New and Interesting Fungi. 6.

Three new genera, six new species, three combinations, six epitypes, and 25 interesting new host and / or geographical records are introduced in this study. New genera: Neoleptodontidium (based on Neoleptodontidium aquaticum), and Nothoramularia (based on Nothoramularia ragnhildianicola). New species: Acremonium aquaticum (from cooling pad water, USA, Cladophialophora laricicola (on dead wood of Larix sp., Netherlands), Cyphellophora neerlandica (on lichen on brick wall, Netherlands), Geonectria muralis (on moss growing on a wall, Netherlands), Harposporium illinoisense (from rockwool, USA), and Neoleptodontidium aquaticum (from hydroponic water, USA). New combinations: Cyphellophora deltoidea (based on Anthopsis deltoidea), Neoleptodontidium aciculare (based on Leptodontidium aciculare), and Nothoramularia ragnhildianicola (based on Ramularia ragnhildianicola). Epitypes: Cephaliophora tropica (from water, USA), Miricatena prunicola (on leaves of Prunus serotina, Netherlands), Nothoramularia ragnhildianicola (on Ragnhildiana ferruginea, parasitic on Artemisia vulgaris, Germany), Phyllosticta multicorniculata (on needles of Abietis balsamea, Canada), Thyronectria caraganae (on twigs of Caragana arborescens, Ukraine), and Trichosphaeria pilosa (on decayed Salix branch, Netherlands). Furthermore, the higher order phylogeny of three genera regarded as incertae sedis is resolved, namely Cephaliophora (Ascodesmidaceae, Pezizales), Miricatena (Helotiales, Leotiomycetes), and Trichosphaeria (Trichosphaeriaceae, Trichosphaeriales), with Trichosphaeriaceae being an older name for Plectosphaerellaceae. Citation: Crous PW, Akulov A, Balashov S, Boers J, Braun U, Castillo J, Delgado MA, Denman S, Erhard A, Gusella G, Jurjevic Z, Kruse J, Malloch DW, Osieck ER, Polizzi G, Schumacher RK, Slootweg E, Starink-Willemse M, van Iperen AL, Verkley GJM, Groenewald JZ (2023). New and Interesting Fungi. 6. Fungal Systematics and Evolution 11: 109-156. doi: 10.3114/fuse.2023.11.09.

Visualization, navigation, augmentation. The ever-changing perspective of the neurosurgeon.

Introduction: The evolution of neurosurgery coincides with the evolution of visualization and navigation. Augmented reality technologies, with their ability to bring digital information into the real environment, have the potential to provide a new, revolutionary perspective to the neurosurgeon. Research question: To provide an overview on the historical and technical aspects of visualization and navigation in neurosurgery, and to provide a systematic review on augmented reality (AR) applications in neurosurgery. Material and methods: We provided an overview on the main historical milestones and technical features of visualization and navigation tools in neurosurgery. We systematically searched PubMed and Scopus databases for AR applications in neurosurgery and specifically discussed their relationship with current visualization and navigation systems, as well as main limitations. Results: The evolution of visualization in neurosurgery is embodied by four magnification systems: surgical loupes, endoscope, surgical microscope and more recently the exoscope, each presenting independent features in terms of magnification capabilities, eye-hand coordination and the possibility to implement additional functions. In regard to navigation, two independent systems have been developed: the frame-based and the frame-less systems. The most frequent application setting for AR is brain surgery (71.6%), specifically neuro-oncology (36.2%) and microscope-based (29.2%), even though in the majority of cases AR applications presented their own visualization supports (66%). Discussion and conclusions: The evolution of visualization and navigation in neurosurgery allowed for the development of more precise instruments; the development and clinical validation of AR applications, have the potential to be the next breakthrough, making surgeries safer, as well as improving surgical experience and reducing costs.

Fusarium and allied fusarioid taxa (FUSA). 1.

Seven Fusarium species complexes are treated, namely F. aywerte species complex (FASC) (two species), F. buharicum species complex (FBSC) (five species), F. burgessii species complex (FBURSC) (three species), F. camptoceras species complex (FCAMSC) (three species), F. chlamydosporum species complex (FCSC) (eight species), F. citricola species complex (FCCSC) (five species) and the F. concolor species complex (FCOSC) (four species). New species include Fusicolla elongata from soil (Zimbabwe), and Neocosmospora geoasparagicola from soil associated with Asparagus officinalis (Netherlands). New combinations include Neocosmospora akasia, N. awan, N. drepaniformis, N. duplosperma, N. geoasparagicola, N. mekan, N. papillata, N. variasi and N. warna. Newly validated taxa include Longinectria gen. nov., L. lagenoides, L. verticilliforme, Fusicolla gigas and Fusicolla guangxiensis. Furthermore, Fusarium rosicola is reduced to synonymy under N. brevis. Finally, the genome assemblies of Fusarium secorum (CBS 175.32), Microcera coccophila (CBS 310.34), Rectifusarium robinianum (CBS 430.91), Rugonectria rugulosa (CBS 126565), and Thelonectria blattea (CBS 952.68) are also announced here. Citation: Crous PW, Sandoval-Denis M, Costa MM, Groenewald JZ, van Iperen AL, Starink-Willemse M, Hernández-Restrepo M, Kandemir H, Ulaszewski B, de Boer W, Abdel-Azeem AM, Abdollahzadeh J, Akulov A, Bakhshi M, Bezerra JDP, Bhunjun CS, Câmara MPS, Chaverri P, Vieira WAS, Decock CA, Gaya E, Gené J, Guarro J, Gramaje D, Grube M, Gupta VK, Guarnaccia V, Hill R, Hirooka Y, Hyde KD, Jayawardena RS, Jeewon R, Jurjevic Z, Korsten L, Lamprecht SC, Lombard L, Maharachchikumbura SSN, Polizzi G, Rajeshkumar KC, Salgado-Salazar C, Shang Q-J, Shivas RG, Summerbell RC, Sun GY, Swart WJ, Tan YP, Vizzini A, Xia JW, Zare R, González CD, Iturriaga T, Savary O, Coton M, Coton E, Jany J-L, Liu C, Zeng Z-Q, Zhuang W-Y, Yu Z-H, Thines M (2022). Fusarium and allied fusarioid taxa (FUSA). 1. Fungal Systematics and Evolution 9: 161-200. doi: 10.3114/fuse.2022.09.08.

Symptomatic Citrus trees reveal a new pathogenic lineage in Fusarium and two new Neocosmospora species.

The diversity of fusaria in symptomatic Citrus trees in Greece, Italy and Spain was evaluated using morphological and molecular multi-locus analyses based on fragments of the calmodulin (CAM), intergenic spacer region of the rDNA (IGS), internal transcribed spacer region of the rDNA (ITS), large subunit of the rDNA (LSU), RNA polymerase largest subunit (RPB1), RNA polymerase second largest subunit (RPB2), translation elongation factor 1-alpha (EF-1α) and beta-tubulin (TUB) genes. A total of 11 species (six Fusarium spp., and five Neocosmospora spp.) were isolated from dry root rot, crown, trunk or twig canker or twig dieback of citrus trees. The most commonly isolated species were Fusarium sarcochroum, F. oxysporum and Neocosmospora solani. Three new Fusarium species are described, i.e., F. citricola and F. salinense belonging to the newly described F. citricola species complex; and F. siculi belonging to the F. fujikuroi species complex. Results of pathogenicity tests showed this new complex to include prominent canker causing agents affecting several Citrus spp. In addition, two new species are described in Neocosmospora, named N. croci and N. macrospora, the latter species being clearly differentiated from most members of this genus by producing large, up to nine-septate sporodochial conidia.

Neocosmospora perseae sp. nov., causing trunk cankers on avocado in Italy.

Trunk and branch cankers are among the most important diseases compromising avocado production worldwide. A novel species, Neocosmospora perseae sp. nov. is described isolated from trunk lesions on Persea americana in the main avocado producing area of Sicily, Italy. The new species is characterised using a polyphasic approach including morphological characters and a multilocus molecular phylogenetic analysis based on partial sequences of the translation elongation factor-1α, the internal transcribed spacer regions plus the large subunit of the rDNA cistron, and the RNA polymerase II second largest subunit. Pathogenicity tests and the fulfilment of Koch's postulates confirm N. perseae as a novel canker pathogen of Persea americana.

High species diversity in Colletotrichum associated with citrus diseases in Europe.

Species of Colletotrichum are considered important plant pathogens, saprobes, and endophytes on a wide range of plant hosts. Several species are well-known on citrus, either as agents of pre- or post-harvest infections, such as anthracnose, postbloom fruit drop, tear stain and stem-end rot on fruit, or as wither-tip of twigs. In this study we explored the occurrence, diversity and pathogenicity of Colletotrichum spp. associated with Citrus and allied genera in European orchards, nurseries and gardens. Surveys were carried out during 2015 and 2016 in Greece, Italy, Malta, Portugal and Spain. A total of 174 Colletotrichum strains were isolated from symptomatic leaves, fruits, petals and twigs. A multi-locus phylogeny was established based on seven genomic loci (ITS, GAPDH, ACT, CAL, CHS-1, HIS3 and TUB2), and the morphological characters of the isolates determined. Preliminary pathogenicity tests were performed on orange fruits with representative isolates. Colletotrichum strains were identified as members of three major species complexes. Colletotrichum gloeosporioides s.str. and two novel species (C. helleniense and C. hystricis) were identified in the C. gloeosporioides species complex. Colletotrichum karstii, C. novae-zelandiae and two novel species (C. catinaense and C. limonicola) in the C. boninense species complex, and C. acutatum s.str. was also isolated as member of C. acutatum species complex. Colletotrichum gloeosporioides and C. karstii were the predominant species of Colletotrichum isolated. This study represents the first report of C. acutatum on citrus in Europe, and the first detection of C. novae-zelandiae from outside New Zealand. Pathogenicity tests revealed C. gloeosporioides s.str. to be the most virulent species on fruits. The present study improves our understanding of species associated with several disease symptoms on citrus fruits and plants, and provides useful information for effective disease management.

First Report of Fusarium Wilt on Philotheca myoporoides Caused by Fusarium oxysporum in Italy.

Philotheca myoporoides (DC.) M.J. Bayly (previously known as Eriostemon myoporoides), commonly called long-leaf waxflower and native to eastern Australia (Rutaceae family), is a hardy compact shrub or small tree occurring in subtropical to cool temperate regions. P. myoporoides is cultivated in Sicily (Italy) for its ornamental appeal. During April of 2010, a widespread wilting was observed on approximately 80% of 2,000 1-year-old, potted long-leaf waxflower plants grown in a commercial nursery near Catania (eastern Sicily, Italy). Internally, symptomatic plants had conspicuous vascular brown discoloration from the crown to the canopy. Diseased crown and stem tissues of 20 plants 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 white or light purple aerial mycelia and violet pigmentation on the underside of the cultures developed after 9 days. On carnation leaf agar, 20 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 aggregate chlamydospores. A PCR assay was conducted on one representative isolate (ITEM 13490) by analyzing sequences of the benA gene (coding ß-tubulin protein) and CaM gene (coding calmodulin protein) using the primers reported by O'Donnell et al. (1). The benA gene sequences of ITEM 13490 (GenBank No. FR828825) exhibited an identity of 100% to Fusarium oxysporum f. sp. radicis-lycopersici strain ATCC 52429 (GenBank No. DQ092480). CaM gene sequences of ITEM 13490 (GenBank No. FR828826) exhibited an identity of 99.6% to F. oxysporum strain ITEM 2367 (GenBank No. AJ560774). Morphological characteristics of the 20 isolates, as well as the PCR assay on a representative strain, identified the isolates associated with disease symptoms as F. oxysporum Schlechtend.:Fr. A pathogenicity test was performed by placing two 1-cm2 plugs of PDA from 9-day-old mycelial cultures near the crown on potted, healthy, 2-month-old cuttings of P. myoporoides. Thirty plants were inoculated with strain ITEM 13490 and 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 25 ± 1°C. Plants were then moved to a greenhouse where temperatures ranged from 23 to 27°C. First symptoms, which were identical to those observed in the nursery, developed on one plant 15 days after inoculation. Wilting was detected on all plants after 30 days. Control plants remained symptomless. F. oxysporum was successfully reisolated from symptomatic crown and stem tissues and identified as described above, fulfilling Koch's postulates. To our knowledge, this is the first report of F. oxysporum causing disease of P. myoporoides worldwide. Moreover, this pathogen was recently reported in the same nursery on Eremophila sp. (2), confirming the presence of Fusarium wilt as a potential threat to ornamental plant production in this area, and necessitates the innovation and development of disinfection methods for alveolar trays, greenhouses, and various propagation materials to reduce future disease outbreaks. References: (1) K. O'Donnell et al. Mycoscience 41:61, 2000. (2) G. Polizzi et al. Plant Dis 94:1509, 2010.

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

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 ß-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.

First Report of Crown and Root Rot Caused by Rhizoctonia solani AG-4 on Banana Passionflower (Passiflora mollissima) in Italy.

The genus Passiflora (Passifloraceae family) contains more than 500 species and several hybrids. In Italy, some of these species and hybrids are grown as ornamental evergreen vines or shrubs. During August and September 2010, a crown and root rot was observed in a stock of approximately 6,000 potted 2-year-old plants of Passiflora mollissima (Kunth) Bailey, commonly known as the banana passionflower, in a nursery located in eastern Sicily (southern Italy). Disease incidence was approximately 20%. Disease symptoms consisted of water-soaked lesions at the crown and a root rot. Successively, older crown lesions turned light brown to brown and expanded to girdle the stem. As crown and root rot progressed, basal leaves turned yellow and gradually became necrotic and infected plants wilted and died. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently isolated from crown lesions and brown decaying roots when plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 µg/ml. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Mycelium was branched at right angles with a septum near the branch with a slight constriction at the branch base. Hyphal cells removed from 10 representative cultures grown at 25°C on 2% water agar were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates on 2% water agar in petri plates (4). Pairings were made with tester strains of AG-1, AG-2, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4 (3). Pathogenicity tests were performed on container-grown, healthy, 3-month-old cuttings. Twenty plants of P. mollissima were inoculated near the base of the stem with five 1-cm2 PDA plugs from 5-day-old mycelial plugs obtained from two representative cultures. The same number of plants served as uninoculated controls. Plants were maintained at 25°C and 95% relative humidity with a 12-h fluorescent light/dark regimen. Wilt symptoms due to crown and root rot, identical to ones observed in the nursery, appeared 7 to 8 days after inoculation with either of the two isolates and all plants died within 20 days. No disease was observed on control plants. R. solani AG-4 was reisolated from symptomatic tissues and identified as previously described, confirming its pathogenicity. Damping-off or crown and root rot due to R. solani were previously detected on P. edulis in Brazil, Africa, India, Oceania, and Australia (2). To our knowledge, this is the first report of R. solani causing crown and root rot on P. mollissima. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) J. L. Bezerra and M. L. Oliveira. Fitopathol. Brasil. 9:273, 1984. (3) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Sclerotinia Stem and Twig Blight Caused by Sclerotinia sclerotiorum on Citrus volkameriana Rootstock in Italy.

Volkamer lemon (Citrus volkameriana Ten. & Pasq., Rutaceae family) is the most commonly used rootstock for some ornamental citrus (oval kumquat and calamondin), improving the aesthetic quality of the plants and their marketable value. During the winter of 2011, symptoms of stem blight were observed on approximately 10% of 12,000 1-year-old potted C. volkameriana seedlings grown in different blocks in a commercial nursery near Catania (eastern Sicily, Italy). In the same nursery, only 1% of 15,000 older seedlings (2-year-old) showed disease symptoms. Initial symptoms included gray lesions on stems and occasionally on twigs. Later, buff lesions and gum exude appeared. Symptomatic stems and twigs were usually girdled and killed. In the lesions, irregular, dark gray sclerotia (1.0 to 5 × 1.0 to 7.0 mm, average 2.5 × 3.9 mm) were produced. In high relative humidity, cottony, white mycelia on the bark surface of infected tissues were also observed. Isolations were performed by transferring approximately 300 fragments of symptomatic tissues from 15 C volkameriana seedlings, surface-sterilized with 1% NaClO for 1 min, on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate. Sclerotinia sclerotiorum (Lib.) de Bary was recovered from all infected plants. Colony type, morphology, and dimensions of sclerotia were examined on PDA at 22 ± 1°C after 10 days in the dark. Sclerotia produced on PDA measured 2.0 to 7.0 × 1.5 to 4.0 mm (average 5.6 × 2.6 mm). DNA isolation was performed with the DNA Purification Kit (Puragene-Gentra, Minneapolis, MN) following the manufacturer's instructions. Amplification and sequencing of the internal transcribed spacer (ITS) region of rDNA was performed with primers ITS1/ITS4 (2). BLAST analysis of the 550-bp segment showed a 98% homology with S. sclerotiorum strain ms85 (GenBank Accession No HQ833450.1), thus confirming identification based on morphology. Koch's postulates were fulfilled by pathogenicity tests carried out on 20 1-year-old potted C. volkameriana seedlings. Each seedling was inoculated with five mycelial agar plugs (6 mm in diameter) and five sclerotia from the edge of 10-day-old colonies on PDA and placed in wounds made with a sterile blade in the bark of stem and twigs. Inoculated wounds (10 for each plant) were wrapped with Parafilm. The same number of control plants were wounded and inoculated with sterile PDA plugs. All inoculated plants were incubated in a growth chamber at 22°C with 80 to 90% relative humidity for 14 days. Blight symptoms and lesions on the stem and twigs identical to those observed in the nursery developed on all plants with both types of inoculum. Noninoculated control plants remained symptomless. S. sclerotiorum was reisolated from all symptomatic tissues and identified by morphology as previously described, completing Koch's postulates. To our knowledge, this is the first report of S. sclerotiorum stem and twig blight on C. volkameriana. Worldwide, Sclerotinia stem and twig blight is considered a minor disease on citrus (1), but this evidence suggests that in eastern Sicily, S. sclerotiorum may be an important pathogen of young C. volkameriana seedlings in nurseries. References: (1) J. A. Menge. Page 35 in: Compendium of Citrus Diseases. 2nd ed. The American Phytopathological Society, St. Paul, MN, 2000. (2) T. J.White et al. Page 315in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.

Reduction of Corky Root Infections on Greenhouse Tomato Crops by Soil Solarization in South Italy.

Five greenhouse experiments were conducted in southeastern Sicily (Italy) from 2000 to 2009 to evaluate the effectiveness of soil solarization in reducing natural infections of tomato corky root caused by Pyrenochaeta lycopersici. Tests were performed with clear, traditional, and innovative plastic films and fumigant applications. In all the trials, soil solarization was effective in controlling corky root disease relative to an untreated control. Although inducing different thermal regimes in the soil, the use of different greenhouse covering and mulching films for solarization proved effective in reducing corky root severity relative to the untreated control. Solarization reduced infections caused by P. lycopersici comparable with methyl bromide fumigation and greater than metham sodium and metham potassium. Among the tested films, green coextruded film may be most attractive because it can be left on after solarization as mulch.

First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Pink Ipê (Tabebuia impetiginosa) in Italy.

Pink ipê or pink lapacho (Tabebuia impetiginosa Martius ex DC., family Bignoniaceae) is one of the most attractive blooming trees in the world. In Europe, pink ipê is widely used as an ornamental tree in landscaped gardens and public areas. In August 2010, a widespread damping-off was observed in a stock of approximately 100,000 potted 2-month-old seedlings in a nursery in eastern Sicily (Italy). The seedlings were being watered with overhead irrigation. More than 5% of the seedlings showed disease symptoms. Initial symptoms were black lesions at the seedling crown that expanded rapidly to girdle the stem. On infected seedlings, leaves turned black and gradually died. Black extended stem lesions were followed by death of the entire seedling in a few days. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently isolated from crown and stem lesions when plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 µg/ml. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Mycelium was branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells removed from cultures grown at 25°C on 2% water agar were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11 on 2% water agar in petri plates (4). Anastomosis was observed only with tester isolates of AG-4, giving both C2 and C3 reactions (2). Pathogenicity tests were performed on container-grown, healthy, 3-month-old seedlings. Forty seedlings of T. impetiginosa were inoculated near the base of the stem with two 1-cm2 PDA plugs from 5-day-old mycelial cultures. The same number of plants only inoculated with PDA plugs served as controls. Plants were incubated in a growth chamber and maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Crown and stem lesions identical to those observed in the nursery appeared 5 days after inoculation and all plants died within 25 days. No disease was observed on control plants. R. solani AG-4 was reisolated from symptomatic tissues and identified as previously described. R. solani AG-4 was previously detected in the same nursery on Chamaerops humilis (3). To our knowledge, this is the first report of R. solani causing damping-off on T. impetiginosa. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (3) G. Polizzi et al. Plant Dis. 94:125, 2010. (4) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

Calonectria spp. causing leaf spot, crown and root rot of ornamental plants in Tunisia.

Calonectria spp. are important pathogens of ornamental plants in nurseries, especially in the Northern Hemisphere. They are commonly associated with a wide range of disease symptoms of roots, leaves and shoots. During a recent survey in Tunisia, a number of Calonectria spp. were isolated from tissues of ornamental plants showing symptoms of leaf spot, crown and root rot. The aim of this study was to identify these Calonectria spp. using morphological and DNA sequence comparisons. Two previously undescribed Calonectria spp., C. pseudomexicana sp. nov. and C. tunisiana sp. nov., were recognised. Calonectria mexicana and C. polizzii are newly reported for the African continent. Pathogenicity tests with all four Calonectria spp. showed that they are able to cause disease on seedlings of Callistemon spp., Dodonaea viscosa, Metrosideros spp. and Myrtus communis.

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

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.

First Report of Crown and Root Rot Caused by Binucleate Rhizoctonia AG-A on Thryptomene saxicola in Italy.

Thryptomene saxicola (Hook.) Schauer is an evergreen shrub native to Western Australia and a member of the Myrtaceae. In Italy, this species was recently introduced as an ornamental plant from abroad. From July of 2008 to September 2009, a new crown and root rot of T. saxicola was observed on several stocks of approximately 20,000 1- to 3-year-old potted plants. Diseased plants were obtained from a commercial nursery in eastern Sicily, Italy. They were propagated from cuttings and grown under drip irrigation. More than 30% of the plants showed disease symptoms. Infected plants were characterized by a lack of vigor. Roots and crowns were partially or completely destroyed, and as a consequence, infected plants were chlorotic and often wilted. Early in the disease development, roots and crowns showed brown lesions. Successively, mature crown lesions turned dark brown. Longitudinal sections of crown tissues revealed a discoloration of the basal stem. Diseased tissues were surface disinfested for 1 min in 1% NaOCl, rinsed in sterile water, plated on potato dextrose agar (PDA) amended with 100 mg/liter of streptomycin sulfate, and then incubated at 25°C. A binucleate Rhizoctonia (BNR) species was consistently isolated from affected tissues of plants. Phytophthora isolates were not recovered from symptomatic tissues plated on BNPRAH (benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol) selective medium. Fungal colonies were white with floccose, aerial hyphae. Hyphal cells were determined to be binucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with five different tester isolates of BNR AG-A on 2% water agar in petri plates (3). Anastomosis was observed with all tester isolates. The rDNA-ITS of one isolate of BNR (DISTEF-TS1) was sequenced (GenBank Accession No. AB514570) (2). The sequence from this isolate exhibited 99% homology with BNR AG-A (GenBank Accession No. AY738628). Pathogenicity tests were conducted on potted, healthy, 1-year-old plants of T. saxicola. Forty plants were inoculated by placing 1/cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem. The same number of plants was treated with 1/cm2 PDA plugs as controls. Plants were kept at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Root and crown rots, identical to those observed in the nursery, appeared 45 days after inoculation, and 80% of the inoculated plants died within 4 months. Control plants remained healthy. Binucleate Rhizoctonia was reisolated from symptomatic tissues, completing Koch's postulates. To our knowledge, this is the first report in the world of BNR causing disease on T. saxicola. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) M. Hyakumachi et al. Phytopathology 95:784, 2005. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Leaf Spot Caused by Cylindrocladium pauciramosum on Dwarf Willow Myrtle in Italy.

Dwarf willow myrtle (Agonis flexuosa (Willd.) Sweet) cv. Nana, an evergreen ornamental shrub belonging to the Myrtaceae, is grown in Italy as an ornamental potted plant. In November 2008, a widespread new leaf spot disease was noticed on ~80% of 5,000 6-month-old potted plants. Plants were obtained from cuttings and produced by a commercial nursery in Catania Province. Symptomatic leaves showed minute, reddish brown spots that enlarged (3 to 5 mm in diameter) and then darkened, presenting a necrotic center defined by a dark purple halo. Leaf spots were surface disinfested with 0.8% NaOCl and plated on potato dextrose agar. Twenty isolates of the fungus that was consistently isolated from the spots were selected and cultured for 8 days at 25°C on carnation leaf agar (CLA). Macroconidiophores consisted of a stipe, a penicillate arrangement of fertile branches, and stipe extension terminating in an obpyriform to ellipsoidal vesicle (6 to 10 µm in diameter). Cylindrical conidia were rounded at both ends, straight, one-septate, and ranged from 44 to 60 × 4 to 5 µm. The fungus was tentatively identified as Cylindrocladium pauciramosum based on these morphological characteristics (2). All single-conidium isolates were mated with tester strains of Calonectria pauciramosa C. L. Schoch & Crous, telomorph of C. pauciramosum, on CLA and produced fertile perithecia (4). Perithecia were solitary or in groups, orange to red-brown, subglobose to ovoid, and ranged from 280 to 400 µm long × 180 to 290 µm in diameter. Further confirmation of species was obtained by amplification and sequencing of the intergenic spacer (IGS) region of rDNA, using M13 Forward (-20) and M13 Reverse primers. On the basis of the complete IGS sequence, two primer sets (218F/218R and 106F/106R) were designed and successfully used in a nested-PCR protocol for the detection of C. pauciramosum from tissues of infected plants (3). On the basis of the combination of morphological characters, mating type, and molecular data, the isolates were identified as C. pauciramosum C.L. Schoch & Crous. One representative isolate (DISTEF-Af1) was deposited at Centraalbureau voor Schimmelcultures open fungi collection (Fungal Biodiversity Centre, Utrecht, the Netherlands; CBS 124659). Pathogenicity tests were performed by adding sterile water to CLA cultures of C. pauciramosum from a single-conidium isolate (DISTEF-Af1) and spraying the resulting spore suspension (105 conidia per ml) on the leaf surface of 20 6-month-old A. flexuosa cv. Nana potted plants. The same number of plants served as noninoculated controls. Following inoculation, plants were kept in plastic bags in a growth chamber at 25 ± 1°C. All inoculated plants developed circular, brown leaf spots identical to those observed in the nursery 5 to 7 days after inoculation. Control plants remained symptomless. C. pauciramosum was always reisolated from the infected plants and identified as previously described. Leaf spotting in seedlings of A. flexuosa was previously associated with infections by C. scoparium in Australia (1). To our knowledge, this is the first record in the world of leaf spots caused by C. pauciramosum on A. flexuosa. References: (1) A. L. Bertus. Agric. Gaz. N. S. W. 87:22, 1976. (2) P. W. Crous. Taxonomy and Pathology of Cylindrocladium (Calonectria) and Allied Genera. The American Phytopathological Society, St. Paul MN, 2002. (3) F. Nigro et al. J. Plant Pathol. 88:S22, 2006. (4) G. Polizzi and P. W. Crous. Eur. J. Plant Pathol. 105:407, 1999.

First Report of Damping-Off Caused by Rhizoctonia solani AG-4 on Mediterranean Fan Palm in Italy.

Mediterranean fan palm (Chamaerops humilis L.), one of just two autochthonous European palms, is native to the western Mediterranean Region in southwestern Europe and northwestern Africa. It can be found growing wild in the Mediterranean area. In Europe, this species is very popular as an ornamental plant. In March 2009, a widespread damping-off was observed in a stock of approximately 30,000 potted 1-month-old plants of C. humilis cv. Vulcano in a nursery in eastern Sicily. Disease incidence was approximately 20%. Disease symptoms consisted of lesions at the seedling shoot (plumule). Stem lesions were initially orange, turned brown, and followed by death of the entire plumule or eophyll. A fungus with mycelial and morphological characteristics of Rhizoctonia solani Kühn was consistently isolated from lesions when plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 µg/ml. Fungal colonies were initially white, turned brown with age, and produced irregularly shaped, brown sclerotia. Mycelium was branched at right angles with a septum near the branch and a slight constriction at the branch base. Hyphal cells removed from cultures grown at 25°C on 2% water agar were determined to be multinucleate when stained with 1% safranin O and 3% KOH solution (1) and examined at ×400. Anastomosis groups were determined by pairing isolates with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11 on 2% water agar in petri plates (3). Anastomosis was observed only with tester isolates of AG-4, giving both C2 and C3 reactions (2). One representative isolate obtained from symptomatic tissues was deposited at the Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (CBS No. 125095). Pathogenicity tests were performed on container-grown, healthy, 1-month-old seedlings. Twenty plants of C. humilis cv. Vulcano were inoculated near the base of the stem with two 1-cm2 PDA plugs from 5-day-old mycelial cultures. The same number of plants served as uninoculated controls. Plants were incubated in a growth chamber and maintained at 25°C and 95% relative humidity on a 12-h fluorescent light/dark regimen. Symptoms identical to those observed in the nursery appeared 5 days after inoculation and all plants died within 20 days. No disease was observed on control plants. A fungus identical in culture morphology to R. solani AG-4 was consistently reisolated from symptomatic tissues, confirming its pathogenicity. To our knowledge, this is the first report in the world of R. solani causing damping-off on Mediterranean fan palm. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) D. E. Carling. Page 37 in: Grouping in Rhizoctonia solani by Hyphal Anastomosis Reactions. Kluwer Academic Publishers, the Netherlands, 1996. (3) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

First Report of Southern Blight on Silverbush (Convolvulus cneorum) Caused by Sclerotium rolfsii in Italy.

The genus Convolvulus has more than 200 species that are encountered in temperate to tropical climates all around the world. Convolvulus cneorum L., also known as silverbush, is a perennial shrub native to southern Europe (Sicily and Croatia) with dense, silver foliage and masses of large, circular, white flowers. During July of 2009, a widespread blight was observed on approximately 10% of 12,000 4-month-old potted silverbush plants. The plants were obtained from cuttings and produced by a commercial nursery in eastern Sicily, Italy. Symptomatic plants initially had sunken, tan lesions at the ground level that developed into typical southern blight. Circular and crescent-shaped patches were observed on the masses of weeds on the surface of the containers where silverbush were grown. At the soil line, white mycelia and small (1 to 2 mm in diameter), brown, spherical sclerotia with internally differentiated rind, cortex, and medulla characteristic of Sclerotium rolfsii Sacc. were observed. Crown and stem lesions were surface disinfested (1% NaOCl) for 1 min, rinsed in sterile water, and placed on potato dextrose agar. Isolation consistently yielded colonies of Sclerotium rolfsii (teleomorph Athelia rolfsii (Curzi) Tu & Kimbrough) with typical sclerotia produced within 6 to 7 days (2). Pathogenicity tests were performed on 20 plants by placing 10 sclerotia obtained from 10-days-old cultures in the soil below the crown portion on each of 5-month-old healthy cuttings of silverbush. The same number of plants served as noninoculated controls. All plants were maintained in a growth chamber at 25 ± 1°C and enclosed for 7 days in polyethylene bags. Plants were then moved to a greenhouse where temperatures ranged from 24 to 28°C. Symptoms of southern blight developed after 7 to 20 days on all inoculated plants. Control plants remained symptomless. S. rolfsii was reisolated from symptomatic plants. S. rolfsii was reported for the first time in Sicily in 2004 in an ornamental nursery (1). To our knowledge, this is the first report in the world of S. rolfsii on silverbush and it is the first outbreak of southern blight on Convolvulus species. The high susceptibility of silverbush to the pathogen could be a limiting factor for the cultivation in nursery of this indigenous wildflower plant. References: (1) G. Polizzi et al. Plant Dis. 88:310, 2004. (2) Z. K. Punja and A. Damiani. Mycologia 88:694, 1996.

First Report of Crown Rot and Stem Rot Caused by Rhizoctonia solani AG-4 on Marmalade Bush in Italy.

Marmalade bush (Streptosolen jamesonii (Benth.) Miers), also known as fire bush, is an evergreen, perennial shrub in the family Solanaceae, which is native to South America (Colombia, Ecuador, and Peru). In Italy, this species is cultivated as an ornamental creeper or bush. During September 2009, a new disease was observed in a stock of ~10,000 pot-grown, 2-month-old plants of marmalade bush in a nursery in eastern Sicily, Italy. More than 50% of the plants exhibited symptoms of disease. Disease symptoms consisted of extensive water-soaked, dark brown lesions at the crown level that girdled entire stems and an internal brown discoloration of cortical tissue. Infected plants died within a few days. Diseased tissue was disinfested for 10 s in 1% NaOCl, rinsed with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate at 100 mg/liter. Fungal colonies were initially white, turned brown after 2 to 3 days, and produced irregularly shaped, brown sclerotia. Microscopic examination showed mycelium consistent with Rhizoctonia solani Kühn that branched at right angles, constricted at the base of the branch originating from primary hyphae, and septate near the constriction. The number of nuclei per hyphal cell was determined on cultures grown at 25°C on 2% water agar in petri plates by staining with 1% safranin O and 3% KOH solution (1) and examined at ×400. The hyphal cells were all multinucleate. Anastomosis group was determined by pairing isolates on 2% water agar in petri plates (2). Pairings were made with tester strains AG-1 IA, AG-2-2-1, AG-2-2IIIB, AG-2-2IV, AG-3, AG-4, AG-5, AG-6, and AG-11. Anastomosis was observed only with tester isolates of AG-4. Pathogenicity tests were performed by placing 1-cm2 plugs of PDA from 5-day-old mycelial cultures near the base of the stem on 25 potted, healthy, 2-month-old rooted cuttings of marmalade bush. The same number of plants treated with 1-cm2 PDA plugs served as controls. Following inoculation, all plants were maintained for 20 days at 25°C and 95% relative humidity under a 12-h fluorescent light/dark regimen. Crown and stem symptoms, identical to those observed in the nursery, developed 5 days after inoculation on all inoculated plants. Control plants remained symptomless. R. solani was consistently reisolated from symptomatic tissues and identified as previously described. To our knowledge, this is the first report of R. solani causing disease on marmalade bush. References: (1) R. J. Bandoni. Mycologia 71:873, 1979. (2) C. C. Tu and J. W. Kimbrough. Mycologia 65:941, 1973.

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

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.