First Report of Fenhexamid-Resistant Botrytis cinerea Causing Gray Mold on Heurchera in a North American Greenhouse.

Greenhouse-grown Heuchera plants, treated with fenhexamid (Decree, SePRO, Carmel, IN; FRAC group 17 hydroxyanilide), with active gray mold were submitted to the Penn State Plant Disease Clinic in December 2010 from a commercial operation in north-central Pennsylvania. Genetic and phenotypic analyses identified the isolate as Botrytis cinerea Pers. (teleomorph Botryotinia fuckeliana (de Bary) Whetzel), HydR3 phenotype (2) and not B. pseudocinerea (previously Botrytis group I) (4), naturally resistant to fenhexamid (phenotype HydR1) (1). While 0.2 µg of fenhexamid per ml or less is required to slow mycelial growth and germ tube elongation of sensitive isolates by 50% (EC50), the radial growth EC50 of the Heuchera isolate was approximately 2,000 µg of fenhexamid per ml in culture. Five cucumber seedlings receiving 25 µl of 0.1 M dextrose containing the label rate of Decree (1,800 µg/ml) on the growing tip were inoculated with colonized agar in the drop. Five check plants received 25 µl of 0.1 M dextrose. B. cinerea from silica gel storage since 1988 was also tested. This experiment was repeated three times. The 1988 isolate killed all fungicide-free but no fenhexamid-treated plants. The Heuchera isolate killed all fungicide-free and fenhexamid-treated plants within 4 days. To our knowledge, this is the first report of B. cinerea from a greenhouse in North America with fenhexamid resistance. Resistance occurs in U.S. fields (3). The Heuchera isolate's HydR3 resistance phenotype (2) has been detected in Germany, Japan, and France and has mutations affecting the 3-keto reductase protein, encoded by the erg27 gene, the specific target of fenhexamid and involved in Botrytis sterol biosynthesis. The Decree label states that it is to be used only twice on a crop before switching to a different mode of action. Greenhouses have resident Botrytis populations that are likely to be exposed to any fungicide applied in the structure. Growers should consider using fenhexamid only twice in a particular greenhouse, rather than on a particular crop, before switching to a different mode of action. References: (1) P. Leroux et al. Crop Prot. 18:687, 1999.(2) P. Leroux et al. Pest Manag. Sci. 58:876, 2002. (3) Z. Ma and T. J. Michailides. Plant Dis. 89:1083, 2005. (4) A.-S. Walker et al. Phytopathology 101:1433, 2011.

First Report of Phytophthora cinnamomi on Ilex glabra in Virginia.

During 2002, two nurseries in southeastern Virginia reported losses exceeding 75% of container-grown inkberry holly (Ilex glabra) cv. Shamrock. The development of necrotic leaf spots and blotches followed initial symptoms of leaf yellowing and wilting. Affected leaves rapidly turned brown and fell. Dark brown-to-black roots were washed and plated on agar media. Phytophthora cinnamomi Rands was consistently isolated and identified on the basis of its morphology (2) and single-stranded conformational polymorphism fingerprint (1). The organism had nonpapillate, internally proliferating, noncaducous, ovoid to ellipsoid sporangia that formed only in water. It did not grow at 35°C and had abundant botryose hyphal swellings, coralloid hyphae, and grape-like clusters of chlamydospores. The isolate, determined to be the A2 mating type, produced elongate cylindrical, amphigynous antheridia and oogonia with a tapered base. A pine bark potting mix amended with V8 juicetreated vermiculite colonized by the suspected pathogen was placed in 12-liter containers. Two inkberry holly cv. Shamrock liners were planted in each of three containers and two 1-yr-old plants were planted in each of three additional containers during April 2004. An identical set of six containers of noninoculated plants was also established. During June 2004, inoculated plants exhibited symptoms identical to those observed in nurseries, and P. cinnamomi was isolated. Noninoculated check plants did not develop symptoms. Japanese holly (I. crenata) was previously known as a host, but to our knowledge, this is the first report of inkberry holly (I. glabra) susceptibility. References: (1) P. Kong et al. Fungal Genet. Biol. 39:238, 2003. (2) D. J. Stamps et al. Mycol. Pap. No. 162. CAB International Mycological Institute, Wallingford, UK, 1990.

Species of Pythium from Greenhouses in Pennsylvania Exhibit Resistance to Propamocarb and Mefenoxam.

Pythium isolates from commercial greenhouses in Pennsylvania were tested in vitro and in vivo for sensitivity to propamocarb. Isolates of Pythium aphanidermatum, P. irregulare, and P. ultimum consistently infected geranium seedlings that had been treated with propamocarb. P. ultimum and P. irregulare isolates exhibited dual fungicide resistance by overcoming propamocarb, mefenoxam, and a mixture of propamocarb and mefenoxam to infect seedlings. The sensitivity of isolates to propamocarb in vitro was not a good predictor of in vivo sensitivity. This is the first report of Pythium spp. resistance to propamocarb and dual resistance to propamocarb and mefenoxam.

Ralstonia solanacearum Race 3, Biovar 2, the Causal Agent of Brown Rot of Potato, Identified in Geraniums in Pennsylvania, Delaware, and Connecticut.

The Plant Disease Diagnostic Laboratory of the Pennsylvania Department of Agriculture received diseased geranium (Pelargonium × hortorum) samples from several Pennsylvania (PA) greenhouses in 1999 and 2000 and from one Delaware (DE) greenhouse in 1999. Originating from Guatemala, plants exhibited yellowing, wilting, stunting, and bacterial oozing from the vascular tissues. Isolations on yeast dextrose-CaCO3 (YDC) and triphenyl-tetrazolium-chloride (TTC) agars resulted in off-white mucoid colonies and white, fluidal colonies with pink centers, respectively. Such colonies are typical of Ralstonia solanacearum (1). The disease was similar to a bacterial wilt of geranium caused by an unidentified biovar of R. solanacearum (3). Preliminary tests using Biolog MicroLog 3 (Hayward, Ca; 4.01A) and enzyme-linked immunosorbent assay (ELISA) (Agdia Inc., Elkhart, IN; BRA 33900/0500) identified the organism as R. solanacearum. For pathogenicity tests, a 10-µl droplet of water suspension containing 1 × 106 CFU of each of five geranium strains (PDA 22056-99, 81849-99, 81862-99, 51032-00, and 64054-00) per milliliter was placed on a stem wound made by cutting off the terminal growth of each of 4 6-leaf stage plants of geranium 'Orbit Scarlet', tomato 'Rutgers', potato 'Russet Norkotah', and eggplant 'Black Beauty' in a growth chamber at 28°C, 86% relative humidity, and 12 h light/dark cycle. Water was included as a control. The five strains caused severe yellowing and wilting within 10 days. Colonies typical of R. solanacearum were reisolated from symptomatic tissue on YDC and TTC. To determine the specific biovar, 20 pathogenic geranium strains from PA and DE plus a strain of R. solanacearum originally isolated from a geranium plant of Guatemalan origin received from Connecticut in 1995 were grown up to 28 days on Ayers mineral medium supplemented with a 1% final concentration of D-cellobiose, dextrose, meso-inositol, lactose, maltose, D-ribose, trehalose, mannitol, sorbitol, or dulcitol (1). Acid was produced by 21 test strains from the first five carbohydrates only. Such carbohydrate utilization is typical of bv 2 (1). Bv 2 identification was confirmed by real-time polymerase chain reaction using bv 2-specific primers and probes (N. Schaad, unpublished) designed from a bv 2-specific DNA fragment (2). All tested strains were positive using ELISA. In contrast, strains of bv 2 from geraniums in Wisconsin and South Dakota were reported to be negative using ELISA (4). From our results, it appears that bv 2 was introduced into the United States on geraniums from Guatemala in 1995 and 1999. This cool climate bv 2, a regulated agent by the Agricultural Bioterrorism Protection Act of 2002, has caused extensive crop loss in potatoes in Europe, but has not been found in potatoes in the United States. References: (1) T. P. Denny and A. C. Hayward. Ralstonia solanacearum. Pages 151-174 in: Lab Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad et al. eds. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001. (2) M. Fagen et al. Development of a diagnostic test based on the polymerase chain reaction (PCR) to identify strains of R. solanacearum exhibiting the Biovar 2 genotype. Pages 34-43 in: Bacterial Wilt Disease: Molecular and Ecological Aspects. P. H. Prior et al. eds. Springer-Verlag, Berlin, 1998. (3) D. L. Strider et al. Plant Dis. 65:52, 1981. (4) L. Williamson et al. (Abstr.) Phytopathology 91 (Suppl.):S95, 2001.

Identification and Characterization of Pythium Species Associated with Greenhouse Floral Crops in Pennsylvania.

During 1996 to 2001, samples submitted to clinics from commercial greenhouses involved 11 species and two unidentified isolates of Pythium from 110 plant samples, five potting soil tests, and five tests of irrigation water. Pythium irregulare was found in 45% of the plant samples, four of the five water samples, and three of the five potting soils. Pythium aphanidermatum accounted for 29% of all plant but 77% of the poinsettia samples. The Pelargonium samples received were infected with P. aphanidermatum, P. dissotocum, P. heterothallicum, group F, P. irregulare, P. myriotylum, and P. ultimum. The base pair sequence of the ITS1, 5.8S, and ITS2 regions of ribosomal DNA effectively differentiated the species encountered. The ras-related protein gene sequence did not differentiate P. aphanidermatum, P. arrhenomanes, and P. deliense from one another. One isolate each of P. cylindrosporum, P. dissotocum, P. heterothallicum, P. splendens, and P. ultimum exhibited resistance to the phenylamide fungicide mefenoxam, an isomer of metalaxyl, while 38% of the P. aphanidermatum and 37% of the P. irregulare isolates were resistant.

Branch Dieback of Savin Juniper in Pennsylvania Caused by Diplodia mutila.

Branch dieback of savin juniper (Juniperus sabina L.) was observed on a tree in Dauphin County, PA, in May 1996. The symptomatic tree was in an ornamental planting that had been established approximately 10 years previously. Branches were cankered and girdled, causing yellowing and death of foliage beyond the cankers. Black pycnidia occurred in necrotic bark of cankers. Dark, two-celled conidia obtained from these pycnidia produced pure cultures of Diplodia mutila (Fr.:Fr.) Mont., the anamorph of Botryosphaeria stevensii Shoemaker. The fungus was identified based on pycnidial, conidial, and cultural characteristics, and comparison with known isolates provided by N. A. Tisserat (2). Pathogenicity of a single conidial isolate from Pennsylvania was tested in a greenhouse by wounding and inoculating twigs of potted eastern red cedar (Juniperus virginiana L.) with water agar plugs colonized by mycelium. Cankers formed and enlarged to girdle and kill the inoculated shoots, from which the pathogen was reisolated. No symptoms developed on, nor was the pathogen isolated from, control twigs. B. stevensii has been reported more frequently on angiosperms, such as apple (Malus Mill.) and oak (Quercus L.), than on gymnosperms. However, a canker disease caused by B. stevensii previously has been reported to affect J. scopulorum Sarg. and J. virginiana in Kansas, Missouri, and Iowa (1,2). References: (1) P. H. Flynn and M. L. Gleason. Plant Dis. 77:210, 1993. (2) N. A. Tisserat et al. Plant Dis. 72:699, 1988.

The formation of histotypic structures from monodisperse fetal rat lung cells cultured on a three-dimensional substrate.

Enzymatically dissociated lungs from rat fetuses at 19-days gestation yield single cells which reaggregate to form alveolar-like structures when cultured on gelatin sponge discs. These structures form within 2 days and have been maintained in vitro for as long as 6 weeks. They are composed primarily of type II pneumonocytes as characterized by large, lightly stained nuclei and cytoplasmic inclusion bodies. The lamellar structure of these inclusion bodies has been confirmed by electron microscopy. The dynamic formation of inclusion bodies is suggested by the presence of lamellar bodies in the extra-cellular space and the appearance of new inclusions in the cytoplasm of the type II pneumonocytes. The formation and long-term maintenance of histotypic lung structures in vitro provides a model system for the study of lung development and synthesis of surfactant by type II alveolar pneumonocytes.