First Report of Phaeoacremonium parasiticum Causing Petri Disease of Grapevine in Peru

Since 2005, symptoms of grapevine decline have been observed on 4- to 8-month-old grapevines (cvs. Red globe and Crimson) grafted onto 1103 P rootstock in Ica and Pisco valleys in southern Peru. Affected plants exhibited weak growth, interveinal chlorosis, necrosis and wilting of leaves, and death. Dark brown-to-black streaking of the xylem was seen when transverse or longitudinal cuts were made in the trunk and shoots. Symptomatic plants were collected and sections (5 cm long) were cut from the zone between the rootstock and the scion, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally, and small pieces of discolored tissues were placed onto potato dextrose agar (PDA) supplemented with oxytetracycline (500 mg liter–1). Plates were incubated at 25°C in the dark for 15 days. A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and malt extract agar (MEA) in the dark at 25°C for 3 weeks until colonies produced spores (3). Colonies were brown on PDA and olive brown on MEA. Conidiophores were branched, 27.5 to 67.5 (42.5) μm long, and often consisting of a single phialide. Conidia were hyaline, oblong ellipsoidal, 2.5 to 4.5 (3.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium parasiticum (Ajello, Georg & C.J.K Wang) W. Gams, Crous & M.J. Wingf. (teleomorph Togninia parasitica L. Mostert, W. Gams & Crous) (2,3). Identity of isolate Ppa-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the beta-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. FJ151015). The sequence was identical to the sequence of P. parasiticum (GenBank Accession No. AY328379). Pathogenicity tests were conducted using the isolate Ppa-1. Approximately 20 μl of a suspension containing 103 conidia ml–1 was injected into the pith of four nodes on each of 10 dormant, unrooted, 15 cm long cuttings of cv. Red Globe. Four nodes on each of 10 cuttings were used as controls and injected with an equal volume of sterile distilled water. Inoculation points were covered with Parafilm. The cuttings were planted in plastic pots and maintained at 24 ± 3°C in diffuse light, watering as needed. Within 2 months of inoculation, all P. parasicitum-inoculated cuttings exhibited shoots with very poor growth with small leaves and short internodes. In the xylem vessels, black streaks identical to symptoms observed in declining vines in the vineyard were observed. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of symptomatic shoots of all inoculated cuttings but not from the control shoots. To our knowledge, this is the first report of P. parasiticum causing young grapevine decline in Peru. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) P. W. Crous et al. Mycology 88:786, 2006. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Phaeoacremonium mortoniae Causing Petri Disease of Grapevine in Spain

Plant Disease ◽

10.1094/pdis-91-9-1206a ◽

2007 ◽

Vol 91 (9) ◽

pp. 1206-1206 ◽

Cited By ~ 8

Author(s):

D. Gramaje ◽

S. Alaniz ◽

A. Pérez-Sierra ◽

P. Abad-Campos ◽

J. García-Jiménez ◽

...

Keyword(s):

Tap Water ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Fungal Isolation ◽

Centraalbureau Voor ◽

First Report ◽

Grapevine Decline

In May 2006, symptoms of grapevine decline were observed on 4-year-old grapevines (cv. Cabernet Sauvignon) grafted onto 110 R rootstock in Daimiel (Ciudad Real Province, central Spain). Affected vines had low vigor, reduced foliage, and chlorotic leaves. Cross or longitudinal sections of the rootstock trunk showed black spots and dark streaking of the xylem vessels. Five symptomatic plants were collected and analyzed for fungal isolation. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g L–1 of streptomycin sulfate. Plates were incubated at 25 to 26°C in the dark for 14 to 21 days and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were yellowish white on PDA and white-to-pale gray on MEA. Conidiophores were short and unbranched, 12.5 to 37.5 (20.5) μm long, and often consisting of a single subcylindrical phialide. Conidia were hyaline, oblong to ellipsoidal or reniform, 2.5 to 7.5 (4.6) μm long, and 1.2 to 1.9 (1.6) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (2,3). Identity of isolate Pmo-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region (Phaeoacremonium-specific primers Pm1-Pm2) with the restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EF517921). The sequence was identical to the sequence of P. mortoniae (GenBank Accession No. DQ173109). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using two isolates, Pmo-1 and a reference isolate of P. mortoniae (CBS-101585) obtained from the Centraalbureau voor Schimmelcultures (Utrecht, the Netherlands). Seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL–1) harvested from 21-day-old cultures grown on PDA. Controls were inoculated with sterile distilled water. There were 20 replicates for each isolate with an equal number of uninoculated plants. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months after inoculation, symptoms developed as reduced growth, chlorotic leaves, severe defoliation, and finally wilting. Control plants did not show any of these symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. mortoniae causing young grapevine decline in Spain. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) M. Groenewald et al. Mycol. Res. 105:651, 2001. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Phaeoacremonium scolyti Causing Petri Disease of Grapevine in Spain

Plant Disease ◽

10.1094/pdis-92-5-0836b ◽

2008 ◽

Vol 92 (5) ◽

pp. 836-836 ◽

Cited By ~ 7

Author(s):

D. Gramaje ◽

S. Alaniz ◽

A. Pérez-Sierra ◽

P. Abad-Campos ◽

J. García-Jiménez ◽

...

Keyword(s):

Tap Water ◽

Restriction Enzymes ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Fungal Isolation ◽

First Report ◽

Petri Disease

In May 2007, a survey was conducted to evaluate the phytosanitary status of grapevine propagating materials in a commercial nursery located in Valencia Province (eastern Spain). Fungal isolation was performed on 25 grafted plants (1-year-old grapevines cv. Tempranillo grafted onto 110 R rootstock) because they showed reduced root biomass and black discoloration of the xylem vessels. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were placed onto malt extract agar (MEA) supplemented with streptomycin sulfate (0.5 g L–1). Plates were incubated at 25°C in the dark for 14 to 21 days after which all colonies were transferred to potato dextrose agar (PDA). Togninia minima (Tul. & C. Tul.) Berl. (anamorph Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai) and another Phaeoacremonium sp. were consistently isolated from necrotic tissues. Single conidial isolates of this Phaeoacremonium sp. were grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were grayish brown on PDA and pinkish white on MEA. Conidiophores were mostly short and unbranched, 15 to 30 (mean 20.8) μm long, often consisting of an elongate-ampuliform phialide. Conidia were hyaline, oblong-ellipsoidal occasionally reniform or allantoid, 2.5 to 5.6 (mean 3.8) μm long, and 1 to 2.1 (mean 1.4) μm wide. On the basis of these characteristics, these isolates were identified as Phaeoacremonium scolyti L. Mostert, Summerb. & Crous (2,3). Identity of isolate Psc-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region using Phaeoacremonium-specific primers Pm1-Pm2 and restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EU260415). The sequence showed high similarity (98%) with the sequence of P. scolyti (GenBank Accession No. AY579292). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using the isolate Psc-1. Ten seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL–1) harvested from 21-day-old cultures grown on PDA. Ten controls plants were inoculated with sterile distilled water. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months of inoculation, symptoms developed on all of the inoculated plants as crown necrosis, chlorotic leaves, severe defoliation, and wilting. Control plants did not show any symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. scolyti causing Petri disease in Spain. References: (1) A. Aroca and R. Raposo. Appl. Environ. Microbiol. 73:2911, 2007. (2) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Phaeoacremonium krajdenii Causing Petri Disease of Grapevine in Spain

Plant Disease ◽

10.1094/pdis-11-10-0808 ◽

2011 ◽

Vol 95 (5) ◽

pp. 615-615 ◽

Cited By ~ 3

Author(s):

D. Gramaje ◽

M. I. Aguilar ◽

J. Armengol

Keyword(s):

Tap Water ◽

Vitis Vinifera L ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

First Report ◽

Worldwide Distribution ◽

Black Spots ◽

Pathogenicity Tests ◽

Weak Growth ◽

Human Infections

In September 2009, symptoms of grapevine (Vitis vinifera L.) decline were observed on 3-year-old grapevines in a vineyard in Roquetas de Mar (Almeria Province, southern Spain). Affected vines were weak with reduced foliage and chlorotic leaves. Black spots and dark streaking of the xylem vessels could be seen in cross- or longitudinal sections of the rootstock trunk. Symptomatic plants were collected and sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g liter–1 of streptomycin sulfate. Dishes were incubated at 25 to 26°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (2). Colonies were grayish brown on PDA and dark brown on MEA. Conidiophores were short and unbranched and 11.5 to 46 (25.5) μm long. Phialides were often polyphialidic. Conidia were hyaline, oblong-ellipsoidal or allantoid, 2.5 to 5 (4.2) μm long, and 1 to 1.7 (1.2) μm wide. On the basis of these characters, the isolates were identified as Phaeoacremonium krajdenii L. Mostert, Summerb. & Crous (1,2). DNA sequencing of a fragment of the beta-tubulin gene of the isolate (Pkr-1) using primers T1 and Bt2b (GenBank Accession No. HM637892) matched P. krajdenii GenBank Accession No. AY579330. Pathogenicity tests were conducted using isolate Pkr-1. Ten 1-year-old callused and rooted cuttings of 110 R rootstock grown in pots with sterile peat were wounded at the uppermost internode with an 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 3 months, shoots on all Phaeoacremonium-inoculated cuttings had weak growth with small leaves and short internodes and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 1.2 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated cuttings, completing Koch's postulates. P. krajdenii has a worldwide distribution, although these reports are from human infections (1). P. krajdenii was first reported as a pathogen of grapevines in South Africa (1). To our knowledge, this is the first report of P. krajdenii causing young grapevine decline in Spain or any country in Europe. References: (1) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (2) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Phaeoacremonium mortoniae Associated with Grapevine Decline in Iran

Plant Disease ◽

10.1094/pdis-05-11-0361 ◽

2011 ◽

Vol 95 (8) ◽

pp. 1034-1034 ◽

Cited By ~ 6

Author(s):

H. Mohammadi

Keyword(s):

Fungal Pathogens ◽

Vascular Tissue ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Fars Province ◽

Distilled Water ◽

Conidial Suspension ◽

Single Spore ◽

First Report ◽

Grapevine Decline

In July 2009, a survey was conducted in individually owned rooted vineyards in Iran to determine fungal pathogens associated with grapevine decline. Symptoms of grapevine decline such as slow dieback, stunted growth, small chlorotic leaves, and reduced foliage were observed on 7-year-old grapevines (cv. Askari) in Bavanat (Fars Province, southwestern Iran). Internal wood symptoms such as black spots and dark brown-to-black vascular streaking were observed in cross and longitudinal sections of stems and trunks. Wood samples were collected from symptomatic trunks and cordons. The bark of each fragment was removed and 10 thin cross sections (2 to 3 mm thick) were cut from symptomatic vascular tissue of the samples. These disks were immersed in 1.5% sodium hypochlorite solution for 4 min, washed thrice with sterile distilled water, and plated onto malt extract agar (MEA) supplemented with 100 mg liter–1 of streptomycin sulfate. Plates were incubated at 25°C in darkness. All colonies were transferred to potato dextrose agar (PDA) and incubated at 25°C. Five isolates of a Phaeoacremonium sp. were obtained. Single-spore isolates were transferred to PDA, MEA, and oatmeal agar (OA) media and incubated at 25°C for 8 to 16 days in the dark (2). Colonies reached a radius of 9.5 to 12 mm after 8 days of incubation. Colonies were flat and yellowish white on PDA and OA and white-to-pale gray after 16 days of incubation on MEA. Conidiophores were short and unbranched, 14 to 38.5 (23.5) μm long, and often ending in a single terminal phialide. Phialides were terminal or lateral and mostly monophialidic. Conidia were hyaline, oblong to ellipsoidal or reniform, 2 to 6.5 (4.9) μm long, and 1.1 to 1.7 (1.4) μm wide. On the basis of these characteristics, the isolates were identified as Phaeoacremonium mortoniae (1,2). Additionally, identity of the PMH1 isolate was confirmed by sequencing a fragment of the -tubulin gene with primers T1 and Bt2b (GenBank Accession No. JF831449). The sequence of this isolate was identical to the sequence of P. mortoniae (GenBank Accession No. HM116767). Pathogenicity tests were conducted on 2-month-old grapevine seedlings of cv. Askari by watering the roots with 25 ml of a conidial suspension (107 conidia ml–1) harvested from 21-day-old cultures grown on MEA. Controls were inoculated with 25 ml of sterile distilled water. Fifteen replicates were used for each isolate with an equal number of noninoculated plants. All plants were grown under greenhouse conditions (25 to 30°C). Two months after inoculation, inoculated seedlings showed reduced growth, chlorotic leaves, epinasty, severe defoliation, and finally wilting, while control seedlings remained healthy. The fungus was reisolated from internal tissues of the stems of inoculated seedlings. To my knowledge, this is the first report of P. mortoniae causing grapevine decline in Iran. References: (1) M. Groenewald et al. Mycol. Res. 105:651, 2001. (2) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Campylocarpon fasciculare Causing Black Foot Disease of Grapevine in Spain

Plant Disease ◽

10.1094/pdis-02-11-0105 ◽

2011 ◽

Vol 95 (8) ◽

pp. 1028-1028

Author(s):

S. Alaniz ◽

C. Agustí-Brisach ◽

D. Gramaje ◽

M. I. Aguilar ◽

A. Pérez-Sierra ◽

...

Keyword(s):

Tap Water ◽

Peat Moss ◽

Vitis Vinifera L ◽

Sodium Hypochlorite Solution ◽

Distilled Water ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Foot Disease ◽

Black Foot ◽

Black Foot Disease

In May 2008, symptoms of black foot disease were observed on 8-year-old grapevines (Vitis vinifera L.) cv. Garnacha in Albuñol (Granada Province, southern Spain). Affected plants showed delayed budding with low vigor. Roots showed black discoloration and necrosis of wood tissues. Root fragments were cut, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small pieces of discolored or necrotic tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter–1 of streptomycin sulfate. Plates were incubated at 25°C in the dark for 10 days and all colonies were transferred to PDA. A Cylindrocarpon-like fungus was consistently isolated from necrotic root tissues. Single conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer Agar (SNA) and incubated at 25°C for 10 days in darkness. On PDA, the isolates developed white, thick, and cottony to felty abundant mycelium. On SNA, all isolates produced slightly to moderately curved one-septate (22.5-) 25.6 (-27.5) × (5-) 5.63 (-6.25) μm, two-septate (30-) 36.1 (-45) × (6.25-) 7.08 (-7.5) μm, three-septate (37.5-) 47.9 (-52.5) × (6.25-) 7.5 (-8.75) μm, four-septate (47.5-) 53.3 (-62.5) × (7.5-) 7.89 (-8.75) μm, and five-septate (52.5-) 61.8 (-67.5) × (7.5-) 8 (-8.75) μm macroconidia. Microconidia were not observed. DNA sequence of the rDNA internal transcribed spacer region (ITS) was obtained for isolate Cf-270 and deposited in GenBank (Accession No. HQ441249). This sequence showed high similarity (99%) to the sequence of Campylocarpon fasciculare Schroers, Halleen & Crous (GenBank Accession No. AY677303), in agreement with morphological features (1). Pathogenicity tests were conducted with inoculum produced on wheat (Triticum aestivum L.) seeds that were soaked for 12 h in flasks filled with distilled water. Each flask contained 300 ml of seeds that were subsequently autoclaved three times after excess water was drained. Two fungal disks of a 2-week-old culture of C. fasciculare (isolate Cf-270) grown on PDA were placed aseptically in each flask. The flasks were incubated at 25°C for 4 weeks and shaken once a week to avoid clustering of inoculum. Plastic pots (220 cm3) were filled with a mixture of sterilized peat moss and 10 g of inoculum per pot. One-month-old grapevine seedlings were planted individually in each pot and placed in a greenhouse at 25 to 30°C in a completely randomized design. Control plants were inoculated with sterile uninoculated seeds. Six replicates (each one in individual pots) were used, with an equal number of control plants. The experiment was repeated. Symptoms developed on all plants 20 days after inoculation and consisted in reduced vigor, interveinal chlorosis and necrosis of the leaves, necrotic root lesions with a reduction in root biomass, and plant death. The fungus was reisolated from the roots of affected seedlings and identified as C. fasciculare, completing Koch's postulates. No symptoms were observed on the control plants. Black foot disease of grapevines can be caused by different species of Cylindrocarpon and Campylocarpon. C. fasciculare was first reported in South Africa in 2004 (1). To our knowledge, this is the first report of C. fasciculare causing black foot disease of grapevine in Spain as well as other countries in Europe. Reference: (1) F. Halleen et al. Stud. Mycol. 50:431, 2004.

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First Report of Marasmiellus palmivorus Causing Post-Emergence Damping Off on Coconut Seedlings in Malaysia

Plant Disease ◽

10.1094/pdis-07-12-0627-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 143-143 ◽

Cited By ~ 5

Author(s):

B. S. A. Almaliky ◽

M. A. Zainal Abidin ◽

J. Kader ◽

M. Y. Wong

Keyword(s):

Oil Palm ◽

Tap Water ◽

Leaf Growth ◽

Large Subunit ◽

Post Inoculation ◽

Malt Extract ◽

Distilled Water ◽

Damping Off ◽

First Report ◽

Pathogenicity Tests

In April and June 2010, coconut seedlings with symptoms of very slow growth, yellowing of leaves, and general abnormal leaf growth were observed in germination beds in Teluk Intan, Perak, Malaysia. The roots were soft, rotten, and brown, extending upward and downward from these lesions. Rhizomorphs and basidiocarps were produced on coconut seeds near the germination eye and identified as Marasmiellus palmivorus according description by Turner (2). Three isolates were obtained by plating surface sterilized symptomatic roots and basidiocarp on malt extract agar (MEA) amended with 85% lactic acid (1 ml added to 11 of the medium). To confirm the identity of the fungus, genomic DNA was extracted from mycelia and basidiocarps of isolates and the large subunit (LSU) region was amplified and sequenced using LR0R/LR7 primers (3). All isolates had identical LSU sequences (GenBank Accession No. JQ654233 to JQ654235). Sequences were identical to each other and 99% similar to a M. palmivorus sequence deposited in the NCBI database (Accession No. AY639434).To confirm pathogenicity, three isolates of M. palmivorus that were obtained from symptomatic plant tissue was inoculated onto seeds of Malaysian Red Dwarf variety. Each isolate was grown in 100 ml of malt extract broth in 250 ml Erlenmeyer flasks and incubated at 27 ± 2°C for 5 days on an orbital shaker (125 rpm). The resulting culture was passed through two layers of sterile cloth. Mycelial suspension was obtained by blending mycelia in 100 ml of sterile water. Seeds were sterilized by soaking in 10% v/v sodium hypochlorite in distilled water for 3 min. The seeds were then rinsed three times over running tap water. The calyx portion of the seed was removed and five holes were made around the germination eye. The seeds were inoculated by injecting 2 ml of suspension into each hole. The control seeds were inoculated with sterile distilled water only. The seeds were transferred to 40-cm diameter plastic pots containing a mixture of sand, soil, and peat in the ratio of 3:2:1, respectively, and steam treated at 100°C for 1.5 h. Pots were placed in the glasshouse with normal exposures to day-night cycles, temperatures of 29 ± 4°C, and high relative humidity (85 to 95%) achieved by spraying water twice daily. After 2 months, 75% of the inoculated seeds failed to germinate. It was speculated that the artificial inoculum was higher than under germination bed conditions. Rhizomorphs and basidiocarps were produced on husk seeds near the germination eye. Seedlings that emerged successfully developed symptoms similar to those observed in the germination bed. No symptoms developed in the noninoculated seeds and seedlings. At 80 days post inoculation, basidiocarps were observed emerging from three diseased seedlings near the germination eye. Three reisolations were made on MEA from root lesions surface sterilized. Pathogenicity tests and LSU sequence analyses indicated that M. palmivorus is the causal agent of the symptoms observed on coconut seedlings. M. palmivorus was first recorded on coconuts and oil palm in the 1920s (1) and attacks the fruit and the petiole on oil palm (2). To our knowledge, this is the first report of M. palmivorus causing post-emergence damping off on coconut seedlings. References: (1) K. G. Singh. A check-list of host and diseases in Malaysia. Ministry of Agriculture and Fisheries, Malaysia, 1973. (2) P. D. Turner. Oil palm diseases and disorders. Oxford University Press. 1981. (3) R. Vilgalys et al. J. Bacteriol. 172:4238, 1990.

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First Report of Cylindrocarpon pauciseptatum Associated with Grapevine Decline from Castilla y León, Spain

Plant Disease ◽

10.1094/pdis-10-10-0750 ◽

2011 ◽

Vol 95 (3) ◽

pp. 361-361 ◽

Cited By ~ 5

Author(s):

M. T. Martin ◽

L. Martin ◽

M. J. Cuesta ◽

P. García-Benavides

Keyword(s):

Morphological Characteristics ◽

Vascular Lesions ◽

Width Ratio ◽

Morphological Identification ◽

Malt Extract ◽

Internal Transcribed Spacer Region ◽

Current Season ◽

First Report ◽

Grapevine Decline ◽

Agar Plug

During a survey for grapevine decline, 10 young grapevines (cvs. Tempranillo and Verdejo) with low vigor and little foliage were collected between June 2008 and August 2009. Small pieces of vascular and brown wood were placed onto malt extract agar supplemented with 0.25 g/liter of chloramphenicol and incubated at 25°C. Fifteen resulting colonies were transferred to potato dextrose agar in petri dishes (90 mm). Colonies with white mycelium covered the dishes after 10 days of incubation at 25°C in darkness; mycelium gradually became yellowish with some brownish aspect. Macroconida were predominantly three septate (40 to 45 to 50 × 8.6 to 9 to 9.5 μm with a length and width ratio of 4.7 to 5 to 5.4), straight, and cylindrical with both ends broadly rounded. Chlamydospora and ovoidal microconidia were observed on synthetic nutrient-poor agar (1). Cylindrocarpon pauciseptatum was not easy to distinguish from other Cylindrocarpon species based on morphological characteristics. Ribosomal internal transcribed spacer region sequences of single-spore cultures confirmed the morphological identification and revealed 100% genetic identity with other isolates of C. pauciceptatum present in GenBank (EF607090), a sequence of the fragment was deposited with Accession No. EU983277. Pathogenicity tests were conducted with two isolates. The inoculations were done on 110R rootstock wood of four different young plants and 15 detached canes of current-season growth (cv. Tempranillo). Plants were inoculated with an agar plug containing C. pauciceptatum; controls were treated with agar only. Grapevines were maintained in a greenhouse at 20 to 25°C. After 3 to 4 months, C. pauciceptatum was reisolated from brown tissues and internal vascular lesions in 45% of inoculated samples, fulfilling Koch's postulates. Control plants were asymptomatic and C. pauciceptatum was not recovered. To our knowledge, this is the first report implicating C. pauciceptatum as a cause of grapevine black foot disease in Spain with potentially significant impact on grapevine nurseries. Reference: (1) H. J. Schroers et al. Mycol. Res. 112:82, 2008.

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First Report of Phaeoacremonium venezuelense Associated with Wood Decay of Apricot Trees in Spain

Plant Disease ◽

10.1094/pdis-12-13-1198-pdn ◽

2014 ◽

Vol 98 (7) ◽

pp. 1001-1001 ◽

Cited By ~ 3

Author(s):

D. Olmo ◽

D. Gramaje ◽

C. Agustí-Brisach ◽

M. León ◽

J. Armengol

Keyword(s):

Tap Water ◽

Wood Decay ◽

Prunus Armeniaca ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Fungal Isolation ◽

First Report ◽

Bitter Almond ◽

Weak Growth ◽

Mallorca Island

In July 2011, a survey was conducted to evaluate the phytosanitary status of apricot trees (Prunus armeniaca L.) in an orchard in Binissalem (Mallorca Island, Spain). Fungal isolation was performed on a 40-year-old apricot trees (cv. Galta Vermella, double-grafted onto bitter almond and Japanese plum) showing a collapse of branches, chlorosis of leaves, and shoot dieback. These symptoms appeared in approximately 10% of the trees. Black spots and dark streaking of the xylem vessels were observed in cross- or longitudinal sections of the branches. Symptomatic branches were collected and wood sections (10 cm long) were cut, washed under running tap water, surface-disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally, and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g liter−1 of streptomycin sulfate. Dishes were incubated at 25°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues (more than 50% of the isolations). Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were brownish orange on PDA and MEA. Conidiophores were short and occasionally branched, and 26 to 35 (avg. 29) μm long. Phialides were terminal or lateral, mostly monophialidic. Conidia were hyaline, oblong-ellipsoidal or fusiform-ellipsoidal, 3 to 4 (avg. 3.9) μm long, and 1 to 1.5 (avg. 1.2) μm wide. Based on these characters, the isolates were identified as Phaeoacremonium venezuelense L. Mostert, Summerb. & Crous (2,3). DNA sequencing of a fragment of the beta-tubulin gene of the isolate 9.3 using primers T1 and Bt2b (GenBank Accession No. KF765487) matched P. venezuelense GenBank accession HQ605026. Pathogenicity tests were conducted using isolate 9.3. Ten 2-year-old apricot trees of cv. Galta Rotja grown in pots were wounded in two branches with a 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm non-colonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 5 months, shoots on all Phaeoacremonium-inoculated branches had weak growth with chlorosis of leaves and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 0.6 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was re-isolated from discolored tissue of all inoculated cuttings, completing Koch's postulates. P. venezuelense was reported as a pathogen of grapevines in Algeria (1) and South Africa (2) and, to our knowledge, this is the first report of P. venezuelense associated with wood decay of apricot trees in Spain or any country in the world. References: (1) A. Berraf-Tebbal et al. Phytopathol. Mediterr. 50:S86, 2011. (2) L. Mostert et al. J. Clin. Microbiol. 43:1752, 2005. (3) L. Mostert et al. Stud. Mycol. 54:1, 2006.

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First Report of Anthracnose of Papaya (Carica papaya L.) Caused by Colletotrichum siamense in China

Plant Disease ◽

10.1094/pdis-10-20-2154-pdn ◽

2021 ◽

Author(s):

Yujie Zhang ◽

Wenxiu Sun ◽

Ping Ning ◽

Tangxun Guo ◽

SuiPing Huang ◽

...

Keyword(s):

Carica Papaya ◽

Disease Incidence ◽

Aerial Mycelium ◽

Postharvest Disease ◽

Distilled Water ◽

First Report ◽

Surface Samples ◽

Initial Symptoms ◽

Colletotrichum Siamense ◽

Pathogenicity Tests

Papaya (Carica papaya L.) is a rosaceous plant widely grown in China, which is economically important. Anthracnose caused by Colletotrichum sp. is an important postharvest disease, which severely affects the quality of papaya fruits (Liu et al., 2019). During April 2020, some mature papaya fruits with typical anthracnose symptoms were observed in Fusui, Nanning, Guangxi, China with an average of 30% disease incidence (DI) and over 60% DI in some orchards. Initial symptoms of these papayas appeared as watery lesions, which turned dark brown, sunken, with a conidial mass appearing on the lesions under humid and warm conditions. The disease severity varied among fruits, with some showing tiny light brown spots, and some ripe fruits presenting brownish, rounded, necrotic and depressed lesions over part of their surface. Samples from two papaya plantations (107.54°E, 22.38°N) were collected, and brought to the laboratory. Symptomatic diseased tissues were cut into 5 × 5 mm pieces, surface sterilized with 2% (v/v) sodium hypochlorite for 1 minute, and rinsed three times with sterilized water. The pieces were then placed on potato dextrose agar (PDA). After incubation at 25°C in the dark for one week, colonies with uniform morphology were obtained. The aerial mycelium on PDA was white on top side, and concentric rings of salmon acervuli on the underside. A gelatinous layer of spores was observed on part of PDA plates after 7 days at 28°C. The conidia were elliptical, aseptate and hyaline (Zhang et al., 2020). The length and width of 60 conidia were measured for each of the two representative isolates, MG2-1 and MG3-1, and these averaged 13.10 × 5.11 μm and 14.45 × 5.95 μm. DNA was extracted from mycelia of these two isolates with the DNA secure Plant Kit (TIANGEN, Biotech, China). The internal transcribed spacer (ITS), partial actin (ACT), calmodulin (CAL), chitin synthase (CHS), β-tubulin 2 (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) regions were amplified by PCR and sequenced. The sequences were deposited into GenBank with accessions MT904003, MT904004, and MT898650 to MT898659. BLASTN analyses against the GenBank database showed that they all had over 99% identity to the type strain of Colletotrichum siamense isolate ICMP 18642 (GenBank accession numbers JX010278, GQ856775, JX009709, GQ856730, JX010410, JX010019) (Weir et al., 2012). A phylogenetic tree based on the combined ITS, ACT, CAL, CHS, TUB2 and GAPDH sequences using the Neighbor-joining algorithm also showed that the isolates were C. siamense. Pathogenicity tests were conducted on 24 mature, healthy and surface-sterilized papaya fruits. On 12 papaya fruits, three well separated wounded sites were made for inoculation, and for each wounded site, six adjacent pinhole wounds were made in a 5-mm-diameter circular area using a sterilized needle. A 10 µl aliquot of 1 × 106 conidia/ml suspension of each of the isolates (MG2-1 and MG3-1) was inoculated into each wound. For each isolate, there were six replicate fruits. The control fruits were inoculated with sterile distilled water. The same inoculation was applied to 12 non-wound papaya fruits. Fruits were then placed in boxes which were first washed with 75% alcohol and lined with autoclaved filter paper moistened with sterilized distilled water to maintain high humidity. The boxes were then sealed and incubated at 28°C. After 10 days, all the inoculated fruits showed symptoms, while the fruits that were mock inoculated were without symptoms. Koch's postulates were fulfilled by re-isolation of C. siamense from diseased fruits. To our knowledge, this is the first report of C. siamense causing anthracnose of papaya in China. This finding will enable better control of anthracnose disease caused by C. siamense on papaya.

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First Report of Seedborne Fusarium thapsinum and its Pathogenicity on Soybean (Glycine max) in the United States

Plant Disease ◽

10.1094/pdis-08-14-0806-pdn ◽

2014 ◽

Vol 98 (12) ◽

pp. 1745-1745 ◽

Cited By ~ 2

Author(s):

R. Pedrozo ◽

C. R. Little

Keyword(s):

United States ◽

The United States ◽

Soybean Plant ◽

Sodium Hypochlorite Solution ◽

Distilled Water ◽

Seedling Mortality ◽

Beta Tubulin ◽

First Report ◽

Significant Difference ◽

Germinated Seeds

A three-year survey from 2010 to 2012 was conducted in Kansas to investigate the identity and diversity of seedborne Fusarium spp. in soybean. A total of 408 soybean seed samples from 10 counties were tested. One hundred arbitrarily selected seeds from each sample were surface-sterilized for 10 min in a 1% sodium hypochlorite solution to avoid contaminants and promote the isolation of internal fusaria. Seeds were rinsed with sterile distilled water and dried overnight at room temperature (RT). Surface-sterilized seeds were plated on modified Nash-Snyder medium and incubated at 23 ± 2°C for 7 days. Fusarium isolates were single-spored and identified by morphological characteristics on carnation leaf agar (CLA) and potato dextrose agar (PDA) (3). From 276 seedborne Fusarium isolates, six were identified as F. thapsinum (2). On CLA, F. thapsinum isolates produced abundant mycelium and numerous chains of non-septate microconidia produced from monophialides. Microconidia were club-shaped and some were napiform. No chlamysdospores were found. On PDA, three of the isolates presented characteristic dark yellow pigmentation and three were light violet. Confirmation of the isolates to species was based on sequencing of an elongation factor gene (EF1-α) segment using primers EF1 and EF2 and the beta-tubulin gene using primers Beta1 and Beta2 (1). Sequence results (~680 bp, EF primers; ~600 bp, beta-tubulin primers) were confirmed by using the FUSARIUM-ID database (1). All isolates matched F. thapsinum for both genes sequenced (Accession No. FD01177) at 99% identity. Koch's postulates were completed for two isolates of F. thapsinum under greenhouse conditions. Soybean seeds (Asgrow AG3039) were imbibed with 2.5 × 105 conidia ml−1 for 48 h. After inoculation, seeds were dried for 48 h at RT. One isolate each of F. equiseti and F. oxysporum were used as the non-pathogenic and pathogenic inoculation controls, respectively. In addition, non-inoculated seeds and seeds imbibed in sterile distilled water (mock) were also used. Twenty-five seeds from each treatment were planted in pots (500 ml) with autoclaved soil and vermiculite (1:1). The experiment was a completely randomized design with three replicates (pots) per isolate. The entire experiment was repeated three times. After 21 days, aggressiveness of both F. thapsinum isolates was assessed using initial stand (%), final stand (%), and seed mortality (% of non-germinated seeds). Both seedborne F. thapsinum isolates caused reduced emergence and final stand, and increased seedling mortality when compared to the non-inoculated and F. equiseti controls (P< 0.0001). No significant difference was observed between F. thapsinum isolates and F. oxysporum. F. thapsinum isolates were re-isolated from wilted seedlings and non-germinated seeds, but not from the control treatments. Typically, F. thapsinum is considered a pathogen of sorghum, but it has also been recovered from bananas, peanuts, maize, and native grasses (3). However, its presence on soybean plant tissues and its pathogenicity has never been reported. To our knowledge, this is the first report of seedborne F. thapsinum and its pathogenicity on soybean in the United States. References: (1) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004. (2) C. J. R. Klittich et al. Mycologia 89:644, 1997. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Oxford, UK, 2006.

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