First Report of Colletotrichum acutatum Causing a Leaf Spot on Myrica cerifera in Florida

Wax myrtle (Morella cerifera (synonym Myrica cerifera) (L.) Small) is a native tree used in Florida landscapes. In the summer of 2005 and spring of 2006, small necrotic spots were observed on young leaves in two commercial nurseries in central Florida. Lesions were dark brown-to-black and eventually coalesced to form large, irregular necrotic areas. Leaves with large lesions abscised prematurely, defoliating the entire plant. Conidia formed on acervuli were observed on the surface of the largest lesions and were tentatively identified as a Colletotrichum sp. Isolations from the edges of lesions were made on potato dextrose agar (PDA) after surface disinfestation of leaf pieces in 0.6% NaOCl for 30 sec. Red chromogenic colonies developed after 5 days of incubation at 24°C. Colonies produced hyaline, oblong conidia with pointed ends averaging 14 × 4 μm and were identified as Colletotrichum acutatum J.H. Simmonds (1). The sequence from internal transcribed spacer regions 1 and 2 and the 5.8s rRNA gene of the rDNA repeat for an isolate (GenBank Accession No. DQ839609) was 100% identical to sequence from the same region of 36 C. acutatum isolates in the NCBI database. These isolates came from at least 16 different hosts, including seven ornamental hosts. There were three isolates from blueberry among the matches (Accession Nos. AB219029, AJ301911, and AJ301905), and the rDNA sequence was also identical to the sequence obtained in our laboratory for a chromogenic C. acutatum isolate from blueberry. Three single-spore isolates were tested for pathogenicity on potted plants in the greenhouse. Two young shoots were spray inoculated with a suspension (1 × 106 conidia/ml) of each isolate. Shoots were covered with a plastic bag for 24 h and maintained at 26.5°C. Two shoots were sprayed with sterile water as a control and similarly covered. All isolates produced brown spots on the youngest leaves 3 to 5 days after inoculation; no symptoms developed on control shoots. The fungus was reisolated from all inoculated shoots. To our knowledge, this is the first report of C. acutatum on wax myrtle in Florida. The disease has a potential to spread and become a significant problem for the cultivation of this species in ornamental nurseries in Florida. Reference: (1) J. H. Simmonds. Qld. J. Agric. Anim. Sci. 22:437, 1965.

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First Report of Anthracnose on Pepper Fruit Caused by Colletotrichum scovillei in Brazil

Plant Disease ◽

10.1094/pdis-04-14-0426-pdn ◽

2014 ◽

Vol 98 (10) ◽

pp. 1437-1437 ◽

Cited By ~ 10

Author(s):

N. P. Caires ◽

D. B. Pinho ◽

J. S. C. Souza ◽

M. A. Silva ◽

D. O. Lisboa ◽

...

Keyword(s):

Colletotrichum Acutatum ◽

Correct Identification ◽

Single Spore ◽

Transparent Plastic ◽

First Report ◽

Molecular Approaches ◽

Single Spore Isolate ◽

Pepper Fruit ◽

Plastic Bag ◽

Capsicum Spp

Anthracnose is major disease of pepper (Capsicum annum) in the tropics and causes severe damage both in the field and postharvest. In Brazil, this disease is caused by Colletotrichum acutatum, C. boninense, C. capsici, C. coccodes, and C. gloeosporioides, where the first species is responsible for 70% of all occurrences (3). Recently, C. acutatum has been considered a species complex (1); thus, the aim of this study was to verify the etiology of anthracnose on peppers using a morphological and molecular approaches. In 2011, pepper fruits with typical symptoms of anthracnose (dark, sunken spots with concentric rings of orange conidial masses) were collected in Viçosa, Minas Gerais, Brazil. A single spore isolate was obtained on potato dextrose agar (PDA), and the derived culture was deposited in the Coleção de Culturas de Fungos Fitopatogênicos “Prof. Maria Menezes” (code CMM-4200). The upper side colonies on PDA were gray, cotton-like, and pale gray to pale orange. Conidia were hyaline, aseptate, smooth, straight, cylindrical with round ends or occasionally with end ± acute, 12.5 to 17 μm long and 3.5 to 4 μm wide on synthetic nutrient deficient agar. The isolate was morphologically typical of species belonging to the C. acutatum complex. Molecular identification of the pathogen was carried out and sequences of the regions internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β-tubulin (βt) were obtained and deposited in GenBank (Accession Nos. KJ541821 to KJ541823). A search in the Q-bank fungi database using the ITS, βt, and GAPDH sequences retrieved C. scovillei with 100% identity for all three genes. This pathogen was previously reported in Capsicum spp. only in Thailand, Indonesia, and Japan (1,2). To confirm pathogenicity, drops with 105 spores/ml were deposited in 10 artificially wounded fruits (cv. Itapuã 501 and Melina). In control fruits, drops of sterilized water were deposited onto wounds. The fruits were covered for one day with a transparent plastic bag with moisture supplied by a wet filter paper. The fruits were detached and mature. The bags were removed, and the fruits were incubated for 10 days in a growth chamber at 25°C with a photoperiod of 12 h. After 4 days, gray-brown to black sunken spots with concentric rings were observed on 100% of the wounded fruits that had been inoculated. No disease was observed on the control fruits. The fungus C. scovillei was successfully re-isolated from symptomatic fruits to fulfill Koch's postulates. To our knowledge, this is the first report of anthracnose on pepper fruit caused by C. scovillei in Brazil. Due to the diversity of species that cause anthracnose in Capsicum, future studies using morphological and molecular tools are essential for the correct identification of Colletotrichum spp. on pepper in Brazil. References: (1) U. Damm et al. Stud. Mycol. 73:37, 2012. (2) T. Kanto et al. J. Gen. Plant. Pathol. 80:73, 2014. (3) M. J. Z. Pereira et al. Hortic. Bras. 29:569, 2011.

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First Report of Fusarium Wilt on Eremophila spp. Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-08-10-0597 ◽

2010 ◽

Vol 94 (12) ◽

pp. 1509-1509 ◽

Cited By ~ 1

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Morphological Characteristics ◽

Gene Sequences ◽

Single Spore ◽

First Report ◽

Potted Plants ◽

Plastic Bags ◽

Gene Coding ◽

Affected Plant ◽

Tubulin Protein

Eremophila spp. (Myoporaceae family), endemic to Australia, are evergreen shrubs or small trees occurring in arid, semi-arid, tropical, or temperate regions. In Europe, Eremophila spp. are grown for their horticultural appeal. During 2009 and 2010, extensive wilting was observed on 2-month to 1-year-old potted plants of Eremophila laanii F. Muell., E. glabra subsp. carnosa Chinnock, and E. maculata (Ker Gawl.) F. Muell. grown in a commercial nursery near Catania (southern Italy). Internally, symptomatic plants had conspicuous vascular discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissues. Colonies with purple mycelia and violet reverse colors developed after 9 days. On carnation leaf agar, single-spore isolates produced microconidia on short monophialides, macroconidia that were three to five septate with a pedicellate base, and solitary and double-celled or aggregated chlamydospores. A PCR assay was conducted on two representative isolates (ITEM 12591 and ITEM 12592) by analyzing sequences of the partial CaM gene (coding calmodulin protein) and benA (coding beta-tubulin protein) using the primers as reported by O'Donnell et al. (1). Calmodulin sequences of ITEM 12951 and ITEM 12952 isolates (GenBank Nos. FR671157 and FR671158) exhibited 99.8 and 99.5% identity with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), respectively, and had 99.5% homology between them. BenA gene sequences of ITEM 12951 (GenBank No. FR671426) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain CC-612-3 (GenBank No. AY714092.1), and benA gene sequences of ITEM 12952 (GenBank No. FR671427) exhibited an identity of 100% to F. oxysporum f. sp. vasinfectum strain LA 140 (GenBank No. FJ466740.1), whereas the homology between the two strains is 99.5%. Morphological characteristics, as well as CaM and benA sequences, identified the isolates as F. oxysporum Schlechtend:Fr. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 3-month-old cuttings of E. laanii, E. glabra subsp. carnosa, and E. maculata. Twenty plants for each species were inoculated with each isolate. The same number of plants served as noninoculated controls. All plants were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. Symptoms identical to those observed in the nursery developed 20 days after inoculation with both strains. Crown and stem discoloration was detected in all inoculated plants after 45 days. Wilting was detected on 15% of plants. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously above. To our knowledge, this is the first report of F. oxysporum causing disease of Eremophila spp. worldwide. Reference: (1) K. O'Donnell et al. Mycoscience 41:61, 2000.

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First Report of Fruit Rot Disease on Pomelo Caused by Lasiodiplodia theobromae in China

Plant Disease ◽

10.1094/pdis-03-11-0214 ◽

2011 ◽

Vol 95 (9) ◽

pp. 1190-1190 ◽

Cited By ~ 5

Author(s):

M. Luo ◽

Z. Y. Dong ◽

S. Y. Bin ◽

J. T. Lin

Keyword(s):

Its Region ◽

Fruit Rot ◽

Economic Losses ◽

Single Spore ◽

Lasiodiplodia Theobromae ◽

First Report ◽

Potted Plants ◽

Diseased Fruit ◽

Long Time ◽

Water Plants

Pomelo (Citrus grandis) is widely cultivated in MeiZhou Guangdong Province of China. In 2008, a disease on pomelo fruit caused significant economic losses by affecting fruit quality. Diseased fruit was collected in December 2008 from MeiZhou Guangdong, surface sterilized in 75% ethanol for 1 min and internal necrotic tissue was transferred to potato dextrose agar (PDA) and incubated at 28°C for 5 days. Three single-spore isolates were obtained from different fruit and identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. (synonyms Diplodia natalensis Pole-Evans and Botryodiplodia theobromae Pat.; teleomorph Botryosphaeria rhodina (Cooke) Arx) on the basis of morphological and physiological features. The fungus produced dark brown colonies (initially grayish) on PDA. Young hyphae were hyaline and aseptate, whereas mature hyphae were septate with irregular branches. Cultures of L. theobromae produced globular or irregular pycnidia abundantly on PDA (pH 3.5) at 28°C after 1 month. Mature conidia of L. theobromae were 20 to 26 × 12 to 15.5 μm, subovoid to ellipsoid-ovoid, initially hyaline and nonseptate, remaining hyaline for a long time, and finally becoming dark brown and one septate with melanin deposits on the inner surface of the wall arranged longitudinally giving a striate appearance to the conidia. The internal transcribed spacer (ITS) region of the rDNA was amplified from gDNA using primers ITS1 (5′-TCCGATGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (1). Amplicons were 542 bp long (GenBank Accession No. JF693024) and had 100% nucleotide identity with the corresponding sequence (GenBank Accession No. EU860391) of L. theobromae isolated from a Pinus sp. (2). To satisfy Koch's postulates, six asymptomatic fruit on potted plants were sprayed until runoff with a spore suspension (1 × 106 spores/ml) prepared from 30-day-old cultures of one isolate. Control fruit received water. Plants were covered with sterile wet gauze to maintain high humidity. Fruit spot symptoms similar to those on diseased field fruit appeared after 15 days on all inoculated fruits. L. theobromae was reisolated from all inoculated test fruit. No symptoms were observed on the fruit of control plants. To our knowledge, this is the first report of L. theobromae causing disease on pomelo fruit in China. This pathogen has also been previously reported to be economically important on a number of other hosts by mostly affecting the leaves. References: (1) J. C. Batzer et al. Mycologia 97:1268, 2005. (2) C. A. Pérez et al. Fungal Divers. 41:53,2010.

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First Report of Acidovorax avenae subsp. avenae Causing Bacterial Leaf Stripe of Strelitzia nicolai

Plant Disease ◽

10.1094/pdis-03-11-0160 ◽

2011 ◽

Vol 95 (11) ◽

pp. 1474-1474 ◽

Cited By ~ 2

Author(s):

T. E. Seijo ◽

N. A. Peres

Keyword(s):

Pathogenic Bacteria ◽

Fatty Acid Analysis ◽

South Florida ◽

Metabolic Phenotype ◽

Bacterial Suspension ◽

Rrna Gene ◽

First Report ◽

Plastic Bags ◽

Young Leaves ◽

Symptomatic Tissue

White bird of paradise (Strelitzia nicolai Regel & K. Koch) is a commonly grown ornamental in central and south Florida. Each summer of 2004 to 2007, a reoccurring disease was observed at a commercial nursery in central Florida. Diseased plants had brown, necrotic stripes between the lateral leaf veins, which usually appeared along the midvein and spread toward the leaf edge. Lesions developed on the youngest leaves as they emerged from the central whorl. During 2004 and 2005, 20 symptomatic leaves were sampled. A white, nonfluorescent bacterium was consistently isolated from symptomatic tissue. It induced a hypersensitive response (HR) on tomato, grew at 41°C, and was identified as a Acidovorax sp. based on fatty acid analysis and as Acidovorax avenae subsp. avenae by Biolog metabolic phenotype analysis (similarity 0.76 to 0.86). A partial 16S rRNA gene sequence (1,455 bp) (Accession No. EF418616) was identical to four sequences in the NCBI (National Center for Biotechnology Information) database: one from A. avenae subsp. avenae and three from A. avenae of undetermined subspecies. To confirm pathogenicity, a bacterial suspension (O.D590 = 0.1) was applied to fill the central whorl (~0.5 to 1 ml) of potted S. nicolai. Plants were incubated for 7 to 10 days inside plastic bags at ambient temperature. Plants were inoculated individually with five strains of A. avenae subsp. avenae, four from S. nicolai, and one from corn (ATCC19860). Two to nine plants per strain were inoculated in each experiment. All strains were tested at least twice and noninoculated control plants were included. Symptoms were reproduced on the emerging leaf of 50 to 100% of inoculated plants with all five A. avenae subsp. avenae strains. No symptoms were observed on the controls. The bacteria recovered from symptomatic tissue were confirmed to be A. avenae subsp. avenae. Corn seedlings were inoculated as described above, except that entire seedlings were sprayed. Water-soaked lesions along the length of older leaf blades developed in 4 to 7 days. Only the corn strain was pathogenic (>80% of seedlings symptomatic), indicating host specificity. To our knowledge, this is the first report of A. avenae subsp. avenae infecting S. nicolai. In 1971, Wehlburg (2) described the same symptoms on orange bird of paradise (S. reginae) as being caused by a nonfluorescent Pseudomonas sp. This report likely describes the same disease since the published description is consistent with symptoms caused by A. avenae subsp. avenae. The pathogen reported by Wehlburg (2) had one polar flagellum, reduced nitrate, produced oxidase and a HR, and utilized arabinose, but not sucrose or arginine, characteristics consistent with those of A. avenae subsp. avenae (1). The only difference was A. avenae subsp. avenae has a delayed positive starch hydrolysis (1), whereas Welhburg's strain was negative. This disease occurs mainly on young leaves when plants receive daily overhead irrigation. Incidence can be as high as 40%, occasionally causing mortality, but even mild symptoms affect appearance and reduce marketability as an ornamental. References: (1) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) C. Wehlburg. Plant Dis. Rep. 55:447, 1971.

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First Report of Damping-Off on Strawberry Tree Caused by Colletotrichum acutatum and C. simmondsii in Italy

Plant Disease ◽

10.1094/pdis-07-11-0567 ◽

2011 ◽

Vol 95 (12) ◽

pp. 1588-1588 ◽

Cited By ~ 9

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Gene Sequence ◽

Morphological Characteristics ◽

Colletotrichum Acutatum ◽

Commonwealth Mycological Institute ◽

Damping Off ◽

Pcr Assay ◽

Single Spore ◽

First Report ◽

Strawberry Tree ◽

Colletotrichum Spp

In June 2010, a widespread damping-off was noticed in a commercial nursery in eastern Sicily on ~20,000 potted 2-month-old strawberry tree (Arbutus unedo L.) seedlings. More than 40% of the seedlings showed disease symptoms including brown lesions at the seedling crown above and below the soil line that expanded rapidly to girdle the stem. Stem lesions were followed by death of the entire seedling in a few days. Diseased stem and crown tissues of 20 seedlings were surface disinfested for 2 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C in the dark. Fungal isolates with mycelial and morphological characteristics of Colletotrichum spp. were isolated from all seedlings. Fungal colonies were pale orange or gray without carmine pigments. On carnation leaf agar (CLA), single-spore isolates produced many orange masses of hyaline, aseptate conidia with a cylindrical to ellipsoidal shape, rounded apex, and 11 to 15 μm long and 3 to 4.5 μm wide (average 13.2 × 3.7 μm). The pointed conidia of 10 isolates were morphologically similar. DNA isolation was performed with the Wizard Magnetic DNA Purification Kit (Promega, Madison, WI) following the manufacturer's instructions with some modifications. A PCR assay was conducted on two representative isolates (ITEM 13492 and ITEM 13493) by analyzing sequences of gene benA (coding β-tubulin protein) using the primers T1 and T10 reported by O'Donnell and Cigelnik (1). BenA gene sequence of ITEM 13492 exhibited an identity of 99.8% to C. simmondsii strain BRIP 4704 (GenBank No. GU183277), while BenA gene sequence of ITEM 13493 exhibited an identity of 100% to C. acutatum strain BRIP52695 (GenBank No. GU183314). The identification of these two species was made by comparing the internal transcribed spacer region and BenA sequences of these two strains with that deposited by Shivas and Tan (2). Morphological characteristics, as well as the PCR assay, identified the isolates as Colletotrichum acutatum J.H. Simmonds and C. simmondsii R.G. Shivas & Y. P. Tan (2,3). Pathogenicity tests were carried out on 2-month-old seedlings of strawberry tree grown on alveolar trays. Conidial suspensions of two isolates (ITEM 13492 and ITEM 13493) were obtained from 14-day-old single-spore colonies on CLA, then adjusted to 105 conidia per ml and sprayed on seedlings. Fifty seedlings for each isolate were used. The same number of seedlings was mock inoculated with sterile distilled water. All seedlings were enclosed for 4 days in plastic bags and placed in a growth chamber at 24 ± 1°C for 45 days. Identical symptoms to those observed in the nurseries appeared 30 days after inoculation, and after 45 days, 80% of the plants were dead. No difference in virulence between the two isolates was observed and no symptoms were detected on the control plants. C. acutatum and C. simmondsii were successfully reisolated from all symptomatic tissues and identified as previously described, completing Koch's postulates. To our knowledge, this is the first report in the world of C. acutatum and C. simmondsii on strawberry tree. This suggests that Colletotrichum spp. may be important pathogens of young seedlings of strawberry tree in nurseries. References: (1) K. O'Donnell and E. Cigelnik. Mol. Phylo. Evol. 7:103, 1997. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009. (3) B. C. Sutton. Page 523 in: The Coelomycetes. Commonwealth Mycological Institute, Kew, Surrey, England, 1980.

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First Report of Leaf Blight on Rubus brasiliensis Caused by Colletotrichum acutatum in Brazil

Plant Disease ◽

10.1094/pdis-06-10-0441 ◽

2010 ◽

Vol 94 (11) ◽

pp. 1378-1378 ◽

Cited By ~ 1

Author(s):

U. P. Lopes ◽

L. Zambolim ◽

H. S. S. Duarte ◽

P. G. C. Cabral ◽

O. L. Pereira ◽

...

Keyword(s):

Native Species ◽

Morphological Characteristics ◽

Minas Gerais ◽

Leaf Blight ◽

Colletotrichum Acutatum ◽

Conidial Suspension ◽

Specific Primer ◽

First Report ◽

Young Leaves ◽

Necrotic Spots

There are more than 300 blackberry (Rubus) species worldwide. Rubus brasiliensis Mart. is a native Brazilian species found in tropical forests. In January 2009, samples of R. brasiliensis with severe leaf blight were collected from an area of rain forest in the city of São Miguel do Anta, State of Minas Gerais, Brazil. Dark spots began developing in the young leaves and progressed to necrotic spots with occasional twig dieback. From the spots, a fungus was isolated with the following morphology: acervuli that were 20 to 50.0 × 50 to 125.0 μm and hyaline amerospores that were ellipsoid and fusiform and 7.5 to 23.75 × 2.5 to 5.0 μm. On the basis of these morphological characteristics, the fungus was identified as Colletotrichum acutatum. In Brazil, C. acutatum is reported in apple, citrus, strawberry, peach, plum, nectarine, olive, medlar, and yerba-mate, but it was not reported as the causal agent of leaf blight in R. brasiliensis. A sample was deposited in the herbarium at the Universidade Federal de Viçosa, Minas Gerais, Brazil (VIC 31210). One representative isolate, OLP 571, was used for pathogenicity testing and molecular studies. Identity was confirmed by amplifying the internal transcribed spacer (ITS) regions of the ribosomal RNA with primers ITS4 (3), CaInt2 (a specific primer for C. acutatum [2]) and CgInt (a specific primer for C. gloeosporioides [1]). Isolates of C. acutatum (DAR78874 and DAR78876) and C. gloeosporioides (DAR78875) obtained from Australian olive trees were used as positive controls. The primers ITS4 and CaInt2 amplified a single DNA product of 500 bp expected for C. acutatum. OLP 571 was grown for 7 days on potato dextrose agar. Young leaves of R. brasiliensis were inoculated with a conidial suspension (106 conidia/ml) on young leaves. Inoculated plants were maintained in a moist chamber for 2 days and subsequently in a greenhouse at 25°C. Necrotic spots similar to those described were detected on young leaves 3 days after the inoculation. Control leaves, on which only water was sprayed, remained healthy. The same fungus was reisolated from the inoculated symptomatic tissues. To our knowledge, this is the first report of C. acutatum causing leaf blight in the native species of R. brasiliensis in Brazil. References: (1) P. R. Mills et al. FEMS Microbiol. Lett. 98:137, 1999. (2) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 1990.

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First Report of Atypical Scab Caused by Venturia asperata on Apple in China

Plant Disease ◽

10.1094/pdis-11-20-2431-pdn ◽

2021 ◽

Author(s):

Yang Zhou ◽

Haidong Bu ◽

Chingchai Chaisiri ◽

Qin Tan ◽

Li Wang ◽

...

Keyword(s):

Venturia Inaequalis ◽

Disease Incidence ◽

Large Subunit ◽

Apple Scab ◽

Agar Plate ◽

Single Spore ◽

First Report ◽

Sequence Identity ◽

Blast Analysis ◽

Plastic Bag

Apple cv. ‘Huangtaiping’ (Malus pumila Mill.) is grown widely in northern China for the production of jellies, preserves, and cider. In 2018, atypical scab symptoms were observed on fruits of Huangtaiping in Heilongjiang Province of China. The disease incidence was estimated at approximately 0.4%. Symptoms were scab-like black spots (3 to 5 mm diam.) distinct from scab caused by Venturia inaequalis. Conidia were generally produced on lesions and using a modified microscope (Goh 1999), a single spore was picked up from each sample on water agar plate with a glass needle and then transferred to PDA amended with lactic acid (0.50 ml/L) and sulfate streptomycin (0.20 g/L). Fifteen isolates were obtained and incubated at 21°C for 6 weeks in darkness on PDA. The colonies on PDA were gray-black with circular morphology and floccose texture, which were similar with the characteristics of V. asperata described previously (Turan et al. 2019). The conidia were cylindrical to fusiform, 0 to 1 septate, yellowish and 19.7 (13.5 to 25.8) × 5.7 (3.6 to 6.9) μm (n = 10) in size, which were larger than previously described ones (Turan et al. 2019). DNA of three randomly selected isolates were extracted by a modified SDS method (Ping et al. 2004). The internal transcribed spacer (ITS) region of rDNA of the three selected isolates was amplified with the primers ITS4/ITS5 (White et al. 1990), sequenced and deposited in GenBank (MN958665, MN95866 and MN958667). BLAST analysis showed that the amplified sequences were identical and had 99.3% sequence identity with V. asperata (AF333447, MT459450 and MT459451), 95.4% sequence identity with V. cerasi (MK810963 and MK810964) and 94.3% sequence identity with V. carpophila (MN958609, MN958610 and MN958611). In addition, the complete large subunit ribosomal RNA gene (LSU) was amplified with the primers LROR/LR5 (Vilgalys and Hester 1990), sequenced and deposited in GenBank (MT845787, MT845788 and MT845789). BLAST analysis showed that the amplified sequences were identical and had 99.7% sequence identity with V. asperata (EF114711), 99.2% identity with V. carpophila (MT772296, MT845732 and MT845733 ) and 98% identity with V. cerasi (MK810848 and MK810849). Phylogenetic analysis based on concatenated ITS and LSU sequences showed that the tested isolates grouped with V. asperata strain 2349 in the same clade and the closest species with V. asperata was V. carpophila, followed by V. cerasi. In July 2019, pathogenicity of the isolate VAHLJ3-1-1 was evaluated on Huangtaiping. A conidia suspension with a concentration of 5×105/ml was sprayed evenly on the surface of six fruits. In order to maintain high humidity, inoculated fruits were wrapped with a plastic bag (a cotton ball with water was placed in the plastic bag) to maintain wetness for 3 days. Six fruits sprayed with water were used as a control. Four weeks after inoculation, similar symptom of atypical scab was observed on fruits of Huangtaiping, and V. asperata was isolated again from six inoculated fruits with reisolation frequency of 100% by the single spore isolation, while no symptom was observed on the control fruits. Based on the morphological and molecular identifications, the causal agent of atypical scab on Huangtaiping was identified as V. asperata. Apple scab is usually caused by V. inaequalis (Shen et al. 2020). However, apple scab has also been caused by V. asperata in Italy and France (Caffier et al. 2012; Turan et al. 2019). To the best of our knowledge, this is the first report of V. asperata associated with apple scab-like lesions in China. This information augments our knowledge of the spectrum of Venturia species associated with disease on apple fruit and will be a valuable foundation underpinning management strategies for this cultivar.

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First Report of Fusarium Wilt of Paper Flower (Bougainvillea glabra) Caused by Fusarium oxysporum in Italy

Plant Disease ◽

10.1094/pdis-94-4-0483a ◽

2010 ◽

Vol 94 (4) ◽

pp. 483-483 ◽

Cited By ~ 3

Author(s):

G. Polizzi ◽

D. Aiello ◽

V. Guarnaccia ◽

A. Vitale ◽

G. Perrone ◽

...

Keyword(s):

Fusarium Oxysporum ◽

Fusarium Wilt ◽

Disease Incidence ◽

Elongation Factor ◽

Gene Sequences ◽

Single Spore ◽

Potential Threat ◽

First Report ◽

Potted Plants ◽

Bougainvillea Glabra

Paper flower (Bougainvillea glabra Choisy), native to Brazil, is the most widely and intensively cultivated species of bougainvillea as a potted plant in Sicily (Italy). During 2008 and 2009, a wilting of vegetatively produced B. glabra cv. Sanderiana was observed in several nurseries in eastern Sicily (Catania and Messina provinces). Disease incidence was higher (~10 to 30%) in the tree-shaped potted plants (standards). Occasionally, wilting was detected on plants that were not tree shaped. Internally, symptomatic plants showed conspicuous vascular orange discoloration from the crown to the canopy. Diseased crown and stem tissues were surface disinfested for 30 s in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and incubated at 25°C. A Fusarium sp. was consistently isolated from affected plant tissue. Colonies with light purple or purple mycelia and violet reverse colony colors developed after 10 days. On carnation leaf agar, single-spore isolates produced microconidia in false heads on short monophialides, macroconidia that were 3-septate with a pedicellate base, and solitary and double-celled or aggregate chlamydospores. A PCR assay was conducted on two representative strains (DISTEF-BGS1 and DISTEF-BGS2) by analyzing sequences of the parzial translation elongation factor alpha gene (TEF-1α) and CaM gene (coding calmodulin protein). The primers used are previously used by O'Donnell et al. (1,2). Calmodulin sequences of BGS1 and BGS2 strains (GenBank Nos. FN645740 and FN645741, respectively) exhibited 99% homology with Fusarium oxysporum strain ITEM 2367 (GenBank No. AJ560774), and have homology of 99.6% between them. TEF-1 gene sequences of BGS1 (GenBank No. FN645739) exhibited an identity of 100% to F. oxysporum f. sp. lycopersici MUCL 22544 GenBank No. EF056785.1) and TEF-1α gene sequences of BGS2 (GenBank No. FN655742) exhibited an identity of 100% to F. oxysporum strain NRRL 45954 (GenBank No. FJ985431.1), whereas the homology between the two strains is 98.5%. Both PCR approaches established the identity of the isolates to the F. oxysporum Schlechtend:Fr (1,2). Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 10-day-old mycelial cultures near the crown on 40 potted, healthy, 6-month-old cuttings of paper flower. Twenty plants for each isolate were used. The same number of plants served as noninoculated controls. All plants were enclosed for 5 days in plastic bags and placed in a growth chamber at 24 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 24 to 26°C. Symptoms identical to those observed in nurseries developed 1 month after inoculation with both strains. Crown and stem orange discoloration was detected in all inoculated plants after 2 months. Control plants remained symptomless. F. oxysporum was consistently reisolated from symptomatic tissues and identified as previously described. To our knowledge, F. oxysporum was previously reported on paper flower in Ghana (3). However, this is the first demonstration of the pathogenicity of F. oxysporum on paper flower and it is the first report in Europe of the disease. The presence of Fusarium wilt in Sicily is a potential threat to paper flower production in nurseries. References: (1) K. O'Donnell et al. Proc. Natl. Acad. Sci. USA 95:2044, 1998. (2) K. O'Donnell et al. Mycoscience 41:61, 2000. (3) P. Spaulding. USDA Agric. Handb. 197:1, 1961.

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First Report of Infection of Lygodium microphyllum by Puccinia lygodii, a Potential Biocontrol Agent of an Invasive Fern in Florida

Plant Disease ◽

10.1094/pd-89-0110a ◽

2005 ◽

Vol 89 (1) ◽

pp. 110-110 ◽

Cited By ~ 3

Author(s):

M. B. Rayamajhi ◽

R. W. Pemberton ◽

T. K. Van ◽

P. D. Pratt

Keyword(s):

Lower Surface ◽

Positive Control ◽

Control Agent ◽

New Host ◽

First Report ◽

Central Florida ◽

Potential Biocontrol Agent ◽

The Family ◽

Plastic Bag ◽

Southern Florida

Lygodium microphyllum (Cav.) R.Br. (Old World climbing fern), in the family Schizaeaceae, is one of the most invasive (Category I in Florida) weeds in Florida. It has invaded more than 50,000 ha of wetlands and moist habitats in southern Florida and is rapidly spreading in new areas of the Everglades (3). The search and evaluation of biocontrol agents for this fern is currently in progress. Puccinia lygodii (Har.) Arth. (Uredinales) (1), previously recorded on L. volubile Sw. and L. venustum Sw. in South America (2), attacks foliage and severely damages L. japonicum Thunb. (Japanese climbing fern) vines in northern and central Florida (4). We hypothesized that since L. japonicum occurred mainly in northern and central Florida, P. lygodii did not have opportunity to interact with L. microphyllum, which primarily occurs in southern Florida. Therefore, we used two inoculation methods to test the possible pathogenicity of P. lygodii on the new host, L. microphyllum. Method-I was designed to imitate a seminatural inoculation technique in which three containerized (0.45-L capacity) L. microphyllum test plants (15- to 30-cm-high sporelings) were intermixed among a group of containerized (5.0-L capacity) P. lygodii-infected L. japonicum plants (source of inoculum) in a glasshouse. In Method-II, uredospores obtained from pustules on diseased L. japonicum foliage were adjusted to 1 × 106 uredospores/ml and then misted on three L. microphyllum sporelings (same size as in Method-I) until foliage was completely wet. The plants were then covered individually with a plastic bag for 3 days to facilitate spore germination and infection. In both methods, three L. japonicum sporelings of similar size as L. microphyllum were intermixed among diseased L. japonicum plants as a positive control. All test and infected plants were placed on 6-cm-high trays filled two-thirds with water and exposed to diffused daylight and a temperature range of 20 to 35°C in a glasshouse. These plants were monitored for the development of rust symptoms (halos and rust pustules) development for 8 weeks. Minute cinnamon flakes that developed into eruptive pustules were seen on the lower surface of the pinnules approximately 42 and 28 days after treatment initiation (in both methods) for L. microphyllum and L. japonicum (positive control), respectively. Each method was repeated twice. Dimensions (29.7 [±3.7] × 23.5 [±2.6] μm) and morphology of urediniospores from pustules on inoculated L. microphyllum were similar to those reported for P. lygodii on other host systems (1,2,4). To our knowledge, this is the first report demonstrating the infection of P. lygodii on L. microphyllum. The potential use of P. lygodii as a classical bio-control agent of L. microphyllum in southern Florida will be further investigated. References: (1) J. C. Arthur. Bull. Torrey Bot. Club 51:55, 1924. (2) J. W. McCain et al. Mycotaxon 39:281, 1990. (3) R. W. Pemberton. SIDA 20:1759, 2003. (4) M. B. Rayachhetry et al. Plant Dis. 85:232, 2000.

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First Report of Leaf Spot on Tibouchina semidecandra Caused by Beltrania rhombica in China

Plant Disease ◽

10.1094/pdis-04-12-0363-pdn ◽

2012 ◽

Vol 96 (9) ◽

pp. 1380-1380 ◽

Cited By ~ 1

Author(s):

Z. R. Shi ◽

M. M. Xiang ◽

Y. X. Zhang ◽

J. H. Huang

Keyword(s):

Leaf Spot ◽

Its Region ◽

Control Measures ◽

Control Treatment ◽

Sterile Water ◽

Single Spore ◽

First Report ◽

Potted Plants ◽

Detached Leaves

Tibouchina semidecandra Cogn. is a popular ornamental plant in tropical and subtropical areas (1). In August 2011, a leaf spot was observed on approximately 70% of 5,000 potted plants of T. semidecandra in a nursery in Zhongshan, Guangdong Province, China. Each leaf spot was round with a brown center surrounded by a reddish brown border, and ranged from 8 to 10 mm in diameter. A fungus was isolated consistently from the lesions by surface-sterilizing symptomatic leaf sections (each 3 cm2) with 75% alcohol for 8 s, washing the sections with sterile water, soaking the sections in 3% NaOCl for 15 s, rinsing the sections with sterile water three times, and then placing the sections on potato dextrose agar (PDA) at 28°C. Each of three single-spore isolates on PDA produced gray, floccose colonies that reached 70 mm in diameter after 5 days at 28°C. Setae were dark brown, straight, erect, distantly and inconspicuously septate, and 125 to 193 × 3.0 to 4.5 μm. Conidiophores were light brown, cylindrical, simple or sometimes branched at the base, and 105 to 202 × 3 to 5 μm. Separating cells were hyaline, oval, and 12 to 13 × 4 to 5 μm. Conidia were unequally biconic, unicellular, dark brown with a pale brown or subhyaline band just above the widest part, and 26 to 31 × 8.5 to 12 μm (mean 27.3 × 10.6 μm) with a conspicuous appendage at the apex that was 6 to 14 × 1 to 1.8 μm. These characteristics were consistent with the description of Beltrania rhombica Penz. (3). The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) of one isolate (GenBank Accession No. JN853777) was amplified using primers ITS4 and ITS5 (4) and sequenced. A BLAST search in GenBank revealed 97% similarity to the ITS sequence of an isolate of B. rhombica (GU797390.1). To confirm pathogenicity of the isolate, ten detached leaves from 3-month-old plants of T. semidecandra ‘Purple Glorybush’ were inoculated in vitro with 5-mm diameter, colonized mycelial plugs from the periphery of 5-day-old cultures of the isolated fungus. The agar plugs were put on the leaf surface and secured with sterile, moist cotton. Sterile PDA plugs were similarly used as the control treatment on ten detached leaves. Leaves were placed in petri dishes and incubated in a growth chamber with 12 h of light/day at 28°C. Necrotic lesions appeared on leaves after 2 to 3 days of incubation, whereas control leaves inoculated with sterile PDA plugs remained asymptomatic. B. rhombica was consistently reisolated from the lesions using the same method described above, but was not reisolated from the control leaves. Although there are approximately 77 reported hosts of B. rhombica (2), to our knowledge, this is the first report of B. rhombica causing a leaf spot on T. semidecandra. Because the disease caused foliar damage and reduced the ornamental value of the nursery plants, control measures may need to be implemented for this species in nurseries. References: (1) M. Faravani and B. H. Bakar. J. Food Agric. Env. Pap. 5:234, 2007. (4) D. F. Farr and A. Y. Rossman. Fungal Databases. Systematic Microbiology Laboratory, ARS, USDA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , 30 Mar. 2012. (2) K. A. Pirozyski and S. D. Patil. Can. J. Bot. Pap. 48:567, 1970. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, CA, 1990.

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