Single Conidial Isolates Research Articles

First Report of Damping-Off Caused by Cylindrocarpon pauciseptatum on Pinus radiata in Spain.

In the fall of 2009, damping-off of Pinus radiata seedlings was observed in a pine nursery in Sant Feliu de Buixalleu, Girona Province, northeastern Spain. Plants exhibited needle blight, extensive root necrosis, and root death. Root sections of symptomatic plants were cut, washed under running tap water, surface disinfected for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. Small fragments of discolored tissues were plated onto potato dextrose agar (PDA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Plates were placed at 25°C in the dark for 10 to 14 days, and all fungal colonies were transferred to PDA. A Cylindrocarpon sp. was consistently isolated from necrotic root tissues. Single-conidial isolates were obtained and grown on PDA and Spezieller Nährstoffarmer agar (SNA) (2) at 25°C for 10 days with a 12-h photoperiod. On PDA, the isolates developed abundant mycelium, which varied from white-to-grayish brown or golden brown. On SNA, all isolates produced two-septate, (35-) 39.4 (-40) × (7.5-) 7.7 (-8.75) μm, and three-septate, (32.5-) 40.9 (-52.5) × (7.5-) 7.7 (-8.75) μm, macroconidia. Microconidia, one-septate macroconidia, and chlamydospores were not observed. Identity of these isolates was determined by a multiplex PCR system using a set of three pair of specific primers (Mac1/MaPa2, Lir1/Lir2, and Pau1/MaPa2) (1), which generated a 117-bp product that was characteristic of Cylindrocarpon pauciseptatum Schroers & Crous. Morphological characteristics also supported this identification (4). Internal transcribed spacers regions (ITS1 and ITS4) of rDNA were obtained for isolate 1052 and deposited in GenBank (Accession No. HQ441248). This sequence was identical (100%) with the sequence of C. pauciseptatum (GenBank Accession No. HM036590). Pathogenicity tests were conducted with inoculum produced on wheat kernels that were soaked in distilled water in flasks for 12 h. Each flask contained 200 ml of kernels that were subsequently autoclaved three times after excess water was drained. Two fungal disks from a 2-week-old culture of C. pauciseptatum (isolate 1052) grown on PDA were placed aseptically in each flask. Cultures in flasks were incubated at 25°C for 4 weeks and shaken once a week. A plastic pot (220 cm3) was filled with a mixture of sterilized peat moss and 10 g of inoculum. A 1-month-old seedling of P. radiata was planted in plastic pots and placed in a greenhouse at 25 to 30°C in a completely randomized design with six replications. Controls contained sterile wheat kernels. The experiment was repeated. Symptoms developed 20 days after inoculation and consisted of root lesions, a reduction in root biomass, needle blight, and the death of all seedlings. The fungus was reisolated from affected seedlings. Damping-off was not observed on the control plants. C. pauciseptatum causing black foot disease of grapevine (3) was first found in Spain in 2008, but to our knowledge, this is the first report of C. pauciseptatum causing damping-off of P. radiata in Spain.

Uneven distribution of mating type alleles in Iranian populations of Cercospora beticola , the causal agent of Cercospora leaf spot disease of sugar beet

Normal 0 14 false false false IT ZH-TW X-NONE MicrosoftInternetExplorer4 Cercospora beticola , the causal agent of Cercospora leaf spot disease on sugar beet, is thought to be exclusively asexual because no teleomorph has yet been found. The possibility of a clandestine sexual cycle in the Iranian population of Cercospora beticola was evaluated by analyzing the distribution and frequency of the mating type alleles on a microspatial and a macrogeographical scale. A total of 89 single-conidial Cercospora beticola isolates were obtained from sugar beet fields in the Moghan, the Talesh and the Khoy regions. The isolates were identifed using a Cercospora beticola -specifc primer set in a PCR assay. A multiplex PCR method using previously designed mating type primers was used to study the distribution and the frequency of the mating type alleles. All isolates showed either the 805-bp fragment or the 442-bp fragment of the MAT1-1 and MAT1-2 genes, but no isolate had both fragments. The distribution of the mating type genes in the sampled areas was uneven. From three sugar beet fields sampled in the Moghan region, two fields had only MAT1-1 isolates; while in the third field all isolates had only the MAT1-2 allele. In the Talesh region only MAT1-1 isolates occurred, and in the Khoy region the mating type alleles were uniformly distributed amongst the isolates. The skewed distribution of mating type alleles in Northwestern Iran was in line with the lack of a sexual cycle for this species and may also indicate that sugar fields in the Moghan region were infected by C. beticola populations of different origins. Normal 0 false false false EN-US X-NONE FA

First Report of Phaeoacremonium krajdenii Causing Petri Disease of Grapevine in Spain.

In September 2009, symptoms of grapevine (Vitis vinifera L.) decline were observed on 3-year-old grapevines in a vineyard in Roquetas de Mar (Almeria Province, southern Spain). Affected vines were weak with reduced foliage and chlorotic leaves. Black spots and dark streaking of the xylem vessels could be seen in cross- or longitudinal sections of the rootstock trunk. Symptomatic plants were collected and sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface disinfested for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were plated onto malt extract agar (MEA) supplemented with 0.5 g liter-1 of streptomycin sulfate. Dishes were incubated at 25 to 26°C in the dark for 14 to 21 days, and all colonies were transferred to potato dextrose agar (PDA). A Phaeoacremonium sp. was consistently isolated from necrotic tissues. Single conidial isolates were obtained and grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (2). Colonies were grayish brown on PDA and dark brown on MEA. Conidiophores were short and unbranched and 11.5 to 46 (25.5) μm long. Phialides were often polyphialidic. Conidia were hyaline, oblong-ellipsoidal or allantoid, 2.5 to 5 (4.2) μm long, and 1 to 1.7 (1.2) μm wide. On the basis of these characters, the isolates were identified as Phaeoacremonium krajdenii L. Mostert, Summerb. & Crous (1,2). DNA sequencing of a fragment of the beta-tubulin gene of the isolate (Pkr-1) using primers T1 and Bt2b (GenBank Accession No. HM637892) matched P. krajdenii GenBank Accession No. AY579330. Pathogenicity tests were conducted using isolate Pkr-1. Ten 1-year-old callused and rooted cuttings of 110 R rootstock grown in pots with sterile peat were wounded at the uppermost internode with an 8-mm cork borer. A 5-mm mycelium PDA plug from a 2-week-old culture was placed in the wound before being wrapped with Parafilm. Ten control plants were inoculated with 5-mm noncolonized PDA plugs. Plants were maintained in a greenhouse at 25 to 30°C. Within 3 months, shoots on all Phaeoacremonium-inoculated cuttings had weak growth with small leaves and short internodes and there were black streaks in the xylem vessels. The vascular necroses that developed on the inoculated plants were 5.5 ± 1.2 cm long, significantly greater than those on the control plants (P < 0.01). Control plants did not show any symptoms. The fungus was reisolated from discolored tissue of all inoculated cuttings, completing Koch's postulates. P. krajdenii has a worldwide distribution, although these reports are from human infections (1). P. krajdenii was first reported as a pathogen of grapevines in South Africa (1). To our knowledge, this is the first report of P. krajdenii causing young grapevine decline in Spain or any country in Europe.

OCCURRENCE, MOLECULAR CHARACTERISATION, AND PATHOGENICITY OF NEOSCYTALIDIUM DIMIDIATUM ON CITRUS IN ITALY

During 2008 and 2009, a new disease was noticed in eastern Sicily (Italy) in two re-grafted citrus orchards (Citrus sinensis (L.) Osbeck) on sour orange rootstock. Symptoms consisted of blight of vigorously growing shoots and a sooty canker on shoots and rootstock trunks. A Scytalidium-like fungus was isolated consistently from symptomatic tissues on 2% potato-dextrose agar (PDA). The mycelium was composed of branched, septate, brown hyphae which disarticulated into 0-1-septate phragmospores. In culture it also produced a pycnidial synanamorph, with hyaline conidiogenous cells that were intermingled with paraphyses. Conidia were ellipsoid to ovoid, hyaline, with an acutely rounded apex, truncate base, initially aseptate, becoming brown and 2-septate at maturity, with the central cell darker than the end cells. Genomic DNA was extracted from mycelia of single-conidial isolates cultivated on 2% malt extract agar and the nuclear rRNA operon spanning the 3’ end of 18S rRNA gene, the internal transcribed spacers, the 5.8S rRNA gene, and a part of the 5’ end of the 28S rRNA gene was amplified and sequenced. On the basis of morphological characters and molecular data, the fungal isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers. Koch’s postulates were satisfied and confirmed the pathogenicity of the fungus. N. dimidiatum appears to pose a serious threat to re-grafted citrus orchards.

First Report of Cercospora beticola Causing a Leaf Spot Disease on Acanthus mollis in California.

The genus Acanthus (Acanthaceae) includes ~30 herbaceous, perennial species grown for their attractive foliage and flower spikes. Between June and December 2009 the CDFA Plant Pest Diagnostics Lab in Sacramento, CA received multiple leaf spot disease samples on Acanthus spinosus and A. mollis, commonly known as bear's breeches. Samples were collected four times from two nurseries in Santa Barbara County. Disease was observed in nearly 100% of the plants inspected. Leaf spots were brown, roundish to elliptical, and 1 to 4 mm in diameter. Older spots often developed grayish centers and often coalesced, leading to large necrotic areas. Conidiophores were fasciculate, amphigenous, light brown to olivaceous, multiseptate, geniculate, and had distinctive spore scars. Conidia were hyaline, straight to slightly curved with tapered tips and truncate bases. Conidia were solitary, multiseptate (1 to 10) and 48 to 160 × 2.5 to 5 μm (average 100 × 3.9 μm). Colonies obtained from single conidial isolates were established on acidified potato dextrose agar (APDA). Morphologically, the causal agent was identified as Cercospora diantherae Ellis and Kellerm (1), a species synonymous with C. apii sensu lato (2). The C. apii sensu lato complex includes three morphologically similar taxa, C. apii, C. beticola, and C. apiicola (3). Sequence analysis of the internal transcribed spacer region from the Acanthus isolate confirmed it belongs to the C. apii complex (GenBank HQ328503). Multiplex PCR to distinguish species within the complex was also performed on the isolate (3). A 176-bp fragment was only observed in the PCR reaction containing the C. beticola primers. To confirm pathogenicity, hyphal suspensions were used to inoculate healthy leaves of A. mollis plants potted in 3.7-liter containers. Hyphal suspensions were obtained by grinding 3-week-old colonies grown on APDA with distilled water using a mortar and pestle. Both sides of healthy leaves and petioles were sprayed with ~40 ml of the suspension. Five plants were inoculated with C. beticola and five plants were sprayed with sterile water. Plants were incubated in a dew chamber for 48 h and then transferred to a 25°C growth chamber with a 12-h photoperiod. The experiment was repeated. Five days after inoculation, small necrotic leaf spots developed on the leaves. After 14 days, the spots had enlarged and the leaves began to turn yellow. Over time, the spots coalesced leading to large necrotic areas, especially along the leaf margins. Petiole spots, not seen on field samples, were seen on laboratory inoculated plants. Sporulation of C. beticola occurred within most of the spots and the pathogen was successfully reisolated from all inoculated leaves. No foliar symptoms developed on any of the control plants. Worldwide, C. beticola is a destructive pathogen of sugar beet (4), and has also been reported on a number of other plant hosts (3). To our knowledge, this is the first report of C. beticola causing a leaf spot disease on a host in the Acanthaceae family. This strain has been deposited into the culture collection at Centraalbureau voor Schimmelcultures.

First Report of Leaf Spot Disease of Peony Caused by Seimatosporium botan in China.

Tree peony (Paeonia suffruticosa Andrews), a perennial ligneous deciduous shrub in the Paeoniaceae family, is known for its beautiful and charming flowers. It is regarded as the flower symbol of China and is cultivated throughout the country. In August 2008, a previously unknown leaf spot was observed on peony cultivated in the Mountain Peony Garden located in the Luoyang area of Henan Province, China. In 2009, the leaf spot disease was observed in some gardens in the city of Luoyang, China. Initial symptoms appeared as small, round or irregular, brown, necrotic lesions in the middle of leaves. These lesions gradually enlarged up to 1 cm in diameter and were circular or irregular, brown to dark brown, and brown on the margins. In a humid atmosphere, black, sessile, discoid acervuli developed on the lesions, and the lesions sometimes became waxy-like, eventually coalesced, and nearly covered the entire leaf. Conidia produced in acervuli had two morphologically different types. One type had a single basal appendage, ellipsoid to fusiform, transversely three septate, 16 to 20 × 5 to 7 μm, smooth, basal cell obconic with a truncate base, subhyaline, 3 to 5 μm long; two central cells subcylindrical to dolioform, brown to dark brown, 8 to 10 μm long, apical cell conical with rounded apex, concolorous with the central cells, 4 to 5 μm long, basal appendage filiform, unbranched, excentric, 4 to 8 μm long. The other type had a single appendage at both ends, fusiform to subcylindrical, transversely three septate, 16 to 20 × 4 to 5 μm, smooth; basal cell obconic with a truncate base, subhyaline, 4 to 5 μm long; two central cells subcylindrical to dolioform, pale brown, 8 to 11 μm long; apical cell conical with an acute apex, hyaline to subhyaline, 4 to 5 μm long; basal appendage filiform, unbranched, excentric, 4 to 8 μm long; apical appendage filiform, unbranched, 4 to 8 μm long. Single conidial isolates of both types of conidia yielded identical colonies, which produced both types of conidia on potato dextrose agar (PDA), thus showing that both types of conidia belonged to the same fungus. Colonies on PDA were slimy in appearance, yellow to villous with an irregular taupe margin; reverse brown to grayish brown. Cultural and conidial characteristics of the isolates were similar to those of Seimatosporium botan (1). The DNA sequence for the fungus showed internal transcribed spacer region (ITS1-5.8S-ITS2) sequences (GenBank Accession No. HM067840) with 93% sequence identity to S. discosioides (Accession Nos. EF600970.1 and EF600969.1). This is the first submission of a S. botan sequence to GenBank. To determine pathogenicity, 20 healthy leaves of P. suffruticosa were inoculated by spraying a conidial suspension of S. botan onto the foliage. Ten leaves were sprayed with sterile water and served as controls. Plants were covered with plastic for 24 h to maintain high relative humidity. After 15 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Koch's postulates. On the basis of morphology and ITS region sequences, we conclude that S. botan is the causal agent of leaf spots of P. suffruticosa. There is a report of S. botan on P. suffruticosa stems in Japan (1), but to our knowledge, this is the first report of leaf spot disease of peony caused by S. botan in China.

First Report of Gummosis Disease of Japanese Apricot Caused by Botryosphaeria dothidea in Taiwan.

Japanese apricot (Prunus mume Sieb. et Zucc.) is an economically important fruit crop grown on more than 10,000 ha in Taiwan. During May 2008, twigs of Japanese apricot trees in the commercial farms of Renai Region (Nantou County) showed symptoms of gummosis disease, with 12 to 18% of the trees affected. The disease was more severe on trees weakened by drought stress. Limb and twig infections began around lenticles as small, sunken, discolored lesions at the margins of wounds. Following infection, cortical cells collapsed, bark became depressed, and blisters developed, which were often cracked with whitish gummy exudation. Necrotic areas were seen on the cortical tissues. Leaves showed yellowing and drooping. In winter months, numerous black pycnidia or perithecia formed on infected twigs. Single conidial isolates of the pathogen were obtained from diseased twigs on acidified potato dextrose agar (PDA) incubated at 25 ± 1°C for 3 days. On the basis of morphological characteristics, the fungus was identified as Botryosphaeria dothidea (3). Conidia (17 to 22.6 × 4.3 to 6.0 μm) were hyaline, unicellular, and spindle shaped. Asci (78 to 125 × 15 to 17 μm) were hyaline, bitunicate, clavate, and eight spored. Ascospores (18 to 22 × 7.0 to 8.2 μm) were hyaline and spindle shaped or fusoid. The pathogen identity was further confirmed by PCR amplification and sequencing of ribosomal DNA internal transcribed spacer from the fungus with the primers ITS5: 5'-GGAAGTAAAAGTCGTAACAAGG-3' and ITS4: 5'-TCCTCCGCTTATTGATATGC-3' (4), and a representative sequence was deposited in NCBI GenBank (Accession No. GU594225). The sequence showed 99 to 100% homology with previously characterized strains of B. dothidea (GenBank Accession Nos. EU441944, DQ177876, and AY786320). Pathogenicity tests were conducted with inoculum prepared by culturing the fungus on PDA under a continuous photoperiod of 128 ± 25 μE·m-2·s-1 at 25°C for 3 days. Shallow cuts (3 × 3 × 3 mm) were made on 12- to 15-month-old healthy twigs with a scalpel and inoculated with either a 5-mm mycelial disc or 0.5 ml of conidial suspension (105 conidia/ml) of the fungus. Two twigs on each of six trees were inoculated. Inoculated areas were covered with moist, sterile cotton and the entire twigs were enclosed in plastic bags. Twigs were inoculated with 5-mm PDA discs or sterile water for controls. The symptoms described above were observed on all inoculated twigs 14 days after inoculation, whereas control twigs remained healthy. Reisolation from the inoculated twigs consistently yielded B. dothidea. In Taiwan, B. dothidea has been reported as the causal agent of gummosis of peach (1) and fruit ring rot of pear (2); however, to our knowledge, this is the first report of B. dothidea causing gummosis on Japanese apricot.

Anthracnose of salt-wort (Salsola komarovii) caused by Colletotrichum truncatum

In July 2006, black rot was observed on the leaves of 4-leaf-stage seedlings of salt-wort (Salsola komarovii) in Yamagata Prefecture, Japan. We isolated two single-conidial isolates from the diseased leaves. Although colony appearance of the isolates was different from that of each other, both isolates were identified as Colletotrichum truncatum by morphology and molecular similarity. After inoculation of healthy salt-wort plants with the isolates, the isolates were reisolated from symptomatic plants. We thus propose a new disease, anthracnose of salt-wort.

Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus

Oxalacetate acetylhydrolase (OAH), a member of the phosphoenolpyruvate mutase/isocitrate lyase superfamily, catalyzes the hydrolysis of oxalacetate to oxalic acid and acetate. This study shows that knock-out of the oah gene in Cryphonectria parasitica, the chestnut blight fungus, reduces the ability of the fungus to form cankers on chestnut trees, suggesting that OAH plays a key role in virulence. OAH was produced in Escherichia coli and purified, and its catalytic rates were determined. Oxalacetate is the main OAH substrate, but the enzyme also acts as a lyase of (2R,3S)-dimethyl malate with approximately 1000-fold lower efficacy. The crystal structure of OAH was determined alone, in complex with a mechanism-based inhibitor, 3,3-difluorooxalacetate (DFOA), and in complex with the reaction product, oxalate, to a resolution limit of 1.30, 1.55, and 1.65 A, respectively. OAH assembles into a dimer of dimers with each subunit exhibiting an (alpha/beta)(8) barrel fold and each pair swapping the 8th alpha-helix. An active site "gating loop" exhibits conformational disorder in the ligand-free structure. To obtain the structures of the OAH.ligand complexes, the ligand-free OAH crystals were soaked briefly with DFOA or oxalacetate. DFOA binding leads to ordering of the gating loop in a conformation that sequesters the ligand from the solvent. DFOA binds in a gem-diol form analogous to the oxalacetate intermediate/transition state. Oxalate binds in a planar conformation, but the gating loop is largely disordered. Comparison between the OAH structure and that of the closely related enzyme, 2,3-dimethylmalate lyase, suggests potential determinants of substrate preference.

Leaf Spot of Nymphoides peltata Caused by Septoria villarsiae in Korea

Nymphoides peltata (syn. Limnanthemum nymphoides, Villarsia nymphaeoides) is an aquatic flowering plant in the family Menyanthaceae, and commonly called fringed water-lily or water fringe. The plants are widely cultivated for ornamental purposes in ponds and water gardens in Korea. In June 2008, plants showing typical symptoms of leaf spots with yellowing were first found in a pond in Osan, Korea. Simple microscopic examination suggests that the spots are associated with infections by a species of Septoria (Shin and Sameva, 2004). Further surveys of water gardens in Korea were made in 2008 and 2009, and we confirmed the occurrence of the disease in Daejeon, Incheon, Pocheon, Pyeongchang and Suwon. The leaf spots frequently occurred, detracting from the beauty of glossy leaves of the plant (Fig. 1A). Under certain circumstances, leaves were heavily infected even before they unfold at the water surface, devastating whole host populations. Initial symptoms were circular, pale greenish to brownish, later expanding to form larger lesions without definite margin. Numerous black conidiomata were formed on the surface of the lesion (Fig. 1B). Under humid condition and also by placing the infected leaves in humid chamber, white to cream-colored conidial horns were produced from conidiomata (Fig. 1C). Microscopic examination of the lesions from fresh materials was performed under a microscope. Conidiomata were pycnidial, epiphyllous, scattered to confluent, sometimes 2-3(-5) pycnidia touched with their walls, dark brown to rusty brown, globose, immersed in host tissue or somewhat erumpent, 40-90 μm in diameter, with an ostiole of 15-30 μm in diameter. Conidia were substraight, guttulate, hyaline, 25-55×2-2.5 μm, (0-)1-5-septate (Fig. 1D). Single conidial isolates formed dark greyish colonies on potato dextrose agar (Fig. 1E). Based on the morphological characteristics, the causal fungus was identified as Septoria villarsiae Desm. (Radulescu et al., 1973; Paul and Singh, 2003). Voucher specimens are housed at Korea University (KUS-F23403, F24594) and isolates were deposited in the Korean Agricultural Culture Collection of the National Academy of Agricultural Science (KACC43773, KACC44794). Conidiomata matured after 5 weeks when plates were incubated under fluorescent illumination for 12 hrs photoperiods at 25oC, producing abundant conidia. Pathogenicity was confirmed by inoculating the leaves of three plants with a conidial suspension (ca. 2×105 conidia/ml). Three non-inoculated plants served as controls. The plants were covered with plastic bags for maintaining 100% relative humidity for 48 hrs. After 8 days, typical leaf blotch symptoms, identical to the ones observed in the field, started to develop on the leaves of inoculated plants. No symptoms were observed on control plants. Septoria villarsiae was reisolated from the lesions of inoculated plants, fulfilling Koch’s postulates. The leaf spot diseases associated with S. villarsiae have previously been recorded on N. peltata in Romania (Radulescu et al., 1973), the Netherlands (Van der Velde et al., 1982), and India (Paul and Singh, 2003). To our knowledge, this is the first report of this fungus as well as its host plant in East Asia.

First Report of Colletotrichum boninense, C. capsici, and a Glomerella sp. as Causes of Postharvest Anthracnose of Passion Fruit in Florida.

Anthracnose is an important foliar and fruit disease of passion fruit, Passiflora spp. (3). In 2008, postharvest anthracnose on purple and yellow passion fruits (P. edulis Sims and P. edulis f. flavicarpa O. Degner, respectively) from a commercial planting in Miami-Dade County, FL was examined. Lesions began as light brown areas that became papery, covered much of the fruit surface, and developed pink-to-dark sporulation. Single-conidium isolates from lesions were examined morphologically and with internal transcribed spacer (ITS) sequences. Four taxa were identified: Colletotrichum boninense (GenBank No. GU045516) with felted cream-to-orange colonies and cylindrical conidia; C. capsici (synonym C. truncatum [2]) (GU045515) with sparse, white mycelia, setose acervuli, and falcate conidia; C. gloeosporioides with fluffy white-to-gray colonies and straight, cylindrical conidia; and a Glomerella sp. (GU045517) with darkly pigmented perithecia. In two experiments, four mature, yellow passion fruit were wounded at a single equatorial site with a sterile needle and inoculated with a 15-μl drop of 0.3% water agar that did not contain (noninoculated control) or contained 105 conidia per ml of representative isolates from each taxon. After 21 days at 25°C without light, anthracnose incidence was recorded and the presence of the isolates was confirmed by their recovery from lesion margins on potato dextrose agar. Anthracnose did not develop on noninoculated control fruit. Mean incidences of anthracnose exceeded 50% for isolates of C. boninense (three from passion fruit), C. capsici (two from passion fruit), and a Glomerella sp. (two from passion fruit and one each from papaya and eugenia). Despite its common indictment as a causal agent of anthracnose on passion fruit (3), symptoms developed on only one fruit that was inoculated with an isolate of C. gloeosporioides from passion fruit (13%) and did not develop after inoculation with an isolate from papaya. Work is needed to determine whether host-specific populations of C. gloeosporioides exist on passion fruit that were not assessed during this study or whether the pathogen was misidentified in previous reports on this host. C. boninense was associated previously with postharvest anthracnose of passion fruit in Japan and Colombia, whereas C. capsici was associated with leaf anthracnose of passion fruit in Florida and Japan (4); both species are reported here for the first time as causes of postharvest anthracnose of passion fruit in Florida. Glomerella sp. caused darkly pigmented lesions and produced the teleomorph on symptomatic passion fruit and in single-ascospore cultures. Isolates with ITS sequences that are 99% homologous to those from passion fruit have been recovered in South Florida from eugenia, papaya, and Piper betle (4) and from other locations on several other hosts (GenBank); they are often nonpathogenic endophytes. Almeida and Coêlho (1) reported in Brazil a Glomerella sp. that formed the teleomorph in culture and caused anthracnose on passion fruit, but did not provide ITS sequences. Additional work is warranted on the identity and ecology of these fungi.

First Report of Fruit Rot of Loquat Caused by an Alternaria sp. in Taiwan.

During March 2007, a fruit rot disease was observed in several loquat (Eriobotrya japonica (Thunberg) Lindley) fields located in Taichung, Nantou, and Miaoli counties. Loquat is a valuable fruit crop grown predominantly in central Taiwan, and hence, even a minor yield loss by this new disease is economically significant. Symptoms on fruits initially appeared as small lesions (<1 mm) that later developed into light-to-dark brown, circular, larger (7 mm), sunken lesions, indicating invasion of a pathogen into the fruit. Pieces of rotted fruit tissue (1 × 1 × 1 mm) were immersed for 1 min in 3% commercial bleach, followed by 70% ethanol, cultured on potato dextrose agar (PDA), and incubated under constant fluorescent light (185 ± 35 μE·m-2·s-1) at 24°C for 2 days. Three single conidial isolates (AS1 to AS3) were selected and used in morphological and pathogenicity studies. All three isolates were identified as an Alternaria sp. (1-3) and formed abundant, dark brown mycelium when cultured on PDA with light at 24°C. Conidiophores were 60 to 89 × 3 to 5 μm, densely fasciculate, cylindrical, simple or branched, and had distinct conidial scars. Conidia were 12 to 74 × 6 to 14 μm, golden brown, straight or curved, obclavate with beaks measuring half the length of the conidium, and observed in chains of 10 or more spores with four to seven transverse septa and several longitudinal septa. Pathogenicity tests were conducted twice by inoculating eight surface-sterilized wounded or unwounded fruits with each of the three isolates in each experiment. Two cuts (1 × 1 × 1 mm) were made on each fruit 3 cm apart with a sterile scalpel, and a 300-μl spore suspension (2 × 105 conidia per ml) was placed on each wound. Similarly, a 300-μl spore suspension was placed on unwounded fruits and air dried for 5 min. Control fruits were similarly treated with sterile water. Inoculated fruits were enclosed in a plastic bag and kept at 24 ± 1°C. Symptoms of soft rot were observed on 60% (unwounded) and 100% (wounded) of inoculated fruits 5 days after inoculation, while control fruits did not develop disease symptoms. Reisolation from the symptomatic fruits consistently yielded an Alternaria sp. This fungus previously has been reported as the causal agent of fruit rot or black spot of papaya, mango, kiwifruit, pear, and carambola from Australia, India, Malaysia, South Africa, and the United States (1-3). To our knowledge, this is the first report of fruit rot of loquat caused by an Alternaria sp. in Taiwan. To manage this disease, growers may resort to fungicidal sprays followed by bagging of fruits to reduce pre- and postharvest losses.

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.

Austropleospora osteospermi gen. et sp. nov. and its host specificity and distribution on Chrysanthemoides monilifera ssp. rotundata in Australia

Hendersonia osteospermi was found for the first time in Australia on leaf spots of the introduced invasive plant Chrysanthemoides monilifera ssp. rotundata (bitou bush) in coastal regions of New South Wales. Pathogenicity tests on species from 11 tribes in the family Asteraceae, demonstrated that H. osteospermi caused severe necrosis on leaves and stems of C. monilifera ssp. rotundata and its congener C. monilifera ssp. monilifera (boneseed). Small necrotic spots also developed on Osteospermum fruticosum and Dimorphotheca cuneata in the Calenduleae and on Helianthus annuus (sunflower) in the Heliantheae. None of the other plant species tested developed leaf spots, although H. osteospermi was re-isolated from senescent leaves of Cynara scolymus (globe artichoke) in the Cynareae and Vernonia cinerea in the Vernonieae. Single ascospores from ascomata of a Pleospora-like fungus found on diseased stems of bitou bush produced H. osteospermi in culture, which proved the anamorph/teleomorph connection. The ITS region of both a single-ascospore isolate and a single-conidium isolate were sequenced and found to be identical. The taxonomic status of H. osteospermi is re-examined and Austropleospora osteospermi gen. et sp. nov. is described as its teleomorph based on morphology, host range tests and DNA sequence analysis. The potential of A. osteospermi for the biological control of bitou bush and boneseed in Australia is discussed.

The development of mating type-specific primers forMycosphaerella fijiensis, the causal agent of black Sigatoka of banana, and analysis of the frequency of idiomorph types in Mexican populations

A multiplex-PCR method was developed to identify the distribution and frequency of the mating types of the fungal banana leaf pathogen Mycosphaerella fijiensis in Mexico. PCR amplification identified different sized fragments for mat1-1 and mat1-2 genes. The same primers were also used to amplify fragments of the idiomorphs of Mycosphaerella musicola, another important banana pathogen closely-related to M. fijiensis. These were sequenced and were highly similar to M. fijiensis. Single conidial isolates of M. fijiensis were obtained from different fields in regions of Mexico not previously sampled, and from different banana and plantain cultivars, to determine the frequency and distribution of the mating types within natural populations. The multiplex primers and methods used for population analysis in M. fijiensis could also be applied to M. musicola populations andmay also be applicable to determine mating types in other closely related species, for a better understanding of the Mycosphaerella complex of pathogens and saprophytes associated with banana leaves. Additional keywords: epidemiology, fungal hybridisation, genetic mapping, virulence.

First Report of Blast of Guinea Grass Caused by Pyricularia sp. LS-Group in Japan.

Guinea grass (Panicum maximum Jacq.) is an important C-4 perennial herbage in the southern part of Japan. In February 2002, a blast disease was found on the grass cultivated on the Okinawa Islands, the southern most region of Japan. Early symptoms appeared as small, round or ellipsoid lesions on leaves. Lesions later expanded to 2 to 5 × 1 to 2 mm and were spindle shaped and grayish white in the central area with dark brown margins. We obtained three single-conidia isolates of a Pyricularia-like fungus from the lesions and deposited them in the NIAS Genebank, Japan as MAFF306662, 306671, and 306672. The isolates were grown under near-UV light on V8 juice agar for 7 days to produce conidia, and guinea grass plants of the seven- to eight-leaf stage grown from seeds in a green house, five plants for each isolate, were inoculated by atomizing them with the conidial suspension of 105 conidia/ml. The same number of plants sprayed with sterilized distilled water served as the control. The experiments were repeated twice. All plants were covered with plastic bags for 24 h at 25°C to maintain high relative humidity. After 7 days, all inoculated plants showed symptoms identical to those observed in the field. Controls remained symptom free. The Pyricularia-like fungus was reisolated from lesions on inoculated leaves. The morphologies of the isolates were observed and described from the colonies grown under the condition described above. Conidiophores were pale brown, emerging singly or in small groups, straight or flexuous, geniculate toward the apex, and 36 to 197 × 2 to 5 μm. Conidia were obpyriform, straight, colorless to pale brown, smooth, and 19 to 30 × 5 to 10 μm with two to three septa. The morphologies were the same as those of the description of the genus Pyricularia. Previously, all Pyricularia isolates from Gramineae had been identified as P. grisea, except for those from rice (3,4). However, a new taxonomy of Pyricularia spp. based on DNA analyses was proposed by Couch and Kohn (1). Only the isolates from Digitaria were classified as P. grisea and those from C-3 grasses classified as P. oryzae. However, the species names for the isolates from the other C-4 grasses were not described. We analyzed the sequences of the rDNA-ITS region (ITS1-5.8s-ITS2) and β-tubulin gene of the isolates from guinea grass following Couch and Kohn (1). The sequences of rDNA-ITS (GenBank Accession No. AB512785) and β-tubulin (AB512786) of the isolate MAFF306672 matched the sequences of those of the Pyricularia sp. LS-group (AB274426 and AB274458, respectively) isolated from Leersia oryzoides. Hirata et al. (2) reclassified Pyricularia isolates from Gramineae by multilocus phylogenetic analysis and showed that non-P. oryzae and non-P. grisea isolates could be classified into two groups of the Pyricularia sp., a LS- and a CE-group, corresponding to those isolated from Leersia spp. and Setaria spp. or Cenchrus spp. of grasses, respectively. Since no Magnaporthe teleomorph was produced by the crossing tests using the isolates, we identified the isolates from guinea grass as the Pyricularia sp. LS-group on the basis of their morphology and the molecular phylogenetic analysis. To our knowledge, this is the first report of blast on guinea grass in Japan.

Detection and characterization of benzimidazole resistance of Botrytis cinerea in greenhouse vegetables

From 2003 to 2006, a total of 426 single-conidial isolates of B. cinerea collected from greenhouse vegetables in China were characterized for resistance to benzimidazole fungicides and diethofencarb according to inhibition of mycelial growth. Rapid development of double-resistance to benzimidazoles and diethofencarb was observed. Three types of benzimidazole-resistant isolates, Ben R1, Ben R2 and Ben R3 were detected. A new phenotype, Ben R3, which showed low level of resistance to benzimidazole fungicides and resistance to diethofencarb, was detected with frequencies of 6.8%, 10.0%, 13.2% and 12.4% from 2003 to 2006, respectively. Further studies indicated that Ben R3 was caused by a point mutation from GAG in sensitive(S) isolates to GTG at codon 198 in the β-tubulin gene, predicted to cause a change from glutamic acid to valine. Ben R3 isolates had comparable growth, sporulation and pathogenicity ability as isolates of other phenotypes but were more sensitive at lower temperatures.

First Report of Crown Rot Caused by Cylindrocladium pauciramosum on Scarlet Honey Myrtle in Italy.

Scarlet honey myrtle (Melaleuca fulgens R. Br.), native to Australia, is an evergreen colorful shrub (Myrtaceae) and grown in Italy as an ornamental plant. During November 2008, a widespread disease was noticed on ~90% of 3,000 6-month-old M. fulgens cv. Red potted plants. Plants were obtained from cuttings and produced by a commercial nursery in Catania Province. Symptomatic plants showed a crown rot and longitudinal sections of tissues revealed a brown discoloration of the basal stem. As a consequence, leaves gradually became necrotic and abscised, followed by death of the entire plant. Root rots and leaf spots were not observed. M. gibbosa, M. ericifolia, M. thymifolia, and M. elliptica, cultivated in the same nursery, did not show disease symptoms. A Cylindrocladium sp. was consistently isolated from the crown and basal stem of symptomatic plants on potato dextrose agar (1). Ten Cylindrocladium isolates obtained from infected basal stems and crowns were selected and cultured for 8 days at 25°C on carnation leaf agar (CLA). Macroconidiophores consisted of a stipe, a penicillate arrangement, and a stipe extension terminating in an obpyriform to ellipsoidal vesicle (6 to 10 μm in diameter). Cylindrical conidia were rounded at both ends, straight, 1-septate, and 42 to 60 × 4 to 5 μm. All single-conidial isolates were mated with opposite tester strains of C. pauciramosum on CLA and produced fertile perithecia (3). Perithecia were solitary or in groups, orange to red-brown, subglobose to ovoid, and 270 to 400 μm high × 180 to 290 μm in diameter. Further confirmation of species was obtained by amplification and sequencing of the intergenic spacer (IGS) region of rDNA with the 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 (2). On the basis of morphological characters, mating type, and molecular data, the isolates were identified as C. pauciramosum C.L. Schoch & Crous. One representative isolate (DISTEF-MFR2; CBS 124657) was deposited at Centraalbureau voor Schimmelcultures open fungi collection (Fungal Biodiversity Centre, Utrecht, the Netherlands). Pathogenicity tests were performed by adding sterile water to CLA cultures of C. pauciramosum from a single-conidial isolate and incorporating the resulting spore suspension (105 conidia per ml) on the soil surface of 20 3-month-old M. fulgens cv. Red potted plants. The same number of plants served as uninoculated controls. Following inoculation, plants were well irrigated and maintained in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. All inoculated plants developed crown rot symptoms identical to those observed in the nursery 2 months after inoculation. Control plants remained symptomless. C. pauciramosum was always reisolated from the infected plants and identified as previously described. C. pauciramosum was previously detected in Italy as being responsible for a leaf spot on M. hypericifolia (3). To our knowledge, this is the first record in the world of crown rot of scarlet honey myrtle caused by C. pauciramosum.

First Report of Shoot Blight, Canker, and Gummosis Caused by Neoscytalidium dimidiatum on Citrus in Italy.

In September 2008, a new disease was noticed in eastern Sicily, Italy in a 2-year-old regrafted citrus orchard with approximately 1,500 plants of sweet orange (Citrus sinensis (L.) Osbeck cv. Tarocco Scirè) on sour orange rootstock. Symptoms on the sweet orange scion consisted of blight of vigorously growing shoots and a sooty canker on shoots and rootstock trunks, resulting in shoot dieback to the cankered area. Masses of black fungal spores appeared under the bark and on the canker surface. Abundant gummosis was frequently associated with the affected tissues. Of the 1,500 plants surveyed, 12% were infected. A Scytalidium-like fungus was isolated consistently from symptomatic tissues on 2% potato dextrose agar (PDA). Conidia were ellipsoid to ovoid, hyaline, with an acutely rounded apex, truncate base, initially aseptate, becoming brown and two-septate at maturity, (10-) 12 to 13 (-14) × (4-) 5 (-6) μm. Mycelium was branched with septate, brown hyphae that disarticulated into 0- to 1-septate phragmospores (toruloid state). Genomic DNA was extracted from mycelia of single-conidial isolates cultivated on malt extract agar. Primers V9G and ITS4 were used to amplify the nuclear rRNA operon spanning the 3' end of 18S rRNA gene, the internal transcribed spacers, the 5.8S rRNA gene, and a part of the 5' end of the 28S rRNA gene (1,2). Both PCR primers were used to sequence directly the entire amplicon. DNA sequences of two isolates (CBS 124887 and 124888) were deposited in GenBank (Accession Nos. GQ330902 and GQ330903, respectively). These sequences were 100% identical in more than 545 nt to GenBank Accession Nos. AY213688 and FJ648577. On the basis of morphological characters and molecular data, the fungal isolates were identified as Neoscytalidium dimidiatum (Penz.) Crous & Slippers (2,3). Pathogenicity tests were conducted on five 2-year-old potted plants of sweet orange cv. Tarocco Scirè and lemon cv. Femminello Zagara Bianca, both grafted on sour orange. Eight 5-cm mycelial plugs of a single-conidial isolate were placed in wounds made with a sterile blade in the inner bark of plant stems and branches. Inoculation wounds were wrapped with Parafilm. The same number of plants inoculated with sterile PDA plugs served as controls. Inoculated plants were maintained in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. After 2 weeks, all inoculated plants developed gummosis originating from the inoculation point. Shoot blight and death of the entire plant were observed within 6 months on all inoculated plants. No differences were observed among the two citrus species. Control plants remained healthy. N. dimidiatum was reisolated from the infected plants and identified as described. To our knowledge, this is the first record of a disease caused by N. dimidiatum on citrus in Italy. The pathogen has been previously observed to infect freeze-damaged limbs of citrus in California, inducing a disease named Hendersonula branch wilt (4). This pathogen on citrus is important mainly as a wound-invading pathogen, therefore posing a serious threat to regrafted citrus orchards.

First Report of Leaf Spot and Shoot Blight Caused by Cylindrocladium scoparium on Mallee Honeymyrtle in Italy.

In December of 2008, a widespread disease was observed on several blocks of approximately 15,000 plants (6-month to 2-year-old) of mallee honeymyrtle (Melaleuca acuminata F. Muell.). The plants were grown in two nurseries in eastern Sicily where high diffusion of diseases caused by Cylindrocladium pauciramosum and C. scoparium was previously detected. The plants exhibited leaf spots, defoliation, and apical blight of shoots. Crown rot and root rot were not present. Leaf spots were detected on all plants, whereas shoot blight was observed on approximately 3% of the plants. A Cylindrocladium sp. was consistently isolated from the diseased portions of plants onto potato dextrose agar. To determine the species, 20 single-conidia isolates of the fungus obtained from symptomatic tissues from different blocks and nurseries were cultured on carnation leaf agar (CLA) for 7 days at 25°C under 12-h light/dark conditions. Mycelia and spores growing on the carnation leaves were examined with a light microscope and the isolates were identified as C scoparium Morgan (teleomorph Calonectria morganii Crous, Alfenas & M.J. Wingf.) on the basis of their pyriform to broadly ellipsoidal terminal vesicles, conidiophore branching pattern, and conidia (1). In addition, the ability of the isolates to mate with South African and Italian opposite tester strains of C. scoparium (2,4) confirmed the identification of all the isolates. Koch's postulates were fulfilled by inoculating 30 6-month-old plants of mallee honeymyrtle with a spore suspension (105 conidia per ml) of one isolate of the pathogen (DISTEF-MA1) obtained from 14-day-old single-spore colonies grown on CLA at 24°C under fluorescent cool white lights on a 12-h light/dark regimen. Following inoculation, all plants were maintained in plastic bags in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. The same number of mallee honeymyrtle plants was used as uninoculated controls. Leaf spots, defoliation, and apical shoot blight identical to those observed in the nurseries appeared within 4 to 25 days. No symptoms were detected on the control plants. C. scoparium was reisolated from the artificially infected tissues and was identified as previously described. The isolate used in the pathogenicity proof was deposited at the Fungal Biodiversity Centre, Centraalbureau voor Schimmelcultures (Accession No. CBS 124658). In Italy, C. scoparium was detected for the first time on Pistacia lentiscus in 2005 (3). Another report confirmed the spread of the pathogen in Sicilian ornamental nurseries (4). To our knowledge, this is the first record in the world of C. scoparium causing disease on mallee honeymyrtle. The data demonstrate the high susceptibility of this species to the C. scoparium leaf spot and twig blight especially when environmental conditions (heavy rains and mild temperatures) are conductive to the infections.