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

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 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 parasiticum Causing Petri Disease of Grapevine in Peru

Plant Disease ◽

10.1094/pdis-93-2-0200b ◽

2009 ◽

Vol 93 (2) ◽

pp. 200-200 ◽

Cited By ~ 2

Author(s):

L. C. Romero-Rivas ◽

L. A. Álvarez ◽

D. Gramaje ◽

J. Armengol ◽

C. Cadenas-Giraldo

Keyword(s):

Restriction Enzymes ◽

Sodium Hypochlorite Solution ◽

Malt Extract ◽

Distilled Water ◽

Internal Transcribed Spacer Region ◽

Poor Growth ◽

First Report ◽

Grapevine Decline ◽

Pathogenicity Tests ◽

Weak Growth

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 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 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 Diaporthe hongkongensis Causing Fruit rot on Peach (Prunus persica) in China

Plant Disease ◽

10.1094/pdis-07-20-1505-pdn ◽

2021 ◽

Author(s):

Zhou Zhang ◽

Zheng Bing Zhang ◽

Yuan Tai Huang ◽

FeiXiang Wang ◽

Wei Hua Hu ◽

...

Keyword(s):

Prunus Persica ◽

Fruit Rot ◽

Hunan Province ◽

Post Inoculation ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report ◽

Representative Isolate ◽

Rot Disease

Peach [Prunus persica (L.) Batsch] is an important deciduous fruit tree in the family Rosaceae and is a widely grown fruit in China (Verde et al., 2013). In July and August 2018, a fruit rot disease was observed in a few peach orchards in Zhuzhou city, the Hunan Province of China. Approximately 30% of the fruit in more than 400 trees was affected. Symptoms displayed were brown necrotic spots that expanded, coalesced, and lead to fruit being rotten. Symptomatic tissues excised from the margins of lesions were surface sterilized in 70% ethanol for 10 s, 0.1% HgCl2 for 2 min, rinsed with sterile distilled water three times, and incubated on potato dextrose agar (PDA) at 26°C in the dark. Fungal colonies with similar morphology developed, and eight fungal colonies were isolated for further identification. Colonies grown on PDA were grayish-white with white aerial mycelium. After an incubation period of approximately 3 weeks, pycnidia developed and produced α-conidia and β-conidia. The α-conidia were one-celled, hyaline, fusiform, and ranged in size from 6.0 to 8.4 × 2.1 to 3.1 μm, whereas the β-conidia were filiform, hamate, and 15.0 to 27.0 × 0.8 to 1.6 μm. For molecular identification, total genomic DNA was extracted from the mycelium of a representative isolate HT-1 and the internal transcribed spacer region (ITS), β-tubulin gene (TUB), translation elongation factor 1-α gene (TEF1), calmodulin (CAL), and histone H3 gene (HIS) were amplified and sequenced (Meng et al. 2018). The ITS, TUB, TEF1, CAL and HIS sequences (GenBank accession nos. MT740484, MT749776, MT749778, MT749777, and MT749779, respectively) were obtained and in analysis by BLAST against sequences in NCBI GenBank, showed 99.37 to 100% identity with D. hongkongensis or D. lithocarpus (the synonym of D. hongkongensis) (Gao et al., 2016) (GenBank accession nos. MG832540.1 for ITS, LT601561.1 for TUB, KJ490551.1 for HIS, KY433566.1 for TEF1, and MK442962.1 for CAL). Pathogenicity tests were performed on peach fruits by inoculation of mycelial plugs and conidial suspensions. In one set, 0.5 mm diameter mycelial discs, which were obtained from an actively growing representative isolate of the fungus on PDA, were placed individually on the surface of each fruit. Sterile agar plugs were used as controls. In another set, each of the fruits was inoculated by application of 1 ml conidial suspension (105 conidia/ml) by a spray bottle. Control assays were carried out with sterile distilled water. All treatments were maintained in humid chambers at 26°C with a 12-h photoperiod. The inoculation tests were conducted twice, with each one having three fruits as replications. Six days post-inoculation, symptoms of fruit rot were observed on inoculated fruits, whereas no symptoms developed on fruits treated with agar plugs and sterile water. The fungus was re-isolated and identified to be D. hongkongensis by morphological and molecular methods, thus fulfilling Koch’s Postulates. This fungus has been reported to cause fruit rot on kiwifruit (Li et al. 2016) and is also known to cause peach tree dieback in China (Dissanayake et al. 2017). However, to our knowledge, this is the first report of D. hongkongensis causing peach fruit rot disease in China. The identification of the pathogen will provide important information for growers to manage this disease.

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First Report of Fusarium ipomoeae Causing Peanut leaf Spot in China

Plant Disease ◽

10.1094/pdis-01-21-0226-pdn ◽

2021 ◽

Author(s):

Manlin Xu ◽

Xia Zhang ◽

Jing Yu ◽

zhiqing Guo ◽

Ying Li ◽

...

Keyword(s):

Leaf Spot ◽

Genomic Dna ◽

Block Design ◽

Management Strategies ◽

Morphological Characteristics ◽

Ethanol Solution ◽

Distilled Water ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

First Report

Peanut (Arachis hypogaea L.) is one of the most economically important crops as an important source of edible oil and protein. In August 2020, circular to oval-shaped brown leaf spots (2-6 mm in diameter) with well-defined borders surrounded by a yellow margin were observed on peanut plant leaves in Laixi City, Shandong Province, China. Symptomatic plants randomly distributed in the field, the incidence was approximately 5%. Leave samples were collected consisted of diseased tissue and the adjacent healthy tissue. The samples were dipped in a 70% (v/v) ethanol solution for 30 s and then soaked in a 0.1% (w/v) mercuric chloride solution for 60 s. The surface-sterilized tissues were then rinsed three times with sterile distilled water, dried and placed on Czapek Dox agar supplemented with 100 μg/ml of chloramphenicol. The cultures were incubated in darkness at 25 °C for 3–5 days. Fungal colonies were initially white and radial, turning to orange-brown in color, with abundant aerial mycelia. Macroconidia were abundant, 4 to 7 septate, with a dorsiventral curvature, and were 3.3–4.5 × 18.5–38.1 μm (n=100) in size; microconidia were absent; chlamydospores were produced in chains or clumps, ellipsoidal to subglobose, and thick walled. The morphological characteristics of the conidia were consistent with those of Fusarium spp. To identify the fungus, an EasyPure Genomic DNA Kit (TransGEN, Beijing, China) was used to extract the total genomic DNA from mycelia. The internal transcribed spacer region (ITS rDNA) and the translation elongation factor 1-α gene (TEF1) were amplified with primers ITS1/ITS4 (White et al. 1990) and EF1/EF2 (O’Donnell et al. 1998), respectively. Based on BLAST analysis, sequences of ITS (MT928727) and TEF1 (MT952337) showed 99.64% and 100% similarity to the ITS (MT939248.1), TEF1 (GQ505636.1) of F. ipomoeae isolates. Sequence analysis confirmed that the fungus isolated from the infected peanut was F. ipomoeae (Xia et al. 2019). The pathogenicity of the fungus was tested in the greenhouse. Twenty two-week-old peanut seedlings (cv. Huayu20) grown in 20-cm pots (containing autoclaved soil) were sprayed with a conidial suspension (105 ml−1) from a 15-day-old culture. Control plants were sprayed with distilled water. The experiment was conducted as a randomized complete block design, and placed at 25 °C under a 12-h photoperiod with 90% humidity. Symptoms similar to those in the field were observed on leaves treated with the conidial suspension ten days after inoculation, but not on control plants. F. ipomoeae was re-isolated from symptomatic leaves but not from the control plants. Reisolation of F. ipomoeae from inoculated plants fulfilled Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing peanut leaf spot in China. Our report indicates the potential spread of this pathogen in China and a systematic survey is required to develop effective disease management strategies.

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First Report of Phomopsis longicolla Causing Stem Canker of Eggplant in Guangdong, China

Plant Disease ◽

10.1094/pdis-07-13-0790-pdn ◽

2014 ◽

Vol 98 (3) ◽

pp. 426-426 ◽

Cited By ~ 4

Author(s):

C. Shu ◽

J. Chen ◽

H. Huang ◽

Y. He ◽

E. Zhou

Keyword(s):

Disease Incidence ◽

Guangdong Province ◽

Stem Canker ◽

Universal Primers ◽

Distilled Water ◽

Conidial Suspension ◽

Phomopsis Longicolla ◽

Fungal Isolation ◽

First Report ◽

Absorbent Cotton

Eggplant (Solanum melongena L.) is an economically important vegetable crop worldwide. In August 2012, severe stem cankers were observed on eggplant at the early stage of maturation in several fields in Guangdong Province, China. Diseased plants raised cankers on the stems and branches, which resulted in wilting and stunting. No symptoms developed on eggplant fruit. Disease incidence was as high as 40% within affected fields. By using routine fungal-isolation methods and single-spore purification technique, five single-conidial isolates were obtained from each diseased stem. Colonies were grayish-white, circular, and got yellow pigmentation when placed in acidified potato dextrose agar (PDA) in an incubator at pH 4.5 and 25°C with a 12-h photoperiod. Stromata were black, large, and spreading in a concentric pattern. Conidiomata were pycnidial, and the pycnidia were round, oblate, triangular or irregular, and unilocular. Conidiophores were colorless, separated, dichotomous, and 10.0 to 18.0 × 1.5 to 2.0 μm. Alpha conidia were single-celled, ellipsoidal to fusiform, guttulate, and 6.0 to 8.0 × 2.0 to 2.5 μm. Beta conidia, produced on oat meal agar in 2 weeks at 25°C in the dark, were filiform, hamate, and 16.0 to 28.0 × 0.7 to 1.0 μm. Based on these morphological characters, the fungus was identified as Phomopsis longicolla Hobbs (1). The ITS-rDNA sequence (GenBank Accession No. KC886605) of the isolate EPPL1 of this fungus (P. longicolla EPPL1) was obtained by using universal primers ITS5/ITS4 (1). BLAST searches showed a 98% homology with the sequence of the ITS region of rDNA of P. longicolla. Phylogenetic analysis showed that P. longicolla EPPL1 clustered with P. longicolla SYJM15 and formed a distinct clade distantly related to P. vexans PV3 (GU373630), a well-known pathogen of eggplant. Digestion of PCR-amplified DNA with Alu I yielded two restriction fragments of sizes consistent with those reported for P. longicolla (2). Pathogenicity tests were performed on 30-day-old plants of cv. Yuefengzihongqie grown in a plastic pot (1 liter) in a greenhouse by using mycelial plugs and conidial suspensions of isolate EPPL1 as inocula. A mycelial plug (4 mm in diameter) from a 7-day-old PDA culture was placed on stems of both wounded and non-wounded plants and covered with sterile absorbent cotton moistened with sterile distilled water. Both wounded and non-wounded plants were inoculated with 0.5 ml of conidial suspension (1 × 106 conidia ml–1) dropped onto sterile absorbent cotton covering the stems. Control assays were performed with agar plugs and sterile distilled water only. Inoculated plants were placed in a greenhouse with a 12-h photoperiod at 28°C. Each treatment was replicated on five plants, and the test was repeated. Twenty-five days after inoculation, both wounded and non-wounded plants inoculated with either method showed raised cankers at the points of inoculation and canker lesions similar to those observed in the field expanded up and down the stems to reach lengths of 15 to 30 mm. Later, sparse, small, black pycnidia formed on the surface of the lesions. The inoculated plants exhibited stunting and premature senescence compared to controls. P. longicolla was re-isolated from the infected stems of inoculated plants. Control plants were asymptomatic. To our knowledge, this is the first report of P. longicolla causing stem canker in eggplant in Guangdong, China. Considering the economic importance of eggplant in Guangdong Province and throughout the world, further study of phomopsis stem canker of eggplant is warranted. References: (1) T. W. Hobbs et al. Mycologia 77:535, 1985. (2) A. W. Zhang et al. Plant Dis. 81:1143, 1997.

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First Report of Leaf Blight of Cyperus iria Caused by Fusarium equiseti in India

Plant Disease ◽

10.1094/pdis-07-12-0690-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 838-838 ◽

Cited By ~ 1

Author(s):

V. Gupta ◽

V. K. Razdan ◽

D. John ◽

B. C. Sharma

Keyword(s):

Leaf Blight ◽

Spore Suspension ◽

Commonwealth Mycological Institute ◽

Fungal Culture ◽

Distilled Water ◽

Total Production ◽

Conidial Suspension ◽

Internal Transcribed Spacer Region ◽

Fusarium Equiseti ◽

First Report

In India, rice (Oryza sativa L.) plays a major role in national food security, with total production of 102.75 million t, harvested from 44 million ha during 2011 (1). Weeds are one of the major causes of losses in rice. Cyperus iria, locally known as chatriwala dela (rice flat sedge), is an annual weed in the Cyperaceae that can reach 50 to 60 cm tall. A leaf blight of C. iria was observed during August 2010 in a 20-ha rice field (cv. Basmati 370) at the University Research Farm, Chatha, Jammu (32° 43′ N, 74° 54′ E). Symptomatic plants were scattered randomly in the field and had water-soaked spots on the upper leaf surfaces initially, which turned brown after 4 days and developed a yellow halo, resulting in a blighted appearance. The diseased leaves shriveled and infected plants died. Infected C. iria leaf pieces with adjacent healthy tissue were collected, surface-sterilized in 0.1% mercuric chloride for 20 s, then rinsed three times in sterilized distilled water. The pieces were plated onto potato dextrose agar (PDA) and incubated at 27 ± 1°C for 4 days. A pure fungal culture was obtained by single-spore technique on 2% water agar and maintained on PDA at 10°C. The fungus initially produced white mycelium that became brown with age. Dark brown spots or flecks of pigment formed in the agar. Macroconidia were long and slender, with tapered apical cells that were elongated or even whip-like. Basal cells of macroconidia were prominent, foot shaped, and elongated. Macroconidia were 39.55 to 56.74 × 3.75 to 4.5 μm with 3 to 5 septa. Conidiophores were compact, penicillately branched, and arose from lateral branches which initially were one-celled and bore 2 to 4 phialides at the apex. Chlamydospores were intercalary, solitary, in chains or in knots, globose, and 7 to 9 μm in diameter. On the basis of morphological characteristics (2), the fungus was identified as Fusarium equiseti (Corda) Sacc. and deposited in the Indian Type Culture Collection, New Delhi (8424.11). The ITS (internal transcribed spacer) region of rDNA was amplified by PCR with primers ITS1/ITS2 and sequenced. BLASTn analysis of the sequence showed 100% homology with the ITS sequence of F. equiseti in the NCBI database (JN596252.1), and the sequence was deposited in GenBank (KC434458). To confirm pathogenicity of the F. equiseti isolate, 10 seeds of C. iria were planted in five clay pots (each 38 cm in diameter) filled with sterilized soil. Three seedlings were used for the experiment and the remaining seedlings removed from each pot. A total of 15 seedlings (5 pots × 3 seedlings per pot) at the two-leaf stage were spray-inoculated with a 50-ml conidial suspension of the isolate (105 cfu/ml) using a hand atomizer. The control treatment included three seedlings treated similarly with sterile distilled water. The spore suspension was prepared in potato dextrose broth using a culture of the fungus incubated for 10 days and then homogenized at 140 rpm. Tween 20 (1%) was added to the spore suspension. Small spots developed 4 days after inoculation, and the lesions then coalesced into large necrotic areas, resulting in leaf blight 10 days after inoculation. F. equiseti was reisolated from inoculated leaves using the method described above, whereas no fungus was reisolated from control plants, fulfilling Koch's postulates. The isolated fungus displayed the same morphological and cultural features as the original isolate. F. equiseti has been reported to infect Echinochloa spp. in Iran (3), but to our knowledge, this is the first report of F. equiseti infecting C. iria in India. Thus, F. equiseti represents a potential biocontrol agent for managing C. iria in rice fields. References: (1) Anonymous. Direct. Rice Res. Newslett. 10:2, 2012. (2) C. Booth. The Genus Fusarium. Commonwealth Mycological Institute, Kew, Surrey, England, p. 157, 1971. (3) M. R. S. Motlagh. Austral. J. Crop Sci. 4:457, 2010.

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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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