chloramphenicol peptone agar (DCPA), and on malt agar medium supplemented with 100 ppm chloramphenicol. Homogeneous mycelium of a Fusarium species developed from both types of tissue and on both media, and was transferred to potato dextrose agar (PDA) and to spezieller nährstoffarmer agar (SNA) media for morphological examination. Microscope slides were then prepared by pressing gently a clear self-adhesive tape onto the surface covered by mycelium and sporodochia, which was further stained with lactic acid/methylene blue. Typical multiseptate (often three septa), hyaline, slightly curved Fusarium macroconidia 29.2 to 41.8 (32.5) × 3.6 to 4.5 (4.3) μm were collected in sporodochia. In the aerial mycelium, long and short conidiophores with mono- or polyphialidic cells bearing false heads of ellipsoidal microconidia were observed. In addition, a pungent distinctive odor was produced by the mycelium grown on PDA. These features were consistent with F. foetens (2). To support the diagnosis, total DNA was further extracted from the pure culture and a partial region of the translation elongation 1 (tef1) gene was amplified by PCR using EF1-EF2 primer pair (4). Nucleotide sequence was determined and deposited on GenBank (Accession No. JX298790). Analysis of the sequence by BLAST showed that it was 100% identical with all the available F. foetens sequences, which confirmed our morphological diagnosis. To our knowledge, this is the first official report of F. foetens in France. Since this first detection, F. foetens was again identified in 2010 in another nursery located in the Pays de la Loire on collapsed B. elatior. Approximately 15 to 20% of the Begonia plants showed typical Fusarium wilt symptoms and the infected lots were systematically destroyed. The origin of these infections could not be traced back since the mother plants tested negative. The disease is considered as eradicated in France but causes major economic losses to Begonia growers and marketers in regions where the disease is established (2). References: (1) H. Huvenne et al. Eur. J. Plant Pathol. 131:705, 2011. (2) H. J. Schroers et al. Mycologia 96:393, 2004. (3) X. L. Tian et al. Plant Dis. 94:1261, 2010. (4) D. Geiser. Eur. J. Plant Pathol. 110:473, 2004.

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First Report of Phytophthora alni subsp. uniformis on Black Alder in Spain

Plant Disease ◽

10.1094/pdis-10-11-0891-pdn ◽

2012 ◽

Vol 96 (4) ◽

pp. 589-589 ◽

Cited By ~ 5

Author(s):

C. Pintos Varela ◽

C. Rial Martínez ◽

O. Aguín Casal ◽

J. P. Mansilla Vázquez ◽

A. Ares Yebra

Keyword(s):

Mitochondrial Gene ◽

Rapid Evolution ◽

Alnus Glutinosa ◽

Pcr Primers ◽

Soil Extract ◽

Selective Medium ◽

Causal Organism ◽

Black Alder ◽

Plant Pathol ◽

First Time

Phytophthora alni is the causal organism responsible for devastating losses occurring on riparian alders stands in Europe. This emergent hybrid pathogen has multiple variants that have been placed in three subspecies (1). P. alni subsp. uniformis and P. alni subsp. multiformis are reported to be less aggressive than P. alni subsp. alni, though all are considered pathogenic. In Spain, P. alni subsp. alni was detected for the first time in 2009 in Galicia (northwestern Spain) causing root and collar rot on riparian alder populations (3,4), but other subspecies had not been identified. In April 2011, a survey along the Deza River in Galicia was carried out to clarify the Phytophthora sp. associated with the alder decline. Thirty riparian Alnus glutinosa stands, from both sides of the river, were surveyed. Samples of bark and roots of 18 alder stands that showed symptoms of Phytophthora rot and soil from all 30 stands were collected. Roots and tissue from fresh, active, inner bark lesions from 54 trees were transferred to selective medium V8-PARPH agar and incubated for 7 days at 22°C in the dark. P. alni subsp. alni (1) was isolated from roots, bark, or soil in five alder stands. Another Phytophthora sp. was isolated from the bark of one symptomatic tree located in Silleda (Pontevedra), transferred to carrot agar (CA), and incubated in the dark. On CA, the isolate produced irregular and appressed colonies with an optimum growth temperature of 22 to 23°C. The isolate was homothallic with smooth-walled oogonia with a diameter ranging from 36 to 50 μm and two-celled, amphigynous antheridia (1). In soil extract, noncaducous, nonpapillate, ellipsoid-to-ovoid sporangia were produced. Average sporangium were 43.4 × 30.1 μm with a length/breadth ratio of 1.43. Internal proliferation occurred. Amplification of DNA was accomplished by sequence characterized amplified region (SCAR)-PCR primers (2). The amplicon sizes obtained were identical to P. alni subsp. uniformis. Internal transcribed spacer (ITS) (DC6-ITS6/ITS4) and nadh1 (NADHF1/NADHR1) mitochondrial gene regions were also amplified and deposited in GenBank (Nos. JN880411 and JN880410). Comparison of the sequences showed 100% homology with P. alni subsp. uniformis (GenBank Nos. GU259293 and DQ202489). Pathogenicity was tested on 10 3-year-old black alder plants grown in pots. A shallow wound was made with a scalpel at the root collar level of each plant. A 5-mm-diameter mycelia plug, taken from the margin of a 7-day-old culture grown on CA, was inserted in every wound and sealed with Parafilm. Five black alder control plants received only sterile CA agar plugs. Plants were kept at 24°C and 80% humidity. After 3 months, wilting of shoots, dead leaves, and dark stained necroses of the bark tissue varying in length from 0.8 to 5 cm were observed on inoculated plants. Control plants remained healthy. P. alni subsp. uniformis was recovered from inoculated plants, but not from controls. To our knowledge, this is the first time that P. alni subsp. uniformis has been reported in Spain. The presence of a new subspecies in a new region can result in hybridization between individuals of different species or subspecies. This process may allow the rapid evolution and adaptation of these species to new hosts or environmental conditions. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (3) C. Pintos et al. Plant Dis. 94:273, 2010. (4) A. Solla et al. Plant Pathol.59:78, 2010.

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First Report of Grapevine Cankers Caused by Lasiodiplodia crassispora and Neofusicoccum mediterraneum in California

Plant Disease ◽

10.1094/pdis-94-6-0785b ◽

2010 ◽

Vol 94 (6) ◽

pp. 785-785 ◽

Cited By ~ 10

Author(s):

J. R. Úrbez-Torres ◽

F. Peduto ◽

W. D. Gubler

Keyword(s):

Dna Sequences ◽

Vascular Tissue ◽

Elongation Factor ◽

Fungal Culture ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Consensus Sequences ◽

Fast Growing ◽

Agar Plug ◽

Vascular Discoloration

Several species in the Botryosphaeriaceae family cause perennial cankers in the vascular tissue of grapevines and are responsible for the disease known as bot canker in California (3). Tissue from grapevine vascular cankers from samples submitted to our laboratory in the summer of 2009 were plated onto potato dextrose agar (PDA) amended with 0.01% tetracycline hydrochloride. Lasiodiplodia crassispora (Burgess & Barber) and Neofusicoccum mediterraneum (Crous, M.J. Wingf. & A.J.L. Phillips) were identified based on morphological and cultural characters as well as analyses of nucleotide sequences. L. crassispora isolates were characterized by a fast-growing, white mycelium that turned dark olivaceous with age on PDA. Conidia from pycnidia formed in cultures were thick walled and pigmented with one septum and vertical striations when mature. Conidia measured (25.8–) 27.5 to 30.5 (–33.4) × (12.1) 14.3 to 16.8 (–18.2) μm (n = 60). Pycnidia contained septate paraphyses. N. mediterraneum was characterized as having moderately fast-growing, light green mycelia on PDA. Pycnidia formation was induced with pine needles placed on 2% water agar. Conidia from pycnidia were hyaline, ellipsoidal, thin walled, unicellular, and measured (18.2–) 20.5 to 27.8 (–29) × (5.1) 5.9 to 6.5 (–7.2) μm (n = 60). DNA sequences of the internal transcribed spacer region (ITS1-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from L. crassispora (UCD23Co, UCD24Co, and UCD27Co) and N. mediterraneum (UCD695SJ, UCD719SJ, UCD720SJ, UCD749St, and UCD796St) grapevine isolates from California were amplified and sequenced. Consensus sequences from L. crassispora and N. mediterraneum from California showed 99 to 100% homology with L. crassispora and N. mediterraneum isolates previously identified and deposited in GenBank (1,2). Sequences from the examined DNA regions of all isolates were deposited at GenBank (GU799450 to GU799457 and GU799473 to GU799488). Pathogenicity tests using three isolates per species were conducted on detached dormant canes of cv. Red Globe. Ten canes per isolate were inoculated by placing a 7-day-old 5-mm-diameter agar plug from each fungal culture into a wound made with a drill on the internode (4). Twenty shoots were inoculated with noncolonized PDA plugs for negative controls. Six weeks after inoculations, necrosis was measured from the point of inoculation in both directions. One-way analysis of variance was performed to assess differences in the extent of vascular discoloration and means were compared using Tukey's test. L. crassispora isolates caused an average necrotic length of 21.1 mm, which was significantly lower (P < 0.05) than the average necrotic length of 35.6 mm caused by the N. mediterraneum isolates. Reisolation of L. crassispora and N. mediterraneum from necrotic tissue was 100% for each species. The extent of vascular discoloration in infected canes was significantly greater (P < 0.05) than in control inoculations (8 mm) from which no fungi were reisolated from the slightly discolored tissue. To our knowledge, this is the first report of L. crassispora and N. mediterraneum as pathogens of Vitis vinifera and as a cause of grapevine cankers in California. References: (1) T. I. Burgess et al. Mycologia 98:423, 2006. (2) P. W. Crous et al. Fungal Planet. No. 19, 2007. (3) J. R. Úrbez-Torres and W. D. Gubler. Plant Dis. 93:584, 2009. (4) J. R. Úrbez-Torres et al. Am. J. Enol. Vitic. 60:497, 2009.

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First Report of Colletotrichum clavatum Causing Quince Anthracnose in Serbia

Plant Disease ◽

10.1094/pdis-01-14-0052-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1272-1272

Author(s):

S. Živković ◽

V. Gavrilović ◽

T. Popović ◽

N. Dolovac ◽

N. Trkulja

Keyword(s):

Management Practices ◽

Morphological Characteristics ◽

Aerial Mycelium ◽

Fruit Trees ◽

Morphological Identification ◽

Distilled Water ◽

First Report ◽

Mediterranean Countries ◽

Wide Range ◽

Plant Pathol

Quince (Cydonia oblonga Mill.) tree is traditionally grown in Serbia. The fruits are used for compote, marmalade, and brandy production. In December 2012, quince fruits cv. Leskovacka with symptoms of postharvest anthracnose were collected in a storage facility in the area of Sabac, western Serbia. The symptoms were observed on fruits approximately 2 months after harvest. The incidence of the disease was about 3%, but the symptoms were severe. Affected fruits showed sunken, dark brown to black lesions with orange conidial masses produced in black acervuli. Small pieces (3 to 5 mm) of necrotic tissue were surface sterilized for 1 min in 1% NaOCl, washed twice with sterile distilled water, and placed on potato dextrose agar (PDA). Macroscopic and microscopic morphology characteristics of three isolates were observed after growth on PDA for 7 days at 25°C under a 12-h photoperiod. Fungal colonies developed white to gray dense aerial mycelium with orange conidial masses in the center of the colony. Conidia were hyaline, aseptate, clavate with rounded distal apices, 15.2 (12.8 to 16.8) × 4.5 (4.0 to 5.2) μm (mean L/W ratio = 3.3, n = 100). Morphological characteristics are consistent with the description of Colletotrichum clavatum (2). Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA region using ITS1/IT4 primers and β-tubuline 2 gene using T1/T2 primers. The nucleotide sequences were deposited in GenBank (ITS Accession Nos. KF908866, KF908867, and KF908868; β-tubuline 2 gene KF908869, KF908870, and KF908871). BLAST analyses of ITS and β-tubuline 2 gene sequences showed that isolates from quince were 100% identical to other C. clavatum in GenBank (ITS JN121126, JN121130, JN121132, and JN121180; β-tubuline 2 gene JN121213 to 17, JN121219, JN121228, JN121261 to 62, and JN121266 to 69), thus confirming the morphological identification. To fulfill Koch's postulates, asymptomatic fruits of quince cv. Leskovacka (five fruits per isolate) were surface sterilized with 70% ethanol, wounded with a sterile needle, and inoculated with 50 μl of a spore suspension (1 × 106 conidia/ml). Five control fruits were inoculated with 50 μl of sterile distilled water. The experiment was repeated twice. After 10 days of incubation in plastic containers, under high humidity (>90% RH) at 25°C, typical anthracnose symptoms developed on inoculated fruits, while control fruits remained symptomless. The isolates recovered from symptomatic fruits showed the same morphological features as original isolates. C. clavatum previously indicated as group B (3), or genetic group A4 within the C. acutatum sensu lato complex (4), is responsible for olive anthracnose in some Mediterranean countries (1,2), and has been reported as causal agent of anthracnose on a wide range of other hosts including woody and herbaceous plants, ornamentals, and fruit trees worldwide (4). To our knowledge, this is the first report of C. clavatum in Serbia, and the first report of quince anthracnose caused by this pathogen in Europe. Anthracnose caused by C. clavatum can endanger the production and storage of quince in the future, and may require investigation of new disease management practices to control this fungus. References: (1) S. O. Cacciola et al. J. Plant Pathol. 94:29, 2012. (2) R. Faedda et al. Phytopathol. Mediterr. 50:283, 2011. (3) R. Lardner et al. Mycol. Res. 103:275, 1999. (4) S. Sreenivasaprasad and P. Talhinhas. Mol. Plant Pathol. 6:361, 2005.

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First Report of Phytophthora pseudosyringae Associated with Ink Disease of Castanea sativa in Italy

Plant Disease ◽

10.1094/pdis-94-8-1068b ◽

2010 ◽

Vol 94 (8) ◽

pp. 1068-1068 ◽

Cited By ~ 8

Author(s):

B. Scanu ◽

B. T. Linaldeddu ◽

A. Franceschini

Keyword(s):

Plant Protection ◽

Castanea Sativa ◽

Pond Water ◽

Culture Collection ◽

Selective Medium ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Representative Sequence ◽

Agar Plug ◽

Ink Disease

Since December 2008, a severe outbreak of ink disease has been observed in a chestnut grove in the Sardinia Region in Italy (40°01′N, 9°13′E, 1,200 m above sea level). Trees have shown symptoms such as microphylly and yellowish foliage as well as necrosis on the main roots and collar. Isolations were made from infected roots and soil using green apples as baits. Small pulp pieces were cut from the lesions that developed in the apples and plated on Phytophthora selective medium (1). In addition to Phytophthora cambivora, another Phytophthora sp. was detected from 60% of 25 symptomatic trees sampled. Colonies subcultured onto carrot agar (CA) were generally appressed and stellate. Growth occurred from 2 to 26°C with an optimum at 20°C (mean radial growth rate of 4.5 mm/day). Sporangia were produced abundantly in unsterile pond water; they were semipapillate, rarely bipapillate, limoniform or ovoid, occasionally caducous with short pedicels (<5 μm), and 35.2 to 58.1 (46.3) × 22.1 to 35.3 (31.9) μm, with a length/breadth ratio of 1.5:1. Catenulate hyphal swellings were frequently present, whereas no chlamydospores were observed. Isolates produced numerous homothallic oogonia with diameters from 23.7 to 31.7 (27.3) μm. Antheridia were predominantly paragynous. Cultural and morphological features were in close agreement with those described for P. pseudosyringae (2). Identity was confirmed by analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. BLAST searches at GenBank showed 100% identity with reference sequences of P. pseudosyringae (Accession Nos. AY230190 and EU074793). The representative sequence of one P. pseudosyringae strain (CST2A), stored in the culture collection of the Department of Plant Protection-University of Sassari, was submitted to GenBank (Accession No. GU460375). Koch's postulates were fulfilled by inoculating 10 5-month-old chestnut seedlings grown in pots. One shallow cut was made into the bark on the main stem and an agar plug colonized by P. pseudosyringae was inserted beneath the flap. Seedlings were kept at the laboratory at temperatures varying from 16 to 22°C and watered as necessary. After 20 days, extensive, sunken, necrotic lesions measuring 27.2 ± 1.9 mm (mean + standard error) developed around the inoculation sites. Control plants inoculated with sterile CA plugs did not show any disease symptoms. The pathogen was consistently reisolated from infected tissues. P. pseudosyringae has recently been reported as the causal agent of stem necroses on chestnut seedlings in a nursery in Spain (3). To our knowledge, this is the first report of P. pseudosyringae on Castanea sativa in Italy. References: (1) C. M. Brasier and S. A. Kirk. Plant Pathol. 50:218, 2001. (2) T. Jung et al. Mycol. Res. 107:772, 2003. (3) C. Pintos Varela et al. Plant Dis. 91:1517, 2007.

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First Report of Botryosphaeria iberica and B. viticola Associated with Grapevine Decline in California

Plant Disease ◽

10.1094/pdis-91-6-0772c ◽

2007 ◽

Vol 91 (6) ◽

pp. 772-772 ◽

Cited By ~ 27

Author(s):

J. R. Úrbez-Torres ◽

W. D. Gubler ◽

J. Luque

Keyword(s):

Dna Sequences ◽

Aerial Mycelium ◽

Width Ratio ◽

Optimum Growth ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Grapevine Decline ◽

San Luis ◽

Length Width ◽

Dark Green

Grapevine decline symptoms in California include dead spurs and cordon and trunk dieback due to canker formation in the vascular tissue. Seven Botryosphaeria spp. are known to be associated with grapevine cankers in California, viz. Botryosphaeria australis, B. dothidea, B. lutea, B. obtusa, B. parva, B. rhodina, and B. stevensii (3). Recently, B. iberica and B. viticola also were isolated from grapevine cankers in a field survey that was conducted throughout California. Identification was based on morphological comparisons along with DNA analyses with previously identified isolates from Spain (1,2): B. iberica (CBS115035, ex-type) and B. viticola (CBS117006 and CBS117009, ex-type). DNA sequences of the rDNA internal transcribed spacer region (ITSI-5.8S-ITS2), part of the β-tubulin gene (BT2), and part of the translation elongation factor 1-α gene (EF1-α) from B. iberica and B. viticola isolates from California were amplified using primers ITS4/ITS5, Bt2a/Bt2b, and EF-728F/EF-986R, respectively. All DNA sequences of B. iberica and B. viticola from California showed 99 to 100% homology with those previously identified and deposited in GenBank. B. iberica, isolated from grapevine cankers from San Luis Obispo County (central coast), formed colonies on potato dextrose agar (PDA) that were dark green with aerial mycelium, optimum growth at 20 to 25°C, and formed pycnidia after 15 days of incubation at 25°C. Conidia were brown, one-septate, oblong to ovoid with a rounded apex, and measured (20.1-) 22.5 to 23.5 (-27.1) × (8.1) 9.3 to 9.8 (-11.2) μm, length/width ratio = 2.4 (n = 60). B. viticola, isolated from grapevine cankers in Sonoma (north coast), San Luis Obispo, Santa Barbara (south coast), Riverside (southern California), and Yolo (Sacramento Valley) counties, formed colonies on PDA that were dark green to grayish with aerial mycelium, optimum growth at 25°C, and formed pycnidia after 2 weeks. Conidia were brown, one-septate, oval to oblong, and measured (16.6-) 19.3 to 20.3 (-23.5) × (8.1) 9.3 to 9.6 (-11.1) μm, length/width ratio = 2.1 (n = 60). Two isolates of each species were used to complete pathogenicity tests (B. iberica: ATCC MYA-4110, ATCC MYA-4111; B. viticola: ATCC MYA-4115, ATCC MYA-4116). Ten fresh pruning wounds on 15-year-old cv. Zinfandel vines were inoculated per isolate using 50 μl of a 5 × 106 conidia per ml suspension. Twenty control pruning wounds were inoculated with the same amount of sterile water. Twelve months after inoculation, all wood inoculated with B. iberica and B. viticola showed internal necrosis extending 35 to 50 and 30 to 35 mm from the point of inoculation, respectively. Necrosis and extent of vascular discoloration in infected wounds was significantly greater (P < 0.05) than in control inoculations (6.5 mm). B. iberica and B. viticola were reisolated from the necrotic region surrounding all inoculation sites. Representative isolates of B. iberica and B. viticola from California were deposited at the American Type Culture Collection (B. iberica: MYA-4110, MYA-4111; B. viticola: MYA-4112 to MYA-4116). Sequences from the studied DNA regions of all isolates were deposited at GenBank. To our knowledge, this is the first report implicating either species as a cause of grapevine decline in California and B. iberica as a pathogen of Vitis vinifera anywhere in the world. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) A. J. L. Phillips et al. Mycologia 97:513, 2005. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006.

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