First report of a sexual state in an ambrosia fungus: Ambrosiella cleistominuta sp. nov. associated with the ambrosia beetle Anisandrus maiche

Genera of ambrosia beetles in the tribe Xyleborini with large, mesonotal mycangia host unique fungal symbionts in the genus Ambrosiella. The symbiont of a recent invasive to the USA from Asia, Anisandrus maiche Stark, had not been previously characterized. We found the mycangium anatomy of An. maiche collected in Ohio to be similar to that of Anisandrus dispar and consistently isolated a novel fungus, Ambrosiella cleistominuta sp. nov., from An. maiche mycangia and galleries. The fungus was distinguished from other named Ambrosiella by morphological characters and DNA sequences (ITS rDNA and tef-1α). The mycangial symbionts of ambrosia beetles had been assumed to be strictly asexual, but A. cleistominuta produces cleistothecious ascomata with ascospores in beetle galleries and in culture. In contrast to ascomata of other Ceratocystidaceae, the relatively small ascomata of A. cleistominuta are neckless and without ostioles. The ascospores are relatively large, and single-ascospore colonies produced ascomata and ascospores in culture, showing that A. cleistominuta is homothallic.

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First Report of Phytophthora citricola Occurring on Fagus sylvatica in Slovenia

Plant Disease ◽

10.1094/pdis-91-7-0907c ◽

2007 ◽

Vol 91 (7) ◽

pp. 907-907 ◽

Cited By ~ 6

Author(s):

A. Munda ◽

M. Žerjav ◽

H.-J. Schroers

Keyword(s):

Fagus Sylvatica ◽

European Beech ◽

Pond Water ◽

Morphological Characters ◽

Phytophthora Species ◽

Its Rdna ◽

First Report ◽

5.8S Rdna ◽

Phytophthora Citricola ◽

Crown Dieback

During 2005 and 2006, eight declining, mature Fagus sylvatica trees with severe crown dieback were encountered at two stands near Ljubljana and Kamnik in central Slovenia. Bleeding cankers were seen mostly in the lower parts of the stem. Fresh cankers showed orange brown necroses of the inner bark and cambium. Older cankers were dark brown and surrounded by cracks and shedding bark. Small pieces of marginal tissue were excised from fresh necrotic lesions and incubated in the dark at 20°C on P5ARP and cornmeal (CMA) agar plates. Soil samples collected from around the base of these declining trees were submerged in sterile distilled water. Leaves of Rhododendron catawbiense were used as bait to test for the presence of Phytophthora spp. Several similar isolates were obtained from trees and soil from both stands. Colonies were narrowly petaloid, slow growing on P5ARP, and fast growing on CMA. The colonies produced abundant sporangia when submerged in pond water. Semipapillate, noncaducous sporangia were ovoid to obpyriform, but were occasionally distorted, and 30 to 69 (49) μm long and 23 to 44 (34) μm wide. Neither hyphal swellings nor chlamydospores developed. Isolates were homothallic showing paragynous antheridia, spherical oogonia of 22 to 31 (25) μm diameter, and plerotic oospores of 20 to 27 (23) μm diameter. The morphological characters resembled those described for Phytophthora citricola (1). ITS rDNA, spanning ITS 1 and 2, plus the 5.8S rDNA were generated using primers ITS4 and ITS5. Isolates from symptomatic trees and the soil yielded identical sequences and were the same as various sequences deposited for P. citricola at GenBank. The ITS rDNA of one representative strain was deposited at GenBank (Accession No. EF423556). Mycelial plugs from one of the P. citricola strains grown on CMA were used to inoculate stem wounds of seven potted seedlings and wounds made on four freshly cut, healthy branches of F. sylvatica. Sterile agar plugs were used as controls. The test was carried out over 4 weeks at 20°C. Extensive necrotic lesions developed around inoculation points on seedlings and branches, whereas the controls showed no symptoms. P. citricola could also be reisolated from margins of these lesions. During the past decade, declining F. sylvatica trees were observed in an increasing number of stands in Germany (2). Several Phytophthora species appeared to be involved in this decline, but P. citricola was the most frequently recovered species (2,3). To our knowledge, this it the first report of P. citricola found associated with a decline of European beech in Slovenia. References: (1) D. C. Erwin and O. K. Ribeiro. Pages 282–287 in: Phytophthora Diseases Worldwide. The American Phytopathological Society, St. Paul, MN, 1996. (2) T. Jung. Forst Holz. 60:131, 2005. (3) T. Jung et al. Mycologist 19:159, 2005.

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First Report of Phytophthora pini Causing Foliage Blight and Shoot Dieback of Rhododendron pulchrum in China

Plant Disease ◽

10.1094/pdis-07-20-1422-pdn ◽

2020 ◽

Author(s):

Yue Xu ◽

Xiao Yang ◽

Yaxing Li ◽

Zhenpeng Chen ◽

tingting Dai

Keyword(s):

Morphological Traits ◽

Control Plant ◽

Its Region ◽

Morphological Characters ◽

Public Parks ◽

First Report ◽

Landscape Plant ◽

Whole Plant ◽

Foliage Blight ◽

The Usa

During a 2019–2020 survey for plant pathogenic oomycetes in Nanjing, China, severe foliage blight and dieback were observed on approximately 20 Rhododendron pulchrum plants at three public parks and gardens. Approximately 25% of leaves and shoots were affected. Symptoms included brown to black lesions on leaves and stems, dieback of shoot tips, and wilting. Diseased tissues were collected from a five-year-old shrub with typical disease symptoms at Xuanwuhu Park. They were cut into 10×10 mm2 squares, immersed in 70% ethanol for 30 sec, and placed onto fresh clarified V8 juice agar (cV8A) containing pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene. Phytophthora-like hypae were transferred to new cV8A plates daily. A total of five isolates were obtained after five days of incubation at 25°C. After approximately 20 days, all isolates were identical in morphological traits including semi-papillate sporangia and gametangia (homothallic). Thirty sporangia of a representative isolate Ppi were randomly selected and examined. They were mostly ovoid and sometimes obpyriform, averaging 41.0 ± 3.9 × 24.8 ± 3.2 µm. Antheridia of 30 randomly selected gametangia were paragynous, averaging 16.7 ± 0.7 × 12.4 ± 1.5 µm. Average diameters of oogonia and plerotic oospores were 29.2 ± 0.3 µm and 26.4 ± 1.6 µm, respectively. Chlamydospores were not observed. The above morphological traits suggested the causal agent belonging to the “P. citricola-complex”. Isolate Ppi was subjected to sequencing of the rDNA internal transcribed spacer (ITS) region and the ras‐related GTP‐binding protein 1 (Ypt1) gene. ITS sequence of Ppi (GenBank ACN. MT672594) has 100% identity to that of P. pini (MG865565). It has a 3-nt difference from the ITS sequences of P. acerina (MG518642) and P. citricola (MG865475) and a 4-nt difference from that of P. plurivora (FJ665225). Ypt1 sequence of Ppi (MT680000) has 100% identity to that of P. pini (MK058416). Pathogenicity of Ppi on R. pulchrum was tested using both detached-leaf and whole-plant assays. In the former assay, each of six asymptomatic leaves was symmetrically wounded at both sides using a sterile inoculation needle. A 5×5 mm2 Ppi-colonized cV8A plug was placed on each wound of five leaves. Sterile agar plugs were used for a control leaf. All six leaves were placed on a wet filter paper in a closed container at 25°C. This assay was repeated twice. On the fifth day, all inoculated leaves had necrotic tissues around the wounds, while the control leaves remained asymptomatic. In the whole-plant assay, 20-inch-tall plants were used. Five attached leaves and the twig base of each plant were wounded. A control plant was inoculated in the same manner above, while sterile agar plugs were used. Each plant was covered with a plastic bag and maintained at 25°C. Wet cotton balls were placed in the bags to maintain humidity. After two days, the bag containing cotton balls was removed. This assay was repeated three times. After two weeks, all three inoculated plants in the three replicated trials had severe foliage blight and dieback, whereas control plants remained healthy. Phytophthora isolates recovered from artificially inoculated tissues were identical to isolate Ppi in morphological characters. Rhododendron diseases caused by P. pini were reported in the USA and Finland . This is the first report of P. pini causing foliage blight and dieback on R. pulchrum, an important nursery and landscape plant in China. Additional surveys are ongoing to determine the distribution of this pathogen in Nanjing. Management programs are under development to contain the spread of P. pini and treat diseased plants.

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Phylogenetic Relationship of Indonesian Water Yam (Dioscorea alata L.) Cultivars Based on DNA Marker Using ITS-rDNA Analysis

Journal of Agricultural Science ◽

10.5539/jas.v9n2p154 ◽

2017 ◽

Vol 9 (2) ◽

pp. 154

Author(s):

Purnomo Purnomo ◽

Budi Setiadi Daryono ◽

Hironobu Shiwachi

Keyword(s):

Phylogenetic Relationship ◽

Dna Sequences ◽

Phylogenetic Trees ◽

Morphological Characters ◽

Its Rdna ◽

28S Rdna ◽

Dioscorea Alata ◽

Cultivar Groups ◽

Pcr Products ◽

Water Yam

Research on genetic diversity and intra-species classification of Indonesian Dioscorea alata L. based on morphological characters has been done, and the result shows that there are 4 sub-groups of green cultivars group, and 5 sub-groups of purplish-red cultivars group. The objectives of this study are to determine the phylogenetic relationship cultivars of D. alata Indonesia compared to D. bulbifera as nearest species as well as 3 cultivars from GenBank. The young leaves of 18 water yam cultivar accessions were collected from Java, Madura, South Sumatera, South Kalimantan, Centre Celebes (Sulawesi), Ternate, West Papua, and Nusa Tenggara islands of Indonesia. DNA Isolation was conducted using Phytopure reagent. DNA amplification was conducted using thermocycler, predenaturation at 95 oC 5 minutes, denaturation at 95 oC 1 minutes, annealing at 60 oC 3 minutes, and elongation at 72 oC 2 minutes along with 30 cycles. The PCR products were electrophoresed in 1.5% agarose and visualized under UV transiluminator. Fifty-five µl of PCR products with positive targeted band between 700-800 bp were sent to 1st Base Singapore for purification and sequencing of 18S, ITS-1, 5.8S, ITS-2, and 28S rDNA. The DNA sequences were compared and aligned by BioEdit program (version 7.0.5.2) and MEGA programs. Comparison of entire sequences of the tested samples were aligned by software ClustalW (version 1.83). Phylogenetic trees were based on hierarchical clustering of the alignments of 18S, ITS-1, 5.8S, ITS-2, and 28S rDNA and produced by Neigbor-Joining using MEGA 5 software of the bootstrap values (1000 replicates). The study result shows that D. alata cultivars have high genetic variability on ITS-1, 5.8S, and ITS-2 rDNA region. Groups of green and purplish red cultivars formed based on morphological characters are not formed based on ITS-rDNA markers. Sub-groups were formed based on ITS-rDNA molecular markers derived from both the green and purplish-red cultivar groups. This result revealed that two cultivar groups are not similar with RAPD and morphological characters.

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First Report of Stachybotrys chartarum Causing Leaf Blight of Tillandsia tenuifolia in China

Plant Disease ◽

10.1094/pdis-94-9-1166b ◽

2010 ◽

Vol 94 (9) ◽

pp. 1166-1166

Author(s):

G.-H. Zhao ◽

D.-W. Li ◽

J.-H. Jiang ◽

J. Peng

Keyword(s):

Dna Sequences ◽

Basal Area ◽

Severe Infection ◽

Its Region ◽

Morphological Characters ◽

Culture Collection ◽

Leaf Blight ◽

Stachybotrys Chartarum ◽

Diseased Plant ◽

First Report

Tillandsia tenuifolia L. is an air plant that is native to Bolivia, Brazil, and Venezuela. It was introduced to China in 2006. In September 2008, a leaf blight of T. tenuifolia ‘Bronze Tip’ was observed in a greenhouse in Jurong, Jiangsu. Severe infection led to death of foliage, shoot rot, and eventual mortality of whole plants. The pathogen was isolated from the diseased leaves on potato dextrose agar and subsequently further cultured on a slide culture for 7 days. Anamorphic structures were examined under a compound microscope. Diseased plant parts were covered with abundant conidia, phialides, conidiophores, and mycelia of the pathogen. Conidiophores were simple or branched, zero to two septate, hyaline, smooth at base, brown, smooth to rough at upper portion, and 47.9 ± 7.6 × 4 ± 0.4 μm (n = 30). Phialides were one-celled, obovate, hyaline to pale brown, 8.8 ± 0.9 × 5.3 ± 0.3 μm (n = 20), and in whorls. Conidia were one-celled, ellipsoid to subglobose, dark brown to black, rough to ridged, 10.4 ± 1.4 × 6.2 ± 0.9 μm (n = 30), and in slimy masses. Using morphological characters, the pathogen was identified as Stachybotrys chartarum (Ehrenb.) S. Hughes (1). The living culture was deposited in the China General Microbiological Culture Collection Center (CGMCC 3.13634). S. chartarum, a saprophyte with a worldwide distribution, grows on various substrates such as soil, paper, dry walls, and wood. (2). It was also isolated from moldy sorghum seed and soybean root lesions (3,4). Pathogenicity on T. tenuifolia was confirmed by Koch's postulates in the laboratory. Ninety-six leaves on four plants were pricked with a sterilized needle and inoculated with a suspension of 6.35 × 106 conidia ml–1. Forty-seven leaves on two plants were pricked and sprayed with sterile water as controls. All the plants were kept in a growth chamber with a 12-h photoperiod, 28 ± 1°C, and relative humidity of 70 ± 3%. Initial lesions, which were water soaked, slightly sunken, and pinhead size, appeared on the inoculated plants in 7 days and expanded to 1.5 to 2.0 mm in 21 days. Necrotic spots subsequently coalesced and caused the death of the leaves. Infected shoots were rotten and shed leaves from the basal area. The plants died within 45 days. Symptoms were similar to those observed in the greenhouse. S. chartarum was reisolated from infected leaves. Control plants remained healthy. To confirm the identity of our isolate, DNA sequence was obtained from the internal transcribed spacer (ITS) region and deposited in GenBank (GU945205). The ITS sequence was 100% identical to S. chartarum strains whose DNA sequences were deposited in GenBank. To our knowledge, this is the first report of S. chartarum causing leaf blight on T. tenuifolia. The popularity of T. tenuifolia continues to grow in China. The disease should be monitored to determine its risk and economic significance in China and other regions. References: (1) S. C. Jong and E. E. Davis. Mycotaxon 3:409, 1976. (2) P. M. Kirk. Mycopathologia 115:149, 1991. (3) S. Li et al. Mycopathologia 154:41, 2002. (4) J. Y. Liang and J. K. Bai. J. Shenyang Agric. Univ. 19:27, 1988.

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Systematics, Biogeography, and Morphological Character Evolution of the Hemiepiphytic Subfamily Monsteroideae (Araceae)

Annals of the Missouri Botanical Garden ◽

10.3417/2018269 ◽

2019 ◽

Vol 104 (1) ◽

pp. 33-48 ◽

Cited By ~ 4

Author(s):

Alejandro Zuluaga ◽

Martin Llano ◽

Ken Cameron

Keyword(s):

Dna Sequences ◽

Pacific Islands ◽

R Package ◽

Morphological Characters ◽

Ancestral State ◽

Seed Shape ◽

Long Distance ◽

The Pacific ◽

And Migration ◽

The Tropics

The subfamily Monsteroideae (Araceae) is the third richest clade in the family, with ca. 369 described species and ca. 700 estimated. It comprises mostly hemiepiphytic or epiphytic plants restricted to the tropics, with three intercontinental disjunctions. Using a dataset representing all 12 genera in Monsteroideae (126 taxa), and five plastid and two nuclear markers, we studied the systematics and historical biogeography of the group. We found high support for the monophyly of the three major clades (Spathiphylleae sister to Heteropsis Kunth and Rhaphidophora Hassk. clades), and for six of the genera within Monsteroideae. However, we found low rates of variation in the DNA sequences used and a lack of molecular markers suitable for species-level phylogenies in the group. We also performed ancestral state reconstruction of some morphological characters traditionally used for genera delimitation. Only seed shape and size, number of seeds, number of locules, and presence of endosperm showed utility in the classification of genera in Monsteroideae. We estimated ancestral ranges using a dispersal-extinction-cladogenesis model as implemented in the R package BioGeoBEARS and found evidence for a Gondwanan origin of the clade. One tropical disjunction (Monstera Adans. sister to Amydrium Schott–Epipremnum Schott) was found to be the product of a previous Boreotropical distribution. Two other disjunctions are more recent and likely due to long-distance dispersal: Spathiphyllum Schott (with Holochlamys Engl. nested within) represents a dispersal from South America to the Pacific Islands in Southeast Asia, and Rhaphidophora represents a dispersal from Asia to Africa. Future studies based on stronger phylogenetic reconstructions and complete morphological datasets are needed to explore the details of speciation and migration within and among areas in Asia.

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First Report of Flyspeck Caused by Zygophiala wisconsinensis on Sweet Persimmon Fruit in Korea

Plant Disease ◽

10.1094/pdis-12-10-0917 ◽

2011 ◽

Vol 95 (5) ◽

pp. 616-616 ◽

Cited By ~ 1

Author(s):

J. Kim ◽

O. Choi ◽

J.-H. Kwon

Keyword(s):

Disease Incidence ◽

Morphological Characteristics ◽

Its Rdna ◽

Control Treatment ◽

Diospyros Kaki ◽

Distilled Water ◽

First Report ◽

Persimmon Fruit ◽

Fungal Isolates ◽

Agar Disc

Sweet persimmon (Diospyros kaki L.), a fruit tree in the Ebenaceae, is cultivated widely in Korea and Japan, the leading producers worldwide (2). Sweet persimmon fruit with flyspeck symptoms were collected from orchards in the Jinju area of Korea in November 2010. The fruit had fungal clusters of black, round to ovoid, sclerotium-like fungal bodies with no visible evidence of a mycelial mat. Orchard inspections revealed that disease incidence ranged from 10 to 20% in the surveyed area (approximately 10 ha) in 2010. Flyspeck symptoms were observed on immature and mature fruit. Sweet persimmon fruit peels with flyspeck symptoms were removed, dried, and individual speck lesions transferred to potato dextrose agar (PDA) and cultured at 22°C in the dark. Fungal isolates were obtained from flyspeck colonies on 10 sweet persimmon fruit harvested from each of three orchards. Fungal isolates that grew from the lesions were identified based on a previous description (1). To confirm identity of the causal fungus, the complete internal transcribed spacer (ITS) rDNA sequence of a representative isolate was amplified and sequenced using primers ITS1 and ITS4 (4). The resulting 552-bp sequence was deposited in GenBank (Accession No. HQ698923). Comparison with ITS rDNA sequences showed 100% similarity with a sequence of Zygophiala wisconsinensis Batzer & Crous (GenBank Accession No. AY598855), which infects apple. To fulfill Koch's postulates, mature, intact sweet persimmon fruit were surface sterilized with 70% ethanol and dried. Three fungal isolates from this study were grown on PDA for 1 month. A colonized agar disc (5 mm in diameter) of each isolate was cut from the advancing margin of a colony with a sterilized cork borer, transferred to a 1.5-ml Eppendorf tube, and ground into a suspension of mycelial fragments and conidia in a blender with 1 ml of sterile, distilled water. The inoculum of each isolate was applied by swabbing a sweet persimmon fruit with the suspension. Three sweet persimmon fruit were inoculated per isolate. Three fruit were inoculated similarly with sterile, distilled water as the control treatment. After 1 month of incubation in a moist chamber at 22°C, the same fungal fruiting symptoms were reproduced as observed in the orchards, and the fungus was reisolated from these symptoms, but not from the control fruit, which were asymptomatic. On the basis of morphological characteristics of the fungal colonies, ITS sequence, and pathogenicity to persimmon fruit, the fungus was identified as Z. wisconsinensis (1). Flyspeck is readily isolated from sweet persimmon fruit in Korea and other sweet persimmon growing regions (3). The exposure of fruit to unusual weather conditions in Korea in recent years, including drought, and low-temperature and low-light situations in late spring, which are favorable for flyspeck, might be associated with an increase in occurrence of flyspeck on sweet persimmon fruit in Korea. To our knowledge, this is the first report of Z. wisconsinensis causing flyspeck on sweet persimmon in Korea. References: (1) J. C. Batzer et al. Mycologia 100:246, 2008. (2) FAOSTAT Database. Retrieved from http://faostat.fao.org/ , 2008. (3) H. Nasu and H. Kunoh. Plant Dis. 71:361, 1987. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, Inc., New York, 1990.

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Nothing in (sponge) biology makes sense – except when based on holotypes

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315415000521 ◽

2015 ◽

Vol 96 (2) ◽

pp. 305-311 ◽

Cited By ~ 10

Author(s):

Dirk Erpenbeck ◽

Merrick Ekins ◽

Nicole Enghuber ◽

John N.A. Hooper ◽

Helmut Lehnert ◽

...

Keyword(s):

New Species ◽

Reference Material ◽

Species Identification ◽

Dna Sequences ◽

Morphological Plasticity ◽

Morphological Characters ◽

Molecular Techniques ◽

Reference Sequences ◽

Species Descriptions ◽

Preservation History

Sponge species are infamously difficult to identify for non-experts due to their high morphological plasticity and the paucity of informative morphological characters. The use of molecular techniques certainly helps with species identification, but unfortunately it requires prior reference sequences. Holotypes constitute the best reference material for species identification, however their usage in molecular systematics and taxonomy is scarce and frequently not even attempted, mostly due to their antiquity and preservation history. Here we provide case studies in which we demonstrate the importance of using holotype material to answer phylogenetic and taxonomic questions. We also demonstrate the possibility of sequencing DNA fragments out of century-old holotypes. Furthermore we propose the deposition of DNA sequences in conjunction with new species descriptions.

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Sinalliaria, a new genus of Brassicaceae from eastern China, based on morphological and molecular data

Phytotaxa ◽

10.11646/phytotaxa.186.4.2 ◽

2014 ◽

Vol 186 (4) ◽

pp. 188 ◽

Cited By ~ 6

Author(s):

Ying-Ying Zhou ◽

HONG-WEI ZHANG ◽

JIANG-QIN HU ◽

Xiao-Feng Jin

Keyword(s):

Dna Sequences ◽

New Genus ◽

Eastern China ◽

Distinct Group ◽

Morphological Characters ◽

Molecular Data ◽

New Combinations ◽

The Family ◽

Morphological And Molecular Data ◽

Seed Testa

Sinalliaria is described here as a new genus of the family Brassicaceae from eastern China, based on the morphological characters and molecular sequences. Sinalliaria differs from the related genus Orychophragmus in having basal leaves petiolate, simple or rarely with 1‒3 lateral lobes (not pinnatisect); cauline leaves petiolate, cordate at base (not sessile, auriculate or amplexicaul at base); petals obovate to narrowly obovate, claw inconspicuous (not broadly obovate, with a claw as along as sepal); siliques truncate (not long-beaked) at apex. The microscopic characters of seed testa also show significant differences between Sinalliaria and Orychophragmus. Phylogenetic evidence from DNA sequences of nuclear ribosomal ITS and plastid region trnL-trnF indicates that Sinalliaria is a distinct group related to Orychophragmus and Raphanus, but these three genera do not form a clade. The new genus Sinalliaria is endemic to eastern China and has only one species and one variety. The new combinations, S. limprichtiana (Pax) X. F. Jin, Y. Y. Zhou & H. W. Zhang and S. limprichtiana var. grandifolia (Z. X. An) X. F. Jin, Y. Y. Zhou & H. W. Zhang are proposed here.

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First Report of Leaf Blight of Japanese Yew Caused by Pestalotiopsis microspora in Korea

Plant Disease ◽

10.1094/pdis-08-13-0821-pdn ◽

2014 ◽

Vol 98 (5) ◽

pp. 691-691 ◽

Cited By ~ 2

Author(s):

Y. H. Jeon ◽

W. Cheon

Keyword(s):

Dna Sequences ◽

Apical Cell ◽

Morphological Characteristics ◽

Leaf Blight ◽

Economic Damage ◽

Leaf Margin ◽

Conidial Suspension ◽

First Report ◽

Pestalotiopsis Microspora ◽

Large Trees

Worldwide, Japanese yew (Taxus cuspidata Sieb. & Zucc.) is a popular garden tree, with large trees also being used for timber. In July 2012, leaf blight was observed on 10% of Japanese yew seedling leaves planted in a 500-m2 field in Andong, Gyeongsangbuk-do Province, South Korea. Typical symptoms included small, brown lesions that were first visible on the leaf margin, which enlarged and coalesced into the leaf becoming brown and blighted. To isolate potential pathogens from infected leaves, small sections of leaf tissue (5 to 10 mm2) were excised from lesion margins. Eight fungi were isolated from eight symptomatic trees, respectively. These fungi were hyphal tipped twice and transferred to potato dextrose agar (PDA) plates for incubation at 25°C. After 7 days, the fungi produced circular mats of white aerial mycelia. After 12 days, black acervuli containing slimy spore masses formed over the mycelial mats. Two representative isolates were further characterized. Their conidia were straight or slightly curved, fusiform to clavate, five-celled with constrictions at the septa, and 17.4 to 28.5 × 5.8 to 7.1 μm. Two to four 19.8- to 30.7-μm-long hyaline filamentous appendages (mostly three appendages) were attached to each apical cell, whereas one 3.7- to 7.1-μm-long hyaline appendage was attached to each basal cell, matching the description for Pestalotiopsis microspora (2). The pathogenicity of the two isolates was tested using 2-year-old plants (T. cuspidata var. nana Rehder; three plants per isolate) in 30-cm-diameter pots filled with soil under greenhouse conditions. The plants were inoculated by spraying the leaves with an atomizer with a conidial suspension (105 conidia/ml; ~50 ml on each plant) cultured for 10 days on PDA. As a control, three plants were inoculated with sterilized water. The plants were covered with plastic bags for 72 h to maintain high relative humidity (24 to 28°C). At 20 days after inoculation, small dark lesions enlarged into brown blight similar to that observed on naturally infected leaves. P. microspora was isolated from all inoculated plants, but not the controls. The fungus was confirmed by molecular analysis of the 5.8S subunit and flanking internal transcribed spaces (ITS1 and ITS2) of rDNA amplified from DNA extracted from single-spore cultures, and amplified with the ITS1/ITS4 primers and sequenced as previously described (4). Sequences were compared with other DNA sequences in GenBank using a BLASTN search. The P. microspora isolates were 99% homologous to other P. microspora (DQ456865, EU279435, FJ459951, and FJ459950). The morphological characteristics, pathogenicity, and molecular data assimilated in this study corresponded with the fungus P. microspora (2). This fungus has been previously reported as the causal agent of scab disease of Psidium guajava in Hawaii, the decline of Torreya taxifolia in Florida, and the leaf blight of Reineckea carnea in China (1,3). Therefore, this study presents the first report of P. microspora as a pathogen on T. cuspidata in Korea. The degree of pathogenicity of P. microspora to the Korean garden evergreen T. cuspidata requires quantification to determine its potential economic damage and to establish effective management practices. References: (1) D. F. Farr and A. Y. Rossman, Fungal Databases, Syst. Mycol. Microbiol. Lab. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ (2) L. M. Keith et al. Plant Dis. 90:16, 2006. (3) S. S. N. Maharachchikumbura. Fungal Diversity 50:167, 2011. (4) T. J. White et al. PCR Protocols. Academic Press, San Diego, CA, 1990.

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Clostridium difficilewith Moxifloxacin/Clindamycin Resistance in Vegetables in Ohio, USA, and Prevalence Meta-Analysis

Journal of Pathogens ◽

10.1155/2014/158601 ◽

2014 ◽

Vol 2014 ◽

pp. 1-7 ◽

Cited By ~ 15

Author(s):

Alex Rodriguez-Palacios ◽

Sanja Ilic ◽

Jeffrey T. LeJeune

Keyword(s):

Antimicrobial Resistance ◽

Meta Analysis ◽

Green Pepper ◽

First Report ◽

Iceberg Lettuce ◽

Vegetable Products ◽

Fresh Vegetables ◽

The Usa ◽

Enrichment Protocol ◽

Health Relevance

We (i) determined the prevalence ofClostridium difficileand their antimicrobial resistance to six antimicrobial classes, in a variety of fresh vegetables sold in retail in Ohio, USA, and (ii) conducted cumulative meta-analysis of reported prevalence in vegetables since the 1990s. Six antimicrobial classes were tested for their relevance as risk factors forC. difficileinfections (CDIs) (clindamycin, moxifloxacin) or their clinical priority as exhaustive therapeutic options (metronidazole, vancomycin, linezolid, and tigecycline). By using an enrichment protocol we isolatedC. difficilefrom three of 125 vegetable products (2.4%). All isolates were toxigenic, and originated from 4.6% of 65 vegetables cultivated above the ground (n=3; outer leaves of iceberg lettuce, green pepper, and eggplant). Root vegetables yielded noC. difficile. TheC. difficileisolates belonged to two PCR ribotypes, one with an unusual antimicrobial resistance for moxifloxacin and clindamycin (lettuce and pepper; 027-like, A+B+CDT+;tcdC18 bp deletion); the other PCR ribotype (eggplant, A+B+CDT−; classictcdC) was susceptible to all antimicrobials. Results of the cumulative weighted meta-analysis (6 studies) indicate that the prevalence ofC. difficilein vegetables is 2.1% and homogeneous(P<0.001)since the first report in 1996 (2.4%). The present study is the first report of the isolation ofC. difficilefrom retail vegetables in the USA. Of public health relevance, antimicrobial resistance to moxifloxacin/clindamycin (a bacterial-associated risk factor for severe CDIs) was identified on the surface of vegetables that are consumed raw.

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