Tim Schubert

Detection and Characterization of a New Strain of Citrus Canker Bacteria from Key/Mexican Lime and Alemow in South Florida

In the Wellington and Lake Worth areas of Palm Beach County, FL, citrus canker appeared on Key/Mexican lime (Citrus aurantiifolia) and alemow (C. macrophylla) trees over a period of about 6 to 7 years before detection, but nearby canker-susceptible citrus, such as grapefruit (C. × paradisi) and sweet orange (C. sinensis), were unaffected. Colonies of the causal bacterium, isolated from leaf, stem, and fruit lesions, appeared similar to the Asiatic group of strains of Xanthomonas axonopodis pv. citri (Xac-A) on the nutrient agar plate, but the growth on lima bean agar slants was less mucoid. The bacterium produced erumpent, pustule-like lesions of typical Asiatic citrus canker syndrome after inoculation into Key/Mexican lime, but brownish, flat, and necrotic lesions on the leaves of Duncan grapefruit, Madame Vinous sweet orange, sour orange (C. aurantium), citron (C. medica), Orlando tangelo (C. reticulata × C. × paradisi), and trifoliate orange (Poncirus trifoliata). The bacterium did not react with the Xac-A specific monoclonal antibody A1 using enzyme-linked immunosorbent assay (ELISA) and could not be detected by polymerase chain reaction (PCR)-based assays using primers selected for Xac-A. DNA reassociation analysis confirmed that the pathogen, designated as Xac-AW, was more closely related to Xac-A and Xac-A* strains than X. axonopodis pv. aurantifolii or the citrus bacterial spot pathogen (X. axonopodis pv. citrumelo). The strain can be easily differentiated from Xac-A and Xac-A* using ELISA, PCR-based tests, fatty acid analysis, pulsed-field gel electrophoresis of genomic DNA, and host specificity.

Invasive Pathogens in Plant Biosecurity. Case Study: Citrus Biosecurity

Most of the world’s major citrus production areas were developed outside the citrus centres of origin, separated from many co-evolved natural enemies (pests and pathogens), but progressive globalisation has reunited some pests with their citrus hosts. Additionally, some ‘new-encounter’ pathogens have not co-evolved with citrus. The movement of major citrus pathogens of biosecurity concern is discussed with particular emphasis on tristeza, leprosis, huanglongbing and citrus variegated chlorosis. The chapter details recent attempts to eradicate citrus canker in Florida (USA) and Emerald (Australia) and focusses on the processes and impediments encountered to achieve eradication under very different climatic, legislative and industry conditions. The impact of citrus black spot in areas climatically conducive to the disease and a discussion of fruit as a pathway for introduction of the disease to new areas are discussed. The experience and learning acquired from managing and eradicating these citrus pests will be of value to other countries and regions that are faced with similar pest incursions.

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 Report of Pseudomonas cichorii Causing Leaf Spot of Stevia Detected in Florida

During a routine nursery inspection in Hernando County, Florida in July 2011, leaf spot symptoms were observed on the popular sugar substitute stevia (Stevia rebaudiana). Spots were roughly circular to irregular in shape, variable in size, and dark brown with a yellow halo. White to cream-colored, circular, convex, gram-negative bacterial colonies were isolated on nutrient agar and identified as Pseudomonas cichorii based on the LOPAT scheme (2). DNA from four individual colonies inoculated in nutrient broth was extracted using DNeasy columns (Qiagen Inc., Valencia, CA). The PCR-amplified product from four different genes, 16S rRNA (1.5 Kbp), gyrB (0.9 Kbp), rpoB (1.2 Kbp), and rpoD (0.7 Kbp), was sequenced (GenBank Accession Nos. JQ994483, JQ994484, JQ994485, and JQ994486). Nucleotide and translated amino acid sequences for each gene were compared to the nucleotide and protein databases, respectively. The best matches were always with P. cichorii with nucleotide identities ranging from 98 to 99% and amino acid identities from 99 to 100%. Four healthy stevia plants were spray inoculated with 20 ml of a 108 CFU ml-1 suspension prepared from a 24-hour-old culture, of which two were sprayed with carborundum (silicon carbide) immediately prior to inoculation. Two additional plants were sprayed with carborundum only or sterile tap water only and served as healthy controls. Symptoms began to develop 4 days after inoculation. Spots originated at the tips or edges of the leaves and enlarged over time. Enlarging lesions progressed to encompass the entire leaf, accompanied by water soaking, curling, and necrosis. Blighting typically spread down to the stem and caused dieback. The pathogen was successfully reisolated from the lesions and produced identical LOPAT scheme results. Based on the information collected, it is believed that this is the first confirmed report of bacterial leaf spot caused P. cichorii on stevia worldwide. This find is significant due to the rising popularity of stevia cultivation for its sweetness and medicinal properties (1). To our knowledge, stevia is not currently being grown commercially in Florida; however, there is commercial acreage being developed elsewhere in the United States. References: (1) D. Patil et al. Asian J. Pharm. Clin. Res. 5:1, 2011. (2) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd Edition. The American Phytopathological Society. St. Paul, MN, 2001.