Regional Spatial-Temporal Spread of Citrus Huanglongbing Is Affected by Rain in Florida.

Citrus huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus' (Las), disseminated by Asian citrus psyllid (ACP), has devastated citrus in Florida since 2005. Data on HLB occurrence were stored in databases (2005 to 2012). Cumulative HLB-positive citrus blocks were subjected to kernel density analysis and kriging. Relative disease incidence per county was calculated by dividing HLB numbers by relative tree numbers and maximum incidence. Spatiotemporal HLB distributions were correlated with weather. Relative HLB incidence correlated positively with rainfall. The focus expansion rate was 1626 m month-1, similar to that in Brazil. Relative HLB incidence in counties with primarily large groves increased at a lower rate (0.24 year-1) than in counties with smaller groves in hotspot areas (0.67 year-1), confirming reports that large-scale HLB management may slow epidemic progress.

Molecular Detection of the Laurel Wilt Fungus, Raffaelea lauricola.

The laurel wilt disease fungus, Raffaelea lauricola, is killing redbay trees, spreading rapidly in the U.S. southeastern coastal plain forest, and posing a serious threat to the avocado industry in Florida. A molecular tool is urgently required to facilitate detection of this pathogen. The 5' region of the large ribosomal RNA (28S) gene is highly variable among Raffaelea spp. and ideal for this purpose but amplification of this sequence from R. lauricola has been difficult. Different amplification conditions were tested and a high-fidelity polymerase chain reaction (PCR) procedure utilizing a dNTP mix containing 7-deaza-dGTP was found to reliably amplify 28S sequences from R. lauricola. Sequencing the amplified products or cloned inserts also turned out to be difficult and required using a custom-blended sequencing mix containing 1 M betaine, 5% dimethyl sulfoxide, and dGTP-BigDye v3.1. Three GC-rich stem and loop or cruciform secondary structures were discovered, which may have interfered with amplification. This improved protocol made it possible to partially characterize the internal transcribed spacers sequence from R. lauricola, which also has interfering secondary structures. A TaqMan real-time PCR assay was designed using the species-specific 28S sequences and this allowed detection of R. lauricola from wood tissues or cultures. Wood tissues from symptomatic redbay, avocado, and sassafras trees in Florida were screened using this TaqMan assay and several were found to test positive for R. lauricola. Results were further confirmed by performing Koch's postulates for avocado specimens collected from commercial grooves.

First Report of Guignardia citricarpa Associated with Citrus Black Spot on Sweet Orange (Citrus sinensis) in North America.

In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 µm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 µm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 µm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km2. The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown 'Valencia' sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.

First Report of a 16SrIV-D Phytoplasma Associated with Texas Phoenix Palm Decline on Pigmy Date Palm (Phoenix roebelenii) in Florida.

The pigmy date palm (Phoenix roebelenii O'Brien) is used as an ornamental in Florida and is popular and lucrative within the nursery trade. Severe decline of several pigmy date palms was observed at a residence in Hillsborough County, FL. Several other palm species, including P. canariensis (Canary Island date palm), P. sylvestris (wild date palm), P. dactylifera (date palm), Syagrus romanzoffiana (queen palm), and Sabal palmetto (cabbage palm), in Florida are known to be affected by Texas Phoenix palm decline (TPPD), a disease associated with a phytoplasma subgroup strain 16SrIV-D (2,3). Moreover, the location of the affected pigmy date palms was in the proximity of many other diseased cabbage palms that were identified in previous surveys and subsequently rogued. Genomic DNA was extracted from 100 mg of ground-up palm trunk tissues containing phloem cells with a DNeasy Plant Mini kit column (QIAGEN Inc., Valencia, CA) from four specimens. A high-fidelity PCR (Hf-PCR) procedure was used in preference to standard PCR because it was 10,000 to 100,000 times more sensitive (1,4). The Hf-PCR (50 µl) utilized two DNA polymerases; Taq (five units) and ACCUZYME (one unit), 350 µM dNTP, a buffer (50 mM Tris pH 9.2, 16 mM ammonium sulfate, and 1.75 mM magnesium chloride), a higher concentration of primers (200 pM) (2,3), and palm DNA templates (>10 ng) or no DNA negative control. Hf-PCR was performed using three linked profiles: (i) 94°C (2 min) (1 cycle); (ii) 94°C (10 s), 50°C (30 s) for P1m/P7 or 57°C for LY16Sf/LY16Sr and 68°C (2 min) (10 cycles); and (iii) 94°C (10 s), 50°C (30 s) for P1m/P7 or 57°C for LY16Sf/LY16Sr and 68°C (2 min plus 20 s added for every consecutive cycle) (20 cycles) (1). The genomic DNA extracted from P. roebelenii specimens was used as template for amplification by Hf-PCR. Expected 1.8- and 1.4-kb DNA bands for each primer combination were readily amplified. The Hf-PCR products were sequenced (GenBank Accession No. JF791816) and a BLAST search revealed a 100% similarity with a phytoplasma subgroup strain 16SrIV-D (EF042899 and AF434989), which is known to cause severe palm decline (TPPD) in other hosts (2,3). To our knowledge, this is the first report of TPPD from P. roebelenii, and therefore, expands the host range of this pathogen. In areas where TPPD is present, the landscape industry may need to identify alternative nonhost palm species or resistant varieties for disease management. References: (1) W. M. Barnes. Proc. Natl. Acad. Sci. USA 91:2216, 1994. (2) N. A. Harrison et al. Plant Dis. 86:676, 2002. (3) N. A. Harrison et al. Ann. Appl. Biol. 153:85, 2008. (4) A. Jeyaprakash and M. A. Hoy. Insect Mol. Biol. 9:393, 2000.

Gladiolus Rust Caused by Uromyces transversalis Makes First Nearctic Appearance in Florida.

The most serious rust pathogen of gladiolus (Gladiolus × hortulanus), Uromyces transversalis, has been listed as an exotic pathogen of concern for the United States for more than 80 years (4). Native to South Africa, the pathogen was reported in the Western Hemisphere for the first time in Brazil (2) and Argentina (1). Reports of gladiolus rust in several central Mexican states from 2004 to 2005 (3; http://www.pestalert.org/espanol/oprDetail.cfm?oprID=138 ) and interceptions at Mexican border stations and in Brazilian imports in 2005 at the port of Miami, FL collectively raised the alert level in the United States to high. In April 2006, the Hawaii Department of Agriculture notified the USDA of rust-infected gladiolus in a cut-flower shipment that was traced back to a 1,400-acre (565 ha) farm in Manatee County, FL. Inspection at the farm yielded samples that were quickly confirmed as U. transversalis by FDACS-DPI and USDA plant pathologists. The disease was identified in eight residential gardens near the commercial find and in another 700-acre (285 ha) farm in remote Hendry County, 100 miles to the southeast. In May 2006, gladiolus rust was detected in residential and commercial gladiolus in San Diego County, CA (see companion publication). On the advice of a USDA-assembled panel of experts, strict rust management guidelines and fallow host-free periods were implemented with the ultimate goal of eradication. Subsequent summer, fall, and now winter surveys in the infested commercial and residential areas have uncovered diminishing amounts of rust, with last traces detected on 9 September 2006. Commercial planting resumed at both farms in late summer, and crops remained rust free under weekly inspection until 15 February 2007 in Manatee County and 29 March 2007 in Hendry County. To insure a rust-free product, cut flowers are carefully inspected and foliage stripped at the packinghouse. Eradication will be attempted once more with a fallow host-free period before the 2007 season. U. transversalis is an autoecious rust that mainly infects Gladiolus spp., but has been known to infect other members of the Iridaceae: Anomatheca, Crocosmia, Melasphaerula, Tritonia, and Watsonia. Amphigenous uredinia form in transverse lines across gladiolus foliage and also on flower spikes under heavy disease pressure. The isolate present in Florida fits the literature description of U. transversalis in every respect (uredinia 0.5 to 1.5 mm in diameter, subglobose to ellipsoid verruculose yellow-amber urediniospores, 15 to 28 × 14 to 20 µm with wall 1.5 to 2.5 µm thick; telia also amphigenous, 0.5 to 1.3 µm in diameter, dark brown-black, subglobose to pyriform smooth amber teliospores, 20 to 30 × 15 to 20 µm with wall 1.5 to 2.0 µm thick, 4 to 6 µm thick at apex, pale brown to hyaline pedicel 30 to 40 µm long, yellow-brown paraphyses in pustule) ( http://nt.ars-grin.gov/fungaldatabases/new_allView.cfm?whichone=all&thisName=Uro myces%20transversalis&organismtype=Fungus ). Urediniospores initiated typical foliar lesions on transplanted gladiolus samples kept in the FDACS-DPI quarantine greenhouse during the diagnostic process. References: (1) J. R. Hernandez and J. F. Hennen. Sida 20:313, 2002. (2) G. P. B. Pitta et al. Biologica 47:323, 1981. (3) G. Rodriguez-Alvarado et al. Plant Dis. 90:687, 2006. (4) J. A. Stevenson. Page 82 in: Foreign Plant Diseases. USDA Fed. Hortic. Board Bureau Plant Ind. Government Printing Office, Washington DC, 1926.

First U.S. Report of Pseudocercospora paederiae Leaf Spot on the Invasive Exotic Paederia foetida.

Paederia foetida L., commonly referred to as skunk vine, is a native of eastern and southern Asia and was introduced into the United States prior to 1897. By 1916 it was already a troublesome weed in central Florida. It is a fast growing perennial twining vine (up to 7 m) with a woody rootstock adapted to a wide range of light, soil, water, and salt conditions (4). Naturalized in Florida, Georgia, Hawaii, Louisiana, North Carolina, South Carolina, and Texas, it occurs most often in disturbed areas. In Florida, where it is listed by the Florida Department of Agriculture and Consumer Services as a noxious weed, it invades various native plant communities including sandhills, flood plains, and upland mixed forests, where it creates dense canopies leading to injury or death of native vegetation and structural alteration of the native plant community (2,4). Current work underway to find biological control agents for invasive weeds led to the discovery in central Florida of a skunk vine plant with irregular to angular, sunken leaf spots ranging in color from shiny black to dark brown, some with tan centers and dark brown borders. Leaf spots had coalesced in some areas, blighting portions of leaves. Pseudocercospora paederiae (Sawada ex) Goh & Hsieh (1,3) was recovered from these leaf spots. Fruiting was amphigenous (chiefly epiphyllous) with globular or subglobular stromata, formed singly or coalesced, 37.2 µm wide (range = 19.9 to 62.3 µm). Conidia were hyaline to faintly olivaceous, with up to 6 septa, straight to mildly curved, measuring 49.6 µm (range = 18.8 to 72.3 µm) × 4 µm (range = 3 to 5 µm). To confirm Koch's postulates, a healthy, vigorous P. foetida plant in a 12 liter pot was spray-inoculated with 47 ml of a conidial suspension (13,000/ml) of P. paederiae. The plant was covered with a clear plastic bag to create a moist atmosphere and kept at room temperature (25°C) for 3 days after which it was uncovered and moved into a greenhouse. The greenhouse temperature fluctuated between 15°C (nighttime) and 29°C (daytime). Symptoms started appearing after 2 weeks, becoming more prominent by the third and fourth week. The inoculated plant showed irregular to angular dark brown to black leaf spots with dark brown borders. Necrosis along veins was observed and severely infected leaves abscised. The fungus was consistently recovered from inoculated symptomatic leaf tissue. Continued incubation of the plant under greenhouse and outdoor raised bench conditions eventually resulted in the secondary infection and leaf spotting of new foliage. P. paederiae was recovered from these secondary lesions. P. paederiae has been previously reported from Taiwan, China, and Japan. This represents the first report of the pathogen in the Western Hemisphere. Pathogenicity tests suggest possible application as a mycoherbicide. References: (1) C. Chupp. 1953. A Monograph of the Fungus Genus Cercospora. Cornell University Press. Ithaca, New York. (2) G. Gann and D. Gordon. Natural Areas J. 18:169, 1998. (3) W. H. Hsieh and T. K. Goh. 1990. Cercospora and Similar Fungi from Taiwan. Maw Chang Book, Taiwan, Republic of China. (4) K. A. Langeland and K. C. Burks, eds. 1998. Identification & Biology of Non-Native Plants in Florida's Natural Areas. University of Florida Press, Gainesville, FL.

Outbreaks of Stem and Leaf Blight of Eustoma grandiflorum Caused by a Phomopsis sp. in Florida.

Between November 1997 and May 1998, numerous lisianthus (Eustoma grandiflorum) cultivars exhibited severe stem and leaf blight at two pot-flower production sites in Hillsborough and Dade counties, FL. Blight occurred in mature plants and ranged in incidence from 3 to 5% in Dade County and from 40 to 80% in Hillsborough County. Initial stem necrosis was rapidly followed by leaf blight and production of numerous dark pycnidia in diseased tissue. As stem blight progressed, infected plants collapsed and died. Pycnidia contained biguttulate, 7.2 × 2.2-µm spores typical of Phomopsis alpha conidia; beta conidia were not observed. A Phomopsis sp. was isolated consistently when pycnidia from symptomatic stems were placed on acidified 25% potato dextrose agar after surface-disinfestation in 0.5% NaOCl; only alpha conidia were observed in culture. Pathogenicity was confirmed using a suspension of hyphae, pycnidia, and conidia made by comminuting one 95-mm-diameter acidified carnation leaf agar plate containing a 4-week-old colony of the Phomopsis sp. in 100 ml of deionized water. Six plants of lisianthus 'Maurine Blue' (three wounded at the crown, three nonwounded) each were inoculated with 10 ml of the fungal suspension. An equal number of noninoculated lisianthus (three wounded, three nonwounded) served as controls. After inoculation, plants were maintained in a greenhouse with average high and low temperatures of 38 and 25°C, respectively. Stem and leaf blight symptoms were observed in two of three wounded plants and in all nonwounded plants within 11 and 15 days after inoculation, respectively. Infection by a Phomopsis sp. was confirmed by reisolation from symptomatic tissue. Although this Phomopsis sp. has been detected previously in lisianthus exhibiting leaf and stem lesions (1), this report establishes the ability of the of the fungus to act as a primary pathogen and to cause extensive losses in this crop. Reference: (1) Alfieri et al. 1994. Diseases and Disorders of Plants in Florida. Bull. No. 14. Division of Plant Industry, Gainesville, FL.

Expanded Range of Cylindrocladium colhounii in the United States.

Plant pathologists in Florida and Oregon have recently found Cylindrocladium colhounii for the first time on two new ornamental plant species. Brown, pinpoint leaf spots were observed on Callistemon rigidus (stiff bottlebrush) in a Florida nursery. C. colhounii was isolated consistently from these lesions. To confirm Koch's postulates, 25 ml of aconidial suspension at 96,000 conidia per ml was used to spray a 38.1-cm branch of C. rigidus. Plants were maintained in a moist chamber at room temperature (25 ± 2°C). Symptoms appeared within 3 days, and included brown, pinpoint spots (1 mm or less) occurring on both leaf surfaces, sunken blotches, and blight. The fungus was consistently reisolated from symptomatic tissue. In Oregon, the first detection of C. colhounii was from leaf spots on Gaultheria procumbens (wintergreen) in a nursery. No proof of pathogenicity was done in Oregon on G. procumbens. C. colhounii has now been reported on 14 host genera in 10 families from Australia, India, Mauritius, South Africa, and the U.S. (FL, HI, LA, NC, OR, SC) (1-4). References: (1) P. W. Crous and M. J. Wingfield. Mycopathologia 122:45, 1993. (2) A. Peerally. Mycotaxon 40:323, 1991. (3) A. Y. Rossman. Mycol. Pap. No. 150, Commonw. Mycol. Inst., Kew, Surrey, England, 1983. (4) J. Y. Uchida and M. Aragaki. Plant Dis. 81:298, 1997.

[Consequences of a surgical correction of an insufficient closure of the vulva on genital flora and conception rate in mares]. / Auswirkungen der chirurgischen Korrektur eines mangelhaften Schamschlusses auf die Genitalflora und die Konzeptionsrate bei Stuten.

In this thesis the influence of pneumo-vagina on the microbiological colonization of the genital tract and their manifestation in cytological smears was examined. For mares with poor vulval conformation a comparison of the bacterial growth before and after plastic surgery of the vulva and vestibulum was carried out, as well as the registration of conception rates after operation and insemination. The biggest reduction of the bacterial content in the reproductive tract was found between vestibulum and cranial section of the vagina. The increased number of contaminant bacteria in the cranial section of the vagina during abnormal vulval conformation could be reduced significantly by the plastic surgery of the vulva and vestibulum (p < 0.05). Assuming that an 18-20 days pregnant uterus is free from a colonization with virulent bacteria, a high significant reduction of the increased number of contaminant bacteria in the uterus during vulval incompetence was found after surgical correction (p < 0.01). In mares with poor conformation of the vulva, which had not been operated, the contamination of the reproductive tract had increased at the time of checkup in comparison to the first swab specimens, taken 18-20 days before. The share of mares with a negative bacteriological and positive cytological result was in case of vulval incompetence twice as large as if they had a proper vulval conformation. A decrease of these latent infections was found after plastic surgery of the vulva and vestibulum. From the operated mares a total of 72.9% became pregnant.