Stipe Extensions Research Articles

Calonectria eucalyptorum sp. nov., a new leaf blight pathogen of Eucalyptus from India.

In this study, Calonectria eucalyptorum sp. nov. is described from the blighted leaves of Eucalyptus in Indiausing morphological and multi-locus phylogenetic analyses. The new species belongs to the Calonectria cylindrospora species complex, and its unique microscopic features and DNA sequence information enable clear separation from the12 currently accepted species in this complex. Conidia of the new taxon are slightly longer than those of its phylogenetic neighbors. Additionally, this species produces central as well as lateral stipe extensions, which is a feature not known for the other members ofthe C. cylindrospora species complex. Analyses of the combined partial calmodulin, histone, translation elongation factor-1α, and β-tubulingene regions revealed a distinct phylogenetic position for C. eucalyptorum. Recombination analysis provided additional support for the new species hypothesis. Koch's postulates for the new taxon as a foliar pathogen of Eucalyptus were fulfilled. The discovery of novel and pathogenic Calonectria species is important because it sheds light on species diversity, potential threats, and disease control.

First Report of Calonectria ilicicola causing Leaf spot on Camellia sinensis in Yunnan province, China.

Tea (Camellia sinensis (L.) Kuntze) is among the most significant industrial crops due to its distinctive fragrance and flavor generated (Bag et al. 2022). From October to December in 2021, a leaf spot disease affected the quality and yield of tea (C. sinensis var. assamica cv. Yunkang 10), in Pu'er (100.57°E, 22.45°N), Yunnan province, China. Based on the survey, the incidence was approximately 15% in a plantation of 4500 m2 (2050 tea trees approximately). The symptoms on leaves were regular circular, dark brown lesions with black conidiomata in gray centers. Twenty symptomatic leaves were collected from 10 trees. After rinsing and surface sterilization (75% ethanol for 30 s and 3% NaClO for 90 s, rinsed 3 times with sterile distilled water), diseased tissues (5 × 5 mm) were cut at the junction of infected and healthy site and placed on potato dextrose agar (PDA) (3 pieces per plate) and incubated in the dark at 28℃ for 5 days (Mao et al. 2023). Three single-spore isolates 6a-H-1, 6a-H-2 and 6a-H-3 were obtained, which showed identical in morphology and molecular analysis. Therefore, the targeted isolate 6a-H-2 was used for further study. Fungal colonies were white, then gradually turning into goose yellow (Fig.2. A-C). Chlamydospores were dark brown and oval (Fig.2. G). Asci produced after 30 days approximately, were orange-red, nearly spherical, rough-surface, and measured as 470 µm ± 11.68 µm (n = 50) (Fig.2. H). Ascospores were released from the asci orifice (Fig.2. I) which were hyaline, fusoid with rounded ends, straight to slightly curved, two septate, slightly constricted at the septum, and ranged from 48.77 ± 2.76 µm × 6.22 ± 0.41 µm (n = 50) (Fig.2. D-F). Macroconidia were cylindrical (Fig.2. J), rounded at both ends, straight, with an average length of 63.5 ± 0.31 μm × 2.62 ± 0.03 μm without septa (n=50) (Fig.2. M-O). Stipe extension terminated in sphaero-pedunculate vesicles (Fig.2. K-L). The morphological features were consistent with the descriptions of Calonectria ilicicola (Pei et al. 2015; Polizzi et al. 2012). The pathogen was confirmed to be C. ilicicola by amplification and sequencing of the histone (HIS3), translation elongation factor 1-alpha (TEF1) and calmodulin (CAL) genes using primers H3-3F/H3-3R, EF1-728F/EF1-986R and CAL-228F/CAL-2Rd, respectively (Crous et al. 2004). The sequences of PCR products were deposited in GenBank with accession numbers OR188222 (HIS3), OR188223 (TEF1) and OR188221 (CAL). BLAST searches of the obtained sequences revealed 99.22% (510/514 nucleotides), 98.37% (241/245 nucleotides) and 99.58% (472/474 nucleotides) homology with those of C. ilicicola (CBS 190.50) in GenBank (AY725676, AY725726 and AY725764), respectively. Phylogenetic analysis (MEGA 7.0) using the Maximum Likelihood method placed the isolate 6a-H-2 in a well-supported cluster with C. ilicicola. The pathogenicity of 6a-H-2 was tested through a pot assay. Five healthy plants had their leaves scratched with a sterilized needle, then inoculated by spraying 20 mL of spore suspension (105 spores mL-1) of 6a-H-2. Five additional tea plants sprayed with sterile distilled water served as controls. All plants were placed in a growth chamber at 28℃, with 70% relative humidity. The symptoms developed on all inoculated leaves but not on the control leaves. The lesions were first visible 72 h after inoculation, and typical lesions similar to those observed on field plants appeared after 10 days. The same fungus was reisolated and identified based on the morphology and molecular analyses (HIS3, TEF1 and CAL) from the infected leaves but not from the non-inoculated leaves. To our knowledge, this is the first report of leaf spot on tea caused by C. ilicicola in China. This study provides valuable information for the identification and control of the leaf spot on tea.

First report of leaf spot and stem blight on blueberry (Vaccinium corymbosum cv. Bluerain) caused by Calonectria pseudoreteaudii in China.

Blueberry has high nutritional value and is one of the five healthy fruits. In 2018, leaf spots and stem blights were observed on Vaccinium corymbosum cv. Bluerain in Guangzhou, Guangdong Province, China. Up to 80% of the plants were affected. Initial symptoms of affected leaves were red-brown, irregular, small spots, which gradually coalesced and formed larger irregular necrotic patches. The affected stems showed red-brown and irregular large lesions. Diseased tissues were surface sterilized with 75% alcohol for 15 s, followed by 2.5% NaClO for 30 s, and rinsing three times in sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 25 C. Representative strains, ZHKUCC 21-0021 from diseased leaves and ZHKUCC 21-0073 from diseased stems, were selected for further studies. Colonies grew slowly at 25 C on malt extract agar (MEA) (average 5.68 mm/d), producing white aerial mycelium and red-brown color on the underside after 7 days. Macroconidiophores were hyaline, smooth, consisting of a stipe bearing fertile branches, and a stipe extension terminating in a vesicle. Each terminal branch produced 2-4 phialides, 8-13 × 3-6 μm, reniform or doliiform; Stipe extensions were septate, terminating in a narrowly clavate vesicle, 2-6 μm. Macroconidia were hyaline, straight cylindrical, round at both ends, 83-100 × 7-11 μm (average = 94 × 8 μm; n = 50), with 5 septa. These morphological characteristics were similar to the description of Calonectria pseudoreteaudii (Lombard et al., 2010). The partial calmodulin (cmdA), beta-tubulin (β-tubulin), and translation elongation factor 1-alpha (tef1-α) genes of the two isolates were respectively amplified using primers CAL-228F/CAL-737R (Carbone et al., 1999), EF1-728F/EF2 and T1/CYLTUB1R (Lombard et al., 2015), and sequences were deposited in GenBank (cmdA: MZ516854 and MZ516855; β-tubulin: MZ516858 and MZ516859; tef1-α: MZ516856 and MZ516857). BLAST analysis of three gene sequences showed 100% similarity to those of C. pseudoreteaudii. In the maximum likelihood (ML) tree of the concatenated sequences of the three genes, the two isolates from this study were clustered with C. pseudoreteaudii with 100% bootstrap support. Five-mm-diameter hyphal plugs of two representative isolates grown on PDA for five days were used in the pathogenicity test. Leaves were inoculated with ZHKUCC 21-0021, and stems were inoculated with ZHKUCC 21-0073 with five replicates. As controls, sterile PDA plugs were used. All inoculated plants were maintained at 25 C . After 7 days, inoculated leaves and stems developed symptoms similar to field samples, whereas the control plants remained asymptomatic. The pathogen was reisolated from inoculated plants and confirmed to be C. pseudoreteaudii by morphological characteristics. Five Calonectria species (C. canadensis, C. colhounii, C. ilicicola, C. kyotensis and C. pyrochroa), have been reported associated with blueberry (Farr and Rossman, 2022; Fei et al, 2017). Calonectria canadensis and C. ilicicola have been reported to cause stem blight and stem rot in Vaccinium spp. in China (Fei et al, 2017 and 2018). Calonectria colhounii has been reported to cause stem blight in V. angustifolium and V. corymbosum in the United States (Sadowsky et al, 2011). However, this is the first report of C. pseudoreteaudii causing leaf spot and stem blight on Vaccinium spp. worldwide. These results will provide a foundation for future research on prevention and control of this disease.

Curvicladiellapaphiopedili sp. nov. (Hypocreales, Nectriaceae), a new species of orchid (Paphiopedilum sp.) from Guizhou, China.

BackgroundAn asexual fungus, collected from diseased leaves of Paphiopedilum sp. from Guizhou Province, China, and based on the phylogenetic analyses and morphological characters, it was identified as a new species in Curvicladiella. The genus Curvicladiella are recorded for the first time for China.New informationThe morphology of Curvicladiellapaphiopedili sp. nov. is characterised by penicillate conidiophores with a stipe, dull, tapering towards the apex, the curved stipe extension and cylindrical conidia. In the phylogenetic analyses of combined cmdA, his3, ITS, LSU, tef1 and tub2 sequence data, this taxon was clustered as sister to Curvicladiellacignea within Nectriaceae.

First Report of Root Rot Caused by Calonectria montana on Sugar Beet in Heilongjiang Province, China.

Sugar beet (Beta vulgaris L.) is an important crop that has significant economic value in northern regions of China, especially in Heilongjiang Province. In October 2019, root rot was discovered on the sugar beet cultivar HDW09 in Hulan (126°64' E, 46°00' N), Heilongjiang Province, China. Typical symptoms included lesions on root tissues, which were initially small and dark brownish, then gradually turned into irregular shapes and black in color. As the disease progressed, the extent of necrosis penetrated from external layers into inner tissue. Root tissues suffered from severe decay, which resembles symptoms of several previously reported root rot diseases of sugar beet(Harveson 2006). To identify the pathogen, pieces of the transition zones (3-5mm) between asymptomatic and symptomatic tissues were surface sterilized for 15 seconds in 1% NaClO, rinsed twice with sterilized distilled water， plated on corn meal agar supplemented with penicillin G (50 mg/L), and incubated at 25 ± 2°C in the dark. Isolates belonging to a Calonectria sp. were recovered and purified using the hyphal tipping technique. Four isolates including A1, A5, A6, and A7 were used for morphological characterization and identification by DNA sequencing. The seven-day-old colonies on malt extract agar produced buff and wooly aerial mycelia. They were sienna to umber in color. Chlamydospores and microsclerotia were produced abundantly throughout the medium. For further identification, the isolates were cultured on potato dextrose agar (PDA) at room temperature(25°C) under near-ultraviolet light irradiation. Macroconidiophores comprised of a stipe, a penicillate arrangement of fertile branches, a stipe extension, and a terminal vesicle. Stipe extension were septate, straight to flexuous, 61-117 μm long, 2-4 μm wide at the apical septum, terminating in a sphaeropedunculate vesicle, 5-9 μm diam. Conidiogenous apparatus were 31-177 μm long, and 16-110 μm wide(n=30). Primary branches of conidiogenous apparatus were aseptate or 1-septate, 16-51 × 3-7 µm; secondary branches aseptate, 6-31 × 2-7 µm; tertiary branches aseptate, 8-19 × 2-6 µm, each terminal branch producing 1-6 phialides. Conidia cylindrical were rounded at both ends, straight, 32-53 × 3-5 µm, (mean = 47 × 4 µm), 1-septate, lacking a visible abscission scar, held in parallel cylindrical clusters by colorless slime(n=100). Partial sequences of calmodulin (Carbone et al. 1999), histone H3, the translation elongation factor 1-alpha, and beta-tubulin 2 (Crous et al. 2004) genes of the four isolates were obtained and deposited into GenBank under accession numbers MW118652 to MW118667. BLAST results showed that the calmodulin, histone H3, the translation elongation factor 1-alpha and beta-tubulin 2 sequences of A1, A5, A6 and A7 were highly identical to the sequences of Ca. montana strain CERC 8957 MF527082.1 (CAL) (99-100%), CERC 8930 MF527061.1 (HIS) ( 98-99%), HSP4 MN356465.1 (EF1-alpha) (100%) and HSP4 MN356460.1 (tub2) (98-99%), respectively. A phylogenetic tree using the maximum likelihood algorithm and sequences of the four concatenated genes was reconstructed in RAxML and revealed that the four isolates clustered in the clade of Ca. montana. The pathogen was identified as Ca. montana based upon these morphological and molecular traits(Liu et al. 2017; Stępniewska et al. 2020). Ten eight-week-old sugar beet plants without root rot symptoms were selected for pathogenicity test. The roots near the ground were carefully cleaned with hands. One mycelial plug (5 mm in diameter) from a seven-day-old colony of isolate A1 were used to inoculate each sugar beet root. Ten plants inoculated with plugs of noncolonized PDA served as the control plants. Pathogenicity tests were repeated three times. Plants were incubated under greenhouse conditions at 25 ± 2°C and watered when the surface soil appeared dry. All inoculated plants showed symptoms that resembled those in the field after 30 days, while the control plants remained healthy. The symptomatic tissues were plated in corn meal agar for 7 days at 25°C. Ca. montana was reisolated from 100% of the inoculated tissues, and identification was confirmed by molecular sequencing, validating Koch's postulates. This is the first report of Ca. montana in China causing root rot on sugar beet. The study suggests its broader host range and wider geographical distribution than ever known and lays a basis for further monitoring and managing this important pathogen.

First Report of Ficus carica Bot Rot Caused by Botryosphaeria dothidea in China

HomePlant DiseaseVol. 104, No. 6First Report of Ficus carica Bot Rot Caused by Botryosphaeria dothidea in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Ficus carica Bot Rot Caused by Botryosphaeria dothidea in ChinaXiaoqiang Wang, Xia Zhang, Miaomiao Li, Xiuting Ji, Chao Feng, and Fenglong WangXiaoqiang Wang†Corresponding author: X. Wang; E-mail Address: [email protected]http://orcid.org/0000-0002-2173-6442Chinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100Search for more papers by this author, Xia ZhangShandong Peanut Research Institute, Qingdao, Shandong, China, 266100Search for more papers by this author, Miaomiao LiChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100Search for more papers by this author, Xiuting JiChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100Search for more papers by this author, Chao FengChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100Search for more papers by this author, and Fenglong WangChinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100Search for more papers by this authorAffiliationsAuthors and Affiliations Xiaoqiang Wang1 † Xia Zhang2 Miaomiao Li1 Xiuting Ji1 Chao Feng1 Fenglong Wang1 1Chinese Academy of Agricultural Sciences Institute of Tobacco Research, Qingdao, Shandong, China, 266100 2Shandong Peanut Research Institute, Qingdao, Shandong, China, 266100 Published Online:25 Mar 2020https://doi.org/10.1094/PDIS-09-19-2039-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Ficus carica L., belonging to the Moraceae family, is one of the earliest cultivated fruit trees. Fig is an important crop for dry and fresh consumption worldwide because it contains an abundant source of minerals and vitamins. Moreover, fig fruit has been a typical component in health-promoting diets for millennia. A previously uncharacterized disease was observed on fig fruit during 2016 and 2017 in Qingdao, Shandong Province, China (N 36°08′49″, E 120°25′5″). Approximately 5 to 8% of fruit on greater than 500 trees was lost due to the disease during the 2016 and 2017 seasons. The symptoms were first observed on green fruit, and infected fruit initially showed soaked symptoms on the surface, which subsequently became covered with fluffy mycelia that ultimately caused fruit decay. Infected fruit were collected, and small pieces of discolored or necrotic tissues were rinsed with 70% alcohol for 10 s followed by 1% NaOCl for 60 s, after which they were washed with sterile distilled water (Wang et al. 2015). Pieces were subsequently plated in Petri dishes containing potato dextrose agar (PDA) and incubated for 4 days at 28°C in the dark. The obtained isolates were purified through single-spore culture. Colonies of all isolates on the PDA plates were identical, producing mycelia that were initially white and fluffy but that subsequently turned gray and then black as they matured. Mycelia were hyaline, smooth, septate, 110.5 to 260 × 3 to 4 μm; stipe extension septate, erect to flexuous, 123.5 to 179 μm long, and 2 to 3.5 μm wide at the apical septum. Hyphae were hyaline and septate, consistent with the description of Botryosphaeria dothidea. The pycnidia contained hyaline, nonseptate, fusiform conidia measuring 13.5 to 26.5 × 4.8 to 8.0 μm. Perithecia contained clavate asci, which averaged 130.5 × 17 μm. The internal transcribed spacer region, partial translation elongation factor 1-alpha, and β-tubulin genes of the rDNA of the isolate were sequenced using primers ITS4/ITS5 (GenBank accession no. MN428793), EF1-728F/EF1-986R (MN478488), and Bt2a/Bt2b (MN508257), respectively (Sun et al. 2014). Based on morphological features and phylogenetic analysis, we concluded that the isolates belonged to B. dothidea. To test the pathogenicity, F. carica fruit were collected, surface sterilized with 1% NaOCl, and rinsed with sterilized distilled water. Plugs (5 × 5 mm) of B. dothidea from the PDA plate were placed on the surface of the fruit (n = 10) and incubated at 28°C and 60% humidity in the dark. Four days after inoculation, bot rot symptoms appeared, and the surfaces of the fruit were covered with fluffy mycelia. In contrast, fruit inoculated with PDA plugs that did not contain B. dothidea remained symptom-free. B. dothidea was reisolated from diseased fruit, fulfilling Koch’s postulates. To our knowledge, this is the first report of B. dothidea causing bot rot in F. carica in China.The author(s) declare no conflict of interest.

First Report of Gliocephalotrichum bulbilium Causing Fruit Rot of Posthavest Mangosteen in China.

Mangosteen (Garcinia mangostana L., Guttiferae) is a tropical fruit renowned for its pleasant taste, rich nutrition, and medicinal value. Little research about mangosteen diseases during storage and transport has been reported. In June of 2012, fruit rots on mangosteens imported from Thailand were observed in Guangzhou, China. In infected fruits, pericarps showed an increased firmness, were discolored to deep pink, and the edible aril became brown and rotten. In order to search for the etiological agent of this rot symptom, infected mangosteens were analyzed. Diseased mangosteen tissues were surface-sterilized with 70% alcohol, then with 0.1% HgCl2, dipped in sterilized water three times, and placed onto potato dextrose agar (PDA) at 26°C. The fungi isolated from tissues of the pericarp and aril were similar in morphology and grew rapidly, covering the plate surface (9 mm diameter) after 2 to 3 days of incubation at 26°C. The morphological characters of 10 single-spore isolates were observed. These isolates showed light yellow to light brown fertile colonies on PDA. On corn meal agar (CMA), conidiophores were erect, arising from wide hyphae; they were composed of a basal stipe ending in a penicillate conidiogenous apparatus with directly subtending sterile stipe extensions ranging from 74.5 to 195.0 μm long. Conidia were unicellular, smooth, oblong to elliptical, 6.3 to 8.5 × 2.5 to 3.0 μm, and accumulated in a mucilaginous mass. Chlamydospores were multicellular, dark brown, regular in shape and thick-walled, and 40.0 to 52.5 μm in diameter. On the basis of these morphological characters, these isolates were identified as Gliocephalotrichum bulbilium (2). To confirm the identity of this fungus, genomic DNA of two isolates was extracted, and fragments of ITS region and β-tubulin gene were amplified by PCR, sequenced, and compared with sequences of Gliocephalotrichum species available in NCBI GenBank. Both DNA regions (GenBank Accession Nos. KF716166 and KF716168) had sequence similarities of 99% and 97%, respectively, to other G. bulbilium sequences at GenBank. Pathogenicity tests were conducted on three detached fruits for two isolates. Fruits were inoculated using 5-mm mycelial disks with conidia taken from 3-day-old cultures of G. bulbilium isolate Gb1 and Gb10 grown on PDA. Controls were inoculated with PDA disks only. All treated fruits were kept individually in a humid chamber at 26°C. Tests were repeated twice. Three days after inoculation, white mycelial growth for Gb was observed at inoculation sites. Eight days after inoculation, mycelium of Gb nearly covered the fruit, causing fruit rot, and the pericarp became hard and light in color. The control fruit did not rot. G. bulbilium was re-isolated from diseased plant tissue, thus fulfilling Koch's postulates. G. bulbilium has been reported causing postharvest fruit rot of rambutan (Nephelium lappaceum) and guava (Psidium guajava) in some locations (3,4). Moreover, the fungus caused cranberry fruit rot in the United States (1). To our knowledge, this is the first report of G. bulbilium causing postharvest fruit rot of mangosteen in China. It is uncertain whether the fungus infected mangosteen in Thailand and was carried to China due to commercial relationship.

First Report of Boxwood Blight Caused by Calonectria pseudonaviculata in Delaware, Maryland, New Jersey, and New York.

Boxwood (Buxus spp.) are commercially important evergreen ornamental plants with an annual market value of over $103 million in the United States. The recent U.S. incursion of boxwood blight disease caused by the fungus Calonectria pseudonaviculata (syn. Cylindrocladium pseudonaviculatum, Cy. buxicola) threatens the health and productivity of boxwood in both landscape plantings and nurseries. The first confirmed U.S. reports of the disease were made from Connecticut and North Carolina in November 2011 (2,4), followed by diagnoses in 10 additional states during 2012 and 2013. By August 2013, symptoms consistent with boxwood blight had been observed from B. sempervirens in Delaware, Maryland, New Jersey, and southeastern New York. Affected plants showed rapid onset of disease symptoms: dark brown to black spots or diffuse dark areas on leaves, followed by defoliation. Narrow, elongate black cankers also formed on current season shoots. Symptomatic stems and leaves were placed in petri dishes with moistened filter paper at 22°C for 3 days under continuous light. Conidiophores were excised, then placed on potato dextrose agar amended with streptomycin and neomycin (0.3 g/l). Resultant colonies showed dark brown pigmentation at the colony center surrounded by tan to reddish brown rings with white mycelia at the advancing edge. Conidia (n = 30 per isolate) were hyaline, cylindrical, rounded at both ends, with a single septum (45 to 76 × 4 to 6 μm; avg. 63 × 5 μm). Conidiophores (n = 20 per isolate) comprised a stipe, a hyaline septate stipe extension (length 119 to 192 μm; avg. 150 μm) and a terminal ellipsoidal vesicle (diameter 4 to 10 μm; avg. 7 μm). Based on morphological characteristics, the causal agent was identified as C. pseudonaviculata (1,4). Voucher specimens were deposited in the U.S. National Fungus Collections (BPI 892698 to 701). To verify morphological diagnosis, genomic DNA was extracted from fungal biomass grown in liquid cultures of yeast extract peptone dextrose media. A portion of the β-tubulin gene (TUB2) was PCR amplified and sequenced bi-directionally using primers Bta/Bt2b (3). BLASTn searches of NCBI GenBank databases using the TUB2 sequences (Accession Nos. KF785808 to 11) demonstrated 96 to 100% sequence identity with other C. pseudonaviculata isolates. To confirm pathogenicity, 5-month-old B. sempervirens and B. microphylla seedlings were spray-inoculated with a spore suspension of 1 × 104 conidia/ml. One isolate from each state was independently tested with four replicates each. Non-inoculated water-sprayed plants served as negative controls. Plants were maintained in growth chambers at 22°C under constant light. Blight symptoms developed 4 to 5 days post inoculation. C. pseudonaviculata was re-isolated from inoculated plants; no symptoms or signs were observed from control plants. To our knowledge, this is the first report of C. pseudonaviculata in the states of Delaware, Maryland, New Jersey, and New York. This report demonstrates that C. pseudonaviculata is now widespread across the United States eastern seaboard, and represents a substantial threat to boxwood plants in North American landscapes and nurseries.

Leaf Blight of Buxus sempervirens in Northern Forests of Iran Caused by Calonectria pseudonaviculata.

Buxus sempervirens subsp. hyrcana (Pojark.) Takht. (boxwood) is an evergreen shrub/tree in Caspian hyrcanian forests covering the Alborz mountain range of northern Iran. During the summer of 2012, a sudden leaf and twig blight disease of boxwood was observed throughout the northern forests of Iran. Disease symptoms included circular dark spots on leaves leading to defoliation, and longitudinal brown-black streaks on the shoots. Diseased plant material was collected from the Guilan and Mazandaran areas, placed in moist chambers, and incubated at 20°C to induce sporulation. Single conidia were plated onto half-strength potato dextrose agar supplemented with 250 mg/L streptomycin and incubated at 25°C under near-ultraviolet light. Isolates were transferred to carnation leaf agar and incubated at 25°C under near-ultraviolet for morphological characterization, and representative isolates were deposited into the culture collection of the CBS-KNAW Fungal Biodiversity Centre under accession numbers CBS 134431 and CBS 134432. Gross morphological characters were determined by mounting fungal structures in lactic acid and 50 measurements at 1,000× magnification were made for all taxonomically informative characters. The observed macroconidiophores consisted of a stipe bearing a penicillate suite of reproductive branches and a stipe extension, terminating in a naviculate vesicle. The stipe extensions were septate, hyaline (85 to 160 × 2 to 4 μm), terminating in a naviculate vesicle, 6 to 11 μm in diam. Conidia were cylindrical, rounded at both ends, straight, with one septum (55 to 68 × 4 to 6 μm). These morphological observations agreed to those provided for C. pseudonaviculata Lombard, M. J. Wingf. & Crous (1,2). To confirm morphological identification, DNA sequence data were generated for the ITS1-5.8S-ITS2 region of the rDNA, and a fragment of the translation elongation factor 1-alpha gene region (3). These sequences were compared to other sequences of C. pseudonaviculata in GenBank (100% similarity for both loci), which confirmed the morphological observations. Sequences were submitted to GenBank under the accession numbers KC736850 and KC736851 for ITS, and KC736852 and KC736853 for TEF. Koch's postulates were proven by spraying a 3 × 106 conidia/ml conidial suspension of isolate CBS 134431 onto 1-year-old B. sempervirens subsp. hyrcana plants until run-off, and covering them for 24 h with a plastic bag to maintain high humidity. Control plants were sprayed with sterile water. Ten plants were used for each treatment and maintained in a greenhouse at 20 to 22°C with 95% relative humidity. Symptoms similar to those observed in nature developed within 4 days of inoculation and the test fungus was successfully reisolated from the inoculated plants. No symptoms were observed on the control plants. Boxwood blight caused by C. pseudonaviculata, was first reported in the United Kingdom in the mid-1990s and has since become widespread, causing epidemics globally (1,2,4). To our knowledge, this study represents the first report of boxwood blight in its native environment and in Iran.

First Report of Dieback on Euonymus fortunei Caused by Cylindrocladiella parva in Germany.

In August 2011, a severe shoot dieback was observed on several hundred plants of 1-year-old Euonymus fortunei cv. Emerald 'n Gold in a nursery in Lower Saxony and on a cemetery in Berlin. Single shoots or the whole plant were affected. Chocolate brown lesions around the shoots spread primarily acropetally to be followed by wilting of the shoot tip, reddish discoloration, dropping of leaves, and finally plant death. Two fungal isolates, JKI 2187 and JKI 1288, forming white mycelium on 2% malt extract agar (MEA) were obtained from symptomatic shoots. Both were identified by their morphology as Cylindrocladiella parva (P.J. Anderson) Boesewinkel (syn. Cylindrocladium parvum). After incubation for one week at 25°C in the dark, the reverse side of the colony became buff to ochreous and this was associated with development of long chains of chlamydospores. Microsclerotia and fruiting bodies were not observed. Morphological characteristics were determined on synthetic nutrient agar (SNA) after 7 days at 25°C under near-ultraviolet light. The conidiophores were penicillately branched. The stipe extensions were thick-walled with clavate to naviculate vesicles. Conidia measured 12.7 to 17.1 (14.9) × 2.2 to 3.3 (2.7) μm. The molecular studies confirmed the morphological identification. Genomic DNA was isolated from the mycelia. The rDNA internal transcribed spacer (ITS) region was amplified with the primers ITS1 and ITS4 and a part of the β-tubulin gene with the primers Bt2a and Bt2b (2). The sequences generated in this study were compared with sequences obtained from GenBank. A BLAST analysis showed that the ITS sequence had a 99% similarity with that of C. parva GenBank Accession No. AY793454 and the β-tubulin gene had a 100% similarity with AY793489. So far, pathogenicity of C. parva has been demonstrated for only a few plant species. Its pathogenicity was confirmed on grapevine (Vitis vinifera) in New Zealand (3), on common oak (Quercus robur) in Italy (4), and on eucalyptus in South Africa (1). To fulfill Koch's postulates for the pathogen on E. fortunei, the isolate JKI 2188 of C. parva was inoculated on 40 two-year-old plants of cv. Emerald 'n Gold. The leaves around one node were removed on five shoots per plant. After wounding the nodes with a needle, colonized agar plugs were placed on them. The plugs were covered with moist cellulose swabs and sealed with Parafilm. To act as negative controls, 20 plants were treated with sterile agar plugs. All the plants were incubated in a growth chamber at 21/16°C (day/night), with a day length of 12 h and a relative humidity of 90 to 100%. Seven weeks after inoculation, all inoculated plants showed symptoms identical to those of the diseased plants from which C. parva was originally isolated. The negative controls remained healthy. The strains reisolated were identical to the original isolates. To our knowledge, this is the first report of C. parva as a pathogen of Euonymus. Since 2011, there were no further reports of this disease. At present, the disease is not of economic importance.

First Report of Gliocephalotrichum bacillisporum Causing Fruit Rot of Rambutan (Nephelium lappaceum) in Malaysia.

Rambutan (Nephelium lappaceum L.) is among the tropical fruit grown in Malaysia and the demand for export rose in 2011. A fruit rot was observed between August and December 2011 from several areas in the states of Pulau Pinang and Perak, Malaysia. The symptoms initially appeared as light brown, water-soaked lesions that developed first in the pericarp and pulp, later enlarging and becoming dark brown. Greyish brown mycelia were observed on infected areas that turned yellowish at later stages of infection. Gliocephalotrichum bacillisporum was isolated from infected fruit by surface sterilization techniques. Conidia were mass-transferred onto potato dexstrose agar (PDA) plates and incubated at 27 ± 1°C. Tissue pieces (5 × 5 mm) excised from the margins between infected and healthy areas were then surface sterilized in 1% sodium hypochlorite for 3 to 5 min before being rinsed with distilled water, plated on PDA, and incubated at 27 ± 1°C for 7 days. Ten isolates of G. bacillisporum were obtained. Colonies on PDA were initially white before turning yellow with a feathery appearance. Microscopic characteristics on carnation leaf agar (CLA) consisted of hyaline conidia that were slightly ellipsoid to bacilliform with rounded apex ranging from 6.0 to 8.5 μm long and 2.0 to 2.5 μm wide. Conidiophores (70 to 130 μm long) were mostly single arising from large hypha approximately 13 to 16 μm. The conidiogenous structures were mostly quadriverticillate with dense, short, penicillate branches. The phialides were cylindrical and finger-like. Chlamydospores were present singly, in groups of 2 to 4, or in occasionally branched short chains and were brown in color with thick walls ranging from 11 to 13 μm. The cultural and morphological characteristics of G. bacillisporum isolates in the present study were very similar to previously published descriptions (1) except the conidiophores formed without sterile stipe extensions. All the G. bacillisporum isolates were deposited in culture collection at the Plant Pathology Lab, University Sains Malaysia, Penang. Molecular identification was accomplished from the ITS regions using ITS1 and ITS2 primers, and the β-tubulin gene using Bt2a and Bt2b primers (2). BLAST results from the ITS regions showed a 98 to 99% similarity with sequences of G. bacillisporum isolates reported in GenBank. Accession numbers of G. bacillisporum ITS regions: JX484850, JX484852, JX484853, JX484856, JX484858, JX484860, JX484862, JX484866, JX484867, and JX484868. The identity of G. bacillisporum isolates infecting rambutan was further confirmed by β-tubulin sequences (KC683909, KC683911, KC683912, KC683916, KC683919, KC683920, KC683923, KC683926, and KC683927), which showed 92 to 95% similarity with sequences of G. bacillisporum. Pathogenicity tests were also performed using mycelial plug (5 mm) and sprayed conidial suspensions (20 μl suspension of 106 conidia/ml) prepared from 7-day-old cultures. Inoculated fruits were incubated at 27 ± 1°C and after 10 days, similar rotting symptoms appeared on the fruit surface. The pathogen was reisolated from fruit rot lesions, thus fulfilling Koch's postulates, and tests were repeated twice. To our knowledge, this is the first report of G. bacillisporum causing fruit rot of rambutan (N. lappaceum L.) in Malaysia.

First Report of Calonectria hongkongensis Causing Fruit Rot of Rambutan (Nephelium lappaceum).

Fruit rot of rambutan is a pre- and post-harvest disease problem of rambutan orchards. In 2011, fruit rot was observed at USDA-ARS orchards in Mayaguez, Puerto Rico. Infected fruit were collected and 1 mm2 tissue sections were surface disinfested with 70% ethanol followed by 0.5% sodium hypochlorite. Infected fruit were rinsed with sterile, deionized, double-distilled water and transferred to acidified potato dextrose agar (APDA). Plates were incubated at 25 ± 1°C for 6 days. Three isolates of Calonectria hongkongensis (Cah), CBS134083, CBS134084, and CBS134085, were identified morphologically using taxonomic keys (2,3). In APDA, colonies of Cah produced raw sienna to rust-colored aerial mycelial growth. Conidiophores of Cah had a penicillate arrangement of primary to quaternary branches of 2 to 6 phialides. Conidia (n = 50) were cylindrical, hyaline, 1-septate, rounded at both ends, and 44 to 52 μm × 3.5 to 4.5 μm. Conidiophores produced terminal and lateral stipe extensions with terminal sphaeropedunculate vesicles that were 8 to 12 μm wide. Subglobose to ovoid perithecia, 300 to 500 μm × 200 to 350 μm and orange to red-brown, were produced in groups of 3. Asci were clavate and contained 8 ascospores aggregated at the top of the ascus. Ascospores (n = 50) were hyaline, guttulate, fusoid with rounded ends, straight to curved, 1-septate with constriction at the septum, and 28 to 36 μm × 4 to 7 μm. For molecular identification, the ITS rDNA, fragments of β-tubulin (BT), histone H3 (HIS3), and elongation factor (EF1-α) genes were amplified by PCR, sequenced, and compared using BLASTn with Calonectria spp. submitted to the NCBI GenBank. The sequences of Cah submitted to GenBank include accessions KC342208, KC342206, and KC342207 for ITS; KC342217, KC342215, and KC342216 for BT; KC342211, KC342209, and KC342210 for HIS3; and KC342214, KC342212, and KC342213 for EF1α. The sequences were >99% or identical with the ex-type specimen of Cah CBS 114828 for all genes used. Pathogenicity tests were conducted on 5 healthy superficially sterilized fruits per isolate. Both scalpel-wounded and unwounded fruit tissues were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C under 12 h of fluorescent light. The test was repeated once. Three days after inoculation (DAI), white mycelial growth was observed on the fruit. Five DAI, the fruit changed color from red to brown and yellowish mycelia colonized 50 to 62% of the fruit surface. Eight DAI, all the fruit turned brown, the mycelium growth covered the entire fruit, and conidiophores were produced on spinterns (hairlike appendages). Fruit rot of spinterns, exocarp (skin), endocarp (aril), and light brown discoloration were observed inside the fruit. Untreated controls showed no symptoms of fruit rot and no fungi were reisolated from tissue. Cah was reisolated from diseased tissue, fulfilling Koch's postulates. Calonectria spp. (or their Cylindrocladium asexual states) have been associated with lychee decline syndrome in North Vietnam (1). Both fruits belong to the Sapindaceae family. To our knowledge, this is the first report of Cah causing fruit rot of rambutan.

First Report of Calonectria Leaf Spot on Ohelo in Hawaii.

Ohelo, Vaccinium reticulatum (Smith), is an endemic Hawaiian shrub, less than 1 m tall, which grows between 640 and 3,700 m elevation on disturbed volcanic sites on the islands of Maui and Hawaii (3). Ohelo berries are made into jams, jellies, and pie filling and are also a food source for the endemic nene goose, the state bird of Hawaii (3). In the summer of 2010, Ohelo berry plants grown in a greenhouse nursery located in Hilo, Hawaii, exhibited severe disease symptoms including leaf spots, stem lesions, and defoliation. The leaf spots appeared rapidly and were fairly severe. Subsequent field surveys of areas of naturally growing Ohelo within Hawaii Volcanoes National Park and along the roadside of Saddle Road were negative. An anamorphic state of Cylindrocladium was consistently isolated from the diseased portions of plants on potato dextrose agar (PDA). To determine the species, single-conidial isolates of the fungus were cultured for 14 days at 25°C under 12 h of light/dark conditions. Conidia were produced on penicillately branched condiophores having a stipe extension of 101.5 to 231.3 × 1.9 to 3.5 μm, terminating in a narrowly clavate vesicle, 2.4 to 3.5 μm. Conidia were hyaline, cylindrical, rounded at both ends, straight, three septate, and 58.6 to 77.77 × 4.29 to 5.72 μm. The nucleotide sequence of the partial β-tubulin gene was determined for strain Vr1 and a BLAST analysis of the β-tubulin sequence (GenBank Accession No. JX852715) revealed 99% similarity (337/341 bp) with the sequence of Calonectria pseudocolhounii strain CMW27213 (HQ285789) (1). Based on morphology and molecular sequencing, the fungus was identified as Calonectria irrespective of the teleomorphic stage in accordance with Lombard et al. (2). Koch's postulates were fulfilled by spray inoculating eight Ohelo seedlings and eight Ohelo variety N06-7 ('Kilauea') plants with a spore suspension (105 conidia per ml) of one isolate of the pathogen obtained from 14-day-old single-spore colonies grown on PDA at 25°C. Following inoculation, all plants were maintained in plastic bags in a growth chamber at 25 ± 1°C and 90 to 95% relative humidity. Four plants were used as a control. After 5 to 7 days, foliar symptoms resembling those seen in the nursery were detected on inoculated plants; leaf drop was first observed after day 7. No symptoms were detected on the control plants. The Calonectria sp. was reisolated from the artificially infected tissues. To our knowledge, this is the first report of a Calonectria sp. causing disease on Ohelo berry in Hawaii.

First Report of Box Blight Caused by Cylindrocladium pseudonaviculatum (C. buxicola) in British Columbia, Canada.

Boxwoods (Buxus spp.) are common woody ornamental hedging plants in Europe and North America, typically propagated by cuttings. In October 2011, shoot dieback and defoliation was observed on Buxus sempervirens 'Suffruticosa' (dwarf English boxwood) and 'Green Balloon' in outdoor, 10-cm pots at a wholesale nursery in Chilliwack, British Columbia. Circular leaf spots with black rings occurred on leaves and black, water-soaked, cankers girdled the stems and petioles. Leaf and stem samples were collected on November 21, 2011, and incubated for 48 h in a moist chamber at room temperature. In addition to Volutella buxi, a Cylindrocladium species producing conidia on white sporodochia was observed on host tissue under the microscope. Leaves with lesions were surface-sterilized in 10% bleach for 30 to 60 s, rinsed in sterile water, and lesions were cut out and plated on PDA and carnation leaf media. The species was identified as Cylindrocladium pseudonaviculatum Crous, J.Z. Groenew. & C.F. Hill 2002 by comparison of conidia and phialide morphology to published descriptions. Conidia were hyaline, one-septate, cylindrical with rounded ends and 38 to 76 μm (mean 51 μm) × 4 to 6 μm on carnation leaf media and 41 to 66 μm (mean 52 μm) × 4 to 6 μm on B. sempervirens 'Suffruticosa' leaves, comparable to the reported range of 40 to 75 × 4 to 6 μm (1,2,3,4). Conidia were produced in clusters on terminal, ellipsoid vesicles at the tips of penicillate conidiophores. Vesicles were 10.2 (7.6 to 12.8 μm) at the widest point, consistent with the 6 to 11 μm reported in (2,3) and tapered to a rounded point; stipe extensions were septate and measured an average of 130 μm (107 to 163 μm) in length to the tip of the vesicle, consistent with the 95 to 155 μm reported in (1), 89 to 170 μm reported in (2), and 95 to 165 μm in (3). Chlamydospores were not observed on host tissue but appeared in older PDA cultures as dark brown microsclerotia. DNA was extracted from single-spore colonies on PDA and the internal transcribed spacer (ITS) region of rDNA was amplified with primers ITS1 and ITS4. The ITS sequence (GenBank Accession No. KC291613) was 100% identical to C. buxicola strain CB-KR001 (HM749646.1) and Calonectria pseudonaviculata strain ATCC MYA-4891 (JX174050.1). In early December 2011, box blight was identified on container-grown B. sinica var. insularis × B. sempervirens 'Green Velvet,' 'Green Gem', and 'Green Mountain' and B. sempervirens L. (common or American boxwood). The pathogen was identified by microscopic examination at three wholesale nurseries in the eastern Fraser Valley and one landscape planting. The isolate has been deposited in the Canadian Collection of Fungal Cultures in Ottawa, Canada (DAOM 242242).

First Report of Gliocephalotrichum bulbilium and G. simplex Causing Fruit Rot of Rambutan in Puerto Rico.

Post-harvest disease losses of rambutan (Nephelium lappaceum L.) have been reported worldwide and several pathogens have been associated with fruit rot (3,4). In 2011, fruit rot of rambutan was observed on 11-year-old trees at the USDA-ARS Tropical Agriculture Research Station in Mayaguez, Puerto Rico. Infected fruit sections (1 mm2) were surface-sterilized, rinsed with sterile deionized-distilled water, and transferred to acidified potato dextrose agar (APDA). Gliocephalotrichum bulbilium J.J. Ellis & Hesseltine (Gb) and G. simplex (J.A. Meyer) B. Wiley & E. Simmons (Gs) were identified using a taxonomic key (1). In corn meal agar (CMA), five isolates of Gb were light yellow-to-light brown. Conidiophores had sterile stipe extensions ranging from 120 to 150 μm long and were produced contiguous to the erect conidiogenous penicilli. Conidia were unicellular, smooth, oblong to elliptical, and 5.5 to 7.5 μm long by 2.0 to 2.5 μm wide. Bulbilloid aggregates were observed and averaged 70 μm long. In CMA, five isolates of Gs were light brown-to-chestnut brown. Conidiophores had sterile stipe extensions 130 to 180 μm long that were produced approximately 15 to 30 μm away from the conidiogenous penicilli. Conidia were unicellular, smooth, cylindrical to elliptical, and with slightly curved ends ranging from 6.5 to 8.5 μm long by 2.0 to 2.5 μm wide. Chlamydospores were unicellular, brown, smooth and thick-walled, averaging 35 μm long. Pathogenicity tests were conducted on five detached fruits per isolate. Five isolates of each Gliocephalotrichum spp. were inoculated on fruits using 5-mm mycelial disks of 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Inoculated fruit was kept in a humid chamber for 8 days at 25°C under 12 hours of fluorescent light. Test was repeated once. Five days after inoculation (DAI), white mycelial growth for Gb and golden mycelial growth for Gs were observed on rambutan fruits. Eight DAI, fruit rot, and aril (flesh) rot symptoms were observed on fruits inoculated with isolates of Gb and Gs. Infected fruit changed in color from red to brown, and, on average, mycelia of Gb and Gs covered 50 and 60% of the fruit, respectively. Conidiophores were observed on spintems (hair-like appendages). Control fruit did not rot. Both species were reisolated from diseased plant tissue, thus fulfilling Koch's postulates. For molecular identification of these species of Gliocephalotrichum, the ITS1-5.8S-ITS2 region of the rDNA and a fragment of the β-tubulin gene were amplified by PCR and aligned with other Gb and Gs sequences in NCBI GenBank for comparison. The sequences submitted to GenBank included Gs Accession Nos. JQ688045 and JQ688046 and Gb Accession Nos. JQ688044 and JQ68847 for the ITS sequences. For the β-tubulin gene, Gs Accession Nos. JQ688049 and JQ688050 and Gb Accession Nos. JQ688048 and JQ688051. Both DNA regions had 99.9 to 100% sequence identity to other isolates of Gb and Gs reported in GenBank (1). Gliocephalotrichum spp. have been associated with rambutan fruit rot in Hawaii, Sri Lanka and Thailand (2,4). To our knowledge, this is the first report of G. bulbilium and G. simplex causing fruit rot of rambutan in Puerto Rico.

First Report of Boxwood Blight Caused by Cylindrocladium pseudonaviculatum in the United States.

In September and October 2011, a new disease was observed on Buxus spp. in North Carolina and Connecticut, respectively. In North Carolina, over 10,000 containerized Buxus sempervirens (American boxwood) were affected at one location. A few weeks later, the disease was found in Connecticut on entire plantings of B. sempervirens 'Suffruticosa' (English boxwood) at two residential properties, and shortly thereafter on over 150,000 plants at two production nurseries. Initial foliar symptoms appeared as light to dark brown spots, often with dark borders. Spots enlarged and coalesced, often with a concentric pattern, and black streaks or cankers developed on stems. Infected leaves became brown or straw colored and dropped quickly after foliar symptoms were visible. Branch dieback and plant death were also observed in Connecticut. Cultures were isolated from symptomatic leaves and stems and identified as Cylindrocladium pseudonaviculatum Crous, Groenewald & Hill 2002 (1) (syn. Cylindrocladium buxicola Henricot 2002 [2]) on the basis of morphological characteristics. Macroconidiophores were single or in groups of up to three and comprised a stipe, stipe extension, and a penicillate arrangement of fertile branches. The stipe extension was septate, hyaline (89 to 170 × 2 to 4.5 μm), and terminated in an ellipsoidal vesicle (6 to 11 μm diameter) with a papillate or pointed apex. Conidia were cylindrical, straight, hyaline, and one septate (48 to 62 × 4 to 6 μm), occurring in slimy clusters. No microconidiophores were observed. Chlamydospores were medium to dark brown, thick walled, and smooth to rough. Microsclerotia were observed on PDA (1). A portion of β-tubulin gene sequence from two Connecticut (Genbank Accession Nos. JQ866628 and JQ866629) and two North Carolina isolates showed 100% similarity with only C. pseudonaviculatum strains. USDA-APHIS-PPQ confirmed this new United States record on October 24, 2011. Pathogenicity was confirmed by inoculating three 1-gallon container plants of B. sempervirens 'Suffruticosa' in North Carolina and four liners of B. sinica var. insularis × B. sempervirens 'Green Velvet' in Connecticut with a spore suspension of approximately 5.0 × 106 conidia (North Carolina) or 1.0 × 106 conidia (Connecticut) on the foliage of each plant; untreated control plants were sprayed with water. After incubation at ambient temperature, all inoculated plants developed foliar and stem lesions within 3 to 4 days and blighting occurred within 2 weeks; control plants remained asymptomatic. C. pseudonaviculatum was reisolated from inoculated plants. To our knowledge, this is the first report of C. pseudonaviculatum in the United States. C. pseudonaviculatum causes a serious disease of Buxus spp. in the United Kingdom and several other European countries as well as New Zealand (1). Confirmation of boxwood blight in the United States is significant because of the popularity of boxwood as a landscape plant, and because of the potential economic impact this disease may have on commercial growers; boxwood production in the United States has an annual wholesale market value of approximately $103 million (3).

Phylogeny and taxonomy of the genus Cylindrocladiella

The genus Cylindrocladiella was established to accommodate Cylindrocladium-like fungi that have small, cylindrical conidia and aseptate stipe extensions. Contemporary taxonomic studies of these fungi have relied on morphology and to a lesser extent on DNA sequence comparisons of the internal transcribed spacer regions (ITS 1, 2 and 5.8S gene) of the ribosomal RNA and the β-tubulin gene regions. In the present study, the identity of several Cylindrocladiella isolates collected over two decades was determined using morphology and phylogenetic inference. A phylogeny constructed for these isolates employing the β-tubulin, histone H3, ITS, 28S large subunit and translation elongation factor 1-alpha gene regions resulted in the identification of several cryptic species in the genus. In spite of the 18 new Cylindrocladiella species described in this study based on morphological and sequence data, several species complexes remain unresolved.

First Report of Stem Blight Caused by Calonectria colhounii (Anamorph Cylindrocladium colhounii) on Greenhouse-Grown Blueberries in the United States.

Necrotic stems and leaves were observed on 2- to 4-month-old, rooted microshoot plants (Vaccinium corymbosum L. 'Liberty' and 'Bluecrop', V. angustifolium Aiton 'Putte', and V. corymbosum × V. angustifolium 'Polaris') in a Michigan greenhouse in 2008 and 2009. As the disease progressed, leaves fell off and 80 to 100% of the plants died in some cases. Root rot symptoms were also observed. A fungus was isolated from stem lesions. On potato dextrose agar (PDA), cultures first appeared light tan to orange, then rusty brown and zonate with irregular margins. Chains of orange-brown chlamydospores were abundant in the medium. Macroconidiophores were penicillately branched and had a stipe extension of 220 to 275 × 2.5 μm with a narrowly clavate vesicle, 3 to 4 μm wide at the tip. Conidia were hyaline and cylindrical with rounded ends, (1-)3-septate, 48 to 73 × 5 to 7 (average 60 × 5.5) μm and were held together in parallel clusters. Perithecia were globose to subglobose, yellow, 290 to 320 μm high, and 255 to 295 μm in diameter. Ascospores were hyaline, 2- to 3-septate, guttulate, fusoid with rounded ends, slightly curved, and 30 to 88 × 5 to 7.5 (average 57 × 5.3) μm. On the basis of morphology, the fungus was identified as Calonectria colhounii Peerally (anamorph Cylindrocladium colhounii Peerally) (1,2). The internal transcribed spacer region (ITS1 and ITS2) of the ribosomal DNA and the β-tubulin gene were sequenced (GenBank Accession Nos. HQ909028 and JF826867, respectively) and compared with existing sequences using BLASTn. The ITS sequence shared 99% maximum identity with that of Ca. colhounii CBS 293.79 (GQ280565) from Java, Indonesia, and the β-tubulin sequence shared 97% maximum identity with that of Ca. colhounii CBS 114036 (DQ190560) isolated from leaf spots on Rhododendron sp. in North Carolina. The isolate was submitted to the Centraalbureau voor Schimmelcultures in the Netherlands (CBS 129628). To confirm pathogenicity, 5 ml of a conidial suspension (1 × 105/ml) were applied as a foliar spray or soil drench to four healthy 'Bluecrop' plants each in 10-cm plastic pots. Two water-sprayed and two water-drenched plants served as controls. Plants were misted intermittently for 2 days after inoculation. After 7 days at 25 ± 3°C, drench-inoculated plants developed necrotic, sporulating stem lesions at the soil line, while spray-inoculated plants showed reddish brown leaf and stem lesions. At 28 days, three drench-inoculated and one spray-inoculated plant had died, while others showed stem necrosis and wilting. No symptoms were observed on control plants. Fungal colonies reisolated from surface-disinfested symptomatic stem, leaf, and root segments appeared identical to the original isolate. Cy. colhounii was reported to cause a leaf spot on blueberry plants in nurseries in China (3), while Ca. crotalariae (Loos) D.K. Bell & Sobers (= Ca. ilicicola Boedijn & Reitsma) causes stem and root rot of blueberries in North Carolina (4). To our knowledge, this is the first report of Ca. colhounii causing a disease of blueberry in Michigan or the United States. Because of its destructive potential, this pathogen may pose a significant threat in blueberry nurseries.

Root and Crown Rot of Anthurium Caused by Calonectria ilicicola in Iran.

In the autumn of 2008, a severe disease of Anthurium andraeanum with wilting and root and crown rot symptoms was observed in a greenhouse in the Varamin area of Tehran. A species of Calonectria was isolated consistently from symptomatic tissues on 2% potato dextrose agar (PDA). The fungus produced perithecia and a Cylindrocladium anamorph when incubated on carnation leaf agar under near-ultraviolet light at 25°C. Perithechia were reddish brown, subglobose to ovoid, and 300 to 400 μm in diameter. Asci were clavate, hyaline, 90 to 140 × 12 to 19 μm, and tapering to a long thin stalk. Ascospores were fusoid, straight to slightly curved, 1- (-3) septate, and (30-) 37 to 50 (-65) × (4-) 5 to 6.5 (-7) μm (mean = 45 × 6 μm; n = 30). Penicillate conidiophores gave rise to stipe extensions that terminated in sphaeropedunculate vesicles (6-) 7 to 10 (-12) μm in diameter. Conidia were hyaline, cylindrical, rounded at both ends, straight, (45-) 70 to 82 (-90) × (4-) 5 to 6.5(-7) μm (mean = 62 × 6 μm; n = 30), and (1-) 3-septate. On the basis of morphology, the fungus was identified as Calonectria ilicicola Boedijin & Reitsma. Koch's postulates were fulfilled by spray inoculating 1-month-old seedlings with a conidial and mycelial suspension (105 particles per ml) of the fungus obtained from 14-day-old single-spore colonies grown on PDA at 25°C. Following inoculation, all plants were maintained in plastic bags in a glasshouse at 25 ± 1°C. After 15 to 25 days, symptoms resembling those seen in the diseased glasshouse were detected on inoculated plants. C. ilicicola was reisolated from the artificially infected tissues. No symptoms were detected on the control plants. Nucleotide sequences of the internal transcribed spacer (ITS) regions of the nrDNA operon and the partial histone H3 gene were determined for derived strain CPC 16334 as described previously (1,3). The ITS sequence (GenBank Accession No. GU057378) matched 100% (644/644 bp) with the sequence of C. ilicicola strain CBS 463.76 (GenBank AF493963) and the histone H3 sequence (GenBank GU057379) matched 99% (456/458 bp; due to two versus three AC repeats in the sequence) with that of C. ilicicola strain CBS 112217 (GenBank AY725686). To our knowledge, this is the first report of Calonectria and Cylindrocladium genera and the disease caused by C. ilicicola from Iran.

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.