First Report of Phytophthora megasperma Associated with Decline and Death of Common Walnut Trees in Italy

Common walnut (Juglans regia L.) is an important nut crop in Italy, which is the fifth largest producer of walnut in Europe. In recent years, walnut decline and subsequent death has increased in many Italian commercial orchards. In the summer of 2010, several declining trees were present in waterlogged area of a walnut orchard located in the Veneto region. Symptoms included sparse foliage, wilting, and shoot and branch dieback. By the next year, a larger area of about 1 ha with 20% of dead trees was present, and soil/root samples were subjected to azalea leaf baiting and successively cultured on PARBH medium (3). Isolates were identified as Phytophthora megasperma based on morphological characteristics (2) and DNA sequence analysis. Sporangia were 35.0 to 62.0 × 12.0 to 30.0 μm, nonpapillate, and noncaducous when produced in soil extract solution. Oogonia had an average diameter of 36 μm with mostly paragynous antheridia. Identity was confirmed by sequence comparison in NCBI database with 99% and 100% identity for internal transcribed spacer (ITS) and mitochondrial partial COI (4) for cytochrome oxidase subunit 1, respectively. The sequences of the isolate AB199 were deposited in GenBank with the accession nos. HE805270 and HE805269 for ITS and COI, respectively. Pathogenicity tests were conducted in the greenhouse on six 1-year-old walnut shoots 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 to the phloem. Controls were treated as described above except for inoculation with sterile PDA plugs. After inoculation, shoots were incubated in test tubes with sterile water for 1 week in the dark at 22 ± 2°C. Lesions were evident at the inoculation points. P. megasperma was consistently reisolated from the margin of symptomatic tissues. Controls remained symptomless. P. megasperma is a polyphagous, ubiquitarious Phytophthora species that attacks many crops and fruit species including walnut. Although several other species of Phytophthora have been reported from Italy (1), to the best of our knowledge, this is the first report of Phytophthora decline on common walnut in Italy caused by P. megasperma. References: (1) A. Belisario et al. Acta Hort. 705:401, 2006. (2) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (3) G. C. Papavisas et al. Phytopathology 71:129, 1981. (4) G. P. Robideau et al. Mol. Ecol. Resour. 11:1002, 2011.

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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 Leaf Spot Disease on Walnut Caused by Colletotrichum fioriniae in China

Plant Disease ◽

10.1094/pdis-09-14-0938-pdn ◽

2015 ◽

Vol 99 (2) ◽

pp. 289-289 ◽

Cited By ~ 9

Author(s):

Y. Z. Zhu ◽

W. J. Liao ◽

D. X. Zou ◽

Y. J. Wu ◽

Y. Zhou

Keyword(s):

Dna Sequences ◽

Leaf Spot ◽

Juglans Regia ◽

Morphological Characteristics ◽

Leaf Spot Disease ◽

Koch’S Postulates ◽

First Report ◽

Spot Disease ◽

Leaf Spots ◽

Koch's Postulates

In May 2014, a severe leaf spot disease was observed on walnut tree (Juglans regia L.) in Hechi, Guangxi, China. Leaf spots were circular to semicircular in shape, water-soaked, later becoming grayish white in the center with a dark brown margin and bordered by a tan halo. Necrotic lesions were approximately 3 to 4 mm in diameter. Diseased leaves were collected from 10 trees in each of five commercial orchards. The diseased leaves were cut into 5 × 5 mm slices, dipped in 75% ethanol for 30 s, washed three times in sterilized water, sterilized with 0.1% (w/v) HgCl2 for 3 min, and then rinsed five times with sterile distilled water. These slices were placed on potato dextrose agar (PDA), followed by incubating at 28°C for about 3 to 4 days. Fungal isolates were obtained from these diseased tissues, transferred onto PDA plates, and incubated at 28°C. These isolates produced gray aerial mycelium and then became pinkish gray with age. Moreover, the reverse of the colony was pink. The growth rate was 8.21 to 8.41 mm per day (average = 8.29 ± 0.11, n = 3) at 28°C. The colonies produced pale orange conidial masses and were fusiform with acute ends, hyaline, sometimes guttulate, 4.02 to 5.25 × 13.71 to 15.72 μm (average = 4.56 ± 0.31 × 14.87 ± 1.14 μm, n = 25). The morphological characteristics and measurements of this fungal isolate matched the previous descriptions of Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan (2). Meanwhile, these characterizations were further confirmed by analysis of the partial sequence of five genes: the internal transcribed spacer (ITS) of the ribosomal DNA, beta-tubulin (β-tub) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, chitin synthase 3(CHS-1) gene, and actin (ACT) gene, with universal primers ITS4/ITS5, T1/βt2b, GDF1/GDR1, CHS1-79F/CHS1-354R, and ACT-512F/ACT-783R, respectively (1). BLAST of these DNA sequences using the nucleotide database of GenBank showed a high identify (ITS, 99%; β-tub, 99%; GAPDH, 99%; CHS-1, 99%; and ACT, 100%) with the previously deposited sequences of C. fioriniae (ITS, KF278459.1, NR111747.1; β-tub, AB744079.1, AB690809.1; GAPDH, KF944355.1, KF944354.1; CHS-1, JQ948987.1, JQ949005.1; and ACT, JQ949625.1, JQ949626.1). Koch's postulates were fulfilled by inoculating six healthy 1-year-old walnut trees in July 2014 with maximum and minimum temperatures of 33 and 26°C. The 6-mm mycelial plug, which was cut from the margin of a 5-day-old colony of the fungus on PDA, was placed onto each pin-wounded leaf, ensuring good contact between the mycelium and the wound. Non-colonized PDA plugs were placed onto pin-wounds as negative controls. Following inoculation, both inoculated and control plants were covered with plastic bags. Leaf spots, similar to those on naturally infected plants, were observed on the leaves inoculated with C. fioriniae within 5 days. No symptoms were observed on the negative control leaves. Finally, C. fioriniae was re-isolated from symptomatic leaves; in contrast, no fungus was isolated from the control, which confirmed Koch's postulates. To our knowledge, this is the first report of leaf disease on walnut caused by C. fioriniae. References: (1) L. Cai et al. Fungal Divers. 39:183, 2009. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009.

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First Report of Chalara fraxinea on Common Ash in Italy

Plant Disease ◽

10.1094/pdis-94-1-0133a ◽

2010 ◽

Vol 94 (1) ◽

pp. 133-133 ◽

Cited By ~ 20

Author(s):

N. Ogris ◽

T. Hauptman ◽

D. Jurc ◽

V. Floreancig ◽

F. Marsich ◽

...

Keyword(s):

Morphological Characteristics ◽

Aluminum Foil ◽

Malt Extract ◽

First Report ◽

Chalara Fraxinea ◽

Ash Trees ◽

Mycelial Plug ◽

Northeastern Italy ◽

Xylem Discoloration ◽

Branch Dieback

In many European countries, the anamorphic Chalara fraxinea Kowalski (teleomorph Hymenoscyphus albidus [Roberge ex Desm.] Phillips; 1–3) is responsible for a severe and rapidly spreading dieback of common ash (Fraxinus excelsior L.) since it was first reported in Poland. Recently, this disease was added to the EPPO Alert List and the NAPPO Phytosanitary Alert System. Symptomatic trees were observed in a 1.8-ha ash-maple forest in northeastern Italy (Fusine, UD; 46°30′N, 13°37′E; 782 m above sea level) along the Italo-Slovenian border in July 2009. Symptoms were found on approximately 10% of mature common ash and 70% of seedlings. Main symptoms were shoot, twig, and branch dieback, wilting, and bark cankers (1). Fungal fruiting bodies were not found on or near the canker surface. Furthermore, longitudinal and radial sections through the cankers revealed gray-to-brown xylem discoloration. One symptomatic 3-year-old plant was randomly selected and from the necrotic margin of one canker previously surface-sterilized with 3% sodium hypochlorite and rinsed, four 2-mm-wide chips were placed on malt extract agar (MEA) and incubated at 21 ± 1°C in the dark. Among a variety of microorganisms, after 19 days, slow-growing colonies (mean radius of 12 mm) appeared that were effuse, cottony, and often fulvous brown but sometimes dull white with occasional gray-to-dark gray patches. The purified isolate was then transferred to the same medium at 4 ± 1°C in the dark, and after 11 days, hyaline-to-dark gray phialides were observed producing numerous conidia in slimy droplets and sometimes in chains. Phialophores measured 8.6 to 21.0 (15.1) μm long (n = 20), 4.2 to 13.4 (8.8) × 3.6 to 5.5 (4.7) μm at the base, and 5.2 to 8.7 (6.5) × 2.5 to 3.1 (2.8) μm at the collarette; conidia measured 2.8 to 4.2 (3.4) × 1.9 to 2.5 (2.2) μm (n = 40); and first formed conidia measured 5.5 to 6.5 (5.9) × 1.8 to 2.5 (2.1) μm (n = 20). These morphological characteristics matched Kowalski's (1) description of C. fraxinea. In August of 2009, the fungal isolate was used to test pathogenicity with current year shoots of 25 6-year-old (150 to 210 cm high) asymptomatic common ash trees under quarantine conditions (Slovenian Forestry Institute's experimental plots). For every plant, the bark of the main shoot (10 to 13 mm in diameter) was wounded with a 6-mm-diameter cork borer. Twenty saplings were inoculated with one 6-mm-diameter mycelial plug obtained from the margin of a 26-day-old culture (MEA), while five saplings were inoculated with sterile MEA plugs. All wounds were sealed with Parafilm and aluminum foil. After 28 days, all plants inoculated with the C. fraxinea showed bark lesions (2 to 39 mm long, mean 7 mm) and wood discoloration (6 to 85 mm long, mean 22 mm) from which the pathogen was reisolated. These symptoms were absent from controls and the pathogen was never reisolated. To our knowledge, this is the first report of C. fraxinea in Italy. Investigations on its presence in all Fraxinus species naturally growing in the investigated area and in the nearest regions are in progress. The obtained isolate is preserved in both Padova and Ljubljana herbaria as CFIT01. References: (1) T. Kowalski. For. Pathol. 36:264, 2006. (2) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:304, 2009.

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First Report of Walnut Canker Caused by Fusarium incarnatum from India

Plant Disease ◽

10.1094/pdis-04-11-0352 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1587-1587

Author(s):

B. Singh ◽

C. S. Kalha ◽

V. K. Razdan ◽

V. S. Verma

Keyword(s):

Fusarium Species ◽

Juglans Regia ◽

Infected Plant ◽

Distilled Water ◽

Conidial Suspension ◽

State University ◽

Single Spore ◽

Jammu And Kashmir ◽

First Report ◽

Branch Dieback

While screening newly introduced cultivars of walnut (Juglans regia) at Bhaderwah (Mini Kashmir), Jammu and Kashmir, India in September 2008, 60% of grafted plants were found to be dying because of a cankerous growth observed on seedling stems. Later, these symptoms extended to lateral branches. In the surveyed nurseries, cvs. SKU 0002 and Opex Dachaubaria were severely affected by the disease. Cankers were also observed in all walnut nurseries in the area with several wild seedlings also being observed to be exhibiting similar cankerous symptoms on stem and branches. Necrotic lesions from cankerous tissues on seedling stems were surface disinfested with 0.4% NaOCl for 1 min and these disinfected cankerous tissues were grown on potato dextrose agar (potato-250 g, dextrose-15 g, agar-15 g, distilled water-1 liter). A Fusarium sp. was isolated consistently from these cankerous tissues, which was purified using single-spore culture. Carnation leaf agar was used for further culture identification (2,3). The fungal colony was floccose, powdery white to rosy in appearance when kept for 7 days at 25 ± 2°C. Macroconidia were straight to slightly curved, four to eight septate and 30 to 35 × 3.5 to 5.7 μm. These are characteristics consistent with Fusarium incarnatum (3). Pathogenicity was confirmed by spraying a conidial suspension (1 × 106 conidia/ml) onto bruised branches of 1-year-old walnut plants (cv. Opex Dachaubaria) while sterile distilled water sprays were used for the controls. Inoculated plants were incubated at 20 ± 2°C and 85% relative humidity for 48 h. Fifty days following inoculation, branch dieback followed by canker symptoms developed on inoculated plants. Control plants remained healthy with no symptoms of canker. F. incarnatum (Roberge) Sacc. was repeatedly isolated from inoculated walnut plants, thus satisfying Koch's postulates. Infected plant material has been deposited at Herbarium Crytogamae Indiae Orientalis (ITCC-6874-07), New Delhi. To our knowledge, this is the first report of walnut canker caused by F. incarnatum (Roberge) Sacc. from India. This fungus was previously reported to be affecting walnut in Italy (1) and Argentina (4). References: (1) A. Belisario et al. Informatore Agrario 21:51, 1999. (2) J. C. Gilman. A Manual of Soil Fungi. The Iowa State University Press, Ames, 1959. (3) P. E. Nelson et al. Fusarium Species. An Illustrated Manual for Identification. The Pennsylvania State University Press, University Park, 1983. (4) S. Seta et al. Plant Pathol. 53:248, 2004.

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First report of Nigrospora sphaerica causing fruit dried-shrink disease in Akebia trifoliata from China

Plant Disease ◽

10.1094/pdis-11-20-2471-pdn ◽

2021 ◽

Author(s):

Xiujing Hong ◽

Shijia Chen ◽

linchao Wang ◽

Bo Liu ◽

Yuruo Yang ◽

...

Keyword(s):

Elongation Factor ◽

Morphological Characteristics ◽

Its Region ◽

Average Diameter ◽

Ethanol Solution ◽

Likelihood Method ◽

Conidial Suspension ◽

Multiple Sequence ◽

First Report ◽

Pure Cultures

Akebia trifoliata, a recently domesticated horticultural crop, produces delicious fruits containing multiple nutritional metabolites and has been widely used as medicinal herb in China. In June 2020, symptoms of dried-shrink disease were first observed on fruits of A. trifoliata grown in Zhangjiajie, China (110.2°E, 29.4°N) with an incidence about 10%. The infected fruits were shrunken, colored in dark brown, and withered to death (Figure S1A, B). The symptomatic fruits tissues (6 × 6 mm) were excised from three individual plants, surface-disinfested in 1% NaOCl for 30s and 70% ethanol solution for 45s, washed, dried, and plated on potato dextrose agar (PDA) containing 50 mg/L streptomycin sulfate in the dark, and incubated at 25℃ for 3 days. Subsequently, hyphal tips were transferred to PDA to obtain pure cultures. After 7 days, five pure cultures were obtained, including two identical to previously reported Colletotrichum gloeosporioides causing leaf anthracnose in A. trifoliata (Pan et al. 2020) and three unknown isolates (ZJJ-C1-1, ZJJ-C1-2, and ZJJ-C1-3). The mycelia of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 were white, and formed colonies of approximate 70 mm (diameter) in size at 25℃ after 7 days on potato sucrose agar (PSA) plates (Figure S1C). After 25 days, conidia were formed, solitary, globose, black, shiny, smooth, and 16-21 μm in size (average diameter = 18.22 ± 1.00 μm, n = 20) (Figure S1D). These morphological characteristics were similar to those of N. sphaerica previously reported (Li et al. 2018). To identify species of ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3, the internal transcribed spacer (ITS) region, β-tubulin (TUB2), and the translation elongation factor 1-alpha (TEF1-α) were amplified using primer pairs including ITS1/ITS4 (Vilgalys and Hester 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995), and EF1-728F/EF-2 (Zhou et al. 2015), respectively. Multiple sequence analyses showed no nucleotide difference was detected among genes tested except ITS that placed three isolates into two groups (Figure S2). BLAST analyses determined that ZJJ-C1-1, ZJJ-C1-2 and ZJJ-C1-3 had 99.73% to N. sphaerica strains LC2705 (KY019479), 100% to LC7294 (KY019397), and 99.79-100% to LC7294 (KX985932) or LC7294 (KX985932) based on sequences of TUB2 (MW252168, MW269660, MW269661), TEF-1α (MW252169, MW269662, MW269663), and ITS (MW250235, MW250236, MW192897), respectively. These indicated three isolates belong to the same species of N. sphaerica. Based on a combined dataset of ITS, TUB2 and TEF-1α sequences, a phylogenetic tree was constructed using Maximum likelihood method through IQ-TREE (Minh et al. 2020) and confirmed that three isolates were N. sphaerica (Figure S2). Further, pathogenicity tests were performed. Briefly, healthy unwounded fruits were surface-disinfected in 0.1% NaOCl for 30s, washed, dried and needling-wounded. Then, three fruits were inoculated with 10 μl of conidial suspension (1 × 106 conidia/ml) derived from three individual isolates, with another three fruits sprayed with 10 μl sterilized water as control. The treated fruits were incubated at 25℃ in 90% humidity. After 15 days, all the three fruits inoculated with conidia displayed typical dried-shrink symptoms as those observed in the farm field (Figure S1E). The decayed tissues with mycelium and spores could be observed on the skin or vertical split of the infected fruits after 15 days’ inoculation (Figure S1F-H). Comparably, in the three control fruits, there were no dried-shrink-related symptoms displayed. The experiment was repeated twice. The re-isolated pathogens were identical to N. sphaerica determined by sequencing the ITS, TUB2 and TEF-1α. Previous reports showed N. sphaerica could cause postharvest rot disease in kiwifruits (Li et al. 2018). To our knowledge, this is the first report of N. sphaerica causing fruits dried-shrink disease in A. trifoliata in China.

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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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First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China

Plant Disease ◽

10.1094/pdis-10-21-2138-pdn ◽

2021 ◽

Author(s):

Shucheng Li ◽

Liuhua Xiao ◽

Fan Wu ◽

Yinbao Wang ◽

Mingshu Jia ◽

...

Keyword(s):

Leaf Spot ◽

Juglans Regia ◽

Morphological Characteristics ◽

Effective Control ◽

Bootstrap Support ◽

Sterile Water ◽

First Report ◽

Myrica Rubra ◽

Initial Symptoms ◽

Colletotrichum Fructicola

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.

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First report of Neofusicoccum parvum causing branch dieback on Juglans regia in Turkey

Journal of Plant Pathology ◽

10.1007/s42161-020-00662-8 ◽

2020 ◽

Author(s):

Merve Kara ◽

Emine Mine Soylu ◽

Soner Soylu ◽

Aysun Uysal ◽

Şener Kurt

Keyword(s):

Juglans Regia ◽

Neofusicoccum Parvum ◽

First Report ◽

Branch Dieback

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First Report of Fruit Rot and Associated Branch Dieback of Almond in California Caused by a Phomopsis Species Tentatively Identified as P. amygdali

Plant Disease ◽

10.1094/pdis.1999.83.11.1073c ◽

1999 ◽

Vol 83 (11) ◽

pp. 1073-1073 ◽

Cited By ~ 7

Author(s):

J. E. Adaskaveg ◽

H. Förster ◽

J. H. Connell

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

The United States ◽

Tree Canopy ◽

Fruit Rot ◽

Control Treatment ◽

Field Inoculation ◽

First Report ◽

Branch Dieback ◽

Summer Fruit

A fruit rot of almond (Prunus dulcis (Mill.) D. Webb.) was observed in an orchard in Durham, CA (Butte County), in June of 1998 after an unusually wet spring with a total precipitation of 17.2 cm for April and May. Disease incidence on fully developed fruit of almond cv. Sonora was nearly 90% in the lower tree canopy by July. Almond cv. Nonpareil grown in alternate rows in the same orchard was much less affected. Fruit symptoms included extensive grayish brown discolored and shriveled hulls, often associated with a clear gum secretion and shriveled kernels. Affected fruit frequently abscised. Leaf symptoms and branch dieback were not associated with the disease in 1998. In May of 1999, however, extensive twig dieback was observed on almond cv. Sonora in the same orchard. Isolations from more than 100 symptomatic fruit were conducted from 9 sampling sites in the 9-ha orchard. Based on morphological characteristics, the same fungus was isolated from 93% of the fruit. The fungus also was isolated consistently from samples exhibiting twig dieback. During a major disease survey conducted in 1998, the fungus was only incidentally isolated from almond fruit from other California orchards. Ascomata were not observed in vivo or in vitro. The fungus produced alpha and beta spores in pycnidia when cultured on potato dextrose agar. Spore measurements were obtained from 10 spores for each of 3 isolates obtained from fruit or twig dieback of almond cv. Sonora. Conidial dimensions of fruit and twig isolates were very similar. Based on spore sizes, with alpha spores measuring 5.3 to 7.5 (to 8) × 1.7 to 2.5 μm and beta spores measuring12.8 to 29.8 × 0.6 to 0.7 μm, the fungus was tentatively identified as Phomopsis amygdali (Del.) Tuset & Portilla (2). Previous reports on this fungus (2), however, indicated that beta spores are not produced in culture, and disease symptoms have not been observed on fruit. The fungus was morphologically different from other species of Phomopsis reported from almond and other Prunus species, including P. mali Roberts, P. padina (Sacc. & Roum.) Died., P. parabolica Petrak, P. perniciosa Grove, P. pruni (Ellis & Dearn.) Wehm., P. prunorum (Cooke) Grove, P. ribetejana Camara, and P. stipata (Lib.) Sutton (3). Field inoculation studies were performed in May of 1999 on almond cvs. Carmel and Mission. Almond fruit were wounded (2 × 2 × 2 mm) or left unwounded and were sprayed with water (control) or a suspension of alpha spores (105 spores per ml). Branches were bagged for 4 days to maintain high humidity. Fruit symptoms on cv. Carmel were observed after 4 weeks on wounded and nonwounded inoculated fruit, and P. amygdali was successfully reisolated from diseased tissue. No symptoms were observed in the control treatment for almond cv. Carmel or in any treatment for cv. Mission. This is the first report of P. amygdali causing a late spring and summer fruit rot and associated branch dieback of almond in North America (1). References: (1) D. F. Farr et al. 1989. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN. (2) J. J. Tuset and M. T. Portilla. Taxonomic status of Fusicoccum amygdali and Phomopsis amygdalina. Can. J. Bot. 67:1275, 1989. (3) F. A. Uecker. 1988. A World List of Phomopsis Names with Notes on Nomenclature, Morphology, and Biology. Mycologia Memoir No. 13. J. Cramer, Berlin.

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