Leaf Spot of Arugula, Caused by Alternaria japonica, in California.

Arugula (Eruca vesicaria subsp. sativa (Mill.) Thell. is a Cruciferous plant used for culinary purposes. From 2012 to 2013, a foliar disease seriously impacted the growth and quality of about 0.1 ha of hydroponically grown arugula at a Santa Barbara County nursery. Samples of affected arugula seedlings exhibited adaxial and abaxial symptoms of mottling with circular to oval, water soaked, dark green leaf spots, each 1 to 3 mm in diameter, and some of which coalesced. Conidia of an Alternaria sp. were observed on the foliage. Symptomatic leaf pieces were disinfested with 0.6% NaOCl, blotted dry, and plated on acidified potato dextrose agar (APDA). Cultures were incubated under near-UV lights for 24 h/day. Olivaceous-grey colonies of the same Alternaria species observed on the leaves grew after 7 days. After 21 days on carrot-piece agar (3), the fungus produced beakless conidia with longitudinal and constricted transverse septa that measured 30.0 to 69.0 × 12.5 to 20.0 µm and were borne singly or in short chains of 2 to 3 conidia. In addition, knots of dark, thick-walled micro-chlamydospores were produced by the hyphae. The fungus was identified morphologically as Alternaria japonica Yoshii (2), and the species confirmed by sequence analysis. A portion of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) was amplified using ITS1 and ITS4 primers (4). The sequence (GenBank Accession No. KJ126846) was 100% identical to the ITS rDNA sequence of an isolate of A. japonica (KC584201) using a BLASTn query. A. japonica was also detected in seeds of the lot used to grow the affected arugula crop. Pathogenicity of a single isolate was tested by inoculating four 37-day-old plants each of arugula, cabbage (Brassica oleracea L. var. capitata), and broccoli (B. oleracea L. var. botrytis L.). Inoculum was obtained from 11-day-old cultures of the isolate grown at 24°C on half-strength APDA. Half of a 2.5 cm diameter agar plug containing hyphae and conidia was ground in 2 ml of sterilized water, and the volume of water increased to 45 ml. Leaves of four plants/host species were sprayed with 3.5 to 4.0 ml of inoculum. The inoculated plants and four control plants of each species treated similarly with sterilized water were immediately incubated in a dark dew chamber at 23°C. After 72 h in the dew chamber, inoculated plants of all three hosts produced similar symptoms of wilting, water soaking, and dark green leaf spotting as the original symptomatic field plants. Conidia formed in the leaf spots on both sides of inoculated leaves. A. japonica was re-isolated from all of the inoculated plants but from none of the symptomless control plants using the method previously described. Pathogenicity tests were repeated, with similar results. Although reported in Italy in 2013 (1), to our knowledge, this is the first report of A. japonica on arugula in the United States. References: (1) G. Gilardi et al. Acta Hort. 1005:569, 2013. (2) E. G. Simmons. Page 368 in: Alternaria, An Identification Manual. CBS Fungal Biodiversity Centre, Utrecht, 2007. (3) S. Werres et al. Z. Planzenkr. Pflanzensh. 108:113, 2001. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990.

First Report of Neofusicoccum nonquaesitum Causing Branch Cankers on Giant Sequoia (Sequoiadendron giganteum) in North America.

Between 2001 and 2007, samples from three California native plants showing canker symptoms were submitted to the California Department of Food and Agriculture's Plant Pest Diagnostics laboratory. Giant sequoia (Sequoiadendron giganteum) and coast redwood (Sequoia sempervirens) showed branch cankers and dieback, whereas tanoak (Lithocarpus densiflora) had bleeding bole cankers. Samples were collected from mature trees in private landscapes in El Dorado, Sacramento, and Alameda counties in California. A fungus was isolated on one-half strength acidified potato dextrose agar (APDA) from the canker margins of all three hosts. Colonies were moderately fast growing, initially white, later turning olivaceous black. Pycnidia developed singly or in small groups and contained conidia that measured 18 to 29 × 6 to 8 µm (average 21.5 × 6.8 µm). Conidia were aseptate, hyaline, and fusiform, with truncate bases. rDNA sequences of the internal transcribed spacer (ITS) region of the isolates (GenBank JQ282157 through JQ282159), amplified using primers ITS1 and ITS4 (2), were 100% identical to the holotype isolate of Neofusicoccum nonquaesitum Inderb., Trouillas, Bostock & Michailides (1) by a BLAST query (GenBank GU251163). Pathogenicity of the N. nonquaesitum isolate from giant sequoia (CDFA4) was tested on five saplings using cultures grown on APDA for 14 days. A single wound was made approximately 2 cm above the soil line on the cambium of each plant using a 3-mm cork borer. One 3-mm colonized agar plug was placed on each wound and secured with Parafilm. Plugs of APDA were placed onto wounds of five plants as controls. All plants were kept in a growth chamber at 23°C with a 12-h photoperiod. After 4 days, Parafilm was removed to reveal dark brown cankers measuring 12 to 43 mm long on the inoculated plants. Fourteen days after inoculation, cankers were black, sunken, and measured 79 to 117 mm (average 91.4 mm) long. Most of the inoculated plants were wilted with chlorotic to necrotic foliage. Mature pycnidia with cirri developed in most of the cankers. N. nonquaesitum was reisolated on APDA from all of the cankers. No symptoms developed on the control plants. The experiment was repeated once with similar results. Botryosphaeria dothidea, also in the Botryosphaeriaceae, has been reported to cause similar cankers on giant sequoia and coast redwood in California (3). However, rDNA sequencing of the ITS region of this isolate obtained from the American Type Culture Collection (ATCC 60344) (GenBank JQ284384) showed it matched the type specimen of Neofusicoccum australe (GenBank GU251219), not our isolate. To our knowledge, this is the first report of N. nonquaesitum as a pathogen of giant sequoia in North America. This study expands the host range of N. nonquaesitum from almond (Prunus dulcis), California bay (Umbellularia californica), and blueberry (Vaccinium spp.) (1) to include giant sequoia, coast redwood, and tanoak, which are economically important trees in California forests and landscapes. References: (1) P. Inderbitzin et al. Mycologia 102:1350, 2010. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (3) J. J. Worrall et al. Plant Dis. 70:757, 1986.

First Report of Botryotinia Blight of Asiatic Hybrid Lily Caused by Botryotinia sphaerosperma in North America.

In November 2002, a disease was observed on eight cultivars of Asiatic hybrid lilies (Lilium sp.) in two fields in Carpinteria, CA. The same disease was observed on greenhouse grown Asiatic hybrid lilies in Nipomo, CA in an adjacent county. 'Monte Negro' was the most severely affected. Symptoms consisted of necrotic leaf tips as well as 5- to 10-mm ellipsoidal necrotic spots on stems and the abaxial and adaxial leaf surfaces. Lesions were initially dark brown and water soaked, becoming lighter as they dried. Although leaf spot symptoms usually began at the leaf tip, eventually the entire leaf would become blighted. Disease symptoms were not observed on Oriental hybrid lilies in the same nurseries. A Botrytis sp. was consistently associated with the disease symptoms. In pure culture, the fungus matched the description of Botryotinia sphaerosperma (Gregory) Buchw. (anamorph Botrytis sphaerosperma (Gregory) Buchw.) (1). The fungus is distinguished by its large, spherical conidia that average 23 to 25 µm in diameter at maturity. The conidia are born in compact heads on unbranched conidiophores. Conidia and conidiophores are hyaline when young. At maturity, the conidia turn dark brown, whereas the conidiophores turn blackish brown. The fungus formed abundant sclerotia on potato dextrose agar when incubated at room temperature with 12 h per day of fluorescent lighting. The sclerotia were black, ovate to sphaeroidal, had a rough surface, and were 1.5 to 2.5 × 1 to 2.5 mm. Apothecia were not observed. Pathogenicity was tested on 12 Asiatic hybrid and 12 Oriental hybrid lilies. Inoculum consisting of mycelial fragments and conidia (1 × 105 conidia per ml) was produced on lily leaf agar (500 ml of H2O, 7.5 g of agar, and 25 g of healthy, macerated lily leaves). Plants were inoculated by rubbing leaves and stems with the spore and mycelial fragment suspension using cotton swabs. Plants were maintained in a greenhouse at 25°C. After 7 days, characteristic lesions were observed on the leaves and stems of Asiatic hybrid and Oriental hybrid lilies, from which the same Botrytis sp. was consistently isolated. Three control plants each of Asiatic hybrid and Oriental hybrid lilies treated with water as a check, remained symptomless. Although studies to determine potential fungicides for controlling this pathogen have not yet been undertaken, good sanitation, a lily-free period, and steam sterilization of beds should be considered in the management of this disease. In Europe, this fungus has been reported as a pathogen on Lilium regale (2). To our knowledge, this is the first report of this pathogen in North America. Herbarium specimens were submitted to the USDA APHIS mycology laboratory in Beltsville, MD (Accession No. BPI 842231). Cultures were also submitted to the American Type Culture Collection (MYA-2890). References: (1) P. H. Gregory. Trans. Br. Mycol. Soc. 25:26, 1941. (2) G. L. Hennebert. Friesia 9:52, 1969.

First Report of Mimosa Wilt Disease of Silk Tree (Albizia julibrissin) in California Caused by Fusarium oxysporum f. sp. Perniciosum.

A Fusarium sp. was isolated from a 12-year-old Silk Tree (Albizia julibrissin) in a residential area of Redlands, CA. The scaffold branches and trunk exhibited gummosis, the sap oozing from fissures or intact bark. Internally the wood exhibited brown to black broad streaks of discoloration from the scaffold branches down into lateral roots below the root crown, similar to symptoms observed in Virginia (2). Wilted and dried foliage remained on the scaffold branches. Two-week-old cultures of the isolate grown on Komada (1) and acidified potato dextrose agar media developed short conidiophores, macroconidia, and colony morphology typical of Fusarium oxysporum. To complete Koch's postulates, 1-month-old seedlings were root-dip inoculated with a water suspension of macro- and microconidia (106 per ml). Two weeks after inoculation, typical Fusarium wilt symptoms developed in all inoculated seedlings. The fungus was reisolated from symptomatic seedlings. This is the first report of mimosa wilt disease in California. The disease has the potential to adversely impact California's nursery and landscape industry. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) R. J. Stipes and P. M. Phipps. Phytopathology 65:188, 1975.