First Report of a Leaf Spot Disease of Golden Dewdrop (Duranta erecta) Caused by Pseudomonas cichorii and a Xanthomonas Species in Florida.

Duranta erecta (Verbenaceae) is used extensively in southern states as an ornamental shrub and has replaced boxwood as the most common short hedge accenting flower beds. Over the past 2 years, during warm wet periods, dark necrotic leaf spots have been observed on golden dewdrop plants in Florida. Isolations from these spots on Difco nutrient agar (NA) consistently yielded two types of bacterial colonies that were not always simultaneously present: 1) round butyrous, bright yellow and 2) flat cream-colored. Both were 2 mm in size after 48 h, gram-negative, and produced a hypersensitivity reaction (HR) on tobacco cv Hicks. Yellow colony bacteria were oxidase negative and non-fluorescent on King's medium B (KMB) (1). Cream-colored colony bacteria were oxidase positive and fluorescent on KMB. Three isolates of both types were selected for further study. Partial 16S rDNA sequencing and fatty acid analysis (FAME) MIDI Microbial Identification System (Microbial ID, Inc., Newark, DE) were used for identification of strains. The 16S rDNA primers used were; forward primer AMB14 5'-TCCAGCAATGCCGCGTGTGT-3' and reverse primer AMB13 5'-CATCCACCGCTTGTGCGGGT-3'. The PCR program consisted of an initial denaturing cycle of 95°C for 2 min followed by 30 cycles of denaturing at 95°C for 30 s, annealing at 60°C for 40 s and extension at 72°C for 1 min and one final extension at 72°C for 10 min. Using FAME analysis, the three strains of the cream-colored colony type were identified as Pseudomonas cichorii with high similarity values (0.907, 0.961, 0.819) and this corresponded well with the 16S rDNA sequences where 99% sequence identity was observed with P. cichorii strain JBC1 16S ribosomal RNA gene, partial sequence GenBank Accession No. JF951725. Two of the three yellow colony strains were identified by MIDI FAME profiles as Xanthomonas axonopodis pv. manihotis with similarity coefficients of 0.767 and 0.826. The third strain had a low similarity match to X. a. pv. carotae (0.541). The 16S rDNA sequencing of these strains showed 98% sequence identity to X. citri subsp. citri strain SA1 16S ribosomal RNA gene only, partial sequence identity JQ890091.1, thus indicating a possible undescribed X. axonopodis pathovar. To satisfy Koch's postulates, three golden dewdrop 'Golden Mound' plants were sprayed with a suspension of 108 CFU/ml of a 2-day NA culture of each strain, bagged for 24 h to raise humidity, and placed in a greenhouse. A strain of P. cichorii (P409) isolated from chrysanthemum was used as a positive control when comparing cream-colored strains. A saline buffered control was used as a negative control. Within 3 weeks, leaf spots developed on plants sprayed with each of the six strains, including positive control strain of P. cichorii. Reisolations yielded the same type of colony as the originally inoculated strain. Inoculation experiments were repeated three times with a minimum of three plants per isolate with similar results. To our knowledge, this is the first report in the United States of bacterial leaf spot caused by P. cichorii and X. axonopodis on golden dewdrop. An earlier morphological and physiological description of a Xanthomonas sp. was done on Duranta in India in 1962 (2). Due to the difficulty in controlling bacterial diseases and the popularity of Duranta spp. in the landscape, these diseases may present a problem in ornamental trade. References: (1) E. O. King et al. J. Lab. Clin. Med. 44:301, 1954. (2) M. C. Srinivasan et al. Proc. Indian Acad. Sci. 56:88, 1962.

Control of Bacterial Wilt of Geranium with Phosphorous Acid.

Various bactericides were screened for efficacy in protecting geranium plants (Pelargonium hortorum) from Ralstonia solanacearum infection. Many of these bactericides were found to slow the disease progress; however, they were not able to protect the plants from infection and subsequent death. Potassium salts of phosphorous acid were found to be effective in protecting plants from infection when applied as a drench. The active portion of the potassium salts was found to be phosphorous acid (H3PO3). Phosphorous acid was found to inhibit in vitro growth of R. solanacearum. It is thought to be protecting plants from infection by acting as a bacteriostatic compound in the soil. The plants, however, are not protected from aboveground infection on wounded surfaces. Phosphorous acid drenches were shown to protect geranium plants from infection by either race 1 or 3 of R. solanacearum. Other phosphorous-containing products commonly used in the industry, such as phosphorus pentoxide (P2O5) and phosphoric acid (H3PO4), were not able to protect plants from bacterial wilt infection.

Characterization of Erwinia Populations from Nursery Retention Ponds and Lakes Infecting Ornamental Plants in Florida.

Water shortages in Florida are occurring due to intense utilization of the aquifer system by municipalities and agriculture, and because of continued deficits in annual rainfall. Water districts therefore, are, recommending the use of recycled irrigation water, stormwater runoff, reclaimed municipal sewage water, and lakes for agricultural use. With recycled water, however, there is potential for both introducing and concentrating plant pathogens. In Florida, Erwinia soft-rot bacteria (synonym Pectobacterium) cause extensive crop losses in ornamental plant production. To determine Erwinia spp. population levels, samples were taken monthly for 1 year from four hypereutropic lakes and eight nursery retention ponds. Seventy-seven Erwinia strains were collected by both direct plating and by an enrichment process. With the direct plating method, 0 to 29 CFU/ml were detected on sodium polypectate medium. Significantly higher populations of Erwinia were detected in retention ponds of nurseries that were actively reutilizing their water. Erwinia strains were identified to species by fatty acid analysis and biochemical tests. Strains were further characterized by repetitive element-polymerase chain reaction (rep-PCR) and compared with 120 strains of Erwinia collected from ornamentals over a 17-year period in Florida. Using rep-PCR, most strains were clustered into two heterogeneous populations of E. chrysanthemi and E. carotovora subsp. carotovora in a 1:2 and a 1:4 ratio for isolates from ornamentals and from water, respectively. Within each population of E. chrysanthemi and E. carotovora, genetically different subpopulations could be identified that contained high percentages of Erwinia strains from water sources. Even though genetic differences exist, 99% of the strains from water sources were found to be pathogenic on dieffenbachia. Without water treatment of irrigation and stormwater runoff, there is a potential for both introducing and concentrating Erwinia populations within these water sources.

First Report of Xanthomonas axonopodis Infecting Agapanthus in Florida.

Agapanthus praecox subsp. orientalis, commonly called African lily or lily-of-the-Nile, bears large, round, blue or white flowers above attractive dark green foliage. Because of these horticultural features, this member of the family Liliaceae, has become a popular perennial bedding plant. For the past 2 years during warm wet periods, symptoms of chlorotic, water-soaked, leaf-streaks have been observed on agapanthus production in Florida. Round butyrus, bright yellow colonies were consistently isolated on nutrient agar. Bacteria were characterized as gram negative, catalase positive, motile, strictly aerobic, and not hydrolytic on starch. Using fatty acid analysis (FAME) and the MIDI Microbial Identification System with software version TSBA 3.90 (Microbial ID, Inc., Newark DE), three strains were further characterized and identified as Xanthomonas axonopodis with similarity coefficients to X. axonopodis pv. dieffenbachiae (0.907, 0.915, and 0.944) and to X. axonopodis pv. poinsetticola (0.912, 0.922, and 0.916). The three isolates were each inoculated on three plants each of agapanthus cv. Blue African lily, Dieffenbachiae maculata cv. Camille, and poinsettia, Euphorbia pulcherrima cv. PeterStar Red. Plants were sprayed with a suspension of each isolate at 1 × 108 CFU/ml, bagged for 24 h to raise humidity, and placed in a glasshouse for symptom development. Strains of X. axonopodis pv. poinsetticola (NZTCC 5779) and X. axonopodis pv. dieffenbachiae (X1718) were used as positive controls. Within 3 weeks, isolates from agapanthus produced leaf streaks on agapanthus plants, small, scattered, water-soaked lesions on dieffenbachia leaves, and no symptoms on poinsettia. No symptoms developed on the agapanthus plants when inoculated with either control strain. Both control strains formed lesions on leaves of their original host species. Xanthomonas was reisolated from treatments with symptomatic leaves. Plant inoculations were repeated with similar results. Although the agapanthus isolates were highly similar in FAME profiles to X. axonopodis pv. dieffenbachiae, symptoms produced on dieffenbachia were mild as compared with those produced by the dieffenbachia isolate. Therefore, these isolates may represent a distinct pathovar.

First Report of Ralstonia (Pseudomonas) solanacearum Infecting Pot Anthurium Production in Florida.

Xanthomonas campestris pv. dieffenbachiae is a common pathogen of pot anthurium production in Florida. While X. campestris pv. dieffenbachiae was isolated from systematically infected plants with chlorotic, necrotic, and wilted leaves, a fluidal, beige bacteria was occasionally isolated on nutrient agar (Difco, Detroit, MI), as opposed to the common, yellow pigmented Xanthomonas sp. Distinction in the symptomology of plants systematically infected with a Xanthomonas sp. or this new bacterium could not be made. Three isolates were obtained of this unidentified bacterium from leaves and stems of three separate plants. With FAME (fatty acid methyl esters) analysis, using MIDI (Microbial Identification System, software version TSBA 3.90 [Newark, DE]), these isolates were classified as Ralstonia (Pseudomonas) solanacearum (syn. Burkholderia solanacearum) with a mean similarity indice of 0.895. Isolates were found to be gram negative, oxidase negative, catalase positive, motile, strictly aerobic, and metabolically classified as biovar 1; they accumulated poly-ß-hydroxybutyrate and produced a hypersensitive response on tobacco within 24 h. A characteristic fluidal, white growth with a distinctive, red, swirling, egg-shaped, pigmentation pattern was observed on triphenyltetrazolium chloride medium. Further confirmation of identity as R. solanacearum was obtained by polymerase chain reaction amplification and electrophoretic analysis with species-specific primers (2), which in all cases produced a 148-bp product along with control strains. The three isolates were inoculated onto three plants of anthurium, tomato, triploid banana, and pothos. Inoculations were done at least twice; plants were inoculated either by stabbing the plant stems with a needle dipped in a suspension of bacteria or by applying 10 ml of a 1 × 108 CFU/ml suspension to the soil of the test plants. Chlorosis, necrosis, and wilt symptoms appeared within 2 weeks on all plant species tested. Recently, pothos (Epipremnum aureum) cuttings imported to Florida from Costa Rica have been implicated as a source of R. solanacearum (1). Imported cuttings of pothos were being grown in hanging baskets over the infected anthuriums. Although no R. solanacearum infections were detected in the pothos, these imported plants are the probable source of the initial inoculum for this disease outbreak on anthuriums. References: (1) D. J. Norman and J. M. F. Yuen. Phytopathology 87:S70, 1997. (2) S. E. Seal et al. Appl. Environ. Microbiol. 58:3751, 1992.

New Disease on Ornamental Asparagus Caused by Xanthomonas campestris in Florida.

From dark, water-soaked lesions on stems of asparagus tree fern (Asparagus virgatus) in commercial nurseries in Florida, 33 xanthomonad strains were isolated. Strains formed large, round, butyrus, bright yellow colonies on yeast dextrose calcium carbonate medium, and were gram negative, oxidase negative, catalase positive, motile, strictly aerobic, and did not hydrolyze starch. Strains were further characterized by carbon substrate utilization patterns (Biolog), and by fatty acid methyl esters (FAME) analyses. The metabolic fingerprints of most strains were similar to Xanthomonas campestris pv. vitians, and X. campestris pv. dieffenbachiae from Xanthosoma or Syngonium. Representative strains from A. virgatus were not pathogenic on Dieffenbachia. X. campestris pv. dieffenbachiae strains that did not hydrolyze starch produced scattered lesions on A. virgatus stems. However, starch-hydrolyzing strains of X. campestris pv. dieffenbachiae did not produce symptoms when inoculated onto A. virgatus. FAME analysis indicated the strains were X. campestris pv. vitians or X. campestris pv. translucens; however, low similarity indices ( x = 0.461) indicated that the asparagus strains were not represented in the MIDI library database. FAME analysis profiles were also compared to the University of Florida database, which contains 1,048 X. campestris strains of which 200 are X. campestris pv. dieffenbachiae. Similarity indices were again low with 15 strains matched to X. campestris pv. secalis (x = 0.412), seven strains to X. fragariae (x = 0.224), six strains to X. campestris pv. translucens ( x = 0.437), and five strains matched < 0.20 to other pathovars. Five representative strains were tested on six Asparagus species or cultivars: A. virgatus, A. setaceus, A. macowanii, A. densiflorus 'Sprengeri' , A. densiflorus 'Myers', and A. officinalis. All five strains were pathogenic on A. virgatus but were less virulent on A. setaceus and A. densiflorus 'Sprengeri'.