First Report of Alternaria petroselini Causing Leaf Blight of Fennel in Spain.

Hackberry (Celtis australis L.) is widely used for reforestation and as shade tree in parks and roadside plantings in southern Europe (4). In autumn 2011, a foliar disease was observed affecting several trees planted in a garden area located in Alzira (Valencia province, eastern Spain). Symptoms appeared on lower leaf surfaces as reddish to dark brown velvety irregular spots, later becoming grayish brown on the upper surface. Most of the infected trees were prematurely defoliated. Spots on lower leaf surfaces were covered by mycelium, conidiophores, and conidia. Fungal isolates were recovered directly from the structures present on the lesions and by surface-disinfecting small fragments of symptomatic leaf tissue in 0.5% NaOCl, double-rinsing the sections in sterile water, and plating the sections onto potato dextrose agar (PDA) amended with 0.5 g of streptomycin sulfate per liter. Single conidium cultures made onto PDA were maintained for 2 months at 25°C in darkness for morphological examination. Conidia were thick walled, dark reddish brown, often markedly curved or coiled, cylindrical to obclavate, smooth, wrinkled, or verrucose, typically multicellular, 2 to 40 transversely septate and occasionally with 1 to 3 longitudinal or oblique septa that were often constricted, 20 to 96 (44.9) × 6 to 9 (7.1) μm, with an inconspicuous scar at the base. Morphological characters corresponded to the description of Sirosporium celtidis (Biv. ex Spreng) M. B. Ellis published in 1963 (3). The internal transcribed spacer (ITS) region of the rDNA was amplified with the primers ITS1 and ITS4 from DNA extracted from the isolate AL1, and sequenced (GenBank Accession No. JX397963). The sequence was identical to that obtained from an isolate of S. celtidis from the Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (CBS 289.50). Pathogenicity tests were conducted on five 2-year-old hackberry trees by spraying onto the upper and lower leaf surfaces a conidial suspension of S. celtidis (approximately 50 ml/plant, 106 conidia/ml of water). Five control plants were sprayed with sterile water. Plants were covered with clear plastic bags and incubated in a growth chamber for 72 h at 25°C with a 12-h photoperiod. First leaf spots were visible on inoculated plants after 7 days, but symptoms were not observed on control plants. The fungus was reisolated from leaf lesions on inoculated plants, confirming Koch's postulates. S. celtidis was first described in Sicily in 1815 (3) and has been recorded on various hackberry species in Mediterranean countries and the USA (1,2). To our knowledge, this is the first report of the disease in Spain. The economic and ecological significance of the pathogen in natural ecosystems in Spain remains to be determined but it could certainly become a serious problem for nurseries and urban plantings.

Turnip (Brassica rapa subsp. rapa L.) is an annual vegetable crop cultivated for consumption of its succulent root. In July 2011, symptoms consisting of leaf spots 1 to 8 mm in diameter with a dark brown color surrounded by a yellow halo and black sunken lesions in the swollen storage root were observed in production areas in Alicante Province in east-central Spain. Disease incidence was approximately 20% in fields of about 3 ha where infection was highest. Symptomatic leaves and roots collected from turnip cv. Virtudes-Martillo in three affected fields were surface disinfected with 0.5% NaOCl for 2 min, and small fragments from necrotic lesions were plated on potato dextrose agar (PDA) amended with 0.5 g streptomycin sulfate per liter. Alternaria colonies were consistently isolated from affected leaves and roots after 7 days of incubation at 24°C, and were transferred to V-8 with autoclaved turnip cv. Virtudes-Martillo leaves. Two isolates from leaves and two isolates from roots were included in the study. Plates were incubated for 15 days at 24°C with an 8-h fluorescent light period and a 12-h dark period for morphological examination. Conidia produced in culture were mostly solitary or in short chains of 2 to 3 spores, beakless, ovoid to ellipsoid, and light brown. Conidia were 32 to 78 × 13 to 24 μm, with 3 to 7 transverse septa and 1 to 2 longisepta. Aggregated hyphal chains of dark, thick-walled ornamented cells distinctive of Alternaria japonica Yoshii (3) were observed. The 5.8S, ITS2, and 28S ribosomal RNA (rRNA) regions were amplified using the primers ITS3 and ITS4 (4) and sequenced from DNA extracted from the isolate designated as IVIA-A070, obtained from turnip leaves cv. Virtudes-Martillo in Alicante Province (GenBank Accession No. JX983044). The sequence had 100% identity (total score 302, 73% coverage) with that of A. japonica strain ATCC 13618 (2) (AY376639). Pathogenicity tests were performed twice on two 3-month-old plants of turnip cv. Virtudes-Martillo and cv. Blanco-Globo, and cabbage (B. oleracea var. capitata L.) cv. Brunswick. Plants were inoculated by spraying a conidial suspension of the isolate IVIA-A070 (10 ml/plant, 104 conidia/ml water) using manual pressure sprayer. Two plants of each host sprayed with sterile distilled water were used as controls in each experiment. Plants were covered with black plastic bags and incubated in a growth chamber for 48 h at 25°C. Leaf spots similar to those observed in affected plants in the field were visible on all turnip and cabbage plants 4 days after inoculation with the fungus. No symptoms were observed on control plants. Fungal colonies morphologically identified as A. japonica were reisolated from leaf lesions on inoculated turnip and cabbage plants, but not from asymptomatic leaves of control plants. Based on these results, the disease was identified as black spot of turnip caused by A. japonica. In Spain, black spot of brassicas was previously associated only with A. brassicae (Berkeley) Saccardo and A. brassicicola (Schw.) Wiltshire (1).

The production of spinach (Spinacia oleracea L.) in Spain has increased 50% since 2009, mainly due to the commercialization of fresh-cut spinach leaves packaged in modified atmosphere containers. In October 2012, light brown leaf spots 1 to 2 cm in diameter with dark concentric rings were observed in a commercial spinach production area in Valencia Province, Spain. The initial outbreak comprised an area of about 3 ha with a 20% disease incidence. Symptomatic leaves from spinach cv. Apollo were collected in the affected area and were surface disinfected with 0.5% NaOCl for 2 min. Small fragments from lesions were placed onto potato dextrose agar (PDA) amended with 0.5 g streptomycin sulfate/liter. Fungal colonies developed after 3 days of incubation at 23°C from about 90% of the infected tissues plated. Isolates were transferred to oatmeal agar (OA) (1) and water agar (WA) amended with autoclaved pea seeds (2). Plates were incubated for 30 days at 24°C with 13 h of fluorescent light and 11 h of dark for morphological examination. Colonies were olivaceous grey in OA and pycnidia developed in WA were globose to subglobose, olivaceous black, and 100 to 200 μm in diameter. Conidia were globose to ellipsoidal, hyaline, aseptate, and 3.8 to 7.7 × 2.4 to 3.9 μm. Swollen cells were observed. Isolates showed a positive reaction to NaOH (1). Partial 18S, ITS1, 5.8S, ITS2, and partial 28S ribosomal RNA (rRNA) regions were amplified using the primers ITS1 and ITS4 (4) and sequenced from DNA extracted from the isolate designated as IVIA-V004 (GenBank Accession No. KF321782). The sequence had 100% identity (e-value 0.0) with that of Pleospora betae (Berl.) Nevod. (syn. Phoma betae A.B. Frank) representative strain CBS 523.66 (1). Pathogenicity tests were performed twice by inoculating 4-month-old plants of spinach cv. Apollo, table beet (Beta vulgaris L.) cv. Detroit, and Swiss chard (B. vulgaris subsp. cicla) cv. Verde de Penca Blanca. Plants were inoculated by spraying a conidial suspension of isolate IVIA-V004 (10 ml/plant, 105 conidia/ml water) using a manual pressure sprayer. Plants were immediately covered with black plastic bags and incubated in a growth chamber at 23°C. In each experiment, four plants of each host were inoculated with the fungus and four additional plants sprayed with sterile distilled water were used as controls. Plastic bags were removed after 48 h and leaf spots similar to those observed in affected spinach plants in the field were visible on all spinach, table beets, and Swiss chard plants 3 to 5 days after inoculation. No symptoms were observed on control plants. Fungal colonies morphologically identified as P. betae were re-isolated from leaf lesions on inoculated plants, but not from asymptomatic leaves of control plants. To our knowledge, this is the first report of leaf spot caused by P. betae on spinach in Spain, where it was previously described affecting sugar beet (3). The disease reduces the quality of spinach leaves and proper control measures should be implemented.

In April 2005, serious seedling damping-off was noted on fennel (Foeniculum vulgare Mill. cv. Rondo) in a transplant greenhouse facility in Maasdijk, the Netherlands. Symptoms appeared 3 to 4 weeks after sowing and included black, sunken lesions aboveground on the stem and belowground on the hypocotyls. Mortality of seedlings was 6 to 10% (10 to 15 seedlings per 150-plant tray). Following removal of diseased seedlings, further transplant mortality in the field was not evident. Samples of diseased tissue were collected, surface disinfested, and placed in petri dishes containing water agar. After 7 to 10 days of incubation at 25°C under fluorescent lights, an Alternaria sp. was growing from each sample. Single conidium cultures were obtained from representative colonies and cultured on potato dextrose agar (PDA) and potato carrot agar (PCA) for morphological examination. On PDA, colonies were dark olive brown, cottony, subsurface microsclerotia production abundant, and no production of pigments in the medium. On PCA, conidia were darkly pigmented, broadly ellipsoidal to subsphaerical, and produced singly. Mature conidia were 28 to 45 × 20 to 25 μm with two to four transepta and one to three longisepta. Characteristics were consistent with those of Alternaria petroselini (2,3). In subsequent freezer-blotter assays (ISTA blotter method; www.seedtest.org ) of seed lot used in the original planting, the same fungus was recovered at an infestation level of 30%, confirming that it was seedborne. To confirm pathogenicity on fennel, pathogenicity tests were conducted on a common fennel cultivar (Floro F1) in the greenhouse and on fennel stalks in the laboratory. Fennel seeds were soaked in a conidia suspension (106/ml in sterile H2O) for 10 min. Control seeds were soaked in sterile H2O. Seeds were dried on paper, sown in soil plugs, and grown in the greenhouse at 16 to 20°C. After 4 weeks, black lesions were observed on the fennel stems and symptoms were similar to those observed on the original infected material. Control plants remained healthy. A fungus was reisolated from the lesions of symptomatic plants and was identical to the fungus isolated from the original infected material. For the fennel stalk assay, two surface-sterilized fennel stalks were sliced longitudinally and three 4-mm plugs from a 10-day-old culture of each isolate were placed along the fennel stalks. Sterile agar plugs were used as negative controls. After 7 to 10 days of incubation at 25°C in plastic boxes, test isolates grew extensively from agar plugs and resulted in extensive black necrosis of the fennel stalks. No necrosis resulted from control plugs. DNA was extracted from field isolates, and the nuclear internal transcribed spacer region was sequenced using protocol previously described (1). A representative sequence was deposited in GenBank (Accession No. EF636901). A BLAST search of the NCBI database revealed A. petroselini Accession No. AY154685 as the closest match (total score = 1,014, 100% coverage, 99% sequence identity). The next closest match was A. radicina Accession No. DQ394074 (total score 987, 100% coverage, 98% sequence identity). To our knowledge, this is the first report of A. petroselini causing disease of fennel and the fungus being seedborne on fennel seed. An isolate has been deposited at the Centraalbureau voor Schimmelcultures (Accession No. 118228).

Summer Cypress (Bassia scoparia) is a large annual herb belonging to the family Amaranthaceae native to Eurasia. It has been introduced in many other countries of the world. In Pakistan, summer cypress is also known as kochia and grown as an ornamental plant for its red fall foliage for landscapes. During October, 2017 a survey was conducted in Punjab Province, Pakistan, where 100 wilted plant samples were collected from 30 different plantations of Faisalabad district. Up to 50% loss of plantation was noted in all visited locations. Lower parts of the plants were affected first presenting with necrosis of leaf tips surrounded by a chlorotic zone (Fig. 1. A). Then necrosis of apical margins of the plant parts occurred, followed by stem discoloration and wilting of entire herbaceous branches, leading to the partial wilting of the plants. Ultimately, whole plant wilted and died, (Fig. 1. B) appearing as though they had been scorched. Diseased tissues from lower stem (crown portion) were sampled, surface sterilized in 70 % ethanol for 30 s, and cultured on to Potato Dextrose Agar (PDA) medium. Petri dishes were incubated at 25 ˚C with alternating 12-hour periods of light and dark. Frequently observed, fast growing whitish grey fungal cultures with black pin head points were obtained after 7 days (Fig. 1. C). Young conidia were one-celled, yellow to orange in color and turned brown to black (Fig. 1. D & E), ranged in size from 11 μm to 16 μm x 9.5 μm to 12 μm (Fig. 1. F), and were ellipsoidal at maturity (Fig. 2. A). Hyphae were branched, septate and dark brown in color while conidiophores were flexuous, branched and ranged between 3.5 μm to 4.5 μm in diameter and 14.5 μm to 26.5 μm in length. Based on morphology (Ellis, 1971), the pathogen was identified as Nigrospora oryzae and submitted to the Westerdijk Collection of Fungi, Netherland (CBS 146145/RNOEG30). Total DNA of isolate EG30 was extracted and portions of the Internal transcribed spacer (ITS) region and beta-tubulin (βt) gene were amplified using the universal primers ITS1F and ITS4 (White et al., 1990) and βt2a and βt2b (Glass and Donaldson, 1995). The generated ITS (GenBank Accession No. MG745331.1 491 bp) and βt (GenBank Accession No. MN629896 408 bp) sequences were searched against GenBank using BLASTn and were 99% homologous to ITS (KX986074 525 bp ; MN341493 550 bp) and 100% homologous to βt (MK262852 409 bp) gene region from Nigrospora oryzae (Wang et al., 2017; Zhang, 2019). For pathogenicity tests, ten healthy two-month-old summer cypress plants were inoculated by soil drenching of a spore suspension (106-107 spores/mL) of the fungal isolate EG30 while five plants were treated with sterilized water and used as control treatments. Plants were incubated at 60 to 75% relative humidity (RH) and 25 ˚C in a greenhouse. Leaf necrosis and partial to whole plant witling (Fig. 2. B & C) were observed in the inoculated plants after 21 days. No symptoms appeared in control plants. A fungus was re-isolated from the lower stem (crown portion) parts of the inoculated plants that was identical in morphology to isolate EG30. No fungus resembling EG30 was isolated from the control plants. To the best of our knowledge, this is the first report of summer cypress wilt caused by Nigrospora oryzae (Berk. and Broome) Petch, a known pathogen of several important crops in China, Australia, India, Canada, and Pakistan (Sharma et al., 2013).

In April 2005, an Alternaria sp. was isolated from carrot (Daucus carota) roots harvested in the fall of 2004 and held at 1 to 3°C in a storage facility in Newaygo County, MI. The pathogen was readily isolated on water agar from root tissue exhibiting grayish black, sunken lesions. Morphological characteristics were noted 5 to 7 days after single-conidium cultures were established on potato dextrose agar (3). Sixteen Alternaria sp. isolates were recovered. Cultures were dark olive brown, and conidia were pigmented, ellipsoidal, and produced singly or in chains of two. Conidia were 35 to 45 μm long and 15 to18 μm in diameter, usually with three to eight transverse and one to four longitudinal septa. Pathogenicity of isolates was tested on carrot roots in the laboratory and carrot seedlings (cv. Goliath) in the greenhouse. In the laboratory, four surface-sterilized, whole carrot roots were sprayed until runoff with 2 × 106 conidia/ml of each isolate and incubated at 23 to 25°C in a moist chamber for 10 days. Controls were sprayed with sterile distilled water. Ten to fifteen days after inoculation, inoculated carrots exhibited grayish black, sunken lesions, and an Alternaria sp. was reisolated from the margin of the lesions. Controls remained healthy. In the greenhouse, seven pots containing one 2-week-old carrot seedling were watered to saturation and plants were sprayed until runoff with 2 × 106 conidia/ml for each isolate. Control plants were sprayed with sterile distilled water. After inoculation, plants were enclosed in clear plastic bags, placed under 63% woven shade cloth and watered regularly. Black lesions were observed on the foliage 7 days after inoculation, and wilt and death of plants were observed 15 to 30 days after inoculation. Alternaria sp. was reisolated from the foliage of symptomatic plants. Control plants remained healthy. DNA was extracted from all isolates, and the nuclear ribosomal internal transcribed spacer (ITS) region amplified with primers ITS4 and ITS5 and sequenced. A portion of the ITS sequence has been deposited in the NCBI database (GenBank Accession No. DQ394073). A BLAST search of the NCBI database with the ITS sequences revealed A. radicina, Accession No AY154704, as the closest match with 100% sequence similarity. In September 2005, an Alternaria sp. was isolated from black lesions on carrot roots, crowns, and foliage that were collected from fields in Newaygo and Oceana counties, MI. The recovered isolates were morphologically similar to A. radicina isolates obtained from stored carrots in April 2005. First isolated and identified on stored carrots in New York (3), A. radicina is also present in other carrot-producing areas of the United States (1) and was isolated not only from stored carrots but also from carrots in the field (2) and carrot seeds (4). To our knowledge, this is the first report of A. radicina on stored and field carrots in Michigan, which signifies a serious risk to a carrot industry that ranks among the top five in the United States.

In May 2007, a survey was conducted to evaluate the phytosanitary status of grapevine propagating materials in a commercial nursery located in Valencia Province (eastern Spain). Fungal isolation was performed on 25 grafted plants (1-year-old grapevines cv. Tempranillo grafted onto 110 R rootstock) because they showed reduced root biomass and black discoloration of the xylem vessels. Sections (10 cm long) were cut from the basal end of the rootstocks, washed under running tap water, surface sterilized for 1 min in a 1.5% sodium hypochlorite solution, and washed twice with sterile distilled water. The sections were split longitudinally and small pieces of discolored tissues were placed onto malt extract agar (MEA) supplemented with streptomycin sulfate (0.5 g L-1). Plates were incubated at 25°C in the dark for 14 to 21 days after which all colonies were transferred to potato dextrose agar (PDA). Togninia minima (Tul. & C. Tul.) Berl. (anamorph Phaeoacremonium aleophilum W. Gams, Crous, M.J. Wingf. & Mugnai) and another Phaeoacremonium sp. were consistently isolated from necrotic tissues. Single conidial isolates of this Phaeoacremonium sp. were grown on PDA and MEA in the dark at 25°C for 2 to 3 weeks until colonies produced spores (3). Colonies were grayish brown on PDA and pinkish white on MEA. Conidiophores were mostly short and unbranched, 15 to 30 (mean 20.8) μm long, often consisting of an elongate-ampuliform phialide. Conidia were hyaline, oblong-ellipsoidal occasionally reniform or allantoid, 2.5 to 5.6 (mean 3.8) μm long, and 1 to 2.1 (mean 1.4) μm wide. On the basis of these characteristics, these isolates were identified as Phaeoacremonium scolyti L. Mostert, Summerb. & Crous (2,3). Identity of isolate Psc-1 was confirmed by PCR-restriction fragment length polymorphism of the internal transcribed spacer region using Phaeoacremonium-specific primers Pm1-Pm2 and restriction enzymes BssKI, EcoO109I, and HhaI (1). Additionally, the β-tubulin gene fragment (primers T1 and Bt2b) of this isolate was sequenced (GenBank Accession No. EU260415). The sequence showed high similarity (98%) with the sequence of P. scolyti (GenBank Accession No. AY579292). Pathogenicity tests were conducted on 2-month-old grapevine seedlings (cv. Tempranillo) using the isolate Psc-1. Ten seedlings were inoculated when two to three leaves had emerged by watering the roots with 25 mL of a conidial suspension (106 conidia mL-1) harvested from 21-day-old cultures grown on PDA. Ten controls plants were inoculated with sterile distilled water. Seedlings were maintained in a greenhouse at 23 to 25°C. Within 2 months of inoculation, symptoms developed on all of the inoculated plants as crown necrosis, chlorotic leaves, severe defoliation, and wilting. Control plants did not show any symptoms. The fungus was reisolated from internal tissues of the crown area and the stems of all inoculated seedlings, completing Koch's postulates. To our knowledge, this is the first report of P. scolyti causing Petri disease in Spain.

Polygonatum cyrtonema Hua is one of the most important traditional Chinese medicine herbs used for treating stomach weakness, palpitation, diabetes, and dry cough and is commonly planted in the mountain area of southeast and southwest China. During June of 2018 and 2019, necrotic lesions on the leaf apex and leaf margin of P. cyrtonema were observed in seven greenhouses, affecting approximately 2 ha, in Jinzhai County, Anhui Province, China. Disease incidence averaged approximately 30% among all affected greenhouses. The symptoms initially appeared on the leaf apex and leaf margin as small dark brown spots surrounded by light yellow halos. Over time, the lesions enlarged and coalesced to form large areas of necrotic lesions on leaves. To identify the pathogen, 15 symptomatic leaves were collected and surface disinfested with 2% sodium hypochlorite for 2 min followed by 70% ethanol for 30 s, rinsed in sterile water, and dried on filter paper. Plant tissues were then transferred onto potato dextrose agar (PDA) and incubated at 28°C for 3 days in the dark. Five fungal isolates were obtained from the diseased P. cyrtonema leaves by a single-spore isolation method. Colonies of the isolates had round margins and thick fluffy aerial mycelia with olive-green to brown coloration after 5 days on PDA. Conidiophores were pale brown, straight or flexuous, septate, single or in clusters. Conidia were obclavate or oval, multicellular, pale brown, and ranged from 4.91 to 15.83 × 17.29 to 47.32 μm (n = 50), with longitudinal and transverse septa varying from zero to three and from one to six, respectively. Conidia beaks were cylindrical, pale brown, and measured 2.00 to 7.46 × 2.00 to 5.28 μm. On the basis of morphological characteristics, the fungal isolates were preliminary identified as Alternaria spp. (Simmons 2007). To further identify the fungal isolates, the internal transcribed spacer (ITS) region of rDNA of each isolate was amplified and sequenced using the primers ITS4/ITS5 (Guo et al. 2012). The ITS sequence (accession no. MN249629) was 99.45% similar to the reference sequences of Alternaria alternata (KU527784) and A. tenuissima (KX664408) in GenBank, respectively. Partial regions of β-tubulin 2 (TUB2) and histone 3 (H3) genes were sequenced using primers T1/T2 (O’Donnell and Cigelnik 1997) and H3-1a/H3-1b (Glass et al. 1995), respectively. Sequences of TUB2 and H3 (MN256456 and MN256455) of the isolate were 100 and 99.80% similar to the fungus A. tenuissima (JQ811941 and MH647724), respectively. To complete Koch’s postulates, a pathogenicity test of the A. tenuissima isolates was performed on sterile leaves of P. cyrtonema. Ten milliliters of conidial suspension (5 × 10⁵ spores/ml) from each of the five isolates was sprayed on five 6-month-old plants. As a control, five plants were sprayed with sterile water. All treated plants were placed in a moist chamber at 26°C, with 90% relative humidity and an 8-h photoperiod of fluorescent light. Ten days later, 90% of the inoculated plant leaves developed brown spots and necrotic lesions similar to those observed in the field, whereas the control plants were asymptomatic. The same pathogens were again isolated from the necrotic leaves and confirmed as A. tenuissima by DNA sequencing. A. tenuissima is known to cause leaf spot and necrosis on many economically important crops, including lotus (Nelumbo nucifera) and kiwifruit (Actinidia spp.), in China (Li et al. 2019; Zhang et al. 2019). To our knowledge, this is the first report of A. tenuissima causing leaf necrosis on P. cyrtonema in Jinzhai, China.

In northeastern China, Asian ginseng (Panax ginseng) roots exhibited reddish brown lesions of various sizes, irregular shapes, and diffuse margins, typical of rusty root disease. The lesions remain superficial, smooth, and limited to the epidermal and peridermal tissues. In September 2013, 10 symptomatic roots were collected from each of three fields in Jilin and Heilongjiang provinces. One piece of symptomatic skin tissue from each root was excised, surface-disinfested in 1% NaClO for 3 min, rinsed three times with sterile water, and then placed on tetracycline-amended (50 μg/ml) potato dextrose agar. After incubation at 22 ± 1°C in the dark for a week, small olivaceous black colonies developed from the symptomatic tissue from five of the 30 samples. No spores were observed. A single hyphal tip of each colony was transferred to a fresh V8 agar plate to purify the culture. Two-week-old colonies on V8 agar were olivaceous gray, and 42 to 46 mm in diameter with an outer white margin (3 to 5 mm wide). Conidia produced in V8 broth after 3 weeks with a 12-h photoperiod were straight and hyaline, cylindrical or subcylindrical with no or one septum. Mature conidia were 12.8 to 21.8 × 2.2 to 4.5 μm (mean 18.2 × 3.0 μm, n = 100 conidia for each of three isolates). Three isolates selected randomly were further identified by analyzing the partial sequences of the ITS region of rDNA with primers ITS4 and ITS5 (5), and partial sequences of β-tubulin with the primers tub2F and tub2R (1). Sequences of the three isolates (GenBank Accession Nos. KJ149287, KJ149288, and KJ149290 to 93) showed 99% to 100% homology with previously identified and deposited Rhexocercosporidium panacis isolates (DQ2499992 and DQ457119) for both loci (3). Therefore, the three isolates were identified as R. panacis and deposited in China General Microbiological Culture Collection Center (CGMCC3.17259 to 61). Pathogenicity of R. panacis in Asian ginseng was investigated using these three isolates as described previously with slight modifications (4). Bare roots of 3-year-old Asian ginseng were surface-disinfested as described above, and inoculated with mycelial plugs (4 mm diameter) cut from the margin of actively growing colonies of the isolates on V8 agar. Three mycelial plugs were placed on each root at 3-cm intervals and four roots (replicates) were inoculated for each isolate. Four additional roots were inoculated with non-colonized agar plugs as control. The treated roots were placed on moist filter paper in an enamel tray. The plates were sealed with plastic wrap to prevent desiccation and incubated in the dark at 18 ± 1°C. Four weeks post inoculation, all the inoculated ginseng roots showed red-brown lesions, which turned to dark red or black over time. R. panacis was recovered from symptomatic roots for all isolates and confirmed by ITS sequence analysis. The mock-inoculated control roots remained symptomless and no R. panacis was isolated. The inoculation experiment was repeated and showed the same results. R. panacis was reported in 2006 to infect roots of Panax quinquefolius (2,3,4). To our knowledge, this is the first report of R. panacis causing rusty root of P. ginseng.

In July 2007, a severe rot was observed on Phoenix dactylifera and P. canariensis palms in the vicinity of Heraklion (Crete), Greece. Initial symptoms were pale, elongated spots that gradually turned to dark brown streaks extending along the leaf base and rachis. In early stages, the upper parts of the leaves usually remained unaffected. Eventually decay and premature death of leaves occurred, followed by terminal bud necrosis. Shoot blights and stalk rots were also observed. A filamentous fungus was consistently isolated onto potato dextrose agar (PDA) from leaf base necrotic lesions. Immersed pycnidial conidiomata on pine needles in culture were multiloculate and dark brown to black. Pycnidial paraphyses were absent. Conidiogenous cells were hyaline, cylindrical, and swollen at base. Conidia were thick-walled, ovoid to ellipsoid, with rounded apex and base; initially hyaline and aseptate, 15.2 ± 0.4 × 11.7 ± 0.3 μm, later becoming dark brown and 1-septate, 21.3 ± 0.4 × 11.8 ± 0.3 μm, with a striate appearance. Total DNA was extracted and used for PCR amplification and sequencing of the ITS1-5.8S-ITS2 region, together with parts of the flanking 18S and 28S rRNA genes (1). The sequence (GenBank Accession No. JX456475) was found 99% identical to Neodeightonia phoenicum ITS sequences (GenBank Accession Nos. EU673338 to EU673340), and was clustered together as a single group with the above sequences with good support by phylogenetic analysis that included representatives of other Neodeightonia species and several other Botryosphaeriaceae members. Based on the morphological, molecular, and phylogenetic analyses, the pathogen was identified as N. phoenicum A. J. L. Phillips & Crous (2) (syn. Diplodia phoenicum (Saccardo) H. S. Fawcett & Klotz), formerly also known as Macrophoma phoenicum Saccardo and Strionemadiplodia phoenicum (Saccardo) Zambettakis. To prove pathogenicity, the petioles of the older leaves of seven 2-year-old seedlings of each of three palms, P. canariensis, P. theophrasti, and Washingtonia filifera were wounded with a sterile scalpel (shallow cuts 0.5 to 1.0 cm wide, made parallel to the surface) and inoculated with agar discs from a 1-week-old PDA culture of the fungus. For controls, PDA discs without fungal mycelium were placed on the wounds of four seedlings of each host. Petiole rot, blight, and leaf necrosis were evident on all inoculated plants 6 weeks post inoculation and the pathogen was consistently reisolated from all three inoculated palm species, whereas no symptoms were observed on control plants. N. phoenicum has repeatedly and globally been reported on P. dactylifera (3). To the best of our knowledge, this is the first report of the occurrence of N. phoenicum infecting Phoenix species in Greece. Palms are extensively used as ornamental trees throughout Greece. A potential spread of palm rot caused by N. phoenicum might have a substantial economic impact and should be urgently addressed through appropriate disease management programs.

Lavender cotton, Santolina chamaecyparissus, is an evergreen shrub growing primarily in dry, calcareous habitats and is grown in rock gardens and mixed borders mainly for its ornamental and aromatic foliage. During 2004, several commercial nurseries in Valencia Province (eastern Spain) reported high mortality of lavender cotton. The foliage of the diseased plants turned brown, wilted, and died. A Phytophthora sp. was isolated consistently from the soil and roots of infected plants using apple baits and the selective medium PARBH (1), respectively. Four pure cultures (PS-31, PS-32, PS-33, and PS-34) were established from hyphal tips and characterized. Colony morphology on potato dextrose agar (PDA) at 24°C was stoloniferous (short stubby branches) with a growth rate of 2.2 mm per day. Sporangia, chlamydospores, and oospores were produced on V8 agar. The sporangia were ovoid to obpyriform, 27.5 to 64.8 (48.3) × 25 to 52.5 (37.5) μm, length/breadth ratio of 1.3:1, and papillate, from which 20% were caducous with a short pedicel (<5 μm). Hyphal swellings and chlamydospores (22 to 38 μm in diameter) were present. Isolates were homothallic, oogonia were globose, mostly terminal 27.5 to 40 (36.2) μm in diameter, 88% of the antheridia were paragynous, monoclinous, or diclinous, and occasionally with two paragynous antheridia per oogonium. Amphigynous antheridia (12%) were also observed. Oospores were aplerotic, 25 to 35 (32.3) μm in diameter, and thin walled. These characteristics and measurements conformed to the description of P. tentaculata described by Kröber and Marwitz (2). Sequencing the internal transcribed spacer region of Santolina isolates PS-32 and PS-34 and comparison of these sequences with other sequences available in GenBank revealed that they were identical to P. tentaculata (AF266775). Pathogenicity tests used 10 4-to-5-month-old potted lavender cotton and two methods. In the first method, inoculum was prepared on a media of 200 g of oats and 120 ml of V8 juice to 1 liter of distilled water. The medium was inoculated with P. tentaculata grown on PDA and incubated in the dark at 20°C for 4 weeks. Inoculum was buried into the compost mixture around the roots at a rate of 3% (w/v). The second method applied a zoospore drench of 50 ml per plant (1 × 104 zoospores per ml) obtained by inducing zoospores in sterile soil extract from cultures of V8 juice agar. The control plants were inoculated with sterile media and sterile distilled water. The following day, the pots were flooded for 2 days, plants were maintained in a glasshouse at 24 ± 5°C, and watered twice a week. All plants inoculated with the first method had wilted foliage and died within 2 months after inoculation, while plants inoculated with zoospores died after 3 months. P. tentaculata was reisolated and the test was repeated twice. The control plants did not show any symptoms of the disease. P. tentaculata was first reported causing root and stalk rot on Chrysanthemum frutescens hybrids, C. leucanthemum, Delphinium ajacis, and Verbena hybrids in Germany (2). It has also been reported on Verbena hybrids in Spain (3). To our knowledge, this is the first report of P. tentaculata causing root rot on lavender cotton.

Small, circular, necrotic spots with yellow border, present on ca. 50% of the leaves were observed in 2012 on Judas trees (Cercis siliquastrum L.) in the urban area of Thessaloniki (Greece). Tissue fragments excised from the margin of the lesions were surface- disinfected for 1 min in 1% sodium hypochlorite and plated on potato dextrose agar (PDA). Two single-spore cultures grown on potato carrot agar (PCA) and incubated at 24°C with a 12 h photoperiod, gave rise to initially white colonies which turned grayish-black due to abundant sporulation. Conidiophores were short, septate, branched or unbranched, green to brown. Mature conidia, produced in long, single or more often branched chains, were obpyriform, with a conical or cylindrical beak, ovoid or ellipsoidal, had 1 to 5 transverse and 0 to 3 longitudinal septa and measured 9.4-30.8 x 5.6 -15.4 μm (average 20.1 x 10.7 μm). These morphological traits tally with those of Alternaria alternata (Fr.) Keissl. (Simmons, 2007). The ITS1-5.8S-ITS2 region of the two PCA isolates was amplified with primers ITS1 and ITS4 and sequenced (GenBank accession Nos. KP780092, KP780093). A BLAST search revealed 100% homology with the sequences of various A. alternata isolates (e.g. KM233278). Spraying leaves of 10 healthy 2-year-old greenhouse-grown C. siliquastrum plants with a 106 conidia/ml spore suspension resulted in the production, 20 days post inoculation, of leaf spots similar to those observed in the field. Controls plants sprayed with sterile distilled water remained healthy A. alternata was reisolated from artificially inoculated leaves fulfilling Koch’s postulates. This is the first report of A. alternata as the cause of a leaf spot disease on C. siliquastrum in Greece and elsewhere in the world.

Aloe vera (L.) Burm. f. is a perennial succulent plant that is grown worldwide mainly for medicinal and cosmetic uses. In the USA, it is mainly cultivated in some southern states to produce aloe gel for the cosmetic industry (3), and in Louisiana it is also sold commercially as an ornamental. During the summer of 2011, several A. vera plants infected with leaf spots were observed on the campus of Louisiana State University, Baton Rouge. Large, necrotic, sunken, circular to oval, dark brown spots were present on both surfaces of the leaves. Infected leaf tissue pieces were surface disinfested with 1% NaOCl solution for 1 min and plated on potato dextrose agar (PDA). Plates were incubated at 28°C in the dark for 4 days. A dark olivaceous fungus with profuse golden brown, branched, and septate hyphae was consistently isolated from the infected tissue on PDA. The fungus produced conidia with longitudinal and transverse septa, and was morphologically identified as an Alternaria sp. (4). Conidia were produced in long chains, pale to light brown, obpyriform, with a beak (6.0 μm long), one to seven transverse and up to three longitudinal septa, and measured 10 to 45 μm long × 7 to 18 μm wide. Conidiophores were straight, septate, light to olive golden brown with conidial scar, and measured 35 to 100 μm long × 2 to 5 μm wide. Genomic DNA from a single-spored isolate was extracted and the internal transcribed spacer (ITS1-5.8s-ITS2) regions were amplified and sequenced using primers ITS1 and ITS4. BLASTn analysis of a 486-bp sequence (GenBank Accession No. JQ409455) resulted in 100% homology with A. alternata strain DHMJ16 (GenBank Accession No. JN986768) from China and several other Alternaria spp. The fungus was identified as A. alternata based on mycelial and conidia characters after being grown under standard, previously described conditions (4). Pathogenicity tests were carried out by inoculating six potted aloe plants with 0.5-cm diameter discs taken from a 6-day-old culture grown on PDA. Four discs were placed on the upper surface of each of the bottom leaves of every plant. Inoculated plants were individually covered with a plastic bag and maintained in a greenhouse for 1 week at 25 ± 2°C. Six control plants received only agar plugs. Seven days after inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated from these spots. No leaf spots were observed on control plants. To the best of our knowledge, this is the first report of leaf spot caused by A. alternata on A. vera in Louisiana. Several outbreaks of the disease have been reported in Pakistan and India as damaging aloe gel production in those countries (1,2). An outbreak of this disease in Louisiana could represent a serious issue for the state's A. vera ornamental commerce.

HomePlant DiseaseVol. 100, No. 7First Report of Anthracnose Caused by Colletotrichum spaethianum on Fragrant Plantain Lily in Korea PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Anthracnose Caused by Colletotrichum spaethianum on Fragrant Plantain Lily in KoreaW. Cheon and Y. JeonW. CheonSearch for more papers by this author and Y. JeonSearch for more papers by this authorAffiliationsAuthors and Affiliations W. Cheon Y. Jeon , Department of Bioresource Sciences, Andong National University, Andong 760-749, Korea. Published Online:28 Mar 2016https://doi.org/10.1094/PDIS-11-15-1341-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In autumn 2012, fragrant plantain lily (Hosta plantaginea Aschers) plants in Andong Region, Korea, were found to be showing leaf blight symptoms. Voucher specimens were deposited in the Herbarium of Mycology, Agricultural Science and Technology Research Institute, Andong National University, as Accession No. ANU-APEC0025F. Disease symptoms included brown necrotic lesions 30 to 100 mm in diameter. At the initial stage of the infection, lesions on the outer edge of the leaves were round to elliptical, and 5 to 15 mm in diameter. Gradually, the lesions became irregular, dark brown, and frequently surrounded by a pale yellow halo that later became necrotic and spread toward the petioles. Small sections (5 to 10 mm2) of diseased areas were excised from the margins of the lesions and placed on potato dextrose agar (PDA) after surface sterilization with 70% ethanol and 1% NaOCl for 1 min, and incubated for 3 days at 25°C. The fungus isolated using this procedure produced whitish mycelia when grown on PDA, and these mycelia later became gray to dark gray and produced tufts of aerial mycelia. Analysis of light micrographs showed that the fungus had falcate, hyaline, aseptate conidia 13.78 to 22.81 μm × 2.85 to 4.04 μm. These conidia were observed from both naturally infected leaves and PDA-cultured colonies. Appressoria were found either singly or in loose groups of dark brown, irregular shapes, and 6.3 to 11.1 × 5.3 to 7.7 μm. The acervular conidiomata were covered with abundant setae that were straight and dark brown, and 75.23 to 131.3 × 2.53 to 4.52 μm. The fungus was identified as Colletotrichum spaethianum on the basis of its morphology (Damm et al. 2009) and by sequence analysis of conserved ribosomal RNA (rRNA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sequences. Specifically, the rRNA internal transcribed spacer (ITS) region was amplified using a polymerase chain reaction (PCR) to form rDNA for in vitro sequencing using the primers ITS1 (forward) and ITS4 (reverse) with the GenBank Accession No. KU572437, and the GAPDH region was amplified using the GDF (forward) and GDR (reverse) primer set, as described previously (Templeton et al. 1992). Nucleotide BLAST analysis of the two PCR products showed 100% sequence similarity with the nucleotide sequences of C. spaethianum (GQ856747 and GU228200). In our study, the sequences of C. spaethianum were deposited as GenBank Accession Nos. KT321124 and KT321125. To confirm pathogenicity, H. plantaginea plants grown in greenhouse were inoculated by foliar spray with 2-week-old cultures grown on PDA. Control plants were sprayed with sterile water. Plants were covered with plastic bags for 72 h to maintain high relative humidity (∼80 to 90%) in a growth chamber (26°C). Seven days after inoculation, the small, circular, pale brown spots developed on pathogen-inoculated leaves, and gradually expanded, developing a yellow halo. Based on the observed symptoms, morphological characteristics, molecular data, and pathogenicity against the host plant, it can be concluded that C. spaethianum is the causal agent of anthracnose of H. plantaginea in Korea. Colletotrichum spaethianum was isolated from H. sieboldiana in Germany (White et al. 1990) a different species of the same genus. To our knowledge, this is the first documented report of C. spaethianum causing anthracnose on fragrant plantain lily in Korea.

HomePlant DiseaseVol. 103, No. 10First Report of Black Root Rot Disease on Morinda officinalis Caused by Lasiodiplodia pseudotheobromae in China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Black Root Rot Disease on Morinda officinalis Caused by Lasiodiplodia pseudotheobromae in ChinaZhangyong Dong, Yongxin Shu, Mei Luo, Weili Zhang, Xinyu Chen, Yongxin Xiao, and Meimei XiangZhangyong Dong†Corresponding authors: Z. Dong; E-mail Address: dongzhangyong@hotmail.com and M. Xiang; E-mail Address: mm_xiang@163.comhttp://orcid.org/0000-0001-7524-0226Zhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, Yongxin ShuZhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, Mei Luohttp://orcid.org/0000-0002-1950-4204Zhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, Weili ZhangZhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, Xinyu ChenZhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, Yongxin XiaoZhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this author, and Meimei Xiang†Corresponding authors: Z. Dong; E-mail Address: dongzhangyong@hotmail.com and M. Xiang; E-mail Address: mm_xiang@163.comZhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of ChinaSearch for more papers by this authorAffiliationsAuthors and Affiliations Zhangyong Dong † Yongxin Shu Mei Luo Weili Zhang Xinyu Chen Yongxin Xiao Meimei Xiang † Zhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of China Published Online:7 Aug 2019https://doi.org/10.1094/PDIS-04-19-0857-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Morinda officinalis is a wildly cultivated medicinal plant in Zhaoqing, Guangdong Province of China. The annual production of M. officinalis in Deqin county of Zhaoqing accounts for 90% of China’s total production. In 2018, a disease on M. officinalis caused significant economic losses by affecting medicinal plant quality. Symptomatic plants exhibited blackened necrotic discoloration of roots. A diseased plant was collected in June 2018 from Zhaoqing, Guangdong, surface sterilized in 75% ethanol for 1 min and then in 2% NaClO for 3 min, and rinsed three times in sterile distilled water; internal necrotic tissue was then transferred to potato dextrose agar (PDA) and incubated at 28°C for 3 days. The fungal colonies were initially white and then became green to dark green with sporulation. Some small black pellets were found on the edge of the colony cultured on PDA medium for 25 days. The morphology of black fungal pellets was examined by light microscopy. The conidia were ellipsoidal, initially hyaline, unicellular, becoming dark brown, and developing a thick wall and a central septum. Conidia measured 16.01 to 21.74 μm long and 8.83 to 11.43 μm wide (n = 60). The conidial morphology matched that of Lasiodiplodia, a member of the Botryosphaeriaceae family (Alves et al. 2008). Moreover, the internal transcribed spacer region of the ribosomal RNA was amplified by using primers ITS1 and ITS4 (White et al. 1990) and sequenced (GenBank accession no. MK090538). The 542-bp sequence was compared with the GenBank database using nucleotide BLAST, and the isolate sequence was 100% similar to the sequence of Lasiodiplodia pseudotheobromae (GenBank accession no. MH663527). The β-tubulin gene region of the ribosomal DNA was amplified by using primers T1 (O’Donnell and Cigelnik 1997) and CYLTUB1R (Crous et al. 2004) and sequenced (GenBank accession no. MK328528). The 459-bp sequence was compared with the GenBank database using nucleotide BLAST, and the isolate sequence was 100% similar to the sequence of L. pseudotheobromae (GenBank accession no. KY583260.1). On the basis of morphological characteristics and nucleotide homology, the isolate was identified as L. pseudotheobromae, a member of the Botryosphaeriaceae family. To satisfy Koch’s postulates, both roots and leaves of M. officinalis were inoculated by placing a mycelium plug from the growing margin of 7-day-old colonies upside down directly into a fresh wound. After 3 days, the root xylem of inoculated plants turned brown and gradually became dark, similar to symptoms observed in the field. The leaves also turned brown and gradually became dark brown after 4 days. The disease spots were round or nearly circular. No symptoms were observed on the control plants. The pathogen was reisolated from root lesions, and its identity was confirmed by morphological characteristics. To our knowledge, this is first report of L. pseudotheobromae causing black root rot of M. officinalis in China.The author(s) declare no conflict of interest.