First Report of Sclerotium rolfsii on Ascocentrum and Ascocenda Orchids in Florida.

Southern blight caused by Sclerotium rolfsii is known to occur on several economically important orchid hosts, including Vanda species and hybrids (1-3). In the summer and fall of 2008, an outbreak of southern blight on Vanda orchids was seen in several commercial nurseries and landscapes throughout South Florida. More than a dozen orchids were affected at one of the locations, and symptoms of S. rolfsii were observed on Ascocentrum and Ascocenda orchids, which are also common in the trade and demand a resale value ranging from $20 to $150 for specimens in bloom. Affected Ascocentrum and Ascocenda orchids were found severely wilted at the apex, while around the base of the plants, tan, soft, water-soaked lesions were present. As the lesions progressed, leaves around the base of the plants began to fall off, leaving the stems bare. After 2 days, white, flabellate mycelium was seen progressing up the stem and numerous, tan-to-brown sclerotia were present. Leaves and portions of the stems were plated on acidified potato dextrose agar (APDA) and grown at 25°C. White, flabellate mycelium and tan sclerotia approximately 2 mm in diameter were produced in culture and microscopic examination revealed the presence of clamp connections. The fungus was identified as S. rolfsii and a voucher specimen was deposited with the ATCC. A PCR was performed on the ITS1, 5.8S rDNA, and ITS2 and the sequence was deposited in GenBank (Accession No. GQ358518). Pathogenicity of an isolate was tested by placing 6-mm plugs taken from APDA plates directly against the stem of five different Ascocentrum and Ascocenda orchids. Five Ascocentrum and Ascocenda orchids were inoculated with 6-mm plugs of plain APDA and five were untreated controls. Plants were housed under 50% shade, 60 to 95% humidity, and temperatures ranging from 75 to 88°F. Within 7 days, all inoculated plants developed symptoms that were identical to those observed on original plants and S. rolfsii was consistently reisolated from symptomatic tissue. Ascocentrum and Ascocenda were previously reported under miscellaneous orchid species and hybrids as hosts for S. rolfsii (1). However, this report was highly ambiguous and the most current edition does not report the host fungus combination (2). To our knowledge, this is the first report of S. rolfsii affecting Ascocentrum and Ascocenda orchids. References: (1) S. A. Alfieri, Jr., et al. Diseases and Disorders of Plants in Florida. Bull. No. 11. Division of Plant Industry, Gainesville, FL, 1984. (2) S. A. Alfieri, Jr., et al. Diseases and Disorders of Plants in Florida. Bull. No. 14. Division of Plant Industry, Gainesville, FL, 1994. (3) D. F. Farr et al. Fungi on Plants and Plant Products in the United States. The American Phytopathological Society, St. Paul, MN, 1989.

First Report of a Bacterial Soft Rot on Tolumnia Orchids Caused by a Dickeya sp. in the United States.

Tolumnia orchids are small epiphytic orchids grown for their attractive flowers. In the fall of 2008, approximately 100 Tolumnia orchids with soft, brown, macerated leaves were brought to the University of Florida Extension Plant Diagnostic Clinic in Homestead. Ten plants were randomly selected and bacteria were isolated from the margins of symptomatic tissues of each of the 10 plants on nutrient agar according to the method described by Schaad et al. (2). Four reference strains were used in all tests, including the molecular tests: Erwinia carotovora subsp. carotovora (obtained from J. Bartz, Department of Plant Pathology, University of Florida, Gainesville), E. chrysanthemi (ATCC No. 11662), Pectobacterium cypripedii (ATCC No. 29267), and Acidovorax avenae subsp. cattleyae (ATCC No. 10200). All 10 of the isolated bacteria were gram negative, grew at 37°C, degraded pectate in CVP (crystal violet pectate) medium, grew anaerobically, produced brown pigment on NGM (nutrient agar-glycerol-manganese chloride) medium (1), were sensitive to erythromycin, and produced phosphatase. Three of the strains were submitted for MIDI analysis (Sherlock version TSBA 4.10; Microbial Identification, Newark DE) (SIM 0.732 to 0.963), which identified them as E. chrysanthemi. A PCR assay was performed on the 16S rRNA gene with primers 27f and 1495r described by Weisburg et al. (3) from two of the isolates and a subsequent GenBank search showed 99% identity of the 1,508-bp sequence to that of Dickeya chrysanthemi (Accession No. FM946179) (formerly E. chrysanthemi). The sequences were deposited in GenBank (Accession Nos. GQ293897 and GQ293898). Pathogenicity was confirmed by injecting approximately 100 µl of a bacterial suspension at 1 × 108 CFU/ml into leaves of 10 Tolumnia orchid mericlones. Ten plants were also inoculated with water as controls. Plants were placed in a greenhouse at 29°C with 60 to 80% relative humidity. Within 24 h, soft rot symptoms appeared on all inoculated leaves. The water controls appeared normal. A Dickeya sp. was reisolated and identified using the above methods (biochemical tests and MIDI), fulfilling Koch's postulates. To our knowledge, this is the first report of a soft rot caused by a Dickeya sp. on Tolumnia orchids. Although 16S similarity and MIDI results suggest the isolated bacteria are D. chrysanthemi because of its close similarity with other Dickeya spp., these results are not conclusive. Further work should be conducted to confirm the identity of these isolates. Through correspondence with South Florida Tolumnia growers, it appears this disease has been a recurring problem, sometimes affecting international orchid shipments where plant losses have been in excess of 70%. References: (1) Y. A. Lee and C. P. Yu. J. Microbiol. Methods 64:200, 2006. (2) N. W. Schaad et al. Erwinia soft rot group. Page 56 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. American Phytopathological Society. St. Paul, MN, 2001. (3) W. G. Weisburg et al. J. Bacteriol. 173:697, 1991.

First Report of Sclerotium rolfsii on Brassidium Hybrid Orchid.

Brassidium hybrid orchid leaves were sent to the University of Florida Plant Disease Clinic in the summer of 2002. Symptoms on leaves were a spreading, cottony, white mass of mycelium with necrotic centers 3 to 5 × 5 to 9 mm. Approximately 1% of leaves submitted were affected. Sclerotium rolfsii Sacc. was isolated from the affected leaves using acid potato dextrose agar (APDA) (1). Two plastic boxes were lined with wet paper towels and 6 uninoculated leaves were placed in each. Three leaves in each box were treated as controls and received 3 mm2 of APDA, while the other three leaves in each box received 3 mm2 of S. rolfsii culture on APDA. All leaves were misted with water and enclosed to maintain humidity. Twenty-four hours later, infection was visible on two treated leaves. Within 72 h, significant infection was visible on three of the six inoculated leaves. All control leaves remained uninfected. S. rolfsii was consistently reisolated from the inoculated leaves, fulfilling Koch's postulates. Over the past 12 months, S. rolfsii has been detected in 30% of commercial orchid nurseries as well as homeowner orchid collections in Miami-Dade County, Florida. No resistance to this disease in orchids has been reported. To our knowledge, this is the first report of S. rolfsii on this orchid hybrid. Reference: (1) J. Tuite. Media and nutrient solutions used by plant pathologist and mycologist. Page 53 in: Plant Pathological Methods Fungi and Bacteria. Burgess Publishing Company, Minneapolis, MN, 1969.

First Report of Dieback Caused by Verticillium dahliae on Blighia sapida in the United States.

Mature akee trees, Blighia sapida K. Koenig, in a local south Florida commercial orchard had wilt and dieback symptoms during spring 1999. A fungus isolated from the gray xylem root tissue on V8 agar was identified as Verticillium dahliae Klebahn at the Division of Plant Industry of the Florida Department of Agriculture and Consumer Services. Twenty akee seedlings were transplanted into 3.85-liter plastic pots and grown in a greenhouse at a daytime temperature of 28°C and nighttime temperature of 23°C. When plants were approximately 25 cm high, a 15-cm knife was used to sever roots in the four quadrants of each pot. Inoculum was made from a 2-week-old culture of V. dahliae on V8 agar and blended with 160 ml of sterile water, and 15 ml of this slurry was poured into the disturbed soil of each of 10 treated plants. A plate of uninoculated V8 agar was applied, as above, to 10 control plants. Plants were kept in the greenhouse. After 6 weeks, inoculated plants showed symptoms of leaf wilt, dieback and plant death. No symptoms were seen on control plants. V. dahliae was isolated directly from the gray vascular tissue of inoculated plants. The inoculation experiment was repeated three times, fulfilling Koch's postulates. To our knowledge, this is the first report of Verticillium dieback on B. sapida in the United States.

First Report of Aristastoma Leaf Spot on Desert Rose.

In the spring of 1995, a species of Aristastoma was isolated from foliar lesions of Adenium obesum that originated in a commercial nursery in Dade County, FL, where 100% of the crop was affected. Plant foliage had irregular, oval to circular, rusty brown, necrotic lesions 5 to 15 mm in diameter. Large leaf spots developed tan centers. An undescribed species of Aristastoma was isolated consistently from symptomatic plant material. The identity of the fungus was confirmed by the International Mycological Institute, Egham, England. Both in vivo and in vitro, the fungus has immersed mycelium, pale brown to hyaline, branched and septate. Pycnidia are 160 to 300 µm in diameter with prominent setae 75 to 125 µm surrounding the ostiole, with hyaline, 1 septate, conidia that are 25 to 30 × 3 to 4 µm. Pathogenicity was tested on shadehouse-grown, 8-month-old desert rose plants by spraying plants with a conidial suspension containing 104 conidia per ml. Inoculated plants developed foliar symptoms within 5 to 10 days that were consistent with those originally observed on the diseased plant samples. The pathogen was isolated from symptomatic tissue of inoculated plants. Species of Aristastoma are reported to cause foliar symptoms mostly on leguminous crops, and in Florida Aristastoma oeconomicum causes lesions on Vigna. This is the first report of this or any species of Aristastoma on Adenium obesum or any other member of the Apocynaceae.

The determination of volatile fatty acids in the caecum of the conscious rabbit.

1. A method of obtaining dialysed samples from the caecum of the conscious rabbit is described. 2. Values for total volatile fatty acid content and for molar proportions of individual volatile fatty acids in dialysate samples were in good agreement with those obtained from caecal material. 3. The volatile fatty acid level in the caecum throughout the day was determined using two groups of animals, one group fed ad lib. and the other group on a restricted food intake. These results indicated a marked diurnal fluctuation in volatile fatty acid level in the caecum of rabbits fed once/d which was not evident in those fed ad lib.