Half-strength Potato Dextrose Agar Research Articles

First Report of Panicle Wilting on Rice (Oryza sativa L.) Caused by Fusarium kyushuense in Heilongjiang Province, China.

Heilongjiang is the largest rice-producing province in China, with annual yield of 28.9 million tons cultivated on 3.8 million hectares (Liu et al. 2021). During field surveys from July to August (2021-2022), symptoms of wilting were observed on rice panicles across Baoqing county (46.32°N, 132.20°E), Shuangyashan city, Heilongjiang province, China. Disease incidence ranged from 10 to 35%, and yield losses were estimated to be 5 to 20% over 7 surveyed fields of 18.5 ha in total. Initially, infected panicles exhibited carmine to brownish spots at the flowering and early grain-filling stages, which gradually merged into large and irregular lesions and spread to the entire panicle surface. Eventually, panicles became wilting and decayed at the ripening stage. To identify the etiological agent, thirty-five symptomatic panicles were collected randomly from 35 plants at different positions in 7 fields. The fragments (approximately 3 mm2) were dissected from margins of individual lesions, surface-disinfested with 70% ethanol for 30 s followed by 2% sodium hypochlorite for 2 min, and rinsed three times in sterilized water. The pieces were then dried and placed onto half-strength potato dextrose agar (PDA) supplemented with 50 μg/mL of streptomycin sulfate. After incubation at 28°C for 4 days, nineteen cultures were obtained and purified using the single-spore isolation method. On PDA plates, the colonies produced fluffy and cottony aerial mycelia and were white to yellowish with deep-yellow to red-brown pigments. The microconidia were hyaline, elliptical or clavate, zero to one septum, measuring 6.3 to 19.2 × 2.6 to 5.1 μm in size (n = 50). On carnation leaf agar (CLA), the macroconidia were thick-walled, falcate to almost straight, three to five septa, apical cell hooked to tapering, basal cell foot-shaped, measuring 27.4 to 47.8 × 3.6 to 5.4 μm in size (n = 50). No chlamydospore was observed. The internal transcribed spacer (ITS) region of ribosomal RNA, translation elongation factor (TEF-1α) gene, and β-tubulin (β-TUB) gene were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1/EF2 (O'Donnell 2000), and T1/T22 (O'Donnell and Cigelnik 1997) from three representative isolates (PJ58, PJ69 and PJ83), respectively. The obtained sequences were deposited in GenBank (accession nos. ON527509, OQ772202 and OQ777725 for ITS; ON573222, OQ784926 and OQ784927 for TEF-1α; ON573223, OQ784928 and OQ784929 for β-TUB, respectively). BLASTn analysis revealed 99.8 to 100% homology with the corresponding sequences of Fusarium kyushuense (MH892849 for ITS, AB674297 for TEF-1α, and GQ915442 for β-TUB, respectively) in GenBank. Maximum likelihood phylogeny based on the concatenated sequences of ITS, TEF-1α and β-TUB grouped three representative isolates in the F. kyushuense clade. Combined with the morphological and molecular characteristics, the fungus was identified to be F. kyushuense. Pathogenicity of the three isolates of F. kyushuense was evaluated on a susceptible rice cultivar Nanjing 46 at the booting stage. The upper part of a healthy panicle was inoculated by injecting 2 ml of a conidial suspension (1 × 106 spore/ml) obtained from a 7-day-old PDA culture of each isolate. The negative control was treated with sterile distilled water. The experiment was performed thrice with ten replicated plants for each treatment. All plants were placed in a humid chamber at 25°C with a 12-h photoperiod and 80% relative humidity. Twenty days after inoculation, it was found that the inoculated panicles showed typical reddish to brownish lesions, whereas control plants remained symptomless. Pathogens were reisolated from the artificially inoculated panicles and confirmed by morphological and molecular tests, fulfilling Koch's postulates. In recent years, this species has been associated with stalk rot and ear rot of maize (Cao et al. 2021; Wang et al. 2014) and wilt of tobacco (Wang et al. 2013). Also, it was mentioned as a producer of mycotoxins, especially trichothecenes and HT-2 toxin (Varga et al. 2016). To our knowledge, this is the first report of F. kyushuense causing panicle wilting on rice in China. The appropriate control strategies should be made to reduce the risk of disease due to food security concerns and potential threats to rice production.

First Report of New Guinea Impatiens (Impatiens hawkeri) Leaf Spot Caused by Alternaria burnsii - A. tomato species complex in Taiwan.

New Guinea impatiens (Impatiens hawkeri) is a common ornamental crop usually planted in pots and planters, flower beds, home gardens, or parks in Taiwan. In June 2021, leaf spots on 87.1% (27/31) of potted I. hawkeri plants on National Chung Hsing University (NCHU) campus were observed. Initially, tiny chlorotic leaf spots were found, which aged into brown to grayish white necrotic spots with reddish-purple margins. The necrotic spots enlarged, coalesced, and formed concentric rings. To isolate the pathogen, diseased leaves were surface-disinfected with 70% ethanol for 15 seconds and blotted dry with a paper towel. Small pieces (~2×6 mm2) of tissues were excised from the junction of the lesions and healthy areas, placed onto 2% water agar, and incubated at 25°C with 12-h photoperiod for three days. Individual hyphal tips growing out of diseased tissues were transferred onto potato dextrose agar (PDA). Three isolates, OM10, OM43, and OM45, were obtained and grown on half-strength PDA at 28°C in the dark for at least two weeks. Conidia of each isolate produced on the half-strength PDA were washed off in sterile water with 0.01% of Tween 20. Pathogenicity tests were performed by spraying leaves of 2- to 3-month-old potted healthy I. hawkeri plants with 5 ml of conidial suspension (1 × 105 conidia/ml) of the three isolates, respectively. Control plants were sprayed with sterile water. There were four plants per treatment and the experiments were conducted twice. Inoculated plants were covered with plastic bags for two days and incubated in a greenhouse with a temperature range of 19 to 31°C. Leaf spots similar to those observed in the field were observed at 7 to 14 days after inoculation in both trials. The same fungus was isolated from inoculated plants, whereas control plants showed no symptoms. Thereafter, the three isolates were subjected to morphological and molecular identification. Colonies were brown to gray in the center and white in the border with abundant aerial mycelia. Conidia were brown, obclavate to ovoid, produced in single or branched chains, one to seven transverse and zero to three longitudinal septa. Conidial size of the three isolates ranged between 11.2 to 43.1 × 6.0 to 12.7 μm (n = 50 for each isolate). Conidiophores of the three isolates were dark-brown, septate, branched or unbranched, and measured 27.0 to 147.65 × 2.71 to 4.54 μm (n = 50 for each isolate). Based on the morphological characteristics, the three isolates were identified as Alternaria spp. (Simmons 2007). For molecular identification, the internal transcribed spacer (ITS) region, RNA polymerase II second largest subunit (RPB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and major allergen Alt-a-1 gene (Alt-a-1) were amplified using primer pairs ITS1/ITS4 (White et al. 1990), RPB2-5F2/RPB2-7cR (Sung et al. 2007), gpd1/gpd2 (Berbee et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequence analyses of isolates OM10 (ITS: GenBank Accession no. OP358436; RPB2: OP377483; GAPDH: OP377468; Alt-a-1: OP377471), OM43 (ITS: OP358437; RPB2: OP377484; GAPDH: OP377469; Alt-a-1: OP377472), and OM45 (ITS: OP358438; RPB2: OP377485; GAPDH: OP377470; Alt-a-1: OP377473) showed 100%, 99.61 to 100%, 99.65%, and 100% identities with a reference strain CBS 107.38 of A. burnsii for ITS (KP124420), RPB2 (KP124889), GAPDH (JQ646305), and Alt-a-1 (KP123967), respectively. They also showed 100%, 99.61 to 100%, 99.65%, and 99.58% identities with an A. tomato strain CBS 103.30 for ITS (KP124445), RPB2 (KP124915), GAPDH (KP124294), and Alt-a-1 (KP123991), respectively. Based on the morphological and sequence characteristics, the pathogen causing New Guinea impatiens leaf spot was identified as a member of the Alternaria burnsii - A. tomato species complex. The diseased plants on NCHU campus were destroyed. There have been no reports of the disease in other landscape areas or nurseries. To our knowledge, this is the first report of A. burnsii - A. tomato species complex causing New Guinea impatiens leaf spot in Taiwan. Since the pathogens in the species complex have been documented causing diseases on several important economic crops and the New Guinea impatiens is widely planted in nurseries and landscapes, the host range and the significance of the pathogen in agro-ecosystem may warrant further investigations.

In Vitro Antagonistic Action by Bacillus Velezensis Strain LP16S Against Cotton Wilt Pathogens

Cotton productivity and profitability are hampered by several biotic stresses, including the two most destructive wilt pathogens Verticillium dahliae and Fusarium oxysporum f. sp. vasinfectum (FOV). In this study, an in vitro assay was conducted to determine the activity of Bacillus velezensis LP16S against three V. dahliae isolates and six F. oxysporum f. sp. vasinfectum isolates. Among the fungal isolates, the response when exposed to the B. velezensis LP16S strain in a half-strength potato dextrose agar plate varied markedly. Fusarium oxysporum f. sp. vasinfectum isolates FOV11, FOV 944, and FOV Tx8 were tolerant, while FOV Tx39, FOV 1073, and the three V. dahliae isolates were highly sensitive to B. velezensis LP16S. In conclusion, this Bacillus sp. strain has potential for use in managing these damaging cotton diseases.

Sugar beet root rot caused by oomycetous pathogens in Turkey and their control by seed treatmen

The aim of this study was to determine the oomycetous pathogens causing root rot on sugar beet and test their control by seed treatment in Konya Province, Turkey. Oomycetous fungus-like pathogens of sugar beet were investigated using 866 plant samples collected at 2 growth stages, early seedling and late root, from fields in the Konya region of Turkey and 1 sample from the Thrace region. Herein, 10 oomycetous species belonging to 3 genera: Aphanomyces cochlioides, Phytophthora cryptogea, Ph. pseudocryptogea, Ph. megasperma, Ph. inundata, Pythium aphanidermatum, Py. helicoides, Py. heterothallicum, Py. sylvaticum, and Py. ultimum var. ultimum (Globisporangium ultimum var. ultimum) were discovered at various times with in the 2 growth periods, all of which were the first records for Turkey. A. cochlioides was the most serious pathogen, both in terms of its wide distribution and aggressiveness. The pathogen produced more than 90% disease severity when tested by soil infestation at the seedling stage, although it also occurred at the late root growth stage. Pythium species were also ascommon, such as A. cochlioides, the majority of which were very aggressive, producing more than 84% disease severity at the seedling stage, except for Py. aphanidermatum. Half-strength potato dextrose agar medium was found to be very useful for the isolation of all of the pathogens from the plant samples at both stages. Morphological features of all of the pathogens were abundantly produced when the pathogens were grown on amended grated carrot agar medium and culture disks of fungal growth of this medium were submerged in sterile and nonsterile soil extracts. Out of the 15 fungicide mixes tested, 2 mixes, thiram+metalaxyl+hymexazole and thiram+metalaxyl+hymexazole+ pyraclostrobin reduced seedling root rot caused by both A. cochlioides and Pythium ultimum var. ultimum, while the standard seed treatment fungicide mix of thiram+hymexazole was not effective against either of the pathogens.

First report of basal rot of dragon fruit caused by Fusarium oxysporum in Bangladesh.

Dragon fruit (Hylocereus polyrhizus) is a high value newly introduced fruit crop in Bangladesh. It has drawn considerable public attention due to its appealing flesh color, sweet taste and fruit qualities. Recently, basal rot of dragon fruit plants was observed in several farmer's fields, nurseries and in the research field of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) where about 10-15% of plants were infected in each location. Initially, the symptoms appeared in the basal part near the soil as brown lesions which gradually extended to the upper stem and finally becoming soft and watery (Figure 1a). Infected plants were collected from Kapasia of Gazipur district (Latitude 24.266 and Longitude 90.633) to isolate the causal organism. Isolations were carried out following the procedure reported by Briste et al. (2019). Briefly, infected plant parts were surface sterilized in 2% NaOCl for 1 min followed by 70% ethanol for 5 min and rinsed 3 times with sterile double distilled water. A large piece of a surface sterilized plant was cut into small pieces (2 mm × 2 mm) from the margin of the necrotic lesion and placed on half strength potato dextrose agar (PDA) and incubated for 7 days at 25 °C. The BTFD1 and BTFD4 isolates were purified from single spores resulting in white colonies with a growth rate of 1cm/day on PDA (Figure 1b). Colonies produced single celled microconidia from unbranched, short monophialidic conidiophores and septate macroconidia as well as chlamydospores in PDA which is consistent with Fusarium oxysporum (Figure 1c). To confirm the identity of the isolates, the internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) and translation elongation factor-1alpha (EF-1α) were amplified using primers ITS-1/ ITS-4 and EF1-728F/ EF1-986R, respectively (Surovy et al. 2018). The ITS sequences of the isolates BTFD1 and BTFD4 (GenBank accession # MN727096 and MN727095, respectively) showed 100% similarity with the sequence from F. oxysporum strain JJF2 (MN626452). Sequence identity for EF-1α (GenBank accession # MN752123 and MN752124, respectively) was 100% with the sequence from F. oxysporum strain CAV041_EO (MK783088). The isolates (BTFD1 and BTFD4) were identified as F. oxysporum based on the aligned sequences of ITS and EF-1α, molecular phylogenetic analyses by maximum likelihood tree (Figure 2a) and maximum parsimony tree methods (Figure 2b). The isolates were stored at 4°C on dried filter paper as well as in an ultra-low temperature freezer (-80°C) at IBGE, BSMRAU, Bangladesh and are available on request. To ensure pathogenicity, isolate BTFD1 was grown on PDA, incubated at 25°C for 7 days and 250 ml conidial suspension (with 1 × 105 conidia/ml) was prepared. Twelve,three-month-old healthy dragon fruit plants were inoculated. Pathogenicity tests were carried out in two sets using three replications in each set. In one set, only the basal part of the plants was dipped into the conidial suspension and in another set the whole plant was dipped into the conidial suspension for two hours. Sterile distilled water was also used in another set of plants as a control. The inoculated plants were placed on wet tissue in a plastic box (31cm × 24cm × 8cm) covered and incubated at 25°C. After 10 days, all inoculated plants in both sets developed rot symptoms similar to those observed in the field, while the control plants remained healthy (Figure 1d). The pathogen was successfully re-isolated from the inoculated symptomatic parts on half strength PDA medium and had morphology as characterized before, thus fulfilling Koch's postulates. This disease has been reported in Argentina and Malaysia (Wright et al. 2007; Hafifi et al. 2019). To the bet of our knowledge, this is the first report of Fusarium basal rot of dragon fruit in Bangladesh caused by F. oxysporum.

First Report on Fusarium oxysporum f. sp. niveum Causing Watermelon Fusarium Wilt in Bangladesh

Watermelon (Citrullus lanatus) is a popular summer fruit in Bangladesh. Production of this fruit was 293,956 and 253,661 metric tons in 2014–2015 and 2016–2017, respectively (Bangladesh Bureau of Statistics 2017). Watermelon fields were affected by pest and diseases before harvest in 2018 (Star Country Desk 2018). During February to March 2018, 30 to 70% of watermelon plants wilted were observed in commercial fields of Subornachar (22.662586° N, 91.094281° E), Noakhali, Bangladesh. The infected stems and roots (2- to 3-cm pieces) were surface sterilized with 1% NaOCl for 5 min, rinsed three times with sterile distilled water, and placed on half-strength potato dextrose agar (PDA). The culture plates were incubated at 25 ± 2°C for 7 days and purified to obtain single-spore isolates. The single-spored isolates produced flat, round, light purple mycelial growth on the surface and reverse of PDA plates. Hyphae were smooth, hyaline, branched, and septate. Macroconidia were mainly fusiform with three septa. Single-celled oval microconidia were produced in monophialidic conidiophores that looked like false heads. Fungal inoculum of two isolates, NSWF001 and NSWF004, were prepared with a mixture of sterilized broken maize, wheat bran, and grass pea pod seed coat (1:10:5 w/w) for soil inoculation. Thirty-five pieces (5 mm in diameter) of PDA grown with 7-day-old mycelium of each isolate were used to inoculate into 1 kg of mixture and incubated for 10 days at room temperature with a 12-h photoperiod. To confirm their pathogenicity, 15-day-old seedlings (10 seedlings/isolate, repeated three times) of watermelon cultivar Sugar Emperor were transplanted into 9-cm-diameter pots filled with soil (treated with 10 g of inoculum/kg of soil). After 2 weeks, the transplanted seedlings in the inoculated soil showed stunted growth and wilt symptoms similar to those observed in the farmer’s fields. The control seedlings remained healthy. Light orange discoloration was observed on crown tissues in longitudinal cut of stems and roots of inoculated seedlings. Control seedlings remained symptomless. The fungus was successfully reisolated from stems and roots of infected seedlings on half-strength PDA with the same morphological characters as before, which fulfilled Koch’s postulates. The internal transcribed spacers (ITS) of the two isolates were amplified by using primers ITS-1/ITS-4 (White et al. 1990). The ITS sequences of the two isolates NSWF001 (481 bp) and NSWF004 (479 bp) (GenBank accession nos. MN148629 and MN148630) showed 100% similarity with e-value of 0 to the reference sequences of Fusarium oxysporum (MN633363) and F. oxysporum f. sp. niveum (KX275296). In Fusarium MLST database, both NSWF001 and NSWF004 isolates revealed 100% similarity with isolate CBS 159.57 of F. oxysporum f. sp. cyclaminis. O’Donnell et al. (2009) found F. oxysporum f. sp. forming a cluster with different groups of the F. oxysporum species complex because of its polyphyletic nature. Based on the aligned sequences of ITS, molecular phylogenetic analysis by the maximum likelihood method, the isolates NSWF001 and NSWF004 were identified as F. oxysporum f. sp. niveum. The isolates are stored on dried filter paper at –20°C and archived at the Plant Pathology Division, Bangladesh Agricultural Research Institute. To the best of our knowledge, this is the first report of watermelon Fusarium wilt caused F. oxysporum f. sp. niveum in Bangladesh.

First Report of Red Crown Rot of Soybeans Caused by Calonectria ilicicola (Anamorph: Cylindrocladium parasiticum) in Illinois

HomePlant DiseaseVol. 103, No. 7First Report of Red Crown Rot of Soybeans Caused by Calonectria ilicicola (Anamorph: Cylindrocladium parasiticum) in Illinois PreviousNext DISEASE NOTESFirst Report of Red Crown Rot of Soybeans Caused by Calonectria ilicicola (Anamorph: Cylindrocladium parasiticum) in IllinoisN. Kleczewski, D. Plewa, C. Kangas, E. Phillippi, and V. KleczewskiN. Kleczewski†Corresponding author: N. Kleczewski; E-mail Address: nathank@illinois.eduhttp://orcid.org/0000-0001-5671-6727University of Illinois, Department of Crop Sciences, Urbana, IL 61801Search for more papers by this author, D. PlewaUniversity of Illinois, Department of Crop Sciences, Urbana, IL 61801Search for more papers by this author, C. KangasUniversity of Illinois, Department of Crop Sciences, Urbana, IL 61801Search for more papers by this author, E. PhillippiUniversity of Illinois, Department of Crop Sciences, Urbana, IL 61801Search for more papers by this author, and V. KleczewskiGrowmark Inc., Bloomington, IL 61701Search for more papers by this authorAffiliationsAuthors and Affiliations N. Kleczewski1 † D. Plewa1 C. Kangas1 E. Phillippi1 V. Kleczewski2 1University of Illinois, Department of Crop Sciences, Urbana, IL 61801 2Growmark Inc., Bloomington, IL 61701 Published Online:7 May 2019https://doi.org/10.1094/PDIS-01-19-0105-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In September 2018 soybean (Glycine max) disease was observed in a commercial field located near Pittsfield, Illinois. Signs and symptoms resembled those of red crown rot, caused by Calonectria ilicicola (Hartman et al. 2015). Red to brown lesions were observed at the stem base, and red, raised perithecia were observed on the lesion surface. The root systems of affected plants were black and severely rotted, often with only taproots remaining after removal from the soil. The inner pith of the lower stem was light gray in appearance. Foliar symptoms included interveinal chlorosis and defoliation. Disease incidence was estimated at 75%, and yield loss was estimated at approximately 25%. Affected plants were harvested, placed in a cooler on ice, and transported to the University of Illinois. Stems were surface sterilized in 10% bleach (30 s), 75% ethanol (30 s), followed by two rinses in sterile distilled water bleach, and allowed to air dry under a biosecurity cabinet. Isolations were made from sections of symptomatic stems on Petri plates containing half-strength potato dextrose agar placed under a light bank equipped with cool fluorescent lights set on a 12-h light/dark cycle at 20°C. Fungi were isolated from 100% of sections plated, and individual isolates were collected via hyphal tip technique. Isolated colonies initially produced white, aerial mycelia that later turned burnt red to brown with age. Conidia were hyaline, cylindrical with zero to three septae, and measured 28.5 to 87.1 × 3.9 to 7.6 μm. Perithecia were reddish orange, oval to globose, 202.6 to 424.7 μm tall, and 100.3 to 353.8 μm in width. Asci were hyaline and clavate and measured 111.0 to 206.1 × 10.5 to 28.6 μm, and each contained eight falcate ascospores with one to two septa. Morphological characteristics identified this organism as C. ilicicola (anamorph: Cylindrocladium parasiticum [Hartman et al. 2015]). A 600-bp fragment of the β-tubulin gene was amplified using the T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers. Thermocycler conditions were as follows: initial denaturation for 10 min at 96°C followed by 30 cycles of 96°C for 15 s, 55°C for 35 s, and 75°C for 35 s, with a final elongation step of 75°C for 2 min (Crous et al. 1999). Polymerase chain reaction products were purified and sequenced at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. A sequence of the representative isolate 3A was submitted to GenBank (MK370992). Comparisons with GenBank showed 99% similarity with multiple sequences of C. ilicicola (top match JX0693831.1, bit score 1,107, E = 0.0, 607/610 bp). Pathogenicity was confirmed by inoculating 3-week-old seedlings of soybean cultivar Pioneer 93Y25 grown in in 15-cm-diameter pots containing autoclaved greenhouse potting media. Six plants were present in each pot, and three pots were used per treatment. Inoculation was conducted by drenching with a 105 conidia/ml suspension acquired from 14-day-old, single-spore fungal isolates. Three unamended pots were used as controls. Plants were watered daily and maintained in a greenhouse set to 23°C and a 14-h/10-h light/dark cycle. After 14 days, inoculated plants showed similar basal stem symptoms and after 30 days developed interveinal chlorosis on leaves. C. ilicicola was reisolated from diseased plants and confirmed through microscopy. This pathogen may pose a threat to >440,000,000 ha of soybean in Illinois, not including surrounding states. Our understanding of impact and management under Midwest production practices is currently unknown.The author(s) declare no conflict of interest.

First Report of Neofusicoccum parvum Causing Pod Rot on Cacao in Hawaii

HomePlant DiseaseVol. 103, No. 6First Report of Neofusicoccum parvum Causing Pod Rot on Cacao in Hawaii PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Neofusicoccum parvum Causing Pod Rot on Cacao in HawaiiA. S. Puig, J. P. Marelli, T. K. Matsumoto, L. M. Keith, and O. A. GutierrezA. S. Puig†Corresponding author: A. S. Puig; E-mail Address: alina.puig@ars.usda.govhttp://orcid.org/0000-0002-4662-4658Subtropical Horticultural Research Station, USDA-ARS, Miami, FL; Search for more papers by this author, J. P. MarelliMars/USDA Cocoa Laboratory, Miami, FL 33158; and Search for more papers by this author, T. K. MatsumotoDaniel K. Inouye U.S. Pacific Basin Agriculture Research Center, USDA-ARS, Hilo, HISearch for more papers by this author, L. M. Keithhttp://orcid.org/0000-0001-9974-1818Daniel K. Inouye U.S. Pacific Basin Agriculture Research Center, USDA-ARS, Hilo, HISearch for more papers by this author, and O. A. GutierrezSubtropical Horticultural Research Station, USDA-ARS, Miami, FL; Search for more papers by this authorAffiliationsAuthors and Affiliations A. S. Puig1 † J. P. Marelli2 T. K. Matsumoto3 L. M. Keith3 O. A. Gutierrez1 1Subtropical Horticultural Research Station, USDA-ARS, Miami, FL; 2Mars/USDA Cocoa Laboratory, Miami, FL 33158; and 3Daniel K. Inouye U.S. Pacific Basin Agriculture Research Center, USDA-ARS, Hilo, HI Published Online:2 Apr 2019https://doi.org/10.1094/PDIS-10-18-1719-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Cacao (Theobroma cacao) production is a rapidly expanding industry in Hawaii, but little information exists regarding diseases present in the area. A trip was conducted in January 2018 to assess disease problems on Hawaii Island, and pods with firm, dark brown/black lesions were observed on three of the four farms visited. Symptomatic tissue was sampled from lesion margins and inserted into a healthy, green cacao pod to maximize recovery of primary pathogens (Chee and Foong 1968). After 3 days, tissue was excised from the margins of emerging lesions, surface disinfested in 70% ethanol for 20 s, and plated on half-strength potato dextrose agar (1/2 PDA). Samples from two of the three farms yielded Phytophthora palmivora, the causal organism of black pod rot, one of the most economically important diseases of T. cacao. However, all samples from the third farm yielded a fast-growing fungus with white, aerial mycelia that turned dark gray within 4 days. Disease incidence at this farm was less than 1% (four affected trees), with all infections emanating from the peduncle. A single organism was recovered from three of the five pods sampled, with no organisms being recovered from the other two. A single representative isolate (H23) was used for molecular analysis and pathogenicity tests. DNA was extracted from 10-day-old mycelium, using the Qiagen DNeasy Plant Mini Kit, and the organism was identified by amplifying and sequencing, and by depositing in GenBank sequences of the internal transcribed spacer region (ITS; primers ITS1F/ITS4; MH449672), the translation elongation factor 1-α gene (TEF1; primers EF1-728F/EF1-986R; MH93602), and a partial sequence of the β-tubulin gene (BT; primers Bt2a/Bt2b; MH936021). BLASTN analysis identified the organism as Neofusicoccum parvum based on the ITS (99% match to MF631021), TEF1 (100% match to FJ150710), and BT (100% match to KX464975) sequences. All matching sequences were generated from organisms isolated in Hawaii from a range of host plants such as Metrosideros polymorpha, Pipturus albidus, T. cacao, and Leucadendron cultivar Safari Sunset. Owing to recent additions and reorganization of the Neofusicoccum genus, searches were limited to entries published or modified between 2012 to 2018 to ensure accuracy. To confirm pathogenicity, artificial inoculations were done using 4- × 15-cm2 pieces of immature cacao pods (clones SCA 6, EQX 3348-52, and GNV 164). Plugs of 1/2 PDA with actively growing mycelia were inoculated into wounds made with a 6-mm-diameter cork borer. A P. palmivora isolate from cacao served as a positive control and 1/2 PDA as a negative control. Four replications were inoculated with N. parvum, four with P. palmivora, and three with 1/2 PDA, and they were then kept at 25°C in the dark for 5 days. Necrotic lesions identical to those originally seen on the field-collected samples developed on all pathogen-inoculated pieces. A Welch’s ANOVA with posthoc t tests for mean separation determined that lesion radii were significantly higher for N. parvum (33.9 ± 5.8 mm) and P. palmivora (29.4 ± 1.6 mm) versus the negative control (1.3 ± 0.6 mm) (P = 0.0006). The pathogens were reisolated and identified based on colony morphology. Although N. parvum has been reported to cause dieback on ‘ōhi‘a trees in Hawaii (Hughes et al. 2018) and blueberry in Chile (Espinoza et al. 2009), to our knowledge, this is the first report of N. parvum causing pod rot on cacao. Hawaii has a burgeoning cacao industry, and thus detailed knowledge of pathogens targeting the crop is critical.The author(s) declare no conflict of interest.

First Report of Fusarium Wilt Caused by Fusarium oxysporum on Strawberry in Bangladesh

Strawberry (Fragaria × ananassa) is a newly introduced small fruit crop in Bangladesh, which has gained considerable interest from both growers and consumers. Increasing incidence of wilt and crown rot diseases is a challenge for strawberry growers. In March 2016, wilted foliage and dead strawberry plants of cv. Strawberry Festival were found in a strawberry nursery in Rajshahi district of Bangladesh affecting 10% of the plants. Symptoms included wilting of foliage, drying and withering of older leaves, stunted growth, and reduced fruit production, leading to eventual plant collapse. A lengthwise cut through the crown of diseased plants showed distinct brown to reddish discoloration similar to that described by Koike et al. (2009). The crowns were surface sterilized in 2% NaOCl for 1 min followed by 70% ethanol for 5 min. Small pieces (2 × 2 mm) of the internal discolored tissue were placed on half-strength potato dextrose agar (PDA) medium and incubated for 7 days at 25°C. Single-spore isolates (BRSP-02 and BRSP-12) were obtained and plated to PDA, cornmeal agar, and V8 juice agar. Isolates had a growth rate 0.8 cm/day on PDA medium and produced sporodochia with septate macroconidia. Colonies also produced single-celled microconidia from unbranched, short, monophialidic conidiophores on PDA consistent with Fusarium oxysporum (Nelson et al. 1983). The internal transcribed spacer (ITS1, 5.8S rRNA, and ITS2), actin (ACT), and translation elongation factor 1-alpha (EF-1α) of the isolates were amplified using primers ITS-1/ITS-4, ACT-512F/ACT-783R, and EF1-728F/EF1-986R, respectively (Carbone and Khon 1999; White et al. 1990). ITS sequences of the isolates BRSP-02 and BRSP-12 (GenBank accession nos. LC383470 and LC383471, respectively) showed 99% similarity with the sequence from F. oxysporum strain SHBV2 (KY090783). Sequence identity for EF-1α (GenBank accession nos. LC383472 and LC383473, respectively) was 98 to 99% with the sequence from F. oxysporum isolate BRIP59170 (KX434915). The ACT sequences (GenBank accession nos. LC383784 and LC383785, respectively) showed 97 to 98% similarity with the sequence from F. oxysporum isolate C-11 (KY798318). Based on the aligned sequences of ITS and EF-1α, molecular phylogenetic analysis by the maximum likelihood method and maximum parsimony method, the isolates (BRSP-02 and BRSP-12) were named as F. oxysporum. The isolates are archived in the Department of Biotechnology at Bangabandhu Sheikh Mujibur Rahman Agricultural University in Bangladesh on dried filter paper at 4°C and are available on request. To confirm the pathogenicity of the fungus, five 3-month-old tissue-cultured ‘Strawberry Festival’ plants were inoculated by dipping the roots into either 400 ml of conidial suspension (with 5 × 10⁶ conidia/ml) of the isolate BRSP-12 or into sterile distilled water (control) for 15 min. The inoculated plants were transplanted into plastic pots with artificial peat-based soil mix and maintained in the greenhouse with a high daytime temperature of 25°C and low nighttime temperature of 20°C. After 3 weeks, all plants inoculated with isolate BRSP-12 were stunted and developed wilt symptoms similar to those observed in the field, whereas the control plants remained healthy. Plants were then dug out, and crowns were cut longitudinally to check for internal discoloration. Light orange discoloration of crown tissues was observed in inoculated plants, but all the control plants remained symptomless. The fungus was successfully reisolated on half-strength PDA medium from 100% of crowns and had morphology as characterized before, fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of Fusarium wilt of strawberry in Bangladesh. This disease has been reported from a number of other countries including Argentina, Australia, China, South Korea, Spain, the United States, and Japan (Koike et al. 2009; Williamson et al. 2012).

First Report of a Leaf Anthracnose on Rohdea japonica (Japanese Sacred Lily) Caused by Colletotrichum liriopes (Glomerella Species) in the United States.

HomePlant DiseaseVol. 102, No. 11First Report of a Leaf Anthracnose on Rohdea japonica (Japanese Sacred Lily) Caused by Colletotrichum liriopes (Glomerella Species) in the United States PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of a Leaf Anthracnose on Rohdea japonica (Japanese Sacred Lily) Caused by Colletotrichum liriopes (Glomerella Species) in the United StatesR. N. Trigiano, S. L. Boggess, E. C. Bernard, and A. S. WindhamR. N. Trigiano†Corresponding author: R. N. Trigiano; E-mail: E-mail Address: rtrigian@utk.eduhttp://orcid.org/0000-0002-7264-1822Search for more papers by this author, S. L. BoggessSearch for more papers by this author, E. C. BernardSearch for more papers by this author, and A. S. WindhamSearch for more papers by this authorAffiliationsAuthors and Affiliations R. N. Trigiano † S. L. Boggess E. C. Bernard , Department of Entomology and Plant Pathology, University of Tennessee, Knoxville, 37996-4560 A. S. Windham , Department of Entomology and Plant Pathology, University of Tennessee, Nashville, 37211. Published Online:24 Aug 2018https://doi.org/10.1094/PDIS-03-18-0388-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Rohdea japonica (Asparagaceae: Japanese sacred lily) is an ornamental garden plant in the United States but is native to eastern Asia. The species is typically evergreen in warm growing areas but can be deciduous in colder climates, has inconspicuous flowers, and produces bright red berries that are persistent in winter. In May of 2016 and 2017, anthracnose-like, circular lesions with red borders surrounded by chlorotic tissues were observed on leaves of R. japonica at the University of Tennessee, Knoxville. Signs included numerous cushion-like, dark acervuli with prominent, long, black setae formed on necrotic tissue. Conidia were single celled, colorless, fusiform to linear, and had a mean (n = 20) length of 24.8 µm (21.3 to 27.3 µm) and a mean width of 4.4 µm (4.0 to 5.1 µm). The description of the disease and the fungus agreed well with the reports of Colletotrichum liriopes infecting R. japonica (Kwon and Kim 2013) and Liriope muscari (Oo and Oh 2017) in Korea, except the conidia in these reports were slightly narrower. A similar disease of R. japonica caused by a Colletotrichum sp. was reported from California (French 1989). Lesions with a border of healthy tissue were excised and dipped in 70% ethanol for 30 s, immersed in 1.3% NaOCl for 10 min, and rinsed three times in sterile water. Tissues from the margin of lesions were transferred to half-strength potato dextrose agar augmented with 10 mg of rifampicin/liter and incubated in the dark. Two colony morphotypes were apparent after 1 week: a dark, black, flocculent mycelium with conidiomata and conidia, without setae, and a light brown, appressed slimy mycelium with numerous red-brown perithecia formed on orange-pigmented mycelium. The eight ascospores/ascus had a mean (n = 20) length of 18.5 µm (14.4 to 20.8 µm) and a mean width of 6.8 µm (5.8 to 7.6 µm). A Glomerella sp. sexual stage is often associated with Colletotrichum spp., but none has been reported for C. liriopes (Réblová et al. 2011). Sclerotia (Kwon and Kim 2013; Oo and Oh 2017) were not observed in our cultures. DNA was extracted from both colony morphotypes and amplified with ITS1-S4 primers (White et al. 1990), and the amplicons were sequenced. Sequences from both colony morphotypes were identical (MH005034) and aligned perfectly with GenBank accession HM585396 for C. liriopes. Sequences for five other C. liriopes entries in GenBank were the same as our sequence except there was a single nucleotide variant (C to A) at bp 494. Leaves of 10 plants were sprayed with a 1 × 105 conidia/ml suspension to runoff, and 10 control plants with sterile water. Plants in containers were placed in plastic bags and maintained in high humidity at room temperature for 2 days and then grown for 28 days exposed to natural light in our lab. Anthracnose symptoms appeared after 10 to 14 days and were identical to the original disease profile; control plants were symptomless. Only the dark black mycelium was reisolated from lesions. We concluded that the disease on R. japonica was caused by C. liriopes. The disease did not kill plants in Tennessee but did affect their aesthetic quality, and therefore, control measures should be developed and employed.

First Report of Cercospora apii s. lat. Causing a Leaf Spot on Cardinal Flower (Lobelia cardinalis) in the United States

Cardinal flower (Lobelia cardinalis L.: Campanulaceae) is a wildflower common throughout much of the eastern United States and grows in a variety of habitats. It is used as an ornamental plant because its bright red flowers appear in the fall and attract hummingbirds. The foliage is generally free of disease when the plant is not flowering, although a few leaf and stem lesions may be present during the growing season. In August/September 2016 and 2017, plants established in eastern Tennessee developed numerous spots on stems and leaves. The round- to lenticular-shaped lesions had dry, white, papery necrotic centers surrounded by reddish-purple host tissue. There were no signs of a pathogen. Leaves were washed in running water for 5 min, surface disinfested with 1.05% NaClO for 15 min, and rinsed three times in sterile water. Lesions and some green tissue from leaves were transferred to half-strength potato dextrose agar (PDA) augmented with 10 mg rifampicin/liter, and incubated in the dark. Mycelium was i...

First Report of Forsythia Shoot Blight Caused by Phytophthora nicotianae in Connecticut

Blighting of Forsythia × intermedia 'Showoff' was first observed affecting several hundred plants in a commercial nursery in Connecticut in September 2012. Symptoms included wilting, leaf and stem blight, and dieback progressing to plant death. A Phytophthora sp. was isolated from symptomatic tissues on half-strength potato dextrose agar (½PDA). Colonies were white and cottony on ½PDA, reaching 9 mm in 15 days at 25°C, but colorless and inconspicuous on pimaricin, ampicillin, rifampicin, pentachloronitrobenzene agar (PARP) with sparse and limited aerial mycelium, reaching 60 mm in 15 days at 25°C. The characteristics of the pathogen were observed and measured from a 3-month-old colony on ½PDA. Sporangia were abundant, various in shape, ovoid, ellipsoid to pyriform or limoniform, occasionally gourd shaped or irregular; (17.9) 27.2 to 41.4 (47.3) × (12.6) 19.1 to 30.5 (36.7) μm (n = 30), length/breadth ratio 1.4 ± 0.2, papillate and noncaducous. Papillae measured 2.9 ± 0.8 × 3.4 ± 0.8 μm (n = 10). Chlamydospores were present, 23.4 ± 3.1 × 22 ± 3.3 μm (n = 10). Oogonia and oospores were not observed. Arachnoid mycelia were present. These morphological characteristics are consistent with Phytophthora nicotianae Breda de Haan (1). The identity of the pathogen was confirmed as P. nicotianae by BLAST analysis of ITS, Cox II, and beta tubulin gene sequences (99% match for the three sequences, E value = 0). Pathogenicity tests were conducted four times on healthy liners of Forsythia × intermedia 'Showoff' grown in 10-cm-diameter pots. Leaves and stems were wounded by pricking with a sterile needle and six plants were inoculated with 0.25 cm2 plugs of the pathogen growing on ½PDA placed on three leaves and in three stem nodes and covered with Parafilm. Controls consisted of an equal number of plants wounded and inoculated with ½PDA alone. All plants were placed inside high humidity chambers for 24 h and then transferred to a greenhouse for up to 1 month. Typical symptoms developed within 1 week of inoculation and the pathogen was re-isolated from diseased tissue. Disease incidence was nearly 100% of inoculated leaves and stems and not observed in control plants without the pathogen. Three replicate 6-week-old broadleaf tobacco 'C9' plants were each inoculated with tobacco or forsythia isolates of P. nicotianae or sterile media alone, by wounding stems and placing colonized 0.25 cm2 ½PDA plugs into wounds and covering with Parafilm. After 1 week, stems were split and the length of internal necrosis in the stem measured. Disease resulted from inoculation with both the tobacco and forsythia isolates and stem necrosis averaged 43 and 23 mm for tobacco or forsythia isolates, respectively. No necrosis was observed in the pathogen-free controls. P. nicotianae has been reported from the basal stem and roots of F. viridissima in Italy (2) and from shoots of Forsythia × intermedia in Virginia (3). To our knowledge, this is the first report of P. nicotianae causing shoot blight on Forsythia in the northeastern United States.

First Report of Fusarium proliferatum Causing Dry Rot in Michigan Commercial Potato (Solanum tuberosum) Production

Fusarium dry rot of potato (Solanum tuberosum L.) is a postharvest disease caused by several Fusarium spp. Thirteen Fusarium spp. have been implicated in dry rot of potatoes worldwide. Among them, 11 species have been reported causing potato dry rot of seed tubers in the northern United States (1). Historically, Fusarium sambucinum was the predominant species in Michigan potato production (3). Dry rot symptomatic tubers (n = 972) were collected from Michigan commercial potato storage facilities in 2011 and 2012 to determine the composition of Fusarium spp. Sections were cut from the margins of necrotic tissue with a sterile scalpel and surface disinfested in 0.6% sodium hypochlorite for 10 s, rinsed twice in sterile distilled water, and dried on sterile filter paper. The tissue sections were plated on half-strength potato dextrose agar (PDA) amended with 0.5 g/liter of streptomycin sulfate. Dishes were incubated at 23°C in the dark for 7 days. Putative Fusarium isolates were transferred onto water agar and hyphal tips from the margin of actively growing cultures were removed with a sterile scalpel and plated to carnation leaf agar (CLA) and half-strength PDA to generate pure cultures. Seven hundred and thirty Fusarium isolates were collected using these techniques. Preliminary identification of the 730 isolates was based on colony and conidial morphology on PDA and CLA, respectively. While F. oxysporum and F. sambucinum were isolated as expected from prior reports (3), three isolates of F. proliferatum were also identified. On CLA, macroconidia of F. proliferatum were sparse, slender, and mostly straight, with three to five septae (4). Microconidia were abundant, usually single celled, oval or club-shaped in short chains or false heads on monophialides and polyphialides (4), and chlamydospores were absent. On PDA, abundant white mycelium was produced and turned violet with age. Koch's postulates were confirmed through pathogenicity testing on disease-free potato tubers cvs. Atlantic and Russet Norkotah. Tubers were surface disinfested for 10 min in 0.6% sodium hypochlorite and rinsed twice in distilled water. Three tubers of each cultivar per isolate were wounded at the apical end of the tuber to a depth of 4 to 10 mm with a 4 mm diameter cork-borer. Tubers were inoculated by inserting a mycelial plug from a 7-day-old culture grown on PDA into the wound and incubating the tubers at 20°C for 21 days. All Fusarium isolates were tested. Control tubers were inoculated by inserting a water agar plug. Pathogenicity and virulence testing were replicated three times and repeated. Tubers inoculated with F. proliferatum developed typical potato dry rot symptoms but no dry rot symptoms were observed on control tubers. Fusarium proliferatum was re-isolated from symptomatic tubers, confirming Koch's postulates. To our knowledge, this is the first report of F. proliferatum causing potato dry rot in Michigan.

First Evidence of a Binucleate Rhizoctonia as the Causal Agent of Dry Rot Canker of Sugar Beet in Nebraska.

Sugar beet (Beta vulgaris L.) is the primary source of domestic sucrose in the United States. In 2011, a sugar beet field in Morrill County, NE, was noted with wilting and yellowing symptoms suggestive of Rhizoctonia root and crown rot (RRCR), an important disease of sugar beet primarily caused by Rhizoctonia solani anastomosis group 2-2 (4). While the foliar symptoms were consistent with RRCR, the symptoms on the root were not. Root symptoms consisted of localized, dry, sunken lesions covering brown spongy tissue penetrating deeply into taproots. The surface tissues of the cankers distinctively produced a series of concentric circles. These root symptoms are inconsistent with RRCR, but are suggestive of a rarely occurring disease known as dry rot canker (DRC). DRC was first identified from Utah in 1921 (1), and assumed at the time to be caused by an uncharacterized strain of R. solani. It has since been sporadically but empirically noted from most western sugar beet growing states (4), but little is known about the pathogen or disease due to its infrequent appearances. To investigate the etiology of this disease, necrotic lesion borders were excised from diseased taproots, surface disinfested in 1% (v/v) sodium hypochlorite for 90 s, rinsed with distilled water for 90 s, and after drying on sterile tissue paper, placed on half-strength potato dextrose agar (½PDA) and incubated at 25 to 27°C. After 24 to 36 h, Rhizoctonia-like fungal growth was readily observed emerging from tissue pieces. Resulting colonies were tan to light brown. The ITS region of the rDNA was amplified from 4 isolates obtained from 4 distinct lesions and roots using the ITS1 and ITS4 primers (3) with standard PCR conditions, and sequenced (GenBank KC842197 to KC842200). The ITS regions were 100% identical between the 4 isolates and 96% (E-value = 0.0) identical to binucleate Rhizoctonia and Ceratobasidium sp. AG-F (e.g., JF519832, FR734295, JF705217). Hyphal cells were observed to be binucleate after staining 48-h-old cultures with lactophenol blue. Therefore, these isolates were identified to be a binucleate Rhizoctonia group AG-F based on morphological and molecular characteristics. Although distinct from DRC, a similar phenomenon has been recently reported from China implicating binucleate Rhizoctonia species with seedling disease in sugar beets (2). To determine pathogenicity of DRC isolates, 1- and 2-month-old sugar beet plants grown in 10 cm pots (5 plants per pot with 4 replications per isolate) were inoculated with all 4 isolates by placing 3 mycelial plugs (8 mm diameter) taken from the leading edge of ½PDA plates onto the soil surface of each pot. PDA plugs were utilized as controls. After ~3 weeks, root lesions resembling DRC were observed and isolates were recovered and identified from diseased plants as described above. No symptoms developed on control plants. To our knowledge, this is the first formally confirmed report of DRC on sugar beets in more than 75 years from the Western Hemisphere. The original investigator suspected that the isolates he found inducing this disease were different from typical R. solani isolates based on different symptoms (1). Our results, based on different symptoms but also with distinct molecular, biological, and pathogenicity traits, validate those suspicions while also fulfilling Koch's postulates with binucleate Rhizoctonia AG-F pathogenic to sugar beet that is distinct from the more common R. solani.

First Report of Colletotrichum phormii Causing Anthracnose on New Zealand Flax in the United States.

Phormium colensoi Hook.f. (syn. P. cookianum), New Zealand flax, (family Xanthorrhoeaceae) is popular in ornamental landscapes in the United States because of its sturdy blade-like foliage available in diverse colors. In February 2012, the Oregon State University Plant Clinic received three potted plants of P. colensoi 'Black Adder' from a commercial nursery in Santa Cruz County, California. The margins and midribs of several leaves had brown lesions that were variable in size, and fusiform to ellipsoidal in shape. Embedded in the lesions were black acervuli without setae that exuded salmon-colored spore masses under moist conditions. Conidia were hyaline, cylindrical to fusiform, straight to slightly curved, and 22.4 to 35.2 × 4.0 to 6.4 (average 24.7 × 4.9) μm. Based on morphology, the fungus was confirmed by USDA-APHIS National Identification Services to be Colletotrichum phormii (Henn.) D.F. Farr & Rossman (2). In March 2012, the California Department of Food and Agriculture Plant Pest Diagnostic Lab received additional samples from the same nursery lot (25% disease incidence) from which a similar fungus was recovered. rDNA sequences of the internal transcribed spacer (ITS) region from the California isolate (GenBank KC122681), amplified using primers ITS1 and ITS4 (2), were 100% identical to multiple species of Colletotrichum, including C. phormii by a BLAST query (JQ948446 through JQ948453). ITS sequence similarity alone is not sufficient to address Colletotrichum taxonomy and must be used in combination with host range and morphology (1). Pathogenicity of C. phormii (isolate CDFA986) was tested on three 'Black Adder' plants, which were inoculated with 6-mm agar plugs from a 14-day-old culture grown on half strength potato dextrose agar (PDA). Leaves were wound-inoculated along the midrib using colonized plugs (4). Five leaves per plant were inoculated with C. phormii plugs and five leaves per plant were treated with uncolonized PDA agar plugs as controls. Plants were sprayed with water and incubated in plastic bags at 22°C with a 12-h photoperiod. After 48 h, the bags and caps were removed and plants were kept under the same conditions. Two weeks later, water-soaked lesions had developed on the inoculated leaves. Lesions expanded along the midrib and became fusiform in shape after 21 to 28 days. C. phormii was isolated from lesion margins of all the inoculated leaves, but not from control leaves. This experiment was repeated once with similar results. Another Colletotrichum species, C. gloeosporiodes, also occurs on Phormium spp., but differs from C. phormii in morphology and symptom expression. Subsequent nursery and landscape surveys showed that anthracnose caused by C. phormii occurs on several P. colensoi cultivars as well as on P. tenax in five California counties including Santa Cruz, Yolo, Sacramento, San Luis Obispo, and Solano. C. phormii is also reported to infect P. colensoi and P. tenax in New Zealand, Europe, the United Kingdom, Australia, and South Africa (2,3). To our knowledge, this is the first report of C. phormii causing anthracnose on Phormium in North America. This disease could impact the American nursery trade and New Zealand flax production due to crop loss and increased production costs for pest management.

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 Verticillium Wilt on Lettuce (Lactuca sativa) in Washington Caused by Verticillium tricorpus.

Symptoms of Verticillium wilt were observed on lettuce (Lactuca sativa L.) harvested from high tunnel and open field experimental plots in annual, consecutive spring plantings in western Washington from 2010 to 2012. Leaves had v-shaped, chlorotic lesions, and yellow or brown vascular tissue was noted in the crowns. Total disease incidence increased from 0.2% in 2010 to 1.9% in 2011 and to 14.4% in 2012. Verticillium spp. obtained from infected crown tissues and cultured on half-strength potato dextrose agar medium produced yellow pigment, black microsclerotia, white mycelia, tan chlamydospores, and uniseptate conidia averaging 10.6 × 3.7 μm. Isolates were identified tentatively as Verticillium tricorpus I. (3). Three isolates, Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2, were evaluated for pathogenicity on 4-week-old 'Coastal Star' seedlings in two greenhouse trials. In Trial I, four replicates of two duplicate plants per each isolate, and in Trial II, five replicates of one plant per each isolate were inoculated with conidial suspensions adjusted to 2.0 × 106 and 5.0 × 106 conidia/ml, respectively. Additionally, in each trial, two sets of control treatments of five plants each were inoculated with either an isolate of V. dahliae at the same conidial concentration or with sterile water. Root tips were cut and exposed to the suspensions for 5 s, then seedlings were transplanted into Sunshine Mix #1 (SunGro Horticulture Distribution Inc., Bellevue, WA), and kept in a greenhouse at 17.7 ± 3.4°C. Plants were harvested 8 to 9 weeks post-inoculation, and symptoms were rated visually. Vt.Ls.2010, Vt.Ls.2011-1, and Vt.Ls.2011-2 caused chlorosis and vascular discoloration on 25, 13, and 13% of the plants in Trial I; and 40, 60, and 20% of plants in Trial II, respectively. V. dahliae caused similar symptoms on 25 and 40% of the plants in the two trials, respectively, but these plants had greater intensity and length of vascular discoloration compared with the three test isolates. None of the water control plants were symptomatic. All V. tricorpus isolates were recovered from inoculated plants, and colony morphologies were similar to the original isolates. The internal transcribed spacer (ITS) rDNA of isolate Vt.Ls.2010 was amplified with ITS4 and ITS6 primer sets. ITS rDNA sequences between Vt.Ls.2010 and two isolates of V. tricorpus in GenBank (Accession Nos. FJ900211 and AB353343) were 100% identical. V. tricorpus is considered a weak pathogen of lettuce crops in California (2), but authors in Japan recently reported pathogenic isolates of V. tricorpus on lettuce (4). To our knowledge, this is the first report of Verticillium wilt caused by V. tricorpus in Washington. Lettuce is the number two crop grown in high tunnels in the United States (1), and cropping lettuce continuously in them can increase the risk of this and other soilborne pathogens.

First Report of Verticillium Wilt Caused by V. dahliae on Grafted Solanum aethiopicum in Washington.

Solanum aethiopicum L., previously S. integrifolium Poir. (4), has been used as a rootstock for commercial, grafted eggplant production throughout Asia (3). In August 2010 and 2011, symptoms of Verticillium wilt were observed on 'Epic' eggplant (S. melongena L.) grafted onto S. aethiopicum at two sites with a history of the disease: one in the irrigated, dryland Columbia Basin of eastern Washington near Eltopia, and the other in maritime western Washington near Mount Vernon. Interveinal chlorosis, V-shaped necrotic lesions, and wilting were evident at both sites in both years. Each year, stems of 20 symptomatic plants from each field site were cut at the soil line to a 20-cm length, surface sterilized for 5 min in a 10% bleach solution, rinsed in tap water for 30 s, cut longitudinally, and incubated in moisture chambers for 4 weeks at room temperature in the dark. Microsclerotia that formed in the stems were typical of those produced by V. dahliae. One isolate, 'MVEgg301', from an infected stem at Mount Vernon, developed dark microsclerotia and verticillate conidiophores in a radiating pattern, typical of Verticillium on half-strength potato dextrose agar (1/2 PDA) medium. The internal transcribed spacer (ITS) region of rDNA amplified by PCR assay using primers ITS6 and ITS4 from mycelia sampled directly from 1/2 PDA media revealed a 100% match with ITS rDNA sequences of >50 V. dahliae accessions in GenBank (1). Pathogenicity of 'MVEgg301' was assessed in two tests. In both, 12 each of non-grafted and grafted (with cv. Epic) S. aethiopicum plants were inoculated with 'MVEgg301' by cutting approximately 5 mm off the root tips and dipping the remaining roots in a suspension of 106 conidia/ml for 5 s. Similarly, 12 each of non-grafted and grafted S. aethiopicum plants were cut and dipped similarly in sterile water as controls. Chlorosis, necrosis, and wilting were observed in 11 of the 12 inoculated, non-grafted plants and 8 of the 12 inoculated, grafted plants in Test I. The same symptoms were observed in 10 of the 12 inoculated, non-grafted plants and 10 of the 12 inoculated, grafted plants in Test II. V. dahliae was reisolated and confirmed from symptomatic, inoculated non-grafted and grafted plants using the stem assay and direct PCR assay described above. Chlorosis, necrosis, and wilting were observed in one non-grafted control plant in Test I, and two non-grafted and four grafted control plants in Test II. The symptoms were mild and likely due to nutritional deficiencies; microsclerotia were not observed in any assayed water-inoculated plant stems. Although there are several reports of V. albo-atrum infecting S. aethiopicum in the United States (2), to our knowledge, this is the first report of V. dahliae causing Verticillium wilt on this eggplant species. This finding is significant because S. aethiopicum is used as a rootstock for control of soilborne diseases like Verticillium wilt in commercial grafted eggplant production (3).

First Report of Target Spot of Flue-cured Tobacco Caused by Rhizoctonia solani AG-3 in China.

In early August 2006, a disease caused severe losses in a 1,400-ha field of 5-month-old tobacco plants in Kuandian and Fengcheng Counties, Dandong City of Liaoning in northeast China. Symptoms were observed on almost every plant. Disease symptoms were subsequently observed at nearly 100% incidence in 2,000 ha of fields from three towns in Kaiyuan County and two towns in Xifeng County, Tieling City, Liaoning Province in the second half of August 2006. Symptoms first appeared on leaves as small (2 mm) water soaked spots, and developed into expanded, dark brown lesions (2 cm) on the middle to lower leaves. Each lesion exhibited concentric rings, a necrotic center, and a tear in the center and margin that often resulted in a shot-hole appearance. Fungal isolates were obtained from the margins of lesions that were surface-sterilized by dipping each leaf section into 75% ethyl alcohol for 3 sec, then in 0.1% HgCl2 for 15 sec, rinsing in sterilized distilled water three times, and plating the leaf section onto half-strength potato dextrose agar (PDA). Six isolates were identified as Rhizoctonia solani Kühn on the basis of mycelial characteristics: multinucleate cells, septate hyphae constricted at the junction of hyphae, and hyphal branching at approximately right angles (3). The sequence of the internal transcribed spacer (ITS) 1-5.8S-ITS2 region of rDNA from each of six isolates was amplified by PCR assay using universal primers ITS1 and ITS4. The sequences (GenBank Accession Nos. JQ219152 to JQ219157) matched 100% with the ITS sequence of an isolate of R. solani AG-3 (GQ885147). Koch's postulates were conducted for each of the six isolates by wound-inoculating six tobacco leaves (cv. NC89) detached from a total of three 8-week-old plants. Each tobacco leaf was first surface-sterilized in 0.5% NaOCl for 30 sec, rinsed in sterilized distilled water, and wounded at each of four locations by inserting a needle into the leaf. Each leaf was inoculated by depositing a PDA plug (0.5 cm diameter) colonized with R. solani onto each of the four wounds; wounded control leaves (six tobacco leaves from a total of three plants) were inoculated similarly with non-colonized PDA plugs. Inoculated leaves were incubated at 28°C in natural light within a plastic container covered with a hyaline cap to maintain high relative humidity. Symptoms similar to those observed on the original plants developed on inoculated leaves within 3 days, but not on the control leaves. The pathogen was reisolated from symptomatic leaves but not from control leaves and showed morphological characteristics consistent with those of R. solani. Tobacco target spot has been recorded in South America (1), South Africa (4), Argentina, and the USA (2). However, to our knowledge, this is the first report of target spot caused by R. solani AG-3 on flue-cured tobacco in China.

First Report of Leaf Curl on Celery Caused by Colletotrichum acutatum in the United States.

In June 2010 and July 2011, celery (Apium graveolens) samples cv. Tango were submitted to the Penn State Plant Disease Clinic from Franklin and Dauphin Counties, PA, respectively. Plants exhibited curling and twisting of leaves and petioles and dark, brownish-black necrotic lesions at the base of the plant, extending up the petioles. A fungal organism with morphology consistent to Colletotrichum acutatum J.H. Simmonds was isolated from plant lesion tissue excised from the Dauphin Co. sample. Grown on half strength potato dextrose agar (PDA), the colony had gray aerial mycelium and a pink reverse. Conidia were 5.1 to 14.5 × 2.6 to 5.1 μm, aseptate, hyaline, elliptical, with one or both ends slightly pointed, and formed from the mycelium or in dense orange masses of acervuli on the aerial surface of the culture. Setae were not present. To test pathogenicity, five 23-week-old plants of the cv. Sonora and five 11-week-old plants each of the cvs. Tango and Tall Utah were sprayed until runoff with a conidial suspension (1.3 × 106 conidia/ml and 1.4 × 106 conidia/ml, respectively) and 0.025% Tween. One plant of each cv. was sprayed with milliQ water and 0.025% Tween as a control. Plastic bags were sprayed with the conidial suspension (milliQ water for the control), and secured over the individual plants for 24 h to create a humidity chamber. Plants were incubated in a growth chamber with a 16-h photoperiod, 25°C day/18°C night temperatures, and 70% humidity. Post-inoculation, all of the cv. Tango plants exhibited leaf cupping and curling after 7 days and most plants had dark stem lesions after 3 weeks, consistent with celery leaf curl symptoms. Plants of cvs. Tall Utah and Sonora developed malformed leaves and leaf curl symptoms 16 days and 10 days post-inoculation, respectively. None of the control plants developed symptoms. Infected tissue was excised from diseased plants, surface disinfested in 0.5% sodium hypochlorite for 45 s and plated on half strength PDA. Fungal colonies consistent with C. acutatum were recovered from all inoculated celery tissues (except two of the five inoculated cv. Tall Utah plants and the negative controls). To verify morphological identification, the internal transcribed spacer (ITS) rDNA region was amplified and sequenced for our original isolate and those recovered from the inoculated plants using ITS1 and ITS4 primers (2) (GenBank Accession No. JQ794875). Sequence homology revealed 99 to 100% similarity to accessioned isolates of C. acutatum, which included the holotype and a paratype of C. acutatum (Accession Nos. AF411700 and AF411701, respectively). Celery leaf curl has been reported to have caused devastating crop losses on celery in Australia (1, 3) and to our knowledge, C. acutatum causing leaf curl of celery has not been officially reported in the United States. Infected celery plants are unmarketable because of the leaf malformation and eventual plant necrosis caused by C. acutatum. As such, this disease could have serious negative implications for celery growers in the United States.