First Report of Wilt and Dieback on Pokeweed (Phytolacca decandra) Caused by Phytophthora nicotianae

Pokeweed (Phytolacca decandra, synonym Phytolacca americana) is a root perennial plant that produces a succulent annual stem. In late June 2001, a severe dieback occurred on a group of pokeweed plants being grown as ornamentals in a garden in Rome. Disease symptoms consisted of leaf wilting followed by collapse of the plant. Stem collars and roots had dark brown-to-black water-soaked lesions. A wet rot was observed on plants with advanced disease symptoms. Isolations, from sections of roots and stems previously washed in running tap water, were made on PARBhy selective medium (10 mg of pimaricin, 250 mg of ampicillin [sodium salt], 10 mg of rifampicin, 50 mg of hymexazol, 15 mg of benomyl, 15 g of malt extract, and 20 g of agar in 1,000 ml of H2O) (2), followed by incubation at 20°C. A species of Phytophthora identified based on morphological and cultural characteristics (1) was isolated consistently from rotted roots and collars of diseased plants. All isolates produced papillate, spherical, ovoid to obturbinate, noncaducous sporangia and terminal and intercalary chlamydospores. Hyphal swellings with hyphal outgrowths were present. Observed characteristics were similar to those described for P. nicotianae. Isolates were mating type A2 with amphigynous antheridia in paired cultures with the A1 tester isolate of P. nicotianae. Identification was confirmed by comparing restriction fragment length polymorphism patterns of the internal transcribed spacer region of ribosomal DNA with those obtained from previously identified Phytophthora species. Pathogenicity tests were conducted on 10 2-month-old potted pokeweed plants. Inoculum was prepared by inoculating sterilized millet seeds moistened with V8 broth with plugs of mycelium and growing for 4 weeks. The inoculum was added to potting soil at 3% (wt/vol), and sporulation was induced by flooding the soil for 48 h. Five uninoculated plants were used as controls. Plants were maintained outdoors and assessed for symptoms within 2 months after inoculation. Wilting, root rot, and dark brown lesions on the collar developed on inoculated plants. The pathogen was reisolated from the inoculated plants and morphologically identical to the original isolates, which confirmed P. nicotianae as the causal agent of the disease. Few diseases have been reported on Phytolacca decandra. This species is not only an invasive weed, but is also cultivated as an ornamental and medicinal plant. In addition, antiviral (PAP) and antifungal (Pa-AFP) proteins that are used as a remedy for several human and plant infections have been extracted from the plant. To our knowledge, this is the first report of P. nicotianae on pokeweed. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) A. M.Vettraino et al. Plant Pathol. 50:90, 2001.

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First Report of Phytophthora nicotianae and P. citricola Associated with English Walnut Decline in Europe

Plant Disease ◽

10.1094/pdis.2003.87.3.315b ◽

2003 ◽

Vol 87 (3) ◽

pp. 315-315 ◽

Cited By ~ 1

Author(s):

A. Belisario ◽

M. Maccaroni ◽

A. M. Vettraino ◽

A. Vannini

Keyword(s):

Lateral Roots ◽

Juglans Regia ◽

The United States ◽

Phytophthora Nicotianae ◽

Malt Extract ◽

Veneto Region ◽

Internal Transcribed Spacer Region ◽

Campania Region ◽

First Report ◽

English Walnut

English (Persian) walnut (Juglans regia), among the most widely cultivated species of Juglans worldwide, is cultivated primarily for fruit production but also for timber. In the last 10 years, walnut decline causing leaf yellowing, sparse foliage, overall decline, and plant death has increased in Italian commercial orchards. In Italy, Phytophthora cactorum, P. cambivora, P. cinnamomi, and P. cryptogea are associated with this disease (1,4). Over the last 5 years, P. cinnamomi was the most widely isolated and destructive species (1). Recently, a different species of Phytophthora was isolated from diseased roots and soil from around lateral roots of 10 declining trees in two orchards in the Veneto Region of northern Italy. Another species of Phytophthora was isolated consistently from rotted roots of declining walnut trees in two orchards in the Campania Region of southern Italy. Phytophthora spp. were isolated directly from plant material or Rhododendron spp. leaf baiting on soil samples with PARBhy selective medium (10 mg of pimaricin, 250 mg of ampicillin [sodium salt], 10 mg of rifampicin, 50 mg of hymexazol, 15 mg of benomyl, 15 g of malt extract, 20 g of agar in 1,000 ml of H2O). Two species of Phytophthora were identified based on morphological and cultural characteristics (2). The species from trees in the Veneto Region was identified as P. nicotianae. All isolates produced papillate, spherical to obturbinate, occasionally caducous sporangia with short pedicels, terminal and intercalary chlamydospores, and were mating type A2. The species isolated from trees in the Campania Region was identified as P.citricola. Isolates were homothallic, produced semipapillate, persistent, obclavate to obpyriform sporangia, occasionally with two apices, and antheridia paragynous. Identifications were confirmed by comparing restriction fragment length polymorphism patterns of the internal transcribed spacer region of rDNA with those obtained from previously identified species of Phytophthora. Pathogenicity of two isolates each of P. citricola and P. nicotianae was tested on 2-year-old potted walnut seedlings. Inocula were prepared by inoculating sterilized millet seeds moistened with V8 broth with plugs of mycelium and incubated for 4 weeks at 20°C in the dark. Infested seeds were added to potting soil at a rate of 3% (wt/vol). One day later, pots were flooded for 48 h to promote sporulation. Ten noninoculated seedlings were used as the control. Symptoms were assessed 2 months after inoculation. Seedlings inoculated with P. nicotianae developed necrosis of feeder and lateral roots, but only limited infection of taproots. Seedlings inoculated with P. citricola developed necroses at the insertion points of lateral roots. All four isolates produced visible damage to lateral roots on inoculated plants. P. nicotianae and P. citricola were reisolated from respectively infected roots. Results from these inoculations confirmed P. nicotianae and P. citricola as root pathogens of English walnut. Both species were associated with walnut decline as reported in the United States (3). To our knowledge, this is the first report of P. nicotianae and P. citricola on J. regia in Europe. References: (1) A. Belisario et al. Petria 11:149. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) M. E. Matheron and S. M. Mircetich. Phytopathology 75:977, 1985. (4) A. M. Vettraino et al. Plant Dis. 86:328, 2002.

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First Report of Phytophthora spp. as Pathogens of Pandorea jasminoides in Italy

Plant Disease ◽

10.1094/pdis-92-2-0313b ◽

2008 ◽

Vol 92 (2) ◽

pp. 313-313 ◽

Cited By ~ 3

Author(s):

A. Pane ◽

S. O. Cacciola ◽

A. Chimento ◽

C. Allatta ◽

S. Scibetta ◽

...

Keyword(s):

Growth Temperature ◽

Root Rot ◽

Selective Medium ◽

Phytophthora Nicotianae ◽

Optimum Growth ◽

Internal Transcribed Spacer Region ◽

Phytophthora Root Rot ◽

First Report ◽

Optimum Growth Temperature ◽

Phytophthora Spp

In the summer of 2005, approximately 5% of a nursery stock of 12-month-old potted plants of bower vine (Pandorea jasminoides (Lindl.) K. Schum.) in Sicily (Italy) showed wilt, leaf chlorosis, defoliation, root rot, and collapse of the entire plant. Three Phytophthora spp. (20, 50, and 30% of the isolations of the first, second, and third species, respectively) were isolated from rotted roots on BNPRAH selective medium (2). Single-hypha isolates of the first species formed petaloid colonies on potato dextrose agar (PDA) and had an optimum growth temperature of 25°C (9.3 mm/day); on V8 juice agar, they produced uni- and bipapillate, ovoid to limoniform sporangia with mean dimensions of 45 × 30 μm and a mean length/width (l/w) ratio of 1.4:1. They did not produce gametangia when paired with A1 and A2 isolates of Phytophthora nicotianae. The second species formed arachnoides colonies on PDA, had an optimum growth temperature of 30°C (6.9 mm/day) and produced sporangia that were uni- and bipapillate, ellipsoid, ovoid, or pyriform to spherical (dimensions 44 × 34 μm; l/w ratio 1.3:1). All isolates were A2 mating type and produced amphyginous antheridia and spherical oogonia with smooth walls. The third species formed rosaceous colonies on PDA, had an optimum growth temperature of 28 to 30°C (11.9 mm/day), and produced uni- and bipapillate, ellipsoid or limoniform, caducous sporangia (dimensions 52 × 26 μm; l/w ratio 2.1:1) with a tapered base and a long pedicel (as much as 150 μm). All isolates were A1 type and produced amphigynous antheridia and spherical oogonia with smooth walls. The three species were identified as P. citrophthora, P. nicotianae, and P. tropicalis, respectively. The electrophoretic analysis of the mycelial proteins and four isozymes (1) confirmed the identification. Blast analysis of the sequence of the internal transcribed spacer region of the rDNA of a P. tropicalis isolate from bower vine (GenBank Accession No. EU076731) showed 99% similarity with the sequence of a P. tropicalis isolate from Cuphea ignea (GenBank Accession No. DQ118649). The pathogenicity of three isolates from bower vine, IMI 395552 (P. citrophthora), IMI 395553 (P. nicotianae), and IMI 395346 (P. tropicalis), was tested on 3-month-old potted bower vine plants (10 plants for each isolate) by applying 10 ml of a suspension (2 × 104 zoospores/ml) to the root crown. The plants were maintained at 24°C and 95 to 100% relative humidity. All inoculated plants wilted after 4 weeks. Noninoculated control plants remained healthy. The three Phytophthora spp. were reisolated from symptomatic plants. To our knowledge, this is the first report of Phytophthora root rot of bower vine in Italy. References: (1) S. O. Cacciola et al. Plant Dis. 90:680, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996.

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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 Phytophthora ramorum on Viburnum bodnantense in Belgium

Plant Disease ◽

10.1094/pdis.2003.87.2.203c ◽

2003 ◽

Vol 87 (2) ◽

pp. 203-203 ◽

Cited By ~ 11

Author(s):

D. De Merlier ◽

A. Chandelier ◽

M. Cavelier

Keyword(s):

The Netherlands ◽

Plant Protection ◽

Morphological Characteristics ◽

Pcr Primers ◽

Selective Medium ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Pathogenicity Test ◽

First Report ◽

Root Necrosis

In the past decade, a new Phytophthora species inducing shoot canker on Rhododendron and dieback of Viburnum has been observed in Europe, mainly in Germany and the Netherlands, and California. This new pathogen has been named Phytophthora ramorum (3). In May 2002, a diseased Viburnum plant (Viburnum bodnantense) from the Plant Protection Service (Ministry of Agriculture, Belgium) was submitted to our laboratory for diagnosis. Symptoms included wilting, leaves turning from green to brown, discolored vascular tissues, and root necrosis. The plant came from a Belgian ornamental nursery that obtained supplies of stock plants from the Netherlands. Pieces of necrotic root tissue were excised, surface-disinfected, and transferred aseptically to a Phytophthora selective medium. P. ramorum was identified based on morphological characteristics, including the production of numerous, thin-walled chlamydospores (25 to 70 µm in diameter, average 43 µm) and deciduous, semi-papillate sporangia arranged in clusters. Radial growth after 6 days at 20°C on V8 juice agar was 2.8 mm per day. Random amplified microsatellite markers (RAMS) (2) from the total genomic DNA of the Belgian strain (CBS 110901) were similar to those of P. ramorum reference strains (CBS 101330, CBS 101332, and CBS 101554). Using PCR primers specific for P. ramorum, the identification was confirmed by W. A. Man in't Veld (Plantenziektenkundige Dienst, Wageningen, the Netherlands) (1). A pathogenicity test was carried out on three sterile cuttings of Rhododendron catawbiense (3). Brown lesions were observed on the inoculated cuttings after 6 to 7 days. None of the three uninoculated cuttings showed symptoms of infection. P. ramorum was reisolated from lesion margins on the inoculated cuttings. To our knowledge, this is the first report of the fungus from Belgium. Since our initial observation, we have found P. ramorum in other Belgian nurseries on R. yakusimanum. References: (1) M. Garbelotto et al. US For. Ser. Gen. Tech. Rep. PSW-GRT. 184:765, 2002. (2) J. Hantula et al. Mycol. Res. 101:565, 1997. (3) S. Werres et al. Mycol. Res. 105:1155, 2001.

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First Report of Phytophthora hydropathica Causing Wilting and Shoot Dieback on Viburnum in Italy

Plant Disease ◽

10.1094/pdis-03-14-0308-pdn ◽

2014 ◽

Vol 98 (11) ◽

pp. 1582-1582 ◽

Cited By ~ 5

Author(s):

S. Vitale ◽

L. Luongo ◽

M. Galli ◽

A. Belisario

Keyword(s):

Natural Infection ◽

Morphological Characteristics ◽

Leaf Tissue ◽

The United States ◽

Selective Medium ◽

Phytophthora Species ◽

Phytophthora Ramorum ◽

Width Ratio ◽

Morphological Identification ◽

First Report

The genus Viburnum comprises over 150 species of shrubs and small trees such as Laurustinus (Viburnum tinus L.), which is one of the most widely used ornamental plants in private and public gardens. Furthermore, it commonly forms stands of natural woodland in the Mediterranean area. In autumn 2012, a survey was conducted to determine the presence of Phytophthora ramorum on Viburnum in commercial nurseries in the Latium region where wilting, dieback, and death of twigs were observed on 30% of the Laurustinus plants. A Phytophthora species was consistently recovered from soil rich in feeder roots from potted Laurustinus plants showing symptoms. Soil samples were baited with rhododendron leaves. Small pieces of leaf tissue cut from the margin of lesions were plated on P5ARPH selective medium (4). Pure cultures, obtained by single-hypha transfers on potato dextrose agar (PDA), were petaloid. Sporangia formation was induced on pepper seeds (3). Sporangia were almost spherical, ovoid or obpyriform, non-papillate and non-caducous, measuring 36.6 to 71.4 × 33.4 to 48.3 μm (average 53.3 × 37.4 μm) with a length/width ratio of 1.4. Chlamydospores were terminal and 25.2 to 37.9 μm in diameter. Isolates were considered heterothallic because they did not produce gametangia in culture or on the host. All isolates examined had 30 to 35°C as optimum temperatures. Based on these morphological characteristics, the isolates were identified as Phytophthora hydropathica (2). Morphological identification was confirmed by internal transcribed spacer (ITS), and mitochondrial partial cytochrome oxidase subunit 2 (CoxII) with BLAST analysis in the NCBI database revealing 99% identity with ITS and 100% identity with CoxII. The sequences of the three isolates AB234, AB235, and AB236 were deposited in European Nucleotide Archive (ENA) with the accession nos. HG934148, HG934149, and HG934150 for ITS and HG934151, HG934152, and HG934153 for CoxII, respectively. Pathogenicity tests were conducted in the greenhouse on a total of six 1-year-old shoots cut from V. tinus plants with two inoculation points each. Mycelial plugs cut from the margins of actively growing 8-day-old cultures on PDA were inserted through the epidermis into the phloem. Controls were treated as described above except that sterile PDA plugs replaced the inoculum. Shoots were incubated in test tubes with sterile water in the dark at 24 ± 2°C. After 2 weeks, lesions were evident at the inoculation points and symptoms were similar to those caused by natural infection. P. hydropathica was consistently re-isolated from the margin of lesions, while controls remained symptomless. In the United States in 2008, P. hydropathica was described as spreading from irrigation water to Rhododendron catawbiense and Kalmia latifolia (2). This pathogen can also attack several other horticultural crops (1), but to our knowledge, this is the first report of P. hydropathica causing wilting and shoot dieback on V. tinus. References: (1) C. X. Hong et al. Plant Dis. 92:1201, 2008. (2) C. X. Hong et al. Plant Pathol. 59:913, 2010. (3) E. Ilieva et al. Eur. J. Plant Path. 101:623, 1995. (4) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986.

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First report of Eutypa lata causing cankers and dieback in sweet cherry in Chile

Plant Disease ◽

10.1094/pdis-02-21-0240-pdn ◽

2021 ◽

Author(s):

Daina Grinbergs ◽

Javier Chilian ◽

Andrés France

Keyword(s):

Fungal Pathogens ◽

Sweet Cherry ◽

Prunus Avium ◽

Tap Water ◽

Central Area ◽

Wood Decay ◽

Internal Transcribed Spacer Region ◽

Indole Butyric Acid ◽

First Report ◽

Eutypa Lata

Sweet cherry (Prunus avium) is one the most important fruit crops in Chile. Its production has significantly grown in recent years, reaching 228,448 tons exported in 2019/2020, to 47 countries. One of the main threats for this expanding crop are fungal pathogens, especially those that cause wood diseases. Cherry orchards (n=35) located in the central area of Chile, from Curicó (34°58'58''S 71°14.366'W) to Angol (37°47'42.7''S 72°42.982'W), were surveyed during 2020. Wood samples were collected (n= 72) from living branches and trunks showing dieback, cankers and dark necrosis, mostly wedge shaped. Small wood sections (0.5-cm) were cut off from the margin of the necrosis and surface disinfected using 0.5% v/v sodium hypochlorite. Sections were plated on a quarter-strength potato dextrose agar amended with 1mg/L tetracycline (PDA-tet). Plates were incubated at 25°C until mycelial development and subsequently the isolates were purified transferring excised fungal tips to PDA. Colonies (n=21) developed white cottony mycelia, which turned slightly greyish and flatter after 10-days at 25°C. Isolates developed black pycnidia which released beige conidial matrixes after subsequent 15-days at 25 +/-2°C and 12-h photoperiod. Conidia were hyaline, curved and filiform, measuring 19.8-(27.9)-36.7 μm length (lineal) x 1.2-(1.7)-1.9 μm width (n=70), according to Eutypa lata (Rappaz, 1984). DNA was extracted from mycelia of the representative isolates HMCe30a, HMCe41a, HMCe109c and HMCe110a. The partial β-tubulin gene was amplified using bt2A/bt2B primers (Glass & Donaldson 1995) and the internal transcribed spacer region was amplified using ITS1/ITS4 primers (White et al. 1990). Sequences were BLAST analyzed, finding that ITS shared 99% and βTUB 100% identity with E. lata strain CBS 208.87 (Rolshausen et al. 2006). Sequences were accessioned to GenBank (MW363035, MW363034, MW363033 and MW363032 [ITS], and MW366820, MW366819, MW366818and MW366817 [βTUB]). The isolates were inoculated on sweet cherry healthy plants cv. Kordia, produced by rooting scions in tap water amended with 500 ppm of indole-butyric acid, for 30 days. An injury was made in the upper third of the shoot using a sterile 0.5-cm diameter corkborer. Mycelial plugs were placed on the injuries and covered with plastic film, using sterile agar for controls (n=25). Plants were incubated in aerated tap water for 60 days at 23 +/-3 °C. After incubation, plants were cut exposing dark-brown necrotic lesions, while control plants remained asymptomatic. Moreover, 2-year old potted plants cv. Lapins were inoculated (n=3 per isolate) with mycelial plugs, on fresh cuts of their main lateral branches, in January 20th, and remained under partial shade for 72-days. After incubation, bark was removed from inoculated branches and the necrotic lesions length was measured. HMCe109c was the most virulent isolate (3.6 cm), followed by HMCe30a (2.1 cm), HMCe41a (1.9 cm) and HMCe110a (1.1 cm), while symptoms were not reproduced in controls. Fulfilling Koch’s postulates, fungi were reisolated from all inoculated plants in both pathogenicity tests and no fungus was recovered from controls. To our knowledge this is the first report of Eutypa lata causing wood decay in sweet cherry in Chile. The pathogen was recently reported causing dieback of grapevines in Chile (Lolas et al. 2020). These are significant findings due to the frequent proximity of sweet cherry orchards and vineyards, which facilitates cross infections.

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First Report of Phytophthora megasperma Causing Decline and Death on Celtis australis in Italy

Plant Disease ◽

10.1094/pdis-05-14-0534-pdn ◽

2015 ◽

Vol 99 (1) ◽

pp. 155-155 ◽

Cited By ~ 2

Author(s):

L. Luongo ◽

A. Haegi ◽

M. Galli ◽

S. Berti ◽

S. Vitale ◽

...

Keyword(s):

Sensu Stricto ◽

Biological Species ◽

Selective Medium ◽

Phytophthora Species ◽

Universal Primers ◽

Term Survival ◽

Phytophthora Megasperma ◽

First Report ◽

Umbria Region ◽

Celtis Australis

European hackberry (Celtis australis L.) is a popular shade tree mainly planted in southern Europe and known to be tolerant to dry and poor soils. In early autumn 2013, hackberry plants grown in soil in a commercial nursery located in the floodplain in Umbria region showed symptoms of wilting, dieback, and death. Up to 100% of the canopy was affected, and over 60% of the plants were symptomatic or dead. A Phytophthora species was consistently isolated from symptomatic 6-year-old plants by plating small pieces of collar and root tissues, cut from the margin of dark-brown necrotic lesions, onto P5ARPH selective medium (4). Pure cultures were obtained by single-hyphal transfers on potato dextrose agar (PDA). Sporangia, produced on pepper seeds in soil extract solution (3), were nonpapillate and noncaducous, measuring 34.0 to 85.0 × 22.0 to 50.0 μm. Oospores had an average diameter of 44 μm with mostly paragynous antheridia. On the basis of morphological features, the isolates were identified as P. megasperma Drech. (2). The identity was confirmed by sequencing the cytochrome c oxidase subunit II (Cox II) (5), which gave 100% identity with P. megasperma sequences available in GenBank (GU222070), and by sequencing the internal transcribed spacer (ITS) using the universal primers ITS4 and ITS6, which gave 99% identity with the AF266794 sequence from Cooke et al. (1). The sequences of one isolate (AB239) were deposited in the European Nucleotide Archive (ENA) with accession numbers HG973451 and HG973450 for Cox II and ITS, respectively. Pathogenicity tests were conducted in the greenhouse with isolate AB239 on eight 2-year-old potted European hackberry plants. Mycelial plugs (5 mm diameter) cut from the margins of actively growing 8-day-old cultures on PDA were inserted through the epidermis to the phloem at the collar level. Two plants were used as controls and treated as described above except that sterile PDA plugs replaced the inoculum. Inoculated plants were kept for 4 weeks in a greenhouse at 24 ± 2°C. During that period, inoculated plants showed wilting symptoms similar to those observed in the field. Lesions were evident at all the inoculation points progressing downward to the roots. Colonies of Phytophthora were isolated from the margins of lesions and identified as P. megasperma, thus fulfilling Koch's postulates. Controls remained symptomless. P. megasperma taxonomy is rather complex since it embraces different subgroups, including host specialized forms (formae speciales), some of which are recognized as biological species. Based on morphological and molecular data presented here, the Phytophthora isolates from hackberry belong to P. megasperma sensu stricto, which is included in the “pathogenic to a broad range of hosts” (BHR) group (1). This pathogen is rather polyphagous, attacking mainly fruit and ornamental woody plants, commonly Prunus spp., Malus spp., and Actinidia deliciosa. Like other homothallic Phytophthora species, it is particularly dangerous due to its abundant production of thick-walled resting oospores that enable long-term survival in the soil. To our knowledge this is the first report of P. megasperma sensu stricto (1) on C. australis and its family Ulmaceae/Cannabaceae. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) D. C. Erwin and O. K. Ribeiro, American Phytopathological Society, St. Paul, MN, 1996. (3) E. Ilieva et al. Eur. J. Plant Path. 101:623, 1995. (4) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (5) F. N. Martin and P. W. Tooley. Mycologia 95:269, 2003.

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Shoot Blight of Forsythia × intermedia in Virginia Nurseries Caused by Phytophthora nicotianae

Plant Disease ◽

10.1094/pd-89-0430c ◽

2005 ◽

Vol 89 (4) ◽

pp. 430-430 ◽

Cited By ~ 2

Author(s):

C. X. Hong ◽

P. A. Richardson ◽

P. Kong

Keyword(s):

Single Strand Conformation Polymorphism ◽

The United States ◽

Morphological Characters ◽

Selective Medium ◽

Shoot Tips ◽

Phytophthora Nicotianae ◽

First Report ◽

Plastic Bags ◽

Shoot Blight ◽

Polymorphism Pattern

A severe blighting of shoots on Forsythia × intermedia cv. Lynwood Gold plants was observed at several commercial nurseries in Virginia from 2001 to 2004. Crop losses ranged from 10 to 35%. Symptoms first occurred at the tips of shoots, including those that were trimmed and not trimmed, and then progressed downward. Diseased shoots wilted quickly and usually turned black, and foliage on these shoots withered and became necrotic. With PARP-V8 selective medium (2), a species of Phytophthora was isolated consistently from symptomatic shoots (including tissues from shoot tips, leaves, and stems) as well as from apparently healthy roots. These isolates produced arachnoid mycelia and numerous noncaducous, papillate sporangia but did not produce sexual structures on isolation plates; these morphological characters are consistent with those of Phytophthora nicotianae. All isolates produced a single-strand conformation polymorphism pattern typical of P. nicotianae (3). To test pathogenicity, 1-year-old, healthy-appearing cv. Lynwood Gold forsythia plants (canopy size = 100 cm × 60 cm) in four 12-liter containers were sheared. Two plants were inoculated by spraying each plant with 200 ml of a zoospore suspension (1.6 × 104 spores per ml, prepared from one isolate), and the other two plants were not treated and served as controls. Plants were covered with plastic bags overnight to encourage infection and then were grown in a field (temperature range = 20 to 33°C). Severe blight developed on trimmed shoots and new shoot tips of inoculated plants within 1 week after inoculation. The same pathogen was isolated from all blighted leaf and stem pieces assayed. Blight symptoms were not observed on control plants during a 1-month observation period. Phytophthora nicotianae has been reported to attack F. viridissima in Italy (1) causing root and collar rot but not shoot blight. To our knowledge, this is the first report of shoot blight on Forsythia spp. caused by P. nicotianae and the first report of P. nicotianae on Forsythia spp. in the United States. References: (1) S. O. Cacciola et al. Plant Dis. 78:525, 1994. (2) A. J. Ferguson and S. N. Jeffers. Plant Dis. 83:1129, 1999. (3) P. Kong et al. Fun. Gen. Biol. 39:238, 2003.

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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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