R. A. Cating

Silwet L-77 Improves the Efficacy of Horticultral Oils for Control of Boisduval Scale Diaspis boisduvalii (Hemiptera: Diaspididae) and the Flat Mite Tenuipalpus pacificus (Arachnida: Acari: Tenuipalpidae) on Orchids

ABSTRACT Pest management of insects and mites on orchids has been based on the use of synthetic organic pesticides. However, less-toxic chemical control is needed by hobbyists and small growers. Orchids from 8 genera were treated with Silwet L-77 alone and no evidence of phytotoxicity was seen. Subsequently, 3 petroleum oils in combination with Silwet L-77 were tested to determine efficacy in suppressing Boisduval scale Diaspis boisduvalii Signoret (Hemiptera: Diaspididae) and the flat mite Tenuipalpus pacificus Baker (Arachnida: Acari: Tenuipalpidae). The results indicate Silwet L-77 increased toxicity of the oils and increased removal of the scales and mites from foliage over oil alone, yet no phytotoxicity was observed in any of the plants treated. Petroleum oil + Silwet L-77 is considered low-toxic, may be less expensive than some pesticides, and may conserve natural enemies, making it a potential addition to an integrated pest management program for orchids.

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

A Comparison of Standard and High-Fidelity PCR: Evaluating Quantification and Detection of Pathogen DNA in the Presence of Orchid Host Tissue

20 to

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

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 Uredo manilensis in the Western Hemisphere

The polymerase chain reaction (PCR) has been used with increasing frequency for detecting and identifying plant pathogens. Although PCR is sensitive, research has shown that amplification of target microbial DNA from within another organism, such as an arthropod or plant, can be inhibited by the presence of host DNA. In this study, the sensitivity of standard and high-fidelity PCR, which incorporates a second DNA polymerase with proofreading ability, to detect and amplify DNA from the fungal pathogen Pseudocercospora odontoglossi while in the presence of Cattleya orchid DNA, was compared. Different dilutions of plasmids containing internal transcribed spacer (ITS)1, 5.8S, and ITS2 rDNA from P. odontoglossi were spiked with Cattleya orchid plant DNA. The high-fidelity PCR could detect and amplify as few as 207 plasmids containing the fungal DNA, whereas the standard PCR required over 200 million copies. The high-fidelity PCR was more efficient than conventional PCR in detecting Sclerotium rolfsii and a Dickeya sp. from freshly inoculated orchid plants, demonstrating its increased sensitivity in early detection of fungal and bacterial pathogens that are difficult to discriminate early in disease development.

First report of tar spot on orange geiger, Cordia sebestena, caused by Diatractium cordianum in Florida.

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 Kochs 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 Downy Mildew on Gynura aurantiaca Caused by Plasmopara halstedii sensu lato in Florida

Crepe jasmine, Tabernaemontana divaricata (L.) R. Br. ex Roem. & Schult. (Apocynaceae), is a popular flowering shrub in South Florida. A native of Southeast Asia, it is one of approximately 100 ornamental species in the genus. In December 2007, rust was observed on the leaves of landscape plants in Key West and Miami. The rust has become prevalent and severely affects young and old leaves of plants in the landscape and in commercial nurseries. Leaf lesions begin as chlorotic flecks that expand into necrotic spots with orange-to-reddish brown, subepidermal uredinia; brown telia develop on the abaxial side of leaves. Urediniospores are one-celled, initially hyaline, minutely echinulate and spherical, turn dark orange, and measure (22) 24 to 29 (32) × (19) 21 to 24 (26) μm. Teliospores are (26) 29 to 36 (38) × (20) 22 to 26 (28) μm, two-celled, ellipsoidal to ovoid, echinulate, constricted at the septum, reddish brown, and have 0.8-μm thick spore walls; pedicels are 25 × 5.6 μm, persistent, and hyaline. Attributes for urediniospores are consistent with those from the original description of Uredo manilensis Syd. & P. Syd. on T. coronariae in Manila (2); however, there are no reports of a telial stage for this rust. Attributes for urediniospores of the South Florida fungus were also consistent with those on herbarium specimens of U. manilensis from the U.S. National Fungus Collection, also collected in Manila but from T. polygama (BPI Accession Nos. 0155269 and 0155270). Notably, these specimens contained telia that matched those found in South Florida. Subsequent comparisons were made with herbarium specimens of the three Puccinia spp. that have been reported on Tabernaemontana spp. (the U.S. National Fungus Collection or the Arthur Herbarium, Purdue University, West Lafayette, IN). Puccinia engleriana (five specimens from India, New Guinea, and the Philippines) differs from the BPI specimens of U. manilensis and the South Florida fungus by its bigger teliospores (32) 35 to 41 (45) × (21) 22 to 24 μm. P. tabernaemontana (six specimens from Uganda) has bigger urediniospores ([45] 34 to 41 × [34] 26 to 32 μm) and yellow-brown, poorly echinulated to almost smooth teliospores. The revised material of P. morobensis (type) was poor, but according to the original description (1) and notes found in the herbarium specimen, the teliospores (24 to 29 × 33 to 45 μm) and urediniospores are larger (23 to 28 × 29 to 35 μm) and the teliospores walls are finely and sparsely echinulated to sometimes smooth, and the pedicels are very short and fragile. A specimen of the South Florida fungus was deposited with the U.S. National Fungus Collections (BPI Accession No. TBA). To our knowledge, this is the first report of U. manilensis in the Western Hemisphere and the first time a telial stage (provisionally P. manilensis) has been recognized for the fungus. This disease has become a concern in South Florida for gardeners as well as producers who must now treat the crop with fungicides. References: (1) G. B. Cummins. Mycologia. 33:148, 1941. (2) H. Sydow, and P. Sydow. Ann. Mycol. 8:36, 1910.

First report of Phytophthora palmivora causing foliar blight of Pachira aquatica in Florida.

In July 2007, tar spot symptoms were observed on the leaves of orange Geiger, Cordia sebestena L. (Boraginaceae), in the landscape and a commercial nursery in Homestead, FL. The disease appears to be spreading and is locally severe. Symptoms were circular, slightly hypertrophied spots approximately 5 to 8 cm in diameter, which were slightly chlorotic on the abaxial surface and had numerous circular blackened stroma, 0.2 to 0.4 mm in diameter, on the adaxial surface. As leaves aged and yellowed, the areas around the spots remained pale green. Embedded in the stroma were numerous perithecia, 173 to 312 μm in diameter, circular to irregular in shape, with lateral necks as much as 200 μm long and 73 to 104 μm in diameter. Asci, 77 to 92 × 11 to 13 μm, contained elongate, two-celled ascospores, 50 to 61 × 3 to 5 μm that had a conspicuous constriction at the dividing cell wall. These dimensions and the pathogens appearance matched closely with those published for Diatractium cordianum (Ellis & Kelsey) Syd (1). Young, symptomless leaves of C. sebestena were sprayed to runoff with a suspension of ascospores approximately 104 ml-1 that were harvested from affected leaves. Inoculated leaves were placed on water-saturated paper towels in petri plates and maintained in a growth chamber at 25°C with fluorescent light at 10 h day-1. Symptoms similar to those observed on affected trees in the landscape began to develop after 21 days and perithecia were evident after 28 days. An ITS 1, ITS 2, and 5.8s rDNA sequence was deposited in GenBank (Accession No. EU541488). A herbarium specimen was deposited at the U.S. National Fungus Collections (BPI No. 878441). This is a new host record for D. cordianum and is the first time the pathogen has been reported in the United States. Previous records were from Venezuela and several Caribbean islands, including Cuba and Jamaica. Symptoms of this disease have not been observed on Texas wild olive, Cordia boissieri, in close proximity to affected C. sebestena. P. F. Cannon (1) indicated that the disease had no economic impact. However, the conspicuous nature of symptoms on C. sebestena and the importance of this tree in the South Florida ornamental trade (2) suggest that this disease may become significant on the latter host. References: (1) P. F. Cannon. Mycol. Res. 92:327, 1989. (2) E. F. Gilman and D. G. Watson. Fact Sheet ST-182. Univ. Fla, Fla Coop Ext. Serv., 1993.

Bacterial Soft Rot of Oncidium Orchids Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in Florida

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Neofusicoccum parvum causes a lethal dieback of Syzygium paniculatum in Florida

Pachira aquatica Aubl. is a member of the Bombaceae, indigenous to Central America and northern South America. Known as the money tree within the ornamental plant industry, this tropical species is well adapted to landscapes in south Florida, Hawaii, and milder areas in southern California. Recently, its become more popular as a potted plant for use in the interiorscape. During August 2011, several local nurseries submitted P. aquatica samples to the Florida Extension Plant Diagnostic Clinic in Homestead, FL. The foliage exhibited dark brown to black water soaked spots that became papery as the disease progressed, and rapidly enlarged and coalesced, resulting in severe leaf blight. Both young and mature leaves were affected. Phytophthora was initially confirmed by serological testing with a commercially available ImmunoStrip test (Agdia, Elkhart, IN). On closer examination, the pathogen was further identified as Phytophthora palmivora by the presence of numerous papillate, deciduous, ellipsoidal to ovoid sporangia with short pedicels. The sporangia averaged 53 × 32 μm with ranges of 48 to 59 × 29 to 35 μm (1). Phytophthora species-specific primers (pal1s and pal2a) targeting part of the 18S rRNA gene, the ITS 1, the 5.8S rRNA gene, and the ITS 2 resulted in a PCR product of 648 bp, testing positive for P. palmivora (2). The PCR product was cleaned (Qiagen Purification Kit) and sequenced (GenBank Accession No. JQ354937). The sequence from our isolate was nearly identical (exhibited 99% nucleotide identity) to an isolate of P. palmivora (GenBank Accession No. HE580280) collected from diseased cassava in China. To further support identification, phylogenetic analysis by the maximum likelihood method (Tamura-Nei model) was performed using the obtained sequence and several other Phytophthora and Pythium species from GenBank (MEGA 5.05). Our isolate grouped with other P. palmivora isolates with high support (100% bootstrap values, 1,000 replicates). Pathogenicity of the sequenced isolate was evaluated in shade house experiments. Six-month-old Pachira aquatica plants were inoculated with sporangial suspensions (1 × 106 conidia/ml) of P. palmivora. Inoculum or autoclaved water was sprayed over the foliage until runoff. Six plants were sprayed per treatment, and the experiment was repeated twice. Inoculated plants were placed in a shade house (70% shade) when temperatures ranged from 25 to 32°C with 78 to 98% relative humidity. Plants were observed for disease development, which occurred within 7 days of inoculation. No symptoms developed on the control plants. Foliar lesions closely resembled those observed in the affected nurseries and P. palmivora was reisolated from symptomatic leaf tissue. To our knowledge, on the basis of serological testing, molecular analysis, and distinguishing morphological characters, this is the first report of P. palmivora causing foliar blight of Pachira aquatica in Florida. The high incidence and severity of this disease may seriously influence local tropical foliage producers in the future. References: (1) D. C. Erwin and O. K. Ribeiro. Phytophthora Diseases Worldwide. American Phytopathological Society, St Paul, MN, 1996. (2) Tsai et al. Botanical Studies 47:379, 2006.